AgriculturA l And food science - MTT · 2013-06-18 · AgriculturA l And food science special issue of the XVI International silage conference 2-4 July 2012, Hämeenlinna, finland

AgriculturAl And food science

special issue of the XVI International

silage conference 2-4 July 2012, Hämeenlinna, finland

The Scientific Agricultural Society of Finland w w w . afsci.fi

Vol. 22, no. 1, 2013

AgriculturAl And food scienceAgricultural and food science publishes original reports on agriculture and food research related to primary production. The papers, which are of international interest but feature a northern perspective, cover a wide range of topics in basic and applied research. Submissions are internationally refereed. Review articles and research notes will also be con-sidered. Readers are welcome to send their comments to the journal (Letter to the Editor).

PublisherThe Scientific Agricultural Society of Finland

editorial boardJuha Helenius - editor in chief

Tapani AlatossavaXavier Irz

Kaisa KuoppalaMervi Seppänen

eila turtolaPekka uimari

Erja Rappe - Managing Editor

AddressAgricultural and food science

Editorial Officedepartment of Agricultural sciences

P.o. Box 27FI-00014 University of Helsinki, Finland

http://www.afsci.fi

From volume 19 Agricultural and Food Science is available as an Open Access journal, free of charge to the user. The issues and articles published since volume 11 have also been trans-ferred to the Open Access mode. Publishing continues in electronic format only, available on the journal's web-pages (www.afsci.fi), or via database links or where the journal is indexed. The printing of special issues will still be considered if the applicant offer to provide the resources.

Single printed issues up to volume 18 may be obtained from Bookstore Tiedekirja, Kirkkokatu 14, FI-00170 Helsinki, Finland, e-mail: [email protected].

The printing of this issue was funded by University of Helsinki/Department of Agricultural Sciences and MTT Agrifood Research Finland/Animal Production Research.

Front cover images courtesy of Eeva Saarisalo and Erja Rappe.

Printed by Unigrafia Oy, Helsinki 2013

AGRICULTURAL AND FOOD SCIENCEA. Vanhatalo (2013) 22: 1–2

Preface Aila VanhataloGuest Editor

Department of Agricultural Sciences, University of Helsinki

e-mail: [email protected]

The XVI International Silage Conference (ISC) was held in Hämeenlinna, Finland, 2-4 July 2012. Ever since the first ISC took place in Edinburgh, Scotland, UK, in 1970, this conference has been held in every two to four years. For the first twenty years the series of conferences took place in the UK, but since the conference held in Dublin, Ireland, in 1993, it has also been organized by other countries such as Sweden (Uppsala 1999) and USA (Madi-son 2009). A total of 320 delegates from around 40 countries attended the XVI ISC in Finland demonstrating that silage indeed is an important issue worldwide.

The programme of the silage conferences has traditionally covered all core topics of developments in silage science and technology. It has also been customary to document invited and volunteered presentations as abstracts or more extended papers in the Proceedings volume. This was also the practise in the XVI ISC held in Finland last year. However, with a high hope that the best offerings of the conference would be thoroughly documented for future use the authors of the XVI ISC were invited to contribute to a peer-reviewed Special issue of Agricultural and Food Science (AFS) to be published in due course after the conference. Now it is a pleasure to announce that our call was very positively responded by the contributors of the ISC and resulted in this AFS Special issue totalling seventeen papers, which comprise six review articles, seven full papers and four research notes.

The Special issue begins with a review of Richard Muck focusing on recent advances in silage microbiology. He points out how the new techniques available in silage microbiology helps us to gain better understanding of si-lage microbiology and allow us to better manage ensiling process and develop better additives. Silage is, how-ever, a potential source of microbiological and chemical contaminants in the dairy chain. Therefore, the paper on silage microbiology is followed by a comprehensive review of Frank Driehuis summarizing current knowledge about silage and the safety and quality of dairy foods. The full papers and research notes concerning this topic deal with research issues such as mode of action of inoculants or efficacy of inoculants on herbage of various plant species ensiled in various conditions. Aerobic stability is one of the key words common to almost all of these pa-pers emphasising the importance of efforts to improve aerobic stability of silages made of various forage crops. Some of these papers seek for methods for quantifying and controlling dry matter and nutrient losses from silos during and after the ensiling period.

Silage has been an invaluable innovation especially in the dairy cow feeding in Northern areas such as Finland, where grazing period of cows is very short and winter period indoors long. Pekka Huhtanen et al. reviewed silage research made in Finland starting from the Nobel Prize winner A.I. Virtanen, who showed the importance of low pH and inhibition of plant and microbial enzymes in silage preservation already 80 years ago. The thorough paper of Huhtanen et al. reviews more recent achievements of the Finnish silage research with the special emphasis on ensiling, feed evaluation, feed intake and milk production. Richard Dewhurst in his review extends the topic of milk production from grass silage to other ensiled forages especially to legume and maize silages and their mix-tures in dairy cow feeding. He predicts that mixtures of maize and legume silages relative to grass silage in dairy cow rations may have potential to reduce both N and methane emissions without loss of milk production. Tim Keady et al. focuses on the factors affecting the utilization of ensiled forages by beef cattle, dairy cows, pregnant ewes and finishing lambs in their review paper, which summarizes data on research conducted in this field in Ire-land and UK during recent 30 years.

AGRICULTURAL AND FOOD SCIENCEA. Vanhatalo (2013) 22: 1–2

The research articles focusing on animal production cover various aspects of feeding silage to different species of animals. Pesonen et al. investigated the effects of concentrate supplementation on the product quality of beef bulls fed grass silage-barley based diets. Gerlach et al. conducted an interesting study to monitor how changes in maize silage fermentation products during aerobic deterioration affects preferences of goats in terms of silage dry matter intake. Sarria and Martens showed that silages made from some legume forages available in tropics have potential to serve as feed supplement in pig diets. This is an interesting finding demonstrating that ensiling technology may be applied to local feed resources and utilized also in feeding of monogastrics.

The review by Tom Misselbrook et al. compiles topical research on opportunities for reducing environmental emissions from forage-based dairy farms. While there is a need to increase animal production to meet increas-ing global demand, it is fundamental to minimize inevitable environmental emissions. These authors review the sources and impacts of emissions to atmosphere and water in the context of a forage-based dairy farms considering potential mitigation strategies and giving also examples using a farm-scale modelling approach. They show that much can be achieved with systematic improvements in the efficiency of production in dairy systems includ-ing for instance means such as improvements in dairy herd fertility, dietary modifications, use of nitrification in-hibitors with fertiliser and slurry applications as well as through attention to the quantity, timing and method of application of nutrients to forage crops.

This Special issue includes seventeen scientific papers, of which a few were briefly introduced here to highlight its leading themes. As a chair of the scientific committee of the XVI ISC I wish to thank all the authors and contribu-tors as well as the Editorial board of the Journal for making this Special issue possible. Especially, the invaluable work of Dr. Kaisa Kuoppala from MTT Agrifood Research Finland and Dr. Seija Jaakkola from University of Helsinki in various stages of this Special issue is greatly appreciated. It is probably not an overstatement to say that silage is needed more than ever today. Silage made of a wide range of plant species and various plant by-products is in-creasingly used for ruminant production animals worldwide, but it is important to note that silage may also have increasing potential in feeding of monogastrics in future. I hope that this Special issue of AFS will be a tool and an inspiration for future progress in the field of silage science and technology.

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AGRICULTURAL AND FOOD SCIENCER. Muck (2013) 22: 3–15

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Recent advances in silage microbiologyRichard E. Muck

USDA, Agricultural Research Service, US Dairy Forage Research Center, Madison, Wisconsin, United States


Recent advances in silage microbiology are reviewed. Most new techniques in silage microbiology use the poly-merase chain reaction (PCR) to make copies of a portion of the DNA in microorganisms. These techniques allow us to identify and quantify species as well as do community analysis. The PCR-based techniques are uncovering new species, both bacteria and fungi, during storage and feeding. Silage inoculants are widely available, but of greater interest has been research investigating why inoculants are so successful. Various inoculant strains have been found to produce bacteriocins and other compounds that inhibit other bacteria and fungi, improving their chances for success. In vitro ruminal fermentation research is showing that some inoculated silages affect rumen microorgan-isms, reducing methane in some cases and increasing microbial biomass production in others. Better understanding of silage microbiology will allow us to better manage silos and develop better inoculants to improve silage quality.

Key words: lactic acid bacteria, PCR, aerobic stability, inoculants

Introduction

Recently, dramatic changes have occurred in how we study the microbiology of ensiling. Until about 10 years ago, our knowledge of what occurred in the silo was limited by what would grow on various selective media, and iden-tification of species from those agar plates was tedious work. API 50 strips allowed us to grow strains on 50 dif-ferent substrates simultaneously. That did speed the identification of lactic acid bacteria, but the strips were not infallible. We struggled to know cause and effect in the silo. Were the acids and other products that we measured during fermentation due to this strain or that strain?

Today, we still have issues with understanding cause and effect in the silo. However, we have much better tools to know which microorganisms have been involved in the ensiling process. We also have a better understand-ing regarding how microorganisms affect silage quality over beyond production of lactic acid, volatile fatty acids, alcohols and carbon dioxide. In this paper, I will discuss recent developments in the measurements of microbial dynamics during ensiling, our current knowledge of the species that contribute to ensiling as well as the species that spoil silage, the extent to which microbial additives modify fermentation and the utilization of silages by live-stock, and efforts to find new microbial additives.

Recent microbial techniques

The ability to extract microbial DNA from silages, amplify portions of DNA, and then separate those portions by the strains of microorganisms that have produced them has been at the core of the changes that have occurred recently in silage microbiology. These developments have allowed us to enumerate strains that do not grow on agar and reveal new species in silages. These techniques can be divided into two groups: 1) identification and quantification of specific species/strains and 2) community analysis.

Identification and quantification of specific species/strainsMost of these techniques use the polymerase chain reaction (PCR) to make many copies of a portion of the DNA in microorganisms. The most commonly amplified portion of the DNA from bacteria is the 16S ribosomal RNA gene, which is the primary basis today for classifying bacteria. The PCR primer binding sites for this gene are highly conserved across bacteria while other portions of the 16S sequence generally are more variable between species, which permits classification. Some techniques use other portions of the DNA as will be discussed.

Manuscript received July 2012


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For identification, DNA can be extracted, for example starting from a colony on an agar plate where there is one strain of microorganism present. The 16S rRNA gene can be amplified by PCR and then sequenced. Once the se-quence is known, a program such as BLAST may be used to compare the sequence of the unknown strain with those of known species. Consequently, one can enumerate lactic acid bacteria or other types by standard plating techniques and then use PCR to identify the species on those plates.

A related technique is real-time PCR (RT-PCR) or quantitative real-time PCR. This analytical technique allows one to quantitate specific species present in a sample. Primers are selected from the region of a gene that is specif-ic to the species of interest. A portion of the 16S rRNA gene has been most commonly used (e.g., Schmidt et al. 2008), but other genes such as the recA gene have been used with lactic acid bacteria (e.g., Stevenson et al. 2006) because it was easier to find sequences to separate species that have very similar 16S rRNA genes. This method is very useful for following species that you expect to be in the environment such as comparing the level of Lac-tobacillus plantarum in silages that are untreated vs. those inoculated with L. plantarum. Initial studies using RT-PCR in silages have compared their primers against other known species to be sure that all known strains of the species of interest react with the primers whereas known strains of other species do not. There still may be spe-cies not tested, particularly unknown species, that provide false positives or strains of the species of interest with slight differences in the sequence in the region of the primers causing a false negative. The latter is not a problem if you are following an inoculant strain that reacts with the primers.

Quantification in RT-PCR is based on how many cycles of amplification are needed to reach a target number of copies. The advantage of this technique is that you can enumerate a species that is present at a low level. For ex-ample, by standard techniques that begin by picking colonies from an agar plate, one is unlikely to detect a species that is present at less than 1% of the total population. With RT-PCR, we may be able to enumerate a species that is at 100 cfu g-1 silage even though the total LAB population is 109 cfu g-1. Thus RT-PCR allows us to follow known species more rapidly and at a much lower detection limit than previously possible.

Community analysisEven with PCR and RT-PCR, investigating the effects of various factors on the microbial community in the silo is laborious. Fortunately, a variety of techniques have been developed that allow us to get a snapshot of the bacte-rial community and then using statistical techniques like principal component analysis to determine if there are significant differences in communities. If there are, then we can use PCR and RT-PCR to document the differences. At least four techniques have been used to study microbial communities in silages: length heterogeneity PCR (LH-PCR), terminal restriction fragment length polymorphism (T-RFLP), denaturing gradient gel electrophoresis (DGGE) and automated ribosomal intergenic spacer analysis (ARISA). All four techniques use PCR to amplify a portion of the microbial DNA and then use various methods to separate the amplified DNA. It is beyond the scope of this pa-per to discuss these methods in detail. It is more important to know general differences between the techniques.

Length heterogeneity PCR uses the variation in the length of a gene between different microbial species to deter-mine how many species may be active in an environment. Separation of the amplified DNA is by capillary elec-trophoresis. Brusetti et al. (2006) investigated this technique using the differences in the length of a region of the 16S rRNA gene to follow the development of various LAB species during the ensiling of maize. The technique was successful in following most of the species identified in the silages. However, two species identified, Weissel-la confusa and W. kimchii, had identical fragment lengths of 379 base pairs and thus could not be differentiated by LH-PCR. Similarly the Enterobacter species identified had the same fragment lengths. A substantial number of peaks (29−58%) were not identified.

Terminal restriction fragment length polymorphism operates on a similar principal to LH-PCR. However, a restric-tion enzyme is added to the amplified DNA cutting it in two. The DNA is separated by either gel or capillary elec-trophoresis according to the length of the fragments. McEniry et al. (2008) investigated bacterial community dy-namics in wilted grass silage using T-RFLP. The 16S rRNA genes were amplified, then digested with the restriction endonuclease MspI, and separated by electrophoresis. The technique did show that there were shifts in the spe-cies with time (0, 2, 6, 14, 35 and 98 d) and ensiling method (baled vs. precision-chop silage). However, identifica-tion of species based on the length of a fragment using a database and simulated digest of the 16S rRNA gene by the restriction enzyme was often not possible because multiple species had the same fragment length.


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In denaturing gradient gel electrophoresis, the movement of each DNA segment is determined by the composi-tion of the nucleotides in the DNA rather than just the length of the DNA sequence. Normally this is used where the amplified DNA is relatively similar in length. For example, Li et al. (2011) amplified the V3 region of the bac-terial 16S rRNA gene and the fungal 18S rRNA gene and analysed these by DGGE. An advantage of this procedure was that they could excise prominent bands in the gel and then use PCR techniques/BLAST to identify the species that produced each band.

Automated ribosomal intergenic spacer analysis amplifies the region between the 16S rRNA gene and the 23S rRNA gene in bacteria. Separation is similar to T-RFLP, using fluorescent primers and by fragment length. Brusetti et al. (2008) used both ARISA and LH-PCR to assess bacterial communities in maize silage. ARISA gave 12 peaks on average compared with 9 peaks with LH-PCR, and the range of base pairs was much larger for ARISA. These suggest more sensitivity in the ARISA measurements because the ARISA is amplifying a much more variable re-gion. They used PCR to identify 388 isolates taken from MRS agar plates, falling into 11 known species. However, no attempt was made to match these isolates with the peaks from either ARISA or LH-PCR. So the authors were not able to determine which community method was more accurate in describing differences between silages.

All of the community techniques allow a relatively rapid comparison of communities between treatments. Of the three techniques that separate by length of DNA (LH-PCR, T-RFLP and ARISA), it would appear that ARISA is the least likely to have multiple species with fragments of the same length due to the heterogeneity of the intergenic spacer region in bacteria. However, it is possible for a strain to produce more than one peak in ARISA so that com-munity diversity may appear greater than it is. Because DGGE is performed by gel electrophoresis, the results are more qualitative and variable from one gel to the next compared with using capillary electrophoresis in the other three techniques, where their results are easily imported into statistical software for principal component analy-ses, etc. On the other hand, DGGE has the advantage mentioned earlier that bands can be excised and cloned by PCR for species identification.

New microbial species in silage

These new techniques have made it possible to more easily find new species and understand the dynamics of the microbial populations with time in the silo. However, these new techniques have often verified that the tra-ditional species associated with ensiling are the predominant species. Species that have been recently isolated in silages are summarised in Table 1. Specifics about these species are as follows. Brusetti et al. (2006) using LH-PCR reported the presence of Bacillus megaterium early in the ensiling of maize (day 0 and 1), Weissella kimchii (d 6) and Enterococcus flavescens (d 13). While these species had not been reported in silage, other more common spe-cies dominated the silages. Rossi and Dellaglio (2007) surveyed farm silages, primarily lucerne, maize and Italian ryegrass as well as mixtures of maize silage and maize grain. The LAB isolates matched known silage species with the exception of Lactobacillus zeae. Anaerobic spore formers found were largely known silage clostridial species with the exception of Clostridium baratii and Paenibacillus macerans. Three yeasts species were identified: Can-dida mesenterica, Candida apicola and Pichia fermentans. This was the first report of the two Candida species in silages even though other Candida species are commonly found in silage.

Several new species have been isolated from silage and had names proposed. These include Lactobacillus taiwan-ensis (Wang et al. 2009) and Pediococcus lolii (Doi et al. 2009).

Parvin et al. (2010) analysed laboratory silages made with Italian ryegrass, maize, guinea grass and rhodes grass ensiled with and without L. plantarum or Lactobacillus brevis inoculants. DGGE analysis followed by cloning and sequencing of prominent bands found mostly well known silage species. However, the untreated Italian ryegrass had 6 prominent bands, all but one (L. plantarum) was unusual: Pediococcus dextrinicus, Paralactobacillus selan-gorensis, Burkholderia spp., Serratia spp. and an uncultured bacterium.

Pang et al. (2011b) identified strains isolated from maize stover, and 86% of the LAB consisted of L. plantarum, Lactobacillus pentosus and L. brevis, three species commonly reported in silages. On the other hand they report-ed the presence of Leuconostoc lactis, Enterococcus mundtii and Weissella cibaria, the latter a recently described species. The same group isolated LAB strains from maize, rice, sorghum and lucerne silages (Pang et al. 2011a) and found that W. cibaria and Weissella confusa were the dominant species observed in maize silage. In the other silages, L. plantarum was the dominant species.


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Table 1. Microbial species recently isolated from silages

Species Silage Type ReferenceLactic Acid BacteriaEnterococcus flavescens Maize Brusetti et al. (2006)Entercoccus mundti Maize Stover Pang et al. (2011b)Lactobacillus acetotolerans Maize Li and Nishino (2011b)Lactobacillus panis Maize Li and Nishino (2011b)Lactobacillus reuteri Maize Li and Nishino (2011b)Lactobacillus taiwanensis Unknown Wang et al. (2009)Lactobacillus zeae Lucerne Rossi and Dellaglio (2007)Leuconostoc lactis Maize Stover Pang et al. (2011b)Paralactobacillus selangorensis Italian Ryegrass Parvin et al. (2010)Pediococus dextrinicus Italian Ryegrass Parvin et al. (2010)Pediococcus lolii Ryegrass Doi et al. (2009)Pediococcus parvulus Maize Li et al. (2011)Weissella cibaria Maize, Maize Stover Pang et al. (2011a,b)Weissella kimchii Maize Brusetti et al. (2006)Weissella paramesenteriodes Maize Li et al. (2011)Anaerobic Spore FormersClostridium baratii Maize Rossi and Dellaglio (2007)Paenibacillus macerans Maize Rossi and Dellaglio (2007)BacillusBacillus megaterium Maize Brusetti et al. (2006)EnterobacteriaErwinia persicina Italian Ryegrass Li and Nishino (2011a)Pantoea agglomerans Italian Ryegrass Li and Nishino (2011a)Rahnella aquatilis Italian Ryegrass Li and Nishino (2011a)Acetic Acid BacteriaAcetobacter pasteurianus Maize Li and Nishino (2011b)YeastsCandida apicola Maize, Italian Ryegrass Rossi and Dellaglio (2007)Candida intermedia Maize Li et al. (2011)Candida glabrata Maize Li et al. (2011)Candida magnolia Maize Li et al. (2011)Candida mesenterica Maize Rossi and Dellaglio (2007)Candida quercitrusa Maize Li et al. (2011)Saccharomyces martiniae Maize Li et al. (2011)Pichia deserticola Maize Li et al. (2011)Pichia fermentans Maize Rossi and Dellaglio (2007)Pichia kudriavzevii Maize Li et al. (2011)

Li et al. (2011) using DGGE identified the dominant bacterial and fungal species in maize silage. Pre-ensiling and later in the silage, L. brevis, Pediococcus parvulus, W. confusa and Klebsiella pneumoniae were reported. Addi-tionally in the silage, Weissella paramesenteriodes, L. plantarum and Lactobacillus lactis were observed. Yeasts in pre-ensiled maize and maize silage were predominantly Candida species and Cryptococcus flavus. However, the Candida species they reported (magnolia, intermedia, glabrata and quercitrusa) have not been found previ-ously in silages. When the maize silages were subjected to aerobic exposure, Saccharomyces and Pichia species appeared, genuses commonly found in silage, but some of the species were newly reported as being in silages (S. martiniae, P. deserticola, P. kudriavzevii).

Li and Nishino (2011b) sampled maize silage from bunker silos. They detected a number of uncommon silage LAB species, Lactobacillus acetotolerans, Lactobacillus panis and Lactobacillus reuteri, as well as other new species in silages: Acetobacter pasteurianus, Stenotrophomonas maltophilia, an Acinetobacter species and a Rahnella spe-cies. Li and Nishino (2011a) studied wilted Italian ryegrass silages in mini-silos, finding enterobacteria such as Er-winia persicina, Pantoea agglomerans and Rahnella aquatilis in untreated silages. Known LAB species accounted for the majority of the other species detected.


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Overall, these studies have indicated that the new PCR based techniques are uncovering some new species. In most cases, traditional silage LAB species have been the dominant bacterial species present. However, we still have a difficult time knowing how significant the new species are to silage preservation because these PCR meth-ods do not indicate the contribution of the various species to the overall fermentation. If we could isolate any of the newly detected species, grow it and inoculate silage with it, we could have an insight of its role/effect on si-lage fermentation.

Effects of various factors on silage microbial populations

The new techniques have also been useful to study how various factors affect fermentation. The most common factor is the use of a bacterial inoculant, which will be discussed later. However, there have been a few recent studies that have investigated the effects of other factors on the course of fermentation.

McEniry et al. (2010) compared the microbial community in perennial ryegrass in baled vs. precision chop ensiling at two DM concentrations (185 and 406 g DM kg-1) over 0 to 14 d using T-RFLP. Using a T-RFLP database, they as-signed fragment lengths they observed to general groups, e.g., LAB, enterobacteria, etc. The most abundant frag-ment was associated with enterobacteria and was most prevalent early in ensiling and in the drier silages whereas the most prevalent fragments associated with LAB behaved in an opposite direction with those two factors. Only three minor species were affected by the ensiling system. In a second experiment, unchopped, unwilted perenni-al ryegrass was ensiled with or without compaction and with and without air infiltration for 100 d. For the top 20 fragments, most were not affected by either compaction or air infiltration. Six fragments were affected by compac-tion, having a negative effect on LAB and enterobacteria and a positive effect on clostridia. Only 4 fragments were affected by air infiltration: two Clostridium negatively, and one Clostridium and one Bacillus species each positively.

Naoki and Yuji (2008) compared the microbial community in vacuum-packed bag Italian ryegrass silage with wrapped bale silage using DGGE. Specific bands were not excised and identified, but the band pattern in the out-er portions of one bale was similar to the pattern in the vacuum-bag silage. However, there were differences in microbial community between bales and locations within a bale, suggesting field variability was affecting, in part, the dominant species.

Brusetti et al. (2006) investigated the usefulness of LH-PCR by following the progression of fermentation in maize silage up to 30 d. Pediococcus pentosaceus and W. confusa were the most prevalent species present at ensiling. Both species were present throughout the 30 d. Lactococcus lactis subsp. lactis was also present at ensiling, reach-ing its highest level at 6 d. Lactobacillus species were reported at various times during the course of the 30 d: L. plantarum, minor levels throughout; L. brevis, d 6-30; L. paraplantarum, d 13, d 20.

Parvin and Nishino (2009) used DGGE to study microbial changes with storage time (15 to 180 d) from the ensil-ing of guinea grass at two DM concentrations (286, 443 g kg-1). In the wetter silage, Lactococcus lactis and L. bre-vis were the dominant species at 15 d but by 180 d, Lc. lactis was a faint band and L. pentosus was more preva-lent. This shift coincided with a reduction in lactate to acetate ratio in the silage with time. In the drier silage, two strains of L. plantarum were observed in addition to Lc. lactis and L. brevis. The Lc. lactis band did not diminish with time, but an L. pentosus band did appear at later time points. In the drier silage, lactate to acetate ratio did not change with time, but both acids increased with time.

Parvin and Nishino (2010) measured the changes in microbial community with storage time (15 to 180 d) in rho-des grass silage using DGGE, and prominent bands were sequenced. L. brevis, L. plantarum and L. pentosus were present at all time points although the L. plantarum strain in the early time points appeared to be a different strain than that at later times. Lactococcus lactis had a strong band at 15 d that became fainter with each succeeding time point. There was a faint band of Escherichia coli through 90 d.

Ávila et al. (2010) investigated microbial population changes in 5 cultivars of sugarcane silage at 10, 20, 30 and 40 d. Of particular interest were the yeast populations that varied by cultivar and time. For 3 cultivars, the high-est yeast counts occurred at 10 d with yeast counts being significantly lower at 40 d. For the other 2 cultivars, the highest yeast counts occurred at 30 d with counts dropping significantly at 40 d. Colonies were picked, and iden-tified by PCR techniques. Of the culturable yeasts, only 4 species were present in the 10 d silages (Torulaspora delbrueckii, Pichia anomala, Saccharomyces cerevisiae and Candida glabrata), which were the dominant species across all time points. Five other species were identified. The sugarcane cultivars varied in the yeast species ob-served: one cultivar with just two species (Torulaspora delbrueckii, Pichia anomala), three with five species of yeast, and one with seven species of yeast.


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Villa et al. (2010) compared the ensiling of two maize varieties, one grown in a warm climate and one grown in a cool climate in Colombia. The maize from the warm climate had a higher initial count of LAB, leading to a more rapid decline in pH. The fermentation of the cool climate variety was dominated by Lactobacillus and Pediococ-cus species with populations of both genuses peaking upon reaching pH 3.8 at 7 d. In the warm climate variety, both genuses peaked at 2 d when the pH was below 4.0, but Leuconostoc species also contributed, peaking at 5 d when pH had reached 3.7.

The dynamics of microbial groups in Danish stack silos of maize silages were observed every other month begin-ning in January and continuing until September (Storm et al. 2010). Yeasts and lactic acid bacteria declined with time over this period whereas moulds were highest in the March and May sample periods. The filamentous fungi were identified and the primary species found were Penicillium roqueforti, Zygomycetes (primarily Mucor spp.), Penicillium paneum and Aspergillus fumigatus. The percentage of stack silos with these species was highest either in March or May. By September, there was a greater diversity across the 20 silos.

Several recent studies have investigated the effects of low temperature on ensiling. They have not used PCR or other sophisticated techniques, but their results are of interest. Wang et al. (2011) ensiled reed grass at 0 and 4 °C and sampled at weekly intervals from 3 to 8 weeks. By week 5, pH had decreased significantly at both temper-atures. At week 5, the pH was 4.20 in the silage at 4 °C, significantly lower than the silage at 0 °C. Also acetic acid was lower and lactic acid higher at 4 °C. Pauly and Spörndly (2011) investigated maize silage made at 6, 12 and 18 °C in the first year and 2.6, 6, 12 and 20 °C in the second year. The pH was below 4.0 by 20 d in the two warmest temperatures. Fermentation at 6 °C appeared to have stabilized by 60 d at a pH of 4.1 in both years with a fer-mentation that was lower in lactic and acetic acids and higher in ethanol than the fermentations at warmer tem-peratures. When some of the silages stored at 6 °C were raised to 18 °C after 45 d and stored for an additional 61 d, more fermentation occurred reducing pH similar to that of the 18 °C silages but with higher concentrations of lactic acid, acetic acid and ethanol than the 18 °C treatments.

Drawing general conclusions across these studies is difficult. It would appear that type of silo (even field-scale vs. laboratory) or density has only small effects on the dominant species during ensiling. There are considerable dif-ferences in the dominant species from one trial to the next, but in most studies, common Lactobacillus, Pedio-coccus and Lactococcus species were the most prevalent. The biggest deviation appeared to be in maize silage in warm climates where Weissella and Leuconostoc species contributed to early stages of fermentation. Finally, silage fermentation can occur at near freezing temperatures, but active fermentation continues over weeks, not days.

Aerobic deterioration of silage

When oxygen is introduced to silage, aerobic microorganisms begin to grow, initially respiring soluble substrates and then more complex compounds. This reduces the digestibility and feeding value of silage. The general pat-tern of spoilage has been known for approximately two decades. Yeasts are generally the initiators of aerobic de-terioration, consuming sugars and fermentation acids and raising silage temperature and pH (Pahlow et al. 2003). With increased pH, bacilli and other aerobic bacteria grow, increasing temperature further. Finally, moulds com-plete the deterioration of the silage. In maize silage, acetic acid bacteria have been found to be initiators of aero-bic deterioration in some cases.

Much of the recent work on aerobic deterioration has been in the study of inoculants to inhibit the rate of spoil-age. However, there are a number of recent studies that contribute to our understanding of microbial dynamics in spoiling silages. Dolci et al. (2011) studied microbial dynamics during aerobic exposure of inoculated (L. buch-neri, L. plantarum, E. faecium) maize silages stored in polyethylene or oxygen barrier film bags for 110 d. The lat-ter had a permeability to oxygen that was 5% of that of the polyethylene film. The silages were sampled 6 times between opening and 14 d of aerobic exposure. DGGE was used to study both the bacterial and fungal communi-ties, and prominent bands were excised and cloned for identification of species. At opening, bands identified as L. buchneri were dominant in both treatments. With aerobic exposure, the L. buchneri band diminished within one week in the polyethylene treatment where the silage began to heat in 3 d. At day 5 and continuing through day 14, the dominant band in that treatment belonged to Acetobacter pasteurianus, and a fainter band identified as Bacillus subtilis was present. In the silage made with the oxygen barrier film, these species did not appear until day 9 and 14, respectively, where heating did not begin until 9 days. The two fungal species present at opening (Kazachstania exigua, Aureobasidium pullulans) were unusual species for silages. Both bands disappeared after 5 days in the polyethylene treatment. Later Pichia kudriavzevii and Aspergillus fumigatus appeared in the poly-


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ethylene treatment whereas A. fumigatus and an unknown species appeared in the oxygen barrier film treat-ment. The unknown species appeared tied to the beginning of heating, being the dominant band at 9 d. Overall, it is interesting that in the film with higher oxygen permeability it appeared that Acetobacter was the initiator of aerobic deterioration whereas an unknown fungal species initiated spoilage in the other treatment. Bacillus and mould species became more prevalent after heating and increases in pH had occurred, as expected.

Borreani and Tabacco (2010) cored the faces of 54 maize silage bunker silos measuring fermentation products, yeasts, moulds, clostridial spores and also taking temperatures at 200 mm behind the feed out face. At least 18 core samples were taken from each silo spanning the width and height of each bunker. They compared these temperatures with the temperature in the middle of the face at 400 mm depth, where temperature is relatively constant, similar to temperatures deeper in the bunker. The 200 mm temperature at a specific location minus the temperature at 400 mm in the middle of the bunker was positively correlated with pH, yeast and mould counts. This suggests that temperature measurements at the farm could be used to rapidly estimate fungal counts and assess the aerobic stability of silage. This survey was done in northern Italy. It would be interesting to know if dif-ferences in silage temperature will be a good predictor of fungal counts in more severe climates.

Tabacco et al. (2011) surveyed 42 farm silos with maize silage, half of them treated with an L. buchneri inoculant. Aerobic stability was negatively related to yeast count, and it appeared that improved aerobic stability in the in-oculated silages was due to the reduction in yeast count. Pearson correlation coefficients between yeast count and various chemical and management variables indicated the strongest relationship was a negative correlation with feed out rate (−0.579). Also highly correlated (p< 0.01) with yeast count were lactic acid (0.549), pH (−0.456), silo DM density (−0.451) and lactic-to-acetic acid ratio (0.437). Acetic acid was correlated at p< 0.05 (−0.331). These results confirm that in the real world susceptibility to aerobic losses is a function of both the fermentation in the silo and management factors (density and feed out rate) that influence the exposure of the silage to oxygen prior to removal from the silo.

Perhaps one of the more puzzling observations in the real world has been the appearance of clostridia and bu-tyric acid in or near spoiled layers in silos. Given that clostridia are anaerobic bacteria, such observations seem to contradict logic. A recent study by Tabacco et al. (2009) adds some new information to help explain what may be happening. Maize and sorghum silages were made with (L. plantarum or L. buchneri) and without inoculant in 30 l silos. After 90 d ensiling, the silages were tested for aerobic stability and analysed for chemical and microbial changes after 0, 5, 7, 9 and 14 d aerobic exposure. In the maize silage, heating began after approximately 2 d in the untreated and L. plantarum treatments. At 5 d, the pH was above 5.0 in these two treatments, but clostridial spore counts were approximately 2 log10 (cfu g-1 silage). At 7 d, the pH was above 6.5, temperatures were more than 20 °C above ambient, and clostridial spore counts had risen to >6 log10 (cfu g-1 silage). Coincidentally nitrates in those treatments, which were at approximately 1000 mg kg-1 herbage at opening, had fallen linearly to unde-tectable levels by day 7. Nitrate content is indirectly related to inhibition of clostridia when reduced in anaerobic environments to nitrite (Pahlow et al. 2003). These results suggest that a combination of factors together are al-lowing clostridia to grow near spoiling layers: an increase in pH and temperature due to aerobic microorganisms, a return to anaerobic conditions due to spoilage microorganisms exhausting the oxygen supply closer to the oxygen source, and the utilization of nitrates that would normally inhibit clostridial growth under anaerobic conditions. This loss of nitrate suggests that enterobacteria (or possibly some LAB) may be proliferating prior to the clostridia because these bacteria are associated with nitrate reduction early in ensiling.

Inoculants

Microbial inoculants have become the dominant silage additive type in most parts of the world and have been available in many countries for decades. Until recently, most of these products were strains of facultative hetero-fermentative LAB (commonly called homofermenters) such as L. plantarum, L. casei, various Pediococcus species and Enterococcus faecium. The goal was to have a rapid and efficient fermentation that produced mostly lactic acid, minimizing DM losses and attempting to keep nutritive value similar to that of the crop at ensiling. The best of these products have not only enhanced silage fermentation and DM recovery but also improved animal per-formance: milk production, gain, feed efficiency (Weinberg and Muck 1996). However, these products have had a negative effect on aerobic stability in whole-crop maize and small grain silages, presumably because of the re-duction in acetic acid.


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In the late 1990’s, a new class of inoculants, based on obligate heterofermenters such as L. buchneri, entered the market. These strains grow slowly even after the active fermentation period is finished, producing acetic acid from sugars or lactic acid. The primary goal is to increase acetic acid so that yeast and mould growth is inhibited and aerobic stability is improved. However, these products appear to have limited effects on animal performance other than by keeping silage cool. Today we have a third class of inoculants that combine L. buchneri with more traditional strains attempting to get the DM recovery and animal performance of the facultative heterofermenta-tive strains along with the aerobic stability improvements provided by L. buchneri.

There are many papers that report on the testing of various inoculants across a wide variety of forages. To review all of those studies properly is a manuscript by itself. Certainly this work is important in allowing scientists in vari-ous parts of the world to provide good recommendations to producers. However, from a more broad scientific perspective it is more interesting to understand why these products work and to look at current innovative ef-forts to find a new crop of inoculants.

How inoculants dominate silage fermentationRecent studies are helping us understand why inoculants are often successful in the silo and how they may alter si-lage quality in a way that affects livestock response. The facultative heterofermentative LAB inoculant strains have been selected for rapid, homofermentative growth under a wide range of temperatures and DM concentrations. We expect that these strains will be highly competitive and produce largely lactic acid, reducing pH compared to an untreated silage with its mixture of obligate and facultative heterofermenters. However, there are a substan-tial number of incidences (e.g., Muck 1989) where the inoculant was applied at less than 10% of the epiphytic LAB population and still affected silage fermentation. Such instances suggest that at least some inoculant strains are not just faster but also have other competitive advantages over their fellow LAB as well as other epiphytic bacteria.

Some inoculant LAB strains produce antimicrobial compounds. Gollop et al. (2005) investigated whether 10 in-oculants/commercial strains produced antibacterial activity. Nine of 10 inoculants when grown on MRS broth did produce compounds in the broth that inhibited the growth of Micrococcus luteus, a bacterial species susceptible to bacteriocins and other antibacterial compounds. Extracts of inoculated silages were also tested for inhibition of M. luteus. In 15 of 27 cases, the silage extracts from crop inoculated with one of the nine positive strains showed inhibition of M. luteus whereas none of the silage extracts from untreated control or the negative strain inhibited M. luteus (0 of 6 cases). Others (e.g., Marcinakova et al. 2005, Ratanapibulsawat et al. 2005) have isolated LAB strains from silage that produce inhibitory activity against a variety of bacterial species: Staphylococcus aureus, Salmonella sp., Bacillus sp., Listeria sp. and Escherichia coli.

Vazquez et al. (2005) studied the effects of bacteriocins from 6 strains of LAB (L. brevis, L. casei, L. helveticus, Lac-tococcus lactis, Leuconostoc mesenteroides, P. acidilactici) on the growth of those strains. The bacteriocin from a particular strain generally promoted the growth of that strain when added to the culture as well as increased bacteriocin production. When that bacteriocin was added to cultures of the other LAB strains, one would expect reduced growth. That was true particularly with the bacteriocins from L. brevis and Lc. lactis. However, the L. ca-sei and L. helveticus bacteriocins enhanced growth in the other five strains. Similarly bacteriocin production in one strain was most often reduced, but in some cases increased, by the presence of bacteriocin from another strain. The largest increase in bacteriocin production across species (50%) was in L. casei when bacteriocins from P. acidilactici were added.

There is also evidence that some LAB produce antifungal compounds. Broberg et al. (2007) inoculated grass silage with two L. plantarum strains, one isolated from silage, that produce antifungal compounds in MRS broth. The an-tifungal compounds identified in laboratory culture, 3-phenyllactic acid and 3-hydroxydecanoic acid, were found at higher concentrations in the inoculated silages compared with those in the untreated silage. Other antifungal compounds, largely acids associated with lignin synthesis, were also elevated in the inoculated silages. It was not known what caused the increased concentrations of these compounds. Recently, Prema et al. (2010) also isolated a L. plantarum strain from grass silage that produced 3-phenyllactic acid and demonstrated that the acid inhibited a wide range of mould species common to silage.

These studies show that some LAB strains are capable of inhibiting a considerable spectrum of bacteria and fun-gi. The results of Gollop et al. (2005) indicate that it is relatively common to find antibacterial activity in inoculant strains. However, it is likely that much more could be done to select inoculant strains that are not only capable of dominating silage fermentation but also inhibiting undesirable anaerobic and aerobic microorganisms and so po-tentially reducing losses in quality and DM beyond that attained from an efficient fermentation.


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How inoculants alter animal performanceThe effects of inoculants on gain or milk production in livestock have been greater than expected (Weinberg and Muck 1996). In fact, there are a significant number of reported cases where animal performance has been in-creased even though there was either no or only minor changes in pH or silage fermentation products. This is certainly intriguing. However, beyond scientific curiosity, improvements in animal performance provide a bigger return to the farmer than improvements in DM recovery. So there is incentive both scientifically and in helping farmers choose effective inoculants to understand how LAB silage inoculants affect livestock.

In some cases, there is an apparent linkage between changes in silage quality and animal performance. For ex-ample, Ando et al. (2006) found that guinea grass silage treated with L. rhamnosus had higher DM and organic matter digestibility and higher voluntary intake in wethers than untreated silage. When digestibility is improved, livestock should eat more if intake of the diet is limited by rumen fill. But we are still left wondering why an inocu-lant that consumes soluble portions of the crop should affect DM digestibility, which is primarily a function of the digestibility of insoluble structural polysaccharides.

In the past 10 years, we have begun to get important clues as to what may be happening at least with some inocu-lants. These clues point to changes occurring in the rumen of ruminant livestock. Weinberg et al. (2003) found that inoculant LAB could survive in rumen fluid, and some of the strains appeared to buffer pH, keeping it from drop-ping as much as pH in unamended rumen fluid. Given that cellulolytic activity decreases at low rumen pH, perhaps this may be a key to improved digestibility. Following up on these results, Muck et al. (2007) made silages using a wide range of inoculants. In vitro analysis was performed in serum bottles, measuring gas pressure. Surprisingly, some of the inoculated silages had reduced gas production compared with the untreated silages. Because digest-ibility has not been depressed by inoculants, these results suggested that in vitro fermentation was being shifted from gas production to another product – volatile fatty acids or rumen microorganisms.

Recent research has indicated that in vitro fermentation is altered by some inoculant strains. Cao et al. (2010) in-vestigated the effect of a L. plantarum strain on an ensiled total mixed ration (TMR) based on whole crop rice. In vitro analysis of the inoculated TMR silage showed reduced methane production (p=0.065) at 6 h of incubation compared with that of untreated TMR silage. Dry matter digestibility was not affected nor was the production of volatile fatty acids with the exception of butyrate being higher in the in vitro fermentation of the untreated silage. Cao et al. (2011) found similar results with the same inoculant strain in vegetable residue silage with the inocu-lated silage having the highest in vitro DM digestibility and lowest methane production. Contreras-Govea et al. (2011) performed in vitro analysis of maize and lucerne silages inoculated without or with one of four inoculants. While the inoculants produced only minor changes in silage fermentation, in vitro results were affected by treat-ment. At 9 h incubation, three of the four inoculated silages produced more microbial biomass yield as estimated by true minus apparent digestibility as compared with the untreated silages. At 48 h, two of the inoculated silages had higher microbial biomass yield than the untreated silages. At both times, gas and volatile fatty acid production were not affected by treatment, and there were no inoculant by crop interactions. These results together suggest that some, but not all, inoculants are altering in vitro ruminal fermentation, whether by reduced methane pro-duction or increased microbial biomass production, in ways that should lead to increased animal performance.

One of the inoculants that increased microbial biomass yield in the Contreras-Govea et al. (2011) study was one with considerable published animal data (L. plantarum MTD/1), showing positive effects even in some cases where silage fermentation was not affected (Weinberg and Muck 1996). An animal trial has been performed to investigate whether this inoculant can improve rumen microbial biomass production (Muck et al. 2011). Milk production on inoculated lucerne silage was increased compared to the untreated silage. This was accompanied by a significant reduction in milk urea nitrogen that suggests better nitrogen utilisation and most likely more rumen microbial protein production. However, we are awaiting the results of the omasal samples to confirm that. Using ARISA to look at the rumen microbial community, we did not observe significant differences due to treatment, but real-time PCR did find elevated levels of L. plantarum in the rumens of cows on the treated silage (Mohammed et al. 2012).

Certainly there is considerably more research to be done in this area. Even if in the case of L. plantarum MTD/1 we can confirm that there is improved rumen microbial protein production that in turn explains increases in milk production, we still do not understand why that may be happening. Fortunately, it appears that in vitro analyses and our new PCR-based tools may be helpful in uncovering the secrets of how inoculation of silages by particular LAB strains affects silage utilisation by ruminants.


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Strides to find new inoculants

Most of the published efforts to develop new inoculant strains have been in Asia, South America and Africa. This might be expected. The major international companies producing inoculants are based in Europe and North Amer-ica. So more of the inoculant research from these parts of the world is involved in testing commercial products. In addition, these products have been developed for cool-season grasses, whole-crop maize and lucerne, the domi-nant crops ensiled on those continents. These inoculants may or may not be as effective when used on warm-season grasses and tropical legumes.

One of the most complete, recently published screening procedures focused on identifying potential homofer-mentative strains with antimicrobial properties for ensiling cool-season grasses in Finland (Saarisalo et al. 2007). They began by selecting LAB strains from various sources, not just silages, based on antimicrobial activity against a wide range of microorganisms (coliforms, clostridia, Bacillus spp., Listeria spp., yeasts and moulds). Second, they grew the candidate strains on a grass extract medium, measuring fermentation products, growth rate, am-monia N and pH. They also grew the strains on an API 50 CHL test kit to determine the range of substrates each strain could ferment. They selected four strains that grew rapidly on a wide range of sugars, producing high levels of lactic acid, low pH and low ammonia N. These four strains were then tested in a mini-silo trial with a timothy-meadow fescue mixture and with silos being opened at 1, 3, 5, 7, 14, 21, 63 and 84 d. Fermentation characteris-tics, gas production, and microbiological changes were measured. The ensiling trial confirmed the results of the grass extract screening. While they did not proceed further, additional winnowing of strains could be carried out, investigating the range of DM concentrations and temperatures under which the candidate strains could grow.

The approach that one takes in selecting strains depends upon the goals. In the Finnish research, the goal was to find a strain that could actively suppress non-LAB microbial species while producing an efficient fermentation largely of lactic acid and little breakdown of amino acids to ammonia. Similarly, Marcinakova et al. (2008) studied an E. faecium isolate that produces bacteriocins as a potential inoculant. More commonly the goal is to find spe-cies that rapidly and efficiently ferment sugars to lactic acid with little or no ammonia production. Recent such research includes for example: Penteado et al. (2007) in Panicum maximum silage, Kim et al. (2008) in whole-crop rice silage, Kim et al. (2009) in whole-crop barley silage, Yan et al. (2011) in maize silage.

Other goals depend upon the particular issues with a crop or its use. With sugar cane silage, the two major con-cerns are high ethanol concentrations and aerobic instability. Ávila et al. (2009, 2010) identified an L. buchneri strain that was better than commercial strains in reducing ethanol concentration and yeast counts while improving aerobic stability. In guinea grass silage, the targets for Pasebani et al. (2011) beyond low pH and ammonia were high crude protein and low fibre concentrations. Today, there can be alternate uses for silage as seen in the utili-sation of silage to produce methane via anaerobic digestion. Banemann et al. (2010) investigated the potential of an inoculant to increase methane yield from maize silage. Consequently the selection goal will change the strain that will be most effective in the preservation or utilisation of a silage.

The most difficult target today is improvement in animal performance. Animal trials are expensive and time-con-suming. This means that one has to narrow the field of candidates to two or three strains without having an ef-fective measure to know how livestock will respond. Hopefully as we understand how inoculants affect animal performance we will be able to develop in vitro techniques that will allow us to more easily find strains that will be beneficial to livestock.

Conclusions and future directions

Over the past decade we have seen a marked increase in the use of PCR-based techniques in silage research. These techniques are greatly enhancing our ability to detect and monitor the species involved in ensiling. Often, com-mon species like L. plantarum have been found across diverse crops and different continents, making it appear that perhaps we do not need to spend more time looking at the species in silages. In other cases such as with maize in warm climates, it appears that obligate heterofermenters such as Weissella and Leuconostoc species may play more significant roles than in temperate climates. This may or may not be significant to the utilisation of maize silage by livestock or the value of an inoculant. At this time, we do not know.

More studies of the microbial ecology of ensiling using these new PCR-based techniques are needed. Currently we have a limited number of snapshots of microbial dynamics over a wide variety of crops and locations. In some studies, not enough snapshots have been taken in the first week of ensiling to capture the dynamics when the


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major changes in fermentation products and pH are occurring. So we may be missing species that are keys to si-lage quality. We need to have more pictures of major silage crops in various locations so that we can build a glob-al picture of whether those populations vary substantially by location or climate or not, and thus what inoculant characteristics may be most important.

The studies so far have primarily focused on ensiling under good conditions and on the spoilage of silages after silo opening. What would be of particular value is to better understand the microbial ecology of silage fermenta-tions that go wrong. For example in the U.S., farmers occasionally get high acetic acid silages that have reduced intake, but other high acetic acid silages like those inoculated with L. buchneri are consumed at a level expected based on standard nutritive characteristics. The difference is likely the microbial species that produced the acetic acid, and the factor affecting intake is probably not acetic acid but some other product not measured. As we bet-ter understand the species that are causing such problems, we will be able to devise management strategies or additives to prevent those problems.

In concert with our ability to identify species in silage, we need to strive to do more than analyse for the major fermentation products. Some of the recent inoculant research discussed above has found various antimicrobial compounds at low levels, suggesting there are many minor compounds that may influence the course of fermen-tation in the silo and possibly rumen fermentation in cattle and other ruminants. There are concerns about vol-atile organic compounds coming from silages and their effect on the environment, and we need to understand whether those compounds are directly caused by microorganisms or indirectly by chemical interactions during storage. Metabolomics is just entering agricultural research (Ametaj et al. 2010) allowing us to identify many more minor compounds. It along with the PCR-based techniques may be keys to bringing us to a new level of under-standing of what occurs in the silo and how these processes affect livestock and the environment. Armed with this new understanding, we will hopefully be able to improve the quality of the silage that we deliver to our livestock.

AcknowledgementsThe author wishes to express his appreciation to F.E. Contreras-Govea and D.M. Stevenson in reviewing the man-uscript.

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AGRICULTURAL AND FOOD SCIENCEF. Driehuis (2013) 22: 16–34

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Silage and the safety and quality of dairy foods: a reviewFrank Driehuis

NIZO food research, P.O. Box 20, NL-6710 BA Ede, the Netherlands


Silage contains a number of potential hazards to the safety and quality of milk and dairy products. This paper re-views the present knowledge about silage as a source of (1) spores of anaerobic spore-formers (Clostridium species) and aerobic spore-formers (mainly Bacillus and Paenibacillus species), (2) the zoonotic pathogenic bacteria Listeria monocytogenes and Escherichia coli, and (3) mycotoxins. A distinction is made between field-derived mycotoxins, i.e. mycotoxins that are formed during growth of crops in the field, and ensilage-derived mycotoxins, i.e. mycotox-ins that are formed after ensiling. The routes of transmission of these hazards from feed to milk, the effect of pas-teurization of milk, and reduction strategies are discussed. Aerobic deterioration of silages is a major factor influ-encing levels of spores of both aerobic and anaerobic spore-formers, L. monocytogenes, and certain mycotoxins.

Key words: bacterial spores, milk quality, mycotoxins, pathogens, silage quality

IntroductionFood producers are responsible for the safety and quality of their products for consumers (European Commission 2002, European Commission 2005). Quality assurance of food products requires an integrated approach that as-sures safety and quality at all stages of the production chain. The safety and quality of milk and dairy products depend on the quality of raw milk produced at dairy farms, the quality of any other ingredients, processing con-ditions, and distribution and storage conditions. As suppliers of raw milk, dairy farms have an important role in the dairy production chain. Therefore, milk production at dairy farms needs to meet the demands and criteria with respect to animal health, feed quality and milking hygiene. The objective of dairy farm quality assurance is to prevent contamination of raw milk by residues of veterinary medicines and agricultural chemicals, environmen-tal contaminants from for instance feed or soil and by harmful micro-organisms arising from feed, the housing system or the animals themselves. Feed is an important source of chemical and microbiological contaminants of milk (McEvoy 2002, Vissers and Driehuis 2009). The diet of high-yielding dairy cattle consists of two main classes of feedstuffs: forages and concentrates. Fresh, dried or ensiled forages generally constitute the largest fraction of the diet, usually 50 to 75%. Forage preserved as silage is the most popular form of forage in many countries. For example, grass and maize silage represented on average 67% of dry matter dietary intake of Dutch dairy cows in autumn and winter 2005 (Driehuis et al. 2008b).

This paper summarizes the present scientific knowledge about silage as a source of microbiological and chemical contaminants in the dairy chain. The paper focuses on three groups of safety or quality hazards of milk and dairy products: (1) spores of endospore-forming bacteria, such as Clostridium and Bacillus species, (2) the zoonotic pathogenic bacteria Listeria monocytogenes and Escherichia coli, and (3) silage-associated mycotoxins.

Spores of endospore-forming bacteriaContamination pathway from silage to raw milk

Endospore-forming bacteria are an important group of contaminants of raw milk because of the resistance of the spores to heat and other adverse environmental conditions. Spores of many species survive pasteurization of milk and some even survive sterilization conditions. Important sources of bacterial spores are soil, silage and bedding materials. The main contamination pathway of spores from these sources to milk is shown in Figure 1. The origi-nal source of spores occurring in silage is often soil. Contamination of a crop by soil occurs during growth in the field or during harvesting. In crops that are ensiled, this soil contamination usually determines the initial spore concentration (Vissers et al. 2006, Vissers et al. 2007b). Whether a spore population will increase in concentration during ensilage depends on the properties of the micro-organism and the conditions prevailing in the silage. This will be discussed further on in this paper. Spores occurring in silage or other feeds that are consumed by a cow pass the gastrointestinal tract of the animal unaffected and are excreted with the faeces. There is evidence that spore concentrations increase during passage through the intestinal tract (Ali-Yrkkö and Antila 1975, Vissers et al.



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2007b), which can be explained by digestion of feed components. Vissers et al. (2007b) measured concentrations of spores of butyric acid bacteria in mixed grass and maize silage, faeces, bedding materials and raw milk from 24 Dutch dairy farms and observed that the concentration in faeces was on average about three times higher than the concentration in silage. Bedding materials that are used in barns where cows are housed usually become con-taminated by excreted faeces. Under normal dairy farming practices, for instance when cows are lying in the barn, it is inevitable that bedding and faeces attach to the surface of the cow’s udder and teats.

Crop

Soil

Silage

BeddingOther feeds

Faeces

Cows eatingdiet withspores

Cows with dirtattached to

udder and teats Raw milk

Environment, field, farmyard Barn Milking

Good dairy farming practice requires that teats are cleaned before milking (FAO and IDF 2011). However, since teat-cleaning methods are relatively inefficient from a microbiological perspective, a fraction of bacteria and spores from dirt and faecal matter remains attached to the teat surfaces and is rinsed off during milking operations. An evaluation of different manual teat-cleaning methods revealed that spore concentrations in milk were reduced by 45% to 96% when compared to milking without teat-cleaning (Magnusson et al. 2006). Some teat-cleaning methods involve treatment with solutions, foams or wetted towels containing disinfectants. These methods to some extent inactivate vegetative bacteria, but no evidence is currently available that spores are inactivated. Other theoreti-cal contamination pathways, such as aerial contamination of raw milk by spores from silage and direct contami-nation of milk by silage, are insignificant under normal production conditions (Vissers et al. 2006, Vissers 2007).

Vissers et al. (2006) developed a predictive model of the contamination of raw milk by spores of butyric acid bac-teria from feed or other sources in the farm environment, for instance soil. The model was based on a mathe-matical translation of the contamination pathway described above. It was used to evaluate the most important variables and to identify effective and non-effective strategies to control levels of spores of butyric acid bacteria in farm tank milk. It was concluded that the variation of the concentration of spores in silage is, by far, the most important variable, and significantly more important than, for instance, teat-cleaning efficiency and barn hygiene (see also Vissers 2007).

Clostridium speciesClostridium species that occur in silage have been summarized by Pahlow et al. (2003). These authors divided the most common species into three groups, based on their protein and carbohydrate fermentation properties (Table 1). The first group consists of so-called proteolytic clostridia, of which Clostridium sporogenes is the predominant species in silage. Species of this group derive their energy from fermentation of both proteins and carbohydrates. The second group was named the Clostridium butyricum group. Species of this group typically ferment a wide range of carbohydrates but are unable to ferment proteins. Klijn et al. (1995) showed that many strains originating from silage, the farm environment and milk and originally identified as C. butyricum on the basis of phenotypic char-acteristics, genetically belonged to the species Clostridium beijerinckii. The third ‘group’ is formed by Clostridium tyrobutyricum, which ferments a limited number of carbohydrates but in addition has the ability to ferment lac-tic acid to acetic acid and butyric acid at low pH. This type of fermentation is known as butyric acid fermentation and the bacteria responsible for it are referred to as butyric acid bacteria. In addition to the above-mentioned species, Rossi and Dellaglio (2007) detected Clostridium saccharolyticum and Clostridium baratii in silages with high counts of clostridial spores. Using a cultivation-independent DNA-based method (PCR-denaturing gradient gel electrophoresis; DGGE), Julien et al. (2008) identified Clostridium disporicum as another predominant mem-ber of clostridial populations in silage.

Fig. 1. Contamination pathway of bacterial spores from silage and other feeds to raw milk.


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C. tyrobutyricum is a species that is studied most in relation to silage quality for two reasons. Firstly, because C. tyrobutyricum is the main cause of butyric acid fermentation in silage due to its tolerance to low pH conditions in combination with its ability to use lactic acid as a substrate for growth (Pahlow et al. 2003, Vissers 2007). This can have large negative effects on the preservation quality, nutritive value and palatability of silage. The second reason is that spores of C. tyrobutyricum that are present in silage are transferred to milk, and their presence in cheesemilk can lead to a defect called late-blowing in semi-hard and hard cheese types, such as Gouda, Emmental and Gruyère. Late-blowing is caused by butyric acid fermentation that takes place during cheese ripening and results in off-flavours and excessive gas formation leading to texture defects. Late-blowing may cause significant loss of product. Interest-ingly, the factors that are important for growth of C. tyrobutyricum in silage and cheese are the same: low pH, low water activity, use of lactic acid as a substrate and a low concentration of nitrate. In both silage and cheese a high level of nitrate inhibits the germination of spores and outgrowth of C. tyrobutyricum (Klijn et al. 1995, Pahlow et al. 2003). Studies by Klijn et al. (1995) showed that late-blowing in Gouda cheese is exclusively associated with growth of C. tyrobutyricum. Cheese made from milk in which spores of other silage-associated Clostridium species, such as C. beijerinckii and C. sporogenes, were added showed no signs of late-blowing. Moreover, growth of C. tyrobutyri-cum was detected in all experimental and commercial cheeses showing obvious signs of late-blowing. Since C. ty-robutyricum is not harmful to man and animals, its occurrence in silage and cheese is only of economic importance.

Table 1. The predominant Clostridium species occurring in silage and their characteristics. Adapted from Pahlow et al. 2003. Based on data from Ali-Yrkkö and Antila 1975, Bühler 1985, Klijn et al. 1995, Rossi and Dellaglio 2007, Julien et al. 2008.

Characteristic Proteolytic group C. butyricum group C. tyrobutyricum

Species C. sporogenes C. butyricum C. tyrobutyricum

C. bifermentans C. beijerinckii

C. baratii C. acetobutyricum

C. saccharolyticum

C. disporicum

Minimum pH allowing growth >5 >4.5 >4.2

Substrates fermented:

Proteins + - -

Carbohydrates + + +

Monosaccharides variable many few

Lactate weak - +

The pathogen Clostridium botulinum, the causative agent of botulism, is rarely found in silage. Botulism is caused by highly potent neurotoxins produced by C. botulinum (botulinum toxins). Occurrence of C. botulinum and bot-ulinum toxins in silage can be associated with the presence of carcasses of birds or small mammals, for instance due to killing of the animals during harvesting of the crop (Cobb et al. 2002). Poultry manure is a notorious source of spores of C. botulinum. Silage crops may become contaminated with C. botulinum spores when contaminated poultry manure is used as a fertilizer (Livesey et al. 2004). C. botulinum is more sensitive to low pH values than for instance C. tyrobutyricum and C. beijerinckii (Pahlow et al. 2003). For that reason, C. botulinum does not grow in silage under normal ensiling conditions. Occurrence of C. botulinum in silage and its relevance in cattle have been reviewed previously (Kehler and Scholz 1996, Lindström et al. 2010). Foodborne botulism occurs when foods are consumed in which botulinum toxins have been formed. Foods associated with foodborne botulism include canned vegetables and low-acid foods (in particular home-canned foods), sausages, meat products and seafood products (Sobel et al. 2004). Because of their occasional occurrence in silage, transfer of C. botulinum spores to raw milk cannot be excluded. However, historically, dairy products have not been associated with outbreaks of foodborne botulism (Shapiro et al. 1998, Sobel et al. 2004).

The concentration of spores of butyric acid bacteria is traditionally determined by most probable number (MPN) methods, using (1) a medium containing lactic acid and incubation conditions that are selective for anaerobic, gas-forming bacteria, and (2) pasteurization of sample dilutions before inoculation of the medium to inactivate vegetative bacteria (Bergère and Sivelä 1990). These methods are useful for enumeration of C. tyrobutyricum spores, but they are not specific for this species. Other species, for instance C. beijerinckii, are sometimes detect-ed as well. Detection of butyric acid bacteria spores in raw milk is part of the milk quality systems of a number of dairy companies, for instance Dutch dairy companies. Apart from not exclusively detecting C. tyrobutyricum, MPN


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methods for enumeration of butyric acid bacteria spores have several other disadvantages: the analysis time is long (4 to 7 days) and the results have a high uncertainty (which is inherent to most MPN procedures). Alterna-tive methods for detection of C. tyrobutyricum are available, for instance methods based on qPCR (Herman et al. 1995, Lopez-Enriquez et al. 2007) or immunological techniques (Nedellec et al. 1992, Lavilla et al. 2010). How-ever, these methods are currently not used for routine analyses in the dairy sector, presumably because they are relatively laborious and costly.

Concentrations of butyric acid bacteria spores in silage vary from 10 to 100 spores g-1 fresh matter, which repre-sents the initial contamination level of fresh crops arising from soil contamination at harvest, to 106 to 107 spores g-1 in silages with extensive butyric acid fermentation (Stadhouders and Spoelstra 1990, Pahlow et al. 2003, Viss-ers et al. 2007b). Information about concentrations of butyric acid bacteria spores in farm-scale silages is rather scarce. Studies in France conducted in the 1970s showed that about 20% of grass silages contained more than 105 butyric acid bacteria spores g-1, a level that can be described as ‘poor quality’ (ITEB/ITG 1980). In a survey conducted in the Netherlands in 1982, 44% of grass silages exceeded the level of 105 butyric acid bacteria spores g-1 and the average concentration was 4.9 log10 g

-1 (Spoelstra 1984, 1990). More recent data from the Nether-lands show that the quality of grass silage with regard to butyric acid bacteria spores has significantly improved since the 1980s: in 5% of grass silages produced between 2002 and 2004 the concentration of butyric acid bac-teria spores exceeded 105 g-1 and the average concentration was 3.2 log10 g

-1 (Table 2). These results were based on samples taken from unopened silages sampled, on average, eleven weeks after ensiling. The improvement of silage quality with regard to butyric acid bacteria spores in the Netherlands since the 1980s is in agreement with information from Dutch dairy companies, who experienced a decreasing trend in the level of butyric acid bacte-ria spores in raw milk delivered by Dutch farmers between 1980 and 2000. The survey conducted in the Nether-lands between 2002 and 2004 also included samples from unopened maize silages. The average concentration of butyric acid bacteria spores in maize silage were approximately 0.5 log10 unit lower than in grass silage and maize silages, with a high level of butyric acid bacteria spores almost absent: none of 197 tested maize silages ex-ceeded 105 spores g-1 and only 0.5% exceeded 104 spores g-1 (Table 2). The findings were in accordance with the knowledge that, due to the low buffering capacity of the maize, lactic acid fermentation in maize silage is gen-erally fast and the final pH low (pH 3.8 to 4.0), conditions that do not favour outgrowth of butyric acid bacteria.

Table 2. Concentration of butyric acid bacteria spores in unopened and opened grass and maize silages and mixed silage at commercial dairy farms in the Netherlands. From unopened silages, only core samples were analyzed. From opened silages, samples from the core, surface layer and areas with visible moulds were analyzed. Mixed silage consisted of grass and maize silage and was sampled in the barn where it was offered to dairy cows. Data from Vissers et al. (2007a), Vissers et al. (2007b) and Vissers and Driehuis, unpublished results.

Sample type Number of samples

Average concentration

(log10 spores g-1)

Percentage of samples containing(spores g-1):

<103 103-104 104-105 >105

Grass silage, unopened 460 3.2 48% 32% 15% 5%

Grass silage, opened

Core 22 3.0 50% 41% 9% 0%

Surface layer 22 3.1 64% 23% 9% 5%

Area with visible moulds 14 3.9 21% 29% 29% 21%

Maize silage, unopened 197 2.7 79% 21% 0.5% 0%

Maize silage, opened

Core 21 3.0 62% 19% 14% 5%

Surface layer 21 3.6 43% 24% 14% 19%

Area with visible moulds 15 5.5 7% 13% 13% 67%

Mixed silage in barn 122 4.2 17% 24% 41% 18%

Until recently, the generally accepted view was that high concentrations of butyric acid bacteria spores are asso-ciated with anaerobic instability of silage due to insufficient pH decline during the primary fermentation phase and that growth of clostridia in silage depends on the contents of dry matter, water soluble carbohydrates and nitrate and the buffering capacity of the crop before ensiling (McDonald et al. 1991, Kaiser et al. 2002, Pahlow et al. 2003). A different view on the issue of butyric acid bacteria spores in silages came from a study by Vissers et al. (2007a), who showed that, on Dutch dairy farms, increased concentrations of butyric acid bacteria spores were often related to aerobic instability problems rather than to anaerobic instability problems. In that study, samples


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were taken at 21 commercial dairy farms from various locations in clamp silos of grass and maize silage and from mixed grass and maize silage that was offered to dairy cows in the barn. It was found that the samples of mixed silage in the barn had an average concentration of butyric acid bacteria spores that was more than 10-fold high-er than samples taken from the core of the grass and maize silage clamps, which represented the major fraction of the silage that was offered to cows. In addition, it was found that a high percentage of samples of mixed si-lage (18%) contained butyric acid bacteria spores in a concentration exceeding 105 spores g-1 (Table 2). The study showed that the total quantity of butyric acid bacteria spores consumed by cows was determined by only a small fraction of silage that contained a high concentration of above 105 spores g-1 (‘hot spots’). Further analysis of the silages that were used at the farms revealed that high spore concentrations were detected particularly in samples from areas showing signs of aerobic deterioration, i.e. areas with a high concentration of yeasts and moulds and increased temperature and pH. High concentrations of butyric acid bacteria spores were found most often in sur-face layers and in particular in areas with visible moulds (up to 107 spores g-1). Unexpectedly, high concentrations of butyric acid bacteria spores were detected more often in maize silage than in grass silage (Table 2). The data showed that at the surveyed farms, which were representative for dairy farming in the Netherlands, maize silage contributed more to the total intake of butyric acid bacteria spores by dairy cows than grass silage. The results by Vissers et al. (2007a) confirmed earlier observations by Jonsson (1989, 1991), who showed that C. tyrobutyricum has the ability to grow and produce spores in silage that is exposed to air. Also recent studies from Italy indicated that high levels of clostridia spores in maize silage are associated with air penetration and aerobic deterioration processes (Borreani and Tabacco 2008, 2010).

The growth of the strictly anaerobic bacterium C. tyrobutyricum in aerobically deteriorated areas of silage may seem contradictory. However, microbial ecosystems with aerobic and anaerobic zones are found in many envi-ronments, for example in sediments and intestines (Brune et al. 1995, Fourcans et al. 2004). The occurrence of anaerobic niches in aerobically deteriorating silage was postulated for the first time by Jonsson (1989). The oc-currence of these niches may be explained as follows. Aerobic deterioration in silage areas that are exposed to air is usually initiated by growth of acid-tolerant, lactate-assimilating yeasts that oxidize residual sugars and or-ganic acids, leading to an increase in pH. Since the concentration of oxidizing yeasts is relatively low during the early phases of aerobic deterioration, the consumption rate of oxygen is also low and oxygen penetrates relatively deep into the silage. However, as the concentration of the yeasts increases, the consumption rate of oxygen also increases. As a result, oxygen penetrates less deeply into the silage, and deeper parts of the silage return to an-aerobic conditions (Muck and Pitt 1994). Consequently, anaerobic niches with an increased pH may develop close to air-exposed areas and growth of C. tyrobutyricum and possibly other clostridia are no longer inhibited due to the increased pH in these niches.

Bacillus cereus and other aerobic spore-forming bacteriaSpores of aerobic spore-forming bacteria are ubiquitous and can be isolated from a wide variety of sources in the dairy farm environment, including soil, silage, concentrate feeds, bedding and faeces. Contamination of raw milk by spores from these sources occurs during milking via contaminated udders and teats, as described earlier in this paper. After the initial contamination, the concentration of spores may increase further during storage of the milk at the farm, for instance when the storage temperature is not low enough and spores of psychrotrophic spore-forming bacteria germinate. Another possible route of contamination is via insufficiently cleaned milking equip-ment. Certain spore-formers are known to be capable of forming biofilms which can attach to stainless steel and release high numbers of spores into surpassing milk (Eneroth et al. 2001, Simoes et al. 2010).

Aerobic spore-formers with particular relevance for dairy products are Bacillus cereus and the highly heat-resist-ant spore-formers Bacillus sporothermodurans and Geobacillus stearothermophilus. B. cereus is a major spoilage organism of pasteurized milk and milk products stored at refrigeration temperature (Griffiths 1992, Te Giffel 1997, Heyndrickx and Scheldeman 2002). B. cereus spores also occur in milk powder and in infant formulae that contain milk powder. Spores of psychrotrophic strains of B. cereus are capable of germination and the bacteria can grow in pasteurized milk and milk products at temperatures as low as 5°C. The content of spores of psychrotrophic B. cereus often limits the shelf life of these products as high levels may cause off-flavours and curdling. B. cereus is also a concern for food safety as it can produce different types of toxins and is a potential food poisoning agent (Stenfors Arnesen et al. 2008). Therefore, the organism is generally regarded as a pathogen. Dairy products are only sporadically involved in outbreaks of foodborne illness caused by B. cereus. B. sporothermodurans and G. stearo-thermophilus are thermophilic bacteria producing highly heat-resistant spores and can cause non-sterility prob-lems in ultrahigh-temperature (UHT) processed or sterilized milk products (Huemer et al. 1998, Scheldeman et al. 2006). The section below focuses on the role of silage as a source of spores of aerobic spore-formers in raw milk.


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Aerobic spore-formers isolated from silage belong to the taxonomic families Bacillaceae and Paenibacillaceae (Table 3). Species frequently isolated include B. cereus, B. licheniformis, B. coagulans, B. pumilus, B. sphaericus and Pae-nibacillus polymyxa. Populations of heat-resistant spore-formers that are isolated from silage show high diversity and also include B. sporothermodurans, the species associated with spoilage of UHT-products. The primary source of most aerobic spore-formers is soil (Claus and Berkeley 1986). Concentrations of spores of aerobic spore-formers and spores of B. cereus in soil vary from 105 to 107 spores g-1 and 101 to 106 spores g-1, respectively, depending on soil type, sampling site and season (Rammer et al. 1994, Slaghuis et al. 1997, Christiansson et al. 1999, Vissers et al. 2007c, Vissers and Driehuis, unpublished data). The actual levels of spores of aerobic spore-formers and spores of B. cereus on crops prior to ensiling depend on the amount of soil that contaminates the crop during growth in the field and during harvesting. They also depend on whether the soil or crop has been fertilized with cattle manure, since spore concentrations can be high in cattle faeces. Slaghuis et al. (1997) detected a concentration of spores of aerobic spore-formers in grass and maize prior to ensiling of 102 to 104 spores g-1. Several studies, summarized by Pahlow et al. (2003), have reported on concentrations of spores of aerobic spore-formers occurring in farm-scale silages. The reported spore concentrations vary considerably between the silos. For wilted grass silages these con-centrations ranged from 103 to 108 spores g-1, for whole crop maize silages from 102 to 109 spores g-1 and for sugar beet pulp and brewers’ grain silages from 103 to 107-108 spores g-1. Concentrations in core samples of silages were generally on the lower side of these concentration ranges: 104 to 105 spores g-1 in grass silages and 103 to 104 spores g-1 in maize silages. These data are in line with the view that in well-fermented silage germination of spores and outgrowth of vegetative bacteria does not occur. Growth of aerobic spore-formers probably occurs during the later phases of aerobic deterioration, i.e. after aerobic deterioration has been initiated by yeasts or acetic acid bacteria (Pahlow et al. 2003). High levels of spores of aerobic spore-formers have been detected in the surface layers of grass and maize silage (Slaghuis et al. 1997, Driehuis et al. 2009). Table 4 summarizes results from studies in the Nether-lands on concentrations of spores of aerobic spore-formers in unopened and opened farm-scale grass and maize silages and on the distribution of spores in opened silages. The highest levels were detected in surface layers and areas with visible moulds of opened silages and in mixed grass and maize silage offered to cows in the barn. These data confirm that high concentrations of spores of aerobic spore-formers relate to aerobic deterioration problems.

Table 3. Species of aerobic spore-forming bacteria isolated from silage. Data from Lindgren et al. 1985, Jonsson 1989, McDonald et al. 1991, De Silva et al. 1998, Inglis et al. 1999, Pettersson et al. 2000, Te Giffel et al. 2002, Driehuis et al. 2009.

Species

Heat-resistance not specified Bacillus cereus, Bacillus licheniformis, Bacillus coagulans, Bacillus pumilus, Bacillus sphaericus, Bacillus firmus, Bacillus lentus, Bacillus circulans, Paenibacillus polymyxa, Paenibacillus validus, Paenibacillus pabuli, Brevibacillus chosinensis

Highly heat-resistant species Bacillus cereus, Bacillus licheniformis, Bacillus subtilis, Bacillus sporothermodurans, Bacillus oleronius, Bacillus siralis, Brevibacillus borstelenis, Aneurinibacillus spp.

Table 4. Average pH and microbiological composition of samples from unopened and opened grass and maize silages and from mixed grass and maize silage offered to cows at commercial dairy farms in the Netherlands (Driehuis et al. 2009). The data were collected between 2002 and 2005.

Sample type Number of samples

Spores of aerobic spore-formers (log10 spores g-1)

Yeasts & moulds (log10 cfu g-1) pH

Grass silage

Unopened, core 460 4.8 3.8 4.8

Opened, core 22 5.1 3.3 4.8

Opened, surface 22 5.7 4.9 5.3

Opened, area with visible moulds 14 8.0 6.8 7.1

Maize silage

Unopened, core 197 3.3 6.0 3.8

Opened, core 21 4.5 5.8 3.9

Opened, surface 21 5.1 7.0 4.2

Opened, area with visible moulds 15 7.7 7.8 6.6

Mixed silage in barna 122 6.2 6.4 4.8a Mixed grass and maize silage offered to dairy cows in the barn


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As described previously, spores of B. cereus occur in silages. However, this species does not increase in numbers to the same extent as other aerobic spore-formers. Vissers et al. (2007c) monitored B. cereus spore concentra-tions in different feeds, faeces, bedding, soil and raw milk at 24 commercial Dutch dairy farms and detected aver-age concentrations of B. cereus spores of 2.2 to 2.8 log10 spores g-1 in mixed silage offered to cows with maximum concentrations of 4.0 log10 spores g-1. Although feed in general, and silages in particular, were found to be impor-tant sources of B. cereus spores in raw milk, it was concluded that the concentrations detected in silage were not critical with respect to the quality and safety of dairy products. The authors indicated that soil and insufficiently cleaned milking equipment are more critical potential sources of B. cereus spores at dairy farms. In studies con-ducted in Sweden, soil was identified as the major source of contamination of raw milk by B. cereus during grazing of cows, whereas used sawdust bedding material, in particular in free-stalls with deep sawdust beds, was a major source when cows were kept indoors (Christiansson et al. 1999, Magnusson et al. 2007).

Listeria monocytogenes

The facultatively anaerobic Gram-positive bacterium Listeria monocytogenes is an important food-borne patho-gen because it is the causative agent of listeriosis. Due to the severity of this disease, the high mortality rate and the increasing incidence, L. monocytogenes is of great concern to public health (European Food Safety Authority 2011). The bacterium is widely distributed in the environment and has been isolated from a variety of sources, in-cluding soil, surface water and vegetative materials. It occurs at low numbers in many raw and ready-to-eat foods. Consequently, humans are commonly exposed to low numbers of L. monocytogenes from various types of food. Generally, this is not considered a serious health hazard (Food and Drug Administration 2001). However, ingestion of food contaminated with high numbers of L. monocytogenes may result in disease, in particular in populations with an increased risk of listeriosis, such as immunocompromised patients, elderly and neonates. High numbers of L. monocytogenes in foods usually arise from growth during storage of contaminated food products that sup-port the growth of the bacterium. Foods particularly linked to L. monocytogenes contamination include raw and smoked fish, raw and cooked meat, and soft and semi-soft cheeses produced from unpasteurised milk. For these and other ready-to-eat foods that are able to support the growth of L. monocytogenes the internationally applied food safety criterion is absence in 25 g throughout their shelf life (also referred to as zero-tolerance). For foods that are unable to support the growth of L. monocytogenes and foods in which limited growth can occur a com-monly applied criterion is 100 cfu g-1 during their shelf life (see for instance EU regulation on microbiological cri-teria for foodstuffs; European Commission 2005).

An important feature of L. monocytogenes is its psychrotolerance. The bacterium has the ability to grow at tem-peratures as low as 0 °C, and therefore can grow during refrigerated storage of foods (Wilkins et al. 1972). The bac-terium also has considerable osmotolerance and acid tolerance, although it is unable to grow at pH levels lower than 4.4. Due to its high tolerance to stress conditions, L. monocytogenes is capable of survival for extended pe-riods in environments in which it is unable to grow. As a vegetative bacterium, L. monocytogenes is fairly sensi-tive to heat inactivation and the organism is effectively killed by pasteurization processes of milk that are used in the dairy industry. Therefore, heat treatment is an effective processing tool in the control of L. monocytogenes in foods. Contamination of processed food products by L. monocytogenes often results from recontamination during the manufacturing process or packaging, and environments inside food processing plants have been recognized as important potential sources of L. monocytogenes (Wiedmann 2003).

Outbreaks and sporadic cases of listeriosis in cattle, sheep and goats have been associated with feeding of silage contaminated with L. monocytogenes (Fenlon, 1988, Ho et al. 2007). Different studies have shown a high diversi-ty of L. monocytogenes strains in silages and in faeces shed by cows that were fed silage (Nightingale et al. 2004, Borucki et al. 2005). Not only animals with clinical signs of listeriosis shed L. monocytogenes in their faeces. Asymp-tomatic animals from farms with an outbreak of listeriosis and healthy animals from farms without a record of lis-teriosis cases can shed the bacterium also (Unnerstad et al. 2000, Nightingale et al. 2004, Vilar et al. 2007). Con-tamination of raw milk by L. monocytogenes has been linked to the occurrence of high levels of L. monocytogenes in silage (Sanaa et al. 1993, Tasci et al. 2010). Transmission of L. monocytogenes to raw milk is most likely taking place via faeces and bedding that is contaminated by faeces, as described previously for bacterial spores. Another transmission route is shedding of L. monocytogenes in milk by cows with mastitis caused by this bacterium (Bour-ry et al. 1995). However, the incidence of bovine mastitis caused by L. monocytogenes is low (Fedio et al. 1990).

The degree of anaerobiosis and the pH are important factors determining survival and growth of Listeria spp. in silage. L. monocytogenes added to grass at ensiling rapidly disappeared under strictly anaerobic conditions and


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at a pH lower than 4.4. However, at an oxygen tension of 0.5% (v v-1) survival was prolonged, and growth was ob-served even at a pH as low as 4.2. Higher oxygen tensions strongly encouraged L. monocytogenes growth (Donald et al. 1995). High numbers of L. monocytogenes and other Listeria species have been detected in different types of silage. For instance, L. monocytogenes levels in excess of 106 cfu g-1 were detected in surface layers of big bale grass silages that were visibly infested by moulds (Fenlon 1986). Different studies have shown that the incidence of Listeria species in silage increases with increasing pH (Ryser et al. 1997, Vilar et al. 2007, Tasci et al. 2010). For instance, Vilar et al. (2007) detected Listeria spp. in 30% of silage samples with a pH ≥4.5 and in 6% of samples with a pH <4.5. These data are in line with the view that the occurrence of Listeria species in silage is associated with aerobic deterioration problems. The relatively high pH values that generally exist in aerobically deteriorated areas, in combination with the presence of oxygen lead to conditions that favour growth of Listeria. Silages with a greater likelihood of aerobic surface spoilage are more susceptible to contamination by Listeria, for example si-lage with low packing density, silage that is inadequately sealed and big bale silage (Fenlon et al. 1989).

In conclusion, L. monocytogenes has frequently been detected in silages and has been associated with occurrence of aerobic spoilage. Its presence in silage has been linked to contamination of raw milk. However, since L. mono-cytogenes is effectively inactivated by pasteurization used in milk processing, the food processing plant environ-ment appears to be the major source of finished product contamination.

Enterobacteriaceae and Escherichia coli

Several species of the facultatively anaerobic Enterobacteriaceae belong to the epiphytic microflora of most for-age crops. Erwinia herbicola and Rahnella aquitilis often dominate the fresh crop, but after ensiling these spe-cies are rapidly superseded by other species, such as Hafnia alvei, Escherichia coli and Serratia fonticola (Heron et al. 1993). The most important species in this group from the viewpoint of human health risks is E. coli. Most E. coli strains are harmless and are part of the normal intestinal microbiota of humans and many animals. How-ever, some types of E. coli cause severe gastrointestinal diseases. Among the pathogenic E. coli, the group of Shi-ga toxin-producing E. coli (STEC), also called verocytotoxin-producing E. coli (VTEC) or enterohaemorrhagic E. coli (EHEC), is of serious public health concern.

The gastrointestinal tract of healthy ruminants, including cattle, is recognized as the main natural reservoir of STEC, in particular for E. coli O157:H7. Major sources of E. coli O157:H7 and other STEC strains for human infec-tion are (raw) meat products, faecally contaminated vegetables and drinking water, and direct contact with ani-mals. In addition, raw milk and unpasteurized dairy products have been implicated in outbreaks caused by infec-tion with E. coli O157:H7 (Hussein and Sakuma 2005). The presumed route of transmission to raw milk is faecal contamination during milking, as described previously for bacterial spores and L. monocytogenes. Fortunately, E. coli O157:H7, like other E. coli, is sensitive to heat and is effectively killed by pasteurization of milk used in the dairy industry. Therefore, as described previously for L. monocytogenes, heat treatment is an effective processing tool in the control of E. coli O157:H7 and other STEC strains in dairy products.

During the early stages of silage fermentation, Enterobacteriaceae compete with the lactic acid bacteria and oth-er bacterial groups for nutrients. Most Enterobacteriaceae do not grow and lose viability at pH values lower than 4.5 to 5.0. A fast pH decline therefore decreases growth and survival of Enterobacteriaceae in silage (Heron et al. 1993). However, the presence of oxygen prolongs their survival in silage and some enterobacteria that survive the storage phase may start growing again and reach numbers in excess of 108 cfu g-1 when silage pH increases during aerobic deterioration (Lindgren et al. 1985, Donald et al. 1995). No studies are known to the author that have shown the presence of E. coli O157:H7 and other STEC strains in silage. In a number of studies the growth and survival of E. coli O157:H7 in grass, maize and barley silage, inoculated with this bacterium prior to ensiling, was investigated. These studies showed that E. coli O157:H7 does not survive in well-fermented silage with a fast pH decline and low pH (Byrne et al. 2002, Bach et al. 2002, Pedroso et al. 2010). The same result was achieved for STEC serotype O26 in maize silage (Dunière et al. 2011). However, other studies showed that E. coli O157:H7 po-tentially can survive and grow in poorly fermented silage and in aerobically deteriorated silage (Fenlon and Wil-son 2000, Pedroso et al. 2010).

In conclusion, E. coli O157:H7 and other STEC strains do not survive normal ensiling conditions and no data show-ing occurrence of these bacteria in silages are currently available.


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Mycotoxins

This section summarizes scientific knowledge about the major mycotoxins occurring in silages, the conditions un-der which they are formed and prevention of their formation. Mycotoxins in silage are of dual concern. Firstly, they can have adverse effects on animal health and cause production losses. Secondly, they may jeopardize the safety of food products of animal origin. Of the major mycotoxins in silage crops, the second concern holds true for afla-toxin only, as is described further on in this paper. The metabolism of mycotoxins in ruminants and their carry-over into milk are briefly described. Toxic effects of mycotoxins in animals and man, analytical methods for detection of mycotoxins and legislative aspects are not described in this paper. Information about these topics can be found elsewhere (Council for Agricultural Science and Technology [CAST] 2003, Krska et al. 2008, Driehuis et al. 2010).

Mycotoxins are a large, diverse group of toxic metabolites of fungi. Currently, more than 300 mycotoxins have been identified (CAST 2003). Mycotoxins can be found in a wide variety of crops all around the world, including crops that are commonly fed as silage, such as maize, wheat and grasses (CAST 2003, Driehuis et al. 2010). Moulds and mycotoxins of relevance for silage that is produced from these crops are listed in Table 5. A distinction is made between mycotoxins that are formed before ensiling and those that are formed after ensiling. It is important to make this distinction because different types of moulds, different types of mycotoxins and different types of ag-ricultural factors influencing mycotoxin levels are involved. Mycotoxins that are formed before ensiling are asso-ciated with moulds that infect a crop during its growth in the field or with endophytic moulds that live as symbi-onts in for instance grasses or cereals (field-derived mycotoxins). Field-derived mycotoxins include trichothecenes, zearalenone, fumonisins, aflatoxins and ergot alkaloid mycotoxins. Mycotoxins that are formed after ensiling are associated with moulds that develop in silage during storage or feeding-out (ensilage-derived mycotoxins), usu-ally as a result of poor silage management practices. These mycotoxins include mycotoxins formed by Penicillium roqueforti and Penicillium paneum and a diverse group of mycotoxins formed by Aspergillus fumigatus. Table 5. Major mycotoxigenic moulds and mycotoxins in silage crops and silages.

Mycotoxin group Major toxin(s) Mould species Crop(s)Field- or ensilage-derived

Aflatoxins Aflatoxin B1 (M1), B2, G1, G2 Aspergillus flavus, A. parasiticus Maize Field

Trichothecenes Type A: T2, diacetoxyscirpenol Fusarium langsethiae, F. poae, F. sporotrichioides

Maize, Sg cereals1 Field

Type B: DON, nivalenol F. graminearum, F. culmorum Maize, Sg cereals, grass

Field

Fumonisins Fumonisin B1, B2 F. verticillioides, F. proliferatum Maize Field

Resorcylic acid lactones

Zearalenone F. graminearum, F. culmorum Maize, Sg cereals, grass

Field

Ochratoxins Ochratoxin A A. ochraceus, Penicillium verrucosum Sg cereals Field

Ergot alkaloids Clavines, lysergic acid amide, ergotamine

Claviceps purpurea Sg cereals Field

Lolitrem B, ergovaline Neotyphodium lolii, N. coenophialum Grass Field

P. roqueforti toxins Roquefortine C, mycophenolic acid

P. roqueforti, P. paneum All types of silages Ensilage

A. fumigatus toxins Gliotoxin, fumigaclavines A. fumigatus All types of silages Ensilage

M. ruber toxins Monacolin K, citrinin Monascus ruber All types of silages Ensilage1 Sg cereals: Small grain cereals (wheat, triticale, rye, barley).

Field-derived mycotoxinsThe major toxinogenic moulds capable of producing field-derived mycotoxins are Fusarium species, Aspergil-lus flavus and Aspergillus parasiticus and endophytic Claviceps and Neotyphodium species. Detailed information about these moulds and mycotoxins can be found elsewhere (CAST 2003, Barug et al. 2006, Driehuis et al. 2010). The most frequently occurring mycotoxins produced by Fusarium species are trichothecenes, zearalenone and fumonisins. These moulds occur world-wide, but seem to be particularly prevalent in temperate climates. The de-velopment of Fusarium mycotoxins is strongly influenced by weather conditions. Infection of plants by Fusarium can take place via kernels, leaves, the stalk or infected seeds. Soil and decaying plant residues in the field are the


25

main sources of Fusarium spores and conidia. A high level of mechanical or insect damage of the plant increases the risk of infection and is often associated with higher mycotoxin levels.

Examples of plant diseases associated with Fusarium infection include ear rot and stalk rot in maize and ear blight in wheat. The predominant species causing these diseases are Fusarium graminearum and Fusarium culmorum. These species are capable of producing zearalenone and different types of trichothecenes, including deoxyniva-lenol (DON; synonym for vomitoxin), nivalenol, diacetoxyscirpenol and T-2 and HT-2 toxin. DON is the most com-monly occurring trichothecene. DON and zearalenone often co-occur in contaminated crops. An important and often unrecognized feature of DON and zearalenone contamination of maize and wheat is that these mycotoxins occur not only in the grains and kernels but also in the green parts of the plant, i.e. the leaves and stalk. This is of significance because these crops are often fed as whole crop silage. The limited information that is available on this topic indicates that DON and zearalenone levels can be even higher in leaves and stalk of maize than in the cob (Oldenburg et al. 2005). Fumonisins are formed by Fusarium verticillioides (syn. Fusarium moniliforme) and Fusar-ium proliferatum, species associated with pink or white ear rot disease in maize. Fumonisins are found exclusively in maize. It is generally assumed that DON, zearalenone and other Fusarium mycotoxins are not produced in silage (Driehuis et al. 2010). Fusarium species do not survive the acidic and anaerobic conditions of silage and usually have a lower prevalence in silage than for instance Aspergillus, Penicillium and Monascus species. However, a few studies have reported development of Fusarium mycotoxins in silage. An example is a study conducted in Italy in which zearalenone was detected in high concentrations in extensively aerobically deteriorated peripheral areas of maize silage. Concentrations in these areas were up to 40 times higher than concentrations in non-deteriorated central areas of the silage, which were similar to the concentration of the forage at ensiling (Cavallarin et al. 2004).

Aflatoxins are produced by A. flavus and, to a lesser extent, A. parasiticus. Aflatoxins are highly toxic and carcino-genic to man and animals. Aflatoxin B1 is the most prevalent and most toxic form. Aflatoxin B1 is transformed into aflatoxin M1 in the liver of cattle. In this form it is (partially) excreted into milk. With respect to the risks of myco-toxins in feed aflatoxin M1 is the only mycotoxin of concern for the safety of dairy products to consumers. This re-lates to its significant feed-to-milk carry-over rate and its high toxicity. Although Aspergillus is generally classified as a mould associated with mycotoxin production during storage of commodities, it can infect crops in the field under favourable conditions, especially in subtropical and warm temperate climates. A. flavus and A. parasiticus are associated with aflatoxin production in a number of crops, including maize, sunflower, peanut and several tree nuts. Maize plants can become infected by Aspergillus conidia from the environment, usually soil or insects. A high level of insect damage increases the risk of infection. If conditions are favourable, the mould colonizes the cobs and penetrates into the kernels. Aflatoxin development in the kernels occurs within narrow ranges of mois-ture content and temperature. Drought stress generally increases aflatoxin development in maize.

Ergot alkaloid mycotoxins are produced by Claviceps purpurea in rye and barley and some grasses and by endo-phytic Neotyphodium moulds in perennial grasses. Detailed information about ergot alkaloid mycotoxins can be found elsewhere (Wyss et al. 1997, CAST 2003, EFSA 2012). C. purpurea infects the plant when flowering. It pro-duces a resting structure, called sclerotia or ergots, that is comparable in size to grain kernels and allows the mould to survive adverse conditions. These ergots contain high concentrations of alkaloids (e.g. clavines and lysergic acid amide). Several grasses, such as perennial ryegrass (Lolium perenne) and tall fescue (Festuca arundinacea), can harbour endophytic Neotyphodium species capable of producing similar alkaloid mycotoxins (e.g. lolitrem B and ergovaline). The plant benefits from this symbiosis through increased drought tolerance and resistance to insects. Endophytic Neotyphodium are highly prevalent in ‘wild’ grass populations in natural or extensively managed pas-tures in the North America, Australia, New Zealand and Europe. The prevalence of mycotoxin-producing endo-phytes in intensively managed pastures is generally low. Grass cultivars selected for grazing or silage production often do not contain these types of endophytes.

Ensilage-derived mycotoxinsSince the majority of mould species are obligate aerobic micro-organisms, they do not develop in well-preserved, anaerobic silage. However, in practice, silages are not completely anaerobic. Firstly, because silage covering mate-rials are generally not fully airtight. Secondly, because of unintended damage to the silage covering during storage (for instance caused by rodents or birds). Moreover, exposure to air becomes inevitable after the silo is opened for feeding. Growth of moulds and development of mycotoxins in silage are associated with the duration and ex-tent of air infiltration. The extent of infiltration of air into the silage mass is mainly dependent on the porosity and density of the silage and the rate of silage removal after opening. The occurrence of moulds in silage is usu-ally highest in surface layers.


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Moulds that are commonly detected in silage are P. roqueforti and P. paneum, Monascus ruber, A. fumigatus, Bys-sochlamys nivea, Mucoraceae (in particular Rhizopus nigricans) and Chrysonilia sitophila (Scudamore and Livesey 1998, Pahlow et al. 2003). No mycotoxins from Mucoraceae and C. sitophila have been documented. The pre-dominant mould species in silages is P. roqueforti, which is tolerant to acidic conditions and able to grow at oxy-gen levels as low as 0.1% (v v-1) (Lacey 1989, Pahlow et al. 2003). At silage surfaces it usually forms white to grey coloured spots or layers. Occasionally, P. roqueforti forms typical green to blue coloured balls or lumps of mouldy silage approximately 50 to 100 cm below the top surface. This occurs particularly in maize silage. P. paneum is closely related to P. roqueforti and the occurrence of these species in silage cannot be differentiated visually (Boy-sen et al. 1996). P. roqueforti and P. paneum are capable of producing a wide range of mycotoxins in vitro under laboratory conditions, including for instance different roquefortines, mycophenolic acid, PR-toxin, festuclavine and agroclavine (Nielsen et al. 2006, O’Brien et al. 2006). P. paneum additionally produces patulin. However, a number of these mycotoxins are probably not formed in silage or may not be stable under conditions prevailing in silage (as discussed later in this paper). A. fumigatus is a mould species that is particularly detected in heavily moulded parts of silage and capable of producing a large number of different toxic metabolites, including gliotoxin, verruc-ulogen, fumitremorgens, fumigaclavines and trypacidin (Richter et al. 2009). Apart from production of mycotox-ins, the occurrence of A. fumigatus in silage is considered a health risk because inhalation of spores of this mould can cause lung disease (aspergillosis) in animals and man. M. ruber forms red-purple spots on silage surfaces and is a producer of monacolin K and citrinin (Schneweis et al. 2001). B. nivea is a producer of patulin (Escoula 1975).

Stability of mycotoxins in silageInformation about the stability of mycotoxins in silage is not fully conclusive and for some mycotoxins is contradic-tory. Possibly, this relates to the heterogeneity of silage and the fact that the conditions change over time. There are data indicating that certain field-derived and ensilage-derived mycotoxins are degraded in silage.

Zearalenone is generally regarded as stable in silage. No effect of ensiling on the zearalenone concentration was detected in studies in which the level of this mycotoxin was monitored for nine months of ensilage (Lepom et al. 1988, Garon et al. 2006). This finding is consistent with data showing that the average and range of zearalenone concentrations in maize silage and unfermented maize products used as feed ingredient are similar (Dänicke et al. 2000). With respect to the stability of DON in silage there is contradictory information. In a study investigating DON stability in wheat and maize silage it was concluded that ensiling induced a strong reduction of DON (Rich-ter et al. 2009). However, no effect of ensiling on DON concentration was detected in other studies (Lepom et al. 1990, Garon et al. 2006). Furthermore, surveys in Europe and United States of America have shown a high inci-dence of DON in maize silages (see next section) and the average and range of DON concentrations are similar in maize silage and unfermented maize products used as feed ingredient (Dänicke et al. 2000). These data indicate that DON is stable in silage under most conditions or may be degraded to a limited extent. Aflatoxin B1 produced in maize in the field has been found to be degraded slowly in maize silage (Kalac and Woolford 1982). This obser-vation was confirmed in a recent French study, in which a 3-fold decline of aflatoxin B1 was detected during nine months storage of maize silage (Garon et al. 2006). Likewise, partial degradation of ochratoxin A, a mycotoxin that is associated with small grain cereals, has been observed in ensiled barley (Rotter et al. 1990). No information is available about the stability of fumonisins in silage, but probably these mycotoxins are stable.

Contradictory information is available about the stability of ergot alkaloids produced by Claviceps and Neotypho-dium species in silage. Health problems of cattle have been associated with high concentrations of ergovaline in silage from endophyte infected perennial ryegrass (Lean 2001) and with high concentrations of ergocryptine in silage from maize that was contaminated with a weed containing Claviceps ergots in the field (Naude et al. 2005). This indicates that these substances were at least partially stable in silage. On the other hand, the concentration of C. purpurea ergot alkaloids (ergometrine, ergotamine and ergocryptine) in extensively managed grasslands were strongly decreased when the grass was ensiled (Wyss et al. 1997). The Penicillium mycotoxins roquefortine C and mycophenolic acid appear to be stable in silage, whereas PR-toxin and patulin are presumably unstable. In con-trast to roquefortine C and mycophenolic acid, PR-toxin and patulin are rarely detected in silage. Experiments with blue-veined cheeses manufactured with P. roqueforti strains showed that PR-toxin was degraded and detoxified as a result of a chemical reaction with ammonia and free amino acids (Scott and Kanhere 1979). Many types of silage contain relatively high concentrations of ammonia and free amino acids, so reaction with these compounds may be the reason that PR-toxin is often undetectable. For patulin a similar mechanism may apply. Patulin is known to react with SH-groups of cysteine and other sulphur containing amino acids in protein-rich environments and is known to be inactivated in fermented foods, such as wine, beer and cheese (Ciegler et al. 1976, Scott 1984).


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Occurrence of mycotoxins in silage

Information about the incidence and concentrations of mycotoxins in silages is relatively scarce, in particular for silages other than maize silage. Table 6 gives an overview of results from surveys in Europe and the United States of America for DON and zearalenone in silage, conducted between 1989 and 2007. The data show that the inci-dence of DON in maize silage was high: in six out of seven surveys the incidence was 72 to 100%, in one it was 42%. The average DON concentration of positive samples in these surveys varied between 0.60 and 1.85 mg kg-1. The incidence of zearalenone in maize silage was also high, but generally lower than that of DON: in five out of six surveys the incidence was 32 to 59%, in one it was 96%. The average zearalenone concentration of positive samples varied between 0.05 and 0.45 mg kg-1. Information about the occurrence of DON and zearalenone in si-lages other than maize silage are scarce. In a survey in the Netherlands between 2002 and 2004, DON was not detected in 120 grass silage samples and in 3 of 30 (10%) wheat silages, whereas zearalenone was detected in 7 of the grass silages (6%) and none of the wheat silages (Driehuis et al. 2008a). Fumonisin contamination of maize is widespread, as indicated by the high incidence of fumonisins in maize and maize by-products intended for use in animal feed (Binder et al. 2007). Incidence of fumonisins in maize silage is also likely to be high, since evidence indicating degradation of fumonisins in silage is lacking. This is confirmed by the results of a survey in Midwest-ern USA in 2001 and 2002, in which fumonisin B1, B2, and B3 were detected in, respectively, 97%, 72% and 57% of maize silages and average concentrations in positive silages were 0.615, 0.093, and 0.051 mg kg-1, respectively (Kim et al. 2004). In contrast, in the survey in the Netherlands described earlier, fumonisin B1 and B2 were detected in only 1.4% of the maize silages (Driehuis et al. 2008a). This low incidence probably reflects that the environmen-tal conditions of forage maize growth in the Netherlands are not favourable for infection by fumonisin producing moulds (F. verticillioides). Aflatoxin B1 has been detected in maize silages in some surveys, but in most surveys this mycotoxin was undetectable in silages (Scudamore and Livesey 1998, Whitlow and Hagler 2005, Storm et al. 2008, Driehuis et al. 2008a). The occurrence of aflatoxins in silage is associated with geographical regions with a tropical or sub-tropical climate and is generally field-derived. However, there are reports indicating development of aflatoxins in poorly preserved silage with extensive mould infestation (Gonzalez Pereyra et al. 2008, Gonzalez Pereyra et al. 2011). Table 6. Incidence and average and maximum concentrations of the Fusarium mycotoxins DON and zearalenone (ZEA) in silage in different surveys.

Myco-toxin

Silage crop Location Year(s)

Percentage positive (total number)1

Concentration (mg kg-1)2

ReferenceAverage (of positive samples)

Maximum

DON Maize North Carolina, USA 1989-1993 76% (106) 1.85 - Whitlow and Hagler 2005

DON Maize Austria 1995-1999 91% (418) 0.75 2.8 Hochsteiner and Schuh 2001

DON Maize Germany 1998 79% (24) 1.61 9.86 Dänicke et al. 2000

DON Maize Pennsylvania, USA 2001-2002 42% (62) 0.6 3.7 Mansfield et al. 2005

DON Maize Netherlands 2002-2004 72% (140) 0.85 3.14 Driehuis et al. 2008a

DON Maize Netherlands 2005 100% (16) 0.93 2.39 Driehuis et al. 2008b

DON Maize Denmark 2007 100% (20) 1.06 5.09 Storm et al. 2010

DON Wheat Netherlands 2002-2004 10% (30) 0.62 1.17 Driehuis et al. 2008a

ZEA Maize North Carolina, USA 1989-1993 32% (93) 0.45 - Whitlow and Hagler 2005

ZEA Maize Germany 1993-1995 38% (44) 0.05 0.17 Dänicke et al. 2000

ZEA Maize Austria 1995-1999 59% (149) 0.07 0.6 Hochsteiner and Schuh 2001

ZEA Maize Germany 1998 96% (24) 0.13 1.07 Dänicke et al. 2000

ZEA Maize Netherlands 2002-2004 49% (140) 0.17 0.94 Driehuis et al. 2008a

ZEA Maize Netherlands 2005 50% (16) 0.15 0.48 Driehuis et al. 2008b

ZEA Grass Netherlands 2002-2004 6% (120) 0.09 0.31 Driehuis et al. 2008a

ZEA Grass Netherlands 2005 13% (16) 0.13 0.21 Driehuis et al. 2008b1 The percentage of positive samples and total number of samples analysed.2 Concentration in dry matter.


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As described earlier, the occurrence of ensilage-derived mycotoxins produced by P. roqueforti and P. paneum, A. fumigatus and M. ruber relates to the preservation quality of silage and depends on infiltration of oxygen during storage or during feeding-out. Very low incidences of roquefortine C and mycophenolic acid in maize and grass silages were found in a survey in the Netherlands, in which samples were analysed that were taken relatively shortly after ensiling (3 to 6 weeks) from completely sealed silages that were not yet in use for feeding purposes. Roquefortine C was detected in none of 140 maize silages and in one of 120 grass silages, and mycophenolic acid was detected neither in maize nor in grass silages (Driehuis et al. 2008a). In contrast, samples taken from opened silages at 16 Dutch dairy farms showed high incidences of roquefortine C and mycophenolic acid in surface layers of maize silages (50%) and grass silages (19%). Concentrations of both mycotoxins were highest in surface areas with visible moulds. For example, the average roquefortine C concentration in samples of visibly moulded maize silage was 16 times higher than that in silage surface samples and 270-fold higher than that in silage centre sam-ples (Driehuis et al. 2008b). Similar observations were made in studies conducted in Germany, not only with re-spect to the incidence and distribution in silages of roquefortine C and mycophenolic acid but also with respect to the incidence and distribution of the M. ruber mycotoxins monacolin K and citrinin and the A. fumigatus my-cotoxins gliotoxin, verruculogen and fumigaclavin C (Richter et al. 2009).

DON, zearalenone, roquefortine C and mycophenolic acid were identified as the mycotoxins with the highest in-cidence in a survey of mycotoxins occurring in the total diet of high-yielding dairy cows at 24 dairy farms in the Netherlands (Driehuis et al. 2008b). As expected, roquefortine C and mycophenolic acid were detected in ensiled feeds only. DON and zearalenone were detected in compound feed, feed commodities and ensiled feeds. Maize silage was found to be the most important source of all of these four mycotoxins in the diet. Maize silage repre-sented on average 30% of the total daily feed intake of the animals, but contributed about 80% of the total dietary intake of DON and zearalenone and more than 95% of that of roquefortine C and mycophenolic acid.

Metabolism of mycotoxins in ruminants and impact on food safetyThe significance of a mycotoxin occurring in feed with respect to animal health and the safety of animal food prod-ucts for consumers depends on its metabolism in the animal, its toxicological effects in man and animals, and its carry-over from feed into milk, meat or organs. After intake via silage or another feed, mycotoxins, like other xe-nobiotics, follow the typical pharmacokinetic cascade of uptake from the gastro-intestinal tract to the blood, in-ternal distribution, metabolism, storage/remobilization and excretion. The rumen has an important function in the metabolism of mycotoxins in ruminants. It contains a complex and dense microflora with a high biodegradative power. Some mycotoxins are rapidly metabolized in the rumen into less toxic metabolites, some are transformed into equally toxic or more toxic metabolites, while some are not transformed at all (Driehuis et al. 2010). DON and ochratoxin A are examples of mycotoxins that are transformed into less toxic metabolites in the rumen. For that reason cattle are less sensitive to these mycotoxins than non-ruminant animals such as pigs. Zearalenone is transformed in the rumen into different metabolites, with varying toxic activities. Fumonisins and aflatoxin B1 are not metabolized in the rumen. Aflatoxin B1 is transformed into aflatoxin M1 in the liver of ruminants. Aflatoxin M1 is less mutagenic and genotoxic than aflatoxin B1, but the cytotoxicity of aflatoxin M1 and B1 is similar. Information concerning the metabolism of Claviceps and Neotyphodium ergot alkaloid mycotoxins and A. fumigatus mycotoxins is lacking. Research on the metabolism of roquefortine C and mycophenolic acid in cattle is currently in progress.

Aflatoxin B1 is the only mycotoxin with significant carry-over into milk: between 1 and 6 percent is excreted in milk (as aflatoxin M1) (EFSA 2004). Sixty countries now have regulations for aflatoxin M1 in milk. The two most prevail-ing limit concentrations for aflatoxin M1 in milk are 0.05 µg kg-1 and 0.5 µg kg-1 (FAO 2004). Based on a quantitative risk assessment the Codex Alimentarius established a limit concentration for aflatoxin M1 in milk at 0.5 µg kg-1 (FAO 2001). Carry-over rates of DON, zearalenone, fumonisin B1, ochratoxin A and the alkaloid ergovaline appear to be at least about 100-fold lower than that of aflatoxin B1/M1 (Driehuis et al. 2010). Carry-over rates of other mycotox-ins frequently occurring in silage have not been assessed experimentally. However, there are no indications that significant transfer of these mycotoxins into milk occurs. More detailed information on the metabolism of myco-toxins in ruminants and their carry-over into milk can be found elsewhere (Spahr et al. 1999, Barug et al. 2006).


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Prevention of mycotoxins in silageRegarding prevention strategies, a distinction is made between field-derived and ensilage-derived mycotoxins. Pre-vention of field-derived mycotoxins focuses on two areas: reduction of the infection pressure of moulds and reduc-tion of the susceptibility of the plant to fungal infections (Council for Agricultural Science and Technology 2003).

The most important prevention strategy for ensilage-derived mycotoxins is to restrict exposure of silage to oxy-gen. At ensiling, oxygen is entrapped in the ensiled mass, but this is rapidly consumed (within hours) by respira-tory activity of the plant and (facultative) aerobic microorganisms. Once the silo is filled, the material should be protected from oxygen as quickly as possible, for instance by sealing with sheets of plastic or foil. Where appro-priate, measures should be taken to prevent damage to the seal. However, since in practice the sealing of silos is never completely airtight, it is inevitable that surface layers will be exposed to air and some air will penetrate the silage during storage. A high packing density of the silage is important because it restricts air ingress during stor-age and after opening of the silo for feeding, when exposure to air becomes inevitable. Another factor of impor-tance is the silage removal rate during feeding. Maintaining a high silage removal rate minimizes ingress of air into the material behind the silage face. Finally, when preventive measures have not been successful, visibly moulded silage should be discarded before feeding, since these areas are hot-spots of ensilage-derived mycotoxins, as dis-cussed previously. More information on the impact of oxygen on silage spoilage and mould growth can be found elsewhere (Honig 1991, Pahlow et al. 2003).

In conclusion, silage can be contaminated with a variety of mycotoxins, originating from infection of the crop by moulds in the field or from growth of moulds in silage during storage or feeding-out. Prevention of mycotoxin contamination of silage requires different strategies. Field-derived mycotoxins can be reduced by application of recommended agricultural practices in crop production, whereas ensilage-derived mycotoxins can be reduced by application of adequate silage management, with emphasis on prevention of aerobic spoilage. DON and zearale-none are the mycotoxins with the highest incidence in silages and maize silage is the most important source of these mycotoxins. Based on current knowledge, aflatoxin B1 is considered the only silage-associated mycotoxin of potential concern for the safety of milk and dairy products, due to its high carry-over rate into milk as aflatox-in M1 and the high toxicity of this toxin. However, the incidence of aflatoxin B1 in silages is very low in most parts of the world and national and international surveys of aflatoxin M1 in milk indicate a high degree of compliance with existing legislation (World Health Organisation 2001, European Food Safety Authority 2004). Relatively little information is available about the effects that the ensilage-derived mycotoxins produced by Penicillium species and A. fumigatus can have on animal health and productivity. This subject should be investigated in more depth in future research.

AcknowledgementsThe author acknowledges the Dutch Dairy Organisation (NZO, Zoetermeer, the Netherlands) and the Dutch Dairy Board (PZ, Zoetermeer, the Netherlands) for their financial support of parts of the research described in this pa-per. The author also expresses his grateful thanks to Cor van den Boogaard (FrieslandCampina, Amersfoort, the Netherlands), Margreet Hovenkamp (NZO) and Marjon Wells-Bennik (NIZO food reasearch) for their comments on the manuscript.

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AGRICULTURAL AND FOOD SCIENCEP. Huhtanen et al. (2013) 22: 35–56

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An overview of silage research in Finland: from ensiling innovation to advances in dairy cow feeding

Pekka Huhtanen1, Seija Jaakkola2 and Juha Nousiainen3

1Department of Agricultural Research for Northern Sweden, Swedish University of Agricultural Sciences,

S-901 83 Umeå, Sweden 2Department of Agricultural Sciences, PO Box 28, FI-00014 University of Helsinki, Finland

3Valio Ltd., Farm Services, PO Box 10, FI-00039 Valio, Finland


Because of the climatic conditions, the Finnish milk production research has focused to improve the utilisation of grassland, mainly as conserved forages. The main research areas have been ensiling, evaluation of the forage feeding value, predicting nutrient supply from grass silage-based diet and the effects of forage quality and concen-trate supplementation on milk production responses. Due to changes in ensiling technologies and variety of forage crops new silage additives have been adopted. A centralized system for the analysis of forage energy value is based on NIRS calibration. It was calibrated against in vitro pepsin-cellulase solubility method that was validated against in vivo digestibility. The concentration of indigestible neutral detergent fibre was found to be a useful parameter both in empirical models predicting forage digestibility and mechanistic rumen models predicting the amounts of absorbed nutrients. Models predicting relative intake potential of forages and total diet were developed, and an intake model combining animal and diet effects independently of each other was developed. Using meta-analysis approaches a nutrient response model was developed for dairy cows for milk, energy corrected milk and protein yield. Feed evaluation, intake and nutrient response models form now the basis of practical Finnish ration formula-tion system that can optimize diets according to maximum income over feed cost in addition to minimum feed cost.

Key words: grass silage, ensiling, feed evaluation, nutrient intake, milk production

Introduction

Milk production systems in different climatic zones have developed to utilize local feed resources. Due to the short grazing period (100–120 days) in Finland grazed grass cannot contribute more than 20–25% of total feed energy intake for dairy cows. This has increased the importance of conserved forages in dairy cow rations. Relative com-petitiveness of grass in Finland is high, since in the main milk production regions grass dry matter (DM) yields are more than two-fold compared with cereal grains (Kangas et al. 2010). Grasses can utilize efficiently the long days in early summer, and daily DM growths exceeding 200 kg are common (e.g. Kuoppala et al. 2008). The nutritive value of forages in terms of digestibility is high due to the relatively cool climate and long day length which de-lay the lignification of cell walls (Van Soest et al. 1978, Deinum et. al. 1981). Earlier high concentrate costs and a shortage of protein supplements favoured forage-based feeding systems, but since Finland joined the EU in 1995 subsidised grain and protein prices have reduced the competitiveness of grassland production.

Because of the climatic conditions, Finnish milk production research has focused to improve the utilisation of grassland, mainly as conserved forages. The main research areas have been ensiling, evaluation of the forage feed-ing value, predicting nutrient supply from grass silage-based diets and the effects of forage quality and concen-trate supplementation on milk production responses. More recently, environmental aspects of milk production and product quality, mainly milk fatty acid composition, have been important research subjects. Finnish silage re-search was previously reviewed by Lampila et al. (1988) and Huhtanen (1998). The objectives of this paper are to review the recent achievements of the Finnish silage research in the areas of silage fermentation, evaluation of silage feeding value, feed intake and milk production relative to international literature with the special emphasis on ensiling, feed evaluation, feed intake and milk production.



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Ensiling

The control of major preservative factors of silage (e.g. pH, water activity, epiphytic flora), and their interactions, is the basis for biologically and economically efficient silage production. Virtanen (1933) was first to show system-atically the importance of low pH and inhibition of plant and microbial enzymes in silage preservation. By using hydrochloric and sulphuric acids he introduced the A.I.V.-method and established the principle of rapid achieve-ment of pH 4 to suppress respiration of plant cells, to prevent degradation of proteins and vitamins and to avoid clostridial fermentation. He also showed that different crops, e.g. leguminous plants vs. grasses, require different amounts of acids to achieve target pH.

Ensilability of silage crops Ever since the innovation of A.I. Virtanen, the control of silage fermentation by silage additives has been the core of ensiling in Finland. In the late 1960’s, combinations of inorganic acids and organic acids, mainly formic acid (FA), and additives containing formaldehyde were in the focus of research (Ettala et al. 1975). The corrosive nature of inorganic acids and other hazardous effects of formaldehyde were reasons to abandon these products later.

The research done in Norway (Saue and Breirem 1969) demonstrated the effectiveness of FA which became the most commonly used silage additive also in Finland. Direct acidification using a relatively high application rate of FA (approximately 4 L t-1, expressed as 100% w/w) has facilitated that relatively wet and low sugar crops, predomi-nantly timothy, meadow fescue and some legumes, can be ensiled successfully. The climatic conditions in Finland exclude the more easily ensiled crops like perennial ryegrass and fodder maize. This highlights the importance of adjusting harvesting and ensiling management according to crop characteristics and local conditions (Lampila et al. 1988). The most important ensilability factors of crops were clearly presented by Weissbach et al. (1974) in an equation predicting anaerobic stability and clostridial development from crop dry matter (DM), buffering capacity (BC) and water soluble carbohydrates (WSC).

Increasing size of Finnish farms and demand for high labour efficiency in the ensiling systems have been the ma-jor reasons for the technological development, like pre-wilting and harvesting techniques related to it. Although some of the techniques, e.g. chopping with harvesters and additive applicators, have had some important posi-tive effects on silage quality the biological efficiency has not necessarily increased. Gordon (1989) concluded in Northern Ireland that a harvesting system based on wilting decreased the output of animal product per hectare by 13% as compared to a direct-cut system. The increasing popularity of wilting, a concomitant decrease of ap-plication rate of FA and a shift to using biological additives have all changed the challenges of ensiling. Effluent losses and the risk of clostridial fermentation decreases with increasing DM content but at the same time wilt-ing may increase nutrient losses during drying, impair the microbiological quality of crop and expose the silage to aerobic deterioration.

Wilting grass to DM content of 300 g kg-1 did not alone prevent clostridia (Ettala et al. 1982) but in favourable harvesting conditions it supports achievement of good fermentation quality and feeding value without additives (Heikkilä et al. 2010). However, an ensiling system based on baling of high DM grass without additive is more sus-ceptible to unfavourable harvesting conditions and to lower feeding value of silage as compared to ensiling in bunker silo with lower DM content and FA-based additive (Jaakkola et al. 2008). In spite of the low butyric acid and ammonia N content of untreated bale silage having relatively high DM content (380 g DM kg-1), the use of inoculants or FA improved milk production and sensory quality of milk (Heikkilä et al. 1997). This demonstrates that fermentation parameters of high DM silage insufficiently describe the value of silage in animal production. The unpredictability of weather conditions and variation in crop DM and WSC concentration and epiphytic flora are important factors to be considered in the risk management of ensiling and when making decision on the use of additives. Currently 50–60% of the Finnish farm samples analysed in the laboratory of Valio Ltd are from silag-es treated with acid based additives, 25–30% from silages treated with biological additives and 10–15% from un-treated silages (J. Nousiainen, personal communication).

A risk of undesirable fermentation is higher when forage and grain legumes with high BC are ensiled as compared to grass species. Slight wilting of lucerne, galega, red clover and lotus to 250 g DM kg-1 alone was not sufficient to avoid poor fermentation in research made in Germany, Sweden and Finland (Pahlow et al. 2002). Wilting to 400 g DM kg-1 prevented the production of butyric acid, but silage quality was further improved by the use of ad-ditives. The challenging ensiling characteristics of forage legumes are alleviated in a mixture with grass species having lower BC. Similarly, when whole-crop field beans and field peas were ensiled without an additive, inclu-sion of 0.25 to 0.50 of wheat ensured a good fermentation (Pursiainen and Tuori 2008). However, common vetch with a high BC and a low WSC concentration was best ensiled using FA to prevent extensive protein degradation.


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Preservation of small grain cereal crops has been successful in our conditions when harvested at the dough stage (300–400 g DM kg-1) and when ensiling is based on a low pH generated by fermentation and/or acid based addi-tives (Vanhatalo et al. 1999b, Jaakkola et al. 2009). Ensiling of cereal crops either untreated or treated with urea resulted in clostridial fermentation (Alaspää 1986). The low DM content of whole crop cereals even at a late ma-turity in our conditions does not support alkaline preservation. Extensive research in 1970’s in Finland demon-strated that ensiling of high moisture grain is an efficient storage method as an alternative to grain drying. Early harvest, crimping and treatment with an additive diminishes the challenges of short growing season, increases the grain yield and reduces the use of fossil fuels. In the later studies the use of dry barley and ensiled barley re-sulted in the same animal performance in growing cattle (Huhtanen 1984) and dairy cows (Jaakkola et al. 2005).

Restriction of fermentation The variation in crop characteristics and application rate of FA, and probably the variation in the evenness of FA application, in different experiments explains the inconsistent results obtained in the fermentation quality of FA-treated silage and consequently in animal responses (Harrison et al. 2003, Kung et al. 2003). A high application rate of FA restricts fermentation resulting in lower content of total acids (TA; lactic acid plus volatile fatty acids [VFA]) and ammonia N, and higher content of residual WSC in silage as compared with extensively fermented untreated or inoculated silage (Chamberlain et al. 1992, Heikkilä et al. 1998, Shingfield et al. 2002a). With lower FA applica-tion rates the differences in fermentation profiles are smaller. The low ammonia N content in silage reveals that FA treatment inhibits the conversion of herbage protein to non-protein-nitrogen (NPN) and increases the propor-tion of peptide N in silage NPN as compared with untreated silage (Nagel and Broderick 1992, Nsereko and Rooke 1999). The extent of silage fermentation thus dictates the amount and type of nutrients available for animals. Consequently, the nutritive value of restrictively fermented silages can be equal to that of respective barn dried forages (Jaakkola and Huhtanen 1993).

The effects of increasing level of FA on silage fermentation pattern have been linear (Jaakkola et al. 2006a) or cur-vilinear (Chamberlain and Quig 1987, Jaakkola et al. 2006b). This indicates that the balance and survival of desir-able and undesirable microorganisms may differ with the characteristics of ensiled material and the additive. Due to the corrosive nature and handling problems of pure FA the commercial additives generally contain salts of FA like ammonium and sodium formate. Ammonium tetraformate maintains good silage quality if applied accord-ing to the molar concentration of acid (Randby 2000). Replacing FA (5.1 kg t-1) with increasing proportion of am-monium formate up to 45% delayed the drop of pH in unwilted (210 g DM kg-1) and wilted (406 g DM kg-1) grass silage while the quality of silage was not compromised (Saarisalo and Jaakkola 2005).

Even a low application rate of FA disrupts cell membranes and releases soluble cell contents (Kennedy 1990, Jaakkola et al. 2006a). As a result, in wet material increased effluent losses partly offset the advantages of reduced fermentation losses. As a positive effect, cell wall degradation leads to efficient consolidation and may increase storage density as compared with untreated silage. This could partly explain why the use of a high rate of FA may result in good aerobic stability and low yeast count despite restricted fermentation and high residual WSC content in silage (Saarisalo et al. 2006) which often have been considered risk factors for aerobic stability.

Formic acid has a selective bactericidal effect but it is not specifically effective against yeasts (McDonald et al. 1991). More antifungal alternatives applied in a combination with FA have sometimes improved (Heikkilä et al. 2010) but sometimes not (Lorenzo and O’Kiely 2008) the aerobic stability as compared to untreated silage. The increased risk of aerobic deterioration concerns mainly wilted FA silages since low-DM or minimum wilted FA-treated grass silages have been shown to be more stable than untreated and inoculated silages (Pessi and Nousiainen 1999). As underlined already in the studies of Ettala et al. (1982) the feeding rate and good silo management are the key issues in preventing aerobic deterioration. However, even a small amount of oxygen may start the growth of yeasts and moulds responsible for aerobic deterioration. The use of combinations of hexamethylene-tetraamine, sodium nitrite, sodium benzoate and sodium propionate has improved the quality and storage stability of silage made from wilted grass (Lingvall and Lättemäe 1999, Knicky and Spörndly 2009).

Stimulation of fermentation The interest on enzymes and inoculants as silage additives increased in Finland in the late 1970’s (Vaisto et al. 1978, Poutiainen and Ojala 1982). Compared to early products, the improvements in inoculants and better un-derstanding of the conditions in which inoculants are effective have generally improved the results (Kung et al. 2003). Inoculants alone are unable to produce enough lactic acid to lower the pH to an acceptable level if the WSC


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content of the original crop is a limiting factor (Seale et al. 1986). A content of 25–30 g kg-1 in fresh material has been suggested to ensure sufficient production of fermentation acids in untreated silage (Wilkinson et al. 1983, Pettersson 1988). Accordingly, a high WSC content of grass (32 g kg-1) resulted in minor differences in the fermen-tation of untreated silage and silages treated with inoculants or enzymes (Rauramaa et al. 1987). The amount of fermentable substrate can be increased by using efficient enzymes as an additive or the ensilability can be in-creased by wilting which increases the content of WSC on a fresh basis. The decreased rate of N fertilization has also enhanced ensilability by increasing WSC content of grass. However, the concomitant lower nitrate content may have an opposite effect since nitrite and nitric oxide, the reduction products of nitrate, effectively inhibit clostridia (Spoelstra 1985, McDonald et al. 1991).

Another purpose of using cell-wall degrading enzymes as an additive was to increase the rate and/or extent of digestion of cell wall carbohydrates in the rumen. The degradation of fibre in the silo was shown to increase with increasing cellulase level (Vaisto et al. 1978, Huhtanen et al. 1985). However, enzyme treatment had no consistent effect on organic matter digestibility but it decreased fibre digestibility in cattle (Jaakkola and Huhtanen 1990, Ja-akkola et al. 1990) and in sheep (Jaakkola 1990; Table 1). Enzymes clearly affected the most easily degradable frac-tion of fibre which is also completely degraded in the rumen. On the other hand, with a successful combination of cell-wall degrading enzymes even a high-moisture (172 g kg-1) and low-WSC (16 g kg-1) grass was well preserved (Jaakkola et al. 1991). Generally the ensiling results with enzymes have been inconsistent. Kung et al. (2003) sug-gested that e.g. the lack of synergistic activities of enzyme complexes or environmental factors (pH, temperature) may be the potential reasons for failures in improving silage fermentation with enzymes.

Table 1. The effect of enzyme treatment on silage NDF or crude fibre concentration and digestibility, and on organic matter digestibility.

Reference Animal Silage treatment 1) NDF or crude fibre, g kg-1 DM 2)

NDF or crude fibre digestibility 2)

Organic matter digestibility

Jaakkola (1990) Sheep Untreated 666 0.707 0.678

Formic acid 619 0.695 0.683

Cellulase 200 ml t-1 609 0.672 0.677

Cellulase 400 ml t-1 596 0.666 0.675

Cellulase 800 ml t-1 579 0.619 0.644

Jaakkola and Huhtanen (1990) Cattle Formic acid 301 0.655 0.739

Cellulase 400 ml t-1 272 0.583 0.724

Jaakkola et al. (1990) Cattle Formic acid 303 0.600 0.701

Cellulase 150 ml t-1 284 0.540 0.6881) Formic acid application rate 4 L t-1 (as 100%), enzyme treatments: glucose oxidase 50 000 IU t -1+ cellulase produced by Trichoderma reesei, activity 25 000 nanokatal HEC (hydroxyethyl cellulose) ml-1.2) NDF (Jaakkola 1990), crude fibre (Jaakkola and Huhtanen 1990, Jaakkola et al. 1990)

Selection of effective bacteria strains for the use as inoculants is crucial for successful ensiling. A screening meth-od using grass extract proved to be useful in strain selection (Saarisalo et al. 2007). Lactobacillus plantarum strain (VTT E-78076) having a broad-spectrum antimicrobial activity against gram positive and gram negative bacte-ria, and Fusarium moulds, was originally isolated from beer (Niku-Paavola et al. 1999, Laitila et al. 2002) but was shown to be also efficient in producing lactic acid, lowering pH rapidly and especially decreasing the ammonia-N production in grass silage (Saarisalo et al. 2006, Saarisalo et al. 2007). However, the antimicrobial properties were not efficient enough to improve aerobic stability (Saarisalo et al. 2006).

One possibility to overcome the inability of lactic acid to prevent yeast and mould growth is to use chemical addi-tives in combination with the inoculants (Weissbach et al. 1991). Skyttä et al. (2002) showed that a combination of a selected inoculant, potassium sorbate and sodium benzoate inhibited in vitro the growth of four spoilage yeast strains isolated from grass silage. In two ensiling trials the combination of lactic acid bacteria and sodium benzoate (0.3 g kg-1) had variable effects on the aerobic stability of wilted grass silage showing that the minimum effective application rate of sodium benzoate varies (Saarisalo et al. 2006). As shown in the meta-analysis of Kleinschmit and Kung (2006) improved aerobic stability has been observed in different types of forages when acetic and pro-pionic acid production in silage fermentation is increased with L. buchneri inoculation. In our experiment, buffered propionic acid and a combination of L. plantarum and sodium benzoate were more efficient than a combination of L. plantarum and L. buchneri to prevent heating of high DM silage (Jaakkola et al. 2010).


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Feed evaluationSilage fermentation quality

Practical on-farm silages show wide variation in the fermentation quality due to e.g. crop characteristics, additives used and ensiling technologies. In-silo fermentation can influence the profile of absorbed nutrients and especially intake potential compared with fresh herbage (Huhtanen et al. 2007). Silage quality assessment with traditional wet chemistry for on-farm feeds is too expensive. For the analysis of farm samples Moisio and Heikonen (1989) developed a rapid electrometric titration method (ET). From the titration curve the concentrations of lactic acid, VFA, WSC, amino acid carboxyl groups and the protein degradation products (ammonia, amines) can be predicted (Moisio and Heikonen 1989). Later work revealed that ET over-predicted WSC, especially for extensively fermented or very dry samples. The system has been used for on-farm silage assessment for more than 20 years, with the ex-ception that WSC are currently determined with the NIRS from dried samples. A comparable ET system has been also studied in UK (Porter et al. 1995) as an alternative or an additional silage measurement to either wet or dry NIRS (Park et al. 1998). However, direct comparisons between dry or wet NIRS and ET have shown that ET can be more accurate especially for VFA and ammonia-N (M. Hellämäki, personal communication).

Silage composition with reference to nutrient availability The first step in a successful feed chemistry system is to divide forage DM into (1) cell contents that can be digest-ed by mammalian enzymes and (2) a cell wall fraction that can only be digested by anaerobic microbial fermenta-tion. The proximate feed analysis (Weende system) has been available for over 100 years, and it divides feed OM into crude protein (CP; 6.25 × N), crude fat (EE), crude fibre (CF) and nitrogen free extracts (NFE). Within the sys-tem, CF should represent the least available and NFE readily available feed components with a high true digest-ibility. The primary problems associated with NFE and CF fractions (Van Soest 1994, Huhtanen et al. 2006b) were realised by Paloheimo (1953), who initiated research to develop improved analytical methods for plant cell wall. In the pioneering work, Paloheimo and co-workers (Paloheimo and Paloheimo 1949, Paloheimo and Vainio 1965) used a weak hydrochloric acid and a two-stage ethanol extraction to remove cellular contents to describe vege-table fibre. Despite the correct criticism against fractionating feed carbohydrates into CF and NFE, these methods were too laborious, not applicable to faecal samples and the fibre residue was contaminated with protein. Based on these ideas, Van Soest (Van Soest 1967, Van Soest and Wine 1967) introduced the neutral detergent (ND) frac-tionation, which mainly resolved these drawbacks. The evaluation based on a wide dataset of silages (Huhtanen et al. 2006b) clearly demonstrated the biological weaknesses of the proximate feed analysis.

Neutral detergent (ND) fractionation (Van Soest 1967) divides forage DM into neutral detergent fibre (NDF) and neutral detergent solubles (NDS). Originally NDS was calculated as DM – NDF, but because ash does not provide energy, expressing NDS as organic matter (OM – NDF) may be preferable. True digestibility of the NDS fraction is close to unity (Van Soest 1994, Weisbjerg et al. 2004) when estimated by the Lucas test. The Lucas test allows es-timation of ideal nutritional entities that have a uniform digestibility across a wide range of feedstuffs by plotting the digestible nutrient concentration in DM against the nutrient concentration in DM. The slope of regression pro-vides an estimate of the true digestibility and the intercept is an estimate of the metabolic and endogenous fae-cal matter (M). Huhtanen et al. (2006b) reported a value of 0.963 for true NDS digestibility for different forages. Regrowth silages had a lower true NDS digestibility (0.925), the reasons for which are not known. Based on the Lucas principles the concentration of digestible OM (DOM; g kg-1 DM) can be expressed as:

DOM (g kg-1 DM) = NDS + dNDF - M [1]

Given that digestible NDF (dNDF) = NDF × NDF digestibility coefficient (NDFD), NDS = OM - NDF, M = 100 and di-gestibility of NDS = 1.00, the equation [1] can be written as:

DOM (g kg-1 DM) = 1.00 × (OM - NDF) + NDF × NDFD - 100 [2]

The equation [2] indicates that variation in DOM and OMD (OM digestibility) of forages is primarily a function of the concentration and digestibility of NDF, implying that the main emphasis in the evaluation of forage feeding value should be focused to the NDF.


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A fraction of NDF in forages is completely indigestible even if it is subjected to digestion for an infinite time. This fraction can be defined as indigestible NDF (iNDF), and it can be determined e.g. by extended incubations in situ (Huhtanen et al. 1994) or in vitro (Van Soest et al. 2005). We have used a 12-d in situ incubation using bags with a small pore size (6 – 17 µm) to avoid particle losses. Potentially digestible NDF (pdNDF) is then calculated as:

pdNDF (g kg-1 DM) = NDF - iNDF [3]

Since iNDF is by definition a uniform nutritional entity with constant zero digestibility, equation [2] can be rewrit-ten as:

DOM (g kg-1 DM) = (OM - NDF) + pdNDF × pdNDFD - 100 [4]

where pdNDFD is pdNDF digestibility. This equation indicates that variation in DOM is a function of iNDF concen-tration and pdNDFD. The smaller coefficient of variation (4.1 vs. 11.4%) and range (0.79–0.94 vs. 0.48–0.87) in pdNDF digestibility compared with total NDF digestibility for the wide range of silages (Huhtanen et al. 2006b) indicates that pdNDF is a more ideal nutritional entity than total NDF. Digestibility of pdNDF was on average 0.85 with a mean faecal pdNDF output of 60 (sd 23; range 13–105) g kg-1 DM intake (Huhtanen et al. 2006b). Faecal pdNDF can be defined as updNDF (= faecal NDF - iNDF) that represents the loss of potentially digestible OM in addition to obligatory losses of M.

Prediction of silage digestibility Digestibility measured in sheep fed at maintenance still forms the basis of many feed evaluation systems. How-ever, this method is not applicable for on-farm silages, and even not often for research samples. Hence, much re-search has been conducted to develop OMD prediction systems that are suitable for extension purposes i.e. that are rapid, accurate, precise and inexpensive. For this purpose, empirical models based on silage composition, in vitro methods using either rumen fluid or commercial fibrolytic enzymes and several in situ incubation procedures have been studied. In Finland, a database (n = 86) including grass and legume silages harvested at different maturity with detailed chemical analysis and in vivo digestibility in sheep has been collected (see Huhtanen et al. 2006b) to standardize in vitro or in situ OMD prediction models. In carefully conducted in vivo trials measurements of OMD are associated with a SD of 0.02 units (Van Soest 1994). For studies conducted according to Latin square designs the residual SD (RSD) was 0.014 units (Nousiainen 2004); i.e. determination of forage in vivo OMD in 4 × 4 Latin squares would be associated with a minimum inherent error of 0.007 units. However, the development of any prediction model for silage OMD should take in account inter- and intra-laboratory variation in both in vivo and in vitro OMD measurements and laboratory analyses. To tackle this problem in Finland, we adopted a strategy that in vivo and in vitro determinations as well as laboratory analyses and NIRS calibration are conducted only in one or two forage laboratories with standardized methods. Supporting this strategy, Hall and Mertens (2012) reported relatively high 95% probability limits for within-lab repeatability and between-lab reproducibility (0.102 and 0.134, respectively) for in vitro forage NDFD as determined according to the method by Goering and Van Soest (1970).

Many attempts have been made in developing regression equations that relate various chemical components to forage OMD, but without success owing to large interspecies and environmental variation (Van Soest 1994). In the Finnish silage dataset statistically significant relationships between chemical components and OMD were identified, but the prediction error using CP, NDF and ADF as independent variables was not markedly lower than the SD for in vivo OMD (Huhtanen et al. 2006b). Lignin was the best single predictor of OMD, but this entity could only account for proportionately 0.43 of observed variation, whilst the prediction error (0.042) is too high for practical feed evaluation. Van Soest et al. (2005) suggested a universal and constant relationship between lignin and iNDF over several types of forages (iNDF = 2.4 × Lignin). However, evidence from the Finnish forage dataset does not support this, suggesting that biological methods are required for predicting forage iNDF and OMD (Huhtanen et al. 2006b).

Several in vitro laboratory methods have been used for estimating forage OMD. The two-stage rumen fluid in vitro technique by Tilley and Terry (1963) and Goering and Van Soest (1970) are the most widely used methods. Tilley and Terry (1963) demonstrated a close correlation between DMD determined in vivo and in vitro and reported that the values determined in vitro were almost the same as those determined in sheep. However, even with a good lab practice it is important to calibrate any in vitro method using in vivo data to derive reliable prediction equations (Weiss 1994, Nousiainen 2004).


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Due to several practical difficulties in conducting the rumen fluid in vitro method enzymatic in vitro procedures for the determination of forage digestibility have been studied (Jones and Theodorou 2000, Nousiainen et al. 2003a and 2003b). In principle, the methods include removing cell solubles with HCl-pepsin or ND followed by incuba-tion in a buffered enzyme solution. This procedure results OM solubility (OMS) that differs from in vivo OMD in at least two key respects; no metabolic and endogenous matter is produced and the capacity of commercial en-zymes to degrade NDF is substantially less than that of rumen microbes (McQueen and Van Soest 1975, Nousi-ainen 2004a). In predicting in vivo OMD from OMS the coefficient of determination (R2) was 0.804 and RSD 0.025 digestibility units (n = 86, Huhtanen et al. 2006b). Because the relationship was highly dependent on forage type, using a forage specific correction equation increased R2 to 0.925 and decreased RSD to 0.015. With a mixed model regression analysis, RSD was further decreased to 0.010 units, indicating that OMS predicted OMD within a study very accurately. The reduction in RSD can be attributed to differences between sheep used in digestibility trials and/or the contribution of between-year variation in the relationship between OMS and OMD. Using the general OMS correction underestimated the OMD of primary growth grass silages but overestimated OMD in regrowth grass and whole-crop cereal silages (Huhtanen et al. 2006b). The OMS method was also successfully used in pre-dicting OMD for herbage samples taken before ensiling, provided that silages are well-preserved (Huhtanen et al. 2005). Owing to the problems in standardizing OMS method in different laboratories (Nousiainen 2004a), it is recommended that each laboratory should develop their own forage specific correction equations. In conclusion, the OMS method provides a reliable basis for OMD prediction, but caution should be directed to forage specific-ity. A recent comparison (Jančík et al. 2011) of different laboratory methods in predicting OMD revealed that OMS gave substantially higher OMD estimates than empirical iNDF equation or mechanistic model using gas in vitro production kinetics, especially for Lolium perenne. This suggests that specific OMS correction equations may be needed even for different grass species.

Equation [4] suggests that iNDF should correlate closely to forage OMD. Indeed, the evaluation of the Finnish da-taset showed that iNDF correlated with in vivo OMD for silages made from 1st cut and regrowth grass (Nousiain-en et al. 2003b), and over a wider range of silage types (Huhtanen et al. 2006b). The relationship between iNDF and in vivo OMD was more uniform compared with OMD equation based on OMS. Mean square prediction error of OMD was 0.010 for a mixed regression model (within study) and 0.019 for a fixed regression model. A reliable prediction of OMD can be attributed to a more consistent digestibility of pdNDF compared with total NDF and the inverse relationship between iNDF content and the rate of pdNDF digestion. However, iNDF seems to under-estimate the digestibility of legume silages, particularly lucerne, probably because of their higher rate of pdNDF digestion relative to iNDF concentration (Rinne et al. 2006). Precision of OMD estimates was slightly improved when the concentrations (g kg-1 DM) of iNDF and NDF were used:

OMD = 0.882 - 0.00121 × iNDF - 0.00011 × NDF [5]

Prediction error of this model was 0.0174 and 0.0090 for the fixed and mixed model regression, respectively, and the respective parameter estimates were biologically sound. The more recent work with a wider range of forage types (Krizsan et al. 2012) confirmed that empirical OMD equation based on forage iNDF forms a relatively uni-versal basis for NIRS, especially for a more heterogeneous sample population. Under-prediction of OMD for lu-cerne silages by iNDF (Rinne et al. 2006, Krizsan et al. 2012) suggests that this assumption is not always true. An additional advantage of iNDF in forage evaluation is that it can be predicted with a relatively good accuracy by NIRS either on scans from dried feed (Nousiainen et al. 2004a) or faeces (Nyholm et al. 2009). In our digestibil-ity dataset, in vivo OMD could be predicted almost as accurately from iNDF determined by NIRS as with iNDF de-termined by 12-d in situ incubation. However, it must be highlighted that both feed and faecal iNDF calibrations are based on reference values obtained from two laboratories that have standardized in situ procedure with no substantial inter-lab bias in the iNDF values and scans from only one NIRS lab. Evidence from the iNDF ring-test (Lund et al. 2004) suggests that a reliable reference database for NIRS cannot be established by simply compiling data from several labs.

NIRS applications in forage evaluation

Since Norris et al. (1976) first introduced NIRS equations for predicting forage quality, considerable progress has been made to implement NIRS applications for silage analysis. The development of computers, optical devices and calibration soft wares has facilitated this process (Deaville and Flinn 2000). Although individual wavelengths in the NIR spectrum lack specificity to important feed parameters, especially being non-specific for functional proper-ties of feeds (e.g. NDF, digestibility, intake potential), quantitative analysis of forage quality by NIRS is possible by


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calibrating the reflectance spectrum against biologically sound reference methods (Deaville and Flinn 2000, Nousi-ainen 2004). NIRS applications for forage evaluation include quantitative analysis of both cell wall (NDF, iNDF) and cell content (CP, WSC, silage fermentation products) characteristics (Deaville and Flinn 2000, Nousiainen 2004). The scans may be obtained from dried and finely ground or coarse wet samples, although the latter may be less accurate. Interpretation of published NIRS equations reveal that OM digestion and cell wall lignin bonding of for-ages is associated to spectral regions near to 1650–1670 and 2260–2280 nm (Deaville and Flinn 2000). In agree-ment with this, Nousiainen et al. (2004a) demonstrated that the absorbance in these regions was negatively cor-related with the iNDF content of grass silages.

The precision and repeatability of NIRS are known to be much better than any feed chemistry method (Deaville and Flinn 2000). Consequently, within a single lab NIRS calibration statistics often suggests very accurate predic-tion of any feed trait. When several chemical, in vitro and in situ reference methods in calibrating silage OMD were compared (Nousiainen 2004), the calibration statistics for all of them showed high R2 and a low standard error of calibration (SEC) and cross validation (SECV). However, the total error of prediction (in vivo vs. NIRS) was highly dependent on the biological validity of the reference method used. Therefore caution should be used in the choice of calibration method for NIRS. A relatively high correlation (R2 0.23) between the residuals of OMD estimates based on iNDF or OMS in the Finnish dataset (Huhtanen et al. 2006b) suggests that in vivo reference values include some random error. Therefore it is likely that with NIRS the true errors may be smaller than appar-ently estimated. For commercial laboratories OMS method may be the most practical choice to calibrate the NIRS for the prediction of OMD (Nousiainen 2004a, Huhtanen et al. 2006b).

By using forage specific corrections for OMS and a sufficiently diverse range of reference samples, total prediction performance can be considered satisfactory. The standard error of prediction (SEP) for D-value using OMS based calibrations was circa 17–20 g kg-1 DM (Huhtanen et al. 2006b), consistent with a RSD of 14 g kg-1 DM for measure-ments of OMD in digestion trials (Nousiainen 2004). Alternatively iNDF can be used for OMD or D-value calibration for NIRS in one of two ways; (1) predict digestibility with a direct regression equation (Nousiainen 2004) or (2) use a summative method of uniform feed fractions (Huhtanen et al. 2006b). In the future, NIRS may be used to predict forage traits for use in dynamic digestion models. The digestion rate of pdNDF can be calculated from OMD, NDF and iNDF using the Lucas principle for the NDS fraction and constant passage kinetic parameters at maintenance intake (Huhtanen et al. 2006a). Incubation of isolated NDF in automated in vitro gas production system resulted in similar digestion rate of pdNDF as estimated from the in vivo data (Huhtanen et al. 2008c).

y = 1.05x - 1.52R2 = 0.929

RMSPE = 11.5

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Fig. 1. The relationship between NIRS predicted and in situ determined iNDF (A) with residual analysis (B) and predictions of in vivo OMD from iNDF determined in situ (C) or by NIRS (D). In residual analysis each NIRS predicted value is centred by subtracting of mean predicted from each predicted value (n = 80). (Data from Huhtanen et al. 2006b).


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Digestibility at production intake

Digestibility determined in sheep fed at maintenance describes the intrinsic digestibility of the diet, i.e. in vivo di-gestibility under optimal conditions (Mertens 1993). Feed values for cattle diets are traditionally computed using these digestibility coefficients by summing up individual dietary components. In general, the digestibility coeffi-cients for a given feed are similar in sheep and cattle (Yan et al. 2002). Because diet digestibility decreases with increased feed intake, energy values are adjusted for the level of feeding in many feed evaluation systems. In a re-cent meta-analysis based on the evaluation of 497 diets in lactating cows, OMD was on average 0.038 units lower in dairy cows fed at production levels of intake compared with OMD estimated at maintenance intake (Huhtanen et al. 2009). Digestibility in cows was shown to decrease with DM intake, the extent of depression being greater for highly digestible diets (Huhtanen et al. 2009). Dietary CP concentration had a positive effect on OM and NDF digestibility, while OMD decreased in a quadratic manner with increases in the proportion of whole-crop silage in the diet and linearly with concentrate fat intake (Table 2). The RSD of a multivariate mixed regression model was 0.007 indicating that the differences in OMD between the diets of lactating cows could be predicted accu-rately from digestibility at maintenance, feed intake and diet composition (Huhtanen et al. 2009). Interestingly, there was no difference in the accuracy of OMD prediction in cows when OMD at maintenance were determined either in vivo with sheep or based on predictions from various in vitro measurements. The variation in OMD in dairy cows was almost completely related to the concentration and digestibility of NDF (Huhtanen et al. 2009). This indicates that the negative associative effects of feeding level and diet composition on OMD at the produc-tion level of intake are mainly associated with decreased NDF digestibility. It is therefore important to distinguish between iNDF and uNDF. Indigestible NDF is not digested by ruminants, whereas uNDF represents faecal output of pdNDF per kg DM intake. Total faecal NDF also includes a proportion of pdNDF (i.e. uNDF) that is not digested because the retention time in the fermentation compartments is not long enough for complete pdNDF digestion. In dairy cows fed at production level of intake pdNDFD was substantially lower than in sheep fed at maintenance (0.75 vs. 0.85) resulting in a greater loss of potentially digestible NDF in faeces.

Table 2. The best-fit equation for multiple regression of OM or NDF digestibility (OMD or NDFD, respectively) in lactating dairy cows1; adjusted RMSE for OMD 7.1 g kg-1 (n= 497) and for NDFD 12.4 g kg-1 (n = 394) (Huhtanen et al. 2009).

Effect UnitEstimate (g kg-1)

OMD NDFD

Intercept 18.4 -285

OMDm2 g kg-1 DM 0.651

pdNDF3/NDF g kg-1 NDF 0.647

DMI kg d-1 -2.72 -4.85

Ln CP4 g kg-1 DM 53.7 101

Wcrop5 22.2 -28

WCrop × WCrop -61.4 -70

(NFC6/NDF) × (NFC/NDF) -55

Cfat7 g kg-1 DM -17.7 -331) All values are adjusted for the random study effect. 2) OM digestibility determined at maintenance level of feeding in sheepor with a corresponding in vitro method. 3) pdNDF = potentially digestible NDF. 4) Natural logarithm of crude protein concentration.5) Proportion of whole crop cereal silage in forage (kg kg-1). 6) Concentration of non-fibre carbohydrates in the diet (g kg-1 DM).7) Concentrate fat

Nutrient supply Feed intake

Accurate prediction of DM intake (DMI) is a prerequisite for the formulation of economical dairy cow diets. Despite intensive research, no generally accepted intake model has been developed. Limited success is at least partly due to complicated interactions between the animal and feed factors, and difficulties in distinguishing and quantify-ing these factors. Many intake models include observed milk yield as a predictor of intake. However, these mod-els are primarily useful in predicting intake required to sustain a given level of milk production, as stated by Keady et al. (2004a). It should also be remembered that the yield can only be known retrospectively after the diet has been fed (Ingvartsen 1994). Several attempts have been made to develop prediction equations for practical ra-tion formulation using multiple regression equations for individual animal data. However, these models usually


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have large residual errors, and consequently the effects of e.g. silage fermentation characteristic were non-signif-icant.This is probably due to large between animal variations in intake within a diet and study, and large between study variations both in the intake and composition of diets. Mixed model regression analysis with random study effects allows estimation of quantitative relationship between dietary variables and DMI and the relative intake potential of diets.

The first relative silage DMI index (SDMI-index) model included D-value (g digestible OM in DM), a quadratic nega-tive effect of total acid (TA) concentration and a logarithmic of ammonia N (Huhtanen et al. 2002a). Volatile fatty acids, especially propionic acid, had a stronger negative effect on intake than lactic acid. Digestibility was a much better predictor of SDMI than CP and NDF. The effects of D-value and fermentation quality were combined into a single index by defining standard silage (SDMI-index = 100) and that 0.10 kg DM is one index point. Root mean squared prediction error (RMSE) adjusted for the random study effect was 0.41 kg d-1, i.e. the model predicted precisely the differences in the intake potential of silages within studies. The model was revised to include other variables that significantly influence SDMI (Huhtanen et al. 2007). In addition to D-value and fermentation char-acteristics, the revised model includes the concentrations of silage DM and NDF, harvest of grass silage (primary vs. regrowth) and forage type (grass, legume and whole-crop). Silage DM concentration influenced quadratically SDMI with maximum intake with a DM concentration of 350–400 g kg-1. Intake of regrowth silages was 0.4 kg DM d-1 less than that of primary growth silages when the differences in other variables were taken into account. Both legume and whole-crop silages displayed positive associative effects on SDMI, i.e. the intake of silage mixtures was greater than the mean of the two silages when fed alone. Maximum NDF intake was observed at a D-value of 640 g kg-1 DM suggesting that the cows do not use the full rumen capacity when fed high D silages. Indeed, the rumen NDF pool has reduced with increased silage digestibility (Bosch et al. 1992, Rinne et al. 2002) despite increased SDMI. These observations do not support the bi-phasic intake regulation theory (e.g. Mertens 1994); it rather sug-gests that DMI is regulated by interplay between physical and metabolic factors. In the revised model fermenta-tion variables were simplified to the linear negative effect of TA concentration. However, with silages displaying secondary fermentation the intake predictions can be improved by including acetic acid or VFA in the model (Eis-ner et al. 2006). The adjusted RMSE of the revised model was 0.34 kg d-1 and it explained 0.85 of the variation in SDMI within a study. D-value, fermentation quality and DM concentration were the three most important variables.

It is well-known that both the amount and composition of the concentrate supplements influence SDMI. There-fore the next step in developing the intake prediction model was to include concentrate factors in the model (Huh-tanen et al. 2008a). Total DMI increases with increased concentrate DMI (CDMI) but the increases diminished at high levels of supplementation; i.e. substitution rate increased. Substitution rate also increased with increased in-take potential (SDMI-index) of silages. Interestingly, SDMI explained the variation in substitution rate better than any single component of it. The interaction between forage intake potential and concentrate supplementation is also included in the Feed into Milk model, presented by Keady et al. (2004b). In their model silage intake poten-tial is determined by NIRS calibrated against standardized intake data by cattle. In addition to CDMI, the model of Huhtanen et al. (2008a) includes the quadratic effect of supplementary protein intake, negative linear effect of fat and positive linear effect of concentrate NDF. The adjusted RSME of the CDMI model in studies in which dif-ferent concentrate treatments were used with the same silage was 0.27 kg. The two indexes were combined to a single total DMI index (TDMI-index) that describes quantitative differences in DMI within a study by assuming the effects are additive. In the model evaluation the observed DMI response at 0.095 kg/index point was close to default value of 0.100 and the adjusted RMSE of the TDMI-index model was 0.37 kg DM d-1.

Evaluation of the TDMI-index model indicated that quantitative differences in the intake potential of the diets can be estimated accurately. The modelling was based on an assumption that within a study the animal factors (e.g. yield, live weight [LW]) are similar for all diets. However, in practical ration formulation in addition to relative in-take potential related to diet characteristics, accurate predictions of actual intake including animal factors is re-quired. Most intake prediction models use milk yield and live weight as animal variables. Because milk yield is a function of both cow’s genetic potential and diet characteristics, it is important that animal and diet variables are modelled independently of each other to avoid double-counting. It is important to note that cow’s genetic intake potential does not increase when she is fed a better diet; the intake response is entirely due to the diet effect. To avoid this double-counting and to have unbiased estimates of diet effects in the model, we used standardised en-ergy corrected milk (sECM) rather than observed yield to describe the production potential of the cow (Huhtanen et al. 2011b). Observed ECM was adjusted for days in milk, TDMI-index and dietary metabolizable protein (MP) concentration, i.e. to predict how much the cow would produce at a given stage of lactation when fed a standard diet. An advantage of this approach is that all data is available at the time of prediction, in contrast to observed


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ECM yield. The final model comprised sECM, LW, days in milk as animal factors and TDMI-index to describe the dietary intake potential. The regression coefficient of TDMI-index (0.088) remained close to the default value sug-gesting that the true animal and diet effects were separated properly.

Rumen fermentation Typically the molar proportion of propionate is low in cattle fed diets based on restrictively fermented grass si-lages with moderate levels of concentrate supplementation; for example in the review of 34 diets fed to growing or lactating cattle the molar proportion of propionate was only 165 mmol mol-1 (Huhtanen 1998). Water soluble carbohydrates are fermented to lactic acid and VFA during ensilage with the extent and type depending on ensil-ing characteristics of forages and additives used. These changes have a strong influence on ruminal fermentation pattern. Increased concentration of silage lactic acid increases propionate in rumen VFA. Intraruminal infusions of lactic acid demonstrated that propionate is the main end-product of lactate fermentation (Chamberlain et al. 1983, Jaakkola and Huhtanen 1992). Jaakkola and Huhtanen (1992) calculated that propionate comprised about 50% of the end-product of lactate fermentation in the rumen. Consistently, increased lactic acid concentration in silage has consistently increased propionate in rumen VFA (van Vuuren et al. 1995, Harrison et al. 2003). In contrast to lactic acid, the effects of silage WSC on rumen fermentation pattern have been inconsistent: sometimes butyrate (Jaakkola et al. 1991, 2006a) and sometimes acetate (Cushanan et al. 1995, Huhtanen et al. 1997) has increased.

Rumen fermentation pattern in cattle fed grass silage-based diets appears to be rather resistant to increased con-centrate supplementation. The effect of dietary starch concentration on the proportion of propionate in rumen VFA was not significant in multiple regression models derived from the Nordic dataset (107 diets in 29 studies) (Sveinbjörnsson et al. 2006). In this dataset, dietary lactic acid concentration had the strongest effect on rumen propionate suggesting that silage lactic acid is a more important factor influencing rumen fermentation pattern than starch. Mixed model analysis of an unpublished Finnish dataset (106 diets) indicated that dietary starch con-centration influenced rumen propionate in a quadratic manner with a minimum at 200 g kg-1 DM. In the same dataset, molar proportion of acetate decreased quadratically and that of butyrate increased linearly with increased starch concentration. The results suggest that at low levels of concentrate (starch) supplementation silage lactate dominates the rumen fermentation pattern, whereas at moderate levels of dietary starch concentrations the role of rumen protozoa becomes more important. The number of rumen protozoa increases with increased starch sup-plementation (Rooke et al. 1992, Jaakkola and Huhtanen 1993) and this may explain the changes in rumen fermen-tation pattern with increased concentrate supplementation in cattle fed grass silage-based diets.

As for increased starch supplementation, the effects of fat supplementation on rumen fermentation pattern are also rather small in cattle fed grass silage-based diets. In the analysis of the Finnish dataset there was a quadratic positive response in rumen propionate to increased dietary concentration of concentrate fat. The model predicts 10 – 15 mmol mol-1 increases in rumen propionate for dairy cows fed 500 g d-1 of supplementary fat as plant oils. Only at high inclusion rates of plant oils quantitatively important changes in rumen fermentation pattern can be expected in animals fed grass silage-based diets (Tesfa 1993, Shingfield et al. 2008).

Protein supply Microbial protein synthesised in the rumen comprise the major part of the supply of amino acids (AA) absorbed from the small intestine. Regression coefficients of bivariate regression model predicting milk protein yield were five times greater for bacterial metabolizable protein (MP) compared with feed MP both in North American and North European dairy cow trials covering a wide range of dietary ingredients (in total >1 700 diets) emphasizing the importance of microbial protein (Huhtanen and Hristov 2009). It has generally been believed that the efficien-cy of microbial protein synthesis (MPS) is lower in animals fed grass silage-based diets than in those fed dried or fresh forages, but there is little experimental evidence to support this. Three reasons have been suggested for the lower efficiency of MPS: silage fermentation products provide less ATP for microbial growth than WSC (Chamber-lain 1987), the nature of N constituents (more ammonia and NPN) and asynchronous energy and N release from the silage (Thomas and Thomas 1985). Microbial protein production in the rumen increased when silage fermen-tation was restricted using formic acid based additives (e.g. Jaakkola et al. 1991, 2006a, Huhtanen et al. 1997). In addition to increases in measured MPS, increased plasma concentrations of AA, particularly branched-chain AA, (Nagel and Broderick 1992, Huhtanen et al. 1997) indicated a greater amount of absorbed AA in response to restricting in-silo fermentation. There were no differences in the total or microbial protein flow at the duode-num between diets based on dried hay or restrictively fermented silage harvested simultaneously from the same sward (Jaakkola and Huhtanen 1993, Table 3). All these results suggest that the preservation method per se does


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not influence MPS and that the extent, and possibly type, of the in-silo fermentation are more important factors influencing the protein value of forages than preservation method.

The asynchrony between energy and N supply, often assumed to be a main reason for the low efficiency of MPS, has attempted to be minimized by feeding soluble carbohydrates. Feeding sugar supplements has decreased rumen am-monia N concentration (Syrjälä 1972, Chamberlain et al. 1985). However, the marginal increases in MPS with sugar supplements have not been greater than those predicted from the increased supply of fermentable energy (Cham-berlain and Choung 1995), i.e. there were no extra benefits from a better synchrony. In line with this, Khalili and Huhtanen (1991) reported significant increases in microbial protein flow with different sucrose supplements in cattle fed a grass silage-based diet. However, the continuous infusion of sucrose decreased rumen ammonia N and increased microbial N flow numerically more than feeding sucrose twice daily, despite a better synchrony of energy and N release with the latter. Similar conclusions can be drawn from the studies of Henning et al. (1993) and Kim et al. (1999); continuous supply of energy stimulated MPS more than attempts to catch high post-pran-dial ammonia concentrations by pulse doses of rapidly fermentable carbohydrates.

Table 3. The effect of forage preservation method (silage vs dried hay) and the application rate of formic acid in silage on the flow of nitrogen at the duodenum (g day-1).

Reference Treatment Ammonia-N Non-ammonia N Microbial N Feed N

Jaakkola and Huhtanen (1993) Silage (formic acid 4 L t-1) 5.0 148.2 83.4 52.8

Dried hay 4.8 142.8 73.1 57.8

Jaakkola et al. (2006a) Untreated 3.9 114.5 49.0 53.4

Formic acid 2 L t-1 4.2 126.1 57.3 56.7

Formic acid 4 L t-1 4.5 128.4 58.4 57.9

Formic acid 6 L t-1 5.1 136.9 65.4 59.4

Formic acid application rate expressed as 100% solution

Despite their rather small contribution to the total MP supply, forage factors influencing the supply of rumen un-degraded protein (RUP) have been investigated more intensively than factors influencing MPS. Studies conducted with the in situ method have suggested large differences in ruminal degradability of forage protein, but seldom these differences have been realized as production responses. Two reasons can be suggested for this discrepancy: the differences in RUP supply are overestimated by the current methods and/or that the value of forage RUP is low. In the analysis of omasal flow data the slope between the predicted (NRC 2001) and measured feed N flow was 0.76 (Broderick et al. 2010) suggesting that the differences in ruminal degradability of dietary CP are small-er than the model predictions based on the tabulated in situ data. The models computing ruminal degradability from the kinetic data assume that the immediately disappearing fraction (buffer/water soluble N) is degraded at infinite rate. However, there is a plenty of evidence that soluble non-ammonia N (SNAN) fractions can escape from the rumen in the liquid phase (e.g. Choi et al. 2002, Reynal et al. 2007). Ahvenjärvi et al. (2007) reported using 15N labeled silage buffer soluble N that approximately 15% of SNAN fraction escaped ruminal degradation in dairy cows. Consistent with these results, a meta-analysis based on 253 diets showed no negative influence of the proportion of SNAN in silage on milk protein yield when a silage MP values were calculated using a constant CP degradability irrespective of the proportion of soluble N (Huhtanen et al. 2008b). The meta-analysis of milk production data (Huhtanen et al. 2010) silage D-value and especially intake potential were more important deter-minants of milk protein yield than silage CP or ammonia concentrations.

Production responsesSilage digestibility

The effects of silage quality on feed intake and production responses can be attributed to intrinsic nutritive value of grass at the time of harvest and changes in the composition of grass during ensilage. In the northern latitudes the digestibility of primary growth grasses decreases very rapidly (0.65 %-units d-1; in the dataset of Huhtanen et al. 2006b) with concomitant rapid increases in grass DM yield. Therefore the timing of the harvest of primary growth of grass is one of the most important management decisions in a dairy farm. Improved silage digestibility, expressed as D-value, clearly increases intake and ECM yield (Fig. 2). The average increases in silage DMI and ECM yield were


47

0.027 and 0.045 kg per one g kg-1 in D-value. In the studies of Kuoppala et al. (2008) and Randby et al. (2012) intake of grass (mixtures of timothy and meadow fescue) silage was 17 kg DM d-1 when fed with 8 kg d-1 of concentrates. These results indicate a high intake potential of restrictively fermented grass silages harvested at early stages of maturity and wilted to DM concentration of approximately 300 g kg-1. The effects of silage digestibility on milk fat concentration have been variable and usually small, whereas milk protein concentration has increased with improved digestibility (Rinne et al. 1999a, Kuoppala et al. 2008), probably reflecting an increased energy supply. In all stud-ies (Fig. 2) the silages were supplemented with different levels of concentrate allowing calculation of concentrate sparing effects of improved silage digestibility. The average ECM yield response was 0.48 (SE = 0.04) kg ECM per kg increase in concentrate DMI. The average “concentrate sparing effect” was 0.81 (SE = 0.12) kg DM per 10 g kg-1 DM increase in silage D-value. Assuming that silage D-value decreases 5 g kg-1 DM per day, one day delay in harvest cor-responds to 0.22 kg decrease in ECM yield or 0.45 kg DM greater concentrate requirement to maintain ECM yield.

Silage fermentation

In a meta-analysis of data from silage fermentation studies (47 studies, 234 diets), both the extent and type of in-silo fermentation influenced milk production variables (Huhtanen et al. 2003). In this dataset the silages were harvested at the same stage of maturity and ensiled with different additive treatments. The yields of milk, ECM and milk components decreased with increased concentrations of lactic acid and VFA in silage. Numerically the effects of VFA were stronger than those of lactic acid. Proportional decreases in the yield of milk components with increasing extent of in-silo fermentation were the smallest for lactose and the highest for milk fat. When si-lage DMI was included in the prediction models, the effect of TA concentration on milk yield was not significant. However, increased silage TA concentration reduced the ECM yield even when silage DMI was included in the model, but the regression coefficient was much smaller (-5.8 vs. -18.6 g per 1 g TA kg-1 DM). It can be concluded that the effects of in-silo fermentation on the production of milk and milk components are mainly derived from the changes in feed intake.

Milk fat and protein concentrations decreased with increased in-silo fermentation (Huhtanen et al. 2003). Reduced milk protein concentration can be attributed to decreased feed intake and the lower efficiency of MPS, whereas the lower fat concentration is most likely related to the reduced proportion of lipogenic VFA in the rumen. The effects of silage TA concentration remained negative even at fixed DMI indicating that the changes in the composition of absorbed nutrients influenced milk composition beyond the responses related to DMI. Decreases in milk protein yield with increased in-silo fermentation were not greater than those predicted from reduced intake, even though negative effects of high TA concentration in silage on the efficiency of MPS are well-documented (Harrison et al. 2003). It is possible that increased propionate production from silage lactate increases hepatic gluconeogenenis thereby sparing AA from being used for glucose production. Higher plasma glucose concentration in cows fed ex-tensively fermented silages compared with those fed restrictively fermented silages (Heikkilä et al. 1998, Shingfield et al. 2002b) support this hypothesis. However, the lack of responses to dietary supplementation of propylene glycol of cows given restrictively fermented silages (Shingfield et al. 2002a, Jaakkola et al. 2006b) do not support the hypothesis that the diets based on restrictively fermented silages are specifically limited by the glucose supply.

Fig. 2. The effects of the concentration of digestible organic matter (DOM) on silage dry matter intake (DMI) and energy corrected milk (ECM) yield. The values are means over 2 or 3 concentrate levels in each study.

20.0

24.0

28.0

32.0

36.0

600 640 680 720 760

DOM (g/kg DM)

ECM

(kg/

d)Rinne et al. (1999)

Kuoppala et al. (2008)

Randby et al. (2012)

Sairanen, unpublished

10.0

12.0

14.0

16.0

600 640 680 720 760

DOM (g/kg DM)

Sila

ge D

MI (

kg/d

)

Rinne et al. (1999)

Kuoppala et al. (2008)

Randby et al. (2012)

Sairanen, unpublished


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Improved silage fermentation can be realized as increased yield or as “concentrate sparing effect”. Compared with formic acid-treated silage the cows given untreated silage required an additional 2.9 kg concentrate per cow per day to produce the same amount of milk fat plus protein (Shingfield et al. 2002a). The “concentrate sparing effect” of formic acid treatment was greater than reported by Mayne (1992) and Keady and Murphy (1996). The greater value in the study of Shingfield et al. (2002a) may be related to the higher levels of concentrate feeding, and therefore smaller marginal responses to supplements attained.

Concentrate supplementation It is well-known that concentrate supplementation decreases silage DMI but increases total DMI. Silage DMI de-creased by 0.45 kg and total DMI increased by 0.55 kg per 1 kg increase in concentrate DMI in our data-set from milk production trials (233 treatment means from concentrate supplementation studies, Huhtanen et al. 2008a). The effects of concentrate DMI on total DMI were strongly curvilinear with decreasing responses at high levels of supplementation. When the data was divided into two groups according to the relative silage DMI index (<100 [mean 91] and >100 [mean 107]) the total DMI increased less (0.51 vs. 0.61 kg per kg increase in concentrate DMI) for silage of high compared with low intake potential, respectively. As a result of interactions between the forage quality and the level of concentrate supplementation substitution rates can be high, even close to 1.0, in cows fed high quality grass silages with moderate to high amounts of concentrates (Kuoppala et al. 2008, Randby et al. 2012).

The mean linear ECM yield response to increased concentrate allocation was 0.71 kg kg-1 concentrate DM, but it decreased with the increasing supplementation level (Huhtanen et al. 2008a). With high quality silages, margin-al production responses to increased concentrate allocation were small (Kuoppala et al. 2008) or even negative (Randby et al. 2012). Small production responses are related to the high substitution rate, negative associative ef-fects in digestion and possibly repartitioning nutrient towards body tissues with high concentrate levels. Although the digestibility of concentrates at maintenance level is greater than that of forages, diet digestibility in dairy cows at production level was not related to the concentrate intake (Nousiainen et al. 2009). Interestingly, when the data-set from concentrate supplementation studies were divided according to mean milk yield (<27 kg d-1 and >27 kg d-1) the linear ECM responses were greater (0.76 vs. 0.63 kg kg-1 concentrate DM) at low (mean 23 kg d-1) compared with high (31 kg d-1) production level. This is mainly because total DMI responses were greater (0.65 vs. 0.47 kg per kg concentrate DM) at low production level. In the analysis of a larger dataset ECM yield respons-es to increased ME intake did not depend on the production level of the cows (Huhtanen and Nousiainen 2012).

Protein supplementation Proper determination of animal protein requirements is critically important for maximizing production and mini-mizing N input in dairy production systems. Efficiency of N utilization in milk production is relatively low at 25–28% (Huhtanen and Hristov 2009). Although increasing N input usually increases milk protein yield, conversion of di-etary N to milk N will decrease. Earlier, when the feed protein evaluation was based on digestible CP the strategy in Finland was to increase CP concentration in grass silage by high levels of N fertilization and early harvest (Hil-tunen 1979). As discussed before, maturity stage at harvest has a strong influence on intake and milk production. However, when CP concentration in grass silage was increased from 120 to 150 g kg-1 DM by greater application rate of N fertilizer feed intake or output of milk and milk protein were not influenced, while provision of additional N in concentrate supplements improved all of these parameters (Shingfield et al. 2001).

Inclusion of protein supplements such as soybean and rapeseed meals in grass silage-based diets increased milk pro-tein yield, but at the same time reduced the efficiency of N utilization (Huhtanen and Hristov 2009). The increases in milk protein yield ranged from 98 (soybean meal) to 136 g kg-1 increase in CP intake (untreated rapeseed meal) in recent meta-analysis by Huhtanen et al. (2011a). Similar differences were reported in a single study by Shingfield et al. (2003), who compared soybean mean and rapeseed expeller at four graded isonitrogenous levels (Table 4).

Plasma AA profiles suggested that rapeseed increased the supply of histidine and branched-chain AA compared with soybean meal (Shingfield et al. 2003). Positive production responses to supplementary protein in cows fed grass silage-based diets are partly associated with increased ME intake resulting from a greater silage DMI (Huh-tanen et al. 2008a) and improved diet digestibility (Nousiainen et al. 2009). Marginal responses to incremental ME (0.16–0.18 kg ECM MJ-1 ME) in protein studies (Huhtanen et al. 2011a) were greater than usually obtained with increased inclusions of concentrate feeds (about 0.10). This may indicate that a greater AA/ME ratio in absorbed nutrients can improve the efficiency of ME utilization for milk production. Data from a whole lactation study (Law et al. 2010) indicated that calculated ME balance was greater for cows fed low vs. medium and high protein diets,


49

but the differences in blood metabolites, body condition score or live weight change did not indicate any true dif-ferences in energy balance.

Table 4. The effects of graded levels of rapeseed expeller (R) and soybean meal (S) supplementation on milk production and plasma amino acids (AA) in cows fed grass silage based diets (Shingfield et al. 2003). The number refers to crude protein concentrations in concentrates.

Control R150 R180 R210 S150 S180 S210

Intake (kg DM day-1)

Silage 11.6 11.9 12.3 12.4 12.0 12.2 12.5

Total 20.0 20.6 20.9 20.8 20.3 20.4 20.5

Production

Milk (kg day-1) 26.2 28.2 29.3 30.1 26.9 27.0 28.5

ECM (kg day-1) 29.8 31.2 31.6 32.8 30.3 30.4 32.1

Protein (g kg-1) 33.5 33.9 33.7 33.4 34.3 33.6 33.8

Protein (g day-1) 859 930 967 993 902 889 954

Milk N / N intake (g kg-1) 307 295 280 271 290 262 262

Plasma AA (µmol L-1)

Lysine 76 80 92 93 73 88 90

Methionine 20 21 23 25 19 22 20

Histidine 18 28 41 51 24 29 34

Two main strategies, reducing ruminal CP degradability of supplementary protein and balancing profile by ab-sorbed AA by using AA supplements or balancing dietary ingredients, to improve milk N efficiency have widely been investigated. In the meta-analysis (Huhtanen et al. 2011a) untreated and heat-treated rapeseed meal elicited similar milk protein yield responses. This is consistent with the meta-analysis by Ipharraguerre and Clark (2005), who did not find any differences in milk production between soybean meal and different RUP sources. Accord-ing to the meta-analysis of Huhtanen and Hristov (2009), ruminal CP degradability had a significant effect on milk protein yield, but calculated marginal responses to MP derived from reduced degradability was only 6–8%. It has been suggested that the protein supplements treated to reduce ruminal protein degradability have not increased milk yield as the untreated supplements already met the cow’s MP requirements. To test this hypothesis Rinne et al. (1999b) fed untreated and heat-treated rapeseed meal at four different levels. Both supplements increased milk and protein yields linearly, but no differences between untreated and treated rapeseed feeds were observed.

Methionine and lysine are often considered as limiting and/or co-limiting AA in dairy cows, but there is no evi-dence that these AA limit milk protein production in cows fed grass silage-based diets (Choung and Chamberlain 1992 and 1995, Varvikko et al. 1999). Vanhatalo et al. (1999a) infused post-ruminally histidine alone or in com-binations with methionine, lysine or both (Table 5). Histidine increased significantly milk protein yield, whereas lysine, methionine or lysine + methionine did not produce any further response. Later studies (Huhtanen et al. 2002b, Korhonen et al. 2000) confirmed that histidine was the first limiting AA in cow fed low CP grass silage based diets. Attempts to identify methionine, lysine and branched-chain AA as the second limiting AA were not success-ful (Vanhatalo et al. 1999a, Huhtanen et al. 2002b, Korhonen et al. 2002). A recent study by Lee et al. (2012) sug-gested that histidine could also be a limiting AA in cows fed low CP diets based on maize silage. It is possible that after the first-limiting AA the differences between the next limiting AAs are small in cows fed grass silage–based diets, and that the ranking of these AA can vary between experiments.

Analysis of data from milk production trials clearly indicated that dietary CP concentration was the best single variable predicting milk N efficiency (Huhtanen and Hristov 2009). Intake of N has often been used as a predictor of milk N efficiency (e.g. Castillo et al. 2000), but the adverse effect of increased N intake is much stronger when derived from increased dietary CP concentration rather than from increased DMI. As could be expected from its relatively small effects on milk production, ruminal protein degradability had a relatively small influence on milk N efficiency (Huhtanen and Hristov 2009). Milk urea concentration is closely related to dietary CP concentration and it predicted the differences between diets in milk N efficiency and calculated urinary N output accurately (Nousi-ainen et al. 2004b) suggesting that it can be used as a farm diagnostic tool.


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Table 5. Effects of postruminal infusions of amino acids (AA) on milk production and plasma AA concentrations. The amounts of amino acids and glucose (Gluc) infused (g day-1) are given in brackets.

DMI Milk Protein Protein Plasma AA (µmmol L-1)

(kg day-1) (kg day-1) (g kg-1) (g day-1) Lys Met His

Vanhatalo et al. (1999)

Control 16.1 22.9 30.4 695 82 21 18

His (6) 16.3 23.6 30.6 721 77 17 53

His + Met (6.5) 16.3 23.7 31.0 728 90 33 57

His + Lys (19) 16.2 24.2 29.8 717 120 18 47

His + Met + Lys 16.4 23.7 31.1 729 115 30 38

Korhonen et al. (2000)

Control 17.8 27 31.9 861 72 21 23

His (2) 18.2 28.1 31.3 877 82 23 28

His (4) 17.9 28.1 32.2 907 86 21 51

His (6) 17.9 28.8 32 919 91 23 64

Huhtanen et al. (2002b)

Control 16.6 23.6 29.4 691 79 18 21

His (6.5) 16.7 24.4 29.7 715 73 17 52

Gluc (250) 16.6 24.2 29.3 706 69 19 17

His + Gluc 17.1 25.0 30.2 748 69 18 52

His + Leu (12) 17.0 24.7 29.9 736 76 19 52

His + Gluc + Leu 17.0 24.9 30.2 751 72 19 49

Conclusions

Silage research in Finland during the latest 30 years has systematically focused on the production and ensiling of grass and legume silages with special reference to the utilization and supplementation of silages in cattle produc-tion. This work has facilitated the development of ration formulation systems based on meta-analyses of large and comprehensive datasets that has been compiled mainly from Finnish and North European studies. Success-ful economical dairy cattle ration optimization requires (1) a well-performing feed evaluation system, (2) accurate and cheap feed analyses for on-farm produced silages, (3) DM intake prediction models integrating independent-ly dietary and animal constraints and (4) equations to estimate true nutrient supply and marginal production re-sponses to changes in nutrient intake. Based on these principles Huhtanen and Nousiainen (2012) presented milk production response models that are currently used in practical feed ration planning in Finland.

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Wilkinson, J.M., Chapman, P.F., Wilkins, R.J. & Wilson, R.F. 1983. Inter-relationships between pattern of fermentation during ensi-lage and initial crop composition. In: Smith, J.S. and Hays, V.W. (eds.). Proceedings of the 14th International Grassland Congress, Lexington, USA. p. 631-634.

Yan, T.. Agnew R.E. & Gordon, F.J. 2002. The combined effects of animal species (sheep versus cattle) and level of feeding on di-gestible and metabolizable energy concentrations in grass silage-based diets of cattle. Animal Science 75: 141–151.

AGRICULTURAL AND FOOD SCIENCER.J. Dewhurst (2013) 22: 57–69

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Milk production from silage: comparison of grass, legume and maize silages and their mixtures

Richard J. DewhurstTeagasc, Animal and Grassland Research & Innovation Centre, Grange, Dunsany, Co. Meath, Ireland


The high rates of rumen fermentation, physical breakdown and passage rates from the rumen of legume silages lead to higher intakes than for grass silages of comparable digestibility. Although total tract digestibilities for leg-ume silages and maize silages are often lower than for grass silages, milk yields are usually higher. A further ben-efit of legumes and maize is the reduced rate of decline in digestibility. Legume silages often lead to a reduction in milk fat concentration and increased levels of polyunsaturated fatty acids, 18:2 n-6 and 18:3 n-3. This latter effect is related to reduced rumen biohydrogenation as a consequence of increased rumen passage rates or the effects of polyphenol oxidase. There is quite a wide range of maturities (300 – 350 g kg-1 DM) that leads to maximum dry matter intakes and milk production from maize silage; milk production is reduced with immature or over–mature maize crops. Forage chop length exerts a number of effects, both in the silo and in the rumen, but effects on ru-men function, feed intake and milk production have been inconsistent. The high protein content and high N deg-radability of most legume silages is associated with a low efficiency of converting dietary N into milk N, with a con-comitant increase in urine N. Reducing N intake by inclusion of maize silage in mixtures with legume silages leads to a marked reduction in urine N without loss of production potential. It is predicted, on the basis of their chemical composition and rumen kinetics, that legume silages and maize silages would reduce methane production relative to grass silage, though in vivo measurements are lacking. Extensive fermentation in the silo reduces the amount of fermentable substrate, and reduced methane production in comparison with grass silage where fermentation had been restricted by high levels of acid additive.

Key words: clover silage, feed intake, grass silage, maize silage, milk quality

Introduction

This paper focuses on silages used for milk production in the maritime region of North–West Europe. Following the transition from hay to silage over the last half century, the main forage on many dairy farms is often grass si-lage, typically based on timothy (Phleum pratense) and meadow fescue (Festuca pratensis) in cooler areas, and ryegrasses (Lolium spp.) in more temperate areas. There are areas where red clover (Trifolium pratense) and maize (Zea mays) are important, and experience in these areas has prompted renewed interest in alternative forages in other areas. Maize is a tropical crop and so has not always achieved an adequate level of maturity in cooler parts of the region; the use of other whole–crop cereals has expanded in some of these areas. Plant breeders have made considerable advances in achieving earlier maturing maize varieties that are more reliable in areas such as Northern Britain and Ireland.

The main focus of the paper is on feed intake, milk production and milk composition when cows are offered di-ets based on silages prepared from grass, red clover, white clover (Trifolium repens) or maize. The high produc-tion potential of legume silages has long been recognised – both for white clover (Castle et al. 1983), red clover (Thomas et al. 1985), and lucerne (Medicago sativa; Hoffman et al. 1998). Some early studies evaluated milk pro-duction potential with silage as sole feed. Castle (1982) obtained milk yields of between 13.3 and 16.0 kg day-1 from high–digestibility grass silage as sole feed in mid-lactation. Rae et al. (1987) conducted a multi-year study with spring-calving cows offered just grass silage and grazed grass. At one location, with high-quality silage, they obtained lactation yields of 4700 kg without feeding concentrates. Mean silage dry matter (DM) intakes were 13.2 kg day-1 and mean milk yields were 21.1 kg day-1 for cows and 16.1 kg day-1 for heifers in the 3-month period prior to turnout. Particularly impressive were the DM intakes (19.3 and 17.7 kg day-1) and milk yields (26.8 and 19.6 kg day-1) achieved by Castle et al. (1984) and Cohen et al. (2006) respectively with white clover silage as sole feed. Steinshamn and Thuen (2008) recorded milk yields of 22 kg day-1 when they offered diets based on grass silage with either white clover silage (0.28 of DM) or red clover silage (0.42 of DM) as sole-feed to cows in early lacta-tion (average 74 days in milk). In addition to considering the effects of different silages on productivity and milk composition, this paper also considers effects on nutrient efficiency and emissions.



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Chemical composition and digestibility

Table 1 uses mean feed values taken from INRA (2007) to illustrate the mean and range for grass, legume and maize silages. These relate to conditions in France, but the pattern of greater variation in grasses in comparison with legumes is evident in other areas (Dewhurst et al. 2003b, Hetta et al. 2004).

Table 1. Range of composition of different silages in feed tables from France (INRA 2007).

Unité Fourragère Lait† (per kg DM)

Crude protein (g kg-1 DM)

Perennial ryegrass 1st cut, prior to 10% ear emergence 1.01 151

1st cut, 10% ear emergence 0.97 141

1st cut, end of heading 0.83 112

2nd cut, stemmy, heading 0.83 131

Range (% of highest value) 17.8 13.2

Lucerne 1st cut, 10% budding 0.82 190

1st cut, 50% budding 0.77 182

2nd cut, stemmy, budding 0.76 187


Red clover 1st cut, 10% budding 0.90 178

1st cut, 50% budding 0.86 171

2nd cut, stemmy, budding 0.81 181


Maize Milk-dough (250 gDM kg-1) 0.90 86

Dough-flint (300 gDM kg-1) 0.90 84

Flint (350 gDM kg-1) 0.90 82

Range (% of highest value) 0 4.6

†Unité Fourragère Lait (UFL) is a Net Energy for Lactation Unit in the French Feeding System and is expressed relative to barley, which has a value of 1 UFL.

Legumes generally contain more protein and less fibre than grasses, whilst maize contains less protein and less fibre than grass. Early work suggested that the rate of decline in digestibility is less for legumes than grasses (Uly-att 1970, Thomas et al. 1981). Rinne and Nykänen (2000) showed a more rapid decline in the digestibility of timo-thy than red clover during primary growth. Hetta et al. (2004) showed that the rate of decline in digestibility was greater for timothy than for red clover during spring growth, but not during summer growth. Phipps et al. (2000) evaluated maize silages of widely divergent maturity, varying from 230 to 380 gDM kg-1. Starch replaced fibre as the crop matured from 230 to 330 gDM kg-1 (NDF: 570 vs. 430 g kg-1 DM; starch: 110 vs. 310 g kg-1 DM).

Digestibility of silages is affected both by rates of fermentation in the rumen, and residence time within the diges-tive tract. The higher rates of fermentation for legumes in comparison with grasses (Smith et al. 1972), as well as higher rates of physical breakdown and passage from the rumen (Wilson and Kennedy 1996) have long been rec-ognised. The higher rates of fermentation of clover silages have been confirmed in more recent work (Dewhurst et al. 2003a). White clover has relatively low fibre content and an inherently high rate of fermentation, so that despite a much lower retention time than ryegrass (and consequent higher intake), it remains more digestible than ryegrass (Dewhurst et al. 2003a).


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Rumen function

Despite the differences in rumen fermentation rates, Dewhurst et al. (2003a) found no effect of legume silages (white clover, red clover or lucerne) on rumen pH or VFA concentrations, though rumen ammonia-N concentra-tions were significantly higher than for diets based on grass silage. Whilst Vanhatalo et al. (2009) confirmed the increased rumen ammonia-N when feeding red clover silage (in comparison with grass silage), they also observed higher VFA concentrations and a higher molar proportion of acetic acid.

Whilst legumes may be attractive diet components for other reasons, their high crude protein content can lead to wastage of nitrogen, so there has been interest in reducing the rumen degradation of N. Table 2 summarises some data from nylon bag studies of N degradation with grasses and legumes at different growth stages. Overall, estimates for legumes were higher than for perennial ryegrass, though Hoffman et al. (1993) found higher values for perennial ryegrass when comparisons were made with immature herbage. The values in Table 2 were calcu-lated using an estimated rumen outflow rate of 0.05 per hour, though as has been noted this may be higher for the legumes, which would reduce the differences.

Table 2. Estimates of nitrogen degradability (g g-1) of forage legumes, assuming a rumen outflow rate of 0.05 hour-1.

Perennial ryegrass White clover Red clover Lucerne

Fresh herbage: early season1 0.70 0.83 ––– –––

Fresh herbage: mid season1 0.67 0.79 ––– –––

Fresh herbage: late season1 0.67 0.75 ––– –––

Dried herbage: vegetative2 0.89 ––– 0.88 0.85

Dried herbage: bud/boot2 0.87 ––– 0.82 0.79

Dried herbage: flowering2 0.69 ––– 0.73 0.73

Dried silage: mixed cuts3 0.76 0.83 0.77 –––

1Beever et al. 1986, 2Hoffman et al. 1993, 3Dewhurst et al. 2003a

Dewhurst et al. (2003a) showed similar soluble (’a’) and insoluble but potentially degradable (’b’) fractions for N in grass silage and white clover silage, but a much higher degradation rate for white clover silage (0.063 vs. 0.031 hour-1). For red clover silage, the ’a’ fraction was reduced (’b’ fraction increased) and the degradation rate inter-mediate (0.046 hour-1). Broderick et al. (2004) evaluated a wide range of accessions of red clover and lucerne, using an in vitro system, in order to obtain a comprehensive description of the range of N degradability for these forages. They confirmed that N degradability was lower for red clover than for lucerne. This appears to be related to the action of PPO in producing protein-quinone complexes that are resistant to rumen degradation (Lee et al. 2008). Condensed tannins present in other forage legumes, such as sainfoin, remain activ in silages and have al-tered the partitioning of dietary N between urine and faeces in sheep (Theodoridou et al. 2012). However, these crops are not agronomically suited to typical silage production systems for dairy cows in North-West Europe and red clover remains the most promising crop with reduced rumen N degradability. Recent work has demonstrated PPO activity in cocksfoot (Lee et al. 2006) and plant breeders are now considering ways to introduce this benefi-cial activity into grasses that are used more frequently for silage production (Kingston-Smith et al. 2013).

Results based on urinary excretion of purine derivatives provide little evidence for the effects of different silages on N-use efficiency (NUE) being mediated via effects on microbial protein synthesis. The low NUE with diets based on legume silages, particularly lucerne silage (Dewhurst et al. 2003b), were associated with increased microbial efficiency (g microbial N per kg digested organic matter; Dewhurst et al. 2003a). Further, there were no consist-ent effects of microbial efficiency associated with increased NUE for legume/cereal silage mixtures (Dewhurst et al. 2010, Cheng et al. 2011).

Rumen methanogenesis

High-forage diets have long been known to lead to production of more methane per unit of energy intake than high-concentrate diets (Blaxter and Clapperton 1965) and this is a particular challenge to production systems based on high levels of forage, including silage. The high fibre, high rumen pH and low rumen passage rates all favour rumen methanogenic archaea (Beauchemin et al. 2008).


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There are a number of reasons to predict that methane production should be less from legumes than from grass, including lower fibre content, higher DM intakes and an increased passage rate from the rumen (Beauchemin et al. 2008). A number of early studies with fresh herbage showed reduced methane from legumes (McCaughey et al. 1999, Waghorn et al. 2006). We have used a methanogen marker (archaeol) to show a reduced archaeal popu-lation after a meal of white clover (McCartney et al. 2012), presumably reflecting the difficulty that archaea have in surviving when intake and rumen passage rates are high. However, evidence from feeding studies with silages does not confirm this effect. Van Dorland et al. (2007) found no difference between grass silage, red clover silage and white clover silage in their effects on methane output, whether expressed per day, per kg of milk, or per kg digested organic matter, though clover silages only made up 0.40 of forage mixtures which also contained 0.60 ryegrass silage.

Extensive fermentation in the silo leaves a reduced proportion of dietary energy available for rumen fermenta-tion, so it would be expected that methane production is reduced. Cushnahan et al. (1995) demonstrated this effect with significantly less methane production from extensively-fermented grass silage, prepared with a bac-terial inoculant, in comparison with grass silage prepared from the same herbage, with a high rate of application of an acid-based additive. Unfortunately, the overall reduction in methane production was counteracted by an increase in urine N, so there may be no net effect on greenhouse-gas emissions when methane and potential ni-trous oxide are taken into account.

Whilst it is envisaged that the higher starch content and lower fibre content of maize silage will lead to reduced methane production in comparison with diets based on grass silage, this has not been verified experimentally (Beauchemin et al. 2008). Further, Vellinga and Hoving (2011) caution that reductions in methane production from feeding maize silage may be offset by the loss of soil carbon associated with ploughing permanent pasture to grow maize.

Feed intake

The lower intakes of grass silages are the most remarkable feature when comparisons are made across silage types. Legume silages generally lead to higher intakes than grass silages of comparable digestibility. Huhtanen et al. (2007) in a meta analysis showed a curvilinear effect with increasing intakes as legume silages replaced grass silages up to 0.80 inclusion. The same situation applied when Cheng et al. (2011) compared grass silages with mixtures of legume and cereal silages – despite lower digestibilities, the latter led to higher intakes. This effect is not confined to legume silages – total intakes and short-term intakes of total-mixed rations based on maize silage where higher than those based on grass silage (Abrahamse et al. 2008).

Rinne et al. (2002) investigated feed intake and rumen function for a series of timothy/meadow fescue silages of increasing maturity. The increased intakes of early-cut grass silage were associated with a reduction in rumen fill, suggesting that rumen fill is not solely responsible for control of feed intake. The effects of the level and compo-sition of nutrients derived from silages, and their interaction with animal potential, are also important in regula-tion of silage intake. There have been few studies of the effects of stage of maturity on intake and milk produc-tion from red clover silage. Hoffman et al. (1997) showed reductions in intakes of red clover silage in two sepa-rate years (milk yields were also reduced in one of the studies), whilst Vanhatalo et al. (2009) showed a significant increase in intake with a more mature red clover silage. Vanhatalo et al. (2008) showed reductions in intake with more mature red clover silages, whether from primary growth or regrowths. They found significantly higher in-takes of red clover silages prepared from regrowths as opposed to primary growths.

With the exception of diets that contain less than 250 g NDF kg-1 DM and are likely to be associated with rumen acidosis, there is a general negative relationship between diet NDF and DM intake (Allen et al. 2000). The fibre content of white clover silage is much lower than in other silages (Dewhurst et al. 2003b) and this explains the high intake characteristics, both as sole forage (Castle et al. 1984, Cohen et al. 2006), and with concentrate feeding (Dewhurst et al. 2003b). However, the fibre content of silages from grass, lucerne, red clover and maize are more similar and other mechanisms must explain differences in feed intake. The higher intake of these silages relative to grass silage relates to their more rapid fermentation and physical breakdown within the rumen.

Differences in intake have been attributed to both faster rates of fermentation (Beever and Thorp 1996) and more rapid particle breakdown and clearance from the rumen (Moseley and Jones 1984, Waghorn et al. 1989, Jamot and Grenet 1991). Dewhurst et al. (2003a) suggested that fermentation rate may be more important for white clover


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silage, whilst rapid particle breakdown may be more important for lucerne silage. Bosch and Bruining (1995) com-pared grass silages at differing maturities and suggested that the control of rumen fill is related to the disappear-ance rate of small particles (0.07 to 1.25 mm) from the rumen. In comparison with the elongated vein structure of grasses, the reticular vein structure of legumes breaks down into small particles more readily (Wilman et al. 1996, Wilson and Kennedy 1996). Since fermentation may contribute to the buoyancy of rumen particles, which limits the ability to leave the rumen (Baumont and Deswysen 1991), more rapid fermentation would also increase pas-sage rates from the rumen. Some aspects of intake regulation of legume silages remain to be elucidated; Kuoppala et al. (2009) could not explain the low intakes of early-cut red clover silage in terms of silage digestibility, fermen-tation quality or rumen fill and concluded that it must be related to some other aspect of nutrient composition.

Intakes of mixtures of grass and legume silages were usually intermediate to intakes of the grass and legume si-lages separately (Dewhurst et al. 2003b). High intakes were also achieved with mixtures of red clover silage and maize silage (Dewhurst et al. 2010). Intakes of maize silage increase with increasing maturity, up to the optimal 300–350 gDM kg-1 (Phipps 1990).

There has been a lot of work, particularly in North America, over the last decade evaluating effects of varying chop length in a range of forages, including lucerne, maize and oat silage. Part of the reason for this interest is the bet-ter compaction and ensiling that can be achieved with shorter chop material. At the same time, the short forage particles will increase problems with sub-acute rumen acidosis because of increased fermentation rates and re-duced chewing, rumination and saliva production. Whilst a number of studies showed effects on rumen fermen-tation measures, there were no consistent effects on feed intake, milk production or milk composition (Bhandari et al. 2007, 2008). Working with dairy steers, Rustas et al. (2010) showed an effect of chopping fermented whole-crop barley silage on DM intake when the crop was harvested at the mid-dough stage (Zadoks code 85), but not when it was harvested three weeks earlier at the heading stage (Zadoks code 59).

The basis for differences in DM intake between well preserved grass silages is less clear. Kuoppala et al. (2009, 2010) compared silages prepared from primary growth and regrowth of timothy/meadow fescue at two growth stages. They found no explanation for the higher intake and milk production from first-cut silages, despite exten-sive studies of feed chemistry, kinetics of rumen digestion and passage, and the protein/energy ratio of absorbed nutrients. The lack of explanation for differences in intake characteristics between silages from different herbage species and managements is an important area for further research because these effects drive the differences in milk production and nutrient utilisation described below. Whilst we understand some of the mechanisms involved in regulating silage intake, and have some analytical tools, new insights are needed. For example, Huhtanen et al. (2007) speculated that microbiology of herbage and the proportion of dead material may explain some of the dif-ference between first-cut and later silages.

Milk production

Diets based on legume silages, maize silage, or mixtures of the two often lead to higher milk production than di-ets based on grass silage (e.g. Thomas et al. 1985, Phipps et al. 1988, 1992, R.J. Dewhurst, unpublished observa-tions). However, in most situations diets often involve mixtures of these silages with grass silage. With average-quality grass silage, Phipps et al. (1992) found that a 50/50 mixture of grass silage and maize silage maximised intakes and milk production, whilst with a poorer grass silage, the optimal mixture was 75/25 maize silage/grass silage. Comparisons of milk production from lucerne silage and red clover silage are equivocal (Hoffman et al. 1997, Broderick et al. 2000, 2001, Dewhurst et al. 2003b).

Grasses and legumes tend to contain adequate levels of crude protein, so that production is limited by their in-take characteristics and energy content. Red clover and lucerne silage can often be of lower digestibility than grass silage, but performance can be higher as a result of higher DM intakes (lucerne: Hoffman et al. 1998; red clover silage: Dewhurst et al. 2003b, Moorby et al. 2009).

Although legume silages generally lead to higher intakes and milk production than grass silages, there remains considerable variation due to weather conditions and the success of ensilage, so there are examples of legume silages with lower intake and production characteristics (Bertilsson and Murphy 2003).

Milk yields were maximised using maize silage of 330 gDM kg-1 (310 g starch kg-1 DM) in the study of Phipps et al. (2000), which compared a range from 230 to 380 gDM kg-1. There was no further increase in production with


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higher maturity (380 gDM kg-1; 350 g starch kg-1 DM). These authors suggest that milk production will be opti-mised across a relatively wide range of maize silage maturity (300–350 gDM kg-1), though this optimal range may be different for special maize varieties such as ‘stay green’ maize.

Milk fat and protein content

Phipps et al. (1992) found no effect of varying the ratio of grass silage to maize silage from 100/0 to 75/25 in the diet on fat concentration or protein concentration. Similarly, there appears to be little difference be-tween grass silage and lucerne silage in their effects on milk fat concentration and protein concentra-tion. Legume silages have often led to reductions in milk fat concentration and/or milk protein concentra-tion and this is summarised in Table 3. The reduction in milk fat concentration with clover silages is most consistent, with reductions in milk protein concentration often small and occasional significant increas-es in milk protein concentration when clover silages with exceptionally high intake characteristics were fed.

Table 3. Effects of clover silages on milk fat concentration and milk protein concentration

Reference(†) Comparison Effect of clover silage on milk fat concentration

Effect of clover silage on milk protein concentration

Al-Mabruk et al. 2004 Red clover vs. grass No effect Small increase

Bertilsson & Murphy 2003 (1) Red clover vs. grass No effect No effect

Bertilsson & Murphy 2003 (2) Red clover vs. grass No effect Reduction

Dewhurst et al. 2003a (1) Red clover vs. grass No effect No effect

Dewhurst et al. 2003a (2) Red clover vs. grass No effect Small reduction

Moorby et al. 2009 Red clover vs. grass Reduction Reduction

Thomas et al. 1985 Red clover vs. grass Reduction No effect

Vanhatalo et al. 2009 Red clover vs. grass Reduction Reduction

Bertilsson & Murphy 2003 (1) White clover vs. grass No effect No effect

Bertilsson & Murphy 2003 (2) White clover vs. grass Reduction Reduction

Dewhurst et al. 2003a (1) White clover vs. grass No effect No effect

Dewhurst et al. 2003a (2) White clover vs. grass Reduction Increase

Broderick et al. 2000 Red clover vs. lucerne Reduction Small reduction

Broderick et al. 2001 (1) Red clover vs. lucerne No effect No effect

Broderick et al. 2000 (2) Red clover vs. lucerne Reduction No effect

Hoffman et al. 1997 (1) Red clover vs. lucerne No effect No effect

Hoffman et al. 1997 (2) Red clover vs. lucerne No effect Reduction

Steinshamn & Thuen (2008) Red clover vs. white clover Tendency for reduction Small reduction

†(1) and (2) refer to separate experiments reported within the same paper.

Dewhurst et al. (2010) and Cheng et al. (2011) found no differences in milk fat concentration and milk protein concentration when comparing diets based on grass silages and diets based on a series of mixtures of red clover silage with either maize silage or whole-crop oat silage. Phipps et al. (2000) found no consistent effect of maize silage maturity on milk fat concentration or protein concentration.

Milk fatty acids

In comparison with milk from cows fed grass silages, clover silages had only small and inconsistent effects on pro-portions of the various saturated fatty acids, as well as conjugated linoleic acid, in milk (Dewhurst et al. 2006). In contrast, both red clover and white clover silages led to highly significant increases in the proportion of the n-3 fatty acid α–linolenic acid (18:3 n-3) in milk. Figure 1 summarises the results of a number of comparisons of ef-fects of grass silage and red clover silage on proportions of 18:3 n-3 in milk fat. There was a three-fold increase in this fatty acid when red clover silage made up a high proportion of the diet (concentrate level was 4 kg/day) in


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the studies reported by Dewhurst et al. (2003b) and Moorby et al. (2009). Other studies have confirmed the in-crease in 18:3 content of milk from cows fed red clover silage (Al-Mabruk et al. 2004, Vanhatalo et al. 2007, Van Dorland et al. 2008) and white clover silage (Van Dorland et al. 2008). Vanhatalo et al. (2007) showed a greater increase in 18:3 content of milk fat for early–cut red clover silage in comparison with late–cut red clover silage. Dewhurst et al. (2003b) observed intermediate levels of 18:3 in milk when feeding mixtures of grass silage and red clover silage; Arvidsson et al. (2012) found only small but statistically significant increases when feeding a mix-ture of timothy and red clover silage.

The effect of clover silages is probably the basis for increased levels of 18:3 in organic milk, at least in the UK where red and white clover are important components of organic systems (Dewhurst, 2003, Ellis et al. 2006, But-ler et al. 2011; Figure 2). Steinshamn and Thuen (2008) compared diets based on grass silage with either white clover silage (0.28 of DM) or red clover silage (0.42 of DM), either as sole–feed or with 10 kg day-1 concentrates, and showed higher 18:3 content milk from cows fed red clover silage (0.87 vs. 0.73 g 100g-1 of milk fatty acids).

The mechanism for these effects relates to a reduction in rumen biohydrogenation of 18:3 from red clover silage and white clover silage. They cannot be explained as a dilution effect since yields of milk fat from cows fed clover silages were increased in comparison with yields from grass silage (Dewhurst et al. 2003a). In the case of red and white clover silages, this may be related to the higher rate of passage from the rumen (Dewhurst et al. 2003a), whilst in the case of red clover silage, it appears also related to the action of PPO (Lee et al. 2008).

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Fig. 1 Effect of replacing grass silage with red clover in the diet of lactating dairy cows on the proportion of 18:3 n-3 fatty acid in milk fat (g 100g-1)(†).

†(1) and (2) refer to separate experiments reported within the same paper.

Fig. 2 Comparisons of proportions of n-3 polyunsaturated fatty acids in milk from cows fed conventional or organic diets – based on surveys of farms or retail milk conducted in the UK.

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Other milk attributes

The increase in polyunsaturated fatty acids, both 18:2 and 18:3, in milk from cows fed red clover silage increases the likelihood of problems with oxidation. Al-Mabruk et al. (2004) demonstrated a reduction in the shelf-life of milk from cows fed diets based on red clover silage.

Bertilsson and Murphy (2003) observed an increased deviation from good organoleptic characteristics of milk when grass silage was replaced with silage made from white, and particularly, red clover silage. It seems likely that higher levels of dietary protein are one cause of off-flavours since compounds such as methyl sulphide and skatole are derived from degradation of amino acids. Ethanol is another silage component that can lead to off-flavour in milk (Randby et al. 1999).

Moorby et al. (2009) showed changes in the physical appearance of milk produced from red clover silage in com-parison with grass silage. Milk from cows fed red clover silage had a reduced whiteness score and a ’thinner’ tex-ture; it was suggested that this relates to lower levels of β–carotene in the milk. Feeding cows diets based on red clover silage increased levels of flavonoids (Steinshamn et al. 2008), which may affect human health.

Utilisation of dietary N and urinary N output

Kebreab et al. (2001) analysed a series of five N Balance studies conducted at the Centre for Dairy Research in Reading, with 30 diets based on grass silage and concentrates and obtained the following relationship (equation 1) for urine N output:

Urine N (g day-1) = 0.003 N intake (g day-1)1.8 (r2=0.67 based on individual values) (1)

Huhtanen et al. (2008) obtained a relationship (equation 2) that produces very similar predictions within the range of N intake encountered in practice. This relationship was based on a large number of treatment means taken from the literature and was based largely on grass silage-based diets:

Urine N (g/day) = –126 +0.676 N intake (g day-1) (n=515, RMSE=12.3) (2)

As might be predicted from equations (1) and (2), the high N content of legume silages can lead to low efficien-cies of conversion into milk N (NUE) and particularly high urinary N output (Cohen et al. 2006, Dewhurst et al. 2003b, 2009). Urine N has been reduced by offering low protein supplements, such as barley (Cohen et al. 2006) or maize silage (Margan et al. 1994, Auldist et al. 1999, Dewhurst et al. 2010) alongside legume silages. There is no evidence that the inefficiency is driven by asynchronous supply of energy and N to rumen micro-organisms since meal patterns had no effect on NUE (Cohen et al. 2006).

Whilst the high urine N from diets based on legume silages as sole forage are as expected, it is important not to miss two attributes of legume silages that may improve NUE and reduce urine N relative to expectations. The first mechanism relates to the action of the enzyme polyphenol oxidase (PPO) in red clover. PPO produces quinones that bind to proteins and reduce N degradability in the rumen (Broderick and Albrecht 1997, Cassida et al. 2000). Red clover silage contains approximately 0.3 to 0.4 (proportionately) less non-protein-N than lucerne silage (Al-brecht and Muck 1991, Owens et al. 1999). This may explain observations of reduced rumen ammonia, reduced milk urea N, reduced urine N, and increased NUE with diets based on red clover silage in comparison with those based on lucerne silage (Broderick et al. 2000, 2001, 2007). We have used mixtures of red clover silage and maize silage to achieve the production benefits of legume silages, including increased milk protein yield, whilst reduc-ing urine N production (Fig. 3).


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In two studies comparing grass silage–based diets with diets based on mixtures of legume silages and maize si-lage or other cereal silages, we noticed that urine N was 50 to 100 g day-1 less than predicted using equations (1) or (2) (Dewhurst et al. 2010, Cheng et al. 2011). Further analysis revealed that this effect was related to effects on DM intake. Further statistical analysis was conducted using treatment means from our studies (21 treatment means from Dewhurst et al. 2010, Cheng et al. 2011 and two unpublished studies with diets based on legume si-lages, grass silages and mixtures with maize silage). There was no significant relationship between DM intake and N intake for these treatment means (r2=0.07; p>0.1), so in addition to a simple regression (equation 3), we were able to conduct a multiple regression analysis (equation 4).

Urine N (g day-1) = –122 + 0.614 N intake (g day-1) (r2=0.60, p<0.001) (3)

Urine N (g day-1) = 105.5 + 0.769 N intake (g day-1) – 16.86 DM intake (kg day-1) (r2=0.91, p<0.001) (4)

The interpretation of equation 4 is that diets affect urine N both through the supply of N, and through effects on DM intake – presumably affecting the ability of the animal to utilise feed N in the rumen or tissues. In fact, the same effect was noted in the analysis by Huhtanen et al. (2008, equation 5), so there is a general principle that achieving higher intakes without increasing N intakes will increase NUE and reduce urine N.

Urine N (g day-1) = 27 +0.844 N intake (g day-1) – 13.0 DM intake (kg day-1) (n=515, RMSE=9.28) (5)

The effect described in equations (4) and (5) is the basis for the success of diets based on mixtures of legume and cereal silages. However, there are examples of this effect operating with other forages. Cushnahan et al. (1995) demonstrated a significantly higher proportion of N intake going to urine N with extensively-fermented grass si-lage of low intake characteristics, despite its having a lower N concentration. Cammell et al. (2000), comparing diets based on maize silages of varying digestibility, showed a reduction in urine N, relative to N intake, with diets based on the maize silage (330 gDM kg-1) that maximised intake of digested organic matter, and thus, ME.

Future research

The effects of legume and maize silages in increasing feed intake and milk production are well-recognised, as are the effects of legume silages on milk fatty acids. It is likely that the use of legumes for silage will increase and there is a need to follow up on the increasing evidence of effects on the physical and organoleptic properties of milk. We need to properly describe the range and frequency of effects, as well as identifying the mechanisms involved, as a basis for amelioration strategies.

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Grass silage 40% red clover 60% maize

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Fig. 3 Effect of replacing grass silage with mixtures of red clover silage and maize silage on outputs of milk N and urine N (g day-1). Based on results of Dewhurst et al. 2010.


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Many of the benefits of legume silages in comparison with grass silages – increased intakes and milk production, increased polyunsaturated fatty acids in milk, and a reduction in urine N at a given N intake – relate to the in-creased rumen passage rates. In addition to seeking to change the chemical composition of grasses and silages, further work should investigate potential to achieve differences in physical and chemical breakdown characteris-tics of grasses (e.g. cell structure and lignification).

Whilst differences in rumen fermentation rate, particle breakdown, and passage from the rumen explain some of the higher intake with legume silages, there are clearly other facets of the control of silage intake that remain to be understood. Ammonia-N has long been recognized as merely a proxy for some aspects of poor fermentation quality in silages, which are not fully characterized. Ammonia and amines directly added to the diet have not re-duced silage intake. Further studies are required to understand the complex balance between fermentation qual-ity, effects on rumen kinetics and fill, as well as the balance between nutrient supply and animal requirements, in their effects on silage intake.

The observation that diets based on high intake characteristic forages leads to an increased NUE and a reduction in urine N should be investigated further. In particular, there is a need to understand the basis for the independent effects of N intake and DM intake since this offers potential to increase productivity without increasing problems linked to urine N output. Many aspects of good grassland management result in high levels of herbage N, and the search for low N/high intake forages may be limited by the requirement to have protein present in the plant pho-tosynthetic machinery. Differences in rumen microbial efficiency appear to explain only a small proportion of the effect, which appears to be more based at the tissue level.

The lack of data on methane production from alternative silages is an important gap to fill, particularly in the case of legume silages that have the additional benefit of reducing carbon intensity of production by replacing inor-ganic N fertilizer.

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Production and utilization of ensiled forages by beef cattle, dairy cows, pregnant ewes and finishing lambs: a review

Tim W.J. Keady1, James P. Hanrahan1, Christina L. Marley2 and Nigel D. Scollan2

1Animal and Grassland Research and Innovation Centre, Teagasc, Athenry, Co. Galway, Ireland2Institute of Biological Environmental and Rural Sciences, Aberystwyth University, Gogerddan,

Aberystwyth, Wales, UK, SY23 3EB


This paper reviews the production of, and factors affecting the performance of dairy cows, beef cattle and sheep offered silage based diets in Ireland and UK. Digestibility is the most important factor influencing the feed value of grass silage and consequently animal performance. Each 10 g kg-1 increase in digestive organic matter in the dry matter (DOMD) increases milk yield of dairy cows by 0.33 kg d-1, carcass gain of beef cattle by 23.8 g d-1 , carcass gain of finishing lambs by 9.3 g d-1, lamb birth weight by 52.3 g and ewe weight post lambing by 1.3 kg, respectively. Factors influencing feed value of grass silage are discussed including harvest date, wilting, fertilizer management, chop length and use of additives at ensiling. Maize silage increases the performance of cattle and sheep whilst whole crop wheat silage has no beneficial effect. Advances in silage technology, has enabled the ensiling high protein for-ages, such as red clover, lucerne and kale.

Key words: milk yield, carcass gain, grass silage, maize silage, whole crop wheat silage, red clover, lucerne, kale

Introduction

Grass silage is the basic component of beef, milk and sheep production systems, in Ireland, UK, Scandinavia, many other parts of Europe, New Zealand, Australia and North America, during the winter feeding period. Levels of ani-mal performance achieved from grass silage are variable reflecting its feed value. Grass silage feed value is a re-flection of the stage of maturity of herbage at ensiling, management at ensiling and the fermentation process all of which impact on digestibility (the major factor influencing feed value) and subsequently animal performance. High feed-value grass silage can deliver high levels of animal performance. However in practice the preparation of high feed-value grass silage is often difficult due to a wide variety of factors, including prevailing weather conditions.

Given the increase in costs of concentrate inputs and availability and costs of major protein sources, such as soy-abean meal, there is much renewed emphasis on maximising production from both grazed and ensiled forages. As silages differ in feed value cattle and sheep are normally supplemented with concentrates to achieve commer-cially optimum production levels. In recent years other ensiled forages, including maize (Zea mays), whole crop wheat (Triticum aestivum), kale (Brassica oleracea) and legumes such as red clover (Trifolium pratense) and lu-cerne (Medicago sativa), have partially replaced grass silage in the diet of growing and lactating ruminants. The objective in this paper is to investigate factors influencing the feed value of grass silage and effects on animal performance. The effects of including maize and whole crop wheat silages in grass silage-based diets on the per-formance of lactating dairy cows, finishing beef cattle, pregnant ewes and finishing lambs is discussed along with recent progress in the ensiling of high protein forage legumes. Whilst much of the literature cited in this paper is from Ireland and the UK, it is relevant to most regions with temperate climate, and the principles apply to most silage production and its utilization world wide.

Manuscript received September 2012


71

Effects of ensiling on forage intake

It is often assumed that ensiling results in a reduction in forage intake and animal performance, as in practice cattle and sheep grazing outdoors have higher intakes than those indoors receiving silage. However this is not a valid comparison as the animals are usually at different stages in their production cycle, grazing animals can select the forage, whilst those offered silage (particularly if precision chopped) cannot select, and other management, animals and feed factors differ. Keady and Murphy (1993) reviewed data from 75 and 14 comparisons undertaken with sheep and beef cattle and showed a mean reduction in silage dry matter (DM) intake of 37% and 6% relative to the parent herbage respectively. However, the fermentation characteristics of the silages offered in these studies differed dramatically. Silage intake characteristics are different for the ovine and bovine (Cushnahan et al. 1994). Keady and Murphy (1993) reviewed 7 comparisons of the effects of ensiling on forage intake of heifers and sheep and reported that whilst offered the same forages, ensiling reduced forage intake by sheep whilst having no ef-fect when offered to heifers. More recently Keady et al. (1995) and Keady and Murphy (1998) reported that when silage is produced using good ensiling management then ensiling per se had no effect on forage intake (Table 1), but reduced animal performance due to changes in the nitrogenous components and reduced energy value of volatile fatty acids as energy sources for the rumen microflora.

Table 1. The effects of ensiling on herbage composition and animal performance

TreatmentFresh grass Silage (70 days ensiled)

Forage composition Dry matter (g kg-1) 172 184 pH 6.41 3.89 Crude protein (g (kg DM)-1) 176 173 Water soluble carbohydrate (g (kg DM)-1) 130 33 Dry matter digestibility (g (kg DM)-1) 739 752Animal performance Forage dry matter intake (kg d-1) 13.6 13.0 Milk yield (kg d-1) 14.1 12.3 Fat plus protein yield (kg d-1) 0.98 0.84Keady et al. 1995, Keady and Murphy 1998

During the ensiling process, major changes occur in the chemical composition of herbage. Two major changes are the conversion of water-soluble carbohydrate (WSC) primarily to lactic and volatile fatty acids, and secondly an increase in the rapidly soluble component of crude protein due to proteolysis and deamination processes (McDonald et al. 1991). Supplementation of silage with sucrose to replenish carbohydrate lost during the ensil-ing process doesn’t compensate for the reduced animal performance due to ensiling per se (Keady and Murphy 1998). However, supplementation with fishmeal, a known source of undegraded dietary protein, increased animal performance probably due to improved efficiency of rumen microbial protein synthesis as protein in silage is ex-tensively degraded in the rumen (Keady and Murphy 1998).

Grass silage feed value

To obtain the optimum level of performance from finishing beef cattle and lactating dairy cows, finishing lambs and pregnant ewes grass silages are normally supplemented with concentrates. The level of concentrate supple-mentation is dependent on the feed value of the silage and the stage of the production cycle of the animals being offered the silage. The feed value of grass silage is a combination of its intake potential and nutritive value, which is determined primarily by digestibility.


72

Silage digestibilityDigestibility is the most important factor influencing feed value of grass silage and, consequently, the performance of animals offered diets based on grass silage (Keady 2000). Data from studies, undertaken using lactating dairy cows, finishing beef cattle, pregnant ewes and finishing lambs that were offered grass silages differing in digest-ibility, were collated to evaluate the effects of increasing silage digestibility on animal performance. Where not available, the digestible organic matter in the dry matter (DOMD, D-value) of the grass silage was determined using the equation of Keady et al. (2001):

DMD = 49.1 + 0.988 DOMD , R2 = 0.95

The available data were categorised according to the source and least squares procedures were used to fit a model with source as a fixed effect and the proportion of forage in the diet as a covariate; the linearity of the effect of forage proportion was tested in all cases by fitting a quadratic term but this was not significant in any case except for live-weight gain of lambs. The R2 value for the variation attributable to regression relative to the variation that remained after fitting source was calculated as a guide to the explanatory value of the regressor.

There is a substantial body of evidence to indicate that increasing silage digestibility increases silage DM intake (DMI) and milk yield. The effects of digestibility on food intake and performance of lactating dairy cows from 23 comparisons are presented in Table 2. The mean responses to an increase of 10 g kg-1 in DOMD were: increased silage DMI 0.22 ± 0.071 kg d-1, milk yield 0.33 ± 0.041 kg d-1, protein concentration 0.087 ± 0.0292 g kg-1 and yield of fat plus protein 0.026 ± 0.0027 kg d-1, and a reduction in fat concentration -0.019 ± 0.0548 g kg-1. Whilst the mean response in milk yield was 0.33 kg for each 10 g kg-1 increase in silage DOMD the response varied from −0.26 to 0.85 kg. The variation in milk yield response to a change in silage digestibility may be due to many factors in-cluding cow genotype, forage:concentrate (F:C) ratio. When the responses in Table 2 were analysed with a model that included effects for data source and the proportion of forage in the diet, the regression on forage proportion was not significant for any performance variable except protein concentration (p<0.03) and approached formal significance for milk yield (R2 = 0.23, p<0.09). The estimates of the response in silage DMI, the yields of milk and of fat plus protein, and the concentrations of fat and protein are displayed in Table 3 for F:C ratios of 80:20, 60:40, and 40:60. The milk yield response per 10 g kg-1 increase in silage DOMD was 0.58, 0.37 and 0.16 kg d-1 when the F:C ratio was 80:20, 60:40 and 40:60 respectively. These estimates show that whilst the response declined as pro-portion of concentrate increased there was still a significant response in terms of fat plus protein yield and pro-tein concentration when high levels of concentrate (60% of DM intake) were offered.

The effects of silage digestibility on food intake and performance of finishing beef cattle from 34 comparisons are summarised in Table 4. The mean responses to each 10 g kg-1 increase in silage DOMD were: increased silage DMI 0.07 ± 0.007 kg d-1, live-weight gain 30.5 ± 2.66 g d-1 and carcass gain 22.8 ± 2.00 kg d-1. Whilst the mean re-sponse in carcass gain was 22.8 kg d-1 for each 10 kg d-1increase in silage DOMD the response estimates varied significantly with F:C ratio (p<0.01, R2 = 0.70); the estimates for F:C ratios from 100:0 down to 40:60 are given in Table 3 for silage DMI, live-weight gain and carcass gain The carcass gain response per 10 g kg-1 increase in silage DOMD was 35, 26, 17 and 8 when the F:C ratio was 100:0, 80:20, 60:40, and 40:60, respectively. Whilst the re-sponse to increased silage DOMD declined as concentrate proportion in the diet increased, a significant response to silage DOMD was still evident when concentrate accounted for 60% of the total DMI. Steen et al. (2002) and Keady and Kilpatrick (2006) concluded that high feed-value grass silage can sustain high levels of beef cattle per-formance. Steen et al. (2002) offered high feed-value grass silage (DOMD 750 g [kg DM ] -1 ) to finishing steers and reported no increase in carcass gain (0.78 kg d-1) when concentrate accounted for more than 40% of the diet. Keady and Kilpatrick (2006) showed that bulls offered high feed-value grass silage (DOMD 775 g [kg DM ]-1 ) as 50% of the diet sustained the same live-weight gain (1.6 kg d-1) as bulls offered concentrate for ad libitum consumption.


73

Tabl

e 2.

The

effe

cts o

f sila

ge d

iges

tibili

ty o

n th

e pe

rfor

man

ce o

f dai

ry c

ows

Refe

renc

eSi

lage

DO

MD(

D) (g

kg-1

)Si

lage

DM

inta

ke

(kg

d-1)

F:Cb

Milk

yie

ld

(kg

d-1)

Milk

fat

(g

kg-1

)M

ilk p

rote

in

(g k

g-1)

Lo

w

High

Low

-DHi

gh-D

Chan

gea

Low

-DHi

gh-D

Chan

gea

Low

-DHi

gh-D

Chan

gea

Low

-DHi

gh-D

Chan

gea

Cast

le a

nd W

atso

n (1

971)

632

719

7.7

8.0

0.04

0.58

15.7

18.0

0.27

44.8

41.5

-0.3

832

.533

.30.

0960

968

76.

97.

40.

060.

5515

.517

.20.

2344

.842

.7-0

.27

32.0

32.3

0.04

Bulte

r TM

(197

7)63

9†71

1†16

.719

.50.

3838

.536

.0-0

.34

29.9

29.7

-0.0

3Go

rdon

(198

0a)

617

699

8.1

10.0

0.23

0.55

23.3

25.0

0.21

36.5

36.3

-0.0

230

.731

.40.

0961

772

68.

110

.70.

240.

5523

.326

.20.

2736

.537

.50.

0930

.732

.50.

17Go

rdon

(198

0b)

624

707

8.6

9.3

0.08

0.49

22.0

23.6

0.19

40.8

39.9

-0.1

134

.734

.70.

00St

een

and

Gord

on (1

980)

64

2†69

5†9.

29.

90.

130.

5821

.825

.00.

6037

.538

.10.

1132

.632

.3-0

.06

642†

695†

7.8

8.9

0.21

0.44

24.2

27.2

0.56

36.1

36.1

0.00

31.5

32.4

0.17

Thom

as e

t al.

(198

1)63

974

89.

59.

90.

040.

6024

.728

.00.

3041

.036

.1-0

.45

29.4

31.5

0.19

Kead

y et

al.

(199

9)68

672

411

.210

.8-0

.11

0.57

27.6

29.0

0.37

44.7

43.9

-0.2

133

.934

.20.

0855

172

410

.710

.80.

010.

5527

.129

.00.

1142

.543

.90.

0831

.834

.20.

14Ke

ady

et a

l. (2

003)

650

680

9.9

10.2

0.10

0.62

25.9

27.3

0.47

38.6

38.9

0.11

31.8

31.5

-0.1

065

068

09.

914

.41.

510.

6229

.532

.00.

8540

.638

.5-0

.71

32.6

32.8

0.08

650

730

9.3

10.1

0.09

0.49

25.9

30.4

0.57

38.6

41.5

0.37

31.8

34.6

0.35

650

730

9.3

12.2

0.35

0.49

29.5

33.1

0.46

40.6

40.5

-0.0

232

.635

.00.

31Ke

ady

et a

l. (2

008a

)61

772

18.

713

.10.

420.

5924

.828

.80.

3840

.339

.6-0

.06

30.4

32.7

0.22

617

721

8.2

10.8

0.25

0.46

28.3

31.5

0.31

39.4

38.4

-0.1

032

.033

.50.

14Ra

ndby

et a

l. (2

012)

647

708

14.4

14.5

0.02

0.80

25.8

26.7

0.15

38.8

42.6

0.62

32.2

32.0

-0.0

364

770

813

.314

.40.

180.

6627

.629

.40.

3039

.541

.20.

2832

.831

.8-0

.16

647

708

11.9

12.9

0.16

0.55

30.8

29.2

-0.2

638

.939

.60.

1132

.233

.60.

2364

774

714

.417

.00.

260.

8025

.829

.10.

3338

.841

.30.

2532

.232

.20.

0064

774

713

.316

.70.

340.

6627

.632

.80.

5239

.540

.90.

1432

.832

.80.

0064

774

711

.914

.20.

230.

5530

.831

.60.

0838

.939

.70.

0832

.233

.20.

10

Mea

n (n

=23)

635

716

10.1

11.6

0.22

**0.

5825

.027

.40.

33**

39.8

39.8

0.01

9 N

S32

.032

.80.

087*

*a Ch

ange

per

10

g kg

-1 in

crea

se in

sila

ge D

OM

Db Fo

rage

:Con

cent

rate

ratio

for t

he lo

w D

OM

D sil

age

diet

† Base

d on

equ

atio

n of

Kea

dy e

t al.

2001


74

Table 3. Responses in silage intake and performance of lactating dairy cows, and finishing beef cattle and lambs to a change of 10 g kg-1 in grass silage DOMD at various forage:concentrate ratios

Animal type

Performance trait Forage: concentrate ratio100:0 80:20 60:40 40:60

Dairy Milk yield (kg d-1) - 0.58 ± 0.144 0.37 ± 0.050 0.16 ± 0.100Fat (g kg-1) - -0.01±0.220 -0.07 ± 0.076 -0.13 ± 0.152Protein (g kg-1) - 0.14 ± 0.093 0.06 ± 0.032 0.26 ± 0.065Fat +Protein yield (kg d-1) - 0.037±0.0101 0.026±0.0035 0.015±0.0070Silage DM intake (kg d-1) - 0.33 ± 0.277 0.20‡ ± 0.096 0.07 ± 0.192

Beef Live-weight gain (kg d-1) 47 ± 5.4 30 ± 3.3 12 ± 4.0 -5 ± 6.7Carcass gain (g d-1) 35 ± 4.0 26 ± 2.5 17 ± 2.9 8 ± 4.8DM intake (kg d-1) 0.12 ± 0.019 0.09 ± 0.006 0.07 ± 0.014 0.04 ± 0.024

Lamb Carcass gain (g d-1) 16 ± 2.3 13 ± 1.3 9 ± 0.9 6 ± 1.5DM intake (kg d-1) 0.08 ± 0.007 0.07 ± 0.004 0.05 ± 0.003‡ 0.03 ± 0.005

Responses in bold are significantly different from zero (p<0.05)‡P + 0.057

The effects of silage digestibility on ewe weight at lambing and lamb birth weight, from 9 comparisons, are pre-sented in Table 5. The mean response to each 10 g kg-1 increase in silage DOMD was an increase in ewe weight post lambing of 1.3 ± 0.08 kg and an extra 52.3 ± 11.41 g in lamb birth weight. Whilst the mean response in lamb birth weight per 10 g kg-1 increase in silage DOMD was 52.3 g it varied from -20 to 101.8 g d-1. When the 9 comparisons in Table 5 were analysed for the effect of concentrate input in late pregnancy (as a proxy for the proportion of for-age in the diet) there was no evidence of any association between silage D-value and forage:concentrate ratio of the diet. Lamb birth weight is positively related to weaning weight and an increase of 1 kg in lamb birth weight re-sults in an increase of 3.2 kg in weaning weight (based on Keady et al. 2007 and Keady and Hanrahan 2009b and c).

The effects of silage digestibility on food intake and performance of finishing lambs from 10 comparisons are pre-sented in Table 6. The mean responses in silage DMI, live-weight gain and carcass gain to each 10 g kg-1 increase in silage DOMD were 0.05 ± 0.006 kg d-1, 14.4 ± 2.74 g d-1and 9.3 ± 1.32 g d-1, respectively. The response to silage DOMD varied significantly with F:C ratio (R2 = 0.61: p<0.03 for carcass gain). The estimates of response as a func-tion of F:C ratio are given in Table 3 and show that a significant response was observed for F:C ratios as low as 0.4. The carcass gain responses per 10 g kg-1 increase in silage DOMD were 16, 13, 9 and 6 g d-1 for F:C ratios of 100:0, 80:20, 60:40 and, 40:60, respectively.

In summary, silage digestibility is positively correlated with carcass gain of beef cattle and finishing lambs, milk yield and composition of dairy cows, and lamb birth weight and ewe weight post lambing.

Effects of silage digestibility on concentrate sparing effect

The level of concentrate supplementation required for silage-based diets to ensure target performance levels is dependent on the feed value of the silage and the stage of the production cycle of the animals being offered the silage. When concentrate price is high relative to product price (milk or meat) one of the potential benefits of in-creasing silage digestibility is the opportunity to maintain animal performance whilst reducing concentrate input.

As the evidence indicates that increasing silage digestibility increases silage intake and animal performance the latter can be maintained by increasing silage digestibility whilst reducing concentrate feed levels. Animal perfor-mance data from the 23, 34, 9 and 10 comparisons summarized in Tables 2−6 were analysed with a linear model having source as a fixed effect and silage DOMD and concentrate intake as covariates; the non-linearity of the co-variate effects was tested in all cases.

The resulting regression equations describing the effects of silage digestibility and concentrate feed level on the performance of lactating dairy cows, finishing beef cattle, finishing lambs and pregnant ewes are presented in Table 7. There was an interaction between the linear responses of beef cattle to changes in DOMD and concen-trate intake as well as a significant quadratic response to concentrate intake. There was no interaction (p>0.05) between silage DOMD and concentrate feed level, or any quadratic effects, for the response in the performance of lactating dairy cows, finishing lambs or pregnant ewes. Each increase of 5 percentage units in silage DOMD enabled the yields of milk and of fat plus protein from dairy cows, carcass gain by finishing lambs and lamb birth weight to be maintained whilst concentrate feed level (DM basis) was reduced by 2.35 kg d-1, 2.80 kg d-1, 0.30 kg d-1and 19.2 kg during late pregnancy, respectively.


75

Tabl

e 4.

The

effe

cts o

f sila

ge d

iges

tibili

ty o

n th

e pe

rfor

man

ce o

f fin

ishin

g be

ef c

attle

Refe

renc

eSi

lage

DO

MD

(g k

g-1)

Sila

ge D

M in

take

(kg

d-1)

TDM

Ic (kg

d-1)

F:Cb

Live

wei

ght g

ain

(g d

-1)

Carc

ass

gain

(g

d-1

)Lo

w

H

igh

Low

-DHi

gh-D

Chan

gea

Low

-DHi

gh-D

Low

-DHi

gh-D

Chan

gea

Low

-DHi

gh-D

Chan

gea

Flyn

n (1

981)

671†

720†

9.5

10.3

0.16

9.5

10.3

1.00

0.67

0.75

16.5

0.45

0.57

24.7

671†

720†

8.0

8.8

0.15

10.6

11.3

0.76

0.78

0.72

-12.

40.

580.

6514

.4St

een

and

McI

lmoy

le (1

982a

)62

466

15.

55.

80.

065.

55.

81.

000.

370.

5240

.50.

090.

2337

.862

466

15.

04.

8-0

.04

6.7

6.6

0.74

0.82

0.77

-13.

50.

310.

4024

.3St

een

and

McI

lmoy

le (1

982b

)60

764

65.

05.

10.

035.

05.

11.

000.

190.

3848

.760

764

64.

94.

7-0

.04

5.7

5.6

0.84

0.41

0.60

48.7

607

646

4.8

4.8

-0.0

26.

56.

50.

740.

670.

7417

.960

764

64.

24.

2-0

.01

6.8

6.7

0.63

0.79

0.95

41.0

607

684

5.0

5.6

0.08

5.0

5.6

1.00

0.19

0.56

48.1

607

684

4.9

4.8

-0.0

15.

75.

60.

840.

410.

7139

.060

768

44.

84.

5-0

.04

6.5

6.2

0.74

0.67

0.84

22.1

607

684

4.2

4.1

-0.0

26.

86.

60.

630.

790.

9014

.3St

een

(198

4b)

644

733

5.8

6.3

0.06

5.8

6.3

1.00

0.47

0.72

28.1

0.27

0.46

21.3

644

733

4.7

5.1

0.05

6.6

7.0

0.71

0.75

0.89

15.7

0.48

0.60

13.5

Dren

nan

and

Kean

e (1

987)

562

676

6.8

8.2

0.13

6.8

8.3

0.99

0.38

0.85

41.4

0.18

0.44

22.4

562

676

6.1

7.3

0.11

8.6

9.9

0.71

1.02

1.40

33.0

0.60

0.81

18.9

562

676

4.7

5.6

0.08

9.6

10.5

0.49

1.34

1.60

22.7

0.84

0.97

11.4

613

693

7.8

9.4

0.21

7.8

9.5

0.99

0.66

1.35

86.3

0.44

0.87

53.8

613

693

7.2

7.6

0.05

9.8

10.2

0.73

1.23

1.58

43.8

0.83

1.01

22.5

613

693

6.0

6.6

0.08

11.2

11.8

0.54

1.49

1.67

22.5

0.97

1.17

25.0

Stee

n (1

992)

643

671

6.6

6.6

0.00

6.6

6.6

1.00

0.62

0.74

42.9

0.42

0.46

14.3

643

717

6.6

7.1

0.07

6.6

7.1

1.00

0.62

0.94

43.2

0.42

0.60

24.3

640

682

6.7

7.3

0.14

6.7

7.3

1.00

0.57

0.82

59.5

0.32

0.54

52.4

640

709

6.7

7.2

0.07

6.7

7.2

1.00

0.57

0.90

47.8

0.32

0.54

31.9

636

699

6.0

5.7

-0.0

57.

77.

40.

780.

990.

92-1

1.1

0.51

0.56

7.9

636

732

6.0

6.4

0.04

7.7

8.1

0.78

0.99

1.12

13.5

0.51

0.66

15.6

663

686

5.0

5.5

0.22

6.7

7.2

0.75

0.76

0.88

52.2

0.48

0.55

30.4

663

734

5.0

6.0

0.14

6.7

7.7

0.75

0.76

1.14

53.5

0.48

0.68

28.2

Stee

n et

al.

(200

2)64

374

36.

37.

50.

128.

29.

40.

770.

601.

0141

.00.

380.

6729

.064

374

35.

36.

20.

099.

310

.20.

570.

781.

0931

.00.

480.

7830

.064

374

33.

84.

20.

0410

.110

.40.

381.

001.

044.

00.

640.

7713

.064

374

31.

92.

30.

0410

.110

.20.

191.

161.

12-4

.00.

770.

792.

0Ke

ady

et a

l. ( 2

008b

)67

173

04.

65.

80.

208.

19.

30.

570.

831.

1045

.80.

470.

6529

.567

173

03.

14.

20.

199.

39.

70.

331.

031.

1215

.20.

560.

6719

.3

Mea

n (n

=34)

628

698

5.55

6.05

0.0

7***

7.56

8.03

0.76

0.75

0.95

30.6

***

0.49

0.66

23.8

***

†Bas

ed o

n eq

uatio

n of

Kea

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D sil

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l dry

mat

ter i

ntak

e


76

Tabl

e 5.

The

effe

cts o

f sila

ge d

iges

tibili

ty o

n th

e pe

rfor

man

ce o

f pre

gnan

t ew

es

Refe

renc

eSi

lage

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MD

(D)

(g k

g-1)

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e of

pre

gnan

cy

Conc

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ate

in

late

pre

gnan

cy(k

g ew

e-1)

Diffe

renc

e in

w

eigh

t b (kg)

Lam

b bi

rth

wei

ght (

kg)

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high

Chan

gea

Low

-DHi

gh-D

Chan

gea

Apol

ant a

nd C

hest

nutt

(198

5)63

474

8la

st 1

4 w

ks

167.

40.

65.

205.

5328

.963

073

0la

st 1

4 w

ks

172.

60.

34.

955.

4853

.0Bl

ack

and

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tnut

t (19

90)

689

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last

14

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2.0

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y an

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y an

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154.

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25

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5.10

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and

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ahan

(201

2a)

675

725

last

14

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21

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3.48

3.78

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in e

we

wei

ght a

t lam

bing

due

to 1

0g k

g-1 in

crea

se in

sila

ge D

OM

D

Tabl

e 6.

The

effe

cts o

f sila

ge d

iges

tibili

ty o

n th

e pe

rfor

man

ce o

f fin

ishin

g la

mbs

Refe

renc

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MD(

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kg-1

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tal D

M in

take

(kg

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F:Cb

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-1)

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High

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Low

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Low

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gea

Low

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Fitz

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ld (1

987)

685†

732†

0.56

0.79

0.04

90.

560.

790.

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00-1

124

7.5

-13

217.

366

4†73

2†0.

440.

790.

052

0.44

0.79

0.05

1.00

-58

2412

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021

9.0

685†

732†

0.59

0.74

0.03

20.

850.

990.

030.

7059

998.

644

685.

266

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2†0.

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0.72

0.99

0.04

0.65

3299

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1968

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d Ha

nrah

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688

719

0.56

0.89

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741.

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7644

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(201

3)68

871

90.

440.

690.

081

0.88

1.13

0.08

0.50

108

177

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9.7

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719

0.31

0.52

0.06

81.

001.

210.

070.

3116

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012

.987

115

9.0

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y an

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6753

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(201

2b)

706

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0.63

0.68

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01.

281.

320.

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4915

918

510

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976.

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004

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3620

821

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912

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0

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n (n

=10)

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728

0.52

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0***

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***

0.64

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128.

414

.4**

37.8

73.0

9.3*

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ange

per

10g

kg-1

incr

ease

in si

lage

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MD

b Fora

ge:c

once

ntra

te ra

tio o

n th

e lo

w D

OM

D sil

age

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† Base

d on

equ

atio

n of

Kea

dy e

t al.

2001


77

Table 7. Regression equations for the relationship of animal performance with silage digestibility and concentrate feed level

Animal type

Performance trait Constant

Regresssion coefficient (s.e.) for R2

Sig¶

DOMD† Conc§ D-value*Conc Conc2

Dairycows

Milk yield (kg d-1) 4.85 +0.260 (0.0462) +0.554 (0.1265) 0.61 ***

Fat & Protein (kg d-1) -0.034 +0.023 (0.0036) +0.041 (0.0098) 0.64 ***

Beef cattle

Carcass gain (kg d-1) -1.90 +0.033 (0.0037) +0.333 (0.0661) -0.0036 (0.00100) -0.0038 (0.00199) 0.75 ***

Finishinglambs

Carcass gain (g d-1) -632 +8.6 (1.21) +142.4 (13.60) 0.91 ***

Pregnantewes Lamb BW (kg) 0.73 +0.050 (0.0115) +0.013 (0.0120) 0.67 ***

†In units of 10 g kg-1; §Concentrate dry matter (kg d-1) ¶Significance of the regression equation.

An analysis of the relationship between the delay in harvest date and the corresponding change in digestibility was undertaken using data from studies that involved at least 3 harvest dates (Gordon 1980a, Steen 1984b, Dren-nan and Keane 1987, and Keady et al. 2000). The model used had source as a fixed effect and number of days as a covariate. This analysis yielded a significant linear relationship between the number of days by which harvest date was delayed and the corresponding change in D-value (g kg-1). The regression coefficient was 4.8 ± 0.68 g kg-1 per 1 day delay (p<0.001, R2 0.86). Thus, silage digestibility declines, on average, by 3.3 percentage units for each 1 week delay in harvest date. Consequently, for each 1 week delay in harvest an extra 1.55 kg d-1, 0.20 kg d-1and 12.7 kg (in late pregnancy) of concentrate (DM) is required to maintain milk yield of dairy cows, carcass gain of lambs and lamb birth weight. For finishing beef cattle offered silage with a DOMD of 670 or 710 g kg-1and a daily concentrate supplement of 4 kg DM, each 1 week delay in harvest date requires an additional concentrate DM input of 0.99 and 1.30 kg d-1 to maintain carcass gain (Table 8).

Table 8. Effects of a change ( ±50 g kg-1) in silage DOMD on the concentrate sparing effect with beef cattle offered silages differing in DOMD and supplemented with different levels of concentrate

Concentrate DM (kg day-1)DOMD (DMD) 2 4 6670 (711) 1.67 1.50 1.21690 (731) 1.85 1.70 1.44710 (751) 2.07 1.97 1.77730 (771) 2.35 2.34 2.32

Major factors affecting digestibility of grass silage

Harvest dateMost of the factors that effect silage digestibility can be controlled by the producer. Harvest date is the most im-portant factor affecting silage digestibility. Silage digestibility declines as harvest date is delayed. An analysis of the relationship between the delay in harvest date and the corresponding change in digestibility was undertaken using data from studies that involved at least 3 harvest dates (Gordon 1980a, Steen 1984, Drennan and Keane 1987, Steen et a.l 1992 and Keady et al. 2000). The model used had source as a fixed effect and number of days as a covariate. This analysis yielded a significant linear relationship between the number of days by which harvest date was delayed and the corresponding change in D-value (g kg-1). The regression coefficient was 4.8 ± 0.68 g kg-1 per 1 day delay (p<0.001, R2 0.86). Thus, silage digestibility declines, on average, by 3.3 percentage units for each 1 week delay in harvest date. The rate of decline in herbage digestibility from the primary regrowth is similar to that for the primary growth. Therefore for each one week delay in harvesting of grass for ensilage, to sustain milk yield of dairy cows, carcass gain of beef cattle and finishing lambs and lamb birth weight from pregnant ewes, an additional 0.8-1.55 kg d-1, (depending on silage feed value and concentrate feed level, Table 8) 0.20 kg d-1 and 12.7 kg (during late pregnancy) of concentrate DM must be offered to lactating dairy cows, finishing beef cattle, finishing lambs, and pregnant ewes, respectively.


78

Whilst date of harvest is negatively correlated with silage digestibility it is positively correlated with herbage yield. Keady and O’Kiely (1998) and Keady et al. (2000) reported that each one day delay in harvest increased herb-age yield of the primary growth of predominantly perennial ryegrass swards by 145 and 151 kg DM respectively.

Lodging, or flattening, of the grass crop prior to harvest accelerates the rate of decline in herbage digestibility as harvest date is delayed. This accelerated decline in digestibility is due to the accumulation of dead leaf and stem at the base of the sward. Digestibility may decline by as much as 9 percentage units per week in severely lodged crops (O’Kiely et al. 1987).

Sward typeNormally, it is assumed that silage produced from old permanent pastures has a lower digestibility than silage produced from a perennial ryegrass sward. However, the negative impact of old permanent pasture on silage di-gestibility is dependent on botanical composition. If old permanent pastures are harvested at the correct stage of growth silage with a high feed value can be consistently produced.

A 2 year study was undertaken by Keating and O’Kiely (2000), using 4 harvests per year, to evaluate the effects of sward type on grass silage feed value. In the first year of the study, beef carcass output (kg ha-1) was similar for si-lage produced from old permanent pasture (45% Poa spp, 26% Agrostis spp, 10% Lolium perenne, 6.5% Alopecu-rus protensis, 2% Dactylis glomerate, 10.5% other) and a perennial ryegrass sward. Carcass output was lower for the silage from the old permament pasture in the second year of the study, but this was attributable to the fact that the silage produced from the first harvest of this pasture had a lower digestibility than that from the peren-nial ryegrass sward (swards closed the previous October) (Keating & O’Kiely 2000).

The effects of sward type on feed value of silage harvested from the second re-growth (third harvest) (Keady et al. 1994) are presented in Table 9. Silage produced from an old permanent pasture (52% L perenne, 28% Agrostis stolonifera, 10% Poa spp, 10% Holcus lanatus) and that from a predominantly perennial ryegrass (L perenne) pas-ture resulted in silages that had similar (high) feed value, as determined by metabolisable energy (ME) concen-trations (determined in-vivo) and intake when offered to growing cattle. Consequently, high feed-value silage can be produced from old permanent pasture provided it has a moderate level of perennial ryegrass and is ensiled at the correct stage of maturity using good ensiling management. Table 9. Effect of sward type on silage composition, digestibility and intake

Sward typeOld permanent pasture Perennial ryegrass SE sig.

Silage Composition pH 3.97 4.07 0.025 NS Ammonia nitrogen (g kg-1 N) 75 74 2.4 NS Metabolisable energy (MJ [kg DM]-1) 12.0 11.7 0.08 *Silage DM intake (kg d-1) 3.66 3.56 0.17 NS

Keady et al. 1994

Perennial ryegrass varieties are classified according to heading date. Whilst the general recommendation is to harvest swards at approximately 50% ear emergence, the actual date of emergence depends on the grass varie-ties in the sward and thus on their heading date. The effects of heading date (intermediate or late) of perennial ryegrass varieties, and date of harvest on the performance of beef cattle were evaluated in two studies by Steen (1992); the main results are presented in Table 10. The intermediate- and late-heading swards each consisted of 3 different varieties (with similar heading date) of perennial ryegrass. Whilst the mean heading date of the in-termediate- and late-heading swards differed by 24 days (19 May and 12 June), herbage from the late-heading swards had to be ensiled within 8 days of that from the intermediate-heading swards to give the same silage di-gestibility and daily carcass gain of finishing beef cattle. If the harvest of the late-heading sward was delayed un-til 50% ear emergence the resulting silage DOMD would be 51 g kg-1 lower than the silage from the intermediate-heading sward, consequently reducing silage DMI and carcass gain (from 0.63 to 0.40 kg d-1). Steen (1992) also reported no significant effect of either date of harvest of the primary growth or heading date (grass variety type) of the sward on total animal DM production.


79

Table 10. Effect of sward heading date and harvest date on silage digestibility and animal performance

Performance Heading date × Harvest dateIntermediate (19 May) Late (12 June)

20 May 28 May 5 June 28 May 5 June 13 JuneSilage DOMD (g [kg DM]-1 ) 725 685 640 722 679 652Silage DM intake (kg d-1) 6.8 6.2 6.3 6.6 6.4 5.9Carcass gain (kg d-1) 0.63 0.51 0.46 0.61 0.55 0.40

DOMD: digestibility of organic matter, DM: dry matter Steen 1992

Similarly, results from studies using small scale silos show that herbage from late-heading varieties (heading date 10 June) must be ensiled on 31 May to produce similar silage digestibility as that for intermediate-heading varieties (heading date 22 May) (Humphreys and O’Kiely 2007). However, these authors also noted that the rate of decline in digestibility with harvest date was not as rapid for late-heading varieties as for intermediate-heading varieties.

Silage fermentationRelative to well-preserved silage, poorly preserved untreated silage with low lactic acid concentrations and high concentrations of ammonia nitrogen normally has lower digestibility. The reduction in DOMD in untreated silag-es due to deterioration in silage fermentation can be as high as 70 to 80 g kg-1 (Keady and Steen 1995). However for silages which are treated with an effective bacterial inoculant at ensiling, but which have poor fermentation characteristics (at feed out) there is no negative impact on digestibility or on subsequent animal performance (Keady and Steen 1995, Keady 1998).

Fertilizer N application and wiltingApplication of excess fertilizer N alters silage digestibility. Increasing the rate of fertilizer N from 72 to 168 kg ha-1 for the primary growth of predominantly perennial ryegrass swards reduced silage DOMD by 13 g kg-1 (Keady et al. 2000). The reduction in digestibility due to increased N fertilizer application is probably due to increased con-centrations of acid detergent fiber and acid detergent lignin both of which are negatively correlated with digest-ibility (Keady et al. 2000). Increasing N fertilizer application increases herbage yield. Long et al (1991), Keady and O’Kiely (1998) and Keady et al. (2000) reported increased herbage yields of 10.2, 5.2 and 7.9 kg DM per kg increase in N fertilizer application, respectively.

Wilting reduces silage digestibility. From reviews of the literature, Wilkins (1984) and Rohr and Thomas (1984) re-ported average proportional reductions in silage DM digestibility, as assessed through sheep, of 0.031 and 0.041, respectively. More recently Steen (1984a) and Gordon et al. (1999), using beef cattle, and Yan et al. (1996) and Keady et al. (1999), using dairy cows, reported proportional reductions in total diet digestibility of 0.03, 0.045, 0.02 and 0.02, respectively. The decline in digestibility due to wilting is due to a loss of available nutrients and an increase in ash concentration. The rate of decline in digestibility due to wilting depends on the length of time be-tween mowing and ensiling the herbage, and on soil contamination due to mechanical treatment. Rates of loss in digestibility vary from 2.3 to 9.0 g kg-1 per 10 hour wilting period. Thus each day (24 hours) of wilting will reduce silage DOMD by between 6 and 22 g (kg DM)-1.

Wilting

Wilting herbage prior to ensiling has many advantages including reduced effluent production, improved ensila-bility characteristics, reduced quantities of material for transport during ensiling and feed out, reduced freezing in cold climates and reduced straw requirement for bedding livestock. When wilting, a rapid wilt is desirable to minimize the decline in digestibility. The rate of water loss during wilting is primarily related to solar radiation and the weight, per unit area, of herbage in a swath (Wright 1997) and prevailing wind speed. Furthermore the lower the initial DM concentration of herbage at mowing the more water that has to be removed to increase the DM concentration by 100 g kg-1, e.g., if herbage is mowed at a DM concentration of 150 g kg-1 and dried to 250 g kg-1 then an extra 1 kg water per 1 kg DM is lost than is lost when herbage with an initial DM concentration of 200 g kg-1 is dried to 300 g kg-1 (Wright et al. 2000). Reducing the density of the cut herbage involves covering the to-tal ground area with herbage, which results in a higher drying rate. Herbage mown in auto-swaths (two swaths placed into one) has a much higher density than when the herbage is tedded out, thus management practices


80

have a big impact on herbage drying rate (Table 11). The data in Table 11 show that to increase herbage DM from 160 g kg-1 to 250 g kg-1 required 65, 30 and 14 hours, respectively, for herbage that was mown in auto-swaths (6 m width of herbage in one swath), single swaths (3 m width of herbage in one swath) or tedded out, to cover the total ground area, immediately post mowing, respectively.

Table 11. Effects of swath treatment and wilting period on herbage dry matter concentration (g kg-1) (Yield = 29.4 t ha-1)

Wilting period (hours)

0 24 48

Swath treatment Auto-swathed+ 160 192 228 Single swath 160 229 317 Tedded out 160 304 500+ two swaths placed into one Wright 1997

Many studies have been undertaken on the effects of wilting on animal performance. Steen (1984a), from a re-view of 40 comparisons in the literature, Steen (1984a), from the mean of four studies, and O’Kiely (1994), from one study, reported that wilting herbage prior to ensiling resulted in an 18%, 5% and 13% increase in silage DMI, 41, -30 and -56 g change in daily live-weight gain and 30, 40 and 31 g reduction in daily carcass gain of beef cat-tle, respectively. Using pregnant ewes, Chestnutt (1989) reported that wilting herbage at ensiling increased silage DMI by 7.4% whilst having no beneficial effect ( –0.05 kg) on lamb birth weight. Similarly, using finishing lambs, Fitzgerald (1986) reported that wilting herbage at ensiling increased silage DMI by 26% but had no effect on daily carcass gain. More recently, data from dairy cows, from the mean of 11 comparisons (Patterson et al. 1996 and 1998), summarized by Keady (2000) show that rapid wilting of herbage from a DM concentration of 160 g kg-1 to 320 g kg-1 increased silage DMI by 17% and milk solid output by 3% but reduced cow feeding days per hectare by 174 days and milk output by 3074 l ha-1.

Many producers delay harvesting in showery weather conditions, with the intention of getting dry weather for wilting. However, in a prolonged period of showery weather crop digestibility is declining, whilst there may be opportunities to harvest and ensile as direct cut (unwilted). The effects of direct cutting, ensiling following water application (equivalent to rainfall) and wilting on the performance of dairy cows have been evaluated (Keady et al. 2002) and a summary is presented in Table 12. The wilted herbage was ensiled at a DM concentration of 277 g kg-1 following a 30 hour wilting period. Wilting increased silage DMI but had no effect on milk yield or composi-tion. Application of water at ensiling reduced herbage DM concentration at ensiling (131 v. 187 g kg-1) but had no effect on silage DMI or on milk yield or composition, illustrating that ensiling herbage direct cut (unwilted) during showery conditions has no negative impact on animal performance.

Table 12. Effect of herbage dry matter at ensiling on dairy cow performance

Herbage dry matter at ensiling (gkg-1)131 187 277 SE sig.

Silage dry matter intake (kg d-1) 9.7 9.6 13.6 0.026 ***Milk yield (kg d-1) 20.1 20.0 20.0 0.14 NSFat (g kg-1) 39.9 40.1 41.3 0.53 NSProtein (g kg-1) 33.2b 32.8a 34.2c 0.13 ***Keady et al. 2002

The data clearly show that whilst wilting reduces effluent production, wilting increases daily silage DMI and re-duces the number of animal feeding days and the output of animal product per hectare.


81

Fertilizer managementNitrogen

To achieve the maximum response to fertilizer N, soil P, K and pH need to be at optimum levels. The response in herbage yield to inputs of fertilizer N is presented in Table 13. The response varied from 5.2 to 10.2 kg herbage DM per 1 kg N. The response varies depending on the base level of nitrogen applied, prevailing weather condi-tions and harvest date. Fertilizer N also affects herbage composition. Increasing the rate of fertilizer N applied increases herbage crude protein concentration and reduces herbage DM concentration (Keady and O’Kiely 1996, 1998; Keady et al. 2000) thus presenting a greater challenge for silage preservation. Furthermore, increasing N fertilizer application can reduce the concentration of WSC (Wilson and Flynn 1979, Keady et al. 2000), probably associated with the synthesis of WSC to plant structural components with increased herbage yield causing a re-duction in the capture of solar radiation per unit of plant mass. Therefore, applying excess fertilizer N can have a negative impact on herbage ensilability. However, an inadequate level of fertilizer N reduces herbage yield and the crude protein concentration of the subsequent silage. Furthermore, inadequate N fertilizer application may result in a clostridial fermentation, as determined by butyric acid, due to inadequate concentrations of nitrate in the silage (Jaakkola et al. 1999).

The optimum level of N for the first, second and third harvests from predominantly perennial ryegrass swards are 120, 100 and 80 kg ha,-1 respectively (Keady et al. 1998). When paddocks are closed for silage after being grazed it should be assumed that between 20 and 30% of the N applied for the most recent grazing is available for the silage crop. Table 13. Effect of fertilizer (N) application on herbage dry matter (DM) yield in the primary growth

Source Range in N application (kg ha-1)

kg DM per kg nitrogen

Long et al. (1991) 100–150 10.2Keady and O’Kiely (1998) 120–168 5.2Keady et al. (2000) 72 −168 7.9

PotassiumLarge quantities of K are required, and removed, by silage crops. Up to 26 kg of K are removed per tonne of herb-age dry matter (Keady and O’Kiely 1998). It has been suggested previously that there is a strong correlation be-tween soil K concentration and the subsequent buffering capacity of Lucerne (M sativa) (Muck and Walgenbach 1985) and that this may have a negative impact on the composition and feed value of the resultant silage. A study was undertaken by Keady and O’Kiely (1998) to evaluate the effects of fertilizer K on herbage yield, composition and feed value. Increasing the level of fertilizer K increased herbage yield at both the first and second harvests (Table 14). The mean response, between the two harvests, was 4.5 kg herbage DM per 1 kg K applied. Applica-tion of excess K can result in luxury uptake of K by the crop. However excess K application has no effect on herb-age composition or ensilability (Table 14) or on predicted feeding value of the resultant silage (Keady and O’Kiely 1998). The quantity of fertilizer K that should be applied for silage production depends on the soil potassium index and expected herbage yield (thus crop requirements).

Table 14. Effect of potassium (applied on 2 March) on herbage yield at the first and second harvest (index = 3 K in soil) and composition of the herbage from the first harvest

Potassium applied (kg ha-1)0 60 120 180 240 SE sig.

Herbage dry matter yield (t ha-1) - first harvest 6.31 6.57 6.74 6.93 6.93 0.091 *** - second harvest 2.56 2.73 2.83 2.94 2.99 0.056 ***Dry matter (g kg-1) 179 170 169 171 169 3.1 NSBuffering capacity ( mEq [kg DM-1]) 430 442 454 445 442 9.7 NSWater soluble carbohydrate (g[kg DM-1]) 101 93 94 100 96 2.7 NSNitrate (mg [kg DM-1]) 35 18 18 13 15 9.1 NS

Keady and O’Kiely 1998


82

Chop length

Whilst chop length has no effect on silage DMI or on the performance of beef cattle (Steen 1984a) or dairy cows (Gordon 1982), chop length affects the intake characteristics of silage when offered to pregnant ewes (Chestnutt 1989) and finishing lambs (Fitzgerald 1996). Reducing silage chop length, determined by harvester type, increased silage DMI and live-weight gain of finishing lambs by up to 34% and 242%, respectively (Fitzgerald 1996). When offered to pregnant ewes, reducing silage chop length by use of a precision chop harvester relative to single chop-ping, increased silage DMI and, consequently, increased lamb birth weight by 0.25 kg and reduced weight loss by ewes during pregnancy by 4.9 kg (Chestnutt, 1989).

In a recent study (Keady and Hanrahan 2008) big-bale and precision-chop silage systems were compared in both the first and second harvests using herbage that had been ensiled at a mean dry matter concentration of 249 g kg-1 (Table 15). Results showed that system of ensiling had little impact on silage DMI or on lamb birth weight. However, weaning weight was 1.8 kg higher for lambs from ewes that were offered the precision-chop silage dur-ing pregnancy and was due to higher daily live weight gain between birth and 10 weeks of age. Table 15. Effect of silage harvest system on ewe performance

Harvest systemBig bale Precision chop

Silage dry matter intake (kg d-1) 0.95 0.97Lamb weight - birth (kg) 4.7 4.8 - weaning (kg) 32.5 34.3Lamb weigh gain (g d-1) - 0 to 5 weeks 314 338 - 5 to 10 weeks 314 332Keady and Hanrahan 2008

Additive management

In the past the principal objective in applying a silage additive was to improve silage fermentation under difficult ensiling conditions. This was achieved be applying acid or sugar-based additives. However, more recent research has shown that the use of effective bacterial inoculants can substantially improve animal performance without necessarily altering the fermentation quality of the silage at the time of feeding (Gordon 1980; Keady and Steen 1994, 1995).

Animal performance is the most important measure of the efficacy of a silage additive as producers are paid for animal product, and not for the preservation quality of silage as measured by conventional laboratory analysis. When applying additives it is important to apply them at the correct rate, taking account of changes in the mois-ture concentration in the herbage being ensiled. For example, if the dry matter concentration of the herbage is increased from 180 to 250 g kg-1, the fresh weight of grass will be reduced from 29.5 to 21 t ha-1 consequently re-ducing additive requirement per hectare by 40%.

Many studies have been undertaken to evaluate different classes of additives with respect to the performance of beef cattle and lactating dairy cattle. From a review of 11 published studies in which molasses (mean application rate of 15.8 l t-1 of herbage) and formic acid treated silages were compared with untreated silages Keady (1996) concluded that whilst molasses treatment improved silage fermentation it did not increase animal performance. In the same review Keady (1996) concluded that formic acid treatment increased animal performance by 17%. From a review of 95 comparisons in which silages made with different types of additives were compared to un-treated silages Keady (1998) concluded that use of proven effective bacterial inoculants, under a wide range of ensiling conditions, or formic acid, under difficult conditions, increased the performance of lactating dairy cows and finishing beef cattle. Whilst use of molasses, sulphuric acid and enzyme-based additives improved silage fer-mentation, they had no significant effect on animal performance. Patterson et al. (1998) reported similar improve-ments in dairy cow performance from inoculant treatment of direct cut (unwilted) and wilted herbage at ensiling.


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Mode of action of inoculantsWhilst inoculant treatment may have little effect on silage fermentation at the time of feed out, numerous stud-ies (Gordon 1989a, Keady and Steen 1994, 1995) have shown that inoculant treatment results in a more rapid fermentation, as measured by the fall in pH immediately post-ensiling relative to untreated herbage. This more rapid fermentation due to inoculant treatment suppresses proteolysis and deamination processes of herbage protein (Heron et al. 1987) and results in higher retention of soluble components. From a review of 40 compar-isons in the literature, in which the organic matter digestibility of inoculated and untreated silages were com-pared, Keady (1991) concluded that inoculant treatment increased organic matter digestibility by 2%. In a more recent series of five comparisons Keady et al. (1994) and Keady and Steen (1994, 1995) concluded that the use of an effective inoculant containing a single strain of L. plantarum substantially increased silage digestibility (organic matter digestibility) by 33 g kg-1, particularly where silage was from difficult-to-ensile herbage, primarily due to dramatic improvements in the digestibility of the fibre fractions. The same authors concluded that the improve-ment in animal performance following treatment with an effective inoculant can be attributed to the retention of greater proportions of the more soluble components of the plant due to a more rapid and efficient fermenta-tion process within the silo. This subsequently results in increased silage digestibility and a reduction in the ex-tent of protein break down.

Alternative forages

Traditionally in many parts of Europe, Australia, New Zealand and North America, grass silage was offered to cat-tle and sheep during the indoor feeding period. However in recent times other ensiled forages, such as maize (Z. mays) and whole crop wheat (T. aestivum), have increased in popularity and have partially replaced grass silage in the diet.

Maize silageMajor developments, both in plant breeding and in agronomic practice, have enabled the consistent production of high yields of maize forage in areas in which it was not possible to grow the crop 20 to 30 years ago. For example, the dry matter yield of maize crops produced in Northern Ireland have increased by 300%, to 12.2 t ha-1, primar-ily due to plant breeding efforts (Keady 2005). Developments in agronomic practices, particularly the complete cover plastic mulch system (CCPM), which was developed in Ireland, have further increased yield potential and the degree of maturity attained by crops grown in more temperate climates. The CCPM system involves cover-ing the crop with a thin clear film (6 to 9 microns) through which the plant emerges at approximately the 6 leaf stage. Use of this system increases forage yield by up to 50% as it enables later maturing varieties to be planted at earlier sowing dates (Keady 2005, Keady and Hanrahan 2013). Keady (2005) concluded that silage produced from maize sown under the CCPM system, because of its higher yield potential, could be produced at the same cost as grazed grass, but if sown in the open the cost of silage from maize was 30% and 20% greater than that of grazed grass and 3-cut grass silage, respectively.

Effects of maize on beef cattle performance

Keady (2005) concluded, from the mean of 34 comparisons in which grass silage was partially or totally replaced with maize silage, that maize silage significantly increased forage DMI (1.5 kg d-1) and performance of lactating dairy cows, as determined by milk yield (+1.4kg d-1), fat concentration (+0.6 g kg-1) and protein concentration (+0.8 g kg-1). Keady (2005) also concluded, from the mean of 9 comparisons in which grass silage was partially or totally replaced with maize silage, that maize silage significantly increased forage DMI (1.5 kg d-1) and performance of beef cattle, as determined by live-weight gain (+0.23 kg d-1), carcass gain (+0.11 kg d-1) and carcass weight (+12 kg). In the same review Keady (2005) concluded, based on dairy cow studies, that in order to achieve optimum levels of performance from dairy cattle the maize crop should be harvested and ensiled at a dry matter concentration of approximately 300 g kg-1. More recently, using beef cattle, Keady et al. (2013) concluded that the response to the inclusion of maize silage in the forage component of the diet, in terms of daily carcass gain, was dependant on stage of maturity of the maize at harvest and level of inclusion in the diet. Keady et al. (2013) concluded that maize silage with a dry matter of 304 g kg-1, when offered ad libitum as the sole forage, increased daily carcass gain by 31% due to a combination of increased ME intake and improved efficiency of ME utilization and produced carcasses with whiter fat. However, when offered as 50% of the forage component of the diet, stage of maturity (maize silage with dry matter concentrations of 217 and 304 g kg-1, respectively) had no significant effect on beef cattle performance.


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As outlined by Keady (2005) and Keady et al. (2007, 2008a and 2013) when the cost of concentrate is high rela-tive to the price of animal product, one of the potential benefits of including an alternative forage in diets based on grass silage is the potential to maintain animal performance whilst reducing concentrate feed level. Keady (2005) and Keady et al. (2007, 2013) reported potential concentrate sparing effects of up to 2.4 kg per animal daily when maize silage was included in the diet of beef cattle; the effect depended on level of maize inclusion in the diet and crop maturity at harvest. For lactating dairy cows Keady (2005) concluded including maize silage in grass silage based diets had a potential concentrate sparing effect of up to 5.0 kg per cow daily

Effects of maize on performance of pregnant ewes

In many parts of Ireland, the UK and other sheep producing regions ewes are normally housed during the winter feeding period and offered ensiled forage. Whilst many studies have shown that including maize silage in the for-age component of diets offered to beef cattle and dairy cows increases animal performance (Keady 2005, Keady et al. 2007, 2008a, 2013) few studies have been undertaken to evaluate the effects on the performance of preg-nant ewes or finishing lambs. One of the characteristics of maize silage is its low crude protein concentration (consistently less than 100 g (kg DM) -1, which declines as maturity increases (Keady et al. 2003, 2008a, 2013) and may impact on its ability to meet the protein requirements of ewes in early and mid pregnancy. Robinson (1983) concluded that forages offered during early and mid pregnancy should contain a minimum crude protein concen-tration of 10 g per MJ of ME, otherwise the forage needs to be supplemented with protein to meet requirements. Keady and Hanrahan (2008) evaluated the effects of replacing grass silage with maize silages differing in maturi-ty at harvest and supplemented with either 0 or 200 g soyabean meal during mid and late pregnancy. Replacing grass silage with maize had no effect on ewe performance at lambing or on lamb birth or weaning weights. Sup-plementation of maize silage with soyabean meal during mid and late pregnancy increased ewe condition at lamb-ing but did not alter lamb performance. More recently Keady and Hanrahan (2009a) evaluated the effects matu-rity of maize at harvest, grass silage feed value, soyabean meal supplementation during mid and late pregnancy, and concentrate supplementation during late pregnancy on ewe and lamb performance (Table 16). Increasing maturity of maize significantly increased ewe weight at lambing. Whilst supplementation with soyabean meal in-creased ewe weight at lambing and lamb birth weight, there was no effect on lamb weight at weaning (14 weeks). Both studies (Keady and Hanrahan 2008, 2009a) indicated that increasing maturity of maize at harvest tended to increase lamb weaning weight by about 1 kg. Keady and Hanrahan (2009d) offered pregnant ewes maize silage as the basal forage and concluded that there is no benefit to supplementing ewes with protein in mid pregnancy and that total concentrate supplementation during late pregnancy for twin bearing ewes could be reduced to 10 kg soyabean (with mineral and vitamin supplementation) meal without having any negative impact on ewe or subsequent lamb performance.

These studies show that maize silage can replace high feed-value grass silage in the diet of ewes in mid and late pregnancy. Maize silage, whilst low in crude protein, can be offered as the sole forage without protein supple-mentation until late pregnancy (final 6 to 7 weeks). Increasing the maturity of maize at harvest tended to increase lamb weaning weight by 1 kg. Table 16. The effects of maturity of maize silage, grass silage feed value and concentrate level on animal performance

Maize silage (MS) dry matter Grass silage feed value (GS)Low High Low High sig.3

Soya (S)1/conc (C)2 0/15 200/15 0/15 200/15 0/15 0/25 0/5 0/15 0/25 sem MS S GSEwe weight (kg)4 63.0 68.6 68.2 76.6 61.2 61.6 70.4 73.6 73.6 2.15 ** ** ***Litter size (lambs/ewe) 2.09 1.74 2.04 2.03 1.95 1.80 1.62 1.65 1.81 0.171 NS NS NSLamb weight (kg)

- birth 4.62 4.92 4.65 5.29 4.60 4.56 4.85 5.13 5.13 0.168 NS ** *** - weaning 33.4 32.8 34.1 34.3 33.6 32.1 34.0 35.0 34.3 1.05 NS NS *Lamb weight gain (g d-1) 296 289 303 299 300 284 301 309 302 10.17 NS NS NSAge at slaughter (days) 162 160 159 141 170 175 170 154 164 9.0 NS NS p=0.08Dressing proportion 0.43 0.43 0.42 0.43 0.43 0.43 0.42 0.44 0.43 0.009 NS NS NSCarcass weight (kg) 18.9 18.2 19.1 18.7 18.8 19.1 19.4 19.6 19.2 0.43 NS NS NSFat classification 5 3.1 2.8 3.0 2.9 2.8 3.0 2.9 3.0 2.9 0.12 NS p=0.07 NS

1Soya = g d-1 for duration of the study. 2Total concentrate(kg) in late pregnancy. 3There were no significant concentrate (C) effects or maize silage x soyabean meal or grass silage x C level interactions. 4Weight post lambing. 5Fat classification (scale 1 to 5; where 1 = thin, 5 = fat)Keady and Hanrahan 2009a


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Effects of maize silage on performance of finishing lambs

Prime lamb production is seasonal and grass-based with lambing normally targeted to coincide with the start of grass growth in spring (Keady and Hanrahan 2013). However, in Ireland 20% of the lamb kill occurs during the first 3 months of the year, which helps to maintain seasonality of supply of lamb to the market. As grass growth rate during the winter declines to as low as 0 kg d-1 a large proportion of these lambs are finished on high concentrate diets, or diets containing conserved forages and concentrates.

Keady and Hanrahan (2013) reported that increasing the maturity of maize at ensilage, as indicated by starch con-centration (277 v. 33 g [kg DM]-1 ), improved carcass gain of finishing lambs. The effects of maturity of maize at ensilage, grass silage feed value and concentrate feed level on the performance of finishing lambs from two studies (Keady and Hanrahan 2012b, 2013) are presented in Table 17. Maize silage replaced high feed-value grass silage and was better than medium feed-value grass silage (DOMD 688g kg-1) in the diet of finishing lambs, as indicated by daily carcass gain, regardless of concentrate feed level. As concentrate feed level increased the response to forage feed-value declined, but was still evident when concentrates accounted for up to 70% of total DMI. Keady and Hanrahan (2012b and 2013) reported that replacing medium feed-value grass silage with maize silage result-ed in a potential concentrate sparing effect of up to 0.48 kg per lamb daily. Table 17. Effects of forage type and concentrate feed level on lamb performance

ForageGrass silage Maize

Conc (kg d-1) Medium HighTotal DM intake (kg d-1) 0.3 0.9 1.1 1.1

0.7 1.1 1.2 1.11.0 1.2 1.3 1.3

ad libitum 1.4Carcass weight (kg) 0.3 17.3 20.6 20.2

0.7 21.1 22.4 22.11.0 22.8 24.2 23.3

ad libitum 26.6Carcass gain (g d-1) 0.3 14 62 59

0.7 72 91 871.0 98 119 107

ad libitum 150Keady and Hanrahan 2012b, 2013

Whole crop wheatThere has been increased interest in the production of whole-crop cereal silage for feeding to beef and dairy cat-tle in recent years. The increased interest in this crop is due primarily to the similar cost of production relative to grass silage and the perceived potential benefits in forage DMI and subsequently animal performance. Whole crop wheat is predominantly ensiled and fermented at DM concentrations ranging from 250 to 450 g kg-1. How-ever, whole crop wheat can also be ensiled at high DM concentrations, ranging from 550 to 800 g kg-1, and treated with either urea or a urea-based additive to encourage an alkaline environment. Recent developments in the en-siling of whole crop cereals involves the ensiling of crops at high DM concentrations (700−800 g kg-1), harvested through a forage harvester fitted with a grain processor and ensiled with a urea-based additive.

Effect of whole crop wheat inclusion on animal performance

From a review of 20 comparisons involving dairy cows and 7 comparisons involving finishing beef cattle Keady (2005) concluded that whilst partially or totally replacing grass silage with whole crop wheat, either fermented or urea treated, increased forage DMI it had no beneficial effect on the yields of milk or of fat plus protein from dairy cows, or on carcass gain of beef cattle. Similarly, O’Kiely (2011) noted that replacing low feed-value grass silage with triticale reduced carcass gain of finishing beef cattle. However, Walsh et al. (2008) noted that replac-ing a poorly preserved, low feed-value grass silage (pH and concentrations of DM, ammonia nitrogen and DOMD of 4.3, 174 g kg-1 155 g kg-1 N and 634g (kg DM-1), respectively) with whole crop wheat increased performance of finishing beef cattle.


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Forage legume and kale silagePreviously legume forages, e.g., red clover (T. pratense) and lucerne (M. sativa), were regarded as being unsuitable for ensiling due to having a low concentration of WSC and a high buffering capacity. However, advances in silage technology have enabled these forages to be ensiled as high-protein forage for livestock. Results from a recent study (Marley et al. 2007) of the effects of forage type (red clover, lucerne, kale, peas) showed that live-weight gain, food conversion and nitrogen use efficiency were higher in lambs offered the red clover, lucerne and kale (B oleracea) silages compared with those offered low feed-value hybrid-ryegrass silage (ME 10 MJ kg-1 DM). As ru-minants are typically viewed as inefficient converters of dietary protein, the data presented in Table 18 show the potential of using these forages to enable more sustainable and nutrient-efficient livestock systems. Table 18. Effect of forage type on lamb live-weight gain (LWG g d-1), food conversion efficiency (FCE) [kg live-weight gain (DM consumed-1); kg kg-1] and nitrogen use efficiency (NUE) [live-weight gain (N consumed) -1; kg kg-1] of finishing lambs fed different silages.

Trait Ryegrass Red clover Lucerne Kale s.e.d. Level of significanceLWG 36 135 135 100 8.9 ***

FCE

0.053b 0.133a 0.130a 0.136a

0.0081 ***NUE 2.9c 4.4b 3.7bc 5.3a 0.36 ***

Marley et al. 2007

Despite the potential for these ensiled alternative forages to contribute to livestock systems there has been rela-tively little research into their effects on the performance of beef cattle or sheep. However a number of studies have been undertaken to evaluate the effects of legumes on the performance of dairy cows which have been re-viewed by Dewhurst (2012).

Red clover and lucerne

Typically, legumes have a higher buffering capacity than grasses (Pitt 1990), and the pH of lucerne silage tends to be higher than that of red clover silage (McDonald et al. 1991) due to its higher buffering capacity (typical buffer-ing capacities of red clover and lucerne are 560 and 480 mEq (kg DM) -1 respectively (Wilkinson 1978)) and lower amounts of fermentable substrate (Raguse and Smith 1966). The conservation of red clover and lucerne as silage in areas within the EU that have high rainfall often necessitates harvesting at a DM concentration less than 300 g kg-1. The dry matter concentration of legumes at ensiling can affect the fermentation and quality of the silage produced. Fychan et al. (2002a) wilted red clover and lucerne for either 27, 51 or 75 hours and reported that in-creasing the wilting period increased pH and WSC concentration and reduced the concentrations of ammonia-N and lactate in the silages, and that the wilting rate of lucerne was higher than that of red clover.

Legumes for ensiling should be harvested at the stage of maturity that maximises crude protein concentration while ensuring adequate sugars for fermentation. As legumes mature, the concentration of crude protein de-clines. The different stages of maturity can be determined using the formula and phenological-staging scheme described by Kalu and Fick (1981) for red clover, and modified for lucerne by Ohlsson and Wedin (1989). Research on the effect of stage of maturity at harvest on the yield and chemical composition of red clover during three har-vest years showed that allowing red clover to mature to early-late bud stage before taking the first harvest gave a 20% increase in dry matter yield and a 32% increase in protein yield per hectare compared to harvesting at a late vegetative stage for all harvest years (Fychan et al. 2002b). A study on the effect of stage of maturity at harvest on the yield and chemical composition of lucerne during three harvest years showed that it was advantageous to allow the first harvest to be taken when the stage of growth is between early and late bud (Fychan et al. 2002b).

Proteolysis during ensiling is influenced by forage species. Protein degradation is lower when red clover is ensiled compared to lucerne (Papadopoulos and McKersie 1983, McKersie 1985, Jones et al. 1995a) as red clover contains an enzyme, polyphenol oxidase, that inhibits proteolysis, both in the silo and in the rumen (Jones et al. 1995b, Broderick and Albrecht 1997). Fychan et al. (2002c) showed that ensiling lucerne and red clover in a clamp silo, when compared to round bales, resulted in lower pH, and lower concentrations of free amino acids and WSC, and higher concentrations of lactate in both lucerne and red clover silages. Marley et al.,(2003) concluded that the op-timum ratio to sow red clover lucerne bi-crop, based on the resultant silage analysis, was 50:50 red clover:lucerne due to the improved wilting properties of lucerne and the reduced proteolysis of ensiled red clover.


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Ensiled legumes can increase growth rate in lambs due to higher forage DMI and improved nitrogen utilisation efficiency relative to grass silage (Fraser et al. 2000, Speijers et al. 2005a). However Speijers et al. (2005a) report-ed that the intake and growth rate by finishing lambs offered ensiled lucerne was the same as that of lambs fed ryegrass silage. Using twin bearing ewes Speijers et al. (2005b) reported higher food intake for lucerne silage compared to ryegrass silage.

Incorporating red clover silage into the winter diet of an upland beef system increased live-weight gain relative to animals offered low feed value grass silage (DOMD 586 g kg-1) (Fraser et al. 2007). In the same study, meat from the steers fed on red clover silage over winter had a higher lipid oxidation than steers grazing permanent pasture supplemented with grass silage (previously ensiled from that pasture) probably related to the vitamin E content of the loin muscle, however there were no differences between treatments in the sensory properties (texture, juici-ness or flavor) of the beef. With respect to vitamin E concentrations, Beeckman et al. (2010) reported that treat-ing ryegrass, red clover or white clover with formic acid or lactic acid bacteria at ensiling did not affect vitamin E concentration. Furthermore Beeckman et al. (2010) showed that ryegrass had higher concentrations of vitamin E than clovers and that still difference remained following wilting and ensiling. Studies with beef cattle offered ensiled lucerne showed higher intakes for beef cattle fed ensiled Lucerne relative to grass silages (Pocknee and Campling 1981, Doyle and Thomson 1985, Han et al. 2006). The higher intakes recorded for ruminants offered legume silages relative to grass silage have been attributed to legume silages having a higher passage rate due to higher rumen outflow rate (Dewhurst et al. 2003b).

One constraint to the use of red clover in sheep systems is the presence of phyto-oestrogen compounds, which reduce ewe fertility (Barrett et al. 1965). Phyto-oestrogens, e.g., formononetin, are a group of naturally-occurring plant-derived non-steroidal compounds that have the ability to cause oestrogenic and/or anti-oestrogenic effects in livestock (Benassayag et al. 2002). Thomson (1975) concluded that feeding ensiled red clover could still reduce ewe fertility when offered at rates as low as 25% of the diet. In a more recent study, ewes being offered a diet containing 50% red clover silage were found to have isoflavones in body tissues (Urpi-Sarda et al. 2008).Sarelli et al. (2003) reported that the oestrogen content of red clover silage was higher than the raw material and that the type of silage inoculant used during ensiling affected phyto-oestrogen levels in the resultant silage. There are con-tradictory reports on the effect of red clover silage on cattle. Kallela et al. (1984) reported that plant oestrogens in a pure red clover silage (with 0.56% formononetin in the dry matter) were the most likely cause of infertility problems in cows. However Austin et al. (1982) conclude that there was no evidence to indicate that herd fertil-ity was suppressed by red clover silage.

Kale

The introduction of big bales for silage production provided the opportunity to conserve kale (Brassica oleracea) more effectively than had been previously achieved in clamps, despite its low dry matter concentration. Kale, which has high crude protein concentrations, can be grown as an early-sown catch crop or a late-sown main crop (Martyn et al. 1997). Other advantages of kale are its high DM yield relative to other brassica crops (Drew et al. 1974;) and digestibility (Young 1997a,b). The crude protein concentration of kale declines as the plant matures. Forage kale contains anti-metabolites, such as S- methyl cysteine sulphoxide and glucosinolates, which break down in the ru-men and lead to various toxicity symptoms in ruminants (Coxganser et al. 1994). However, the work of Fales et al. (1987) showed that ensiling forage brassicas can effectively reduce the potential toxicity of fresh brassica forages.

Marley et al. (2007) and Vipond et al. (1998) reported that relative to medium feed value grass silage ensiled kale increased live weight gain of finishing lambs. Furthermore, kale significantly improved nitrogen use efficiency in growing lambs compared to red clover, lucerne and ryegrass (Marley et al. 2007).

Conclusions

It is concluded that evidence from Ireland and UK show that in relation to grass silage digestibility is the most im-portant factor influencing feed-value and consequently the performance of animals offered grass silage based diets. Each 10 g kg-1 increase in DOMD increases daily milk yield of lactating dairy cows by 0.33 kg, daily carcass gain of beef cattle by 23.8 g head-1, daily carcass gain of finishing lambs by 9.3 g head-1,lamb birth weight by 52.3 g, and ewe weight post lambing by 1.3 kg. Harvest date is the main factor effecting silage digestibility. Each one week delay in harvest reduces digestibility by 3.3 % units. To sustain animal performance due to delay of harvest by one week requires an additional 12.7 kg concentrate DM per ewe in late pregnancy, 0.20 kg concentrate DM


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daily per finishing lamb, 1.2 kg concentrate DM daily per finishing beef animal (depending on silage feed value and concentrate feed level), and 1.55 kg concentrate DM daily per lactating dairy cow. Solar radiation and swath density are the major factors effecting rate of water loss from herbage during wilting. Wilting results in increased silage DMI and reduced animal output per hectare. Potassium fertilizer impacts on herbage yield, but does not affect herbage ensilability or silage feed value. Chop length has no effect on silage DMI by dairy and beef cattle. However reducing chop length increases silage DMI by pregnant ewes and finishing lambs.Use of bacterial inoc-ulants across a wide range of ensiling conditions and formic acid under difficult ensiling conditions increase ani-mal performance.

In relation to alternative forages maize when produced using the CCPM system, can be produced at a similar cost as grazed grass (under Irish and UK conditions). Optimum stage to harvest maize is at approximately 300 g kg-1. Partially or totally replacing grass silage with maize silage in grass silage based diets increases the performance of finishing beef cattle, finishing lambs and pre-weaned lambs sucking their dams (which had been offered maize si-lage during pregnancy). Partially or totally replacing grass silage with whole crop wheat silage in grass silage based diets increases forage intake without any beneficial effect on animal performance. Advances in silage technology have opened up opportunity for ensiling high protein legume-based forages such as red clover, lucerne and kale. Offering red clover, lucerne and kale silages to lambs can improve live-weight gain relative to that with medium feed-value grass silage.

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Opportunities for reducing environmental emissions from forage-based dairy farms

Tom Misselbrook1, Agustin del Prado2 and David Chadwick1

1Rothamsted Research, North Wyke, Okehampton, Devon EX20 2SB, UK 2BC3-Basque Centre for Climate Change, Alameda Urquijo 4, 48008 Bilbao, Spain


Modern dairy production is inevitably associated with impacts to the environment and the challenge for the in-dustry today is to increase production to meet growing global demand while minimising emissions to the environ-ment. Negative environmental impacts include gaseous emissions to the atmosphere, of ammonia from livestock manure and fertiliser use, of methane from enteric fermentation and manure management, and of nitrous oxide from nitrogen applications to soils and from manure management. Emissions to water include nitrate, ammonium, phosphorus, sediment, pathogens and organic matter, deriving from nutrient applications to forage crops and/or the management of grazing livestock. This paper reviews the sources and impacts of such emissions in the context of a forage-based dairy farm and considers a number of potential mitigation strategies, giving some examples using the farm-scale model SIMSDAIRY. Most of the mitigation measures discussed are associated with systemic improve-ments in the efficiency of production in dairy systems. Important examples of mitigations include: improvements to dairy herd fertility, that can reduce methane and ammonia emissions by up to 24 and 17%, respectively; diet modification such as the use of high sugar grasses for grazing, which are associated with reductions in cattle N ex-cretion of up to 20% (and therefore lower N losses to the environment) and potentially lower methane emissions, or reducing the crude protein content of the dairy cow diet through use of maize silage to reduce N excretion and methane emissions; the use of nitrification inhibitors with fertiliser and slurry applications to reduce nitrous oxide emissions and nitrate leaching by up to 50%. Much can also be achieved through attention to the quantity, timing and method of application of nutrients to forage crops and utilising advances made through genetic improvements.

Key words: ammonia, diffuse water pollution, farm-scale model, greenhouse gas, mitigation

Introduction

The dairy sector, in common with other agricultural sectors, currently faces a great challenge to meet rising global food demands, particularly for livestock-derived food products, in a sustainable way (Godfray et al. 2010). There are important interactions between food production and other ecosystem services, including climate regulation, air and water quality, nutrient cycling, soil erosion, biodiversity and landscape quality, as discussed by Pilgrim et al. (2010) for temperate grassland systems, and the sustainable intensification of production relies on a good un-derstanding of these interactions and our ability to identify potential ‘win-win’ strategies.

The assessment of such interactions for given management or mitigation scenarios on forage-based dairy farms was the primary aim of the development of the farm-scale SIMSDAIRY model (del Prado et al. 2011). SIMSDAIRY inte-grates all of the major components of a dairy farm into a modelling framework using a system-based approach. It consists of modules dealing with overall farm management, herd nutrition and performance, field-scale flows of nitrogen (N) and phosphorus (P), livestock manure, economics and sustainability attributes. Specifically, SIMSDAIRY quantitatively simulates the effect of interactions between farm management, climate and soil characteristics on losses of N, P and carbon (C), including effects on farm profitability and giving a more qualitative indication of ef-fects on biodiversity, milk quality, soil quality and animal welfare. While developed for UK dairy systems, and not-ing that outputs can vary depending on the model-scenario farm characteristics (particularly soil and climate), this can be used more generically as a useful tool in providing an assessment at the whole farm system level of the introduction of single or multiple mitigation methods, showing trade-offs between production and environ-mental effect, or between different environmental effects and identifying win-win scenarios. It is important that environmental effects are expressed per unit of production (e.g. litre of milk), i.e. an emission intensity metric, such that strategies leading to sustainable intensification of production can be identified as distinct from those which may reduce environmental impact at the expense of production.



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The aims of this paper are to give an overview of the potential environmental impacts to air and to water of pre-dominantly forage-based dairy systems, to discuss some of the most promising potential mitigation strategies and to assess the impacts of a number of these using the farm-scale model SIMSDAIRY.

Environmental impacts of dairy farmsEmissions to the atmosphere

The key emissions to the atmosphere of environmental concern from dairy farms are ammonia (NH3) and the greenhouse gases methane (CH4) and nitrous oxide (N2O). Other potential emissions of environmental concern include non-methane volatile organic compounds, fine particulates and heavy metals (Misselbrook et al. 2011), and while these may be of local importance in some instances such as around very intensive feedlots (e.g. Shaw et al. 2007), agriculture is generally not considered to be a major source for these species and they are not dis-cussed further here.

Agriculture is the major source of NH3 emissions to the atmosphere, accounting for >80% of total anthropogenic emissions in the UK (Passant et al. 2011), with the dairy sector accounting for approximately one third of total agricultural NH3 emissions. In a dairy farm context, NH3 emissions arise predominantly from the urea content of urine excreted by dairy cows, the urea being readily hydrolysed to ammonium in the presence of the ubiquitous enzyme urease. Emissions will therefore occur from wherever cattle urine is deposited, at grazing, in housing and yards, and from manure storage and spreading. In addition, emissions occur from urea- and ammonia-based in-organic fertilisers applied to land. Ammonia is of concern because of potential damage to sensitive ecosystems through acidification and eutrophication, and also because of its role in the formation of secondary particulates in the atmosphere (ammonium nitrate and ammonium sulphate) and their negative implications regarding hu-man health (Erisman et al. 2007).

The NH3 flux from an emitting surface depends on a number of factors, including the NH3 concentration at the emitting surface, pH, total exposed surface area (and surface area to volume ratio), temperature and the air flow above the emitting surface. Management, in addition to environmental conditions, can therefore have a great influence on emissions from livestock housing and manure storage (Sommer et al. 2006), from manure applica-tion to land (Sommer et al. 2003) and from fertiliser applications (Sommer et al. 2004). Mitigation strategies are therefore generally aimed at reducing the overall emitting surface area, reducing the NH3 concentration at the emitting surface or reducing air flow at the emitting surface.

Agriculture is a significant source of anthropogenic CH4 emissions to the atmosphere, accounting for c. 40% of emissions in the UK (MacCarthy et al. 2011), with the dairy sector estimated to account for approximately one third of total agricultural emissions. Methane is a greenhouse gas, with a global warming potential of 25 times that of CO2, over a 100 year lifetime (Forster et al. 2007). The major source of CH4 emissions from the dairy sec-tor is enteric fermentation in the rumen of cattle, whereby CH4 is a by-product of microbial carbohydrate degra-dation. Enteric emissions are influenced by the gross energy intake of the animal and the digestibility of that en-ergy, with the energy intake in turn being dependant on the energy requirements of the animal for maintenance, production (milk and/or growth), pregnancy and activity. Mitigation strategies are aimed at directly inhibiting the methanogenic bacteria in the rumen, manipulating the microbial breakdown pathways in the rumen, manipulat-ing the digestibility of the diet or maximising the proportion of energy intake over the lifetime of an animal ulti-mately being used for milk production.

Methane emissions also arise from manure management, deriving from the microbial breakdown of excreted volatile solids under anaerobic conditions. Key driving factors are temperature, manure composition and degree of anaerobicity, which will be influenced by management (Chadwick et al. 2011). Mitigation strategies are aimed at reducing storage duration and/or temperature, minimising anaerobic conditions or through capturing and uti-lising produced CH4.

Agriculture is also a major source of N2O emissions, accounting for c. 80% of emissions in the UK (MacCarthy et al. 2011), with the dairy sector estimated to account for approximately one fifth of total agricultural emissions. Ni-trous oxide is a potent greenhouse gas, with a global warming potential of 297 times that of CO2, over a 100 year lifetime (Forster et al. 2007). Nitrous oxide emissions arise as products, or partial products, of the microbial pro-cesses of nitrification (conversion of ammonium to nitrate [NO3

-]) and denitrification (conversion of NO3- to dinitro-

gen gas [N2], with intermediary products as nitrite [NO2-], nitric oxide [NO] and N2O). Nitrification is essentially an


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aerobic process, while denitrification occurs under anaerobic conditions. The key direct sources of N2O emission from dairy farming are from N amendments to the soil, either as inorganic fertiliser, manure applications, graz-ing excretal returns or crop residues, and the management of livestock manure during housing and during stor-age. Major influencing factors are the availability of N and C, anaerobicity and, to a lesser extent, temperature. A proportion of NO3

- leached and N deposited to land is re-emitted as N2O. These indirect losses of N2O are signifi-cant. Mitigation strategies for direct N2O emissions are aimed at reducing the availability of N, particularly under anaerobic conditions (e.g. wet soils), and at impeding the microbial processes through the use of inhibitors. Miti-gation of indirect losses of N2O, for example via NO3

- leaching, are aimed at optimising N supply for crop demand and minimising the risk of excess N in the soil.

Emissions to ground and surface watersSources of diffuse water pollution on dairy farms include the farm steading (uncollected seepage from buildings, manure stores, yards frequented by dirty equipment and livestock), tracks, and the land itself (via fertiliser and manure applications, livestock grazing on the grassland, and nutrient applications to and cultivation of maize or cereal land). The principal diffuse water pollutants are NO3

- , ammonium, P, sediment, pathogens and organic mat-ter (which generates at oxygen demand in the water course) (Chadwick and Chen 2003).

As much as 60% of the NO3- found in UK watercourses is thought to come from agriculture. It arises from excess

N input from fertiliser, applied manure and excreta from grazing livestock that is not utilised by the grass or crop. Rainfall then leaches the NO3

- through the soil profile to drains and into watercourses.

Ammonium is a cation and hence can be immobilised in the soil profile. It is also readily nitrified to NO3-, so is

generally only found in low concentrations below grasslands. However, it can be lost following rainfall events that result in rapid overland flow, or movement of slurry through cracks in the soil to drains. The effect of excess N in watercourses is to provide nutrients to algae and other aquatic plant life (eutrophication), resulting in excessive growth and potential algal blooms. Nitrite and NH3 are also found in drainage water and are toxic to freshwater fish.

Phosphorus is another nutrient that contributes to eutrophication of watercourses. The relative impact of the N or P leached to surface waters depends on their nature; in some ecosystems N is the first limiting nutrient for algae growth whereas in other systems P may be limiting. Phosphorus is immobilised on soil surfaces and complexes with organic matter and metals such as iron, and is held strongly within the soil profile. Most of the P applied to grasslands is found in the top soil layer, so it’s main pathway to watercourses is via detachment of soil particles and colloids in storms followed by overland flow (or again via cracks in the soil to drains following a slurry appli-cation), in contrast to movement of N which can be vertical and horizontal. In general, temperate grassland may lose 1−3 kg total P ha-1 year-1 (depending on inputs), but an individual rainfall event following a slurry application could result in ‘incidental’ losses as high as this in just one storm (Preedy et al. 2001).

Phosphorus losses are associated with sediment transfers from agricultural land. Sediment is a pollutant per se, as it affects the spawning grounds of salmonids. Although arable land is known to be a large source of agricultural sediment, grasslands are also a source (Granger et al. 2010), which is exacerbated by grazing livestock under wet soil conditions. On dairy farms, land used for forage maize is a potential critical source for sediment (and P) ero-sion and transfer to watercourses, especially if late harvests coincide with wet soil conditions.

The organic matter in dairy slurry and dirty water can generate an oxygen demand if it finds its way into a water-course. This biological oxygen demand (BOD) results in rapid proliferation of micro-organisms in the watercourse which respire rapidly, removing oxygen from the water – resulting in asphyxiation of aquatic life. The BOD of a typical dairy slurry is ca. 10,000 mg l-1 (Chadwick and Chen 2003), and that of dirty water ranges from 200−1000 mg l-1 (Cumby et al. 1999). The recommendation for treated effluent entering a watercourse is 20 mg l-1 (HMSO 1980), so any significant loss of slurry or dirty water into a river will have negative environmental impacts.

Livestock manures applied to agricultural land and faeces deposited during grazing are sources of a range of patho-genic organisms. For dairy farming, the key pathogens include Cryptosporidium and Campylobacter. Whilst specific pathogens are of key interest in terms of human health, it is the indicator species of E. coli and Intestinal Entero-cocci, known as faecal indicator organisms (FIOs) on which legislation is based (CEC 2006). The risks of FIO losses from livestock farms to watercourses has been explored (Chadwick et al. 2008b), and are greater from farms with a greater number of livestock, steeply sloping land, and limited slurry storage capacity (Oliver et al. 2009). How-ever, risks are also affected by farmer attitudes. In some instances, the topography of a farm can act as a ‘safety net’, e.g. where flat land reduces the risk of transfers, even if a farmer has limited slurry storage capacity or is un-aware of the consequences of injudicious management of livestock and their manures.


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Haygarth et al. (2005) introduced the concept of pollutant movement from source to the watercourse, via the source-mobilisation-delivery-impact model. This model lends itself to addressing mitigation of diffuse water pol-lutants at each stage. Thus the source can be reduced, either through e.g. application of less fertiliser nitrogen, or by applying it in frequent doses and not all at once – thus reducing the risk of excess nitrogen in the soil at risk of loss. Mobilisation is the process by which a pollutant starts its journey towards the watercourse, and can occur via detachment or solubilisation. So mitigation methods suitable to reduce mobilisation would include the use of a nitrification inhibitor with an ammonium-based fertiliser, the incorporation of slurry into a maize field, rather than leaving it on the soil surface, or the injection of slurry into grassland soils to avoid surface run-off, reduce NH3 emissions and avoid potentially harmful bacteria from coming into contact with the crop. Finally, delivery can be reduced by intercepting pollutant rich drainage or overland flow via e.g. a constructed wetland.

Guidance is supplied to farmers to protect watercourses from diffuse water pollution, e.g. the UK Joint Code of Practice (Defra 2009). In some countries legislation is in place to reduce the impact of agriculture on water quality, e.g. the EU Nitrates Directive (EC 1991) has resulted in individual member states developing action plans to reduce the NO3

- concentrations of vulnerable watercourses. The action plans include closed periods for the spreading of high available N content manures, e.g. dairy slurry, and set a maximum N loading for a farm, thus introducing a stocking rate limit. The EU has also introduced the Water Framework Directive (CEC 2000) to protect the ecologi-cal status of watercourses. This covers a wider range of pollutants than just NO3

-, and governments are putting in place guidance to farmers to help them comply with strict targets on future ecological status of watercourses.

Through a greater knowledge of the behaviour of different water pollutants, mitigation methods can be devel-oped that are method-centric and can tackle multiple pollutants, rather than addressing just one individual pol-lutant (Granger et al. 2010). It is also essential that guidance on choice of mitigation methods takes account of any secondary impacts, e.g. pollution swapping. Cuttle et al. (2006) produced a Mitigation Manual for Diffuse Water Pollutants. The 44 methods included management of land use, soil, fertilisers, manures, livestock and farm infra-structure, e.g. provision of bridges to allow livestock to ford streams to reduce sediment and pathogen transfers to water. This Mitigation Manual was provided to Catchment Sensitive Farming Officers to provide advice on practical methods which could be introduced on farms, and at what cost. It has recently been updated to include mitigation methods for greenhouse gas and NH3 emissions. Both guidance documents highlight the relevance of each method to different farming systems, expresses the potential effectiveness in reducing the target pollutant(s) and the sec-ondary impacts on other pollutants, the indicative cost of introducing the method, its practicality and likely uptake.

Potential mitigation methods

Considerable research effort in recent years has been aimed at developing mitigation methods and strategies to reduce the environmental impact of agricultural production practices. Specifically for dairy farms, these include animal health, diet, crop nutrient management, grazing management and genetic improvement in both livestock and crops. These are discussed in more detail below, with some specific scenarios assessed using the SIMSDAIRY model. Changing the intensity of production (defined by milk yield per cow) may be considered as a potential mitigation strategy; Hagemann et al. (2011) cite level of intensity as the main reason for differences in GHG emis-sion per kg energy corrected milk across 45 dairy regions in 38 countries. However, this is not considered explic-itly within this review as it is recognised that a range of dairy production systems and levels of intensity will exist for reasons of climate, resource availability and socio-economic considerations. Rather, it is the scope to which potential mitigation measures can be implemented, the efficiency of production improved and the environmen-tal impact per litre of milk production reduced that is important across all of these systems.

Livestock healthProduction losses as a consequence of animal ill health and/or poor fertility result in an increase in the environ-mental emissions per litre of milk produced. In particular, the proportion of replacement animals required in a herd (related to the average number of lactations per dairy cow) can have a significant effect on emission inten-sity. Garnsworthy (2004) showed that significant reductions in CH4 and NH3 emissions could be made through im-provements to dairy cow fertility, by up to 24% and 17%, respectively.


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Dietary strategies

Dairy diet manipulation can lead to reductions in enteric CH4 emissions, and in N and P excretion, while having no detrimental effect on productivity. Potential dietary manipulations include the use of dietary additives with spe-cific inhibitory effects on rumen CH4 production, manipulation of the in-house diet composition, particularly with respect to protein content and form, and manipulation of the grazed sward composition.

A number of dietary additives have been assessed for their effectiveness in reducing enteric CH4 emissions, either by direct inhibition or depopulation of rumen methanogens or through encouraging alternative microbial path-ways of removing rumen hydrogen (e.g. Martin et al. 2010, Cottle et al. 2011), but in vitro effects are often diffi-cult to replicate in vivo (van Zijderveld et al. 2011) or are short-lived (Guan et al. 2006).

Dairy cow dietary P intake is often in excess of requirements (e.g. Powell et al. 2002, O’Rourke et al. 2010) and improved matching of requirement in the diet can result in significant reductions in P excretion (Dou et al. 2002) without compromising production or fertility (Wu and Satter 2000). The subsequent reduction in environmental impact of excreted P was reported by O’Rourke et al. (2010), who observed a 63% reduction in manure total P con-tent from a 43% reduction in dietary P, and a significant reduction in the P concentration in overland flow follow-ing manure application for manure from the low dietary P treatment. However, the same authors also concluded that the time interval between manure application and the generation of overland flow has a greater impact on P losses than does varying the dietary P content.

Manipulating the protein content of the diet, both in terms of the amount and forms of the protein has been shown to have significant effects of the amount of N excreted by cattle. Many studies have shown the potential that reducing the crude protein (CP) content of the diet can have on N excretion (e.g. Kulling et al. 2001, Broder-ick 2003) and therefore subsequent losses of N to the environment. Misselbrook et al. (2005b) showed this for a lactating dairy cow diet with a CP content of 14% compared with one of 19% (with the same proportion and type of forage), but also showed the influence of including condensed tannins in the diet (through manipulation of forage type), with significant reductions in NH3 emissions from the cattle excreta without negatively impacting on milk production. Both dietary strategies had the effect of reducing urinary N excretion by the cattle, which is more susceptible to environmental losses, at least in the shorter term, than faecal N.

Dietary manipulation at grazing relies on management of the sward composition. For example, the use of grass varieties with a high content of water soluble carbohydrate, so called high sugar grasses (HSG), can reduce N ex-cretion by almost 20% (urinary N excretion by 29%) in cattle through more efficient utilisation of the feed N in the rumen and enhance productivity (Miller et al. 2001, Moorby et al. 2006). In a recent trial, CH4 emissions from growing lambs grazing HSG were also shown to be reduced, by an absolute value of 20%, when compared with lambs grazing a conventional ryegrass sward, and also showed increased intake values and live weight gain (IBERS 2010). Inclusion of red clover in the sward, with the protein-binding action of the polyphenol content (Jones et al. 1995), has been hypothesized to reduce N excretion, as shown empirically by Powell et al. (2009) particularly for urine N excretion, although results from a study by van Dorland et al. (2007) were less supportive.

Model scenarios for diet manipulation Farm scale modelling enables the impact of dietary (and other) strategies on a number of potential production and pollutant outputs to be assessed and, in particular, highlight where trade-offs in impacts may have to be made. The farm scale model, SIMSDAIRY (del Prado et al. 2011) was used to assess the impact of two dietary strategies: i) growing and feeding HSG (i.e. replacing conventional grass cultivars); and ii) restricting CP intake either through the increased use of forage maize produced on-farm or by just reducing N concentration in the concentrates diet (depending on intensity of dairy system). Given a user-defined herd structure and type, milk production target and diet profile (as proportion of the diet coming from grass silage, maize silage and concentrates) SIMSDAIRY simulates through several iterations the metabolic processes and N and C pathways at the animal level (this includes DM intake, energy and true protein requirements, rumen CH4 and N excretion in urine and dung), the N and P flows and N and CH4 losses at the manure handling level, including the stages from the excretion on a barn floor or open lot surface where it remains until it is removed, the storage of manure and the application of manure to the soils in the farm. Subsequently, SIMSDAIRY simulates on a monthly time-step the N turnover in soil after application of animal manure and slurry (or urine and dung deposited whilst grazing), which in combination with mineral fertil-izer N management and soil and weather conditions affects both productivity and quality of grass or maize and losses of N. Forage area is adjusted according to the total grass and maize required and the productivity of grass


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and maize per hectare (once silage making and feeding losses are accounted for). Herd typologies were defined for a set of locations and intensity of production systems (intensive/fully-housed, medium, extended), with full details given in IBERS (2010). New (from associated experimental work in IBERS 2010) and existing (Miller et al. 2001) experimental information at the animal level were incorporated on the effect of different diets on enteric CH4 output, milk production and N excretion. The main changes were carried out to simulate the effect of HSG intake on milk yield and voluntary dry matter intake, both of which are enhanced by HSG (Miller et al. 2001). The empirical equations relating enteric CH4 production to dry matter intake (del Prado et al. 2011) were modified for the forage maize portion of the diet according to empirical evidence from IBERS (2010).

For the HSG scenarios (Fig. 1), overall greenhouse gas emissions were reduced by up to 19% per litre of milk, through reductions in both CH4 and N2O. Ammonia emissions per litre of milk were reduced by up to 22%, mainly due to the combination of fewer hectares required to produce 1 litre of milk and also due to reductions in excreted N (particularly urine N). Reductions in N excretion were also associated with reductions in NOx emissions, because of the smaller pool of inorganic N subject to nitrification. Nitrate leaching was not significantly affected. Despite the potential beneficial effect of HSG on greenhouse gas emissions, if reseeding is required more frequently than for conventional grass varieties (to ensure persistence of effect), then the reduction in emissions described above could be offset by an increase in soil N2O emissions, CO2 emissions from fossil fuel use associated with reseeding and a decrease in potential soil C storage.

Fig. 1. Change (%) in greenhouse gases (GHG), soil C storage, NH3, NOx and NO3- leaching for high

sugar grasses (HSG) and reduced crude protein (CP) mitigation measures compared with baseline scenarios. Range reflects the model outputs across the range of locations and intensities of production.


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For lower CP intake scenarios (Fig. 1), overall greenhouse gas emissions were reduced by up to 11% per litre of milk. The details of the reduction depended on the intensity of the dairy system. For example, for the intensive-fully housed system there was no reduction in enteric CH4 as the starch to fibre ratio was not altered in the diet, whereas N2O emissions were reduced because of reductions in N excretion, particularly in urine. For medium and extensive systems, enteric CH4 was reduced through a higher starch to fibre concentration in the diet (e.g. Beauchemin et al. 2008, Cottle et al. 2011), but soil N2O emissions were increased mainly caused by the replace-ment of grass with forage maize and the changes in manure application rates and timing. The proportion of land use change from grassland to maize determined the extent of the potential soil C loss. Results for NH3 and NOx emissions very much depended on the intensity of the system, with a balance between reductions in N losses as-sociated with lower N excretion and increases in N losses through indirect management changes after grassland conversion to maize. The main effect on NO3

- leaching losses was the conversion of grassland to maize and the associated changes in manure application timing.

Crop nutrient managementThe soil nitrogen cycle is complex and potential crop uptake and losses to water and the atmosphere are very de-pendent on the form, rate and timing of the nitrogen inputs to the soil, soil texture and water status, and subse-quent environmental conditions.

For inorganic nitrogen fertilisers, much can be achieved by attention to the type, timing and rate of application, ensuring that nitrogen supply matches crop requirements and is not applied in excess. Urea fertiliser, in particular, can be associated with large NH3 emissions of up to almost 50% of the applied N (Misselbrook et al. 2004), par-ticularly if used under hot, dry conditions. Use under cooler conditions, at low application rates will be associated with much lower emissions (Misselbrook et al. 2004), and the incidence of rainfall soon after application will also reduce emissions, by up to 90%, by ensuring rapid dissolution and transport of the urea into the soil matrix (Sanz-Cobena et al. 2011). Smith et al. (2012) give some evidence that direct N2O emissions are less from urea fertilis-er applications than from other fertiliser types, but indirect emissions associated with the greater NH3 emissions from urea would have been greater, so on balance there was no overall difference between fertiliser types. Emis-sions of N2O may increase disproportionately with fertiliser application rate, as shown for fertiliser applications to grassland at three sites in England by Cardenas et al. (2010) where the annual emission factor (proportion of total fertiliser N applied during the year lost as N2O) was greater for higher cumulative annual application rates.

The use of forage legumes, such as clover in grass leys, offers the potential to offset applied inorganic N with bio-logically fixed N. Perceived disadvantages with the use of white clover are year to year variation in sward content and persistence (Frame et al. 1986). With greatly increasing fertiliser prices in recent years, there is a growing resurgence of interest in forage legumes, and a combination of improved traits through breeding and improved management practices may overcome some of these main perceived disadvantages (Parsons et al. 2011). Whilst the clover is growing, soil N2O emissions are generally smaller than those from inorganic fertilised soils as N origi-nating from biological fixation is generally less available for nitrification and subsequent denitrification. Bacteria fix the N2 gas from the air into the NH4

+ ion that is largely used by the clover to form protein compounds. Once the legume crops are harvested, however, the protein compounds in residues are susceptible to decomposition and mineralisation to NH4

+, which can then be nitrified and denitrified, leading to N2O emissions (Snyder et al., 2009). Nitrate leaching losses have been shown to be lower from grass-clover pastures than from fertilized grass (e.g. Hooda et al. 1998, Stopes et al. 2002), although may be similar for equivalent levels of N input (Sprosen et al. 1997, Scholefield et al. 2002).

Urease and nitrification inhibitors offer potential to reduce nitrogen emissions from fertiliser applications. Urease inhibitors, such as N-(n-butyl) thiophosphoric triamide (NBPT), delay the hydrolysis of urea to ammonium (Gill et al. 1999), thus delaying the opportunity for NH3 emissions to occur. Significant reductions (40–70%) in NH3 emis-sions from urea fertiliser have been demonstrated using NBPT (e.g. Sanz-Cobena et al. 2008, Zaman et al. 2008, Chambers and Dampney 2009).

Nitrification inhibitors block the conversion of ammonium to NO3- (Amberger 1989), thus the N is retained in the

soil for longer in the ammonium form, thereby being less susceptible to losses via NO3- leaching and denitrifica-

tion. A recent meta-analysis of literature research results by Akiyama et al. (2010) suggested a mean reduction in N2O emissions of c. 40% through the use of nitrification inhibitors over a range of soil types and climatic condi-tions. A significant body of research has been conducted in New Zealand over the past 7−8 years assessing the


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use of nitrification inhibitors to reduce NO3- leaching and N2O emissions from pasture systems, assessing reduc-

tions in emissions from urea fertiliser applications and urine returns by grazing livestock through the use of dicy-andiamide (DCD). Reductions in N2O emission of up to 90% have been reported (de Klein and Eckard 2008), al-though Clough et al. (2007) proposed a more conservative 50% reduction to be applied to the emission factors used within the New Zealand inventory. Pasture yield increases are also reported from some studies, but not con-sistently (de Klein and Eckard 2008).

When using nitrification inhibitors with urea fertiliser or urine, there is the potential to reduce N2O emissions and NO3

- leaching at the expense of increased NH3 emissions, as the N is being retained in the ammonium form for longer. The use of a double inhibitor (urease and nitrification) may prevent such trade-offs, but this has not been shown consistently (Zaman and Blennerhassett 2010).

There are opportunities to mitigate environmental impacts from manure management throughout the manage-ment continuum of housing, storage and spreading (Sommer and Hutchings 2001, Sommer et al. 2006, Chadwick et al. 2011). Opportunities are limited during the cattle housing phase, and depend also on choice of system. In general, a slurry-based system is associated with greater NH3 emissions throughout the management continuum than a straw-bedded deep litter system (Thorman et al. 2003). For a slurry-based housing system, there may be some potential in the rapid removal of excreta from fouled concrete areas to storage and in the use of urease inhibitors to reduce NH3 emissions (e.g. Varel et al. 1997, Misselbrook et al. 2006). For straw-bedded deep litter systems, NH3 emissions can be reduced through the targeted use of straw bedding to ensure sufficient bedding is supplied particularly to key locations which may be associated with higher emissions, such as near water troughs or feed areas (Gilhespy et al. 2009).

Options for reducing gaseous emissions during slurry storage include covering the store, the effectiveness of which will depend on the nature of the cover (e.g. Sommer et al. 1993, Blanes-Vidal et al. 2009, van der Zaag et al. 2010b) with natural crust formation providing some mitigation (Misselbrook et al. 2005a, Petersen et al. 2005). Anaerobic digestion of slurries can reduce CH4 emissions if the gas is properly captured and utilised, but increased availability of N in the digestate may increase losses of NH3, N2O and NO3

- leaching during subsequent storage and application to land if not properly managed. Minimising slurry storage during warmer months will reduce CH4 emissions (van der Zaag et al. 2010a) and NH3 emissions (Sommer et al. 2006). Covering and compac-tion of farmyard manure heaps can decrease gaseous emissions (Chadwick 2005), although may not be widely viewed as a practical measure.

As with inorganic fertilisers, rate and timing of application are important in managing the environmental impact of manure applications to land. Smith et al. (2002) showed a very clear relationship between NO3

- leaching, crop N uptake and timing of application for slurry applications to freely draining soils in England, with up to 50% of applied N being lost via leaching and largest losses from applications in the September to November period. Ap-plication technique has a large effect on NH3 losses following slurry application, and significant reductions can be achieved through using slurry application techniques designed to minimise the emitting surface area and/or en-courage slurry transfer to the soil matrix. Compared with surface broadcast application, reduction in emission of the order of 50−80% can be achieved using shallow injection, 40–60% using trailing shoe (designed for applica-tions to grassland) and 10−40% using band spreading (more suitable for use in growing crops) (e.g. Misselbrook et al. 2002). Emissions may be further reduced by applying slurry beneath a more developed crop canopy, using band spreading (to arable crops) or trailing shoe (to grassland) application, where the combined effects of re-duced air speed and temperature at the ground surface and the direct uptake of emitted NH3 by the crop canopy reduce emissions significantly compared to slurry applied to a bare surface (Thorman et al. 2008). Slurry applica-tion by trailing shoe to grassland can increase the window of opportunity for applications to be made; Laws and Pain (2002) and Laws et al. (2002) showed that grazing or silage harvesting could be made sooner after slurry ap-plication with this technique, compared with surface broadcast application, with no detrimental effects. The ef-fect of slurry application technique on N2O emissions is less clear, with some reports of increasing emissions (e.g. Flessa and Beese 2000, Wulf et al. 2002, Velthof et al. 2003), which might be expected in particular for slurry in-jection where the anaerobic conditions in the injection slots with high available nitrogen and carbon concentra-tions would favour denitrification, and other reports of no net increase when compared with surface broadcast application (e.g. Sommer et al. 1996, Vallejo et al. 2005).


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Model scenarios for nutrient management

Nutrient management scenarios, specifically aimed at mitigating greenhouse gas emissions through improvements in fertilisation management, were evaluated in a modelling study using SIMSDAIRY (del Prado et al. 2010). The sce-narios consisted of firstly, optimisation of mineral fertiliser N application rates and timing, and secondly, the use of nitrification inhibitors. Mineral fertilizer N use (rate and timing) was optimised using the in-built routine within SIMSDAIRY according to one of three criteria: (i) to maximise the efficiency ratio (defined as kg N in herbage per kg N loss (Brown et al. 2005); (ii) to maximize annual herbage N production; or (iii) to meet a field-specific target for annual herbage N production equal to that of the baseline farm. Values were averaged for a range of farms dif-fering in site conditions and nutrient use intensity.

Tactically matching the plant N requirements to the rate and temporal distribution of mineral N fertiliser through SIMSDAIRY´s optimisation led to a reduction in overall N losses. For example, NH3 emissions were reduced by about 10%, NOx by 97% and NO3

- leaching by 6−14% per litre of milk produced. Denitrification losses were also de-creased but site conditions greatly influenced the form of N loss (i.e. as N2O or N2). Nitrogen optimisation for the drier site with light soils was carried out favouring fertilisation applications at weather conditions that promoted smaller N2 losses but large N2O:N2 ratios. As observed in a previous study by del Prado and Scholefield (2008), the optimised fertiliser distributions were achieved by lower annual rates of inorganic N fertilisers and higher relative rates in early spring. Lowering the total annual fertiliser rate also reduced the indirect pre-farm CO2 emission due to fertiliser manufacture.

Use of white clover in grass leys as a substitute for inorganic fertiliser N was one of the main differences between a conventional and organic dairy farm in a simulation by del Prado et al. (2011) using SIMSDAIRY. Greenhouse gas emissions per litre of milk were lower by 11−25%, although differences in C sequestration, with the organic system assumed to be ploughed and reseeded every 5 years to ensure persistence of clover in the sward, were not taken into account. Ammonia emissions and concentration of NO3

- in leachate were also lower for the organic system.

Nitrification inhibitors (e.g. DCD) added to both mineral N and manures applied to land reduced most forms of soil N losses. Whereas N2O and NO3

- leaching were reduced up to 55 and 40%, respectively, emissions of NOx and NH3 were not substantially affected. Nitrous oxide, for example, was greatly reduced as a consequence of a simulated increase in plant N use efficiency and a reduction in the rate of nitrification (and, therefore, subsequent denitrifi-cation). Greater reductions in emissions were achieved for drier soil conditions. The mitigation of N2O emissions was also greater in light-textured soils than in heavy-textured soils, which reflects, at least indirectly, the more effective nitrification inhibition found by experimental evidence in lighter soils with low organic matter content (e.g. Sahrawat and Keeney 1985).

Grazing managementDairy farms demonstrate a number of different strategies in terms of grazing management, ranging from year round grazing (where climate and soil conditions allow) to year round housing for all or part of the herd. Webb et al. (2005) discussed the trade-off between grazing strategies in terms of NO3

- leaching losses, expected to be greater from grazing livestock from the high N intensity urine patches, and NH3 emissions, expected to be greater from housed livestock through the manure management continuum. They concluded that for a conventional UK system of approximately 6 months housing, extending the grazing season by one month in each of the spring and autumn periods reductions in NH3 emissions would be more than offset by increases in NO3

- leaching in terms of total N loss. Recent research has indicated that increasing the housing period can reduce N2O emissions at the farm level, both from indirect and direct emissions by about 10% (e.g. de Klein et al. 2006, Luo et al. 2008). However, pre-farm CO2 emissions from mineral fertiliser manufacture increased substantially due to a shift to-wards more forage area needed for grass for conservation and hence more total mineral fertiliser needed. Using SIMSDAIRY, del Prado et al. (2010) suggested that reducing grazing during the wetter parts of the season (by c. two months) reduced GHG emissions per litre of milk. Increasing the housing period can reduce N2O emissions, espe-cially through a more uniform return of excreta via managed manure compared with very localized urine returns deposited by grazing (Oenema et al. 2006). There is also more potential for improved ration formulation when animals are housed and there is greater control over diet (Chadwick et al. 2008a), although there may be nega-tive impacts on welfare and fertility (Marley et al. 2010, Mee 2012).

Model outputs are very dependent on system conditions (production system, soil and climatic conditions). For example del Prado et al. (unpublished data) showed that simulations of UK dairy farms under projections of fu-ture climate change scenarios resulted in more productive farms for most future time-slices and for most regions


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of the UK, mainly caused by a longer grass growing season. One proposed potential adaptation measure is to in-crease the grazing season by this extra growing season time (e.g. one month). The implications on other pollu-tion N and C losses were not consistent across all regions. For example, for the South West UK region in the 2020s time-slice this adaptation measure implied pollution swapping between N emissions to water and to air (Fig. 2). There were much larger NO3

- leaching losses than in the un-adapted scenarios and slightly larger N2O emissions and enteric CH4 emissions. Methane from manure management would be greatly reduced by requiring smaller storage volumes of manures. Overall net greenhouse gas emissions (as kg CO2eq l-1 milk) were reduced by increas-ing the grazing season, despite a small increase in enteric CH4 emissions, as were NH3 and NOx emissions. The net farm income and the other socio-economic scores all improved. Milk quality, for example, improved because of the shift to a larger proportion of fresh grass (grazed) over silage in the forage diet, associated with a better pro-file of polyunsaturated fatty acids in the milk. Animal welfare scores improved because of implied reduction in lameness and on the social structure of the cattle. Feeding cows mainly on fermented herbage (silage) also poses increased risks, which are principally generated by undesirable microorganisms (e.g. Listeria), undesirable chemi-cals (mycotoxins), and excess acidity (Wilkinson 1999).

Genetic potentialGenetic improvement of livestock is a particularly effective technology, producing permanent and cumulative changes in performance. Wall et al. (2010) discuss the use of genetic selection tools for breeding schemes with the aims of improving productivity and efficiency and, potentially, selecting for inherently low CH4 emitting animals, although it is important that this selection is on the basis of multiple traits including feed efficiency, particularly for predominantly forage-fed animals, and yield to ensure that gains are realised as reduced emissions per unit product. Improvements in fertility would lead to a reduction in the required number of replacement animals, as discussed previously. However, it should be noted that dairy cows must breed to lactate and a reduction in total livestock numbers can only be achieved with improved fertility in dairy cows if a greater proportion of the dairy-bred calves can replace beef-cow calves, i.e. through the use of a beef bull.

Improved N use efficiency by grass varieties is an on-going aim of breeding programmes. However, while this may result in lower N losses though reduction in fertiliser requirement, an enhanced grass CP content could increase N excretion by cattle, thereby leading to increased losses from grazing returns and manure management (del Prado et al. 2010). Breeding for increased polyunsaturated fatty acid content, potentially decreasing enteric CH4 emissions may be another aim, although Dewhurst et al. (2001) noted that genetic variation in this trait is small compared with variation through the growing season. Other plant changes may involve traits in the shoot to root biomass

Fig. 2. Comparison between simulated results in terms of N, C pollution and other socio-economic parameters (e.g. biodiversity, animal welfare, soil quality, economics) between un-adapted and adapted (one extra grazing month) dairy farm in the South West UK (2020). Values for the adapted scenario <1 indicate an improvement over the baseline scenario.


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ratio or plants with exudates capable of altering the mineralisation rate from decaying biomass remaining after harvest or grazing. Both measures have potential trade-offs between N forms lost (del Prado and Scholefield 2008).

Combinations of measuresDel Prado et al. (2010), using SIMSDAIRY, investigated the potential errors incurred if we estimate the effectiveness of GHG mitigation measures in combination compared with studies where the effectiveness of each method ap-plied singly is simply added together. This latter, linear approach obviously ignores many of the potential syner-gies that may occur when applying different methods affecting soil, plant and/or animal components of the farm system. For example, the additive effect on farm level GHG emissions of a dietary measure to reduce N excretion by cattle and the use of a nitrification inhibitor to reduce N2O emissions from manure application to land will be greater than if the two are used in combination, as the dietary measure will reduce the size of the N pool on which the inhibitor is acting. Assessing measures singly also ignores the fact that some mitigation options may be mutu-ally exclusive. The extent to which mitigation methods target processes that are interrelated is key to estimating the effectiveness of combined mitigation methods. The results from the del Prado et al. (2010) study indicated that for the measures considered in the scenario, the overall impact of applying a combination of measures was less than the simple addition of the effect of the measures applied singly.

Conclusions

Dairy production undoubtedly impacts upon the environment, particularly through emissions of NH3 and green-house gases to the atmosphere and transfers of pollutants to water. Research has improved our knowledge of the pollutant transfer processes and enabled the (on-going) development of a range of mitigation measures. How-ever, it must be accepted that within the complex biological systems involved in dairy production, the complete elimination of environmental impacts is impossible.

Most of the mitigation measures discussed in this paper are associated with systemic improvements in the ef-ficiency of production in dairy systems, rather than specific technological fixes (although these may also have a place). Much can be achieved through attention to livestock health, matching dietary requirements with supply, attention to the quantity, timing and method of application of nutrients to forage crops and utilising advances made through genetic improvements. The relative impact of many of the mitigation measures is specific to the genetic potential, soil, climate and management system of a particular dairy farm and therefore the use of deci-sion support tools to explore alternative scenarios, and identify site-specific optimum practices are recommended.

Areas where further research and development are required include on-going genetic improvements in livestock and plant traits, development of diets or additives which have a consistent and persistent inhibitory effect on CH4 production in the rumen, assessment of alternative plant species and varieties for inclusion in grazed and ensiled forages, cost-effective delivery mechanisms for using urease and nitrification inhibitors, and a more complete ac-counting for the effects of silage production and management on forage quality in existing farm-scale models.

Acknowledgements

Agustin del Prado is funded by the projects from the Spanish National R+D+i Plan (CGL2009-10176) and the De-partment of Education, Universities and Research of the Basque Country (PC2010-33A).

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AGRICULTURAL AND FOOD SCIENCER.E. Muck et al. (2013) 22: 108–114

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Silage extracts used to study the mode of action of silage inoculants in ruminants

Richard E. Muck1, Zwi G. Weinberg2 and Francisco E. Contreras-Govea3

1USDA, Agricultural Research Service, US Dairy Forage Research Center, 1925 Linden Drive, Madison, Wisconsin, 53706 United States

2The Volcani Center, Bet Dagan, Israel3University of Wisconsin-Madison, Madison, Wisconsin, United States


Lucerne and two maize crops were ensiled with and without Lactobacillus plantarum and fermented for 4 or 60 d to assess the effect of inoculant on in vitro rumen fermentation of the resulting silages. Water and 80% ethanol ex-tracts of the silages were also analysed for effects on in vitro rumen fermentation. The inoculant affected lucerne silage characteristics but had little effect on the maize silages. In vitro fermentation of the silages showed few ef-fects except increased microbial biomass yield (MBY) at 24 h in the inoculant-treated lucerne silages. In vitro fer-mentation of the lucerne silage water extracts produced no differences due to treatment except for reduced MBY in the inoculant-treated extracts. The ethanol extracts produced results inconsistent with the in vitro results of the silages. Consequently it appears that the factor in in vitro fermentation of inoculated silages causing increased MBY was in neither the water nor ethanol extracts.

Key words: inoculant, in vitro fermentation, silage, microbial biomass yield, gas production

Introduction

Microbial silage inoculants are additives used to improve silage fermentation (Muck and Kung 1997). These ad-ditives also may increase milk production or daily gain in livestock, but the mechanisms are unknown (Weinberg and Muck 1996, Kung and Muck 1997). The most common silage inoculants contain facultative heterofermentative lactic acid bacteria (LAB) that shift fermentation toward lactic acid production, reducing acetic acid and ethanol. Based on analysis of data from 47 experiments, a 10 g kg-1 DM increase in lactic acid and 10 g kg-1 DM reduction in acetic acid should increase energy-corrected milk by 0.12 kg d-1 (Huhtanen et al. 2003). Inoculants often reduce ammonia in silages. That is also correlated to higher milk production; a 10 g kg-1 N reduction in ammonia would be expected to increase energy-corrected milk by 0.19 kg d-1 (Huhtanen et al. 2003). Sometime inoculants reduce the amount of fermentation products, and that should have a positive effect on milk production (Huhtanen et al. 2003, Jaakkola et al. 2006). Unfortunately, the expected improvement in milk production from all of these shifts in silage composition are much less than the observed average improvement in milk production (1.4 kg d-1) from feeding inoculated silage (Kung and Muck 1997). Consequently, changes in common silage characteristics due to silage inoculant use cannot explain the magnitude of improvements in milk production observed.

Recent research is providing evidence, suggesting possible means by which inoculants may alter animal responses to treated silages. Two studies indicated that lactic acid bacteria survived in rumen fluid and resulted in small but consistent increases in pH (Weinberg et al. 2003, Weinberg et al. 2004), which should be beneficial to cell wall-degrading microorganisms in the rumen. In addition, nine of ten inoculant LAB exhibited antimicrobial activity when grown in broth, and the majority of extracts of silages treated with these inoculants also had antimicrobial activity (Gollop et al. 2005). In experiments comparing 14 inoculants, none of the inoculants had a positive effect on in vitro dry matter digestibility (IVDMD) (Filya et al. 2007). In contrast, in vitro gas production (GP) on undried silages from those experiments was reduced in many of the inoculated silages compared to untreated control si-lages while only minor effects on volatile fatty acid (VFA) production were observed (Muck et al. 2007). The re-duced GP suggested that inoculated silages were producing more rumen microbial biomass yield (MBY) than un-treated silage. Recently, Contreras-Govea et al. (2011) reported on three ensiling experiments (lucerne and two whole-crop maizes), comparing untreated and four different microbial inoculants. In this study, there were few differences in silage fermentation characteristics among inoculated and untreated silages, but in the in vitro rumen



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fermentations, silages from two of the inoculants consistently produced more MBY than the corresponding un-treated silages. Furthermore the increase in MBY was of an order that could explain the 3 to 5% increase in milk production observed in cow studies (Kung and Muck 1997). These studies suggest that the effects of inoculated silages on animal performance are due to changes in rumen microbial fermentation by an unknown mechanism.

The objective of the current experiment was to determine if extracts from silages treated with Lactobacillus plan-tarum would affect GP, MBY and VFA production from in vitro fermentation similarly to the in vitro fermentation of the silages. The hypothesis was that the factor in inoculated silage that enhances rumen microbial growth should be extractable from silage.

Materials and methods

Third cut of a second year lucerne (580 g dry matter (DM) kg-1) was harvested on 31 August 2010 after field wilting for approximately 24 h, and two maize crops were harvested, one with low DM concentration (< 300 g DM kg-1, Maize-LDM) on 27 August 2010 and one with high DM concentration (~ 500 g DM kg-1, Maize-HDM) on 7 Septem-ber 2010. The forages were chopped with a conventional precision-chop forage harvester and ensiled individu-ally in 1-l glass jar mini-silos (Weck, Wher-Oftlingen, Germany) at a density of 500 g l-1 with two treatments: un-treated control and Lactobacillus plantarum (LP, Ecosyl MTD/1, Ecosyl, North Yorkshire, UK) at 106 cfu g-1 of fresh weight, six mini-silos per treatment. During ensiling three samples of each crop and a sample of the inoculant were taken for enumeration of LAB by Rogosa SL agar (Muck and Dickerson 1988). Three mini-silos of each treatment were frozen (−20 °C) after 4 and 60 d of fermentation until analysed. At opening, each mini-silo was poured into a disinfected plastic pan and mixed to uniformity. A 20 g sample was taken, diluted 10-fold with distilled water, and macerated for 30 s in a high-speed blender. Silage extract was filtered through 4 layers of cheesecloth, and pH was measured immediately using a pH meter. One 20 ml aliquot sample was placed in a 50 mL polypropylene tube, centrifuged for 20 min at 25,100 × g at 4 °C, and the supernatant decanted into a 20 ml scintillation vial and frozen at −20 °C for later analysis of fermentation products. Fermentation products (succinate, lactate, acetate, propionate, butyrate, and ethanol) were performed using high performance liquid chromatography (Muck and Dickerson 1988). Two silage sub-samples of approximately 50 g each were taken for moisture analysis by freeze-drying. The freeze-dried silage samples were ground to 1 mm and used for the determination of neutral detergent fibre (aNDF) analysis, with heat stable amylase and sulphite, using an ANKOM fibre analyser (Ankom Technology Corp., Fairport, NY, USA).

The remaining fresh silage from each mini-silo was chopped to a particle size of 1−4 mm using a commercial food processor (Robot Coupe, Inc., Joliet, IL, USA) for 30 s and stored at −20 °C for later analysis of in vitro rumen fer-mentation. The MBY and VFA production in 50 ml polypropylene plastic tubes and GP in 160 ml bottles were de-termined on these wet-ground silages, as described previously (Contreras-Govea et al. 2011). The rumen fluid was collected from four rumen cannulated lactating cows in the morning before feeding, following the procedure de-scribed by Weimer et al. (2005). Donor cows were fed a TMR diet of 50:50 forage (corn silage and alfalfa silage): concentrate. Gas production, VFA and MBY were measured after 9 and 24 h incubation at 39 °C. The MBY was cal-culated by the difference of in vitro apparent digestibility and in vitro true DM digestibility (Blümmel et al. 1997).

In addition, 1:1 aqueous and 80% ethanol extracts of wet-ground control and inoculated silages were prepared to study their effects on in vitro ruminal MBY and GP. Ethanol was removed from the ethanol extracts by vacuum centrifuge, and the extract reconstituted with Type 1 water. Rumen fluid was prepared as above. Each tube or bot-tle contained 12 ml rumen fluid, 17 ml buffer, 1 ml extract and 2 mg ml-1 glucose, which was the primary carbon source for the rumen microorganisms. In an additional in vitro treatment, L. plantarum suspension was added in place of extract to the buffered rumen fluid at 106 cfu ml-1.

The silage fermentation data were analysed as a 3 × 2 × 2 factorial experiment (crop × treatment × fermentation time) and the silage extract data were analysed as a 3 × 2 × 2 × 2 factorial experiment (crop × treatment × fermen-tation time × extract treated) using the PROC Mixed procedure of SAS (SAS Inst. Inc., Cary, NC, USA). The direct application of L. plantarum to in vitro fluid was analysed separately from the silage extract results by a one-way analysis of variance with crop as the fixed effect. Differences among means were tested by using the LSMEANS statement with the PDIFF option with significance declared at p<0.05.


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ResultsSilage fermentation

The inoculant was applied at slightly above the intended rate, 6.06 log10(cfu g-1 crop). This rate was similar to the epiphytic population on Maize-LDM ( 6.28 log10 [cfu g-1 crop]), but more than ten-fold higher than the LAB on the other two crops (4.81 log10[cfu g-1 crop]). There were crop by treatment and crop by fermentation time interac-tions with regard to silage characteristics (Tables 1 and 2), but no crop by treatment by fermentation time inter-actions were found (p > 0.05), except for pH and ethanol concentration. These interactions were significant be-cause the difference in pH between the two treatments in lucerne was much greater at day 4 (0.478) than at day 60 (0.139) whereas there were no differences in pH between treatments on a given day for either maize except at day 4 in Maize-HDM (0.049). In the ethanol, LP-treated Maize-LDM at 4 d was similar to control (5.07 and 4.90 g kg-1 DM, respectively), but at 60 d it was greater than control (15.09 and 9.69 g kg-1 DM, respectively); in con-trast in the other two crops, no effects of treatment were observed at day 4 or 60. The pH was lower in LP than control in lucerne but not the two maize silages (Table 1). Inoculant treatment effects on lactic acid concentra-tion differed by crop. In lucerne, lactic acid concentration was 45% greater in LP than control while in both maize experiments, lactic acid was similar for both treatments (Table 1). Acetic acid concentration was different among crops (p < 0.001) and reduced by inoculant treatment. Maize-LDM had the greatest acetic acid concentration fol-lowed by Maize-HDM and lucerne (Table 1). Ethanol concentration in Maize-LDM was higher in LP than control while there was no effect of treatment on ethanol in the other two crops.

Table 1. Mean silage characteristics (g kg-1 DM except as noted) by treatment across silage fermentation times.

Lucerne Maize-HDM1 Maize-LDM SEM p-value

Constituent Control LP Control LP Control LP C×T C T C×T

Dry matter(g kg-1) 575 570 494 490 306 296 4.9 <0.001 0.13 0.81

aNDF 345 350 401 393 376 360 11.7 0.001 0.55 0.66

pH 5.30 4.99 4.19 4.18 3.79 3.77 0.008 <0.001 <0.001 <0.001

Lactic Acid 24.4 35.3 33.6 31.7 46.4 42.3 1.75 <0.001 0.25 0.001

Acetic Acid 4.63 4.38 7.28 5.95 10.80 9.14 0.429 <0.001 0.01 0.25

Ethanol 0.92 0.58 3.17 3.49 7.29 10.10 0.368 <0.001 0.01 0.011 HDM=high dry matter, LDM=low dry matter, LP=treated with Lactobacillus plantarum, SEM=standard error of the mean, C=crop, T=treatment, C×T=crop×treatment interaction, aNDF=neutral detergent fibre analysed using heat-stable amylase

Silage fermentation was also affected by fermentation time (Table 2). In lucerne and Maize-LDM, the aNDF was similar between 4 and 60 d whereas there was a decrease in aNDF with time in Maize-HDM. In all three crops, the pH was lower at 60 d. Lactic and acetic acid concentrations were higher at 60 d for lucerne and Maize-HDM, but similar between 4 and 60 d for Maize-LDM. Ethanol was higher in Maize-LDM at 60 d than 4 d.

Table 2. Mean silage characteristics (g kg-1 DM except as noted) by silage fermentation time across treatments.


Constituent 4 d 60 d 4 d 60 d 4 d 60 d C×D C D C×D

Dry matter (g kg-1) 575 570 491 494 297 304 4.9 <0.001 0.67 0.52

aNDF 343 351 425 368 378 359 11.7 0.001 0.03 0.04

pH 5.66 4.63 4.36 4.00 3.82 3.74 0.008 <0.001 <0.001 <0.001

Lactic Acid 10.4 49.3 23.8 41.5 42.7 46.0 1.75 <0.001 <0.001 <0.001

Acetic Acid 1.11 7.91 4.76 8.47 9.83 10.11 0.429 <0.001 <0.001 <0.001

Ethanol 0.59 0.92 2.82 3.85 4.99 12.39 0.368 <0.001 <0.001 <0.0011 HDM=high dry matter, LDM=low dry matter, SEM=standard error of the mean, C=crop, D=days of fermentation, C×D=crop×day interaction, aNDF=neutral detergent fibre analysed using heat-stable amylase


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In vitro rumen fermentation

There were few effects on the 9 and 24 h in vitro rumen fermentations of the wet-ground silages relative to treat-ment (Table 3). At 9 h with Maize-HDM, IVDMD was higher in LP than control. At 24 h in lucerne, MBY was great-er in LP than control (p = 0.074). No other effects of treatment were observed. The highest IVDMD occurred in Maize-HDM and lowest in lucerne, both at 9 and 24 h. At 9 h, propionate was highest in Maize-LDM and lowest in lucerne whereas butyrate was highest in Maize-HDM and lowest in lucerne. At 24 h, all three VFA measured were highest in Maize-HDM and lowest in lucerne. At 9 h, MBY was highest in Maize-HDM. By 24 h, MBY was highest in lucerne and lowest in Maize-LDM. There was no effect of silage fermentation time on any of these in vitro fer-mentation characteristics (data not shown).

Table 3. In vitro rumen fermentation profile and microbial biomass yield (MBY) of wet-ground silages at 9 h and 24 h averaged across silage fermentation times.


Constituent Control LP Control LP Control LP C×T C T C×T

9 h

IVDMD2 9h 619 609 701 729 677 656 9.3 <0.001 0.88 0.03

Acetate (mM)

49.8 50.6 49.4 48.9 49.0 47.3 1.25 0.29 0.65 0.61

Propionate (mM)

13.5 13.7 16.4 16.6 18.3 17.6 0.62 <0.001 0.87 0.68

Butyrate (mM)

6.7 6.6 12.1 12.2 10.1 10.3 0.31 <0.001 0.86 0.95

MBY (mg/g DM)

329 325 418 429 315 268 17.4 <0.001 0.36 0.24

24 h

IVDMD 24h 645 661 846 836 768 760 11.5 <0.001 0.94 0.47

Acetate (mM)

53.8 52.7 73.0 71.8 60.9 62.2 1.18 <0.001 0.72 0.50

Propionate (mM)

14.6 15.0 26.2 26.5 20.7 21.2 0.71 <0.001 0.52 1.00

Butyrate (mM)

8.0 8.6 20.6 21.0 13.7 13.9 0.55 <0.001 0.35 0.93

MBY (mg/g DM)

314 347 241 241 196 193 8.3 <0.001 0.15 0.07

1 HDM=high dry matter, LDM=low dry matter, LP=treated with Lactobacillus plantarum, C=crop; T=treatment, C×T=crop×treatment interaction, SEM=standard error of the mean2 IVDMD=In vitro dry matter digestibility, MBY=Microbial biomass yield estimated by the difference of in vitro true digestibility and in vitro apparent digestibility (Blümmel et al. 1997).

In vitro rumen fermentation of the water and ethanol silage extracts had an effect on MBY and GP, but the effect was different among crops and between treatments (Table 4). The MBY of water extracts was greater in control than LP in lucerne, while no effect of treatment was observed in either maize. In contrast, the MBY of ethanol ex-tracts was unaffected by treatment, but there were trends for greater MBY in LP than control in lucerne and the opposite in the two maizes. Averaging across crops and treatments, the MBY was 4.7% greater in the ethanol ex-tract than the water extract (Table 4). The GP in the water extracts was not affected by treatment (Table 4). In the ethanol extracts, GP was higher in LP than control in lucerne and Maize-LDM.

Effects of treatment on the VFA profiles of the in vitro rumen fermentations of silage extracts were observed in the lucerne extracts (Table 5). The acetate concentrations from ethanol extracts of the LP treatment were higher than the LP water extract and both control extracts. Propionate concentrations were reduced in the ethanol ex-tracts compared to the water extracts. Butyrate concentration was lower in the ethanol extracts of the control compared to the other extracts. There was no effect of silage fermentation time on VFA concentrations. However, there were significant interactions with treatment (data not shown); acetate and butyrate concentrations were lower in the control extracts at 4 d than at 60 d and than in the LP extracts at 4 and 60 d, which were all similar.


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Table 4. Microbial biomass (MBY, mg g-1 DM) and gas production (GP, mL g-1 DM) from water or ethanol silage extracts and glucose after 24 h in vitro rumen fermentation.


Extract Control LP Control LP Control LP C×T C T C×T

MBY

Water extract

20.7 15.7 15.9 16.5 16.6 15.9 1.32 0.21 0.20 0.02

Ethanol extract

16.4 18.8 19.9 18.1 17.1 15.7 1.32 0.21 0.20 0.02

LP-DA2 13.5 13.3 12.0 1.31 0.70

GP

Water extract

35.2 36.0 19.8 21.3 25.4 27.2 1.06 <0.001 0.02 0.08

Ethanol extract

33.7 37.3 21.9 19.7 24.5 28.2 1.06 <0.001 0.02 0.08

LP-DA 24.7 16.8 21.6 1.49 0.011 HDM=high dry matter, LDM=low dry matter, LP=treated with Lactobacillus plantarum, C=crop, T=treatment, C×T=crop×treatment interaction, SEM=standard error of the mean2 LP-DA= direct addition of LP to rumen inoculum, no silage extract.

Table 5. Volatile fatty acid concentrations (mM) from water or ethanol lucerne silage extracts and glucose after 24 h in vitro rumen fermentation.

Control LP1

Water Ethanol Water Ethanol SEM p-value

Acetate 37.2 37.9 36.1 41.4 0.38 0.001

Propionate 11.3 9.3 11.0 10.1 0.17 0.005

Butyrate 7.2 6.6 7.0 7.1 0.10 0.0041 LP=treated with Lactobacillus plantarum, SEM=standard error of the mean

The direct application of LP to the in vitro rumen inoculum produced less MBY and GP than the silage extracts (Table 4). Extracts for each crop were performed in separate in vitro runs and tubes with LP added directly to ru-men fluid were included in each run. So, similar MBY and GP values for direct application of LP would be expect-ed across crops. That was true for MBY but not GP, where GP was lower in the Maize-HDM in vitro runs than in the other two crops.

Discussion

A probiotic effect of silage microbial inoculants on animal performance was proposed by Weinberg and Muck (1996). Even though the mechanism by which silage microbial inoculants enhance animal performance has not been elucidated, better preservation of nutrients in the silage could explain in part the animal performance effect (Muck et al. 2007, Contreras-Govea et al. 2011). In the current study, the hypothesis was that a factor in inocu-lated silage that enhances rumen microbial activity should be extractable from silage. Therefore, three different crops harvested separately were inoculated with a specific strain of LP that has usually shown a positive effect on silage fermentation and animal performance (Kung et al. 2003).

Silage fermentation characteristics of the three crops with or without LP were typical of silages ensiled at high and more typical DM concentrations. Because of their high DM concentrations, the pH and fermentation products of lucerne and Maize-HighDM silages were different between day 4 and day 60, indicating that active fermentation by LAB was not complete after 4 d (Table 2). In contrast, there were no differences in silage fermentation charac-teristics in Maize-LDM between the two days with the exception of an increase in ethanol. Given that lactic acid did not increase between days 4 and 60, the increase in ethanol in the Maize-LDM silages may have been due to the activity of yeasts (McDonald et al. 1991).


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The LP treatment affected silage fermentation, compared to control, in lucerne but not appreciably in the two maize experiments. In lucerne, LP decreased pH and increased lactic acid compared to control (Table 1), and the effect was evident at both days 4 and 60 (data not shown). This is what would be expected when an inoculant like L. plantarum is used. For example, Filya et al. (2007) also reported lower pH and higher lactic acid in the inoculated lucerne silage compared with control when ensiled at high DM concentrations. In contrast, the only effect of LP on silage fermentation in Maize-HDM was a lower pH at day 4 (4.339 vs. 4.388), and in Maize-LDM a higher ethanol concentration at day 60 (15 vs. 10 g kg-1 DM). The lack of an inoculant effect is not uncommon. Muck and Kung (1997) in a survey of published studies reported that 60% of the time silage microbial inoculants decreased pH compared to uninoculated treatments. So, there are a substantial numbers of instances where an inoculant has not affected silage fermentation. In the case of Maize-LDM, the epiphytic LAB population was similar to the inoculant application rate and may have competed effectively with the inoculant. In Maize-HDM, the epiphytic LAB popula-tion was lower than the inoculant application rate. Perhaps other epiphytic bacteria such as enterobacteria domi-nated the early fermentation (Pahlow et al. 2003). Unfortunately the only microbial group analysed was the LAB.

The absence of a consistent effect of LP across crops on the in vitro rumen fermentation at 9 h and 24 h (Table 3) was not unexpected. Muck and Kung (1997) reported that a positive effect of inoculant treatment on in vitro true DM digestibility occurred in only 30% of the studies reviewed. In addition, Muck et al. (2007) reported no consist-ent effect of microbial inoculants on VFA composition of in vitro rumen fermentations of inoculated and untreated lucerne silages. The MBY at 9 h and 24 h in the current study did not show a consistent effect of LP across crops in contrast to our earlier study (Contreras-Govea et al. 2011). In Contreras-Govea et al. (2011), MBY was greater in the LP-treated silages than control across lucerne, maize and brown-midrib maize, but this effect was not consistent across the other inoculants tested. In our study, the MBY at 9 h was unaffected by inoculant treatment in any of the crops (Table 3). At 24 h, MBY was higher in LP-treated lucerne silages than control silages (p = 0.074) whereas MBY was unaffected by treatment in the maize silages. Because the inoculant only affected the ensiling of the lu-cerne, the lack of treatment effects in the in vitro fermentations, particularly in the maize silages, is not surprising.

The hypothesis of this study was that if the mechanism that improves animal performance is produced by the mi-crobial inoculant during fermentation, it should be extractable from the silage. The 80% ethanol extract was ex-pected to remove more complex carbohydrates and N compounds than the water extract. Averaging across crops and treatments, MBY was 4.7% greater in the ethanol silage extract than water silage extract (Table 4). MBY from the extracts in maize were unaffected by treatment, but given the few effects of treatment in silage fermentation and in vitro fermentation of these silages, the lack of effect was expected. In the lucerne, the water extract of the control produced a higher MBY than the water extract of the LP silage and the ethanol extract of the control. These results suggest that the factor causing higher MBY in the LP-treated lucerne silage at 24 h in vitro fermen-tation is not water-soluble. While there was only a numerical trend for MBY to be higher in the ethanol extract of LP-treated lucerne silages compared to control, the ethanol extracts from the LP-treated lucerne silages produced more GP, acetate, propionate and butyrate than the control ethanol extracts (Tables 4 and 5). Such results stand in contrast to the in vitro rumen fermentations of the silages in this study and earlier ones (Muck et al. 2007, Con-treras-Govea et al. 2011) where LP-treatment had no effect on or reduced GP and had little or no effect on VFA production. Consequently these results suggest that the substances causing increased MBY in in vitro fermenta-tions of LP-treated silages were in neither the water nor ethanol extract.

Finally, MBY and GP from direct addition of LP to in vitro rumen fluid were lower than silage extracts (Table 4). These results suggest that the inoculant LAB are not having any direct effect on in vitro rumen fermentation or at least that the extracts are providing more fermentable substances than the bacteria themselves.

Conclusion

In this study, Lactobacillus plantarum MTD/1 appeared to dominate in only one of the three crops ensiled, a lu-cerne. In that crop, in vitro rumen fermentation of the silages produced more MBY at 24 h in the LP-treated silag-es, consistent with earlier studies. In vitro fermentation of the water extracts of the lucerne silages produced no significant treatment differences in fermentation products with the exception of reduced MBY in the LP-treated extracts, indicating the factor affecting in vitro fermentation of the inoculated silages was not in the water extract. The ethanol extracts produced in vitro results that were also not consistent with the in vitro results of the silages. Consequently it appears that the factor in in vitro fermentation of inoculated silages causing increased MBY was in neither the water or ethanol extracts. However, there is the need for further research to confirm these results.


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Acknowledgements

The authors wish express their appreciation for the technical assistance of U.C. Hymes-Fecht and J.A. Boyd.

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AGRICULTURAL AND FOOD SCIENCEJ.L. De Boever et al. (2013) 22: 115–126

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The effect of a mixture of Lactobacillus strains on silage quality and nutritive value of grass harvested at four growth stages and

ensiled for two periodsJohan L. De Boever1, Elien Dupon2, Eva Wambacq3 and Joos Latré2

1ILVO, Institute for Agricultural and Fisheries Research, Animal Sciences Unit, Scheldeweg 68, B-9090 Melle, Belgium, 2University College Ghent, Faculty of Science and Technology, Experimental Farm Bottelare, Diepestraat 1,

B-9820 Merelbeke, Belgium, 3University College Ghent, Associated Faculty of Applied Bioscience Engineering, Valentijn Vaerwyckweg 1,

B-9000 Ghent, Belgium


The effect of adding an inoculant containing Lactobacillus buchneri, L. plantarum and L. casei to wilted perennial ryegrass, harvested at four growth stages and ensiled for either 60 or 150 d on silage fermentation quality, chemi-cal composition, rumen degradability of neutral detergent fibre (NDF) and organic matter (OM) and in vitro OM digestibility (OMd) was studied. Compared to the control silage, more sugars were fermented to lactic and acetic acid with the inoculant, resulting in a lower pH, less dry matter losses and protein degradation and a better aero-bic stability. The effects of the additive on fermentation quality were more pronounced after 150 than after 60 d of ensiling, because the quality of the control silage was worse after the long ensiling period. The treatment lowered NDF content of grass harvested at the first two growth stages by degrading cell walls to complex sugars, but had no effect on NDF degradability of the silage. The inoculant had no effect on rumen OM degradability nor on OMd after the short ensiling period, but increased the rumen OM degradability for the first two growth stages and OMd for all growth stages after the long ensiling period.

Key words: cell wall degradability, ferulate esterase, Lactobacillus, grass silage

Introduction

Grass silage is an important component in the ration of cattle especially during the winter time. The quality of grass silage depends to a large extent on the growth stage of the grass at harvest, the weather conditions during field wilting and the ensiling practices. In case of unfavourable ensiling conditions, silage additives may be used. Currently mostly inoculants containing living micro-organisms are used. Recently an inoculant for ensiling grass was introduced with the aim to improve not only silage fermentation quality and aerobic stability, but also to en-hance digestibility and nutritive value of the silage. This so-called multipurpose inoculant consists of three Lacto-bacillus strains: L. plantarum, L. casei and L. buchneri. The former two strains are facultative heterofermentative, whereas the latter is strict heterofermentative (Holzer et al. 2003). The production of acetic acid besides lactic acid explains the improvement of aerobic stability, observed with the use of L. buchneri (Kung and Ranjit 2001). Moreover, L. buchneri is able to produce ferulate esterase (FE), an enzyme which breaks down the linkages be-tween (hemi)cellulose and lignin (Donaghy et al. 1998).

The objective of this study was to examine if the above-mentioned aims are reached by investigating the effects of the inoculant on the fermentation characteristics, the chemical composition, the in situ rumen degradability and in vitro digestibility of grass silage. Although it is recommended to mow grass not too late for making good quality silage, the efficacy of the inoculant was studied at four growth stages. It would answer the question if the claimed positive effect of the additive on digestibility offers farmers more flexibility to postpone harvest in case of bad weather conditions or to obtain higher grass yields. The effect of inoculant additive was studied after 60 and 150 days of ensiling to examine if a long ensiling period could have an additional effect compared to a more common ensiling period. This additional effect is expected as L. buchneri is known as a slowly growing bacterium (Schmidt et al. 2009).



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Material and methods

The grass used in the experiment originated from a parcel perennial ryegrass (Lolium perenne) sown in April 2008, which received 120 kg N ha-1 in March 2010. The first cut of the grass was weekly harvested between the end of April and early June 2010. Then, 4 stages were selected for further study based on distinct contents of NDF. At each harvest a representative batch of about 150 kg grass was mown with a Haldrup harvester (Inotec, Løgstør, Den-mark) around 1100 h. Dry matter (DM) yield was estimated from the fresh weight of the harvested area and the DM-content of the grass. Then, grass was wilted on the field to obtain a DM-content of about 35%. Wilting lasted for 25, 29, 73 and 24 h, respectively; the longer wilting period after the third harvest was because of rain on the second day. Subsequently, the grass was cut with a field chopper (Sperry New Holland, Zedelgem, Belgium) set at a theoretical length of 24 mm. Samples of the wilted grass were taken, dried in a ventilated oven at 65 °C, ground to pass a 1-mm screen and analysed for residual moisture, neutral detergent fibre (NDF) and sugar content. NDF was determined with the filter bag method using α-amylase and sodium sulphite (Van Soest et al. 1991). Sugars were extracted with 40% ethanol (EC 1971a).

The wilted grass was ensiled in cylindrical plastic tubes with a volume of 2.75 l (height 35 cm, diameter 10 cm) and provided with a CO2-lock at a density of about 180 kg DM per m³. Ten micro-silos were made for both the control and the treatment. For making the treated silos, 20 kg wilted grass spread on a plastic sheet was sprayed with 0.2 l of distilled water in which 20 mg of inoculant additive was dissolved (recommended dose: 1 g per ton wilted grass) and thoroughly mixed. The studied inoculant Pioneer ® 11GFT is a commercial product (Pioneer Hi-bred Northern Europe) containing the strains L. buchneri LN40177, L. plantarum LP24011 and L. casei LC32909 at concentrations of >1.0 x 1011, >2.0 x 1010 and >1.0 x 1010 cfu per g product, respectively. Before filling the control silos, 0.2 l distilled water was added to another 20 kg of wilted grass. The number of lactic acid bacteria (LAB) in the inoculant as well as in the control and inoculated silage material was counted according to ISO 15214 (1998).The filled micro-silos were stored at ambient temperature in an enclosed barn. Half of the tubes were ensiled for 60 d, the other half for 150 d.

Micro-silos were weighed weekly to determine fermentation losses. Eighteen days before opening, aerobic stress was induced to all micro-silos by removing the tape from openings at the bottom and top of the tubes during 24 h. At the opening of the silos, 4 out of 5 tubes were selected for further study by eliminating the micro-silo with visible most mould growth. The remaining tubes were individually sampled to determine silage quality. Aerobic stability was measured according to Honig (1990) for a maximum of 170 h and expressed as the time in hours until temperature of silage raised 3 °C above ambient temperature. To determine fermentation characteristics an ex-tract was made by soaking 100 g of silage in 1 l distilled water at 4 °C during 16 h. On the extract pH, ammonia-N (KjeldahI without previous destruction, ISO 5983-2 2005), lactic acid (Noll 1966, Gawehn 1984), acetic acid, pro-pionic acid, butyric acid and alcohols with gas chromatography (Jouany 1981) were determined.

The effect of additive on rumen degradability of NDF and OM was studied on 3 out of 4 randomly selected mi-cro-silos per control/treatment. Also, DM, crude ash and NDF were analysed on each micro-silo. The DM-content was determined by drying a sample in a ventilated oven at 65 °C and analysis of residual moisture in a ground subsample by drying at 103 °C during 4 h (EC 1971b). Crude ash content was obtained by incineration at 550 °C (ISO 5984, 2002). The rumen degradation characteristics of OM and NDF were determined by means of the nylon bag technique. Therefore, bags (8 x 10 cm, pore size 37 µm) were filled with 2.5/5.0 g DM-equivalent of frozen and finely cut silage (particles ≤ 10 mm) and incubated in two rumen-cannulated cows for 8, 24, 48, 72 and 336 h (a two-fold sample weight was used for the two long incubation times). Per time four bags, two per cow, were incubated. Besides, 3 bags were filled with sample but underwent no rumen incubation to determine the wash-out fraction (W). The lactating cows received a basal ration consisting of maize and grass silage (50/50 on DM-ba-sis) supplemented with concentrates. After incubation, bags with residues were rinsed under running tap water, frozen, machine-washed (Zanussi, Frankfurt/Main, Germany) with cold water for 50 min without spin cycle and freeze dried. Residues were pooled per incubation time and ground to pass a 1-mm screen. Incubation residues were analyzed for residual moisture, crude ash and NDF. The potentially degradable fraction (D) was calculated as 100 - W - U, with U being the undegradable fraction after 336 h of incubation.


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The degradation rate (kd) of D was derived by iteration using the exponential model d(t) = W + D x (1 -e(-kdxt)) with d(t) the disappearance at time t (Ørskov and McDonald 1979). Then, the rumen fermentable NDF fraction (FNDF) was calculated as:

FNDF (%) = DNDF x [kdNDF/(kdNDF + kpNDF)]

with kpNDF being the passage rate of NDF derived from the equation: kpNDF = 0.1775 x kdNDF + 1.39 and assuming WNDF = 0 (Tamminga et al. 2007).

The rumen fermentable OM (FOM) fraction was calculated as:

FOM (%) = WOM + DOM x [(kdOM/(kdOM + kpOM)]

with kpOM the passage rate of OM, equaling 4.5 % h-1 (Tamminga et al. 2007).

Finally, the remaining material of the 3 micro-silos was pooled per treatment, dried and ground to pass a 1-mm screen. Crude protein (CP; ISO 5983-2, 2005), sugars and crude fat (ISO 6492, 1999) were determined and total OM digestibility (OMd) was estimated by an in vitro cellulase technique (De Boever et al. 1986). The content of residual non starch polysaccharides (RNSP), as a measure of pectins and complex sugars like arabans, xylans and beta-glucans (Tamminga et al. 2007) was calculated as:

RNSP = 1000 - NDF - CP - crude ash - sugars - crude fat - FP

with FP being the silage fermentation products calculated as the sum of lactic, acetic, propionic and butyric acid as well as the alcohols.

The DM-content and chemical composition were corrected for losses of volatile substances according to Dulphy and Demarquilly (1981).

Statistical analysis was carried out by means of SAS for Windows version 9.3. The normality for the repetitions within control/treatment was examined according to Kolmogorov-Smirnov. If normal, an ANOVA was done to in-vestigate the effect of the additive treatment and the interaction between additive treatment and growth stage; control and treatment means within growth stage and ensiling period were compared by a t-test. If not normal, a non-parametric Kruskall-Wallis analysis was carried out to investigate the effect of treatment and to compare control and treatment means; this was only the case for pH after 60 and 150 d and for butyric acid content after 150 d. For the parameters determined on a pooled silage sample, CP, sugars, crude fat and OMD as well as the sum of the fermentation products and RNSP, the overall effect of treatment was examined across the 4 growth stages within ensiling period using ANOVA.

Results

From the first to the last harvest date DM-yield increased from 3100 to 5100 kg and NDF content from 365 to 535 g kg-1 DM (Table 1). Sugar content varied in the range of 150 g kg-1 DM at the third growth stage to 225 g kg-1 DM at the first growth stage.

Table 1. Dry matter yield and chemical composition of grass harvested at 4 growth stages

Harvest date Yield(kg DMa ha-1)

DM(g kg-1)

NDFb

(g kg-1 DM)Sugars

(g kg-1 DM)

28 April 2010 3100 364 365 225

17 May 2010 3450 336 422 184

25 May 2010 4010 365 505 150

2 June 2010 5100 360 535 201a dry matterb neutral detergent fibre


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The number of LAB in the applied product amounted to 1.3 x1010 cfu g-1, which was somewhat lower than the number mentioned on the label (1.1 x 1011 cfu g-1). Following the recommendation of the manufacturer 1.3 x 104 cfu was effectively added per kg of wilted grass. Compared to the control silage addition of the inoculant in-creased the number (cfu g-1 wilted grass) of lactic acid bacteria (ISO 15214) from 6.0 x 102 to 3.2 x 104 at the first stage, from 4.8 x 102 to 3.0 x104 at the second, from 2.0 x 103 to 1.5 x 104 at the third and from <10 to 1.2 x 104 at the last growth stage.

Chemical composition and in vitro OM digestibilityThe DM-content of the control silage either after 60 d or 150 d of ensiling (Table 2) corresponded fairly well with that of the wilted grass at ensiling (Table 1). The addition of the inoculant significantly (p ≤ 0.01) increased DM-content at all stages and for both ensiling periods. The increase was most pronounced for stages 1 and 2 after both 60 and 150 d of ensiling.

Compared with the grass at ensiling, the NDF content of the control silages after both 60 and 150 d of ensiling was somewhat lower. Treatment with the inoculant significantly (p ≤ 0.01) decreased NDF content at the first two growth stages after 60 d of ensiling and the first three stages after the long ensiling period.

The ash content of the control silage was significantly (p ≤ 0.05) higher than that of the treated silages. The differ-ence was smallest at the last growth stage.

The protein content of the control silage decreased with later harvesting from 238 g kg-1 DM for stage 1 to 132 g kg-1 DM for stage 4. Treated silages had a lower CP content than the control silages; averaged over the four growth stages the difference amounted to 4 and 10 g kg-1 DM for periods of 60 and 150 d of ensiling, respectively, but significantly (p ≤ 0.05) only after 60 d.

The sugar content of the control silage after 60 d was clearly lower compared with that of the grass at ensiling except for stage 2; longer ensiling further decreased sugar content. All treated silages had a lower sugar content than the control silage. Averaged over the four growth stages the sugar content of the control and treated silages after the 60 d of ensiling amounted to 95 and 35 g kg-1 DM, respectively, and after the 150 d of ensiling to 45 and 23 kg-1 DM, respectively. The difference was significant (p ≤ 0.05) only for the short ensiling period.

The content of fermentation products (FP) of the control silage after 60 d varied between 78 g kg-1 DM at stage 2 to 98 g kg-1 DM at stage 4. Longer ensiling further increased FP for stages 1 and 2. Compared with the control silage, the inoculant significantly increased FP content from 91 to 125 g kg-1 DM after 60 d and from 103 to 131 g kg-1 DM after 150 d.

Crude fat content of all silages varied between 38 and 49 g kg-1 DM and was not affected by the treatment.

The content of RNSP tended to increase with later harvesting and longer ensiling. The inoculant increased the RNSP content in all cases, except for the fourth growth stage after 150 d of ensiling. Averaged over the four growth stages the RNSP content of the control and treated silage after the 60 d of ensiling amounted to 77 and 124 g kg-1 DM, respectively, and after the 150 d of ensiling to 102 and 124 kg-1 DM, respectively. The difference was signifi-cant (p ≤ 0.05) only after the short ensiling period.

The cellulase OM digestibility of the control silage (averaged for the two ensiling periods) decreased gradually from 91.3% at growth stage 1 to 73.4% at growth stage 4. The OMd was always higher with the inoculant than with the control, except at growth stage 2 after 60 d of ensiling. The mean OMd for the four growth stages of the control and the treated silage after 60 d of ensiling amounted to 82.6 and 83.6%, respectively, and after 150 d of ensil-ing to 81.6 and 83.9%, respectively. The difference was significant (p ≤ 0.05) only after the long ensiling period.


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Table 2. The effect of the inoculant (control C versus treatment T) on the chemical composition and in vitro OMd of grass harvested at 4 growth stages and after an ensiling period (EP) of either 60 or 150 d (means of 3 micro-silos for DM, NDF and crude ash; single values for the other parameters).

EP Stage 1 Stage 2 Stage 3 Stage 4 SEMe Tf TxSg

(d) C T C T C T C T

Dry matter 60 387 397** 342 362** 370 380** 359 365** 2.9 ** **

(g kg-1) 150 378 404** 334 356** 367 375** 352 358* 3.5 ** **

NDFa 60 344 317** 397 377** 491 484ns 513 506ns 15.3 ** **

(g kg-1DM) 150 344 313** 410 379** 492 483* 504 514ns 15.3 ** **

Crude ash 60 111 108* 94 86** 81 75** 81 78ns 2.7 ** **

(g kg-1DM) 150 117 110** 97 87** 82 78** 80 79* 2.9 ** **

Crude protein 60 231 226 169 167 141 138 135 129 13.5 * nd

(g kg-1DM) 150 245 225 186 167 144 142 129 129 14.7 ns nd

Sugars 60 119 70 153 38 58 17 52 14 16.1 * nd

(g kg-1DM) 150 48 47 65 26 36 10 32 9 6.3 ns nd

FPb 60 96 132 78 136 91 120 98 112 6.8 * nd

(g kg-1DM) 150 118 141 108 143 93 120 94 120 6.3 ** nd

Crude fat 60 41 46 41 46 39 38 41 41 1.0 ns nd

(g kg-1DM) 150 45 49 46 47 40 43 41 44 1.0 ns nd

RNSPc 60 57 101 70 149 100 127 82 120 10.2 * nd

(g kg-1DM) 150 83 115 88 150 115 124 120 105 7.0 ns nd

OMdd 60 91.8 92.4 88.8 88.1 76.9 79.1 73.1 74.6 2.64 ns nd

(%) 150 90.7 93.0 86.8 88.6 75.2 79.0 73.6 74.9 2.61 * nda neutral detergent fibre b fermentation products c residual non starch polysaccharidesd in vitro cellulase digestibility of the OM e standard error of the meanf significance of treatment effectg significance of interaction between treatment (T) and growth stage (S) ns: not significant (p > 0.05), * significant at p≤ 0.05, ** significant at p ≤ 0.01nd: the interaction treatment x growth stage could not be determined because of limited degrees of freedom

Silage fermentation quality

The weekly DM-losses of all micro-silos increased linearly (p ≤ 0.01) from week 1 to the opening of the silos af-ter both 60 and 150 d of ensiling. For the control silages of 60 d ensiling losses increased gradually up to 6 weeks after ensiling but with a steeper slope after the induction of aerobic stress (Fig. 1). The losses were higher with advancing harvest stage. Initially, treated silages had higher DM-losses than control silages, the difference be-ing significant (p ≤ 0.05) up to weeks 4, 4, 3 and 2 after ensiling for stages 1, 2, 3 and 4, respectively. With longer ensiling, DM-losses for treated silages were no longer different (p > 0.05) from control silages and became even smaller (p ≤ 0.05) after 8, 7 and 4 weeks for stages 1, 3 and 4, respectively.


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The DM-losses of the control silages ensiled for 150 d (Fig. 2) increased gradually for stages 3 and 4, whereas loss-es for stages 1 and 2 showed a steep increase after about 8 weeks and were higher than those of the later stages at the opening of the silos. Initially, treated silages had higher DM-losses than control silages, the difference be-ing significant (p ≤ 0.01) up to weeks 3, 3, 2 and 1 after ensiling for stages 1, 2, 3 and 4, respectively. With longer ensiling, DM-losses for treated silages were no longer different (p > 0.05) from control silages and became even smaller (p≤ 0.01) after 12, 12, 7 and 6 weeks for stages 1, 2, 3 and 4, respectively.

The final weight loss of the control silages after 60 d of ensiling varied from 1.4% for growth stage 2 to 2.2% for growth stage 4 (Table 3). Longer ensiling increased weight losses particularly for growth stages 1 and 2, when losses more than doubled. The inoculant significantly (p ≤ 0.01) decreased losses, the effect being significant for stages 1, 3 and 4 after 60 d of ensiling and for all stages after the long ensiling period.

Fig. 1. Weekly DM-losses of control (C) and treated (T) grass silage at four growth stages during a 60 d ensiling period.

Fig. 2. Weekly DM-losses of control (C) and treated (T) grass silage at four growth stages during a 150 d ensiling period.

0,0

0,5

1,0

1,5

2,0

2,5

3,0

3,5

0 5 10 15 20

DMlo

sses

(%)

Weeks

C stage 1 T stage 1 C stage 2 T stage 2


0,0

0,5

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1,5

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DMlo

sses

(%)

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Table 3. The effect of the inoculant (control C versus treatment T) on silage quality of grass harvested at 4 growth stages and after an ensiling period (EP) of either 60 or 150 d (means of 4 micro-silos).

EP Stage 1 Stage 2 Stage 3 Stage 4 SEMa Tb TxSc

(d) C T C T C T C T

Final weight 60 1.6 0.8** 1.4 1.2ns 1.9 1.4** 2.2 1.5** 0.08 ** **

loss (%) 150 3.3 1.2** 3.2 1.4** 2.4 1.7** 2.6 1.8** 0.14 ** **

pH 60 4.93 3.93** 4.60 3.84** 4.41 3.93** 4.42 4.04** 0.066 ** nd

150 5.92 3.95** 5.07 3.85** 4.62 4.01** 4.82 4.07** 0.121 ** nd

Lactic acid 60 32 87** 40 83** 46 71** 45 52ns 3.5 ** **

(g kg-1 DM) 150 29 89* 26 87** 34 65** 33 55** 4.5 ** **

Acetic acid 60 26 24ns 11 32** 11 27** 11 34** 1.6 ** **

(g kg-1 DM) 150 16 29** 8 33** 16 29** 10 40** 2.0 ** **

Butyric acid 60 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.00 ns ns

(g kg-1 DM) 150 0.0 0.0ns 0.0 0.0ns 2.3 0.0* 3.3 0.1** 0.26 ** nd

Alcohols 60 38 21** 27 21* 34 22** 42 26** 1.4 ** **

(g kg-1 DM) 150 73 24** 74 24** 41 26** 48 25** 3.7 ** **

NH3-N/N 60 4.5 2.7** 6.3 3.8** 7.6 6.3** 8.3 5.5** 0.32 ** **

(%) 150 4.8 3.6** 6.7 4.5** 7.1 6.4** 8.6 6.0** 0.27 ** **

Aerobic stability 60 30 127* 24 153** 31 150** 32 >170** 12.0 ** ns

(h)d 150 94 >170** 43 >170** 76 >170** 39 >170** 12.8 ** **a standard error of the meanb significance of treatment effectc significance of interaction between treatment (T) and growth stage (S) d aerobic stability was measured for a maximum of 170 hns: not significant (p > 0.05), * significant at p ≤ 0.05, ** significant at p ≤ 0.01nd: not determined because of non-parametric Kruskall-Wallis analysis

The pH of the control silage after 60 d of ensiling varied between 4.41 for stage 3 to 4.93 for stage 1. Longer ensil-ing increased pH with 0.2 to 1.0 units; the greatest increase was observed for stage 1. Treating grass significantly (p ≤ 0.01) decreased pH at all stages and for both ensiling periods. The effect varied from 0.4 to 2.0 units and was most pronounced for the long ensiling period. The difference decreased with later harvest date.

Lactic acid content of the control silage after 60 d varied between 32 g kg-1 DM for stage 1 to 46 g kg-1 DM for stage 3 and was lower at each stage after 150 d of ensiling. The inoculant significantly (p ≤ 0.01) increased lactic acid content for both ensiling periods. The effect clearly decreased with later harvest date.


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Acetic acid content of the control grass silage after 60 d varied between 11 g kg-1 DM for stages 2, 3 and 4 to 26 g kg-1 DM for stage 1. Longer ensiling decreased the content for stage 1 and increased the content for stage 3. The addition of the inoculant significantly (p ≤ 0.01) increased acetic acid content for both ensiling periods. For stage 1 after 60 d of ensiling no difference was observed.

Butyric acid was not detected in the control nor in the treated silages after 60 d. After 150 d of ensiling butyric acid was present in the control silage of stages 3 and 4, but not in the treated silages.

Propionic acid was not detected in any of the control or treated silages.

The total alcohol content of the control silages after 60 d varied from 27 g kg-1 DM for stage 2 to 42 g kg-1 DM for stage 4. Longer ensiling increased alcohol content, particularly for stages 1 and 2. Treatment significantly (p ≤ 0.01) lowered alcohol content; the decrease was most pronounced for stages 1 and 2 after long ensiling.

The ammonia fraction of the control silage after 60 d increased gradually with later harvesting from 4.5% for stage 1 to 8.3% for stage 4. Longer ensiling increased the ammonia fraction a little more except for stage 3 when a small decrease was observed. Treatment with the inoculant significantly (p ≤ 0.01) decreased the ammonia fraction for all stages and both ensiling periods with 0.7 to 2.8%-units.

Aerobic stability of the control silage after 60 d was fairly constant among stages amounting to about 30 h. Longer ensiling increased aerobic stability in all stages. Treatment with the inoculant significantly (p ≤ 0.01) improved aer-obic stability. After 150 d of ensiling, the silage with the inoculant remained stable at all stages for more than 7 d.

Rumen degradability of NDF and OM

The potentially degradable fraction as well as the degradation rate of NDF in the rumen decreased with later har-vest date (Table 4). As a result, the rumen fermentable NDF fraction decreased from 67.6% for the first stage to 52.5% for the last stage. The inoculant had no effect on DNDF nor on kdNDF of the grass silage after 60 d. On the oth-er hand after 150 d of ensiling, treatment increased DNDF, the effect being significant (p ≤ 0.01) at stages 2 and 4, whereas it decreased kdNDF, but the difference was only significant (p ≤ 0.01) at stage 2. Treatment had no effect on the rumen degradable NDF fraction at all growth stages and for the two ensiling periods.

Later harvesting decreased the washable OM fraction and the degradation rate of OM and had no clear effect on the potentially degradable OM fraction. As a result the rumen fermentable OM fraction averaged for both ensil-ing periods decreased from 74.8% for the first stage to 56.5% for the last stage. Treatment did not affect any of the degradation characteristics after 60 d of ensiling. On the other hand after longer ensiling, treatment signifi-cantly (p≤ 0.05) increased WOM at stages 1 and 2, had no effect on DOM and decreased kdOM, but only significantly (p ≤ 0.05) at the last stage after 150 d. As a result, treatment had no effect on FOM% after 60 d of ensiling, where-as it increased %FOM at stages 1 and 2 after 150 d of ensiling.


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Table 4. The effect of the inoculant (control C versus treatment T) on rumen degradation characteristics of NDF and OM of grass harvested at 4 growth stages and after an ensiling period (EP) of either 60 or 150 d (means of 3 micro-silos)

EP Stage 1 Stage 2 Stage 3 Stage 4 SEMh Ti TxSj

(d) C T C T C T C T

DNDFa 60 92.9 90.4 87.7 86.6 81.9 82.1 78.2 76.0 1.21 ns ns

(%) 150 89.5 91.2ns 86.9 89.7** 81.8 82.9ns 77.6 81.6** 0.96 ** ns

kdNDFb 60 6.87 6.63 6.51 5.74 3.81 4.06 4.34 4.34 0.257 ns ns

(% h-1) 150 7.76 7.31ns 6.36 5.50** 4.47 4.18ns 4.70 4.36ns 0.282 ** ns

FNDFc 60 67.3 65.2 63.0 61.0 53.1 53.9 52.2 50.7 1.30 ns ns

(%) 150 65.9 66.7ns 62.3 62.7ns 54.9 54.9ns 52.7 54.5ns 1.11 * ns

WOMd 60 43.6 44.9 41.6 39.8 30.2 30.3 28.7 27.5 1.43 ns *

(%) 150 40.6 46.0** 36.5 40.2** 31.1 30.1ns 29.2 28.1ns 1.29 ** **

DOMe 60 51.1 48.9 47.3 49.4 54.6 54.9 53.2 52.6 0.58 ns *

(%) 150 50.4 48.3 52.4 51.5 53.9 56.1 52.6 56.1 0.55 ns **

kdOMf 60 7.45 7.79 7.35 7.17 4.22 4.58 4.72 5.17 0.302 ns ns

(% h-1) 150 8.96 8.49ns 7.10 6.45ns 4.78 4.56ns 5.11 4.69* 0.356 * ns

FOMg 60 75.4 75.9 70.9 70.2 56.5 57.9 55.9 55.6 1.79 ns ns

(%) 150 74.1 77.5* 68.5 70.5* 58.8 58.4ns 57.1 56.7ns 1.65 ** **a potentially degradable NDF fractionb degradation rate of DNDFc rumen fermentable NDFd washable OM fractione potentially degradable OM fractionf degradation rate of DOMg rumen fermentable OM h standard error of the meani significance of treatment effectj significance of interaction between treatment (T) and growth stage (S) ns: not significant (p > 0.05), * significant at p ≤ 0.05, ** significant at p ≤ 0.01

Discussion

Control silage

The effect of the inoculant was studied with a first cut grass harvested at four distinct growth stages. The evolu-tion in growth stage was clearly reflected by the increase in NDF content (from 344 to 509 g kg-1 DM) and the de-crease in CP content (from 238 to 132 g kg-1 DM) as well as in OMd (from 91.3 to 73.4%) of the control silage. The growth stage had not only an effect on the quantity of NDF but also on its quality. From the rumen degradation characteristics of NDF it appeared that later harvesting resulted in an increase of the undegradable fraction from about 10% at the first stage to more than 20% at the last stage and means a decreasing potentially degradable fraction. Moreover, the degradation rate of the latter almost halved in the period from the first to the last growth stage. Later harvesting also increased the rumen undegradable OM fraction, whereas the potentially degradable OM fraction remained almost constant because of the decrease of the washable OM fraction. This later tendency can be explained by the decrease of sugars and of soluble protein in silage of a later growth stage.

The inoculant was applied to grass wilted at a DM content of about 35%. Wilting grass is already a good measure to improve silage quality because epiphytic lactic acid bacteria are relatively more tolerant to low moisture avail-ability than the vegetative forms of undesirable clostridia (Woolford 1984). According to the latter there is no more


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advantage to wilt further than 300−340 g DM kg-1, because from then on oxidation could increase losses. Indeed the control silage of all 4 stages after 60 d of ensiling showed small DM-losses (1.6 to 2.2%), a low pH (4.4 to 4.9), no butyric acid and a low ammonia fraction (4.5 to 8.3%). Despite the proper fermentation characteristics, aero-bic stability of the control silage at all stages was low (about 30 h), considering that the target for potential aero-bic stability is 7 d (Wilkinson and Davies 2013).

For testing the efficacy of silage additives, EFSA (2006) recommends an ensiling period of 90 d or longer. In this study a shorter period of 60 d was chosen, because it is a common practice that farmers open their silo after 2 months of ensiling in the assumption that silage fermentation is finished. However, comparison of the control si-lages after 60 and 150 d of ensiling in the present study showed worse quality after longer ensiling at all stages. Compared with 60 d of ensiling, longer ensiling resulted in higher DM-losses (2.4 to 3.3%), a lower DM content, a higher pH, particularly for the first growth stage, a reduced lactic acid content and an increased alcohol con-tent and the presence of butyric acid at stages 3 and 4. These changes were accompanied by a further decrease of sugar content and indicate that the fermentation process was still ungoing after 60 d of ensiling. Considering the low aerobic stability observed after 60 d of ensiling, the oxidation of the control silages during longer ensiling may have been caused by some air ingression through the tape covering the openings in the tube. Another expla-nation is the higher risk for aerobic deterioration at a density of 180 kg DM per m³, as applied in our experiment. Such a density also prevails in practice, but is lower than the recommended minimum density of 210 kg DM per m³ (Wilkinson and Davies 2013).

The effect of the inoculant on chemical composition and silage quality The number of LAB counted in the inoculated grass silage corresponded fairly well with the number added with the inoculant, whereas the number in the control silages was low to very low. Notwithstanding the proper quality of the control silage after 60 d of ensiling, treatment clearly improved almost all fermentation characteristics. The inoculant decreased weight losses, resulting in a higher DM content of the silage. It increased lactic acid content; the effect was most pronounced for the first two harvest dates. It also increased acetic acid content at all stages except the first. The increase in the production of acids was reflected in a decrease of pH. On the other hand, the addition of the inoculant decreased the formation of alcohols, which is an indication that yeasts and moulds were inhibited, which is also reflected by the better aerobic stability of the treated silage. That the inoculant reduced the activity of undesirable organisms appears also from the lower ammonia fraction, meaning less protein degra-dation in the silo. Similar effects by inoculating wilted perennial ryegrass (330 g DM kg-1) with L. buchneri plus a mixture of Pediococcus pentosaceus and L. plantarum on pH, lactic acid, the ammonia fraction, DM loss and aero-bic stability were observed by Driehuis et al. (2001).

The production of more acetic acid besides more lactic acid proves the activity of the heterofermentative bacte-rium L. buchneri in the inoculant. Acetic acid inhibits yeasts and moulds, which was clearly reflected by the lower alcohol production and better aerobic stability. The higher acid production by the added living Lactobacilli was possible through the fermentation of more sugars. The addition of the inoculant also lowered NDF content of the silage, particularly at the early growth stages. The decrease of NDF content is interesting for the nutritive value of the silage as it should result in a higher feed intake. Indeed, cell wall content highly affects feed intake by con-tributing to rumen fill (Jung and Allen 1995).

The decrease in NDF content was accompanied by an increase of RNSP content, a measure of complex sugars. This is another indication of the activity of L. buchneri, which was shown to produce ferulate esterase (Donaghy et al. 1998). This enzyme seems able to attack young cell walls, but not lignified ones.

The improvement of silage quality by addition of the inoculant was even more pronounced after 150 d of ensiling. This greater effect is rather due to the worse quality of the untreated silage after 150 d of ensiling than to a pro-longed effect of the inoculant. The lower contents of CP and crude ash in the DM of the treated grass silage at all stages after both ensiling periods, although small, may be explained by a dilution effect. Indeed, when expressed per kg of silage, CP and ash contents were similar for the control and the treated silage.


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The effect of the inoculant on NDF and OM degradability

Because of the presence of L. buchneri, which is able to produce ferulate esterase (FE), the studied inoculant is claimed to improve the nutritive value of the resulting grass silage through a better cell wall degradability in the rumen. Weinberg et al. (2004) have shown that lactic acid bacteria consumed with silage enter the rumen and may survive there. Nsereko et al. (2008) examined the activity of 8 FE producing Lactobacilli by in situ incubations and found that they all increased 48 h rumen NDF degradability of perennial ryegrass by 9 to 11%. The studied strains did not affect NDF content of the silage and one strain increased NDF content, whereas we found a decrease of NDF content in the treated silage of growth stages 1 and 2. In their study however, the control grass silage contained 571 g NDF kg-1 DM, which is even more than the cell wall content of the last stage in our study. Thus, it agrees with our finding that the inoculant has no effect on cell wall content in silage from older grass. But also Driehuis et al. (2003) found no effect of L. buchneri, with or without homofermentative lactic acid bacteria, on NDF con-tent of perennial ryegrass with 438 g NDF kg-1 DM, similar to that of the second growth stage in our experiment. In agreement with our results, Van Vuuren et al. (1989) found a decrease of NDF content when herbage was treat-ed with cell wall degrading enzymes; the effect decreased with increasing maturity and DM content of the grass.

In contrast with Nsereko et al. (2008), who found an increase of NDF degradability with the same L. buchneri strain as present in the inoculant of our study, we did not find an effect of treatment on the rumen fermentable NDF fraction at any of the growth stages either after 60 or 150 d of ensiling. Considering the clear positive effects on silage fermentation quality, particularly the increase in acetic acid and the better aerobic stability, one may con-clude that L. buchneri has worked in our experiment. Moreover, it seems that L. buchneri has developed FE ac-tivity during the ensiling process by degrading easily degradable cell walls, and so leaving cell walls which were more difficult to degrade in the rumen. Similar observations were done by Van Vuuren et al. (1989) treating herb-age with cell wall degrading enzymes.

The percentage of rumen FOM is another interesting nutritive parameter because it determines microbial pro-tein production. The inoculant had no effect on %FOM at any growth stage after 60 d of ensiling, but increased %FOM of the silage from the first and second growth stage after 150 d of ensiling. This increase was mainly due to a higher WOM fraction, which can be explained by the increase in fermentation products as well as complex sug-ars. The treatment effects for rumen FOM are confirmed by those for in vitro OM digestibility. The latter mimics digestibility over the whole digestive tract and treatment increased OMd after 150 d of ensiling with on average 2.3%-units over the four growth stages.

Conclusions

By treating wilted grass with the inoculant more sugars were fermented to lactic and acetic acid, resulting in a lower pH, less DM-losses and protein degradation and a better aerobic stability. The effects on silage quality were more pronounced after 150 d of ensiling than after 60 d, but this was rather due to the worse quality of the con-trol silage than to a prolonged effect of the inoculant. Although wilting grass is a good measure to obtain good quality silage, quality can still further be improved by using the inoculant.

The treatment lowered NDF content of grass harvested at the early growth stages, probably by degrading NDF to complex sugars but had no effect on NDF degradability in the rumen. The inoculant improved rumen fermenta-bility as well as in vitro digestibility of OM after 150 d of ensiling. Postponing harvest and correcting the nutritive value of the silage by applying inoculant additive seems however no option.

Acknowledgements

The authors highly appreciate Dr.ir. A. De Vliegher from the Plant Sciences Unit of ILVO for caring and harvesting the grass parcel.


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AGRICULTURAL AND FOOD SCIENCEE. Wambacq et al. (2013) 22: 127–136

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The effect of Lactobacillus buchneri inoculation on the aerobic stability and fermentation characteristics of alfalfa-ryegrass,

red clover and maize silageEva Wambacq1, Joos P. Latré2 and Geert Haesaert1

1University College Ghent – Associated Faculty of Applied Bioscience Engineering, Valentijn Vaerwyckweg 1, 9000 Ghent, Belgium

2University College Ghent – Faculty of Science and Technology, Brusselsesteenweg 161, 9090 Melle, Belgium


Aerobic spoilage of silages occurs frequently and is undesirable because it reduces both its nutritive and hygienic quality. Silage inoculants containing heterofermentative lactic acid bacteria, like Lactobacillus buchneri, have already been proven to improve aerobic stability by augmented production of acetic acid, which inhibits yeasts. In this study, the effect of L. buchneri on fermentation characteristics and aerobic stability of alfalfa-ryegrass silage, red clover silage and maize silage was assessed using microsilos. Two dosages, 1×105 and 3×105 cfu g-1 of fresh matter, were compared to untreated control silage. Inoculation with L. buchneri clearly altered the fermentation characteristics of alfalfa-ryegrass and red clover silage, resulting in a significantly higher aerobic stability at both dosages. The ef-fects of L. buchneri inoculation on maize silage were less clear, but nevertheless the aerobic stability of maize silage inoculated with 1×105 cfu g-1 of fresh matter was significantly higher compared to the untreated silage.

Key words : silage additive, Lactobacillus buchneri, fermentation, heating

Introduction

Aerobic spoilage and heating of ensiled fodder crops occurs frequently (Wilkinson 2005, Stryszewska and Pys 2006). This process is usually initiated by acid-tolerant yeasts and occasionally by acetic acid bacteria when oxygen is able to penetrate between the plant particles, during feed-out or due to damage of the silo coverage (Spoelstra et al. 1988, Woolford 1990, Driehuis et al. 1999). Yeasts and acetic acid bacteria aerobically break down residual water-soluble carbohydrates and organic acids like lactic acid and acetic acid in a first phase of aerobic deteriora-tion (Oude Elferink et al. 2006). This not only lowers the nutritive value of the silage, but also triggers a raise of the silage-pH, allowing growth of opportunistic bacteria and moulds in a second phase of aerobic spoilage (Weinberg and Muck 1996, Scudamore and Livesy 1998, Stryszewska and Pys 2006). Mould development in silages is unde-sired since inhalation of high concentrations of certain airborne mould spores can affect the respiratory system and cause allergenic reactions in animals and humans, but also because some mould genera can produce myco-toxins during silage conservation (Bui et al. 1994, Auerbach et al. 1998). Furthermore, mould growth decreases the nutritive value of the silage even more and also lowers its palatability (DiConstanzo et al. 1995, Wilkinson 2005). Mould species that regularly have been isolated from silage belong to the genera Penicillium, Aspergillus, Mu-cor, Byssochlamys, Fusarium, Alternaria, Geotrichum, Monascus, Paecilomyces and Trichoderma (Nout et al. 1993, Auerbach et al. 1998, Samson and Frisvad 2004, Samson et al. 2004, O’Brien et al. 2005, Mansfield et al. 2008, Declerck et al. 2009). Fusarium, Aspergillus and Penicillium are the most important mycotoxin producing mould genera. In animals, mycotoxins are factors contributing to chronic problems like a higher incidence of disease and lower production efficiency (Scudamore and Livesey 1998, Barug et al. 2006, Fink-Gremmels 2008).

Application of a silage inoculant consisting of heterofermentative lactic acid bacteria (HeLAB) has already been proven to reduce heating and increase aerobic stability of silages of maize, grass and other fodder crops (Driehuis et al. 1999, Driehuis et al. 2001, Adesogan et al. 2003, Filya et al. 2006, Kleinschmit and Kung 2006). While homo-fermentative lactic acid bacteria (HoLAB) improve silage fermentation by very efficient production of lactic acid, heterofermentative lactic acid bacteria produce both lactic acid and acetic acid - the latter having an inhibiting effect on yeasts and moulds (Moon 1983, Oude Elferink et al. 2001, Filya et al. 2006). Therefore, HeLAB inoculants should decrease aerobic spoilage of silages (Kung and Ranjit 2001, Danner et al. 2003).

The aim of this study was to assess the effect of HeLAB inoculation with Lactobacillus buchneri on aerobic stabil-ity and fermentation characteristics of three silage types : red clover (Trifolium pratense), whole crop maize (Zea mays) and a mixture of alfalfa (Medicago sativa) and Italian ryegrass (Lolium multiflorum).



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Material and methodsEnsiling trials

In 2008, trials were performed with a mixture of alfalfa and Italian ryegrass (approx. 50/50 w/w) and with red clo-ver, while the trial with whole crop maize was conducted in 2009. Microsilos with a content of 2.75 litre (height 35 cm, diameter 10 cm) equipped with a CO2-valve were used. The microsilos had two openings (diameter 0.5 cm) covered with plastic tape, to be able to provide aerobic stress by removing the tape.

The alfalfa-ryegrass was from the first cut, the red clover from the second cut. Both crops were harvested at flow-ering with a Haldrup mower (Inotec, Løgstør, Denmark), spread in a thin layer and wilted to approximately 350 gram dry matter per kilogram fresh matter (quick estimation by microwave drying) during a few hours. Both crops were chopped to approximately 5 cm with a New Holland chopper (Sperry New Holland, Zedelgem, Belgium). The maize variety ‘Lafortuna’ was chopped to approximately 5 mm with a Mengele chopper (Mengele Agrartechnik, Standort Waltstetten, Germany).

A representative sample of untreated chopped material was taken for determination of dry matter (DM), wa-ter-soluble carbohydrates (WSC), crude ash (CA), crude protein (CP), crude fat (CF), in vitro digestibility, neutral detergent fibre (NDF) and acid detergent fibre (ADF). The Net Energy for Lactation (NEL) was estimated (formulas in Debrabander 2011). Counts of epiphytic yeasts, moulds, lactic acid bacteria and enterobacteria were also per-formed. The results of the analyses on the herbage prior to ensiling are summarized in Table 1.

Table 1. Chemical composition, nutritive value and microbial counts of raw materials prior to ensiling

Alfalfa- ryegrass Red clover Maize

Dry matter (g kg-1 FM) 383 371 351

Water-soluble carbohydrates (g kg-1 FM) 14 26 25

Crude ash (g kg-1 DM) 149 95 44

Crude protein (g kg-1 DM) 219 209 64

Crude fat (g kg-1 DM) 23 14 24

Neutral-detergent fibre (g kg-1 DM) 441 516 636

Acid-detergent fibre (g kg-1 DM) 258 299 241

Net energy for lactation (MJ) 4.58 4.70 6.53

In vitro digestibility of OM (%) 81.0 69.5 71.2

Yeasts (log cfu g-1 FM) < 2 < 2 5.7

Moulds (log cfu g-1 FM) 4.0 4.2 4.8

Lactic acid bacteria (log cfu g-1 FM) 6.7 7.0 5.5

Enterobacteria (log cfu g-1 FM) < 1 < 1 4.8

FM: fresh matter, DM: dry matter, MJ: mega-Joule, OM: organic matter, cfu: colony-forming units

Silages were inoculated with L. buchneri NCIMB 40788 (Lallemand sas). In each trial, the following treatments were included: a control treatment (C) of sterile demineralized water, a low dosage of L. buchneri (LD) and a high dosage of L. buchneri (HD). LD-solution was dosed at 1×105 cfu g-1 of fresh matter, while the dosage of the HD- solution was 3×105 cfu g-1 of fresh matter. On the L. buchneri inoculant powder, counting of lactic acid bacteria was performed prior to ensiling to ensure correct dosage. Inoculants were suspended in sterile demineralized water with 0.85% sodium chloride just before ensiling. All solutions were applied homogeneously on chopped material by hand held sprayers in a ratio of 10 ml kg-1of fresh matter. A different sprayer was used per treatment. Six micro-silos were ensiled per treatment, at a mean silo density of respectively 165, 154 and 196 kg DM per cubic meter for alfalfa-ryegrass, red clover and maize silage. Microsilos were weighed empty and immediately after filling, which was continued on a weekly basis. All microsilos were subjected to aerobic stress during 24 hours at 71–72 days after ensiling. At silo opening after 90 days, samples were taken from five microsilos per treatment for chemical analyses (dry matter, crude protein, ammonia, ethanol, pH, lactic acid, acetic acid, butyric acid and propionic acid), microbial analyses (counts of yeasts, moulds and lactic acid bacteria) and determination of the aerobic stability.


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Chemical analyses

DM content was determined by air drying at 65 °C during 170 hours. On dried material, WSC content was ana-lysed by the Luff-Schoorl method, while CA, CP, CF, in vitro digestibility, NDF and ADF were determined accord-ing to ISO 17025 as follows: CA by 71/250/EG, CP by the Dumas combustion method according to NF ISO 15670 and CF by 71/393/EG (European Economic Community 1971), in vitro digestibility according to De Boever et al. (1986), NDF and ADF according to Van Soest et al. (1991). Fresh silage material was stored at –20 °C immediately after sampling for subsequent analyses. Ethanol content was determined on an aqueous extract of fresh ma-terial by NIR absorption by an adaptation of the method described by Sørensen (2004). Measurement of pH was done on a 1/10 (w/w) aqueous extract of fresh material (Muck et al. 1999). Also on fresh material, ammonia and crude protein were determined according to Kjeldahl (1883), while lactic acid, acetic acid, butyric acid and propi-onic acid were determined by HPLC (Ohmomo et al. 1993). DM content at silo opening was corrected (indicated as “cDM”) for volatile compounds (Dulphy and Demarquilly 1981).

Microbial countsSamples were stored at 4 °C prior to counting, which was started within one day after sampling. Counts of lactic acid bacteria and yeasts and moulds were conducted according to resp. ISO 15214 (1998) and ISO 21527-1 (2008), while enterobacteria were counted with Petrifilm® Afnor 3M 01/6-09/97.

Aerobic stabilityThe aerobic stability was determined based on the protocol suggested by Honig (1990). The equivalent of 100 g DM was placed loosely into polystyrene boxes and allowed to deteriorate aerobically at 20 °C ± 1 °C. The top and bottom of the boxes had an opening to allow gas exchange. Per microsilo, two polystyrene boxes were filled. A double layer of cheesecloth allowing air penetration was placed over each container to prevent drying. A ther-mocouple probe was placed into the geometric centre of each silage mass. Silage temperature was recorded every 10 minutes and averaged over 2-hour periods. Silages were not disturbed during testing. Monitoring was carried out over a period of 7 days for the maize silage and of 15 days for the alfalfa-ryegrass and red clover silages. An increase of silage temperature of 3 °C above surrounding temperature was taken as cut-off for aerobic stability.

Statistical analysesData were statistically analyzed with SAS 4.1 (SAS 2006). Normality was tested by Kolmogorov-Smirnov, while equality of variances was checked by Levene’s test. Normally distributed, homoscedastic data were subjected to one-way ANOVA with Tukey as post hoc test. Otherwise, data were subjected to non-parametric one-way ANOVA with Wilcoxon as post hoc test, applying Bonferroni correction. Significance was declared at p < 0.05, with p-values of < 0.001 marked as “***”, < 0.01 as “**” and < 0.05 as “*”. Non-significance was indicated as “n.s.”.

ResultsFermentation losses

Fermentation losses were calculated based on the fresh weight losses of the microsilos during the ensiling pe-riod. The evolution of the fermentation losses is presented in Table 2 (five observations per treatment for each silage type).


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Table 2. Evolution of fermentation losses (g kg-1 fresh matter) during ensiled period

Fermentation losses (g kg-1 FM) after

Alfalfa-ryegrass Red clover Maize

mean SD sign. mean SD sign. mean SD sign.

7 days

C 4.45 0.13 a

***5.39 0.24 n.s.

7.28 0.19n.s.LD 8.07 0.32 b 5.69 0.54 7.19 0.32

HD 8.41 0.34 b 5.94 0.74 7.22 0.09

14 days

C 6.17 0.10 a

***

6.54 0.32 a

***

7.88 0.34

n.s.LD 10.7 0.30 b 7.98 0.64 b 8.03 0.70

HD 10.8 0.29 b 8.17 0.85 b 7.97 0.20

21 days

C 6.68 0.23 a

***

7.26 0.33 a

***

8.33 0.42

n.s.LD 11.6 0.34 b 8.96 0.67 b 8.56 0.84

HD 11.6 0.15 b 9.16 1.00 b 8.54 0.22

28 days

C 7.12 0.22 a

***

7.42 0.42 a

***

8.66 0.45

n.s.LD 12.2 0.33 b 9.33 0.81 b 9.00 0.79

HD 12.4 0.19 b 9.44 1.16 b 8.95 0.26

35 days

C 7.47 0.16 a

***

7.65 0.41 a

***

8.83 0.55

n.s.LD 12.8 0.43 b 9.59 0.79 b 9.13 0.78

HD 12.9 0.19 b 9.77 1.12 b 9.13 0.24

42 days

C 8.11 0.27 a

***

8.05 0.51 a

***

8.97 0.63

n.s.LD 13.6 0.41 b 10.1 0.84 b 9.31 0.82

HD 13.7 0.21 b 10.4 1.17 b 9.34 0.24

49 days

C 8.52 0.22 a

***

8.19 0.44 a

***

9.07 0.66

n.s.LD 14.0 0.35 b 10.3 0.85 b 9.43 0.81

HD 14.1 0.21 b 10.5 1.29 b 9.48 0.22

56 days

C 8.70 0.27 a

***

8.26 0.42 a

***

9.16 0.68

n.s.LD 14.2 0.34 b 10.4 0.87 b 9.51 0.81

HD 14.3 0.17 b 10.7 1.32 b 9.60 0.20

63 days

C 9.36 0.25 a

***

8.46 0.32 a

***

9.32 0.70

n.s.LD 14.9 0.34 b 10.8 0.91 b 9.66 0.87

HD 15.0 0.17 b 11.0 1.32 b 9.73 0.22

70 days

C 9.90 0.27 a

***

8.67 0.47 a

***

9.40 0.72

n.s.LD 15.4 0.31 b 10.9 0.98 b 9.77 0.86

HD 15.5 0.17 b 11.2 1.33 b 9.84 0.20

aerobic stress provided at 71–72 days after ensiling

77 days

C 11.0 0.34 a

***

9.21 0.46 a

***

10.5 0.32

n.s.LD 15.9 0.10 b 11.8 0.59 b 10.5 0.52

HD 16.0 0.16 b 12.0 1.04 b 10.4 0.13

84 days

C 11.0 0.34 a

***

9.23 0.48 a

***

10.5 0.30

n.s.LD 15.9 0.10 b 11.8 0.59 b 10.6 0.59

HD 16.0 0.16 b 12.0 1.04 b 10.5 0.13

90 days

C 11.2 0.38 a

***

9.41 0.41 a

***

10.6 0.26

n.s.LD 16.0 0.09 b 12.0 0.61 b 10.7 0.47

HD 16.1 0.15 b 12.2 1.10 b 10.7 0.12SD: standard deviation, sign.: significance, n.s.: not significant, ***: p < 0.001FM: fresh matter C: control LD: low dosage of L. buchneri (1×105 cfu g-1 of fresh matter) HD: high dosage of L. buchneri (3×105 cfu g-1 of fresh matter)


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For the alfalfa-ryegrass silage inoculated with L. buchneri, either at high or low dosage, the fermentation losses were significantly higher compared to the untreated control. A similar pattern was detected in the red clover si-lage from 14 days after ensiling on till the end of the ensiling period. High dosage of L. buchneri resulted in slightly higher fermentation losses than low dosage, but the differences between low dosage and high dosage of L. bu-chneri were not significant. No significant differences in fermentation losses between treatments were observed for the maize silage.

Fermentation characteristicsBased on the water-soluble carbohydrate content of the herbage prior to ensiling, the alfalfa-ryegrass mixture was considered as difficult to ensile (< 15 g kg-1 fresh matter of water-soluble carbohydrates) according to the EFSA opinion on silage additives guidelines (European Food Safety Authority 2008), while the red clover and whole crop maize were considered to be moderately easy to ensile (15–30 g kg-1 fresh matter of water-soluble carbohydrates). Due to the evidently higher crude protein content of the alfalfa-ryegrass silage and red clover silage, a higher buffering capacity was expected for these silages compared to maize silage (McDonald et al. 1991, Wilkinson 2005).

The fermentation characteristics of the silages are listed in Table 3 (five observations per treatment for each silage type).

Table 3. Effect of Lactobacillus buchneri inoculation on fermentation characteristics of alfalfa-ryegrass, red clover and maize silage



cDM (g kg-1 FM)

C 419 3.3 n.s.

371 2.0 a *

356 2.6 a

***LD 417 4.2 369 4.9 ab 344 3.1 b

HD 421 4.3 364 4.4 b 349 1.8 c

Crude protein (g kg-1 cDM)

C 209 4.2

*

198 4.7 n.s.

60.6 1.02LD 212 1.7 a 199 7.5 61.5 1.15 n.s.

HD 205 3.1 b 191 4.7 59.7 0.95

Ammonia (g kg-1 cDM)

C 5.79 0.1 a

***

5.86 0.28 n.s.

0.69 0.05

LD 6.31 0.1 b 5.96 0.29 0.73 0.04 n.s.

HD 6.21 0.2 b 5.89 0.26 0.75 0.03

Ammonia nitrogen (g kg-1 nitrogen)

C 138 3.9 a

***

145 7.7 n.s.

52.3 3.1 a

*LD 148 2.8 b 148 7.1 54.1 2.7 ab

HD 151 3.4 b 152 8.8 57.2 2.4 b

Ethanol (g kg-1 cDM)

C 17.3 1.2 a

***

18.5 3.30 n.s.

19.7 3.11

LD 21.6 0.4 b 19.7 0.66 20.9 2.72 n.s.

HD 21.7 1.3 b 20.0 2.32 21.2 1.45

pH

C 4.54 0.01 a

***

4.36 0.02 a

***

3.77 0.01 ab

*LD 4.58 0.01 b 4.46 0.02 b 3.77 0.02 a

HD 4.59 0.02 b 4.47 0.06 b 3.74 0.01 b

Lactic acid (g kg-1 cDM)

C 48.3 1.32 a

**

64.9 1.44 a

*

41.3 3.06 a

*LD 41.2 2.48 b 56.0 3.49 ab 45.0 2.38 b

HD 40.8 4.27 b 50.9 11.3 b 45.4 1.71 b

Acetic acid (g kg-1 cDM)

C 18.5 2.99 a

***

22.1 1.06 a

*

15.9 2.51 a

**LD 30.2 3.70 b 30.7 3.66 ab 19.5 2.55 ab

HD 31.1 4.06 b 34.1 9.46 b 21.3 1.58 b

Butyric acid (g kg-1 cDM)

C 0.12 0.27 n.s.

0.00 0.00 -

0.00 0.00

-LD 0.00 0.00 0.00 0.00 0.00

HD 0.00 0.00 0.00 0.00 0.00

SD: standard deviation, sign.: significance, n.s.: not significant, ***: p < 0.001, **: p < 0.01, *: p < 0.05C: control LD: low dosage of L. buchneri (1×105 cfu g-1 of fresh matter) HD: high dosage of L. buchneri (3×105 cfu g-1 of fresh matter) FM: fresh matter cDM: corrected DM content, according to Dulphy and Demarquilly (1981)


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The corrected dry matter content of the silages did not differ significantly between treatments for alfalfa-ryegrass silage, however inoculation with L. buchneri lowered the DM content of red clover and maize silage compared to untreated control silage. In case of red clover silage, a significant difference in DM content between the untreat-ed control (371 g kg-1 FM) and the high dosage of L. buchneri (364 g kg-1 FM) was observed, while for maize silage the DM content of all treatments (C : 356, LD : 344, HD : 349 g kg-1 FM) differed significantly.

The crude protein content of the alfalfa-ryegrass silage and the red clover silage was within normal range, while the maize silage was relatively low in crude protein (Hoffman and Broderick 2001, Debrabander 2011). No sig-nificant differences in crude protein content between treatments were observed in red clover silage and maize silage. The crude protein content of alfalfa-ryegrass silage treated with low dosage of L. buchneri (212 g kg-1 cDM) was significantly higher compared to high dosage (205 g kg-1 cDM), while the untreated control silage had an in-termediate crude protein content (209 g kg-1 cDM).

As expected, the ammonia content of maize silage was lower than the ammonia content of alfalfa-ryegrass silage and red clover silage. Red clover silage and maize silage showed no significant differences in ammonia content between treatments. The ammonia content of L. buchneri inoculated alfalfa-ryegrass silage (LD: 6.31, HD: 6.21 g kg-1 cDM) was significantly higher than that of untreated silage (5.79 g kg-1 cDM), and the same observation can be made for the ammonia nitrogen fraction (C: 138, LD: 148, HD: 151 g ammonia nitrogen kg-1 total nitrogen). The fraction of ammonia nitrogen to total nitrogen of untreated maize silage (52.3 g ammonia nitrogen kg-1 to-tal nitrogen) was significantly lower compared to maize silage with high dosage of L. buchneri (57.2 g ammonia nitrogen kg-1 total nitrogen), while no significant differences in ammonia nitrogen fraction between treatments were observed in red clover silage.

In all three silage types, the ethanol content was lower in untreated silage compared to L. buchneri inoculated silage. These differences between treatments were only significant in alfalfa-ryegrass silage (C: 17.3, LD 21.6, HD :21.7 g kg-1 cDM).

Looking at the pH values, it can be noticed that the maize silage had clearly lower pH values than the alfalfa-ryegrass silage and the red clover silage - the latter two silage types having a higher buffering capacity due to a higher crude protein content. In alfalfa-ryegrass silage, inoculation with L. buchneri at both low and high dosage (LD: 4.58, HD: 4.59) resulted in a significantly higher pH compared to untreated control silage (4.54). The same pattern was observed in red clover silage (C: 4.36, LD: 4.46, HD: 4.47). In maize silage, the lowest pH values were observed after inoculation with high dosage of L. buchneri (3.74), which resulted in a significantly lower pH than after inoculation with low dosage of L. buchneri (3.77); the untreated control silage had an intermediate pH (3.77). It should be noted that in absolute value the differences between treatments were low for all three silage types.

The alfalfa-ryegrass silage inoculated with L. buchneri at low dosage and at high dosage had a significantly lower lactic acid content (LD: 41.2, HD: 40.8 g kg-1 cDM) and a significantly higher acetic acid content (LD: 30.2, HD: 31.1 g kg-1 cDM) than untreated control silage (lactic acid: 48.3 - acetic acid: 18.5 g kg-1 cDM). Butyric acid was only detected in untreated alfalfa-ryegrass silage; not in the red clover and maize silages. High dosage of L. buchneri in red clover silage resulted in a significantly lower lactic acid content (50.9 g kg-1 cDM) and significantly higher acetic acid content ( 34.1 g kg-1 cDM) compared to no inoculation (lactic acid: 64.9, acetic acid: 22.1 g kg-1 cDM). Red clover silage inoculated with low dosage of L. buchneri contained intermediate levels of lactic acid (56.0 g kg-1 cDM) and acetic acid (30.7 g kg-1 cDM). In maize silage, significantly higher lactic acid levels were observed after inoculation with L. buchneri (LD: 45.0, HD: 45.4 g kg-1 cDM). Also acetic acid level was increased significantly after inoculation with high dosage of L. buchneri (21.3 g kg-1 cDM) compared to untreated maize silage (15.9 g kg-1 cDM).

Microbial counts and aerobic stabilityThe results of the microbial counts at silo opening and of the aerobic stability determination are presented in Ta-ble 4 (five observations per treatment for each silage type).

In all three silage types, yeast counts in untreated silage at silo opening were slightly higher compared to inocu-lated silage, but the differences were not significant.

No significant differences in mould counts between treatments were observed in red clover silage and maize si-lage, but L. buchneri inoculated alfalfa-ryegrass silage contained significantly less mould spores (LD: 2.03, HD: 2.21 log cfu g-1 FM) than untreated silage (3.25 log cfu g-1 FM).


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Table 4. Effect of Lactobacillus buchneri inoculation on microbial counts and aerobic stability of alfalfa-ryegrass, red clover and maize silage



Yeasts (log cfu g-1 FM)

C 2.03 0.15 n.s.

2.58 0.57 3.10 1.17

LD 1.96 0.02 2.06 0.23 n.s. 2.72 1.72 n.s.

HD 1.95 0.00 2.26 0.69 2.31 0.51

Moulds (log cfu g-1 FM)

C 3.25 1.00 a

*

2.21 0.58 n.s. 1.95 0.00

LD 2.03 0.15 b 2.27 0.66 2.17 0.49 n.s.

HD 2.21 0.22 b 1.97 0.03 2.21 0.57

Lactic acid bacteria (log cfu g-1 FM)

C 6.93 0.28 n.s.

6.78 0.62 a ***

6.59 0.35 a

LD 7.17 0.24 8.21 0.45 b 7.36 0.24 b ***

HD 7.24 0.28 7.90 0.31 b 7.41 0.22 b

Aerobic stability (hours)

C 145 97.2 a

***

296 22.2 a

***

85.9 15.6 a

LD 3601 0.0 b 3601 0.0 b 127 28.7 b *

HD 3601 0.0 b 3601 0.0 b 115 16.0 abSD: standard deviation, sign.: significance, n.s.: not significant, ***: p < 0.001, *: p < 0.05 C: control LD: low dosage of L. buchneri (1×105 cfu g-1 of fresh matter) HD: high dosage of L. buchneri (3×105 cfu g-1 of fresh matter) cfu: colony-forming units FM: fresh matter 1 no heating within 15 days after desiling: aerobic stability of 360 hours as fixed value

Inoculation with L. buchneri at ensiling elevated the lactic acid bacteria numbers, with the differences between inoculated treatments and untreated control being significant for red clover silage (C: 6.78, LD: 8.21, HD : 7.90 log cfu g-1 FM) and maize silage (C: 6.59, LD: 7.36, HD: 7.41 log cfu g-1 FM).

In alfalfa-ryegrass silage (C: 145, LD: 360, HD: 360 h) and red clover silage (C: 296, LD: 360, HD: 360 h), inoculation with L. buchneri significantly increased the aerobic stability at both dosages. In these two silage types, inoculat-ed silage did not heat up to 3 °C above surrounding temperature within 15 days of aerobic stability testing. The aerobic stability of maize silage was clearly lower compared to alfalfa-ryegrass silage and red clover silage. The untreated maize silage (85.9 h) was significantly less stable than the maize silage inoculated with low dosage of L. buchneri (127 h). High dosage of L. buchneri resulted in maize silage with intermediate aerobic stability (115 h).

Discussion

Lactobacillus buchneri belongs to the heterofermentative lactic acid bacteria and thus exerts the HeLAB metabolism: hexose and pentose sugars are converted into lactic acid, acetic acid, carbon dioxide, etha-nol etc. (Kandler 1983, McDonald et al. 1991). HeLAB are less efficient in producing lactic acid than HoL-AB, usually resulting in higher fermentation losses after HeLAB inoculation (Driehuis et al. 1999, Filya et al. 2006). This was confirmed statistically in the trials with alfalfa-ryegrass silage and red clover silage, and the same trend could be remarked for the maize silage. Aerobic stress was provided to all microsilos at 71–72 days after ensiling. Ingression of air caused secondary fermentations, which further increased the fermentation losses (Woolford 1990).

The expected effects of HeLAB inoculation on the fermentation characteristics of silage are higher DM losses during the ensiling period, production of less lactic acid and more acetic acid, higher pH and higher ethanol com-pared to untreated silage (Driehuis et al. 2001, McDonald et al. 1991, Filya et al. 2006, Oude Elferink et al. 2006). Since L. buchneri is a slow-growing bacterial species, the pH might drop slowly in L. buchneri inoculated silage, allowing enterobacteria more time for protein breakdown, possibly resulting in elevated ammonia levels and higher ammonia nitrogen fraction (Driehuis et al. 2001). The elevated acetic acid levels slow down the first phase of aerobic deterioration by inhibiting yeasts and thus increase aerobic stability. Lower yeast counts are expected after inoculation with HeLAB, which might reduce also mould development in the second phase of aerobic spoil-age (Moon 1983, Lindgren et al. 1985, Driehuis et al. 1999). Many of these expectations were confirmed statisti-cally in the ensiling trials that were performed.


134

The corrected dry matter content of the silage was significantly higher in untreated red clover silage than in red clover silage inoculated with high dosage of L. buchneri. In maize silage, inoculation with L. buchneri resulted in significantly lower dry matter contents both at low and high dosage. It should however be noted that these dif-ferences in mean dry matter content are relatively small, with high standard deviations. Ammonia contents were systematically higher in L. buchneri inoculated silage for all three silage types, but this was confirmed statistically only in the alfalfa-ryegrass silage. Ammonia nitrogen fraction followed the same trend as ammonia content, being significantly higher after inoculation with L. buchneri for alfalfa-ryegrass silage at both low and high dosage and significantly lower for untreated maize silage compared to maize silage inoculated with high dosage of L. buch-neri. The alfalfa-ryegrass silage and the red clover silage had a high ammonia nitrogen content, reflecting a mod-erate to poor silage conservation quality. The maize silage on the other hand was conserved well based on its low ammonia nitrogen fraction (Wilkinson 2005). These observations are in accordance with the expectations based on the chemical composition of the raw material. On the basis of the water-soluble carbohydrate content, the al-falfa-ryegrass mixture was considered to be difficult to ensile, while the red clover silage and maize silage should be moderately easy to ensile. Moreover, the protein content of red clover silage was much higher compared to maize silage, which should result in a higher buffering capacity. The higher conservation quality of maize silage compared to the other two silage types was thus expected. Ethanol content of untreated silage was systematically lower than ethanol content of L. buchneri inoculated silage; this difference was significant for alfalfa-ryegrass silage.

It should be noted that for all three silage types the pH values did not differ much in absolute value and the pH values reflected a good silage conservation (Wilkinson, 2005). Inoculation with L. buchneri, either at low or high dosage, resulted in significantly higher pH values in alfalfa-ryegrass silage and red clover silage. The significantly higher pH values after inoculation in alfalfa-ryegrass silage and red clover silage can be attributed to lower lac-tic acid (pKa 3.85) contents in combination with higher acetic acid (pKa 4.75) levels of inoculated silage compared to untreated control silage. Contradictory to the expectations, these trends were not observed likewise in maize silage: L. buchneri inoculated silage did contain more acetic acid than untreated control silage, but also more lac-tic acid. This could be attributed to the characteristics of the epiphytic lactic acid bacteria, which were not deter-mined. Only inoculation of maize silage with high dosage of L. buchneri resulted in significantly higher acetic acid levels compared to untreated control. The lowest pH was observed in maize silage inoculated with high dosage of L. buchneri, the highest pH in maize silage inoculated with low dosage of L. buchneri. Furthermore, the aerobic stability of the maize silage after inoculation with low dosage of L. buchneri was significantly higher compared to the untreated control, with the high dosage of L. buchneri having an intermediate aerobic stability. This observa-tion was quite surprising, regarding the non-significant difference in acetic acid content between maize silage in-oculated with low dosage of L. buchneri and the untreated control silage. In none of the three silage types stud-ied inoculation with L. buchneri resulted in significantly lower yeast counts, but the absolute numbers of yeasts were lower after inoculation. For red clover silage and maize silage, mould counts did not differ either between inoculated silage and control silage. Alfalfa-ryegrass silage did contain less moulds after inoculation with L. buch-neri at both dosages.

Differences between the two dosages of L. buchneri in fermentation quality and aerobic stability of the three silage types were rather limited. High dosage of L. buchneri compared to low dosage did not result in significant differ-ences in fermentation losses, pH at silo opening and aerobic stability for the alfalfa-ryegrass and red clover silage. For alfalfa-ryegrass, both L. buchneri dosages did not differ significantly in ammonia content, ammonia fraction, ethanol content, lactic acid content and acetic acid content. However for red clover silage, only high dosage of L. buchneri caused a significantly lower lactic acid and higher acetic acid level compared to untreated control silage. Driehuis et al. (2001) found in one experiment that L. buchneri type activity was higher in case of inoculation with 1×105 cfu g-1 FM compared to 3×105 cfu g-1 FM. This was attributed to augmented competitiveness of L. buchneri to-wards epiphytic lactic acid bacteria at lower DM content (approx. 255 g kg-1 FM) than at higher DM content (approx. 366 g kg-1 FM). In the three ensiling trials performed, the DM content at ensiling was 383, 371 and 351 g kg-1 FM for alfalfa-ryegrass, red clover and maize silage, respectively. These values are near the higher DM content as referred to by Driehuis et al. (2001), so it should not be surprising that only few significant differences were observed be-tween high and low dosage of L. buchneri in these ensiling trials.

Conclusion

Overall, it can be concluded that inoculation with the HeLAB L. buchneri did succeed in altering the silage fermen-tation characteristics, resulting in better aerobic stability. The effect is however less clear in maize silage, which typically has a good ensilability, than in alfalfa-ryegrass and red clover silages, which have a higher buffering ca-pacity due to a higher crude protein content.


135

Acknowledgements

All three ensiling trials were conducted by the University College Ghent as contractual research for Lallemand sas.

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AGRICULTURAL AND FOOD SCIENCEJ. Jatkauskas et al. (2013) 22: 137–144

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The effects of three silage inoculants on aerobic stability in grass, clover-grass, lucerne and maize silages

Jonas Jatkauskas1*, Vilma Vrotniakiene1, Christer Ohlsson2 and Bente Lund2

1Institute of Animal Science of Lithuanian University of Health Sciences, Baisogala, Lithuania 2Chr-Hansen, Hørsholm, Denmark

*e-mail: [email protected]

The objective of the study was to investigate the effects of homofermentative and heterofermentative lactic acid bacteria (LAB) inoculants on fermentation and aerobic stability in a variety of crops and dry matter concentrations. The experiments were conducted with lucerne, ryegrass, ryegrass-timothy, red clover-ryegrass and whole crop maize using three additives in laboratory scale conditions. Each treatment and crop was replicated five times when deter-mining the chemical composition and aerobic stability in the silage. The data were statistically analyzed as a rand-omized complete block by using the GLM procedure of SAS. Additive application reduced pH and formation of bu-tyric acid, alcohols and ammonia-N in all crops compared with the untreated silage (p < 0.05). The use of additives increased the content of lactic acid except heterofermentative LAB in maize with 276 g kg-1 DM and increased the content of acetic acid except homofermentative LAB in ryegrass-timothy and maize with 276 g kg-1 DM compared with the untreated silage (p < 0.05). It was observed that the aerobic stability of silages was improved significantly (p < 0.05) by using homofermentative and heterofermentative LAB inoculants.

Key- words: ryegrass, lucerne, red clover, maize, lactic acid bacteria, fermentation, aerobic stability

IntroductionEfficient fermentation of sugar to lactic acid and minimal proteolysis are crucial for silage preservation (Nadeau et al. 2000). It is well documented that the fermentation quality of silages can be improved by lactic acid bacteria based additives (McDonald et al. 1991, Kung et al. 2003, Filya et al. 2007). The mode of action of the additives ap-plied to herbage during silage making can include limiting respiration or proteolysis by plant enzymes, manipulat-ing fermentation, inhibiting the activity of clostridia and aerobic micro-organisms such as yeast and mould (Mc-Donald et al. 1991, Laitila et al. 2002, Rooke and Hatfield 2003, Kung et al. 2003). Driehuis et al. (1999) showed that LAB affected the activity of yeasts in two ways. Firstly during anaerobic conditions, the survival of yeasts is reduced, and secondly, during the aerobic exposure, yeast growth is reduced. Silages treated with inoculants con-taining various strains of Lactobacillus plantarum had lower yeast, moulds, ethanol, and ammonia-N concentra-tions than did untreated silages. The silage inoculated with a moderate rate of Lactobacillus buchneri (1 × 105 cfu g-1 of forage) or the strains of L. plantarum enhanced the aerobic stability of the corn silage. The silage treated with L. buchneri 1 × 106 cfu g-1 of fresh forage underwent a very extensive heterolactic fermentation that resulted in a marked enhancement in aerobic stability (Ranjit and Kung 2000). Inoculating forages at harvest with a heterofer-mentative LAB species L. buchneri has improved the aerobic stability of the silages (Weinberg et al. 1999), because

this organism converts lactic acid to acetic acid under anaerobic conditions (Oude Elferink et al. 2001). Moreo-ver, this inhibits fungi and thus preserves silages susceptible to spoilage upon exposure to air (Filya et al. 2007).

The combination of different cultures of lactic acid bacterial species as a silage inoculant may be more beneficial than using a single species alone due to the differences in growth pattern and positive interaction among bacte-ria. Recently, L. buchneri has been marketed in combination with homofermentative lactic acid bacteria, which are commonly added into silages to increase lactic acid production, rapidly drop pH, and decrease DM losses (Kung et al. 2003, Jaakkola et al. 2010). The lactic acid bacteria inoculant strains have been selected for rapid growth under wide range of temperatures and dry matter concentrations. These strains are highly competitive and pro-duce largely lactic acid, reducing pH compared to untreated silage (Muck 2012). Recent studies have shown that L. buchneri inhibits yeast and mould growth and increases aerobic stability of silages and these effects are re-tained when L. buchneri is added in combination with homofermentative lactic acid bacteria (Kleinschmitt et al. 2005). In the study of Jatkauskas and Vrotniakiene (2011) the selected lactic acid bacteria and selected lactic acid bacteria in combination with sodium benzoate or enzime xylanase were efficient in improving red clover-per-ennial ryegrass silage characteristics in terms of lower pH, ammonia-N concentrations, reduced DM losses and



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populations of yeasts and mould compared with control silages. Homofermentative LAB in combination with L. buchneri or sodium benzoate shifted fermentation towards acetate and had a great effect on the aerobic stability of slightly wilted red clover-ryegrass silage.

The objective of the present study was to investigate the efficacy of the variety of LAB strains on the fermenta-tion characteristics and aerobic stability of lucerne, ryegrass, ryegrass-timothy, red clover-ryegrass and whole crop maize silages.


Eight trials were conducted in 2009−2011 at the Institute of Animal Science of Lithuanian University of Health Sciences according to the DLG (Deutsche Landwirtschafts-Gesellshaft e.V./ internationally acknowledged German Agricultural Society) Guidelines for the testing of silage additives and to the Guidelines on the assessment of the safety and efficacy of silage additives, on a request from the Commission under Article 7(5) of Regulation (EC) No 1831/2003 (EFSA-Q-2004-088). Adopted on 20 April 2006. In each experiment comparisons were made between untreated silages and inoculated silages.

Test materialThe following LAB combinations were tested: Lactobacillus buchneri CCM 1819 (TA); Lactobacillus buchneri CCM 1819, Enterococcus faecium NCIMB 11181 and Lactobacillus plantarum DSM 16568 (TB); and Enterococcus fae-cium NCIMB 11181, Lactobacillus plantarum DSM 16568 and Lactococcus lactis DSM 11037 supplemented with sodium benzoate at 400 g-1 ton forage (TC). Lactic acid bacteria strains rather than formulations were registered as silage additives in the EU. Therefore, trade names are not provided in Table 1.

Table1. Blend of bacterial strains or/and other components that were used as test material

Inoculant Bacterial srains or/and other components Bacterial proportion, %

Actual count in product, cfu g-1

Application rate, cfu g-1 fresh forage

TA Lactobacillus buchneri CCM 1819 100 2.4 E + 11150 000–L, R, RC:R (70:30), R:T (70:30), M1, M2, M3

TBLactobacillus buchneri CCM 1819, Enterococcus faecium NCIMB 11181, Lactobacillus plantarum DSM 16568

50: 30: 20 2.8 E + 11 150 000–RC:R (50:50), M3

TC

Enterococcus faecium NCIMB 11181, Lactobacillus plantarum DSM 16568, Lactococcus lactis DSM 11037 + NaB at 400 g-1 ton forage

40: 30: 30 2.29 E + 11 150 000–R:T (70:30), RC:R (50:50), M3

NaB = sodium benzoate, cfu = colony-forming units, L = lucerne, R = ryegrass, RC:R = red clover:ryegrass, R:T = ryegrass:timothy, M1 = whole crop maize (DM 328 g kg-1), M2 = whole crop maize (DM 295 g kg-1), M3 = whole crop maize (DM 276 g kg-1)

The inoculants were supplied by Chr. Hansen A/S (Hørsholm, Denmark) as freeze-dried powders, sealed aluminium pouches clearly labelled. The test materials were stored at temperatures below 5oC until used. Opened pouches were discarded after use.

Crop material and micro-silo preparationLucerne (Medicago sativa L.) two-years-old, second cut, at early bloom stage of maturity (L), perennial ryegrass (Lolium perenne L.) 1-year-old, second cut, at midbloom stage of maturity (R), a mixture of red clover (Trifolium pretense L.) and perennial ryegrass (Lolium perenne L.) with two-years old, second cut, at early bloom stage of maturity of red clover (RC:R, 70:30 on a fresh weight basis), a mixture of perennial ryegrass (Lolium perenne L.) and timothy (Phleum pratense L.) 1-year-old, second cut, when the timothy was 50–75 % headed (R:T, 70:30 on a fresh weight basis), a mixture of red clover (Trifolium pretense L.) and perennial ryegrass (Lolium perenne L.) with two-years-old, second cut, at the early bloom stage of maturity of red clover (RC:R, 50:50 on a fresh weight ba-sis) and whole crop maize (Zea mays L.) at the dough stage of maturity (M1, 328 g kg-1 DM) and milk dough stage of maturity (M2, 295 g kg-1 DM and M3, 276 g kg-1 DM) were used in the present study. The herbages were cut


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by a mower conditioner (Kverneland Taarup) and wilted for 8–12 hours. Wilted crops were chopped by a forage harvester under farm conditions to 2–3 cm length. Whole crop maize was harvested and chopped with a maize harvester (Class Jaguar 840) to a length of <2> cm. Chopped forage was transported in a polyethylene bag to the laboratory. Laboratory experiments started within 2 h from crop preparation. Three silage inoculants were used in the experiments.

The application rates of the inoculants were in accordance with the level of LAB in the inoculants as determined by manufacturer and in accordance with Table 1. The test materials were suspended in chlorine free H2O immedi-atly prior to application targeting in a concentration of cells of 1.5 × 108 cfu ml-1 suspension. Additionally, 400 g so-dium benzoate for TC was diluted in 1 litre of distilled water. 1.00 ml of each suspension was used per 1 kg forage (added 3 ml of chlorine-free water for uniform spraying). The same volume (4 ml g-1 of fresh forage) of chlorine-free water was used instead of the suspension in the control treatment (for spontaneous fermentation). Subse-quently, the additives and water were sprayed into the fresh forage using a spray bottle and the forage was thor-oughly mixed. The number of viable bacteria in each suspension was counted on DeMan-Rogosa-Sharpe (MRS) agar after incubation anaerobically at 37 °C for 48 h. (ISO 15214, Leuschner et al. 2003).

The 3-litre mini-silos (glass jars) were filled with chopped lucerne (L), ryegrass (R), red clover:ryegrass (RC:R, 70:30), ryegrass-timothy (R:T, 70:30), red clover-ryegrass (RC:R, 50:50), and whole crop maize (M1, M2 and M3). Each treatment and crop was replicated five times. The density of forage in the silage was in compliance with DLG rec-ommendations, 1 kg DM per 5 litre volume. The silos were closed immediately with caps, with a potential to vent gas, 30 min after being filled. Ensiling lasted for 90 days at a constant temperature of 20 °C. At the end of the en-siling period, the silages were subjected to chemical analysis and to aerobic stability test.

Sampling and chemical analysisBefore ensiling five representative samples (> 500 g each) of each fresh chopped forage were collected for the subsequent chemical analysis. At the sampling time of silages on day 90 of the ensiling period, five micro-silos per treatment were weighed for determination of DM loss and subsequently opened and sampled to analyze the DM content, pH, fermentation products and ammonia-N. The DM content of forage and silage was determined by oven-drying at 105 °C for 24 h. For the analysis of the chemical composition of herbage, the samples were oven-dried (1 h at 102 °C and 48 h at 50 °C) and then ground to pass a 1-mm sieve. Silage DM content was corrected for volatile alcohols and fatty acids during oven drying as described by Weissbach (2009). The total nitrogen was determined by Kjeldahl-AOAC 984.13 (AOAC 1990). Crude protein (CP) content was calculated by multiplying the total nitrogen content by a factor of 6.25. The NDF and ADF concentrations were determined according to Van Soest et al. (1991) by using an Ankom200 fiber analyzer (“Ankom Technology”, USA). Water soluble carbohydrates (WSC) were determined using the anthrone reaction assay from the herbage or silage extracts obtained from steep-ing fresh herbage or silage in water (Faithfull 2002). Ash concentration was determined by ashing the samples in a furnace at 600 °C for 15 h. Buffering capacity of the forage was determined according to Playne and McDonald (1966), expressed as mEq of alkali required to change the pH from 4 to 6 per 1 kg of DM. Lactic acid, volatile fatty acid, alcohol, and ammonia N concentrations and pH were determined in silage extracts, prepared by adding 270 g of demineralized, deionized water to 30 g of silage and homogenizing for 5 min in a laboratory blender. Lactic acid, volatile fatty acids and alcohol concentrations were determined by gas-liquid chromatography. Gas-liquid chromatograph “GC-2010 Shimadzu” with capillary column (Stabilwax®-DA 30m, 0.53mm, ID, 0.5µm) was used according to Gas Chromatography and Biochemistry Analyzer official methods. Ammonia-N concentration was determined by direct distillation using the “Kjeltec Auto System 1030” (AOAC 1990 941.04). The pH of silage was measured by using “Thermo Orion Posi-pHlo SympHony” electrode and “Thermo Orion 410” meter. Dry matter losses were estimated by measuring differences in silo weights after ensiling (on day 0 after ensiling) and at the end of the ensiling period (on day 90 after ensiling).

Aerobic stability measurementAfter opening the micro-silos, all silages were subjected to a 19-day aerobic stability test. A 1000 ± 10 g sample from each silo (five silos from each treatment) was loosely placed into a polystyrene box according to recommen-dations from DLG and allowed to aerobically deteriorate at constant room temperature (~ 20 °C). The top and bottom of the boxes contained a 2-cm-diameter hole to allow air to enter and CO2 to leave. A transducer was placed in the centre of the silage mass through a hole in the cover of the box, which exposed the silage to air. These silages were not disturbed during the period of recording the temperatures. Ambient temperature and the temperature of each silage was recorded every 6 h by a data logger. Ambient room temperature was measured


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by using an empty control box. Aerobic stability of silages was examined by calculating the differences between silage temperature and ambient room temperature adjusted for base ambient temperature. Aerobic stability was defined as the number of hours the silage remained stable before rising more than 3 °C above the ambient tem-perature (Moran et al. 1996).

Statistical analysesSilage composition data and aerobic stability data (hours for a 3 oC increase in temperature) for each herbage type separately were analyzed using Proc GLM of SAS (Statistical Analysis System SAS®, version 8.02, 2000) and LSD tests were used to indicate significant differences between untreated and additive treatment. Significance was declared at p < 0.05.

ResultsCharacteristics of the forages

The data on the composition of the herbages at the time of silo filling are presented in Table 2. The lucerne culti-var was wilted up to 328 g kg-1 and had a very low WSC concentration and high crude protein concentration and buffering capacity. Consequently, L was characterised as difficult to ensile. The ryegrass was wilted up to 308 g kg-1 DM concentration . The concentrations of WSC (85 g kg-1 DM) and crude protein (153 g kg-1 DM) were medium. The buffering capacity was typical for R herbage. The red clover: ryegrass sward (70:30) was wilted up to 317 g kg-1 of DM concentration. The concentration of WSC was medium (92 g kg-1 DM) and crude protein concentration was high (199 g kg-1 DM). A mixture of ryegrass:timothy (70:30) was wilted to 265 g kg-1 DM concentration. The concentrations of WSC (102 g kg-1 DM) and crude protein (172 g kg-1 DM) were medium. The buffering capacity was typical for R:T herbage.

Table 2. Chemical composition (g kg-1 DM unless otherwise stated) and microbial parameters of herbage prior to ensiling. Means of five samples.

Variable L R RC:R (70:30) R:T (70:30) RC:R

(50:50) M1 M2 M3

Dry matter (g kg-1) 328 308 317 265 266 276 295 328Crude protein 229 153 199 172 174 89 90 89Neutral detergent fibre 414 484 363 493 361 466 443 447WSC 49 85 92 102 89 99 95 110Crude ash 84 79 65 87 99 50 52 47BC, mE kg-1 DM 564 242 328 355 365 217 152 228pH 6.4 6.5 5.9 6.3 6.6 5.7 5.6 5.7Nitrate, mg kg-1 DM 840 146 187 153 243 841 436 746Clostridia, log cfu g-1 <0.1 <0.1 <0.1 <1.0 <0.1 <1.0 <0.1 <0.1Yeasts, log cfu g-1 3.2 3.5 3.3 3.8 5.1 4.3 4.5 5.6Moulds, log cfu g-1 3.7 4.4 4.1 4.9 5.5 4.6 4.8 6.0

WSC = Water soluble carbohydrates, BC = Buffering capacity, L = lucerne, R = ryegrass, RC:R = red clover:ryegrass, R:T = ryegrass:timothy, M1 = whole crop maize (DM 328 g kg-1), M2 = whole crop maize (DM 295 g kg-1), M3 = whole crop maize (DM 276 g kg-1)

The red clover:ryegrass (RC:R) sward (50:50) was wilted up to 265 g kg-1 DM concentration. The concentration of WSC was medium (89 g kg-1 DM) and that of crude protein was high (172 g kg-1 DM). Whole crop maize (M1, M2 and M3) contained medium DM concentration (276–328 g kg-1), WSC concentration was medium (95–110 g kg-1 DM) and crude protein low (89–90 g kg-1 DM). The buffering capacity was typical of whole crop maize herbage. Consequently, R, RC:R, R:T, M1, M2 and M3 forages were characterized as moderately difficult to easy to ensile.

Silage fermentation and aerobic stabilityChemical composition, microbial parameters and aerobic stability of the untreated and treated with TA, TB, and TC additives grass and clover-grass silages are presented in Table 3.

Addition of inoculants TA, TB and TC lowered (p <0.05) the pH, increased (p <0.05) lactic acid and acetic acid for-mation (except R:T (70:30) inoculated with TC) and decreased (p <0.05) dry matter losses in R , RC:R (70:30), R:T (70:30) and RC:R (50:50) silages compared to the untreated control. Lactic acid was the predominant fermentation


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product detected in R:T (70:30) and RC:R (50:50) silages inoculated with TC. Marked increases (p <0.05) in lactic acid contents in TC- inoculated R:T (70:30) and RC:R (50:50) resulted in the highest lactic to acetic acid ratios in comparison with the other treatments. Ammonia-N, butyric acid and ethanol concentrations were decreased (p <0.05) in all inoculated silages compared with the corresponding untreated control.

Aerobic stability expressed as the number of hours the silage remained stable before temperature rising more than 3 °C above the ambient temperature was significantly increased (p <0.05) in the R, RC:R (70:30), R:T (70:30) and RC:R (50:50) silages inoculated with TA, TB and TC as compared with corresponding UT silages.

The number of yeasts and moulds were significantly lower (p <0.05) in TA treated R and R:T (70:30) silages, and numerically lower in TC inoculated R:T (70:30) silages as compared with UT silages.

Chemical composition, microbial parameters and aerobic stability of the untreated and treated with TA, TB, and TC additives lucerne and maize silages are presented in Table 4.

Addition of inoculants TA, TB and TC lowered (p <0.05) the pH, increased (p <0.05) lactic acid concentration (ex-cept M3 treated with TB), increased (p <0.05) acetic acid formation (except M2 treated with TA) and decreased (p <0.05) dry matter losses in L, M1,M2 and M3 silages compared with the corresponding untreated control (UT).

Table 3. Chemical composition (g kg-1 DM unless otherwise stated) and aerobic stability of the grass and clover-grass silages

Item R RC:R (70:30) R:T (70:30) RC:R (50:50)UT TA SEM UT TA SEM UT TA TC SEM UT TB TC SEM

Dry matter, g kg-1 292 301* 1.8 294 299* 1.4 250 252 253 0.9 239 250* 252* 1.8Dry matter loss, % 7.0 4.1* 0.53 10.2 6.7* 0.57 6.3 5.8 4.9* 0.21 12.3 6.7* 5.7* 0.83pH 5.1 4.3* 0.13 4.7 4.3* 0.07 4.6 4.2* 3.9* 0.07 5.6 4.7* 4.4* 0.134NH3 N, g kg-1 N 59 45* 2.3 57 40* 3.7 60 51* 44* 2.0 92 52* 52* 5.7

Lactic acid 23 40* 3.3 27 32* 2.1 31 34 72* 5.1 14 38* 57* 5.2Acetic acid 22 35* 2.3 29 38* 2.1 28 46* 19* 3.1 18 32* 33* 2.3Butyric acid 4.7 0.4* 0.87 6.5 0.3* 1.34 2.5 1.9 0.7* 0.26 37.5 1.9* 0.3* 4.63Ethanol 9.0 7.0* 0.48 11.0 7.0* 0.85 7.0 6.0* 5.0* 0.26 14.0 6.0* 4.0* 1.32AS hours 62 257* 19.6 96 168* 5.9 104 312* 182* 15.7 198 450* 370* 14.8Yeasts, log cfu g-1 2.9 1.0* 0.33 - - - 3.2 1.3* 1.5 0.26 - - - -Moulds,log cfu g-1 4.1 1.3* 0.50 - - - 3.0 1.3* 1.4 0.22 - - - -

R = ryegrass, RC:R = red clover:ryegrass, R:T = ryegrass:timothy, UT = untreated, TA = Lactobacillus buchneri CCM 1819, TC = Enterococcus faecium NCIMB 11181, Lactobacillus plantarum DSM 16568 and Lactococcus lactis DSM 11037 supplemented with sodium benzoate at 400 g-1 ton forage, SEM = standard error of mean, TB = Lactobacillus buchneri CCM 1819, Enterococcus faecium NCIMB 11181 and Lactobacillus plantarum DSM 16568, * = denotes significant compared to untreated control at level 0.05.

Table 4. Chemical composition (g kg-1 DM unless otherwise stated) and aerobic stability of the lucerne and maize silages

Item L M 1 M 2 M 3UT TA SEM UT TA SEM UT TA SEM UT TA TB TC SEM

Dry matter, g kg-1 313 319* 3.3 312 314 0.8 282 287* 1.4 264 268 269 269* 1.3Dry matter loss, % 6.8 4.6* 0.26 7.4 5.8* 0.37 5.9 3.8* 0.43 5.5 3.8* 3.7* 3.6* 0.22pH 5.4 5.0* 0.04 4.0 3.7* 0.05 3.9 3.7* 0.04 3.7 3.6* 3.6* 3.6* 0.11NH3 N, g kg-1 N 102 79* 2.9 61 52* 2.3 80 57* 4.7 58 48* 46* 47* 1.9Lactic acid 17 39* 2.5 28 34* 1.5 40 50* 2.32 56 50* 56 74* 2.2Acetic acid 34 49* 2.3 26 36* 1.8 19 18 0.4 26 38* 33* 22* 1.5Butyric acid 14 1.0* 1.68 1.9 0.2* 0.37 0.5 0.2* 0.09 0.5 0.1* 0.1* 0.0* 0.04Ethanol 12 7.0* 0.77 27.1 18.4* 1.68 11.2 8.1* 0.51 10.3 7.5* 6.0* 7.0* 0.36AS, hours - - 66 142* 5.4 46 76* 3.8 61 151* 143* 145* 11.7Yeasts, log cfu g-1 - - 3.4 2.9* 0.13 2.2 1.4* 0.12 1.9 1.1 1.2 1.3 0.13Mould, log cfu g-1 - - 4.1 3.5* 0.15 1.8 1.1* 0.15 2.0 1.0 1.1 1.3 0.12

L = Lucerne, M1 = whole crop maize (DM 328 g kg-1), M2 = whole crop maize (DM 295 g kg-1), M3 = whole crop maize (DM 276 g kg-1), UT = untreated, TA = Lactobacillus buchneri CCM 1819, SEM = standard error of mean, TC = Enterococcus faecium NCIMB 11181, Lactobacillus plantarum DSM 16568 and Lactococcus lactis DSM 11037 supplemented with sodium benzoate at 400 g-1 ton forage, TB = Lactobacillus buchneri CCM 1819, Enterococcus faecium NCIMB 11181 and Lactobacillus plantarum DSM 16568, * = denotes significant compared to untreated control at level 0.05.


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Lactic acid was the predominant fermentation product detected in M3 TC inoculated silages which resulted in the highest lactic to acetic acid ratios relative to other treatments. Ammonia-N, butyric acid and ethanol concentra-tions were decreased (p <0.05) in all inoculated silages compared with the corresponding untreated control (UT).

Aerobic stability expressed as the number of hours the silage remained stable before temperature rising more than 3 °C above the ambient temperature was significantly increased (p <0.05) in the M1 and M2 TA treated silag-es and in M3 TA, TB and TC inoculated silages as compared with corresponding UT silages. The numbers of yeasts and moulds were significantly lower (p <0.05) in the M1 and M2 TA inoculated silages, and numerically lower in M3 silages inoculated with TA, TB and TC.

Discussion

The LAB strains heterolactic Lactobacillus buchneri CCM 1819, heterolactic Lactobacillus buchneri CCM 1819 in combination with homolactic Enterococcus faecium NCIMB 11181 and Lactobacillus plantarum DSM 16568, and homolactic Enterococcus faecium NCIMB 11181, Lactobacillus plantarum DSM 16568 and Lactococcus lactis DSM 11037 in combination with sodium benzoate as silage inoculants were investigated in difficult to ensile crop lu-cerne, in moderately easy to ensile crops ryegrass, red clover:ryegrass and ryegrass:timothy, and in easy to ensile crop whole crop maize.

The crop material used in this experiment had a low dry matter concentration and in this respect the raw mate-rial was demanding for the inoculants. It was expected that LAB will increase the fermentation rate, causing the pH to decline lower. By using heterolactic LAB strain or heterolactic LAB strain in combination with homolactic LAB strains products of fermentation are increased, resulting in more lactic acid and more acetic acid. It was ob-served that using homolactic LAB strains in combination with sodium benzoate increased lactate concentration, but decreased (except RC:R 50:50) acetate concentration. In the studies in which L.buchneri was used as an in-oculant for silage fermentation, an anaerobic degradation of moderate amounts of lactic acid to acetic acid un-der anaerobic conditions was observed (Driehuis et al. 1999). Oude Elferink et al. (2001) concluded that the lac-tic acid conversion rate can be influenced by the temperature of the silage, the number of L. buchneri or relatives present, and the strain used.

The importance of a rapid drop in pH and increasing of lactic acid for silage quality is apparent when the UT and LAB-inoculated silages are compared. Inoculation reduced ammonia-N production compared with corresponding untreated control. Multiplication of clostridia and enterobacteria, which are responsible for protein degradation resulting in the formation of ammonia, stops at pH 4.5 (Pahlow et al. 2003). The positive effects of LAB inoculants on nitrogen fractions can be explained either by domination of the fermentation resulting in the rapid achieve-ment of a low pH or via the low proteolytic activity of the strains (Winters et al. 2000). In the present study the use of inoculants reduced fermentation losses by 2.7% units (variation from 0.5 to 6.6% units) compared to the corresponding UT silages. Filya et al. (2007) and Wyss and Rubenshuh (2012) concluded that main effect of silage inoculants was the increased production of lactic acid which in connection with significant reduction of pH value, improved the silage quality and minimised dry matter losses.

All the additives improved the fermentation quality compared with the corresponding UT silages, especially in terms of the ammonia-N, which was 9–40 g kg-1 N lower in the inoculated than in the corresponding UT silages. Re-garding the used LAB inoculants, the results agree with the laboratory scale experiments (Saarisalo et al. 2006a) in which inoculation resulted in a drop in pH, increased lactic acid production and restricted ammonia-N production.

The results of the present study indicate clearly that heterolactic L. buchneri CCM 1819 or heterolactic L. buch-neri CCM 1819 in combination with homolactic Enterococcus faecium NCIMB 11181 and Lactobacillus plantarum DSM 16568 improved the aerobic stability of silages. The explanation for the aerobic stability increasing effect of L. buchneri can be that the activity of yeasts is impaired. Yeasts are generally the initiators of aerobic deteriora-tion, consuming sugars and fermentation acids and raising silage temperature (Pahlow et al. 2003). Finally, moulds complete the deterioration of the silages (Dolci et al. 2011). In the present experiment another indicator showing that yeast activity was suppressed was lower ethanol concentration of the inoculated silages when compared with the corresponding UT silages. Acetic acid is a fungicidal agent and can inhibit the growth of yeasts and moulds, in response to the increasing aerobic stability of silages (Danner et al. 2003, Schmidt et al. 2009). Kleinschmitt and Kung (2006) indicated that the improved aerobic stability has been observed in different types of forages when acetic and propionic acid production in silage fermentation increased with L. buchneri inoculation. However, König


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et al. (2012) suggest that the use of a pure L. buchneri additive on forages with a low dry matter content is not ap-propriate due to the excessive fermentation and furthermore, high acetic acid concentration is not necessary for aerobic stability improvement. The improvement of aerobic stability was observed in a RC:R (50:50), R:T (70:30) and M3 silages by using homolactic LAB strains in combination with sodium benzoate. Sodium benzoate inhib-its spore-forming bacteria, yeasts and moulds (Woolford 1975). A beneficial effect of sodium benzoate on silage quality was demonstrated by Kleinschmit et al. (2005). Jaakkola et al. (2010) observed that the combination of L. plantarum with sodium benzoate was more efficient than the combination of L. plantarum and L. buchneri. In the experiment of Saarisalo et al. (2006b) the aerobic stability of silages was increased by inoculation with L. plan-tarum and was distinctly increased by treatment with L. plantarum in combination with sodium benzoate com-pared with untreated silage. Some inoculant lactic acid bacteria strains produce anti-microbial compounds that inhibit mould growth or undesirable bacterial species like Salmonella sp., Listeria sp. and Escherichia coli (Gollop et al. 2005). The mixture of Lactobacillus plantarum and Enterococcus faecium used inhibited the development of yeast and mould populations in barley silage, both during ensiling and upon aerobic exposure and increased aerobic stability (McAlister et al. 1995).

Conclusions

In conclusion, the novel LAB strains or LAB strains in combination with sodium benzoate were effective in improv-ing fermentation quality, and especially in reducing ammonia-N formation, dry matter losses and number of yeasts and moulds in legume, grass, grass:legume or whole crop maize silages compared with the untreated control.

The aerobic stability was significantly improved compared to control in silages inoculated with L. buchneri, L. bu-chneri in combination with homolactic LAB, and homolactic LAB in combination with sodium benzoate.

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Dolci P., Tabacco, E., Cocolin L. & Borreani, G. 2011. Microbial dynamics during aerobic exposure of corn silage stored under oxy-gen barrier or polyethylene films. Journal of Applied and Environmental Microbiology 77: 7499–7507.

Driehuis, F., Oude Elferink, S. & Spoelstra, S. 1999. Anaerobic lactic acid degradation during ensilage of whole crop maize inoculat-ed with Lactobacillus buchneri inhibits yeast growth and improves aerobic stability. Journal of applied Microbiology 87: 585–594.

Faithfull, N. 2002. Methods in Agricultural Chemical Analyse. A Practical Handbook. CABI Publishing. Institute of Rural Studies, University of Wales, Aberrystwyh, UK. 206 p.

Filya, I., Muck, R. & Contreras-Govea, F. 2007. Inoculant effects on alfalfa silage: fermentation products and nutritive value. Jour-nal of Dairy Science 90: 5108–5114.

Gollop, N., Zakin, V. & Weinberg, Z. 2005. Antibacerial activity of lactic acid bacteria included in inoculants for silage and in silages treated with these inoculants. Journal of Applied Microbiology 98: 662–666.

Jaakkola, S., Saarisalo, E. & Heikkilä, T. 2010. Aerobic stability and fermentation quality of round bale silage treated with inocu-lants or propionic acid. Grassland Science in Europe 15: 503–505.

Jatkauskas, J. &Vrotniakiene, V. 2011. The effects of silage inoculants on the fermentation and aerobic stability of legume-grass silage. Žemdirbystė-Agriculture 98: 367–374.

Kleinschmitt, D., Schmidt, R. & Kung, L. 2005. The effects of variuos antifungal additives on the fermentation and aerobic stabil-ity of corn silage. Journal of Dairy Science 88: 2130–2139.

Kleinschmitt, D. & Kung, L. 2006. A meta-analysis of the effects of Lactobacillus buchneri on the fermentation and aerobic stabil-ity of corn and grass and small-grain silages. Journal of Dairy Science 89: 4005–4013.

König, W., Puhakka, L. & Jaakkola, S. 2012. The effects of lactic acid bacteria-based additives and wilting on grass silagefermenta-tion characteristics. In: Kuoppala, K. et al. (eds.) Proceedings of the 16th International Silage Conference, 2-4 July, MTT Agrifood Research Finland, University of Helsinki, Hämeenlinna, Finland. p. 378–379.

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Laitila, A., Alakomi, H.L., Raaska, L., Mattila-Sandholm, T. & Haikara, A. 2002. Antifungal activities of two Lactobacillus plantarum strains against Fusarium moulds in vitro and in malting barley. Journal of Applied Microbiology 93: 566–576.

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Wyss, U. & Rubenshuh, U. 2012. Efficacy of three different silage inoculants on the fermentation quality and aerobic stability of ryegrass ensiled with three different prewilting degrees. In: Kuoppala, K. et al. (eds.) Proceedings of the 16th International Silage Conference, 2-4 July, MTT Agrifood Research Finland, University of Helsinki, Hämeenlinna, Finland. p. 386–387.

AGRICULTURAL AND FOOD SCIENCEB. Köhler et al. (2013) 22: 145–150

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Dry matter losses of grass, lucerne and maize silages in bunker silos

Brigitte Köhler1, Michael Diepolder2, Johannes Ostertag1, Stefan Thurner3 and Hubert Spiekers1,Bavarian State Research Center for Agriculture, Vöttinger Str. 38, D-85354 Freising, Germany

1Institute for Animal Nutrition and Feed Management

2Institute for Agricultural Ecology, Organic Farming and Soil Protection3Institute for Agricultural Engineering and Animal Husbandry


An efficient feed management is important for a sustainable and economic agricultural production. One of the main points for improving the efficiency is the reduction of feed losses. In the present investigation the dry matter (DM) losses of grass, lucerne and maize silages in farm scaled bunker silos were analysed. The method of determining DM losses was the total-in versus total-out DM mass flow of the silos, including the determination of DM content and other silage parameters via manual sampling. The results taken from 48 silos showed on average for all investi-gated crops 9–12% of DM losses. Density and feed out rate showed a negative correlation to DM losses in maize si-lages. According to the applied method for determining DM losses on farm scale, a guideline of 8% can be suggested for maximum DM losses in bunker silos for grass and maize silages. The described method seems to be applicable for improving the feed management by using largely automated measurements on the harvest and feeding side.

Key words: farm scale, feed management, fodder mixer wagon, silage management, total-in vs. total-out

Introduction

Agricultural production faces worldwide challenges due to climate change, population growth and loss of agricul-tural land. Therefore, the efficiency optimisation in feed management is highly relevant for a sustainable and eco-nomic feed production (Humphreys et al. 2009). One major possibility to improve the efficiency is the reduction of feed losses in the silos. An efficient silage system for feed production should ensure a low level of losses and a high silage quality. In respect of the losses at storage, investigations indicated DM losses on average, established during ensiling and storage, of approximately 16% (Watson and Nash 1960) and 32% (Bastiman and Altman 1985), respectively. However, only 7% of energy losses during the ensiling process are supposed to be unavoidable (Zim-mer 1980). Because those results were obtained from measurements in bench scale silos, transferability of these test results to practice is questionable. Mayne and Gordon (1986) reported 6% DM losses of wilted grass silages in silos of 100 t capacity. However, aerobic deterioration and reheating as discussed by Spiekers et al. (2009) as a major source of DM losses has not been taken into account. Therefore, concerning DM losses of farm scale bun-ker silos, reliable values are still not available. For this reason, in the present study DM losses at storage for 48 si-los were examined. The silos were investigated by the total-in versus total-out procedure to obtain reliable infor-mation about acceptable dimensions of DM losses for farm scale bunker silos. The aim of the study was to define promising starting points in order to improve the efficiency in feed management and furthermore, to develop a useful tool to control DM losses in farm scale bunker silos.


The mass flow of silages was examined on one organic and two conventional farms. These farms belong to the Bavarian State Research Center for Agriculture with a total livestock of between 70 to 190 dairy cows. The long-term mean precipitation ranges from 800–1100 mm and the annual mean temperature from 7–8 °C, respective-ly. Data were collected over a period of four years from 2008 to 2011. The DM losses were determined from 26 grass, 4 lucerne and 18 maize silos. The feed sources of the farms involved included permanent grassland, maize (all farms) and further forage crops such as grass-clover mixture, lucerne or annual ryegrass on two farms. Per-manent grassland was dominated by grass species (approximately 80 % contribution to total DM), in particular by Alopecurus pratensis. Only the organic farm had a proportion of 40–50 % of grass-red clover-mixture, which is



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analysed with the grass silos. The fertilisation level ranged from 100–290 kg N ha-1 for grassland and from 150–200 kg N ha-1 for maize. On all farms five cuttings per year from permanent grassland are the usual practice. In most cases, the wilting period did not exceed 24 hours for grass and 48 hours for lucerne. Annual yields were on average 6–10 t DM ha-1 for grass, 8 t DM ha-1 for lucerne and 10–17 t DM ha-1 for maize, respectively. The good practice of ensiling on the farms was in accordance with the guidelines recommended by the German research and advisory group feed preservation (DLG 2011). At harvest time the ensiled materials were sampled and anal-ysed for their characteristics (Table 1).

All crops were harvested with a self-propelled forage harvester, cut to a theoretical chopping length of 20–50 mm for grass, grass-clover and lucerne or 4–9 mm for maize. Silage additives were used for lucerne, partly for grass, but not for maize. For lucerne, a chemical additive was applied consisting of a mixture of sodium nitrate and hexa-methylenetetramine (2 l t-1 FM). On the organic farm, molasses (30 l t-1) and homofermentative lactic acid bacteria (L. plantarum, 3 * 105 cfu t-1) were added to grass and grass-clover mixture. On one conventional farm, a mixture of L. plantarum, L. rhamnosus, P. pentosaceus, L. buchneri and L. brevis was used for grass. On the second conven-tional farm no silage additives were applied. The grass and maize silages were conserved in side walled bunker silos using silo pit foils, underlay films, silage films and protective covers and weighed down with gravel bags. The bunker silos capacities ranged from 170 m³ to 690 m³. The harvested mass per hour did not exceed the fourfold weight of vehicles used for compaction. All silos were kept closed for at least six weeks.

The total-in versus total-out procedure is defined as the method of determination for the DM losses in the farm scale bunker silos. Figure 1 shows the principle behind the measurements from harvesting to feeding. During the harvest, every wagonload was weighed on a 40 t cart scale (measurement accuracy ±10 kg). In general, four sam-ples per ha were retrieved from the harvested ensiling materials. The samples were taken from each wagonload after unloading with a random sampling of 5–10 single samples. Pooled samples were used for analysing DM con-tents and crude nutrients. Results of the crude nutrient content determinations were transferred to the respec-tive silos. The analysis of sugars was performed by NIRS. The buffering capacity reveals the quantity of lactic acid in g, which had to be added to lower the pH of 1 kg DM to 4.0.

Fig. 1. Principle of measurements used for the total-in vs. total-out method

During the period of removal (three months on average), all feedstuffs were taken out of the silos by a fodder mix-er wagon equipped with a digital weighing system. Therefore each feed portion was weighed separately. The DM content of the silages was determined weekly, using a core drill for sampling (PioneerTM -drilling jig; drilling depth 40 cm). Three samples were taken at different heights from the face of the silos. The DM samples were dried in a cabinet dryer and subsequently, corrected for volatiles (Weißbach and Kuhla 1995). The applied equations for grass/lucerne were DMcorrected (%) = 2.08 + 0.975 * DMuncorrected (%) and for maize DMcorrected (%) = 2.22 + 0.96 * DMun-

corrected (%). Silo controllings (as described by Spiekers et al. 2009) were undertaken once, when half of the silo had been removed. As parameters of the silages the fermentation quality, DM density and temperature were anal-ysed (Table 2). The mean density in the silos was calculated using three measurements (top, middle, bottom) at


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the silo face, using a special core drill (r = 4.60 cm; drilling depth approximately 20 cm). Temperatures were mea-sured at six points at a depth of 40 cm. The feed out rate from each silo was calculated from the feed removal on a weekly basis.

The determination of DM losses was calculated by subtracting the removed DM masses from the ensiled masses, summing up all single values. Spoiled materials, which were not used for feeding, were regarded as losses. Evalu-ation of all data was carried out by statistical analysis (SAS 9.2). The normal distribution was tested, correlations were calculated with the Pearson correlation coefficient and for boxplots a SAS macro program (Friendly 2005) was used. The correlations between silage density and feed out rate to DM losses were analysed.

Results and discussion

With reference to the nutrient values and fermentation coefficients, we observed a wide range of the investi-gated ensiled materials (Table 1). However, on average a good ensilability was achieved according to Weißbach et al. (1974). Nevertheless, the wide range of the material gives a better transferability of the results to practical conditions. In accordance with the predicted ensilability, good fermentation qualities could be obtained (Table 2). Major differences were found for grass silages. In 7 of 19 grass silages butyric acid was analysed and is a sign for an unfavourable ensiling process.

Table 1. Chemical composition, feeding value and fermentation coefficients of three different types of ensiled material

Grass (n=26) Maize (n=18) Lucerne (n=4)

unit mean min−max mean min−max mean min−max

DM g kg-1 DM 316 229−503 356 295−451 316 222−385

CP1 g kg-1 DM 176 137−237 72 61−79 199 152−225

ADFom2 g kg-1 DM 300 213−338 250 189−294 369 356−382

NEL MJ kg-1 DM 5.9 5.5−6.7 6.5 6.2−7.0 5.7 5.5−5.9

FC3 - 42 33−55 54 44−69 36 26−451CP: crude protein, 2ADFom: acid detergent fibre, without residual crude ash; 3FC: fermentation coefficient = DM % + 8 * (sugar/buffering capacity) according to Weißbach et al. 1974.

Table 2. Parameters for preservation quality and feed management for the three different types of silage material

Grass Maize Lucerne

Parameter unit n mean min-max n mean min-max n mean min-max

Dry matter (DM) g kg-1 22 293 223−419 16 356 286−441 4 352 268−409

Lactic acid g kg-1 DM 19 55 23−129 14 48 26−69 4 72 46−94

Acetic acid g kg-1 DM 19 22 7−50 14 17 7−38 4 30 18−50

Propionic acid1 g kg-1 DM 5 3 2−5 0 − − 2 4 2−5

Butyric acid1 g kg-1 DM 7 10 3−23 0 − − 2 8 2−14

pH -- 19 4.4 3.8−4.8 14 3.9 3.7−4.3 4 4.7 4.4−5.3

Density kg m-3 DM 22 194 155−278 16 246 215−299 4 244 203−288

Feed out rate m week-1 19 2.1 1.1−3.4 18 2.1 1.0−3.6 4 1.9 1.3−2.4

Temperature °C 22 − 3−37 16 − 2−23 4 − 5−23

1Propionic and butyric acid: n = number of analysis which exceeded the limit of detection


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By the measurement of the temperature, a reheating was determined in 2 maize and 5 grass silages in the upper layers of the evaluated silos. The wide range of the compaction quality of the silages was in agreement with find-ings of Richter et al. (2009). In some cases compaction was lower than recommended for maize silages, primarily (DLG 2011). Altogether a high feed out rate on average could be realised due to the convenient proportion be-tween silo face and feed removal per day. The general requirements for ensiling material, silage making and feed management were achieved at all farms. Hence transferability of the results to practice is justifiable.

The DM losses determined for 48 farm scale silos are presented in Figure 2. The collected data followed a normal distribution. For maize silages an average of 10% DM losses was observed, for grass silages 9% and for lucerne si-lages 12%, respectively. The average DM losses from these silage types seem to be low. This was unexpected, in particular of the grass silages, because DM losses of grass silages in laboratory scale exceeded those of maize si-lages (Thaysen et al. 2007). Grass silages, which were shown to be most heterogeneous, revealed the largest range of DM losses from –2% to 26%, followed by maize silages from –4% to 19% and lucerne silages from 6% to 15%.

-5

0

5

10

15

20

25

30

maize silage grass silage lucerne silage

n = 18 n = 26 n = 4

DM

loss

es [%

]

Silage type and number of evaluated silos (n)

Legend boxplots:

Brackets = highest + lowestvalue of the non outside observations

(max. 1.5 interquartile ranges); Box: values between upper (75 %) and lower (25 %) quartil; Bar = median; Cross = mean

With respect to other characteristics of silage quality the grass silages showed the highest variability for DM con-tents and densities. Nevertheless, for one grass silo 26% losses of DM were determined. For this silo an intense reheating could be observed, however, the appearance of butyric acid was found. The high DM losses could be explained with the occurrence of this parameters and the ascertained low feed out rate.

Further tests showed, that the feed out rate had a negative significant correlation coefficient with the DM losses for both silages (maize –0.555, grass –0.570) (Table 3). A negative correlation with the density was significant for the maize silos (–0.625). Furthermore, no correlation between the density and the DM losses of grass silos was found.

Table 3. Correlation coefficients of density and feed out rate with the dry matter losses of the maize and grass silos

DM losses of maize silos (n=18) DM losses of grass silos (n=26)

Variable n correlation n correlation

Density, kg m-3 DM 15 –0.625* 18 –0.039

Feed out rate, m week-1 22 –0.555* 19 –0.570*

*significance 0.05>p>0.01

Fig. 2. Dry matter losses (%) of silages determined by the total-in vs. total-out method


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These relations should be taken into account, because in most cases maize silages did not reach the recommend-ed density levels. However, besides density there are several other parameters causing DM losses in silos. Refer-ring to the farm scale study of Borreani and Tabacco (2012), who investigated maize silages for aerobic deteriora-tion, the influence of the recommended feed out rate was proven. Moreover, the mean feed out rate in the cited study was lower than in the present investigation. Our findings also exhibited low levels of DM losses in general. Compared to the values of other studies (McGechan 1989), it is probably attributed to the good ensiling practice and the partial high feed out rate on the participating farms.

As negative values for DM losses are impossible, they have to be ascribed to the precision of the method, which strongly depends on the accuracy of the data collection and their susceptibility to errors. Potential sources of er-ror are an inadequate distribution of control points for DM determination, as well as inaccurate weighing by the fodder mixer wagon. The correctness and frequency of DM determination is crucial, especially for the investiga-tion of the heterogeneous grass silages (Thurner et al. 2011). Therefore, progress of DM determination by the im-plementation of online NIRS systems on the fodder mixer wagon (Twickler et al. 2012) could be an improvement for the proposed method. Supported by technical measurements of the permanent process the method appears to be applicable for farms in spite of different procedures for feeding. In the present study, fifty percent of the ascertained values were below 8% of DM losses. Thus, according to the present results a maximum value of 8% unavoidable DM losses can be set as a guideline for bunker silos, independent of the crop. This method of control could be implemented by using technical measurements with justifiable effort on practical farms.

Conclusions

A method for the determination of DM losses in bunker silos has been described. The DM losses in grass silages seem to be comparable with those for maize, assuming there is a good practice of ensiling. The density and feed out rate showed a negative correlation to DM losses in maize silages. As a guideline for maximum DM losses in bunker silos, 8% emerged to be an adequate value. In spite of problems concerning data collection, the method seems to be adaptive and useful for commercial farms as a tool of control.

AcknowledgementsThis study was financed by the Bavarian State Ministry of Food, Agriculture and Forestry”, as part of the project “Efficient Feed Management and Nutrient Flows on Dairy Farms” (A/08/01).

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AGRICULTURAL AND FOOD SCIENCEM. Pesonen et al. (2013) 22: 151-167

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Effects of concentrate level and rapeseed meal supplementation on performance, carcass characteristics, meat quality and valuable cuts of Hereford and Charolais bulls offered grass silage-barley-based rations

Maiju Pesonen1, Markku Honkavaara2, Helena Kämäräinen3, Tiina Tolonen4, Mari Jaakkola4, Vesa Virtanen4 and Arto K. Huuskonen1

1MTT Agrifood Research Finland, Animal Production Research, FI-92400 Ruukki, Finland2Finnish Meat Research Institute, P.O. Box 56, FI-13101, Hämeenlinna, Finland

3University of Eastern Finland, Department of Biosciences, P.O. Box 1627, FI-70211 Kuopio, Finland4University of Oulu, Kajaani University Consortium, CEMIS-Oulu, Salmelantie 43, FI-88600 Sotkamo, Finland


The objectives of this experiment with Hereford (Hf) and Charolais (Ch) bulls offered grass silage-based diets were to determine the effects on performance, carcass traits and meat quality of the proportion of concentrate in the diet, and the inclusion of rapeseed meal (RSM) in the barley-based concentrate. The two concentrate proportions were 200 and 500 g kg-1 dry matter, fed without or with RSM. The Ch bulls tended to achieve higher gain, produced less fat, had a higher percentage of meat from high-priced joints and had a lower degree of marbling in their meat compared to the Hf bulls. Dry matter and energy intakes, growth performance and carcass conformation improved with increasing concentrate level. Intake parameters and conformation improved more with the Ch bulls than with the Hf bulls as a consequence of increased concentrate allowance. RSM had only limited effects on the perfor-mance, carcass traits or meat quality.

Key words: beef production, bulls, concentrate level, supplementary protein, performance, eating quality, meat fatty acids

Introduction

Although beef production in Finland is based mainly on raising dairy bulls, production from beef breed calves is increasing at present. In total, 12 beef breeds are currently kept, and Charolais (Ch) and Hereford (Hf) are the two most frequently used beef breeds. The decrease in the number of dairy cows has diminished the supply of calves for beef production originating from dairy herds. Because the supply of domestic beef has been decreasing, there is nowadays a clear discrepancy between the demand for and supply of domestic beef. Consequently, slaughter-house pricing favours heavy carcasses and the average carcass weights of slaughtered animals have clearly increased in Finland during recent years. For example, the average carcass weight of slaughtered bulls (including both dairy and beef breeds) increased from 275 kg (1996) to 335 kg (2008) in twelve years (Karhula and Kässi 2010). Current-ly, it is typical that bulls of British beef breeds (Hereford, Angus) are slaughtered in carcass weights near 400 kg and late maturing beef breeds (Charolais, Simmental) in carcass weights above 400 kg (Huuskonen et al. 2012).

In intensive beef production, grass silage is typically supplemented with grain to increase the energy and nutrient intake of growing bulls. Rapeseed meal (RSM) is the most important protein feed used in concentrates for cat-tle. Nowadays many beef producers use protein supplements with grass silage grain- based feedings (Huuskonen 2009a) even though the price of RSM is high compared to those of grain or forages and feeding extra protein in-creased the N and P excretion to the environment (Klopfenstein and Erickson 2002).The effects of both concen-trate level and protein supplementation on the performance of growing cattle have been extensively studied. It is well established that good quality silage can support high levels of performance with moderate concentrate supplementation. However, increasing the allowance of concentrate has often improved the growth rate and de-creased the days until slaughter (e.g. Huuskonen et al. 2007, Randby et al. 2010). With dairy bulls it was concluded that concentrate with a higher protein concentration than barley grain is not needed when the animals are fed high- or medium-digestibility and restrictively fermented grass silage and barley-based concentrate (Huuskonen et al. 2007, 2008b, Huuskonen 2009b, 2011). Relative to dairy bulls, much less research has been carried out on the feeding of beef bulls and, in fact, there is lack of information on the effects of concentrate proportions and protein supplementation on the performance, carcass traits and meat quality of beef-breed bulls offered typical Finnish grass silage-barley-based rations and slaughtered with high carcass weights.



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Meat quality aspects are receiving considerable attention among consumers. For example, meat colour is an im-portant determinant of the visual appearance of meat, with light coloured beef often being preferred, although some consumers may favour dark beef associating this appearance with a more natural production method (Razminowicz et al. 2006). Tenderness and marbling are important properties of beef for consumers and have been studied widely. Nevertheless, studies on the effects of concentrate level and protein supplementation with grass silage-barley-based rations on eating quality are scarce. In addition, the fatty acid composition of beef has received considerable attention in view of its implications for human health and for meat quality characteristics (De Smet et al. 2004). The objectives of the present experiment with growing Hf and Ch bulls were to determine the effects on animal performance, carcass characteristics, valuable cuts, meat quality parameters and fatty acid composition of the Longissimus muscle of (1) the proportion of concentrate in the diet, and (2) the inclusion of RSM in the barley-based concentrate fed in total mixed rations (TMR) when animals are slaughtered at typical Finnish carcass weights.

Materials and methodsAnimals and housing

The feeding experiment was conducted in the experimental barn of the North Ostrobothnia Research Station of MTT Agrifood Research Finland (Ruukki, 64°44’N, 25°15’E) and included three trials. The first trial started in De-cember 2008, the second in January 2010 and the third in January 2011. The experimental procedures were eval-uated and approved by the Animal Care and Use Committee of MTT Agrifood Research Finland. The three feed-ing trials comprised in total 48 purebred Hf bulls (Hf dams sired by Hf bulls) and 48 purebred Ch bulls (Ch dams sired by Ch bulls) in order that there were 32 bulls per trial. Diet in vivo digestibility, animal performance (intake and gain) and carcass characteristics (carcass weight, dressing proportion, conformation score and fat score) were determined in all three trials, the meat quality parameters and valuable cuts were measured in the second and third trial. Two bulls were excluded from the study due to several occurrences of bloat, one due to pneumonia and three bulls due to hoof problems. There was no reason to suppose that the diets had caused these problems. The records of the six removed animals were not included in the results.

All the animals, initial live weight (LW) 306 ± 97.9 kg (Hf) and 333 ± 63.1 kg (Ch), on average, were spring-born calves purchased from commercial suckler herds. During their first summer all the calves had been kept on pas-ture together with their dams. At the start of the experiment the animals were 195 ± 55.6 days old, on average, and there was no difference between breeds. During the feeding experiment the bulls were placed in an insulated barn in adjacent tie-stalls. The width of the stalls was 70–90 cm for the first four months and 113 cm until the end of the experiment. The bulls were tied with a collar around the neck, and a 50 cm long chain was attached to a horizontal bar 40–55 cm above the floor. The floor surface was solid concrete under the forelegs and metal grids under the hind legs. No bedding was used on the floor. Each bull had its own water bowl.

Feeding and experimental designThe bulls were fed a TMR ad libitum (proportionate refusals of 5%). A 2×2×2 factorial design was used to study the effects of concentrate proportion and RSM inclusion in the barley-based concentrate. Both Hf and Ch bulls were randomly allotted to the experimental feeding treatments. The two concentrate proportions were 200 (L) and 500 (M) g kg-1 DM, fed without RSM (RSM−) or with RSM (RSM+). The concentrate used was rolled barley. Rapeseed meal was given so that the crude protein (CP) content of the concentrate was raised to 160 g kg-1 DM in the RSM+ diets. Therefore the amount of RSM supplement depended on the CP content of the barley which was measured by chemical analyses. In the RSM− diets the average CP content of the concentrate was 126 g kg-1 DM, so the content increased 27% with RSM supplementation.

The grass silages in all three trials were growth from mixed timothy (Phleum pratense) and meadow fescue (Fes-tuca pratensis) stands and were cut using a mower conditioner, wilted for 5 h, and harvested using a precision-chop forage harvester. The grass silages were ensiled in bunker silos and treated with a formic acid-based additive (AIV-2 Plus: 760 g formic acid kg-1, 55 g ammonium formate kg-1, supplied by Kemira Ltd., P.O. Box 171, FI-90101 Oulu, Finland) applied at a rate of 5 litres t-1 of fresh grass. The daily ration for the bulls included also 150 g of a mineral mixture (A-Rehu Ltd., P.O. Box 908, FI-60061 Atria, Finland: KasvuApeKivennäinen: Ca 260, P 0, Na 70, Mg 35 g kg-1). A vitamin mixture (Suomen Rehu Ltd.: Xylitol ADE-Vita: A 2,000,000 IU kg-1, D3 400,000 IU kg-1, E DL-a-to-copheryl acetate 1,000 mg kg-1, E DL-a-tocopheryl 900 mg kg-1, Se 10 mg kg-1) was given at 50 g per animal weekly.


153

Feed and faecal sampling and analysis

Silage sub-samples for chemical analyses were taken twice a week, pooled over periods of four weeks and stored at –20°C. Thawed samples were analysed for DM, ash, crude protein (CP), ether extracts, neutral detergent fibre (NDF), indigestible NDF (iNDF), starch, silage fermentation quality (pH, water-soluble carbohydrates [WSC], lactic and formic acids, volatile fatty acids, soluble and ammonia N content of N) and digestible organic matter (DOM) in DM (D value). Concentrate sub-samples were collected weekly, pooled over periods of eight weeks and analysed for DM, ash, CP, ether extracts, NDF, iNDF and starch. The analyses were performed as described by Huuskonen et al. (2008a).

The metabolizable energy (ME) contents of the feeds were calculated according to the Finnish feed tables (MTT 2012). The ME value of the silage was calculated as 0.016 × D value. The ME values of the concentrates were calcu-lated based on concentrations of digestible crude fibre, CP, crude fat and nitrogen-free extract described by MAFF (1984). The digestibility coefficients of the concentrates were taken from the Finnish feed tables (MTT 2012). The supply of amino acids absorbed from the small intestine (AAT) and the protein balance in the rumen (PBV) were calculated according to the Finnish feed tables (MTT 2012).

Because the grass silages used in the feeding experiment came from three different harvests, the chemical com-positions and feeding values are also given separately for the three silages in Table 1. The silages used were of good nutritional quality as indicated by the D value as well as the AAT and CP contents (Table 1). The fermentation characteristics of the silages were also good as indicated by the pH value and the low concentration of ammonia N and total acids. The silages used were restrictively fermented with high residual WSC concentration and low lactic acid concentration. Because the chemical compositions and feeding values of the barley grain and RSM were very uniform throughout the experiment, only the mean values over the trials are given for barley and RSM in Table 1.

Table 1. Chemical composition and feeding values of barley, rapeseed meal and grass silages.

Silage trial 1 Silage trial 2 Silage trial 3 Silage mean (trials 1,

2, 3)

Barley Rapeseed meal

N a 16 13 9 38 19 19Dry matter (DM), g kg-1 feed 252 300 343 298 885 881Organic matter (OM), g kg-1 DM 937 936 918 930 975 927Crude protein, g kg-1 DM 164 128 161 151 126 341Neutral detergent fibre (NDF), g kg-1 DM 558 574 523 552 241 331Indigestible NDF, g kg-1 DM 60 51 56 56 43 133Ether extract, g kg-1 DM 39 35 38 37 16 44Starch, g kg-1 DM 14 7 8 10 524 30Metabolizable energy, MJ kg-1 DM 10.8 10.5 10.9 10.7 13.1 11.7AAT c, g kg-1 DM 85 79 84 83 101 151PBV d, g kg-1 DM 20 -1 37 19 -32 111Digestible OM in DM, g kg-1 DM 678 654 683 672 ND b NDFermentation quality of silage pH 4.06 4.04 4.56 4.22 Volatile fatty acids, g kg-1 DM 18 18 17 18 Lactic + formic acid, g kg-1 DM 53 48 30 44 Water soluble carbohydrates, g kg-1 DM 47 67 101 72 In total N, g kg-1

NH4N 69 73 65 69 Soluble N 534 540 511 528

a Number of feed samples. Silage: values of three trials are given separately. Other feeds: only mean values over the trials are given because the chemical compositions and feeding values were very uniform throughout the experiment.b Not determined.c Amino acids absorbed from small intestine.d Protein balance in the rumen.


154

Diet digestibility was determined for all animals when the bulls were 580 ± 61 kg LW, on average. Feed and fae-cal samples were collected twice a day (at 7:00 a.m. and 3:00 p.m.) during the collection period (5 d) and stored frozen prior to analyses. The samples were analyzed for DM, ash, CP and NDF as described above. The diet digest-ibility was determined using acid-insoluble ash (AIA) as an internal marker (Van Keulen and Young 1977).

Live weight, slaughter procedures and meat quality measurementsThe animals were weighed on two consecutive days at the beginning of the trials and thereafter approximate-ly every 28 days. Before slaughter they were weighed on two consecutive days. The target for average carcass weight in the experiment was 380 kg for Hf bulls and 420 kg for Ch bulls which are nowadays the average slaugh-ter weights for Hf and Ch bulls in Finland (Huuskonen et al. 2012). The animals were selected for slaughter based on LW and assumed dressing proportions (0.530 for Hf bulls and 560 for Ch bulls) which were assessed based on earlier studies (unpublished data) in Finland with beef-breed bulls. The LWG was calculated as the difference be-tween the means of initial and final live weights divided by the number of growing days. The estimated rate of carcass gain was calculated as the difference between the final carcass weight and the carcass weight in the begin-ning of the experiment divided by the number of growing days. The carcass weight at the start of the experiment was assumed to be 0.50 × initial LW, which was assessed based on earlier studies (unpublished data).

The animals were slaughtered in the Atria commercial slaughterhouse in Kuopio, 265 km from the Research Sta-tion. After slaughter the carcasses were weighed hot. The cold carcass weight was estimated as 0.98 of the hot car-cass weight. Dressing proportions were calculated from the ratio of cold carcass weight to final LW. The carcasses were classified for conformation and fatness using the EUROP quality classification (EC 2006). For conformation, the development of the carcass profiles, in particular the essential parts (round, back, shoulder), was taken into consideration according to the EUROP classification (E: excellent, U: very good, R: good, O: fair, P: poor) and for fat cover degree, the amount of fat on the outside of the carcass and in the thoracic cavity was taken into account using a classification range from 1 to 5 (1: low, 2: slight, 3: average, 4: high, 5: very high). Each level of the confor-mation scale was subdivided into three sub-classes (O+, O, O-) to produce a transformed scale ranging from 1 to 15, with 15 being the best conformation.

After classification carcasses were chilled overnight below 7 °C. Day after slaughter the right side of carcasses were commercially cutted. Primal cuts were forequarter, back, side and round. The right side of each carcass was cut into valuable cuts [outside round (Musculus semitendinosus), inside round (Musculus semimembranosus), corner round (Musculus quadriceps femoris), roast beef (Musculus gluteus medius), tenderloin (Musculus psoas major), loin (Musculus longissimus lumborum) and entrecote (Musculus longissimus thoracis)], subcutaneous fat and bones as described by Manninen et al. (2011). All cuttings, subcutaneous fat and bones were weighed and their yields were expressed as percentages of the cold carcass weight (0.98 × hot carcass weight, 50 min post mortem). Forequarter was cutted into subcutaneous fat, bones, trimmings and entrecote (Musculus longissimus thoracis between the 4th and the 7th rib). Back was cutted into fat, bones, trimmings and loin (Musculus longissimus lum-borum between the 7th rib and the 5th lumbar vertebra). Loin was cutted at the level of the 1st lumbar vertebra, and the achieved 2 kg loin sample between the 1st and the 5th lumbar vertebra was used for further analysis. The marbling score of entrecote (at the 7th rib) and loin (at the 1st lumbar vertebra) were evaluated by using a six-point scale (0=devoid to 5=abundant).

pH-value of the loin was measured with a Knick 651 instrument with Inlab Solid electrode (Mettler Toledo) at the level of the 1st lumbar vertebra. Meat color of the loin was measured after a bloom time of half an hour (Warris 1996) with a Minolta Cr-200 handheld chroma meter (Minolta Camera Co., Ltd., Osaka, Japan). The chroma me-ter had an 8 mm diameter measuring area, used diffuse illumination and 0º viewing angle geometry to provide accurate readings in a wide variety of color control applications. Before measurements Cr-200 was calibrated to a standard white plate, and CIE Standard Illuminant D65 conditions were used for the measurements. Readings were displayed in L*a*b* (L* luminance from 0 to 100; a* green to red from −60 to 60, respectively; and b* blue to yellow from −60 to 60, respectively). Each sample was measured three times and a mean value was calculated.

During cutting, a 2 kg loin sample was taken and vacuum packed. These samples were sent to the Finnish Meat Research Institute (LTK) for further analyses. Total ageing time of samples was 8 days at 4 °C. Thereafter sam-ples were analysed for drip loss, moisture, protein and fat concentrations, Warner-Bratzler shear force and for tenderness, juiciness and beef flavour (sensory analysis). Drip loss was determined by the amount of water loss from the 2 kg loin sample after ageing. Moisture, protein and fat concentrations were determined as described by Huuskonen et al. (2010). For shear force measurements, loin samples were heated in a water bath at 85 °C


155

until the core temperature of the meat was 70°C. After chilling for 24 hours (4 °C), loin samples about 6 cm long (parallel to the myofibres), 1 cm high and 1 cm wide (square probe of 1 cm × 1 cm surface area) were placed in a Warner-Bratzler shear blade to be sheared perpendicular to the longitudinal axis of the muscle fibres in an In-stron testing machine. The maximum force was recorded and results were expressed as kg (cm-2)-1 (Honkavaara et al. 2003) because the sheared meat sample had a height of 1.0 cm and a width of 1.0 cm and a length of 6.0 cm. Thus the shear force is expressed as kg per sheared surface area of 1.0 cm2.

For the sensory analysis, surface fat was removed and trimmed loin was cut into four slices with thickness of 1.5 cm. After that these four samples were heated simultaneously up to internal temperature of 68 °C in a rolling grill (Palux Rotimat, Germany). Heated samples were served immediately in a sensory panel room with white light-ning and temperature of 24 °C. Six trained sensory panelists evaluated the samples for tenderness, juiciness and beef flavour. These traits were scored on a seven-point scale (1 = very tough/very dry/very non beef like,…, 7 = very/tender/very juicy/very beef like).

Fatty acids were extracted from loin samples according to a slightly modified AOAC standard method (AOAC 2002) and methylated to corresponding fatty acid methyl esters (FAMEs) in hexane with 2M sodium hydroxide and 1M hydrochloride acid in methanol. FAMEs were analyzed using a gas chromatograph (Agilent 6850 Series) equipped with flame ionization detector by a previously published method (Jaakkola et al. 2012) with modified tempera-ture program: the temperature was increased 25°C min-1 from 35°C to 190°C, and then by 3°C min-1 to 205°C and then to 220°C with 8°C min-1, and finally held there for 22 min. FAMEs were identified by comparing samples with fatty acid standards GLC 461, UC-60M, U-48M, U-69M, U-99M, U-101M and U-84M (Nu-chek Prep Inc., Elysian, MN, USA). Methyl stearate (Sigma-Aldrich) was used for quantification purposes.

Statistical methodsThe results were analysed across all three trials (results of meat quality and valuable cuts across two trials) and are shown as least squares means. The normality of analysed variables was checked using graphical methods: box-plot and scatter plot of residuals and fitted values. The data were subjected to analysis of variance using the SAS MIXED procedure (version 9.1, SAS Institute Inc., Cary, NC). The statistical model used was

yijklm = µ + δl + αi + βj + γk + (α×β)ij + (α×γ)ik + (β×γ)jk + (α×β×γ)ijk + (δ×α×β×γ)lijk + eijklm

where μ is intercept and eijklm is the random error term associated with mth animal. αi, βj and γk are the fixed ef-fects of ith breed (Hf, Ch), jth concentrate level (200, 500) and kth RSM supplementation (RSM−, RSM+), respectively. δl is random effect of lth trial (l=1,2,3). (δ×α×β×γ)lijk is random effect of trial-by-treatment which is used as an er-ror term when differences between treatments (=breed, concentrate level, RSM supplementation) were tested.

Results

The average chemical compositions of the TMR used are presented in Table 2. Because of the higher energy and AAT contents of the concentrate, increasing the concentrate proportion increased the calculated energy and AAT values of the rations. Increasing the proportion of the concentrate also increased the starch content, but decreased the NDF content of the rations. The CP content of the L and M rations increased 8 and 12% with RSM supplemen-tation, respectively (Table 2).

Diet digestibility, feed intake and growth performanceSignificant, but numerically small, breed × concentrate level × RSM supplementation three-way interactions were observed for the DM, OM and NDF digestibilities (Table 3). Other interactions for digestibility variables between breed, concentrate level and RSM supplementation were not observed. Breed had no effects (p>0.05) on the diet digestibility coefficients (Table 3). Increasing the concentrate proportion led to improved DM (p<0.001), OM (p<0.001) and CP (p<0.01) apparent digestibilities. The digestibility of NDF decreased 3% with increasing concen-trate proportion (p<0.001). Rapeseed meal supplementation had no effect on the DM, OM and NDF digestibilities, but the CP digestibility was 7% higher for the RSM+ diets than for the RSM−diets (p<0.001).


156

Table 2. Chemical compositions and nutritional values of total mixed rations used (mean values of three trials).

Concentrate proportion, g kg-1 dry matter (DM) a L (200) L (200) M (500) M (500)Rapeseed meal supplementation - + - +DM, g kg-1 feed 344 344 446 446Organic matter, g kg-1 DM 939 937 953 949Crude protein, g kg-1 DM 146 157 139 155Neutral detergent fibre (NDF), g kg-1 DM 490 494 397 403Ether extract, g kg-1 DM 33 34 27 28Starch, g kg-1 DM 113 88 267 230Metabolizable energy, MJ kg-1 DM 11.2 11.1 11.9 11.8Amino acids absorbed from small intestine, g kg-1 DM 87 89 92 96Protein balance in the rumen, g kg-1 DM 9 18 -7 7a L = low concentrate proportion (200 g kg-1 DM); M = medium concentrate proportion (500 g kg-1 DM).

There were no differences in average DM, energy and CP intakes between Hf and Ch bulls (Table 3). Instead, in-creasing the level of concentrate led to higher DM (p<0.001), energy (p<0.001) and CP (p<0.01) intakes by the bulls whereas the supply of NDF decreased (p<0.001) with increasing concentrate level. There were also interac-tions (p<0.05) between the breed and the concentrate level for DM, energy and CP intakes. Intake increased more with the Ch bulls than with the Hf bulls as a consequence of increased concentrate level. The average supply of CP (p<0.001), AAT (p<0.05) and PBV (p<0.001) were higher when RSM was included in the diet, but RSM supple-mentation had no effect on the average DM or energy intake.

There were no significant interactions for live weight or gain variables between breed, concentrate level and RSM supplementation (Table 3). The mean final LW of the Hf and Ch bulls were 726 and 754 kg, respectively. The live weight gain and carcass gain of the Ch bulls were 10 and 22% higher than those of the Hf bulls, respectively (p<0.001). Increasing the proportion of concentrate led to an improvement of daily LWG and carcass gain of the bulls (p<0.001). The RSM supplementation had no effect on growth performance, but LWG and carcass gain of the bulls tended to be 5% lower on RSM− diets than on RSM+ diets (p=0.08).

There were no interactions for feed conversion variables between breed, concentrate level and RSM supplemen-tation. Feed conversion (kg DM kg-1 carcass gain) and energy conversion rates (MJ ME kg-1 carcass gain) improved 14 and 7%, respectively, with increasing concentrate proportion (p<0.001 and p<0.01, respectively). Both feed and energy conversion rates were poorer with Hf than with Ch bulls (13.5 vs. 11.3 kg DM kg-1 carcass gain and 155 vs. 131 MJ kg-1 carcass gain, respectively, p<0.001). The RSM supplementation had no effect on feed or energy con-version, but both variables tended to be 5% poorer on RSM-diets than on RSM+ diets (p<0.1).

Carcass characteristics and valuable cutsThere were no interactions for carcass weight or dressing proportion between breed, concentrate level and RSM supplementation. The mean carcass weights of the Hf and Ch bulls were 386 and 426 kg, respectively, and close to the pre-planned carcass weight (Table 3). The carcass weight of the M bulls was 5% higher than that of the L bulls (396 vs. 416 kg, p<0.01). The RSM supplementation had no effect on carcass weight. The dressing propor-tion of the Ch bulls was 6 % higher than that of the Hf bulls (531 vs. 562 g kg-1, p<0.001). The dressing proportion of the M bulls was 1% higher than that of the L bulls (543 vs. 550 g kg-1, p<0.05), but the RSM supplementation had no effect on dressing proportion.

The carcass conformation score of the Ch bulls was 32% higher than that of the Hf bulls (6.5 vs. 8.6, p<0.001) and the conformation of the M bulls was 17% higher than that of the L bulls (6.9 vs. 8.1, p<0.001). There was also an interaction (p<0.01) between the breed and the concentrate level for carcass conformation. The conformation score of the Ch bulls improved more than that of the Hf bulls as a consequence of increased concentrate level (Table 3). The RSM supplementation had no effect on carcass conformation score. Carcass fat score of the Hf bulls was 55% higher than that of the Ch bulls (4.5 vs. 2.9, p<0.001). The concentrate level had no effect on carcass fat score, but it tended to be 7% higher on M diets than on L diets (3.8 vs. 3.6, p=0.06). The RSM supplementation had no effect on carcass fat score, and there were no interactions for carcass fat score between breed, concen-trate level and RSM supplementation (Table 3).


157

Tabl

e 3.

Effe

cts

of b

reed

(B),

conc

entr

ate

leve

l (C)

and

rape

seed

mea

l sup

plem

enta

tion

(RSM

) on

daily

dry

mat

ter (

DM) i

ntak

e, a

ppar

ent d

iet d

iges

tibili

ty, g

row

th p

erfo

rman

ce a

nd c

arca

ss c

hara

cter

istic

s of

gr

owin

g bu

lls.

Bree

d (B

) aHF

CHSE

Mb

Conc

entr

ate

leve

l (C)

cL

(200

)M

(500

)L

(200

)M

(500

)p-

valu

eRS

M su

pple

men

tatio

n (R

SM)

-+

-+

-+

-+

BC

RSM

B×C

B×RS

MC×

RSM

B×C×

RSM

Num

ber o

f ani

mal

s11

1212

1111

1211

10Du

ratio

n of

the

expe

rimen

t, d

356

355

310

327

336

342

306

299

19.2

0.03

0.00

10.

640.

990.

750.

260.

75In

take

DM

inta

ke, k

g d-1

8.91

9.07

9.31

9.76

8.94

8.61

10.0

410

.01

0.25

80.

21<0

.001

0.59

0.03

0.13

0.76

0.80

DM

inta

ke, g

kg-1

W0.

7582

.883

.686

.788

.981

.378

.188

.887

.41.

880.

16<0

.001

0.88

0.11

0.11

0.56

0.97

Met

abol

izabl

e en

ergy

, MJ d

-110

110

211

111

710

196

120

119

3.1

0.36

<0.0

010.

770.

020.

120.

780.

66 C

rude

pro

tein

, g d

-112

9214

1112

8214

4712

8913

4013

9014

9140

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340.

002

<0.0

010.

010.

130.

950.

78 A

AT d , g

d-1

775

811

856

923

772

765

925

945

24.0

0.40

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010.

040.

020.

130.

710.

76 P

BV e , g

d-1

6412

1-7

4-1

662

120

-73

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60.

01<0

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010.

080.

53<0

.001

0.17

Neu

tral

det

erge

nt fi

bre,

g d

-141

3542

5634

5336

1842

3841

0437

3237

3611

1.0

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<0.0

010.

450.

100.

130.

840.

89

Dige

stib

ility

coe

ffici

ents

dry

mat

ter

0.75

0.75

0.79

0.78

0.76

0.76

0.77

0.79

0.00

60.

72<0

.001

0.87

0.05

0.22

0.51

0.04

org

anic

mat

ter

0.77

0.77

0.81

0.80

0.78

0.77

0.79

0.80

0.00

60.

80<0

.001

0.91

0.06

0.17

0.42

0.03

cru

de p

rote

in0.

720.

770.

740.

790.

730.

770.

730.

800.

010

0.47

0.00

5<0

.001

0.29

0.44

0.39

0.26

neu

tral

det

erge

nt fi

bre

0.72

0.72

0.71

0.70

0.73

0.72

0.68

0.70

0.00

70.

09<0

.001

0.81

0.13

0.87

0.34

0.03

Initi

al li

ve w

eigh

t, kg

311

311

308

305

327

318

325

331

18.0

0.01

0.74

0.84

0.50

0.91

0.30

0.91

Fina

l liv

e w

eigh

t, kg

712

726

716

749

730

741

769

784

18.9

0.02

0.03

0.12

0.24

0.67

0.61

0.75

Live

wei

ght g

ain,

g d

-111

0211

9313

3713

8312

1312

5614

9915

4355

.1<0

.001

<0.0

010.

080.

260.

680.

670.

75Ca

rcas

s gai

n, g

d-1

605

649

746

775

728

768

934

939

30.3

<0.0

01<0

.001

0.08

0.13

0.69

0.45

0.76

Feed

con

vers

ion

Kg

DM k

g-1 c

arca

ss g

ain

15.0

14.0

12.5

12.6

12.5

11.3

10.7

10.7

0.48

<0.0

01<0

.001

0.07

0.16

0.74

0.13

0.91

MJ k

g-1 c

arca

ss g

ain

171

158

149

151

140

126

128

128

5.8

<0.0

010.

004

0.06

0.17

0.76

0.15

0.80

Carc

ass c

hara

cter

istic

s C

arca

ss w

eigh

t, kg

375

383

382

402

406

418

438

439

11.3

<0.0

010.

008

0.12

0.33

0.62

0.76

0.46

Dre

ssin

g pr

opor

tion,

g k

g-152

652

853

353

855

556

356

956

05.

0<0

.001

0.02

0.57

0.57

0.71

0.55

0.18

Con

form

atio

n sc

ore

f6.

36.

16.

76.

77.

57.

89.

89.

20.

33<0

.001

<0.0

010.

630.

002

0.85

0.28

0.18

Fat

scor

e g

4.5

4.5

4.5

4.7

2.7

2.7

3.0

3.2

0.22

<0.0

010.

060.

450.

340.

990.

560.

84a H

F =

Here

ford

; CH

= Ch

arol

ais.

b S

tand

ard

erro

r of m

ean

(for n

=10)

. c L =

low

con

cent

rate

pro

port

ion

(200

g k

g-1 D

M);

M =

med

ium

con

cent

rate

pro

port

ion

(500

g k

g-1 D

M). d

Am

ino

acid

s abs

orbe

d fr

om sm

all i

ntes

tine.

e Pro

tein

bal

ance

in th

e ru

men

. f Co

nfor

mat

ion:

(1 =

poo

rest

, 15

= ex

celle

nt).

g Fat

cov

er: (

1 =

lean

est,

5 =

fatt

est)

.


158

Tabl

e 4.

Effe

cts o

f bre

ed (B

), co

ncen

trat

e le

vel (

C) a

nd ra

pese

ed m

eal s

uppl

emen

tatio

n (R

SM) o

n va

luab

le c

uts (

half

carc

ass)

of g

row

ing

bulls

.

Bree

d (B

) aHF

CHSE

Mb

Conc

entr

ate

leve

l (C)

cL

(200

)M

(500

)L

(200

)M

(500

)p-

valu

eRS

M su

pple

men

tatio

n (R

SM)

-+

-+

-+

-+

BC

RSM

B×C

B×RS

MC×

RSM

B×C×

RSM

Num

ber o

f ani

mal

s7

88

77

88

7Va

luab

le c

uts

Ten

derlo

in, k

g2.

22.

12.

22.

22.

62.

72.

72.

80.

11<0

.001

0.63

0.88

0.76

0.41

0.99

0.56

F

rom

yie

ld, %

1.1

1.1

1.1

1.1

1.2

1.2

1.2

1.3

0.05

<0.0

010.

730.

310.

170.

760.

340.

44 L

oin,

kg

5.1

5.3

5.5

5.7

6.1

6.2

6.6

6.7

0.24

<0.0

010.

004

0.44

0.74

0.84

0.70

0.98

F

rom

yie

ld, %

2.6

2.8

2.8

3.0

2.8

2.9

3.1

2.9

0.10

0.07

0.00

80.

180.

580.

070.

340.

33 E

ntre

cote

, kg

3.0

3.1

3.1

3.0

3.2

3.5

3.5

3.7

0.13

<0.0

010.

120.

110.

160.

210.

300.

93

Fro

m y

ield

, %1.

41.

51.

61.

61.

61.

61.

61.

70.

060.

040.

030.

110.

240.

410.

630.

30 O

utsid

e ro

und,

kg

10.3

10.5

10.5

10.8

12.2

12.9

14.1

13.6

0.44

<0.0

010.

010.

460.

100.

920.

320.

27

Fro

m y

ield

, %5.

35.

55.

75.

55.

85.

96.

36.

20.

21<0

.001

0.03

0.89

0.46

0.88

0.16

0.83

Ins

ide

roun

d, k

g5.

85.

76.

06.

07.

07.

47.

97.

40.

26<0

.001

0.06

0.91

0.65

0.70

0.28

0.18

F

rom

yie

ld, %

3.0

3.1

3.1

3.1

3.3

3.3

3.6

3.4

0.14

0.00

20.

170.

940.

370.

410.

330.

73 C

orne

r rou

nd, k

g5.

85.

75.

96.

07.

17.

37.

57.

60.

24<0

.001

0.09

0.51

0.70

0.65

0.83

0.55

F

rom

yie

ld, %

3.0

3.1

3.2

3.1

3.3

3.3

3.3

3.5

0.13

<0.0

010.

100.

730.

750.

670.

850.

18 R

oast

bee

f, kg

2.8

2.8

2.9

2.7

3.7

3.9

3.9

3.7

0.16

<0.0

010.

880.

720.

770.

530.

180.

50

Fro

m y

ield

, %1.

41.

51.

51.

51.

61.

71.

81.

60.

100.

002

0.67

0.62

0.94

0.80

0.23

0.41

Sub

cuta

neou

s fat

, kg

26.6

21.6

21.2

20.7

16.2

16.8

14.7

15.4

2.33

<0.0

010.

090.

380.

580.

170.

300.

41

Fro

m y

ield

, %13

.111

.010

.610

.28.

38.

46.

77.

01.

17<0

.001

0.02

0.40

0.92

0.23

0.38

0.55

Bon

es, k

g36

.836

.136

.835

.438

.138

.140

.741

.41.

54<0

.001

0.11

0.63

0.06

0.47

0.90

0.66

F

rom

yie

ld, %

18.1

18.5

18.0

17.3

19.5

19.0

18.8

19.0

0.55

0.00

20.

160.

630.

650.

890.

600.

15a H

F =

Here

ford

; CH

= Ch

arol

ais.

b Sta

ndar

d er

ror o

f mea

n (fo

r n=7

). c L

= lo

w c

once

ntra

te p

ropo

rtio

n (2

00 g

kg-1

DM

); M

= m

ediu

m c

once

ntra

te p

ropo

rtio

n (5

00 g

kg-1

DM

).


159

There were no significant interactions for carcass cuts between breed, concentrate level and RSM supplementa-tion (Table 4). Breed had a clear effect on the amount (kg) and yield (%) of valuable cuts. The yields of tenderloin (p<0.001), loin (p=0.07) and entrecote (p<0.05) were 13, 4 and 5% higher with Ch than with Hf bulls, respectively. In addition, the yields of outside round (p<0.001), inside round (p<0.01), corner round (p<0.001) and roast beef (p<0.01) were 9, 10, 10 and 14% higher with Ch than with Hf bulls, respectively. The yield of subcutaneous fat was 49% higher (p<0.001) from the carcasses of the Hf bulls than those of the Ch bulls. On the contrary, the yield of bones was 6% higher (p<0.01) from the carcasses of the Ch bulls than those of the Hf bulls. Concentrate level af-fected the yields of loin, entrecote, outside round and subcutaneous fat (Table 4). The yields of loin (p<0.01), en-trecote (p<0.05) and outside round (p<0.05) were 6, 5 and 5% higher with M bulls than with L bulls, respectively. The yield of subcutaneous fat was 18% higher (p<0.05) from the carcasses of the L bulls than from those of the M bulls. The RSM supplementation had no effect on the amount and yield of valuable cuts.

Meat evaluationThe treatments had no effects on the pH of the carcasses, but significant breed × concentrate level × RSM supple-mentation three-way interaction was observed (Table 5). The loin sample of the Ch bulls had higher moisture (739 vs. 726 g kg-1, p<0.001) and protein (213 vs. 210 g kg-1, p<0.05) and lower fat (35 vs. 51 g kg-1, p<0.001) contents than that of the Hf bulls. Concentrate level and RSM supplementation had no effects on the chemical composi-tion of loin, and there were no significant interactions between breed, concentrate level and RSM supplementa-tion. Breed affected the marbling score of loin and entrecote (Table 5). The loin (p<0.001) and entrecote (p<0.01) of the Hf bulls had 39 and 44% higher marbling scores than those of the Ch bulls, respectively. Concentrate level and RSM supplementation had no effects on the marbling score of the loin or entrecote. Interactions for marbling scores between breed, concentrate level and RSM supplementation were not observed.

There were no interactions for shear force value, drip loss, colour or sensory characteristics between breed, con-centrate level and RSM supplementation (Table 5). The treatments had no effects on the drip loss, but the shear force value of the Ch bulls was 13% higher than that of the Hf bulls (9.9 vs. 8.8 kg cm-1, p<0.05). Concentrate level and RSM supplementation had no effects on the shear force value. The muscle lightness (L value) of the Ch bulls was 8% higher than that of the Hf bulls (p<0.001), but there were not differences in redness (a value) or yellow-ness (b value) between breeds. In addition, the muscle lightness was 3% higher with M bulls than with L bulls, but concentrate level did not affect the muscle redness or yellowness. The RSM supplementation had no effects on any of the measured meat colour parameters. Treatments had no effects on the sensory characteristics (tender-ness, juiciness, beef flavour) of the loin, but there was a tendency (p=0.08) for tenderness to be 6% better for the meat of the Hf bulls than that of the Ch bulls.

The n-6/n-3 fatty acid ratio of the longissimus muscle (LM) of the Ch bulls was 20% higher than the correspond-ing value for the Hf bulls (p<0.01) (Table 6). In addition, the LM of the Ch bulls contained a higher proportion of polyunsaturated fatty acids (PUFA) compared to that of the Hf bulls (p<0.001). On the contrary, the LM of the Hf bulls contained a higher proportion of monounsaturated fatty acids (MUFA) compared to that of the Ch bulls (p<0.01). Breed had no effect on the proportion of saturated fatty acids (SFA). The LM of the Hf bulls had a higher proportion of 10:0 (p<0.05), 18:1 cis-9 (p<0.001) and 20:1 cis-11 (p<0.05) fatty acids compared to that of the Ch bulls. On the contrary, the LM of the Ch bulls contained a higher proportion of 15:0 (p<0.01), 16:0 (p<0.05), 16:1 cis-9 (p<0.001), 18:1 cis-11 (p<0.001), 18:2 cis-9,cis-12 (p<0.001), 18:3 cis-9,cis-12,cis-15 (p<0.001), 18:3 cis-6,cis-9,cis-12 (p<0.001) and 20:3 cis-8, cis-11, cis-14 (p<0.01) fatty acids compared to that of the Hf bulls.

The n-6/n-3 fatty acid ratio of the LM increased 59% with higher concentrate level (p<0.001) and the LM of the M bulls also tended (p=0.05) to contain a 5% higher proportion of MUFA compared to that of the L bulls (Table 6). On the contrary, the LM of the L bulls tended (p=0.06) to have a 4% higher proportion of SFA compared to that of the M bulls. Concentrate level had no effect on the proportion of PUFA. The increasing concentrate level de-creased the relative proportion of 15:0 (p<0.001), 17:0 (p<0.001), 18:1 cis-11 (p<0.05) and 18:3 cis-9,cis-12,cis-15 (p<0.001) fatty acids of the LM and increased the relative proportion of 18:1 cis-9 (p<0.05) and 18:2 cis-9,cis-12 (p<0.01) fatty acids of the LM. In addition, the LM of the M bulls tended (p=0.09) to have a higher proportion of 10:0 fatty acid compared to that of the L bulls (Table 6).


160

Tabl

e 5.

Effe

cts o

f bre

ed (B

), co

ncen

trat

e le

vel (

C) a

nd ra

pese

ed m

eal s

uppl

emen

tatio

n (R

SM) o

n m

eat q

ualit

y of

gro

win

g bu

lls.

Bree

d (B

) aHF

CHSE

Mb

Conc

entr

ate

leve

l (C)

cL

(200

)M

(500

)L

(200

)M

(500

)p-

valu

eRS

M su

pple

men

tatio

n (R

SM)

-+

-+

-+

-+

BC

RSM

B×C

B×RS

MC×

RSM

B×C×

RSM

Num

ber o

f ani

mal

s7

88

77

88

7pH

5.51

5.57

5.55

5.51

5.55

5.53

5.55

5.55

0.02

20.

670.

910.

970.

300.

450.

190.

03Ch

emic

al c

ompo

sitio

n, g

kg-1

Moi

stur

e72

273

472

073

174

474

273

573

46.

1<0

.001

0.11

0.16

0.59

0.09

0.86

0.90

Pro

tein

210

211

209

210

211

213

213

214

2.0

0.04

0.89

0.44

0.26

0.99

0.90

0.90

Fat

5442

5846

3232

3939

6.9

<0.0

010.

140.

120.

890.

220.

930.

99Sh

ear f

orce

val

ue, k

g cm

-19.

38.

98.

78.

39.

710

.49.

410

.30.

770.

010.

370.

730.

770.

210.

910.

89Dr

ip lo

ss, %

0.80

0.73

1.22

1.03

1.63

0.76

1.16

1.68

0.35

80.

160.

130.

360.

820.

680.

230.

10Co

lour

at 1

4 d

“L”

(lig

htne

ss)

37.0

36.4

37.9

37.9

38.8

39.8

39.8

42.2

0.77

<0.0

010.

007

0.25

0.76

0.08

0.38

0.61

“a”

(red

ness

)24

.624

.024

.524

.225

.223

.524

.722

.91.

120.

790.

950.

120.

600.

290.

880.

89 “

b” (y

ello

wne

ss)

7.3

6.5

6.7

6.7

7.5

7.2

7.2

6.7

0.75

0.36

0.74

0.40

0.72

0.86

0.92

0.65

Sens

ory

anal

ysis

d

Ten

dern

ess

6.0

5.9

5.9

5.6

5.5

5.4

5.8

5.7

0.28

0.08

0.74

0.40

0.19

0.95

0.57

0.83

Jui

cine

ss5.

85.

85.

45.

35.

45.

65.

65.

40.

230.

560.

110.

930.

090.

930.

370.

78 B

eef f

lavo

ur5.

55.

55.

75.

55.

65.

65.

45.

50.

190.

810.

970.

810.

500.

640.

740.

65M

arbl

ing

scor

e e

Loi

n1.

971.

782.

051.

581.

171.

191.

521.

430.

218

<0.0

010.

440.

220.

220.

260.

580.

78 E

ntre

cote

1.74

1.38

1.43

1.25

0.83

0.91

1.01

1.21

0.23

80.

003

0.92

0.62

0.14

0.16

0.53

0.97

a HF

= He

refo

rd; C

H =

Char

olai

s. b S

tand

ard

erro

r of m

ean

(for n

=7).

c L

= lo

w c

once

ntra

te p

ropo

rtio

n (2

00 g

kg-1

DM

); M

= m

ediu

m c

once

ntra

te p

ropo

rtio

n (5

00 g

kg-1

DM

).d Sens

ory

anal

ysis:

scal

e fr

om 1

to 7

. Te

nder

ness

: 1=v

ery

toug

h, 7

=ver

y te

nder

. Jui

cine

ss: 1

=ver

y dr

y, 7=

very

juic

y. B

eef f

lavo

ur: 1

=ver

y no

n be

ef li

ke, 7

=ver

y be

ef li

ke.

e M

arbl

ing

scor

e: sc

ale

from

0 to

5 (0

=dev

oid,

5=a

bund

ant)

.


161

Tabl

e 6.

Effe

cts o

f bre

ed (B

), co

ncen

trat

e le

vel (

C), a

nd ra

pese

ed m

eal s

uppl

emen

tatio

n (R

SM) o

n fa

tty

acid

pro

file

(% o

f tot

al fa

tty

acid

s) o

f the

Lon

giss

imus

mus

cle

of g

row

ing

bulls

.

Bree

d (B

) aHF

CHSE

Mb

Conc

entr

ate

leve

l (C)

cL

(200

)M

(500

)L

(200

)M

(500

)p-

valu

eRS

M su

pple

men

tatio

n (R

SM)

-+

-+

-+

-+

BC

RSM

B×C

B×RS

MC×

RSM

B×C×

RSM

Num

ber o

f ani

mal

s7

88

77

88

78:

00.

000.

000.

010.

000.

010.

000.

010.

000.

004

0.29

0.45

0.06

0.85

0.25

0.40

0.87

10:0

0.03

0.02

0.03

0.04

0.03

0.01

0.02

0.02

0.00

90.

010.

090.

040.

220.

110.

001

0.96

12:0

0.06

0.03

0.06

0.05

0.06

0.06

0.07

0.05

0.01

80.

380.

550.

230.

430.

790.

910.

5014

:03.

052.

662.

962.

753.

272.

852.

872.

900.

189

0.14

0.47

0.02

0.46

0.58

0.25

0.50

14:1

cis-

90.

590.

430.

460.

560.

560.

490.

590.

490.

070

0.46

0.85

0.15

0.89

0.82

0.16

0.10

15:0

0.42

0.39

0.36

0.35

0.54

0.53

0.34

0.37

0.04

70.

003

<0.0

010.

760.

030.

490.

660.

7816

:029

.94

28.0

929

.02

28.1

931

.03

28.9

930

.16

28.8

00.

697

0.02

0.24

<0.0

010.

900.

760.

350.

9016

:1 c

is-9

3.71

3.13

3.53

3.45

3.98

3.87

4.12

3.73

0.20

8<0

.001

0.89

0.02

0.76

0.29

0.80

0.14

17:0

0.98

1.04

0.93

0.92

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13, c

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80.

460.

660.

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980.

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SFA d

51.9

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FA f

3.24

2.97

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4.87

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0.82

0.29

0.34

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32.

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774.

634.

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264.

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735.

180.

557

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0.61

0.91

0.44

0.25

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a HF

= He

refo

rd; C

H =

Char

olai

s. b S

tand

ard

erro

r of m

ean

(for n

=7).

c L

= lo

w c

once

ntra

te p

ropo

rtio

n (2

00 g

kg-1

DM

); M

= m

ediu

m c

once

ntra

te p

ropo

rtio

n (5

00 g

kg-1

DM

).d Sa

tura

ted

fatt

y ac

ids.

e M

onou

nsat

urat

ed fa

tty

acid

s.f Po

lyun

satu

rate

d fa

tty

acid

s.


162

There were no effects of RSM supplementation on the n-6/n-3 fatty acid ratio of the LM or on the proportion of SFA, MUFA or PUFA. However, RSM supplementation decreased the relative proportion of 10:0 (p<0.05), 14:0 (p<0.05), 16:0 (p<0.001) and 16:1 cis-9 (p<0.05) fatty acids of the LM. There were also interactions (p<0.05) be-tween breed and concentrate level for the relative proportion of 15:0 and 18:1 cis-11 fatty acids, and between the breed and RSM supplementation for the relative proportion of 18:1 cis-11 fatty acid (Table 6). In addition, there were interactions between concentrate level and RSM supplementation for the relative proportion of 10:0, 18:2 cis-9,cis-12 and 18:3 cis-6,cis-9,cis-12 fatty acids. Significant breed × concentrate level × RSM supplementation three-way interactions were observed for the relative proportion of 18:2 cis-9,cis-12, 18:3 cis-6,cis-9,cis-12, 20:1 cis-11 and 22:2 cis-13,cis-16 fatty acids and for the proportion of PUFA (Table 6).

DiscussionAnimal performance

The increased apparent digestibility of DM (DMD) and OM (OMD) of grass silage-based diets due to increasing concentrate feed level has been well documented (Huuskonen et al. 2007, Keady et al. 2007, 2008). The substi-tution of silage with barley improved the digestibility, because the digestibility of barley is generally higher than that of grass silage (MTT 2012). The reduction in fibre digestibility due to increased concentrate level has been reported previously Steen et al. (2002), Huuskonen et al. (2007) and Keady et al. (2007, 2008). The negative as-sociative effect is attributed to a depression in fibre digestibility in the rumen and in the total digestive tract from inclusion of rapidly fermentable carbohydrates such as barley-based (starch) concentrate (Huhtanen and Jaak-kola 1993) and sucrose (Khalili and Huhtanen 1991) in grass silage-based diets. In accordance with Huuskonen et al. (2007, 2008b) and Huuskonen (2009b), the apparent CP digestibility increased with protein supplementation. Some of the increased apparent digestibility of the CP in the RSM supplemented diets may have reflected the better digestibility of RSM protein. Most of this increase was, probably, only apparent, related to the decreased proportion of faecal metabolic nitrogen recovered in faeces when the CP content increased (Minson 1982). Simi-larly, as reported by Huuskonen et al. (2007) and Huuskonen (2009b), RSM supplementation had no effect on diet apparent DMD or OMD when barley grain was partly replaced by RSM. Most of the experiments in which protein supplementation resulted in positive effects on fibre digestion, have been conducted with poor quality rough-ages (Huuskonen 2009a).

The concentrate proportion had a positive effect on the total DMI which is in accordance with results of beef steers in grass silage-based diets (Caplis et al. 2005, Keane et al. 2006). The substitution rate (SR, decrease in silage DMI per kg increase of concentrate DMI) in the current experiment was 0.81 and 0.60 for Hf and Ch bulls, respective-ly. These results are in line with grass silage-based feedings reported by Keane (2010) with crossbred steers (SR 0.82), Manninen et al. (2010) with Hf bulls (SR 0.71 and 0.53 for farm-made concentrate mixture and commercial compound, respectively) and Randby et al. (2010) with dairy bulls (SR 0.75). McNamee et al. (2001) reported that concentrate feed level and silage feed value are major factors affecting the concentrate substitution rate. For ex-ample, Keady and Kilpatrick (2006) (beef-breed bulls) and Steen et al. (2002) (beef-breed steers), using high-feed value grass silages, reported substitution rates of 0.91 and up to 1.00, respectively. The concentrate protein con-centration did not affect DMI, which was consistent with the results observed with heavy dairy bulls (Huuskonen et al. 2007, Huuskonen 2009b), finishing Hereford bulls (Manninen et al. 2011) or suckled continental-cross bulls (Drennan et al. 1994) with grass silage-based diets.

The higher growth capacity of the Charolais breed compared to the Hereford breed has been demonstrated in nu-merous studies (e.g. Gregory et al. 1994, Aass and Vangen 1998, Bartoň et al. 2006). In the present experiment, the observed increase in LWG was 75 and 91 g d-1 per 1 kg increase in concentrate DMI for Hf and Ch bulls, respec-tively. These outcomes are consistent with grass silage feeding experiments reported by Martinsson (1990) with dairy bulls (84 g d-1) and Manninen et al. (2010) with Hf bulls (85 and 90 g d-1, for farm-made concentrate mixture and commercial compound, respectively), but sometimes responses have been even smaller (dairy bulls, 27 g d-1) (Huuskonen et al. 2007). The improved growth rate in the present experiment was probably due to improved diet digestibility and increased DM and energy intakes with increasing concentrate proportion.

In the present experiment, the growth rate of the bulls tended to be slightly lower on RSM− diets than on RSM+ diets, which disagrees with the findings by Huuskonen et al. (2007, 2008b) and Huuskonen (2009b, 2011) with dairy bulls. The observed response in LWG due to RSM supplementation was slightly higher with lower concen-trate proportion (67 vs. 45 g d-1 for L and M feedings, respectively). This is in line with Huuskonen (2009a) who concluded that the responses to protein supplements seem to be related also to the level of concentrate supple-ment, greater effects being observed with small amounts of concentrates. Hagemeister et al. (1980) reported a


163

tendency towards lower rumen protein synthesis with rations containing very low (0–20%) or very high (70–100 %) proportions of concentrate, and according to Aronen (1992), a medium level of concentrates together with well preserved grass silage may sustain efficient microbial protein production. Therefore, it is likely that a greater response to protein supplementation is to be expected when small rather than large amounts of concentrates are fed to growing cattle on grass silage-based feeding.

In general, the responses to protein supplements are also related to the quality of grass silage used. There is evi-dence that growing cattle are likely to respond to supplementary protein in barley-based concentrates when the digestibility of the grass silage is moderate to low (Huuskonen 2009a). In addition, results with grass silage are dependent on the quality of silage that may vary considerably with the ensiling technique. With poorly preserved silage the response in animal performance to protein supplementation is greater than with well-preserved silage (Hussein and Jordan 1991). There are also differences between extensively and restrictively fermented silages, which both may be well-preserved. Jaakkola et al. (1990) reported that the gain response of growing cattle to fishmeal was greater when enzyme solution (cellulose–glucose oxidase) was used as a silage additive instead of formic acid. Furthermore, Jaakkola et al. (2006) observed that restriction of silage fermentation by formic acid is positively related to the synthesis of microbial protein in the rumen. In the present experiment the fermentation quality of the silages was good and the silages were restrictively fermented with high residual WSC concentration and low lactic acid concentration. Possibly, the responses to protein supplementation may have been greater with untreated and/or poorly preserved silage.

Carcass characteristics and valuable cutsThe superiority of the Ch bulls for the dressing proportion and carcass conformation corresponded to the results reported by Polách et al. (2004) and Bartoň et al. (2006). The lower dressing proportion and conformation score of the Hf bulls in the present experiment can also be explained partly by their lower average slaughter weight compared to Ch bulls because it is established that these traits increased with increasing slaughter weight (Kemp-ster et al. 1988). However, according to Lawrence et al. (2012) the body composition of beef breeds is not only dependent on carcass weight. For example, when early maturing Hf and late maturing Blonde d’Aquitaine breeds were compared, the relative fatness of both breeds remained quite similar at different weights. Both breeds in-creased in fatness as the carcass weight increased but the differential remained quite constant (Lawrence et al. 2012). The different breed bulls are in different stages of their growth path from the beginning of the growing till the end of finishing. The mature weight of the Ch bulls is larger than the Hf bulls but also the tissue composition is different (Alberti et al. 2008). In this regard the Ch bulls will not reach the similar body composition (fat vs. lean) as adult animals such as the Hf bulls. It is justifiable to suppose that the apparently higher carcass efficiency of the Ch bulls compared to the Hf bulls observed in the present experiment was real, and the differences were not only an effect of differences in biological maturity.

The increasing effect of concentrate level on dressing proportion agrees with previous reports (Caplis et al. 2005, Keane et al. 2006). In the present experiment, increasing concentrate proportion also improved the carcass confor-mation, consistent with Keane and Fallon (2001) and Caplis et al. (2005), but contrary to Huuskonen et al. (2007), Manninen et al. (2010) and Randby et al. (2010). Increasing the concentrate level has usually increased the carcass fat score (Patterson et al. 2000, Keane et al. 2006) as in the present experiment. Also higher slaughter weights with increasing concentrate level probably explained the increased fat score, because measures of fatness generally increase with higher carcass weight (Keane and Allen 1998). In accordance with many earlier studies (Huuskonen et al. 2007, 2008b, Manninen et al. 2010, Huuskonen 2009, 2011), there were no effects of protein supplementa-tion on the dressing proportion, carcass conformation score or carcass fat score.

Manninen et al. (2011) reported a similar carcass share of valuable cuts in Hf bulls to those obtained in the pre-sent paper. A number of studies have confirmed a higher share of the most valuable cuts in the carcasses of Ch bulls compared to Hf bulls (e.g. Bartoň et al. 2006, Kaminiecki et al. 2009). Also Kempster et al. (1982) reported a lower saleable meat proportion from carcasses of Hf-sired steers than from carcasses of Ch steers compared at 16 months of age. Similarly to our findings, Bartoň et al. (2006) observed that Hf bulls had a lower percentage of bones compared to Ch bulls. The effects of concentrate proportions on the yields of valuable cuts were quite small and there were only few differences between the different concentrate levels which agree with the findings by Patterson et al. (2000), Caplis et al. (2005) and Keane et al. (2006). Patterson et al. (2000) speculated that the absence of any effect of concentrate proportion on the content of saleable meat in the carcass was considered to reflect the high growth potential of the animals (Blonde d’Aquitaine and Ch bulls). Caplis et al. (2005) observed that bone proportion decreased in growing beef steers with increasing concentrate level which disagrees with


164

our observation. In accordance with the present study, Manninen et al. (2011) reported that the protein supple-mentation had no effect on the amount and yield of valuable cuts in beef bulls.

Meat quality measurementsThe meat colour differences between the breeds corresponded to those reported by Aass and Vangen (1998) who reported meat from Ch to be lighter than meat from Hf. Some studies associated increased lightness with reduced pigment content in the meat of Ch, which suggests the presence of breed differences in relative muscle fibre proportion. Such physiological changes may be related to high genetic growth capacity and increased mus-cularity (Ashmore and Vigneron 1988). In accordance with Bureš et al. (2006), the meat samples from Hf bulls had higher DM and lipid contents and a lower protein content than the samples from Ch bulls. These results in-dicate that the increase in lipid concentrations was associated with the increased DM content and the decreased protein content, which is in accordance with the findings by Van Koevering et al. (1995). Similarly to the present results, greater intramuscular fat deposition and lower moisture in Hf steers compared with Ch steers were re-ported by Gregory et al. (1994).

In agreement with our findings, Bureš et al. (2006) reported no significant effects on sensory characteristics (juici-ness, beef flavour) between Hf and Ch bulls. In our study, however, there was a tendency for the tenderness to be 6% better in the meat of the Hf bulls than that of the Ch bulls. Similarly, poorer tenderness was achieved by large, late maturing Ch steers than by small, early maturing, and fatter Aberdeen Angus steers (Sinclair et al. 2001). The superiority of Hf in tenderness and shear force has been related to a higher marbling level. Several authors (e.g. Gregory et al. 1994, Wheeler et al. 1996) have reported a favourable relationship between intramuscular fat con-tent and shear force/tenderness scores. Aass and Vangen (1998) concluded that a superiority of Aberdeen Angus in intramuscular fat content of the meat has been demonstrated in many studies, and Hf was generally ranked similar or somewhat lower than Angus for this trait, while Ch had the lowest degree of marbling in the meat. This statement agrees with our finding that the loin and entrecote of the Hf bulls had a clearly higher marbling score than those of the Ch bulls. In the present study, the bulls were slaughtered at high CW and high shear force val-ues obtained correspond with tough meat. It is suggested that the taste of beef will strengthen when animals get older and heavier, but meat will become also tougher due to the strengthening of collagen structure (Lawrie & Ledward, 2006). Therefore, longer ageing period would be necessary in meat from animals slaughtered at high LW.

In general, the feeding treatments had no important effects on meat quality characteristics of the Longissimus muscle. These results are broadly in agreement with those reported by Keady et al. (2007, 2008) that concentrate level has no remarkable effect on meat quality of finishing cattle. It is well established that muscle colour is gen-erally darker in forage-fed than in concentrate-fed animals (e.g. Caplis et al. 2005). However, in the present study the muscle lightness (L value) was higher for the M bulls than for the L bulls and the explanation for this effect is not clear. In accordance with our results, Caplis et al. (2005) reported no effects on muscle redness (a value) and yellowness (b value) between concentrate proportions 310 and 550 g kg-1 DM. In agreement with the present study, Manninen et al. (2011) reported that protein supplementation had no effect on the shear force value, pH or sensory characteristics in beef bulls.

The present results suggest that Hf bulls produced healthier meat with a lower n-6/n-3 fatty acid ratio and higher MUFA concentration compared to Ch bulls. Breed differences and associated effects of maturity or growth po-tential on the subcutaneous or intramuscular fatty acid composition of beef are extensively discussed in the re-view by de Smet et al. (2004). It is possible that the differences in carcass fat score between breeds in the present experiment (4.5 vs. 2.9 for Hf and Ch bulls, respectively) affected also the differences in the fatty acid composi-tion of the longissimus muscle. According to de Smet et al. (2004), carcass fat score affects the fatty acid profile of the meat, and breed differences reported in the literature are often confounded by differences in fatness as in the present experiment. Nevertheless, specific breed differences in the n-6/n-3 fatty acid ratio and in the lev-els of longer chain fatty acids that probably could not be attributed to differences in the fat level have also been reported (de Smet et al. 2004), but many of these breed differences are relatively small and are, although often statistically significant, probably of little value from a nutritional viewpoint.

Our results are mainly in accordance with Daley et al. (2010) who concluded that increasing the concentrate level generally increases the n-6/n-3 fatty acid ratio of the longissimus muscle. A healthy diet should consist of roughly one to four times more omega-6 fatty acids than omega-3 fatty acids. The review by Daley et al. (2010) shows sig-nificant difference in the n-6/n-3 fatty acid ratio between grass-fed and grain-fed beef, with an overall average of 1.53 and 7.65 for grass-fed and grain-fed, respectively, for all the studies reviewed. In our study, n-6/n-3 fatty acid


165

ratios for L and M feedings were 3.16 and 5.03, respectively. Furthermore, Daley et al. (2010) reported that grain-fed beef consistently produces a lower concentration of 18:3 cis-9,cis-12,cis-15 fatty acid and higher concentra-tions of MUFAs as compared to grass-fed beef, which includes fatty acids such as 18:1 cis-9, the primary MUFA in beef. These findings are in line with the present results.

Limitation of present study is that the breed effects are partly confounded with carcass weight because the tar-get for average carcass weight was different for Hf and Ch bulls. However, the targeted carcass weights are nowa-days the average weights for slaughtered bulls of these breeds in Finland. Therefore, the present results are valid from a practical point of view.

Conclusions

In conclusion, breed differences in growth performance and carcass traits were observed when the bulls were slaughtered at typical Finnish carcass weights; 380 and 420 kg for Hf and Ch bulls, respectively. The later matur-ing Ch bulls tended to achieve higher weight gain, produced less fat and had a higher percentage of their meat in high-priced joints compared to the earlier maturing Hf bulls. On the other hand, Ch had a lower degree of mar-bling in their meat compared to Hf. The growth performance of the bulls increased with increasing concentrate level and increasing the concentrate allowance also improved carcass conformation. However, also higher slaugh-ter weights with increasing concentrate level probably partly explained some differences in carcass traits between the concentrate proportions. In general, rapeseed meal supplementation had limited effects on the performance, carcass traits or meat quality. According to this study, the choice of breed and feeding can affect the composition of the intramuscular fat. The results indicate that Hf bulls produced healthier meat with a lower n-6/n-3 fatty acid ratio and higher MUFA concentration compared to Ch bulls. In addition increasing the concentrate level increased the n-6/n-3 fatty acid ratio of the longissimus muscle.

AcknowledgementsThis study was partially funded by the Centre for Economic Development, Transport and the Environment for Northern Ostrobothnia. We would like to thank Mr. Lauri Jauhiainen for advice on the statistical analyses. We wish to express our gratitude also to Mr. Matti Huumonen and his personnel for technical assistance and their ex-cellent care of the experimental animals. The personnel of the Slaughterhouse of Atria in Kuopio and the staff of the Finnish Meat Research Institute in Hämeenlinna are thanked for their help in slaughter procedures and meat evaluation. The personnel at Animal Production Research in Jokioinen are thanked for the laboratory analyses.

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AGRICULTURAL AND FOOD SCIENCEK. Gerlach et al. (2013) 22: 168–181

168

Changes in maize silage fermentation products during aerobic deterioration and effects on dry matter intake by goats

Katrin Gerlach1*, Fabian Roß2, Kirsten Weiß3, Wolfgang Büscher2 and Karl-Heinz Südekum1

1Institute of Animal Science, University of Bonn, Endenicher Allee 15, 53115 Bonn, Germany2Institute for Agricultural Engineering, University of Bonn, Nußallee 5, 53115 Bonn, Germany

3Faculty of Agriculture and Horticulture, Humboldt University, Invalidenstraße 42, 10115 Berlin, Germany*e-mail: [email protected]

Chemical and microbiological changes occurring during aerobic exposure of maize silages and their influence on dry matter (DM) intake and preference by goats were evaluated. Eight maize silages differing in DM content, chopping length and compaction pressure were used for the study. After opening, silages were exposed to air for 8 days (d). In 2-d intervals, silage was stored anaerobically for use in preference trials. During the experimental phase, each possible two-way combination of the five silages (d0, d2, d4, d6 and d8) and one standard lucerne hay, was offered as free choice to six goats. Generally, a significant decline occurred in DM intake after 4 d of aerobic exposure. After 8 d, mean decrease in intake was 53% in comparison to the fresh silages. Preference when expressed as DM intake was negatively correlated to silage temperature (as difference to ambient), ethanol and ethyl lactate.

Key words: forage, preference trial, ruminant, volatile organic compound

IntroductionMaize is used as a major forage source for ruminants due to its high yields, nutritional value and good ensiling properties (Allen et al. 2003). Maize silage (like all silages) deteriorates on exposure to air, as a result of aerobic microbial activity (Jonsson 1989). Well preserved silages without butyric acid and low contents of acetic acid are particularly susceptible to aerobic deterioration (Cai et al. 1999).

Aerobic deterioration is a significant problem affecting profitability and feed quality throughout the world (Tabac-co et al. 2011). Caused by the activities of bacteria, yeasts and moulds, there are changes in the chemical compo-sition of the silage (Lindgren et al. 1985) with resulting loss of dry matter (DM) and nutritional components like residual sugars, lactic acid, acetic acid and ethanol that are used as substrates for oxidation. Additionally, there is an increasing risk of proliferation of potentially pathogenic or otherwise undesirable microorganisms. Mycotoxin-producing moulds, Bacillus cereus and Listeria monocytogenes, for example, can pose serious threats to the qual-ity and safety of milk and animal health (Driehuis and Oude Elferink 2000).

It has long been believed that aerobic deterioration depresses DM intake (DMI), caused by an accumulation of degradation products (Lindgren et al. 1988) or changes in volatile compounds. Data on the effects of volatile com-pounds like alcohols, acids, esters, aldehydes and ketones on DMI or product quality (e.g. carry-over to milk) are insufficient (Kalač 2010). Silages that have been exposed to air for several days led to a decrease in roughage in-take of about 10–20% in comparison to fresh silage (Wichert et al. 1998, Bolsen et al. 2002). However, in most studies no indication was given as to which substances or properties of the silages were responsible for selective feeding or at which point of deterioration the decline in DMI began.

The aim of the present study was to determine the changes taking place during eight days of aerobic exposure of maize silages and to characterize the effect on DMI and preference by goats.

Manuscript received August 2012


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Materials and methodsSilage preparation and experimental design

Maize (Zea mays, dual-purpose hybrid ‘Amadeo’, KWS Saat AG, Einbeck, Germany) was planted on May 5, 2009, at a planting rate of 110,000 plants ha-1 at the research station Frankenforst of University of Bonn (Königswinter, Germany, 7°12´E and 50°42´N; 2009 average temperature 10.6 °C, annual precipitation 690 mm, average humid-ity 71.4%). Before planting, the soil was fertilized with about 25 m3 ha-1 of swine manure, and at planting, 200 kg ha-1 of diammonium phosphate was applied. At May 29, 2009, 5 l ha-1 of Zintan Gold Pack (active components: ter-buthylazine, metolachlor and mesotrione; Syngenta AG, Basel, Switzerland) was applied as herbicide. Maize was harvested as whole-crop (cutting height 20 cm) and chopped at two stages of maturity in 2009 (September 9 and 24). The study was arranged in a 2 × 2 × 2 factorial design consisting of DM content (33 and 40% DM), chopping length (10 and 21 mm) and packing density (compaction pressure 0.1 and 0.2 MPa) in the silo (Table 1).

Table 1. Details about the silage treatments used in the trials

Trial DM (%) Chopping length (mm) Compaction pressure (Mpa)

Abbreviation of treatment

Month of opening Temperature (°C)

1 33 10 (short) 0.2 (high) S33hi Feb 2010 13

2 40 10 (short) 0.2 (high) S40hi Feb 2010 13

3 33 10 (short) 0.1 (low) S33lo Apr 2010 17

4 40 10 (short) 0.1 (low) S40lo Apr 2010 17

5 33 21 (long) 0.2 (high) L33hi Jun 2010 22

6 40 21 (long) 0.2 (high) L40hi Jun 2010 22

7 33 21 (long) 0.1 (low) L33lo Aug 2010 22

8 40 21 (long) 0.1 (low) L40lo Aug 2010 22

DM = dry matter, temperature = mean ambient temperature during eight days of aerobic exposure

After harvesting, each crop was immediately ensiled in six 120-l plastic barrels (48 barrels in total; mean density at low and high compaction pressure 235 and 270 kg DM m-3, respectively) and stored anaerobically for at least three months. In February 2010, the barrels containing the first two treatments were opened; the silages were taken out, silage from all six barrels was stirred completely for homogenization and stored aerobically on a heap (ground area 3 m × 3 m) for eight days. The aerobic exposure trials were conducted indoor with a continuous measure-ment of ambient temperature (data logger 175-T1, Testo AG, Lenzkirch, Germany). At the day of opening (d0) and at two-day intervals (d2, d4, d6 and d8 after opening), temperature of the silages was measured at three differ-ent points (middle, left, right) at a depth of 20 cm using a digital probe thermometer (TFA Dostmann GmbH & Co KG, Wertheim, Germany). Aerobic stability was defined as the number of days the silage remained stable before rising more than 3.0 K above the ambient temperature (Honig 1990). For chemical analyses, a composite sample (1000 g) of each homogenized silage was taken at the respective sampling days and frozen immediately (−18 °C).

For the preference trials with goats, silage samples from each day of the aerobic exposure (d0, d2, d4, d6 and d8) were stored anaerobically in polyethylene bags (170 µm, 400 mm x 600 mm, Innovapac GmbH, Durach, Ger-many) and sealed with a chamber vacuum-packing machine (MAX-F 46, Helmut Boss Verpackungsmaschinen KG, Bad Homburg, Germany). A single bag filled with about 1.5–2.0 kg silage was offered to each goat per meal. Bags were stored in a dark, dry and cool room (15 °C) until used in the preference trial. Storage time of the silages in the vacuum bags ranged from five to 26 days depending on the day when fed.


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Preference trials

For each of the eight silage treatments, a preference trial was done at the Institute of Animal Science, University of Bonn, starting in February 2010 (trial 1 and 2), May 2010 (trial 3 and 4), June 2010 (trial 5 and 6) and August 2010 (trial 7 and 8). All trials were conducted with a total of twelve Saanen wethers (German Improved White Goat breed, mean (± SD) body weight 85.8 kg ± 13.9 kg), that were divided into two groups (six goats per group) to conduct two trials concurrently. Goats were allocated to groups such that average body weight was the same. Two animals shared an indoor pen of approximately 2 m × 3 m bedded with straw. They were tied up for the du-ration time of experimental feeding with the possibility of lying down and accessing water and salt-licks.

Preference trials were carried out according to Buntinx et al. (1997). Each trial started with an adaptation period (Kyriazakis et al. 1990), where single meals of each silage (d0–d8) and lucerne (Medicago sativa L.) hay as stand-ard forage were offered to the animals to associate the silage with postingestive metabolic response, taste and smell produced by the forage. The adaptation period lasted six days and forages were offered in a randomized or-der. The standard forage was used to compare the different trials. During the experimental phase, each possible 2-way combination of the five aerobic stability treatments and the standard forage (n = 15) was presented to each of the six goats. Each forage was offered in a plastic feeding box and the silage pairs were presented side by side. The order of presentation of the pairs and the left-right position of the silages in the pair were randomized in all trials. The weight of the silages was determined before, 30 min after offering and after feeding to calculate the initial and total DMI after 3 h. During all trials, consumption of total amount of one preferred type was prevented; so there was always a choice between the two forages in the pair. This was guaranteed by offering additional ma-terial as soon as the silage fell below 300 g. Each trial lasted 21 days, consisting of six days for adaptation and 15 days for the experiment. Each day, the experimental meal was offered for 3 h, starting at 0730 h. Grass hay was offered for ad libitum consumption at 1530 h and removed the following morning at 0700 h.

For laboratory analyses, a subsample (1000 g) of each treatment and each stage of aerobic deterioration (d0–d8) was taken out of the polyethylene bag and frozen immediately at the end of each preference trial.

Laboratory analysesGeneral analyses

The silage samples were freeze-dried (Jumo Imago 500, Jumo GmbH & Co KG, Fulda, Germany) in triplicate repli-cates. The DM of the silages was then estimated by oven-drying of a duplicate subsample at 105 °C overnight. A correction of DM (DMcor) for the loss of volatiles during drying was conducted according to Weißbach and Strubelt (2008) using the following equation:

DMcor = DM + 0.95 × sum of fatty acids (C2−C6) + 0.08 × lactic acid + 0.77 × 1,2 propanediol + 1.00 × other alcohols (C2−C6 including butanediol) [g kg-1].

Proximate analyses were done according to the German Handbook of Agricultural Research and Analytic Meth-ods (VDLUFA 2012) and method numbers are given. Ash and crude lipids (CL) were analysed using methods 8.1 and 6.1.1., respectively. Crude protein (CP) was determined by Dumas combustion (4.1.2, FP328, Leco 8.1, Leco Instrumente GmbH, Mönchengladbach, Germany).

Neutral detergent fibre (aNDFom, 6.5.1; assayed with heat stable amylase), acid detergent fibre (ADFom; 6.5.2) and acid detergent lignin (ADL, 6.5.3) were analyzed using Ankom 2000 Fiber analyzer (Ankom Technology, Mac-edon, USA). The aNDFom and ADFom values are expressed exclusive of residual ash.

The Hohenheim gas test (VDLUFA 2012, method 25.1) was conducted for measuring the 24-h in vitro gas production (GP) and estimating the content of metabolizable energy (ME) using the equation of Menke and Steingass (1987):

ME (MJ kg-1 DM) = 0.136 × GP (ml 200 mg-1 DM) + 0.0057 × CP (g kg-1 DM) + 0.000286 × CL² (g kg-1 DM) + 2.20.

Starch was quantified after enzymatic hydrolysis of starch to glucose as described by Brandt et al. (1987).


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Chemical analyses of fermentation products

A subsample (50.0 g) of each silage was used for determination of lactic acid, pH, volatile fatty acids, alcohols (methanol, ethanol, propanol, 1,2 propanediol, 2,3 butanediol), acetone, ammonia and water-soluble carbohy-drates (WSC). Furthermore, silages were also analysed for two ethyl esters; ethyl lactate and ethyl acetate.

Cold-water extracts were prepared by blending the frozen samples with a mixture of 300 ml distilled water and 1 ml toluol, kept overnight in a refrigerator and afterwards filtered using a folded filter paper. Determination of pH in the extract was done potentiometrically by using a calibrated pH electrode. Lactic acid was analyzed by HPLC (RI-detector, Shimadzu Deutschland GmbH, Duisburg, Germany) according to Weiß and Kaiser (1995). Volatile fatty acids, alcohols and esters were determined by gas chromatography (flame ionisation detector, Shimadzu) as de-scribed by Weiß (2001). Ammonia concentration was analysed colorimetrically based on the Berthelot reaction using a continuous flow analysator (Skalar Analytical B.V., Breda, Netherlands). Concentration of WSC was deter-mined by anthrone method according to von Lengerken and Zimmermann (1991).

Microbiological analyses

At the day of silage opening (d0) and at the fourth (d4) and eighth day (d8) of aerobic exposure, samples of each silage treatment were taken for determination of microbiological status. A composite sample (500 g) was taken using sterile gloves and polyethylene bags, then sealed anaerobically, cooled immediately and sent directly to a laboratory (Wessling Laboratorien GmbH, Altenberge, Germany), where all microbiological analyses were con-ducted the next morning. Aerobic mesophilic bacteria, yeasts and moulds were determined according to VDLUFA (2012, method 28.1.1-28.1.4). All microbial counts were log10-transformed to obtain log-normal distributed data and presented on a wet weight basis. The values below detection level were assigned as value corresponding to half of the detection level to calculate the averages (Tabacco et al. 2009).

Statistical analysesAll data were analyzed using SAS 9.2 (SAS Institute Inc., Cary, North Carolina, USA). The experimental design al-lowed statistical analysis by multidimensional scaling (Buntinx et al. 1997) and by traditional analyses. Multidimen-sional scaling (MDS) is used to develop a spatial arrangement representing the differences expressed as selective forage intake by the animals. For MDS, the difference in preference between a pair of silages was expressed by subtracting the amount of the least preferred forage from the most preferred forage and dividing the difference by the sum of both intakes. In this way, preference was expressed numerically as a relative difference or distance. If an animal consumed equal quantities in one pair, the difference ratio is equal to zero and no preference or dis-tance between the silages was expressed. If only one of the pairs was consumed, the difference ratio is equal to one and the maximum difference in preference between forages is expressed (Buntinx et al. 1997). PROC MDS is an iterative fitting procedure for data with the aim to express distances or relative differences between stimuli (e.g., forages) in an unknown number of orthogonal dimensions, as described by Burns et al (2001). A least squares fit is approximated using an array of points representing the different stimuli. The coordinates of the points are adjusted iteratively until the reduction in residual sum of squares is below a specified level. The residual sum of squares is calculated by comparing the “distance” between the points representing the stimuli and the observed distances or differences between the stimuli. Subsequently, a map is developed with points representing each stimulus. (Burns et al. 2001). Forages with coordinates that are similar in the dimensional space are modelled as similar in preference and, conversely, coordinates being far-of from each other in the dimensional space indi-cate forages differing in preference (Buntinx et al. 1997). The order of fit is dimension one first, which will gener-ally include the most important variables (most sums of squares), followed by dimension two (Burns et al. 2001).

Each preference trial was also tested by analysis of variance after averaging DMI of each forage (averaged across each combination, n = 6). The analysis of variance only included terms for animal and forage. Within the forage treatments, means were separated using the minimum significant difference (MSD) from the Waller-Duncan k-ratio t-test (Burns et al. 2001). Simple linear regression was used to examine the relationship between DMI and silage temperature during the days of aerobic exposure (expressed as difference to ambient temperature). Fur-thermore, correlation coefficients between silage composition and DMI were calculated. Significance was defined at p<0.05, whereas a trend towards a significant effect was noted when 0.05≤p≤0.10.


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ResultsComposition of silages

The chemical composition of whole-crop maize before ensiling is shown in Table 2. Results of chemical composi-tion ranged within expected values.

Table 2. Chemical composition of experimental maize crops before ensiling

Treatment DM Ash CP CL aNDFom ADFom ADL ME

g kg-1 g kg-1 DM MJ kg-1 DM

S33 339 35 71 26 409 198 28 10.3

L33 341 37 79 30 379 218 21 10.5

S40 374 32 72 35 330 182 27 10.1

L40 367 39 78 33 329 173 26 10.3

DM = dry matter, S = short chopping length, L = long chopping length, 33 = 33% DM, 40 = 40% DM, CP = crude protein, CL = crude lipids, aNDFom = neutral detergent fibre assayed with heat stable amylase and expressed exclusive residual ash, ADFom = acid detergent fibre expressed exclusive residual ash, ADL = acid detergent lignin, ME = metabolizable energy

When the barrels were opened, all silages were free of visible moulds or signs of malfermentation. The chemical composition of the eight silages is given in Table 3.

Table 3. Chemical composition of silages (g kg-1 DM unless otherwise stated) at silo opening, lucerne hay (standard forage) and grass hay (fed for ad libitum intake in the afternoon)

Silage Lucerne Grass

Variable S33lo S33hi L33lo L33hi S40lo S40hi L40lo L40hi hay hay

Dry matter (g kg-1) 317 330 315 340 392 379 398 391 908 909

Ash 37 36 35 32 37 33 34 36 91 76

Crude protein 78 76 75 78 72 71 71 77 153 93

Crude lipids 31 26 24 33 24 29 35 32 27 16

aNDFom 384 373 333 357 397 345 302 341 464 592

ADFom 206 214 198 209 231 201 173 194 346 352

ADL 25 27 30 26 35 35 24 27 83 52

24-h gasproduction 299 282 290 302 276 288 306 292 225 221

(ml g-1 DM)

ME (MJ kg-1 DM) 11.1 10.5 10.7 11.2 10.3 10.7 11.3 10.9 9.4 8.3

Starch 351 392 374 383 433 421 362 426

pH 3.9 3.9 3.9 3.9 4.0 4.0 4.0 4.0

Lactic acid 59 63 68 56 57 51 54 54

Acetic acid 16 15 17 14 11 11 13 9

Butyric acid n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d.

Ethanol 7.4 6.4 7.6 5.5 4.7 6.0 8.1 5.9

Ethyl acetate (mg kg-1 DM) 347 479 173 273 138 400 157 177

Ethyl lactate (mg kg-1 DM) 138 157 180 116 176 184 181 161

NH3-N (g kg-1 of total N) 72 66 100 80 96 79 97 90

WSC 18 17 20 27 13 9 8 18

Yeasts (log10 cfu g-1) 4.5 3.8 4.3 4.5 5.3 4.6 3.8 5.5

Moulds (log10 cfu g-1) 2.4 2.4 2.4 2.4 2.4 2.4 2.4 2.4Aerobic mesophilic bacteria (log10 cfu g-1)

5.5 5.0 3.7 5.6 4.5 5.0 3.4 4.7

S = Short chopping length, L = Long chopping length, 33 = 33% DM, 40 = 40% DM, lo = low packing density, hi = high packing density, n.d. = below detection limit (0.03% fresh matter), aNDFom = neutral detergent fibre assayed with heat stable amylase and expressed exclusive residual ash; ADFom = acid detergent fibre expressed exclusive residual ash, ADL = acid detergent lignin, ME = metabolizable energy, Butyric acid = iso-butyric acid + n-butyric acid, iso-valeric acid + n-valeric acid + n-caproic acid, WSC = water-soluble carbohydrates, cfu = colony-forming units


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All silages were well fermented with lactic acid concentrations ranging between 51 and 68 g kg-1 DM, moderate levels of acetic acid and no butyric acid. Regarding proximate constituents and fibre fractions, all values were within expected ranges. Ethyl acetate and ethyl lactate could be detected in all silages at concentrations ranging from 138 to 479 mg kg-1 DM and 116 to 184 mg kg-1 DM, respectively.

Silage samples from each day of aerobic exposure (d0–d8) were analyzed and chemical composition is shown in Table 4. Regarding the concentration of fermentation variables, strong changes occurred during the aerobic ex-posure. Degradation of lactic acid and acetic acid (p<0.001) led to elevated pH value (3.9 to 5.8). Mean content of ethanol and WSC decreased during the eight days of aerobic exposure (p<0.001). In contrast, concentration of oth-er compounds increased or emerged from below detection limit (propionic acid, iso-butyric acid, iso-valeric acid).

Microbiological analysisAt opening, all silages had low concentrations of yeasts, moulds and aerobic mesophilic bacteria (Table 4). Under aerobic conditions, a rapid development of yeasts occurred resulting in high concentrations at d4 and d8. The stagnation after d4 can be explained by the standard method of analysis that did not allow yeast counts exceed-ing 2 × 107 cfu g-1. Growth of moulds started later and was limited to long cut silages. At d8 of aerobic exposure, it has passed over the orientation value of 5 × 103 cfu g-1. Short cut silages did not contain numbers of moulds that exceeded orientation values. A similar development was noted in the numbers of aerobic mesophilic bacteria, that were also mainly restricted on the long cut silages.

Table 4. Composition of silages during eight days (d0–d8) of aerobic exposure, (g kg-1 DM unless otherwise stated; n = 8)

d0 d2 d4 d6 d8 SE

Dry matter (g kg-1) 360 366 371 389 395 14

Ash 35 37 35 35 35 0.7

Crude protein 75 73 76 75 76 1.8

aNDFom 354 370 358 356 362 11.9

ADFom 203 209 217 208 206 10.3

WSC 17a 18a 15 a 9b 11b 1.6

Starch 387 399 408 438 434 16.6

24-h gasproduction (ml g-1 DM) 292 293 292 288 285 3.0

ME (MJ kg-1 DM) 10.8 10.8 10.9 10.7 10.6 0.1

Lactic acid 58a 61a 49a 15b 8b 3.3

Acetic acid 13a 12a 9b 6b 3b 1.1

iso-butyric acid n.d. n.d. n.d. 0.4 0.4 0.1

n-butyric acid n.d. n.d. n.d. n.d. n.d. 0

iso-valeric acid n.d. n.d. n.d. 0.6 n.d. 0.1

n-valeric acid n.d. n.d. n.d. n.d. n.d. 0

n-caproic acid n.d. n.d. n.d. n.d. n.d. 0

Propionic acid n.d. n.d. n.d. 0.1 0.5 0

Ethanol 6.2a 5.5a 4.3b 0.6c 0.1c 0.4

Methanol n.d. n.d. n.d. n.d. n.d. 0

Acetone n.d. n.d. n.d. n.d. n.d. 0

NH3-N (g kg-1 of total N) 83 99 73 62 55 7.8

Ethyl acetate (mg kg-1 DM) 284a 221a 114b 7c n.d.c 46

Ethyl lactate (mg kg-1 DM) 159a 126a 73b 10c n.d.c 10

pH 3.9c 4.0c 4.2b 5.4a 5.8a 0.2

Yeasts (log10 cfu g-1) 4.6b n.a. 7.2a n.a. 7.3a 0.9

Moulds (log10 cfu g-1) 2.4b n.a. 2.8b n.a. 4.2a 0.5

Aerobic mesophilic bacteria (log10 cfu g-1) 4.7c n.a. 5.7b n.a. 6.7a 0.7a,bMean values within rows having different superscripts differ (p<0.05), n.d. = below detection limit (0.03% fresh matter), aNDFom = neutral detergent fibre assayed with heat stable amylase and expressed exclusive residual ash, ADFom = acid detergent fibre expressed exclusive residual ash, WSC = water-soluble carbohydrates, ME = metabolizable energy, n.a. = not analyzed.


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Some differences were observed when comparing fresh silages and samples that had been stored in vacuum bags for use in preference trials. Vacuum-sealed silages contained more ethanol, ethyl lactate and ethyl acetate (p<0.01), possibly due to anaerobic yeast activity (data not shown). For calculation of correlation coefficients between si-lage characteristics and DMI in preference trials, data of vacuum-stored samples were used.

TemperatureDifferences in silage temperature during aerobic exposure are shown in Table 5. Because a constant ambient temperature could not be provided exactly during all trials (see Table 1), silage temperature is expressed as dif-ference to ambient temperature (∆T). In most silages, a strong increase of ∆T was measured between d4 and d6 after opening, three of them heated between d2 and d4 in accordance to their high number of yeasts (long cut silages). Only one silage treatment (S33lo) kept a constant temperature for more than four days. All other silages were already aerobically instable at the fourth day of aerobic exposure, which means they showed an increase in temperature of more than 3.0 K above ambient temperature.

Table 5. Silage temperature (expressed as difference to ambient temperature T∆, in K) during eight days (d0–d8) of aerobic exposure

Silage treatment d0 d2 d4 d6 d8

S33lo -1.5 -1.8 1.0 20.5 22.4

S33hi 0.3 1.3 4.3 22.7 22.7

L33hi -2.0 0.6 13.7 12.4 28.2

L33lo 0.9 1.3 16.0 26.5 35.0

S40lo -1.5 -1.6 4.7 21.2 33.2

S40hi 0.1 1.2 4.3 21.8 33.1

L40hi -1.7 0.0 16.4 19.2 31.1

L40lo 0.9 0.5 6.6 15.3 23.7

S = Short chopping length, L = Long chopping length, 33 = 33% DM, 40 = 40% DM, lo = low packing density, hi = high packing density.

Animal preference and dry matter intakeThe results of MDS showed that selection between forages was associated with two dimensions. The coordinates for the different silages from all preference trials are shown in Table 6.

Exemplarily, the results for one trial with 30-min and 3-h DMI are depicted in Figure 1. A forage with two positive coordinates is generally strongly preferred while two negative coordinates give evidence for strong avoidance (Burns et al. 2001). For the given trial, there was a strong preference for d0 (located in upper right sector in Figure 1), while lucerne hay and d8 were avoided (located in lower left-hand sector). The others (d2, d4 and d6) had one negative dimension and were generally of intermediate preference. The pattern for 30 min and 3 h is very simi-lar, for most treatments values lying close together or at least within one quarter. During all trials, d0 was highly preferred in five and d2 in seven cases. d8-silages were highly avoided in four of eight trials and never preferred.

According to the MSD calculated with the Waller-Duncan k-ratio t-test, DMI did not differ between silages from d0, d2 and d4 but decreased at d6 (p<0.001) in six trials. In all trials, DMI was lowest for d8-silages (p<0.001). In the trial with silage L40hi, DMI decreased after two days of aerobic exposure (p<0.001). In contrast, DMI for S33lo was constant for silages d0–d6; only dropping significantly at d8 (p<0.001). Intake of lucerne hay was at the same level as fresh silages. All goats were of good health throughout the study.

Regression analysis showed that 3-h DMI by goats (y) was negatively related to ∆T during aerobic exposure, which is shown graphically in Figure 2.


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Table 6. Dry matter intake and stimulus coordinates for the two-dimensional solution to the preference among goats, n = 40

Silage treatment d0 d2 d4 d6 d8 Lucerne

hayMean d0−d8 MSD

S33lo Meal (3 h), g 651a 657a 650a 625a 464b 575a,b 609 118

Meal (30 min), g 345a, b 338a, b 332a, b 365a 264b 305a, b 329 99

Dimension 1 0.82 -0.49 1.38 0.60 -2.0 -0.31

Dimension 2 1.41 0.64 -0.09 -1.46 0.0 -0.50

S33hi Meal (3 h), g 650a 610a 633a 380b 136c 680a 481 128

Meal (30 min), g 400a 312b 339a, b 182c 73d 299b 261 67

Dimension 1 -0.54 0.85 0.57 -1.39 0.43 0.08

Dimension 2 0.81 0.20 1.53 -0.27 -2.33 0.06

L33lo Meal (3 h), g 580a 597a 641a 373b 223c 602a 483 92

Meal (30 min), g 324a 339a 394a 160b 108b 368a 265 77

Dimension 1 1.62 0.38 0.71 -1.06 -1.45 -0.20

Dimension 2 -0.28 -1.45 0.97 0.85 -0.95 0.86

L33hi Meal (3 h), g 609b, c 700a, b 720a 585c 284d 732a 580 104

Meal (30 min), g 295a,b 364a 361a 269b 104c 317a,b 279 73

Dimension 1 0.31 0.70 1.49 0.66 -2.3 -0.87

Dimension 2 -0.45 -1.14 0.44 0.61 -0.24 0.78

S40lo Meal (3 h), g 723a 779a 752a 490b 294c 588b 608 121

Meal (30 min), g 370a,b 425a 437a 215c 123d 326b 314 83

Dimension 1 -0.29 1.86 -0.03 0.56 -1.54 -0.55

Dimension 2 1.37 0.23 0.52 -1.66 -0.67 0.21

S40hi Meal (3 h), g 644a 620a 607a 518b 334c 684a 545 97

Meal (30 min), g 358a,b 384a 301b,c 272c 156 d 314b,c 294 66

Dimension 1 0.66 0.54 1.37 -1.75 -1.00 0.19

Dimension 2 -0.56 0.61 0.23 1.19 -1.75 0.28

L40lo Meal (3 h), g 598a,b 569a,b 542b 349c 247d 635a 461 82

Meal (30 min), g 291b 295b 318b 215c 119d 392a 248 73

Dimension 1 -1.35 -0.28 -1.00 0.10 2.11 0.42

Dimension 2 -0.63 -1.12 0.55 1.54 -0.37 0.02

L40hi Meal (3 h), g 715b 657b 467c 444c 256d 816a 508 101

Meal (30 min), g 364a 342a 245b 186b 114c 344a 250 66

Dimension 1 0.80 0.77 -0.54 0.61 -2.17 0.52

Dimension 2 -0.2 -0.96 -1.29 1.37 0.44 0.64

a-d = Means within a row with different superscripts differ, MSD = Minimum significant difference (Waller Duncan k-ratio t-test), d0−d8 = days of aerobic exposure after opening of the silo, S = short chopping length, L = long chopping length, 33 = 33% DM, 40 = 40% DM, lo = low packing density, hi = high packing density.


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Silage characteristics influencing dry matter intakeCorrelation coefficients were calculated between silage characteristics (of vacuum-stored samples used in prefer-ence trials) and DMI of goats (Table 7). A differentiation was made between data referring to silages at all stages of aerobic exposure and the corresponding DMI (n = 40) on one hand and only data connected with the fresh si-lages (d0) to disregard the spoilage process (n = 8) on the other hand. Across all silages, the strongest and nega-tive correlation was between DMI and ∆T. The DMI had also a weak negative relationship with ethanol, ethyl lac-tate and pH. In vitro 24-h gas production and ME were positively associated with DMI.

When using only the fresh silages (d0) that had not undergone aerobic deterioration, DMI was negatively corre-lated with acetic acid. With an average of 12.9 g kg-1 DM, concentrations of acetic acid were generally low. The pH of these fresh silages showed a trend towards a positive relationship with DMI. Generally, fewer significant cor-relations were found, most likely due to the lower number of observations.

-10 0 10 20 30 400

200

400

600

800

1000

∆T

DMI

Fig. 2. Relationship between dry matter intake (DMI, g 3 h-1) of goats and silage temperature during aerobic exposure (expressed as difference to ambient temperature, ∆T (K)); n = 40; y = 662 - 11.69 x; R² = 0.681; p<0.0001.

-2 2

-2

-1

1

2

d0

d2 d4

d6

d8hay

d0

d2

d4

d6

d8

hay

3 hο 30 min

Fig. 1. Multidimensional scaling of the mean preference shown by six goats for five silages (d0–d8) and one lucerne hay (hay) in one preference trial (silage with short chopping length, 33% dry matter and high compaction pressure) after 30 min and 3 h (d = number of days of aerobic exposure).


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Table 7. Correlation coefficients between dry matter intake of goats (g 3 h-1) and characteristics of eight maize silages at day 0–8 of aerobic exposure and the day of opening (d0) respectively.

Variable r (d0–d8) p r (d0) p

DM -0.334 0.035 0.509 0.198

Ash -0.181 0.264 0.352 0.393

Crude protein -0.329 0.038 0.096 0.821

Crude lipids -0.038 0.817 0.172 0.683

aNDFom 0.150 0.362 -0.175 0.679

ADFom -0.248 0.123 -0.247 0.555

ADL 0.062 0.706 -0.044 0.917

ME 0.415 0.008 -0.063 0.882

24-h gas production 0.513 0.001 -0.114 0.789

Starch -0.020 0.902 0.143 0.736

pH -0.433 0.005 0.681 0.063

Lactic acid 0.387 0.014 -0.562 0.147

Acetic acid -0.023 0.888 -0.723 0.043

Ethanol -0.332 0.036 0.293 0.481

Propanol -0.363 0.021 0.004 0.992

WSC -0.072 0.658 -0.453 0.260

Ethyl acetate -0.097 0.552 0.475 0.235

Ethyl lactate -0.327 0.039 0.427 0.291

∆T -0.835 <0.0001

Probabilities of r based on n of 40 (d0–d8) and 8 (d0)aNDFom = neutral detergent fibre assayed with heat stable amylase and expressed exclusive residual ash,ADFom = acid detergent fibre expressed exclusive residual ash, ADL = acid detergent lignin, ME = metabolizable energy, WSC = water-soluble carbohydrates, ∆T = silage temperature expressed as difference to ambient

DiscussionComposition of silages

This study was conducted to describe the changes occurring during aerobic exposure in maize silages and to eval-uate their impact on preference and DMI. Strong shifts were observed in the composition of fermentation prod-ucts, which is consistent with literature, as reviewed by Pahlow et al. (2003). The concentration of lactic acid de-creased significantly in all maize silages during aerobic exposure being nearly depleted after eight days. This can be ascribed to the intense activity of lactate assimilating yeasts, whose population rose above target values within four days of aerobic exposure. A similar decline could be observed in the concentration of acetic acid and WSC. Lactic acid, acetic acid and WSC are the main energy sources for the microorganisms involved in the first phase of aerobic deterioration (McDonald et al. 1991). The microbiological results showed that deterioration was initiated by yeasts followed by moulds and aerobic mesophilic bacteria after the fourth day of aerobic exposure. Moulds have often been observed in advanced stages of aerobic deterioration (Woolford 1990, Pahlow et al. 2003). With reference to suggested target values (VDLUFA 2012), all silages were already spoiled after four days of aerobic exposure. The activity of these organisms leads to the oxidation of fermentation acids and is connected with pro-duction of carbon dioxide and water resulting in evolution of heat (McDonald et al. 1991). The ∆T was strongly correlated with pH, lactic acid, acetic acid and WSC (r = 0.804, -0.882, -0.796, -0.538, respectively; p<0.001) which is consistent with previous literature (McDonald et al. 1991).

Increase in silage temperature is seen as a convenient indicator for the extent and intensity of aerobic deteriora-tion (Borreani and Tabacco 2010). In our experiment, ∆T rose drastically after two (long-cut silages) or four days (short-cut silages) of aerobic exposure, due to rapid colonization of lactate assimilating yeasts which have often


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been shown to be capable of rapid growth and are initiators of aerobic deterioration. Maximum silage tempera-ture was measured in silage L33lo with ∆T reaching 35.0 K at d8. It has to be considered that ambient temperature was 22 °C during this trial which gives good conditions to spoilage organisms like aerobic yeasts mostly being ac-tive at 20–30 °C (Ashbell et al. 2002). Due to different ambient temperatures during aerobic exposure periods, no further conclusions concerning the impact of different treatments (chopping length, DM, compaction pressure) on aerobic deterioration are drawn.

Dry matter intake and preferenceThe DMI of different maize silages decreased significantly after four days of aerobic exposure. After eight days it was more than halved, with reductions ranging between 29% and 79% in comparison to the fresh silages (d0). In the trial with silage L40hi, 3-h DMI decreased after two days of aerobic exposure. Long cut silages with higher contents of DM are especially prone to deterioration after opening, due to restricted fermentation and increased porosity and therefore movement of oxygen into the silage causing more rapid and extensive growth of aerobic microorganisms (Muck et al. 2003). The pH of L40hi rose from 4.0 to 4.5 within four days, giving evidence of a strong and fast spoilage process. This was also supported by an increase of temperature of 16.4 K during these four days. In contrast, DMI was constant for six days in the trial with silage S33lo. When looking at the ∆T in this treatment it can be assumed that the spoilage process started later, therefore temperature remained steady up to the sixth day after opening the silo. This prolonged aerobic stability might be due to the relatively high content of acetic acid in this treatment.

Few other studies dealing with the topic also reported a strong (Wichert et al. 1998) or slight decline (Bolsen et al. 2002) in feed intake after some days of aerobic exposure. Since oxygen can penetrate the silage for 1 to 2 m when still being in the silo (Weinberg and Ashbell 1994), air contact is not restricted on face and feed-out, thus days with air contact can easily exceed time interval of four days under field conditions. As DMI is one of the most important factors determining productivity in milk or beef production, care should be taken to avoid air contact and consequently aerobic deterioration in maize silages. Unfortunately, DM losses have not been calculated in these studies. With reference to literature (McGechan 1990, Bolsen et al. 1993, Tabacco et al. 2011), losses can account for up to 20% of the total stored DM and up to 70% in the peripheral areas and near the sidewalls of the bunkers. When adding these losses to the decline in DMI that occurred in the preference trials reported above, the negative consequences of aerobic deterioration are detrimental. However, data presented here are based on preference experiments. Keady and Murphy (1998) observed that differences in DMI were much stronger when cows were having the possibility to choose between two or more feedstuffs in comparison with single-choice ex-periments. Nevertheless, results give an impression of the potential in DMI that is lost when feeding spoiled si-lages in comparison to fresh ones. Low preference for deteriorated silages may probably result in greater feed sorting and lower intakes when animals have a choice of different feedstuffs. It might be interesting for studies to examine the impact of deterioration in single-choice experiments.

Silage characteristics influencing dry matter intakeThe impact of deterioration on DMI and preference was strongly negative, but it was difficult to attribute the de-cline to a single compound. Some fermentation products (ethyl lactate, ethanol) were negatively related to silage intake, but correlation coefficients were weak.

With restriction to the fresh silages without aerobic deterioration, DMI was strongly negatively correlated to ace-tic acid, which is in agreement with the findings of Buchanan-Smith (1990) where concentrations of acetic acid were shown to be responsible for a decrease in DMI by sheep in a linear manner. In meta-analyses on the effect of fermentation quality on DMI by dairy cows (Eisner et al. 2006, Eisner 2007), acetic acid was the strongest sin-gle predictor of DMI when silages and concentrates were offered separately. Though, the dataset used was mainly based on grass silages or mixtures of grass and maize silages. New findings of Krizsan et al. (2012) showed that an addition of acetic acid to wilted grass silages fed to growing steers reduced silage DMI. However, the reduction equalled the amount provided by the added substances, so no differences in total DMI were observed. From our point of view, a lower DMI caused by slightly increased amounts of acetic acid in fresh silages is compensated by the better aerobic stability and therefore a smaller decline in DMI as a consequence of aerobic deterioration, as seen with silage S33lo.


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Another component negatively related to DMI in this study was ethanol. Huhtanen et al. (2002) and Krizsan and Randby (2007) did not find a negative impact of ethanol on DMI, while results of Hetta et al. (2007) showed a positive effect that eventually could be an associative effect due to the negative correlation between concentra-tions of ethanol and ammonia-N in that study.

Correlation of pH with DMI was ambiguous, with different results for fresh and aerobically stored silages. For the fresh silages (that had not undergone aerobic deterioration), there was a positive relationship between pH and DMI. This is consistent with literature, which reported similar positive relationship for fresh silages (Erdman 1988, Dulphy and Van Os 1996, Eisner 2007). In well fermented silages with a low pH, DMI increases when silage pH in-creases. This positive effect implies a decrease in acidity caused by less fermentation without excessive formation of ammonia-N and fermentation acids (Dulphy and Van Os 1996). Otherwise, correlation of the complete silage dataset (fresh as well as spoiled silages) with DMI shows a negative relationship. Here, the effect of silage pH on intake seems to be a direct consequence of the spoilage processes. Steen et al. (1998) observed a quadratic re-lationship between pH and DMI with a slight positive value at low pH followed by a negative relationship at high pH. Huhtanen et al. (2002) proposed that this might be caused by the contrary influence of acidity at low pH and poor fermentation with high concentrations of ammonia N and volatile fatty acids at high pH.

Ethyl lactate had a weak negative influence on DMI. In other trials, esters were the most abundant class of vola-tile compounds in red clover silages (Figueiredo et al. 2007) as well as in grass silages (Mo et al. 2001) with ethyl esters being the predominant subclass of all esters (Figueiredo et al. 2007). Since esters are known to be odorant, they could have an effect on the taste of a silage and consequently on feed intake (Mo et al. 2001). Also Kristensen et al. (2010) expected them to contribute to the silage flavour due to their volatility. Many esters have low odour thresholds, so they can already be noticed in the parts per million ranges. To our knowledge, the effect of different ethyl esters in silages on voluntary feed intake by ruminants has not been studied previously. There may be need for further studies, since they have recently been observed in considerable amounts in fresh and well fermented silages (Weiß et al. 2011, Weiß and Auerbach 2012), where ethyl acetate and ethyl lactate showed a strong cor-relation with ethanol, which was also confirmed in our study (r = 0.868 and r = 0.918, p<0.001, data not shown).

By far the strongest correlation was between DMI and ∆T. The fact that temperature measured in the silage was a better predictor than any other analyzed constituent emphasizes the difficulty to identify chemical reactions be-ing responsible for decreases in preference caused by aerobic spoilage. Nevertheless, it also underlines the suit-ability of temperature measurement for daily use, as recommended by Borreani and Tabacco (2010) to improve silage management. The target value of 5 °C for maximum ∆T given by Spiekers et al. (2009) for practical use was proven to be appropriate.

In the present study, goats were used as model animals for cattle. Strong evidence can be found in literature that their feeding and preference behaviour are very similar (Squires 1982, Burns et al. 2001). Nevertheless, continuative studies with dairy cows and beef cattle dealing with the topic presented here are needed to verify that assumption.

In conclusion, this study demonstrated that strong changes concerning the fermentation products of maize silage occurred during eight days of aerobic exposure. Counts of spoilage organisms, especially yeasts rose above tar-get values within four days. There was a strong impact of deterioration on feed intake and preference by goats, marked by a decrease of DMI after four days of exposure shown by goats in choice situations. Temperature meas-ured in the silage was the best predictor for DMI in comparison with any single silage constituent. It can be rec-ommended to limit exposure of silages to oxygen during storage and feed-out as much as possible because of its detrimental effects on DMI.

AcknowledgementsThis study was supported by the “Deutsche Forschungsgemeinschaft” (DFG, German Research Foundation, SU 124/22-1 and BU 1235/5-1). We thank Nadja Wahl and Petra Jacquemien, University of Bonn and the team of Central Analytical Laboratory, Humboldt University Berlin, for support in chemical analyses and Dr. Young Anele for language editing.


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AGRICULTURAL AND FOOD SCIENCES. Orosz et al. (2013) 22: 182–188

182

Microbial status, aerobic stability and fermentation of maize silage sealed with an oxygen barrier film or standard

polyethylene filmSzilvia Orosz1, John M. Wilkinson2, Simon Wigley 3, Zsolt Bíró4 and Judit Galló4

1Szent István University, Department of Nutrition, H-2100 Gödöllő, POB 3, Hungary 2 School of Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough, LE12 5RD, UK

3Bruno Rimini Ltd., 309 Ballards Lane, London N12 8NP, UK4Szent István University, Institute of Wildlife Conservation, H-2100 Gödöllő, POB 3, Hungary


An experiment was conducted to compare a bunker silo sealing system comprising an oxygen barrier film (OB: 45μm thickness) with protective woven polypropylene with one comprising standard black polyethylene film (S, 125μm thickness) with protective tyres. Analysis of samples taken to 30 cm depth after 365 days of storage showed no dif-ferences in pH or lactic acid between the two sealing systems. There were no differences in aerobic bacterial count between silages. Whilst 2.56 log10 CFU moulds g-1 fresh weight were found in samples of silage sealed with S, no moulds were found in samples of silage sealed with OB. Aerobic stability, averaged 249 hours and 184 hours for OB and S, respectively. The OB system probably inhibited the development of the micro-organisms responsible for the initiation of aerobic deterioration to a greater extent than the standard silo sealing system.

Key words: maize silage, oxygen barrier film, fermentation, moulds, aerobic stability

Introduction Many factors can affect the deterioration and loss of nutrients during the conservation and feed-out of silage, in-cluding crop maturity, the use of additive, particle size, rate of silo filling, packing density, type of plastic sealing and the fermentation profile of the ensiled material (Johnson et al. 2002, Holmes and Bolsen 2009). Maize silage is particularly susceptible to aerobic deterioration when it is exposed to oxygen in the silo or in the feed bunk (Ash-bell and Weinberg 1992, Kung et al. 1998). According to Pahlow et al. (2003) and Uriarte-Archundia et al. (2002) yeasts that metabolize lactic acid are the primary spoilage microorganisms in maize silage, although the acetic acid bacteria and moulds can also cause aerobic spoilage (Spoelstra et al. 1988).

Borreani et al. (2007) found that loss of dry matter from the upper 40 cm layer was 10% for maize ensiled with no additive treatment and covered with a coextruded oxygen barrier (OB) film (125 mm thickness, 100 cm-3 m-2 per 24 h oxygen permeability at 1 bar, 23 °C, 85% relative humidity) under farm-scale conditions in Italy. Comparable loss of dry matter was higher, averaging 38%, for the same crop ensiled under standard polyethylene film (180 mm thickness, 990 cm3 m-2 per 24 h oxygen permeability at 1 bar, 23 °C, 85% relative humidity). Coextruded oxy-gen barrier (OB) film with thickness of 45mm is 100 times more of a barrier to oxygen than a standard 125 mm polyethylene film (oxygen transmission rate: 3 vs. 400 cm-3 m-2 per 24 h 21% O2) due to its special chemical com-position and physical structure. Studies on thinner, 45mm thickness, OB film have shown positive effects on both grass (Wilkinson and Rimini 2002) and maize silages (Berger and Bolsen 2006).

Wilkinson and Rimini (2002) reported virtually no visible surface mould or spoilage and a lower percentage of inedible silage for triple co-extruded OB film sealed small-scale silos compared to single and double standard 125 mm thickness polyethylene film-sealed silos. Kuber et al. (2008) found that OB film was more effective than standard polyethylene film in preventing the entry of oxygen into large silos of ensiled maize. Bolsen and Bolsen (2006) found that maize silage and high mositure maize grain in the top 0 to 45 cm under the OB covering had better fermentation profiles and lower estimated additional spoilage losses of OM compared to the crops stored under standard plastic film.

The aim of this large-scale experiment was to compare the effect of a standard sealing system comprising a sin-gle layer of standard plastic film with a system comprising an oxygen barrier film and protective woven polypro-pylene tarpaulin, on fermentation characteristics, microbial status and aerobic stability of the top layer (30 cm), under conditions found in commercial practice in Hungary.



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Materials and methodsEnsiling

The trial was carried out on a commercial large-scale farm in Hungary (47°10'29'' N, 19°18'25''E, altitude: 99 m). The size of the bunker silo used in the trial was 25 wide and 70 m in length, with walls along both sides (3 m height) made from pre-cast concrete blocks. Forage maize was harvested without additive on 16 September 2009 with a precision chop harvester fitted with a kernel processor (Claas Jaguar 840). Stubble height was in the range of 30 to 40 cm above the soil level. Consolidation was carried out using two tractors with a weight of 10 tonnes per tractor (Raba Steiger). Sealing was completed within 240 minutes after terminaton of consolidation. The average height of the silage after filling was 3.5 m.

The oxygen barrier (OB) silo sealing system consisted of a transparent thin co-extruded plastic film of 45 μm thick-ness (“Silostop”, 2Gamma, Mondovi, Italy), a close weaved dark green anti-UV polypropylene net (190 g m-2) to protect the film (UV light protection), and gravel bags. The net designed with a woven structure which allows wind to pass through rather than lifting the sheet. The special woven structure also ensures that the nets do not ac-cumulate heat and do not cause the silage the heat. Moreover the net protects the OB film from many types of physical damage (birds, dogs and hail). The gravel bags not only sealed the edges but they also prevented the film and net blowing off the silage. The standard (S) sealing system comprised a single black coloured standard plastic sheet (thickness of 125 μm, 1020 cm3 m-2 per 24 h oxygen permeability) covered with an average of 1.6 used car tyres m-2 (edge-to-edge). A central area of 10 m length was sealed with overlapping OB and S films (Fig. 1) and gravel bags were placed around its periphery. The peripheral edges of the entire silo were sealed with gravel bags. Both film sheets were laid transversally across the silo. The layout of the two silo sealing treatments and the sam-pling protocol are shown in Figure 1.

Width 25 m Oxygen Barrier film

1-1 1-1

Approx.40 m

1-1 1-1

1-11-1

1-1 1-1

1-1 1-1

Overlapping central area - Not sampled

10 m

1-1 1-1

Approx.15 m

1-1 1-1

1-11-1

1-1 1-1

1-1 1-1

Width 25 m Standard film

Sampling Ten initial samples, each of 2.5 kg fresh matter (FM) were taken by corer in both the OB and S areas of the silo before sealing, as shown in Figure 1, after the completion of consolidation but before sealing, to a depth of 30 cm from the top surface, for laboratory analyses and density estimation. A further set of ten samples were taken on the 16 September 2010 (12 months after filling and first sampling) from both the OB and S areas, according to the Figure 1, within 50 cm of the place where the initial samples were taken. These samples, also 2.5 kg FM, were also taken to a depth of 30 cm from the top surface for laboratory analyses, density estimation and assess-ment of aerobic stability.

Fig. 1. Sampling design in split bunker silo. One sample derived from fresh, packed material and one sample from silage after 12 months.


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Physical measurements

Temperature measurements were undertaken on the whole sample to depth of 30 cm with a digital infra-red thermometer immediately after the samples were taken. Fresh matter density was determined by the following method: Each sample was unloaded from the sample corer (approximately 30 cm depth ×15 cm × 15 cm). The vol-ume of each core was then determined by filling the corer with water to the same depth as that of the core and the weight of the core determined using a digital mobile scale. The samples were then divided, with 1 kg retained for laboratory analyses and 1.5 kg for aerobic stability.

Aerobic stability was measured in a special model silo system in a constant temperature room at Szent István Uni-versity, in Gödöllő. Each sample of silage was mixed and put into a model silo (density 120 g DM m-3) leaving a 5 cm air layer above the top of the silo into which air was passed continually through a 10 mm diameter hole. Tem-perature sensors were built into the model silos, which were indvidually insulated with a polyethylene coat and stored in a controlled temperature room at 20 ± 1 ⁰C. Aerobic stability was defined as the number of hours the silage remained stable before rising by 2 oC above the ambient temperature (Ranjit and Kung 2000).

Laboratory analyses Laboratory analyses were executed according to the Hungarian National Standards: dry matter HNS ISO 6496:1993, crude protein HNS 6830-4:1981, crude ash HNS ISO 5984, lactic and volatile fatty acids: HNS 6830-39: 1986 (Hungar-ian Feed Codex 2004). Fiber fractions were determined according to Van Soest (1963). Aerobic bacteria were ana-lysed by the method of STRF-MIKR-LB-1: 1999 (Hungarian Feed Codex, 2004), moulds were detected by LACTOAC-MIKR-LB-1:1999 (Hungarian Feed Codex, 2004). Clostridium perfringens were identified by HNS EN 13401:2000. (Hungarian Feed Codex, 2004).

Statistical analyses The chemical compositional data of the fresh chopped whole crop maize and the silage samples (n=10, S vs. OB samples), fermentation profile and microbial counts (except mould counts) of the silage samples (n=10, S vs. OB samples) were analyzed for their statistical significance with IBM SPSS (version PASW Statistics 18). All microbial counts were log10 transformed to obtain log-normal distributed data. Chemical composition, fermentation pro-file, microbial counts (except mould counts) and aerobic stability (number of hours at 2 oC above the ambient tem-perature, n=10, S vs. OB samples) were analyzed for their statistical significance by ANOVA. Significant differences between variances were identified by the P-values of ANOVA (Levene’s test for equality of variances), and the ef-fects were considered significant at p≤0.05. When calculated values of F were non-significant (equal variances), Student t-test for equality of means was used (p≤0.05), when calculated values of F were significant (non-equal variances), Welch’s t test (p≤0.05) was used to interpret any significant differences among the mean values. Mould counts were analyzed for their statistical significance by the Wilcoxon test (n=8, 36 T+ and 0 T-).

Results

The dry matter and nutrient content of samples of fresh maize in the upper 30 cm layer under either OB or S films are shown in Table 1. Table 1. Composition of the consolidated chopped fresh crop (top 30 cm) under either standard (S) or oxygen barrier (OB) film (n=10)

Consolidated fresh crop (top 30 cm) Silage

Sealing system S OB SED Sig. S OB SED Sig.DM g kg-1 367 376 7.36 NS 359 362 5.82 NSCrude protein g kg-1 DM 77 76 1.48 NS 74 75 0.49 NSCrude ash g kg-1 DM 43 42 1.23 NS 42 44 0.91 p =0.042NDF g kg-1 DM 443 442 10.71 NS 431 417 7.28 NSADF g kg-1 DM 214 208 6.30 NS 238 227 5.50 NSADL g kg-1 DM 25 24 1.51 NS 25 25 2.16 NS

DM = dry matter, SED = standard error of difference. NS = not significant p >0.05, NDF = neutral detergent fiber, ADF = acid detergent fiber, ADL = acid detergent lignin

The physical parameters of the fresh crop and silages sealed with either the OB or S sealing systems are shown in Table 2.


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Table 2. Physical parameters of the consolidated chopped fresh crop (top 30 cm) and of silage under either standard (S) or oxygen barrier (OB) film (n=10)

Consolidated fresh crop (top 30 cm) Silage

Sealing system S OB SED Sig. S OB SED Sig.Fresh matter density kg m-3 528 517 35.2 NS 546 540 30.8 NSDry matter density kg m-3 194 195 15.3 NS 196 196 12.2 NSTemperature oC 23.3 23.0 0.6 NS 25.5 25.0 0.3 NS

SED = standard error of difference, NS -not significant p>0.05

Fermentation characteristics, microbiological composition and aerobic stability of the silages in the top 30 cm stored under the two sealing systems are shown in Table 3.

Table 3. Fermentation characteristics, microbiological composition and aerobic stability of silage under standard (S) or oxygen barrier (OB) film (n=10)

Sealing system S OB SED Sig.pH 3.73 3.80 0.038 NSLactic acid g kg-1 DM 45.0 47.1 3.28 NSAcetic acid g kg-1 DM 32.3 24.7 2.11 p =0.002Propionic acid g kg-1 DM 0.9 0.4 0.19 p = 0.017Butyric acid g kg-1 DM 0.0 0.0 - -Ethanol g kg-1 DM 11.3 6.5 1.01 p=0.005Volatile fatty acids g kg-1 DM 33.2 25.1 2.06 p =0.001Total organic acids g kg-1 DM 78.2 72.2 3.62 NSTotal fermentation products g kg-1 DM 88.7 78.7 4.69 p =0.042Lactic acid/acetic acid 1.4 2.0 0.23 p =0.024Lactic acid/ total fermentation products 0.5 0.6 0.02 p =0.001

AEMB log10 CFU g-1 FM 4.71 4.12 0.54 NS

Moulds log10 CFU g-1 FM 2.56 0.0 0.43 p =0.008

YeastsNumber of positive samples

1 3 - -

- - - -

Clostridium perfringens log10 CFU g-1 FM 1.93 0.56 0.46 p =0.008

Aerobic stabilityNo. hours to +2 oC above ambient 184 249 16.63 p =0.002

DM = dry matter, SED = standard error of difference, NS = not significant p>0.05, FM - fresh material, AEMB = aerobic mesophilic bacteria

Temperature profiles of the aerobic stability phase are shown in Figure 2.

0,0

0,5

1,0

1,5

2,0

2,5

3,0

3,5

4,0

1 17 33 49 65 81 97113 129 145 161 177 193 209 225 241 257 273 289 305

Aerobic hours

Tem

pera

ture

(Deg

rees

C a

bove

am

bien

t)

Conventional film

Oxygen Barrier film

Fig. 2. Temperature changes of maize silages during exposure to air (n=3)


186

Discussion

There were no significant differences in the concentrations of dry matter, crude protein, crude ash, neutral deter-gent fibre (NDF), acid detergent fibre (ADF) or acid detergent lignin (ADL) in the initial fresh maize crop at the point of ensilement under either the OB or the S sealing systems. This indicated that the trial design had been success-ful at ensuring that the starting point for maize quality was the same across the two treatments.

After 12 months storage in the silo there were no significant differences in the concentrations of dry matter, crude protein, NDF, ADF or ADL in samples of silage from in the upper 30 cm layer under either the OB or S sealing sys-tems. However, the crude ash content was higher, by 2.1 g kg-1 dry matter, in the maize silage covered by the OB sealing system than in maize silage stored under the S sealing system. Whilst this was statistically significant the difference was numerically small and probably of little importance biologically or chemically.

There were no significant differences between the two sealing treatments in either the density of the top 30 cm or the temperature at 30 cm depth. Both fresh matter and dry matter densities were similar between the two treat-ments which indicated consistency across the two treatments with respect to compaction during filling.

Silage density is important for the exclusion of air from the silo to ensure an anaerobic environment where nu-trients are preserved. In the present experiment, the average fresh matter density of the silage in the top 30 cm layer was 196 kg DM m-3, reflecting the relatively high tractor packing capacity of 20 tonnes, but was lower than the recommendation of Muck and Holmes (1999) of maize silage of 225 kg DM m-3, This indicates the difficulty in achieving high silage density in the uppermost layer of the silo even when the tractor packing capacity is consid-ered to be adequate for consolidation.

There were no significant differences in pH or lactic acid between the two sealing treatments. Further, the low pH values indicate that the overall silage quality irrespective of treatment was generally good indicating acceptable silo management and forage preservation. In contrast, Borreani et al. (2007) and Kuber et al. (2008) found more lactic acid and lower pH values in silage stored under OB film than under a standard sealing system.

In the present study, the concentration of acetic acid was lower in silage sealed with the OB system than in silage covered with the S system. The concentration of propionic acid was also lower in the silage stored under the OB than in that stored under the S sealing system. Butyric acid was not found in any of the samples. In contrast, Bor-reanni et al. (2007), found a higher concentration of propionic acid in untreated maize silage in the peripheral area of one farm bunker silo sealed with OB than with S, but not in another silo where the maize had been treated with an additive containing Lactobacillus buchneri.

Total concentrations of volatile fatty acids and fermentation products (FP) and the ratios of lactic to acetic acid and lactic acid to FP were higher for silage stored under OB than S, indicating that the fermentation had been enhanced by the OB method of sealing. Berger and Bolsen (2006) also reported a better fermentation profile in maize silage in the top 0 to 46 cm under a 45 μm OB film compared with maize silage stored under a 150 μm polyethylene.

The concentration of ethanol was lower in the silage stored under the OB than under the S sealing system, in-dicating lower yeast activity in the material stored under OB than S film (Pahlow et al. 2003, Rooke and Hatfield 2003). Borreani et al. (2007) and Borreani and Tabacco (2008) found no differences in concentrations of ethanol and yeast counts of less than 1 log10 cfu g-1 in the peripheral areas of untreated maize ensiled under either OB or S sealing systems in a similar split-bunker silo design to that of the experiment reported here. In the present study, counts of yeasts were positive in three of the ten samples of silage covered by the S system (mean 4.91 log10 cfu g-1 fresh matter, FM), whereas only one sample of silage covered by the OB system contained a measurable count of yeasts (4.31 log10 cfu g-1). Yeast counts lower than 3.1 log10 cfu g-1 indicate a likelihood of aerobic stability in ex-cess of 50 hours of in maize silage (Muck 2004). Considering that the majority of the samples were negative for yeasts, good aerobic stability would be expected.

There was no significant difference in aerobic bacteria count between silages. However, a mean mould count of 2.56 log10 cfu g-1 FM were found in silage stored under the S system while there were no moulds found in any of the ten samples of silage stored under the OB system. Borreani and Tabacco (2008) also found that the lower ox-ygen permeability of the OB film was reflected in a lower mould count in the peripheral areas of untreated maize silage stored in a farm bunker silo under OB than under S.


187

Lower populations of Clostridium perfringens were found in the silage stored under OB than under S. C. perfrin-gens is both saccharolytic and proteolytic (Woolford 1984), and is one of a group of undesirable species of silage bacteria, the Clostridia, which can degrade amino acids to ammonia, ferment lactic acid to butyric acid and cause the “late blowing” of cheese (Pahlow et al. 2003). Borreani and Tabacco (2008) found much higher counts of bu-tyric acid bacteria (BAB) spores in untreated maize silage in the peripheral areas of a farm bunker silo. The authors found that the higher counts of BAB spores were associated with higher mould counts, lower concentrations of nitrate in silage and higher temperature differences between the silage and ambient, indicating that clostridial sporulation can be encouraged in silage which is exposed to oxygen, as Kwella and Weissbach (1991) observed.

The time taken for the temperature of the silage samples to rise by 2 oC above ambient averaged 249 hours for material stored under the OB system compared to 184 hours in the case of silage stored under the S system - a difference of 65 hours. Aerobic stability data should be considered as relative values obtained under laboratory conditions. Borreani et al. (2007) found an average aerobic stability of untreated maize silage of 72 and 69 h (+2 °C above ambient) for OB and S systems, respectively. The values observed here, although greater than those re-ported by Kleinschmit and Kung (2006) for uninoculated corn silage (25 h), were similar to those recorded by Bor-reani et al. (2007) with maize silage treated with an additive containing L. buchneri (355 and 178 h for OB and S, respectively). The higher aerobic stability of silage stored under OB than under S can be explained by the signifi-cantly lower mould count in the OB silage.

Johnson et al. (2002) stated that the aerobic stability of maize silage can be increased by kernel processing which in turn is reflected in increased FM packing density compared with unprocessed corn silage. The greater FM packing density limits the exposure of processed silage to oxygen during the initial period of the storage phase compared with unprocessed silage. Therefore, the growth of aerobic microorganisms is minimal in processed corn silage be-fore the opening of the silo. In the present study, the crop was processed and there was no significant difference in the density of the silages between the two sealing treatments, so the differences in aerobic stability must be attributed to differences in oxygen exposure of the upper layers of the silages due to the different oxygen perme-ability of the two plastic films. The significantly higher aerobic stability in the silage sealed by the OB system can-not be due to differences in concentrations of either acetic acid or propionic acid (significantly higher in S than in OB) which are both known to inhibit yeasts and moulds (Woolford 1975, Pahlow and Muck 2009). Therefore, the improved aerobic stability must be a consequence of less aerobic microbial activity (yeasts and moulds) during the storage phase before silo opening.

It can be summarized that the OB silo sealing system had a beneficial effect on the hygienic status of the top 30 cm of silage with lower counts of moulds, yeasts and Clostridium perfringens. Possibly most importantly, the OB system improved the aerobic stability of the maize silage. These results are particularly interesting as it would generally be expected that the silage with the higher acetic acid concentration would provide a greater degree of stability. However, in this experiment the silage under the OB system contained less acetic acid than that under the S sealing system. Whilst oxygen concentrations in the silo were not measured, and no differences were found in aerobic bacterial numbers, differences in the number of samples containing moulds and yeasts were found. Therefore, it is likely that the increased aerobic stability in the upper layer of silage stored under the OB system was due to reduced oxygen permeation through the silo seal during the storage period.

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Van Soest P. J. 1963 Use of detergents in the analysis of fibrous feeds. II. A rapid method for the determination of fiber and lignin. Journal of the Association of Official Analytical Chemists 46: 829−35.

Wilkinson, J. M. & Rimini, R. 2002. Effect of triple co-extruded film on losses during the ensilage of ryegrass. In: Gechie, L. & Thomas, C. (eds.). Proceedings of the XIII International Silage Conference, 11−13 September, Auchincruive, Scotland. p. 168−169.

Woolford, M.K. 1975. Microbiological screening of the straight chain fatty acids (C1-CI2) as potential silage additives. Journal of the Science of Food and Agriculture 26: 219−228.

Woolford, M.K. 1984. The Silage Fermentation. New York: Marcel Dekker. 350 p.

AGRICULTURAL AND FOOD SCIENCER. Latsch & J. Sauter (2013) 22: 189–193

189

Comparison of methods for determining the density of grass silage

Roy Latsch1 and Joachim Sauter1 1Agroscope Reckenholz-Tänikon Research Station ART, Tänikon 1, 8356 Ettenhausen, Switzerland


In practice the only way to determine the bulk density of grass silage is to measure and weigh silage blocks. This study was carried out to compare this variant with four other measuring methods. “Big blocks” are inherently relatively heterogeneous and hence cannot be used for the fast, precise determination of density. “Small blocks” represent density well, but their handling makes them unsuitable for quick sampling. The three measuring methods - “drill-ing jig”, “inclined drilling cylinder” and “vertical drilling cylinder” - gave comparable results. The “inclined drilling cylinder” was identified as the preferred variant on the basis of results and manageability.

Key words: grass-silage, bulk-density, measuring-methods, drilling-cylinders, block-cutter

Introduction

Good forage compaction is essential for the production of high quality grass silage. It minimises reheating and the energy loss accompanying the opening of a silo. Good compaction reduces oxygen diffusion in the forage pile, which should not exceed 20 l m-2 h-1 after opening the silo (Honig 1987). Under these conditions the area of silage favouring the activity of harmful aerobic organisms such as acetic acid bacteria and mould fungi is minimal, and the silage remains stable.

Thus far there have been no methods of determining bulk density during silo filling. Therefore, an assessment of compaction quality can only be made after a silo is opened. Silage blocks cut by a tractor-mounted block cut-ter are used to determine compaction as they are easy to extract, weigh and measure. Normally this type of sampling is not used at problem zones like the inclined end surfaces of a silage pile or, the silage edges along the wall sides because of their asymmetry. Drilling cylinders such as those used to determine the density of maize si-lage can be used for this purpose (Bundesarbeitskreis Futterkonservierung 2012). Due to the fibrous structures of grass silage, however, this method produces mechanical disturbance in the samples. Especially when the cutting process begins, the grass at the surface can be twisted due to its interwoven fibres and therefore loosened by ro-tating forces. At the first centimetres inside the cylinder, the shearing forces of the rotating cylinder work against the compound structure of the grass silage and can also loosen up and disturb the drilling core of the silage. An existing “Apparatus for obtaining an undisturbed core of silage” (Rees et al. 1983) has not become widespread to date. Moreover, there is no standard sampling procedure available. The relationship between bulk density and silage quality is therefore being studied in a research project at the Swiss Federal Research Station Agroscope Reckenholz-Tänikon (ART).

Within this framework different sampling methods were compared to identify the best possible sampling variant to evaluate the density of grass silage. The aim of this study was to compare different hand-held samplers with the “silage block” method and determine a preferred method.



190

Material and methodsFor this investigation, two horizontal silos were available. The stored green material originated from both natural grassland and temporary ley. The theoretical cutting length of the loader wagon involved was 40 mm. Compac-tion was carried out by a standard ballasted tractor with a laden weight of 10230 kg and an internal tyre pressure of 2.5 bar. The mean overall bulk density of each silo investigated was recorded on the basis of the harvested pro-duct introduced and the measured overall volume of the forage pile. Unavoidable dry matter (DM) losses during the fermentation process that can amount up to 8−18% (Bundesarbeitskreis Futterkonservierung 2012, Köhler et al. 2012) and the settlement of the silage pile were not regarded when calculating this mean silo bulk density.

One silage “big block” was taken from each horizontal silo for comparison. The mean DM content of the blocks was 26.6 and 30.7%. The blocks were extracted with a Trioliet type TU 145 block cutter. Samples were subsequently taken from these two blocks with hand-held devices (Fig. 1).

Fig. 1. Sampling devices used in the trial: 1: Silage block cutter (variant “big block”), 2: Drilling cylinder of ART (variants “drilling cylinder inclined” and “drilling cylinder vertical”), 3: Electric silage cutter (variant “small block”), 4: PioneerTM drilling jig (variant “drilling jig”)

A hand-held electric silage cutter (OMC AS/85) was used to cut out “small blocks”. A preliminary test showed, that the work quality of both tested drilling cylinders differed depending on the drilling direction. Due to the small coarse teeth of the drilling jig (PioneerTM), that caused disturbances in the silage when drilling vertical and slid away when drilling inclined, the drilling jig was only used horizontally in this trail. The cone-shaped form of the jig prevented the silage from being pulled out of the cylinder at horizontal drilling direction. The volume of the samples taken with the drilling jig was determined by the drill hole diameter and the measured drill hole depth.

In contrast, the volume of the stainless steel drilling cylinder developed by ART was calculated, core drill-ing being limited to a defined length of 100 mm by slots in the drilling cylinder. This way to determine the volume of the sample should reduce the measurement error caused by loosened silage in the drilling cylin-der port. Because the straight cylinder form could not prevent the silage from being pulled out of the hori-zontally used cylinder, this sampler was only used in vertical and inclined drilling direction. The ART drill-ing cylinder was driven electrically at 120 rpm. Coarse teeth were notched into the chamfered cutting edge in order to chop the grass silage thoroughly. Further technical data of samplers used are listed in Table 1.

Table 1. Technical data of samplers used

variant sampler manufacturer type sample dimensions volume

(w × d × h) (m) (m3)

“silo” sensor bridge ART (Ettenhausen, CH) ultrasonics 6 × 25.6 × 1.4 215

“big block” block cutter Trioliet (Oldenzaal, NL) TU 145 1.75 × 0.75 × 1.2 1.575

“small block” electric silage cutter OMC (Correggio, IT) AS/85 0.2 × 0.15 × 0.2 0.006

“drilling jig” core drill Pioneer (Buxtehude, DE) Hi-Bred Ø 45 mm × depth variable

“drilling cylinder” core drill ART (Ettenhausen, CH) Ø 56 mm × 0.1 0.00025


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As silage blocks can expand vertically when extracted from the pile, the layers for testing (each 0.2 m) were pre-marked in the undisturbed silage. The height of the silage block was limited to 1.2 m for the trial. The precise mea-surements and weight of the silage blocks were determined following extraction.

Figure 2 shows the 18 designated sampling locations at six different levels and in three repetitions. The volume and weight of all the samples were calculated to determine density. The trial was supplemented by pairwise com-parisons of each of the sampling devices effected in the same manner directly in the silage pile.

Statistical analysis was carried out using a pairwise linear regression model (Tibco Spotfire S+, Somerville, MA, USA).


The densities determined from the two silage blocks and the small blocks and core drillings taken from them, as well as the extra samples, are summarised in Table 2.

Table 2. Data to measured bulk density (kg FM m-3)

big block extra samples

silo 1 silo 2 silo 1

Mean SD n Mean SD n Mean SD n

variant (kg FM m-3) (qty) (kg FM m-3) (qty) (kg FM m-3) (qty)

“silo” 690 1 756 1

“big block” 857 1 880 1

“small block” 800 123 15 824 82 15 791 143 11

“drilling jig” 694 95 15 811 88 15

“drilling cylinder inclined” 769 117 15 768 101 15

“drilling cylinder vertical” 807 116 15 816 81 15 737 129 11

SD: standard deviation, n: sample size

Compared with the target values put forward by Honig (1991) (800 kg FM m-3 at 20% DM content; 560 kg FM m-3 at 40% DM content) the densities of both “big blocks” (27 and 31% DM content), and hence of the overall silos, may be considered high. By comparison, the density of the “big block” from Silo 1 was 24% higher, the “big block” from Silo 2 16% higher than that of the respective overall silo. The comparison of “big blocks” and “small blocks” demonstrates the enormous heterogeneity of silage blocks (Fig. 3). For example: the black squares in Fig. 3 repre-sent the “small block” samples, that were taken from “big block 1” (mean bulk density: 857 kg FM m-3) which had its source from silo 1 (mean bulk density: 690 kg FM m-3). The data show a huge heterogeneity not only in the dif-ferent heights of the “big block”, but also within the three repetitions of the same height.

AB C

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Fig. 2. Scheme of the silage block with the spatial allocation of the four different sampling methods in three repetitions.


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An explanation for the not inconsiderable difference between “silo” and “big blocks” may be that the “big blocks” were taken from well compacted positions in the silage pile and not from problematic silo zones like the beginning and end, the wall areas or silage surfaces. Evaluation of the measurements confirms observations whereby den-sity decreases as distance from the base plate increases (Craig and Roth 2005, D’Amours and Savoie 2005). Whole silage blocks are therefore only suitable for a quick assessment of the mean overall density of horizontal silos. The “small block” method was subsequently used as a reference for the comparability of selectively drawn samples.

Figure 4 shows the values for the three drilling variants with reference to the “small block” variant. The residual standard error (Res. SE), as a measure of the dispersion of the data points around the regression line, is compar-atively close together in the three drilling variants. Here the “inclined drilling cylinder” variant compares favour-ably with the other two variants due to lower dispersion, expressed by a lower Standard Error. But if, for example, the difference in the prediction accuracy of both drilling cylinder variants is calculated, these only differ by be-tween 1 and 2%. Both the gradient and the displacement of the regression lines to the x=yline were calculated for x = 869 kg FM m-3 (mean bulk density of both “big blocks”), but played a subordinate role in the given dispersion range of the values. All three variants underestimated the density of the reference “small block”.

Fig. 3. Mean variation of silage density in big blocks determined by small block variant

Fig. 4. Silage density of the drilling variants with reference to the small block (coef a: Shift of regression line from x=y-line in point x = 869 kg FM m-3, coef b: Difference in inclination between regression line and x=y-line, Res. SE: Residual Standard Error, N: number of samples)


193

Kleinmans et al. (2005) and Thaysen et al. (2006) reported good results with the drilling jig in maize silage. Ana-logously to the results shown here, they also reported that the drilling jig tends to underestimate bulk density.

Horizontal drilling is recommended by Kleinmans et al. (2005) for the extraction of maize with the drilling jig. By comparison, the fibrous structure of grass silage results in the silage being pulled out of the drilling jig again during horizontal sampling and measurement of the drill hole depth reduces the calculated density. Drilling car-ried out at an angle to the horizontal bedding layers of the silage generally effects better separation of the grass silage fibres and the individual layers are no longer pulled out of the drilling jig.

The drilling cylinder used in this trial was driven by an electric drill. This represents a huge saving in labour, particularly when extracting a sizeable number of samples. An inclined drilling direction is preferable to a vertical one, as in this way samples can be taken at the cutting point of the silage.

Conclusions

Samples taken with hand-held devices testified to the enormous heterogeneity of density conditions within the silage blocks. A sizeable number of small silage samples represent the heterogeneous density conditions with-in horizontal silos whereas large-volume samples only express one single average. Drilling should be carried out obliquely or vertically in relation to the bedding direction of the fibres in order to cut the fibrous structure of the grass silage and to obtain good filling of the drilling cylinder. In statistical analysis the drilling variants tested showed only slight differences of between 1 and 2% in density prediction accuracy, the tendency being to underestimate the density compared to “small blocks”. Rather better statistical consistency with the reference “small block” and comparatively easier handling made the “inclined drilling cylinder” variant the preferred variant in this trial.

ReferencesBundesarbeitskreis Futterkonservierung (ed.) 2012. Praxishandbuch Futter– und Substratkonservierung. 8. überarbeitete Auflage 2011. DLG-Verlag. 416 p. (in German).

Craig, P.H. & Roth, G. 2005. Bunker silo density study – Summary report 2004–2005. Penn State University, Dauphin, PA, USA.

D’Amours, L. & Savoie, P. 2005. Density profile of corn silage in bunker silos. Canadian Biosystems Engeneering 47. p. 2.21–2.28.

Honig, H. 1987. Gärbiologische Voraussetzungen zur Gewinnung qualitätsreicher Anwelksilage. Grünfutterernte und -konservier-ung KTBL-Schrift Nr. 318. p. 47–58. (in German).

Honig, H. 1991. Reducing losses during storage and unloading of silage. Landbauforschung Völkenrode Sonderheft 123: 116–128.

Kleinmans, J., Ruser, B., Oetjen, G. & Thaysen, J. 2005. Eine neue Methode zur Bestimmung der Silageverdichtung – Einsatz des Probenbohrers in der Praxis. Mais 32: 134–136. (in German).

Köhler, B., Diepolder, M., Ostertag, J., Thurner, S. & Spiekers, H. 2012. Dry matter losses of grass and maize silage in bunker si-los. In: Kuoppala, K., Rinne, M. & Vanhatalo, A. (eds.). XVI International Silage Conference. Hämeenlinna, Finland. p. 318–319.

Miller, A.M. 2006. Gute, stabile Maissilagen: Verteil- und Walzarbeiten entscheiden über den Erfolg. Milchpraxis 44: 118–119. (in German).

Rees, D.V.H., Audsley, E. & Neale, M.A. 1983. Apparatus for obtaining an undisturbed core of silage and for measuring the poros-ity and gas diffusion in the sample. Journal of Agricultural Engineering Research 28: 107–114.

Thaysen, J., Ruser, B. & Kleinmanns, J. 2006. Dichte Controlling – Bedeutung und Instrumente. In: Gesellschaft für Kunststoffe im Landbau e.V. GKL-Frühjahrstagung 2006 – Siliererfolg auch bei großen Erntemassen. Bonn. p. 14–17. (in German).

AGRICULTURAL AND FOOD SCIENCEJ. McEniry and P. O’Kiely (2013) 22: 194–200

194

The estimated nutritive value of three common grassland species at three primary growth harvest dates following ensiling and

fractionation of press-cakeJoseph McEniry and Padraig O’Kiely*

Animal & Grassland Research and Innovation Centre, Teagasc, Grange, Dunsany, Co. Meath, Ireland*e-mail: [email protected]

In a Green Biorefinery processing green biomass one possible application for the press-cake fraction is as a feedstuff for ruminants. This study investigates the effects of ensiling and fractionation on the estimated nutritive value of three grassland species harvested at different stages of maturity. Perennial ryegrass (Lolium perenne L., var. ‘Gan-dalf’), cocksfoot (Dactylis glomerata L., var. ’Pizza’) and red clover (Trifolium pratense L., var. ‘Merviot’,) were grown in field plots and harvested and ensiled in laboratory silos. These silages were subsequently fractionated into press-cake and press-juice fractions. Loss of soluble, fermentable organic matter during ensiling increased the relative proportions of fibre and crude protein. Fractionation resulted in the substantial reduction of herbage soluble nutri-ent and mineral content, increasing the fibre content and reducing digestibility and crude protein. The low energy and protein content of the press-cake fraction, especially at later harvest dates, will limit its use in ruminant diets.

Key words: Green Biorefinery, grass silage, ruminant feedstuff

IntroductionThe need to develop alternatives to non-renewable fossil fuel-derived products has stimulated an interest in plant biomass to provide renewable energy, chemicals and materials. A ‘Green Biorefinery’ involves the sustain-able processing of green biomass into a spectrum of marketable products and energy (Mandl 2010). Most tech-nological concepts of a Green Biorefinery involve the fractionation of the cell contents (i.e. press-juice) from the plant structural framework (i.e. fibre-rich press-cake) and these separated fractions can be subjected to a series of downstream processes to recover or produce valuable products (McEniry et al. 2011). For example, lactic acid and amino acids can be extracted from the press-juice fraction (Ecker et al. 2012), while Grass (2004) described the production of thermal insulation boards from the separated press-cake fraction. Another possible application for the press-cake fraction is as a feedstuff for ruminants.

Two of the main management factors affecting herbage quality and nutritive value are herbage species and stage of maturity at harvest. Herbage harvested at an early vegetative rather than a later growth stage generally has a lower fibre content, and a higher digestibility and crude protein (CP) concentration (Buxton and O’Kiely 2003). In temperate European grasslands, perennial ryegrass (Lolium perenne L.) is commonly the preferred grass species for animal production because of its high digestibility and reduced fibre concentration (Whitehead 1995). How-ever, other grassland species may have different physical and chemical characteristics which may offer benefits for non-agricultural uses or be more suited to specific management (e.g. response to fertiliser) or environmental (e.g. temperature, water deficit) conditions.

Preservation of herbage as silage so as to ensure year-round availability, and its subsequent fractionation, represent two major process steps in the utilization of green biomass as an industrial feedstock. However, limited information is available on the impact of these two processing steps on the nutritive value of the separated press-cake fraction. It would be expected that the process of fractionation would have a much greater deleterious effect on indices of nutritive value. Thus, the objective of this study was to quantify separately the effects of ensiling and fractiona-tion on the estimated nutritive value of three common grassland species harvested at different stages of maturity.



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Material and methodsExperimental plots, harvesting and ensiling

Triplicate plots of each of two common grass species, perennial ryegrass (PRG; Lolium perenne L., var. ‘Gandalf’) and cocksfoot (Dactylis glomerata L., var. ‘Pizza’), and one leguminous species, red clover (Trifolium pratense L., var. ‘Merviot’), were grown in field plots (each 20 m2) at Grange (53° 52’ N, 06° 66’ W) under an inorganic fertilis-er N input of 125 kg N ha-1 (except red clover which received no fertiliser N input). Separate plots were harvested at three dates (12 May [Harvest 1], 9 June [Harvest 2] and 7 July [Harvest 3]) in the primary growth in 2009 (n = 27 plots), as described previously by King et al. (2012a). On each of these dates, herbage from each plot was har-vested as direct-cut material using a Haldrup forage plot harvester (J. Haldrup, Løgstor, Denmark) to an average 6 cm stubble height, passed through a precision-chop harvester (MEX VI, Pöttinger, Grieskirchen, Austria; nominal chop-length of 19 mm) and a representative 6 kg sample from each plot was ensiled in laboratory silos (O’Kiely and Wilson 1991) for 100 days. Representative samples of each herbage pre- and post-ensiling were stored at -18 ⁰C prior to chemical analyses.

FractionationRepresentative 200 g samples of each silage (i.e. one per plot) were fractionated into press-cake and press-juice fractions, as described previously by King et al. (2012b). Briefly, 600 ml of deionised water at 60 °C, with deter-gent (30 g L-1; sodium dodecyl sulphate, Sigma Aldrich), was added to each representative silage sample. Sodium dodecyl sulphate is used routinely in the neutral detergent fibre assay of feedstuffs to help to dissolve pectins and plant cell components and was included to enhance the fractionation process. These 3:1 (water + detergent: si-lage) mixtures were maintained at 60 °C in a water bath for 30 minutes, with thorough mixing. After 30 minutes, excess liquid was drained off using a sieve and the washing step was repeated. After the second washing step the herbage was mechanically pressed (4.5 MPa) using a hydraulic press to remove the press-juice fraction. The sepa-rated press-cake fractions were stored at −18 ⁰C prior to chemical analyses.

Chemical analysesRepresentative herbage samples pre-ensiling were dried at 98 ⁰C, whilst herbage samples post-ensiling and press-cake fractions were dried at 85 ⁰C, for 16 h in an oven with forced air circulation to estimate dry matter (DM) concentration. The DM concentration of the samples post-ensiling were corrected for the loss of volatiles by the equation of Porter and Murray (2001). Replicate samples were also dried at 40 ⁰C for 48 hours before be-ing milled (Wiley mill; 1 mm screen). Dried, milled samples were used for the determination of neutral detergent fibre (NDF), CP and ash, as described previously by King et al. (2012a). In vitro dry matter digestibility (DMD) was determined for the herbage samples pre- and post-ensiling by the ‘two-stage rumen fluid’ method of Tilley and Terry (1963). The separated press-cake fractions appeared to be deficient in N resulting in the failure of this as-say. Consequently, the DMD of this material was determined using the ‘pepsin-cellulase’ method of Aufrère and Demarquilly (1989). The potential net energy content (i.e. feed unit for maintenance and meat production; UFV kg-1 DM) of the press-cake fractions was estimated using herbage DMD values (UFV = 0.00145773x – 0.27977273; where x = DMD [g kg-1]) according to O’Mara (1996).

Using silage samples taken prior to drying, the pH was determined from an aqueous extract using a handheld pH meter (R 315 pH, Reagecon Diagnostics Ltd., Dublin, Ireland). Further juice was extracted for the analysis of lac-tic acid (LA), volatile fatty acids (i.e. acetic acid and butyric acid), ethanol and ammonia-N as described previously by King et al. (2012c).

Statistical analysisMeans and standard deviations (SD) were calculated for herbage chemical composition pre- and post-ensiling and for the separated press-cake fraction (Table 1). Means (SD) were also calculated for silage fermentation charac-teristics and this data is presented in Table 2. To investigate the true effects of harvest date and species on DMD and CP concentration, data for the herbage pre-ensiling were analysed separately as a split-plot design with har-vest date as the main plot and herbage species as the subplot, and accounting for replicate blocks, using the Proc MIXED procedure of SAS, Version 9.1.2. The changes in DMD and CP concentration as a result of both ensiling and fractionation were calculated by subtracting values for (a) herbage pre-ensiling from herbage post-ensiling and (b) herbage post-ensiling from the press-cake fraction, respectively. These data were analysed as a split-plot de-sign, according to the procedure already described. The Tukey test was used to detect the significant differences between means.


196

ResultsHarvest date and species (herbage pre-ensiling)

On average for the herbage samples pre-ensiling, DMD decreased (p<0.01) with advancing harvest date, with herbage CP concentration being highest (p<0.001) for the early harvest date (Harvest 1, May 12) (Tables 1 and 3). Of the three herbage species investigated, red clover had a higher (p<0.05) DMD than cocksfoot and a higher herbage (p<0.001) CP concentration than both grass species. Of the two grass species investigated, on average cocksfoot had a higher (p<0.01) herbage CP concentration than the PRG.

Table 1. The effect of harvest date and species on mean (SD) herbage dry matter (DM: g kg-1), neutral detergent fibre (NDF: g kg-1 DM) and ash (g kg-1 DM) concentrations pre- and post-ensiling and on the separated press-cake fractionHarvest Species Type DM NDF Ash1 Perennial ryegrass Pre-ensiling 168 (5.8) 496 (12.5) 95 (3.6)(May 12) Perennial ryegrass Post-ensiling 163 (7.5) 512 (4.8) 110 (5.5)

Perennial ryegrass Press-cake 307 (43.0) 873 (29.9) 36 (5.9)Cocksfoot Pre-ensiling 180 (19.9) 518 (23.5) 96 (1.9)Cocksfoot Post-ensiling 159 (2.0) 535 (38.7) 108 (2.0)Cocksfoot Press-cake 262 (22.6) 821 (11.4) 39 (0.5)Red clover Pre-ensiling 142 (5.1) 387 (21.7) 101 (3.0)Red clover Post-ensiling 170 (2.6) 405 (10.5) 101 (1.5)Red clover Press-cake 199 (24.4) 644 (30.5) 59 (3.3)

2 Perennial ryegrass Pre-ensiling 220 (8.4) 609 (22.3) 77 (2.9)(June 9) Perennial ryegrass Post-ensiling 218 (6.5) 628 (23.1) 86 (4.3)


3 Perennial ryegrass Pre-ensiling 233 (20.6) 635 (4.6) 72 (3.3)(July 7) Perennial ryegrass Post-ensiling 234 (9.4) 658 (37.4) 73 (2.4)


Ensiling On average, the decrease in DMD as a result of ensiling did not differ (p>0.05) across the three harvest dates or the three species investigated (Table 3). Similarly, the increase in CP concentration with ensiling did not dif-fer (p>0.05) across the three harvest dates, but this increase was higher (p<0.05) for the PRG compared with the cocksfoot herbage.


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Table 2. The effect of harvest date and species on mean (SD) silage fermentation characteristics (g kg-1 DM, except for pH and unless stated otherwise)

Harvest Species pH LA AA BA EtOH NH3-N1 Perennial ryegrass 4.07 (0.181) 93 (37.7) 37 (11.7) 0.0 36 (5.1) 58 (26.4)(May 12) Cocksfoot 4.37 (0.185) 63 (10.2) 43 (11.7) 3.8 (6.58) 36 (12.5) 72 (23.5)

Red clover 4.15 (0.138) 112 (38.3) 22 (5.8) 1.1 (1.82) 16 (2.4) 61 (30.9)2 Perennial ryegrass 3.81 (0.076) 66 (4.8) 16 (3.6) 0.0 32 (14.3) 49 (7.6)(June 9) Cocksfoot 3.90 (0.030) 64 (6.1) 17 (5.4) 0.0 9 (4.4) 52 (3.7)

Red clover 3.92 (0.101) 86 (8.1) 20 (3.2) 0.0 11 (1.6) 47 (11.6)3 Perennial ryegrass 3.52 (0.081) 76 (4.3) 23 (7.2) 0.0 27 (10.3) 62 (11.1)(July 7) Cocksfoot 4.33 (0.519) 21 (27.3) 27 (9.7) 7.5 (1.26) 19 (6.6) 120 (57.3)

Red clover 4.58 (0.254) 35 (9.1) 54 (21.8) 3.3 (5.78) 40 (19.7) 69 (15.5)Abbreviations: LA, lactic acid; AA, acetic acid; BA, butyric acid; EtOH, ethanol; NH3-N, ammonia-N (g kg-1 N)

FractionationOn average, the numerical decrease in DMD as a result of fractionation was lower (p<0.01) for the later harvest date (Harvest 3, July 7), while no difference (p>0.05) was observed between Harvests 1 and 2 or between the three species investigated (Tables 1 and 3). In contrast, the numerical decrease in CP concentration was highest (p<0.001) for the Harvest 1 herbage compared with the later harvest dates. Finally, the numerical decrease in CP concentration was higher (p<0.01) for the red clover compared with the two grasses. The estimated net energy content (UFV kg-1 DM) of the press-cake fractions from the Harvest 1, 2 and 3 PRG, cocksfoot and red clover were 0.67, 0.48 and 0.40, 0.59, 0.46 and 0.38, and 0.66, 0.58 and 0.49, respectively.

Table 3. The effect of harvest date and species on herbage dry matter digestibility (DMD: g kg-1) and crude protein concentration (CP: g kg-1 DM) pre-ensiling and on the changes (negative value indicates a decrease) in chemical composition as a result of ensiling (herbage post-ensiling – herbage pre-ensiling) and fractionation (press-cake fraction – herbage post-ensiling).

Harvest Species Pre-ensiling Ensiling (changes) Fractionation (changes)DMD CP DMD CP DMD CP

1 Perennial ryegrass 785 141 -1.9 17.0 -133.5 -112.7(May 12) Cocksfoot 738 162 -5.5 7.3 -133.7 -107.7

Red clover 812 195 -52.8 7.3 -117.2 -113.22 Perennial ryegrass 702 102 -40.8 11.2 -140.1 -76.4(June 9) Cocksfoot 653 120 -18.3 4.9 -128.8 -70.5

Red clover 686 160 -44.2 6.5 -49.4 -93.83 Perennial ryegrass 569 88 -16.6 12.8 -83.8 -58.7(July 7) Cocksfoot 531 114 -85.0 -5.4 -6.2 -64.1

Red clover 611 159 -35.8 16.3 -45.8 -111.3

Standard error of the mean

Main plot (Harvest) 19.5 4.3 17.83 3.34 14.56 5.20Sub-plot (Species) 17.3 4.3 17.83 2.99 14.56 5.20Harvest x species 30.0 7.5 30.88 5.17 25.22 9.00

Levels of significance

Harvest ** *** NS NS ** ***Species * *** NS * NS **Harvest x species NS NS NS NS NS NS

Abbreviations: ***, p<0.001, **, p<0.01, *, p<0.05, NS, not significant


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Discussion

The effect of ensiling and fractionation on herbage DM, NDF and ash concentrations has been presented and dis-cussed in detail by McEniry et al. (2012). Similarly, the effect of harvest date and species on silage fermentation characteristics has been discussed in detail by King et al. (2012c). The major focus of this paper is on the effects of ensiling and fractionation on indices of nutritive value and on the suitability of the separated press-cake frac-tion as a feedstuff for ruminants.

Harvest date and speciesThe decrease in DMD and CP concentration with advancing harvest date reflects the general change in herbage chemical composition as a grass plant matures. With advancing stage of maturity, the proportion of cell wall com-ponents increases relative to the proportion of cell contents (e.g. crude protein) (Buxton 1996). The corresponding decline in herbage quality reflects the decrease in the ratio of leaf to stem tissue and the greater rate of decline in digestibility of the stem compared with the leaf due to increased lignification (Hatfield 1993).

Perennial ryegrass is commonly the preferred grass species for animal production because of its high digestibility and reduced fibre concentration (Whitehead 1995), which is apparent in the current study. The DMD of the red clover was closely related to that of the PRG and the higher CP concentration in red clover compared with the two grass species reflects the higher inherent CP concentration in legumes (Frame and Laidlaw 2011).

EnsilingWhile fresh grass can be used as an industrial biomass feedstock for a Green Biorefinery, in most cases it may be necessary to preserve it as silage to ensure year round availability of a predictable quality feedstock. The general decrease in herbage DMD (–33 g kg-1 on average) during ensiling in this study largely reflects the increase in NDF concentration for most silages. Small increases in the relative proportions of NDF, ash and CP occurred during en-siling as a result of a loss in organic matter during fermentation and through effluent production. For example, ef-fluent production from the Harvest 1 PRG, cocksfoot and red clover samples was 117, 50 and 287 g effluent kg-1 fresh material ensiled, respectively. These relatively high levels of effluent production (most evident for the PRG and red clover herbages) and poor preservation for some herbages (evident for the Harvest 3 cocksfoot herbage; where silage pH was 4.33 and lactic acid as a proportion of total fermentation products was 0.27) contributed to the general decrease in herbage DMD. In general, however, the DMD of the Harvest 1 silages (783, 732 and 759 g kg-1 for PRG, cocksfoot and red clover, respectively) reflected excellent leafy silage and the relative changes in herbage nutritive value during ensiling were minor compared to the large negative effect of advancing harvest date on DMD.

FractionationThe various technological options for a Green Biorefinery often involve the essential process of fractionating plant biomass into a fibre-rich press-cake and a nutrient rich press-juice. The viability of such an industrial process will depend on the range of suitable applications identified for the separated fractions (Kromus et al. 2004). The use of the press-cake fraction as a feedstuff for ruminants could be considered a relatively low-value application; nev-ertheless, it could be an important part of an overall biorefining process where, for example, high-value products were being produced from the press-juice fraction (e.g. lactic acid, amino acids).

Wachendorf et al. (2009) reported that fractionation reduces herbage water content and the concentration of minerals, and leads to the enrichment of fibrous constituents in the press-cake fraction. Similarly, McEniry et al. (2012) reported that > 0.55 of the N and ash was removed from the press-cake fraction relative to the fresh ma-terial pre-ensiling. In the current study, the removal of a large proportion of the herbage soluble and mineral con-tent during fractionation increased the proportion of fibrous constituents in the press-cake. Fractionation also resulted in an average 0.66 reduction in CP concentration in the press-cake fraction relative to the fresh material pre-ensiling, and this was higher for the red clover and the Harvest 1 material, reflecting the higher CP concentra-tion in these herbages prior to fractionation. Furthermore, the failure of the ‘two-stage rumen fluid’ DMD assay for some of the press-cake fractions also reflects the reduction in CP concentration and a resulting deficiency in N for rumen microorganisms. In general, the smallest decrease in DMD and herbage CP concentration as a result of fractionation was observed for the Harvest 3 herbages and this likely reflects the relatively high fibre and low CP concentrations in these herbages prior to fractionation.


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Press-cake fraction as a feedstuff for ruminants

Management factors such as harvest date and species impact on the quality and nutritive value of the herbage presented for fractionation and this can subsequently influence the estimated nutritive value of the separated press-cake fraction. For example, the DMD of the press-cake fraction prepared from the Harvest 1 PRG and red clover silages (650 and 642 g kg-1 for PRG and red clover, respectively) could be compared with 2nd cut grass silage (662 g kg-1) on a net energy basis (0.67, 0.66 and 0.68 UFV kg-1 DM, respectively) (O’Mara 1996), and may have some potential for animal maintenance and low rates of production. However, this material would have to be sup-plemented to make up for its relatively low CP concentration (46 and 89 g kg-1 DM for the press-cake fraction from Harvest 1 PRG and red clover, respectively). With the exception of the Harvest 1 red clover, the CP concentration in all the press-cake fractions was below the requirement (< 0.08 of diet) to support adequate rumen function and this may reduce fibre digestion in the rumen, ultimately depressing herbage DM intake (Coleman and Moore 2003).

Furthermore, the net energy content of the press-cake fraction prepared from the Harvest 1 cocksfoot and Har-vest 2 red clover (0.59 and 0.58 UFV kg-1 DM, respectively) could be compared with good quality hay (0.60 UFV kg-1 DM) (Jarrige 1989). In contrast, the press-cake fraction prepared from all other later-harvested herbage had low DMD and UFV values (ranging from 444 – 529 g kg-1 and 0.37 – 0.49 UFV kg-1 DM, respectively) similar to that reported for straw (Jarrige 1989). This suggests that any potential of the press-cake fraction as a feedstuff for ru-minants is lost at later harvest dates.

In conclusion, ensiling only has a limited impact on herbage nutritive value. In contrast, fractionation resulted in a substantial increase in fibre content and a decrease in digestibility and crude protein concentration. The low energy and protein content of the resulting press-cake fraction, especially at later harvest dates, will limit its use in ruminant diets.

AcknowledgementsFunding for this research was provided under the National Development Plan, through the Research Stimulus Fund (RSF 07 557), administered by the Department of Agriculture, Food and the Marine, Ireland. The authors thank Ms. B. Weldon, farm staff and the chemical analysis laboratory staff at Teagasc Grange and Moorepark.

ReferencesAufrère, J. & Demarquilly, C. 1989. Predicting organic matter digestibility of forage by two pepsin-cellulase methods. In: Pro-ceedings of the 16th International Grassland Congress, in October in Nice, France. Versailles, France: Association Francaise pour la Production Fourragère. p. 877–878.

Buxton, D.R. 1996. Quality-related characteristics of forages as influenced by plant environment and agronomic factors. Animal Feed Science and Technology 59: 37−49.

Buxton, D.R. & O’Kiely, P. 2003. Preharvest plant factors affecting ensiling. In: Buxton D.R., Muck R.E. & Harrison J.H. (eds.) Silage Science and Technology Wisconsin, USA: American Society of Agronomy. p. 199−250.

Coleman, S.W. & Moore, J.E. 2003. Feed quality and animal performance. Field Crops Research 84: 17−29.

Ecker, J., Schaffenberger, M., Koschuh, W., Mandl, M., Böchzelt, H.G., Schnitzer, H., Harasek, M. & Steinmüller, H. 2012. Green Bi-orefinery upper Austria-pilot plant operation. Separation and Purification Technology 96: 237−247.

Frame, J. & Laidlaw, A.S. 2011. Improved Grassland Management. Wiltshire, UK: The Crowood Press Ltd. 350 p.

Grass, S. 2004. Utilisation of grass for production of fibres, protein and energy. In: Proceedings of the OECD Workshop on Biomass and Agriculture, in September in Paris, France. Paris, France: OECD Publication Service. p. 169−177.

Hatfield, R.D. 1993. Cell wall polysaccharide interaction and degradability. In: Jung, H.J., Buxton, D.R., Hatfield, R.D. & Ralph, J. (eds.). Forage cell wall structure and digestibility. Wisconsin, USA: American Society of Agronomy. p. 285–313.

Jarrige, R. 1989. Ruminant Nutrition – recommended allowances and feed tables. London, UK: John Libbey Eurotext. 400 p.

King, C., McEniry, J., Richardson, M. & O’Kiely, P. 2012a. Yield and chemical composition of five common grassland species in re-sponse to nitrogen fertiliser application and phenological growth stage. Acta Agriculturae Scandinavica, Section B - Soil & Plant Science 67: 644−658.

King, C., McEniry, J., Richardson, M. & O’Kiely, P. 2012b. The effects of hydrothermal conditioning, detergent and mechanical pressing on the isolation of the fibre-rich press-cake fraction from a range of grass silages. Biomass and Bioenergy 42: 179−188.

King, C., McEniry, J., Richardson, M. & O’Kiely, P. 2012c. Silage fermentation characteristics of grass species grown under two ni-trogen fertiliser inputs and harvested at advancing maturity in the spring growth. Grassland Science (in press).

Kromus, S., Wachter, B., Koschuh, W., Mandl, M., Krotscheck, C. & Narodoslawsky, M. 2004. The green biorefinery Austria - de-velopment of an integrated system for green biomass utilization. Chemical and Biochemical Engineering Quarterly 18: 7−12.


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Mandl, M.G. 2010. Status of green biorefining in Europe. Biofuels, Bioproducts Biorefining 4: 268−274.

McEniry, J., Finnan, J., King, C. & O’Kiely, P. 2012. The effect of ensiling and fractionation on the suitability for combustion of three grassland species harvested at sequential growth stages. Grass and Forage Science 67: 559−568.

McEniry J., King C. & O’Kiely P. 2011. The grass is greener - alternative applications for Irish grass. T-Research 6: 26−27. Cited 30 July 2012. Available on the Internet: http://www.teagasc.ie/publications/2011/1002/TResearch_201106.pdf

O’Kiely, P. & Wilson, R.K. 1991. Comparison of three silo types used to study in-silo processes. Irish Journal of Agriculture and Food Research 30: 53−60.

O’Mara, F. 1996. A net energy system for cattle and sheep. Dublin, Ireland: Department of Animal Science and Production, Uni-versity College Dublin. 113 p.

Porter, M.G. & Murray, R.S. 2001. The volatility of components of grass silage on oven drying and the inter-relationship between dry-matter content estimated by different analytical methods. Grass and Forage Science 56: 405−411.

Tilley, J.M.A. & Terry, R.A. 1963. A two-stage technique for the in vitro digestion of forage crops. Journal of the British Grassland Association 18: 104−111.

Wachendorf, M., Richter, F., Fricke, T., Grab, R. & Neff, R. 2009. Utilization of semi-natural grassland through integrated genera-tion of solid fuel and biogas from biomass. I. Effects of hydrothermal conditioning and mechanical dehydration on mass flows of organic and mineral plant compounds, and nutrient balances. Grass and Forage Science 64: 132−143.

Whitehead, D.C. 1995. Grassland Nitrogen. Wallingford, UK: CAB International. 397 p.

AGRICULTURAL AND FOOD SCIENCEP. I. Sarria & S.D. Martens (2013) 22: 201–206

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The voluntary intake in growing pigs of four ensiled forage species Patricia I. Sarria B.1 and Siriwan D. Martens2

1Universidad Nacional de Colombia, Facultad de Ciencias Agropecuarias, Carrera 32 No. 12-100, AA 237, Palmira, Colombia.2International Center for Tropical Agriculture (CIAT), Tropical Forages Program, Recta Cali-Palmira km 17, Cali, Colombia.


Forage can potentially be food resource for pig feeding in the tropics. The palatability of silages by pigs may be bet-ter than that of fresh forage. Foliage silage contains more dry matter than green forage and has a pleasant smell. Thirty commercial pigs (47.0 ± 4.7 kg live weight LW), were used to assess the silage intake capacity of pigs when feeding the legumes Clitoria ternatea, Centrosema brasilianum, Cratylia argentea and the Brachiaria grass hybrid Mulato II. The silages were offered ad libitum as a supplement to a normal balanced diet based on maize and soy bean meal. A crossover design was applied comprising five treatments, Control and the four silage supplements respectively. Daily consumption of dry matter -expressed in g of DM kg-1 metabolic LW- were similar (p>0.05) for diets containing C. argentea, C. ternatea and the Control. Daily consumption of C. brasilianum and Brachiaria was significantly lower (p<0.001). In conclusion, C. argentea and C. ternatea silages have the potential to serve as feed supplement in pig diets.

Key words: dry matter intake, tropical forages, fibre, monogastric animals, silage

Introduction

In the tropics, conventional feed concentrates are mostly imported, often hardly available and quite expensive for smallholders; therefore, locally available alternatives are needed. Improved grasses and forage legumes in-tegrated into smallholder tropical feeding systems can increase the benefits of livestock, for cattle, small rumi-nants, pigs, and/or poultry (Peters 2009). The humid and sub-humid tropics offer almost year-round growing con-ditions (Ly 2005), with seasonal water deficits and excesses; however these constraints are usually manageable (Martens et al. 2012). Forage crops like Vigna unguiculata, Xanthosoma sagittifolium, Morus alba, Trichanthera gigantea, among other examples, may offer an additional feed source for pigs due to their high protein content (particularly legumes), and yields of biomass (Leterme et al. 2005, Ly 2005 and Sarria et al. 2010). Though, the ef-fect of the inclusion of these fibrous feed into the diets of growing pigs needs to be studied. The results of grow-ing pigs receiving foliage as part of the diet, to date are highly variable according to different authors (Sarria et al. 1991, Leterme et al. 2005 and Ly 2005), and some food like forages, with elevated levels of fibre may have nega-tive effects on food intake, digestibility and weight gain (Santomá 1997). Creative approaches are required to fit forage-based feed solutions for monogastric animals into existing smallholder systems. Furthermore, systematic research is required to define the actual value of some less-common forage species for different animal species (Martens et al. 2012). Ensiling forages allows the crops to be harvested at the optimal time and additionally pre-serve its nutritional quality. Moreover, in pigs, the palatability of ensiled forage may be better than that of fresh herbage (Artiles et al. 2012). In addition, wilting helps to reduce the volume and concentrate the nutrients. The higher the dry matter (DM) content, the better the consumption of forage by pigs: for instance pigs of around 100 kg LW consume 0.5 kg DM in the form of fresh leaves but one kg as dried leaves (Leterme et al. 2005). Silage making requires less energy and time with a target DM content of around 35% DM, in contrast to the production of herbage meal which comprises ≥ 90% DM. In a smallholder context in the sub-humid tropics this fact alone, along with the lack of a powerful mill, can make ensiling the preferred method of choice.

The objective of our study was to assess the palatability of ensiled herbage for its use in the fattening of pigs. Four species of forage silage were evaluated in this experiment (Table 1). Forage species and accessions with a high potential for feeding monogastric animals were selected based on the information available in the SoFT database (Cook et al. 2005) in the literature, in pilot experiments and expert knowledge.

Manuscript received October 2012


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Materials and methods

Forages were grown in Southwest of Colombia: Herbaceous legumes C. ternatea (12 weeks growth, flowering) and C. brasilianum (17 weeks, pre-flowering) at CIAT Palmira (03°31’42” latitude N and 76°27’14” longitude W), the shrub legume C. argentea in Santander de Quilichao, (03°00’34” latitude N and 76°29’06” longitude W), and the Brachiaria sp. grass hybrid Mulato II (regrowth with high percentage of dead material) from Popayan (02°26’18” latitude N and 76°’47” longitude W).

Table 1. Tropical forage accessions of different species evaluated in the present experiment

Clitoria ternatea L. CIAT 20692

Centrosema brasilianum (L.) Benth. CIAT 5234

Cratylia argentea (Desv.) Kuntze CIAT 18516/18668

Brachiaria sp. grass hybrid Mulato II CIAT 36087

Management and ProductionContinuity of production Medium Medium Superior SuperiorAltitude, m above sea level 1600−2000 0−1000 0−1200 0−1800Rain precipitation, mm/year 400−2500 600−3000 1000−4000 ≥ 700

Adapted to low fertility soils Medium Superior Superior Superior

Yield leaf, dry matter/hectare/year 3−10 3−10 13−32 25

Nutritional value:Crude protein, % 17−20 12−20 18−30 12−15Anti-nutritive compounds Medium Superior Superior Not determinedRuminant digestibility, % 80 50−70 60−65 55−62Ruminant voluntary intake Medium Medium Medium Superior

Countries were species are reported

Australia, Kenya, The Philippines and Mexico1, Honduras, Mexico2, East Africa, Uganda, India3

Colombia, Australia2, Venezuela, Nigeria3

Costa Rica, Colombia and Nicaragua2

China, Thailand, Mexico, Central America countries, Panama, Colombia, Venezuela, Bolivia, Uruguay and Brasil2

1Villanueva et al. 2004, 2Peters et al. 2011, 3Cook et al. 2005

The forages were dried to a target DM of 350 g kg-1 fresh matter (FM) and chopped in a forage chopper. Subse-quently, sucrose was applied at 20 g kg-1 FM and lactic acid bacteria were added (105 colony forming units CFU) g-1 FM of a strain of Lactobacillus plantarum (DSM 24624, CIAT S66.7). The material was compacted in 18.9 l plastic buckets which were closed tightly using lids with a rubber gasket. The herbages were ensiled between April and May, 2010, and stored at an ambient temperature of 22−33 °C, until February of 2011. Silage quality was deter-mined considering DM, pH, NH3-N and organic acids. The pH was determined by preparing an extract of 10 g FM with 100 ml distilled water and measured using a pH meter (MP 120 pH meter, Mettler-Toledo, Greifensee, Swit-zerland). After 2 h, DM was determined in duplicate at 105°C for 24 h (DIN 38414-S2, 1985). Ammonia was deter-mined according to Voigt and Steger (1967). VFA, alcohols and lactic acid were detected by High Performance Liq-uid Chromatography (HPLC), column Rezex ROH-Organic Acid H+ (Phenomenex Ltd., Torrance, CA, USA), according to Siegfried et al. (1984). The standards used were lactic acid (LA) (Sigma, L-1750), acetic acid (Chem Service O-4), propionic acid (Chem Service O-25), isobutyric acid ( Chem Service O-6), butyric acid (Chem Service O-5), valeric acid ( Aldrich 240370), 1,2 propanediol (Emeral BioSystems, EBS-250), 2,3 butanediol (MP Biomedicals, 203774), and ethanol (absolute, Merck, 100983).


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In February 2011, 30 commercial pigs (Landrace*Large white 47.0 ± 4.7 kg live weight (LW)), were housed in individual pens (2 × 1.5m), with cement floors, and open feeder and automatic water dispenser. They were used to evaluate the consumption of forage silage species. The experiment was carried out at the experimental farm of the National University in Palmira (Colombia) (03°32’22” latitude N and 72°18’13” longitude W).

A completely randomized experimental design was utilized including five treatments, three replicates and two periods of 14 days each, seven days for adapting and seven for measurements. The treatments were: Control, Cratylia argentea, Centrosema brasilianum, Clitoria ternatea and Brachiaria hybrid Mulato II silage supplement, respectively. The Control consisted of a balanced diet of maize and soybean meal (Table 2). Four other diets were prepared. They were composed of the Control diet supplemented with ad libitum access to silages of C. argentea, C. brasilianum, C. ternatea or Brachiaria sp. as specified below.

The animals were fed five times a day (8:00, 10:00, 12:00, 14:00 and 16:00 hours) in order to ensure a better con-sumption. Pigs of Control treatment received initially 80 g DM kg-1 LW0.75/day. The animals of silage treatments re-ceiving 50 g DM kg-1 LW0.75/day of the Control diet and the silages ad libitum, starting with 20 g DM kg-1 LW0.75/day. Silages and Control diet were mixed before being offered to the animals. In order to reduce refusal, the amount of silage distributed was adjusted every day according to the animal’s appetite. Pigs were weighed each week to adjust the amount of food to provide. Refusals were collected after each feeding, and samples were stored at –20 °C, until analysis. The foods were analyzed for dry matter content using an oven at 105°C for 12 h, crude protein (Kjeldahl method) (AOAC 1990). The neutral acid detergent fibre (NDF and ADF) and acid detergent lignin were determined by means of an ANKOM fibre analyzer (Ankom Technology, Madecon, NY, USA) using nylon bags.

Table 2. Composition of the Control diet (g kg-1)

Yellow maize 593

Wheat bran 150

Soybean meal 46% crude protein 230

L-Lysine HCL 78% 2.5

D-L methionine 99% 3.5

Calcium carbonate 12.0

Bi-calcium phosphate 4.0

Salt (NaCl) 4.0

Mineral and vitamin supplement 1.0

Calculations and statistical analysis

The daily consumption was calculated as g DM offered – g DM rejected / pig / day.

The daily metabolic consumption was calculated as g DM consumed per day / kg LW0.75.

The differences in both consumption variables were determined by analysis of variance using the GLM procedure followed by the multiple comparison test of Duncan, of the SAS version 9.1 for Windows (© 2002−2003 by SAS Institute Inc., NC, USA), using the following model:

Yijk = U + Pi + Dj + Pi×Dj + Ek(j)

Where Yijk is the daily consumption or daily metabolic consumption, U the general mean, Pi the effect of the i pe-riod, Dj the effect of the j diet, Pi × Dj the interaction of the period i × diet j, Ek(j) the effect of pig k within diet j as an error term.


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Chemical composition of the Control diet and the four silages is shown in Table 3. The pH of all 4 silages ranged between 4.0 and 4.3 with DM contents > 370 g kg-1 FM. The ammonia-N of total silage nitrogen was lowest in C. brasilianum and C. ternatea silages (44 and 45 g kg-1 N respectively) and slightly higher in C. argentea and Brachi-aria sp. silages (60 and 74 g kg-1 N respectively). All silages were butyric acid free and the sum of acetic and pro-pionic acid ranged between 4 and 11 g kg-1 DM.

The consumption of experimental diets is shown in Table 4. Pigs receiving C. argentea or C. ternatea silage con-sumed the same amount of dry matter as those fed only on the Control diet. In these feeding regimes, C. argen-tea and C. ternatea silage corresponded to 55 and 50% of total DM consumption respectively, the rest was Con-trol diet. The consumption of Brachiaria sp. and C. brasillianum diets was lower than C. argentea and C. ternatea, possibly due to their poorer nutritional quality. The dry matter content was lower in the silages of Brachiaria sp. and C. brasilianum compared to the other two forage legume silages (C. argentea and C. ternatea) (Table 3).

Table 3. Composition of the Control diet, three legume silages and one grass silage (g kg-1 DM) and fermentation quality.

Parameter Control diet Cratylia argentea

Clitoria ternatea Centrosema brasilianum

Brachiaria sp.

Dry matter 887 438 526 370 379Crude protein 202 192 198 129 59Neutral detergent fibre 188 476 490 463 732Acid detergent fibre 74 349 380 349 468Acid detergent lignin 29 157 109 113 200NH3-N (g kg-1 N) 60.4 45.1 43.6 74.1Lactic acid 43.8 34.5 59.5 27.1Acetic+propionic acid 10.5 7.4 4.7 4.0Butyric acid n.d. n.d. n.d. n.d.pH 4.3 4.5 4.0 4.1

n.d. not detected

Table 4. Consumption of the diets including tropical legumes or grass silage, by growing pigs.

Parameter Control Cratylia argentea1

Clitoria ternatea1

Centrosema brasilianum1

1Brachiaria sp. CV% SE p value

Initial live weight (kg) 48.78 45.70 48.40 45.35 45.17 10.1 4.7 0.43

Consumption (g DM/pig*day)

1752a 1642a 1710a 1395b 1358b 9.70 152 0.0003

Consumption (g DM kg-1 LW0.75)2 94.67a 93.83a 90.00a 80.00b 78.18b 3.68 3.4 0.0006

1Control supplemented with the respective forage silage; 250g DM kg-1 LW0.75 corresponded to Control diet and the rest to each silage, respectively. Different letters within rows indicate significant differences between treatments (p <0.05).

The consumption of the pigs on the Control diet (890 g DM kg-1 FM) was not significantly different from those on the C. argentea and C. ternatea silage diets, which had half of the DM content. The dry matter content of the si-lages was the factor that perhaps best explained the consumption by pigs, with a correlation coefficient of r=0.83 among silage treatments. Possibly growing pigs (≥ 45 kg LW), can ingest bulk food when the DM concentration is ≥440 g DM kg-1 FM without presenting physiological constraints for the animal. A review by Pérez (1997) indi-cates that the large intestine of pigs matures slowly; this explains why the pig tends to digest fibrous feeds better in direct relation to its age.

Results similar to those obtained in the present study were recorded by Leterme et al. (2005), who compared the intake of Trichanthera gigantea herbage in two forms: chopped and fresh or dried and ground. They reported twice the intake when forage was supplied in dry form. Most authors correlate the intake capacity of pigs with the fiber content of the feed. Campbell and Taverner (1986) observed less consumption in diets with high levels of fiber (120 g acid-detergent fibre (ADF) per kg DM) compared to those with low fiber (62 g ADF kg-1 DM), which


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decreased productive performance of the growing pigs. Díaz et al. (2005) recorded that the intake capacity of di-ets containing Gliricidia sepium herbage meal was correlated with the NDF content (r = –0.68). In the present ex-periment NDF, ADF and lignin contents of Brachiaria sp. were higher than of C. argentea, C. ternatea and C. brasi-lianum. Correlation coefficients between metabolic DM consumption and diet fibre content over all treatments were −0.73, −0.65 and −0.58 for NDF, ADF and acid detergent lignin (ADL), respectively.

However, Kyriasakis and Emmans (1995) registered that crude fibre or NDF content could not account for the ef-fects on feed intake, with three bulky foods: wheat bran, citrus pulp and grass. They recommended using the wa-ter-holding capacity (WHC), which explained partially the effects on intake of feeds such as grass which appeared to limit intake through their bulk. Here, WHC was higher in Brachiaria sp. silage (5.7 g of water g-1 DM) than in C. argentea (5.1), C. brasilianum (4.5) and C. ternatea silage (4.1 g g-1). Correlation coefficient between WHC and intake in our experiment was −0.61 among all treatments. Therefore, in this study, the factor that best explained the differences in consumption between the different species of silages by the pigs was the dry matter content.

Conclusions

It is concluded that C. argentea and C. ternatea silages of high DM and good quality have the potential to serve as feed supplement in growing pig diets. Inclusion rates up to 500 g kg-1 diet DM do not affect dry matter intake. Growth performance studies have to reveal the effect on live weight gain. Dry matter content followed by NDF, were the factors that best explained the capacity of metabolic consumption of forage silages in growing pigs.

AcknowledgementThis study was part of the project “More chicken and pork in the pot, and money in pocket: Improving forages for monogastric animals with low-income farmers”, with the financial support of Federal Ministry for Economic Co-operation and Development, Germany.

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207

Acknowledgement of referees

Agricultural and Food Science expresses its sincere thanks to the following referees for their constructive critical reviews of one or more manuscripts during the year 2012.

Berglund, K. Mäkelä, P.

Berthiaume, R. Nousiainen, J.Bertilson, J. Nysand, M.Burger, K. O’Kiely, P.Cone, J. Penn, C.Davies, D. Powell, M.

Dax, T. Pyykkönen, P.

Delin, S. Rinne, M.Dewhurst, R. Rustas, B.-O.Dodig, D. Rykaczewska, K.Driehuis, F. Rødbotten, R.Fast Seefeld, H. Saarisalo, E.Giada de Lourdes Reis, M. Salo, T.Gomez, J.R. Schmitz-Eiberger, M.Gonzalez-Ronquillo, M. Seetharaman, K.Heal, K. Seppälä, A.Hessle, A. Skjelvåg, A.O.Huhtanen, P. Spörndly, R.Huuskonen, A.K. Steingass, H.Hytönen, T. Stoddard, F.Jaakkola, S. Suedekum, K.-H.Jeng, A.S. Sumelius, J.Johansen, T.J. Svensson, B.Johansson, E. Tabacco, E.Jordan, K. Torresen, K.Juracek, M. Trematera, P.Karhu, S. Tuori, M.Kavallieratos, N.G. Turtola, E.Kenjeric, D. Vaishar, A.Knicky, M. Vanhatalo, A. Kokkonen, T. Van Vuuren, A.Kreuzer, M. Weinberg, Z.Krogstad, T. Weissmann, F.Kung Jr., L. Vestberg, M.V.Lindhardt Drejer Storm, I.M. Wyss, U.Marzban, G. Yamamoto, A.McAllister, T.A. Yli-Halla, M.Melliou, E. Ylivainio, K.

Mosenthin, R.

AgriculturAl AND FOOD ScieNceVol. 22, No. 1, 2013

contentsAila Vanhatalo Preface

1

Richard Muck Recent advances in silage microbiology

3

Frank Driehuis Silage and the safety and quality of dairy foods: a review

16

Pekka Huhtanen, Seija Jaakkola and Juha Nousiainen An overview of silage research in Finland: from ensiling innovation to advances in dairy cow feeding

35

Richard Dewhurst Milk production from silage: comparison of grass, legume and maize silages and their mixtures

57

Tim Keady, Seamus Hanrahan, Christina Marley and Nigel D. Scollan Production and utilization of ensiled forages by beef cattle, dairy cows, pregnant ewes and finishing lambs: a review

70

Tom Misselbrook, Agustin Del Prado and David Chadwick Opportunities for reducing environmental emissions from forage-based dairy farms

93

Richard Muck, Zwi G. Weinberg and Francisco E. Contreras-Govea Silage extracts used to study the mode of action of silage inoculants in ruminants

108

Johan De Boever, Elien Dupon, Eva Wambacq and Joos Latré The effect of a mixture of Lactobacillus strains on silage quality and nutritive value of grass harvested at four growth stages and ensiled for two periods

115

Eva Wambacq, Joos Pieter Latré and Geert Haesaert The effect of Lactobacillus buchneri inoculation on the aerobic stability and fermentation characteristics of alfalfa-ryegrass, red clover and maize silage

127

Jonas Jatkauskas, Vilma Vrotniakiene, Christer Ohlsson and Bente Lund The effects of three silage inoculants on aerobic stability in grass, clover-grass, lucerne and maize silages

137

Brigitte Köhler, Michael Diepolder, Johannes Ostertag, Stefan Thurner and Hubert Spiekers Dry matter losses of grass, lucerne and maize silages in bunker silos

145

Maiju Pesonen, Markku Honkavaara, Helena Kämäräinen, Tiina Tolonen, Mari Jaakkola, Vesa Vir-tanen and Arto K. HuuskonenEffects of concentrate level and rapeseed meal supplementation on performance, carcass characteris-tics, meat quality and valuable cuts of Hereford and Charolais bulls offered grass silage-barley-based rations

151

Katrin Gerlach, Fabian Roß, Kirsten Weiß, Wolfgang Büscher and Karl-Heinz SüdekumChanges in maize silage fermentation products during aerobic deterioration and effects on dry matter intake by goats

168

Szilvia Orosz, John Michael Wilkinson, Simon Wigley, Zsolt Bíró and Judit Galló Microbial status, aerobic stability and fermentation of maize silage sealed with an oxygen barrier film or standard polyethylene film

182

Roy Latsch and Joachim Sauter Comparison of methods for determining the density of grass silage

189

Joseph McEniry and Padraig O’Kiely The estimated nutritive value of three common grassland species at three primary growth harvest dates following ensiling and fractionation of press-cake

194

Patricia Sarria and Siriwan D. Martens The voluntary intake in growing pigs of four ensiled forage species

201

Acknowledgements 207

I S S N print e d iti o n 1459- 6067

Documents

AgriculturA l And food science - MTT · 2013-06-18 · AgriculturA l And food science special issue of the XVI International silage conference 2-4 July 2012, Hämeenlinna, finland