Safe andEffective GrainFeeding forHorses

A report for the Rural Industries Researchand Development Corporation

by James Rowe, Wendy Brown, andSimon Bird

November 2001

RIRDC Publication No 01/148RIRDC Project No UNE-62A

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© 2001 Rural Industries Research and Development Corporation.All rights reserved.

ISBN 0 642 58368 4ISSN 1440-6845

Safe and effective grain feeding for horsesPublication No. 01/148Project No. UNE-62A

The views expressed and the conclusions reached in this publication are those of the author and notnecessarily those of persons consulted. RIRDC shall not be responsible in any way whatsoever to any personwho relies in whole or in part on the contents of this report.

This publication is copyright. However, RIRDC encourages wide dissemination of its research, providing theCorporation is clearly acknowledged. For any other enquiries concerning reproduction, contact thePublications Manager on phone 02 6272 3186.

Researcher Contact DetailsProf James RoweUniversity of New EnglandArmidale, NSW 2351Phone: (02) 6773 2225Fax: (02) 6773 3275Email: jrowe@metz.une.edu.au

RIRDC Contact DetailsRural Industries Research and Development CorporationLevel 1, AMA House42 Macquarie StreetBARTON ACT 2600PO Box 4776KINGSTON ACT 2604

Phone: 02 6272 4539Fax: 02 6272 5877Email: rirdc@rirdc.gov.au.Website:http://www.rirdc.gov.au

Published in November 2001Printed on environmentally friendly paper by Canprint

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ForewordThe aim of this research program was to develop safer and more effective ways of feeding grain tohorses. Horses have a limited ability to digest starch in the small intestine and the rapid fermentationof undigested starch in the caecum and colon lead to an accumulation of acidic end products thatinitiate a cascade of adverse effects – the best known being laminitis or founder. The focus of theproject was to develop and calibrate an in vitro method for predicting pre-caecal starch digestion andthen use this to identify the safest grains and best processing methods to reduce the amount of starchpassing undigested to the caecum and colon.

This report covers details of two in vitro assays, one for estimating enzyme starch digestion and thesecond for measuring rate of fermentation. These assays were then used to examine differencesbetween a wide range of feed grains and processing methods. There are descriptions of two in vivoexperiments designed to confirm the in vitro predictions in relation to pre-caecal starch digestion andinvestigate the role of exogenous enzyme preparations to improve digestion of wheat grain. There is areview of the characteristics of oat grain that make its digestibility so variable and a report on thepotential value of triticale as a suitable grain for horses.

This project was funded from industry revenue which is matched by funds provided by the FederalGovernment.

This report, a new addition to RIRDC’s diverse range of over 700 research publications, forms part ofour Equine R&D program, which aims to develop better feeds for growth and development andreduce the incidence of grain related problems of hoof care.

Most of our publications are available for viewing, downloading or purchasing online through ourwebsite:

• downloads at www.rirdc.gov.au/reports/Index.htm• purchases at www.rirdc.gov.au/eshop

Peter CoreManaging DirectorRural Industries Research and Development Corporation

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AcknowledgementsParts of the project described in this final report were conducted in parallel with the Project: “PremiumFeed Grains for Livestock” coordinated by the GRDC and supported by MLA, RIRDC (Chicken Meatand Eggs), DRDC and PRDC. Development of the in vitro assays was conducted as joint activitybetween UNE62A and the GRDC-coordinated feed grains project.

Ben Barwick and Karen Roberts made valuable contributions to the experiments measuring in vivostarch digestion as part of their Bachelor of Rural Science honours projects.

The section on oat digestibility is based on a recent paper by Rowe, J.B., May, P.J. and Crosbie, G.(2001) ‘Knowing your oats’ Recent Advances in Animal Nutrition in Australia, 13 (in press).

Measurement of oat digestibility in the horse form part of a research thesis by Bianca Wilson andaccess to the unpublished results is gratefully acknowledged.

Abbreviations

NSP Non-starch polysaccharide. Insoluble NSPs are mainly the structural carbohydrates that arean important part of the plant cell walls. There are also soluble NSPs and these comprisesugars, fructans and other soluble carbohydrates.

VFA Volatile fatty acids are produced as the main end-products of fermentation that are available to theanimal for energy metabolism. Quantitatively the most important VFA is acetate, followed bypropionate and butyrate.

About the authors

JAMES B. ROWE is Professor of Animal Science at the University of New England and has been thecoordinator for this project. His area of special interest is animal nutrition and he has focussed muchof his research in recent years on problems related to fermentative acidosis. His research on themanagement of acid accumulation during fermentation of starch in the digestive tract of horses andruminant animals was instrumental in the development of the product Founderguard based on theantibiotic virginiamycin. More recently he has studied characteristics of feed grains and feed grainprocessing as complementary strategies to reduce the risk of fermentative acidosis and the subsequentproblems of laminitis and behavioural changes.

WENDY BROWN has been the technical coordinator for this project and managed all aspects of the invivo studies including compilation of data and reporting. The wide range of technical skills and herexpert knowledge of horse management have been essential for success of the project.

SIMON H. BIRD completed his doctoral studies in the field of ruminant nutrition and has developed animpressive range of research achievements on the role of protozoa in the fermentation process and onthe relative importance of dietary nutrients such as protein and lipids. For the last four years hisresearch has focused on the nutritive value of feed grains and he has been the principal post-doctoralResearch Fellow working on this project. He has been directly responsible for development of the invitro assays described in this report and has made a major contribution to the analysis of the in vivostudies.

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ContentsForeword.................................................................................................................................................................iiiAcknowledgements.................................................................................................................................................ivAbbreviations..........................................................................................................................................................ivAbout the authors....................................................................................................................................................ivExecutive Summary................................................................................................................................................vi

Implications .......................................................................................................................................................vii1. Introduction..........................................................................................................................................................12. Starch digestion in the horse ................................................................................................................................2

Processing to improve small intestinal starch digestibility ..................................................................................2Measuring starch digestion in the horse...............................................................................................................2

3. New in vitro assays for measuring starch digestion.............................................................................................4An in vitro assay to simulate digestion in the small intestine ..............................................................................4

Protocol for in vitro enzyme digestion of starch..............................................................................................5An in vitro assay to simulate fermentation in the caecum and colon...................................................................5

Protocol for in vitro fermentation assay...........................................................................................................6Results from in vitro assays – enzyme digestion and fermentation .....................................................................6Effects of processing............................................................................................................................................8Conclusions on in vitro assays.............................................................................................................................9

4. Measuring starch digestibility in horses.............................................................................................................10In vivo study 1 Barley, oats, triticale and sorghum............................................................................................10

Experimental Design......................................................................................................................................10Feeding and management ..............................................................................................................................10Measurements ................................................................................................................................................11Results............................................................................................................................................................11Conclusion In vivo study 1 ............................................................................................................................12

In vivo study 2 - Triticale (two cultivars) and wheat (+/-enzymes)..................................................................13Experimental Design......................................................................................................................................13Feeding and management ..............................................................................................................................14Enzymes.........................................................................................................................................................14Measurements ................................................................................................................................................14Results............................................................................................................................................................14Discussion......................................................................................................................................................15

Ratio of groats to non-groat material .................................................................................................................17Hull lignin as a factor influencing digestibility..............................................................................................18Level of feeding .............................................................................................................................................19Combining hull lignin and level of intake with ADF to predict DE ..............................................................20

Processing oat grain for animal feeding.............................................................................................................21Measuring lignin content of oat hulls ................................................................................................................22Visual appearance of low- and high-lignin oat grain .........................................................................................22Digestibility of low- and high-lignin oat grain by horses ..................................................................................22In vitro assays ....................................................................................................................................................24Triticale as a new feed grain for horses .............................................................................................................24Basis for selecting more digestible cultivars of oat grain ..................................................................................24Exogenous feed enzymes...................................................................................................................................24

8. References..........................................................................................................................................................25

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Executive SummaryCereal grain is an important feed ingredient for most intensively managed horses and although cerealsprovide a valuable source of digestible energy their feeding to horses is always associated with somerisk. The danger in feeding cereal grain to horses lies in the risk of incomplete digestion of starch inthe small intestine and the possibility that significant amounts of starch can pass through to the caecumand colon (large intestine, or hind gut). Starch entering the hind gut is fermented very much morequickly than roughage and this rapid fermentation leads to accumulation of acidic end products andlow pH. Just how the acid build up in the hind gut affects the horse is not clear but there is no doubtthat acid accumulation in the hind gut is the primary cause of laminitis as well as many of thebehavioural changes commonly associated with feeding grain to horses.

At the start of this project there was no simple method for determining the intestinal or pre-caecalstarch digestibility of different grains and/or the effectiveness of various processing methods designedto make cereal grains more digestible and therefore safer for horses. Several researchers have mademeasurements of pre-caecal starch digestion using inert markers as references or mobile nylon bagsremoved from the caecum but these methods are time consuming and subject to variability betweenanimals. A simple and repeatable estimate of pre-caecal digestibility is essential if we are tounderstand and improve the safety of grain feeding and the development of such an assay was aprimary focus for this project. An objective measurement of the starch digestibility characteristics of araw or processed grain sample is a valuable tool in that it is not complicated by animal to animalvariation or the level or method of feeding. Such a tool allows us to quickly build a data base ondifferent feeds and the consequences of different processing techniques.

Two in vitro assays were developed during the project. The first is an enzyme-based assay designed topredict pre-caecal starch digestibility and the second was established to measure the rate offermentation. The theory behind these two assays is that we need to understand the risk of starchentering the hind gut as well as the rate at which that starch is likely to ferment when it comes intocontact with the gut microbes. Once the two assays were developed we analysed a set of 55 samplesselected to represent different grains, a range of different cultivars of each grain and, with selectedcultivars, samples taken from geographically distinct locations. The results were analysed bycomparing the enzyme digestibility of each grain with its potential rate of fermentation. What we werelooking for was grains that were highly digestible in the enzyme assay (low risk of starch reaching thehind gut) or grains that had a reasonable level of enzyme digestibility and low rate of fermentation.

By and large the grains with a high level of intestinal (enzyme) digestibility were also those thatfermented most rapidly but there were a few exceptions. Two cultivars of triticale stood out as havingthe highest enzyme digestibility of all grains tested and far higher than would be expected from theirrate of fermentation. On the basis of this result we went on to examine the digestibility of triticale inhorses to confirm that the cultivar difference observed in vitro was also reflected in actual digestibilityin the horse. We were very pleased that this was the case. Further in vitro testing of triticale hasconfirmed that the cultivars of Madonna and Abacus are significantly more digestible than Tahara.Studies of starch digestibility of the two triticale parents, Durum wheat and rye show that thedigestibility of triticale lies in between that of wheat and rye with the higher digestibility cultivars suchas Abacus being far closer to rye than Durum Wheat. This finding is one that will be of interest toplant breeders, feed manufacturers and to horse owners purchasing grain for supplementary feeding.

The in vitro assays showed that oat grain starch was very highly digestible and also very rapidlyfermented. This suggests that while oats is probably one of the safest grains in terms of a low risk ofstarch entering the hind gut, feeding strategies should still be employed that minimise this risk as anyoat starch finding its way into the hind gut will ferment extremely rapidly.

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Grains were ranked by the in vitro assay for enzyme digestion as follows:

Highly digestible Poorly digestibleOats and Cultivars oftriticale

Cultivars of triticale, barley Wheat Sorghum and maize

A second objective of the project was to examine the potential importance of oat hull digestibility inthe utilisation of oat grain by horses. Studies in sheep and cattle had indicated that lignin content ofthe oat hull is genetically controlled and can have a significant effect (approximately 10 percentageunits) on apparent digestibility. This research has been extensively reviewed as part of this final reportand it is suggested that lignin content is likely to have an important effect on digestibility as well asgut fill and body weight. Unfortunately the results of the experiments on digestibility of oat grainconducted as part of this project were not clear cut and only preliminary results are reported. Theseresults suggest that lignin could influence digestibility in the horse and that processing the oats byrolling could also be important in improving its digestibility.

Exogenous enzymes that target the non-starch polysaccharides that make up the cell walls in the cerealgrain have been found to be most effective in increasing starch digestion in poultry. Wheat isconsistently improved by the use of exogenous enzymes and we therefore conducted an experiment tomeasure the response of a commercial exogenous enzyme mix on pre-caecal starch digestion in thehorse. The enzymes appeared to work very well and significantly improved pre-caecal starchdigestibility.

ImplicationsThis project has provided a new tool in the form of powerful in vitro assays. These assays will beuseful for plant breeders, feed manufacturers and horse owners. It is recommended that the in vitroassays developed as part of this project be made available on a commercial basis for these user groupsto test samples prior to use for horse feeding. It is expected that the in vitro enzyme assay will costaround $85/sample at this stage but could become cheaper (around $45/sample) if an NIR calibrationcan be developed.

The identification of high-digestibility triticale for horses is an exciting new development for the horseindustry as it was previously not known that this grain was potentially suitable for horses. Furtherwork in collaboration with feed manufacturers is indicated to explore the effect of processing methodssuch as steam flaking in combination with selection of the more digestible cultivars of triticale.

Unfortunately it is not possible to make a clear statement about lignin content of oat hulls and itsnutritional value in horses. However, because of the importance of oat grain in the diet of horses, andthe very good digestibility of oat starch, it is recommended that further work be undertaken to provideunequivocal data on this factor.

The use of exogenous enzymes to improve pre-caecal digestion of wheat in the horse was clearlyshown in this project. Exogenous feed enzymes are commercially available and it should be possiblefor feed manufacturers to take immediate advantage of this information.

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1. IntroductionCereal grains are commonly used to supplement the diet of horses when an increase in digestibleenergy intake is required. Cereal grains contain high levels of starch (roughly 60-80% DM) andrelatively low levels of fibrous material and provide approximately 50% more digestible energy (on aweight/weight basis) than forages. While it is recognised that provision of proteins and minerals fromgrains is important, it is the efficiency with which the carbohydrate component of grain is utilised thatis considered to be the major determinant of nutritive value. Apart from nutritive value, the risk ofmetabolic disorders is the other major consideration when feeding grain to horses. To understand whygrain may cause metabolic problems it is necessary to understand how food is digested in the horse.

Two major digestive processes occur in the horse. Food consumed by the horse is first exposed to aciddigestion in the stomach and enzymatic digestion in the small intestine. Undigested material passingfrom the small intestine is then subjected to microbial activity resulting in fermentation of feedmaterial in the caecum and colon. It is important to note that the end products of enzyme digestion andmicrobial fermentation are different. Enzyme digestion of starch in the small intestine gives rise toglucose that is absorbed across the intestine wall. In contrast fermentation of feed material in thehindgut gives rise to organic acids that can be absorbed across the gut wall. While both these digestiveprocesses are normal, excessive or rapid production of acid in the hindgut (acidosis) may causemetabolic disorders. Although the enzymes secreted into the small intestine are capable of digesting alldietary starch, digestion in the small intestine may be incomplete because either a high intake of starchhas exceeded the digestive capacity of the small intestine or the enzymes cannot readily access thestarch in the grain. In this latter situation the microstructure of some grains is believed to restrict theaccess of enzymes to the starch. If significant amounts of starch pass undigested into the hindgutextensive fermentation may lead to the accumulation of acid within the gut. The resulting acidosis isthe primary cause of a wide range of problems for the horse, the most devastating of which islaminitis. While acute laminitis is widely recognised as a major problem in the horse industry, it islikely that chronic acidosis is also responsible for many secondary conditions of lameness and poorperformance. Fermentative acidosis, as a consequence of grain feeding, has also been associated withadverse behaviour in horses.

The major problem associated with feeding grain to horses is the passage of large quantities ofundigested starch to the caecum and hindgut. For this reason, the primary objective of safer grainfeeding strategies is to maximise pre-caecal digestion of the starch component of the grain. Thisobjective can only be achieved when the factors limiting starch digestion in the small intestine areclearly identified. The feeding of grain to horses can only be made safe when the intestinaldigestibility of the starch in raw or processed grain can accurately be predicted for any given feedingregime.

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2. Starch digestion in the horseCereal grains are fed to all classes of domestic livestock and the digestibility of starch in pigs, poultry,sheep and cattle has been intensively studied. Much is known about the site of starch digestion in theseanimals and published results indicate that starch digestibility varies considerably both between andwithin grain type. Published results also indicate that starch digestibility varies between animalspecies, for example poultry can completely digest starch in sorghum whereas digestibility of sorghumstarch in cattle may be as low as 70%. This variability is believed to be due to the microstructure ofthe grain and the composition of the starch itself. Unfortunately there is a paucity of informationavailable on the relative digestibility of the cereal grains in horses. The traditional measurement of invivo starch digestibility involves measurements of intake and faecal output. However thesemeasurements do not tell us anything about the site of starch digestion in the horse and do notdifferentiate between enzymatic digestion in the small intestine and fermentative digestion in thehindgut. Therefore most of the current digestibility data does not provide sufficient information onwhich to select and design more effective grain-feeding systems for horses. One of the primary aims ofthis project was to develop a reliable and accurate assay that will allow us to rank raw and processedgrains for small intestinal digestibility in the horse.

Processing to improve small intestinal starch digestibilityThe selection of grains that have an inherently high intestinal digestibility of starch is one way ofmaking grain feeding safer for horses. However these grains may not always be available or there maybe situations where it is not economic to feed these grains. Grain processing can be used to increasethe digestibility of starch and offers an alternative strategy to grain selection. Grain processing alsoincreases the range of grains that can be considered for horse diets. While the benefits of grainprocessing are well documented for some livestock animals, for example steam flaking of sorghumincreases the digestibility of starch in cattle from 70% to approximately 95%, the benefits ofprocessing grains for horses have not received the same degree of attention. Physical treatments(hammer milling or rolling) are commonly used to process grains for horses. These treatments increasedigestibility through a reduction in particle size of the feed and a subsequent increase the surface areaexposed to enzyme attack. Whilst the established practices for processing grains are recognised, theefficacy of adding exogenous enzymes with the grain was also examined in this project. Enzymes areroutinely used in the pig and poultry industry, and we believe that their use in horses warrantsinvestigation.

Measuring starch digestion in the horse

Measurement of the actual amount of starch digested in the small intestine (pre-caecal) is verydifficult. In vivo methods rely either on surgical modification and the placement of a cannula in theileum or caecum, or alternatively to slaughter horses at various times after feeding test diets tomeasure the amount of starch remaining in the different parts of the digestive tract. Although these invivo methods have produced some valuable information the cost and logistical difficulties haveseverely limited the number of feeds that can be tested. In addition there is generally poor agreementbetween the results of different studies even when the grains are apparently the same (See Figure 1)

More recent studies by Cuddeford (1999), and Pagan (1999) have moved towards techniques forranking grains and processing methods using indirect measurements. The results of Cuddeford and hiscolleagues are based on caecal cannulation and the recovery of mobile bags containing grain samplesthat had been administered directly into the stomach using a naso-gastric tube. Starch digestibility wasestimated by measuring the disappearance of starch from the bag. Results from these studies haveshown quite clearly the benefits of micronisation in increasing small intestine digestibility of barleystarch compared to dry rolled grain. The methods used by Pagan and colleagues are based on theglycaemic response, which is used extensively in human nutrition as a method of determining theeffect of starch rich foods on postprandial blood glucose concentrations. Following the consumptionof a test diet, blood samples are taken at regular intervals from horses to monitor the rise and fall of

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blood glucose concentration. The peak glucose concentration and the area under the curve are relatedto starch digestion and glucose absorption. This technique has been useful in examining differencesbetween grains (Pagan, 1999) as well as investigating the efficacy of grain processing. While theglycaemic index measurements are non-invasive and less expensive than measurements involvingcannulation and the use of markers to measure digesta flow it is still a relatively expensive and time-consuming measurement. A major disadvantage in all of the in vivo methods used to determine theintestinal starch digestion of different grains is the large variation that occurs between animals in theefficiency of starch digestion and glucose absorption. This variation in starch digestion is notunexpected given that the rate of eating, extent of chewing and also the amount of amylase producedin the intestines of horses has been reported to vary between animals (Meyer, 1995). Comparison ofresults reported from different studies is difficult because the amount and nature of the roughagecomponent of the diet, the level of feeding and the number of meals fed per day varies betweenstudies. Although it is important to account for differences between animals when designing feedingprograms it is a source of variation that makes characterization of the diet extremely difficult. In vitrostudies offer an alternative to in vivo studies with the advantages of being cheaper and allowing a largenumber of samples to be compared under a standard set of conditions.

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Figure 1 Intestinal digestibility of starch in the small intestine of horses fed different grainsprocessed in different ways showing the variability between studies in estimated digestibility. FromRowe et al. (2001).

In our studies we have devoted considerable effort to the development of in-vitro assays that simulatethe digestion of starch in the small intestine of the horse. In this project two separate in vitro assayswere developed. The first was designed to predict the digestion of starch in the small intestine. Thesecond assay was designed to emulate the fermentation of grain in the caecum and colon. The methodsfor both of these assays are described in the sections that follow. These two assays were then used totest a variety of grains and their cultivars. From the results of these assays, specific grains wereselected for testing in vivo to establish the validity of the assays for use in horses.

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3. New in vitro assays for measuringstarch digestionAn in vitro assay to simulate digestion in the small intestine

Starch occurs in two forms: α-amylose and amylopectin. Amylose is a linear polymer of α-1,4-linkedglucose units. Amylopectin is a much larger polymer consisting of linear chains of α-1,4-linkedglucose units with α-1, 6 branch points occurring, on average, every 12 glucose units (Lehninger1970). Amylose and amylopectin are hydrolysed into small oligosaccharides of two or three glucoseunits by α-amylase secreted into the duodenum by the pancreas. The oligosaccharides are thenhydrolysed to produce glucose by maltase (amyloglucosidase). The enzyme assay developed for thesestudies is based on these two major enzymes responsible for the digestion of starch in the duodenum,and is conducted under similar physiological conditions of pH (7) and temperature (390C) found in theintestines. Therefore ranking grains according to the amount of starch hydrolysed to glucose in thisassay can be expected to provide a reliable in vivo index of intestinal starch digestibility for the testgrains.

The in vitro enzyme assay developed for these studies was adapted from the established method forstarch determination (McCleary et al., 1997). The method of McCleary et al. (1997) is based on twoenzymes: amyloglucosidase (AMG) and α-amylase (AA) and high temperature treatment to gelatinisethe starch. Starch digestion was determined from the amount of starch hydrolysed to glucose. Variouscombinations of incubation times, pH conditions and temperature were examined. There were cleardifferences between the enzyme digestibility of sorghum, oats and barley and these differences wereevident for all combinations of pH, temperature and incubation time. The conditions selected forstandardisation of our assay were: an incubation time of 1 h; pH of 7; and a temperature of 39o C. Theconcept of this assay is summarised in Figure 2 below.

StarchAmylose Amyloglucosidase Glucose

Barrier between enzymes and

starch

Cell walls Protein matrix Protein bodies

Factors affecting availability of starch

Particle size Gelatinisation

Starch structure

Assay conditions set to limit enzyme activity

Temp 39 C Time 1 hr

pH 7 Grinding 0.5 mm

Figure 2 Summary of the in-vitro test to measure intestinal starch digestion. The extent of glucosereleased is an indicator of the accessibility of starch to the amylolytic enzymes.

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Protocol for in vitro enzyme digestion of starch

Mill sample grain to pass through a 0.5mm sieve.Accurately weigh 100±2mg of sample (in duplicate) into the bottom of pre-weighed 20ml culturetubes (+ magnetic stirrer bar). Add duplicates of wheat starch standard.Add 200µl of 80% ethanol to each tube to whet sample.Add 3.0ml of MOPS/Enzyme solution to each tube*.Add 100µl of AMG to the bottom of each tube.Place tubes in heating/stirring block at 40oC for 1hr (1.5 on the stir speed setting).Remove tubes from block and adjust volume to 10ml (10g) with QH2O.Vortex, and centrifuge samples at 3000rpm for 10mins.Dilute sample supernatant 1 in 10 with QH2O (0.5ml sample + 4.5ml QH2O). Vortex.Transfer 100µl of diluted sample (in duplicate) to the bottom of 10ml culture tubes. Include triplicatesof wheat starch control and glucose standards (0, 50, 100µg).Add 3.0ml of GOPOD reagent to each tube.Vortex, and place in shaking water bath at 50oC for 20mins.Remove from water bath and allow to cool for 20mins. Vortex.Read absorbance at 510nm against QH2O.

*MOPS/Enzyme solution is made by adding 3.0ml of α-Amylase (B.licheniformis 3000U/ml) to MOPS Buffer to a final volume of 90ml. For MOPS buffer preparation refer toMegazyme Total Starch Assay procedural booklet.

Calculations:All results are expressed as %starch of ‘as is’ weight of sample.

% Starch = �A x F x 1000 x 1/1000 x 100/W x 162/180

�A = Absorbance of Sample – Absorbance of Reagent BlankF = 100 / (Absorbance for 100�g glucose) or 100 / (Slope of glucose standards curve)1000 = Volume correction (0.1ml taken from 10ml)1/1000 = Conversion from micrograms to milligrams100/W = Factor to express “starch” as a percentage of sample weightW = Weight of ‘as is’ sample in milligrams162/180 = Adjustment from free glucose to anhydro-glucose (as occurs in starch)

An in vitro assay to simulate fermentation in the caecum and colonInitial work on the development of an in vitro fermentation assay concentrated on the publishedmethod of Opatpatanakit (1994). This method was based on the disappearance of starch and some ofthe end products of fermentation (gas and volatile fatty acids). Incubations were conducted with smallamounts of feed (0.1 g) in a sealed tube containing buffered fluid. Several adaptations to thesemethods were made to meet our requirements for this assay. The size of the fermentation vessel wasincreased to one litre and the sample size to 30 g to allow the system to handle whole processed grainsamples. The second important change was the removal of glucose from the incubation mixture.Published methods generally included glucose, but in our studies it was found that the fermentation ofglucose had a confounding effect on the fermentation of grain.

During the development of this assay it became apparent that the measurement of a single end productof fermentation was unlikely to provide an adequate description of the fermentation of grain.Therefore the in vitro fermentation assay was extended to include the measurements of gas production,change in pH, volatile fatty acid and lactic acid production and the disappearance of starch during 5hours of incubation at 39o C.

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Protocol for in vitro fermentation assay

Dry unprocessed grain samples were first hammer-milled and sieved through a 0.5 mm screen.

A sample of milled grain (30 g) was added to a culture jar (1 L). One hour prior to the commencementof the assay 375 ml of McDougalls buffer and urea (1.2 g/l) were added to the jar and stirred with arod until the grain was completely wet. The jar was sealed and flushed with CO2 (via a three-way gastap) and placed in a shaking water bath at 39o C.

Rumen fluid* (2 L/animal) was collected from two steers (fitted with permanent rumen cannula) priorto feeding. The diet of the cattle (fed once/day am) contained approximately 50% mixed grain (oats,barley, wheat maize and sorghum) and 50% chaffed hay.

The assay was initiated with the addition of 125 ml of rumen fluid to the culture jar. The assays werestarted approximately 30 min after the collection of rumen fluid from the cattle. Measurements of gasproduction and pH were made every hour.

The incubation was stopped after 5 h with the addition of H2SO4 (15 ml 20% w/w) and a liquid samplewas collected for the determination of volatile fatty acids and lactic acid. The remainder of incubationcontents were dried and assayed for starch content.

* Note the bacterial inoculum was obtained from fistulated steers rather than horses. The maintenanceof fistulated horses was considered to be impractical. It is believed that the activity of the microbes inthe rumen of cattle is comparable to the microbial activity in the caecum and colon of horses.

Results from in vitro assays – enzyme digestion and fermentation

Initially a total of 55 grains were tested in both assays. All the major cereals grown in Australia wererepresented in this collection and the individual cultivars were sourced from a range of geographicallocations and growing conditions. The results of the assays are presented in Table 1.The most obviousfeature of these results is the variation both between and within grain types for starch fermentation andenzyme digestion. Some of the differences between grain types were expected, as results from in vivostudies reported in the literature indicate that starch digestibility is influenced by grain type. Forexample barley and sorghum are commonly fed to feedlot cattle and it is well known that dry-rolledbarley is used more efficiently than dry-rolled sorghum (Saba et al., 1964). Incomplete whole-tractdigestion of sorghum starch is the primary reason for the poor utilisation of this grain by cattle. Starchcontent of faeces collected from steers fed either dry-rolled sorghum or barley was 25% and 4%respectively (Watts and Tucker, 1993). Encouragingly the assay data provide an explanation for thesein vivo results. The average in vitro fermentability of barley starch (67%) was clearly superior to theaverage fermentability of sorghum starch (44%). A similar difference in enzyme digestibility of starchbetween these two grains (barley 45% vs sorghum 28%) was also observed. Therefore considering thatboth enzyme digestibility and fermentability of sorghum starch is low it is not surprising that asignificant amount of sorghum starch passes through the digestive tract of cattle undigested. The assayresults for sorghum also suggest that this grain would not be suitable for horses. Oat grain is regularlyincluded in the diet of horses and is believed to be the safest of all the cereal grains. This beliefappears to be well founded, as the in vitro enzyme digestibility of starch in oats was the highest of thegrains tested, with the exception of triticale. Therefore it is unlikely that sufficient oat starch wouldescape digestion in the intestine and reach the hindgut where it may cause metabolic problems. Thedata presented in Table 1 indicates that there is a considerable range in the fermentability and enzymedigestibility of starch within each grain type. These differences, (up to 16 percentage points forbarley), may provide an explanation for some of the metabolic problems that are occasionallyencountered when switching from one batch of feed to another, even though the grain type in eachbatch is the same. These results highlight the importance of developing a test that will allow us topredict the digestibility of cereal starch in horses.

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Table 1 Results from in vitro fermentation and enzyme digestion of starch in 55finely milled (0.5 mm screen) samples of grain.

Starch digestibility (%) (expressed as a percent of original starch content in grain)*

Number Fermentation Enzyme digestionGrain type of cultivars

Mean Range Mean RangeBarley 20 67 52-76 45 37-53Wheat 7 48 35-63 43 37-47Oat 4 72 70-77 61 57-66Sorghum 20 44 35-51 28 23-33Triticale 3 60 52-78 70 65-76Maize 1 42 29

There was a weak positive correlation (r2=0.46) between the enzyme digestibility of starch and starchfermentation (Figure 3). The finding that this correlation is not very strong suggests that the graincharacteristics that influence enzyme digestibility of starch are not necessarily the same characteristicsthat influence microbial fermentation. Therefore the results from the enzyme assay cannot be used topredict fermentability and vice versa. Given the importance of intestinal starch digestion in the horsewe believe that the results from the enzyme assay will be more relevant than the fermentation assay inthe development of successful grain-feeding systems for the horse.

20

30

40

50

60

70

80

30 40 50 60 70 80Fermentation (% starch present)

Enzy

me

dige

stio

n (%

sta

rch

pres

ent)

Wheat

Barley

Triticale

Oats

Sorghum

maize

Figure 3 Relationship between fermentation and enzyme digestibility of starch in finelymilled samples of grain.

In comparison with barley, the in vitro fermentability and enzyme digestibility of starch in triticalevarieties was clearly superior (Table 1). The results for triticale were unexpected. This grain is notcommonly fed to domestic livestock, yet the in vitro starch fermentation and enzyme digestioncharacteristics suggest that triticale may have superior qualities to either wheat or barley. Of particularsignificance is the high enzyme digestibility of starch in triticale (70%) which is clearly superior to

8

that of barley (45%) or wheat (43%). This result implies that the digestion of starch in the smallintestine will be more efficient for triticale than for either barley or wheat. The assay results havehighlighted the potential value of triticale as a suitable grain for inclusion in horse diets. Triticale mayprove to be an even better grain than oats because it contains approximately 40% more starch thanoats.

Effects of processingRolling and hammer milling are the most common methods used to process grains for livestock andthe benefits have been well documented. Physical processing increases digestibility because the grainis broken into smaller pieces increasing the area of starchy endosperm that is exposed to intestinal andmicrobial enzymes. Therefore it was important to determine whether our assays were responsive tochanges in particle size. The relationship between particle size (four screen sizes) and grain type (threegrains) was tested with the assays. Particle size had a significant effect on starch digestibility in thethree grain types tested (Figure 4). As particle size decreased starch digestibility increased, which isconsistent with results reported from in vivo studies.

Figure 4 Effect of processing (particle size) on the in vitro digestibility of starch in oat,barley and sorghum grain

0

20

40

60

80

0.0 1.0 2.0 3.0 4.0 5.0

Screen mesh size (mm)

Star

ch d

iges

tibilit

y (%

) Oats

Barley

Sorghum

9

Conclusions on in vitro assaysThe in vitro assays developed in this project have identified large differences between and within grainswith respect to both susceptibility of grain to microbial fermentation and enzyme digestibility of starch.Encouragingly the results obtained with the assays were consistent with results reported from previousin vivo studies suggesting that these assays may provide the basis for developing a reliable method forevaluating the suitability of grain for horses. Given the importance of intestinal starch digestion in thehorse we believe that the results from the enzyme assay will be more relevant than the fermentationassay in the development of successful grain-feeding systems for the horse. However because there islimited information currently available for grain-feeding studies with horses further in vivo calibrationof the assays was required. Also, due to the wide variations found within grains, it was necessary toconduct these in vivo studies using the exact grains that we had tested in our assays for this calibrationto be meaningful.

The next section describes the first of two in vivo studies in which horses were slaughtered after beingfed test grains for 14 days. Samples of digesta were taken from the digestive tract to measure starchdigestibility.

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4. Measuring starch digestibility in horsesIn vivo study 1 Barley, oats, triticale and sorghum

The aim of the experiment was to investigate both the site and extent of starch digestion in the gastro-intestinal tract of the horse. Oats, barley, sorghum and triticale were the test grains selected for ourinitial in vivo measurements of starch digestion. Results from the in vitro enzyme assay indicated thatintestinal starch digestibility in order from highest to lowest should be oats and triticale high, barleyintermediate and sorghum low. If this hypothesis is correct the amount of starch measured in caecaldigesta of the slaughtered horses will be highest for the sorghum-fed horses and lowest for the horsesfed oats and triticale.

Experimental Design

Fourteen mares and geldings of mixed breed were purchased for this experiment. On their arrival atthe stables all horses were weighed, checked for any signs of abnormality, identifying featuresrecorded, identification collars attached and a broad-spectrum anthelmintic paste administered orally.Any animals that were considered to be unsuitable on the basis of health or temperament wereexcluded from entering the trial. Horses were allowed a seven-day adaptation period foracclimatisation to surroundings and adaptation to basal diets and feeding regimes. During thisadaptation period, 2 horses were excluded from the trial on the basis of poor feed intake. Theremaining 12 horses, ranging in size from 275 kg to 445 kg, were stratified according to body weightand within each stratum were allocated at random to one of four dietary treatments: barley, oats,sorghum or triticale (Table 2). All grains were dry-rolled. Horses were fed these diets for the 14-dayexperimental period prior to slaughter.

Table 2 Summary of Study DesignGroup Number of

AnimalsTest Grain Variety Starch content of

grain (%)1 3 Barley Tantangara 532 3 Oats Yarran 353 3 Sorghum Western Red 644 3 Triticale Tahara 59

Feeding and management

During the 7-day adaptation period all horses were fed lucerne chaff (2% of body weight per day) andincreasing levels of a mixed grain diet starting at 500 grams per day and increasing to 1 % bodyweight per day. The mixed grains consisted of equal quantities of each of the grains to be used in theexperimental diets (oats, barley, triticale, and sorghum). For the 14-day experimental period thatfollowed, all horses were fed lucerne chaff (1.5% of bodyweight per day) and the grain to be tested at1% bodyweight per day. The amount fed each day was divided between two equal feeds offered at 8AM and 4 PM. Horses were fed individually in separate stables, and any refusals were weighed andrecorded. Fresh drinking water was available at all times. Horses were exercised daily in a round yardwhile the stables were cleaned. During the final 5 days of the experiment, ytterbium acetate wasadded to the grain component of the diets as an inert marker for digesta flow. Ytterbium is a rare earthmetal that is not absorbed from the digestive tract so the ratio of ytterbium to starch in digesta can beused to estimate starch digestion.

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Measurements

Horses were weighed on arrival at the stables and at weekly intervals thereafter. Feed intake wasmeasured daily, and fresh faecal samples were collected on days 4, 6 and 8 of the experimental period.Faecal samples were mixed 50/50 by weight with distilled water and the pH of each sample wasmeasured and recorded.

At the conclusion of the experimental feeding period, horses were transported to the local pet foodabattoir. Horses were slaughtered approximately 16 hours after their last feed and immediately afterslaughter the digestive tract was removed and ties placed at the following sites: duodenum, ileum,junction between caecum and colon and between the proximal and distal colon. Each section of thegut was weighed before removing sub-samples for analysis of starch, dry matter, pH, VFA andytterbium. The digestibility of starch in different parts of the digestive tract was estimated from theratio of ytterbium to starch in digesta samples.

Results

Table 3 The apparent digestibility of starch prior to the caecum in horses fed supplements ofcereal grain.

Measurements Treatment dietsBarley Oats Sorghum Triticale

Starch intake g/d 2041 1223 2498 2449Apparent digestibility ofstarch prior to caecum (%)* 96 97 64 97

Apparent digestion of starchprior to caecum (g/d) 1959 1186 1598 2375

Starch entering the caecum(g/d) 82 37 900 74

*Digestibility of starch was estimated from the amount of starch that had disappeared betweenthe mouth and the caecum, and expressed as a % of starch intake.

The results from the slaughter study (Table 3) indicate that the pre-caecal digestibility of sorghumstarch was considerably lower than the pre-caecal digestibility of starch in the other grains tested. Thelow digestibility of sorghum starch was consistent with the in vitro assay results (Table 5). Howeverthe in vitro assay results also predicted that the pre-caecal digestibility of barley starch should be lowerthan the digestibility of starch in either triticale or oats but this difference was not obvious in theslaughter data. The lack of agreement between the enzyme assay and the slaughter results may havebeen due to the length of time that elapsed between the final feed and time of slaughter (16h). Duringthis time, starch reaching the caecum will have been exposed to microbial enzyme attack and so thestarch in the more fermentable grains may have been almost completely fermented. This problem wasanticipated and measurements of acidity (pH) and VFA concentration in caecal digesta were made as itwas expected these results would provide an indirect indication of the amount of starch reaching thecaecum. The results for caecal pH and VFA concentration are presented in Table 4 and clearly indicatethat compared with oats, triticale and barley, more starch was reaching the caecum of horses fedsorghum. Total VFA concentration in the caecum of the sorghum-fed horses was 35% higher thanVFA concentration in the caecum of horses fed the other grains. The pH and concentration of VFA incaecal digesta were comparable for horses fed oats, sorghum and triticale diets but again too muchtime may have elapsed after the final meal for differences to be detected. Further in vivo studies arerequired to determine whether the in vitro enzyme assay can accurately predict the intestinaldigestibility of starch in the more digestible grains.

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Grain supplements were fed as a fixed percent (1%) of bodyweight so the large variation in starchintake between the dietary treatments (Table 3) was due mainly to the different starch contents of thegrains fed in this study (Table 2). The most significant feature of the results reported in Table 3 is thelarge amount of starch that was apparently digested prior to the caecum in horses fed triticale. This isan important result for several reasons. Firstly, the enzyme assay predicted that triticale starch shouldbe highly digestible in the intestine of the horse and the slaughter data has confirmed this prediction.Secondly, triticale is not currently used in horse diets so this finding has important implications for thehorse industry. Finally, the results indicate that provided starch is readily accessible to enzyme attack,the intestines in the horse have a high capacity to digest starch.

The pH of faeces provides an indirect indication of pH conditions in the caecum and so this parameterwas monitored during the grain-feeding period. The results presented in Table 4 show that faecal pHwas highest for the triticale-fed horses which is a further indication that most of the triticale starch wasdigested prior to the caecum.

Table 4 Acidity measurements in caecal and faecal samples collected from horses fedsupplements of cereal grain.

Measurements Treatment dietsBarley Oats Sorghum Triticale

Faecal pH - Day 4 6.63 6.43 5.63 6.63Faecal pH – Day 6 6.33 6.15 5.90 6.78Faecal pH – Day 8 6.53 6.70 6.75 7.15

Caecal pH – at slaughter 6.60 6.70 6.40 6.70Caecal VFA (mmol/L) 67 67 93 69

Table 5 Comparison of in vitro assay estimates of starch digestion and estimates of pre-caecal starch digestion in slaughtered horses.

Grain Type Cultivar Starch digestion (%)In vitro assay In vivoEnzyme Fermentation Pre-caecal

Barley Tantangara 48.1 69.0 95.7Oats Yarran 66.5 73.9 97.3Sorghum Western Red 32.6 44.8 63.7Triticale Tahara 65.0 76.9 97.1

Conclusion In vivo study 1

The predictions from the in vitro assay were largely confirmed by results from the slaughter study.Therefore we believe that the enzyme assay developed in this project will prove to be very importantin the development of successful grain-feeding systems for the horse. Of particular note was theconfirmation that most of the starch in triticale was digested in the intestine. This finding hasimportant implications for the horse industry. More sensitive in vivo studies are required to determinewhether the in vitro enzyme assay can accurately predict the intestinal digestibility of starch in thehorse for more digestible grains.

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In vivo study 2 - Triticale (two cultivars) and wheat (+/-enzymes)

Results from our in vitro assay indicated a large variation in the digestibility of different cultivars oftriticale. For this reason, a second experiment was conducted to investigate the starch digestibility invivo of two cultivars of triticale determined by our in vitro assay as being of high and low digestibilityrelative to each other. If this difference between samples of triticale turns out to be a genetically-linked then it provides a very important lead for plant breeders as well as for-end users keen topurchase grains with high starch digestibility.

The second aspect of this study was to investigate the potential application of exogenous enzymes forimprovement of starch digestion of grains not often used in horses. Wheat is a grain with a relativelylow digestibility even in poultry and the risks associated with feeding this grain to ruminants are wellknown. In poultry the poor digestibility has been linked to the presence of non-starch polysaccharidecomponents of the cell walls increasing viscosity. The digestibility of these “low ME” wheats can beconsiderably improved by the use of exogenous enzymes that break down the NSPs. This method ofadding exogenous enzymes to the diet of poultry to improve starch digestibility is very widespread andit is important to know whether this approach will be effective as a method of improving starchdigestibility in the horse.

The effect of exogenous enzymes active against the NSP fraction is partly through modified physicalcharacteristics of the digesta and it is therefore unlikely that efficacy of exogenous enzyme use can beexamined using the in vitro assay described above. For this reason we included the treatment in thecurrent in vivo experiment.

Experimental Design

Sixteen mares and geldings of mixed breed and aged between 3 and 26 years were purchased for thisexperiment. On their arrival at the stables all horses were weighed, checked for any signs ofabnormality, identifying features recorded, identification collars attached and a broad-spectrumanthelmintic paste administered orally. Two animals that were considered by the examiningveterinarian to be unsuitable on the basis of health were excluded from entering the trial. Horses wereallowed a seven-day adaptation period for acclimatisation to surroundings and adaptation to diets andfeeding regimes. During this adaptation period, 1 horse was excluded from the trial on the basis ofpoor health. The remaining 13 horses, ranging in size from 332 kg to 452 kg, were stratified accordingto body weight and within each stratum were allocated at random to one of four dietary treatments:Triticale Madonna, Triticale Tahara, wheat or wheat with enzyme. All grains were hammer milled,and this was to reduce the risk of feeding wheat grain, to decrease variability of mechanical chewingbetween horses due to age differences (several old horses had worn teeth) and to allow maximumopportunity for the enzyme included to have access to the starch content of the grain. Horses were fedthese diets for the 14-day experimental period prior to slaughter.

Table 6 Summary of sudy deign, starch content of the different grains, enzyme starchdigestibility measured in vitro and rate of fermentation (% starch fermented in 3 hours).

Group Numberof

Animals

Test Grain Variety Starch contentof grain (%)

In vitroenzyme assay

(starchdigested)

In vitrofermentation(% starch)

1 3 Triticale Madonna 55.5 65 522 3 Triticale Tahara 57.2 76 783 4 Wheat Janz 56.3 43 584 3 Wheat +

enzymeJanz 56.3

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Feeding and management

During the 7-day adaptation period all horses were fed lucerne chaff mixed with increasing levels oftest grain starting at 500 grams per day and increasing to 1 % body weight per day. For the 14-dayexperimental period that followed, all horses were fed lucerne chaff (0.65 % of bodyweight per day)and the grain to be tested at 1% bodyweight per day. The amount fed each day was divided betweentwo equal feeds offered at 8 AM and 4 PM. Horses were fed individually in separate stables, and anyrefusals were weighed and recorded. Fresh drinking water was available at all times. Horses wereexercised daily in a round yard while the stables were cleaned. During the final 5 days on theexperimental diets ytterbium was added to the grain component of the diets as an inert marker fordigesta flow.

Enzymes

Water or enzyme solution (0.4% of bodyweight per day) was added to all feeds 30 minutes prior tofeeding. Group 4 horses had enzymes included in their diets whilst horses in groups 1, 2, and 3received water only. The enzyme solution contained a mixture of Bio-Feed Alpha (500 U/g β-glucanase and 150 U/g α-amylase) and Bio-Feed Wheat (1000 U/g xylanase) and was included at arate of 800ml and 600ml per tonne of feed respectively (Novozymes Pty Ltd, North Rocks, NSW,Australia).

Measurements

Horses were weighed on arrival at the stables and at weekly intervals thereafter. Feed intake wasmeasured daily, and fresh faecal samples were collected on days 5, 6 and 9 of the experimental period.Faecal samples were mixed 50/50 by weight with distilled water and the pH of each sample wasmeasured and recorded.

At the conclusion of the experimental feeding period, horses were transported to the local pet foodabattoir. Horses were slaughtered approximately 16 hours after their last feed and immediately afterslaughter the digestive tract was removed and ties placed at the duodenum, ileum, the junctionbetween caecum and colon and between the proximal and distal colon. Each section of the gut wasweighed before removing sub-samples for analysis of starch, dry matter, pH, VFA and ytterbium. Thedigestibility of starch in different parts of the digestive tract was estimated relative to ytterbiumconcentration.

Results

The key results are summarised in Tables 7 and 8. Table 7 shows differences between treatment dietsin the amount of starch remaining in the caecum relative to the inert marker 16 hours after the last feedof grain. The intake of starch and rate of fermentation was similar for all grains. There was morestarch entering the caecum in horses fed the triticale Tahara diet and the wheat diet than in horses fedtriticale Madonna or the wheat with the exogenous enzymes added.

Table 7 The apparent digestibility of starch prior to the caecum in horses fed supplements ofcereal grain.

Measurements Treatment dietsMadonna Tahara Wheat Wheat +

EnzymeStarch intake g/d 2317 2260 2533 2427Apparent digestibility of starchprior to caecum (%)* 99.5 96.5 95.3 99.1

Apparent flow of starchentering caecum (g/d) 12 80 116 22

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*Digestibility of starch was estimated from the amount of starch that had disappeared between themouth and the caecum, and expressed as a % of starch intake.

The faecal pH, caecal pH at slaughter and caecal VFA concentrations all showed differencesconsistent with measured differences of starch flow to the caecum. In the Tahara and wheat treatmentsthere was lower pH in both faeces and caecal digesta than measured in horses fed Madonna and thewheat with enzymes. In those horses and treatments with low pH (Tahara and wheat) there were thehighest concentrations of VFA. There were also higher concentrations of VFA in the caecal digesta ofhorses fed wheat plus enzymes.

Table 8 Faecal and caecal pH measured in horses fed two cultivars of triticale and wheat grainwith or without a mixture of exogenous enzymes active against NSP and starch.

Measurements Treatment dietsMadonna Tahara Wheat Wheat +

EnzymeFaecal pH - Day 5 6.95 6.64 6.57 6.68Faecal pH – Day 6 6.82 6.58 6.46 6.59Faecal pH – Day 9 6.72 6.57 6.36 6.85

Caecal pH – at slaughter 6.93 6.38 6.61 6.75Caecal VFA (mmol/L) 38.2 67.6 70.4 62.4

Discussion

The in vivo measurement of pre-caecal starch digestion and the fermentation characteristics in thecaecal and faecal samples are consistent with the in vitro predictions for Madonna, Tahara and forwheat. That is the in vitro enzyme assay ranked the pre-caecal digestibility in the exact order in whichit was measured for these three grains. In addition there was a close regression relationship betweenthe predicted pre-caecal starch digestibility and measured flow of starch to the caecum. It isencouraging that the in vitro assay accurately predicted differences between the two cultivars oftriticale as well as between the triticale and wheat.

y = -2.55x + 221R2 = 0.74

0

20

40

60

80

100

120

140

40 45 50 55 60 65 70 75 80

In vitro enzyme assay (% starch digested)

Star

ch p

assi

ng to

cae

cum

(g/d

)

Wheat

Barley Triticale(Tahara)

Oats

Triticale (Maddona)

Figure 5 In vivo enzyme starch digestion assay and the amount of starch flowing to the caecummeasured in vivo.

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The in vitro assay is not able to measure likely changes in digestibility due to the action of exogenousenzymes. The effects of exogenous enzymes need to be measured in vivo. It is clear that a similarimprovement in starch digestion is possible in the horse as is now well documented in poultry. Theonly concern about the action of the enzymes is the high VFA concentration. This high VFAconcentration is similar to findings in poultry where enzymes have increased both starch digestion andalso the rate of fermentation in the caecum. The increased fermentation could be due to the effect ofenzymes on the cell wall material increasing starch exposure to microbial fermentation.

When the results of the two in vivo studies are considered together (with the exception of sorghum)there is a very good relationship (R2 = 0.74) between the in vitro prediction of enzyme starch digestionand the amount of starch passing to the caecum measured in vivo. It is clear that sorghum is verymuch less digestible than the other grains with approximately 900 g/d starch passing to the caecumcompared with around 100 g/d for the next highest (wheat). While the in vitro assay correctly showssorghum as a feed of low potential digestibility in the small intestine of the horse the actualdigestibility appears not to be on a linear scale with other grains.

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5. Digestibility of oat grainOat grain has long been recognised as an excellent ingredient in the diet of humans and animals. Shaw(1907) declares that “viewed from the standpoint of adaptation for feeding live stock no cereal graingrown in [the USA] compares with the oat”. However, while oats is widely used as a feed grain forruminants and horses its value is often discounted because of the variability in its nutritional value.Variability in the nutritional value of oat grain has been the subject of numerous studies (e.g. Pickeringet al. 1982; Crosbie et al. 1985; Margan et al. 1987; Rowe and Crosbie 1987; Oddy et al. 1990)which show clearly that there are a number of factors to be considered if we are to accurately predictthe performance of animals given this feed.

This section of the report includes a review of literature and some experiments covering utilisation ofoats in ruminant animals and also discusses an experiment conducted as part of the current project. Itis recognised that ruminants and equids have different digestive anatomy and physiology but webelieve that the common element of fermentative digestion of NSP ‘fibre’ components are likely to besimilar in both animal species.

Ratio of groats to non-groat materialThe dominant factor determining nutritional value of any particular sample of oat grain is the ratio ofgroats to non-groat material. The non-groat fraction consists mainly of the fibrous hull but can alsoinclude variable amounts of other material such as weeds and seeds from other plants as well aschaffed head, leaf and stem material not separated from the grain in the harvesting process. Samplesof oat grain can also contain significant quantities of free groats if the hulls and groats are separatedduring harvesting and when this occurs the nutritive value of the sample can be improvedconsiderably. The amount of non-groat material and the presence of free groats in an oat sample canbe seen easily, but unfortunately it is not always accounted for in ascribing variability to oat grainquality. Increasing amounts of non-groat material tend to decrease the density of an oat sample and isalmost certain to decrease its nutritional value (Pickering et al. 1982). For this reason oat grain issometimes traded on the basis of hectolitre or bushel weight. Oddy et al. (1990) reported that bulkdensity measured as kg/hL or as weight per hundred grains was poorly correlated with acid detergentfibre (ADF) and these authors suggested bulk density is of little or no value in predicting oat grainquality. This conclusion regarding hectolitre weight, and therefore groat to hull ratio, is based on arange of diets for sheep constructed from oat fractions to match the extreme range of chemicalcompositions of oats grain found ‘in the field’ (Oddy et al. 1990) and so overlooks the value of graindensity as a predictor of groat to hull ratio in clean samples of oats.

The study of Oddy et al. (1990) with the constructed diets shows the importance of accounting for thevariable hull to groat ratio. Although the relationship with digestible energy is very good (Figure 6) itis important to realise that the most digestible ‘oat’ diet in this study was in fact pure groats and thatthe least digestible ‘oat ‘ contained approximately 75% oats and 25% additional oat hulls. Thereforewhile this is a very important predictive relationship when buying or selling oats with either a lot offree groats or an unusually high proportion of non-groat material, it does not necessarily provide anaccurate description of differences among ‘normal’ oats with only small contents of free groats or non-groat material. The problem of using ADF to predict the digestibility of ‘normal’ oats is shown inFigure 7. The data have been taken from three experiments with sheep in which the digestibility ofdifferent oat samples were measured; two samples were the true oat grain samples described by Oddyet al. (1990), eight values come from the data of (Margan et al. 1987) and two come from Rowe andCrosbie (1988). The challenge is to understand the causes of the variability shown.

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y = -0.021x + 18.9R2 = 0.94

12

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15

16

17

18

0 50 100 150 200 250 300

Acid detergent fibre (g/kg)

Dig

estib

le e

nerg

y (M

J/kg

DM

)

Figure 6 Relationship between ADF and digestible energy of oat diets measured in sheep feedingexperiments as reported by Oddy et al. (1990).

R2 = 0.18

y = -0.012x + 16.7

12

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14

15

16

17

18

50 100 150 200 250

Acid detergent fibre (g/kg)

Dig

estib

le e

nerg

y (M

J/kg

DM

)

Figure 7 Relationship between ADF and digestible energy of oat grain without added groats orhulls. Two values are the grain samples with 180 g ADF/kg and 25 g ADF/kg ofOddy et al. (1990), eight are taken from the data of Margan et al. (1987) and two fromRowe and Crosbie (1988).

Hull lignin as a factor influencing digestibility

Crosbie et al. (1985) reported that there was considerable variation in hull lignin content betweendifferent cultivars of oat grain and that these differences are predominantly genetically controlled.Most cultivars were found to be either ‘high’ lignin with around 3% lignin in the whole grain andaround 6 – 10 % in the hull fraction, or ‘low’ lignin with around 1% lignin in the whole grain and 1 –3% in the hull. This discovery provided a possible explanation for the experience of many livestockmanagers that certain cultivars of oat grain were better for livestock production than others. Rowe andCrosbie (1988) then showed that the differences in hull lignin content in high- and low-lignin oatcultivars had a very significant effect not only on the digestibility of the hulls but of the whole grain.With ADF, the ratio of hulls to groats, and the concentrations of protein and ash all held constant

19

between two cultivars, Rowe and Crosbie (1988) found that a difference of approximately 20 g ligninper kg grain resulted in an improved digestibility of the order of 15%. An independent study byMargan et al. (1987) reported significant differences in digestibility between the grains from twocultivars, Coolabah and Cooba. They also differed in lignin content, though that was not the reasonthey were chosen for the study, and so their results add considerably to appreciation of the importanceof hull lignin content. Table 8 summarises the effect of lignin content on digestibility of oat grain insheep fed principally oats at a rate of approximately 1.6% of body weight per day.

While the ADF content of grains of the high-lignin cultivars is slightly higher, this difference wouldonly explain approximately one third of the change in DE with ADF indicated by the predictiveequation shown in Figure 6.

Table 8 Analysis of the major components (g/kg dry matter) of four samples of oat grain andthe digestible energy (DE, MJ/kg dry matter) obtained with sheep fed these diets atrates equivalent to approximately 1.6% of body weight per day. Data for ‘Cooba’ and‘Coolabah’ were derived from the study of Margan et al. (1987) and for ‘Murray’ and‘Mortlock’ from Rowe and Crosbie (1988).

Cultivar Lignin ADF DELow-lignin

Murray 8 133 15.6Cooba 10 110 15.4

High-ligninMortlock 23 144 14.0Coolabah 30 150 13.5

Average difference between high- andlow-lignin cultivars

17.5 25.5 1.8

Level of feeding

With all feeds it is accepted that as the level of feeding increases there is a slight decrease in theefficiency of digestion, and DE/kg of feed consumed is reduced. For most feeds the decrease indigestibility with increasing level of feed intake is relatively minor. However, in the case of wholeoats, level of intake appears to be an important factor in sheep and cattle as shown in Figure 8 whichuses data from Margan et al. (1987). Level of feed intake is also likely to be a factor in determiningthe digestibility of oat grain in horses.

y = -0.85x + 15.6R2 = 0.81

12

13

14

15

16

0 0.5 1 1.5 2 2.5 3 3.5

Level of intake (g/100 g body weight)

Dige

stib

le e

nerg

y (k

g/M

J DM

)

Figure 8 Decrease in digestible energy value of oats grain with increasing intakes by sheep.Data are for Coolabah oats as reported by Margan et al. (1987)

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Combining hull lignin and level of intake with ADF to predict DE

When we account for both level of feed intake and lignin content of oat hulls in the prediction ofdigestible energy of oats grain we are able to explain much of the variability shown in Figure 7.Figure 9 illustrates the same data as those used in Figure 7, but level of feed intake has replaced ADFas the independent variable and grains have been separated on the basis of their lignin content. Thetwo data points representing 18% ADF and 25% ADF oats are shown as the filled triangles each with asingle line through them; that they fall either side of the line of best fit suggests that the prediction ofDE based on feed intake and lignin could be further improved by considering ADF as a measure ofgroat to non-groat material. A robust method of predicting DE would be to use the predictive equationbased on the results of Oddy et al. (1990), add 1.5 MJ/kg if the grain is low lignin, and then adjust DEfor level of intake by 0.86 MJ/kg for each 1% of body weight consumed above or below the 1% baselevel used by Oddy et al. (1990).

y = -0.85x + 16.9R2 = 0.95

y = -0.87x + 15.6R2 = 0.67

12

13

14

15

16

17

0 0.5 1 1.5 2 2.5 3 3.5

Intake (g oats/100 g bodyweight)

Dig

estib

le e

nerg

y (M

J/kg

dry

mat

ter)

Figure 9 Prediction of digestible energy of oat grain taking into account level of feeding andgrains as high-lignin (▲) or low-lignin (○). As for Figure 2, eight values (plaintriangles and circles) are taken from the data of Margan et al. (1987), two (closedtriangles with a single horizontal line) are from Oddy et al (1990), and two (trianglesand circles with an X) are from Rowe and Crosbie (1988).

Hull lignin content and gut fill in sheep and cattle

A feeding experiment was conducted in cattle by May et al. (1989) using low- and high-lignin oatcultivars, Murray and Mortlock respectively, and demonstrated a significant effect of high-lignin oatson gut fill and dressing percentage. There was accumulation of low digestibility roughage in therumen and a much larger rumen in cattle fed high-lignin Mortlock grain. Similarly in sheep fedsupplements of high or low-lignin grain there was significantly higher weight of digesta in animals fedthe lower digestibility high-lignin grain (see Table 9). Both sets of data in Table 3 suggest that lignincontent of the oats could make a difference of around 10 kg for a 500kg animal.

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Table 9 Differences in gut fill in cattle and sheep fed high-lignin (Mortlock cultivar) or low-lignin (Murray cultivar). Data taken from May et al. (1989a) and Rowe and Coss(1992)

Mortlock Murray Signif.of diff

(P)

Lignin (g/kg) 24 11In vivo dry matter digestibility (%) Measured in sheep (maintenance) 70.2 82.4 Measured in cattle (intake approx. 2.2% of body

weight)64.1 71.6

Dressing % measured in cattle (Carcase weight as % ofbody weight)

49.8 51.8 0.003

Weight of reticulo-rumen (kg) measured in sheep 4.70 3.71 0.01

The results of Johnson et al., (1998) and Pagan (2001), show that the gut contents and weight of thehorse, like sheep and cattle, increases in response to consumption of lower digestibility feeds. It istherefore almost certain that horses fed lower lignin oat grain will have less gut fill and be lighter thanhorses fed an equivalent amount of high-lignin grain.

Processing oat grain for animal feedingThe question of whether or not there is any advantage to be gained from processing oat grain beforefeeding it to cattle and horses is a very important one in determining its suitability for on-farm use insituations where no grinding and mixing equipment are available. We are aware of three studies incattle examining this. The consistent finding in all three studies was that there is little or noimprovement in digestibility or animal performance in response to processing oat grain by rolling orhammer milling. Toland (1976) reported improvements of between 60 and 100% in the digestibility ofwheat and barley starch as a result of rolling compared to feeding whole grain, but found no significantbenefits in the case of oats. It is therefore safe to conclude that oat grain can be fed whole to cattlewithout any risk that it will be digested or utilized inefficiently for production. The data of May et al.(1989b) even suggest possible benefits in whole grain feeding to achieve slightly higher consumption.As is the case with all cereal grains, there is no benefit in rolling or grinding oats before feeding it tosheep.

A study on the effect of rolling on oats on their subsequent digestibility by horses (Wilson 2001)suggests that there are significant improvements in digestibility in response to this form of processing- 50.5% for rolled oats vs 44.6 % in the case of unprocessed whole grain.

Table 10 Categorisation of oat cultivars tested with a combination of wet chemistry and theCrosbie colour test for lignin content. Information for this table was obtained fromCrosbie et al. (1985), G.B. Crosbie (pers. comm.) and A.G. Kaiser (pers. comm.).

High lignin Low lignin MediumDalyup Irwin WestEchidan MurrayKalgan SwanMoore YilgarnMortlock CoobaCoolabah YarranGraza-50 Graza-70Pallinup MarlooDumont CulgoaBettong AmbyCleanleaf BimbilPanorama-5 Carbeen

Nile

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Measuring lignin content of oat hullsMeasurement of lignin content by wet chemistry is time consuming and expensive, and if the analysisis not made by an experienced technician it can yield uncertain results. Probably due to thepainstaking task of measuring large numbers of lignin concentrations, combined with the difficultprocess of hand separation of hulls from groats, there are as yet no NIR prediction equations for hulllignin. Although not quantitative, a test developed by Crosbie and his co-workers (Rowe et al., 2001)provides a quick and accurate indication of whether samples of oat grain fall into the category of‘high-lignin’ or of ‘low-lignin’. The Crosbie oat hull assay is based on the pink colour that developswhen a solution of phloroglucinol stains the lignin component of the hulls, and is most easily made byadding approximately 2 ml of a phloroglucinol solution to about 10 whole oat grains or to separatedhulls from 3 to 5 grains. The solution is prepared by dissolving 1 g of phloroglucinol in 80 ml 2 MHCl plus 20 ml ethanol, and then filtering. The phloroglucinol solution should be kept in a dark bottlein a refrigerator (4 oC) and can be used for about 1 month if properly stored. Sufficient solution isadded to cover the grains and/or hulls placed in a white ice-block tray; the volume of each well easilyaccommodates the sample and solution. The multiple compartments facilitate inclusion of knownhigh- and low-lignin samples, and the white background enables ready detection of differences incolour.

A list of major cultivars of oat grains categorised by hull lignin content by the colorimetric methoddescribed above is summarized in Table 10. About half of samples of the West cultivar, categorised as‘medium’ had high concentrations of lignin and half had low concentrations. When individual grainswere grown to produce seed the low-lignin seeds produced grains with low hull lignin, and the high-lignin seeds produced high-lignin grain.

Visual appearance of low- and high-lignin oat grain

The visual assessment of all grains for evidence of mould or moisture damage is important becausefungal contamination can have adverse effects on feed intake as well as a range of toxic effects. Thepresence of fungus is most easily identified as darker colorations because mycelium and spores areoften black in colour. There has therefore been a preference amongst buyers for bright, light yellowcolours in oat hulls. It is important to note that the hulls of low-lignin oat grain are often slightlydarker than that of high-lignin grains, possibly due to accumulation of pigmented phenolics in the hullof low-lignin cultivars. This darker coloration of low-lignin hulls is natural and does not signifyfungal contamination. In order for buyers to have complete confidence when purchasing low-ligningrains of slightly darker colour it may be necessary to develop a quick assay for fungal contaminationto complement the rapid colour method for differentiating between high- and low-lignin cultivars.

Digestibility of low- and high-lignin oat grain by horsesAn objective of this project was to investigate the role of lignin in determining the digestibility of oatgrain in horses. There have been some problems in interpreting the results of this study. A partialanalysis of the data using data from 4 horses suggests that there was a slightly higher digestibility ofthe low-lignin Yarran cultivar than the lower lignin mortlock cultivar (50.6% vs 44.4%).

Conclusions - oatsWe consider that breeding and cultivation of low-lignin oat cultivars should be encouraged by grainusers being prepared to pay more for the higher digestibility grain than for the high-lignin lowdigestibility cultivars. There are currently a number of low-lignin oat cultivars grown commerciallyand we are not aware of problems, such as lodging or rust resistance, being linked to lower levels oflignin in the hull. It is essential that we obtain unequivocal data for horses on the importance of hull-lignin, processing and level of intake.

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6. TriticaleIn addition to the in vitro and in vivo studies reported above on the digestibility of triticale we haveconducted further tests on low- and high-digestibility cultivars to determine whether the differencesare consistent between different samples of each cultivar. These results are summarised in Table 11below.

Table 11 In vitro digestibility measured in different samples of three cultivars of triticale.

Tahara Abacus MadonnaAverage in vitro digestibility (%) 52 65 57Standard error of mean 4.8 6.3 4.6Number of assays 5 3 4

Ttriticale is a cross between durum wheat and rye. It is therefore reassuring to find that the in vitrodigestibility of triticale lies between that of durum wheat and rye. The cultivars of Madonna andAbacus have digestibilities closer to that of rye while the digestibility of Tahara is closer to durumwheat (see Figure 10). These results suggest that it will be possible to select for cultivars of triticalethat are highly digestible by the horse. Even the use of cultivars such as Abacus and, to a lesser extentMadonna, offer some immediate opportunities for the horse industry. In the same way as variabilityhas reduced the value of oats it is possible that variability between triticale cultivars may diminish theperceived value of triticale – particularly if the reasons for the variability of performance of horses fedtriticale are not understood by processors and feeders.

0

10

20

30

40

50

60

70

80

Durum Tahara Madonnah Abacus Rye

In v

itro

enzy

me

assa

y (%

sta

rch

dige

sted

)

Figure 10 In vitro digestibility of the parents of triticale (rye and durum wheat) and of threecultivars of triticale.

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7. ImplicationsIn vitro assays

Two new assays for the horse feed industry and, particularly the enzyme assay to predict pre-caecalstarch digestion will be useful in helping to ensure that any person feeding grain to a horse will be ableto do so knowing the relative safety of the grain for the feeding purpose. In its simplest form the assayhas helped to confirm differences between grains with respect to starch digestibility and we suggestthe following ranking:

Highly digestible Poorly digestibleOats and Cultivars oftriticale

Cultivars of triticale, barley Wheat Sorghum and maize

The in vitro assay will also find a place in quantifying the effects due to processing of horsefeeds and providing a comparison of ‘before and after’. As illustrated by Channon and Rowe(2001) processing using the steam flaking can produce variable results due to differences in theextent of cooking and fineness of the flake.

Triticale as a new feed grain for horses

The results of the in vitro and in vivo tests have identified triticale as a grain highly digestibleby the horse. There appear to be certain cultivars of triticale that are more digestible than othersand this information will be of value to plant breeders, horse owners and feed manufacturers toproduce and use the most appropriate grains for horses.

Basis for selecting more digestible cultivars of oat grain

The review of oat digestion and the preliminary study in the horse has identified the possiblerole of hull lignin altering digestibility and utilisation of the grain by horses. The question ofrelative importance of hull lignin, processing (rolling and/or steam rolling) and the level of feedintake in determining gut fill and digestible energy deserves further experimentation. Since oatgrain is so widely used as a feed for horses it is important to have a clear understanding of thefactors influencing its utilisation. If hull lignin content emerges as an important factorinfluencing its digestibility then the rapid test for hull lignin and the fact that many cultivars ofoats are known to have low levels of lignin will assist breeders, growers and users to select anduse the most appropriate oat grain for feeding to horses.

Exogenous feed enzymes

The inclusion of exogenous enzyme preparation active against NSP and starch were shown toimprove pre-caecal wheat starch digestion. This demonstrates the potential for using exogenousenzymes to increase the usefulness of a wider range of grains than are currently used inpreparation of diets for horses. In other species such as poultry exogenous enzymes have been acost-effective and efficacious way to improve nutritive value of wheat and barley. In situationswhere the choice of grain is limited and it is necessary to feed wheat then it is likely thatexogenous enzymes may be an excellent way of improving the safety and effectiveness offeeding grain to horses.

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May, P.J.., Barker, D. J., Jones, W. M., Snowdon, J. M. and McMullen, G. R. (1989a). Oat grain of high or lowlignin content in diets for finishing beef cattle. Division of Animal Production, Department of AgricultureWA, Annual Report 1988/89, pp. 103-105.

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Oddy, V. H., Ewoldt, C.L., Jones,A.W. and Warren,H.M. (1990). Metabolisable energy content of diets based onoat grain. Australian Journal of Experimental Agriculture 30, 503-506.

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Pickering, F. S., Barrett, N. C., Farrell, D. J. and Corbett, J. L. (1982). Physical and chemical attributes of oatsgrain and nutritional value. Animal Production in Australia 14, 675.

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Rowe, J. B. and Coss, L. (1992). The importance of lignin content of oat hulls in ruminant nutrition. Proceedingsof the Nutrition Society of Australia 17, 218.

Rowe, J. B. and Coss, L. (1994). Lignin content of oats - does it affect animal performance when sheep are givenoats as a supplementary feed? Animal Production in Australia 20, 421.

Rowe, J. B. and Crosbie, G. B. (1988). The digestibility of grain of two cultivars of oats differing in lignincontent. Australian Journal of Agricultural Research 39, 639-644..

Shaw, T. (1907). Feeding Farm Animals. Orange Judd Company, London, UK.Toland, P. C. (1976). The digestibility of wheat, barley, or oat grain fed either whole or rolled at restricted levels

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