Nutritional Biotechnology in the Feed and Food Industries

Contents i

Nutritional Biotechnology in the Feed and Food Industries

Contents iii

Nutritional Biotechnology in the Feedand Food Industries

Proceedings ofAlltech’s Twentieth Annual Symposium

Edited by TP Lyons and KA Jacques

iv Contents

Nottingham University PressManor Farm, Church Lane, ThrumptonNottingham, NG11 0AX, United Kingdom

NOTTINGHAM

First published 2004© Copyright Alltech Inc 2004

All rights reserved. No part of this publicationmay be reproduced in any material form(including photocopying or storing in anymedium by electronic means and whether or nottransiently or incidentally to some other use ofthis publication) without the written permissionof the copyright holder except in accordance withthe provisions of the Copyright, Designs andPatents Act 1988. Applications for the copyrightholder’s written permission to reproduce any partof this publication should be addressed to the publishers.

Editor’s note: The opinions expressed herein are those of the authorsand do not imply endorsement of any product by the author or anypolicy or claim on the part of the Symposium sponsor.

ISBN 1-904761-27-5

Typeset by Nottingham University Press, NottinghamPrinted and bound by Bath Press, Bath, England

Contents v

Table of contents

Re-imagining the feed industry: focus on price, perception and policy 1

T. Pearse LyonsAlltech Inc., Nicholasville, Kentucky, USA

Future of the feed/food industry: re-inventing animal feed 11

Charles V. MaxwellAnimal Science Department, University of Arkansas, Fayetteville, Arkansas, USA

Glycomics: putting carbohydrates to work for animal and human health 27

Kyle E. NewmanVenture Laboratories, Inc., Lexington, Kentucky, USA

Focus on Poultry

Selenium sources and selenoproteins in practical poultry production 35

Frank W. Edens and Kymberly M. GowdyDepartment of Poultry Science, North Carolina State University, Raleigh, North Carolina, USA

Alternatives to antibiotics in poultry production: responses, practical experience and 57recommendations

Peter R. FerketDepartment of Poultry Science, North Carolina State University, Raleigh, North Carolina, USA

Facing the realities of poultry health and performance without antibiotics in Europe 69

G.G. Mateos1, J.M. Gonzalez-Alvarado2 and R. Lázaro1

1Departamento de Producción Animal, Universidad Politécnica de Madrid, Madrid, Spain2Departamento de Agrobiología. Universidad Autónoma de Tlaxcala, Tlaxcala, México

Reproductive responses to Sel-Plex® organic selenium in male and female broiler breeders: 81impact on production traits and hatchability

Robert A. RenemaDepartment of Agricultural, Food, and Nutritional Science, University of Alberta, Edmonton,Alberta, Canada

Optimizing the replacement of pronutrient antibiotics in poultry nutrition 93

Gordon D. RosenPronutrient Services Ltd., Wimbledon, London, United Kingdom

Comparative aspects of Fusarium mycotoxicoses in broiler chickens, laying hens and turkeys 103and the efficacy of a polymeric glucomannan mycotoxin adsorbent: Mycosorb®

Trevor K. Smith, Shankar R. Chowdhury and H.V.L.N. SwamyDepartment of Animal and Poultry Science, University of Guelph, Guelph, Ontario, Canada

vi Contents

Pig Science

Creating technical and educational forums that help pig producers meet performance and 113economic goals: the Premier Pig Program™

William H. Close1 and Kim Turnley2

1Close Consultancy, Wokingham, Berkshire, UK2Alltech Inc., Melbourne, Victoria, Australia

Successful feed companies in the future 121

Jim HedgesHubbard Feeds Inc., Mankato, Minnesota, USA

The role of selenium and Sel-Plex® in sow reproduction 131

Don MahanDepartment of Animal Sciences, The Ohio State University, Columbus, Ohio USA

Adding value to pork for producers and consumers: enhancing omega-3 DHA and selenium 141content of meat

Paul PennyJSR Genetics Ltd, Southburn, Driffield, United Kingdom

Reducing the environmental impact of swine production through nutritional means 149

K.J. Stalder1, W.J. Powers1, J.L. Burkett1, and J.L. Pierce2

1Department of Animal Science, Iowa State University, Ames, Iowa, USA2North American Biosciences Center, Alltech, Inc., Nicholasville, Kentucky, USA

Nucleotides and young animal health: can we enhance intestinal tract development and 159immune function?

C.D. Mateo and H.H. SteinDepartment of Animal and Range Sciences, South Dakota State University, Brookings,South Dakota, USA

Dairy and Beef Cattle

Evaluating inoculants for forage crops in Argentine beef and milk grazing systems: 171effects on silage quality and system profitability

Leandro O. AbdelhadiEl Encuentro Livestock farm, Research & Extension in Ruminant Nutrition, Alltech Argentina, Cnel.Brandsen, Argentina

Optigen®®®®® 1200: controlled release of non-protein nitrogen in the rumen 179

Veysel Akay1, Jeff Tikofsky1, Corwin Holtz2 and Karl A. Dawson1

1North American Biosciences Center, Alltech Inc., Nicholasville, Kentucky, USA2Comsen Dairy Consultation, LLC, Dryden, New York, USA

Contents vii

Dairy nutrition models: their forms and applications 187

William Chalupa, Ray Boston and Robert MunsonSchool of Veterinary Medicine, University of Pennsylvania, Kennett Square, Pennsylvania, USA

Gastrointestinal development in dairy calves 195

S. I. Kehoe and A. J. HeinrichsDairy and Animal Science Department, The Pennsylvania State University, University Park,Pennsylvania, USA

Meeting the educational needs of dairy clientele in 2020 205

Michael F. HutjensDepartment of Animal Sciences, University of Illinois, Urbana, Illinois, USA

Redefining selenium nutrition using organic selenium (Sel-Plex®): defining maximal 211acceptable tissue residues in beef

C.J. Richards and H.D. LovedayAnimal Science Department, University of Tennessee, Knoxville, Tennessee, USA

Rumen acidosis: modeling ruminant response to yeast culture 221

D. Sauvant, S. Giger-Reverdin and P. SchmidelyINAPG Département des Sciences Animales – UMR INRA–INAPG Physiologie de la Nutrition etAlimentation, Paris, France

The top ten most frequently-asked questions about mycotoxins, cattle and dairy food products 231

L.W. Whitlow1 and W.M. Hagler, Jr.2

1Department of Animal Science, North Carolina State University, Raleigh, North Carolina, USA2Department of Poultry Science, North Carolina State University, Raleigh, North Carolina, USA

Food, Nutrition and Health

All in good taste: creating natural savory flavorings from yeast 257

John DiehlJohn Diehl Consulting Services, Darien, Illinois, USA

One university’s search for intelligence in a universe of foods for wellness 265

Suanne J. KlahorstCalifornia Institute of Food and Agricultural Research, University of California, Davis, USA

How does diet influence health – could the food chain benefit from a more proactive approach 275to clinical nutrition issues?

John C. MacRaeRowett Research Institute, Bucksburn, Aberdeen, United Kingdom

Functional components of the cell wall of Saccharomyces cerevisiae: applications for yeast 283glucan and mannan

Colm A. MoranNorth American Biosciences Center, Alltech Inc., Nicholasville, Kentucky, USA

viii Contents

Food animal agriculture: a few issues that will impact our future food supply 297

Joe M. RegensteinCornell Kosher Food Initiative, Department of Food Science, Cornell University, Ithaca, New York, USA

Mycotoxins in the food chain: a look at their impact on immunological responses 305

Raghubir P. SharmaDepartment of Physiology and Pharmacology, The University of Georgia, Athens, Georgia, USA

Antioxidant activity of hydrolyzed whey, soy, and yeast proteins 315

Youling L. Xiong,1 Lin Wang,1 E. Aida Peña-Ramos,2 and Changtzheng Wang3

1Department of Animal Sciences, University of Kentucky, Lexington, Kentucky, USA2Animal Derived Food Department, CIAD, Hermosillo, Sonora, Mexico3Human Nutrition Program, Kentucky State University, Frankfort, Kentucky, USA

Equine Topics

A novel, knowledge-based concept for performance diagnosis and training adjustment in horses 325

Matthias BojerInstitut für Natursport und Ökologie, Abteilung Reitsport, Deutsche Sporthochschule Köln, Germany

Physiology and feed formulation: the proper role of carbohydrates in the equine diet 331

Stephen Duren1 and Kathryn Watts2

1Performance Horse Nutrition, LLC, Weiser, Idaho, USA2Rocky Mountain Research and Consulting, Center, Colorado, USA

Relevance of the NRC to today’s horse industry 337

Kevin H. KlineDepartment of Animal Sciences, University of Illinois, Champaign-Urbana, Illinois, USA

Bone biomechanics: a review of the influences of exercise and nutritional management on bone 345modeling in the growing and exercising horse

John R. KohnkeJohn Kohnke Consultancy Pty Ltd, Rouse Hill, New South Wales, Australia

Of caterpillars and horses: recent scientific progress on the cause of Mare Reproductive 355Loss Syndrome

Karen J. McDowellDepartment of Veterinary Science, University of Kentucky, Lexington, Kentucky, USA

Modern Agronomics

The pre-and postharvest application potential for Crop-SetTM and ISR 2000TM on citrus 361

John P. BowerHorticultural Science, University of KwaZulu-Natal, Pietermaritzburg, South Africa

Contents ix

Citrus fruit size and quality: response to Crop-Set™ in North America 369

Lawrence J. Marais and John G. FrankImprocrop Inc., Nicholasville, Kentucky, USA

Integrated efficacy of several Improcrop compounds on bacterial wilt of tomato plants 375under greenhouse conditions

Pingsheng Ji1, Tim Momol1 and Necip Tosun2

1North Florida Research and Education Center, IFAS, University of Florida, Gainesville, FL, USA2Plant Protection Department, Faculty of Agriculture, Ege University, Izmir, Turkey

Alternatives against Alternaria: controlling brown spot on Murcott tangors 379

Tim Johnston1, Lawrence J. Marais2 and L.W. Timmer1

1University of Florida, IFAS, Citrus Research and Education Center, Lake Alfred, Florida, USA2Improcrop Inc., Nicholasville, Kentucky, USA

Seed and soil treatments with a natural fungicide product against some fungal and bacterial 383diseases of vegetables

Necip Tosum and Huseyin TurkusayDepartment of Plant Protection, Faculty of Agriculture, Ege University, Bornova–Izmir, Turkey

Abiotic stresses and plant activators 387

Ismail Türkan1, Tijen Demiral1, A. Hediye Sekmen1 and Necip Tosun2

1Department of Biology, Faculty of Science, Ege University, Izmir, Turkey2Department of Plant Protection, Faculty of Agriculture, Ege University, Izmir, Turkey

Advances in Aquaculture

Opportunities and dilemmas in molecular aquaculture genetics 393

Roger W. DoyleGenetic Computation Limited, Halifax, Nova Scotia, Canada

Feeds for the future: the importance of better broodstock and larval nutrition in successful 407aquaculture

D.F. FeganAlltech Inc., Bangkok, Thailand

European finfish culture: current status, recent advances and future perspectives 421

John W. SweetmanEcomarine Ltd, Cephalonia, Greece

Fish meal and fish oil use in aquaculture: global overview and prospects for substitution 433

Albert G.J. TaconSEALAB Aquaculture Laboratory, Hawaiian Institute of Marine Biology, University of Hawaiiat Manoa, USA

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Creating alternative protein sources for aquafeeds using applied enzyme technologies 449

S. L. WoodgatePDM Group Ltd, Greenleigh, Kelmarsh Rd, Clipston, United Kingdom

From farm to fork: the challenges that fish farming faces as a responsible supplier of global food 457

Rohana P. SubasingheFisheries Department, FAO, Rome

Companion Animals

USA poultry meal: quality issues and concerns in pet foods 467

Greg AldrichPet Food & Ingredient Technology, Inc., Topeka Kansas, USA

The role of yeasts in companion animal nutrition 475

Kelly S. Swanson and George C. Fahey, JrDepartment of Animal Sciences, University of Illinois, Urbana, Illinois, USA

The expanding pet food industry: where are the opportunities? 485

Tim PhillipsPETFOOD INDUSTRY Magazine, Watt Publishing Co., Mt. Morris, Illinois, USA

Using nutritional genomics to study canine obesity and diabetes 495

Kelly S. SwansonDepartment of Animal Sciences, University of Illinois, Urbana, Illinois, USA

A peek into the new NRC for dogs and cats 503

Angele ThompsonThompson PetTech, New Providence, New Jersey, USA

The importance of antioxidant protection: demonstrating and branding benefits in pet food 509

Lucy TuckerAlltech Inc., Stamford, Lincolnshire, United Kingdom

A changing landscape: the pet food market in Europe 517

Jürgen ZentekInstitute of Nutrition, Department of Veterinary Public Health and Food Science, VeterinaryUniversity of Vienna, Austria

Index of topics 523

T.P. Lyons 1

Re-imagining the feed industry: focus on price, perception and policy

T. PEARSE LYONS

Alltech Inc., Nicholasville, Kentucky, USA

Global agricultural is in turmoil. World trade barriersare coming down. The EU Common AgriculturalPolicy and the US supports may become a thing ofthe past, opening up markets and leveling the playingfield. BSE cases in Canada and the US have thrownthese industries into a quandary, while southeast Asiais reeling from the impact of avian flu.

Natural feed technologies such as those offered byAlltech have never been more in favor. Increasingacceptance of natural feeding strategies reflects therealization that there is no going back to previousmethods in today’s consumer-oriented markets. At arecent roundtable discussion sponsored by Alltech inthe aquaculture sector, one attendee described theevent as “the most exciting discussion in which I haveparticipated in the past 10 years”. Such is theenthusiasm in these markets. We believe that everycloud has a silver lining, but for agriculture thedifficult times of these past years can only be turnedaround if we embrace change and recognize the threekey determinants of success: Price competitiveness,Perception of the consumer, and Policies we candepend on to guide us now and in the future.

Success Factor No. 1:Price competitiveness

How can other markets compete given the size ofUS and Brazilian farms? How can a small countrymaintain its position? To be successful we must adoptnew technologies, which is how Brazil made suchremarkable strides forward in a comparatively shortperiod of time.

What are some of the new technologies that canmake food animal production more price-competitive? The two most important aretechnologies that improve the efficiency with which

we use feed ingredient raw materials, and the otheris through improved animal health.

Raw materials Utilize wider availability of cereals, protein sources

New generation SSF enzymes to improve efficiency

Improved Herd longevity: cows, sows

animal health More piglets weaned

More high quality chicks per breeder

Reduce heath costs

Success Factor 1

Lowering production costs

THE KOJI PROCESS OF SOLID STATEFERMENTATION: LOWERING THE COST OFCONVERTING FEED TO MEAT AND EGGS

Nature ensures utilization of its abundant feedstuffsby placing microbes and animals in symbiosis. Inboth ruminants and monogastrics, rumen or hindgutmicrobes digest the structural carbohydrates in fiberto release energy and ultimately to provide proteinfrom microbial cells for animal use. Without themicrobe’s ability to produce enzyme arrays, the hostanimal could not make much use of a vegetable-based diet.

Alltech harnessed this symbiosis with a uniquefermented koji, which is sold under the name ofVegproTM SSF. In the koji fermentation the enzyme-producing microbes are grown on substrates similarto those that food animals consume, which inducesthe microbe to produce the spectrum of enzymes mostappropriate for the job. When added to poultry andpig diets containing oilseed meals such as soya and

2 Re-imagining the feed industry

Table 2. More than 50 experiments and 120 companies confirm that the koji enzyme response has a major impact on production economics.

Results

Weight (kg) Feed conversion

Country No. of birds Age (days) Control Vegpro Control Vegpro

Argentina 4000 4 9 2.56 2.57 2.14 2.154000 5 1 2.75 2.87 2.08 2.02

3200000 53 2.64 2.64 2.23 2.245000 4 9 3.43 3.45 1.79 1.79

Brazil 3292 4 2 2.07 2.05 1.95 1.94Ecuador 1200 4 2 2.50 2.63 1.58 1.51Peru 65600 4 9 2.70 2.79 2.03 1.92

95000 4 7 2.67 2.72 2.08 2.02

canola, seven enzyme activities in VegproTM SSFinteract to boost release of energy and protein.Brazilian experience in formulating poultry and pigdiets with VegproTM SSF has shown that savings ofas much as $10-15 per tonne are possible. As globalreliance on shorter supplies of vegetable proteinsincreases, this technology could be crucial (Table 1).Why? Because the enzymes release the energy in soyaand improve protein digestibility. A summary ofexperience with VegproTM SSF in South America isfound in Table 2.

Table 1. The soybean challenge: usage and production growing atdifferent rates.

2001/2002 2001/2002 2003(Million bushels) (Million tonnes) Change (%)

ProductionUnited States 2,890 78.6 -11Brazil 1,598 43.5 +10Argentina 1,084 29.5 +12China 556 15.1 +9Total 6,751 183.7 +6

Use projection for 2004-2005China +15%Russia +25%

LOWER PRODUCTION COSTS BY IMPROVINGANIMAL HEALTH

At a recent presentation on the future of Americandairy farming, Dr. Steve Koenig pointed out thatanimal health is the key to success in the future. Whilehe used the dairy farm to illustrate his points, theycould be applied to any animal production system.An example illustrating the impact improved animalhealth has on productivity is cow longevity. Dairycows average only two lactations in several regionsof the US, while 60% of all sows are culled afteronly three parities. Given that peak milk productionin the dairy cow and peak sow productivity are wellafter these ages, the amount of lost production isastounding. At an average of 10,000 kg of milk per

lactation and 20,000 per lifetime, this means areplacement cost of $0.06/kg of milk - nearly 24%of the total selling price of milk! Imagine anycompany devoting 20-25% of the sales to replacingthe equipment! They could not survive, and nor canwe. If an extra lactation can be achieved, replacementcost drops to $0.04/kg or 17% of the total cost(Table 3).

Table 3. Calculated cost of replacing a cow based on two or threelactations.

Replacement heifer cost, USD 1400Culled cow price, USD 200Milk per lactation, kg 10,000Milk price, USD/kg 0.20Replacement cost of the cow, USD/kg milk Longevity: 2 lactations 0.06 Longevity: 3 lactations 0.04

Sel-Plex® impact on health

Cows and sows are culled for reasons of health andreproduction, both of which are at risk when seleniumstatus is marginal; and it is this specific area whereSel-Plex®, organic selenium produced by yeast, canhelp. Selenium in Sel-Plex® is present in the idealratio of selenoamino acids. When mastitis/MMAimpact is reduced, and selenium needs forreproduction are met, commercial experience withSel-Plex is that herd longevity can be increased,however an extra lactation is just one of the benefitsnoted. Sel-Plex® has implications for health andreproductive efficiency in all food animal species(Table 4). For sows, commercial and universityreports have indicated more pigs born alive and morepigs weaned; and a review of poultry data in refereedpublications alone demonstrates increased number ofchicks hatched (2-4) per broiler breeder hen.Furthermore, improvements in health make the switchto Sel-Plex® easy. Is this new? No. Dr. Don Mahan

T.P. Lyons 3

at Ohio State predicted this in 1995! The issue is asever – not whether the new technology will lowercosts – it can – but whether the will to make thechange in order to reap the benefits exists. Organicselenium – Sel-Plex® – has truly redefined seleniumnutrition and indeed vitamin E and ‘antioxidant’supplementation in general. However, there can beno half measures. Full replacement of sodiumselenite at all stages of life is required. Health is alifelong requirement.

Table 4. Sel-Plex® impact on herd health and productivity.

Dairy cattle Cow longevity in the herd: lifetime yieldFewer days openFewer services/conceptionNo Se injections neededReduced retained placenta incidenceReduced mastitis incidence/impactLower SCCCalf livabilitySupranutritional vitamin E levels unneeded

Beef cattle Meat qualityCalf livabilityFeed efficiency

Pigs Sow longevity in the herd: lifetime productivityAn extra 0.5-1 pigs weaned/litterReduced MMAPiglet health at birth and weaning

Chickens Breeders More settable eggs produced Fertility (male and female) Hatchability More chicks per hen

Broilers Reduced mortality/culls Improved uniformity Feed efficiency Meat quality

Layers Egg production Egg quality Shell quality

Knowledge about organic selenium is accumulatingat an incredible rate in all disciplines, but agricultureis the sector able to take greatest advantage of it.Still, it is always best to remember how much westill do not know! Not long ago science only knewof the role of selenium in glutathione peroxidase(GSH-Px). Now we know there are six forms ofGSH-Px, and 30-50 selenoproteins. Likewise, wenow know that there are a wide range ofselenocompounds in plants and yeast, and failure todiscount the importance of any of them because wedo not today know their function would be absurd.Nature rarely makes things for no reason. Modernanalytical techniques have revealed one reason

response differs between selenium yeast sources.French researchers noted that the profile of seleniumcompounds differs among commercial seleniumsources (Figure 1). Reasons might include differinggrowth media, pH and temperature conditions and(or)yeast strain. As such, data generated from a productmanufactured by one process cannot be extrapolatedto another. This why in clearing ‘selenium yeast’ foruse in the US following review of Sel-Plex®, FDAdefined an allowable product as one made preciselyby this process. In effect, the regulators are holdingall new products to the standard set by Sel-Plex®.

Key Success Factor No. 2:Perceptions of the consumer

Overcoming the negative perception of the consumeris more difficult to achieve than a reduction in costs.Due to a litany of scares – BSE, Foot and MouthDisease and dioxin contaminations – the public isoften suspicious of modern agriculture.

Success Factor 2

Changing consumer perceptions

Animal feed contains antibiotics used in human medicine

Animal feeds contain recycled 'dangerous' animal proteins

Agriculture pollutes soil and water

Meat, milk and eggs are not 'healthy' foods

The recent mad cow scare in the US illustrates howreluctant as an industry we are to change, and perhapsvalidates consumer skepticism and the demand forgreater scrutiny. Carol Tucker Forman, director ofthe Food Policy Institute of the Consumer Federationof America, was quoted as saying “the damage to theAmerican meat industry, and therefore the feedindustry, costs infinitely more than anything UScattlemen would have to pay to do things right”. Butdoing things ‘right’ is not something we are alwaysperceived to excel at. Least cost formulationsoccasionally overrule common sense, and it seemsincredible that in a time when markets are asking fortotal transparency and traceability that one wouldleave anything to chance, much less take unnecessaryrisks. As we marvel at the apparent “repeating of theEuropean BSE mistakes” in the US, we remindourselves that the perception is that many of ourproblems originate from what and how we feed


livestock. This should not be the case; there are anumber of alternatives in use in all sectors of theindustry. Let’s briefly evaluate ways in which naturalfeeding programs combat perceptions of food animalagriculture.

PERCEPTION 1: ALL ANIMAL FEED CONTAINSGROWTH-PROMOTING ANTIBIOTICS

Less true each year. While there were never antibioticsin ‘all’ feeds, even those sectors where inclusion wasroutine such as grow-finish pigs and broiler diets aresteadily eliminating AGPs and have replaced them withnatural products and programs that promote healthand growth.

Bio-Mos® was introduced to the marketplace atAlltech’s 1992 international feed industry symposium.The past 14 years have seen numerous successful trialsand in the past 12 months meta-analysis summariesof the data in studies with weanling pigs, broilers andturkeys have been published (Pettigrew 2003; Hooge,2003a; Hooge, 2003b). One researcher working onmodeling approaches to use in evaluating Bio-Mos®

confirmed that he has found nearly 300 publicationsin this area (G. Rosen, personal communication). Theresounding conclusion: the product is stable in feed,acceptable to the consumer, and works as well if notbetter than AGPs in comparison studies and oncommercial farms. Analysis of the broiler data showa 2% improvement in FCR, a 2% improvement inweight gain, and a 20% decrease in mortality. It clearlyhas lived up to its motto: Bio-Mos®: Performs.Promise. Its mode of action targets intestinal healthand immune modulation. The mannan fraction of Bio-Mos® carbohydrates provides a ‘decoy’ to whichpathogens adhere, thereby avoiding intestinalepithelial colonization, which in turn leads to healthiervilli and more absorption of nutrients. Immune

responses are modulated (as opposed to stimulateddirectly), leaving the animal more prepared whenexposed to pathogens.

The message with Bio-Mos® is that animal health,beginning with gut health, is the key to success.

PERCEPTION: AGRICULTURE POLLUTES

The latest restriction to be placed on animalproduction in an increasing number of markets isthe mandated reduction in dietary copper and zincin order to prevent accumulation in soil profiles(Figure 2). Supranutritional levels have traditionallybeen included in monogastric diets, especially thosefed pigs, to reduce enteric disorders. Mandatedreductions, however, allow only nutritionalminimums at a time when many are questioningwhether such levels are adequate to meet demandsof modern genetic lines.

Old New

Cu

Pigs: 35 - 175 ppm

All species

Fe: 1250

Mn: 250

25 ppm

250 - 750 ppm

100 - 150 ppm

Co: 10 2 ppm

Zn: 250 150-250 ppm (species-dependent)

Figure 2. Changes in trace mineral allowances for foodanimal diets in Europe.

Can animal health and productivity survive withreductions of critical trace minerals to 20-30% of

Figure 1. Differing proportions of selenium in various fractions of three commercial selenium yeast sources (adapted fromEncinar et al., 2003).

Water soluble

Polysaccharide

bound

Protein bound

Residual protein

bound

Residual

hydrolyzable

Fraction Yeast A Yeast B Yeast C

% of total Se

Water Soluble 12 28 22

Polysaccharide-bound 15 26 72

Protein-bound 18 40 4

Residual protein-bound 39 4 0

Residual hydrolysable 16 2 2

Not all selenium yeasts are alike

AABC

T.P. Lyons 5

Enterocytes

lining the villi

Villus height 600 µm

Gut lumen

Mucus layer (50-100 µm)

Negative charge protects enterocytes against highly charged

or toxic ions such as Al+3. This is why Fe+2 is better absorbed

than Fe+3, and why Bioplexes are more easily absorbed

than inorganic ions!

Blood

Unstirred water layer (600 µm)Increasing pH causes inorganic ions to 'hydroxy-polymerize',

forming non-absorbable complexes that are excreted.

Mucus

Unstirred

water layer

their current levels? The answer is yes, providingthat dietary trace minerals are supplied in forms bestsuited to the intestinal environment and absorptivemechanisms. Before reaching the site of absorption(the enterocyte membrane) ingested minerals firstencounter an unstirred water layer and then a mucuslayer with an intense negative charge (Figure 3). Thismeans that though the enterocyte membrane is verythin, the mineral must first traverse two layers, whichare orders of magnitude thicker than the absorptivesurface itself. For inorganic metal ions such as Cu,Zn, Mn and Fe, an immediate danger is so-called‘hydroxy-polymerization’ whereby the increasing pHin the small intestine, and particularly in the unstirredwater layer, causes them to form large insoluble metalhydroxides that cannot be absorbed.

The negatively charged mucus layer presentsanother barrier against the passage of inorganic metalions and evolved as a protective mechanism againsttoxic elements such as aluminum (Al3+). Because ofthe intense negatively charged nature of this layer,the strength of metal cation binding can be describedas follows; M3+ > M2+ > M+ (where M represents ametal ion). Essentially, toxic elements such as Al3+

are bound so tightly that they rarely manage totraverse this layer and are sloughed off as the layer isreplaced. As the charge on the metal ion decreases,inorganic metal ions (which have avoided hydroxypolymerization) may traverse the layer, but atrelatively slow rates. This is basically why ferric iron(Fe 3+) must first be reduced to ferrous iron (Fe2+)before it can be absorbed.

Feeding essential trace metals in the form ofBioplexes circumvents these problems by a)completely avoiding the risk of hydroxy poly-merization reactions, and b) speeding the rate ofpassage of the metal ion across the negatively chargedmucus layer by presenting it in a reduced charge orelectrically neutral form (Figure 4).

When dietary trace minerals are in this form, thenutritional minimums mandated by environmentallaws are able to meet the needs of modern, highlyprolific genetic lines. In studies comparing Bioplex™and inorganic zinc for grow-finish pigs Fremaut(2003) demonstrated that Bioplex™ Zn supplied at30% of the inorganic Zn level resulted in improveddaily gain while the environmental goal of reducedexcretion was accomplished.

Bioplexes:

Peptide protection from:

Forming insoluble complexes

The negatively charged mucus layer

Figure 4. General structure of a Bioplex trace mineral.

Figure 3. Barriers to absorption of highly charged inorganic cations: formation of unabsorbable hydroxy polymers in the unstirredwater layer and adherence to the negatively-charged mucus layer.


PERCEPTION: RENDERED ANIMAL BY-PRODUCTS IN FEED – DO WE HAVE ANALTERNATIVE?

While the antibiotic issue can be put aside safely witha tried and proven replacement, and bioplexing allowslower trace mineral levels, this cannot be said ofanimal by-products. In the US alone, 35 million cattleare processed every year. What could we possibly dowith the waste protein and fat? Europe has grappledwith this problem, but if the US reduces its use ofanimal by-products, the impact on protein prices willbe enormous.

New plant and yeast protein sources: The‘Biorefinery’

The nutritional, cost and environmental problems ofnot recycling animal by-products has no simplesolution, but perhaps the ‘biorefinery concept’, atwork in the rapidly expanding fuel ethanol industry,can provide a useful alternative protein source. Fuelethanol is produced in either the grain dry milling orwet-milling process, using a variety of starch andsugar substrates across the globe. Grain dry millscurrently produce ethanol, distiller’s dried grains withsolubles (DDGS) and CO2. Removal of the starchfor fermentation to ethanol leaves the protein,minerals and fat concentrated in co-products currentlyused in animal feeding, primarily ruminants butincreasingly in monogastrics. With ~30% CP, energyequal to the original grain owing to concentration offat and ~0.7% phosphorus (90% of which isavailable), these co-products have much to offer thefood animal industry in terms of addressing a proteinshortage, but can we improve them further? The‘biorefinery’ approach to processing starch/sugarsources says yes!

Dry mill ethanol plants using corn produce about30 kgs of DDGS for each 100 kgs of corn ground. A‘biorefinery’, in contrast to an ‘ethanol plant’integrates process streams such that a number ofproducts are produced, with ethanol being only oneof potentially many. Options for further processingof spent grains and solubles include secondaryfermentations to increase protein content, boost lysinecontent as much as 3-fold and decrease the indigestiblefiber. Enzymatic hydrolysis of DDG and/or solublesis another approach to add flexibility. Ethanolproducers seeking to expand the market for distilleryco-products have begun integrating processes that ‘re-ferment’ a portion of the solubles and spent grains toprovide specialty ruminant products such as VA101Figure 5). Such directions go well beyond simplyupgrading a ‘by-product’.

Alltech is essentially a yeast biorefinery (Figure 6),constantly examining ways of utilizing yeast or theircomponents. In applying the biorefinery concept toour use of yeast; so another high quality protein foranimal feeds arises. In addition to a wide range ofspecialty yeast applications from animal feeds toethanol, processes that utilize cell wall fractions inproduction of Bio-Mos® and Mycosorb® yield a formof yeast extract, which includes the highly nutritiouscell contents. It is this extract that is processed intoNuProTM, a yeast protein high in nucleotides withapplication in a broad spectrum of specialty diets,particularly those for neonates of all species.

The lesson of NuPro™, however, is not just thatpossible new proteins are available in increasingquantities; the message is that innovative researchand process results in innovative products if we thinkoutside the box and develop new technologies.

PERCEPTION: ‘MYCOTOXINS ARE NOT INANIMAL FEEDS SO WE ARE DOING NOTHINGABOUT THEM’

Like other food safety issues, mycotoxins are asubject that consumers can be expected to beincreasingly familiar with in upcoming years.Regulators are extending guidelines to includemycotoxins other than aflatoxin as science providesmore and more information about these toxins.

The increasing scientific information about toxinchemistry and function provides us an advantage,however, since it gives us an ability to solve theproblem. Knowledge about mycotoxin structuralchemistry provides clues useful in building adsorbents.The 3-dimensional structure of yeast cell wall glucan,the starting material for Mycosorb®, can bemanipulated to optimize toxin-cell wall interactionmaking a ‘glucan web’ to prevent toxins fromaffecting the animal or its products (Figure 7).

Figure 7. Three dimensional structure of yeast cell glucan.

T.P. Lyons 7

Figure 6. A yeast biorefinery.

Evaporator

Dryer

Separation,Refining

Distillation

Solids

Feedstock

CO2

Milling,

processing,

cooking

Whole stillage

Solids

CO2

Inositol

Plant oils

Solubles

Organic acids

Proteins

Fatty acids

Pharmaceuticals

Heterologous proteins

Glycerol

ETHANOL

Centrifuge

Extraction,Enzymaticprocessing

Fermentor

Specialty

feed

ingredients

Distillers

wet

grains

DDGS Condensed

distillers

solubles

SecondaryFermentation

Figure 5. From distillery to biorefinery.

FERMENTATION Sel-Plex®

BioChromeTM

Yea-Sacc1026

®Fuel ethanol

yeasts

Viable

yeast products

NuProTM

Yeast component products

Cell solubles stream

Separation

Mycosorb®

Bio-Mos®

Mannan stream Glucan stream

Yeast-

biosynthesized products

Cell solubles stream


Comparing commercially available adsorbents hasbecome a necessity for feed manufacturers. Table 5contains a 7-point guideline for evaluating suchproducts.

Table 5. 7-point comparison for mycotoxin adsorbents.

1. Can the product adsorb a wide range of toxins?2. Is inclusion rate sufficiently low (ie. 0.5-2.0 kg/t)?3. Is the adsorbent stable in the pH range of the GIT?4. Is adsorption capacity high (will it not be overwhelmed at

high toxin concentrations)?5. Is adsorption affinity high (is it effective at low toxin

concentrations since mycotoxins are often toxic at lowconcentrations)?

6. Is adsorption sufficiently rapid?7. Are there in vivo data that show protection of production

animals against toxins?

Mycosorb®, with its low inclusion rate and structureadapted to adsorb a range of mycotoxins includingaflatoxin, zearalenone, T-2 and DON, is rapidlybecoming the adsorbent of choice global. Protectedby three patents, Mycosorb® has unlimited potentialas we learn more about its structure and howmodifications can increase adsorption of both knownand newly-identified mycotoxins. Again, theappliance of science to solve a practical problem.The fact is that the technology is available to preventmycotoxins from having an impact at even the animallevel, which means that toxins from this source neednot threaten food safety in either perception or reality.

Key Success Factor No. 3:Designing policies for the future:transparency and innovation

Even if we adequately address price competitivenessand consumer perception, in order to be sustainablewe need policies that maintain transparency and spurinnovation in both products and business strategies.

Meet change Listen and act

head-on Take on new technologies before competitors do

Make transparency standard

Differentiation Avoid the 'sameness' trap

Choose exceptional, passionate people

Innovation Creative products

Creative R&D strategies

Success Factor 3

Designing a sustainable policy

for the future

A key step in defining those policies is deciding wherewe stand with regard to change: are we going to beproactive or reactive? Is it something that is going tohappen to us or will it be something we make happen?

CHANGE IS CONSTANT

Change is inevitable in the dynamic animal feedmarket, and failure to change has been the death knellof many enterprises. Once we accept that change is aconstant, our main decisions revolve around how todeal with change. We can either embrace change andmove forward, or we can ignore it until change isforced upon us.

Two large companies whose strategies for changeare apparent to us all are McDonald’s® andStarbucks®. The fast-food industry ‘re-invented’eating out; and for years seemed immune to recession.Recently they have begun to feel the pinch as theyhave watched consumers ‘re-invent’ what is ‘good’about food. As a result McDonald’s® stopped buyingbeef produced using antibiotic growth promoters, theyrefuse genetically modified potatoes, and in GreatBritain have begun to provide organic milk. USMcDonald’s® franchises offer ‘Atkins-friendly’ mealsfor the growing number of carbohydrate-countingcustomers. Is this a case of McDonald’s® beingproactive about changing menus, or are they beingreactive when forced to change?

Starbucks® ‘re-invented’ stopping for a cup of coffeewith huge success, but now they have begun to add‘Fair Trade’ and environmentally friendly products.With Conservation International they havecollaborated on a project to encourage sustainableagricultural practices and biodiversity through theproduction of shade-grown coffee, which follows theInstitution of Coffee Purchasing’s guidelines. IsStarbucks listening to the consumer or is Starbucks®

being proactive?The changes in McDonald’s® and Starbucks® are

examples of transparency and proactive efforts to offerproducts modern customers are interested in buying.They want customers to know of their commitmentsto food quality, safety and sustainable agriculturalpractices. Is our industry just as proactive? Have welost sight of what Dan Glickman (former USSecretary of Agriculture) advised at the AlltechInternational Feed Industry Symposium in 2000 –“Tell us what you want and we will grow it”?

T.P. Lyons 9

AVOIDING THE SAMENESS TRAP:DIFFERENTIATE WITH PEOPLE AND PRODUCTS

In order to be sustainable, companies must avoid the‘sameness trap’ described in Funky Business byNordström and Ridderstråle (2000). They describean oversupplied world of similar companies,employing similar people, with similar educationalbackgrounds, coming up with similar ideas,producing similar things, with similar prices andsimilar quality. Does this sound like our industry? Itdoes, and it underscores our need to differentiate.We need to create new solutions to problems and indoing so create profit and success for ourselves andour partners. We can make our companies, and henceour industry, different and make them stand out inthe industry.

The people factor: exceptional people helpcompanies differentiate

‘Our people make the difference’ must be more thana well-meaning cliché. Many business commentatorsbelieve that we are entering an era where the ‘warfor talent’ is the most important battle that will befought. When land was the important asset, countriesbattled for it, now that talent is the important assetfor business success, companies will battle for talent.Paul Allaire, former CEO at Xerox®, calls it “thebrawl with no rules”.

What kinds of talents are we looking for? It is oneof the fundamental roles of the leader that he/shedevelop the talent around him/her. Inside rapidly-growing Alltech the need to ensure that the nextgeneration of leaders is in place has been acute. Wehave key questions to ask potential employees – themost important of which is: What are you passionateabout? There is no right or wrong answer, it is simplyimportant to find people with the energy and drivefor accomplishment. We have successfully made thetransition from a small local player into a medium-sized global enterprise. The next challenge for ourpeople and for our industry involves becoming theindustry standard bearer. Part of our future successwill be due to recruiting talented and diverseindividuals from across the world, including a greaterproportion of women, a group whose skills andmanagement styles have been underutilized inagribusiness.

FOSTER INNOVATION

In the ‘over-supplied world’ described by Nordströmand Ridderstråle, ideas are what separate successful

companies (and individuals) from failures. Anotherimportant element of the future viability of ourindustry will be our ability to give consumers notonly what they want, but more importantly what theydid not realize they wanted. The new competitionwill take place not only in terms of market share, butmore importantly in newly created markets.Innovation, while a term vastly overused, is acompetency that Alltech and all companies need toexcel at in order to prosper.

I was once asked how it could be possible to take acommodity item like milk and make it unique – avalue-added product. Is it simple? No. Is it possible?Absolutely. We created a slogan: ‘A milk for all ages’.For the young, a lactoferrin-rich milk for the lactose-intolerant. For teenagers, perhaps higher calciumlevels for growing bones; while low fat, high omega-3 and high cholesterol-blocking statin might formpart of ‘milk for middle ages’. For all ages,enrichment with selenium through Sel-Plex® in thecow’s diet to fight against cancer. A Korean companywent further and changed the name from milk toSELK to emphasize selenium enrichment.

The size of a company is irrelevant when it comes toinnovation. The tiny New Zealand dairy co-operative,


Tatua, with only 30,000 cows is still the world’s mostprofitable, largely due to the value-added dairyproducts it offers such as lactoferrin for infantformulas and lactoperoxidase as a natural sterilant.

Alltech’s Bioscience Centers, where scientistscomplete research toward MSc and PhD degrees whileworking with Alltech’s research group, are at the hubof our innovation. We support these scientists’ effortsand encourage creative thinking. Over time, 9 PhDand 42 MSc students contributed to the research onYea-Sacc1026®, now the world’s No. 1 natural rumenmodifier, which is the reason we understand its modeof action so well. A good example of the impact ofthis work is the recently obtained EU approval forYea-Sacc1026® in horses. In the US alone we supportwork being conducted by 36 doctoral candidates andhave 135 ongoing projects in Europe.

Summary

Re-imagining the feed industry means re-imaginingour companies: our goals and what we stand for, ourpeople and the corporate environment we create. Wemust ask and answer carefully the questions ‘Are wefostering the creativity we need to carry the companyinto the future? Do our products and researchdirections address industry needs for pricecompetitiveness and consumer perception? Are thepolicies sustainable?

At Alltech, we recognize the importance of ongoingdiscussion of these questions in building a dynamiccorporate culture. It has allowed us to focus on corecompetencies to develop a ‘Big 6’ list of productdirections while giving us the freedom to find waysto expand to a ‘Big 8’ or ‘Big 10’.

Another result of this corporate dynamic is thegrowing role of the Bioscience Centers as hubs ofinnovation, both in scientific exploration and in thestructure of modern corporate agricultural research-our relationships with other research groups atuniversities and institutes.

The process is exciting; and it is providing productsthat have increasing importance across the world inthe areas of animal health, performance andreproductive efficiency, and consumer perception offood animal products. Clearly decisions we makesurrounding Price, Perception and Policy defineultimately where each of our companies will be in10, 20 or 30 years’ time.

References

Encinar, J.R., M. ?liwka-Kaszyñska, A. Polatajko,V. Vacchina and J. Szpunar. 2003. Methodologicaladvances for selenium speciation analysis in yeast.Analyt. Chim. Acta 500:171-183.

Fremaut, D. 2003. Trace mineral proteinates inmodern pig production: reducing mineral excretionwithout sacrificing performance. In: NutritionalBiotechnology in the Feed and Food Industries,Proceedings of Alltech’s 19th Annual Symposium(K.A. Jacques and T.P. Lyons, eds). NottinghamUniversity Press, UK, pp. 171.

Hooge, D.M. 2004a. Meta-analysis of broiler chickenpen trials evaluating dietary mannanoligosaccharide, 1993-2003. Intl J. Poult. Sci.3(3):163-174.

Hooge, D.M. 2004b. Turkey pen trials with dietarymannan oligosaccharide: meta-analysis, 1993-2003.Intl J. Poult. Sci. 3(3):179-188.

Nordström, K.A. and J. Ridderstråle. 2000. FunkyBusiness - Talent makes capital dance. FinancialTimes Prentice Hall, New Jersey.

Pettigrew, J.E. 2000. Mannan oligosaccharides’effects on performance reviewed. Feedstuffs 52(December 25).

BioplexesTM Trace mineral proteinates for all species

The Alltech 'Big 6'

Mycosorb® Reduce mycotoxin

impact

VegproTM SSF enzyme for vegetable proteins

Yea-Sacc1026® Viable yeast culture for cattle & horses

Bio-Mos® Mannan oligosaccharide,

non-AGP growth promoter

Sel-Plex® Selenium

in the 'food' form

C.V. Maxwell 11

Future of the feed/food industry: re-inventing animal feed

CHARLES V. MAXWELL

Animal Science Department, University of Arkansas, Fayetteville, Arkansas, USA

Challenges and opportunities facing the internationalfeed/food industry have never been greater than thosefaced by these industries today. It has become apparentthat anything less than a very proactive approach toaddressing current challenges may not be sufficient.Current international events are leading feedcompanies to attempt to move from a traditional roleas a ‘feed company’ to becoming an integral part ofthe ‘Food Supply Chain’. This is driven essentiallyby the fact that although one cannot ensure that foodcompany products eaten by consumers are safe, as aprominent player in the early part of the food chain,it is essential that the feed industry ensure that thefeedstuff supply chain is not the problem.

This new reality has led feed companies to initiationof the concepts of oversight, control, and overallsecurity of their component of the food chain. Feedcompanies are, out of necessity, now becoming ‘FoodSafety Guardians’. The commitment by the feedcompany to provide customers with products of thehighest quality has been deemed vital to the successof the business. Therefore, feed companies mustbecome early leaders in assuring that all products areHazard Analysis and Critical Control Point (HACCP)certified. In addition, it is essential that feed companiesbecome registered in the ISO 9001 quality standardor adopt a similar standard. HACCP is a systematicapproach to identifying and preventing contaminationof food and food products during the manufacturingprocess. ISO certification is an internationallyrecognized quality management system thatemphasizes integrity throughout the manufacturingprocess, using standardized and verifiable proceduresin all aspects of operations from product designthrough manufacturing and distribution. The samelevel of concern has progressed through the livestockand poultry production chain to include producers,integrators, processors, and retailers.

Three key issues asked of the industry today are 1)what are animals consuming, 2) how are animalsbeing cared for, and 3) has the animal been sick?The traditional protein business chain from vegetableto animal protein has changed dramatically.Traditionally, this has been a production-based modelfrom the farmer to the consumer with little oversight.The reality today is that the consumer is providingoversight to the retailer who then places constraintson the production chain to conform to specificstandards of production. Overall confidence in foodsafety was down in the early 1990s, tended to riseand peaked in the mid 1990s and has declined since(Figure 1).

Another issue being addressed by the multinationalfeed companies is globalization. Given all the issuesbeing addressed internationally, specific feeds needto be modified to fit specific country labeling. Thispresents tremendous difficulties in developingbranded products with international acceptance.Solutions to these issues are exacerbated by themultitude of regulatory hurdles that must beovercome. These include differences in the use ofantibiotics, animal proteins, genetically modifiedmaterials, and feed additives. Companies that supplyinputs are required to think as globally as processorsand retailers.

These issues were in the implementation stage priorto the discovery of BSE in North America. Allcomponents of the feed/food industries are beingaffected by this discovery. This is leading to the rapiddevelopment and implementation of a nationalidentification program in the US. The ID programwill likely bring rapid adoption of Radio FrequencyIdentification Devices (RFID) in production andprocessing sectors of the food supply chain and willmatch the drive of Wal-Mart’s RFID technology in

12 Future of the feed/food industry: re-inventing animal feed

the retail sector. There will be public pressure forthe US government to fund the development andimplementation of the national animal ID program,perceived as essential for traceability in acomprehensive food safety program. The need fortraceability has been the greatest cost impediment tothe adoption and implementation of country-of-originlabeling (COOL). The Manitoba Pork Council hasinitiated a pilot swine traceability study as part of anational effort to identify the most cost efficientmethod of tracing swine movement in Canada.

The lack of qualified workers has been and continuesto be a major constraint for those associated with theintegrated livestock and poultry industries. A surveyof students enrolled in swine production in the topswine-producing US states was conducted by DuaneReese (Table 1). This study indicates that interestamong students had declined in 9 out of the top 10swine-producing states over the last five years, withan average decline of 28% in the number of studentsenrolled. Many other states reported that a lack ofinterest among students is resulting in swineproduction not being offered or offered on alternateyears. Similarly, poultry production is only offeredby a limited number of universities and interest in apoultry production major is low. It is worth notingthat this lack of interest comes at a time when worldmeat demands are expected to increase as developingcountries have more disposable income, with aprojected increase of 50% by the year 2025 (Elam,2004). At the same time, the acreage of row crops is

projected to decrease over that same time period.Thus, it is imperative to improve productionefficiency in both the livestock and crop sectors tobe able to meet the rising demands. The other hugearea is the need to revamp production agriculture sothat all components can be brought back to consistentprofitability.

Table 1. Students enrolled in swine production in top 10 swineproducing statesa.

State Fall 1998/ Fall 2002/ ChangeSpring 1999 Spring 2003 (%)

Iowa 97 75 ↓ 22North Carolina 64 30 ↓ 53Minnesota 12 0 ↓100Illinois 15 14 ↓ 6Indiana 30 18 ↓ 40Nebraska 11 6 ↓ 45Missouri 25 22 ↓ 12Oklahoma 37 30 ↓ 15Kansas 47 48 ↑ 2Ohio 14 10 ↓ 28Total 352 253 ↓ 28

aReese, University of Nebraska, personal communication

Evaluation for technology developmentby the livestock industry

The swine and poultry industries have madetremendous progress through the years in terms ofgenetics, nutrition, husbandry and health. Advancesin production and management have provided the

65

70

75

1991 1992 1993 1994 1995 1996 1997 1998 1999 2000

80

85

Perc

enta

ge

Figure 1. Overall confidence in food safety: percentage of consumers completely or mostly confident(Food Marketing Institute, 1991-2000).

C.V. Maxwell 13

marketplace with a high volume, low cost animalprotein. Historically the livestock industries have beenmostly concerned about commodity production thatworks best in a least cost, most efficient productionmodel. There are two schools of thought regardingtechnology evaluation for large systems. The typicalapproach that has served the integrated livestockindustry well in the past is the cost analysis systemthat is used by Agrimetrics and Agristats. Thetechnique used is a comparison-based cost analysisthat compares the cost-of-gain of one company withthe cost-of-gain of other comparable enterprises in amanner that keeps the actual companies involved inthe analysis confidential. These tools have been largelyused to drive systems to ‘least cost’ production withlittle regard to optimal cost and maximal profit. Somehave argued that this system may be overly simplisticand does not effectively address the concept of valuereturned/cost of input. For example, the most valueout of a feeding program may result in a higher costof gain if the increased cost results in improvedperformance. The other system is to look at returnsover feed costs or a return over input cost. Dataanalysis services are available that may moreeffectively address where improvements may be madeusing a more broad-based cost/benefit analysis. Theapproach taken by MetaFarms Inc. is to conduct ananalysis of ‘Process Enablers’, which affect a numberof specific parameters monitored in an enterprise.This leads to a continuing evaluation of the impactthat specific processes have on parameters beingmeasured rather than a single focus on cost of gain.One example of this type of analysis is the effect ofractopamine use in a swine production enterprise onperformance. Ractopamine added 10 lbs of weightper pig sold, which improved the bottom line by $1.50to $2.00/pig. Although cost of gain is improved inpigs fed ractopamine, the improvement is minimalcompared to the value of improved gain and leanyield; but ractopamine may not be considered unlessone analyzes technologies outside a simple cost ofgain model. That leaves us questioning how we shouldevaluate technology. Perhaps the effect a technologyhas on both the cost side of the equation as well asthe revenue side should be evaluated. It is essentialthat models are developed that evaluate profitability,not just cost of production.

It is also interesting to note that once all the factorswith huge effects on performance and efficiency havebeen implemented, this leaves the livestock industrieswith the unenviable task of attempting to determineimpacts of products and(or) systems that have a much

smaller effect on profitability. A 3-4% gain in feedefficiency is almost too small to measure, but theeconomic impact on profitability in the integratedindustry is huge.

Although the challenges are great, much progressis being made in providing alternatives that couldbenefit the feed/food industries tremendously. Thefact that growth-promoting levels of antibiotics areno longer permitted in Europe and the possibility ofrestrictions being imposed elsewhere has led to aplethora of studies investigating replacements. Thesestudies offer the potential of a better understandingof the relationship between the microbiota in theenvironment and improved livestock performance aswell as alternative strategies to control the threat ofspecific microorganisms. This may result in improvedperformance over that observed with growth-promoting levels of antibiotics. Similarly, studies toreplace specific animal proteins may lead to a betterunderstanding of factors associated with reducedperformance with plant proteins in neonatal animals.

Relationship between the gut microbiotaand performance in swine

The gastrointestinal tract of the pig harbors ametabolically active microbiota that stimulates thenormal maturation of host tissues and provides keydefense functions (Gaskins, 2001). Several recentexamples of improved post-weaning performance inthe young pig suggest that much of the improvementobserved in nutritional studies may be through animpact on the intestinal microbiota. A good argumentcan be made that the improved performance observedin the young pig as a result of feeding plasma protein,complex diets, antibiotics or acidifiers might be anindirect effect of altering the gut microbiota.Similarly, the positive effects of popular managementstrategies such as segregated early weaning (SEW)may be mediated through reductions in exposure topathogens.

Segregated early weaning reduces the incidence ofa number of pathogens, thus reducing immunologicalstress, which results in improved growth and higherefficiency of feed utilization (reviewed by Maxwell,1999). This strategy has been successful in reducingthe number of pathogens, but has not been successfulin eliminating all pathogens. The premise is that pigsare removed from the sow while their immunity, asa consequence of maternal antibodies, is still high.This maternally derived passive immunity will


prevent vertical transfer of indigenous pathogens. Pigsreared in isolation have been shown to have reducedimmunological stress (Johnson, 1997) resulting inimproved growth and efficiency of feed utilization.This is consistent with observations in our researchat the University of Arkansas to determine ifdifferences in immune stimulation can explainperformance differences in conventional vs off-sitereared pigs. A total 432 weanling barrows (19 ± 2day of age) were obtained from a local commercialcompany from a single source. One-half the barrowsfrom litters were selected for the off-site nurserystudy (6 pigs/pen) with the remaining pigs staying inthe conventional nursery facilities (approximately 18pigs/pen). Pigs were weighed and serum samplesobtained via venipuncture on days 0, 14, and 34 post-weaning from a total of 72 pre-selected pigs. Thepigs were placed in the conventional facilities (aminimum of 1 pig/litter was sampled) and an off-site nursery (a minimum of two pigs in each of 36pens was sampled). Serum α1-acid glycoproteinconcentrations were determined by a single radialimmunodiffusion method using a commercial kit(porcine α1-acid glycoprotein plate, DevelopmentTechnologies International, Inc., Frederick, MD).Pigs reared in the off-site nursery were 0.89 kgheavier (P<0.01) at day 14 post-weaning and 2.40kg heavier (P<0.01) at 34 days post-weaning. Inaddition, serum α1-acid glycoprotein concentrationwas elevated (P<0.01) in pigs reared in theconventional nursery. This suggests that reducedperformance in a conventional nursery may beassociated with the immunological stress associatedwith production under these conditions.

The swine industry is implementing early weaningfor efficient and economical pig production (Wilson,1995; Patience et al., 1997). The obvious consequenceof weaning is the abrupt change in diet from sow’smilk to solid feed and a change in the environment.There is reduced feed intake during the first weekand associated adverse changes in the animal’s gutanatomy and physiology such as villus atrophy,deeper crypts, and infiltration of the villus tip byimmature enterocytes (Spring, 1999). Villous atrophymeans that there is less absorptive area available fornutrient uptake and deeper crypts represent a largetissue turnover (Spring, 1999). The intestinalmicroflora can be adversely affected during weaning,resulting in higher numbers of potentially pathogenicacid-intolerant coliforms and a decline of favorablelactobacilli (Bolduan, 1999). In addition, since thepiglets are young, their immune systems might not

be totally equipped to deal with such pathogenicchallenges.

Recently there has been a concern about the use ofantibiotics in animal production in part due toantimicrobial resistant bacteria. Over the past twodecades, probiotics (direct-fed microbials), whichinclude Lactobacillus cultures, have been used as analternative to antibiotics in animal production (Jin etal., 1998). Lactobacilli are normal inhabitants of thegastrointestinal tract of pigs. Their beneficial role inthe intestinal tract has been attributed to their abilityto survive the digestive process, attach to the epitheliallining of the intestinal tract, produce lactic acid andother microbial compounds and prevent colonizationby pathogens via competitive exclusion (Savage,1987). To investigate weaning-induced changeswithin the enteric system, two experiments wereconducted to determine the effect of milksupplementation with Lactobacillus brevis (1E-1) onpre- and post-weaning pig performance, and intestinalmicroflora. The 1E-1 isolate was sampled from theintestinal tracts of 10 healthy pigs and five pigs withscours, and it was reported that healthy pigs hadhigher levels of lactobacilli, with the majority ofisolates identified as Lactobacillus brevis (Parrott etal., 1994). In each experiment, litters were allottedto two treatments at farrowing: either a control milksupplement, or the control containing 1E-1 via anin-line system using a cup dispenser for each litter.Coliforms and E. coli were enumerated fromesophageal, duodenal, jejunal, and ileal regions ofthe enteric tracts in Experiment 1. Pigs receiving 1E-1 had lower (P<0.05) jejunal E. coli populationspre-weaning and post-weaning compared to pigsprovided only milk supplement (Table 2). Ileal E.coli populations were lower (P<0.02) during the post-weaning period for pigs receiving 1E-1 compared topigs provided milk replacer without 1E-1. Theadministration of 1E-1 prior to weaning may deterthe detrimental alterations in the microbial populationthat occur at weaning as has been observed in pigsfed zinc oxide (Katouli et al., 1999). During the pre-weaning period (birth to weaning), administrationof 1E-1 tended to increase weight gain (Figure 2,P<0.06). During the first five days post-weaning,pigs fed 1E-1 prior to weaning had greater ADG(Table 3, 277 vs 194 g/d; P<0.05) compared to pigsprovided only milk replacer, and overall ADG wasimproved in pigs fed milk replacer with 1E-1. Pigspreviously fed milk replacer with 1E-1 were 1.93 kgheavier at the completion of the 28-day study whencompared to those receiving the milk replacer alone(Figure 3).

C.V. Maxwell 15

Table 2. Pre- and post-weaning mean E. coli populations in thejejunum and ileum of pigs (CFU/g log10).

Pre-wean Post-wean

Control 1E-1 Control 1E-1

Experiment 1Mean E. coli, CFU/g (log10)

Jejunum 5.53a 3.42b 7.10a 4.80b

Ileum 5.91 4.71 6.63a 4.96b

a,bPre- and post-wean means within a row with different letters differ significantly (P<0.05)

Table 3. Effect of Lactobacillus brevis (1E-1) during lactation onsubsequent nursery pig performance (Experiment 1).

Treatment

Milk Milk + L. brevis (1E-1)

Phase 1 (day 0-5) ADG, g 194b 277a

ADFI, g 91 109Phase 1 (day 0-14) ADG, g 211 258 ADFI, g 211 250

Overall (day 0-28) ADG, g 355 388 ADFI, g 435 461 Gain:feed 0.83 0.85Weight, kg Initial 5.50c 6.31d

Phase 1 8.26 9.88 Phase 2 15.19 17.12

abLetters that differ within the same column are different (P< 0.02).cdLetters that differ within the same column are different (P<0.09).

A second study to confirm these results has recentlybeen completed. Beginning at farrowing, pigs wereprovided milk supplementation either with or withoutthe addition of Lactobacillus brevis (1E-1) via an in-line system using a cup dispenser for each litter. Thesetreatments were continued during the nursery period,in which pigs that were administered 1E-1 via milksupplementation continued to receive 1E-1 throughthe watering system. Pigs supplemented with 1E-1had greater ADG (P<0.05) during Phase 2 and in theoverall nursery period (day 0 to 38), greater ADFI(P<0.05) during Phase 3 and the overall nurseryperiod, and tended to have increased gain:feed(P<0.10) during Phase 3 (Table 4). In this study,1E-1 supplementation resulted in a 1.58 kgimprovement (P<0.01) in body weight at the end ofthe six-week nursery period compared to pigs notreceiving 1E-1 (Figure 4). These data indicate that1E-1 supplementation pre-weaning improves nurseryperformance and may provide a healthier intestinalenvironment.

Studies that I have summarized involve classicalculture techniques. We are adapting procedures thatallow quantitative determination of all the primarymicrobiological species in the gut microbiota usingmolecular techniques (Figure 5). The developmentof molecular-based methods that by-pass the needfor culturing bacteria has led to a renaissance inmicrobial ecology studies. Molecular methods thatutilize the Polymerase Chain Reaction (PCR)exponentially amplify copies of bacterial DNA toliterally produce a billion copies of the originaltemplate without growing the bacteria. These

0

50

100

150

200

250

300

Control L. brevis

Dai

ly g

ain

(g)

P<0.06

Figure 2. Effect of milk supplementation with Lactobacillus brevis (1E-1) on daily gain from birth to weaning.


Table 4. Effect of Lactobacillus brevis (1E-1) during lactation onsubsequent nursery pig performance (Experiment 2).

Trait Milk Milk + P-valueL. brevis (1E-1)

ADG, kgPhase 1 0.092 0.100 0.51Phase 2 0.286 0.332 0.014Phase 3 0.507 0.535 0.26Phase 1-3 0.307 0.336 0.05

ADFI, kgPhase 1 0.125 0.128 0.13Phase 2 0.304 0.347 0.17Phase 3 0.563 0.622 0.03Phase 1-3 0.343 0.380 0.04

Feed:gainPhase 1 1.632 1.482 0.57Phase 2 1.123 1.079 0.64Phase 3 1.109 1.180 0.14Phase 1-3 1.141 1.145 0.93

Weight, kgInitial 7.86 7.88 0.09Phase 1 8.73 8.88 0.24Phase 2 12.72 13.55 0.013Phase 3 19.87 21.45 <0.01

methodologies have created the first opportunity forecologists to gain a true representation of the microbialworld as it would exist in natural environments. Toexamine the bacterial ecology of known and unknownbacteria, the most often used method is to PCRamplify the 16S rRNA/DNA genes from a mixedbacterial community. These highly conserved genesencode a portion of the bacterial ribosome found in

every known bacterium. This amplified mix of genesequences can be separated, sequenced and comparedto the 16S rRNA/DNA database presently containingtaxonomic data of over 85,000 bacterial sequences.

These studies suggest that the impact of gutmicrobes on performance in young pigs is greaterthan once thought. Other researchers havedocumented enhanced performance in growing/finishing pigs associated with improved health andmanagement. Pigs with minimal disease due to SEW,which were fed a series of non-limiting diets andreared in pens of three pigs (2.23 m2/pig), achieved104 kg at 136 days of age and 120 kg at 151 days ofage (Schinckel et al., 1995). Pigs raised on theoriginal commercial farm, conventionally weanedwith all-in, all-out production, required 184 days toattain 104 kg live weight. This dramatic effect ofhealth is likely mediated by inflammatory cytokinesor other systemic inflammatory effects in responseto bacterial toxins. The overall effect is to downregulate muscle synthesis and growth. Schinckel etal. (2003) presented a review of current research onmuscle endocrine, and immune regulation of growth.

Bio-Mos®

Although prohibited in many European countries,the addition of antibiotic growth promoters to swinediets remains a common practice in the US,particularly to the diets of newly weaned pigs.However, there has been increasing pressure on the

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BW (k

g)

Figure 3. Effect of milk replacer supplementation with Lactobacillus brevis (1E-1) on final nursery weight (Trial 1-28 days, kg).

C.V. Maxwell 17

livestock industry to decrease or discontinue theseadditions because of the potential development ofantibiotic resistance. The need for alternative methodsto improve growth and efficiency of livestockproduction and to modulate the animal’s naturalability to fight disease has prompted the scientificinvestigation of several feed additives and their ability

to positively alter immune function (Berg, 1998;Turner et al., 2001).

Supplementation of swine diets with mannanoligosaccharides derived from the yeast cell wall ofSaccharomyces cerevisiae has the potential to providean alternative to growth-promoting antibiotics.Mannan-based supplements have the ability to alter

14

16

18

20

22

Control L. brevis

BW (k

g)

P<0.01

Figure 4. Effect of milk replacer supplementation with Lactobacillus brevis (1E-1) on final nursery weight (Trial 2-42 days).

Figure 5. Procedural outline to quantitatively determine all species in a mixed culture (adapted from Liu et al., 1997).

3'5'3'

5'Extract DNA

from community

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PCR with afluorescently labeled

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Fragment separationin sequencing gel (size)

Recognition of labeled fragments

Restriction digestof PCR product

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5'

3'5'

3'5'3'

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3'5'

3'5'3'

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3'5'3'

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AB

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the microbial population in the intestinal tract. Thismodification seems to be accomplished by the abilityof mannans to attach to mannose-binding proteinson the cell surface of some strains of bacteria, therebypreventing these bacteria from colonizing theintestinal tract by interfering with the binding ofcarbohydrate residues on epithelial cell surfaces(Spring et al., 2000). Mannans have also beenreported to alter immune function in swine (Kim etal., 2000), and this may be an additional mechanismby which mannans improve growth performance.

The effects of Bio-Mos® on pig performance andimmunocompetence was evaluated in five nurserypig trials conducted at the University of Arkansas. Atotal of 412 pigs were included in the evaluation with82 total observations (38 pens fed the basal diet, 15pens fed 0.2% Bio-Mos®, and 29 pens fed 0.3% Bio-Mos®). In four of the five trials, Phase 1, Phase 2,and Phase 3 were defined as day 0 to 10 after weaning,day 10 to 24 after weaning, and day 24 to 38 afterweaning, respectively. The fifth trial consisted of a14-day Phase 1, and a 7-day Phase 2. During Phase1, Bio-Mos® supplementation improved (P<0.02)feed efficiency compared to pigs fed the basal diet,whereas improvement in ADG approachedsignificance (Table 5, P=0.11). Pigs supplementedwith 0.3% Bio-Mos® had improved feed efficiencyfor the overall Phase 1 and 2 periods (P<0.03) whencompared to those fed the control diet. During thefirst week of Phase 3, ADG (P<0.05) and feedefficiency (P<0.05) were improved in pigs fed Bio-Mos® when compared to pigs fed the basal diet. Therewere no differences (P>0.20) in lymphocyteproliferation between pigs fed Bio-Mos® and thosefed the basal diet when data from the five trials werecombined. However, evaluation of immune assaysconducted in the fifth trial revealed that Bio-Mos®-supplemented pigs had a greater proportion oflymphocytes in the peripheral blood (P<0.03), anincrease (P<0.10) in the proportion of macrophagesin the jejunal lamina propria, and an increase

(P<0.05) in the phagocytic capacity of jejunal laminapropria macrophages compared to pigs fed the basaldiet (P<0.05, Table 6). Data compiled from fiveexperiments conducted at the University of Arkansasconclude that Bio-Mos® supplementation improvesweight gain and feed efficiency in nursery pigs.Although the function of lymphocytes derived fromperipheral blood was not affected by Bio-Mos®

supplementation, Bio-Mos® did enhance innateimmune function in the gastrointestinal system.

Table 5. Summary of the effect of Bio-Mos® on growth perfor-mance in 5 trials.

Bio-Mos® (kg/t)

0 0.2 0.3 P-value

ADG, gPhase 1c 147 + 9 168 + 16 166 +10 0.26Phase 2 372 + 12 369 + 22 391 +14 0.56Phase 3d 461 + 14 516 + 23 501 +18 0.07

Feed:gainPhase 1d 1.709 + 0.067a 1.388 + 0.126b 1.493 + 0.080b 0.02Phase 2 1.437 + 0.085 1.428 + 0.160 1.195 + 0.104 0.20Phase 1-2f 1.379 + 0.025a 1.375 + 0.046ab 1.273 + 0.030b 0.03Phase 3d 1.700 + 0.028a 1.586 + 0.046b 1.610 + 0.036ab 0.03

a,bMeans in a row with no letters in common differ (P<0.05).cContrast: 0 vs 0.2 + 0.3% Bio-Mos®; P = 0.11dContrast: 0 vs 0.2 + 0.3% Bio-Mos®; P<0.05eContrast: 0 vs 0.2 + 0.3% Bio-Mos®; P<0.10fContrast: 0.2% vs. 0.3% Bio-Mos®; P<0.10

Our data indicate that Bio-Mos® alters the proportionand function of leukocytes isolated from theperipheral blood as well as the gastrointestinal tractof the weanling pig. One of the questions raised bythe results of these studies with Bio-Mos® is; howcan a non-digestible feed additive alter immuneresponses in the gastrointestinal tract and systemicallyin the pig? One possible mechanism is via the uptakeof Bio-Mos® from the lumen of the gastrointestinaltract by M cells of Peyer’s patches. Peyer’s patchesare organized lymphoid follicles located along theluminal surface of the small intestine. Dispersed

Table 6. Immune responses to Bio-Mos®®®®® supplementation to nursery pig diets.

Control Bio-Mos® SEM P=

Leukocyte, % Neutrophils 52.8 45.3 2.7 0.08 Lymphocytes 42.8 50.7 2.0 0.03Macrophage phagocytosis (lamina propria)Average No. SRBC phagocytosed 2.31 2.63 0.11 0.05CD14+ macrophages, % 6.9 13.5 1.6 0.10

C.V. Maxwell 19

throughout the epithelial layer of the Peyer’s patchare specialized epithelial cells, termed M(membranous) cells, which function to pinocytoseand transport macromolecules from the intestinallumen into the subepithelial tissue, deliveringantigenic molecules to leukocytes within the Peyer’spatch. The extraction of Bio-Mos® from the lumenof the small intestine by the M cells of the Peyer’spatch and its exposure to the immune cells locatedthere, may be the impetus for a cascade ofimmunomodulatory events that develop and enhanceimmune function, both locally in the gastrointestinaltract as well as systemically, as cells migrate out ofthe gastrointestinal tissue into the periphery.

Because mannan oligosaccharides have beendocumented to alter bacterial populations within theintestinal tract (Spring et al., 2000), anotherexplanation for the alterations in immune functionobserved in these studies may be from changeselicited in the enteric microbial population by thepresence of mannans in the luminal environment ofthe intestinal tract. The microflora present in thegastrointestinal tract are known to be a factor in thedevelopment of the young pig’s immune system, bothenterically and systemically (Gaskins, 1997), and thealteration of these microbial populations by Bio-Mos®

could have an impact on the progression of immunesystem development.

NuProTM 2000

Pigs produced in conventional intensively managedswine production systems are routinely weaned at 19to 21 days of age and as early as 10 to 14 days of agein off-site SEW systems. At this age, pigs are verysensitive to the source of dietary protein. Many dietaryproteins produce allergic reactions in which diarrhea,reduced growth and increased mortality can occur(Bimbo and Crowther, 1992). Various protein sourceshave been tested in early-weaned pig diets in anattempt to overcome these problems and to decreasediet cost. Spray-dried plasma protein is a proteinsource that has consistently been shown to improveperformance of early-weaned pigs when included inPhase 1 (day 0 to 14 post-weaning) diets at theexpense of dried skim milk (Hansen et al., 1993;Kats et al., 1994; de Rodas et al., 1995), soybeanmeal (Fakler et al., 1993; Coffey and Cromwell,1995; de Rodas et al., 1995), and whey (Hansen etal., 1993). Select grade menhaden fish meal has alsobeen a widely utilized protein source due to a

combination of consistent quality and competitiveprice. Demand for plasma protein is high and supplyis limited, therefore plasma is an expensive proteinsource for nursery diets. Also, regulatory constraintsthat prohibit the use of plasma protein in manycountries may affect the use of bovine plasma in theUS. Similarly, increased demand and decreasedsupply of fish meal has resulted in increased pricevolatility and relatively high current prices.

Preventing intestinal damage or atrophyimmediately post-weaning caused by reduced feedintake and lack of stimulation of the intestinalepithelium by ingested particles has been suggestedas important in maintaining growth performance innursery pigs (Cera et al., 1988; Dunsford et al.,1989). However, there are many other factors,including removal of beneficial factors from sow’smilk, diet form, stress, invasion by microorganisms,or introduction of allergenic compounds in thenursery diet, that may also contribute to intestinalatrophy. Glutamic acid and nucleotides may beimportant nutrient sources for maintaining gutintegrity during the early nursery period.

NuProTM 2000 is a protein source high in crudeprotein (51 to 55%) and digestible amino acids thathas potential as a possible alternative protein sourcein nursery diets. NuProTM is also high in glutamicacid and is an excellent source of nucleotides. Severalanimal-based specialty feed ingredients have beendeveloped to compete against the animal plasma andfish meal market share. However, NuProTM is avegetable-based peptide product which may havegreater international market appeal compared toproducts originating from animal by-products andthe high level of nucleotides may be uniquelybeneficial to the early-weaned pig

A study has been completed at the University ofArkansas involving a total of 216 pigs to evaluatethe efficacy of feeding NuPro™ as an alternative toplasma protein in nursery pig diets (9 pens/treatment).Three dietary treatments were fed from day 0 to 7after weaning (Phase 1) and day 7 to 21 after weaning(Phase 2) and were comprised of 1) a basal dietconsisting of a complex nursery diet containing spray-dried plasma protein devoid of NuProTM, 2) the basaldiet with 50% of the plasma protein replaced byNuProTM, and 3) the basal diet with 100% of theplasma protein replaced by NuProTM. During Phase3 (day 21 to 42 after weaning) a common diet wasfed to groups previously receiving Treatments 1 and2. Half of the pigs previously fed Treatment 3 werefed the common diet received by the Treatment


groups 1 and 2; while the other half were fed a dietcontaining 1.3% NuProTM during the first week ofPhase 3 (day 21 to 28) followed by the common dietfor the remainder of the phase (day 28 to 42). DuringPhases 1 and 2, no significant differences wereobserved among the four dietary treatments withregard to ADG, ADFI, or G:F (Table 7). During thefirst week of Phase 3, pigs previously fed the basaldiet containing plasma protein and fed NuProTM atthe 50% replacement level had lower (P=0.07) ADFIthan pigs previously fed NuProTM at the 100%replacement level and pigs fed NuProTM at 1.3% ofthe diet during the first week of Phase 3. This studyindicates that NuProTM maybe used as an alternativeto spray-dried plasma protein in nursery pig diets,and the removal of NuProTM from the diet does notresult in decreased feed intake as is often the casewith the removal of plasma protein. This studyindicates that NuPro™ may be an effectivereplacement for plasma protein.

Organic selenium from yeast

Organic selenium offers several major opportunities

to the feed/food industries. Although inorganicselenium (sodium selenite) has routinely been addedto most animal diets, research has shown that about60% of it is excreted in the urine. Organic selenium(selenium enriched yeast) is an effective source ofselenium as it is more effectively retained in muscle,milk, and fetal tissues than inorganic selenium andless is excreted. Accumulation in tissues provides aselenium reserve that can be used under conditionsof stress for additional synthesis of selenoproteinsessential for counteracting adverse effects of freeradicals. Organic selenium is also transferred intothe egg more efficiently and into embryonic tissuesin mammals via improved placental transfer whencompared to sodium selenite. This provides the younganimal with higher selenium stores, which canpromote improved disease resistance. In addition tothe benefits to livestock species, higher tissue levelsof organic selenium may offer health benefits toconsumers who choose Se-enriched animal products.Although somewhat controversial, increasedselenium intake has been associated with reductionsin cancer risks in epidemiological studies (Vogt etal., 2003), animal models (Popova, 2002) andchemopreventive studies (Combs et al., 2001; Clark

Table 7. Efficacy of NuProTM in early-weaned pig diets.

Group 1 Group 2 Group 3 Group 4Trait Control 50% 100% 100% SE P>F

NuProTM NuProTM NuProTM

ADG, g Phase 1 47 45 31 35 10 0.60 Phase 2 399 402 429 404 11 0.19 Phase 1-2 282 283 296 281 9 0.58 Phase 3 623 590 626 621 12 0.17 Phase 1-3 451 437 461 450 8 0.20

ADFI, g Phase 1 118 113 99 116 7 0.25 Phase 2 473 475 497 465 15 0.47 Phase 1-2 355 354 365 349 11 0.78 Phase 3 1009 962 1037 1002 19 0.08 Phase 1-3 680 658 701 675 13 0.18

Gain:feed Phase 1 0.393 0.376 0.266 0.272 0.077 0.53 Phase 2 0.845 0.850 0.868 0.874 0.012 0.25 Phase 1-2 0.795 0.799 0.816 0.810 0.011 0.54 Phase 3 0.615 0.615 0.603 0.613 0.008 0.71 Phase 1-3 0.662 0.665 0.658 0.663 0.006 0.87

Weight, kg Initial 6.45 6.45 6.45 6.45 0.005 0.86 Phase 1 6.78 6.77 6.67 6.69 0.07 0.62 Phase 2 12.37 12.39 12.67 12.35 0.18 0.57 Phase 3 25.44 24.79 25.81 25.51 0.34 0.22

Group 1: days 1-21, basal diet; days 21-42 common diet.Group 2: days 1-21, basal with 50% NuProTM replacement; days 21-42 common.Group 3: days 1-21, basal with 10% NuProTM replacement; days 21-42 common.Group 4: days 1-21, basal with 100% NuProTM replacement; days 21-28 1.3% NuProTM; days 28-42 common.

C.V. Maxwell 21

and Marshall, 2001). Should additional studiesunderway prove conclusive, this presents the livestockand poultry industries with an opportunity to provideadditional health benefits to the public consuminganimal products.

Environmental impact of concentratedanimal feeding units

Another major problem facing the feed/food industryis the concentration of nutrients in modern livestockand poultry production systems. Northwest Arkansascontains the headwaters for two scenic rivers and isalso the location of a major concentration of animalproduction, primarily poultry. Disposal of theconcentrated animal waste, which accumulates inefficient production systems, in a manner thatminimizes odor and optimizes nutrient utilization isan increasing problem facing the livestock and poultryindustries in our state. Animal waste can be a valuableresource as an alternative source of fertilizer nitrogen(N), phosphorus (P), and potassium (K) inmaintaining and restoring soil productivity. In fact,by improving ground cover, runoff volume anderosion may also be reduced. Conversely, applicationof animal manure at rates greater than a crop canutilize has been shown to result in nitrate (NO3)movement through the soil into ground water andcan result in an excessive rise in soil P levels, leadingto increased phosphorus runoff. This can be a problemsince phosphorus is normally the limiting nutrientfor eutrophication in freshwater systems. Odor andnutrient problems can both be exacerbated byexcessive nutrient buildup in lagoons/holding pondsthat have not been dewatered in a timely manner.

With the initial population of the new Universityof Arkansas 2000 head/year finishing facility, adecision was made to demonstrate the use of dietaryphytase addition to substantially reduce phosphorusproduction in swine manure without affecting swineperformance or profitability. Facilities wereconstructed to permit production of two types ofmanure that was stored in holding ponds. Pigs placedin half of the pens received normal phosphorus dietsdevoid of phytase and pigs placed in the other halfof the facility received diets with reduced phosphorussupplemented with phytase. The holding ponds weremanaged by emptying the shallow pit under the pigson each diet on a weekly basis and recharging the pitwith effluent from the top of the holding pond. Thissimulated the management of a pull-plug waste

disposal system and allowed the accumulation of thetwo types of manures for application on watersheds.

Table 8 provides the average total and solublephosphorus analysed in the holding ponds. The 24.8%reduction in total phosphorus is consistent with themagnitude of reduction observed in phosphorusbalance studies with pigs to determine the magnitudeof reduction of phosphorus expected by feedingreduced phosphorus diets with added phytase. Themagnitude of reduction in soluble phosphorus wasonly 8.95%, suggesting that a higher percentage ofthe phosphorus from pigs fed phytase was in the solubleform. This is consistent with other observations thatphytase increases soluble phosphorus in manure.

Table 8. Phosphorus concentration in holding ponds (mg/L).

Item Normal P Phytase P Reduction (%)

Total P 289.7 217.9 24.8Soluble P 138.4 126.0 8.95

N = 6 samples per manure source

The reduced risk of phosphorus runoff fromwatersheds receiving manure from phytase-treatedpig diets in relation to manure from pigs fed normalphosphorus, non-phytase diets was also demonstrated.Concern over water quality near animal productionfacilities is primarily with regard to transport ofexcessive amounts of N and(or) P from the animalwaste.

A third watershed evaluated the efficacy ofaluminum chloride (AlCl3) addition to swine manureon runoff. Shreve et al. (1995), Moore et al. (1995)and Smith et al. (2001) recommended treatment ofmanure with aluminum chloride as a means ofreducing both P and NH3 losses. Runoff of nutrientswas compared to a watershed that received no manureor fertilizer. The watershed sites were designated:

1. No manure or fertilizer application

2. Phytase manure: Low P diet, high N, but low Ploading, lower risk of P runoff.

3. Normal manure: Normal P diet , high N and Ploading on pasture, high risk of P runoff.

4. Phytase manure: Low P diet, high N, but low Ploading, aluminum chloride added to reducesoluble phosphorus and lower risk of P runoff.

Manure was transported from the respective holdingbasins and applied to two separate pastures in multiple


applications at rates equivalent to a target of 150 lbN/acre/year. Manure from pigs fed the reducedphosphorus diet with added phytase was also treatedwith aluminum chloride by adding 0.75% aluminumchloride to the manure prior to application. This wasadded to a third watershed at the same applicationrate used in the other watersheds. A fourth watershedhad no manure or fertilizer added. A total of threeapplications were made during the project. A ‘SmallIn-field Runoff Collector’ system was used to collectrunoff water. Samples from each watershed and stormevent were composited and analyzed for total KjeldahlN, total P, soluble P, NH3-N, NO3-N, copper and zinc.

Total runoff data are presented in Table 9. Thewatershed that received no manure or fertilizerproduced the greatest total runoff with 191,344 liters.This might be expected since reduced forage covermay increase runoff. The watershed receiving thenormal phosphorus manure was next with 179,028liters followed by the watershed receiving AlCl3

treated manure from pigs fed phytase (162,418 liters).The watershed receiving untreated manure from pigsfed phytase had the lowest total runoff of 112,826liters.

Table 9. Total runoff.

Treatment (Liters)

Watershed 1 No manure 191,344Watershed 2 Phytase 112,826Watershed 3 Normal P 179,028Watershed 4 Phytase+AlCl3 162,418

The total nutrients applied to the watersheds in thethree applications are presented in Table 10. Theapplication of total N was approximately 150 lbs oftotal N/acre/year. The addition of aluminum chlorideto the swine manure also substantially reduced thesoluble phosphorus at the time of manure application,as expected.

Table 10. Nutrients applied to soil from manure by treatment(lb/ac).a

Treatment Soluble P Total P Total N

Unfertilized n/a n/a n/aPhytase diet 12.7 31.5 230Normal diet 10.2 30.4 205Phytase + AlCl3 1.6 26.6 240

aRepresents three applications over 1.5 years.

The mass of soluble and total P lost from the watershedfertilized with normal manure was greater whencompared to the runoff in the unfertilized watershed

or watersheds fertilized with phytase manure orphytase manure with AlCl3 (Table 11). This total massof P loss in the watershed fertilized with normalmanure also represented the greatest percentage ofapplied total P lost among the watersheds (5.4 vs 3.6and 4.9% for the watersheds treated with phytasemanure and phytase with AlCl3, respectively).Application of manure from pigs fed phytase, withor without treatment with AlCl3, reduced the mass ofsoluble and total P runoff. In fact, the watershedtreated with phytase manure produced the lowest totalP and percentage of soluble and total P runoff amongthe watersheds, even lower than that observed in theunfertilized watershed. It should be noted, however,that runoff volumes were variable betweenwatersheds, which had an impact on the total massof nutrients lost from the runoff events. Whencomparing the mass of nutrients applied to that whichwas lost through runoff, it is important to note thatthe vast majority of nutrients remained within thewatershed. In general, more than 90% of the nutrientsapplied remained in the watershed. The exceptionsto this are the percentage of soluble P lost from thewatershed receiving the normal P manure (12.7%)and the percentage of soluble P lost from thewatershed receiving the phytase manure with AlCl3

(60.6%). A higher percentage of soluble nutrientswere lost through runoff, because the soluble fractionis fairly dynamic, and is also more susceptible torunoff losses than the total fraction. There was moresoluble P removed from the phytase manure withAlCl3 watershed than was applied from the manure,most likely due to the natural loss of soil P as seen inthe unfertilized watershed. The N lost from thesewatersheds was a very small fraction of what wasapplied, ranging from 0.6% to 1.2%.

Table 11. Mass of nutrients lost from watersheds and percentageof applied nutrients lost.

Treatment Soluble P Total P Total N

(lb/acre) (%) (lb/acre) (%) (lb/acre) (%)

Unfertilized 0.89 1.26 1.84Phytase diet 0.92 7.2 1.12 3.6 1.39 0.6Normal diet 1.30 12.7 1.65 5.4 2.38 1.2Phytase + A1C13 0.97 60.6 1.31 4.9 2.28 1.0

Zinc concentrations from runoff were very low(Figure 6). Manure had no apparent impact on metalrunoff when applied as a fertilizer resource to thewatersheds. Copper concentrations in runoff were alsovery low, in the ppb range, and were not affected bythe addition of manure to the watersheds (Table 12).

C.V. Maxwell 23

Table 12. Concentration of copper lost in runoff by treatment.

Treatment Cu (µg/L)

1. Unfertilized 2.52. Phytase Diet 2.33. Normal Diet 3.64. Phytase + A1C13 3.5

One of the objectives of this project was to conduct aphosphorus and nitrogen budget for the farm. A totalof 8,222 lb of phosphorus was delivered to the farmand an estimated 2,507 lbs (30.5%) was removed inpigs marketed or retained in pigs kept as replacementbreeding stock (Table 13). A total of 1,616 lbs(19.6%) was spread on 88 acres for an averageapplication rate of over 12 lbs of total P/acre/yr,which exceeds the P needed for forage productionfor grazing or hay. The amount of total N deliveredto the farm was 40,839 lb (Table 14). An estimated11,608 lbs (28.60%) was removed in pigs marketedor retained in pigs kept as replacement breeding stock.A total of 3,655 lb. (8.94%) was spread on 88 acresfor an average maximum application of 41.50 lbs ofN/acre. If one obtained the expected ammonia lossfrom volatilization of 25%, then the actual appliedN would be about 31 lb/acre, which is probably belowthe crop needs for either the bermuda or fescuepastures where manure was applied. The residual Nis most likely much less than the calculated residualsince ammonia volatilization from the productionfacility and holding ponds is likely to be substantial.

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

Unfertilized Phytase Normal Phytase withAlCl3

Zinc

(mg/

L)

Figure 6. Effect of manure fertilization on zinc concentration in runoff.

Table 13. Farm phosphorus balance.

lbs %

Total P delivered in feed 8,222P removed in pigs marketed 2,507 30.5P in manure spread 1,616 19.6Residual 4,099 49.8

Table 14. Farm nitrogen balance.

lbs %

Total N delivered in feed 40,839N removed in pigs marketed 11,680 28.60N in manure spread 3,655 8.94Residual 25,504 62.45

This study demonstrates that even with judiciousmanagement, phosphorus in the soil accumulates withapplication of swine manure based on plantrequirements for N in forage-based systems.Construction of new production facilities should onlybe considered after development of nutrientmanagement plans ensuring application of nutrientsthat do not exceed crop needs. Technologies to furtherreduce phosphorus in manure would reduce the landbase needed for concentrated animal productionfacilities.

In areas where nutrient excesses exist, progress isbeing made in developing technologies to addressthe problem and some are even receiving praise fordelivering both environmental and economic benefits.Singled out in the popular press recently is the manuremanagement system in the Chino Basin in Southern


California, which utilizes a digester to convert dairymanure into fertilizer and methane used for powergeneration. Smithfield Foods is constructing a $20million system to convert swine manure into liquidmethanol to be used in the production of biodiesel.Success of these systems is critical for maintaininggood relationships in communities with large livestockproduction facilities. Why would every state not wantto have a $7 billion poultry industry like Arkansas?

Conclusions

In summary, it appears there will be increasedemphasis on food safety that will require dramaticchanges in the way national and international feed/food companies operate. This has benefits not onlyfrom a food safety standpoint, but as we develop abetter understanding of controlling microorganismsin the environment, we may also tremendouslyimprove animal health and performance and shouldhave a number of alternatives to antibiotics. Theimpact of an animal’s interaction with microorganismsin the environment on gain and efficiency is evengreater than once thought. Finally, it is evident thatenvironmental and animal welfare issues will haveincreasing influence on decision making.

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Bolduan, G. 1999. Feeding weaner pigs without in-feed antibiotics. In: Biotechnology in the FeedIndustry. Proceedings of Alltech’s 15th AnnualSymposium. (T.P. Lyons and K.A. Jacques (eds). P223-230

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Clark, L. C. and J. R. Marshall. 2001. Randomized,controlled chemoprevention trials in populationswith very high risk for prostate cancer. Elevatedprostate-specific antigen and high-grade prostaticintraepithelial neoplasia. Urology 57:185-7

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Gaskins, H. R. 1997. Immunological aspects of host/microbiota interactions at the intestinal epithelium.In: Mackie, R. I., White, B. A., Isaacson, R. E.(Eds.), Gastrointestinal Microbes and HostInteractions, Vol. 2, Chapman and Hall, New York,pp. 537-587.

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C.V. Maxwell 25

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Popova, N. V., 2002, Perinatal selenium exposuredecreases spontaneous liver tumorogenesis in CBAmice. Cancer Lett. 179:39-42.

Savage, D. C. 1987. Factors affecting the biocontrolof bacterial pathogens in the intestine. FoodTechnology. 41:82-87.

Schinckel A. P., Clark LK, Stevenson G, Knox K,Nielsen J, Grant A, Turek JJ, and Hancock D. 1995.Effects of antigenic challenge on growth andcomposition of segregated early-weaned pigs.Swine Health and Production 3(6):228-234.

Schinckel A. P., M. E. Spurlock, B. T. Richert, W.M. Muir and T. E. Weber. 2003. ProceedingsEuropean Association for Animal Production, RomeItaly, August, 2003.

Spring, P. 1999. The move away from antibioticgrowth promoters in Europe. In: Biotechnology inthe feed industry. Proceedings of Alltech’s 15th

Annual Symposium. T.P. Lyons and K.A. Jacques(editors) P173-184

Spring, P., C. Wenk, K. A. Dawson, and K. E.Newman. 2000. The effects of dietarymannanoligosaccharides on cecal parameters andthe concentrations of enteric bacteria in the ceca ofsalmonella-challenged broiler chicks. Poult. Sci.79:205-211.

Vogt, T. M., R. G. Ziegler, B. I. Graubard, C. A.Swanson, R. S. Greenberg, J. B. Schoenberg, G.M. Swanson, R. B. Hayes and S. T. Mayne. 2003.Serum selenium and risk of prostate cancer in U.S. blacks and whites. Int. J. Cancer. 103:664-70.

Wilson, M, 1995. Segregated early weaning. Pig Lett.15:17-20.


K.E. Newman 27

Glycomics: putting carbohydrates to work for animal and human health

KYLE E. NEWMAN

Venture Laboratories, Inc., Lexington, Kentucky, USA

Introduction

In October of 2001 bioterrorism came to the UnitedStates in the form of letters containing anthrax spores.Imagine a time in the very near future when simplyeating a carbohydrate fraction from yeast can protectyou from such an attack. It is not as far-fetched asyou may think. A recent trial in mice found that micereceiving yeast glucans for 1 week prior to anthraxinfection had double the survival rate ofunsupplemented animals. As a therapeutic agent, micereceiving yeast glucans had a 90% survival ratecompared to 30% survival for control animals(Kournikakis et al., 2003).

In February of 2003, the magazine TechnologyReviews identified glycomics as one of the “10Emerging Technologies That Will Change the World.”Glycomics is defined as the characterization of thesugars and the structure of these sugars that make upa cell. We are all aware of the quest to define thehuman genome (genomics: the full DNAcomplement) that was recently completed. Many areaware of proteomics, which is the study of the fullset of proteins encoded by a genome, but very fewpeople are aware of glycomics. Putting these sciencesin perspective, genomics was child’s play comparedto the undertaking of proteomics, which is dwarfedin comparison to glycomics.

New roles for carbohydrates

At one time, it was thought that there were threemain roles of carbohydrates in biological systems.The most obvious role of sugar is as an energy sourceor storage component for energy reserves. The secondfunction is as a structural component such as celluloseor chitin. The third function seemed to be to confound

scientists studying proteins and lipids by beingassociated with these compounds and subsequentlyneeding to be stripped away in order to trulyunderstand the function of the protein. However, itturns out that the glycosylation of these compoundscan define their function or serve to stabilize them.A good example of this stabilization is industrial-grade enzymes, where shelf-life and heat stabilityhave been enhanced by glycosylation of the protein.

Unlike amino acids or nucleic acids that have acertain predictability to their structure, there is nosimple code for determining the structure of complexsugars. The biological diversity of these compoundscan be easily demonstrated by examining thedifference between α- and ß-bonded (1-4) glucoseunits (Figure 1). When these two glucose units arebound in the α configuration, the resultingcompound, amylose, is easily degraded by starch-degrading enzymes found in saliva. Conversely,ß-bonded (1-4) glucose represents cellobiose, acompound that is not degraded by any mammalianenzyme system. This exemplifies the difference inbiological activity of the same two glucose moleculesbound together at the same site with the onlydifference being the type of bond between them.Compared to DNA or amino acid linkages, whichare somewhat linear, imagine the complexity whenyou consider that a hexose molecule (like glucose)has six binding sites, can branch and the bonds canorient in different ways (as seen in Figure 1). Thepossibilities for oligo- or polysaccharides can be abit overwhelming.

Complex carbohydrates have become a prominentresearch topic with the realization that distinctcarbohydrate structures can have very specificbiological activities. One need only imagine the

28 Glycomics: putting carbohydrates to work for animal and human health

diverse nature of carbohydrate chemistry tounderstand that the opportunities for novel compoundswith unique biological activity are boundless. In fact,the diversity and complexity of these compounds haskept investigators from fully understanding them untilrecently. Carbohydrates and oligosaccharides are alsonow being utilized as a nutritional means of enhancingimmune function.

Trehalose is a disaccharide of two glucosemolecules linked by α(1,1) linkages (Figure 2). Thiscompound is a non-reducing sugar that does not reactwith amino acids or proteins, making it unaffectedby Maillard reactions. When incorporated intomaterials prior to freezing, trehalose has shown anability to prevent ice crystallization damage to themolecule. This phenomenon has been exploited toincrease the shelf life of materials ranging from foodsto probiotic preparations. The role of trehalose isnot limited to extending shelf life. It has been shownthat mice supplemented with 2% trehalose hadreduced symptoms of Huntington disease (Tanaka etal., 2004). Huntington’s disease is a rare, hereditaryneurological illness characterized by sporadic andinvoluntary muscle movements. The disease affectsapproximately 1 in 10,000 people.

OH

O

OH

OH

CH2OH

H

H

HH

O

H

OH

HOH2C

H OH

HH

O

OH H

Figure 2. Trehalose, a non-reducing disaccharide.

Trehalose has also been examined as a possible therapyin osteoporosis. Ovariectomized mice receiving 100mg/kg of trehalose had suppressed osteoclastdifferentiation compared to unsupplementedovariectomized mice. The improved osteoclast

differentiation from bone marrow in supplementedmice prevented the femoral bone loss that is normallyattributed to estrogen deficiency (Nishizaki et al.,2000). The most striking finding of this study is thatthe improvements seen were not due to boostingestrogen levels (the normal therapy for menopause-induced osteoporosis). This could be of enormousbenefit since the usual therapy to prevent osteoporosisis supplemental estrogen, which has been linked withcertain cancers (Stalhberg et al., 2004).

Carbohydrate-related diseases

The role of carbohydrates in health and disease iscoming out of a blur and into focus, but we are onlyobserving the infancy of this field of study. In 2002,a Harvard researcher proposed that an immuneresponse to the complex carbohydrate glycosamin-oglycan (GAG) is a potential cause of rheumatoidarthritis (Wang and Roehrl, 2002). Rheumatoidarthritis is a systemic autoimmune disease ofconnective tissue; and GAGs are a major componentof that tissue. This study was unique in that itdemonstrated a direct link between human disease,carbohydrate antigens and the immune system.

Congenital disorders of glycosylation (CDG), alsoknown as ‘carbohydrate-deficient glycoproteinsyndrome,’ are a group of inherited disorders wheremany glycoproteins are deficient in the carbohydratefraction of the compound. Adults and children withCDG have varying degrees of disabilities such asspeech and cognitive difficulties, poor balance andimpaired motor skills. Several human diseases are aresult of faulty carbohydrate metabolism, the mostcommon of these being diabetes, a conditioncharacterized by abnormally high levels of bloodglucose from a failure in glucose transport from theblood into the cells. Another condition related toerrors in carbohydrate metabolism is Tay-Sachsdisease, an inherited disorder caused by a recessive

Figure 1. α- and ß-bonded (1-4) glucose units.

O

Amylose

OH

OH

CH2OH

H

H

HH

O

O

OH

OH

CH2OH

H

H

HH

O

O

α-(1-4) linkage

α-D GLUCOSE α-D GLUCOSE

Cellobiose

CH2OH

O

O

OH

H

H

OH

H

H HOH

CH2OH

H

OH

H OH

O

OH

H

H

β-D GLUCOSE

β-(1-4) linkage

β-D GLUCOSE

K.E. Newman 29

defect in the gene encoding for hexosaminidase A.This leads to an unusual accumulation of gangliosideGM2 in the central nervous system. Characteristicsof the disease include blindness, seizures, adegeneration of motor and mental function with earlydeath in childhood. While most of the defined diseasesof sugar metabolism are relatively rare, many of theso-called genetic diseases with unknown causes maybe caused by errors in glycosylation because of theimportance of glycosylation in cell-to-cell interactionand the role of glycomics in the immune system.

Carbohydrates, cell-to-cellcommunications and defense againstpathogens

Carbohydrates are important surface entities ofanimal cells that function in a variety of ways toinfluence cell-to-cell communication, impact theimmune system and allow bacterial attachment to thehost. These complexed molecules project from thecell surface and form the antigenic determinants ofcertain cell types. One of the classical examples ofthis antigenicity is blood type in humans. The ABOblood group antigens are glycoproteins on red bloodcells. Small differences in the terminal sugar residuesdistinguish the A and B blood-group antigens (Kuby,1994; Figure 3) Mannose binding protein (MBP) isan integral part of the immune system. MBP in theserum can bind to terminal mannose groups on thesurface of bacteria and interact with two serine

proteases (MASP and MASP2), which ultimately leadto antibody independent activation of the classicalpathway of the immune system (Roitt et al., 1998).

Bacterial infection is due in many cases to the abilityof the bacteria to recognize host cell surface sugarsand use specific receptors that allow them to attach,colonize, and in the case of pathogens, cause diseasein the animal. Mannose-specific adhesins (the bindingentity on the surface of bacterial cells) are utilizedby many gastrointestinal pathogens as a means ofattachment to the gut epithelium. One way to preventpathogens from causing disease is to prevent themfrom attaching to the epithelial cells in the gut. Earlystudies using mannose in the drinking water of broilerchicks demonstrated that this therapy could reducecolonization rate of Salmonella typhimurium.Purified mannose and a complex sugar called mannanoligosaccharide (MOS) have been successfully usedto prevent bacterial attachment to the host animal byproviding the bacteria a mannose-rich receptor thatserves to occupy the binding sites on the bacteria andprevent colonization in the animal.

Several studies have been conducted examining therole of mannans and their derivatives on binding ofpathogens to epithelial cells in the gastrointestinaltract. E. coli with mannose-specific lectins did notattach to mammalian cells when mannose was present(Salit and Gotschlich, 1977). Spring and coworkers(2000) used a chick model to demonstrate that MOS(Bio-Mos®) could significantly reduce thecolonization of Salmonella and E. coli. Animal trialsin other species show similar benefits in reducing

Figure 3. Differences in the terminal sugar residues distinguish the A and B blood group antigens.

Galactose

N-acetylglucosamine

Glucose

Fucose

Lipid or protein

O antigen

B antigenA antigen

N-acetylgalactosamine


pathogen concentrations. In dogs, as well as in poultry,reductions in fecal clostridial concentrations have alsobeen noted with Bio-Mos® supplementation (Finucaneet al., 1999; Strickling, 1999).

Fructo-oligosaccharides (FOS) have beeninvestigated for nutritional manipulation of thegastrointestinal tract to inhibit pathogens. The principlebehind the use of FOS involves the structure andbonding of the fructose molecules. Purifiedpreparations of FOS have been shown to provide anutrient source for beneficial bacteria such asbifidobacteria and certain lactobacilli. By supportingthe growth of the beneficial bacteria it is thought thatthis will provide an in situ competitive exclusion (CE)effect, thus improving animal health. However, itseems important that the concentration of non-complexed fructose molecules be kept to a minimumin order for this oligosaccharide to be successful.Oyarzabal and coworkers (1995) found thatSalmonella spp. could not use a purified FOSpreparation for growth but were able to utilize acommercial preparation of FOS. The authors suggestthe use of lactic acid bacteria in combination withFOS as a feasible approach to control Salmonella.Other studies have demonstrated a reduction inSalmonella concentrations in birds challenged withS. typhimurium with and without FOS and a CEculture. FOS alone had little effect on Salmonellaexclusion when FOS was administered after infection,but FOS in combination with a defined CE producthad an additive effect on Salmonella exclusionespecially when used as a prophylactic prior toinfection (Bailey et al., 1991). However consistentresponse of animals to FOS supplementation is aproblem and may affect other as yet undefinedinteractions. Waldroup and coworkers (1993) found thatsupplementing broilers with 0.375% FOS had fewconsistent effects on production parameters or carcassSalmonella concentrations. These authors also cautionof possible antagonism between FOS and BMD.

Human data for FOS are much more consistent.Hidaka et al. (1986) found that consumption of 8 gFOS/day increased numbers of bifidobacteria,improved blood lipid profiles and suppressedputrefactive substances in the intestine.

Glycomics also plays a vital role in viral diseases.The influenza virus infects by first attaching to a cellsurface carbohydrate called sialic acid. Thisattachment ‘opens the door’ of the cell and allows thevirus to replicate within. The commercial drugsTamiflu and Relenza shorten the duration of the fluby binding to the active site of an enzyme produced

by the virus that frees the virus from the sialic acid.By tying up this enzyme, the virus cannot easilyspread and infect other cells (Schmidt, 2002). Thereare also data examining a novel anti-humanimmunodeficiency virus (HIV) protein. This protein,called actinohivin, binds to a glycoprotein on variousHIV strains and simian immunodeficiency virus(SIV) inhibiting viral entry into cells by binding tothis envelope glycoprotein. Further investigationshowed that only yeast mannan can inhibit thebinding of actinohivin to these viruses. These resultsdemonstrate that the mannose saccharide chains ofthe virus glycoprotein are the molecular targets ofthe anti-HIV activity of actinohivin (Chiba et al.,2004). Sulfated galactomannans also demonstrate invitro and in vivo activity against the flaviviruses,yellow fever virus and dengue virus (Ono et al.,2003). West Nile virus has also gained a strongfoothold in the United States, affecting birds, horsesand man. N-linked sugars with mannose residues onthe cell membrane protein were found to beimportant in West Nile virus binding to the cell (Chuand Ng, 2003).

The future of the science of glycomics seemsenormous at this time. While mannan oligosaccharideis currently being used to improve health andproduction of animals, there are enormouspossibilities to use other sugars as possible agentsagainst pathogen infection. Table 1 shows a partialsummary of scientific studies examining bacterialadhesins.

Table 1. Carbohydrate adhesins of various bacterial strains1.

Bacterial strain Expressing mannose Other carbohydrateadhesins (% of adhesinsthose examined)

Campylobacter coli 0 GlucoseCampylobacter jejuni2 0 GlucoseClostridium spp.2 0 Galactose, glucose,

lactoseEdwardsiella ictaluri 100 Not knownEnterobacter cloacae 100 Not knownEscherichia coli2 53 Fucose, galactose,

glucoseFusobacterium spp. 0 Galactose, lactose,

raffinoseHaemophilus influenzae 0 Galactose, glucoseKlebsiella pneumoniae 100 GlucoseSalmonella spp.2 64 Fucose, galactoseSerratia marcescens 100 Not knownShigella spp. 75 FucoseStreptococcus bovis 0 GlucoseStreptococcus suis 0 Galactose

1Summarized from Mirelman et al., 1980; 1986; Ofek et al.,20032In vivo data have shown reductions in these populations with Bio-Mos®.

K.E. Newman 31

Conclusions

To say that carbohydrates are involved in virtuallyevery aspect of biology is not an understatement.Finding ways to exploit this knowledge is the currentchallenge. The vast array of possibilities that existwith polysaccharide structure and function makeglycomics a science that may well pass on to futuregenerations. We have only scratched the surface, butwe have a better understanding of arthritis, how theimmune system works in identifying invasion, howcertain congenital disorders debilitate and we haveused our limited knowledge to take advantage of the‘sweet tooth’ of pathogens to control infection. Itbrings a whole new meaning to ‘a spoonful of sugarhelps the medicine go down’.

References

Bailey, J.S., L.C. Blankenship and N.A. Cox. 1991.Effect of fructooligosaccharide on Salmonellacolonization of the chicken intestine. Poultry. Sci.70:2433-2438.

Chiba, H., J. Inokoshi, H. Nakashima, S. Omura andH. Tanaka. 2004. Actinohivin, a novel anti-humanimmunodeficiency virus protein from anactinomycetes, inhibits viral entry to cells bybinding high-mannose type sugar chains of gp120.Biochem. Biophys. Res. Comm. 316:203-210.

Chu, J.J. and M.L. Ng. 2003. Characterization of a105-kDa plasma membrane associated glycoproteinthat is involved in West Nile virus binding andinfection. Virology. 312:458-469.

Finucane, M.C., K.A. Dawson, P. Spring, K.E.Newman. 1999. The effect of mannanoligosaccharide on the composition of themicroflora in turkey poults. Poutry Sci. 78(Suppl.1):77.

Hidaka, H., T. Takizawa, T. Tokunaga, and Y.Tashiro. 1986. Effects of fructooligosaccharides onintestinal flora and human health. BifidobacteriaMicroflora. 5:37-50.

Kournikakis, B., R. Mandeville, P. Brousseau andG. Ostroff. 2003. Anthrax-protective effects ofyeast ß(1,3) glucans. Med. Gen. Med. 5:1-5.

Kuby, J. 1994. In: Immunology. 2nd Edition. W.H.Freeman and Co. New York.

Mirelman, D., G. Altmann, and Y. Eshdat. 1980. J.Clinical Microbiol. 11:328-331.

Mirelman, D. and I. Ofek. 1986. Introduction to

microbial lectins and agglutinins. In: MicrobialLectins and Agglutins-Properties and BiologicalActivity. (D. Mirelman, ed). John Wiley & Sons,Inc. NY.

Nishizaki, Y., C. Yoshizane, Y. Toshimori, N. Arai,S. Akamatsu, T. Hanaya, S. Arai, M. Ikeda and M.Kurimoto. 2000. Disaccharide-trehalose inhibitsbone resorption in ovariectomized mice. Nut. Res.20:653-664.

Ofek, I., D.L. Hasty, R.J. Doyle. 2003. In: BacterialAdhesion to Animal Cells and Tissues. ASM Press.Washington, D.C.

Ono, L., W. Wollinger, I.M. Rocco, T.L. Coimbra,P.A. Gorin, M.R. Sierakowski. 2003. In vitro andin vivo antiviral properties of sulfatedgalactomannans against yellow fever virus (BeH111strain) and dengue 1 virus (Hawaii strain). AntiviralRes. 60:201-208.

Oyarzabal, O.A., D.E. Conner, and W.T. Blevins.1995. Fructooligosaccharide utilization bySalmonellae and potential direct-fed-microbialbacteria for poultry. J. Food Prot. 58:1192-1196.

Oyofo, B.A., J.R. DeLoach, D.E. Corrier, J.O.Norman, R.L. Ziprin, and H.H. Mollenhauer.1989a. Prevention of Salmonella typhimuriumcolonization of broilers with D-mannose. PoultrySci. 68:1357-1360.

Roitt, I., J. Brostoff, and D. Male. 1998. In:Immunology. 5th Edition. Mosby International Ltd.London.

Salit, I.E. and E.C. Gotschlich. 1977. J. Exp. Med.146:1182-1194.

Schmidt, K. 2002. Sugar rush. New Scientist. 176:34-38.

Spring, P., C. Wenk, K.A. Dawson, and K.E.Newman. 2000. The effects of dietary mannanoligosaccharide on cecal parameters and theconcentrations of enteric bacteria in the ceca ofSalmonella-challenged broiler chicks. Poult. Sci.79:205-211.

Stahlberg, C., A.T. Pedersen, E. Lynge, Z.J.Andersen, N. Keiding, Y.A. Hundrup, E.B. Obel,B. Ottesen. 2004. Increased risk of breast cancerfollowing different regimens of hormonereplacement therapy frequently used in Europe. Int.J. Cancer. 109:721-727.

Strickling, J.A. 1999. Evaluation of oligosaccharideaddition to dog diets: Influence on nutrientdigestion and microbial populations. Masters Thesis.University of Kentucky.


Tanaka, M., Y. Machida, S. Niu, T. Ikeda, N.R. Jana,H. Doi, M. Kurosawa, M. Nekooki and N. Nukina.2004. Trehalose alleviates polyglutamine-mediatedpathology in a mouse model of Huntington disease.Nat. Med. 10:148-154.

Waldroup, A.L., J.T. Skinner, R.E. Hierholzer, P.W.Waldroup. 1993. An evaluation of fructo-oligosaccharide in diets for broiler chickens andeffects on salmonellae contamination of carcasses.Poult. Sci. 72:643-650.

Wang, J.Y. and M.H. Roehrl. 2002. Glycosamin-oglycans are potential cause of rheumatoid arthritis.Proc. Natl. Acad. Sci. USA. 99:14362-14367.

56 Selenium sources, selenoproteins and practical poultry production

P.R. Ferket 57

Alternatives to antibiotics in poultry production: responses, practicalexperience and recommendations

PETER R. FERKET

Department of Poultry Science, College of Agriculture and Life Sciences, North Carolina StateUniversity, Raleigh, North Carolina, USA

During the past 50 years, the livestock and poultryindustries have developed in several areas includingnutrition, genetics, engineering, management, andcommunications to maximizing the efficiency ofgrowth performance and meat yield. Now theseindustries must focus more attention on how animalagriculture affects the environment and food safety.As in many other industries, the global paradigm isshifting from an emphasis on productive efficiencyto one of public security. Nothing demonstrates thisparadigm shift more clearly than the issues concerningthe use of antibiotic growth promoters. For the pastfour decades, antibiotics have been used in animalagriculture to improve growth performance andprotect animals from the adverse effects of pathogenicand non-pathogenic enteric microorganisms. Now,antibiotics have come under increasing scrutinybecause of the potential development of antibiotic-resistant human pathogenic bacteria after long use(Phillips, 1999; Ratcliff, 2000). In response to thisapparent ‘threat’, the European Union banned theuse of subtherapeutic levels of antibiotics to preventdisease or promote growth, starting with a ban onavoparcin in 1997 and a ban on virginiamycin,bacitracin, spiromycin, and tylosin in 1999.Antimicrobials scheduled to be banned by 2006include avilamycin, bambermycin, salinomycin andmonensin. In June of 2003, McDonald’s Corp.announced that it would prohibit their direct suppliersfrom using antibiotics that are important in humanmedicine as growth promoters in food animals after2004, and they created a purchasing preference forcompanies that work to minimize antibiotic use.Although banning antibiotic growth promoters maynot be scientifically justified, the tide of publicopinion is forcing animal agriculture to developalternatives, or at least substantially reduce the amountof antibiotics used to maintain production efficiency

and produce safe meat and egg products. Some ofthese alternatives may include significant changes inhusbandry practices or the strategic use of entericmicroflora conditioners, including acidifiers,probiotics, enzymes, herbal products, microfloraenhancers, and immuno-modulators. The objectiveof this paper is to briefly review the use of antibioticgrowth promoters as enteric conditioners and discussthe potential of non-pharmaceutical alternatives.

Benefits of feeding antibiotics

Antibiotic usage in animal feeds has many benefits.It improves food safety by increasing animal healthand reducing or eliminating certain pathogens. Itreduces animal production costs and economicbenefits are distributed along the food chain,including the feed industry, production animalagriculture, food processors, retailers, and consumers.Most of the cost savings attributed to antibiotics isfrom improved feed conversion, and this response ishighest in fast-growing genetically improved animalsreared in intensive production systems. Other costsavings come from faster growth rate, reducedmortality, greater resistance to disease challenge,improved reproductive performance, improvedpigmentation, and better manure and litter quality.Rosen (1995) concluded from his review of 12,153feeding studies that antibiotic growth promoters gavea positive response 72% of the time. The magnitudeof responses was dependent upon the type of animalmanagement, disinfection procedures, age of the farmbuildings, and quality of the feed. Finally, the use ofantibiotic growth promoters has a positive impact ontwo important issues facing animal agriculture: animalwelfare and environmental stewardship. Animalwelfare is definitely improved in animals that are

58 Alternatives to antiobiotics in poultry production

healthier due to the disease-suppressing effects ofantibiotics. The improved utilization of dietarynutrients by supplemental antibiotics results insignificant reduction in nitrogen, phosphorus, andother nutrients excreted into the environment(Cromwell, 1999).

Antibiotic modes of action

Antibiotics are natural metabolites of fungi that inhibitthe growth of bacteria. They function by alteringcertain properties of bacterial cellular metabolismresulting in impaired growth or death. Someantibiotics interfere with the building and maintenanceof the cell wall, while others interrupt proper proteintranslation at the ribosomal level. Because of theirelevated rate of growth and proliferation, bacteriaare vulnerable to antibiotics that target active cellularmetabolism. Limiting the growth and proliferationof certain bacteria and inhibiting the production ofvarious toxins restricts the influence that the microbehas upon the host organism. This enables the host togrow and perform better than if grown under normalchallenge conditions.

The term ‘Growth Promoter’ has been used for yearsto describe the use of subtherapeutic levels ofantibiotics to improve growth performance. It is aninappropriate term to describe this use of antibioticsbecause they do not promote growth as do anabolichormones, such as growth hormone or estrogen-likecompounds. This may be why the general publicconfuses this term with the use of anabolic hormones.The poultry industry does not use anabolic hormonesas do the swine and cattle industries. Instead of callingthem ‘Growth Promoters’, they should be called‘Growth Permitters’ because they allow the animalto express its genetic potential for growth withoutcompromise.

Antibiotics limit the growth of detrimentalmicrobes, such as Clostridium perfringens (Truscottand Al-Sheikhly, 1977). They also limit the growthand colonization of numerous non-pathogenic speciesof bacteria in the gut, including lactobacilli,bifidobacteria, bacteroides, and enterococci (Tannock,1997). Antibiotics reduce the production ofantagonistic microbial metabolites, such as ammonia(Zimber and Visek, 1972), which adversely affectthe physiology of the host animal. Subtherapeuticlevels of antibiotics in the diet also reduce weightand length of the intestines (Visek, 1978; Postma etal., 1999). A thinner intestinal epithelium inantibiotic-fed animals may enhance nutrient

absorption (Visek, 1978) and reduce the metabolicdemands of the gastrointestinal system. Theminimization of gastrointestinal bacteria may alsoease the competition for vital nutrients between thebird and the microbes (Ferket, 1991). Finally,antibiotics may reduce the adverse effects ofimmunological stress on growth performance bylowering the enteric microbial load. Over-stimulationof the host immune system by the resident microfloracould impair the optimum growth and performanceof the bird (Cook, 2000; Klasing, 1988).

The antibiotic resistance debate

During the last 10 years, the use of growth promotingantibiotics has been criticized for their possible rolein the occurrence of antibiotic-resistant microbes.Numerous reports have been issued concerning theeffects of agriculture-related antibiotics on theemergence of antibiotic resistance in human pathogens(SCAN Report, 1999; DANMAP, 2000). Although acomplete ban on the use of subtherapeutic doses ofantibiotics in animal feed has not yet been enforcedin many countries, this day may eventually come.There is some evidence that the use of antibioticgrowth promoters in animal and poultry feeds isassociated with bacterial resistance in human diseasetherapy. Rapid selection for resistant bacteria whensubtherapeutic levels of antibiotics are fed occursbecause of the plethora of bacteria in the gut ofanimals, the high mutation rates among these bacteria,and the frequent transfer of genes including resistancegenes. Mathew et al. (2002) demonstrated thatselection for resistant bacteria can occur in as littleas two days following administration of a feed-basedantibiotic. Wide use of antibiotic growth promotersin poultry is one reason the public is placing someblame for antibiotic resistance of potential pathogenson the poultry industry. Gustafson and Bowen (1997)reported that antibiotic resistance of indigenous E.coli of poultry has remained at a relatively high levelsince the 1950s. However, Lou et al. (1995) reportedthat removing antibiotics from a swine herd for nowover 30 years has not eliminated antibiotic resistance.So, the question that needs to be answered is: Can aban on the use of AGPs reverse the trend in increasingantibiotic resistance of human pathogens? Thisquestion can be answered from recent experiencesafter the European ban on AGPs.

Following the ban of all food animal growthpromoting antibiotics by Sweden in 1986, the

P.R. Ferket 59

European Union banned avoparcin in 1997, andbacitracin, spiramycin, tylosin, and virginiamycin in1999. As a consequence of public pressure followingthe ban in 1999, the use of the two remainingantibiotics, bambermycin and avilamycin is scheduledto be banned in 2006. This ban is logical if theantibiotics banned from prophylactic usage werecommonly used in human medicine, but that is notthe case. Only bacitracin is routinely used in humanmedicine. In order to protect their position in theEuropean market, the Danish government instituteda voluntary ban on the use of AGPs along with apenalty tax for use in 1998. In effect, Denmark hasbecome an ideal laboratory to test the consequencesof a total European Ban on AGPs. By 2000, thecomplete ban on the use of AGPs was in effect inDenmark, but then enteric disease problems andmortality rates began to mount and the therapeuticuse of antibiotics began to rise sharply. By 2001, thetotal consumption of therapeutic antibiotics almostreached the same amount as the total consumption ofAGPs before the ban was instituted. In effect, AGPs(avilamycin, virginiamycin, bacitracin, and tylocin)that are not typically used to treat human disease werereplaced by therapeutic antibiotics (ampicillin,erythromycin, streptomycin, tetracycline, etc) thatare used to treat human disease pathogens (Hayesand Jensen, 2003). For example, tetracycline use inDenmark increased from 12,100 kg in 1998 to 27,000kg in 2001. Now Denmark has mounting tetracyclineresistance in human pathogens, such as Salmonellatyphimurium and Campylobactor jejuni (DANMAP,2001). Isn’t it ironic that the policy against the useof AGPs actually resulted in an increase in resistanceto antibiotics that the public is most concerned about?A simple ban on AGPs will not solve the antibioticresistance problem, but may lead to greater risks tohuman and animal health (Casewell et al., 2003).Therefore we need to strategically use different feedadditives and management practices that willminimize the use of both AGPs and therapeuticantibiotics.

General strategies to control gut healthwithout antibiotics

Effective use of feed additives to manage gut healthis dependent upon some degree of understanding oftheir mechanisms of action. Clearly, the modes ofaction of growth promoting antibiotics and theiralternatives can differ considerably. Subtherapeuticantibiotics work in part by decreasing the microbial

load in the gut, resulting in a reduction in energyand protein required to maintain and nourish theintestinal tissues. Because energy required to maintainthe gut accounts for about 25% of the total basalmetabolic needs of an animal (Croom et al., 2000),any reduction in gut tissue mass can have a significantimpact on the amount of energy available for growthand caloric conversion efficiency. The reducedmicrobial load in the gut by subtherapeutic levels ofantibiotics also reduces immunological stress,resulting in more nutrients partitioned toward growthand production rather than toward mechanisms ofdisease resistance. In contrast, most alternativecompounds do not reduce overall microbial loads inthe gut and thus will not promote growth by amechanism similar to antibiotics. Instead, they alterthe gut microflora profile by limiting the colonizationof unfavorable bacteria while promoting thefermentation of more favorable species. Consequently,alternatives to antibiotics promote gut health by severalpossible mechanisms including: altering gut pH,maintaining protective gut mucins, selection forbeneficial intestinal organisms or against pathogens,enhancing fermentation acids, enhancing nutrientuptake, and increasing the humoral immune response.Strategic use of these alternative compounds will helpoptimize growth provided they are used in a mannerthat complements their modes of action.

SANITATION AND PATHOGEN LOADREDUCTION

There is considerable evidence that subtherapeuticantibiotics or alternative compounds are mosteffective when fed to animals raised in unsanitaryenvironmental conditions. Good barn sanitation, pestcontrol, biosecurity practices, and litter or manuremanagement are necessary to reduce pathogen loadand exposure and minimize the need for antimicrobialtherapy. Water must be clean and drinkers must beproperly maintained to minimize spillage and preventa bloom of pathogens in the litter and environmentof the animals. Implementation of a good sanitationprogram is usually much less costly than any diseasetreatment.

ENHANCE PATHOGEN COLONIZATIONRESISTANCE

Colonization of enteric pathogens is dependent uponthe degree of resistance afforded by the stability of

60 Alternatives to antiobiotics in poultry production

the resident microflora and the integrity of theintestinal mucin barrier in the animal. Older animalsare much less susceptible to the colonization of entericpathogens than young animals because they have amore stable and diverse gut microflora thatcompetitively excludes pathogen colonization. Incontrast, the ability of pathogens to colonize in thegut increases after antibiotic administration becauseof a loss of resident microflora. The stability ofresident microflora can be enhanced by theadministration of competitive exclusion cultures(probiotics) or feeding prebiotic compounds that feedthe beneficial microflora. Hollister et al. (1999)reduced salmonella colonization in chicks by feedinga live cecal culture from salmonella-free poultry.Fedorka-Cray et al. (1999) have shown similarresponse to microbial cultures in young swine. Gram-positive bacteria, including Lactobacillus,Enterococcus, Pediococcus, Bacillus, andbifidobacteria, and fungi of the Saccharomyces (yeast)genus are often fed after antibiotic therapy as a meansof re-introducing a beneficial flora to the gut ofaffected animals. Beneficial bacteria inhibit thecolonization of pathogens by producing volatile fattyacids that reduce the pH of the brush-bordermicroenvironment or they can block the attachmentof pathogens. Organic acids have strong antibacterialeffects, especially to Gram-negative pathogens.Blomberg et al. (1993a) also demonstrated thatundefined compounds in a culture of lactobacilliinhibit the attachment to intestinal components of pigsby pathogenic K88 E. coli. They suggested thatcompounds produced by the lactobacilli or thelactobacilli themselves bound to the receptor of K88E. coli in pig intestine, thereby preventing thecolonization by the E. coli.

Mucins and glycoproteins associated with theintestinal brush border serve as a very importantbarrier protecting the delicate absorptive surface fromthe abrasive action of feedstuffs, bacteria colonization,and toxins. Mucin, produced by goblet cells, issecreted in response to the degree of insult on theabsorptive surface of the gut. Glycoproteins of gutmucins specifically bind pathogens and reduce theircolonization by serving as alternative binding sites toreceptors on host enterocytes. For example, pathogencE. coli K88 adhesins were found to bind to ileal mucusfrom pigs, and Blomberg et al. (1993b) concludedthat the intestinal mucus might intercept thesepathogens before they can attach to intestinal tissuesand cause disease. Dietary factors that result inincreased mucus secretion may thus indirectly enhancean animal’s ability to resist pathogen colonization.

There is a complex balance between the gut ecosystemand intestinal mucins, and this balance can be alteredby enteric health conditions and the diet. Althoughintestinal mucins and glycoproteins have a protectivefunction, they also serve as a nutritional substratefor some bacteria that thrive in a galactose-richenvironment, such as bifidobacteria (Roy et al.,1991). In pigs, Pestova et al. (2000) observed asignificant decrease in intestinal mucins followingweaning, and this was partially prevented by theinclusion of galactose in the post-weaning diet.Apparently, the lack of galactose in the post-weaninghigh starch diet increased the scavenging of galactosylunits in mucins by some microflora, thus promotingthe degradation of the protective mucin barrier.Dietary inclusion of compounds that feed beneficialbacteria, such as bifidobacteria, should alleviate theirattack on the protective mucins. Such compoundsinclude oligosaccharides or enzymes that liberategalactose from galactosyl polymers, such as galacto-mannans. More research must be done in this areaof interest.

IMMUNE RESPONSE AUGMENTATION

The immune system is the primary defensemechanism of the animal against infectious disease.Augmentation of humoral and cell-mediatedimmunity will increase an animal’s ability to resistdisease. Although there is a small nutrient cost inthe production of immunoglobulins, good antibodytiter levels indicate a far more efficient capacity toresist disease by humoral immune responses than anactive inflammatory response (Humphrey et al.,2002). A pro-inflammatory innate immune responseis associated with the mobilization of nutrients awayfrom growth and suppression of feed intake. Thus,dietary immunomodulators or vaccines that enhancehumoral immunity and minimize immunologicalstress will affect growth performance most positively.

Although there is now a considerable amount ofknowledge about systemic immunity, knowledgeabout gut-associated immunity is still primitive. Thegut is a major interface where the immune systemcan sample the potential disease antigens in theanimal’s environment and mount a defensive strategyto resist disease. Therefore, the resident microflorawill have a marked effect on the amount and profileof immune factors, such as immunoblobulins.Perdigon et al. (1991) observed that specificlactobacilli fed to mice resulted in enhancedprotection against S. typhimurium and E. coli by

86 Reproductive responses to Sel-Plex® organic selenium in broiler breeders

The objective of this trial was to characterizespecific effects of dietary selenium source on fertilityand embryo viability aspects in commercial broilerbreeder stocks. A female diet with no added seleniumwas used to identify the impact of dietary selenium.Inorganic and organic dietary selenium sources werecompared to demonstrate the impact of differencesin selenium accessibility and tissue storage onreproductive traits and embryo survival. Higher ratesof production, fertility, and ultimately chick quality,would decrease the number of birds required tomaintain current rates of production, as well as theoverall cost of production.

METHODS

Ross 508 pullets were reared in a light tight facilityfollowing the breeder BW profile (Aviagen Inc).From photostimulation (22 wks of age) pullets werefed a selenium-free laying ration (No added Se), astandard ration containing sodium selenite (0.3 mgSe/kg), or a ration containing selenium yeast (0.3mg Se/kg from Sel-Plex®). Thirty hens per treatmentwere inseminated weekly (from 30 wks) using pooledsemen from males fed a standard, sodium selenitediet or a diet containing the same amount of Se fromSel-Plex®. Individual egg production to 58 wk, eggweight, egg specific gravity, and BW were recorded.At 35 and 57 wk of age, eggs from 2 to 5 days afterinsemination were subjected to the perivitelline spermpenetration assay to measure the number of spermpenetrations near the germinal disk. Eggs wereincubated weekly and the hatch residue broken outto determine fertility, hatchability, and embryonicmortality.

OBSERVATIONS

Sperm management

Perivitelline sperm hole numbers of Sel-Plex® andselenite treatment eggs were similar. Both treatmentshad more sperm holes than eggs from unsupplementedhens by a factor of 2 to 3 (Table 3). Sel-Plex®

supplementation improved maintenance of spermnumbers between the day 2 and the day 5 sampling.By day 5, Sel-Plex® eggs still had an average of 60perivitelline sperm holes compared to 14 in controleggs, while selenite treatment eggs were intermediate(31 holes). These values represented a decline of 31%in apparent viable sperm population in Sel-Plex® birds

between Day 2 and 5 after insemination compared toa 46% and 48% drop within non-supplemented andselenite-fed birds, respectively.

The ability to maintain a viable sperm populationfor as long as possible reduces necessary frequencyof insemination. While selenium appears essential toallow the sperm into the oviduct, organic seleniumin Sel-Plex® may have an advantage over inorganicselenium in keeping the sperm population stable andalive. This is especially important as the hens ageand have a reduced sperm storage capacity at theuterovaginal junction (Goerzen et al., 1996).

Table 3. The effect of dietary selenium level and source onperivitelline sperm holes of eggs from broiler breeder hens.

Number of perivitelline sperm holes2 days1 3 days 5 days

No added Se 83b 46 14b

Selenite2 150a 58 31ab

Sel-Plex®2 119ab 61 60a

a,bMeans within a column with no common superscript differ (P<0.05).1Number of days after insemination.20.3 ppm Se

The males on the Sel-Plex® diet produced greatersemen volume early in production, with an averageof 0.36 ml/bird compared to 0.19 ml/bird in maleson the selenite diet (36 weeks of age). At 56 weeksof age, this difference was no longer significant, butremained at nearly the same magnitude. Thecomparison was complicated at the later ages due toseveral small males dropping out of semen productionpart way through the trial (selenite treatment). Testesof all birds are currently being examined for thepresence of functionally active sperm producing cells.

Egg production and egg quality traits

Birds on the Sel-Plex® diet entered egg productionslightly behind the other feeding treatments (non-significant difference), but caught up within a fewweeks. Early egg production to 29 wk of age wasnot different (Table 4). In fact, the rate of lay wassimilar through most of the production period.However, during the late lay period (49-58 weeks)the hen-housed rate of lay was 68% in Sel-Plex® birdscompared to 61% and 60% in the selenite and non-supplemented treatments, respectively. The Sel-Plex®

birds produced an extra 5 eggs/bird during thisperiod, on average. This is an important time to beproducing more eggs, as egg size is higher than in

R.A. Renema 87

young breeders, which results in a larger chick sizeand ultimately a greater broiler weight. Edens (2002)also indicated that the egg production of Sel-Plex®-fed hens initially lagged behind, but caught up andeven surpassed that of the selenite-fed hens after 5wk.

Ultimately the settable egg production in the dietarygroups was 168.5 (non-supplemented), 168.6(selenite), and 174.6 (Sel-Plex®) eggs/bird (Table 4).Overall, unsettable egg production ranged from3.49% in non-supplemented hens to 1.9% in Sel-Plex® hens and was not significantly different.However, during the late lay period (49-58 wk), theSel-Plex® hens produced significantly fewer unsettableeggs (0.9%) than non-supplemented hens (3.3%),while selenite hens were intermediate (1.7%). Eggweight and shell quality of settable eggs was assessedthroughout the trial and was unaffected. This meansthat if the hen laid a good egg, it also had a goodshell. However, diet affected how many eggs wereproduced with good shells, as shell defects were theprimary egg quality problem in unsettable eggs.Feeding Sel-Plex® organic selenium to laying hensat 80 wk of age has previously been shown to improveshell breaking strength (Paton et al., 2000a).

Interestingly, dietary selenium affected the changein shell weight as the hens aged. Between 36 and 56wk of age, shell weight increased by 0.55, 0.80, and0.76 g in eggs of the non-supplemented, selenite,and Sel-Plex®-fed hens, respectively. During this timeegg size also increased, meaning that the shell wasbeing stretched over more egg, and therefore makingup a smaller percentage of total egg weight. Theproportion of shell weight dropped by 0.84% of eggweight in non-supplemented hens, 0.80% in selenite-fed hens, and 0.57% in Sel-Plex® hens between 36and 56 wk of age. The Sel-Plex® hens weresignificantly less affected by age-related declines inthe proportion of egg shell than the non-supplementedhens. While egg specific gravity was not significantlyaffected, this may be an indicator of increased shell

thickness in the Sel-Plex® treatment (not tested). If thiswere different, there could be implications for incubationsuccess and for defense from contamination in the barn.

Hen body weight followed a similar patternthroughout the production period. However, the non-supplemented hens grew heavier than the othertreatment hens by 42 weeks of age. This differencecarried through to 58 weeks of age. This comparisonis somewhat artificial, as the body weight profile ofthe non-supplemented group was inflated by hensthat dropped out of lay at a fairly young age. Nutrientsthey were no longer allocating to egg production wentinto growth instead. By the end of the trial, 100% ofthe Sel-Plex® hens were still in active productionwhile only 87% and 90% of the non-supplementedand selenite treatment hens remained, respectively.Lack of production was due to either birds ceasingproduction, or to hen mortality (mortality limited tonon-supplemented treatment). Reduced mortality hasbeen linked to selenium supplementation (Arnold etal., 1974), particularly under stress conditions suchas an immune challenge (Edens, 2001). However,this does not explain the increased proportion of birdsstill in active lay at 58 weeks of age. Reproductionin the broiler breeder can be a fragile state and isoften the first thing to go when there is stress, ornutrients are insufficient. The fact that all Sel-Plex®

birds were still in production may relate to animproved efficiency of nutrient uptake. Presumablythe gut benefits from the improved protection fromcell membrane damage afforded by the organicselenium. During the late production period, thesebirds were producing more eggs than hens of theother treatments (Table 4) with no effect on theirbody weight relative to that of hens on the othertreatments.

Fertility, hatchability, and embryonic mortality

Prior to 34 weeks, hatchability averaged 88% in Sel-Plex® treatment eggs compared to 80% in selenite-fed

Table 4. The effect of dietary selenium level and source on egg production traits of broiler breeders.

Hen-housed egg production Total egg Settable egg Unsettable egg24-28 wk 29-38 wk 39-48 wk 49-58 wk production production production1

(%) (%) (%) (%) (No.) (No.) (% of total)

No added Se 48.9 88.9 75.6 60.2b 174.5 168.5 3.49Selenite2 49.7 88.1 73.1 60.1b 172.7 168.6 2.37Sel-Plex®2 46.4 87.7 75.5 67.7a 177.7 174.6 1.90

a,b Means within a column with no common superscript differ (P<0.05).1Includes double-yolked, soft-shelled, membranous, and abnormally-shelled eggs.20.3 ppm Se


birds and 77% in non-supplemented birds, and wassimilar in all treatments after peak production.Overall, fertility, hatchability, and hatch-of-fertileeggs demonstrated the beneficial nature of dietaryselenium, but did not differentiate between seleniumsources (Table 5). Fertility, for example, was 86.9%in non-supplemented hens compared to 90.1% in henson selenite and Sel-Plex® diets. Not including seleniumin the diet did not seriously harm hatchability, which isin contrast with work by Latshaw and Osman (1974)demonstrating a drop in hatchability to 18% in selenium-deficient hens. The current study may have providedmore naturally occurring selenium in the other feedingredients and the non-supplemented dietary treatmentwas not imposed until photostimulation (22 weeksof age).

Embryonic mortality can be a telling identifier ofspecific dietary or genetic effects. Problems with earlyembryonic mortality (1-14 days of incubation) canpoint to nutrient deficiencies. In this study, 5.33%of non-supplemented embryos died during this periodcompared to 3.72% (selenite) and 3.52% (Sel-Plex®)in the selenium-supplemented hens (Table 5). Whileselenium source did not make a difference here,clearly selenium supplementation was shown to beimportant. A beneficial effect of organic seleniumwas expected for the late incubation and hatch period,as this is the time of the greatest oxidative load forthe embryo, and when the protective antioxidanteffects of the Sel-Plex® may be most apparent.Variability among birds reduced the significance ofthis comparison, however, and late embryonicmortality, dead-in-shells, and hatchery culls totaled3.66%, 3.85%, and 3.14% of eggs set for the non-supplemented, selenite, and Sel-Plex® treatments,respectively (Table 5). Based on these numerical

differences, there appears to be a potentiallyprotective effect of Sel-Plex® compared to inorganicselenium in the diet.

Examining this relationship more closely revealedan interesting trend over time. Late embryonicmortality of all treatments was similar at the start ofthe trial, when all birds were still on a fairly highplane of nutrition. As the hens aged, late embryonicmortality stayed almost constant in non-supplementedand selenite hens, while it decreased in Sel-Plex® hens(Figure 1). As feed allocations were reduced withage, the micronutrients would have been in shortersupply. The improved efficiency of selenium uptakein the Sel-Plex® diet may not have made a substantialdifference on hatchability until a nutrient challengewas faced by the flock. This fits with observationsthat Sel-Plex® can demonstrate benefits in stressfulsituations. Heat stress and long-term egg storage areexamples of stress situations where Sel-Plex® has beenshown to help. Surai and Dvorska (2001) indicatethat there are numerous on-farm stress conditions thatcould be alleviated in part by organic seleniumsupplementation.

Ultimately what determines the success of a broilerbreeder management program is chick production.In this trial, chick production was calculated fromthe hatchability of settable eggs. The unsupplementedhens produced an average of 131.3 chicks/hen-housedby 58 weeks of age, while selenite hens produced139.1 chicks/hen, and Sel-Plex® hens produced 145.3chicks/hen (Table 5). Between the selenite and Sel-Plex® selenium source diets, the numerical differencesin settable eggs, embryonic mortality, hatchability,and hatch of fertile culminated in a difference of 5.8chicks in favor of the Sel-Plex® hens. This differenceincreased to 14.1 chicks when compared to the non-supplemented hens.

Table 5. The effect of dietary selenium level and source on fertility, hatchability, embryo mortality and chick production traits of broilerbreeder females.

Embryo mortality and culls Hatch of Chick

Infertile Day1-14 Day 15-hatch1 Fertility Hatchability fertile2 production3

(%) (%) (%) (%) (%) (%) (No.)

No added Se 13.06a 5.33a 3.66 86.9b 77.9 88.6b 131.3b

Selenite4 9.91b 3.72b 3.85 90.1a 82.5 91.5a 139.1ab

Sel-Plex®4 9.87b 3.52b 3.14 90.1a 83.5 92.5a 145.3a

a,b Means within a column with no common superscript differ (P<0.05).1Includes embryo mortality to hatch, dead-in-shell, and hatchery culls.2Hatchability calculated only from fertile eggs set (infertile eggs excluded).3Chick production = hatchability X settable eggs.40.3 ppm Se.

R.A. Renema 89

SUMMARY: STUDY 2

Reproductive traits were improved with the inclusionof dietary selenium, while Sel-Plex® supplementationalso improved sperm survival in the oviduct, as wellas settable egg production late in lay through increasedegg production and reduced shell defects. Ultimately,chick production was improved in the Sel-Plex®

treatment through more successful settable eggproduction and the additive culmination of numericalimprovements in hatchability and embryo viabilitymeasurements. Selenium is essential in the diet for asuccessful reproductive effort. Additional benefits ofusing the Sel-Plex® are also possible. Selenium sourceappears to influence the hen’s contribution to thefertility of the breeder flock and to beneficially affectsemen volume early in production.

Conclusions

Managing the broiler breeder female for optimal chickproduction requires an understanding of reproductivephysiology, nutrition, and their interaction. Besidesa thorough knowledge of everyday management,there must also be an awareness of feed ingredientsand their interactions both with each other and withenvironmental effects. Whereas the basic compositionof the egg is fairly constant, diet and specific feedingredients can affect what and how much of some

of the minor ingredients make it into the egg andultimately the embryo. Specialized feed ingredientsare available that behave differently than traditionalingredients and can enhance egg and chick qualityunder the right conditions. Together these factors canbe used to enhance embryo survival.

References

Arnold, R.L., O.E. Olsen and C.W. Carlson. 1974.Tissue selenium content and serum tocopherols asinfluenced by dietary type, selenium and vitaminE. Poult. Sci. 53:2185-2192.

Bakst, M.R., G. Wishart and J.P. Brillard. 1994.Oviduct sperm selection, transport, and storage inpoultry. Poult. Sci. Rev. 5:117-143.

Bilgili, S.F. and J.A. Renden. 1985. Relationship ofbody fat to fertility in broiler breeder hens. Poult.Sci. 64: 1394-1396.

Bramwell, R.K., H.L. Marks and B. Howarth, 1995.Quantitative determination of spermatozoapenetration of the perivitelline layer of the hen’sovum as assessed on oviposited eggs. Poult. Sci.74:1875-1883.

Burk, R.F. 1989. Recent developments in traceelement metabolism and function: newer roles ofselenium in nutrition. J. Nutr. 119:1051-1054.

Cantor, A.H. 1997. The role of selenium in poultry

0

1

2

3

4

5

6

30 35 40 45 50 55 60

Age (weeks)

Mor

talit

y (%

)

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Figure 1. Linear representation of trends in embryonic mortality between 15 days of incubation and hatch (including dead-in-shelland hatchery culls) of broiler breeder hens fed diets containing no supplemental Se, 0.3 ppm inorganic Se, or 0.3 ppm Sel-Plex®.


nutrition. In: Biotechnology in the Feed Industry,Proceedings of Alltech’s 13th Annual Symposium(T.P. Lyons and K.A. Jacques, eds). NottinghamUniversity Press, Nottingham, UK, pp. 155-164.

Edens, F.W. 2001. Involvement of Sel-Plex inphysiological stability and performance of broilerchickens. In: Biotechnology in the Feed Industry,Proceedings of Alltech’s 17th Annual Symposium(K.A. Jacques and T.P. Lyons, eds). NottinghamUniversity Press, UK, pp. 349-376.

Edens, F.W. 2002. Practical applications forselenomethionine: broiler breeder reproduction. In:Nutritional Biochemistry in the Feed and FoodIndustries, Proceedings of Alltech’s 18th AnnualSymposium (K.A. Jacques and T.P. Lyons, eds).Nottingham University Press, UK, pp. 29-42.

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Fasenko, G.M., R.T. Hardin, F.E. Robinson and J.L.Wilson. 1992. Relationship between hen age andegg sequence position with fertility, viability andpreincubation embryonic development in broilerbreeders. Poult. Sci. 71:1374-1383.

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Gaal, T., M. Mezes, R.C. Noble, J. Dixon and B.K.Speak. 1995. Development of antioxidant capacityin tissues of the chick embryo. Comp. Biochem.Physiol. 112B:711-716.

Goerzen, P.R., W.L. Julsrud and F.E. Robinson. 1996.Duration of fertility in ad libitum and feed-restricted caged broiler breeders. Poult. Sci. 75:962-965.

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R.A. Renema 91

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G.D. Rosen 93

Optimizing the replacement of pronutrient antibiotics in poultry nutrition

GORDON D. ROSEN

Pronutrient Services Ltd., Wimbledon, London, United Kingdom

Introduction

Following recent reviews on the setting and themeeting of standards for the efficient replacement ofpronutrient antibiotics in pig and poultry nutrition(Rosen, 2003b; 2003c), this review concentratesattention on ways and means of improving andoptimizing the use of pronutrient antibiotics in broilerand turkey feeds, with particular reference to themultiplicity of proffered candidates and to themultiplexity and interactivity of influential genetic,environmental, managemental and dietary variables.

Why replace pronutrient antibiotics?

Contentiously but concretely, the search for andvalidation of antibiotic replacements stems from theattitudes and demands of consumers and their retailsuppliers and from legislation based on precautionaryprinciples. Soundly-based estimates of the effects onproduction costs and on retail prices are sparse. Butthe number of products offered as alternatives andthe volume of literature thereon have risen steeplysince 1999. Many hundreds, possibly thousands, ofproducts are on offer. One can envisage several years’work ahead to sort the wheat from the chaff.

Irrespective of questions of right or wrong in theproscription of prescription-free usage of antimicrobialsin food production, the need to deploy replacements isexpanding rapidly, creating a formidable task forscientists and producers in their search for fully-effectivealternatives. The potential value of feed antibiotics wasfirst demonstrated in the US by Moore et al. (1946)before commercialization in 1949. Potential problemscaused by bacterial resistance phenomena were earlysources of concern in human medicine, veterinary

medicine and farm animal nutrition. In the early1950s, opponents of antibiotic routines in poultryfeeds initiated resistance research. Concerns wereheightened by the Japanese discovery in 1959-1960of infectious or transferable (as against natural)antibiotic resistance involving natural selection,mutation and DNA fragment transfer. The SwannCommittee (1969) reported on the use of antibioticsin animal husbandry and veterinary medicine andinitiated legislative restrictions banning the use ofpenicillin and tetracyclines without veterinaryprescription. The European Economic Communityfollowed this lead. The US Food and DrugAdministration did not. In 1998, the European Unioncancelled its approvals of six feed antibiotics, withintent to ban the remainder not later than 31December, 2005.

Which are the candidates?

Table 1 contains a short list of the seven maincategories of antibiotic replacement candidates.Whatever their nature or chemical composition, andno matter how numerous and multifarious are theirknown or hypothesized modes of action, the commonthread in the present context resides in their abilitiesto enhance poultry performance at least as efficientlyas the replaced pronutrient antibiotics in terms offeed conversion efficiency, mortality, liveweight,animal product yield and environmental depollution,measured integrally as net return on investment. Theplethora of proffered candidates is evidenced, by wayof example, in the 2000-01 Direct-Fed Microbial,Enzyme and Forage Additives Compendium (Miller

94 Optimizing the replacement of pronutrient antibiotics in poultry nutrition

Publishing Co.), by its lists of 222 candidate products,90 enzymes, 72 microbials, 40 yeasts, 13 moulds andacids and seven oligosaccharides for use in poultryproduction alone.

Table 1. Major antibiotic replacements.

AcidsAnticoccidials

BotanicalsEnzymesNutrients

MicrobialsOligosaccharides

Nutrients are also included because supplementaryamino acids, minerals, purified energy sources andvitamins overlap performance-wise (Rosen, 1997) andmay well overlap with pronutrients in nutritionalimprovements, irrespective of differences in modesof action.

Is nomenclature satisfactory?

More meaningful, standardized, transparentterminology in this field would benefit scientists,legislators and, above all, consumers. Feed additiveantibiotics have been variously named as growthpromoters, growth permitters, performancepromoters, performance enhancers, production aids,feed economizers, nutrient balancers, efficiencyenhancers, digestive enhancers and nutritionimprovers, which descriptors have often been regardedin several senses as inaccurate, incomplete orinappropriate. Notwithstanding long-term, near-universal usage, the word ‘additive’ is nonethelessill-conceived, being insufficiently descriptive. Inpractice the term ‘additive’ can generate auras ofminority, subsidiarity, afterthought, optional extraand contaminant. It may be suitable for fuels, butnot for food or feed; but it is doubtful whetherlegislators would ever countenance a change.

The term ‘pronutrient’ has been introduced toreplace additive. A pronutrient is defined as asubstance that improves the value of nutrients. Asanticipated, a survey of 100 consumers has confirmedit as unknown to them, but 85 interpreted it as‘something good for nutrition’. Therefore it issuggested that pronutrient could usefully replace theterm “non nutrient additive” used by the NationalResearch Council to distinguish an additive from anutrient. However, “non nutrient additive” is

unsatisfactory because it fails to convey the nutritionalbenefits of additives, which can be tantamount tothose of nutrients.

The role of a pronutrient is portrayed in the schemain Figure 1. This shows the functional relationshipof a pronutrient in nutrition relative to that of anantinutrient.

NUTRIENT MIX

Antinutrient

Pronutrient

Better performance

Worse performance

Figure 1. Effects of pronutrients and antinutrients on diets.

Pronutrients function via a hundred or more differentmodes of action in extending the value of the limitingnutrient in a diet. There is thus an overlap betweenthe provision of a pronutrient and a ‘topping-up’ ofa limiting nutrient. Hence, nutrients are, asaforementioned, included as a Table 1 category forconsideration herein. The choice of a pronutrient ora nutrient in replacing feed antibiotics is simply aquestion of relative cost-effectiveness.

The terms ‘probiotic’ and ‘prebiotic’ are bothplagiarisms, appropriated by scientists and suppliers.The US Food and Drug Administration and theEuropean Union Commission, as regulatoryauthorities, both refused them as vagaries, optingrespectively for direct-fed ‘microbial’ and‘microorganism’. The term probiotic was, as a matterof fact, first coined by Winter (1955) in his researchon botanical antibiotics found in cruciferous plants,when he stated that ‘we can call these substancesprobiotics; they are antibiotic against pathogenicmicrobes and they are therefore probiotic for theinfected organism’. Gibson and Roberfroid (1994)took the ‘prebiotic concept’ from its original andliterally-correct meaning and long-standing usage todefine a chemical involved in the origin of life.Prebiotic is incorrect as an appellation for a

G.D. Rosen 95

biosynthesized molecule, which is biofunctional,which nourishes a live microorganism and whichinhabits a live host, in no sense whatsoever, before life.

The adoption of simple, realistic duplex descriptorsto simultaneously impart nature and function couldbe a useful forward step in this field. For example,we could specify (a) pronutrient cellulase, formate,B. cereus, capsicum, etc.; (b) prophylactic narasin,nifursol, P. acidilactici, mushroom polysaccharide,etc., (c) therapeutic tylosin phosphate, lincomycinhydrochloride, penicillin, trimethoprim, etc. and (d)pro-environmental phytase, carbohydrase, protease,etc. Furthermore, it would be advantageous todiscontinue reference to ‘antibiotic alternatives’ or‘antibiotic substitutes’, thereby avoiding a rekindlingof consumers’ scientifically-unproven, anti-antibioticprejudices.

How are antibiotic replacementscompared?

All available properly-controlled test data need to betaken into account. The use of a handful of tests canillustrate the potential of a product as a starting point,but five tests cannot take account of the wide rangeof genetic, environmental, managemental and dietaryfactors affecting response in praxis. A recent survey

on the current status and future needs of replacementsbased on the view of 50 suppliers, users, consultants,educators, communicators and academics revealed alarge number of problems met in comparing candidateefficacies (Rosen, 2003c). Of the 92 nominated, themain problem areas are variation in response, use ofuncontrolled tests, inadequate test designs, missingfeed compositions, invalid ‘field’ tests and unjustifieddosage recommendations.

Serious shortcomings in commonly-used averagingprocedures are illustrated in Table 2. For example,the comparison of the effects on feed conversion ratio(FCReff) of enzymes and antibiotics, as such or inpercentage terms, cannot be meaningful due to their11-day mean duration difference. It is known thatFCReff diminishes through starter to finisher phases.The same applies to time span (year) differences,averaging 1972 and 1987. The coefficients ofvariation in response of 129-1,449% constitute awarning against comparisons based on small testnumbers.

For some researchers, direct comparisons withintests are fundamental, but they are, more often thannot, too expensive. They require much larger, morecostly experiments to manifest statistically significantdifferences between pairs of candidates, whichdifferences are normally much smaller than thosebetween candidates and negative controls.

Table 2. Superficial comparison of the effects of antibiotics, enzymes and microbials (FDIeff, LWGeff, FCReff and MORTeff) on control feedintake (FDIC), control liveweight gain (LWGC), control feed conversion ratio (FCRC) and control mortality (MORTC) with their coeffi-cients of variation (CV) in broiler nutrition.

Parameter Antibiotics Enzymes Microbials

n 5,159/1,5211 2,557/439* 234/57*FDIC, g 2,478 2,106 2,636FDIeff, g 15.0 (0.6%) 32.4 (1.5%) 6.0 (0.2%)CV, % 970 451 1,449

LWGC, g 1,075 1,043 1,331LWGeff, g 39.8 (3.7%) 54.3 (5.2%) 25.3 (1.9%)CV, % 129 147 192

FCRC 2.16 1.99 1.87FCReff -0.073 (-3.4%) -0.105 (-5.3%) -0.030 (-1.6%)CV, % 164 185 195

MORTC, % 5.77 6.53 3.76MORTeff, % -1.40 (-24%) -1.71 (-26%) -0.40 (-11%)CV, % 535 377 360

DUR*, days 41.0 30.3 35.8YEAR – 1900 71.6 87.0 86.6Improvement frequency2, % 74 75 70

1Mortality.2Percentage of tests with improved FCReff and LWGeff.*DUR = duration of test.


Comparative tests versus a positive control alone arecontraindicated because they provide no evidence ofa beneficial response to either candidate. Table 2 alsostarkly indicates a crucial gap in the large majority oftests, which fail to report mortality, viz. 71%, 83%and 76%, respectively for antibiotics, enzymes andmicrobials. Interestingly, the response improvementfrequencies for these three candidate categories arevirtually equal in the range 70-75%. Frequencies ofbeneficial response and magnitudes of variationtherein merit greater attention than hitherto.

How should we determine efficacy?

In the light of the aforementioned shortcomings offew tests and superficial averaging, the answer to thisquestion resides in the formulation of optimalmultifactorial empirical algebraic models fornutritional effects of each candidate. Such models areused to calculate a requirement for any given set ofcircumstances and conditions, in order to compare anantibiotic and a candidate or to compare two or morecandidates. Essentially, all published data are accepted.Manifestly-uncontrolled tests are excluded. Computerfiling of all relevant data for the hundred or morepotentially-important variables includes routines forelimination of errors in the original reports or in dataabstraction and repeats. Performance must bemeasured from start to finish. If second or later phasedata are required they are obtained, e.g., bysubtracting 0-21 day from 0-42 day values. Standardstatistical packages, e.g. Nie et al. (1975) and multipleregression methodology, e.g. Draper and Smith (1981)are used to determine best-fit models.

Best-fit algebraic models have statistically significantregressions, maximum multiple correlation coefficient

squares, minimum standard deviations aboutregression and significant partial regressioncoefficients for all independent variables. Afterexclusion of aberrant (>3 x SD) responses, theemergent models are used to estimate nutritionalresponses and 95% confidence limits for any requiredset of values of the component independent variables.Differences in these predicted responses are testedfor statistical significance. Nutritional responseestimates are then used to compute and compare netprofits to target liveweight and/or target duration.

These procedures are then used in feed formulationto quantify specific requirements for pronutrientsor nutrient counterparts for target production. Thusone can assess whether a nutrient, e.g. L-methionine,would be a better choice than a pronutrient, e.g.endoxylanase or Bacillus subtilis. In other words,should one top up a limiting nutrient per se or increasethe amount of the dietary limiting nutrient?

Which are key variables?

The 48 factors listed in Table 3 exemplify the rangeof variables potentially relevant in the elaborationof working models. There are also subsets of thesefactors to be considered. Routinely dose is consideredas linear, quadratic, logarithmic or exponential. Forbroilers there are four sex types: male, female, mixed(50/50) and as-hatched. Ten to 20 antibiotics andfive to 10 anticoccidials may be relevant, alone orin admixtures. Different disease challenges can beidentified. The data emanate from more than 100countries, though 10 or so usually furnish most ofthe test data. A few individual brands have numbersworthy of test. Types of oils and fats, animal protein,and vegetable proteins, including admixtures, total

Table 3. Independent genetic, environmental, management, dietary and nutrient variables used in the elaboration of multifactorialnutritional models.

Control performance Feed process Maize2 Gross energy3

Duration Antibiotic Sorghum Net energy3

Year of test Anticoccidial Wheat Crude proteinDose Antihistomonial Barley Crude fatInitial age Metabolic test Oats Crude fibreNot day-old Diet marker Rye CalciumSex Part-purified diet Animal fat PhosphorusPhased dose Disease challenge Vegetable oil LysineFactor 2 dose1 Favson Animal protein MethionineSelected birds Institute test Vegetable protein Methionine + cystineHousing Country Wheat offal ThreonineStocking density Brand Rice bran Tryptophan

1Second antibiotic/enzyme/acid/microbial/other pronutrient/nutrient2Dietary concentrations (columns 3 and 4)3Digestible or metabolizable energy are alternatives.

G.D. Rosen 97

more than 200. For example, in Brozyme (Rosen,2000; 2002) there are, including mixtures of eachtype, 71 animal proteins, 15 vegetable proteins, 81oils and fats and 25 carbohydrate sources.

Based on experience to date, level of negativecontrol performance, duration of feeding and yearof test are basic, accounting for 10-50% of variationin effects. Contrastingly, some highly statisticallysignificant factors can account for 1% or less.Interactions as product terms are normally relativelysmall contributors. In models containing 10 or moresignificant variables, dosage tends to be lessimportant, often accounting for 7.5% or less of totalvariation.

Some independent variables are vital for theassessment of praxis values, compared with researchconditions, namely, feed form, use of not-day-olds,males, cages, part-purified diets and specific diseasechallenges.

If the treated value is used as the dependent variablein modelling instead of the effect, multiplecorrelation coefficient squares (R2) are grossly inflatedby the high correlation of control values and effects,e.g. for virginiamycin models (n=306), LWGeff andFCReff R2 values of 0.14 and 0.44 respectively,become 0.97 for LWG and 0.99 for FCR as treateddependent variables. Such inflation of R2 is misleadingin variation accountancy and for some it harbours adelusion that R2 less than 0.4-0.6 is unworthy.

Incomplete test reports limit the accountancy ofvariation. Recent data on phytase responses in broilersoffer a useful perspective in which models containing

20 significant independent variables account for 64-72% of the sizeable variations in nutritional responseswith time and place (Rosen, 2003a). Further progress,however, is unlikely until editors of peer-reviewedjournals take a lead, for others to follow, in ensuringroutine publication of fundamental variables such astemperature, altitude, lighting pattern, disease statusand mortalities.

As examples, Figure 2 contains basal models for agroup of five important broiler antibiotics in orderto illustrate the magnitudes of the effects of keyvariables, with algebraic signs in accordance withnutrition science and practice.

These models contain 19 significant independentvariables. For LWGeff and FCReff, negativecoefficients quantify inferior and superior respectiveresponse contributions, with increase in LWGC andFCRC. Their positive DURs afford better and worseeffects respectively with age increase. Diagnosed orendemic disease enhances LWGeff and FCReff. Thepartial regression coefficients of LWGC and FCRCmean that each 100 g better control performancewould reduce LWGeff by 1.1 g and that each 10 pointlower value for FCRC would reduce FCReff by1.6 points.

How many tests are needed for a workingmodel?

Hitherto, the notional n = c. 50 controlled tests hasbeen thought of as a minimum required to produce a

FDIeff = -79.5 -0.0380FDIC + 2.98DUR +1.02EXDAT -24.3COCR2 .052 se 38.7 0.006 0.507 0.465 7.07SD 126 p 0.029 0.000 0.000 0.028 0.001n 1709

LWGeff = - 72.4 - 0.0114LWGC +++++1.02DUR + 0.871EXDAT +19.5log(AB+1) -16.8COC +76.1VETR2 0.171 se 15.0 0.004 0.153 0.183 3.22 2.70 5.56SD 47.4 p 0.000 0.009 0.000 0.000 0.000 0.0000 0.000n 1709

FCReff = 0.301 - 0.161FCRC + 0.00306DUR - 0.00159EXDAT - 0.0286log(AB+1) - 0.116VETR2 0.287 se 0.029 0.007 0.000 0.000 0.007 0.012SD 0.100 p 0.000 0.000 0.000 0.000 0.000 0.000n 1709

MORTeff = - 1.13 - 0.647MORTC + 0.0363DUR + 1.08log(AB+1) - 2.12VETR2 0.685 se 0.805 0.021 0.012 0.336 0.475SD 3.12 p 0.000 0.000 0.003 0.000 0.000n 708

Figure 2. Antibiotic models relating feed intake, liveweight gain, feed conversion ratio and mortality effects to level of controlperformance, duration, date, dosage, anticoccidial use and disease status.


useful model to quantify the magnitudes of nutritionalresponses in feed formulation. A recent projectaddressed the question of a minimum platform viarandom fragmentation of a large 1,709/mortality 708test resource of five of the most important feedantibiotics (i.e. two bacitracins, two tetracyclines andvirginiamycin) into smaller subsets down to 34-43for the elaboration of progressively smaller-basedmodels (Rosen, 2004). The parent models are thosein Figure 2. The analysis of a total of 704 fragmentedresource models revealed that (a) no model at allwas afforded in 23% of the random fragments; (b)progressive subset fractionation of the data set sharplyreduced the number of significant variables from 79%in the parent models to 17% in the smallest, whichaveraged 1.2 significant independent variables; (c)the chance of three or more significant variables insubset models was one in eight; and (d) the smallestsets, averaging n = 38/mortality 15, did in toto revealall the significant variables, albeit very patchily,found in the parent models.

How are requirement models used?

Requirements are needed for each and every set ofcircumstances for a defined target objective, fornutrients per se or for pronutrients. In other terms,what response might we expect within whatconfidence limits for a given dosage of supplementarymethionine compared with an optimal dosage ofbacitracin, methylene disalicylate, or 6-phytase or amannan oligosaccharide, etc.? The conjoint (overlap)consideration of the value of a limiting nutrientsupplement or the improved efficiency of a limitednutrient supply with a pronutrient is of particularinterest for the comparison of multifunctionalsubstances. This approach could well resolve theongoing, decades-old dispute on the relativities ofDL-methionine (DLM) and methionine hydroxy-analogues (HMTBA and CaHMTB). The use ofrequirement models based on all-available, unselectedcontrol test data for DLM, HMTBA and CaHMTBwith dose expressed simply as product weight (notmolar or other equivalencies in malnutrition tests)should be decisive. Such methodology is imperativebecause DLM is a mixture of a nutrient and aprenutrient and HMTBA and CaHMTB are bifunctionalas prenutrient and pronutrient.

The mode of application and value of multifactorialmodels for antibiotics and their replacements can nextbe illustrated in four examples as follows.

Using zinc bacitracin models based on 1,164 testsand first-generation phytase models (n=296), Table4 shows that zinc bacitracin at 80 ppm affords a fivepoint conversion improvement compared with the iso-cost level of phytase with no effect. In addition, ahuge increase in phytase dosage (x 20) gives a threepoint conversion improvement, for which 42 ppm ofa zinc bacitracin would suffice.

Table 4. Iso-input cost and iso-feed conversion effect comparisonsof zinc bacitracin (n=1164) and first-generation phytases (n=296)for as-hatched 56 day-old broilers of LWGC=3,266g andFCRC=2.079.

Pronutrient Dosage Basis LWGeff FCReff

Zinc bacitracin (ppm) 80 iso-cost 59 -0.05442 iso-FCReff 47 -0.032

Phytase (u/kg) 500 iso-cost -1 0.00110,000 iso-FCReff 159 -0.032

Secondly, Table 5 provides a model-based perspectiveon the results of a test from 7 to 28 days of age onfemale broiler chicks, which compared a Chineseherbal preparation, virginiamycin and a negativecontrol (Guo et al., 2000). The herbal at 938 ppmafforded an FCReff four points inferior tovirginiamycin at 20 ppm, even though the lattermanifested, in this test, a response three points belowits predicted result.

Table 5. Application of LWGeff and FCReff models to assess theresults of a comparative test on a Chinese herbal formulation vsvirginiamycin for LWGC=1,098 g and FCRC=1.554 on 7-28 day-old female birds.

Product Dosage Basis LWGeff FCReff(ppm) (g)

Chinese herbal 938 tested +15 +0.012Virginiamycin 20 tested -6 -0.027

20 predicted* +3 -0.056

*306-test requirement models

The import and value of a negative control is furtherevidenced in a 39-day broiler test by Messikommeron 50 ppm zinc bacitracin versus a rhubarbsupplement (Wenk, 2000). The predicted zincbacitracin response of -0.074 ± 0.026 illustrates thatthe observed value of -0.049 is as expected; and thatthe botanical has potential value at 2,500 ppm(-0.038), but not at 5,000 ppm (+0.018).

Thirdly, truncated comparative tests without anegative control are preferred by some researchers,even though they are unable to confirm efficacy foreither test product. An assessment of the results of apositive control only test can be made using a model

G.D. Rosen 99

for the test antibiotic. Syrvidis et al. (2003) reporteda test on Digestarom 1317 Poultry Premium (a so-called phytobiotic) versus Flavomycin-80, but failedto include test dosages. Hence, a notional negativecontrol performance of LWGC = 2,125 g and FCRC= 1.970 was calculated from flavomycin models(n=394) for a presumed dosage of 2 ppm. Thecomputed negative control values reveal very largeDigestarom responses of LWGeff = 326 g (15.3%)and FCReff = -0.220 (-11.2%). At today’s productionstandards, however, a feed conversion as-hatched fora 40-day 2,125 LWGC would be about 1.730, i.e.,12.2% less than the computed FCRC of 1.97 at 42days old in this trial. Such a result against a positivecontrol alone should be treated with reserve. Theavailability of further tests against negative controlswould afford a better view on the potential ofDigestarom. The same applies to any product if itclaims value from one or more tests solely against apositive control(s).

Fourthly, even prior to the availability of workingmodels for individual product brands of replacements,test data collections of the latter can be reviewed asto their potential value as antibiotic replacements.Bio-Mos® mannan oligosaccharide collections of 34tests for broilers (Hooge, 2003a) and 27 tests forturkeys (Hooge, 2003b) are used to provide anexample. Table 6 compares mean liveweight gain andfeed conversion responses in broilers and turkeys withcorresponding predictions for equivalentcircumstances, using comprehensive multifactorialmodels for the antibiotic products virginiamycin(n=306) in broilers and zinc bacitracin (n=226) inturkeys.

Table 6. Comparison of mean LWGeff and FCReff in 34 mannanoligosaccharide (Bio-Mos®) broiler tests with predicted responsesfor virginiamycin for LWGC=2,149 g, FCRC=1.879 at 42.2 days-old and in 27 turkey tests with predicted responses for zincbacitracin for LWGC=5,643 g, FCRC=1.981 at 68.7 days.

Species/ Dosage Basis LWGeff FCReffproduct (g)

Broilers Bio-Mos® 1.04 g/kg 34 test mean +40 -0.042 Virginiamycin 20 ppm 305 test model +21 -0.040Turkey Bio-Mos® 1.19 g/kg 27 test mean +127 -0.032 Zn bacitracin 50 ppm 236 test model +148 -0.041

The Table 6 data are encouraging for Bio-Mos® as areplacement when used at a dosage of 1 g/kg in broilerand turkey feed. More searching comparisons,however, must now await the availability of

requirement models for Bio-Mos®, quantifying theinfluences of dosage, level of bird performance,presence or absence of other pronutrients, especiallyanticoccidials, bird sex, feed form and limitingnutrients.

By virtue of extensive 15-year test programmes,exogenous enzymes would appear at present to bethe best characterized replacement category. The dataavailable for organic acids in pigs, microbials andoligosaccharides in poultry and pigs may alreadysuffice for the elaboration of initial working models.But all available data should be used to avoid possibleselection bias, as in the organic acid studies of Partanenand Mroz (1999) and Partanen (2001).

How should we test admixtures?

Problems in the use of admixtures of antibioticreplacements arise from shortcomings innomenclature and posology (dosage science).‘Additivity’ and ‘synergism’ are often misused,usually for sub-additivity. The possibility ofantagonism should always be borne in mind. Forpurposes of definition, the possible effects ofadmixture are classified herein as sub-additive,additive, synergistic, ineffective or antagonistic, asdefined for the admixture of 2+3 of A and Bproviding 4, 5, 6, 2 or 3 and 1 unit(s) of responserespectively. A 2 x 2 factorial test is a good startingpoint, shown in Table 7, which also includes otheriso-cost admixtures for A or B alone or for highersingle dosages of A or B. The acid test for maximalresults uses A+B each at its economic optimum.

Table 7. Scope of admixture tests.

Treatment Feed Investmentconcentration cost

(ppm) (MU*)

Basal feed 0 0Replacement A a 4Replacement B b 8Replacement A + Replacement B a + b 12Replacement A 3a 12Replacement B 1.5b 12Replacement A + Replacement B 0.33a + 0.33b 4Replacement A + Replacement B 0.67a + 0.67b 8

*MU money units

Nutritional models can provide a useful guide towardsmaximum admixture efficiency by pre-determinationof the economic optimal doses of Replacements A


and B. These admixture guidelines are also pertinentin situations where pronutrient antibiotics can stillbe utilized in admixture with nutrients or pronutrientsupplements.

Quo vadimus?

The thesis presented herein essentially advocates thatwe should optimize the choice and dosage of nutrientor pronutrient antibiotic replacements by takingcognizance of all available data expressed inpredictive, empirically-based, multifactorial multipleregression requirement models for feed, gain,conversion and mortality effects, at least. Such modelsshould be updated annually to take account of theaccelerating current flow of scientifically-controlledfeeding tests, as seen for example, in the increase ofexogenous enzyme publications from 1,422 up to theyear 2000 rising to its latest content in the Brozymeresource (Rosen, 2002) of 2,175. It is intended alsoto extend these studies to table egg and breeder hensand at least ducks among the minor species.

Following the lead of this Symposium in ‘re-imagining the feed industry’, should we not also pruneand improve its verbiage to better its science andraise its transparency to consumers, setting a goodexample for all members of the food chain?

In conclusion, it may be apposite, in line with thequestion and answer format of this review, toconclude with a Seven Question Test with which theuser of an antibiotic replacement can assess thepotential value of a supplier’s Product X.

1) How many properly-controlled feeding tests doyou have on the efficacy of Product X?

2) How many of these have no negative controls?

3) Can you supply a bibliography for 1) and 2)?

4) How many times out of ten does Product Ximprove liveweight gain and feed conversion?

5) What are the coefficients of variation in the gainand conversion responses?

6) What dosage of Product X will maximize returnon my investment and why?

7) Can you supply me with a model to predictresponses to Product X under my conditions?

Receipt of answers of 1) 30; 2) five; 3) yes; 4) seven;5) 100-200%; and 6) “x ppm because . . .” should beencouraging. A ‘yes’ to 7) would be even better.

References

Draper, N.R. and H. Smith. 1981. Applied RegressionAnalysis, 2nd Edition. John Wylie and Sons, Inc.,New York.

Gibson, G.R. and M.B. Roberfroid. 1994. Dietarymodulation of the human colonic microbiota:introducing the concept of prebiotics. J. Nutr.125:1401-1412.

Guo, F., R.B. Kwakkel and M.W.A. Verstegen. 2000.The use of Chinese herbs as alternatives for growthpromoters in broiler diets. In: Abstracts andProceedings, XXI World’s Poultry Congress,Montreal, Canada, Aug. 20-24. CD-Rom, pp. 2.

Hooge, D.M. 2003a. Broiler chicken performancemay improve with MOS. Feedstuffs, January 6,pp. 11-13.

Hooge, D.M. 2003b. Dietary MOS may haveapplication in turkey diets. Feedstuffs 75(18):11-13. p.42.

Moore, P.R., A. Evenson, T.D. Luckey, E. McCoy,C.A. Elvehjem and E.B. Hart. 1946. Use ofsulphasuccidine, streptothricin and streptomycin innutrition studies with the chick. J. Biol. Chem.165:437-441.

Nie, N.H., C. Hadlai-Hull, J.G. Jenkins, K.Steinbrenner and B.H. Bent. 1975. In: StatisticalPackage for the Social Sciences, 2nd Edition.McGraw Hill Book Company, New York.

Partanen, K. 2001. Organic acids - their efficacy andmodes of action. In: Gut Environment of Pigs (A.Piva, K.E. Bach Knudsen and J.E. Lindberg, eds).Nottingham University Press, UK, pp. 79-96.

Partanen, K.H. and Z. Mroz. 1999. Organic acidsfor performance enhancement in pig diets. Nutr.Res. Rev. 12:117-145.

Rosen, G.D. 1997. In: Proceedings of the 16th

Scientific Day, Southern African Branch of theWorld’s Poultry Science Association (D.G.Poggenpoel, ed). October 16, 1997. pp. 60-79.

Rosen, G.D. 2000. Enzymes for broilers: amultifactorial assessment. Feed Int’l. 21(12):14-18.

Rosen, G.D. 2002. Exogenous enzymes aspronutrients in broiler diets. In: Recent Advancesin Animal Nutrition 2002. (P.C. Garnsworthy andJ. Wiseman, eds). Nottingham University Press,UK. 89-103.

Rosen, G.D. 2003a. Effects of genetic, managementaland dietary factors on the efficacy of exogenous

G.D. Rosen 101

microbial phytase in broiler nutrition. Brit. PoultrySci. 44S1:S25-S26.

Rosen, G.D. 2003b. Pronutrient antibioticreplacement standards discussed. Feedstuffs75(30):11-13. p.16.

Rosen, G.D. 2003c. Setting and meeting standardsfor the replacement of pronutrient antibiotics inpoultry. Proceedings of the 30th Annual CarolinaPoultry Nutrition Conference, Carolina FeedIndustry Association, Research Triangle Park,October 30. pp. 69-79.

Rosen, G.D. 2004. How many broiler feeding testsare required to elaborate working models for theprediction of nutritional effects of pronutrients inpraxis? Brit. Poultry Sci. In press.

Swann Committee. 1969. Joint Committee on the useof Antibiotics in Animal Husbandry and VeterinaryMedicine Report (Cmnd. 4190). Her Majesty’sStationary Office, London. p. 83.

Syrvidis, V., R. Bobiniene, V. Priudokiene and D.Vencius. 2003. Phytobiotics add value to broilerfeed. World Poultry 19(1):16-17.

Wenk, C. 2000. Herbs, spices and botanicals; ‘Oldfashioned’ or the new feed additives for tomorrow’sfeed formulations? In: Biotechnology in the FeedIndustry, Proceedings of Alltech’s 16th AnnualSymposium (K.A. Jacques and T.P. Lyons, eds).Nottingham University Press, UK, pp. 79-96.

Winter, A.G. 1955. The significance for therapy anddiet of antibiotic substances from flowering plants(with special reference to nasturtiums, cress andhorseradish). Die Medizinische Nr. 2:73-80.


T.K. Smith et al. 103

Comparative aspects of Fusarium mycotoxicoses in broiler chickens, layinghens and turkeys and the efficacy of a polymeric glucomannan mycotoxinadsorbent: Mycosorb®

TREVOR K. SMITH, SHANKAR R. CHOWDHURY AND H.V.L.N. SWAMY

Department of Animal and Poultry Science, University of Guelph, Guelph, Ontario, Canada

Introduction

Fusarium fungi thrive in temperate climates aroundthe world and Fusarium mycotoxins are themycotoxins most commonly found in feed grains andforages (Wood, 1992). There are numerouspathologies characteristic of Fusarium mycotoxicosesand this is due to the several chemically distinct groupsof Fusarium mycotoxins, which have very differenteffects on animal metabolism and behavior. Swineare usually considered to be the most sensitive speciesto feed-borne mycotoxins both with respect to feedrefusal and to reproductive failure. Ruminant animalsare thought to be the most resistant because of thedetoxifying effect of rumen microorganisms. Poultryare generally considered to be less sensitive than swineand are often the recipients of contaminated grainsdiverted from swine feeds.

The Fusarium mycotoxin most often detected inCanadian-grown grains is the trichothecenedeoxynivalenol (DON, vomitoxin) (Scott, 1997).Trichothecene toxicosis is characterized by reducedappetite, lesions of the intestinal tract andimmunosuppression. Another frequently detectedcompound is zearalenone, an estrogenic mycotoxinthat impairs mammalian reproduction.

It has been reported that broiler chicks can tolerateup to 15 ppm DON from naturally-contaminatedwheat and oats without adverse effects onperformance (Hulan and Proudfoot, 1982; Kubenaet al., 1987). Others, however, observed reducedperformance and changes in immune function,hematology and serum chemistry in broiler chicksfed 16 to 18 ppm DON from naturally contaminatedsources (Huff et al., 1986; Kubena et al., 1988; 1989;Harvey et al., 1991). It is clear however, that broilerchickens are far less sensitive than swine to DON-

contaminated feeds, particularly with respect toreduced feed intake (Smith et al., 1997).

It has been reported that the feeding of 100 ppmzearalenone had no effect on layer performance,fertility or hatchability (Marks and Bacon, 1976).The feeding of DON at 5 ppm (Hamilton et al., 1985)or 18 ppm (Kubena et al., 1987) did not affect layerperformance. A combination of DON (2-3 ppm) andzearalenone (0.4–0.5) also resulted in no adverseeffects on layer performance (Kesharvarz, 1993).Layer performance was adversely affected, however,when a corn-based diet was fed containing 17.6 ppmDON and 1.6 ppm zearalenone (Danicke et al.,2002). Adverse effects on layer performance are oftengreater under field conditions (Keshavarz, 1993). Itis often difficult, however, to explain why this occurs(Williams et al., 1992).

There is less information available regarding thefeeding of contaminated grains to turkeys. Hamiltonet al. (1985) indicated that turkey poults can toleratediets containing at least 5 ppm DON. Manley et al.(1988) described feed refusal and high mortality in acommercial turkey flock fed diets containing 81 ppbDON + 2.2 ppm salinomycin. The feeding of 4.4ppm DON + 22 ppm salinomycin had no effect onfeed consumption or viability. Morris et al. (1999)observed that the feeding of 20 ppm DON had noadverse effects on poults and no toxicologicalinteraction was observed between DON andmoniliformin.

The discrepancy between the relative tolerance ofpoultry to Fusarium mycotoxins in literature reportsand the seeming susceptibility of poultry undercommercial conditions may be due to several factors.Many of the experiments conducted in literature

104 Comparative aspects of Fusarium mycotoxicoses in poultry

reports were for fairly short periods. This is oftennecessary to conserve valuable stocks of purifiedmycotoxins or fungal culture materials. The use ofpurified mycotoxins, fungal culture materials orartificially inoculated corn can also bias findingsbecause the toxicological synergism arising from thefeeding of combinations of mycotoxins does not takeplace.

Polymeric mycotoxin adsorbents preventmycotoxicoses by adsorbing mycotoxins in theintestinal lumen and preventing transfer through theblood to target tissues (Ramos et al., 1996). Thecurrent studies were conducted to determine theefficacy of Mycosorb®, a glucomannan polymerextracted from the cell wall of yeast, in preventingthe adverse effects of blends of grains naturally-contaminated with Fusarium mycotoxins on poultry.

Materials and methods

EXPERIMENTAL FEEDSTUFFS

Mycotoxins in the current experiments were providedby a blend of naturally contaminated corn and wheatpurchased from producers in Southwestern Ontario.The complete diets were analyzed for DON, 3-acetyl-DON, 15-acetyl-DON, nivalenol, T-2 toxin, iso T-2toxin, acetyl-T-2 toxin, HT-2 toxin, T-2 triol, T-2tetraol, fusarenon-X, diacetoxyscirpenol, scirpentriol,15-acetoxyscirpentriol, neosolaniol, zearalenone,zearalenol, aflatoxin and fumonisin by gaschromatography and mass spectrometry at theVeterinary Diagnostic Laboratory, North Dakota StateUniversity, Fargo, North Dakota (Raymond et al.,2003). Fusaric acid was determined by the highperformance liquid chromatographic method ofMatsui and Watanabe (1988) as modified by Smithand Sousadias (1993) and confirmed by Porter et al.(1995).

EXPERIMENTAL DESIGN

Broilers

Broiler chicks were fed starter (0 – 3 weeks), grower(3 – 6 weeks) and finisher (6 – 8 weeks) diets in two56-day experiments (Swamy et al., 2002; 2004a).Diets included: 1) control, 2) low level ofcontaminated grains, 3) high level of contaminatedgrains, and 4) high level of contaminated grains +0.2% Mycosorb®. Growth rates and feed consumptionwere monitored weekly. At the end of the study, blood

and tissue samples were taken for hematology andserum chemistry measurements.

Laying hens

One hundred and forty-four, 45-week-old laying henswere fed for 12 weeks diets including: 1) control, 2)contaminated grains, and 3) contaminated grains +0.2% Mycosorb®. Parameters measured included feedconsumption, egg production, egg shell measure-ments, egg quality measurements, relative organweights and plasma chemistry.

Turkeys

Two hundred and twenty-five day-old male turkeypoults were fed corn, wheat and soybean meal-basedstarter (0-3 weeks), grower (3-6 weeks), developer(6 – 9 weeks) and finisher (10 – 12 weeks) diets in a12-week experiment. Diets included: 1) control, 2)contaminated grains, and 3) contaminated grains +0.2% Mycosorb®. Parameters measured includedweight gain, feed consumption, relative organweights and plasma chemistry.

Results

MYCOTOXIN ANALYSES

Of the twenty different mycotoxins analyzed for, onlyDON, 15-acetyl DON, zearalenone and fusaric acidwere found in detectable quantities in all experimentaldiets. DON was found to be approximately 0.5 ppm,5.0 ppm, 9.0 ppm and 10.0 ppm for the fourexperimental broiler diets. The concentrations of 15-acetyl DON were about 0.5 ppm, zearalenone wasabout 0.5 ppm and fusaric acid was about 17 ppm.Diets fed to laying hens which containedcontaminated grains had an average of 12.0 ppmDON with the other three mycotoxins in the sameratios as were seen in the broiler trials. In the turkeytrial, diets containing contaminated grains averaged6.5 ppm, 7.6 ppm, 10.6 ppm and 13.3 ppm DON forthe starter, grower, developer and finisher phases withthe other three mycotoxins present in the sameproportions as were seen for the broiler and layerexperiments.

BROILERS

There was a significant linear decrease in growth rateand feed consumption in the grower period when


increasing levels of contaminated grains were fed torapidly growing broilers (Swamy et al., 2004a; Table1). No significant effect of diet was seen in the starteror finisher periods. When broilers were growingmore slowly, growth depression was observed in thefinisher phase (Swamy et al., 2002). At the end ofthe finisher phase in the earlier study, it was observedthat the feeding of contaminated grains elevated redblood cell count and blood concentrations of hemoglobinand uric acid (Table 2). Biliary concentrations ofimmunoglobulin A were reduced while breast meatredness increased.

The feeding of 0.2% Mycosorb® prevented all ofthe above dietary effects.

LAYING HENS

The feeding of contaminated grains decreased feedconsumption compared to controls in the first month(P<0.05) (Table 3). Feed intake increased, however,in the second and third months. The feeding ofMycosorb® prevented this increase in the third month

and also prevented a decrease in feed efficiency. Atthe end of the experiment, it was observed that hensfed contaminated grains had an increased relativekidney weight compared to controls; this effect wasprevented by the feeding of Mycosorb®.

Egg production and egg mass decreased (P<0.05)compared to controls in the first two months whencontaminated grains were fed. The feeding ofMycosorb® prevented this in the first month. The mostobvious effect of feeding contaminated grains onplasma chemistry was on uric acid concentrations(Table 4). In each month, plasma uric acidconcentrations were significantly increased with thefeeding of contaminated grains. In each case, thisincrease was prevented by the feeding of Mycosorb®.

TURKEYS

The feeding of contaminated grains reduced growthrates in the starter, developer and finisher phases andoverall (Table 5). The feeding of Mycosorb®

prevented these effects. The most obvious effect of

Table 1. Effect of feeding blends of grains naturally-contaminated with Fusarium mycotoxins on weight gain and feed consumption of broilerchickens.1

Diet Feed consumption (g/bird)2 Weight gain (g/bird)3

0 - 21 days 21 - 42 days 42 - 56 days 0 - 21 days 21 - 42 days 42 - 56 days

Control 908 2797 2544 435 1678 1303Low mycotoxins 841 2565 2437 376 1522 1274High mycotoxins 923 2392 2456 386 1479 1319High mycotoxins + 0.2% Mycosorb® 968 2472 2532 392 1538 1348SEM 37 60 27 6 16 13Linear effect NS4 0.05 NS NS 0.04 NS

1From Swamy et al., 2004a.2Values are least square means; n = 3.3Values are least square means; n = 90.4Not significant (P>0.05).

Table 2. Effect of feeding blends of grains naturally-contaminated with Fusarium mycotoxins on hematology, serum chemistry and breastmeat coloration of broiler chickens.1

Diet RBC2 Hb3 Uric acid4 Redness5 Biliary IgA6

Control 2.66 95.0 259 0.45 7.54Low mycotoxins 2.84 101.1 286 0.67 7.28High mycotoxins 2.83 99.2 357 0.80 4.99High mycotoxins + 0.2% Mycosorb® 2.54 91.2 281 0.21 6.54SEM 0.04 1.37 10.9 0.07 0.29Linear effect 0.01 0.01 0.009 0.01

1From Swamy et al., 2002.2Red blood corpuscle counts (1012/L); n = 12.3Hemoglobin concentration (ppm); n = 12.4Uric acid concentration (µmoles /L); n = 12.5Unitless scale, 0 = green, 1 = red; n = 15.6mm precipitate; n = 15.


diet on plasma chemistry was on plasma uric acidconcentrations (Table 6). After 4 and 8 weeks of feedingcontaminated grains, plasma uric acid concentrationswere significantly reduced. This effect was not seen,however, when birds received Mycosorb®. At the end of

the experiment, turkeys fed contaminated grains +Mycosorb® had significantly smaller spleens and kidneysand significantly larger bursas than birds fedunsupplemented contaminated grains.

Table 3. Effect of feeding blends of grains naturally-contaminated with Fusarium mycotoxins on feed consumption and feed efficiency oflaying hens.

Diet Feed consumption1 (g/hd/day) Feed efficiency2 (feed/egg mass)

Wk 0 - 4 Wk 4 - 8 Wk 8 - 12 Wk 0 - 4 Wk 4 - 8 Wk 8 - 12

Control 119 120 117 1.88 1.92 1.90Mycotoxins 106 127 132 1.94 2.29 2.23Mycotoxins + 0.2% Mycosorb® 114 124 121 1.90 2.10 1.94

Pooled SD 9 9 7 0.18 0.27 0.17Control vs mycotoxins 0.008 0.04 0.0001 NS3 0.001 0.001Mycotoxins vs Mycosorb® NS NS 0.0006 NS NS 0.003

1n = 12.2n = 12.3Not significant (P>0.05).

Table 4. Effect of feeding blends of grains naturally-contaminated with Fusarium mycotoxins on organ weights and plasma uric acidconcentrations of laying hens.

Diet Organ weights (g) Uric acid (µmol/L)

Liver Spleen Kidney Wk 4 Wk 8 Wk 12

Control 44.51 2.2 5.9 376 392 390Mycotoxins 44.2 2.4 7.6 1009 1154 1030Mycotoxins + 0.2% Mycosorb® 46.2 2.2 6.6 500 539 487

Pooled SD 7.58 0.66 0.98 188 156 159Control vs mycotoxins NS2 NS 0.002 0.001 0.001 0.001Mycotoxins vs Mycosorb® NS NS 0.02 0.001 0.001 0.001

1n = 12.2Not significant (P>0.05).

Table 5. Effect of feeding blends of grains naturally-contaminated with Fusarium mycotoxins on growth of turkeys (g/bird/week).

Diet Starter Grower Developer Finisher Overall

Control 217 378 748 723 517Mycotoxins 196 492 657 637 496Mycotoxins + 0.2% Mycosorb® 213 548 756 852 592

Control vs mycotoxins 0.01 0.01 0.01 0.05 0.05Mycotoxins vs Mycosorb® 0.05 0.05 0.05 0.01 0.01

Table 6. Effect of feeding blends of grains naturally-contaminated with Fusarium mycotoxins on organ weights and plasma uric acidconcentrations of turkeys.

Diet Organ weights (g) Uric acid (µmol / L)

Bursa Spleen Kidney Wk 4 Wk 8 Wk 12

Control 5.31 5.6 16.1 437 339 276Mycotoxins 5.5 6.1 17.7 299 210 305Mycotoxins + 0.2% Mycosorb® 6.5 5.1 15.8 321 197 235

SEM 0.30 0.33 0.71 27 15 43Control vs mycotoxins NS2 NS NS 0.001 0.001 NSMycotoxins vs Mycosorb® 0.03 0.05 0.05 NS NS NS

1n = 12.2Not significant (P>0.05).


Discussion

Broilers, layers and turkeys are all sensitive toFusarium mycotoxicoses. The adverse effects of thediets fed in the current studies are greater thanliterature reports based on the DON content. This islikely because of the relatively short duration ofpreviously reported trials. The blending of differentnaturally contaminated grains, moreover, also resultsin a more complex mixture of mycotoxins therebyincreasing the chances of toxicological synergybetween mycotoxins.

BROILERS

The observation that feeding contaminated grains tobroilers reduced growth only in the grower andfinisher phases supports the concept that broilers donot exhibit feed refusal in a manner similar to swinefed Fusarium mycotoxin contaminated diets (Smithet al., 1997). The reason for this species differencehas been shown to be differences in the effects onbrain neurochemistry (Swamy et al., 2004b). Thefeeding of contaminated grains to pigs elevated brainserotonin concentrations. In broilers, such treatmentselevated both serotonin and catecholamines therebycanceling the effect of serotonin on appetitesuppression. It is likely that mycotoxin-inducedgrowth suppression in broilers was due to gradualalterations in metabolism that occurred with extendedfeeding of contaminated grains.

The mycotoxin-induced elevation in red blood cellcount and hemoglobin is similar to the changes seenin ascites. It is possible that the hypotensive effect offusaric acid may be reducing blood flow to the lungsresulting in an increased need for oxygen trappingcapacity of blood. Elevations in blood uric acidconcentrations were likely due to the inhibition ofprotein synthesis caused by trichothecenes such asDON and 15-acetyl DON. This would result inincreased hepatic oxidation of amino acids andincreased uric acid excretion. Red discoloration ofbreast meat has been reported in turkeys fed Fusariumculture filtrates (Wu et al., 1994). The discolorationseen in the current study was likely due to increasedred blood cell count and hemoglobin concentrationsas well as to edema arising from the hypotensiveeffects of fusaric acid. The reduced biliaryimmunoglobulin A concentrations may have arisenfrom trichothecene-induced inhibition of proteinsynthesis.

LAYING HENS

The effect of feeding contaminated grains on feedintake of laying hens is in contrast to that seen inbroilers. It would seem that after an initial reductionin feed intake and egg production, layers increasedfeed intake perhaps in an attempt to boost eggproduction. The result, however was a very dramaticdeterioration in feed efficiency (feed consumed/eggmass). This may be due, in part, to increased hepaticamino acid oxidation due to the trichothecene-inducedreduction in protein synthesis. It is notable that themycotoxin-induced elevation in blood uric acidconcentrations is similar to, but greater in magnitudethan, the response seen in broilers. The increasedkidney weight seen when contaminated grains werefed is likely due to the increased metabolic burdenof excretion arising from increased uric acidsynthesis.

TURKEYS

Feeding contaminated grains significantly reducedweight gain of turkey poults as early as the secondweek of feeding. This is a more rapid effect thanwas seen in broilers but little effect of diet was seenon feed consumption. It would appear that turkeysare more sensitive to this mycotoxin challenge thanbroilers. The significant depression in blood uric acidconcentrations after 4 and 8 weeks of feeding is inmarked contrast to the responses seen in broilers andlaying hens. The metabolic reason for this remainsto be determined as other indices of blood chemistrywere largely unaffected by diet.

EFFECTS OF MYCOSORB®

Mycosorb® proved to be a very effective preventativetreatment for Fusarium mycotoxicoses in broilers,layers and turkeys. The mode of action of Mycosorb®

is to prevent intestinal uptake of mycotoxins andsubsequent transfer of mycotoxins to sensitive targettissues such as liver, kidney, brain and reproductivetract. It is clear from the efficacy of Mycosorb® inthese trials that it is capable of adsorbing acombination of Fusarium mycotoxins and minimizingthe potential for toxicological synergism.


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