Nutrition ^ v; 0 anddigestiv e physiology inmonogastri c farmanimal s

Reviews presented at the symposium on the occasion of the retirement of Dr Ir E.J. van Weerden, Wageningen, Netherlands, 26 May 1989

E.J. van Weerden & J. Huisman (Editors)

Pudoc Wageningen 1989

!-"' i~* \ T>Y\ CIP data, Royal Netherlands Library, The Hague

Nutrition anddigestiv e physiology in monogastric farm animals. Reviews presented at the symposium onth e occasiono f the retirement of Dr Ir E.J.va nWeerden ,Wageningen , Netherlands, 26 May 1989. / E.J. van Weerden &J. Huisman (Editors). - Wageningen: Pudoc. - x+IOI pp. ISBN 90-220-1011-2 UDC 636.2/.5.084:59l.l32 NUGI 835 Subject heading: animal nutrition

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BIBLIOTHEEK1 LANDBOUWUNIVERSITEIT ' ''.GENINGEN ^ÉÉf

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Dr Ir E.J. van Weerden PREFACE

Dr Ir E.J. vanWeerde n together with Dr Ir P. van derWal , founded the institute now known as ILOB. Dr vanWeerde n started work there in 1960 andwa s responsible for nutritional and physiological research until 1984whe n ILOB became part of the TNO organization. Dr van der Wal then left ILOB.Upo n the departure of Dr van derWal ,D r van Weerden became also responsible for the general management of ILOB. TNO is an organization for applied scientific research and ILOB is an institute within TNO responsible for animal nutrition and physiology. It is partly subsidised by the government,mainl y through the Ministry of Economic Affairs, but the major part of thewor k is concerned with applied contract research. This is often confidential, implying inman y cases that the results can only be published with some delay; in some cases, no publications are allowed atall . Research at ILOB is concerned with various topics, such as: efficacy and safety of rawmaterials ,antibiotics ,probiotics ,ne w growth- promoting agents such as steroid hormones,/2-agonist san d somatropin; digestive physiology and nutrition ofmonogastri c farm animals, pharmacokinetics and biotransformation of additives and veterinary drugs. Dr.va nWeerde n had the ultimate responsibility for this research. Topics inwhic h hewa s especially involved were amino acid requirement in poultry, pigs and veal calves,digestiv e physiology in pigs and veal calves,efficac y and safety of single-cell protein, new growth-promoting agents,antibiotics , probiotics, replacement of skimmed milk powder by other protein sources. / Dr vanWeerde n is an excellent scientist, internationally recognized as a specialist in the fields mentioned who, in the period after 1984, also proved himself to be a good manager.Alway s sensitive to both sense and nonsense in research, he did much of the preliminary work on any new techniques or fields of research himself to seewhic h points may be critical. Hewa s invited to present his work atman y scientific congresses and had considerable influence onman y new developments in the fields of digestive physiology and nutrition of farm animals, advising various industries on the development of new products of application in the feed industry. A symposiumwa s organised inhonou r of Dr vanWeerde n to acknowledge all the effort he had put into the research carried out at ILOB for twenty-nine years and intomanagemen t for the last five years. Two specialists from abroad were invited to present papers on digestive physiology in pigs and veal calves.Tw o of his co-workers contributed papers on developments in amino acids and antinutritional factors.A paper on the effects of somatropin in pigs, resulting from the research that has been done for several years in cooperation with the Agricultural University, was also present. The symposiumwa s closed by Dr vanWeerde n with the presentation of his paper: Past and Future Developments of Protein Supply inMonogastri c FarmAnimals . In this book, the papers presented are published in an extended form as reviews. These contributions are just a small expression of the appreciation felt for Dr vanWeerde n by his colleagues and co-workers from ILOB. The organisers wish to thank Mr Haak,Mr s van den Berg-Volders and Mrs Haak-van den Brink for their excellent assistance in the organization.

J. Huisman, J.B. Schutte, S. Bakker and G.J.M,va n Kempen (Organizing Committee)

Wageningen, Netherlands,Ma y 1989. CONTENTS

Research into the digestive physiology of pigs A.G. Low

Antinutritional factors (ANFs) in the nutrition ofmonogastri c farm animals J. Huisman 17

Research into the digestive physiology of the milk-fed calf R. Toullec, P. Guilloteau 37

Effect of porcine somatotropin onnitroge n gain and energy metabolism in fattening pigs M.W.A. Verstegen,W . van der Hel 57

Practical application of (bio)synthetic amino acids in poultry and pig diets J.B. Schutte /

Present and future developments in the protein/amino acid supply in monogastric farm animals E.J. vanWeerde n °9 RESEARCHINT OTH EDIGESTIV EPHYSIOLOG YO FPIG S

A.G.LO W Pig Department,Agricultura l and Food Research Council Institute for Grasslandan dAnima lProduction ,Shinfield ,Reading ,Berkshir eRG 29AQ , U.K.

Summary

Historical development ofdigestiv e physiology is reviewed briefly. The importance of food intake,an d inparticula r its neuro-endocrine relationship,i sdiscussed . Gastric,biliar yan dpancreati csecretion s and the effects of age and diet are described. The function of the smallintestin ebot hi nterm so fsecretio nan dabsorptio nar ediscusse d with particular reference topractica lmeasuremen t ofprotei nqualit y and carbohydrate digestion and fermentation. The role of the large intestine in nutrient absorption is also discussed. Methods of measuring absorption using blood flow and sampling techniques are discussed. Therevie wend swit hsom estrategie sfo rfutur eresearch .

Keywords /

Pig,Digestion ,Secretion ,Absorption ,Stomach ,Smal lIntestine , LargeIntestine ,Protein ,Carbohydrate .

1. Introduction On the occasion of the retirement of E.J. Van Weerden after a distinguished career, it seems appropriate to review research in digestive physiology in thepig ,becaus e he hasbee n instrumental in several importantdevelopment s inth efield . Atth esam etim eh eha s encouraged others topursu e innovative studies and he has put TNO in theforefron to fresearc ho nthi ssubject . Historically, research into thedigestiv ephysiolog y ofpig swa si n progressa tRothamste d Research Station,Harpenden ,i nEnglan d inth e middleo fth enineteent hcentury ,whe nobservation swer emad eo nusin g anearl yfor mo fcannula . However,majo rdevelopment si nthi sfiel di n pigswer estil lnearl yon ehundre dyear saway . Inth elas tpar to fth e nineteenth century and theearl ypar t of this century Pavlov andhi s colleagues inRussi alai d thefoundation so f thesubjec to fdigestiv e physiology as we know it today. Their many experiments on the physiologyo f food consumption indogs ,an d itsrefle xbasis ,an dth e subsequentserie so fdigestiv ehydrolyses ,involve dth edevelopmen to f methodso f surgeryan d physiological principleswhic hwer e appliedt o pigs by Kvasnitskii and others from the 1930s onwards. Curiously enoughthes e studiesexcite d little interest inothe r countriesunti l the early 1960s when Horszczaruk and colleagues in Poland began to examinehindgu tfunctio ni npigs . Soonafterward sCunningham ,Frien d& Nicholson (1963)bega nt omak estudie si nCanad afollowe db yth eFrenc h groupi nJouy-en-Josa sle db yRéra t(Auffray ,Martine t& Rérat ,1967) , andth eEnglis hgrou pa tShinfiel d formedb yBraud edurin gth e1960s . Fromthe nonward sdevelopmen twa srapi di nal lo fthes ecountries ,an d during the 1970s, further active new groups were formed in the USA, Australia,Cuba ,Belgium ,th eNetherlands ,Denmark ,Sweden ,th eGerma n Democratic Republic and West Germany. Since 1979 there have been meetings on digestive physiology in the pig in Shinfield, England (1979), Jouy-en-Josas, France (1982), Copenhagen, Denmark (1985)an d Jab^onna,Polan d (1988). Thenex tmeetin gwill ,appropriately ,b ehel d during 1991 in Wageningen. Each meeting has been followed by a publication containing important reviews and papers on new research topics. The aim of this review is todiscus s selected topicswhic h have a particular interest for the author,an d tomak e some suggestions for futuredevelopment si nthi sfield .

2. Foodintak e

Thecontro lo f food intakei npigs ,a si nman yothe ranimal san di n man, remains a topic of major interest and much uncertainty. The neuro-endocrinecontro lo ffoo dintak ei sbecomin gprogressivel ybette r understood,a s reviewsb yHoup t (1982)fo r thepig ,an d Forbes (1988) foranimal s ingenera lhav eshown . Butmuc h remainst ob eunderstoo d of what determines the pattern of food intake in relation to the physical, chemical and sensory properties of foods. While we know somethingo fth ewa yi nwhic hth ecentra lnervou ssyste mi sintegrate d for the processes of digestion, through the action of regulatory peptides,glucostati c andgastrointestina l controls,i ti snonetheles s the case that most studies have been made under physiological conditionswhic har edistan t fromthos ewhic h occurwhe nnorma lmeal s areeaten . Thesestudie shav ereveale da nextremel ycomple xsystem ,i n which several ofth e regulatorypeptide sexamine d indetai l appeart o havesevera lcontrastin g roles. Sincefoo dintak ei npractic eappear s to be the main limiting factor in pig production, better specific understanding of the neuro-endocrine regulation is much needed: in particular the roles of cholecystokinin (CCK), motilin, somatostatin and gastric inhibitory peptidemeri t study. One approach to this is specific immunological blocking of individual peptides to compare responsest othei rpresenc eo rabsence ,an dthi si sth eapproac hbein g currentlydevelope da tShinfield . Inaddition ,th ecentra lrol eo fth e vagus nerve in digestive function appears to be very complex and inadequatelyunderstood ,a snote db yLaplac e (1989).

3. Themout han dsalivar ysecretio n

Although the pig is equipped with substantial teeth, under modern farmingcondition sver y little food that requiresmuc hmasticatio n is used,an dthu sfoo dspend slittl etim ei nth emouth . Perhapsfo rthi s reason,th eamount so f saliva secreted are small,an d there islittl e secretion except after meals (Arkhipovets,1956 ;Corring , 1980). It is, however, difficult to measure salivary secretion from the individualduct sbecaus e of their small sizean d their location close to teeth which can easily damage catheters. The ratio of salivary amylaset opancreati camylas ei s1:25 0 000accordin gt oCorrin g (1980) who collected saliva through an oesophageal cannula. Itwoul d beo f interestt okno who wmuc hsaliv ai ssecrete db ywil dpigs ,wh ohav et o chewthei rfoo dextensively ,an db ymatur epigs ,wh ovisuall yappea rt o secrete substantial amounts of saliva, as do pigs under anaesthesia vhileundergoin ggu tsurgery .

4. Gastricfunctio n

Itha sbecom eclea ri nrecen tyear stha tth estomac hi sa nimportan t reservoiro ffood ,providin gth eintestine swit ha relativel yconstan t supply of food, to some extent regardless of the times atwhic h the food was eaten, for 15-18h after a meal (Auffray,Martine t & Rérat, 1967; Zebrowska& Buraczewska ,1972 ;Braude ,Fulfor d& Low ,1976) . The stomach appears to be never completely empty and always displays motilityan dsecretor yactivity . Surgicalmethod so fexaminin ggastri c functionrais eproblems . Most ofth ewor k byKvasnitski i (1951)involve d either a simple stomachi n the fundic regiono r a re-entrant cannula inth eduodenum . A simple gastric cannula may be quite large and apparently does not markedly disturbmotilit y (Cuber,Laplac e& Villiers ,1980 ; Sissons& Rainbird , 1985),whil ere-entran tcannula ema yb eprofoundl ydisturbin g(Laplace , 1980). However it isdifficul t tomak emor etha non eobservatio npe r daywit ha simpl egastri ccannul ai fth eentir econtent sar eremove dt o providea representativ e samplefo ranalysi s (Cubere tal. ,1980 ; Low, Pittman & Elliott, 1985). A further major problem with re-entrant cannulationi stha tth ecannul acanno tb elocate dproxima lt oth ebil e ductan d it isno t easy toplac e itproxima l toth epancreati cduct . Hence the digesta collected from such cannulae inevitably contain post-gastric endogenous secretions in amounts which are considerable butwhic hi ti simpossibl et oseparat ephysicall yfro mgastri cdigesta . The rate of gastric emptying is typically most rapid in the first hour after a meal, when elastic forces as well as physiological mechanisms,appea r tob e important influences; ingenera l thelarge r themea ldr ymatte rsize ,o rth elarge rth evolum eo fwate rgive nwit h themeal ,th efaste r theinitia l rateo fgastri c emptyinga sshow ni n Table1 .(Lo we tal. ,1985) .

Table1 . GastricEmptyin g(g/h )i n40k gPig s Given850 gDie tan dVaryin gLevel so fWate r Dietan dWate r Hoursafte rMea l Intake (g)

2337 958 304 47 70 2975 1220 565 238 -60 3612 1770 469 398 36

It is,however ,no t entirely clearwha t determines the emptying rate froma nutritiona lpoin to fview : Braudee tal . (1976)observe dmuc h more rapid emptying immediately after consumption of semi-purified diets than a diet based on barley,whea t and fishmeal, and thiswa s confirmedb ysevera lothe rgroups . Thedegre eo fosmolarit yo fdigest a inth eduodenu mi sthough tt ob edirectl yproportiona lt oa ninhibitor y effect on gastric emptying, but the putative mechanism has not been demonstrated in pigs. It is appealing to think that this was the mechanism involved in thewor k by Braude et al. (1976)becaus e there was much more osmotically active material in the cereal-based diet. Deliberate modification of thecompositio n of cereal-based diets e.g. byadditio no foil ,sucros eo rcellulos edi dno tlea dt oalteration si n the rateo f gastric emptying inth e studiesb y Lowe t al. (1985)an d these raised a question as to whether gastric emptying is readily manipulated by dietary changes. Subsequently we have examined the effect of a soluble non-starch polysaccharide (NSP), guar gum, on gastric emptying and again sawn oeffec t of theNS Pupo n the rateo f nutrientemptyin gfro mth estomach ,thoug hth erat eo fliqui demptyin g was delayed; this effect could have been due to changes in gastric secretion, or to the hydrophylic and viscous nature of guar gum solutions (Rainbird & Low, 1986). Guar gum also depressed gastro- duodenal motility in corresponding studies (Rainbird & Sissons, unpublished). Themagnitud e of gastric secretion ofwate r containing electrolytes of both basic (in the oesophageal region) and acidic nature (inth e fundic region)i sremarkable . Pigso f 40k gappea r tosecret e10-20 % of their total weight as gastric juice (based on measurement of duodenal digesta flow less separately measured outputs of bile and pancreatic juice)an do famount so fpepsi nwhic h couldhydrolys eman y times more peptide bonds than the diet contains (Zebrowska, Low & Zebrowska, 1983). Yet, in spite of the apparent overproduction of pepsin,ther e is clear evidence of adaptation of thisenzym e todie t typei npiglet s (Cranwell,1985 )an dgrowin gpig s(Low ,1982) . Earlier studiesb yKvasnitski i (1951)provide da nindirec tindicatio no fthis . Secretiono fgastri c acid andwate r alsoappea r tob ediet-dependent : Kvasnitskii (1951) found that the higher the content of NSP in the diet, the greater the level of secretion, and this was confirmed by Zebrowska et al. (1983) and Cranwell, Low & Sambrook (unpublished). The latter authorsprepare d fully-innervated gastric pouches in 30k g pigs. Suchpreparation s dono tpermi t complete collectiono fgastri c secretion; themeasurement s are partlya function of the particular secretorytissu efoun di nth echose nregio no fth estomac han dar eals o unaffected by themechanica l stimulustha t food particles are thought to exert on secretion. Even though the secretions in the gastric region are below pH2,th e contents of the stomach lumen are greatly influenced by meal pH and buffering capacity. During the first 4h aftera mea lwhe na tleas thal fo fth emea lleave sth estomach ,th ep H rarelyfall sbelo wpH3.5 ,whic h isth euppe r optimum forth ecombine d effecto fth evariou spepsin sfound . Insufficiency of gastric secretion, particularly of acid is conventionally thought to occur in piglets: this is a topic which deservesbette runderstandin gi nrelatio nt oimprove drearin gsystems . Finally, iti softe naske dwhethe r there isan y significantgastri c absorptiono fnutrients . Low& Rainbir d (1986)wer eunabl e todetec t 14C or3 H inperiphera l blood following administration ofamin oacid s or 3-o-methyl D-glucose, respectively, into the stomach of anaesthetized pigs with ligated oesophageal and pyloric sphincters. HoweverRéra te tal . (unpublished)measure dincrease dconcentration so f amino acids and glucose in the efferent compared with the afferent gastricbloo dsupply : howeverth econcentration so fnutrient si nthei r workwer ever ymuc hhighe r thanuse db yLo w& Rainbir d (1986). Iti s possible that the transfer across the gastric mucosa seen by Rérat etal . (1989) was the result of passive diffusion, because Low & Rainbird (1986) could not demonstrate the presence of any active transport of amino acids or glucose in isolated gastric mucosa invitro .

5. DuodenalFunctio n

Thedigest a leavingth e stomachhav e anincreasingl y lowp Ha stim e passesafte ra meal : theyals oten dt ocontai na nincreasin gamoun to f soluble matter. However, the proportion of trichloroacetic acid-soluble nitrogen totota lnitroge n rosefro m1 2t o50 %ove r 12h after a meal with casein as the sole protein source,wherea s itwa s approximately 50% throughout this period for pigs receiving a barley-soyadie t(Zebrowsk ae tal. ,1983) . Thisdigest ai simmediatel y mixed with bile and thenpancreati c juice and brought to a pH value between6 an d7 . Measurement ofth econtribution s fromthes e sources isno t easy: while several authorshav eplace d a catheter inon eo r other duct,other shav e prepared a pouch intowhic h theduc t opened, sometimesi nbetwee na re-entran tcannul ai nth ecas eo fth epancreati c duct. Complex feedbackmechanism sappea rt oregulat ethes esecretion s in part and return of the secretions is clearly essential for meaningful measurement (the effects of non-return have been demonstratedb yCorrin g (1974)fo rpancreati c juice). Both bile and pancreatic secretions have been clearly shown to respond to thenatur e of thedie tbu t the relationships appear tob e complex. Forexample ,Alie v (1980) (citedb yJuste ,1982 )foun dtha t dailybil evolum ean dlipi dconten tros ea sdietar ylipi dwa sincrease d to 5% (to 3.5 1 and 14.4 g, respectively) but then fell when the content rose further to12% . Thesean d other observations onbiliar y outputwer e reviewed indetai lb yJust e (1982). Pancreatic outputi n pigsha sbee n examined inmor e detail thanbiliar y output and itha s been found that the volume secreted increases as the meal size increases,o rwhe n themea lNS P content increases. Similarly, iti s generally found thatwhe n thedietar y fato r protein or carbohydrate contenti sincrease dther ei sa correspondin gincreas ei nth esecretio n of theappropriat e enzymes. However, it is interesting tonot e that the amounts of trypsin and carboxypeptidase B did not decrease when pigswer egive na protein-fre edie tafte ra die twit hnorma llevel so f protein,whil e the corresponding chymotrypsin and carboxypeptidase A activitydisappeare dalmos tcompletel yafte r2- 3day s(Zebrowska ,Lo w& Zebrowska,1986) . A remarkableaspec to fpancreati c enzymeoutpu ti s that the amounts secreted are often 10-20 times the amounts required under theoretically optimum conditions for the complete digestiorfo f dietary protein, and yet there are numerous clear indications of adaptation to diet composition, as reviewed by Juste (1982), Simoes-Nunes (1982),o rt otrypsi ninhibitors ,a sreviewe db ySchneema n (1982). it remains an open question as to whether the amounts of enzymesecrete dar eno ti nfac tlarg esurpluses ; under thecondition s withinth egu tlume nver ylarg evariation si noutpu tar esee nfro mda y today ,implyin grelativel yinexac tcontro lo fsynthesi so rsecretiono f the enzymes, and perhaps a consequent need for surplus production. Theneuro-endocrin e regulationo fpancreati csecretio ni npig sha sbee n thesubjec to fmuc h researchi nrecen tyear sb yCorrin gan dhi sgrou p atJouy-en-Josas ,an dth e topicha sbee n recently reviewed indetail , particularly at the levelo fputativ e molecular biological mechanisms (Corring, Juste & Lhoste, 1989). The pig pancreas offers a particularly exciting opportunity tounderstan d thephysiologica l and biochemicalmechanism so fenzymi csynthesi san dsecretio ni neucaryote s since concurrent studies on secretion and on regualtory peptides are relatively straightforward. At a practical level, it is still not cleari fther ear eeve rinstance so fpancreati cinsufficienc yi nnorma l animals, except soon after weaning when amounts of amylase and proteasesma yb elimitin g (thoughthi si shar dt oprove) . Although the total volumes of endogenous inputs into the duodenum varymarkedl ythe yar esubstantia la sshow ni nTabl e2 .

Table2 . Gastric,Biliar yan dPancreati c Secretion(l/24h )i n40k gPig s Diet Gastric Bile Pancreatic Juice Juice Barley-soya (highfibre ) 8 1.8 2.5 Starch-Casein-cellulose (lowfibre ) 4 1.1 1.0 These secretions contain atleas t 6 go fnitroge n per 24h ,variabl e amounts of lipids and considerable quantities of electrolytes. A striking example of mineral secretion concerns the amounts of sodium passingth eduodenu mwhic hwer e400-638 %highe rtha nintak edurin g2 4h periods; correspondingvalue sfo rpotassiu mwer ei nth erang e127-161 % (Partridge, 1978). Th energy demand for synthesis of enzymes, epithelial cells, mucoprotein (formed throughout the gut as a protectiveagent )an dmovemen to fwate ran delectrolyte sha sye tt ob e calculated but it is considerable. The integration of secretions to ensure that homeostasis is maintained in the gut lumen, even though differentdiet s elicitver ydifferen tendogenou s responses,i sa sye t little understood in pigs. Attempts to measure the endogenous contribution to digesta in the duodenum have not always led to consistentanswers ,a snote db yLo w (1979a). Themetho do fcollectio n of digesta appears to have amajo r effect upon the result obtained: attempts to maintain the pressure which exists in the lumen of the duodenum during collection from re-entrant cannulas led to lower nitrogen flows than a free drainage system at room pressure (Low & Zebrowska,1977) . A latersyste mfo rautomati cmeasuremen to fdigest a flow from re-entrant cannulas indicated that theattempt s tomaintai n gut pressure may have inhibited digesta flow (Low, 1979b). These studieshav e led tosom euncertaint y about theexten t towhic hther e mayb eabsorptio ni nth eproxima lduodenu mi npigs ,o ri nth estomach , asdiscusse dearlier . Zebrowskae tal . (1982a,b)conclude d thatther e ismor egastri csecretio ntha nabsorptio ni nth eproxima lduodenum ,bu t themethodolog yuse dmake si tdifficul tt ob ecertai ntha tthi swa sso .

6. Secretionan dAbsorptio nalon gth eSmal lIntestin e

Developments in the field of protein turnover inmammalia n tissues and thewhol ebod y (Waterlow,Garlic k &Millward ,1978 ; Simon,1989 ) have provided a remarkable insight into the complex mechanisms which control the turnover of gut tissue. Nevertheless, the technical complications in such studies, which were discussed in the above reviewsar e formidable. Severaldifferen t approacheshav ebee ntake n insuc hwor kbu ti ti sclea rtha tth eturnove ro fgu ttissu ei so fth e ordero f20 %pe rday ,whil etha to fth epancrea sha sbee nestimate dt o be75 %pe r day,an dmuscl e inth e rangeo f 2-3%pe rday . Evenafte r allowance hasbee nmad e forexperimenta l inaccuracies,i ti sapparen t thatth edigestiv etrac taccount sfo rapproximatel y 25%o fth eprotei n turnover in the whole body of pigs (Simon,Münchmeye r & Zebrowska, 1982). Thisproces s isclearl yassociate dwit ha considerabledeman d forenergy . The wall of the small intestine is a major source of endogenous secretions; Horsczaruk, Buraczewska & Buraczewski (1974) estimated that 70 kg pigs secrete 6 1 of intestinal juice containing 8-12 g nitrogen. Thenitroge ni sa constituen to fshe dmucosa lcells ,plasm a protreinsan durea ; thedail yexcretio no fure a intoth egu tappear s to be similar to the amounts excreted in urine in 24 h (Rérat & Buraczewska, 1986). It seems likely that the nature of the diet influencesth eamoun tan dcompositio no fthes eexcretions ; varyingth e composition of solutions used to perfuse isolated loops of jejunum inviv ole d tomarke d changes inth eoutput ,o fnitroge n (Buraczewska, 1579). Low & Rainbird (1984) found that NSP may also increase excretiono fN int oth ejejunum . Nutrientabsorptio nalon gth esmal lintestin eo fpig sha smainl ybee n measuredusin gre-entran tcannula sa tvariou slocation san dcalculatin g the difference between intake and output from cannulae. Thus Buraczewski,Buraczewsk a& Zebrowsk a (1975)foun d contrastingpattern s ofdisappearanc e ofamin oacid sbetwee ndifferen tdiet sa tseve nsite s alongth esmal lintestine . Informationo fthi skin dma yb ehelpfu li n determiningwhic hpart so fprotein sar emor eo r less readilydigeste d bypigs . Measurementso fth eapparen tabsorptio na tsevera lintestina l siteso f aminoacid s (Low,1979c) , carbohydrates and lipid (Sambrook, 1979a,b)an dmineral s (Partridge,1978 )hav eserve dt odemonstrat eth e complexitieso finter-nutrien tdigestion . Inmos tstudie so fthi skin d no attempt has been made to separate endogenous from dietary constituents of the digesta. The detailed physiological mechanisms involvedi nabsorptio nhav erecentl ybee nreviewe db yFriedric h (1989).

7. AbsorptionMeasure da tth eEn do fth eSmal lIntestin e

Classicalnutritio ndepend scentrall yupo nmeasurement so fth etota l anddigestibl econten to fnutrient si nfeedstuffs ,an di twil lcontinu e to do so in the foreseeable future. Until relatively recently, digestible amounts of nutrients have been measured by subtracting faecal lossfro m intakequantities ,withou t correction for endogenous orbacteria l contributions to faeces. Suchmeasurement s of apparent digestibility have been of major practical value especially for minerals, total carbohydrate and total lipid components of thediet . However,i nth ecas eo fproteins ,thi si sno ts obecaus eo fth erol eo f themicroflor ao fth elarg eintestine . Inwha tha sbecom ea classica l studyi npigs ,Zebrowsk a (1973)maintaine dpig so na protein-fre edie t given orally. During part of the experiment the pigs also received hydrolysed casein either through a duodenal cannula or through a cannula in the terminal ileum. The pigs were in negative nitrogen balance except when they received hydrolysed casein through the duodenalcannul aa sshow ni nTabl e3 .

Table3 . NitrogenBalanc e (g/d)i nPig sInfuse dwit h AminoAcid sint oth eDuodenu mo rTermina lIleu mwhil e Receivinga Protein-Fre eDie tOrall y

Duodenal Ileal NO Infusion Infusion Infusion

! Input 17.34 18.65 1.62 Urine 5.89 18.88 3.21 ' Faeces 1.83 2.96 1.77

Nbalanc e +9.62 -3.19 -3.36

Thisindicate stha twhil eth esmal lintestin eca nabsor bamin oacid sa s such, the large intestine cannot: the metabolic fate of the amino acidsi sdetermine dlargel yb ybacteri awhic hma yincorporat ethe mint o their own protein or theyma y use them as energy or other metabolic fuels, with the release of ammonia and such compounds as diamines, whichca nb epharmacologicall yharmfu lt oth eanimal . Furthermore,i t i^sno weviden ttha tfaeca lnitrogenou scompound sprobabl y includeles s than10 %o fundigeste ddietar ycomponents ; mosto fth eres tappea rt o bemicrobia li nnature . Becauseo fthi smicrobia lactivit yther ehav e beenman ystudie so nth edigestibilit yo fprotein ,usuall yi nterm so f specificamin oacids ,a tth een do fth esmal l intestine: indeedthi s hasbee nth emai nfocu so fapplie ddigestiv ephysiolog y inth epi gi n recent years, and it has been reviewed several times (Just,1980 ; Tanksley& Knabe ,1984 ; Sauer& Ozimek ,1986 ; Sibbald,1987 ; Low& Fisher, 1989). Much effort has been put into designing re-entrant cannulaewhic hd ono tbloc ku pwit hfibrou sfeeds ,by-passin gth elarg e intestinewit hileo-recta lanastomoses ,preservin g thefunctio no fth e ileo-caecal sphincter,an d theus e of simple cannulae coupled witha varietyo fnon-absorbabl ean diner tmarke rsubstances . However,i ti s important to re-assess critically thevalidit y of such approaches in theligh to fcurren tunderstandin go fdigestiv efunction . Inmos t studies on ileal digestibility of amino acids measurements havebee napparen tan di ti sassume dtha tdigest asimpl ycontain samin o acids ofundigeste d dietary origin. However, it is clear thatther e are substantial quantities of endogenous amino acids in this region, and thus several attempts have been made to correct for them using protein-free diets. These raise problems as noted by Van weerden, Slump & Huisman (1980) inpar t because the amount of NSPwhic h they containmarkedl y influences the amino acid content of digesta (Sauer etal. , 1977). Inaddition ,ilea ldigest acontai nsubstantia lamount s ofBacteria ,whic har eabl et oincorporat eure asecrete dint oth esmal l intestine (Bergner et al,, 1986). It therefore seems necessary to removeal lendogenou san dbacteria lcomponent sfro mdigest abefor eth e digestibilityo f thedietar yamin oacid sca nb emeasured ,an d thisi s noeas ytask . Froma practical viewpoint it canb e seen that ileal digestibility values can help to improve the accuracy of diet formulation when unusualingredient sar eused ,o rthos ewhic hhav ebee nbadl yprocesse d (VanWeerde n et al., 1985) but not when feedstuffs used in studies leading to statements of requirements are used, since requirements automatically incorporatedigestibilit y inempirica l systems (Tanksley & Knabe,1974) . Fuller etal . (1981)compare d dietary formulationo n thebasi so filea ldigestibl eo rgros sdietar yamin oacids : freeamin o acids were added to the diet to meet ideal protein requirements and therewer en oclea radvantage so fbasin gthi ssupplementatio no nilea l digestible rather than gross amino acid content. The response was measureda schang ei nnitroge nretention ; whether thiswa sdu et oth e unsuitabilityo filea lmeasuremen to rt oshortcomin gi nth edescriptio n of ideal protein. While measurements of the digestibility of amino acidsi nth eileu mma yb ebette rtha nthos emad ei nfaece sthe yma yno t beparticularl yaccurat eindicator so fnutritiv evalue . Inth ecas eo f proteins containing poorly-available amino acids ileal digestibility values may seriously over-estimate their quality (E.S. Batterham; personalcommunication) . Themarke dmicrobia lactivit yi nth esmal lintestin eha sconsiderabl e implicationsfo rcarbohydrate sa swell . Millard& Chesso n(1984 )firs t demonstratedmajo rdisappearanc eo fNS Pmonomer sfro mswed eanterio rt o the terminal ileum, as a result of microbial activity, or possibly non-enzymichydrolysi s (NSPar eno tdigeste db yhos tcarbohydrases ,b y definition). Graham, Hesselman &Ama n (1986) found substantial NSP monomer disappearance fromwhea tbran ,whol e croppea san dbee tpulp : uronic acids were particularly digestible. This largely microbial activity is associated with substantial production of volatile fatty acids. Longland,Clos e& Lo w (1989)als ofoun dtha tu pt o70 %o fth e uronicacid san d40 %o fth earabinos e fromdiet scontainin gsuga rbee t pulpwer e digested in pigswit h ileo-rectal anastomoses as shown in Table 4. It seems difficult, therefore, to recommend that carbohydratesca nb eassesse di nterm so fdigestibilit ya tan yspecifi c sites within the gut. Faecal output of monomers gives the overall disappearancevi aeithe renzymi co rmicrobia l routes,bu ti ti sa sye t notpossibl et omak ean yaccurat ecorrectio nfo ran ymonomer swhic har e ofendogenou so rbacteria lorigin ,thoug hclearl ysom eo fthe mmus tbe . However,Longlan d & Low (1989)hav e concluded that less than 12%o f faecal microbial dry matter was NSP in form (bacterial dry matter frequentlyexceed s35 %o ftota lfaeca ldr ymatter) , sothi sproble mi s probablyrelativel yminor .

Table4 . Digestibility (%) ofNS PMonomer so fSuga rBee tPul p(SBP ) inIntac tan dIleo-Recta lAnastomose d (IR)Pig s

TotalNS P Ara Xyl Man Gal Glu UAC Intact 0%SB P 59 67 52 84 83 49 87 30%SB P 76 88 45 83 85 65 96 IR 0% 22 40 18 37 67 12 70 30%SB P 29 49 0 29 47 8 77

Ara= arabinos e ; Xyl =xylose ; Man =mannose ; Gal =galactose ; Glu= glucose; UAC= uronicacid s

8. AbsorptionMeasure d UsingBloo dMeasurement s

During the last 20year sRéra tan d his colleagues at Jouy-en-Josas havedevelope d a technique formeasurin g blood flow rate through^th e hepatic portal vein of pigs using an electromagnetic probe, coupled with catheterization of this vein and the carotid artery. The concentration difference of a nutrient between the afferent (to the gut) arterial blood and the efferent venous blood multiplied by the flowrat ei sequa l toth ene tabsorptio nacros sth egut : ittake sn o accounto fth efat eo fnutrient swhich ,thoug habsorbed ,remai nwithi n the gut tissue (Rérat, Vaugelade & Villiers, 1980). Among many publications in this field, those on absorption of glucose and amino acids from wheat and barley (Rérat, Vaissade & Vaugelade, 1979), variouscarbohydrate s (Rérat,Vaissad e &Vaugelade ,1984a,b) ,volatil e fatty acids (Rérat etal. , 1985) and proteins (Rérat, Vaissade & Vaugelade, 1988; Rerat, Jung & Kandé, 1988) are of particular interest. Themetho d isver y time-consuming,technicall y complexan d generates a very large number of samples. Nevertheless, the information which is produced is extremely detailed and presents a kinetic view of diurnal changes in absorption. In addition to measurements of nutrient absorption, this method has been used to provide the most direct measurements available of the kinetics of ammonia and urea exchanges between the gut and blood in the hepatic portal vein (Rérat & Buraczewska, 1986). The great virtue of the method is that it provides a net abosprtion picture from nutrients absorbedthroughou tth egut . The various methods of measuring protein digestion in pigs have recentlybee ncriticall yreviewe di ndetai lb yLo w& Zebrowsk a (1989).

9. SomeThought sfo rth eFutur e

Thepas t2 0year so r sohav esee na majo r increase inou rknowledg e ofth ebasi cmechanism s inth edigestiv ephysiolog y ofpigs ,an dmuc h useful applied information concerning nutrientdigestibilit y hasals o been obtained. On the other hand, there have been few attempts to manipulate digestive function as a result of this new physiological knowledge. It is often said that digestive efficiency should be increased,bu twha tca nb edon ei npractice ? Inth ecas eo fcereal-base ddiet si ti susua lfo ra tleas t20 %o fth e dry matter ingested to be voided in faeces (including 20% of the nitrogen, with a further 25-30% appearing in urine). This faecal matteri slargel ymicrobial ,endogenou so rfibrou si nform . Canthes e components be reduced? Antibiotics tend to increase nutrient digestibility,presumabl ybecaus ethe ylowe rbacteria l fermentationo f dietary compounds, allowing more digestion by host enzymes to absorbablemonomer srathe rtha nbacteria lmass . Theus eo fantibiotic s isalread ylimite di nsom ecountrie san di ti spossibl etha tthe ywil l only be used for therapeutic purposes before long. If so, can microbialactivit yb ereduced ,o rmodified ,b ydietar ychang einstead , orb ymodifyin ggu tphysiolog ye.g .b ychangin gmotilit yi nsuc ha wa y that passage rate is accelerated to an extent that host enzymic digestion isno t impairedbu tmicrobia l fermentation issuppresse db y insufficienttim efo raction ? Asye tw ehav en omean so ffin econtro l ofmotilit yi npig sbecaus ew ed ono tunderstan denoug ho fth ewa ythi s processi scontrolle di na neuro-endocrin econtext ,an dw ed ono tkno w how the latter could be influenced by the nature of the diet. An alternative strategy might be to try and modify the microbial population so that its activities were mainly complete in the small intestine,followe db ydeat han ddigestio no fth ecell san dabsorptio n ofth enutritiona lcomponent sb yth ehost . Butth elarg eintestin eha s evolved to provide a fermentation chamber forundigeste d dietaryan d endogenous residuesan d itma ywel lb ever ydifficul t tochang ethis . Attempts to modify the microbial population by diet appear to have limited successa sfoun db yLi ue tal . (1985)wh ogav ea wid e rangeo f NSP sources topig s and found no significant evidence ofadaptation . It may well be that the metabolic activity of bacteria is more sensitivet ochang etha tspecie sdistribution . The major extent of endogenous secretion into the gut has been discussed in some detail earlier in this review. Iti spertinen tt o examine whether protein turnover and excretion could be reduced, by dietary means, without detriment to the animal. As yet we cannot predictwhethe rthi si seve na possibility . Thedigestibilit y ofdietar y components sucha s starchan dprotein s appears, from various studies, to be essentially completed, unless thesear e closelyassociate d inth edie twit hNS Pan dlignin : lignin inparticula r isknow n todetermin e the rate of fermentation ofman y feedstuffs (Van Soest, 1985) and so any reduction in the "barrier" effects of these compounds would be worthwhile, perhaps by adding exogenousNSP-digestin genzymes ,a si salread ypractise dt osom eexten t commercially,especiall ywit hyoun gpigs . Suchenzymi ctreatmen tmigh t be effective before the diet is eaten in some cases, in order to provideth eanterio rpar to fth edigestiv etrac twit hcomponent swhic h arelikel yt ob edigestible . Thisca nonl yb edon ei na soundly-base d wayi f thephysico-chemica l structureo f feedsi swel l characterised; without such information, tailoring exogenous enzymes, or other physicaltreatments ,i sver yhaphazard . In conclusion, I believe that fundamental research on digestive physiology, combined with better understanding of food structure and composition will continue to play an important role in raising the efficiencyo fpi gproduction . At the same time itca nb ehope d that theenvironmen twil lb e improvedthroug h reducingeffluen tproduction . Inadditio n therear egoo dprospect s for increasing theus e thatpig s can make, as omnivores, of foods that are inedible or at least unattractive forhuma n consumption,t oproduc e ahighl ypalatabl ean d nutritiousfood .

10 r References Arkhipovets,A.I . 1956. Agecharacteristic so fsalivar ysecretio ni n youngpigs . Journalo fPhysiolog yo fUSSR ,42 ,882-886 . Auffray, P., J. Martinet & A. Rérat. 1967. Some aspects of gastrointestinal transit in the pig. Annales de BiologieAnimale , Biochimie,Biophysique ,7 ,261—269 . Bergner,H. ,0 .Simon ,T .Zebrowsk a& R .Münchmeyer . 1986. Studieso n thesecretio no famin oacid sint oth egastrointestina ltrac to fpigs . 3.Secretio no fure adetermine db ycontinuou sintravenou sinfusio no f 1 N-urea.Archi vfü rTierernährung ,36 ,479-490 . Braude,R. ,R.J . Fulford &A.G .Low . 1976. Studieso ndigestio nan d absorption in the intestines of growing pigs. Measurements of the flowo fdigest aan dpH . BritishJourna lo fNutrition ,36 ,497-510 . Buraczewska,L . 1979. Secretiono fnitrogenou scompound si nth esmal l intestineo fpigs . ActaPhysiologic aPolonica ,30 ,319-326 . Buraczewska,L. ,S .Buraczewsk i &T .Zebrowska . 1975. Digestionan d absorptioni nth esmal lintestin eo fpigs . 2.Amin oaci dconten ti n digesta and their absorption. Roczniki Nauk Rolniczych, 97B(1), 103-115. Buraczewska, L., S. Buraczewski, B. Pastuczewska & T. Zebrowska (Editors). 1989. Digestive Physiology inth ePig . PolishAcadem y ofSciences ,Warsaw ,Poland . 407pp . Corring, T. 1974. Regulation of pancreatic secretion by negative feedback in the pig. Annales de Biologie Animale, Biochimie, Biophysique,14 ,487-498 . Corring,T . 1980. Endogenoussecretion si nth epig . In: Low,A.G .& I.G. Partridge (Editors), Current Concepts of Digestion and Absorption in Pigs. National Institute for Research in Dairying, Reading,U.K . Corring,T. ,C .Just e& E.F .Lhoste . 1989. Nutritional regulationo f pancreatic and biliary secretions. Nutrition Research Reviews,2 . (inpress ) Cranwell, P.D. 1985. The development of acid and pepsin (E.C.3.4.23.1) secretory capacity in the pig: effects of age and weaning. BritishJourna lo fNutrition ,54 ,305-320 . Cuber,J.C. ,J.P .Laplac e &P.A .Villiers . 1980. Fistulationo fth e stomachan dresidua lgastri ccontent safte rintak eo fa semi-purifie d maizedie ti nth epig . Reproduction,Nutritio ne tDéveloppement ,20 , 1161-1172. Cunningham,H.M. ,D.W . Friend& J.W.G .Nicholson . 1963. Observations ondigestio ni nth epi gusin ga re-entran tfistula . CanadianJourna l ofAnima lScience ,43 ,215-225 . Forbes,J.M . 1988. Metabolic aspectso f the regulationo fvoluntar y foodintak ean dappetite . NutritionResearc hReviews ,1 ,145-168 . Friedrich, M. 1989. Physiology of intestinal digestion and absorption. In: Bock, H.D., B.O. Eggum, A.G. Low, O. Simon & T.Zebrowsk a (Editors),Protei nMetabolis m inFar mAnimals . Oxford UniversityPress ,Oxford ,U.K . pp.218-272 . Fuller, M.F., B.A. Baird, A. Cadenhead & R. Aitken. 1981. An assessment of amino acid digestibility at the terminal ileum as a measure of the nutritive value of proteins for pigs. Animal Production,32 ,396 . Graham,H. ,K .Hesselma n& P .Aman . 1986. Theinfluenc eo fwheatbra n and sugar-beetpul po n thedigestibilit y ofdietar y components ina cerealbase dpi gdiet . Journalo fNutrition ,116 ,242-251 . Horszczaruk, F., L.Buraczewsk a & S.Buraczewski . 1974. Amountan d composition of intestinal juice collected from isolated intestinal loopso fpigs . RocznikiNau kRolniczych ,95B(4) ,69-77 .

11 Houpt, T.R. 1982. The controls of food intake in the pig. In: Laplace,J.P. ,T .Corrin g& A .Réra t (Editors),Digestiv ePhysiolog y in the Pig. InstitutNationa l de laRecherch eAgronomique ,Paris , France, pp.17-28 . Just,A . 1989. Ilealdigestibilit yo fprotein : appliedaspects . In: Current Concepts of Digestion and Absorption in Pigs. National Institutefo rResearc hi nDairying ,Reading ,U.K . pp.66-75 . Just,A. , H. J0rgensen &J.A .Fernande z (Editors). 1985. Digestive Physiology in the Pig. National Institute of Animal Science, Copenhagen,Denmark . 401pp . Juste,C . 1982. Endogenoussupplie sfro mth edigestiv esecretion si n the pig. In: Laplace, J.P., T. Corring & A. Rérat (Editors), DigestivePhysiolog y inth ePig . InstitutNationa ld e laRecherch e Agronomique,Paris ,France , pp.155-174 . Kvasnitskii,A.V . 1951. Problems of Digestive Physiology in Pigs. Sel'Khozgiz,Moscow ,USSR . 200pp . Laplace,J.P . 1980. Stomachan dsmal lintestin emotilit yi nth epig : electromyography in nutritional studies. In: Low, A.G. & I.G. Partridge (Editors),Curren tConcept so fDigestio nan dAbsorptio ni n Pigs. National Institute for Research in Dairying, Reading, U.K. pp.24-47 . Laplace,J.P. ,M.B .Felix ,0 . Rampin& J.C .Marcilloux . 1989. Food intake- G.I .trac t relationship inth epig . In: Buraczewska,L. , S. Buraczewski,B . Pastuzewska & T. Zebrowska (Editors), Digestive Physiology inth ePig . PolishAcadem y ofSciences ,Warsaw ,Poland . pp.18-35 . Laplace, J.P., T. Corring & A. Rérat (Editors). 1982. Digestive Physiologyi nth ePig .Institu tNationa ld el aRecherch eAgronomique , Paris,France . 317pp . Liu,Y.F. ,K .Fadden ,E.A .Latymer ,A.G .Lo w& M.J .Hill . 1985. The use of the cannulated pig to study the effect of dietary fibre - supplements on the bacterial flora of the porcine hindgut. in: Just, A., H. J0rgensen & J.A. Fernandez (Editors), Digestive Physiology in the Pig. National Institute of Animal Science, Copenhagen,Denmark , pp.300-303 . Longland, A.C. & A.G. Low. 1989. Digestion of diets containing molassedo rplai nsuga rbee tpul pb ypigs . AnimalFee d Sciencean d Technology, (inpress ) Longland, A.C., W.H. Close & A.G. Low. 1989. Digestion of carbohydrates from sugar beet pulp in pigs with ileo-rectal anastomoses and energy balance measured by calorimetry. In: Buraczewska, L., S. Buraczewski, B. Pastuzewska & T. Zebrowska (Editors), Digestive Physiology in the Pig. Polish Academy of Sciences,Warsaw ,Poland , pp.108-119 . Low, A.G. 1979a. Studies on digestion and absorption in the intestineso fgrowin gpigs . 5.Measurement so fth eflo wo fnitrogen . BritishJourna lo fNutrition ,41 ,137-146 . Low,A.G . 1979b. Ane wautomati cmetho d formeasurin gdr ymatte ran d nitrogenflo wthroug h re-entrantcannula si nth eduodenu mo fgrowin g pigs. Proceedingso fth eNutritio nSociety ,38 ,129A . Low, A.G. 1979c. Studies on digestion and absorption in the intestines of growing pigs. 6. Measurements of the flow of amino acids. BritishJourna lo fNutrition ,41 ,147-156 . Low,A.G . 1982. The activity of pepsin, chymotrypsin and trypsin during24 hperiod s inth esmal l intestine ofgrowin g pigs. British Journalo fNutrition ,48 ,147-159 . Low,A.G .& A.L .Rainbird . 1986. Lacko fevidenc e foramin oaci dan d glucoseabsorptio ni nth estomac ho fpigs . Archivfü rTierernährung , 36,327 .

12 Low,A.G .& C .Fisher . 1989. ProteinEvaluatio ni npig san dpoultry . In: Wiseman,J .& D.J.A .Cole ,Feedstuf fEvaluation . Butterworths, London,U.K . (inpress ) Low, A.G. & T. Zebrowska. 1977. Dry matter and nitrogen in the duodenalcontent so fgrowin gpigs : adiscrepanc yexplained . British Journalo fNutrition ,38 ,145-147 . Low,A.G. ,R.J . Pittman &R.J . Elliott. 1985. Gastric emptying of barley-soya bean diets in the pig: effects of feeding level, supplementary maize oil, sucrose or cellulose, and water intake. BritishJourna lo fNutrition ,54 ,437-447 . LoWj A.G. & I.G. Partridge (Editors). 1979. Current Concepts of Digestionan dAbsorptio ni nPigs . NationalInstitut efo rResearc hi n Dairying,Reading ,U.K . 222pp . Millard, P. & A. Chesson. 1984. Modifications to swede (Brassica napusL. )anterio rt oth etermina lileu mo fpigs : someimplication s forth eanalysi so fdietar yfibre . BritishJourna lo fNutrition ,52 , 583-594. Partridge, I.G. 1978. Studies on digestion and absorption in the intestineso fgrowin gpigs . 3.Ne tmovemen to fminera lnutrient si n thedigestiv etract . BritishJourna lo fNutrition ,39 ,527-537 . Rainbird, A.L. & A.G. Low. 1986. Effect of guar gum on gastric emptyingi ngrowin gpigs . BritishJourna lo fNutrition ,55 ,87-98 . Rérat,A .& L .Buraczewska . 1986. Postprandialquantitativ ekinetic s ofure a and ammonia nitrogen exchanges between the digestive tract and the portal blood in conscious pigs receiving a diet with or withouturea . Archivfü rTierernährung ,36 ,252-269 . Rérat,A. ,J .Jun g &J .Kandé . 1988. Absorptionkinetic so fdietar y hydrolysis products in conscious pigs given diets with different amountso ffis hprotein . 2.Individua lamin oacids . BritishJourna l ofNutrition ,60 ,105-120 . Rérat,A. ,M .Fiszlewicz ,P .Herpin ,P .Vaugelad e &M .Durand . 1985. measurement of theappearanc e ofvolatil e fattyacid s inth eporta l veindurin gdigestio ni nth epig . ComptesRendu sd el aAcadémi ede s Sciences,Paris ,300 ,Seri e3 ,467-470 . Rérat,A. ,P .Vaissad e &P .Vaugelade . 1984. Absorption kineticso f some carbohydrates in conscious pigs. 2. Quantitative aspects. BritishJourna lo fNutrition ,51 ,517-529 . Rérat,A. ,P .Vaissad e &P .Vaugelade . 1984. Absorptionkinetic so f some carbohydrates in conscious pigs. 1. Qualitative aspects. BritishJourna lo fNutrition ,51 ,505-515 . Rérat,A. ,P .Vaissad e &P .Vaugelade . 1988. Absoprtionkinetic so f dietary hydrolysis products in conscious pigs given diets with different amounts of fish protein. 1.Amino-nitroge n andglucose . BritishJourna lo fNutrition ,60 ,91-104 . Rérat, A., P. Vaugelade & P. Villiers. 1980. A new method for measuring the absorption of nutrients in the pig: critical examination. In: Low, A.G. & I.G. Partridge (Editors), Current Conceptso fDigestio nan dAbsorptio ni nPigs . NationalInstitut efo r Researchi nDairying ,Reading ,U.K . pp.177-214 . Sambrook,I.E . 1979a. Studieso ndigestio nan dabsorptio n ingrowin g pigs. 7. Measurements of the flow of total carbohydrate, total reducing substancesan d glucose. British Journal ofNutrition ,42 , 267-277. Sambrook,I.E . 1979b. Studieso ndigestio nan dabsorptio n ingrowin g pigs. 8. Measurements of the flow of total lipid, acid detergent fibre and volatile fatty acids. British Journal of Nutrition,42 , 279-287.

13 Sauer,W.C .& L .Ozimek . 1986. Digestibilityo famin oacid si nswine : results and their practical applications. Livestock Production Science,15 ,367-388 . Sauer, W.C., S.C. Stothers, G.D. Phillips & R.J. Parker. 1977. Apparent and true availability of aminoacid s inwhea t andmillin g by-products for pigs. Canadian Journal of Animal Science, 57, 775-784. Schneeman,B . 1982. Digestiveenzym eactivitie sfro mth epancrea si n response to diet. In: Laplace, J.P., T. Corring & A. Rérat (Editors),Digestiv ePhysiolog yi nth ePig . InstitutNationa ld el a RechercheAgronomique ,Paris ,France , pp.125-131 . Sibbald, I.R. 1987. Estimation of bioavailable amino acids for feedingstuffs for poultry and pigs: a review with emphasis on balance experiments. Canadian Journal of Animal Science, 67, 221-300. Simoes-Nunes,C . 1982. Someaspect so fdigestiv eenzym edevelopmen t with age and diet composition adaptation. In: Laplace, J.P., T.Corrin g & A. Rérat (Editors), Digestive Physiology in the Pig. Institut National de la Recherche Agronomique, Paris, France. pp.133-151 . Simon,0 . 1989. Metabolismo fprotein san damin oacids . In: Bock, H.D., B.0. Eggum, A.G. Low, 0. Simon & T. Zebrowska (Editors), ProteinMetabolis mi nFar mAnimals . OxfordUniversit yPress ,Oxford , U.K. pp.273-366 . Simon,0. ,R .Münchmeye r &T .Zebrowska . 1982. Studieso n therang e of tissue protein synthesis in pigs: the effects of thyroid hormones. BritishJourna lo fNutrition ,48 ,571-582 . Tanksley, T.D. & D.A. Knabe. 1984. Ileal digestibilities of amino acids in pig feeds and their use in formulating diets. in: Haresign, W. & D.J.A. Cole (Editors), Recent Advances in Animal Nutrition- 1984 . Butterworths,London ,U.K . Van Soest, P.J. 1985. Definition of fibre in animal feeds. In : Haresign, W. & D.J.A. Cole (Editors), Recent Advances in Animal Nutrition- 1985 . Butterworths,London , pp.55-70 . VanWeerden ,E.J. ,J .Huisman ,P .Va nLeeuwe n& P .Slump . 1985. The sensitivity of the ileal digestibility method as compared to the faecal digestibility to method. In: Just, A., H. Jorgensen & J.A.Fernande z (Editors),Digestiv ePhysiolog yi nth ePig . National Instituteo fAnima lScience ,Copenhagen ,Denmark , pp.392-395 . VanWeerden ,E.J. ,P .Slum p& J .Huisman . 1980. Aminoaci ddigestio n indifferen t parts of the intestinal tract in pigs. In: Oslage, H.J.& K .Roh r (Editors),Protei nMetabolis man dNutrition . Vol.1 , E.A.A.P. Publication No. 27, Braunschweig, West Germany. pp. 207-214. Waterlow,J.C. ,P.J .Garlic k &D.J .Millward . 1978. ProteinTurnove r in Mammalian Tissues and In the Whole Body. North-Holland Publishers,Amsterdam ,Netherlands . 804pp . Zebrowska,T . 1973. Digestionan dabsorptio no fnitrogenou scompound s inth e large intestine of pigs. Roczniki Nauk Rolniczych, 95B(3), 85-90. Zebrowska,T . & L.Buraczewska . 1972. Influenceo f dietary protein level on the rateo fdigestio n inth e small intestine ofpigs . I. Amount and composition of digestion. Roczniki Nauk Rolniczych, 94B(1),71-75 . Zebrowska, T.,A.G . Low & H. Zebrowska. 1983. Studies on gastric digestiono fprotei nan dcarbohydrate ,gastri csecretio nan dexocrin e pancreatic secretion in the growing pig. British Journal of Nutrition,49 ,401-410 .

14 Zebrowska, T., A.G. Low & H. Zebrowska. 1986. The flow and compositiono fduodena ldigest aan dpancreati c juiceo fpig sgive na protein-freediet . Archivfü rTierernährung ,36 ,331-332 . Zebrowska, T., O. Simon, R. Münchmeyer, E. Wolf, H. Bergner & H.Zebrowska . 1982a. Flowo fendogenou samin oacid salon gth egu t ofpigs . Archivfü rTierernährung ,32 ,431-444 . Zebrowska, T., O. Simon, R. Münchmeyer, E. Wolf, H. Bergner & H.Zebrowska . 1982b. Investigationo nth eamin oaci d secretionan d absorption in the stomach of the growing pig. Archiv für Tierernährung,32 ,703-710 .

15

L ANTINUTRITIONAL FACTORS (ANFs)I N THENUTRITIO N OFMONOGASTRI C FARM ANIMALS

J. Huisman

TNO Institute forAnima l Nutrition and Physiology (ILOB), P.O. Box 15, 6700 AA Wageningen

Summary

Many seeds contain ANFs. In soya, beans and peas the most important ANFs are trypsin inhibitors,lectins , tannins and antigenic proteins, Some examples of the effects of these factors inmonogastri c farm animals are presented and discussed. Various points regarding the adequacy of analytical methods for lectins, trypsin inhibitors and antigenic prot eins are discussed. Some assays for lectins and antigenic proteins have b een shown to be inadequate. Examples are given demonstrating that there is a difference in sensitivity toANF s between animal sp ecies. The results show that studies into the effects ofANF s shou Id be carried out in target animals. For future prospects the following po ints are discussed: thewa y ANFs act in the animal, improvement of ana lytical methods and threshold levels for ANFs.

1. Introduction

Many feedstuffs contain factors which produce different deleterious effects in animals (Liener, 1980;1989 ;Marquardt , 1989; Pusztai, 1989). If these factors cause anegativ e effect on growth, feed conversion efficiency and/or health, they are referred to as "antinutritional factors" (ANFs). However, in this definition fibre may also be classified as anANF . Therefore, one restriction is thatANF s have no feeding value. In plants and seeds these factors often act as biopesticides, protecting the seed against moulds, bacteria, and birds (Birk, 1987,1989 ; Bond & Smith, 1989;Liener , 1980; 1989). ANFs can be classified invariou s ways. In the following scheme they are classified on the basis of their effects onnutritiv e value of feedstuffs and biological response in the animal: - factors which have a depressive effect on protein digestion and on the utilization of protein (trypsin and chymotrypsin inhibitors,lectins , polyphenolic compounds, saponins). - factors which have anegativ e effect on digestion of carbohydrates (amylase inhibitors,polyphenoli c coumpounds, flatulence factors). - factors which have anegativ e effect on the utilization of minerals (glucosinolates, oxalic acid, phytic acid, gossypol). - factors which inactivate vitamins or cause an increase in the animals' vitamin requirement (anti-vitamins). In this overview the following pointswil l be discussed: the occurrence ofAN F in different seeds, some effects of ANFs in the animal, adequacy ofAN F analysis, sensitivity of different animal species toANF s and some future prospects.

2- Occurrence in seeds

Many seeds contain ANFs. The levels of different ANFs vary considerably between the different seeds. Table 1 summarizes theANF s in the various seeds.

17 It shows that protease inhibitors and lectins are most important in the legume seeds soya, peas and beans, but some varieties of rye and triticale may also contain moderate levels of trypsin inhibitors. Tannins are mainly present in the coloured flowering varieties of Vicia faba beans and peas, and in sorghum and rapeseed. Glucosinolates are important in rapeseed, alkaloids in lupins, and gossypol in cottonseed. As demonstrated by Liener (1981), one particular seed often contains more than one ANF. For example soya contains: trypsin inhibitors, haemagglutinins (lectins), goitrogens, antivitamins, phytates, saponins, oestrogens, flatulence factors and allergens. This overview will be restricted to the ANFs present in the legume seeds soya, beans and peas.

Table 1. Antinutritional factors in cereals and seeds.

Cereals/seeds Antinutritional factors

Protease Lectins Tannins/ Others inhibitor Polyphenolic compounds

Cereal grains

Wheat -/+ Barley -/+ Rye -/+/++ Triticale -/+/++ Rice -/+ Sorghum -/+ +/++/+++ Corn -/+

Legume see ds

Soya ++/+++ ++ - - Vicia faba bean -/+ + ++/+++ - Phaseolus bean -/+/++ ++/+++ + - Peas +/++ +/++ -

a) Lupins _ _ +/++/+++

Other seeds

b) Rapeseed +/++ +/++/+++

d) Sunflowerseed -/+ +/++

c) Cottonseed -/+ +/++/+++ e) Peanut +/+ +

Below detection limit a)Alkaloid s + Low level b) Glucosinolates ++ Medium level c)Gossypo l +++ High level d) Phenolic compounds (3-3.5%) e)16-18 2 tannins in the skin 3. Some effects ofANF s inmonogastri c farm animals

As mentioned, a seed of one particular species may contain various ANFs. Therefore, the negative effects in the animal is generally not attributed to one particular ANF.Moreover , the various ANFs have their own specific effects. In Table 2a survey is given of the major effects ofANF s on digestion and the utilization of nutrients in monogastric animals.

Table 2.Majo r effects inmonogastri c farm animals of antinutritional factors present in legume seeds

Antinutritional Major inviv o References factor effect

Lectins -damage of gutwal l Donatucci,1983 ; Donatucci et al., 1988; -immunological Greer, 1983;Kik ,1988 ; reaction Liener, 1986;Pusztai , -metabolism toxicity 1987.

Protease inhibitors -trypsin-chymotrypsin -reduction activity Burns, 1987;Liener , inhibitor of (chymo)trypsin 1979; Liener and Kakade, -pancreas hypertrophy 1980; Richardson, 1^80- -decreased digestion 81;Scarbieri &Whitaker , 1982; Birk,1989 .

-«-Amylase inhibitor -forming complexwit h Powers &Whitaker , 1977. amylase in salivary and pancreatic juice -reduces starch avai- labiljt y

Tannins and polyphe­ -forms complex with Griffith, 1981;Philip s noli c compounds enzymes or feed et al., 1981;A w & protein Swanson, 1985;Marquardt , -reduces protein 1989. digestibility

Flatulence factors -gastrointestinal Fleming, 1981;Flemin g discomfort et al., 1988;Saini , -increased maintenance 1989.

Phytic acid -forms complexwit h Reddy et al., 1982. minerals,an d protein -depresses absorption of minerals

Antigenic proteins -gutwal l damage Miller et al., 1984; -immune response Seegraber andMorril , 1982, 1986; Sissons and Smith, 1976;Kilsha w and Sissons, 1979.

Adapted with modifications from Van der Poel (1989).

19 Lectins are characterized by their unique ability to bind to specific sugars. Glycoproteins in the gutwal l contain sugars towhic h lectins have affinity and, as a result,bindin g of lectins to the epithelial cells occur. Due to this binding, a series of effects occur, accumulating in growth depression. Themai n biological effects induced by the binding of lectins are:damag e to the gutwal l (Jaffe,1980 ; Meyer et al., 1982;Pusztai ,1987 ;1989 ;Torres-Pinedo , 1983), impaired transportation of nutrients across the intestinal wall (Donatucci et al., 1988;Jaffe , 1980;Liener , 1986), increased synthesis of mucosal protein (Liener, 1986;Pusztai , 1987), muscle atrophy (Pusztai, 1989), depressed blood insulin levels (Pusztai, 1989), inhibition of brush border hydrolases (Kim et al., 1976;Nakat a &Kimura , 1980), and effects on the immune system (Pusztai, 1989). These effects may result in serious growth depression (Grant et al., 1983;Pusztai et al., 1981; King et al., 1983;Huisma n et al., 1987,1989 ;Huisma n &va n der Poel, 1989,a,b,c,). Protease inhibitors arewidel y distributed in plant seeds. Legume seeds in general contain high levels of these inhibitors. There are various families of plant protein inhibitors (Birk, 1989). The main inhibitors in legume seeds are the trypsin and chymotrypsin inhibitor. These inhibitors are peptides which can form stable, inactive complexes with the proteolytic enzymes from the pancreas (Kakade &Liener , 1980). Due to this complex forming, the activity of the trypsin and chymotrypsin is decreased (Liener &Kakade , 1980;Racki s &Gumbmann , 1981; Rackis et al., 1985). Inactivation of the trypsin in the gut induces the endocrine cells in themucos a to release more of the hormone cholecystokinin (CCK)whic h stimulates the pancreas to produce more digestive enzymes, such as trypsin, amylase and elastase (Birk,1989 ; Liener &Kakade , 1980;Liener , 1989,Schuma n et al., 1983). The net result is an endogenous loss of protein rich in S-containing amino acids which leads to depressed growth (Kakade &Liener , 1980;Liener , 1989). Because of the stimulated production of pancreatic enzymes, the pancreas becomes enlarged in small animal species due tohypertrophi c and hyperplastic changes inmorpholog y (Birk, 1989;Gallahe r & Schneeman, 1986; Gumbmann et al., 1985;Liene r &Kakade , 1980). The growth depressing effect of protease inhibitors ismainl y attributed to the loss of endogenous protein due to the negative feedback mechanism. The oc-amylase inhibitor has been indicated as being responsible for the impaired digestion of starch in red kidney beans (Jaffe &Veg a Letta, 1968). However, addition of this purified inhibitor did not affect the starch availability (Savaiano et al., 1977). Therefore, this factor seems to be ofmino r importance in these beans. Tannins are polyphenolic compounds. Thewa y these compounds act in the animal isno t entirely clear. Tannins form complexes with proteins and carbohydrates in the feed but alsowit h digestive enzymes. Due to this complex forming, the activity of digestive enzymes and the digestibility of nutrients is decreased (Griffiths &Mosely , 1980; Marquardt, 1989). Other antinutritional effectswhic h have been attributed to tannins are damage to the gutwall , toxicity of tannins absorbed from the gut and interference with the absorption of some minerals (Liener, 1989;Mitjavil a et al., 1977). Flatulence factors are related to oligosaccharides which are broken down by intestinal bacteria in the large intestine. These oligosaccharides are not broken down in the small intestine due to the lack of appropriate enzymes,an d so they flow into the large intestine where they are degraded by the action of bacterial a-galactosidae.

20 The cleavage products are converted into carbon dioxide,hydroge n and methane, resulting in flatulence,d iarrhoea,nausea , cramp and discomfort in the animals (Rackis, 1975; Saini, 1989). Antigenic proteins cause gutwa l 1 damage and immunological reactions in the gut linked with disorders in gut function in piglets and veal calves (Kilshaw & Sissons, 1979;Mi lier et al., 1984; Seegraber and Morril, 1982,1986 ; Sissons and Smi th, 1976). Phytic acid forms complexes with divalent and trivalent metal ions resulting in a reduced availability ofmineral s such as Ca,Mg , Zn,C u and Fe. It also inhibits several of digestive enzymes such as pepsin, pancreatin and a-amylase.

In the following some examples of the effects ofANF s in monogastric animals are presented. Table 3 summarizes the growth results of an experiment inwhic h piglets and ratswer e fed a control diet or test diets containing ZOZ raw or toasted Phaseolus vulgaris beans.Al l diets were balanced for contents of protein and amino acids,ne t energy, and vitamins and minerals (for details see Huisman and Van der Poel, 1989b). Phaseolus vulgaris beans were chosen as being representative of a seed containing high levels of toxic lectins.

Table 3.Growt h and feed conversion efficiency in piglets and rats fed Ph. vulgaris bean for 3weeks . ,

Growth Feed conversion efficiency

Diets g/day SD SD

Piglets

control 137.7 19.3 100 1.74 0.12 100

20Z raw beans in the diet -36.0 5.8 -26 negative

201 toasted beans in the diet 111.8C 20.7 81 2.09c 0.11 120

Rats

control 6.68 0.24 100 2.99 0.21 100

2.0Zra w beans in the diet 4.86 0.20 73 3.72 0.32 124

207. toasted beans in the diet 6.32 0.46 95 3.10a 0.18 104

Data in the same column of each specieswit h a different superscript differ significantly (P< 0.05).

21 The results showmarke d negat ive effects due to the inclusion of 20Z raw Phaseolus vulgaris beans inb ' oth animal species.However ,weigh t gain was distinctly more depressed in piglets than in rats. The piglets even lostweigh t when the raw bean swer e fed.Whe n feeding toasted beans, there was still anegativ e ef feet onweigh t gain in both species,whic h may indicate that the ANFs in these beans were insufficiently reduced. The results suggest that ANFs present in Phaseolus vulgaris beans are highly toxic for the animal, Pusztai (1981)demonstrate d that the negative effects may be speci fically related to the lectins present in these beans.

When peas are included in the diets of piglets, reduced weight gain is often observed (Castaing and Grosjean, 1985;Feket e et al., 1984; Freire et al., 1989;Grosjea n and Castaing, 1983;Grosjea n et al., 1986; Grosjean and Gatel, 1989). This effect is possibly related to the presence of antinutritional factors in peas (Griffiths, 1981,1984 ; Grosjean and Gatel 1989;Hov e and King, 1979; Leterme et al., 1989; Savage, 1989; Stickland 1984;Valdebouz e et al., 1980). Table 4, using the results of Freire et al. (1989), illustrates the negative effects due to the inclusion of peas in the diets of young piglets.

Table 4.Weigh t gain and feed conversion efficiency of piglets fed raw peas for 6week s (liveweight 6.7 -25. 2 kg)

Treatment Weight gain Feed conversion efficiency

After After After After 3 weeks 6 weeks 3 weeks 6 weeks

g/day X g/day control diet 395 100 491 00 1 42" 100 1 54" 100 dietwit h 15Z peas 329b 83 443t 90 1 69b 119 1 69bC 110 diet with 30Z peas 310~ b 78 412b 84 1 70b 120 1 72b 112 t diet with 452 peas 311 79 415t 85 1 76b 124 1 75ab 114

Data within the same columnwit h adifferen t superscript differ significantly (P< 0.01)

It is not entirely clear which factor is responsible for the negative effects. Leterme et al. (1989)an d Savage (1989)discusse d the fact that the presence of trypsin inhibitors may be the most important factor in this respect.A n ILOB study also indicated that trypsin inhibitors may play a role in the negative effects. In this study, piglets were fed four diets each containing 30Z from different batches of'Finale 1peas , with different trypsin inhibitor contents.A s a first step, the protein faecal digestibility of the four batches of Finale peaswa s measured. The batchwit h the highest trypsin inhibitor contentwa s the lowest in protein digestibility. The same batches were tested in a growth trial with piglets. The diets of the growth trialwer e balanced for essential amino acids,ne t energy, vitamins andminerals .

22 The diet containing the batchwit h the highest level of trypsin inhibitor showed the lowest growth and highest feed conversion efficiency. The results of the feed conversion efficiency are presented in Figure 1.

Figure 1. Relation of trypsin inhibitor activity in peas and feed conversion efficiency in piglets

feed conversion efficiency

1,75

1,70

1,65

1,60

1,55[ _

0,70 0,75 0,80 0,90 0,95 Trypsin inhibitor activity (mg/g product) in peas

Summarizing the results it can be concluded that there are indications that trypsin inhibitors may play a role in the negative effect when high levels of peas are included in the diets of young piglets.

Vicia faba beans contain different ANFs. It is generally assumed that tannins are the most important ANF from anutritiona l point of view (Marquardt, 1989). Levels of tanninsma y vary considerably between varieties (Cabrera et al., 1986), In one of our studies (Jansman et al., 1989), four varieties with different tannin contents were used in experiments with young piglets and broilers. In piglets, the ileal and faecal digestibilities were measured. Each test group comprised six animals,th emea n liveweigh t during the test period was approximately 16kg . Faeces and ileal chymewer e both collected for five days. The digestibility of the beans was calculated by difference. In chickens a growth trial was performed. At the start of the experiment, the age of the chickswa s 5day s and themea nweigh t per chickwa s 123 g. Each treatment group comprised 90 chicks (6cage s of 15 birds each). The

23 experiment lasted threeweeks . In both piglets and broilers, 302 of the beans were included in the diets. The broiler dietswer e balanced for contents ofmetabolizabl e energy, protein, lysine,methionine+cystine , threonine, tryptophan and arginine,C a and P. Themea n results are summarised in Table 5.

Table 5. Effect of tannins in faba beans onweigh t gain in broilers and protein digestibility in piglets

Diet TanniTannin nconten content t BroilerBroilers s Piglet of beans(2) * Weight gain Protein digestibility beans

(g/day) ileal faecal control - 1039a 302 faba A 0.6 1038a 85a 89a 301 faba B 1.2 1047a 75b 85ab 302 faba C 1.5 1058a 74b 82bC 302 faba D 1.6 1046a 69b 79C

* Folin Denis

Data in the same column with a different superscript differ significantly (P< 0.05)

Inclusion of 302 faba beans with tannin contents (Folin Denis method) ranging from 0.6 to1.6 2i n the diets,ha d no effect onweigh t gain in broilers. The general level ofweigh t gain in the test period can be described as very high for each treatment. In piglets, therewa s a distinct difference in protein digestibility between the varieties. Protein digestibility was depressed with increasing tannin levels.Th e differences were more marked at ileal level than at faecal level.Th e results demonstrate that piglets are sensitive to tannins, but chickens arenot .

Soya beans contain various ANFs such as trypsin inhibitors, lectins and, according to Smith et al. (1982)an d Sissons (1982), also antigenic proteins. When soya flour isheated , themajo r part of the activity of trypsin inhibitors and lectins is reduced. However, some of the protein still remains antigenic. Using an alcohol treatment, the antigenicity of the soya protein is reduced and oligosaccharides are removed (Kilshaw& Slade, 1982;Sissons , 1982;Sisson s et al., 1982). ILOB studies have demonstrated that inclusion of alcohol-treated soya at high levels in diets for fast growing veal calves still resulted innegativ e effects on performance. In order to gain some insight intowhethe r this negative effect is related to digestibility or to othermetaboli c processes,th e following experiment was carried out.A n alcohol-treated soya concentrate with a low titre for antigenicity was tested for digestibility invea l calves. This figure was used to balance a control diet and a dietwit h soya as the sole protein source for digestible amino acids. Both dietswer e fed tovea l calves of approximately 100 kg in aN balance trial. The N balance was measured for two consecutive periods of five days each. The results are given in Table 6.

24 r Table 6. N balance invea l calves of approximately 100 kg live weight

Protein source N balance

g/day SD

Skimmed milk powder 42.8 a 1.4 100 Soya concentrate 34.4l 2.5 80

a -b :significantl y different (P< 0.001 )

The results clearly show that in spite of the alcohol treatment of soya and balancing the diets on digestible amino acids,th e N balance in the soya-fed calves was distinctly lower compared with the control calves fed a diet with skimmed milk powder as the sole protein source. Ina study with calves fed the same batch of soya,gu twal l damage was observed in spite of a low titre for antigenicity. It isno t clear which factor is responsible for the reduced N balance when soya is fed to calves. However, itwa s found that the analytical method commonly used to determine of antigenic compounds was inadequate.Usin g an ELISA method, antigenic compounds were still analysed in this soya (see chapter 4: Discussion points on analytical procedures). Moreover, this soya also contained low levels of trypsin inhibitors and lectins. Itma y be that veal calves are also sensitive to low levels of these factors.

4. Discussion points on analytical procedures

The adequacy of analytical methods forANF s is essential for research into the effects of ANFs in animals and for studies into reducing the negative effects of ANFs. When the analytical procedures are critically evaluated, one comes to the conclusion that themethod s used are often inadequate. •Someo f the most important analytical methods forAN Fwil l be discussed.

4.1 Lectins

Haemagglutination of red blood cells ismos t commonly used to measure lectin activity. This method is based on the the sugar binding properties of lectins to glycoproteins present on the surface of the red blood cells.Du e to this binding, the cells agglutinate and the amount of agglutinated cells is used tomeasur e lectin activity.Marquard t et al. (1975) reported that red blood cells from different animal species haemagglutinate differently with lectins from the same sample. It is not clear which type of red blood cells gives the most reliable results. In our studies,w e also found differences in the haemagglutination activity of different red blood cells with lectins present in the same sample (Table7) . As previously discussed by Huisman et al. (1987), important discussion points concerning thismetho d are: - do the glycoproteins in the red blood cells contain the same sugars as those in the gutwall ? In otherwords : towha t extent does the haemagglutination method predict the binding of lectin to the surface of the gut wall? - not all lectins are pathogenic to the same extent, e.g. lectins from peas are less pathogenic than those in the Phaseolus vulgaris bean. Moreover, one particular seed can contain different types of (iso)lectins. An important question iswhethe r these lectins are all (and to the same extent) pathogenic for the animal.

25 Table 7.Compariso n ofhaemagglutini n activity of three red blood cell origins.

Haemagglutinin activity (units*/mg sample)

Rabbit Pig Horse

Ph. vulgaris cv. Processor - raw 80 20 640 - 20min . steam heated 5 1 100

Ph. vulgaris cv. Procol 20 5 not determined raw soya beanmea l 20 1 0.1

* One haemagglutinin activity unit is defined as the smallest amount of sample required for agglutination under test conditions. Haemagglutinin activity is expressed inhaemagglutini n units per milligram sample (Valdebouze et al., 1980).

- lectins with one binding sitewhic h can originate due to processing, do not haemagglutinate red blood cells.However , they can bind to the gut wall and may therefore cause damage. These discussion points indicate that the haemagglutination method can­ not, in principle be adequate.A first step to improve the analysis of lectins was the development of an ELISA (Enzyme-Linked Immuno-Assay)fo r lectins. In this method, lectin antibodies are raised and used to determine lectins in the samples.Th e advantage of the ELISA method compared to the haemagglutination assay is the higher sensitivity and the fact that all lectins are determined. Using the ELISA method, lectins having no specificity to erythrocytes but to leucocytes,an d the so-called mono-lectins can also be analysed. One disadvantage of ELISA is that no differentiation can bemad e between the different types of lectins, including toxic and non-toxic lectins. A very promising assay for determining of different types of lectins is FLIA (Functional Lectin Immuno-Assay), developed by Hamer et al. (1989). This method is based on the ability of lectins to bind to microtitre plateswhic h have been coated with either a carbohydrate matrix or a gut wall brush border membrane preparation. Principally, this method can be adapted tomeasure ,th e binding ofvariou s lectins from different legumes to the gutwal l of theanimal .

4.2 Antigenic proteins

Soya proteins have been shown to cause immuno-responses accompanied by disorders in gut function in young piglets and veal calves (Kilshaw and Sissons, 1979;Mille r et al., 1984; Seegraber, 1982,1986 ; Sissons & Smith, 1976, Toullec &Guilloteau , this volume). It isno t entirely clear whether these responses should be attributed to the low levels of lectins present in treated soya or to the fact that soya proteins have been proven to be antigenic. Sissons (1982)reporte d that antigenicity of soyawa s eliminated after an alcohol treatment. The antigenicity of the soya tested in that studywa s measured using a passive haemagglutination method. In thismetho d tanned red blood cells are coated with soya. The coated cells aremixe d with blood samples containing soya antibodies. If antigenic compounds are present in the soya, antibodies bind to the coated soya and, as a result, the red blood cells agglutinate (Kilshaw & Sissons, 1979). In one of our studies,w e

26 r used this method to determine the'antigenicity in two batches of soya. One batchwa s split up into two parts.On e partwa s mildly treated with alcohol in order to get a "high-antigen soya" concentrate and the other part treated according to the adequate procedure in order to get a "low- antigen soya" concentrate. The alcohol treatments were carried out by a manufacturer of soya concentrates for veal calves.Bot h batches were analysed by the manufacturer and by our own laboratory according the passive haemagglutination procedure. The mean analysed values of both laboratories for antigenicity of soya were: a titre of <1 for the "low-antigen soya concentrate" and a titre of 6/7 for the "high-antigen soya concentrate". Both batches were tested for ileal and faecal digestibility, immune response and gutwal l damage. There were no differences in ileal and faecal digestibility in either batch,no r was an immune response or gutwal l damage observed. These results did not correspond with the analytical data for antigenicity because no immune response or gutwal l damage should be expected for the "low-antigen batch".A s a follow up,a n ELISA method for determining antigenic proteins was developed. Calves were fed soya and soya protein antibodies were raised. The positive serum of these calves was used to determine antigenic active proteins in the soya concentrates. The titres for both batches using the ELISA method were:7 for the "low-antigen soya" and 8 for the "high antigen soya". These analytical results corresponded well with the results from the animal study. Based on these results, itmus t be concluded that the passive haemagglutination method is inadequate for determining of antigenic proteins. ,,

4.3 Trypsin inhibitors

One of the problems with this analysis is that there are many different methods applied and,moreover , the units inwhic h the trypsin inhibitor activity is expressed are often different. Therefore, it is difficult to compare the results of one laboratory with those from another. Further point is that mainly bovine trypsin is used to determine trypsin inhibitor activity. Boisen (1989)demonstrate d that the use of trypsins from different origins in the assay gives different values. Therefore, itma y be that the use of bovine trypsin gives misleading results when related to the biological response in non-bovine species. Thus, trypsins related to the target animal should be recommended for use in the trypsin inhibitor assay. Another discussion point is that some other factors can inhibit trypsin activity, e.g. tannins.Usin g the common trypsin inhibitor assay (mostly based onKakad e et al., 1974), the true trypsin inhibitor activity may be overestimated. At theAN FWorksho p of 1988 therewa s a strong feeling that an international committee should be set up to develop standardized protocols for determining trypsin inhibitor activity.

4.4 Tannins

Different methods are described to determine tannin contents in feeds and feed ingredients.Mos t are based on the colorimetric principle of determination. However, the results of these assays depend on the colouring agents, reaction conditions and the standard agents used. Furthermore, tannins consist of aheterogeni c group of chemical substances with assumed distinct nutritional effects.Therefore , the colorimetric methods for tannin determination dono t seem to be appropriate. To be able to determine "toxic" tannins,assay s based on the HPLC principle seem promising.Wit h this type of assay, tannins can be separated according to their chemical structure.

27 i_ Further research should clarify the relationship between the presence of different types of tannins in diets for pigs and poultry and their nutritional effects. The results of tannin assay should quantitatively predict the nutritional harm of the tannins involved.

5.Anima l species differences

The mode of action ofANF s in the animal ismainl y studied in rats and chickens.Also , the effects of technological treatments for the inactivation ofANF s are often tested in rats using parameters such as PER, NPU, N balance andweigh t gain.Base d on these results, conclusions are drawn regarding the optimal treatments for inactivating of the ANFs. An important question iswhethe r the results obtained with rats and chickens can be extrapolated to other animal species such as pigs. It is crucial to know towhic h levels theANF s have to be reduced in order tominimiz e their harmful effects. In relation to this, it is also essential to knowwhethe r the animals are equal in their sensitivity to ANFs. There is also hardly any information on threshold levels in target animals. So far only a few data have been published on the effects of ANFs in the different animal species. Studies by Combs et al. (1967)an d Yen et al. (1977) suggest that rats and piglets respond differently when fed raw soyabeans.Visitpanic h et al. (1985) found that the rat and piglet respond differently to the feeding of chickpeas. Liener &Kakad e (1980)demonstrate d that the response of the pancreas to trypsin inhibitors varies between animal species (Table8) .

Table 8. Relationship between size of pancreas of various species of animals and response of pancreas to raw soybeans of trypsin inhibitor.

Species Size of pancreas Pancreatic Reference (Z of bodyweight ) hypertrophy

Mouse 0.6 - 0.8 Schingoethe et al. (1970 ) Rat 0.5 - 0.6 Kakade et al. (1973) Chick 0.4 - 0.6 Lepkovsky et al. (1959) Guinea pig 0 29 Patten et al. (1973) Dog 0.21 -0.24 Patten et al. (1971) Pig 0.10 - 0.12 Yen et al. (1971) Human 0.09 - 0.12 <-)v Calf 0.06 -0.08 Kakade et al. (1976)

Observed in young guinea pigs but not in adults Taken from Long (1961) Predicted response

Table adapted from Liener and Kakade (1980)

As demonstrated in this table,pancrea s hypertrophy due to trypsin inhibitors occurs in smaller animals but not in larger ones such as pigs. However,w e should not concluded from these results that the pancreas in larger animals isno t stimulated to producemor e pancreatic enzymes due to the negative feedback mechanism induced by trypsin inhibitors. In a study by Schuman et al. (1983), increased secretion of

28 pancreas enzymes was found in pigs fed raw soyabean meal. Summarizing this, onemus t conclude that there is a significant lack of knowledge regarding the sensitivity of target animals toANFs . A series of experiments was, therefore, carried out by ILOB-TNO together with theAnima l Nutrition Department of the Agricultural University, to study the effects of ANFs in piglets, rats and chickens. In these studies, the Phaseolus vulgaris beanwa s chosen as representative ofa seed containing high levels of•toxic lectins,an d the pea (Pisum sativum)a s an example of a seed containing moderate levels of trypsin inhibitors and lectins. Moreover, according to Bertrand et al. (1988) the pea lectins are assumed to benon - or low toxic.Wit h Ph. vulgaris beans two experiments were carried out inwhic h 20% raw beans were incorporated into the diets. In the first experiment, the diets were balanced on total protein contents and in the second the diets were balanced on contents of digestible protein (details see:Huisma n &Va n der Poel, 1989b,c). An experiment was also carried outwit h diets containing 301 peas. These diets were balanced on digestible protein content (for details see Huisman &Va n der Poel, 1989d). The results of these studies are summarized in Table 9.

Table 9. Effects of ANFs indifferen t animal species

Inclusion rate Criteria Inviv o effects in the diet piglets ats chickens / 20£ Ph.vulgari s Weight gain /o 0 Pancreas weight* -10 + + Spleenweight * •I- 0 0 Protein digestibility - N.D.

30Z Pisum sativum Weight gain 0 0 Pancreas weight* + + Spleenweight * 0 0

0 =n o effect + = increased weight -/--/ /= decreased weight N.D. = not determined * = 2 of live weight

The results showmarke d differences between piglets on the one hand and rats and chickens on the other.Wit h Ph.vulgari s beans,piglet s did not gain weight, they even lost it.Weigh t losswa s also evident when the diets were balanced on digestible protein, evenwhe n extra casein was added to the diets. In rats, therewa s someweigh t losswit h the diets balanced on total protein, butwhe n balanced on digestible protein therewa s noweigh t loss. In chickens there was no negative effect on weight gain due to feeding thebeans . The pancreas weight was increased in rats and chickens but not in piglets. The increased weight of the pancreas in rats and chickens may be related to the presence of trypsin inhibitors in the beans. In both experiments, spleenweigh t was markedly reduced in the piglets but not in rats or chickens. In the second experiment, the samewa s observed for the thymus weight. These results may indicate that the immune system in piglets ismor e sensitive toANF s than in rats or chickens. Protein digestibility of the dietswa s distinctly more depressed in piglets compared to rats. With respect to the experiment with peas, a reduction inweigh t gain was

29 L observed in piglets. In rats and chickens weight gainwa s not affected. The pancreas weight was increased in the rats and chickens but not in the piglets. The results presented here clearly demonstrate that there is adiffe­ rence in sensitivity between animal species toANF s present in Ph. vul­ garis beans and Pisum sativum. For Vicia faba beans adifferenc e in sensitivity toANF s was also found between piglets and chickens (see chapter 3, Table 5).Piglet s are distinctly more sensitive toANF s in beans and peas than rats and chickens. Thus, the results obtained with rats and chickens cannot be extrapolated to piglets. Therefore, studies into the effects ofANF s should be carried out in the target animal species. 6.Futur e prospects

The use of legume seeds in diets for monogastric farm animals is seriously hampered by the fact that these seeds contain ANFs which have negative effects on animal performance. The nutritional value of these seeds can be improved ifw e are able to reduce these negative effects. However, there is insufficient knowledge about thewa y these factors act in the target animal. For the future itwoul d beworthwhil e to consider the following points: Studies into thewa y ANFs act in the animal An important part of present knowledge is based on results obtained in smaller animals such as rats and chickens. Since itha s been demonstrated here that these results cannot be extrapolated to other animal species,i t is important to carry out more research on the target animal. A difficult problem is that one legume seed contains more than one ANF. Therefore, it is impossible to draw conclusions about a particular ANFwhe n thewhol e seed is fed. In order to understand the effects of each of the various ANFs, it is necessary to isolate and purify them and to study them separately in target animals. This may not always be realistic because large quantities of purified ANFs would be required which, inman y cases,ar e very expensive. Therefore, animal models should be developed inwhic h only small amounts ofANF s are required to study their effects. Promising models, developed by ILOB in cooperation with the Agricultural University inWageninge n and the University of Utrecht, are pancreas- canulated pigs which survive small intestinal biopsy techniques for studying effects of trypsin inhibitors and lectins. In these models only small amounts of ANF are needed. Improvement of analytical methods Adequate analyses are essential for research into the effects of ANFs in animals, for evaluating technological treatments in order to reduce the ANF activity, and for plant breeders todevelo p new varieties low inANFs . As discussed, there ismajo r concern about the analysis for lectins,antigeni c proteins and tannins.Fo r lectins, the newly developed FLIAmetho d looks very promising. Regarding trypsin inhibitors there is anee d to standardize assays. Research into the development of adequate analytical methods should be given high priority because other research depends on the validity of these assays. Threshold levels There is hardly any information concerning threshold levels of ANF in feedstuffs and diets.T o improve the quality of feedstuffs, diet formulation, technology and plant breeding it is important to know which levels ofANF s can be tolerated without causing negative effects m the animal. It is questionable whether the ANF content should be reduced to zero.Ther e are indications that in some animals low levels ofANF s can be tolerated without causing negative effects

30 on performance. However, thiswil l depend on the target animal.A s already demonstrated, piglets are much more sensitive toANF s than chickens. The age of the animalswil l also play a role, e.g. young piglets are more sensitive toANF s than older pigs. Veal calves may be classified as being very sensitive to ANFs. Knowledge of threshold levels is also important for technologists. They need to know towhic h levels ANFs should be reduced. It could be that different products with varying ANF levels can be manufactured for different animal species.Fo r plant breeders, it is important to knowwhic h levels ofANF s are acceptable froma nutritional point of view.Whethe r these levels can be reached by plant breeding is uncertain because seedswit h low levels ofAN F are generally less disease resistant.

8. References

Bertrand, G., Sève,B. , Gallant,D.J . and Tomé,R . (1988). Absence d'effets antinutritionel des lectines de pois, sous forme native ou purifee chez porcelet. Sciences desAliments ,8 ,187-212 . Birk, Y. (1987). Proteinase inhibitors. In:Hydrolyti c enzymes, pp257 - 305. [A.Neuberger and K. Brocklehurst, editors]. Elsevier, Amsterdam. Birk,Y . (1989). In:Recen t advances of research in antinutritional factors in legume seeds,p p 83-94. [J.Huisman,A.F.B , van der Poel and I.E. Liener, editors], Pudoc,Wageningen , The Netherlands. Boisen, S. (1989). Comparative studies on trypsin inhibitors in legumes and cereals. In:Recen t advances of research in antinutritional' factors in legume seeds,p p 118-120. [J.Huisman,A.F.B , van der Poel and I.E. Liener, editors]. Pudoc,Wageningen , The Netherlands. Bond, D.A. and Smith,D.B . (1989). Possibilities for the reduction of antinutritional factors in grain legumes by breeding. In:Recen t advances of research in antinutritional factors in legume seeds,p p 285-296. [J.Huisman, A.F.B, van der Poel and I.E. Liener, editors]. Pudoc,Wageningen , The Netherlands. Cabrera,A. , Lopez-Medina, J. and Martin,A . The genetics of tannin content in faba bean (Vicia faba L.). In:Recen t advances of research in antinutritional factors in legume seeds.,p p 297-300. [J.Huisman, A.F.B, van der Poel and I.E. Liener, editors]. Pudoc,Wageningen , The Netherlands. Castaing, J. and Grosjean, F. (1985). Effet de forts percentages de pois de printemps,dan s de régimes pour porcs charcutiers,a base de maïs ou d'orge et en complément de tourteau de colza.Journée s recherce porcine de France, 17,407-418 . Combs, G.E., Connes,R.G. , Berry, T.H. and Wallace, H.D. (1967). Effect of raw and heated soyabeanmea l on gain,nutrien t digestibility, plasma amino acids and other blood constituents of growing swine. Journal ofAnima l Sciences 26,1067-1071 . Donatucci, D.A., Liener, I.E. and Gross,C.J . (1987). Binding of navy bean (Phaseolus vulgaris) lectin to the intestinal cells of the rat and its effect on the absorption of glucose.Journa l of Nutrition, 117, 2154-2160. Fekete, J., Castaing, J., Lavorel,0. , Leuillet,M . and Quemere, P. (1984). Utilisation des pois protéagineux par le porcelet sevré. Journées recherche porcine en France, 16, 393-400. Freire, J.B., Hulin, J.C., Peiniau, J. and Aumaitre,A . (1989). Effet de la cuisson-extrusion du pois de printemps sur la digestibilite des aliments de sevrage précoce du porcelet et consequences sur les performances jusqu'à l'abattage. Journées recherche porcine en France, 21, 75-82. Gallaher, D. and Schneeman, B.0. (1986). Nutritional and metabolic response to plant inhibitors of digestive enzymes. In:Nutritiona l and

31 Toxicological significance of enzyme inhibitors in foods, pp167-185 . [M.Friedman, editor]. Plenum Press,Ne wYork . Grant, G., More, L.J., McKenzie N.H., Stewart, J.C. and Pusztai,A . (1983). A survey of thenutritiona l and haemagglutination properties of legume seeds generally available in the UK. British Journal of Nutrition, 50,207-214 . Griffiths, D.W. (1981). The polyphenolic content and enzyme inhibitory activity of testas from bean (Vicia faba)an d pea (Pisum Sativum) varieties. Journal of the Science of Food and Agriculture. 32,797 - 804. Griffiths and Mosely, 1980.Th e effect of diets containing field beans of high or low polyphenolic content on the activity of digestive enzymes in the intestines of rats.Journa l of the Science of Food and Agriculture, 31,25 5 -259 . Griffiths, D.W. (1984). The trypsin and chymotrypsin inhibitor activities of various pea (Pisum spp)an d field bean (Vicia faba) cultivars. Journal of the Science of Food and Agriculture, 35,481 - 486. Grosjean, F. and Castaing, J. (1983). Recherche d'amélioration de la valeur alimentaire du pois d'hiver pour le porc charcutier. Journées recherche porcine en France, 15,335-346 . Grosjean, F., Castaing, J. and Gatel, F. (1986). Utilisation comparée de différentes variétés de pois et d'une association pois de printemps- féverole par le porc charcutier. Journées recherche porcine enFrance , 18, 47-56. Grosjean F. and Gatel,F . (1989). Feeding value of Pisum Sativum for pigs: - influence of technology, - influence of genotype (trypsin inhibitor activity). In:Recen t advances of research in antinutritional factors in legume seeds, pp 239-242. [J.Huisman, A.F.B, van der Poel and I.E. Liener, editors]. Pudoc,Wageningen , The Netherlands. Gumbmann,M.R. , Spangler,W.L. ,Dugan ,G.M. , Rackis,J.J . and Liener, I.E. (1985). The USDA trypsin inhibitor study. IV. The chronic effects of soy flour and soy protein isolate on the pancreas in rats after two years. Qualitas Plantanum. Plant Foods for Human Nutrition. 35,275 - 315. Hamer,R.J. , Koninkx, J.F.J.G., Van Oort,M.G. ,Mouwen ,J.M.V.M . and Huisman, J. (1989). New developments in lectin analysis. In: Recent advances of research inantinutritiona l factors in legume seeds, pp 30-33. [J.Huisman,A.F.B , van der Poel and Liener, I.E., editors). Pudoc,Wageningen , TheNetherlands . Hove, E.I.an d King, S. (1979). Trypsin inhibitor content of lupin seeds and other grain legumes.Ne w Zealand Journal ofAgricultura l Research, 22, 41-41. Huisman, J., van der Poel,A.F.B. , ten Haaf,L.A.M. , Smits,C.H.M ,an d de Jong, J. (1987). Species differences:differen t negative effects of antinutritional factors (ANF) in the Phaseolus vulgaris bean in young pigs and rats.Proc . 5th. int. symp. on protein metabolism and nutrition, Rostock, DDR. Huisman, J., van der Poel,A.F.B. ,Verstegen ,M.W.A . and vanWeerden , E.J. (1989). Antinutritional factors (ANF) in pig nutrition. Accepted for publication in:Worl d Review ofAnima l Production. Huisman J. and van der Poel,A.F.B . (1989a). Comparison of effects of antinutritional factors in legume seeds. In:Recen t advances of research in antinutritional factors in legume seeds, pp317-327 . [J.Huisman, A.F.B, van der Poel and I.E. Liener, editors]. Pudoc, Wageningen, TheNetherlands . Huisman, J. and van der Poel,A.F.B . (1989b). Comparison of zootechnical characteristics in piglets and rats fed diets containing Phaseolus vulgaris. In publication.

32 r Huisman, J and van der Poel,A.F.B . (1989c). Effect of variable protein contents in diets containing Phaseolus vulgaris on performance, organ weights and blood parameters in piglets,rat s and chickens. In publication. Huisman, J. and van der Poel,A.F.B . (1989d). Performance and organ weights of piglets, rats and'chickens fed diets containing Pisum Sativum. In preparation. Jaffe,W.G . and Vega Letta, C.V. (1968). Heat stable growth inhibiting fractions in beans (Phaseolus vulgaris). Journal of Nutrition,94 . 203. Jaffe,W . (1980). Hemagglutinins,(lectins) . In: Toxic constituents of Plant Foodstuffs, pp 73-102. (I.E.Liener, editor). Academic Press, New York. Jansman,A.J.M. , Huisman, J. and Van der Poel,A.F.B . (1989). Faba beans with different tannin contents:ilea l and faecal digestibility in piglets and growth in chickens. In:Recen t advances of research in antinutritional factors in legume seeds, pp 176-180. [J.Huisman , A.F.B, van der Poel and I.E. Liener, editors]. Pudoc,Wageningen , The Netherlands. Kakade, M.L., Rackis,J.J. , Mc Ghee,J.E . and Puski,G . (1974). Determination of trypsin inhibitor activity of soy products:A collaborative analysis ofa n improved procedure. Cereal chemistry, 51 376-382. Kilshaw, P.J. and Sissons,J.W . (1979). Gastrointestinal allergy t/> soyabean protein in preruminant calves.Antibod y production and digestive disturbances in calves fed heated soyabean flour. Research inVeterinar y Science, 27,361-365 . Kilshaw, P.J. and Slade, H. (1982). Villus atrophy and crypt elongation in the small intestine of preruminant calves fedwit h heated soyabean flour orwhea t glutin.Researc h inVeterinar y Science, 33,305-308 . Kim, Y.S., Borphy, E.J. and Nicholson,J.A . (1976). Rat intestinal brush border membrane peptidases. II.Enzymati c properties, immunochemistry and interactions with lectins of two different forms of the enzyme. Journal of Biological Chemistry, 251,3206-3212 . King ,T.P. , Begbie,R . and Cadenhead,A . (1983). Nutritional toxicity of raw kidney beans in pigs. Immunocytochemical and cytopathological studies on the gut and the pancreas.Journa l of the Science of Food and Agriculture, 34,1404-1412 . Liener, I.E. Book: Toxic constituents of plant foodstuffs. Academic press, New York,US . Liener, I.E., (1986). Nutritional significance of lectins in thediet . In: The lectins: Properties,function s and applications in Biology and Medicine. [I.E.Liener ,N . Sharon and I.J. Goldstein, editors). Academic press.Ne w York,US . Leterme, P., Beckers,Y . and Thewis,A . Inter- and intravarietal and variability of the trypsin inhibitors content of peas and his influence on apparent digestibility of crude proteins by growing pigs. In: Recent advances of research of antinutritional factors in legume seeds, pp 121-124. [J.Huisman ,A.F.B ,va n der Poel and I.E. Liener, editors]. Pudoc,Wageningen , the Netherlands. Liener, I.E. (1989). Antinutritional factors in legume seeds: State of the art. In:Recen t advances of research in antinutritional factors in legume seeds,p p 6-13. [J . Huisman,A.F.B ,va n der Poel and I.E. Liener, editors]. Pudoc,Wageningen , The Netherlands. Marquardt,R.R . (1989). Dietary effects of tannins,vicin e and convicine. In:Recen t advances of research in antinutritional research in legume seeds, [J . Huisman,A.F.B ,va n der Poel and I.E. Liener, editors). Pudoc,Wageningen , The Netherlands.

33 Meyer, O.M.,Froseth , J.A. and Coon,C.N . (1982). Protein utilization and toxic effects of raw beans (Phaseolus vulgaris) for young pigs. Journal ofAnima l Science, 55,1087-1098 . Miller, B.G., Newby, T.J., Stokes,CR. , Hampson, D.J., Brown, P.J. and Bourne, F.J. (1984). The importance of dietary antigen in the case of postweaning diarrhoea in pigs.America n Journal Veterinary Research, 45, 1730-1733. Mitjavila, S., Lacombe, C, Carrera,G . and Derache,R . (1977). Tannic acid and oxidized tannic acid on the functional state of rat intestinal epithelium. Journal of Nutrition, 107,211 3 -2121 . Nakata, S. and Kimura, T. (1985). Effect of ingested toxic bean lectins on the gastrointestinal tract in the rat.Journa l of Nutrition,115 , 1621-1629. Poel, A.F.B, van der, (1989). Effect of processing on antinutritional factors (ANF)an d protein nutritional value for pigs of dry beans (Phaseolus vulgaris).A review.Anima l Feed Science and Technology, submitted. Pusztai,A. , Clarke,E.M.W. ,Grant ,G . and King, T.P. (1981). The toxicity of Phaseolus vulgaris lectins.Nitroge n and immunochemical studies. Journal of the Science of Food and Agriculture, 32,1037 - 1046. Pusztai,A . (1987). Plant lectins-biological functions.Act a Biochemica et Biophysica, Hungary, 99,355-375 . Pusztai, A. (1989). Biological effects of dietary lectins. In: Recent advances of research inantinutritiona l factors in legume seeds, pp 17-29.[ J. Huisman, A.F.B, van der Poel and I.E. Liener, editors). Pudoc,Wageningen , The Netherlands. Rackis, J.J. (1975). Oligosaccharides o£ food legumes:Alpha - galactosidases activity and flatus problems. In:Physiologica l effects of food carbohydrates, pp 207-222. [J.Alle n and J. Heiige, editors). American Chem. Soc, Washington, DC,U S Rackis, J.J. and Gumbmann,M.R . (1981). Protease inhibitors: Physiological properties and nutritional significance. In: Antinutrients and Natural Toxicants in Foods, pp 203-237. [R.L. Ory, editor]. Food and Nutrition Press,Westport ,Connecticut ,US . Rackis, J.J., Gumbmann,M.R . and Liener, I.E. (1985). The USDA trypsin inhibitor study. I. Background, objectives and procedural details. Qualitas Plantanum Plant Foods for Human Nutrition, 35,213-242 . Saini, H.S. (1989). Legume seed oligosaccharides. In:Recen t advances of research in antinutritional factors in legume seeds, pp 329-341. [J. Huisman,A.F.B , van der Poel and I.E. Liener, editors). Pudoc, Wageningen, theNetherlands . Savage, G.P. (1989). Antinutritive factors in peas. In:Recen t advances of research of antinutritional factors in legume seeds, pp 342-350. [J. Huisman,A.F.B , van der Poel and I.E. Liener, editors]. Pudoc, Wageningen, The Netherlands. Savaiano,D.A. ,Powers ,J.R. , Castello,M.J .Withaker , J.R. and Clifford,A.J . (1977). The effect of ana-amylas e inhibitor on the growth rate ofweanlin g rats.Nutritio n Reports International,15 , 443-449. Schumann,B. , Souffrant,W.B. ,Matkowitz ,R . and Gebhardt,G . (1983). Untersuchungen zur endogenen N-Sekretion im Pankreassekret beim Schwein. Wissenschaftlich Zeitschrift Karl-Marx-Universitat Mathematik Naturwissenschaft, 32,6 ,570-575 . Seegraber, F.J. and Morril,J.L . (1982). Effect of soy protein on calves'intestinal absorptive ability and morphology determined by scanning electronmicroscopy . Journal ofDair y Science,65 ,1962-1970 .

34 r Seegraber, F.J. and Morril, J.L. (1986). Effect of protein source in calf milkreplacres onmorpholog y and absorptive ability of the small intestine. Journal of Dairy Science, 69,460-469 . Sissons,J.W . and Smith,R.H . (1976). The effect of different diets including those containing soya-bean products,o n the passage of digesta movement and water and nitrogen absorption on the small intestine of the pre-ruminant calf.Britis h Journal of Nutrition,36 , 421-438. Sissons, J.W. (1982). Effects of soya-bean products on digestive processes in the gastrointestinal tract of preruminant calves. Proceedings Nutrition Society, 41, 53-61. Sissons, J.W., Smith,R.H. , Hewitt,D . and Nyrup,A . (1982). Prediction of the suitability of soy-bean products for feeding to preruminant calves by an invitro immunochemical method. British Journal of Nutrition, 47,311-318 . Stickland, R.G. (1984). Condensed tannins of pea seeds.Plan t Science Letters, 34,403-410 . Torres-Pinedo,R . (1983). Lectins and the intestine. Journal of the Pediatric Gastroenterology and Nutrition, 2,588-594 . Valdebouze, P., Bergeron, P., Gaborit, T. and Delort-Laval, J. (1980). Content and distribution of trypsin inhibitors and hemagglutinins in some legume seeds.Canadia n Journal of Plant Science, 60,695-701 . Visitpanich, T., Batterham, E.S.an d Norton, B.W. (1985). Nutritional value of chickpea (Cicer arietinum) and pigeon pea (Cajanus cajan) meals for growing pigs and rats. I. Energy content and protein quality. Australian Journal ofAgricultura l Research, 36,327-325 . Yen, J.T., Jensen,A.H . and Simon,J . (1977). Effect of dietary raw soybean and soybean trypsin inhibitor on trypsin and chymotrypsin activities in the pancreas and in the small intestinal juice of growing swine. Journal of Nutrition, 107,156-165 .

35 RESEARCHINT OTH EDIGESTIV E PHYSIOLOGY OFTH EMILK-FE D CALF

R.Toulle c &P .Guillotea u Laboratoired u Jeune Ruminant InstitutNationa l del aRecherch e Agronomique 65ru ed e Saint-Brieuc, 35042Renne s Cedex,Franc e

Summary In thepreruminan t calf,th eactivitie so fman y abomasal (chymosin and lysozyme)an d intestinal (lactasean daminopeptidase s Aan dN )enzyme sar emaximu ma t2 d o fag ean d subsequently decline. In contrast, the activities of most pancreatic enzymes ando f intestinal maltase increase with age,whils t thato fpepsi n doesno tappea r tochange . The useo fseverel yheate d skimmil ko ro freplacemen t protein mayhav edepressiv eeffect so n protease secretion. These adaptations aswel l asgu tmotilit y areprobabl y regulatedb y many gastro-entero-pancreatic hormones whoseplasm a concentrations change withag e andprotei n sourceo rafte r feeding. The specific activitieso fpancreati c enzymes increases with age morerapidl y than therelativ e levelso fth e corresponding mRNAs. Theclosur e of theoesophagea l groove may be impaired by poor management conditions.Th eus eo f non-clotting diets results in faster abomasal emptying rate and absorption of fatan d protein.Replacemen t proteins areles scompletel y digested than milk protein inth esmal l intestine andca nals o induce increased endogenous protein losses.Th edevelopmen to f hypersensitivity reactionsi nth egastro-intestina l tract appearst ob eon e limitingfacto r in theus eo fman yreplacemen tproteins .

Key-words Preruminant calf, digestive secretions, guthormones , pancreatic mRNAs, oesophageal groove,abomasa l emptying,ilea ldigestion ,dietar y allergy.

Introduction The young calf is normally fed colostrum andthe n whole milk or milk substitutes. Usually only liquid diets areuse d for veal calf production until slaughter. Solid food is given early toanimal st ob eweaned ,bu t digestion isessentiall y preruminant until atleas t 4- 5 weeks ofage .Thus ,i nth eabsenc eo frume n function thepreruminan t calf depends on itsdigestiv e secretions for theassimilatio n of dietary fat, carbohydrate andprotein . Whole milk andmil k substitutes based onski m milk andreplacemen t fat are efficiently digested byhealth ypreruminan t calves.However, with theshortag eo fski m milk powder in theEuropea n Community, alternative sources of protein andcarbohydrat e arebein g used more widely. Therefore, it is important to understand how thepreruminan t calf adaptst othes e changes.Th emai n aspectso ffat , carbohydrate andprotei n digestion have been reviewed between 1979-1983 (Thivend et al., 1979; Roy ,198 0; Sissons, 1981 ; Toullec etal. , 1983).Thi s report will deal with recent advances indigestiv e secretions, regulation of digestive function, passage of digesta, ileal digestion and protein intolerance.

37 Digestive secretions

Carbohydrases The carbohydrase level of the newborn calf is relatively weak (Sissons, 1981). Intestinal lactase is the solecarbohydras e whose activity is high at birth. Expressed ona liveweight basis lactaseactivit y ismaximu m at2 d o f age, abruptly declines between 2-7 d and then does not change much with age (table 1) ; however, it appears to stay high enough to digest normal lactoseintake .Ther ei sevidenc e neither of salivary amylaseno r of intestinal sucrase whilst the activities of pancreatic maltase and of intestinal amylase and isomaltase arever y low (Sissons, 1981).Recentl y weobserve d that the activitieso f pancreatic amylasean dintestina l maltase werever y low atbirt h but greadyincrease dwit h ageespeciall y for amylase (table1) . Table 1. Changes with age of digestive enzyme activities, kg liveweight-1 (relative to values at2 d) (I. Le Huërou, C. Wicker, P. Guilloteau, R. Toullec, A. Puigserver &J.H . Burton, unpublished results).

Age(d) 28 119

Abomasum chymosin 0.65 0.22* pepsin 1.21 0.63 lysozyme 0.65 0.39* Pancreas amylase 24* 47*§ trypsin 2.48 1.28 chymotrypsin 2.44* 3.07* elastase 2.43* 2.53* carboxypeptidaseA 2.31* 2.13* carboxypeptidaseB 2.88* 2.27* ribonuclease 1.83* 2.40* lipase 2.15 3.50* colipase 1.61 1.04 phospholipase A2 1.46 1.68 Smallintestin e lactase 0.23* 0.12* maltase 0.83 2.86*§ isomaltase 0.44 0.91 aminopeptidaseA 0.34* 0.47* aminopeptidaseB 0.21* 0.31*

*, § :Differen t from value at2 o r2 8d ,respectivel y (P< 0.05) .

38 Fathydrolase s and bile Salivary lipase largely contributes to the hydrolysis of triglycerides. It is particularly active on triglycerides containing short-chain fatty acids but splits ester bonds of long chain fatty acids too (Edwards-Webb, 1983). The activity of pancreatic lipase per unit liveweight is low at birth but increases thereafter, whilst the activities of colipase and phospholipase A2 do not appear to vary with age (table 1). Little is known about bile secretion ;however , totalbil esalt s secretion during thefirs t 7h afte r amea l wasfoun d to increaseb y36 %whe n sorbitol wasintroduce di n thedie t(Thiven de t al., 1984).

Hydrochloric acid andenzyme sinvolve d inth edigestio n of nitrogenous products

Hydrochloric acid secretion in the abomasum, estimated from CI"- Na + difference in duodenal digesta, was found to rise by 50% between 1-4 weeks of age (Ternouth et al. 1976).I n contrast, between 3-32 weekslittl echang e wasobserve d in thequantit y of H+ secreted per kg liveweight by abomasal pouches (Guilloteau et al., 1980b). Coagulating activity is mainly due to chymosin. The amount of chymosin per kg liveweight is maximum at2 d of age and subsequently decreases (table 1).I n contrast, pepsin activity does not appear to vary with age. Lysozyme is a muramidase which splits the bonds between N-acetylmuramicaci d andN-acetylglucosamin ei n bacterialpeptidoglycan . This enzymei smor e abundant inruminant s thani n non-ruminants andi smainl y concentrated in gastric mucosa (Dobson et al., 1984). In the preruminant calf, lysozyme activity was found tofollo w apatter n similar totha to fchymosi n (table 1).

In contrast, in thepancrea s most proteolytic enzyme activities aswel l asribonuclease activity are minimum at 2 d of age and subsequently increase during the first 2 months (table 1),exhibitin g apatter n which isroughl y therevers eo f that observed for chymosin and lysozyme. Little is known about peptidase activities in the small intestine. The activities of aminopeptidases A and N are maximum at 2 d of age, abruptly decline between 2-7 d andd ono t appear tochang emuc h thereafter. Many digestive secretions involved in protein digestion may be influenced by management practices and dietary factors (teat versus bucket-feeding, excessive heat- treatment of skim milk, use of non-milk protein) (Toullec et al., 1983). Inconsistent results have been reported on theeffect s of non-milkprotei n on gastric secretions.Thes e variationsar eprobabl y duet odifferen t techniquesfo restimatin g secretionsan dtreatment s usedfo r processing thenon-mil k protein. Incontrast ,feedin g replacement protein usually resultsi n areductio n of trypsin and chymotrypsin secretions. Forexample , the activities per kg liveweight present in the pancreatic tissue were found to be depressed by 40% when skim milk was replaced by a soyabean protein concentrate and whey derivatives (Guilloteau et al., 1986a).

Microbialdigestio n In calves given whole milk or diets based on skim milk and replacement fat, fat and lactosedigestio n isapparentl y finished atth een do fileu man donl y 2-4%o f totalnitroge n apparently absorbed in the whole digestive tract disappears in the hindgut (Van Hellemond & Van Weerden, 1973 ; Van Weerden et al., 1977 ; Besle et al., 1980 ; Guilloteau et al., 1986c).Endogenous carbohydrates arestil lpresen t in theilea l digestai n amounts equivalent to about 7-9% of lactose intake (Besle et al., 1980) and are largely fermented in thehindgu t sinceth e apparentdigestibilit y of nitrogen-free-extract isclos et o 0.99.

39 Microbial digestion may be more important for carbohydrates when the enzymes are absent (sucrose and a-galactosides ) orar epresen t atlo w levels (starch).Fo rexample ,i n calves given diets containing 170 g starch per kg dry matter, the proportion of starch digested in thehindgu tvarie d between 7%fo r partially hydrolysed maize up to 61% for banana (Thivend, 1979).Th e volatile fatty acids and lactic acid which are produced are efficiently utilized byth ecal f (Vermorel &Patureau-Mirand , 1978).However ,i fno twel l controlled,microbia l fermentations mayresul ti nloos e faeces. In general, except for abomasal enzymes or intestinal lactase and aminopeptidases A andN ,th edigestiv e capacity of thecal f appears tob eminima ldurin g thefirst day so f life. Therefore, exceptfo r lactose,th eapparen t digestibility of milk constituents increaseswit h age, especially during the first month and beyond for added starch. The digestion of replacement proteins may be limited by their depressive effect on many digestive secretions and theirlesse r sensitivity toenzym ehydrolysi s (Jenkinse t al., 1980).

Vasoactive Cholecystokinin Secretin intestinal peptide

100' 40T 20T

75 30-• 15-

50 20 10-

25 10

04*—H- 01' 'i' i|WW| l' ' I 1 21 91 1 21 91 1 21 91 21 91 Pancreatic Age(d ) polypeptide Somatostatin Motilin

400T b 100T 60 a 300' 75 ' 40-

200" 504 20' 100- 25-

0 0' 0+ 1 21 1 21 1 21 91 Age (d) Figure 1. Changeswit hag ei nth eplasm alevel so fimmunoreactiv egu thormone si nmilk - fedcalve s(Guillotea ue tal. , 1984,1986b & unpublishe dresult s) .a, b:Value swit hunlik e superscriptletter sdiffere d significantly (P <0.0 5 ).

40 Regulation ofdigestiv e function

Hormonal control Thedevelopmen t ofth edigestiv etract ,it ssecretion s andit smoto ractivit y areregulate d by complex neuro-hormonal mechanisms. Forexample , acid secretion in the abomasum is stimulated by gastrin and cholecystokinin (CCK) and inhibited by secretin, whilst pepsin secretion appears to bestimulate d by secretin (McLea y &Bell , 1981).Pancreati c enzyme secretions arefavoure d by CCK and mayb einhibite d bypancreati c polypeptide (PP) (Davicco, 1978).Gastrin , CCK and secretin decrease themyoelectri c activity of the abomasum which results in a slowing down of abomasal emptying (Mc Leay & Bell, 1981).Gastri n has atrophi c effect on thefundi c mucosa and thepancrea s (Wiener et al., 1987) and CCK on the pancreas (Marx et al., 1987). Somatostatin has numerous inhibitory effects ondigestiv e secretions,th ereleas eo f theothe r gastro-entero-pancreatic hormonesan d gutmotilit y (Newman et al., 1987).

In the preruminant calf large changes with age were observed for the basal plasma concentrations of most assayed guthormone s during the first 3week s of life (Guilloteau et al., 1984 & 1986a ; P. Guilloteau, R. Toullec, J.A. Chayvialle & C. Bernard, unpublished results). For example, at 21 d, values were 2.3-, 3.5- and 3.6-fold higher than atbirt h for secretin, CCK and PP,respectivel y and 2.3-, 1.6- and 1.9-fold lower for gastrin, motilin and somatostatin (fig. 1). The decrease was particularly rapid for somatostatin since it occured between the first and the second day. No significant trend was apparent thereafter but,i n anotherexperiment , theplasm a levelo f somatostatin^wa s found to increase by 6.3-fold between 7-70 d of age (P.Guilloteau et al., unpublished results). The early changes of CCK, somatostatin and secretin levels could favour the development ofpancreati c tissuean dit ssecretions .Th edecreas ei n gastrin levelma yhav e resulted in the reduction of chymosin secretion. The decrease of motilin level could be related toth edevelopmen t of gut motilitydu e toentera l feeding. However, littlei sknow n about thechange swit h ageo f thereceptor s corresponding to gutpeptides .Also ,th e effect of agu tpeptid e onth e synthesiso f thedifferen t enzymesi n atissu eeithe rdepend so n the enzyme and/ori saltere d byothe rfactor s : for example,i f CCK favours enzyme synthesis inth epancreas ,amylas eproductio n increases muchmor ewit h agetha n theproductio no f theothe renzymes .

One hour after feeding adie t based on skimmil kpowder , theplasm a levelso f gastrin, CCK and gastric inhibitory peptide (GIP) were increased by 2.6-, 1.8- and 2.3-fold, respectively, compared with the levels before (fig. 2). In contrast, the plasma level of secretin was decreased by 2.1-fold, whilst no systematic trend could be evidenced for motilin, PP and somatostatin. The increase in gastrin and CCK levels after the meal should favour abomasal and pancreatic enzyme secretions. Secretin appears to be particularly involved in theregulatio n of thep H of small intestine digesta by enhancing pancreatic bicarbonate secretion and reducing abomasal acid secretion. Its release is increased when theduodena l pHi s lower than 4.5 (Doylee tal. , 1987).Thi svalue ,whic h is2- 3befor e themea li n thepreruminan t calf,reache s 5-6immediatel y after and2- 3h ar e required before it becomes lower than4.5 . The totalreplacemen t of skim milkprotei n by soya andwhe yprotein sle dt oa decreas e in the basal plasma concentrations of secretin and GIP (fig. 2). After the meal GIP concentration was still reduced whilst CCK concentration was increased. Similar trends were observed for secretin and CCK with fish protein (Guilloteau et al., 1984). These changescoul d bepartiall y related toth efaste r abomasal emptying rateo f protein and fat. CCK release is favoured by products of fat and protein digestion (Marx et al., 1987) which are more abundant within the intestinal lumen during the first after feeding hours with non-coagulating diets than with milk.As far as the soya diet was concerned, the

41 higher plasma level of CCK after the meal could be also a response to the reduction of trypsin production (Guilloteau et al., 1986a). Before feeding, the abomasum of the calves given the non-coagulating diets was moreempt y than that of the control animals. This might result in less acidic chyme flowing to the duodenum which would lead toa lower secretin release. The change observed for GIP could be due to the lower level of carbohydrates producing glucosei nth e soyadiet . Gastrin Cholecystokinin Secretin ng.ml1 150 200 T 40 T

150 30 100 I 100 •• 20 • b 50 50 1

J o 0 1 0 0 10 1 0 10 A1 Time after feeding (h) Gastric inhibitory Pancreatic peptide polypeptide

b 2500 •• 600

2000 400 -1 1500 a a I I [ j Control diet 1000 200 Da §s| Soyadie t 500 0 il JA m 0 10 1 0 10 1 Time after feeding (h) Figure 2. Effects of feeding and protein source (skim milk powder 92.5 and 0%, whey derivatives+ syntheti camin oacid s7. 5an d36. 5% ,ethano lextracte dsoy aconcentrat e0 an d 63.6 % incontro l and soyadiet ,respectivel y )o n theplasm alevel so fimmunoreactiv e gut hormones (Guilloteau et al.,1986a ) . a,b,c :Valueswit h unlike superscript letters differed significantly (P< 0.0 5 ).

Molecularregulatio n ofenzyme s synthesis The development of pancreatic hydrolases in the rat depends on that of the corresponding mRNAs (Han et al., 1986). Little is known about this topic in the calf. Recently, LeHuëro u et al.(1989 )examine d thechang ewit h ageo f both enzyme activities and specific mRNA levelso f chymotrypsin and amylase (fig. 3).Chymotrypsi n specific

42 activity wasfoun d tob e2 0an d50 % higher at 28an d 119 d of agetha n at birth.I n contrast, therelativ e levelo fmRN A was44 %lowe r at2 8d tha n atbirt h butha d almost regainedit sbirt h valuea t11 9d .Chymotrypsi n synthesis appeared tob edetermine d more by some translational and/o rpost-translationa l regulation than byth e transcriptional control mechanism. Amylase activity, which was very low atbirth , was 22 and 43 fold higher, respectively, at 28 and 119 d. During the same time, the level of mRNA increased, butles s than the enzyme activity (by 2.7 and10. 2 fold, respectively). Thus, amylase synthesis was probably regulated at the transcription step as well asb y translation and/orpost-translatio n events. Chymotrypsin Amylase 2,0 50 *§

1,0" 25 *

0,5- *§

* /" 0,0 •i;::.;..;; J, -H —1 28 119 28 119 Age(d ) Figure 3.Change s with ageo fth especifi c activities (|_J ) andmRNA s levels (|j| ) of chymotrypsin and amylase (relativ e tovalue s atth e birth )i nth epancrea s of milk-fed calves (L eHuëro ue tal , 1989& unpublishe d results).*,§ : Different from valuea tbirt h anda t2 8d ,respectivel y (P<0.05) .

Therefore, newmethod s can beuse d toge t abette runderstandin g ofdigestiv e function. After the present descriptive phase,they should lead toothe r ways of interpretingan d improving digestive adaptation.

Passageo fdigest a

Closureo fth eoesophagea l groove Milk andmilk-substitute s normally by-pass therume n thanks toth e closure ofth e oesophageal groove. However some veal calves present problems characterized by inappétence, abdominal distension, alon ghirsut e hair-coat and abundant clay-like faeces (Tadeu dosSanto s et al., 1986 ; Breukink et al., 1988). These symptoms appears2- 4 weeks after thebeginnin g ofth e fattening period. Usingpolyethylen e glycol asa marke r in rumen fistulated calves, Tadeu dosSanto s et al. (1986) found that 57% of the milk ingested fell into the rumen in "abnormal" calves, instead of 3%i n "normal" animals. Breukink et al.(1988 ) observed thatalmos tal lth emil kentere d thereticulorume n andwa s slowly transferred toth eabomasu m in "abnormal" calves. Other characteristics of the "abnormal"calve swer edecrease si n thep Ho fth erume n contents (5.1instea d of6.7, 2h

43 after the meal) and in apparent digestibility (0.73 instead of 0.92 for fat) (Tadeu dos Santose t al., 1986),a swel l asa shortenin go f theintestina lvill i (Breukinke t al., 1988). In an attempt to simulate these phenomenons, Nunes do Prado et al. (1987 & 1988a) introduced 25%o f the milk into therume n via a canula. The pH of the rumen contents decreased whilst theirvolatil efatt y acidsan dlacti caci d concentrationsincreased . Lactose disappeared from therume n within 6h whils t milkprotei n still constituted about 40%o f total protein in rumen contents 16 h after milk introduction. However, there was impairment neither in oesophageal groovefunctio n nori n feed intake even after 3weeks , perhaps because thecanul a could bea noutlet . According toth eobservation s of Breukink et al. (1988), "abnormal" calves exhibit a drinking behaviour different from that of "normal"calve s :the y are "gulpers"instea d of "sippers".Th edeterminan t factor involved in the non-closure of the oesophageal groove is not known but the frequency of the symptoms appears to depend on the management. For example, transportation of the young calves on long distances isunfavourabl e in thisrespect . "Abnormal" calves could be treated by emptying the rumen via a stomach tube and by training the calves to suck small amounts of whole milk or good quality milk-substitute after initiating intensive sucking on the herdsman's fingers (Breukink et al., 1988). However, feed intake and liveweight gain of successfully treatedcalve sremaine d lowertha n thoseo f normalcalves .

Abomasal emptying Incalve s given wholemil kduodena ldigest a contains nointac t casein except duringth e first 10mi n after feeding, indicating that considerable hydrolysis of this protein takes place in the abomasum (Yvon et al., 1984). The phosphopeptides fractions of casein chains, except for the caseinomacropeptide resulting from the hydrolysis of Kcasein ,ar e retained in theabomasu m for alonge r time than thenon-phosphorylate d fractions (Yvon et al., 1986). In contrast, whey protein enters rapidly the duodenum ; a-lactalbumin is largely hydrolysed whilst ß-Iactoglobulin is only slightly split before leaving the abomasum (Yvon et al., 1984). The use of non-casein protein results in a faster abomasal emptying rate for fat and protein duet oabsenc eo f aclot . Thisrapi d emptyingrat ecoul dpartiall y contributet oth e lower digestibility of mosto f thediet scontainin gprotei n substitutes :althoug h not always significant, such atren d has been usually observed for nitrogen and/or fat at the ileal or faecal level by impairing the clotting ability of milk protein-based diets (Toullec et al., 1974 ;Va n Weerden et al., 1977 ;Jenkin s &Emmons , 1982 ;Strudsholm , 1988 ;Peti te t al., 1989).Similarl y with dietsinfuse d intoth eduodenum , arapi d flow rate wasfoun d to depressnitroge n andfa t digestibility (Guilloteaue tal.,1981) .

Fat and amino acid absorption is strongly influenced by abomasal emptying rate (Beynen &Va n Gils, 1983 ;Peti t et al., 1987 ;Nune s doPrad o et al., 1989c). Therefore, with non-clotting diets,fa t and free amino acids accumulate in the blood during the first postprandial hours, although the liver is able to use larger amounts of amino acids (Houlier et al., 1989).

Resultso fprotei n digestion in the smallintestin e

Digestibility of nitrogen andamin oacid s Usually, the apparent digestibilities of totalnitroge n andamin o acid nitrogen arelowe r at the end of the ileum than values obtained for the entire digestive tract (table 2). However, with adie t containing apregelatinize d pea flour, similar values wererecorde d atth etw olevels ,probabl y becauselarg eamount so f starch escaped digestion in thesmal l

44 intestineresultin g inincrease d amountso fbacteri a produced inth ehindgut .Whateve rth e diet,apparen t digestibilities of threonine andcystin ear elowe r than average whilst thato f methionine is higher. The ratios between the faecal digestibilities of total nitrogen of different diets may allow correct estimates of the ratios of the ileal digestibilities of essential amino acids (table 3). However, this assumption is not always confirmed :fo r example, when comparing diets based on skim milk or a hydrolysed fish protein concentrate, theus eo f therati oo f thefaeca l digestibilities of total nitrogen would led to anoverestimatio n ofilea ldigestibilit y of about 10% for histidinefo r thelatte rdiet .

Table 2. Apparent digestibility ofnitroge n (N)an d someamin oacid s (AA) at theen do f theileu m (A) ando f thewhol edigestiv e tract (B) (Nunesd oPrad oe t al., 1989a).

Origin ofdietar yN(%) * Milk (99)§ Pea (34)+ Soya (73)t A B A B A B

TotalN 0.95a 0.97a 0.92b 0.92b 0.91b 0.94b AAN 0.97a 0.98 0.95b 0.94 0.93c 0.95 Threonine 0.94a 0.97 0.90b 0.91 0.87b 0.93 Cystine 0.89 0.93 0.83 0.82 0.87 0.91 Methionine 0.99a 0.99 0.98b 0.96 0.97c 0.96 a, b,c :Value s with unlike superscript letters differed significantly (P< 0.05 ) *Th eremainde r was supplied by skimmil k (+)o rwhe ypowde r (f) an d syntheticA A (§, + and f).

Table 3. Apparent digestibility at the end of the ileum and of the whole digestive tract (relative to control value) (Guilloteau et al., 1980 b (§) ; Guilloteau et al., 1986 c (+) ; Nunes doPrad o et al., 1989a (t) )•

Origin of dietary N(%) * >ria(50)§ Fish(74) + Pea( 3

Ileum totalnitroge n 97 93 97 aminoaci d nitrogen 98 93 98 threonine 101 94 96 histidine 96 88 98 arginine 99 97 99 Faeces totalnitroge n 98 97 95

*The remainder was supplied by skim milk (§ and f) , whey powder (+) and synthetic aminoacid s (§,+ an d f ).

45 m E Asp m Ala Gly Arg ® m^m S2, © Factor 1 (S3 © Leu Met He His Tyr Thr Glu Ser [10 Val ®

Pro u o u ©

Figure 4. Comparison of the amino acid composition (g/100g assayed amino acids) of protein ofilea ldigest a(C~J) collectedfro m preruminantcalve sreceivin gdiet scontainin g different protein sourceswit htha to fdietar y (| |),endogenous(| | )an d gutbacteri a ((_)) proteins.Eac hpoin ti sth eprojectio n of oneprotei n onth eprincipa l plane (1-2)o f thefactoria l correspondance analysis.Th e seventeen assayed amino acids (Ala,Arg ,etc. ) are also projected. Dietary protein source and ileal digesta B, C,F ,M and P :methanol - grown bacteria, casein, hydrolysed fish, milk andpregelatinize d pea flour. Sdiet : heated soyameal ;S digesta :S 1 andS 2alcohol-extracte d soyaconcentrate ,S 3 soyaisolate ,S 4an d S5allergeni cheate dsoy amea lbefor e andafte r sensitizationo fth ecalve s(Guillotea ue t al, 1980b & 1986c;Nune s doPrad oe t al., 1989a, C.Duvaux ,J.W . Sissons,R . Toullec & P. Guilloteau, unpublished results). ( B )'• meancompositio no fpi gan dshee pfaeca l bacteria (Masonetal. , 1976;Mason , 1979).CM:calfmeconium(Grongnetetal., 1981).AFraxeni c lamb faeces (Combe, 1976). Ileal digesta wererepresentativ e of 24 h or 96 h collection periods, except C, S2, S4 and S5 which corresponded to the first 3h after arrival of feedresidue s asindicate d bya pheno l red marker in the effluent.

46 Natureo fprotei nescapin g digestioni nth e smallintestin e Assessment of endogenous protein in ileal digesta can be approached by comparing their amino acid composition to those of calf meconium (Grongnet et al., 1981) and faeces of the germ-free lamb (Combe, 1976).Protei n of bacterial origin can be evaluated by comparisons with compositions of faecal bacteria isolated from pig (Mason et al, 1976) or sheep faeces (Mason, 1979). The amino acid profiles of the ileal digesta corresponding to three control diets based on skim milk and nine experimental diets containing different replacement proteins were submitted to a factorial correspondence analysistogethe r withdietary , endogenous andbacteria lprotein s (fig.4) .

With the control diets the ileal digesta were always near the endogenous products, confirming the high true digestibility of milk protein. The protein of Ml, M2 and M3 digesta could be assumed to be representative of the undigested mixture of endogenous andbacteria l proteins atth een d of ileumi nth epreruminan t calf.B y iterative calculation themea n theoreticalproportion s ofendogenou s andbacteria lprotein si n thatmixtur e were found to be 0.68 and 0.32, respectively. All of the other digesta were farther from the area containing the endogenous products and closer to the dietary proteins than Ml, M2 and M3 digesta (fig. 4). However B, C, F, SI and S2 digesta were not near the line joining Ml, M2 or M3 digesta to the corresponding diet. Therefore, the protein from methanol-grown bacteria, casein, fish and alcohol-extracted soya concentrate which escaped digestion were particular fractions whose amino acid composition was largely different from thato f the whole dietary protein. In contrast, S4an d S5digest a werever y close todietar y protein, suggesting that largeproportion s of little degraded soya protein werepresen t at the end of ileum in calves given heated soya meal.Iterativ e calculations indicated that the proportions of dietary protein could be about 0.58, 0.13, 0.35, 0.18, 0.88 and 0.72 in C,P , S2,S3 ,S 4an d S5digesta , respectively, theres t being the mixture of endogenous and bacterial proteins obtained with milk protein. Calculations of the distance of %^ (Guilloteau et al., 1980a) between the amino acid composition of the theoretical mixtures of dietary, endogenous and bacterial proteins and the actual composition of digesta showed a good fit for P, S3, S4 and S5 but not for C and S2. Theseresult sconfirme d thatth edietar yprotei n escapingdigestio n wasparticula r fractions for C and S2 but not for S4 and S5.Th e proportions of dietary protein with P and S3 wereto olo w todra wconclusions .Replacemen tprotein s areles scompletel y digested than milk protein in the small intestine but can also induce increased amounts of endogenous losses.

Allergeniceffect s of dietaryprotei n

General aspects It is well established that unrefined soya products are unsuitable as major sources of protein in calf milk replacers (Sissons, 1982). Even heated soya flour, in which antinutritionnal factors considered to be harmful for monogastric animals (antitryptic factors and lectins) have been inactivated may cause gastrointestinal hypersensitive reactions involving disturbances in gut motility, digesta movement and nutrient absorption. Tissue inflammatory responses were evidenced in histological studies of the intestinal mucosa ; biopsy samples revealed villous atrophy, oedema and lymphocytic inlfiltration (Roy et al., 1977 ;Barrat t et al., 1979; Seegrabe r &Morill , 1982 ;Pedersen , 1986). Also,a dramati c increase of gut permeability to ß-lactoglobulin was evidenced in calves sensitized to soya and then challenged with heated soya flour mixed with whole milk (Kilshaw & Slade, 1980). These disorders are probably invoked by immuno­ logically active globular proteins, especially glycinin and ß-conglycinin (Kilshaw &

47 Sissons, 1979 a & b) and perhaps a-conglycinin (J.W. Sissons, personal communication), sinceth edenaturatio n of theseprotein sprevent sth etroubles .

Mechanisms Detection of IgG, and more rarely of IgE, antibodies specific to soya proteins in the blood of calves given feeds containing soya products has led to speculation that the deleterious effects could have been due to a gastrointestinal allergic reaction (Smith & Sissons, 1975 ;Barrat t et al., 1979). Dietary allergy couldresul tfro m threedifferen t typeso fhypersensitivit y reactions (type I, III or IV) (Gell & Coombs, 1968). The type Ireactio n or immediate hypersensitivity involves an antigen reacting with mast cells passively sensitized by IgE, inducing the release of histamine orothe rvasoactiv e amines which causes tissuedamage .Th etyp eII I reaction or semi-delayed hypersensitivity is due to the formation of immune complexes between the antigen and systemic IgG,complemen t activation andplatelet s aggregation, leading to thereleas e of vasoactive amines and proteolytic enzymes by leucocytes. The typeI Vo rdelaye d hypersensitivity ismediate d byT -lymphocyte s specific of theantige n which release lymphokines. Porter et al. (1981)reporte d that 50%o f thecalve s exhibited the type III reaction and only 2 - 3%th e type I reaction when given immunologically active products. In contrast, Heppell et al. (1987) observed from cutaneous tests that heated soya flour induced reaginic responses corresponding to type I in all the calves ; however thetyp eI Vreactio ndi d not appeart ooccur .

Disorders similar tothos ereporte d in thecal fhav ebee nevidence d atweanin gi npiglet s with diets containing heated soya flour (Miller et al., 1984). However, piglets rapidly become tolerant to soya whilst preruminant calves do not (Heppell et al., 1987).Failur e of localised immuno-defence mechanisms in the small intestine of the calf against the uptake of soya protein has been ascribed to inadequate synthesis of secretory IgA and IgM (Barratt et al., 1979). Theeffec t of ageo n thedevelopmen t of allergy symptoms isno twel l documented. As far as antibody production against soya protein is concerned, Barratt et al. (1979) found higher titres when the soya diet was introduced at 1 week instead of 4 weeks of age.I n contrast, Srihara (1984) did not show evidence of any differences when the soya diet started at 6, 18o r 30d o f age.

Effects of soyaproduct so n gutmotilit y Studies of intestinal motility have been made in calves given a series of test feeds containing casein, heated soya flour (HSF) or antigen free soya protein (Sissons et al., 1987).Givin g HSFinduce d aprogressiv ereductio n of immediate postprandial abomasal emptying rate and an accelerated passageo fdigest a in the small intestine,togethe r witha reduced antral motility and an increased number and velocity of migrating myoelectric complexes (MMC) along the intestine. These disorders occured with neither casein nor antigen free soya protein. They appeared to develop together with the anti-soya IgG antibodies.Th erapi d effect of ates tfee d containing HSFo n abomasal motility (within 30 min) in sensitized calves is indicative of type I anaphylactic hypersensitivity. The disorders induced by sucrose addition in the control diet were distinct from those observed with HSF (table 4) : the number of MMC was reduced, mainly because the duration of thephas eo f irregular spike activity wasincreased .Therefore , thedisturbance s observed with HSF were probably not due to an osmotic effect of its oligosides. The hypothesis of anallergeni c reaction was strenghtened byth epreventiv eeffec t of ananti ­ allergic drug (Nedocromil sodium, Fisons, pic) given for 3 days before a test feed (Duvaux et al., 1988). In calves which had been given an intraperitoneal injection of

48 serumcontainin g anti-soya IgG butdevoi do f IgEgu tmotilit y was notdisturbe d with the first test feed of HSF : 5 - 6 test feeds were required to obtain net changes in MMC profile. Therefore, IgGdi d notappea r tob edeterminan t ort ob einvolve d withoutIgE . Table 4. Effect of sensitization of calves toheate d soya flour (HSF) and of the addition of sucroseint oth emil k substitute dieto n duodenal motility after ates tmea l (C.Duvaux , J.W. Sissons, L. Heppell, P. Guilloteau &R . Toullec, unpublished results).

Testmea l Casein HSF1* HSFx§ Sucrose

Numbero f MMC+ 6.8a 6.2a 11.8b 3.2c Duration of the first ISA (min)t 83a 106ac 37b 230c

*HSF1 :HS Ffee d given on thefirst occasion . § HSFx :HS Ffee d given onon eo f thefifth-ninth occasion . +Numbe ro fmigratin g myoelectric complexesdurin gth efirst 40 0mi nafte r thetes t feed. t ISA : irregular spikingactivity .

Resultsobtaine d withothe rprotei n sources / Other replacement proteins are capable of inducing gastrointestinal hypersensitive reactions. Kilshaw (1981) and Kilshaw & Slade (1982) reported villous atrophy and serum antibodies towhea t gluten andovalbumi n incalve s challenged with theseproteins . Also raw pea protein was found to lead to antibody production against the whole pea extract and its main two globulins (legumin andvicilin ) (Nunes do Prado et al., 1989b). The amountso fimmunoreactiv e leguminleavin g theabomasu m andth eileu mwer e found to be equivalent to about 24 and 3% of intake, respectively (Nunes do Prado et al., 1989b). Similarly, Sissons & Thurston (1984) detected immunoreactive glycinin at the end of the ileum in calves given heated soya flour. However, as far as legumin was concerned, our preliminary results suggested that the immunoreactive part escaping digestion in the small intestine was mainly partially hydrolysed to fractions whose molecular weights were about 160 and 55kD a instead of 360kD a for the intact protein (R. Bush, R. Toullec, I. Caugant & P. Guilloteau, unpublished observations). Nevertheless, antigenic material can survive along the digestive tract and this extended contact with the intestinal mucosa could favour its absorption and its allergenic effect. Immunoreactive legumin wasdetecte d inbloo dplasm a after the first 3- 4 meals , butno t thereafter in calveschallenge d with adie tcontainin gra wpe aflou r and skimmil k 3time s a week for 4 weeks and then once ada y for 4 weeks (Nunes do Prado et al., 1988b).I n contrast, theplasm a concentration of immunoreactive ß-lactoglobulin increased for 2 -7 weeks andthe n decreased, showing atransien t riseo f gutpermeabilit y to macromolecules probably due to intestinal inflammatory reactions (table 5). Therefore, the earlier disappearance of immunoreactive legumin from the plasma should not be due to a decrease of the amount absorbed but probably to the rapid synthesis of systemic antibodieswhic hhindere d legumin detection byELISA .

Thedevelopmen t of antibodies specific todietar yprotein si s not sufficient to establish an allergenic effect. Forexample ,calve s given adie tcontainin g apregelatinize d pea flour in which about 95%o f the legumin had been denatured developped antibodies against legumin and vicilin (Nunes do Prado et al., 1989c). However there was no symptom of intolerance atth een d ofileu m ;especially , theapparen t digestibility of nitrogen washig h

49 and theamin o acid composition of ileal digesta wasno tmuc h altered (fig. 4), suggesting that thedigestio n of dietary protein wasno timpaired .Beside s themethod s quoted above thepreparatio n of antibodies specific of bovineIg E wouldprovid e atoo lver y helpful to assess allergenic effects (Nielsen &Wilkie , 1977 ;Gershwi n &Dygert , 1983 ;Barra te t al., 1985). Also the changes in the ability of the gut to absorb xylose (Seegraber & Morrill, 1986)o rmannito l andlactulos e (Andrée tal. , 1987) couldb e useful.

Table 5. Effect of thepartia lreplacemen t of skim milk powder by ara w peaflou r on the plasma concentration of immunoreactiveß-lactoglobuli n (Nunes doPrad oe t al., 1989c).

ß-lactoglobulin (ng.ml-1 ) Diet initial minimum § maximum+ final

Pea* 212ad 119b 502a 213cd Control 337a - - 97b

*Th epe adie t wasgive n 3time spe r weekdurin g thefirst 4 week s and then each morning for 4 weeks. § During thefirs t orth e second week. +Betwee n thesecon d and thesevent h week.

Conclusion The influence of age anddie t on abomasal,pancreati c and intestinal secretions is well documented. However little attention has been given to the effect of diet on certain pancreatic proteases, includingcarboxypeptidases , and intestinal aminopeptidases.Als o few data appear tob eavailabl eo nbil e secretion. Studies aredeveloppin g onth ehormona l control of the digestive function and on the molecular control of enzyme synthesis.Th e closureo f theoesophagea l groove can bedisturbe d bypoo r management conditions.Th e effect of protein sourceo n abomasal emptying andit s consequence on digestion arewel l understood. Measurements of ileal digestibility can provide useful informations on the protein fractions resistant to digestion and on the availability of amino acids. The development of hypersensitivity reactions in the gastrointestinal tract with certain replacement proteins has been evidenced. Further work is required to improve the methodso f diagnosisan dt oadap t thetreatment sreducin g theallergenicit y toth e different protein sources.

References

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55 EFFECT OF PORCINE SOMATOTROPIN ON NITROGEN GAIN AND ENERGY METABOLISM IN FATTENING PIGS

M.W.A. Verstegen and W. van der Hel

Agricultural University Wageningen, Department of Animal Nutrition and Department of Animal Husbandry

Summary

From many studies it has become clear that at similar intake of protein growing pigs deposit more protein with porcine somatotropin (pST). This increase can amount to above 30% extra protein gain. This increase seems to be more or less independent of energy intake. However if pST is associated with reduced feed intake as occurs often then the increased protein gain will be less. The resulting effect of pST is increased rate of gain, decreased feed conversion ratio and increased lean % in the carcass. Associated with pST is also a clearly increased metabolic rate. It is not clear to which aspect of energy metabolism this can be attributed. When we assume maintenance is the only reason the increase is about 20%. If pST however is not associated with maintenance but with efficiency the deposition of protein and/or fat will require more metabolizable energy. When all extra heat is associated with the cost of protein the efficiency is lowered from 0.52 to 0.48. When all heat is associated with fat the efficiency is lowered from 0.77 to 0.68. It was also discussed how activity and altered thermal demand may be associated with extra heat production.

Keywords: porcine somatotropin, protein gain, metabolic rate, maintenance, partial efficiency.

Introduction

Exogenous administration of porcine growth hormone increases growth rate and also changes carcass composition markedly (Boyd et al., 1987; Etherton et al., 1987; McLaren et al., 1987). Especially in pigs between 50 and 105 kg these studies have been made. Most of these studies showed that effect of pST - increased rate of gain provided that feed intake increased - decreased feed intake - increased the % of lean tissue in the carcass - reduced the ratio of feed to gain However not all studies showed this effect (see Kanis et al., 1990). More recently Campbell et al. (1988) showed that also in animals from 25-55 kg exogenous administration improved performance. Mostly the earlier studies have been made on castrated male pigs. In later studies with animals of different sexes a similar increase in performance was found. Campbell and King (1989; personal communication) showed that with application of somatotropine to pigs fed various feeding levels the feed/gain ratios between sexes became similar. It was also shown that in general feed intake is diminished in a ratio proportional to the dose

57 Table 2. Protein and fat accretion of animals of 31 days from 60 kg of application. It has also been derived that maintenance feed probably onwards (Campbell et al., 1989). increased (Campbell et al., 1988). In this paper we will discuss some aspects of energy metabolism and N gain. Treatment In this context we will focus on the effects of porcine somatotropin Fat gain Protein gain on protein gain in relation to g/d g/d - pST applied Boars control 317 196 - sex 100 mg/kg d 203 238 - energy intake - protein intake. Gilts control 411 148 Moreover we will focus on aspects of energy metabolism in somatotropin 100 mg/kg d 185 treated pigs. 235 Castrated males control 462 Protein gain 139 100 mg/kg d 223 225 One of the major aspects of pST application is the change in carcass composition in treated animals. Campbell et al. (1988) noted that growth In their experiment animals received ad lib. feed. From this it is not clear how protein gain would have been at different intake levels and hormone reduced body and carcass fat at an increased protein and water in different genotypes. concentration of pigs at 55 kg. In Table 1 some of their data are given. These results suggest that the differences between sexes diminish with pST. It can be derived that probably also different genotypes may react Table 1. Effect of porcine growth hormone (pGH) administration on the differently on pST. In an experiment of the Wageningen group (see composition of the carcass and the empty body at 55 kg (from Campbell Verstegen et al., 1989) pigs of three genotypes Duroc, Pietrain and a et al., 1988). crossbred between Dutch Landrace and Dutch Yorkshire (DYxDL) were treated with pST from 55 kg onwards. During the period of application Feeding Dose pGH Rate of Carcass Empty body animals were placed on metabolism cages during 10 weeks and N balances level g kg-ld-1 gain water protein fat water protein fat were measured during the last 6 weeks. Animals received a feeding level g/d-1 % % % % % % of about 2.6 times maintenance. The diet is given in Table 3.

Ad lib. 0 905 53.8 14.5 28.0 56.6 14.6 25.8 Table 3. Composition of dieta) 100 1052 59.3 16.1 20.0 61.7 16.2 18.8 Ingredient % by weight 80% 0 670 56.0 15.3 23.9 57.7 15.4 23.1 100 842 63.3 17.0 16.0 64.2 16.9 15.3 Yellow corn 3.7 Barley 60% 0 543 58.9 16.2 20.5 11.8 60.2 16.3 19.6 Peas 100 681 64.3 17.3 14.6 65.6 17.3 10 14.1 Extracted soybean meal (48.5% crude protein) 21 Corn gluten feed 5 Tapioca 35 Citrus pulp 0.7 Sugarcane molasses Data on Table 1 show that since rate of gain was increased also rate 5 Animal meal of body protein deposition in these pigs was increased. As a consequence 4 Animal fat lean % can be much higher in pST treated animals (Kanis et al., 1989). 1.2 Mono calcium phosphate It has been shown that the capacity to sustain N gain is highest in boars 0.9 Salt compared to sows. This in turn is higher then in castrated males. From 0.2 Limestone a recent experiment Campbell et al. (1989) reported that from 60 to 100 0.2 Mineral + Vitamin mixture^) kg boars gained 32% more protein then sows and 42% more then castrated 0.5 Mixturec) males. They found that after administration with pST during this period 0.8 protein deposition was raised to the same absolute level regardless of a sex . )Calculated: Dig. lysine, 0.92%; Dig. methionine + cystine, 0.55%; net energy for pigs, 9.0 MJ/kg; analysed dm 88.6%; crude protein, 19.4%; Ca, 0.90%; P, 0.69%.

Results on rate of gain and feed to gain ratio are presented in Table 4.

58 59 Table 4. Rate of gain (kg/d) and feed conversion of animals of three genotypes at constant feeding level during 6 weeks (5-10 weeks after initial administration).

Rate of gain Feed/gain

Pietrain C .87b 2.94b T .99a 2.79c Duroc C .72d 3.40a T .80C 3.19a DYxDL C .92b 2.90b T l.Ofa 2.70d

* Different superscripts in a column means significantly different (P^O.01).

As an average animals with pST increased their rate of gain with about 100 g per day. Since feed intakes were similar ratio of feed to gain was decreased with about 0.17. Results on performance as presented in Table 4 do not give information on alteration of body composition. In the same experiment therefore N gain was also measured. In Table 5 the results in N gain has been recalculated to protein. Since energy balances were also made fat gain could be derived from the data on intake of metabolizable energy, heat production and energy associated with protein gain. This can be done as follows: Metabolizable energy (ME) = Heat production (H) + Retained energy (RE) Moreover retained energy (RE) = energy retained in protein + energy retained in fat. Since energy retained in protein can be obtained from N balance as Energy retained in protein = N balance x 6.25 x 23.7 the resulting fat gain can be calculated. Results have been given in Table 5. The results show that there may be differences between genotypes.

Table 5. Effect of pST on rate of gain in protein and fat (g/d).

Genotype Treatment Protein Fat (g/d) (g/d)

Pietrain Control 142 263 pST 191 249

Duroc Control 119 275 pST 166 215

DYxDL Control 168 280 pST 210 218

On an average protein gain was increased with about 40 g/d and fat gain was decreased with about 40 g/d in these experiments. Detailed data on this have been presented by Van der Hel et al. (1988). Under normal circumstances increases in protein accretion of the magnitudes shown in Table 5 would be expected to require concomitant increases in the levels of dietary protein and amino acids. By using the factorial approach Boyd et al. (1988) predicted an almost two-fold increase in the dietary lysine requirement of pigs administered pST between 55 and 100 kg liveweight. However, this requires accurate information on the

60 rate and amino acid composition of protein gain and relies on numerous assumptions regarding the efficiency with which dietary nutrients are metabolised and integrated into animal tissues. It takes no account of the extent that the increase in protein by pST administration results from relative changes in the rates of protein synthesis and also in the relative breakdown or in the efficiency of amino acid transfer in the intermediary amino acid metabolism. All these factors however, can affect the amount of dietary protein required to support protein growth at the tissue level. In addition the efficiency of amino acid utilisation by a pig is not solely a function of the amino acid composition of the diets. The lipid and carbohydrate content of the diet can be critical factors if energy supply is too limited. In such situations, amino acids can be used for energy supply in preference to protein synthesis. Under conditions where ample energy is available, the amino acids which are surplus to protein synthesis after the requirement for the limiting amino acid has been met, are available for use as energy sources after deamination. The energy available is probably only 5 percent or less of the total energy in a well balanced standard diet. The effect of increasing the energy intake of a diet while maintaining a constant amino acid intake is to increase the synthesis rate of fat. Neither efficiency nor rate of amino acid use appear to be affected. However at low energy intakes especially in young animals protein synthesis may be lower than at adequate levels of energy intake. The consequence will be that lean tissue growth rate will be less then at adequate intakes (see Figure 1). Protein gain g/day 180 160

140 120 100 21.3 MJ ME 80 17.8 MJ ME 60 14.8 MJ ME 40 _ A

-L. 120 200 280 360 Protein intake g/day

Figure 1. Effect of levels of protein and energy intako

From this figure 1, which is derived from data of Campbell and Taverner (1985) with young piglets it is clear that less protein is deposited at low intake of energy compared to higher intakes. At adequate intake of energy however, additional energy does not increase the rate of protein gain. Low (1985) quoted own results in which boars between 20 and 50 kg were given 20% extra gross energy to a standard diet. There appeared to be no increased rate of protein synthesis with extra energy.

61 Fuller and Chamberlain (1985) have discussed the relationship between protein gain and protein intake in young pigs. From this they derived the relation between protein gain and the intake of ideal protein. They defined this as the composition of dietary protein which can not be improved by any substitution of a quantity of one amino acid for the same quantity of another. In our discussion of protein gain we assumed that the protein in the diets contained enough of the ideal protein to given optimum protein gain.

Energy and protein intake

As shown there is evidence which shows that in young pigs protein gain may be limited by insufficient energy intake. Studies of Campbell et al. (1988) showed that at various feeding level growth hormone increased protein gain at each level. This has been depicted in Figure 2. This figure is an extension of Figure 1.

Protein gain g/day

180 |_

160

140 pST / 120 / / t 100 - 21.3 M3 ME 17.8 M3 ME 80 - /, 14.8 M3 ME 60 /.'

40

I -J- J_ J_ 120 200 280 360 Protein intake g/day

Figure 2. Possible effect of pST on protein gain.

Their results obtained from rapid growing animals suggest that at each level of protein intake there is an effect of pST. Energy intake does not seem to limit protein deposition with pST in their experiment. This has been found also by Van Weerden et al. (1989). In a study of Van Weerden et al. (1989) pigs were given 3 protein levels at two levels of energy intake. The diets have been depicted in Table 6.

62 Table 6. Dietary composition in triais of Van Weerden et al. (1989).

Groups Treatment Composition of feed Net energy Crude protein Lysine MJ/kg (%) (%)

I Control 9.1 16.0 0.80 II pST treated 9.1 16.0 0.80

III Control 9.1 18>.0 0.90 IV pST treated 9.1 18.0 0.90

V Control 9.1 20.0 0.98 VI pST treated 9.1 20.0 0.98

VII Control 10.8 18.0 0.92 VIII pST treated 10.8 18.0 0.92

IX Control 10.8 20.0 1.07 X pST treated 10.8 20.0 1.07

The results of their study showed that N gain intake increases N gain and at each level of protein intake this increase is of similar magnitude regardless of energy intake (Table 7). /

Table 7. N balance (g/day) in experiments with various lysine and energy levels (Van Weerden et al., 1989).

Diets Control pST pST as % Energy of control

normal energy, 23.8 29.4 124 Low 16% protein

normal energy 25.4 33.0 130 Medium 18% protein

normal energy 26.9 35.9 134 High 20% protein

high energy 25.4 33.8 133 Medium IS% protein

high energy 26.3 35.6 135 High 20% protein

Thus at each level of increased protein intake we can expect an increased protein gain with pST. This means that N gain from N in feed results in increased efficiency with use of pST. Van Weerden and Verstegen (1989) showed that pST may reduce N output (N feed - N retained in body) in growing finishing pigs (60-110 kg) in the order of 20% due to increased efficiency. In conclusion it can be derived that the application of pST to growing and finishing pigs - improves N gain relative of N in feed, this improvement occurs at each level of N intake. - improves N in gain relative independent on energy in feed

63 - improves N gain to such extent that differences between sexes diminish. As a result Jean % in the carcass may be increased by 0 to 4% (Kanis et al., 1989).

Energy metabolism

In the trials reported by Campbell et al. (1988) they calculated by regression of energy retained on intake of digestible energy that maintenance requirement in energy was increased with pST. This suggests that metabolic rate was increased after application of pST. In the study of Van der Hel et al. (1988) energy metabolism was measured and in Table 8 the heat production and retained energy are given.

Table 8. Heat production (H) and retained energy (RE) in animals of 55 to 100 kg and treated with pST (T) or sham injected (C) (Verstegen et al., 1989).

Heat production (H) Retained energy (RE) kJ/kgO.75 kJ/kgO.75

Pietrain C 632 448 T 653 427

Duroc C 611 469 T 669 418

Crossbred C 623 469 T 682 427

It appears that in all three genotypes heat production is increased with pST. A summary of the results on energy balances is given in Table 9.

Table 9. Energy metabolism (kJ/kg) and pST in growing pigs of 55-100 kg. C = sham injected, pST = treated (Van der Hel et al., 1988).

C pST

Intake (ME) 1092 1084 Energy gain RE 470 416 Heat production 622 668

At similar intake heat production is as an average increased with 44 kJ. Consequently retained energy of the same amount of feed intake will be decreased with 44 kJ. It is not clear why increased energy expenditure is associated with the application of somatotropine to animals. According to Machlin's work (1972) with pigs, pST directs nutrients to the muscle during growth. In understanding the metabolic effect of somato­ tropine on metabolism it is important to note that any intervention that alters the rate of major route of nutrient (or energy) storage will also alter the rate of storage pathways (Reeds, 1987). Therefore it is important to distinguish between the mechanisms which are responsible for a reduction in e.g. fat deposition that might arise from a specific stimulus of protein deposition and that appear to stem primarily from an increase in energy expenditure. It is not clear how the increase in serum glucose and insulin level after somatotropin applications in pigs (Etherton et al., 1986a and Boyd, 1987) is associated with the increased energy expenditure.

64 Some of the possibilities and options to explain this increase have been given by Verstegen et al. (1989). We first assumed that animals are kept at thermoneutral conditions. Heat (or thermal losses) is produced as a result of the many metabolic processes occurring within the animal, the extent to which it occurs is not only characteristic of the animal per se but is dependent upon nutritional, productive, environmental and other related factors. Thus no simple system can be used to describe the contribution made by the various factors to metabolic heat production. In practice, and in order to facilitate the application of energy evaluation systems, it has been customary to partition the thermal losses into those associated with maintenance, on the one hand, and those resulting from the deposition of tissue or products formed within the body, on the other. The former, the maintenance heat loss, represents an animal in a state of energy equilibrium. This means that it is neither losing nor gaining energy, so that the intake of dietary energy exactly balances the animal's heat output. The heat arising from the accretion of tissues or products within the body represents the amount of work done in their deposition and varies with the nutritional state of the animal, so that the higher the level of feed intake, the higher the rate of tissue accretion and the greater the heat output associated with these processes, i.e. the heat increment of feeding (which is synonymous with "specific dynamic action" and "dietary induced thermogenesis"). Both the maintenance heat loss and heat produced as a result of tissue deposition are influenced by a number of factors. As the rate of the animal's heat loss increases at any given level of feed intake, there will be a reduction in the rate at which energy is retained and hence a change in the energetic efficiency of growth. For practical purposes it is important to know to what extent heat production varies in relation to those factors which influence it, since this determined the extent and efficiency of energy utilisation.

It is usual to express the energy retained by an animal as a function of its bodyweight and the quality and quantity of the ration provided. The food can then be described in its capacity to sustain maintenance (MEm) and to promote energy gain (RE). This is illustrated in Figure 3, where metabolizable energy (ME) intake, i.e. the gross energy of the feed minus the energy lost in faeces, urine and methane, is related to energy retained (RE). We assume that partial efficiency (tgC£- in Figure 3) was not altered with increase in ME intake. The inefficiency I-tgoi is for heat increment. Above the basal or fasting level of metabolism, each increment in ME is associated with an increment in heat production (H). However, the increment in ME exceeds the increment in H so that the animal has the capacity to retain energy (RE), although RE only becomes positive at intakes above the maintenance energy requirement, that is MEm = zero RE. The efficiency with which energy is retained is equal to RE/(ME-MEm). As we are working with growing animals feed intake is normally above maintenance. We assumed linearity between energy retention and intake of metabolizable energy. Energy retention can be described as:

RE = kg*ME - b in which: RE = retained energy ME = metabolizable energy kg = dER/(ME-MEm) b/k g = maintenance requirement (MEm)

This procedure was applied by Campbell et al. (1988). They regressed ER on energy intake. They used digestible energy instead of metabolizable energy. This is justified because there is a nearly fixed ratio of digestible

65 Intake of metabolizable energy

Figure 3. The relation between energy retention (kJ/kg0-75 per day) and metabolizable energy (kJ/kg0-75 per day) in the pig at thermoneutral condition, tg is partial efficiency above maintenance. MEm is metabolizable energy needed for maintenance. to metabolizable energy above maintenance. Since there is a very high correlation between metabolizable energy and digestible energy (DE) the formula above also holds for DE. The value (1-kg) represents the increase in energy expenditure with feed intake above maintenance (MEp). The efficiency is representative for total energy gain from ME and does not account for differences in its composition, reflected in the rates of protein and fat deposition. Separate estimates of the energetic efficiency of protein deposition (kp) and fat deposition (k(S) can be calculated with the respective heat increments being MEprot. (1-kp) and MEfat (1-kp. Thus the total thermal loss associated with the metabolism of dietary energy is the sum of the energy costs of maintenance (MEm) and the heat increment associated with the deposition of tissue, calculated as MEp (1-kg) or partitioned into that associated with protein deposition, MEprot. (1-kp) and fat deposition, MEfat (1-kjp. There are thus three options for increased metabolis rate - altered maintenance (MEm) - altered partial efficiency of protein deposition (kp) - altered partial efficiency of fat deposition (k^). In all three cases the result should be a reduced retained energy as shown in Figure 4. Maintenance requirement increased metabolic rate may result in an increased maintenance requirement. Maintenance requirement (MEm) was calculated as MEm = MEintake - En in fat retained/^ - En in prot. retained/kp We used efficiency given by ARC (1981) to calculate MEm. The principle of this calculation is also depicted in Figure 5. It has been assumed that the efficiencies of conversion of ME above maintenance into energy retained in fat and protein are not altered. Resulting maintenance requirement is calculated as follows: Controls: MEm = 393 k3 ME/kg"-^ pST treated: MEm = 491 k3 ME/kgu-/:> 66 RE I • C

• pST

ME

Figure 4. Effect of pST on retained energy RE at the same intake of metabolizable energy.

Figure 5. Effect of pST on maintenance requirement (ME).

Campbell et al. (1988) derived a higher maintenance requirement for pST treated animals from their calculations. Various possibilities can be the reason for increase a maintenance requirement. One reason may be activity. It can be expected that maintenance requirement will be increased by increased activity. However in various discussions it has been suggested that pST treated animals are more lethargic then control animals (Curtis, 1989). Since in studies on N gain (Huisman et al., 1988) animals were kept in metabolic crates it was decided to measure metabolic rate in growing animals which were kept in groups. Van der Hel et al. (1989) studied effect of metabolic rate in two trials with each 2k castrated male pigs of 70 to 90 kg for a period of 3 weeks each. In each trial 12 pigs 67 received daily 4 mg rpST and 12 pigs received a carrier during 38 days. Energy metabolism and activity was measured continuously during 14 days. Activity was measured continuously and heat production associated with activity (Hac) was calculated from the relation between variation in activity and variation in heat production. Animals received a diet with 10.8 Ml net energy per kg and 20% crude protein (about 1% lysine). Animals were fed about 2.4 times maintenance ic. 1100 kJ ME kg0,7*. Metabolizable energy (ME) per kg0,75. In Table 10 results on mean weight, rate of gain and energy balances are given, heat production and heat production associated with activity (Hac) are given (SE within treatment group).

Table 10. Effect of pST (4 mg/day animal) on energy balances per animal and activity.

Mean weight Rate of gain Energy balances in K3/kg0-7-^ (kg) g/d ME Heat prod. Activity related heat production Hac

Trial 1 pST 89.5 902 1083 756 21.5 (=12.4%) controls 84.9 720** 1100 689 15.6 (= 9.8%) SE (31.0) (5.3)

Trial 2 pST 83.0 823 1040 759 15.9 (= 9.1%) controls 80.0 619** 1050 701** 14.3 (= 8.8%) SE (40.0) (3.8)

Results showed that animals fed at the same feeding levels and treated with pST showed increased rate of gain (P^O.01)** and increased metabolic rate. In the same study it was checked whether activity was altered in various part of the day as an example one of the results in trial 1 has been given (Figure 6). Data in this figure 6 show that as a general picture metabolic rate with pST is increased at each time of the day. If the animals are more lethargic as suggested by Curtis (1989) they did not show this in measurements with a burglar device (Wenk and Van Es, 1976). Activity is measured with ultrasound waves during every 6 minute period. The doppler effect (in microvolts) is a measure for activity of animals. Every surface change is associated with a movement of animals. The devices used are a Messl Spacegard Burglar SX15 alarm and also a Solfan microwave intrusion detector, model 3225. Activity is measured by placing activity meters in or above the chambers. The results from the two trials mentioned above do not exclude differences in activity between pST and control animals. Activity in our experiments was measured only as physical movements. They do not reveil various kinds of activity. There may still be differences in behaviour between these two treatments (pST and control). It has been suggested by Whittemore (1983) that protein content of the body may be associated to maintenance. Since in our study differences in metabolic rate were present from the beginning of the treatment (Van der Hel et al., 1989). Therefore difference in body

68 "O 1200 l/> * tooo 2"° 800 .2 D-

O.* *J " 400

200 \\ .„,-. *-<, V/\_/^\,_ — —\— i— 24 to 12 14 18 20 22 Hour of day

Figure 6. Heat production of pST and control pigs during the day (expressed as kJ/kg0-75 day). Control pigs broken line, pST pigs straight line, Upper lines: total heat. Lower lines: activity related heat production. / composition cannot be the reason for the difference in metabolic rate as found in this study. Another possibility of increased maintenance may be by way of extra heat loss from the environment. In experiments of Kanis et al. (1989) it was found that with pST backfat was reduced with about 18%. This may alter heat loss from animals to the environment. Heat transfer from the animal to the environment depends on insulation value of: - Boundary layer of air around the animal. - Hair coat. - Tissue. The first two are termed external insulation. Maximal tissue insulation at vaso constriction depends on the layer of fat around the body. It can be easily calculated what the effect of reduced (backfat) thickness on tissue insulation and thus total insulation will be. Hovell et al. (1977) measured insulation value in thin and in normal sows. They calculated that the tissue insulation of a normal pig is less than half of the total insulation. Therefore it is important to note that Curtis (1989) calculated that a 50% reduction in fat layer reduced the insulation value by more than 10%. This means that one of the avenues of heat transport from the animal's core to the environment is easier. Therefore it depends on the contribution of the tissue insulation to total insulation how much the lower critical temperature is altered (increased) by this. Almost certainly the critical temperature is increased by some degrees by pST depending on the degree of reduction in the fat layer surrounding the body. On the other hand however, as metabolic rate is increased the critical temperature is lowered. It needs to be assessed what the consequences of the combination of the increased metabolic rate and decreased backfat thickness will be. In Figure 7 both situations have been depicted.

69 Heat production

pST

Tl T2 Temperature

Figure 7. Effect of pST on metabolic rate in pigs (horizontal lines) and possible effects on thermal demand. C and pST represents increase of heat production with low temperature. The values Tl and T2 represents lower critical temperature.

The tpcC in Figure 7 is for increased thermal demand below thermoneu- trality and as insulation in pST animals is less than in controls, thermal demand will be probably increased. Traditionally it has been derived that metabolic rate may not be the same in various selected lines. Differences in maintenance requirement have been reported for pigs with different genetic capacities for growth (Campbell and Taverner, 1985) and in pigs selected for different backfat thickness (Sundst^l et al., 1979). Also in mice selected for low body weight Van der Wal et al. (1976) reported a higher maintenance compared to control animals.

Alteration of efficiency of protein synthesis

In Figure 8 it is shown how with similar maintenance differences in efficiency of retained energy can occur. Furthermore this efficiency can be calculated from energy in protein and/or from energy in fat gain. Retained energy

— ME

MEm is similar

Figure 8. Possible effect of pST on efficiency of synthesis of retained energy, assuming constant maintenance.

70 We assume that net protein synthesis is increased with the administra­ tion of rpST. However this does not mean that the partial energetic efficiency of protein accretion is also increased. A decreased protein turnover which is associated with higher protein gain would be expected to yield a lower heat production. It may be that an increase in protein accretion as a result of rpST is additionally associated with a more rapid protein turnover. This would lead to a changed metabolic rate depending on whether synthesis or turnover is changed most. If we assumed the same maintenance (420 k3 ME/kgu-^) in all animals and the same partial efficiency for fat deposition (kf = 0.74) in all animals, then the resulting kp were as follows:

* No change in maintenance 420 kJ ME/kg^-'-\ * No change in energy required per kJ fat deposited (kf = 0.74). * Change in energy required per kJ protein retained: Controls kp = 0.59 Treatment kp = 0.48

The higher metabolic rate with pST may also result from higher rate of protein accretion. Our results showed that the partial efficiency of protein accretion in control animals (kp) was similar to the ARC (1981) estimate. However kp in pST animals was markedly reduced, thus this is similar to the argument of Campbell et al. (1988).

Alterations in efficiency of fat synthesis

There may still be a third reason for the increase in heat production. Increased lypolysis (Etherton et al., 1986a) may increase fat "turnover" and as a result the partial efficiency of fat deposition from metabolizable energy is reduced. This implies that kf in rpST animals should be less than .74 compared to control animals. When we calculated kf by assuming that maintenance is equal in all groups (420 kJ ME/kg^-'-5) and partial protein efficiency was also equal (kp = 0.54) then the resulting kf was as follows. * No change in maintenance 420 kJ ME/kg^-'-'. * No change in energy required per kJ in protein (kp = 0.54). * Change in energy required per kJ in fat: Controls kf = 0.77 Treatment kf = 0.68

Turnover however is found at all levels of metabolism. According to Reeds (1987) at virtually every "key" reaction both forward and reverse reaction (or both) can be increased. The result will be that metabolic control of the cycles will determine what are the changes in heat production. In addition the net result of the forward and reverse cycles. Campbell et al. (1988) argued that the inhibition of lipogenesis may cause the decrease in fat accretion. They however also suggest the reduced amount of energy available for lipogenesis may contribute in a passive way to reduction in fat accretion. Their results of increased fat accretion with extra energy intake and pST showed that the reduced availability of energy above that for protein synthesis and/or maintenance may be responsible for the lower deposition. It may be that energy requirement for in maximum protein gain with pST may be increased. Thus metabolic changes in pST animals need to be assessed with regard to requirements of protein and energy. It can be concluded that pST increased protein gain, reduces fat gain and

71 increases metabolic rate. Until now it is not clear, whether the cause is altered maintenance and/or lower efficiency of energy gain. It need to be studied if the altered metabolic rate associated with pST is of a similar magnitude to the alteration brought about by selection towards a similar combination of protein and fat gain as with pST. In that respect it is also of interest to note that the effects of pST on protein and fat gain are sustained after withdrawal of pST treatment. Campbell et al. (1989) found that after administration during 30-60 kg and withdrawal rate of gain after 60 kg was sustained to be superior compared to control animals. In addition feed intake was similar during the withdrawal period.

It is not clear how pST effects metabolic rate. It would be of interest to know if metabolic rate still remains increased at those conditions. From the discussion on application of pST in growing and fattening pigs it can be concluded that pigs with pST - at same intake more protein gain (?'25% extra) - at same intake less fat gain - at same intake more rate of gain - at same intake more lean % - higher heat production - maintenance and/or efficiencies altered - thermal demand probably altered.

References

ARC, 1980. The nutrient requirement of pigs. Agricultural Research Council Commonwealth Agricultural Bureaux, Slough, England, 341 pp. Boyd, R.D., 1987. Somatotropin and productive efficiency in swine. Animal Health & Nutrition, Febr. 1987, p. 23. Boyd, R.D., D. Way-Cahen, D.E. Bauman, D.H. Beerman, A.F. de Neergard, L. South, 1987. In: Proceedings of the Maryland Nutrition Conference for Feed manufacturers pp. 58-66. Boyd, D.R., D. Way-Cahen and B. Kirk, 1988. Implications of somatotropin on nutrient requirements of growing swine. Proceedings of International Seminar on the Science of Porcine Somatotropin, p. 122-143. Campbell, R.G., N.C. Steele, T.J. Caperna, J.P. McMurtry, M.B. Solomon and A.D. Mitchell, 1988. Interrelationship between energy intake and endogenous porcine growth hormone administration (pGH) on the perfor­ mance body composition and protein and energy metabolism of growing pigs weighing 25 to 55 kg live weight. J.Anim.Sci. 66: 1643-1655. Campbell, R.G., N.C. Steele, T.J. Caperna, J.P. McMurtry, M.B. Solomon and A.D. Mitchell, 1989. Interrelationship between sex and exogenous growth hormone administration on performance, body composition and protein and fat accretion of growing pigs. J.Anim.Sci. 67: 177-186. Campbell, R.G. and M.R. Taverner, 1985. The effects of strain and sex on protein and energy metabolism in growing pigs. In: P.W. Moe and H.F. Tyrell and P.J. Reynolds, Ed. EAAP-publ.no. 31. Proc.En.Met. Virginia, p. 78-81. Roman and Littlefield, USA. Curtis, S.E., 1989. Potential side-effects of exogenous somatotropin in pigs. In: P. van der Wal, G.J. Nieuwhof and R.D. Politiek Eds. Biotechnology for control of growth and product quality in swine. Implications and acceptability, p. 155-158. Pudoc Wageningen.

72 Etherthon, T.D., C.M. Evock, CS. Chung, P.E. Walton, M.N. Sillence, K.A. Magri, R.E. Ivy, 1986a. Stimulation of pig growth performance by long-term treatment with pituitary porcine growth hormone (pGH) and a recombinant pGH. J.Anim.Sci. 63 (suppl. 1): 219 (abstract). Etherton, T.D., J.P. Wiggins, CS. Chung, CM. Evock, J.F. Rebhun, P.E. Walton, 1986b. Stimulation of'pig growth performance by porcine growth hormone and growth hormone releasing-factor. J.Anim.Sci. 63: 1389. Etherton, T.D., 3.P. Wiggins, CM. Evock, CS. Chung, J.F. Rebhun, P.E. Walton, N.C Steele, 1987. Stimulation of pig growth performance by porcine growth hormone: determination of the dose-response relationship. J.Anim.Sci. 64: 433. Fuller, M.F. and A.G. Chamberlain, 1985. Protein requirements of pigs, p. 85-96. In: D.J.A. Cole and W. Haresign, Eds. Recent Developments in pig nutrition. Butterworths, London. Hel, W. van der, M.W.A. Verstegen, J. Huisman, E. Kanis, E.J. van Weerden and P. van der Wal, 1988. Effect of pST treatment on energy balance traits and metabolic rate in pigs. J.Anim.Sci. 66, suppl. 1: 255 abstract 93. Hel, W. van der, M.W.A. Verstegen, E. Kanis, E.J. van Weerden and P. van der Wal, 1989. Effect of pST on metabolic rate and daily pattern of activity. J.Anim.Sci. In press. Hovell, F.D. de, J.G. Gordon and R.M. MacPherson, 1977. Thin sows. 2 Observations on the energy and nitrogen exchanges of thin and normal sows in environmental temperatures of 20 and 5°C J.Agric.Sci. 89: 523-533. Huisman, J., E.J. van Weerden, W. van der Hel, M.W.A. Verstegen, E. Kanis and P. van der Wal, 1988. Effect of rpST treatment on rate of gain in protein and fat in two breeds of pigs and crossbreds. J.Anim.Sci. 66, suppl. 1: p. 255, abstract 93. Kanis, E., G.J. Nieuwhof, K.H. de Greef, W. van der Hel, M.W.A. Verstegen, J. Huisman and P. van der Wal, 1989. Effect of recombinant porcine somatotropin on growth and carcass quality in growing pis. Interaction with genotype and slaughter weight. J.Anim.Sci. In press. Low, A.G., 1985. Amino acid use by growing pigs. p. 108. In: D.J.A. Cole and W. Haresign, Eds. Recent developments in pig nutrition. Butterworths, London. Machlin, L.J., 1972. Effect of porcine growth hormone on growth and car­ cass composition of the pig. J.Anim.Sci. 35: 794. McLaren, D.G., G.L. Grebner, P.J. Bechtel, F.K. McKeith, J.E. Novakofski and R.A. Easter, 1987. Effects of graded levels of natural porcine somatotropin (pST) on growth performance of 57 to 103 kg pigs. J.Anim.Sci. 65 (suppl. 1): 245 (abstract). Reeds, P.J., 1987. Metabolic control and future opportunities for growth regulation. Anim.Prod. 46: 149-160. Sundst{$l, F., N. Standal and O. Vangen, 1979. Energy metabolism in lines of pigs selected for thickness of backfat and rate of gain. Acta Agricul- tura Scandinavica 29: 337-345. Verstegen, M.W.A., W. van der Hel, A.M. Henken, J. Huisman, E. Kanis, P. van der Wal and E.J. van Weerden, 1989. Effect of exogenous porcine somatotropine administration on partitioning nitrogen and energy metabolism in three genotypes of pigs. J.Anim.Sci. In press. Wal, H. van der, M.W.A. Verstegen and W. van der Hel, 1976. Protein and fat deposition in selected lines of mice in relation to feed intake. EAAP publ.no. 19. Proc.En.Metab. Vichy: 125-129.

73 n

Weerden, E.3. van, M.W.A. Verstegen, J.M. Fentener van Vlissingen, W. van der Hel, E. Kanis and P. van der Wal, 1989. Effect of pST on N gain in pigs at various protein and energy intake levels. J.Anim.Sci. In press. Weerden, E.3. van and M.W.A. Verstegen, 1989. Effect of porcine somato­ tropin (pST) on environmental N pollution. In: Biotechnology for control and growth and product quality in swine. Implications and acceptability. P. van der Wal, G.J. Nieuwhof and R.D. Politiek, Eds. p. 237-246. Wenk, C. and A.J.H. van Es, 1976. Eine methode zur Bestimmung des Energieaufwandes für die Körperliche Aktivität von wachsenden Kühen. Monatshefte 54: 232-236. Whittemore, CT., 1983. Development of recommended energy and protein allowances for growing pigs. Agricultural Systems 11: 159-186.

74

IL. PRACTICAL APPLICATION OF (BIO)SYNTHETICAMIN O ACIDS IN POULTRY AND PIG DIETS

J.B. Schutte

TNO Institute of Animal Nutrition and Physiology (ILOB) P.O. Box 15,670 0 AA Wageningen

Summary

In this paper some aspects with regard to the practical application of (bio) synthetic amino acids inpoultr y and pig diets are discussed. - The dietary level of protein in layer and pig diets can be reduced with approximately 2.5 units when the diets are adequately supplied by the first limiting aminoacids . - The replacement of 42 protein from soya by synthetic amino acids did not effect chick performance. In laying hens, however, the substitution of 3.52 soya protein by synthetic amino acids lead toa significantly lower performance. The difference in reaction to dietary supplementation with synthetic amino acids between chicks and layers may be explained by the differences in time during which feed was available to the birds; chickens 24h /day, layers 16 h /day. - D- and DL-methionine are utilized aswel l as L-methionine inchicks , independent of SAA level or nature of the diet. - The D-isomer of tryptophan is only partially utilized by chicks. In pigs indications were found that the utilization of the D-isomer of tryptophan depends partly on the dietary level of L-tryptophan. At a dietary L-tryptophan level of approximately 0.15%, equal utilization was found for DL and L tryptophan. - In pigs themaximu m replacement value of cystine for methionine was found to be approximately 502.

1. Introduction

At present the utilization of protein is an important subject of discussion in the Netherlands because of the serious environmental problems related with the intensive production of pigs and poultry in certain parts of the country. One of the most serious contaminants of the environment originating from animal production isnitrogen ; for example the contamination of the ground waterwit h nitrate and the contamination of the air with ammonia. The principal reasonwh y animal production is largely responsible for this contamination, is the fact that animals are inefficient in converting of feed protein into animal protein. In pigs and poultry on an average only 35 to 451 of the protein cq.nitroge n (N)consume d is used for the production ofmea t and/or eggs. One of the most important factors affecting the utilization of dietary protein is the balance of the amino acids in the protein. The closer the amino acid composition of the diet matches the requirement for maintenance and production of meat and eggs, the less protein the animal needs.However , a typical problem in feed formulation is the achievement of an optimal balance of amino acids. In pig and poultry diets the most critical amino acids in this connection are methionine, lysine, threonine, tryptophan, isoleucine and, in broiler diets also arginine. Therefore, supplementation of a diet with these amino acids provides amean s for increasing the efficiency of the utilization of dietary protein.

75 Recent advances have beenmad e in developing industrial processes for economical production of (bio)synthetic amino acids.A t present lysine, methionine, threonine and tryptophan, are available economically. However, the inviv o utilization of supplemented free synthetic amino acids is still open for discussion.A review of the literature by Bach Knudsen and Jorgensen (1986)pointe d out that the utilization of synthetic amino acids was reported to be less than protein-bound amino acids by some investigators and equal or better by others.Fro m these studies they concluded that the utilization of synthetic amino acids most likely depends on the buffering capacity of the body's metabolic pool of amino acids as the absorption of synthetic amino acids ismor e rapid than that of protein-bound amino acids. In this connection the frequency of feeding and/or the amounts of synthetic amino acids included in the diet would also influence the utilization of synthetic amino acids. This paper dealswit h the use of free synthetic amino acids in pig and poultry diets.Th e topics discussed relate to the inclusion of small and larger amounts of free synthetic amino acids in these diets and the utilization of some individual amino acids.

2. Inclusion of small amounts of synthetic amino acids in poultry and pig diets

2.1 Laying hens

The most critical amino acids in layer diets based on corn and soybean oilmeal are methionine and lysine. Therefore, supplementation of such a diet with small amounts of these amino acids provides amean s for increasing the utilization of dietary protein. Theoretically the dietary level of protein in a corn-soya based layer diet could be reduced from 16.5 (normal practical level) to 14Z,withou t other limitations than lysine and methionine. This is illustrated in figure 1, inwhic h the

Figure 1.Amin o acid profiles of two diets for laying hens in relation to the requirement.

content ofA.A .relativ e to 260 requirement (%) >16,5% c.p.

220 J.4.0%c.p .

180

140

100 requirement =100 %

76 amino acid profiles of a 16.5 and 14.01 crude protein (c.p.) corn-soya diet in comparisonwit h the requirement are presented. The requirement figures for each essential amino acid is set at 1001.Th e amino acid contents of both protein diets are expressed as a percentage of the requirement. If the content of an amino acid in the diet is below1002 , thismean s that the diet isdeficien t in that amino acid, if the content is above 1002,th e diet has a surplus of that amino acid. The requirement figures for amino acids are derived from those presently used in the Netherlands. For some of the most important amino acids these requirement figures (in 2 of the diet) are as follows: SAA (methionine + cystine), 0.682;methionine , 0.341; lysine, 0.762; threonine, 0.502; tryptophan, 0.152 and isoleucine 0.552. These levels hold only when using well digestible diets containing approximately 11.7MJ.ME/kg . The theoretical calculation that by reducing the dietary level of protein from 16.5 to 142, only methionine and lysine will become limiting,wa s tested in a series of four experiments. In these Trials the performance of layers on a 16.52 c.p. diet supplemented with methionine up to a level of 0.652 SAAwa s compared with those on a 142 c.p. diet supplemented withmethionin e (0.652 SAA)an d lysine up toa level of approximately 0.752. Both diets were based on corn and soya. The birds were housed individually in battery cages. The hens were placed in the cages 2-4 weeks before the Trial started at approximately 26week s of age.Group s of 15 adjacent cages containing birds of similar initial body weight were used as replicates. Six replicates of each treatment were used in a randomised block design. Lightwa s provided for a period of 16 hours/day. / The birds were fed the experimental diets ad libitum as dry mash for three months. However, two of the Trials were extended over a full laying period of 52weeks .Th e results achieved in the Trials are summarized in Tables 1an d 2.

Table 1.Layin g henperformanc e (eggmas spe r hen in gpe r day and feed conversion) on a dietwit h 16.5an d 14%protein .Ag e period 26-38weeks .

Experiment Egg mass Feed/egg mass

16.57. c.p1 142 c.p.2 16.52 c.p.1 142 c.p.2

111.03 51.4 50.4 2.37 2.37 111.04 51.1 49.4 2.17 2.25 111.05 55.7 56.1 2.10 2.13 111.07 54.7 53.0 2.07 2.08

Mean 53.2 52.4 2.18 2.21

1 Supplemented with methionine. 2 Supplemented with methionine and lysine.

Innon e of the trialswa s there a significant difference between both diets in egg mass production and feed conversion efficiency. On a com­ posite basis,performanc e of thehen s fed the 16.57. c.p. diet was some­ what better than that of the hens fed the 142 c.p. diet.However , the small difference in favour of the 16.52 c.p. diet was not significant. So, from the results of these trials it can be concluded, that it is

77 Table 2.Layin g hen performance (eggmas spe r hen in gpe r day and feed conversion) on adie twit h 16.5an d 14%protein .Ag e period 21-78weeks .

Experiment Egg mass Feed/egg mass

1 2 1 2 16.52 c.p. 142c.p . 16.5% c.p. 142 c.p.

111.03 51.1 50.3 2.47 2.48 111.04 52.0 51.8 2.25 2.29

Mean 51.6 51.0 2.36 2.38

1 Supplemented with methionine 2 Supplemented with methionine and lysine.

Table 3. Effect of protein level on N excretion.

Layer 1 Layer 2

Crude protein content (2) 16.5 14.0

Intake of N (g/day), 3.04 2.60 N retention (g/day)j 1.12 1.12 N excretion (g/day)~ 1.92 1.48

1 Feed intake 115 g/day 2 1.10 g in eggs and 0.02 g in growth 3 via faeces +urine .

possible to attain nearly the same performance with a 142 c.p. corn soya based diet thanwit h a standard 16.52 c.p. diet, provided the low- protein diet is in addition tomethionin e also supplemented with lysine. The latter will not only improve the utilization of the dietary protein, but as a result also influence N excretion. The consequences of the reduction in dietary protein from 16.5 to 14Z in relation to the excretion of N are illustrated in Table 3. The results presented in this table show that reducing the dietary protein level from 16.5 to 142,result s in a reduction of N excretion of 0.44 g/day. This reduction corresponds with approximately 252.

2.2 Pigs

In practical Dutch pig diets,th e most critical amino acids are lysine,methionine , threonine and tryptophan. Following these amino acids isoleucine,valin e and histidine will most likely become limiting by reducing the dietary protein level. Theoretically the dietary level of protein in a barley, corn,tapioc a and soya bean oilmeal based pig

78 Table 4.Utilizatio n of free synthetic amino acids (FSAA)i n pigs.

Daily weight gain Feed/gain

17.52 c.p. 15.02c.p . + 17.52c.p . 15.01c.p . + FSAA FSAA

707 696 1.905 1.918 711 725 1.919 1.904

Mean 709 710 1.912 1.911

diet could be reduced with 2.5 unitswithou t other limitations thanth e above mentioned amino acids.Thi s assumptionwa s tested intw o Trials with pigs inth e liveweigh t period of 20-40 kg.I nadditio n toa positive control diet containing 17.52crud e protein (normal practical level)a negativ e dietwa s formulated containing 15.02 crude protein. The lowprotei n dietwa s supplemented with lysine,methionine , threonine and tryptophan up toa level present inth e17.5 2protei n diet. Tob e sure that no other amino acids would become limiting inth e lowprotei n diet, also small amounts of isoleucine,valin e andhistidin e were included inthi s diet. Intota l 0.942 of synthetic amino acids were added toth elo wprotei n diet;0.43 2 lysine, 0.142methionine , 0.122 threonine, 0.042 tryptophan, 0.102 isoleucine, 0.052valin e and 0.062 histidine. Each experimental dietwa sfe dt ofou r replicate pens of eight pigs each, twopen s with group-housed barrows and twowit h group-housed gilts. Theexperimenta l dietswer e feda d libitum aspellet s fora period of four weeks (ageperio d of 9-13 weeks). The trialwa s repeated once. The results of this study (Table 4) show thatwit h both diets almost equal performance was achieved. So,th e inclusion of small amounts of synthetic amino acids inth edie t didno taffec t pig performance when the dietswer e feda d libitum aspellets . In conclusion itca nb e stated that theutilizatio n of dietary protein inpig s canb e improved considerably by including the first limiting amino acids inth ediet .A s aresul t thiswil l contribute substantially toth e reduction of theN excretion. The practical potentials, however, depend onth epric e relations between feed components and synthetic amino acids inconnectio n with theamount s they have tob e included inth ediet .Th eamin o acids isoleucine,valin ean d histidine areno t available commercially. So,a nadditio n of these amino acids toth edie t will increase thepric e of feed considerably. Therefore a Trialwa sperforme d to studywhethe r orno tth edie tha st o be supplied with isoleucine,valin e andhistidin e when reducing protein level from 17.5t o15 2i na barley , corn, tapioca and soya bean oilmeal based young pig diet. This studywa s carried outi ncooperatio n withth e Institute forLivestoc k Feeding andNutritio n Research (IVVO). The results of this study pointed out that valine andhistidin e willno t become limiting by reducing thedietar y level of protein with 2.5units . The results with regard toisoleucin e areno tye tavailable .

79 3. Inclusion of larger amounts of synthetic amino acids in poultry diets

3.1 Broiler chicks

To study the effect of larger inclusions of synthetic amino acids on chick performance, two dietswer e formulated and calculated to contain 20 and 162 crude protein (c.p.), respectively. The main components of the diets were corn and soybean oilmeal. Both diets were supplemented with L-lysine and DL-methionine in order to obtain a dietary level of 1.252 lysine and 0.861 SAA. The 162 c.p. diet was fed without and with a supplementation of all other essential amino acids and non-essential amino acids (as glutamic acid and glycine)u p to the levels present in the 202 c.p. diet. Each experimental dietwa s fed to four replicate floor pens of 40bird s each. The dietswer e fed ad libitum as pellets for a period of threeweek s (7-28 days of age).Th e trialwa s repeated once. Reducing the dietary level of protein from 20 to 16%resulte d in both Trials in a significant decrease of chick performance (Table5) . However, this negative effect could completely be overcome by an addition of free synthetic amino acids to the 162 c.p. diet. This suggest that free synthetic amino acids are utilized aswel l as the protein-bound amino acids inchicks . Table 5.Utilizatio n of free synthetic amino acids (FSAA)i nchicks .

Treatment Weight gain (g) Feed/gain

Exp. A Exp. B Exp. A Exp.

202 c.p. diet 810 840 1.61 1.54 1) 162 c.p. diet 666 618 1.92 1.80 2) 162 c.p. diet+FSAA' 813 836 1.59 1.55

1) SAA and lysine at the 202 c.p. level. 2)Al l essential and non-essential amino acids at the 202 c.p. level.

3.2 Laying hens

In the study with laying hens two diets,base d on corn and soybean oilmeal,wer e formulated and calculated to contain 14 and 10.52 c.p., respectively. Both diets were supplemented with L-lysine and DL- methionine in order to obtain dietary levels of 0.732lysin e and 0.65% SAA. The 10.52 c.p. diet was fedwithou t and with an addition of all other essential amino acids and non-essential amino acids up to a level present in the 142 c.p. diet. Each experimental diet was fed to 60 individually caged birds.Th e diets were fed ad libitum as dry mash for a period of 12week s (26-38week s of age).Ligh t was provided for a period of 16 hours/day. Reducing the dietary protein level from 14.0 to 10.52 resulted ina significant decrease of layer performance. The addition of essential and non-essential amino acids to the 10.52 c.p. diet did improve performance significantly. However, egg mass production and feed conversion efficiency were still significantly less favourable than on the 14.02 c.p. diet. These results suggest that Table 6. Utilization of free synthetic amino acids (FSAA) in laying hens.

Treatment Daily Daily Feed/Egg egg'mas s (g) feed intake (g) mass

14.02 c.p. diet 53.8 111 2.06 10.52 c.p. diet } 41.9 102 2.44 10.52 c.p. diet + FSAA ' 50.8 109 2.15

1) SAA and lysine at the 142 c.p. level. 2)Al l essential and non-essential amino acids at the 142 c.p. level. synthetic amino acids are lesswel l utilized than protein-bound amino acids in laying hens. The difference in reaction to dietary supplementation with synthetic amino acids between chicks and laying hens may be explained by the difference in time during which feed was available to the birds,i.e . chicks 24hours/day s and layers 16 hours/day. That the frequency of feeding may influence the utilization of synthetic amino acids is supported by the literature. This was first demonstrated by Batterham (1974), who found that in/pigs free synthetic lysine was utilized much better by feeding the diet six times per day than by feeding the lysine supplemented diet once aday . Buraczewska et al (1980) studied once and four times daily feeding ofa diet supplemented with synthetic lysine on the absorption rate of N and lysine in pigs. When feeding once a day, the rate of passage of nitrogen through the intestine varied from 0.4 to 3.2 g/h,whil e the lysine content of the digesta was 3.2 to 4.5 g/16 gN . When feeding four times a day, the rate of passage of nitrogen was less variable, ranging from 0.7 to 1.8 g/h and the lysine content in protein (Nx 6.25) of the digesta was in the range of 3.4 to 4.1. In the case of laying hens a possible time lag effect might be acting in the change from light to dark. This hypothesis was tested in a study with broiler chicks. Two diets were formulated, a positive control diet containing 202 c.p. and a diet containing 162 c.p. supplemented with all essential and non-essential amino acids up to a level present in the 202 c.p. diet.A t a lighting scheme of 24 h/day, on both diets the same performance was achieved. However,wit h a lighting scheme of 16 h light and 8h darkness/day, feed conversion efficiency of the birds fed the 162 c.p. diet supplemented with synthetic amino acids was significantly less favourable (32) than that of the birds fed the 202 c.p. diet.N o differences inweigh t gain were observed between both groups. So,i n conclusion it can be stated that in broiler chicks a lighting scheme of 16h light and 8h darkness/day may influence the utilization of synthetic amino acids negatively.

4. Utilization of the D-isomer ofmethionin e in broiler chicks

Considering the literature data there still seems to be a lack of agreement on the utilization of the D-isomer ofmethionin e inchicks . Some investigators reported equal utilization of the D- and L-isomer (Leveille et al; 1960;Feathersto n et al;1962 ;Bauriedel , 1963). On the other hand, Brüggemann et al (1962), Smith (1966)an d Baker and Boebel (1980) reported the D-isomer to be inferior to the L-isomer. In

81 m

Table 7. Biological activity of DL-methionine by using a crystalline amino acid basal diet (trial1) .

Treat- Methionine Added SAA Average Average Feed/Ana - ment source methionine level weight daily feed gain ly-? equivalents (Z of ~-jgai- n _•_,._,_intak_ e _J_ (Zo f diet) } diet) (g) (g/bird)

1 - - 0 40 375* 40.4 2 26K 2 L-methionine 0 10 0 50 564b 51.2 1 3 DL-methionine 0 10 0 50 546b 49.8 1 92b 4 - - 0 45 500?. 49.0 2 < 5 L-methionine 0 10 0 55 622b 54.5 1 6 DL-methionine 0 10 0 55 634b 55.1 1 82b 7 - 0 50 564* 51.2 1 90^ 8 L-methionine 0 10 0 60 619b 53.6 1 9 DL-methionine 0.10 0.60 632b 53.8 1 79b

Composite data

L-methionine 602a 53.1 1.85a 4 DL-methionine 604a 52.9 1.84a

1)Th e crystalline amino acid basal diet contained 0.40, 0.45 or 0.501 SAA. 2)Withi n analysis,mean s followed by different superscripts are significantly different (P< 0.05). addition, some authors reported that the utilization of the D-isomer depends on the diet composition. Baker &Boebe l (1980) reported that the D-isomer is aswel l utilized as the L-form using an intact protein diet, but not when tested in a crystalline amino acid diet. Katz and Baker (1975) found that the utilization of the D-isomer depends on the total dietary level of SAA (methionine + cystine). At SAA levels near the requirement they found equal efficacy of D and Lmethionine , but at SAA levels below the requirement, L-methionine was found to be superior to D-methionine. In order to obtain more information about the influence of the diet composition and the total level of SAA on the utilization of the D- isomer, three Trialswit h broiler chickswer e performed. In Trial 1th e biological activity of L and DLmethionin e was compared using a crystalline amino acid diet. The twomethionin e forms were tested in the basal diet at equimolar levels of 0.102methionin e at total SAA levels of 0.40, 0.45 and 0.502, respectively. Trial 2 and 3wer e designed to compare the biological activity of L-, D and DLmethionin e using a semi-purified and a practical type basal diet, respectively. In Trial 2 each of themethionin e formswer e tested at equimolar additions of 0.05, 0.10, 0.15 and 0.20Zmethionine , and in Trial 3a t equimolar additions of 0.05 and 0.102. Each experimental dietwa s fed to six replicates (=cages ) of 13 birds each for a period of 21 days (6 to 27 days of age).Th e diets were fed ad libitum as drymash . The results obtained in the three trials are summarised in Tables 7 8 and 9. In none of the trials were significant differences in performance observed between themethionin e sources. So,i n conclusion it can be stated that the isomers ofmethionin e are equally utilized in broiler chicks, independent of SAA level or nature of the basal diet.

82 Table 8. Biological activity of L, D and DLmethionin e by using a semi- purified basal diet (Trial2) .

Treat­ Methionine Added Average Average Feed/ a ment Source Methionine Weight daily gain flysiT2 )s equivalents gain feed intake (Io f (g) (g/bird) diet ;)

1 D-methionine 0.05 398 43.0 2.27 1 2 L-methionine 0.05 412 44.9 2.29 3 DL-methionine 0.05 428 46.6 2.29

4 D-methionine 0.10 497 49.4 2.09 2 5 L-methionine 0.10 498 49.1 2.07 6 DL-methionine 0.10 494 49.8 2.12

7 D-methionine 0.15 554 52.2 1.98 3 8 L-methionine 0.15 566 52.7 1.96 9 DL-methionine 0.15 564 51.9 1.93

0 D-methionine 0.20 594 52.8 1.87 4 1 L-methionine 0.20 614 54.0 1.85 / 2 DL-methionine 0.20 600 53.8 1.88

Composite data

D-methionine 511 49.4 2.05 5 L-methionine 522 50.2 2.04 DL-methionine 521 50.5 2.06

1) The semi-purified basal diet contained 0.40Z SAA. 2)Withi n analysis,difference s inweigh t gain and feed/gain ratio are not significant (]?< 0.05).

5.Utilizatio n of the D-isomer of tryptophan in chicks and pigs

Data on the biological activity of the D-isomer of tryptophan in chickens and pigs are limited and variable. Ohara and Ariyoshi (1979) found that the biological effectiveness of D-tryptophan relative to the L-isomer was only 15%i n chicks. In a subsequent study (Ohara et al., 1980) they estimated the relative biological utilization of DL and D tryptophan to be 47 and 211, respectively, of that of L-tryptophan. Much higher values are reported by Liebert &Gebhard t (1979). They estimated the biological activity of DL and D tryptophan relative to L-tryptophan in chicks to be 86 and 72Z, respectively. Shelton et al. (1951)an d Thompson et al. (1952)fe d DL-tryptophan to pigs and demonstrated partial use of the D-isomer. Arentsen & Zimmerman (1985) reported that the nutritive value of DL-tryptophan is approximately about 701 of that of L-tryptophan forweanin g pigs. Kirchgessner &Rot h (1985), however, reported an equal biological acti­ vity of DL and L tryptophan for pigs over theweigh t range of 20 to 55kg . At ILOB one trial with chickens and twowit h pigs were carried out to obtain more information about the biological activity of DL-tryptophan. The results of these trials are reported briefly below.

83 Table 9. Biological activity ofD and DLmethionin e by using a practical type basal dietxTria l3) .

Methionine Added Average Average Feed/ Treat­ Ana­ 2) ment source methionine weight daily gain lysis equivalents gain feed intake (1o f (g) (g/bird) diet) ;

1 D-methionine 0.05 657 54.4 1.74 1 2 L-methionine 0.05 669 55.1 1.73 3 DL-methionine 0.05 663 55.2 1.75

4 D-methionine 0.10 679 54.6 1.69 2 5 L-methionine 0.10 683 55.0 1.69 6 DL-methionine 0.10 682 54.6 1.68

Composite data D-methionine 668 54.5 1.72 3 L-methionine 676 55.0 1.71 DL-methionine 672 54.9 1.72

1) The practical type basal diet contained 0.68% SAA.

2) Within analysis,difference s inweigh t gain and feed/gain ratio are not significant (P> 0.05).

5.1 Chickens

In the study with broiler chicks,a semi-purified basal diet was used and calculated to contain 0.08Z tryptophan. The treatments consisted of additions of 0.0, 0.02, 0.04, 0.06 and 0.082 L or DL tryptophan. Each treatment was comprised of 112 birds, subdivided in eight replicates (=cages ) of 14 birds each. The experimental diets were fed ad libitum as dry mash for a period of 18 days (7 to 25 days ofage) . The results of this trial (Table 10) indicate that both tryptophan sources did improve chick performance up to the highest supplemented level of tryptophan. However, at each supplemented levelweigh t gain and feed conversion efficiency of the birds fed the L-tryptophan diets was significantly better than that of the birds fed the DL-tryptophan diets. From the results itwa s calculated that the biological activity ofDL - tryptophan is approximately 65Z of that of L-tryptophan.

5.2 Pies

In the two pig trials,th e biological activity of DL-tryptophan was compared with L-tryptophan by feeding diets severely to moderately deficient in tryptophan (trial 1)an d moderately deficient to adequately supplemented with tryptophan (trial2) . In Trial 1, L- and DL-tryptophan were added in equimolar additions of 0.03 and 0.062 tryptophan to a practical type basal diet containing 0.092 tryptophan. Each experimental dietwa s fed to four replicate pens of 12 pigs each; two penswit h group-housed barrows and two pens with group-housed gilts. The diets were fed twice ada y according toa

84 Table 10.Biologica l activity ofDL-tryptopha n inbroile r chicks.

Treatment Tryptophan Added Weight Feed/gain Source tryptophan gain( g equivalents(Z)

1 - 1) 106 4.21 2 L-tryp 0.02 204 2.84 3 DL-tryp 0.02 158 3.32 4 L-tryp 0.04 427 2.09 5 DL-tryp 0.04 222 2.81 6 L-tryp 0.06 598 1.77 7 DL-tryp 0.06 362 2.14 8 L-tryp 0.08 665 1.68 9 DL-tryp 0.08 490 1.91

Composite data

L-tryp. 474 2.10 DL-tryp. 308 2.54

1)Th e basal diet contained 0.08Z tryptophan.

Table 11.Biologica l activity ofDL-tryptopha n inpig s (trial 1)

Treatment Tryptophan Added Daily Daily Feed/ Feed/gain source tryptophan weight feed gain equivalents (Z) gain (g) intake (g)

1) 37t 300, 8.12, L-tryp. 0 03 91 374 t 3.95 DL-tryp. 0 03 91 359 L-tryp. 0 06 156C 447C 2.87C c DL-tryp. 0 06 177 497C

1)Th ebasa l diet contained 0.09X tryptophan a,b,c, means with the same superscript within the same column didno t differ significantly (P< 0.05).

restricted wet-feeding scheme based upon live weight and expected weight gain fora period of sixweek s (6t o1 2week s ofage) .Th eamoun to f feed supplied was approximately 90Z ofth e estimated ad libitum level. In trial 2,L andD L tryptophan were added in equimolar additions of 0.04, 0.08 and 0.1ZZ of tryptophan toa practica l type basal diet containing 0.132 tryptophan. Sixreplicat e pens of 10pig s each, three with barrows and threewit h gilts,wer e assigned toeac h experimental treatment with the exception of treatment 1 (basal diet). Treatment 1 was comprised of four replicate pens of1 0pig s each; twopen s with barrows and twowit h gilts.Th e experimental diets were feda d libitum in thepellete d form fora period of sixweek s (7t o1 3week s ofage) .

85 Table 12.Biologica l activity ofDL-tryptopha n inpig s (Trial2) .

Treatment Tryptophan Added Daily Daily Feed/gain source tryptophan weight feed equivalents(2) gain (g) intake (g)

1) 1 - 150 391 2.61" 2 L-tryp. 0.04 390f; 680e 1.74 3 DL-tryp. 0.04 343^ 618, 1.80 588 971C 1.64C 4 L-tryp. 0.08 de de 5 605 1008 1.67C DL-tryp. 0.08 de 6 L-tryp. 0.12 619 1029fc 1.66C C 7 DL-tryp. 0.12 633e 1053e 1.66

1) The basal diet contained 0.132 tryptophan. a, b, c, d, e,mean s with the same superscript within the same column did not differ significantly (P < 0.05).

The results of thetw o Trials are summarized in Tables 11 and 12.Th e results show that in Trial 1, almost equal performance was achieved with both tryptophan levels at a dietary level of 0.031, whereas at a level of 0.06%, DL-tryptophan produced slightly better performance than didL - tryptophan. In Trial 2, the supplemental level of 0.042 L-tryptophan produced weight gain and feed intake results which were significantly superior to those of DL-tryptophan. However, at supplemental levels of 0.08 and 0.0122, diets with DL-tryptophan produced somewhat better performance than did L-tryptophan. In summary, in both trials the same trend of increasing utilization of DL-tryptophan by increasing the supplemental level was observed. These results seem to indicate that the utilization of the D-isomer of tryptophan partly depends on the dietary level of L-tryptophan. Based on the results of trial 2,youn g pigs may already have problems with the utilization of the D-isomer when the diet contains less than approximately 0.152 tryptophan. For practical feed formulation, this point is of minor relevance since practical diets for young pigs contain generally more than 0.152natura l tryptophan.

6. Utilization of cystine in pigs

The estimated extent towhic h cystine canb e used to meet the requirement for total SAA ranges from 40 to 702.Mitchel l et al. (1968) reported that cystine can replace at least 702o f the need for total SAA. This value is considerably higher than earlier estimates of 402b y Becker et al. (1955) and 502b y Shelton et al. (1951). Baker etal . (1959) reported that the replacement value of cystine depends onth e feeding system used. They found that when using an ad libitum system, 562o f SAA could be cystine. However, when using restricted feeding conditions, a replacement value of 662wa s found. Recently, Roth & Kirchgessner (1987) reported that considerable losses in performance occur if the proportion of cystine exceeds 552o f the dietary level ofSAA . At ILOB a study was carried out to obtain more information about the replacement value of cystine formethionin e in pigs. For this study two practical type basal diets were formulated containing 0.252 methionine + 0.252 cystine and 0.202 methionine + 0.302 cystine, respectively. Both 86 Table 13.Utilizatio n of cystine in pigs.

Treatment Dietary level (in Z) of: Cystine Daily Feed/gain as Z of weight Meth. Cyst. SAA SAA gain (g)

1 0.20 02 0 0.30.30 0 0.500.5 0 60 60 610* 2.382!* 2 0.20 52 5 0.20.25 5 0.500.5 0 50 50 693 2.238

3 0.20 52 5 0.30.30 0 0.550.5 5 55 55 686 2.264b, 4 0.30 03 0 0.20.25 5 0.550.5 5 45 45 736C 2.136

5 0.30 03 0 0.30.30 0 0.600.6 0 50 50 729° 2.164^° 6 0.30 53 5 0.20.25 5 0.600.6 0 42 42 738° 2.092

a, b, c, d,mean s with the same superscript within the same column did not differ significantly (P< 0.05). diets were supplemented with 0.05 and 0.10Z DL-methionine resulting in dietary SAA levels of 0.55 and 0.602 respectively. Each experimental dietwa s fed to six replicate pens of 9pig s each; three pens with group-housed barrows and threewit h group-housed gilts. The experimental dietswer e fed ad libitum as pellets for a period of sixweek s (8.5 to 14.5week s ofage) . The results of the study are summarized in table 13.Fro m these results it can be deduced that an increase of the dietary SAA levelwit h cystine did not have any effect on pig performance (Treatment 2v s 3, Treatment 4 vs5) . Further, itma y be concluded from the results that in pigs not more than 501 of the total dietary SAA should be furnished by cystine. The 502 replacement value of cystine in pigs agreeswel l with those reported by Schutte et al. (1984)i n layer diets.

7. References

Arentson, B.E. and Zimmerman,D.R . 1985.Nutritiv e value of D-tryptophan for the growing pig. Journal ofAnima l Science 60:474-479. Bach Knudsen, K.E. and Jorgensen, H. 1986.Us e of synthetic amino acids in pig and poultry diets. In:Recen t Advances in animal nutrition. Hazesign,W . and Cole,D.J.A . (Eds.)Butterworths , London, p.215-225. Baker, D.H., Clausing,W. , Harmon, B.G., Jensen, B.G. and Becker,A.H . 1969. Replacement value of cystine formethionin e for the young pigs. Journal of Nutrition 29:581-584. Baker, D.H., and K.P. Boebel, 1980.Utilizatio n of the D- and L-isomers ofmethionin e and methionine hydroxy analogue as determined by chick bioassay. Journal of Nutrition 110:959-964. Batterham, E.S., 1974.Th e effect of frequency of feeding on the utilization of free lysine by growing pigs. British Journal of Nutrition 31:237-242. Bauriedel,W.R. , 1963.Th e effect of feeding D-methionine on the D-amino oxidase activity of chick tissues.Poultr y Science 42:214-217. Becker, D.E., Jensen,A.H. , Terril, S.W. and Norton,H.W . 1955.Th e methionine-cystine need of the young pigs. Journal ofAnima l Science 14:1086-1094. 87 Brüggemann, J.K.,K . Drepper, and H. Zucker, 1962.Quantitativ e deter­ mination of the utilization of D-, L- and DL-methionine andDL-2 - hydroxy-4-methylthiobutric acid-Ca by the chick. Naturwissenschaften 49:344. Buraczewska, L. Lachowicz, J. and Buraczewski, S. 1980.Th e rate of absorption of synthetic lysine and dietary protein in the upper half of the small intestine of pigs. Arch.Tierernährun g 30:751-758. Featherston,W.R. , H.R. Bird and A.E.Harper , 1962.Abilit y of the chick to utilize D- and excess L-indispensable amino acid nitrogen in the synthesis of dispensable amino acids.Journa l of Nutrition 78:95-100. Katz, R.S.,an d D.H. Baker, 1975.Efficac y of D-, L- and DL-methionine for growth of chicks fed crystalline amino acid diets.Poultr y Sei. 54:1667-1674. Kirchgessner, M., and Roth, F.X. 1985.Biologisch e Wirksamkeit vonDL - tryptophan beiMastschweinen . Zeitschrift fur Tierphysiologie, Tierernährung und Futtermittelkunde 54:135-141. Leveille, G.A., R. Shapiro and H. Fischer, 1960.Amin o acid requirements formaintenanc e in the adult rooster. IV. The requirements for methionine, cystine, phenylalanine, tyrosine and tryptophan; the adequacy of the determined requirements.Journa l of Nutrition 72:8-15. Liebert, F. and Gebhardt G. 1979.N-Bilanzuntersuchunge n zur Verwertung von DL-Threonin und DL-Tryptophan am Broilerküken. Archiv Tierernährung 29:581-588. Mitchell, J.R., Becker , D.E., Harmon, B.G., Norton, H.W. and Jensen, A.H. 1968. Some amino acid needs of the young pig fed a semi-synthetic diet. Journal of Animal Science 27(2):1322-1326 . Ohara, J. and Ariyoshi, S. 1979.Nutritiv e value of L-, DL- and D- tryptophan in the chick. Journal of Nutrition Science: Vitaminol. 25:185-193. Ohara, I., Otsuka, S.,Yugari ,Y . and Aryoshi. S. 1980.Compariso n of the nutritive values of L-, DL- and D-tryptophan in the rat and chick. Journal of Nutrition 110:634-640. Roth, F.X. and Kirchgessner,M . 1987.Biologica l efficiency of dietary methionine or cystine supplementation with growing pigs. Journal of Animal physiology and Animal Nutrition 58(5):267-280 . Schutte, J.B., Weerden van E.J. and Bertram, H.L. 1984.Protei n and sulphur amino acid nutrition of the hen during the early stage of laying.Archi v Geflügelkunde 48 (5):165-170. Shelton, D.C., Beeson,W.M . and Mertz, E.T. 1951.Quantitativ e DL- tryptophan requirement of theweanlin g pig. Journal of Animal Science 10:73-79. Smith. R.E., 1966. The utilization of L-methionine, Dl-methionine andmethionine hydroxy analogue by the growing chick. Poultry Science 45:571-577. Thompson. CM., Reber, E.,Whitehair , C.K. andMacVicar, R. 1952. Utilization of D-tryptophan by swine.Journa l of Animal Science 11: 712-720. PRESENT AND FUTURE DEVELOPMENTS IN THE PROTEIN/AMINO ACID SUPPLY OF MONOGASTRIC FARM ANIMALS

E.J. van Weerden

TNO Institute of Animal Nutrition and Physiology (ILOB) P.O. Box 15,670 0A A Wageningen, NL

1. Introduction

In this paper, after a short overview of the present state of affairs of research into protein and amino acids inmonogastri c farm animals, possible developments in this area in the near future are discussed. The emphasis is placed on perspectives and problems of research aimed at solving the technical imperfections inpresent-da y practical feed formulation.

2. Present state of affairs

In the last decades, our knowledge of protein/amino acid nutrition of monogastric farm animals has increased dramatically. These developments will be discussed on the basis of the twomai n criteria relevant to practical nutrition: 1) the contents of available amino acids in feed components 2) the requirements of the animals for these amino acids. '

The data presently available tonutritionists , are summarized below. Contents of available amino acids in feed components - Reliable tables with contents of total amino acids in common feed components - Minimal data on contents of available/digestible amino acids in feed components. Requirement figures -Reasonabl y reliable requirement figures for lysine and sulphur amino acids (SAA) for the most important categories of poultry and pigs. - Insufficiently reliable requirement figures for the other amino acids. - No requirement figures based on determined availability/digestibili­ ty of the amino acids. From the tables it is clear where the gaps are in the present knowledge. First, there is a lack of data on the contents of digestible amino acids in feedstuffs use d in diet formulation for pigs and poultry. The question which of the digestibility parameters, faecal or ileal, is the best estimate for the protein feeding value,wil l be discussed later. Second, our present knowledge on amino acid requirements for the different categories of poultry and pigs is incomplete. This concerns especially the almost complete lack of data from requirement studies in which the digestibility of the amino acids in the test diets is determined rather than calculated. The figures for requirements of digestible amino acids presently used in the Netherlands are all based on data calculated from these incomplete tables. Therefore, the conclusion with regard to the present state of affairs,mus t be that although great progress in amino acid nutrition research has been made, there are still many questions,whic h are also relevant to practical nutrition, left unanswered. In the following, special attention will be paid to these questions and to the possibilities and problems related to further research in thisfield .

89 3. Future developments

3.1 Available amino acids in feedstuffs

This topic will be discussed on the basis of the following three items: -whic h criteria should be used: faecal or ileal digestibility? (3.1.1) - what are the advantages of using the method chosen? (3.1.2) -wha t are the problems, especially with regard to the methods of determination (3.1.3)..

3.1.1 Faecal or ileal digestibility

Many years ago, researchers had already come to the generally accepted conclusion that the criterion "digestible amino acid" isa better measure for "available amino acid" than the criterion "total amino acid" as determined in the standard chemical analysis. However, even since itwa s demonstrated (Zebrowska, 1973;Jus t et al. 1981) that the protein digestion process taking place in the large intestine of pigs does not yield amino acids that can be used by the animal, the question has been raised as towhic h type of parameter for digestibility, faecal of ileal,i s to be preferred. Itwa s realized that especially for feed components and diets inwhic h a considerable part of the protein is broken down by themicroflor a in the large intestine, faecal digestibility isno t a reliable estimate for the availability of amino acids.Afte r approximately ten years of research in different places of theworld , general agreement was reached, stating that in pigs ileal digestibility of amino acids is a better estimate for availability than faecal digestibility. In the Netherlands, a provisional tablewit h ileal digestibility coefficients of lysine,methionin e + cystine, threonine and tryptophan in several of the most important feed components used in pig diets,wil l most probably be published at the end of this year. This table will be based on results of research carried out by ILOB/CIVO during the last eight years, together with data from the literature. The question as towhethe r orno t ileal digestibility of amino acids in poultry is also a better measure of availability than faecal digestibility, cannot yet be answered. Whereas itwa s generally accepted that the caeca and large intestine in fowl play only amino r role in the protein digestion process, recent results of Green &Kiene r (1989)an d of Schutte et al. (1987) created doubts about this concept.Gree n &Kiene r (1989) compared digestibilities of protein and amino acids in intact and caecectomized cocks and found with soya and sunflower seed meal only small differences between both digestibilities. However, inmea t meals, digestibilities with intact birds were higher. Schutte et al. (1987) compared digestibilities of protein and amino acids of Phaseolus vulgaris beans heat-treated in two different ways (105°C for 40minutes , 105°C for 110 minutes) in intact cocks and birds canulated at the terminal ileum. The results are summarized in Table 1. With the 40-min autoclaved beans,th e differences between faecal and ileal digestibilities are relatively small,wit h the beans heated for 110minutes , the digestibilities are lower, especially the ileal digestibilities of threonine and tryptophan. This phenomenon ofa distinct depressive effect of overheating on ileal amino acid digestibility, but not on faecal digestibility, was also observed in pigs by VanWeerde n et al. (1987). Whether these findings indicate that ileal digestibility is in general also the preferable estimate for available amino acids in poultry remains to be seen.

90 Table 1. Faecal and ileal digestibilities of Phaseolus beans incocks .

Beans autoclaved Beans autoclaved at 105 °C, 40mi n at 105 °C, 110 min

faecal ileal faecal ill

Crude protein 68- 71 69 62 Lysine 74 78 65 63 Methionine + cystine 53 58 50 51 Threonine 68 65 62 45 Tryptophan 72 71 66 54

Table 2.Correlation s (r)betwee n N deposition (g/day)an d intake of faecal or ileal digestible protein on amino acids (g/day).

Faecal digestible Ileal digestible

Crude protein 0.75 0.81 Lysine 0.87 0.88 Methionine 0.76 0.78 Threonine 0.89 0.91

Table 3. Correlations (r)betwee nweigh t gain or feed conversion and faecal or ileal digestible protein or organic matter.

Protein Organic Mat ter

faecal dig. ileal dig. faecal dig. ileal dig.

Weight gain 0.34 0.76 0.89 0.80 Feed Conversion -0.65 -0.87 -0.88 -0.77

3.1.2 Advantages of ileal digestibility

Before introducing the concept of ileal digestible amino acids in practical diet formulation, it is desirable to acquire information on the advantages of this new system. For this purpose Just et al. (1985) carried out a series of N balance experiments with 24 diets of different composition (crude fibre contents of 3.9 to 12.5%i n the dry matter, different levels of tapioca and potato starch, etc.). The results are summarized in Table 2an d show that protein deposition is slightly higher when correlated with the intake of ileal digestible protein/ amino acids,bu t the differences with intake of faecal digestible amino acids are small and in general not significant. Dierick et al. (1987) compared diets with an increasing share of the large intestine in the total protein digestion and found (Table 3) thatweigh t gain and feed conversion are correlated considerably higher with ileal thanwit h faecal digestible protein. On the other hand, performance is higher when correlated with faecal rather thanwit h ileal digestible organic matter. According toDieric k et al. (1987) this last finding again stresses the importance of hind gut fermentation for the energy supply of pigs,

91 especially when fed high-fibre diets. The conclusion drawn from both studies could be that it is indeed advantageous to formulate pig diets on the basis of ileal digestible rather than faecal digestible amino acids.Th e extent of the difference between both methods will depend on the type of diet;a s the contribution of the large intestine in the total protein digestion process increases, the differences between bothmethod s will become more marked.

3.1.3 Problems

The method of determining ileal digestible amino acids is more complicated than determining of faecal digestible amino acids and leads, therefore, to a number of problems. Three of these problems will be discussed briefly below.

- Determining of ileal digestibility in pigs, and evenmor e so in fowl, is complex, labourious and hence expensive. The experimental animals must be canulated at the end of the small intestine, the collection of intestinal chyme is time-consuming and indicators are usually necessary to estimate intestinal flow. In the course of the years, several techniques have been developed tomeasur e ileal digestibility in pigs: T-canulaewit h spot-sampling of ileal chyme using indicators, re-entrant canuleswit h quantitative chyme collection, semi-quantitative chyme collection via a large caecum canule, the newly developed ilea-rectal shunt method. In each method, also different modifications are applied. Until now it has not been possible to evaluate all these techniques or to judge the reliability of the results obtained with them. This is because a direct comparison has not yet been carried out. - A second problem, strongly related to the former is that in each technique,modification s have been introduced in order to save time and labour. However, it isno t always clearwhethe r orno t such modifications are justified, especially when the periods of chyme collection are reduced to such an extent that it is questionable whether the figures obtained are reliable.A n extensive evaluation and strict standardization of techniques is a prerequisite to enable a clear comparison of the data obtained from the different research institutes. As an example of the differences obtained from the different research institutes,Tabl e 4give s figures of ileal digestibilities of protein and four important amino acids inmaize ,measure d at institutes in the USA and Canada (four batches) and at ILOB (six batches).

Table 4.Ilea l digestibilities ofmaize .

Source Number of batches Protein Lysine Methionine Threonine Tryptophan X sd X sd X sd X sd X sd

USA/Canada 4 79 2 76 5 89 2 73 4 72 3a

ILOB 6 67 4 54 8 82 3 59 6 45 12

a. For tryptophan the number of batches was two

92 The data from ILOB are in all criteria lower than theAmerica n data. These differences, sometimes more than 20units ,coul d be explained by the difference in origin of themaiz e (ILOB-data are frommaiz e of French origin), but the techniques used may have also played arole . - A third point which is especially relevant for the practical application of ileal digestibility figures,i s the necessity for simple, but reliable screening methods, preferably in vitro techniques, to become available for predicting ileal amino acid digestibilites in feed components and diets.I n this respect it is likely that in the near future a choice can bemad e between the combined vitro-vivo technique of the modified nylon bag method and one of the vitro techniques under development at CIVO-Zeist and IVVO- Lelystad. The first results indicate that a reliable ranking between batches within one feed component will be possible. The conclusion of this paragraph can be summarized as follows. Poultry -A t this stage,n o choice based on experimental data between faecal and ileal digestibility of amino acids possible. - More data needed on both criteria of digestibility. Pigs - Ileal digestibility of amino acids criterion of choice. -Mor e data on ileal digestibility needed. - Standardization of techniques necessary. - Development of simple screening methods for ileal digestible amino acids needed. In the future, research should also solve the problems related to factors responsible for obstructing the optimal process of digestion, especially that taking place in the small intestine. Particularly relevant in this respect are the "antinutritional factors" (ANFs) (Huisman, 1989, this volume) and some of the non-starch polysaccharides (NSPs) that interfere with the normal digestion process in the small intestine. In this last respect it is likely that the application of certain enzymes will be advantageous.

3.2 Amino acid requirements

In this paragraph attention is focussed on two aspects of this broad field of amino acid research, 1) themethod s used in determining amino acid requirements, 2) the effect of the hormonal status of the animal on the amino acid requirements, especially in connection with the effects on environmental N pollution.

3.2.1 Methods of determination

It is evident that the requirement for amino acids should be expressed on the same basis as that used for the contents of available amino acids. This means that in the concept of ileal digestible amino acids, the requirements should also be given as ileal digestible components. Until now amino acid needs in poultry as well as in pigs are almost exclusively determined as "total amino acids".Ver y few results have appeared in the literature with requirement figures based on experimentally determined digestibility data. The recommended levels of faecal digestible amino acids in poultry and pig feeds in the CVE-tables presently used in the Netherlands,ar e based on total amino acid, converted to faecal digestible amino acid using the coefficients mentioned in the tables. It is evident that this situation is very unsatisfactory and it ishighl y desirable that in the near future reliable data on amino acid requirements based on experimentally measured digestibility coefficients of the amino acids should be available. 93 Below, the criteria used in determining amino acid requirements are listed. - Protein deposition: N balance N in carcass -Weigh t gain, feed conversion, slaughter quality -Amin o acids in blood - Urea in blood ,, 14 -Amin o acid oxidation via CO. Whereas, theoretically, themethod s directly determining protein deposition are to be preferred, inyoun g animals weight gain and other performance parameters can also give reliable estimates of amino acid requirement. This is because of the high correlation with protein deposition. The indirect measures of amino acid requirements, blood content of amino acids and blood content of urea,ar e notwidel y applied inmonogastri c animals as the results are, in general,irregular . Methods determining amino acid oxidation bymeasurin g the production of CO.hav e been introduced. Themerit s of this technique are not yet proven, but figures for requirements of young growing pigs, obtained with these methods,ar e definitely too low,a s discussed by Henry et al. (1987). It is generally known that literature data on the amino acid requirements of the different categories of farm animals are rather different. One of the reasons,apar t from the variation inmethod s used, is the different way inwhic h the results of the experiments are analysed. The relevance of this point has been discussed by Fisher et al. (1973) on the basis of the results from a layer experiment described by Bray (1969), inwhic h the effect of lysine intake on egg production was measured. In Figure 1, the relation between lysine intake and egg output as calculated by Fisher et al. (1973) is shown by using four different statistical methods: the linear "broken line"metho d (D)an d three slightly different,non-linea r methods (A,B ,C) . The non-linear methods give a better picture of the real situation than the linear "broken line"mode l (Figure 1).I n ILOB amino acid studies, with poultry aswel l aswit h pigs this is also generally observed. The reason for the non-linearity of the response to graded levels of amino acid intakes is illustrated by Fisher et al. (1973) in Figure 2. In Figure 2, part (a)describe s the reaction of an individual laying hen to increasing intakes of an amino acid. It is assumed that the hen responds in a linear way until a plateau is reached. In part (b)o f the figure the variability in the production level of a small group of laying hens is indicated, resulting in the characteristic S-shape mean response curve of the group. This S-curvewa s generally found in our broiler studies when a broad range of amino acid intake levels was covered. Nowadays, there is general agreement among researchers that an analysis of the results of experiments on amino acid requirement on the basis of the "broken line"mode l usually gives too low figures for the requirement (Figure 1).However ,whe n non-linear models are applied, often too high estimates of the requirement are obtained. Despite many discussions among statisticians, a satisfactory, practical solution to this problem isno t yet available.A t ILOB, the procedure followed is that the results of these types of studies are statistically analysed according to a "best-fitting" model -generall y a non-linear curve, frequently a quadratic -an d the response curve is presented together with the "confidence limits" of the suggested requirement figure. For practical application it is important to give the complete response curve because this gives an indication of the negative effects when feeding below (or above) the optimum. The detrimental effects ofa supply below the optimum can be different for the individual amino acids 94 Figure1 .Typica lse to finput—outpu tdat a (o)fro ma nexperimen ti n whichpullet swer eallowe dfre eacces st odiet swit ha serie so fcontent s ofa nessentia lamin oacid .Afte rBra y (1969).Curv eB (— —) wasfitte d byth eprocedur edescribe di nthi spaper .Th eothe rplot sar ea nexponen ­ tialcurv eA (---), aquadrati ccurv eC (— -— ) andtw ostraigh t linesfitte db yBra yD ( - —). Thecurve swer efitte db yleast-square s procedures.

50 Eggproductio npe rbir d (g/day)

40

30 300 400 500 600 700 Lysineintake ,mg/bir dda y

Figure2 .Mode lpropose d forth erespons eo flayin ghen st oamin oaci d intake,a .Respons eo fa singl ehen ,b ,wit hindividua lan dmea nrespons e ina smal lgroup .Mea nrespons emarke db ybroke nline . Eggproductio npe rbir d (g/day)

Aminoaci dintake ,mg/bir dda y

95 Figure3 .Lysin erequiremen to fyoun gvea lcalves . Nbalance(% ) X X 100- X

1.20 1.40 1.60 1.80 2.00 2.20 analysedlysin econten t(g/10 0feed ) Figure4 .Requiremen to fmethionin ean dcystin efo ryoun gvea lcalves . Nbalance(% ) ^

100L_ ^*

95-

90

85

80-

0.70 0.90 1.10 analysedm+ c content(g/10 0g feed ) asshow ni nFigure s3 an d4 fo rlysin ean dmethionin e+ cystin e inyoun g (four- seve nweeks )vea lcalve s (vanWeerde n& Huisman ,1978) . Whenth elysin econten to fth emil kreplace rbase do nskimme dmil kwa s 10i belowth eoptimum ,N depositio nwa s 51depressed ,bu tfo rmethionin e +cystin eth eN depositio nwa snearl y 10Zlowe rwhe nth edietar y methionine+ cystin econten twa s20 2belo wth eoptimum .

3.2.2Effec to fhormona l statuso namin oaci drequirement s

Inth epape rb ySchutt e (1989,thi svolume )i ti sclearl y showntha t thepossibilitie s ofreducin genvironmenta lN pollutio ncause db y poultryan dpigs ,ar ehighl ydependen to na nexac tknowledg e ofth e

96 amino acid requirements of the animals.Potentiall y a considerable reduction in N pollution is obtainable by replacing part of the dietary protein by one ormor e of the limiting amino acids. A principally different, but very effective method of reducing N pollution is by intervention in the animals'hormona l status. It has been known for many years that the application of anabolic steroids in cattle and pigs results in a drastic improvement in the conversion of dietary protein to body protein, at the same time considerably reducing N output via the urine (vanWeerde n et al., 1981). The same phenomenon was observed in pigswhe n the /3-agonist clenbuterol was administered (vanWeerden , 1987). A third category of hormonal agents effective in improving protein conversion includes the porcine growth hormone (porcine somatotropin, pST).I n Table 5, the effect of recombinant pST (rpST) on Nmetabolis m in fattening castrated male pigs of between 80 and 125 kg liveweight is shown (vanWeerde n &Verstegen , 1989). Protein deposition was increased during this period by 27% and efficiency of protein conversion from 32.91 to 40.6% when rpST was injected. On the basis of these results itwa s calculated that N excretion of the rpST-pigs in the liveweight range from 58 to 110 kg was reduced by approximately 21Z (Table6) .

Table 5. N balance and efficiency of N retention (as X of intake).

Testperio d N Balance Efficiency( %'

Control rpS' Control rpST (g/day) (g/day) (Z(Z .o f control)

PI 27.9 33.8 121 39.1 46.0 P2 30.4 36.4 120 40.1 46.2 P3 26.5 34.6 131 33.4 42.0 P4 26.1 34.5 132 31.3 39.9 P5 23.7 30.7 130 27.2 34.6 P6 23.7 31.0 131 26.5 34.6 mean 26.4 33.5* 127 32.9 40.6

* significantly different from control (P < 0.05)

Table 6. Calculation of N excretion per animal

Property Control rpST

Liveweigh t (kg) 57.7-•11 0 58.5-110 Number of days 59 53 Feed intake (kg) 138 124 N intake (g) 4280 3845 Efficiency of N retention (X of intake) 32.9 40.6 N excretion (Z of intake) 67.1 59.4 N deposition in body (g) 1410 1560 N excretion (g) 2870 2285 Difference (g) -585 Difference (2) -21

* significantly different from control (P< 0.05) 97 P excretion in these animals was reduced by 162 (vanWeerde n &Verste ­ gen, 1989). Whereas it is proven that compounds of the threementione d categories of hormones or hormone-like agents can considerably increase protein deposition and decrease N excretion in pigs, it is still not clear whether orno t the increased amounts of protein gained by the animal require higher levels of amino acids in the diet.A s discussed by Campbell et al. (1989), the factorial approach applied by Boyd et al. (1988) predicting an almost doubling of the lysine requirement of pST- treated pigs, is of doubtful value. This approach is based on a number of assumptions which, especially in the case of agents affecting intermediary metabolism, are of questionable significance. Experimental data on the protein/amino acid requirements of pST- treated pigs published so far,ar e incomplete. They indicate that protein/amino acid requirements may be somewhat, but not drastically, increased after pST treatment (Steele et al. 1989;Campbel l etal . 1989). The results of two ILOB studies,on e with veal calves and one with pigs, tend to confirm this preliminary conclusion. In the study withmilk-replacer-fe d male veal calves of between i25 and 140 kg liveweight, placebos aswel l as calves implanted with oestradiol-17/3+ trenbolone acetate (20m g + 140mg )wer e given diets with increasing contents ofmethionin e + cystine. Each treatment group included six individually fed animals.Th e results for N balance, measured over three consecutive 5-day test periods,ar e given in Table 7. Whereas the number ofmethionin e + cystine levels is only limited and the highest meth. + cyst, contents are apparently too low to enable reliable conclusions on themeth . + cyst, requirement of the steroid- treated calves, the slopes of both response curves are not very different. This suggests that themethionin e + cystine need of the steroidimplanted calves isno t considerably higher than that of the placebo animals, despite the fact that N deposition in the treated calves is,o n average, 281higher . In the experiment with pigs, the effects on N balance in castrated animals of between 70 and 95 kg liveweight were measured of injections of recombinant porcine somatotropine (rpST). Ten groups of six barrows eachwer e fed five test diets: three diets with "normal" energy content (2175 kcal net energy/kg) and different protein contents where lysine was the limiting factor, and two diets with ahig h energy level (2570 kcal/kg)wit h the two higher protein/lysine contents. Each diet was fed on a restricted scheme to a placebo and to a rpST-treated group. The average results of two 5-day test periods are given in Table 8. In the placebo groups fed the "normal" energy diets,protei n deposition aswel l as weight gain and feed conversion were improved when the protein/lysine level in the dietwa s increased. This means that for these fast growing pigs the dietary lysine level of 0.80 -0.90 Z recommended inDutc h practice is already too low. It also stresses the need to regularly retest amino acid requirements in order to cope with genetic and other improvements influencing the animals capacity for protein gain/growth. Increasing the energy content of the diet did not affect protein gain. However,weigh t gain and feed conversion weremor e favourable. Recombinant pST increased protein deposition by 31Z,weigh t gain by 18% and feed conversion was 15Z lower.Whe n dietary protein/lysine was increased, the effect of rpST on protein gainwa s increased, but the differences were not significant; the effects onweigh t gain and feed conversion were irregular. The results of both pig and veal calf studies indicate the tendency for the protein/amino acid requirement aftermanipulatio n of the

98 Table 7.Requiremen to fmethionin e andcystin e invea l calves witha placeboo rimplante d with oestradiol andtrenbolone . Dietary meth. Meth. + cyst. N deposition + cyst. contents (Z) intake (g/day) g/day I

Placebo 0.50 11.9 33.3 86 0.61 14.5 35.4 91 0.69 16.4 37.2 96 0.76 18.1 38.5 99 0.88 21.0 38.8 100

Oestradiol 17/3 0.50 11.9 42.5 85 + trenbolone 0.61 14.5 46.4 93 acetate 0.69 16.4 47.4 95 0.76 18.1 48.6 97 0.88 21.0 50.1 100

asa percentag e ofth ehighes t (m+c)level s

Table8 .Effec to fdietar y protein/lysine andenerg y inpig swit ha placebo ortreate d with rpsT. ,

Energy Protein Lysine Placebo rpST- effect (c.p.,%) (anal.,%) N.E./kg Protein Weight Feed con­ Effect on Effect on Effect on dep. gain version protein dep. weight gain feed conv, (g/day) (g/day) (abs.) (g/day) (g/day) (abs.)

a 813 a a a 2175 16 0.80 149 K 2.84 . 143 -0.43 1 K a a 2175 18 0.90 159?* 852^ 2.72^ 150 -0.42 b a a a 2175 20 0.98 168b 920° 2.50 47 158 -0.38 57a C C 2570 18 0.92 159 924= 52 206° -0.46 C c c 2570 20 1.07 164 1011 2.28 58° 130 -0.28

Values foreac h energy intakei nth esam e line witha differen t index letter differ significantly (]?<0.05) . hormonal status ofth eanima l tob eincreased , butth edifferenc e with the non-treated placebos doesno tappea r tob edramatic . These findings are inagreemen t with theindication s published inth eliterature ,bu t more comprehensive research data areneede d before final conclusionsca n be drawn. Conclusions ofparagrap h 3.ca nb esummarize d asfollows . -Amin o acid requirement data should bedetermine d andcalculate d onth e basis ofavailable/digestibl e amino acids. - Published research data onamin o acid requirements should include confidence (5o r10Z )limit s andcomplet e response curves inorde rt o enable users ofthes e data tocalculat e detrimental effects ofnon - optimal supply. -Determinatio n ofamin o acid requirements should berepeate d regularly in order tofollo w genetic andothe r improvements inth eanimal s capacity forprotei n gain/growth. -Manipulatio n ofth eanimal s hormonal status canb ea ver y effective tool inimprovin g protein conversion andreducin g N excretion. Indications that theamin o acid requirements ofhormone-treate d animals areslightly ,bu tno tdramaticall y increased, need tob e confirmed.

99 4. References

Boyd,D.R. ,Wray-Cahen , D. and Kirk, B., (1988). In: Campbell R.G., Johnson,R.J . and King,R.H . (1989). Implications of biotechnological techniques for manipulating animal growth and development on tissue and dietary nutrient requirements of pigs. Proc. Symp. "Biotechnology for control of growth and product quality in swine, implications and acceptability", p.137-144. (P.va n derWal ,G.J . Nieuwhof, R.D. Politiek, editors). Pudoc,Wageningen , the Netherlands. Bray, D.J., (1969). Studies with Corn-Soya Laying Diets. 8.Requirement s for limiting amino acids. The basal diet and the requirements for isoleucine, lysine and tryptophan. Poultry Science 48,674-684 . Campbell, R.G., Johnson,R.J . and King,R.H. , 1989. Proc. Symp. "Biotechnology for control of growth and product quality in swine, implications and acceptability", Pudoc, Wageningen, p.137 . Centraal Veevoederbureau inNederland , 1986.Veevoedertabel . Centraal Veevoederbureau inNederland , 1984.Voorlopig e tabel verteerbare aminozuren inveevoedergrondstoffe nvoo rvarkens . Dierick, N.A., Vervaeke, J., Decuypere, J., van der Heyde, H. and Henderickx, H., (1987). Correlation of ileal and faecal digested protein and organic matter to production performance in growing pigs. In: European Association forAnima l Production Publication No. 35, Session 3, 50-51. Fisher, C, Morris, T.R. and Jennings,R.C. ,1973 . A model for the description and prediction of the response of laying hens to amino acid intake. British Poultry Science 14,469 - 484. Green, S. and Kiener, T., 1989.Digestibilitie s of nitrogen and amino acids in soya-bean, sunflower,mea t and rapeseed meals measured with pigs and poultry. Animal Production 48,157-179 . Henry, Y., Arnal, M., Obled, C. and A. Rérat,1987 . Protein and Amino Acid Requirements of Pigs. In: European Association for Animal Production Publication No. 35,Sessio n 3, 9-18. Just,A. , Jorgensen, H. and Fernandez,J.A. ,1981 . The digestive capacity of the caecum-colon and the value of the nitrogen absorbed from the hind gut for protein synthesis in pigs. British Journal of Nutrition 46,209-219 . Just, A., Jorgensen, H. and Fernandez,J.A. ,1985 . Correlations of protein deposited in growing female pigs to ileal and feacal digestible crude protein and amino acids. Livestock Production Science 12,145-159 . Schutte, J.B., van Leeuwen, P. and vanWeerden , E.J., 1987. •i: The ileal and faecal digestibility of protein and amino acids of heat treated beans in poultry. In:Europea n Association for Animal jjlji Production Publication No. 35,Sessio n 3,103 . i Steele,N.C. ,Campbell ,R.G. , Caperna, T.J., McMurtry, J.P. and |!• Solomon,M.B. , 1989. PST efficacy inNort h America: management i'i; variables and advantages. In:Proc.Symp . "Biotechnology for j control of growth and product quality in swine, implications and acceptability", pp 51-63. (P.va n derWal ,E.J . Nieuwhof, R.D. Politiek, editors), Pudoc, Wageningen, the Netherlands. VanWeerden , E.J., Berende,P.L.M ,an d Huisman, J., 1981. Application of endogenous and exogenous anabolic agents invea l calves. In: Proceedings E.C.Worksho p "Anabolic agents in beef and veal production", pp. 1-26.

100 VanWeerden , E.J. and Huisman, J., 1978.D e aminozuurbehoefte van het vleeskalf. In:Kalverda g Wageningen, 25 april 1978,pp . 4-21. VanWeerden , E.J., 1987.Effect s of Clenbuterol on N deposition and carcass composition in castrated male pigs. In: Beta-agonists and their effects on animal growth and carcass quality, pp.152-162 . Elsevier Applied Science,Londo n and New York. VanWeerden , E.J. and Verstegen,M.W.A. ,1989 . Effect of PST on environmental N pollution. In: Proc.Symp. "Biotechnology for control of growth and product quality in swine, implications and acceptability", 237-243 ,Pudoc , Wageningen 1989, 237-243. Zebrowska, T., 1973.Digestio n and absorption of nitrogenous com­ pounds in the large intestine of pigs. Rocz. Nauk. Roln., B.95,80 .

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