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Threonine as the second limiting amino acid in barley for rowing-finishing pigs and growing rats Aw-yong, Lai Mon 1974

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THREONINE AS THE SECOND LIMITING AMINO ACID IN BARLEY FOR GROWING-FINISHING PIGS AND GROWING RATS by LAI-MON. AW-YONG B.Sc. (Agr.), M c G i l l U n i v e r s i t y , 1972 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n the Department of ANIMAL SCIENCE We accept t h i s t h e s i s as conforming to the rgjq^ii^ed standard THE UNIVERSITY OF BRITISH COLUMBIA August, 1974 In presenting th is thes is in p a r t i a l fu l f i lment of the requirements for an advanced degree at the Univers i ty of B r i t i s h Columbia, I agree that the L ibrary shal l make it f ree l y ava i lab le for reference and study. I fur ther agree that permission for extensive copying of th is thesis for scho lar l y purposes may be granted by the Head of my Department or by h is representat ives . It is understood that copying or pub l i ca t ion of th is thes is for f i nanc ia l gain sha l l not be allowed without my wri t ten permission. Department of Animal Science The Univers i ty of B r i t i s h Columbia Vancouver 8, Canada Date August 2, 1974 ABSTRACT Supplementation w i t h graded l e v e l s of threonine to an a l l b a r l e y -l y s i n e d i e t (0.75% t o t a l l y s i n e ) improved the d a i l y g a i n , feed e f f i c i e n c y and carcass q u a l i t y of g r o w i n g - f i n i s h i n g p i g s . An a d d i t i o n of 0.10% threo-nine produced the optimum growth response i n the experimental animals. No a d d i t i o n a l improvement was obtained w i t h higher l e v e l s of threonine or threonine p l u s methionine supplementation of the d i e t . The 0.10% l e v e l of threonine supplementation gave performance c r i t e r i a which were comparable to those obtained w i t h the barley-soybean c o n t r o l d i e t , except the former d i e t r e s u l t e d i n s i g n i f i c a n t l y h i g h er backfat measurements. Threonine added at l e v e l s of 0.15% r e s u l t e d i n higher n i t r o g e n r e t e n t i o n than the other b a r l e y - l y s i n e - t h r e o n i n e d i e t s . Nitrogen r e t e n t i o n on t h i s d i e t d i d not d i f f e r s i g n i f i c a n t l y from the c o n t r o l d i e t . Barley-amino a c i d d i e t s r e s u l t e d i n b e t t e r p r o t e i n u l t i l i z a t i o n than barley-soybean c o n t r o l d i e t s . Feeding t r i a l s and metabolism t r i a l s i n d i c a t e d that methionine was not l i m i t i n g i n b a r l e y and that threonine was the second l i m i t i n g amino a c i d . Growth t r i a l s w i t h weanling r a t s confirmed the r e s u l t s obtained i n the p i g n u t r i t i o n a l experiments. Rat experiments i n d i c a t e d that no a d d i t i o n a l b e n e f i c i a l e f f e c t s were obtained when l y s i n e l e v e l s were increased from 0.75% to 0.90% even when supplemented w i t h a d d i t i o n a l threonine. R e sults i n d i c a t e d that supplementation w i t h l y s i n e to a t o t a l l e v e l of 0.75% and threonine at a l e v e l of 0.10% r e s u l t e d i n a h i g h l y balanced amino a c i d r a t i o f o r r a t s , and gave growth r a t e s which approached - i i i -those obtained on the c o n t r o l d i e t . Supplementation of the b a r l e y - l y s i n e d i e t w i t h 0.20% threonine and a l l other e s s e n t i a l amino acid s r e s u l t e d i n growth ra t e s and n i t r o g e n r e t e n t i o n s which resembled the r e s u l t s obtained w i t h the c o n t r o l d i e t . The replacement of the e s s e n t i a l amino a c i d mixture w i t h g l y c i n e on an equal n i t r o g e n b a s i s d i d not r e s u l t i n adequate n i t r o g e n r e t e n t i o n or growth r a t e s . - i v -ACKNOWLEDGEMENTS The author wishes to express h i s g r a t i t u d e to Dr. R.M. Beames of the Department of Animal Science f o r h i s constant encouragement and dynamic guidance throughout the course of study. In a d d i t i o n , h i s h e l p f u l suggestions d u r i n g the p r e p a r a t i o n of the manuscript are g r a t e f u l l y acknowledged. The w r i t e r i s g r a t e f u l to Dr. W.D. K i t t s , Chairman of the Depart-ment of Animal Science, f o r the use of necessary f a c i l i t i e s i n t h i s study. The author a l s o wishes to thank Mrs. L. Mather f o r her a s s i s t a n c e i n c a r r y i n g out the amino a c i d analyses. Thanks are a l s o extended to Dr. J.A. S h e l f o r d and Mrs. A.S. S a i t o f o r t h e i r a s s i s t a n c e s i n data analyses. The w r i t e r i s p a r t i c u l a r l y g r a t e f u l to Mrs. Y.S. Choo who spent c o n s i d e r a b l e time i n the t y p i n g of t h i s manuscript. F i n a n c i a l a s s i s t a n c e provided by the N a t i o n a l Research C o u n c i l i s g r a t e f u l l y a p p r e c i a t e d . - v -TABLE OF CONTENTS Page LIST OF TABLES x i LIST OF FIGURES x i i i I . INTRODUCTION 1 I I . REVIEW OF LITERATURE 3 A. ESSENTIAL AND NON-ESSENTIAL AMINO ACIDS 3 B. DETERMINATION OF AMINO ACID REQUIREMENTS FOR GROWING ANIMALS USING GROWTH PERFORMANCE AND NITROGEN BALANCE 4 C. METHODS OF DETERMINATION OF AMINO ACIDS REQUIREMENTS FOR GROWING PIGS BY DIET, TISSUE AND BLOOD ANALYSIS 6 a. Methods Based on A n a l y s i s of D i e t s 6 (1) A n a l y s i s of adequate d i e t s 6 (2) Determination of the composition of an " i d e a l " p r o t e i n 7 b Methods Based on Tissue and Blood A n a l y s i s 8 D. VARIATION IN AVAILABILITY OF DIETARY PROTEIN 9 E. METHOD OF ASSESSING AMINO ACID AVAILABILITY 10 a. Chemical Methods 10 b. M i c r o b i o l o g i c a l Methods 11 c. Enzymic Methods 12 d. Bioassays 12 F. EFFECT OF PROTEIN DIGESTION AND ABSORPTION IN RELATION TO THE REQUIREMENT 13 G. THE USE OF AMINO ACIDS IN PIG NUTRITION 15 - v i -TABLE OF CONTENTS (Contd.) Page H. AMINO ACID REQUIREMENT FOR GROWING-FINISHING PIGS 17 a. General 17 b. The Requirement f o r L y s i n e 17 c. The Requirement f o r Methionine 20 d. The Requirement f o r Threonine 23 I . AMINO ACID REQUIREMENT OF THE GROWING RAT 25 J . SUPPLEMENTATION WITH AMINO ACIDS OF GRAIN-PROTEIN CONCENTRATE DIETS AND GRAIN ONLY 28 •( K. FACTORS ASSOCIATED WITH AMINO ACID SUPPLEMENTATION OF GRAINS 32 a. V a r i a t i o n of Grain Amino Acids P r o f i l e and A v a i l a b i l i t y 32 b. P r o t e i n L e v e l i n R e l a t i o n to Amino A c i d Requirements 33 c. S i g n i f i c a n c e of E s s e n t i a l and N o n - e s s e n t i a l Amino A c i d R a t i o 35 d. R e l a t i o n s h i p s between P r o t e i n and Energy Content of a Di e t 35 e. Amino Acids Imbalances and I n t e r a c t i o n s 38 f . Animal V a r i a t i o n : Age, Sex and Genetic Factors 39 g. Feeding P r a c t i c e s : Ad L i b i t u m vs R e s t r i c t e d Feeding 42 III. PIG EXPERIMENT I 43 A. EXPERIMENTAL PROCEDURE 43 a. General 43 b. Design 43 - v i i -TABLE OF CONTENTS (Contd.) Page c. Animals 44 d. Diets 44 e. Management 46 ( i ) Housing 46 ( i i ) Feeding method 46 ( i i i ) Feed mixing and storage 46 f. Records 49 g. Chemical Analysis 49 h. Carcass Measurements 49 i . C a l culations 50 j . S t a t i s t i c a l Analysis of Data 50 B. RESULTS 52 C. DISCUSSION 57 D. CONCLUSION 68 IV. PIG EXPERIMENT II 70 A. EXPERIMENTAL PROCEDURE 70 a. General 70 b. Animals 70 c. Diets 71 d. Management 71 (i) Housing • 71 ( i i ) Feeding method 71 - v i i i -Page e. Feces and Urine C o l l e c t i o n 73 f . S t a t i s t i c a l A n a l y s i s of Data 73 B. RESULTS 75 C. DISCUSSION 78 D. CONCLUSION 83 V. RAT EXPERIMENT I 84 A. EXPERIMENTAL PROCEDURE 84 a. General 84 b. Animals 85 c. D i e t s 85 d. Management 85 ( i ) Housing 85 ( i i ) Feeding methods 86 e. Records 86 f. Chemical A n a l y s i s 86 ( i ) Feed 86 ( i i ) Carcass 86 g. C a l c u l a t i o n s 87 h. S t a t i s t i c a l A n a l y s i s 88 B. RESULTS AND DISCUSSION 89 C. CONCLUSION 95 VI. RAT EXPERIMENT I I 96 A. EXPERIMENTAL PROCEDURE 96 a. General 96 - ix -TABLE OF CONTENTS (Contd.) Page b. Animals 96 c. D i e t s 97 d. Management 98 e. Records 98 f. Chemical A n a l y s i s 98 g. C a l c u l a t i o n s 99 h. S t a t i s t i c a l A n a l y s i s 99 B. RESULTS AND DISCUSSION 102 C. CONCLUSION 109 V I I . RAT EXPERIMENT I I I 110 A. EXPERIMENTAL PROCEDURE 110 a. General 110 b. Animals 110 c. D i e t s 111 d. Management 112 e. Records 112 f. Chemical A n a l y s i s 112 g. C a l c u l a t i o n s 112 h. S t a t i s t i c a l A n a l y s i s 112 B. RESULTS AND DISCUSSION 115 C. CONCLUSION 119 - x -TABLE OF CONTENTS (Contd.) Page V I I I . GENERAL CONCLUSIONS 120 IX. LITERATURE CITED 122 X. APPENDIX 142 - x i -LIST OF TABLES E s s e n t i a l amino a c i d requirements of young r a t and growing p i g Percentage composition of d i e t s used i n P i g Experiments I & I I ( a i r dry b a s i s ) Content of e s s e n t i a l amino acids and proximate c o n s t i t u e n t s of b a r l e y and soybean meal on a dry matter b a s i s Content of e s s e n t i a l amino acid s and proximate c o n s t i t u e n t s i n d i e t s (g amino acid/100 g) i n P i g Experiments I and I I Summary of the e f f e c t of the a d d i t i o n of amino acids to b a r l e y on body weight g a i n , feed consumption (D.M.), feed conversion (D.M.) and carcass measurement i n P i g Experiment I Comparison of the e f f e c t s of supplementation w i t h amino acids on mean d a i l y gains and carcass measurements of g i l t s and barrows Summary of the e f f e c t s of supplementation of b a r l e y w i t h amino acids on apparent dry matter d i g e s t i b i l i t y , n i t r o g e n balance, apparent n i t r o g e n d i g e s t i b i l i t y and on v a r i o u s n i t r o g e n r e t e n t i o n i n d i c e s E f f e c t of amino a c i d supplementation of low p r o t e i n b a r l e y on average d a i l y g a i n , feed conversion e f f i c i e n c y and p r o t e i n e f f i c i e n c y r a t i o of r a t s E f f e c t of amino a c i d supplementation of low p r o t e i n b a r l e y on carcass c h a r a c t e r i s t i c s of r a t s Percentage composition of d i e t s used i n Rat Experiment I I ( A i r dry b a s i s ) Content of e s s e n t i a l amino acid s of each d i e t f o r Rat Experiment I I (g amino acid/100 g mixed feed) on a i r dry b a s i s - x i i -Table 12 13 14 15 16 17 LIST OF TABLES (Contd.) Page E f f e c t of amino a c i d supplementation of low p r o t e i n b a r l e y on average d a i l y g a i n , feed conversion e f f i c i e n c y and p r o t e i n e f f i c i e n c y r a t i o of r a t s 103 E f f e c t of amino a c i d supplementation of low p r o t e i n b a r l e y on carcass c h a r a c t e r i s t i c s of r a t s 107 Percentage composition of d i e t s used i n Rat Experiment I I I ( A i r dry b a s i s ) 113 Content of e s s e n t i a l amino acids of each d i e t f o r Rat Experiment I I I (g amino acid/100 g mixed feed) on a i r dry b a s i s 114 E f f e c t of amino a c i d supplementation of low p r o t e i n b a r l e y on average d a i l y g a i n , feed conversion e f f i c i e n c y and p r o t e i n e f f i c i e n c y r a t i o of r a t s 116 E f f e c t of amino a c i d supplementation of low p r o t e i n b a r l e y on carcass c h a r a c t e r i s t i c s of r a t s 117 - x i i i -LIST OF FIGURES Fig u r e Page 1 General view of metabolism cage 72 2 P i g i n the modified a d j u s t a b l e cage, h e l d w i t h canvas b e l t and rubber tubing to minimize u r i n e l o s s 72 - 1 -I . INTRODUCTION Cer e a l p r o t e i n i s w e l l known to be low i n n u t r i t i v e v a l u e , p r i m a r i l y due to a r e l a t i v e l y low l e v e l of s e v e r a l e s s e n t i a l amino a c i d s such as l y s i n e , threonine, methionine and tryptophan. Promising animal growth r e s u l t s have been reported when these l i m i t i n g amino acids are used as supplements f o r c e r e a l d i e t s . Soybean meal and fishmeal are the most common p r o t e i n concentrates used as supplements f o r c e r e a l d i e t s , t h e i r b e n e f i c i a l e f f e c t a r i s i n g from t h e i r overcoming, both the amino a c i d d e f i c i e n c i e s and the t o t a l n i t r o g e n d e f i c i e n c i e s i n the g r a i n . The competition between humans and l i v e s t o c k f o r n a t u r a l p r o t e i n sources has been a cause of concern f o r many years. N a t u r a l d i s a s t e r s such as drought can e s c a l a t e p r i c e s and s e r i o u s l y i n t e r f e r e w i t h the economics of swine p r o d u c t i o n . I t has long been hoped that complete dependence on conv e n t i o n a l p r o t e i n concentrates can be overcome. Hie pro-d u c t i o n of amino acids e i t h e r by fermentation or chemical s y n t h e s i s o f f e r s a p o s s i b l e s o l u t i o n . Although some of these s y n t h e t i c amino acids are s t i l l r e l a t i v e l y expensive at present, an i n c r e a s e i n demand and thus p r o d u c t i o n l e v e l should r e s u l t i n a dramatic r e d u c t i o n i n p r i c e , h o p e f u l l y to the l e v e l where a d d i t i o n to commercial l i v e s t o c k d i e t would be economically a t t r a c t i v e . B a r l e y i s one of the major feed grains i n western Canada. I t i s important t h a t the l i m i t i n g f a c t o r s i n b a r l e y p r o t e i n be e s t a b l i s h e d so that the most economical form of supplementation e i t h e r as conventional p r o t e i n or pure amino acid s can be p r a c t i c e d . - 2 -It i s well established that lysine is the f i r s t limiting amino acid in barley as a feed for pigs. Although methionine has been suggested as the second limiting amino acid by some workers, a large number of experiments have shown either no response to methionine or i n some cases, a growth depression. In contrast, threonine, when tested as the second limiting amino acid, has shown a response with barley, wheat, rice, milo and sorghum. Many recent reports have confirmed threonine as the second limiting amino acid in grain in the nutrition of several monogastric species- including man. The objective of this study was to further evaluate under practical conditions the requirements of threonine as the second limiting amino acid in Peace River barley for growing-finishing pigs. Hie design involved feeding graded levels of threonine in a barley diet in the presence of adequate lysine and measuring growth performance, carcass quality and nitrogen balance. Parallel studies were carried out with rats to further examine the nutritive value of barley with lysine, threonine and a mixture of other amino acids. - 3 -I I . REVIEW OF LITERATURE A. ESSENTIAL AND NON-ESSENTIAL AMINO ACIDS Amino acid s found i n p r o t e i n were c l a s s i f i e d by Rose (1938) i n t o two c a t a g o r i e s , the e s s e n t i a l and the n o n - e s s e n t i a l amino a c i d s , based on growth s t u d i e s w i t h r a t s . E s s e n t i a l are those amino acid s which cannot be s y n t h e s i z e d , at a l l , or at a r a t e f a s t enough to meet the metabolic requirements, and t h e r e f o r e must be s u p p l i e d i n the d i e t . N o n - e s s e n t i a l amino acids are those which can be synt h e s i z e d by animals from normal food c o n s t i t u e n t s v i a transamination r e a c t i o n s . The number of e s s e n t i a l amino acids v a r i e s w i t h species and the type of production d e s i r e d . Those c o n s i -dered to be e s s e n t i a l i n a l l mammalian species are v a l i n e , l e u c i n e , i s o l e u c i n e , threonine, methionine, l y s i n e , phenylalanine and tryptophan. A r g i n i n e and h i s t i d i n e were formerly i n c l u d e d i n t h i s l i s t . I t i s now c l e a r t h a t these amino acid s are synt h e s i z e d i n adequate q u a n t i t i e s to meet minimum metabolic needs. I n some s p e c i e s , f o r example, the r a t , b e t t e r growth does occur when these amino acid s are added to the d i e t . I n marked c o n s t r a s t to the mammalian s p e c i e s , the chick cannot s y n t h e s i z e a r g i n i n e at a l l and must o b t a i n a l l i t s requirements f o r t h i s amino a c i d from the d i e t . In a d d i t i o n , the c h i c k apparently cannot syn t h e s i z e g l y c i n e , s e r i n e , glutamic a c i d and p r o l i n e at a s u f f i c i e n t l y r a p i d r a t e f o r the demands of e a r l y growth (Almquist, 1972) . Cy s t i n e and t y r o s i n e have been considered as s e m i - e s s e n t i a l amino acids as they can be synthesized only from methionine and phenylalanine - 4 -r e s p e c t i v e l y . For example, when c y s t i n e i n the d i e t i s low, the amount of methionine must be s u f f i c i e n t to meet not only the s p e c i f i c methionine requirement, but a l s o p a r t of the c y s t i n e requirement. The b i o l o g i c a l ' v a l u e of a p r o t e i n depends on the l e v e l of each of the e s s e n t i a l amino acids present and f a l l s to a very low l e v e l i f any one i s completely absent. A l l the n o n - e s s e n t i a l amino acid s are found i n animal t i s s u e pro-t e i n , as they are r e q u i r e d i n the formation of body p r o t e i n . They may be regarded as p h y s i o l o g i c a l l y e s s e n t i a l and must be e i t h e r obtained from the d i e t or s y n t h e s i z e d by animals from other amino a c i d s . I t has been reported t h a t glutamic a c i d , a n o n - e s s e n t i a l amino a c i d when e l i m i n a t e d from a d i e t w i t h a low l e v e l of c a s e i n or s y n t h e t i c amino a c i d mixture as the only source of n i t r o g e n , causes a marked depression i n growth r a t e and n i t r o g e n r e t e n t i o n (Harper, 1969; Rama Rao et a l . , 1960). Another r o l e of n o n - e s s e n t i a l amino acids i s t h e i r 'sparing' a c t i o n on the e s s e n t i a l amino a c i d s . The presence of n o n - e s s e n t i a l amino a c i d s i n the d i e t reduces the n e c e s s i t y of s y n t h e s i z i n g them from the e s s e n t i a l amino acids (Rerat and Lougnon, 1968; Almquist, 1972). This s p a r i n g a c t i o n i s of much importance i n p r a c t i c a l n u t r i t i o n . B. DETERMINATION OF AMINO ACID REQUIREMENTS FOR GROWING ANIMALS USING GROWTH PERFORMANCE AND NITROGEN BALANCE The amino a c i d requirements may be estimated i n v a r i e t y of ways. Growth r a t e and feed conversion e f f i c i e n c y are two commonly used c r i t e r i a . - 5 -However, these c r i t e r i a are not without disadvantages p a r t i c u l a r l y w i t h ad l i b i t u m feeding where i t i s d i f f i c u l t to s p e c i f y whether the g a i n f o l l o w i n g the a d d i t i o n of a s y n t h e t i c amino a c i d to a d i e t i s due to an improved appe-t i t e or a b e t t e r amino a c i d composition per se. (R£rat ejt a l . , 1962) . The determination of amino a c i d requirements by the use of growth performance as a c r i t e r i o n , can be improved upon by the a d d i t i o n a l i n f o r m a t i o n obtained from n i t r o g e n balance measurements. The n i t r o g e n balance technique has been w i d e l y used (Oslage et. al., 1966; Thorbek, 1969 and N i e l s e n , 1971). Another method f o r measuring n i t r o g e n r e t e n t i o n i n v o l v e s carcass a n a l y s i s . The amount of n i t r o g e n r e t a i n e d i n a given i n t e r v a l can be e s t i -mated from the d i f f e r e n c e between the t o t a l amount of n i t r o g e n found by a n a l y s i s a t s l a u g h t e r of s p e c i f i c a l l y t r e a t e d animals and that contained i n c o n t r o l animals slaughtered i n i t i a l l y . This technique i s w i d e l y used i n la b o r a t o r y animal experiments (Becker and Harnisch 1958; Rerat, 1961) and has a l s o been employed i n the p i g l e t (Manners and McCrea, 1963; Wood and Groves, 1965) and the g r o w i n g - f i n i s h i n g p i g (Oslage, 1962). Some d i s c r e p a n c i e s have been reported between the r e s u l t s obtained by these two methods of e s t i m a t i n g n i t r o g e n r e t e n t i o n where n i t r o g e n balance has given s l i g h t l y higher r e t e n t i o n f i g u r e s than those obtained by carcass a n a l y s i s ( N i e l s e n , 1971; Oslage, 1962; F u l l e r and Boyne, 1971). - 6 -C. METHODS OF DETERMINATION OF AMINO ACIDS REQUIREMENTS FOR GROWING PIGS BY DIET, TISSUE AND BLOOD ANALYSIS E s t i m a t e s of s p e c i f i c amino a c i d r e q u i r e m e n t s o f a n i m a l s v a r y w i d e l y d e p e n d i n g p a r t l y on the method o f a s s e s s m e n t and p a r t l y on t h e l e v e l s o f o t h e r d i e t a r y c o n s t i t u e n t s e.g. the r e l a t i o n s h i p between the p r o t e i n c o n -t e n t and t h e c a l o r i c c o n t e n t of t h e d i e t . S e v e r a l methods, ba s e d on d i f f e r e n t p r i n c i p l e s , have b e e n employed. Some o f them depend upon t h e a n a l y s i s o f th e d i e t s ( B e c k e r _et a l . , 1 9 5 4 a , b ) w h i c h s u p p l y g r a d e d l e v e l s o f a s p e c i f i c amino a c i d i n c o n j u n c t i o n w i t h adequate l e v e l s o f v i t a m i n s and m i n e r a l s . C o n v e r s e l y , o t h e r methods r e l y upon t i s s u e a n a l y s i s , e.g. W i l l i a m e t a l . (1 9 5 4 ) . a. Methods Based on A n a l y s i s o f D i e t s The methods used f a l l i n t o two groups : (1) A n a l y s i s o f adequate d i e t s and (2) d e t e r m i n a t i o n o f the c o m p o s i t i o n o f an " i d e a l " p r o t e i n . (1) A n a l y s i s o f adequate d i e t s The amino a c i d r e q u i r e m e n t s a r e e s t i m a t e d f r o m t h e a n a l y s i s o f d i e t s on w h i c h s a t i s f a c t o r y growth r a t e has been a c h i e v e d . T h i s i s a r a t h e r i m p r e c i s e method o f e s t i m a t i o n b e c a u s e one may o v e r e s t i m a t e a l l t h e r e q u i r e -ments e x c e p t f o r t h a t o f t h e l i m i t i n g f a c t o r o r f a c t o r s o f t h e p r o t e i n s . However, t h i s method has q u i t e o f t e n been u s e d f o r g r o w i n g p i g s ( E v a n s , 1958; R e r a t and Lougnon, 1 9 6 8 ) . - 7 -(2) Determination of the composition of an " i d e a l " p r o t e i n The p r i n c i p l e on which t h i s group i s based c o n s i s t s of an attempt to b u i l d an i d e a l l y balanced p r o t e i n i n which the amino aci d s p e r f e c t l y meet the requirements w h i l e i n c l u d i n g a minimum amount of a d d i t i o n a l n i t r o g e n . Two methods are used : i n the f i r s t case, the animals are fed a mixture of s y n t h e t i c amino aci d s as the only n i t r o g e n source of the d i e t (Mertz e_t a l . , 1 9 4 9 ) . This method i s very expensive and consequently d i f f i c u l t to apply to la r g e animals. Most of the p r o t e i n sources i n the d i e t are n u t r i t i o n a l l y l i m i t i n g i n one or s e v e r a l amino a c i d s . An attemptwas made to balance the p r o t e i n requirements by the a d d i t i o n of s y n t h e t i c amino acids (Rerat and Henry, 1 9 6 3 , Rosenberg, 1 9 5 9 ) . The requirement can only be determined f o r the l i m i t i n g amino a c i d and i s equal to the amount of t h i s amino a c i d a l l o w i n g the best performance w i t h that d i e t . In t h i s method, the b a s a l d i e t must be w e l l balanced, and adequate i n a l l amino aci d s except the f i r s t l i m i t i n g , o t h e r -wise p o t e n t i a l i t i e s of growth of the p i g cannot manifest themselves, and the requirements d e f i n e d i n t h i s way are too low. This method can only be appl i e d by means of suc c e s s i v e approximation. Another l i m i t i n g f a c t o r may appear when the response to supplementation of the f i r s t l i m i t i n g n u t r i e n t causes t h i s and subsequent d e f i c i t s have to be made up u n t i l an " i d e a l " p r o t e i n i s obtained. The a d d i t i o n a l q u a n t i t y of s y n t h e t i c amino acids must n a t u r a l l y be c a l c u l a t e d according to the other amino aci d s present i n the d i e t and consequently according to n i t r o g e n l e v e l i n order to avoid an imbalance due to excess. - 8 -b. Methods Based on Tissue and Blood A n a l y s i s W i l l i a m e_t a l . (1954) were able to demonstrate the r e l a t i o n s h i p s between amino a c i d requirements and t i s s u e amino a c i d composition of the animals. They showed that the es t i m a t i o n of amino a c i d requirements could be based on t i s s u e a n a l y s i s at d i f f e r e n t ages. The r e l a t i o n s h i p s between amino acids i n the t i s s u e were p o s t u l a t e d as bein g those which should e x i s t i n the "optimum" d i e t . However, there has been c r i t i c i s m of t h i s method. E r i c s o n (1961) po i n t e d out that the method could l e a d to the e r r o r s of underestimation by not ta k i n g i n t o account the d i f f e r e n c e s i n the r a t e of r e l e a s e of d i f f e r e n t amino a c i d s , the r a t e of s y n t h e s i s of p r o t e i n s ; without knowing the p a r t i a l s y n t h e s i s of amino acids by the organism, nor t h e i r u t i l i z a t i o n f o r purposes other than t i s s u e development. The p o s s i b l e s p a r i n g a c t i o n of one amino a c i d on the requirements of others has not been f u l l y e valuated. Plasma amino a c i d (PAA) a n a l y s i s has a l s o been employed as a c r i t e r i o n to estimate the amino a c i d requirements of animals. The p r i n c i p l e i s based on the observations of Morrison e^ t a l . (1961) w i t h the r a t and e s p e c i a l l y of Zimmerman and S c o t t (1965) w i t h c h i c k s . These workers presented evidence that when an amino a c i d i s added i n graded l e v e l s to a d i e t which i s d e f i c i e n t i n t h i s amino a c i d , the plasma c o n c e n t r a t i o n of t h i s amino a c i d remains r a t h e r low and constant u n t i l the d i e t a r y requirement i s reached. There i s a r a p i d and approximately l i n e a r i n c r e a s e i n the c o n c e n t r a t i o n of t h i s l i m i t i n g amino a c i d i n the plasma when i n c r e a s i n g l e v e l s above d i e t a r y requirement are f e d , - 9 -D i e t a r y amino a c i d requirements f o r animals have been estimated by t h i s technique i n c h i c k s (Zimmerman and S c o t t , 1965), r a t s (Stockland e t a l . , 1970) and pigs ( M i t c h e l l et_ a l . , 1968b; Bravo et a l . , 1970; K e i t h et^ a l . , 1972). V a r i a b l e r e s u l t s have been obtained by employing t h i s technique. However, one must take i n t o account the s i g n i f i c a n t v a r i a t i o n i n the c i r c u l a t i n g amino a c i d l e v e l s between animals ( D e v i l a t e t a l . , 1970) and the e f f e c t of feeding regimes and environmental c o n d i t i o n s preceeding the t a k i n g of samples. The i n t e r v a l between the time of samplings and the time of the l a s t feed ( C l a r k e t a l . , 1963; Combs e^ a l . , 1967; M i t c h e l l e t a l . , 1968b; Ostrowski, 1969) i s important. I t i s evident a l s o that the d u r a t i o n of f e e d i n g i s important ( P i c k and Meade, 1970; M i t c h e l l e t a l . , 1968b). Most r e c e n t l y , an i n t e r e s t i n g new c r i t e r i o n was proposed by Brown and C l i n e (1974) . They were able to demonstrated that the t o t a l u r i n a r y n i t r o g e n e x c r e t i o n l e v e l could be used as an i n d i c a t o r of p r o t e i n q u a l i t y to assess the amino a c i d requirements of swine and other non-ruminant animals. The p r i n c i p l e i s based on the decrease of t o t a l u r i n a r y n i t r o g e n output when an animal i s fed w i t h graded l e v e l s of an e s s e n t i a l ..amino a c i d which i s d e f i c i e n t i n the d i e t . D. VARIATION IN AVAILABILITY OF DIETARY PROTEIN The task of p r e c i s e l y d e f i n i n g the amino a c i d requirements of the v a r i o u s animal species has long been complicated by adequately a s s e s s i n g the a v a i l a b i l i t y of the amino acids i n the d i e t . I t has - 10 -been s t r e s s e d that not a l l the amino acids i n a p r o t e i n may be a v a i l a b l e to sy n t h e s i z e the t i s s u e of the animal. Complete u t i l i z a t i o n of the amino a c i d c o n s t i t u e n t s of p r o t e i n s and of those added o c c a s i o n a l l y i n the f r e e form i s only p o s s i b l e under the two f o l l o w i n g c o n d i t i o n s : they must be present at the same time at the s i t e of s y n t h e s i s (Cannon et a l . , 1947); the r e s p e c t i v e p r o p o r t i o n s of the supply must be balanced, a l l excesses w i t h respect to needs r e s u l t i n g i n a corresponding e x c r e t i o n . Consequently, when determining the requirements f o r amino acids from the composition of the d i e t , one must take i n t o account a l l the f a c t o r s which may modify the a v a i l a b i l i t y of amino a c i d s . The a v a i l a b i l i t y of amino aci d s i n the d i e t may be diminished when the d i e t contains substances modifying the d i g e s t i b i l i t y of the dry matter or p r o t e i n or when the p r o t e i n s t r u c t u r e i s changed due to treatment such as the a p p l i c a t i o n of heat during p r o c e s s i n g . Problems i n v o l v e d i n determing a v a i l a b i l i t y of amino aci d s have been discussed by Grau and C a r o l l (1958) and Mauron (1961). Progress has als o been made i n the past decade i n the development of procedures to assay amino a c i d a v a i l a b i l i t y . Despite progress having been made i n some aspects, however, the problem i s s t i l l f a r from r e s o l v e d . E. METHOD OF ASSESSING AMINO ACID AVAILABILITY a. Chemical Methods Chemical, m i c r o b i o l o g i c a l and enzymic methods as w e l l as bioassays have been developed to estimate amino a c i d a v a i l a b i l i t y . The most w i d e l y - 11 -used chemical method i s that developed by Carpenter to determine a v a i l a b l e l y s i n e (Carpenter, 1960; Booth, 1971). The method depends upon the r e a c t i o n of the d i n i t r o f l u r o b e n z e n e w i t h the e-amino group of l y s i n e i n i n t a c t p r o t e i n to produce a col o r e d l y s i n e d e r i v a t i v e which can be estimated a f t e r a c i d h y d r o l y s i s . I n s p i t e of there being a 14-28% ov e r e s t i m a t i o n of l y s i n e a v a i l a b i l i t y when the method i s a p p l i e d to the pure N-a-formyl— N-e-deoxy-f r u c t o s y l - d e r i v a t i v e s of l y s i n e , i t c e r t a i n l y remains the most app r o p r i a t e l a b o r a t o r y method f o r r a p i d l y e v a l u a t i n g heat-damage to p r o t e i n s ( F i n o t , 1973). No chemical methods are a v a i l a b l e f o r the determination of the a v a i l a b i l i t y of the other i n d i v i d u a l amino a c i d s , w i t h the p o s s i b l e exception of methionine, which may be estimated as methionine sulphoxide. Methionine sulphoxide which represents a form of u n a v a i l a b l e methionine can be employed to measure the methionine a v a i l a b i l i t y (Smith, 1972). b. M i c r o b i o l o g i c a l methods M i c r o b i o l o g i c a l method f o r determining a v a i l a b l e amino acids have been w i d e l y used, w i t h the method of Ford (1960) who used Streptococcus  zymogenes bei n g one of the most popular. Ford (1962) was able to determine the a v a i l a b i l i t y of seven amino a c i d s i n t h i s way : methionine, l e u c i n e , i s o l e u c i n e , a r g i n i n e , h i s t i d i n e , v a l i n e and tryptophan. The u l t i m a t e v a l i d i t y of the method r e s t s upon i t s degree of c o r r e l a t i o n w i t h b i o l o g i c a l assays i n v i v o . For methionine good c o r r e l a t i o n s w i t h in_ v i v o r e s u l t s have been obtained (Carpenter e t a l . , 1972). - 12 -c. Enzymic Methods Mauron (1961) has been s u c c e s s f u l i n e v a l u a t i n g heat-damage i n some m a t e r i a l , e s p e c i a l l y m i l k products using enzymic methods. The d i g e s t i o n i s g e n e r a l l y performed with' pepsin, f o l l o w e d by pancreation (Mauron et^ a i l . , 1955). A high c o r r e l a t i o n was obtained between the enzymic i n v i t r o d i g e s t i o n of Mauron e± _ a l . (1955) and d i n i t r o f l u r o b e n z e n e method of Carpenter (1960) w i t h a s e r i e s of m i l k samples (Bujard et al.,1967). However, the enzymic d i g e s t i o n method i s not s a t i s f a c t o r y i n the r o u t i n e determination of a v a i l a b l e amino acids because of incomplete d i g e s t i o n i n  v i t r o n e c e s s i t a t i n g the use of the unheated m a t e r i a l as a c o n t r o l , which i n p r a c t i c e would not always be p o s s i b l e . d. Bioassays The u l t i m a t e standard f o r measuring amino a c i d a v a i l a b i l i t y i s a bioassay w i t h the animal i t s e l f . The most w i d e l y used procedures are based on the growth response of s m a l l animals such as r a t s (Calhoun et a l . , 1960) and chicks (Bragg et_ a l . , 1969; Oh e_t a l . , 1972). Procedures using the r a t or the c h i c k have been developed f o r s e v e r a l amino acid s v i z . l y s i n e , methionine, i s o l e u c i n e and tryptophan ( P e l l e t t , 1963). Oh et a l . (1972) using a s l o p e - r a t i o technique d e s c r i b e d by Firnney (1964) determined the b i o l o g i c a l a v a i l a b i l i t y of methionine f o r the c h i c k i n v a r i o u s p r o t e i n supplements. The a v a i l a b l e methionine content of meat, f i s h , b l o o d , rapeseed, soybean and f e a t h e r meal was estimated to be 91.1, 89.9, 66.3, 87.2, 94.7 and 35.0% r e s p e c t i v e l y . Using the c h i c k to determine a v a i l a b l e l y s i n e , Carpenter e t a l . (1972) concluded that m a t e r i a l s cannot be ranked c o n s i s t e n t l y , - 1 3 -but that absolute estimates of methionine a v a i l a b i l i t y s t i l l vary between assay methods and f u r t h e r improvements are t h e r e f o r e necessary i f bioassays are to be used f o r the c a l i b r a t i o n of i n v i t r o procedures. S e v e r a l s t u d i e s on a v a i l a b i l i t y of p r o t e i n (Olsen et_ JLL. , 1 9 6 8 ; G i o v a n e t t i e t a l . , 1 9 7 0 ; Sauer, 1 9 7 2 ) i n d i c a t e that c o n s i d e r a b l e v a r i a t i o n e x i s t s among amino acids i n t h e i r a v a i l a b i l i t y , although amino a c i d a v a i l a -b i l i t y depends on the type of p r o t e i n being s t u d i e d . I d e a l l y , d i e t s should be formulated according to amino a c i d a v a i -l a b i l i t y r a t h e r than t o t a l amino a c i d content. However, t h i s i s not yet p o s s i b l e i n commercial p r a c t i c e s i n c e no r a p i d method has been e s t a b l i s h e d to estimate a v a i l a b i l i t y . Moreover, the v a r i a t i o n from batch to batch i n d i e t a r y components f u r t h e r complicates the problem. F. EFFECT OF PROTEIN DIGESTION AND ABSORPTION IN RELATION TO THE REQUIREMENT Release of amino acids from p r o t e i n s depends on the h y d r o l y s i s o f p r o t e i n by p r o t e o l y t i c enzyme i n the g a s t r o - i n t e s t i n a l t r a c t . The r a t e at which amino a c i d s are r e l e a s e d from ..pxp.feins may i n f l u e n c e the amino a c i d requirement when determined by the most common techniques of p r o v i d i n g graded l e v e l s of f r e e amino acids to the b a s a l d i e t . The delay of amino a c i d r e l e a s e may cause an underestimate of true amino a c i d a v a i l a b i l i t y provided by the p r o t e i n source. I t i s g e n e r a l l y accepted that i n t e s t i n a l h y d r o l y s i s is the r a t e -- 1 4 -l i m i t i n g step i n the abso r p t i o n of the p r o t e i n . However, the frequent l a c k of c o r r e l a t i o n between plasma amino a c i d l e v e l s and the q u a n t i t i e s of amino acids i n g e s t e d r a i s e s the question of whether r a t e of r e l e a s e of amino acids i s the only f a c t o r a f f e c t i n g the a v a i l a b i l i t y of amino a c i d s . Factors such as molecular weight, a f f i n i t y f o r d i f f e r e n t t r a n s p o r t system, and competition f o r abso r p t i o n on s i t e s have been reported to a f f e c t r a t e of a b s o r p t i o n of i n d i v i d u a l amino acids ( K r a t z e r , 1944; Delhumeau et a l . , 1962; Orten, 1963; A d i b i et: a l . , 1967). However, whether these f a c t o r s apply to a b s o r p t i o n of amino acids r e l e a s e d from p r o t e i n s has been challenged by s e v e r a l researchers (Bergen and Purser, 1968; Coulson and Hernandes, 1970; Nixon and Mawer, 1970a, b ) . Bergen and purser (1968) and Nixon and Mawer (1970a) concluded that there i s no d i f f e r e n t i a l r a t e of a b s o r p t i o n o c c u r r i n g among amino acids from food d i g e s t a i n r a t and man. Nixon and Mawer (1970b) reported that the r a t e s of a b s o r p t i o n of the amino acid s from m i l k p r o t e i n and g e l a t i n p r o t e i n were p r o p o r t i o n a l to the c o n c e n t r a t i o n of amino acid s i n the p r o t e i n s except f o r a r g i n i n e , a l a n i n e , p r o l i n e and g l y c i n e . Glutamic a c i d and methionine were absorbed a t a s i m i l a r r a t e although i t has been reported i n experiments w i t h amino a c i d mixtures (Orten, 1963; A d i b i et_ al., 1967) that glumatic a c i d was p o o r l y absorbed r e l a t i v e to the r a p i d a b s o r p t i o n of methionine. The evidence suggests the r a t e l i m i t i n g f a c t o r i n the d i g e s t i o n and ab s o r p t i o n of a p r o t e i n i s the r a t e of amino a c i d r e l e a s e from the p r o t e i n and not the r a t e of ab s o r p t i o n of i n d i v i d u a l amino a c i d s . Therefore, determinations on the requirements of amino a c i d s should take t h i s i n t o account. - 15 -G. THE USE OF AMINO ACIDS IN PIG NUTRITION Beeson et_ a l . (1948, 1949) were probably the f i r s t workers to s e r i o u s l y attempt to determine the requirements f o r amino acids i n p i g n u t r i t i o n . When they used a p u r i f i e d d i e t i n combination w i t h hydrolyzed f i s h p r o t e i n f o r young pigs (the d i e t was p r a c t i c a l l y f r e e of tryptophan 0.01%) a remarkable response i n growth was observed w i t h the a d d i t i o n of 0.40% DL-tryptophan. During a four-week t r i a l , p i gs fed tryptophan gained 634 g per day whereas the c o n t r o l b a s a l d i e t had zero gain. The r e s u l t c l e a r l y demonstrated the s i g n i f i c a n c e of tryptophan i n the d i e t . Beeson and h i s a s s o c i a t e s u s i n g a s i m i l a r but more p r e c i s e technique found l a t e r on that 0.2% DL-tryptophan was s u f f i c i e n t f o r normal growth of pigs (Shelton, 1951b). At the same time, e x t e n s i v e s t u d i e s on other amino acid s were a l s o c a r r i e d out at Purdue (Beeson et a l . , 1948; Mertz et a l . , 1949; Beeson e t a l . , 1953; Mertz e t a l . , 1955) and at C o r n e l l ( B e l l et_ a l . , 1950; Brin e g a r et_ a l . , 1950a, b; Kroening jet a l . , 1962). Becker and h i s a s s o c i a t e s (Becker et a l . , 1955a, b; Becker, 1959; Becker et_ a l . , 1963) at I l l i n o i s are another group of workers t h a t have done much to evaluate the q u a n t i t a t i v e needs of amino acids i n the growing p i g . A number of other s c i e n t i s t have a l s o c a r r i e d out such s t u d i e s , e.g. Evans (1958, 1960, 1962, 1963) at Cambridge and Clausen e t a l . (1959-1962) i n Copenhagen. Most of the experiments which have been reported have been con-ducted on weanling pigs ( e i g h t weeks of age), although s u c k l i n g pigs have been used to some extent. The method which i s f r e q u e n t l y used to assess - 16 -requirements i s to make up a b a s a l d i e t of p u r i f i e d c a r b o h y d r a t e - r i c h i n g r e -d i e n t s p l u s a p r o t e i n that i s d e f i c i e n t i n one or more of the n a t u r a l l y o c c u r r i n g amino a c i d s . I t has not been too d i f f i c u l t to f i n d p r o t e i n sources low i n p a r t i c u l a r amino a c i d s , such as tryptophan which i s low i n hydrolyzed feather meal, z e i n and g e l a t i n ; l y s i n e which i s low i n z e i n and c e r e a l p r o t e i n ; methionine which i s low i n e x p e l l e r soybean meal and threonine which i s low i n r i c e p r o t e i n . I f the p r o t e i n used i s a poorer source of other amino acids than the one i n q u e s t i o n , a proper supplement of the others must be given. Consequently, a d i e t w i t h a l l ten e s s e n t i a l amino acids ( f o r the r a t ) as the s o l e source of n i t r o g e n , except ammonium c i t r a t e , was success-f u l l y developed as e a r l y as 1950 by Shelton e t a l . (1950) f o r weanling p i g s . Clausen e_t al. (1959-1962) conducted e x t e n s i v e s t u d i e s w i t h the a d d i -t i o n of one or more amino acids to p r a c t i c a l d i e t s and demonstrated the b e n e f i c i a l e f f e c t of amino a c i d supplementation. I t i s w e l l e s t a b l i s h e d that the p r o t e i n q u a l i t y i n feed can be improved by ap p r o p r i a t e supplementation w i t h the f i r s t - l i m i t i n g amino a c i d . However, the amount of supplementation that can be used e f f e c t i v e l y i s governed mainly by the c o n c e n t r a t i o n of the second l i m i t i n g amino a c i d p r e -sent i n the feed and the a v a i l a b i l i t y to the organism. Proper supplementa-t i o n i s achieved when the amount of the f i r s t - l i m i t i n g i s i n balance w i t h the amount of the second l i m i t i n g amino a c i d and.with the r e s t of the p r o t e i n . A l l other n u t r i e n t s must, of course, be presented i n the d i e t to assure f u l l u t i l i z a t i o n of the balanced p r o t e i n . - 17 -H. AMINO ACID REQUIREMENT FOR GROWING-FINISHING PIGS a. General The p r o t e i n requirement of animal can be expressed more p r e c i s e l y i n term of amino a c i d requirement. Animals do not need p r o t e i n per se f o r normal growth but r e q u i r e e s s e n t i a l amino aci d s and an a d d i t i o n a l source of n i t r o g e n f o r n o n - e s s e n t i a l amino a c i d s y n t h e s i s to support normal growth. In g e n e r a l , there i s an i n c r e a s e i n amino a c i d requirements which i s d i r e c t l y p r o p o r t i o n a l to the p r o t e i n and c a l o r i c content of the d i e t (e.g. Bowland, 1962; Becker et_ a l . , 1963; Clawson, 1967). b. The Requirement f o r Lysine Mertz et: a l . (1949) were the f i r s t to demonstrate that l y s i n e was an e s s e n t i a l f a c t o r i n the growing p i g d i e t . B r i n e g a r et_ a l . (1949) concluded t h a t 0.58% l y s i n e was the requirement of weanling p i g s fed a 10.6% crude p r o t e i n d i e t . L a t e r , i t was found t h a t the l y s i n e requirement seemed to vary d i r e c t l y w i t h the p r o t e i n content of the d i e t . The feeding of a 22% crude p r o t e i n d i e t to weanling pigs i n d i c a t e d a requirement based on growth data of 1.20% l y s i n e ( B rinegar et a l . , 1950a, b ) . Change et a l . (1958) report e d a l y s i n e requirement of 0.7% i n a d i e t c o n t a i n i n g 10 or 15% p r o t e i n , and 0.9% i n a d i e t of 20% p r o t e i n content. S i m i l a r l y , McWard jet a l . (1959) showed the requirement of l y s i n e i n a 12.8% p r o t e i n d i e t to be 0.71% and i n a 21% p r o t e i n d i e t to be 0.95% . Becker (1959) r e p o r t i n g experiments based on c h i c k s and p i g s - 18 -concluded that the "requirement f o r each amino a c i d i s l i n e a r , but i s not a f i x e d p r o p o r t i o n of the p r o t e i n content of the d i e t . He i n d i c a t e d that the requirement f o r each amino a c i d , expressed as a percentage of the t o t a l d i e t a r y p r o t e i n decreases as the d i e t a r y p r o t e i n l e v e l i n c r e a s e s . Becker (1963) recommended 0.74% l y s i n e f o r weanling pigs fed a 16% p r o t e i n d i e t . I t has been suggested t h a t d i e t a r y c a l o r i c d e n s i t y can i n f l u e n c e amino a c i d requirements. Pigs eat more of a d i e t low i n c a l o r i c s than of a d i e t of h i g h c a l o r i c d e n s i t y (McWard e t a l . , 1959). A decreased feed i n t a k e r e s u l t s when l i p i d i s added to the d i e t (A.R.C., 1967). I t t h e r e f o r e seems l o g i c a l that the amino a c i d requirements should i n c r e a s e w i t h an in c r e a s e i n d i e t a r y energy c o n c e n t r a t i o n . Results to support t h i s theory have been presented by many workers ( M i t c h e l l et^ a l . , 1965b; Lerner, 1968; B e l l and Voldeng,. 1968; Rerat et a l . , 1970). M i t c h e l l et a l . , (1965b) showed th a t f o r an animal weighing 22 Kg, 0.65% l y s i n e was s u f f i c i e n t i n a d i e t of 2926 K c a l ME/Kg; but i t must be 0.80% i n d i e t of 3,718 K c a l ME/Kg. This was confirmed by Rerat e_t a l . , (1970) and Lerner (1968) by means of d i e t s i n which the energy content was v a r i e d by a d d i t i o n of v e r m i c u l i t e as an i n e r t d i l u e n t . The l y s i n e requirement was c a l c u l a t e d by M i t c h e l l (1965a) to be 2.4 gm/Kcal ME. B e l l and Voldeng (1968) concluded that the l y s i n e requirement f o r 23 to 57 Kg p i g s fed a d i e t c o n t a i n i n g 3,330 K c a l DE/Kg was about 0.70%. B e l l (1965) a l s o demonstrated that i n c r e a s i n g the d i e t a r y l y s i n e l e v e l from 0.55 to 0.67%, was as e f f e c t i v e as i n c r e a s i n g the p r o t e i n l e v e l from 13 to 16%, where the response c r i t e r i a were growth r a t e and e f f i c i e n c y of feed u l t i z a t i o n . - 19 -The l y s i n e requirement v a r i e s according to sex (Henry et a l . , 1971). Henry showed that the l y s i n e requirement f o r female and c a s t r a t e d male p i g s ranging from 20 to 60 Kg was 1.02 and 0.82% r e s p e c t i v e l y of a d i e t c o n t a i n i n g about 3,400 K c a l DE/Kg. The B r i t i s h A.R.C. p u b l i c a t i o n (1967) recommended 0.75-0.8 g d i e t a r y lysine/100 g a i r - d r y feed f o r s a t i s f a c t o r y performance i n young pigs up to 50 Kg and t h e r e a f t e r about 0.6-0.65 g/100 g a i r - d r y feed (0.9-0.95 and 0.7-0.75% of the dry matter i n the d i e t r e s p e c t i v e l y ) . This c o n t r a s t s w i t h the N.A.S.-N.R.C. (1968) recommendation of 0.7% i n the a i r -dry feed f o r 20-35 Kg pigs and 0.5% f o r f i n i s h i n g p i g s . The l y s i n e requirements f o r maximum weight gain and f o r maximum lean meat of f i n i s h i n g pigs have not yet been f u l l y d e f i n e d . Becker et a l . (1966) and N.A.S.-N.R.C. (1968) suggested that a d i e t a r y l e v e l of 0.50% l y s i n e was adequate f o r maximum r a t e of body weight gain i n f i n i s h i n g p i g s . More r e c e n t l y Brown et al.(1973a) reported a requirement of 0.48% l y s i n e f o r maximum r a t e of gain and 0.62% f o r g r e a t e s t feed e f f i c i e n c y , u s i n g a d i e t c o n t a i n i n g 3,501 K c a l ME/Kg and 13.3% crude p r o t e i n . This r e s u l t agrees w i t h the r e s u l t s of many i n v e s t i g a t i o n s on l y s i n e requirements f o r maximum gain ( C a h i l l y ejt a l . , 1963; Lee et a l . , 1967; Smith et a l . , 1967). However, Davidson et a l . (1962) and M i t c h e l l et a l . (1965a) reported that the d i e t a r y l y s i n e l e v e l r e q uired f o r maximum gain was approximately the same as tha t r e q u i r e d f o r maximum feed e f f i c i e n c y . The p r e d i c t e d 0.48 ± 0.02% d i e t a r y l y s i n e requirement reported by Brown (1973a), was consider a b l y lower than 0.55% reported by B e l l and Voldeng (1968). - 20 -C a h i l l y et; a l . (1963) reported that the l y s i n e requirement f o r maximizing percentage of lea n cuts and p r i m a l cuts was greater than f o r maximum gain. However, H i n t z and Heitman (1967) found that l y s i n e supple-mentation of a 10.5% crude p r o t e i n d i e t d i d not a f f e c t carcass c h a r a c t e r i s -t i c s . More r e c e n t l y , Brown et a l . (1973b) concluded that the l y s i n e r e q u i r e -ment f o r maximum carcass l e a n content was 0.51 ± 0.03% and f o r c r o s s -sectioned area of the longissimus muscle was 0 ."60 ± 0.05%. The d i e t s fed were corn-soybean c o n t a i n i n g 13.3% crude p r o t e i n and 3,501 K c a l ME/Kg. From the r e s u l t s reviewed, i t appears that the l y s i n e requirements f o r maximum growth and feed e f f i c i e n c y are s l i g h t l y lower than those f o r maximum carcass l e a n content. c. The Requirement f o r Methionine B e l l e_t a l . (1950) demonstrated the need f o r methionine i n swine r a t i o n s . E a r l y works by Shelton et al.(1951a) i n d i c a t e d that the methionine requirement i n the abscence of c y s t i n e was 0.6% f o r weanling pigs and th a t 50% of the t o t a l s u l f u r amino a c i d requirement can be provided by c y s t i n e i n a d i e t of 21% crude p r o t e i n contents. C u r t i n e t a l . (1952a, b) showed th a t when the c y s t i n e l e v e l was 0.38%, the requirement of methionine d i d not exceed 0.31% i n a d i e t c o n t a i n i n g 22% crude p r o t e i n . Evans (1959, 1960) has repeatedly achieved s a t i s f a c t o r y growth and feed conversion e f f i c i e n c y up to a l i v e w e i g h t of 45 Kg w i t h a low p r o t e i n d i e t c o n t a i n i n g approximately 0.5% methionine plus c y s t i n e made up of lox j - p r o t e i n vegetable foods plus 7% white f i s h meal. The d i e t a r y crude p r o t e i n was 13 to 15%. R6rat e t a l . (1962) suggested that the optimal l e v e l of t o t a l s u l f u r amino acids f o r a h i g h r a t e of growth - 21 -was 0.6 to 0.7% of the d i e t f o r pigs from 20 to 60 Kg l i v e w e i g h t and 0.4 to 0.5% of the d i e t f o r pigs h e a v i e r over 60 Kg. Becker et a l . (1966) i n d i c a t e d 0.5% methionine plus c y s t i n e to be adequate f o r growing pigs of i n i t i a l weight 20 Kg fed a d i e t c o n t a i n i n g 16% p r o t e i n . They found that c y s t i n e could s a t i s f y f o r t y percent of the t o t a l sulphur amino a c i d requirements. R£rat and Henry (1970) demonstrated t h a t the requirement of sulphur amino a c i d v a r i e s w i t h sex. Female and c a s t r a t e d male p i g s between 20 and 60 Kg r e q u i r e d about 0.47% and 0.52% t o t a l sulphur amino a c i d r e s p e c t i v e l y i n a d i e t c o n t a i n i n g 3,420 K c a l DE/Kg. Oestemer et_ a l . (1970) who conducted a s e r i e s of experiments w i t h growing swine to determine the c a p a c i t y of opaque-2 corn to provide methio-nine suggested that the methionine plus c y s t i n e requirement of growing pigs from 21 to 40 Kg was somewhat l e s s than 0.42 to 0.50% of the d i e t as reported by i n v e s t i g a t o r s p r e v i o u s l y c i t e d . The corn d i e t s (crude p r o t e i n 10.85%) contained 0.275, 0.279 and 0.227% methionine plus c y s t i n e . There was no s i g n i f i c a n t improvement i n r a t e of g a i n , gain/feed or p r o t e i n e f f i -c iency r a t i o (PER) as a r e s u l t of supplementing the b a s a l corn d i e t w i t h 0.07, 0.14, 0.21 or 0.28% DL-methionine. A recent r e p o r t by K e i t h e t a l . (1972) i n d i c a t e d the methionine requirement of growing pigs (18 Kg) to be 0.46% of the d i e t . The serum fr e e amino acid s technique was employed i n t h i s experiment. More r e c e n t l y , Braude and Esnaola (1973) i n d i c a t e d that the optimum performance, n i t r o g e n r e t e n t i o n and carcass leaness was obtained w i t h about 4 gm/Kg (0.4%) methionine p l u s c y s t i n e i n the d i e t . The recommended l e v e l s of methionine. - 22 -plus c y s t i n e f o r growing p i g are given as 6 gm/Kg (0.6%) and 5 gm/Kg (0.5%) of the d i e t by A.R.C. (1967) and N.A.S.-N.R.C. (1968) r e s p e c t i v e l y . In an attempt to summarize the work alone i n the l a s t two decades, i t may be concluded that the methionine plus c y s t i n e requirement f o r the grow-i n g p i g i s w i t h i n the ranges of 0.4 to 0.6% of the d i e t when d i e t a r y p r o t e i n ranges from 12 to 18%. The only value o u t s i d e of t h i s range was reported by Oestemer et_ a l . (1970). Braude and Enaola (1973) on the b a s i s of t h e i r s t u d i e s p o inted out that the l e v e l and source of d i e t a r y , p r o t e i n seems to be important i n determining requirements. Therefore, an accurate estimate of requirements i s s t i l l v i r t u a l l y i m p o s s i b l e . Not much work has been done on the s u l f u r amino a c i d requirements of the f i n i s h i n g p i g . Rerat ejt a l . , (1962) reported that t o t a l s u l f u r amino a c i d requirements of f i n i s h i n g pigs (> 60 Kg) was 0.4 to 0.5% of the d i e t . Studies from the Minnesota s t a t i o n (Meade et a l . , 1966a, b) i n d i c a t e d that the methio-nine plus c y s t i n e requirement of the f i n i s h i n g p i g i s equal to or l e s s than the requirement of 0.30% of the d i e t reported by Becker e_t a l . (1966). Welch et a l . (1966) reported that methionine supplementation of a 12% crude pro-t e i n corn-soybean meal d i e t c o n t a i n i n g 0.27% methionine p l u s c y s t i n e f o r -t i f i e d w i t h c r y s t a l l i n e l y s i n e and tryptophan d i d not i n c r e a s e n i t r o g e n r e t e n t i o n of the f i n i s h i n g p i g . Most r e c e n t l y , Brown et^ a l . (1974) i n d i c a t e d that t o t a l s u l f u r amino a c i d requirements f o r maximum n i t r o g e n r e t e n t i o n were 0.17% of a d i e t c o n t a i n i n g 14.1% p r o t e i n and 3,700 K c a l ME/Kg. The values reported by Brown (1974) was s t r i k i n g l y lower than the values reported by the previous i n v e s t i g a t o r c i t e d . The authors p o i n t s out that no estimated value - 23 -of t o t a l s u l f u r amino a c i d lower than 0.17% of the d i e t has been s t u d i e d . I t i s apparent, t h e r e f o r e that more work has to be done to c l a r i f y t h i s e s t i m a t i o n . d. The Requirement f o r Threonine A requirement f o r threonine was f i r s t demonstrated w i t h d i e t c o n t a i n -i n g an amino a c i d mixture i n place of p r o t e i n . Using a p u r i f i e d carbohydrate d i e t supplemented w i t h amino a c i d s , Shelton et_ _ a l . (1950) demonstrated t h a t threonine was e s s e n t i a l as the pigs l o s t weight when threonine was omitted from the d i e t . Beeson e_t al_. (1953) reported that the maximum weight gain and feed conversion e f f i c i e n c y of young pigs were observed when d i e t a r y threonine was provided up to a l e v e l of 0.4% of the d i e t (3% of the crude p r o t e i n ) . The b a s a l d i e t was based on maize and i n c l u d e d nine e s s e n t i a l amino a c i d s . I t provided 13.2% crude p r o t e i n and 0.2% threonine. Mertz e t a l . (1952) a l s o estimated the threonine requirement of pigs to be 0.4% of the d i e t . S ewell e t a l . (1952) i n v e s t i g a t e d the threonine requirement of the 2-3 day o l d baby p i g using s e m i - p u r i f i e d d i e t s (25% crude p r o t e i n ) i n c l u d i n g c a s e i n and washed soybean p r o t e i n . They concluded that the minimum L-threonine requirement of the p i g l e t was 0.90 g/100 g DM d i e t . Becker et a l . (1954a) w i t h a s e m i - p u r i f i e d d i e t based on d r i e d skim m i l k powder p r o v i d i n g 12% crude p r o t e i n c a l c u l a t e d that 0.61% L-threonine was a s a t i s f a c t o r y d i e t a r y c o n c e n t r a t i o n f o r 5 to 9 week-o l d p i g s . However, Evans (1958), using a s i m i l a r method to estimate threo-nine requirements, c a l c u l a t e d the threonine content of d i e t s which had been proven to give s a t i s f a c t o r y performance, namely d i e t s c o n t a i n i n g 7% f i s h - 24 -meal s 20% ground nut meal or 15% soybean meal, to be 0.52, 0.55 and 0.50% r e s p e c t i v e l y . F u r t h e r experiments conducted by the same author (1963) i n d i c a t e d 0.45% d i e t a r y threonine to be adequate f o r weanling p i g s up to 36 Kg l i v e w e i g h t . Less d i e t a r y threonine was needed a f t e r 36 Kg l i v e w e i g h t . Robinson and Lewis(1963) fed a 95% b a r l e y d i e t supplemented w i t h l y s i n e , methionine, threonine, tryptophan and i s o l e u c i n e f o r the growing p i g and noted that the performance of growth was equal to the c o n t r o l d i e t . A l l these d i e t s contained 16.4% crude p r o t e i n and provided 0.46% of t o t a l d i e t a r y threonine. Lougnon and B r e t t e (1971) fed wheat-soybean meal d i e t c o n t a i n i n g 14% crude p r o t e i n supplemented w i t h l y s i n e , methionine and d i f f e r e n t l e v e l of threonine to 17 Kg pigs concluded that the threonine content of the d i e t should not be below 0.50% under r e s t r i c t e d feeding c o n d i t i o n s w i t h d i e t s c o n t a i n i n g 3,125 K c a l ME/Kg. Henry and R.£rat (1970), using a s e m i - p u r i f i e d d i e t w i t h 10% pro-t e i n from a Norwegian h e r r i n g meal, supplemental by a free amino acids mix-ture concluded that the threonine requirement of female p i g s of 20 to 50 Kg l i v e w e i g h t was 0.48% of a d i e t of 3,350 K c a l DE/Kg. Recently, Sowers and Meade (1972a) concluded the threonine requirement of 15 Kg pigs based upon ADG, G/F and plasma f r e e threonine as c r i t e r i a to be 0.39, 0.32 and 0.33 r e s p e c t i v e l y . The b a s a l d i e t c o n t a i n i n g 10.4% crude p r o t e i n was based on corn, s a f f l o w e r meal, d r i e d skim m i l k and c r y s t a l l i n e e s s e n t i a l amino a c i d s except threonine. Glutamic a c i d was added to in c r e a s e the crude p r o t e i n content to 15%. Another - 25 -experiment was conducted by the same author (19 72b) based on Opaque-2 corn and c r y s t a l l i n e amino a c i d s . The b a s a l d i e t was 10.4% crude p r o t e i n . The threonine requirement was evaluated by i n c r e a s i n g the crude p r o t e i n l e v e l by adding glumatic a c i d and keeping the e s s e n t i a l to n o n - e s s e n t i a l amino a c i d r a t i o at 1 : 1 . I t was found that the threonine requirement increased from 0.35 to 0.47% when d i e t a r y p r o t e i n was inc r e a s e d from 10.4 to 15.0%. Sowers and Meade's r e p o r t s i n d i c a t e d that adjustment f o r requirement i s needed w i t h d i f f e r e n t l e v e l of d i e t a r y p r o t e i n . The N.A.S.-N.R.C. (1968) and A.R.C. (1967) recommendations f o r weanling pigs are about 0.45 - 0.50% of the d i e t . I . AMINO ACID REQUIREMENT OF THE GROWING RAT Although the amino a c i d requirements f o r growing r a t s have been i n v e s t i g a t e d very e x t e n s i v e l y and are r e l a t i v e l y w e l l e s t a b l i s h e d , d i f f e r e n c e s s t i l l e x i s t i n the requirements l i s t e d i n v a r i o u s p u b l i c a t i o n s . The most wid e l y used and quoted f i g u r e s f o r the amino a c i d requirements of growing r a t s are those reported by N.A.S.-N.R.C. (1972), and Rama Rao e t a l . (1959). The e s s e n t i a l amino a c i d requirements of growing r a t s presented by N.A.S.-N.R.C. (1972), Rama Rao et a l . (1959) and P i c k and Meade (1971) are summarized i n Table 1 f o r d i s c u s s i o n . The N.A.S.-N.R.C. (1972) recommended values were concluded from many s t u d i e s . The r e p o r t by Rama Rao et a l . (1959) was based on a s e r i e s of t h e i r own experiments i n which they gave the animals a b a s a l d i e t c o n t a i n i n g 5% c a s e i n to which was added an amino a c i d mixtures v a r y i n g i n the content of each e s s e n t i a l amino a c i d . The values presented were the - 26 -Table 1 . E s s e n t i a l amino a c i d requirements of young r a t and growing p i g Rama Rao et a l . N.A.S.-N.R.C." P i c k and Meade N.A.S.-N.R.C. (1959) (1972) (1971) (1968) Rat Rat Rat P i g (20-35 Kg) A r g i n i n e H i s t i d i n e I s o l e u c i n e Leucine Ly s i n e Phenylalanine Phenylalanine + t y r o s i n e Methionine Methionine + c y s t i n e Threonine Tryptophan V a l i n e 0.28 0.21 0.55 0.69 0.90 0.42 0.72 0.50 0.51 0.11 0.56 0.60 0.30 0.55 0.75 0.90 0.80 0.60 0.50 0.15 0.60 0.36 0.70 0.59 0.40 0.43 0.54 0.20 0.18 0.50 0.60 0.70 0.50 0.50 0.45 0.13 0.50 - 27 -minimum l e v e l s on which maximum growth was obtained. The f i g u r e s of P i c k and Meade (1971) were obtained by feeding a d i e t c o n t a i n i n g Opaque-2 maize supplemented w i t h amino a c i d s . From the t a b l e , the values reported by Rama Rao et a l . (1959) and N.A.S.-N.R.C. (1972) are g e n e r a l l y i n agreement w i t h each ot h e r , except that the a r g i n i n e requirement i s con s i d e r a b l y h i g h e r i n the N.A.S.-N.R.C. recom-mendations. However, the values presented by P i c k and Meade (1971) are ge n e r a l l y lower except f o r the v a l i n e requirement which i s i n agreement w i t h that of Rama Rao e t a l . (1959). Stockland e_t a l . (1970 , 1971) reported that the requirement of young r a t s w i t h regard to l y s i n e , p h enylalanine and phenyla-l a n i n e p l u s t y r o s i n e were 0.60, 0.38 and 0.69% of the d i e t . Stockland et j a l . (1970, 1971) agreed w i t h Rama Rao e t a l . (1959) on phenylalanine plus t r y r o s i n e l e v e l s but der i v e d a l y s i n e requirement only two-thirds that of Rama Rao et_ a l . (1959) and N.A.S.-N.R.C. (1972). These are not inconsequen-t i a l d i f f e r e n c e s but i t i s very d i f f i c u l t to say which may be more accurate. S i n c e the amino a c i d requirements of animals are i n t e r r e l a t e d , i t i s s t i l l i m p o s s i b l e , from e x i s t i n g data, to d e f i n e the l e v e l of each e s s e n t i a l amino a c i d f o r o p t i m a l growth. The problem i s a l s o f u r t h e r complicated by other f a c t o r s such as the t o t a l nifcrsgen requirement, the e s s e n t i a l and non-e s s e n t i a l amino a c i d r a t i o and the amino a c i d and energy r e l a t i o n s h i p s . I t i s g e n e r a l l y recognized that the p r o t e i n and amino a c i d r e q u i r e -ments d e c l i n e w i t h age (Forbes and Rao, 1959; Hartsook and M i t c h e l l , 1956). Because of a l a c k of any economic i n c e n t i v e to p r o g r e s s i v e l y change to chea-per d i e t s w i t h advancing age i n most species the problem has not been s t u d i e d - 28 -extensively. Hartsook and Mitchell (1956), by use of a carcass analysis procedure, estimated that the requirements of protein and methionine plus cystine decline from about 28% and 1.3% respectively at 30 days of age to 10% and 0.42% respectively at 50 days of age. The patterns of essential amino acids required by the pig (N.A.S.-N.R.C, 1968) and the rat (N.A.S.-N.R.C, 1972) as shown in Table 1 are similar except for arginine and lysine. However, the arginine requirements of Rama Rao ej: a l . (1959) for the growing rat are similar to those of the pig (N.A.S.-N.R.C, 1968), while the lysine level for the rat given by Pick and Meade (1971) i s similar to that for the pig given by N.A.S.-N.R.C. (1968). The laboratory rat has been accepted as a satisfactory p i l o t animal in swine nutritional research although requirements diff e r in some respects and d i f f i c u l t y i s encountered. J. SUPPLEMENTATION WITH AMINO ACIDS OF GRAIN-PROTEIN CONCENTRATE DIETS AND GRAIN ONLY Conventional pig diets are based mainly on cereal grains such as corn, barley, wheat, oats and sorghum. Cereals generally provide insufficient essential amino acids and sometimes insufficient nitrogen for the support of normal pig growth. As a result, protein supplements such as soybean meal and fishmeal are normally added in order to improve the protein content and to counteract any essential amino acid deficiency. Since the protein concentrates are the most expensive components - 2 9 -i n a p i g d i e t , t h e p o s s i b l e r e p l a c e m e n t o f t h e s e i n g r e d i e n t s w i t h amino a c i d m i x t u r e s i n c o m m e r c i a l p r a c t i c e has r e v i e w e d c o n s i d e r a b l e a t t e n t i o n . A g r e a t d e a l o f e f f o r t has b e e n expended t o i n v e s t i g a t e t h e n u t r i o n a l and e c o n o m i c a l f e a s i b i l i t y o f u s i n g amino a c i d m i x t u r e s i n h i g h g r a i n d i e t s o r d i e t s c o n t a i n i n g o n l y g r a i n p l u s m i n e r a l s and v i t a m i n s f o r p i g f e e d i n g . L y s i n e i s t h e most l i m i t i n g amino a c i d i n c e r e a l g r a i n s w i t h the e x c e p t i o n o f c o r n , where t r y p t o p h a n i s g e n e r a l l y e q u a l l y l i m i t i n g . I n t h e p a s t decade, a v o l u m i n o u s amount o f l i t e r a t u r e has d e m o n s t r a t e d t h a t p e r f o r m a n c e can be i m p r o v e d by s u p p l e m e n t i n g g r a i n - b a s e d d i e t s w i t h l y s i n e ( E v a n s , 1960; Jones et_ a l . , 1962; R e r a t and Lougnon, 1965; Rozman et^ a l . , 1968; O s t r o w s k i , 1969; B r aude e t a l . , 1972). M o r e o v e r , t h e a d d i t i o n o f l y s i n e t o g r a i n d i e t s s u p plemented w i t h w h i t e f i s h m e a l has b e e n r e p o r t e d t o i m p r o v e p i g p e r f o r -mance (Braude and Lerman, 1 9 7 0 ) . B e s i d e s l y s i n e , some o t h e r amino a c i d such as t h r e o n i n e , m e t h i o n i n e and t r y p t o p h a n have been c l a i m e d as l i m i t i n g i n c e r e a l s f o r t h e g r o w i n g p i g when a n a l y s i s a r e compared w i t h N.A.S.-N.R.C. (1968) r e q u i r e m e n t s . A s e r i e s o f s t u d i e s were c o n d u c t e d by M i i l l e r and c o w o r k e r s (1967a, b , c, 1968) t o i n v e s t i g a t e t h e p o s s i b i l i t y o f r e p l a c e m e n t o f p r o t e i n c o n c e n t r a t e w i t h v a r i o u s e s s e n t i a l amino a c i d s t o s i n g l e g r a i n o r g r a i n m i x t u r e s f o r g r o w i n g p i g s . M i i l l e r e t a l . (1967b) were a b l e t o show t h a t p i g s f e d a c e r e a l m i x t u r e s u p p l e m e n t e d w i t h l y s i n e , t h r e o n i n e , t r y p t o p h a n and m e t h i o n i n e , and i n some c a s e s i s o l e u c i n e , gave a p e r f o r m a n c e e q u a l t o t h a t o b t a i n e d w i t h d i e t s c o n t a i n i n g c o n v e n t i o n a l p r o t e i n c o n c e n t r a t e s . R o b i n s o n and L e w i s (1963) r e p o r t e d t h a t t h e growth r a t e and f e e d e f f i c i e n c y i m p r o v e d m a r k e d l y i n g r o w i n g p i g s r e c e i v i n g b a r l e y d i e t s where l y s i n e and m e t h i o n i n e a r e added and t h a t t h e p e r f o r m a n c e was e q u a l t o t h a t o b t a i n e d w i t h a b a r l e y -- 30 -soybean c o n t r o l when there was a f u r t h e r a d d i t i o n of DL-tryptophan, DL-i s o l e u c i n e and DL-threonine. M i i l l e r e_t a l . (1967b) w i t h weanling pigs of 14-18 Kg fed a c e r e a l d i e t of b a r l e y , wheat and oats plus an amino a c i d mixture to 50 Kg l i v e -weight found that the combination of l y s i n e and threonine'had an e x t r a o r d i -n a r i l y f a v o r a b l e e f f e c t upon gains and feed conversion. The supplement of l y s i n e alone enhanced gains i n one case by 19%, i n another case by 22%. The combination of l y s i n e and threonine increased gains by 59% i n the f i r s t and by 92% i n the second t r i a l . The combination of l y s i n e and 0.02% tryptophan showed a p o s i t i v e response, but caused a depression i n the 0.04% tryptophan supplemented l e v e l . A supplement of l y s i n e alone i n c r e a s e d weight gain by 26-28% whereas the combination of l y s i n e and tryptophan i n c r e a s e d gain by 35-42%. Another t r i a l compared l y s i n e alone and l y s i n e plus methio-nine and showed no s i g n i f i c a n t d i f f e r e n c e i n g a i n , i n d i c a t i n g methionine not to be the second l i m i t i n g amino a c i d i n c e r e a l mixtures f o r the growing p i g . Jensen e_t a l . (1965) us i n g the feeding standards of Becker et^ a l . (1963) i n d i c a t e d that l y s i n e and p o s s i b l y methionine were r e q u i r e d as a supplement to sorghum f o r p i g s from 45 to 90 Kg l i v e w e i g h t , whereas l y s i n e and tryptophan were the r e q u i r e d amino aci d s i n the supplementation of corn. The a d d i t i o n of 0.25% l y s i n e to g r a i n improved the growth r a t e but gave no response to the f u r t h e r a d d i t i o n of methionine. Beames et a l . (1968) a l s o i n d i c a t e d that up to 0.2% l y s i n e a d d i t i o n improved growth r a t e and feed e f f i -c iency f o r the f a t t e n i n g p i g but improvement i n carcass q u a l i t y was s l i g h t . - 31 -Bowland (1962) reported that the a d d i t i o n of 0.2% L - l y s i n e to a b a r l e y , wheat and soybean meal d i e t c o n t a i n i n g 13.6% p r o t e i n made the d i e t e q u i v a l e n t to one c o n t a i n i n g 16% p r o t e i n as measured by gain and e f f i c i e n c y of feed u l t i z a t i o n i n pigs over the 14 Kg to 44 Kg body weight range. S o l d e v i l a and Meade (1964) i n d i c a t e d that methionine i s not s e r i o u s l y l i m i t i n g i n b a r l e y f o r they suggested that l y s i n e i s the f i r s t l i m i t i n g amino a c i d and probably threonine i s the second. Recently, Re"rat and Henry (1969) a l s o reported t h a t growth r a t e on b a r l e y only (10.1% crude peotein) was not improved by methionine supple-mentation. Chung (1973) a l s o i n d i c a t e d that no growth improvement was obtained by methionine supplementation of a b a r l e y amino a c i d d i e t (10.3% crude p r o t e i n ) . L y s i n e and threonine have been repeatedly demonstrated to be the f i r s t and second l i m i t i n g amino acids i n r i c e p r o t e i n (Rosenberg et a l . , 1959) and b a r l e y (Sure, 1955) f o r the r a t . Sure (1954) showed a l s o that wheat and rye were d e f i c i e n t i n l y s i n e and threonine and that rye was a l s o d e f i c i e n t i n v a l i n e . L y s i n e and threonine were reported to be l i m i t i n g i n milo a l s o (Pond e t a l . , 1958). Threonine was suggested to be the second l i m i t i n g amino a c i d i n milo f o r r a t s (Pond e_t a l . , 1958). Re c e n t l y , Veum _e_t al_. (1973) reported t h a t normal and opague-2 corn d i e t s supplemented w i t h l y s i n e , methionine and tryptophan apparently were d e f i c i e n t i n i s o l e u c i n e , v a l i n e , threonine and p o s s i b l y phenylalanine when fed to growing r a t s . These r e s u l t s were supported by those of P i c k and - 32 -Meade (1971) who reported that e x c e l l e n t d a i l y gain, gain-feed r a t i o and PER could be obtained when a d i e t c o n t a i n i n g 89.5% opaque-2 corn was supplemented w i t h 0.1% L - i s o l e u c i n e , 0.18 to 0.35% l y s i n e , 0.2% DL-methionine, 0.12% L-phenylalanine, 0.14% threonine and 0.08% L - v a l i n e . The f i n a l amino a c i d l e v e l of t h i s d i e t was 0.70% l y s i n e or l e s s , 0.40% methionine + c y s t i n e , 0.43% threonine, 0.36% i s o l e u c i n e , 0.54% v a l i n e and 0.59 to 0.65% phenylalanine + t y r o s i n e . K. FACTORS ASSOCIATED WITH AMINO ACID SUPPLEMENTATION OF GRAINS a. V a r i a t i o n of Grain Amino Acids P r o f i l e and A v a i l a b i l i t y Maize, B a r l e y , wheat and sorghum are the most commonly used c e r e a l grains i n p i g d i e t s . Although these g r a i n s are commonly l i m i t i n g i n the e s s e n t i a l amino a c i d s , l y s i n e , threonine and methionine, the q u a n t i t i e s of these and other amino acid s vary from one to another. A c c o r d i n g l y , the optimal amino a c i d supplementation of g r a i n w i l l vary from g r a i n to g r a i n . For examples, l y s i n e and tryptophan are e q u a l l y and f i r s t l i m i t i n g i n maize s i n c e the expressed response f o r l y s i n e becomes minimum without tryptophan a d d i t i o n ( G a l l o and Pond, 1968; Pond and Jones, 1964). However, i n b a r l e y , l y s i n e i s the most l i m i t i n g , f o l l o w e d by threonine w i t h methionine p o s s i b l y t h i r d l i m i t i n g (Chung and Beames, 1972, 1974). Tryptophan i s adequate i n b a r l e y according to the N.A.S.-N.R.C. (1968) standards f o r growing p i g s . S i m i l a r to b a r l e y , the f i r s t and second most l i m i t i n g amino acid s of wheat are l y s i n e and threonine r e s p e c t i v e l y (Shimada and C l i n e , 1974). V a r i a t i o n i n a v a i l a b i l i t y of amino acids i n d i f f e r e n t p r o t e i n sources f u r t h e r complicate the amino a c i d - 33 -supplementation problem. Olsen et_ aJL. (1968) reported c o n s i d e r a b l e v a r i a t i o n i n amino a c i d a b s o r p t i o n from wheat by—products. The a b s o r p t i o n was h i g h e s t f o r c y s t i n e , glutamic a c i d , h i s t i d i n e and a r g i n i n e i n a l l products t e s t e d whereas the lowest percentage a b s o r p t i o n was found f o r threonine, l y s i n e , a l a n i n e , g l y c i n e , a s p a r t i c a c i d , methionine and i s o l e u c i n e . Some r e s u l t s ( C a r l s o n and Bayley, 1970; Sauer, 1972) have i n d i c a t e d t h a t the t r u e d i g e s -t i b i l i t i e s of glutamic a c i d , p r o l i n e , •serine, l y s i n e , p h e n y l a l a n i n e , a r g i n i n e and h i s t i d i n e i n soybean meal f o r young pigs are higher than those of a l a n i n e , threonine, methionine, i s o l e u c i n e and g l y c i n e . G i o v a n e t t i e_t aJL. (1970) and Sauer (1972) a l s o reported lower a v a i l a b i l i t i e s f o r l y s i n e , a l a n i n e , threo-nine and methionine i n t r i t i c a l e , wheat and b a r l e y , i n c o n s t r a s t to the h i g h d i g e s t i b i l i t i e s of p r o l i n e , g l u t a m i c a c i d , a r g i n i n e , h i s t i d i n e and p h e n y l a l a -nine . The evidence suggests that amino a c i d requirements should be expressed on an a v a i l a b l e b a s i s and that the content of d i e t a r y components should be given on a s i m i l a r b a s i s . b. P r o t e i n L e v e l i n R e l a t i o n to Amino A c i d Requirements The t o t a l p r o t e i n content of a d i e t may have an important e f f e c t on the amino a c i d requirement. According to Becker (1958) the requirement f o r e s s e n t i a l amino acids i n c r e a s e s w i t h the p r o t e i n content of the d i e t , but the i n c r e a s e i s not i n d i r e c t p r o p o r t i o n . The argument of d i s p r o p o r -t i o n a l r e l a t i o n s h i p i s supported by an experiment i n which the requirement of i s o l e u c i n e , expressed as a percentage of the nitrogenous matter of the d i e t , decreased as the d i e t a r y p r o t e i n i n c r e a s e d (Becker et a l . , 1957). - 34 -This was f u r t h e r confirmed f o r the l y s i n e requirement (McWard _et a l . , 1959) and the tryptophan requirement of growing pigs (Boomgaardt and Baker, 1973) . Kroening et^ al_. (1965) t h i n k that the necessary content of s u l f u r amino acids i n the d i e t v a r i e s i n the same d i r e c t i o n as, but more s l o w l y than the p r o t e i n content,even when growth performances are almost i d e n t i c a l . Boomgaardt and Baker (1970, 1971) i n d i c a t e d that the l y s i n e and tryptophan requirements of young c h i c k s remain constant when expressed as a percentage of the p r o t e i n . These r e s u l t s c o n t r a d i c t the g e n e r a l l y accepted concept that the requirement f o r an e s s e n t i a l amino a c i d decreases l i n e a r l y as a percentage of the p r o t e i n and i n c r e a s e c u r v i l i n e a r l y as a percentage of the d i e t . Rosenberg (1959) i n d i c a t e d that the e f f e c t of a change i n p r o t e i n l e v e l i s a problem of c o n s i d e r a b l e p r a c t i c a l importance because feeds f o r domestic animals range from 10% p r o t e i n f o r the f a t t e n i n g p i g to 30% p r o t e i n f o r the turkey p o u l t . Percentage p r o t e i n i n t h i s corn-soybean d i e t was p l o t t e d a g a i n s t the percentage of amino a c i d i n the p r o t e i n f o r l y s i n e , methionine and the combination of methionine and c y s t i n e i n corn-soybean d i e t s . As the l e v e l of p r o t e i n was i n c r e a s e d , the r e l a t i v e amount of l y s i n e i n the d i e t i n c r e a s e d , w h i l e the r e l a t i v e amount of the s u l f u r - b e a r i n g amino acids decreased. Therefore, methionine d e f i c i e n c y i s l i k e l y to occur as the p r o t e i n l e v e l i n c r e a s e s . Conversely, l y s i n e d e f i c i e n c y i s l i k e l y to occur as the p r o t e i n l e v e l decreases (Rosenberg et a l . , 1959). A c c o r d i n g l y , the adjustment of most e s s e n t i a l amino a c i d . i s needed when the p r o t e i n l e v e l i s a l t e r e d . - 35 -c. S i g n i f i c a n c e of E s s e n t i a l and N o n - e s s e n t i a l Amino A c i d R a t i o Changes i n n i t r o g e n content of the d i e t w i l l g e n e r a l l y be a s s o c i a t e d w i t h an a l t e r a t i o n i n the supply not only of e s s e n t i a l but a l s o of non-e s s e n t i a l amino a c i d s . A d i e t composed of minimum l e v e l s of only the essen-t i a l amino acid s supports a slow r a t e of growth only ( W r e t l i n d , 1949). A d d i t i o n a l sources of n i t r o g e n are r e q u i r e d f o r normal growth and normal p h y s i o l o g i c a l f u n c t i o n . The r a t i o of e s s e n t i a l (EAA) to n o n - e s s e n t i a l amino acids (NEAA) i n a d i e t f o r optimum growth has r e c i e v e d much a t t e n t i o n . S t u c k i and Harper (1962) reported that the NEAA/EAA r a t i o might vary i n the r a t from 4 to 1 without decreasing growth r a t e or n i t r o g e n r e t e n t i o n . They s t a t e that e i t h e r wide or narrow r a t i o s would give u n f a -v o r a b l e r e s u l t s . M i t c h e l l ejt a l . (1968a) reported that the h i g h e s t n i t r o g e n r e t e n t i o n of pigs was obtained when the d i e t contained equal amounts of e s s e n t i a l and n o n - e s s e n t i a l amino a c i d . Recently, Henry and R£rat (1970) i n d i c a t e d that the best growth performance and the g r e a t e s t s p a r i n g of essen-t i a l amino a c i d s was obtained from a w e l l balanced d i e t f u r n i s h i n g approximately 60% of the t o t a l amino acids i n the n o n - e s s e n t i a l form. d. R e l a t i o n s h i p s between P r o t e i n and Energy Content of a D i e t The u l t i z a t i o n of p r o t e i n can only be maximized when there i s s u f f i c i e n t energy i n the d i e t from non-protein sources to s a t i s f y the r e q u i r e -ment of the organism f o r c a l o r i e s . The energy content i n the d i e t i s of c r i -t i c a l importance f o r the s u c c e s s f u l amino a c i d supplementation of animal feeds. Rosenberg _et a l . (1955) reported that no response was observed w i t h the - 36 -a d d i t i o n of methionine to a corn-soybean meal d i e t f o r c h i c k s i n s p i t e of the f a c t that the f i r s t - l i m i t i n g amino a c i d i n the d i e t was considered to be methionine. However, the chicks responded to the supplemental d i e t a r y methionine w i t h improved growth and feed e f f i c i e n c y when f a t was added to the d i e t . This e f f e c t was a l s o shown when f a t was replaced by carbohydrate ( B a l d i n i and Rosenberg, 1957). These s t u d i e s suggest a d i r e c t r e l a t i o n between c a l o r i c d e n s i t y and the amino a c i d requirements i n the d i e t . The st u d i e s a l s o i n d i c a t e d that three c a l o r i c l e v e l s r e q u i r e d three d i f f e r e n t l e v e l s of methionine f o r optimum performance of the c h i c k ( B a l d i n i and Rosenberg, 1955; B a l d i n i and Rosenberg, 1957). B a l d i n i e t a l . (1957), Rosenberg and C u l i k (1955), and W i l l i a m s and Grau (1956) a l s o showed that the c a l o r i c d e n s i t y i s one of the f a c t o r s governing the amino a c i d requirements of growing turkeys, r a t s and c h i c k s . The r e l a t i o n s h i p between the p r o t e i n content and the energy content of a d i e t has a l s o been subjected to many s t u d i e s u s i n g p i g l e t s (Bowland, 1964; Standish and Bowland, 1967) and the g r o w i n g - f i n i s h i n g p i g (Sewell et a l . , 1956; Abernathy et a l . , 1958; McWard ejt a l . , 1959; Henry and Rdrat, 1964; Robinson and Lewis, 1964; Robinson et a l . , 1964 ; Robinson, 1965a; Clawson, 1967; Cooke et a l . , 1972a, b ). Increases i n the energy content of a d i e t reduce feed i n t a k e , but improve feed e f f i c i e n c y and i n some cases a l s o improve growth r a t e . However, the b e n e f i t s of increased energy i n t a k e disappears when p r o t e i n i n t a k e i s i n s u f f i c i e n t . Conversely, an i n c r e a s e d p r o t e i n i n t a k e cannot be u l t i l i z e d e f f e c t i v e l y f o r body p r o t e i n s y n t h e s i s when the d i e t contains i n s u f f i c i e n t energy. - 37 -The s t u d i e s of the r e l a t i o n s h i p s between l i p i d content and amino a c i d content of a d i e t by a number of researchers (Anderson and Bowland, 1967; M i t c h e l l et a l . , 1965b; Re"rat e_t a l . , 1970) showed that the l e v e l of amino acids i n the d i e t have to be i n c r e a s e d w i t h higher d i e t a r y l i p i d content because of the accompanying r e d u c t i o n i n feed i n t a k e . I n terms of e n e r g y - l y s i n e r a t i o , Robinson ej; al_. (1964) concluded that the b e s t growth performance and carcass l e a n content f o r p i g s were recorded when the r a t i o of K c a l DE to Kg d i e t a r y l y s i n e was approximately 3,500 at a l l l e v e l of energy c o n c e n t r a t i o n . Lawrence (1971) i n d i c a t e d that the narrow c a l o r i c / p r o t e i n / l y s i n e 1 2 (CPL) r a t i o d i e t s when compared w i t h wide CPL r a t i o d i e t s gave b e t t e r growth r a t e s and e n e r g e t i c conversion e f f i c i e n c i e s and higher c o l d carcass weights, s m a l l e r back f a t deposits and g r e a t e r percentage of l e a n and bone but s m a l l e r percentage of f a t i n the carcass. The e f f e c t of the i n t e r a c t i o n between p r o t e i n and energy contents of the d i e t on pigs performance and carcass q u a l i t y has been i n v e s t i g a t e d by many workers. In g e n e r a l , i t has been observed that as the c o n c e n t r a t i o n of energy i n the feed i s r a i s e d , d i e t a r y p r o t e i n l e v e l needed f o r b e s t performance i n c r e a s e s . Growth r a t e and e f f i c i e n c y of feed u t l i z a t i o n are g e n e r a l l y 1. D i g e s t i b l e energy : crude p r o t e i n r a t i o = 198 : 1 Crude p r o t e i n : L y s i n e r a t i o = 19.8 : 1 2. D i g e s t i b l e energy : crude p r o t e i n r a t i o = 263 : 1 or 273 : 1 Crude p r o t e i n : Lysine r a t i o = 27.6 : 1 or 26.9 : 1 - 38 -improved by r a i s i n g the n u t r i e n t c o n c e n t r a t i o n w h i l s t carcass q u a l i t y bene-f i t s by an e l e v a t i o n of the p r o t e i n i n t a k e or of the o v e r a l l n u t r i e n t supply. Although energy and p r o t e i n i n t a k e i n f l u e n c e s growth performance and carcass c h a r a c t e r i s t i c s of the growing p i g , many experiments f a i l to demonstrate any s i g n i f i c a n t i n t e r a c t i o n between energy and p r o t e i n i n terms of these c r i t e r i a i n d i c a t i n g that they are independent from each other (Cooke et a l . , 1972a, b; Lodge et a l . , 1972 a). Cooke et a l . (1972b) i n d i c a t e d that p r o t e i n i n f l u e n c e d growth and carcass l e a n content but that the e f f e c t s were overridden by i n t a k e of energy so that there was l i t t l e i n d i c a t i o n of an i n c r e a s e d p r o t e i n i n t a k e c o u n t e r a c t i n g the adverse e f f e c t s of an increased energy i n t a k e on carcass q u a l i t y . e. Amino Acids Imbalances and I n t e r a c t i o n s I t i s g e n e r a l l y recognized t h a t amino a c i d imbalances can r e s u l t i n a decreased growth r a t e and a l o s s of a p p e t i t e . An imbalance may be expressed as a depression i n growth (or any other adverse e f f e c t ) . N itrogen imbalances have been reported i n p i g s (Meade, 1956a, b; Miner e^ t a l . , 1955), however, t h e i r e f f e c t s have been s t u d i e d mostly i n the r a t . Harper (1961) i n d i c a t e d two kinds of imbalances were p o s s i b l e . The f i r s t i s induced by adding s m a l l amount of amino a c i d (but not the most l i m i t i n g one) to a low p r o t e i n d i e t and may be overcome by simply supplementing w i t h the l i m i t i n g amino a c i d s . The second k i n d of imbalances i s due to the p r o t e i n source (or amino a c i d mixture) being t o t a l l y devoid of a given amino a c i d . For example, a d d i t i o n of g e l a t i n may decrease the growth of r a t s unless tryptophan i s added (Harper, 1959). More examples of imbalance s t u d i e s of the r a t can be found - 39 -i n the r e p o r t by Harper (1964). A s e r i e s of s t u d i e s by D'Mello and Lewis (1970a, b, c) using the c h i c k c l e a r l y demonstrated that the requirements f o r amino acid s i n animals are interdependent. The s t u d i e s showed that graded l e v e l s of excess d i e t a r y l y s i n e , l e u c i n e and theronine increased the q u a n t i t a t i v e requirements of a r g i n i n e , i s o l e u c i n e and tryptophan r e s p e c t i v e l y . A subsequent experiment reported by D'Mello (1973) i n d i c a t e d that r a i s i n g the i s o l e u c i n e l e v e l i n a d i e t i n c r e a s e s the requirements f o r l e u c i n e and v a l i n e . These pa t t e r n s of i n t e r a c t i o n s are r e f l e c t e d i n the plasma c o n c e n t r a t i o n of l e u c i n e , i s o l e u -c ine and v a l i n e (D'Mello, 1974). The unique i n t e r a c t i o n between l y s i n e and a r g i n i n e i n c h i c k n u t r i t i o n reported by D'Mello and Lewis (1970a,b,c) supports the observations of some other researchers (O'Dell et_ a l . , 1958; Jones, 1964; Smith and Lewis, 1966). S i m i l a r l y , i n t e r r e l a t i o n s h i p s between s e v e r a l amino acids have been observed i n r a t s . These i n c l u d e i n t e r a c t i o n s between l e u c i n e , i s o l e u c i n e and v a l i n e (Harper et a l . , 1954, Benton j i t a l . , 1956; Rogers j2t a l . , 1967) and between threonine and trytophan (Salmon, 1954; Morrison and Harper, 1960; F l o r e n t i n o and Peason, 1962). The s p e c i f i c i t y of i n t e r a c t i o n among l e u c i n e , i s o l e u c i n e and v a l i n e i s f u r t h e r e x e m p l i f i e d by recent work w i t h the p i g (Oestemer et a l . , 1973). f. Animal V a r i a t i o n : Age", Sex and Genetic Factors D i e t a r y p r o t e i n and amino acids are u t i l i z e d f o r body p r o t e i n s y n t h e s i s ( a n a b o l i c f u n c t i o n ) and f o r maintenance. In the r a p i d l y growing - 40 -animal, the a n a b o l i c f u n c t i o n s account f o r a l a r g e p r o p o r t i o n of the amino a c i d requirements. Conversely, the replacement f u n c t i o n s d i c t a t e the amino a c i d requirements i n the mature animal. As a r e s u l t , g reater abundance of hi g h q u a l i t y amino aci d s are r e q u i r e d by young growing animals than f o r mature animals. I n a d d i t i o n , amino a c i d requirements p r o g r e s s i v e l y decrease as age i n c r e a s e s . Oslage jet a l . (1966) showed that the e f f i c i e n c y of n i t r o g e n r e t e n t i o n decreases w i t h age. In t h e i r experiments, n i t r o g e n r e t e n t i o n e f f i c i e n c y was about 52% i n young animals weighing 25 Kg and decreased to about 35% at 100 Kg and to 22% at 160 Kg l i v e w e i g h t . S i m i l a r r e s u l t s were obtained by some other workers (Rerat and Henry, 1964; Robinson, 1964). Evidence f u r t h e r i n d i c a t e s t h a t amino a c i d allowances should be reduced w i t h age. N i t r o g e n u t i l i z a t i o n and thus n i t r o g e n " r e q u i r e m e n t s ! a r e a f f e c t e d by sex. In g e n e r a l , females grow slower than c a s t r a t e d male and d i f f e r i n body composition. However, at equal r a t e s of growth and feed e f f i c i e n c y , females would have leaner carcasses i n d i c a t i n g h i g h e r n i t r o g e n r e t e n t i o n ' (Bowland and Berg, 1959; Robinson et a l . , 1964; Lodge jet a l . , 1972b). Consequently, the r e s p e c t i v e needs f o r p r o t e i n and energy may vary according to sex (Bowland and Berg, 1959; B l a i r e t a l . , 1969b). S i m i l a r l y , the amino a c i d requirements of the female are d i f f e r e n t from those of the male. Germann c^ t al. (1958) showed that females had optimum growth r a t e w i t h l e s s l y s i n e than was necessary f o r males. R6rat and Henry (1970) i n d i c a t e d that the methionine plus c y s t i n e requirements of growing pigs (20 - 60 Kg) - 41 -v a r i e d w i t h sex. Female pigs needed 0.52% of methionine-cystine i n the d i e t whereas c a s t r a t e d pigs r e q u i r e d only 0.47% i n the d i e t . Bayley and Summers(1968) i n d i c a t e d that boars responded more than g i l t s to increased p r o t e i n l e v e l i n the d i e t . B e l l (1965), B l a i r e t a l . (1969a) and P i e r c e and Bowland (1972) were able to demonstrate t h a t g i l t s were more e f f i c i e n t converters of h i g h p r o t e i n d i e t s than barrows w i t h the d i f f e r e n c e s reducing as the p r o t e i n l e v e l s decreased. I t has been reported that there are important d i f f e r e n c e s between breeds and w i t h i n the same breed as-regards growth performance and body composition (Hetzer et a l . , 1963). The high pressure of s e l e c t i o n to improve the genetic p o t e n t i a l f o r a leaner carcass may create a change of amino a c i d requirements. To date very l i t t l e has been rep o r t e d . The e f f e c t of the g e n e t i c o r i g i n of the animals on n i t r o g e n r e t e n t i o n has been demon-s t r a t e d by Anderson and Bowland (1967) who rep o r t e d t h a t the Large White p i g r e t a i n s n i t r o g e n b e t t e r than the Large White x Lacombe p i g . Bayley and Summers(1968) found a s i g n i f i c a n t i n t e r a c t i o n between s t r a i n of p i g and p r o t e i n l e v e l . Lacombe and Y o r k s h i r e pigs i n c r e a s e d l i v e w e i g h t gain w i t h increased p r o t e i n l e v e l from 13 to 16% whereas no response was found i n the Hampshire x Landrace and Landrace. On the b a s i s of the above r e s u l t s recommendation f o r d i e t a r y n i t r o g e n should take breed and s t r a i n i n t o account. - 42 -g. Feeding P r a c t i c e s : Ad L i b i t u m vs R e s t r i c t e d Feeding L e v e l of feeding i n f l u e n c e s the amino a c i d requirements of p i g s . Ad l i b i t u m feeding i s commonly employed i n North America whereas r e s t r i c t e d feeding i s u s u a l l y p r a c t i c e d i n European c o u n t r i e s . The purpose of r e s t r i c t i n g feed i n t a k e i s to c o n t r o l the energy i n t a k e i n order to improve the carcass q u a l i t y of the animal. A review of energy and p r o t e i n r e l a t i o n -s h i p s , i n d i c a t e s that the l e v e l of e s s e n t i a l amino acid s i n the d i e t must be adjusted according to the degree of feed r e s t r i c t i o n a p p l i e d . According to R6rat e t a l . (1971), when energy i n t a k e i s r e s t r i c t e d to 80% of an ad_ l i b i t u m l e v e l , the i n c r e a s e i n n i t r o g e n and more p a r t i c u l a r l y amino acids content must be about 10% to avoid m o d i f i c a t i o n of the p r o t e i n s y n t h e s i s . I t has been recommended (Rerat, 1972) that amino a c i d r e q u i r e -ment should be expressed i n the form of d a i l y amounts. This recommendation could then be transformed i n t o concentrations when fo r m u l a t i n g d i e t s . - 43 -I I I . PIG EXPERIMENT I A. • EXPERIMENTAL PROCEDURE a. General Many experiments have shown that l y s i n e i s the f i r s t l i m i t i n g amino a c i d i n b a r l e y as a feed f o r p i g s . Although methionine has been suggested as the second l i m i t i n g amino a c i d by some workers a l a r g e number of e x p e r i -ments have shown e i t h e r no response to methionine or i n some cases, a growth depression. In c o n t r a s t , threonine, when t e s t e d as the second l i m i t i n g amino a c i d , has shown a response w i t h b a r l e y ( M i i l l e r ejt a l . , 1967c; Chung, 1973) and w i t h sorghum (Pond et a l . , 1958). S e v e r a l recent review papers (Scrimshaw and A l t s c h u l , 1971) have confirmed the wide-spread f i n d i n g of threonine as the second l i m i t i n g amino a c i d i n s m a l l g r a i n s i n the n u t r i t i o n of s e v e r a l s p e c i e s , i n c l u d i n g man. The present experiment i s designed to evaluate the response to i n c r e a s i n g l e v e l s of threonine i n a d i e t of b a r l e y plus adequate l y s i n e , and to i n v e s t i g a t e the e f f e c t of the a d d i t i o n of methionine to the d i e t c o n t a i n i n g the h i g h e s t l e v e l of added threonine. b. Design The experimental layout was that of a completely randomized t h r e e - f a c t o r ( d i e t — treatment, sex - male or female and p e r i o d — rep) design w i t h a f a c t o r i a l arrangement of treatments and three observations per each treatment combination. (e.g. appendix page 146) - 44 -c. Animals A t o t a l of one hundred and e i g h t Y o r k s h i r e and Y o r k s h i r e x Land-race cross pigs were randomly a l l o c a t e d to s i x d i e t a r y treatments w i t h equal sex numbers (3 c a s t r a t e d males and 3 females) to a pen. These pigs were placed on t r i a l at an average body weight of 20 - 21 Kg and t h e r e a f t e r were weighed weekly and were slaughtered when a body weight o f 85 Kg was a t t a i n e d . The l a s t two pigs i n a pen x^ere sent f o r sl a u g h t e r when the body weight of the heav i e r one exceeded 85 Kg. D i e t s and pens were a l l o c a t e d on a random b a s i s . d. D i e t s The s i x d i e t s were fed i n dry mash form. A d i s c r i p t i o n of the d i e t s i s given below. Ingredients are l i s t e d i n Table 2 1. Barley-soybean meal to supply 0.75% t o t a l l y s i n e ( c o n t r o l d i e t ) 2. B a r l e y + 0.444% L - l y s i n e HC1 to supply 0.75% t o t a l l y s i n e 3. B a r l e y + 0.444% L - l y s i n e HC1 to supply 0.75% t o t a l l y s i n e + 0.05% L-threonine 4. B a r l e y + 0.444% L - l y s i n e HC1 to supply 0.75% t o t a l l y s i n e + 0.10% L-threonine 5. B a r l e y + 0.444% L - l y s i n e HC1 to supply 0.75% t o t a l l y s i n e + 0.15% L-threonine 6. B a r l e y + 0.444% L - l y s i n e HC1 to supply 0.75% t o t a l l y s i n e +0.15% L-threonine + 0.10% DL-methionine - 45 -Table 2. Percentage Composition of D i e t s used i n P i g Experiments I & I I ( A i r dry b a s i s ) Ingredients D i e t No 1 2 3 4 5 6 B a r l e y 81.34 96.38 96.33 96.28 96.23 96.13 Soybean meal (45%) 15.60 - - - - -L - l y s i n e HC1* - 4.44 4.44 4.44 4.44 4.44 L-threonine * - - 0.05 0.10 0.15 0.15 DL-methionine * - - - - - 0.10 D e f l u o r i n a t e d rock phosphate 1.49 1.64 1.64 1.64 1.64 1.64 Limestone 0.57 0.54 0.54 0.54 0.54 0.54 Iod i z e d s a l t 0.50 0.50 0.50 0.50 0.50 0.50 Trace m i n e r a l and v i t a m i n premix 0.50 0.50 0.50 0.50 0.50 • 0.50 The t r a c e m i n e r a l and v i t a m i n premix provided the f o l l o w i n g per kg of r a t i o n : Manganese 44 mg as MnSO^.H^O, Zinc 110 mg as ZnS0 4.7H 20, B.H.T. 500 mg, Vitamin A 3085 I.U., Vi t a m i n D 3 440 I.U., Vitamin 20 ug, R i b o f l a v i n 2.9 mg, N i a c i n 11 mg, Calcium pantothenate 11 mg (45% e q u i v a l e n t ) . * The L - l y s i n e HC1 was feed grade and was 98% pure, c o n t a i n i n g 78% L - l y s i n e . The L-threonine was pure, DL-methionine was 98% pure. A l l were produced by the Ajinomoto Co., Japan, - 46 -The amino a c i d content and chemical composition of the b a r l e y and the soybean meal are shown i n Table 3 . The amino a c i d composition of each d i e t i s l i s t e d i n Table 4 . A l l d i e t s were formulated to c o n t a i n adequate amounts of a l l n u t r i e n t s known to be r e q u i r e d f o r g r o w i n g - f i n i s h i n g pigs except f o r t o t a l p r o t e i n and amino a c i d content. e. Management ( i ) Housing . This experiment was conducted at the Swine Research U n i t , on the U n i v e r s i t y of B r i t i s h Columbia Campus. The b u i l d i n g i s i n s u l a t e d , w i t h a i r exhaust fans f i t t e d w i t h thermostatic c o n t r o l s s et a t 18.5° C. Pens are 2 concrete and p a r t i a l l y s l a t t e d and of t o t a l area 5.6 m . ( i i ) Feeding method Pi g s were fed twice d a i l y at 8:00 A.M. and 1:00 P.M.. Feeding was from troughs on an ad l i b i t u m b a s i s w i t h maximum allowance l i m i t e d to 2.7 Kg per day. Feed consumption records were kept d a i l y . Water was su p p l i e d ad l i b i t u m by d r i n k i n g n i p p l e . ( i i i ) Feed mixing and storage A l l feed i n g r e d i e n t s were purchased and s t o r e d p r i o r to the commencement of the experiment to ensure a uniform d i e t composition through-out. The b a r l e y was s t o r e d i n the whole form. D i e t s were prepared as re q u i r e d i n 450 Kg batches by hammer m i l l i n g (7 mm screen) the b a r l e y - 47 -Table 3. Content of e s s e n t i a l amino acids and proximate c o n s t i t u e n t s of b a r l e y and soybean meal on a dry matter b a s i s . Concentration(g/100g d.m.) Component Ba r l e y Soybean Meal A r g i n i n e 0.539 3.172 H i s t i d i n e 0.247 1.621 I s o l e u c i n e 0.471 1.800 Leucine 0.870 4.089 Lysi n e 0.491 3.041 Methionine 0.200 0.709 (Cystine) 0.241 0.701 Phenylalanine 0.631 2.521 (Tyrosine) 0.338 1.717 Threonine* 0.443 1.743 Tryptophan 0.749 0.126 V a l i n e 0.808 2.052 Crude p r o t e i n % (N x 6.25) 11.4 51.3 Ash % 2.5 6.3 Crude f i b r e % 6.72 5.7 Crude f a t % 3.19 1.49 Uncorrected value from d e s t r u c t i o n of a c i d h y d r o l y s i s (Blackburn, 1968) - 48 -Table 4. Content of e s s e n t i a l amino acids and proximate c o n s t i t u e n t s i n d i e t s (g amino acid/100 g) i n P i g Experiments I and I I . D i e t s Amino a c i d _ (g/100 g A.D.) 1 2 3 4 5 6 A r g i n i n e 0. 82 0 .52 0 .52 0 .52 0 .52 0 .52 H i s t i d i n e 0. 40 0 .24 0 .24 0 .24 0 .24 0 .24 I s o l e u c i n e 0. 58 0 .45 0 .45 0 .45 0 .45 0 .45 Leucine 1. 18 0 .84 0 .84 0 .84 0 .84 0 .84 Lysine 0. 75 0 .75 0 .75 0 .75 0 .75 0 .75 Methionine 0. 24 0 .19 0 .19 0 .19 0 .19 0 .29 (Cystine) 0. 27 0 .23 0 .23 0 .23 0 .23 0 .23 Phyenlalanine 0. 80 0 .61 0 .61 0 .61 0 .61 0 .61 (Tyrosine) 0. 48 0 .33 0 .33 0 .33 0 .33 0 .33 Threonine 0. 56 0 .37 0 .42 0 .47 0 .52 0 .52 Tryptophan 0. 19 0 .11 0 .11 0 .11 0 .11 0 .11 V a l i n e 0. 98 0 .78 0 .78 0 .78 0 .78 0 .78 Dry matter % 88. 55 87 .78 87 .61 88 .90 87 .58 87 .90 Crude p r o t e i n % 17. 58 11 .01 11 .36 11 .36 11 .32 11 .41 - 49 -and then mixing i t w i t h the other i n g r e d i e n t s i n a v e r t i c a l mixer A" f o r 5 to 10 minutes. f. Records A l l p i g s were weighed at the commencement of the experiment and t h e r e a f t e r at weekly i n t e r v a l s . Weekly feed consumption, c o r r e c t e d f o r s p i l t feed, was recorded. g. Chemical A n a l y s i s A.O.A.C. methods (1965) were employed i n the a n a l y s i s of a l l feed f o r moisture, ash and crude p r o t e i n . A c i d detergent f i b r e was d e t e r -mined by the method of Van Soest (1963). For amino a c i d a n a l y s i s , the h y d r o l y z a t e s of b a r l e y and soybean meal were prepared by a c i d h y d r o l y s i s (Kohler and P a l t e r , 1967) except f o r c y s t i n e and tryptophan as these two amino acid s are po o r l y recovered from a c i d h y d r o l y z a t e s . Therefore, c y s t i n e was determined as c y s t e i c a c i d by o x i d a t i o n h y d r o l y s i s (Moore, 1963) . Methionine was determined as methionine s u l f o n e from t h i s chromatogram. Tryptophan was determined by ion-exchange chramotography a f t e r a l k a l i n e h y d r o l y s i s by the method of H u g l i and Moore (1972). h. Carcass Measurements Dressed weight was measured on the hot carcass (with head). The ft K e l l e y Duplex M i l l Machinery, s i z e 220, s e r i a l 57364. The Duplex m i l l m f g. Co., S p r i n g f i e l d , Ohio. - 50 -carcass was then c h i l l e d at 4° C f o r 96 hours and eye muscle area, eye muscle index (length "A" x width "B") and b a c k f a t thickness ("C" without skin) determined on the s u r f a c e exposed by c u t t i n g the carcass at r i g h t angles to i t s l e n g t h at the j u n c t i o n of the 7th and 8th vertebrae ante-r i o r to the d o r s o - s a c r a l j u n c t i o n (Buck e t a l . , 1962). Maximum shoulder f a t , minimum middle f a t and maximum l o i n f a t were measured according to the Canada Department of A g r i c u l t u r e (1968) methods. i . C a l c u l a t i o n s Average d a i l y gain was determined as the b value i n the l i n e a r r e g r e s s i o n equation derived from weekly body weight y = a + bx, where 11 y " i s a e s t i m a t e d body weight, and "x" days on t r i a l . The average d a i l y feed consumption and feed conversion e f f i c i e n c y ( t o t a l feed eaten d i v i d e d by t o t a l body weight g a i n , both expressed i n tha same u n i t s ) were c a l c u l a t e d f o r each pen. Carcass measurements were determined i n d i v i d u a l l y f o r each p i g . j . S t a t i s t i c a l A n a l y s i s of Data The data were subjected to a n a l y s i s of vari a n c e u s i n g UBC-MFAV (Halm and Le, 1974) computer program. Since the program cannot compensate f o r m i s s i n g v a l u e s , carcass measurements of one mi s s i n g value was estimated by missing data c a l c u l a t i o n (Snedecor and Cochran, 1967) . Two d i f f e r e n t analyses were made. For mean d a i l y g a i n , d r e s s i n g percentage, t o t a l back f a t , "C" back f a t , eye muscle index and area, and experimental days, the i n d i v i d u a l treatment combinations ( d i e t , sex, period) - 51 -and t h e i r i n t e r a c t i o n s were i n v e s t i g a t e d (eg. page 147). For mean daily-dry matter feed i n t a k e and feed conversion e f f i c i e n c y , the only f a c t o r was treatment ( d i e t ) w i t h three r e p l i c a t i o n s (three groups of 6 animals each) each (eg. page 145). Mean d a i l y g a i n , feed conversion e f f i c i e n c y , d r e s s i n g percentage and days on t r i a l were analyzed w i t h s t a r t i n g weight as a c o v a r i a b l e . T o t a l back f a t , "C" back f a t , eye muscle area and eye muscle index were analyzed w i t h f i n a l body weight, average d a i l y gain and' carcass l e n g t h as c o v a r i a b l e s . Means from comparisons showing a s i g n i f i c a n t "F" value were test e d u s i n g Tukey's t e s t (1953). - 52 -B. RESULTS N u t r i e n t contents of i n g r e d i e n t s and d i e t s are l i s t e d i n Table 2. The experimental growth and carcass data analyses are summarized i n Table 5. There was no d i f f e r e n c e i n average d a i l y i n t a k e among a l l t r e a t -ments i n d i c a t i n g that the e f f e c t of d i f f e r e n c e s were not due to a p p e t i t e . There was no s i g n i f i c a n t e f f e c t of treatments on d r e s s i n g percentage. The barley-soybean meal d i e t produced a s i g n i f i c a n t l y g r e a t e r growth r a t e than a l l other d i e t s , except f o r the 0.10% threonine supplemented d i e t . There were no s i g n i f i c a n t d i f f e r e n c e s i n growth r a t e s between 0.10% threonine, 0.15% threonine and 0.15% threonine plus 0.10% methionine d i e t s , but a l l gave s u p e r i o r growth to that obtained on the d i e t c o n t a i n i n g no added threonine. The r a t e of growth was s i g n i f i c a n t l y g r e a t e r on the soybean-c o n t r o l d i e t than on any other treatments over the body weight range 20 to 45 Kg whereas no s i g n i f i c a n t d i f f e r e n c e s were observed between d i e t s c o n t a i n i n g 0.10% threonine, 0.15% threonine and 0.15% threonine p l u s 0.10% methionine a d d i t i o n s and the barley-soybean c o n t r o l d i e t f o r l i v e w e i g h t gains from 45 Kg to f i n i s h i n g weight. There were no s i g n i f i c a n t d i f f e r e n c e s i n terms of feed conversion e f f i c i e n c y between the 0.10% threonine, 0.15% threonine, 0.15% threonine p l u s 0.10% methionine and soybean c o n t r o l d i e t s . Feed e f f i c i e n c y was s i m i l a r i n d i e t s c o n t a i n i n g 0.10% or g r e a t e r added _ threonine, a l l of which produced a feed e f f i c i e n c y which was b e t t e r than that obtained on the b a s a l d i e t or the b a s a l plus 0.05% threonine. The a d d i t i o n of 0.10%, 0.15% threonine and 0.15% threonine p l u s 0.10% - 53 -Table 5. Summary of the e f f e c t . o f the a d d i t i o n of amino acids to b a r l e y on body weight g a i n , feed consumption (D.M.), feed conversion (D.M.) and carcass measurement i n P i g Experiment I . Treatment 1 2 3 4 5 6 s o y - c o n t r o l 0.75%lys B + B + B + B + SE (Basal) 0.05%thr 0.10%thr 0.15%thr 0.15%thr+ 0.10%met. Resu l t A of s i g n i -f i c a n c e t e s t * * S t a r t i n g 20.32 wt.(Kg) F i n a l wt.(Kg) 86.58 1.943 Mean d a i l y D.M. Feed i n t a k e (Kg) Mean d a i l y gain (Kg) S t a r t to 45 Kg 45 Kg to f i n i s h S t a r t to f i n i s h 0.648 0.729 0.702 20.41 20.92 20.36 19.94 20.22 85.41 85.50 85.50 84.89 85.94 1.821 1.839 1.911 1.823 1.856 ±0.015 146352 0.466 0.522 0.556 0.536 0.565 ±0.013 164532 0.603 0.619 0.744 0.687 0.686 ±0.018 145632 0.553 0.584 0.675 0.625 0.634 ±0.013 146532 D.M. Feed(Kg) Wt. gain (Kg) /-* 5 J 1 Dressing % T o t a l back f a t (mm) Back f a t 40mm from m i d - l i n e Eye muscle index AxB (rnrn^) 78.59 82.45 14.53 4274 Eye muscle area (mmz) Experimental period(days) 96.9 3.407 3.286 2.951 3.017 3.020 ±0.018 145632 79.37 80.15 78.18 79.04 79.26 ±0.63 326514 96.74 94.93 91.51 88.92 90.95 ±2.00 234651 3432 3694 4001 3902 3740 2414 ' 2645 2758 2658 2649 20.18 18.53 17.14 16.66 18.07 ±0.68 236451 ±78 145632 ±17 145632 121.9 116.8 100.8 ' 106.6 106.5 ±1.83 146532 Standard e r r o r of treatment means. ** Treatment numbers not underscored by the same l i n e are s i g n i f i c a n t l y d i f f e r e n t at the 5% l e v e l of p r o b a b i l i t y (Tukey, 1953). - 54 -methionine to the b a s a l d i e t improved feed conversion e f f i c i e n c y by 15.5%, 12.9 and 12.8% r e s p e c t i v e l y . There was no s i g n i f i c a n t treatment e f f e c t on d r e s s i n g percentage. The barley-soybean c o n t r o l pigs had the lowest t o t a l back f a t t h i c k n e s s which was s i g n i f i c a n t l y d i f f e r e n t from the b a r l e y - l y s i n e b a s a l d i e t (Treat-ment 2) , 0.05% threonine (Treatment 3) and 0.10% threonine (treatment 4) supplemented d i e t s but not s i g n i f i c a n t l y d i f f e r e n t from the hi g h e s t threo-nine (0.15%) (Treatment 5) and 0.15% threonine plus 0.10% methionine (Treatment 6) d i e t . However no s i g n i f i c a n t d i f f e r e n c e s were observed between the v a r i o u s barley-amino a c i d treatments f o r t o t a l back f a t t h i c k -ness. The carcass l e n g t h had a s i g n i f i c a n t negative r e l a t i o n s h i p to t o t a l back f a t t h i c k n e s s . Again the c o n t r o l d i e t produced the lowest back f a t 40 mm from the m i d - l i n e , being s i g n i f i c a n t l y l e s s than that of Treatments 2, 3 and 6 but not s i g n i f i c a n t l y d i f f e r e n t e from Treatments 4 and 5 . No s i g n i f i c a n t d i f f e r e n c e s were observed among the four d i e t s c o n t a i n i n g added threonine. Back f a t 40 mm from the m i d - l i n e i n Treatment 2 d i d not d i f f e r s i g n i f i c a n t l y from measurements f o r Treatments 3,4 and 6 but was s i g n i f i c a n t l y h i g h e r than values f o r Treatments 5 and 1. The cova-r i a n c e a n a l y s i s showed that carcass l e n g t h was s i g n i f i c a n t l y n e g a t i v e l y c o r r e l a t e d w i t h back f a t 40 mm from the m i d - l i n e . There was no d i f f e r e n c e i n eye muscle index between the carcasses from p i g s on the c o n t r o l d i e t and those on Treatment 4 but the former was s i g n i f i c a n t l y h i g her than that f o r a l l other treatments. There were no s i g n i f i c a n t d i f f e r e n c e s among the threonine supplemented d i e t s . The b a s a l - 55 -d i e t showed the lowest eye muscle index though i t was not s i g n i f i c a n t l y d i f f e r e n t from the means f o r Treatments 3 and 6 . S i m i l a r r e s u l t s were observed f o r eye-muscle area except that the c o n t r o l d i e t produced s i g n i f i c a n t l y h i g h er areas than a l l other treatments. Covariance a n a l y s i s i n d i c a t e d that eye muscle index and eye muscle area were s i g n i f i c a n t n e g a t i v e l y c o r r e l a t e d w i t h average d a i l y gain. The mean d a i l y g a in was s i g n i f i c a n t l y higher i n barrows than i n g i l t s . G i l t s had c o n s i s t e n t l y s i g n i f i c a n t l y lower b a c k f a t measurements and l a r g e r eye muscle i n d i c e s and eye muscle areas than barrows. Dressing percentage was not a f f e c t e d by sex. There was no d i e t x sex i n t e r a c t i o n i n mean d a i l y gain or carcass measurements (Table 6 ) . - 56 -Table 6. Comparison of the e f f e c t s of supplementation w i t h amino acids on mean d a i l y gains and carcass measure-§ ments of g i l t s and barrows G i l t Barrow S i g n i f i c a n t F.. Test (sp (s2) J. Mean D a i l y Gain (Kg) 0.612 0.646 ft* Carcass Measurement Dressing % 79.69 77.08 N.S. T o t a l back f a t (mm) 88.09 93.74 ft* Back f a t 40 mm from mid l i n e (mm) 16.74 18.29 ft 2 Eye muscle index (mm ) 3986 3696 ftft 2 Eye muscle area (mm ) 2782 2589 ft* Carcass measurements were analysed w i t h f i n a l body weight, mean d a i l y gain and carcass l e n g t h as c o v a r i a b l e s . * P < 0.05 * * ' -P < 0.01 - 57 -C. DISCUSSION Based on the r e s u l t s obtained i n t h i s feeding t r i a l , i t might be concluded t h a t the g r o w i n g - f i n i s h i n g p i g can perform w e l l based on an a l l -b a r l e y l o w - p r o t e i n d i e t w i t h a d d i t i o n of an optimum l e v e l of l y s i n e and t h r e o n i n e , p r o v i d i n g a d d i t i o n a l v i tamins and minerals are given. With the 0.75% l y s i n e b a r l e y d i e t ( b a s a l d i e t ) , the a d d i t i o n of graded l e v e l s of L-threonine improved markedly the average d a i l y gain and feed conversion e f f i c i e n c y of the growing p i g . The 0.10% threonine a d d i t i o n to the b a s a l d i e t produced r e s u l t s which were not s i g n i f i c a n t l y d i f f e r e n t from the r e s u l t s obtained on the conventional soybean-barley c o n t r o l d i e t i n d i c a t i n g that t h i s l e v e l of threonine (0.47%) i n the d i e t , though l e s s than i n the barley-soybean meal diet,was adequate f o r the growth performance of growing-f i n i s h i n g p i g s . Chung and Beames (1972) reported that an a l l b a r l e y d i e t i s f i r s t l i m i t i n g i n l y s i n e and that threonine i s probably the second-l i m i t i n g amino a c i d . With 0.75% t o t a l l y s i n e i n the l y s i n e - b a r l e y d i e t , Chung (1973) observed a s i g n i f i c a n t response to 0.05% threonine a d d i t i o n i n growth and feed e f f i c i e n c y performance above the l y s i n e - b a r l e y d i e t . The response of threonine i n the present experiment i s i n good agreement w i t h the r e s u l t s of Chung and Beames (1974). However, 0.05% threonine a d d i t i o n to a 0.75% l y s i n e d i e t i n the present experiment improved the p i g p e r f o r -mance but not s i g n i f i c a n t l y . The reason i s probably because the threonine l e v e l of the b a r l e y used by Chung and Beames (1974) was lower than the l e v e l i n the present experiment. The b a s a l d i e t w i t h 0.75% l y s i n e contained 0.24% i n the experiment of Chung and Beames (1974) whereas i n the present experiment the threonine l e v e l was 0.37% . - 58 -Supplementation w i t h l y s i n e of a h i g h c e r e a l - d i e t has been shown by s e v e r a l workers to improve growth and feed e f f i c i e n c y (Evans, 1960; Jones et a l . , 1962; E r i c s o n et a l . , 1962; Ostrowski, 1969; Braude et a l . , 1972). Bloch e t a l . (1972) from an experiment w i t h p i g s r e c e i v i n g an a l l -b a r l e y d i e t showed that b a r l e y p r o t e i n i s p o o r - q u a l i t y without amino a c i d supplementation. Although Chung and Beames (1974) reported t h a t 0.90% l y s i n e content i n a barley-amino a c i d d i e t was s i g n i f i c a n t l y d i f f e r e n t from 0.75% l y s i n e - b a r l e y d i e t as measured by growth r a t e and feed conversion e f f i c i e n c y of g r o w i n g - f i n i s h i n g p i g , the s i g n i f i c a n t d i e t x sex i n t e r a c t i o n i n d i c a t e d that the h i g h e r l e v e l of d i e t a r y l y s i n e content was b e n e f i c i a l only f o r the g i l t s . The a d d i t i o n of 0.05% threonine to 0.75% l y s i n e - b a r l e y d i e t s i g n i f i c a n t l y improved the above performance c r i t e r i a compared to the b a r l e y d i e t s w i t h only 0.75 and 0.90% l y s i n e . This suggested that 0.75% l y s i n e might be the o p t i m a l l e v e l f o r growth and feed e f f i c i e n c y , but t h a t threonine i s l i m i t i n g i n such a d i e t . The l e v e l of d i e t a r y l y s i n e f o r the growing and the f i n i s h i n g p i g x^as recommended by N.R.C.-N.A.S. (1968) to be 0.70 and 0.50% r e s p e c t i v e l y , and 0 . 7 5 - 0 . 8 0 % and 0 . 6 - 0 . 6 5 % by A.R.C. (1967). This appears to support t h a t 0.75% d i e t a r y l y s i n e i s adequate f o r the g r o w i n g - f i n i s h i n g p i g . M i i l l e r e t a l . (1967a) demonstrated that l y s i n e and threonine supplementation of a b a s a l c e r e a l d i e t had a l a r g e f a v o r a b l e e f f e c t upon gains and feed e f f i c i e n c y . With l y s i n e supplementation alone, the improve-ment i n gains was found to be 19% i n one case and 22% i n another case. However, with l y s i n e and threonine supplementation, the gains were improved by 59% i n one t r i a l and 92% i n the other. Respective improvements i n - 59 -feed e f f i c i e n c y were 31 to 34% . M i i l l e r e^ t a l . (1967a) considered that i n growing-pig d i e t s based on c e r e a l s (wheat, b a r l e y and oats) l y s i n e i s the f i r s t l i m i t i n g amino a c i d , f o l l o w e d by threonine, tryptophan and methio-nine. Threonine was the second l i m i t i n g amino a c i d i n b a r l e y i n the present experiment. This supports the f i n d i n g s of previous i n v e s t i g a t i o n s w i t h b a r l e y and other c e r e a l s (Sure, 1955; Pond e_t a l . , 1958; Rosenberg et a l . , 1959) . I n the present experiment, tryptophan was s u f f i c i e n t i n the b a s a l b a r l e y d i e t according to N.A.S.-N.R.C. (1968) Feeding Standards f o r growing-f i n i s h i n g p i g s . Consequently, i t was not i n v e s t i g a t e d . Although t o t a l s u l f u r amino a c i d (methionine + c y s t i n e ) r e q u i r e -ments of growing p i g are widely accepted as being 0 . 5 - 0 . 6 % of the d i e t (N.A.S.-N.R..C., 1968; A.R.C., 1967), many researchers over recent years have i n d i c a t e d t h a t the requirements of s u l f u r amino a c i d of p i g s may be overestimated. Beames and Pepper (1969) i n d i c a t e d that 0.3 - 0.42% i s adequate f o r o p t i m a l growth. Chung and Beames (1974) obtained no response w i t h supplementation of 0.10% methionine to an a l l barley-amino a c i d d i e t c o n t a i n i n g 0.27% methionine + c y s t i n e . S i m i l a r evidence was r e p o r t e d by many other researchers such as Oestemer ort a l . (1970), Jensen et a l . (1965) and Brown e_t aL. (1974) s t r o n g l y supporting the c o n t e n t i o n that the t o t a l s u l f u r amino a c i d requirements of growing and f i n i s h i n g p igs should be lower than N.A.S.-N.R.C. (1968) and A.R.C. (1967) recommended l e v e l s . The f a i l u r e i n response when adding 0.10% methionine to an a l l barley-amino a c i d d i e t c o n t a i n i n g 0.44% methionine + c y s t i n e i n the present experiment - 60 -f u r t h e r confirmed that methionine i s not l i m i t i n g i n b a r l e y f o r growing p i g s . This i s i n agreement w i t h r e s u l t s of S o l d e v i l a and Meade (1964), Rerat and Henry (1969) and Chung and Beames (1974). I t has been reported that feeding c e r e a l mixtures supplemented w i t h l y s i n e , t h r e o n i n e , tryptophan, methionine and i n some experiments a l s o i s o l e u c i n e , r e s u l t e d i n performance equal to that obtained w i t h g r a i n d i e t s c o n t a i n i n g p r o t e i n concentrate (Robinson and Lewis, 1963 ; M i i l l e r e t . a l . , 1967b) . The s i m p l i f i e d d i e t of amino acids added to s i n g l e c e r e a l d i e t s (wheat, b a r l e y or corn) used by M i i l l e r and Malek (1967a, b, c) i n d i c a t e d that t h i s k i n d of d i e t could be employed to r a i s e pigs from weaning (approximately 20 Kg l i v e w e i g h t ) . Many re p o r t s have i n d i c a t e d that the perf mance of animals fed a d i e t w i t h a s i n g l e c e r e a l p l u s amino acids d i e t f a i l to produce performance as good as that achieved w i t h n a t u r a l p r o t e i n concentrate supplemented d i e t (Chung and Beames, 1974). Beames and Pepper (1969) a l s o reported t h a t the use of l y s i n e , e i t h e r w i t h or w ithout methionine, was not completely s u c c e s s f u l as a replacement of h a l f soybean concentrate to sorghum d i e t f o r p i g s l e s s than 45 Kg body weight. However, no d i f f e r e n c e i n performance was observed i n pigs growing from 45 Kg to 90 Kg between the amino a c i d r e p l a c e d d i e t or the o r i g i n a l soybean-sorghum d i e t . R e s u l t s from the present experiment i n d i c a t e d that d i e t s composed of a s i n g l e c e r e a l supplemented w i t h the r e q u i r e d amino acids could induce - 61 -h i g h p i g performance as good as conventional p r o t e i n concentrate supplemented d i e t s . Pigs fed w i t h 0.444% l y s i n e - R C l and 0.10% threonine added to b a r l e y i n a d i e t c o n t a i n i n g adequate minerals and v i t a m i n s were not s i g n i f i -c a n t l y i n f e r i o r to those on the barley-soybean meal d i e t . However, when the crude p r o t e i n , l y s i n e and threonine l e v e l s i n these two d i e t s i s compared, the barley-amino a c i d d i e t i s seen to c o n t a i n l e s s crude p r o t e i n (11.4% vs 17.6%), a s i m i l a r l y s i n e l e v e l (0.75% vs 0.75%) and a lower threo-nine l e v e l (0.46% vs 0.57%). The threonine l e v e l s recommended by N.A.S.-N.R.C. (1968) and A.R.C. (1967) are 0.45% and 0.45 - 0.50% of the d i e t f o r 20 to 45 Kg p i g s . The d i e t a r y threonine l e v e l of 0.47% t h r e o -nine i s the b e s t d i e t of the present experiment which agrees w i t h these recommendations. Henry and R£rat (1970) reported that on a 10% p r o t e i n d i e t , 0.48% d i e t a r y threonine was the optimal l e v e l f o r growth of 20-50 Kg female p i g s . The f u r t h e r i n c r e a s e of threonine l e v e l w i t h or without a d d i t i o n a l methionine s l i g h t l y depressed growth below that obtained w i t h 0.1% threonine a d d i t i o n . This may i n d i c a t e that the a d d i t i o n of 0.05% e x t r a L-threonine created a s l i g h t s t r e s s or an amino a c i d 'imbalance' i n the d i e t . Methionine a d d i t i o n to 0.15% threonine d i e t tended to improve performance s l i g h t l y , p o s s i b l y as a r e s u l t of reducing the imbalance. Although the d i e t c o n t a i n i n g 0.10% added threonine was not s t a t i s -t i c a l l y i n f e r i o r to the b a r l e y soybean meal d i e t by u s i n g Tukey's t e s t , i t appeared t h a t the c o n t r o l soybean-barley d i e t s t i l l gave s l i g h t l y b e t t e r performance. I t i s p o s s i b l e that response to the a d d i t i o n of l y s i n e and threonine to the b a r l e y was l i m i t e d by other f a c t o r s such as the l e v e l of t o t a l n i t r o g e n i n the d i e t , a lower b i o l o g i c a l a v a i l a b i l i t y of l y s i n e and - 62 -threonine or other e s s e n t i a l amino a c i d s , or that the amino a c i d p a t t e r n i n the a l l barley-amino a c i d d i e t was not as w e l l balanced as i n the soybean-barley d i e t although the requirements of a l l e s s e n t i a l amino acids appeared to have been met. The soybean-barley d i e t was s i g n i f i c a n t l y b e t t e r than the d i e t c o n t a i n i n g h i g h e r threonine (0.15%) a d d i t i o n s w i t h or without methionine i n the present experiment. This i s p o s s i b l y as a r e s u l t of a greater imbalance. I t i s p o s s i b l e that b e t t e r growth may have been obtained w i t h an inc r e a s e d l y s i n e l e v e l i n the higher threonine d i e t . The r e s u l t s of Harper (1964) obtained w i t h r a t s i n d i c a t e d that an excess of a p a r t i c u l a r amino a c i d or of an imbalanced p r o t e i n r e q u i r e d f u r t h e r a d d i t i o n of the p r i m a r y - l i m i t i n g amino a c i d . Bayley and Summers (1968) reported that the supplementation of p r a c t i c a l corn-soybean d i e t (14% crude p r o t e i n ) w i t h 0.1% l y s i n e or 0.05% methionine d i d not give a b e n e f i c i a l e f f e c t to growth and feed e f f i c i e n c y . However, there was a p o s i t i v e e f f e c t i f the same l e v e l of both amino acids were supplemented together p a r t i c u l a r l y on the lower p r o t e i n d i e t (12% crude p r o t e i n ) . Although the p i g does not r e q u i r e crude p r o t e i n per se. the crude p r o t e i n l e v e l appears to a f f e c t performance to some extent. Rerat and Henry (1963) i n d i c a t e d that the performance of pi g s r e c e i v i n g a low l y s i n e d i e t could be improved by r a i s i n g the n i t r o g e n l e v e l or by adding l y s i n e to the d i e t . The. high n i t r o g e n l e v e l of the soybean-barley may have c o n t r i b u t e d to the good performance on t h i s d i e t . B l a i r et a l . (1969a) reported that l i v e w e i g h t - 6 3 -gain was not improved s i g n i f i c a n t l y by i n c r e a s i n g the p r o t e i n l e v e l above 16, 14 and 12% f o r the 23-45, 45-68 and 68-90 Kg body weight catego-r i e s , r e s p e c t i v e l y . However, feed conversion e f f i c i e n c y was improved s i g n i f i c a n t l y over the 23-45 Kg weight range by i n c r e a s i n g the l e v e l to 18%. Liveweight gain was not improved s i g n i f i c a n t l y by i n c r e a s i n g the l y s i n e l e v e l above 1.04, 0.74 and 0.70 f o r the 23-45, 45-68 and 68-90 Kg ranges, r e s p e c t i v e l y . Feed e f f i c i e n c y was, however, improved s i g n i f i c a n t l y d u r ing the 23-45 Kg stage by i n c r e a s i n g l y s i n e l e v e l s to 1.22%. I t appears that the p r o t e i n l e v e l (about 11%) and l y s i n e l e v e l i n the barley-amino a c i d d i e t s of the present experiment may have been the f a c t o r s l i m i t i n g the growth and feed e f f i c i e n c y performances of p i g s d u r i n g the e a r l y stage of growth. This was revealed by the growth r a t e d i f f e r e n c e s o c c u r i n g only from 20 to 45 Kg body weight between 0.10% threonine or over d i e t s and the c o n t r o l d i e t . I t i s p o s s i b l e that the growth r a t e can be improved by r a i s i n g the n i t r o g e n l e v e l of the d i e t s through amino a c i d a d d i t i o n during that p e r i o d . The l y s i n e l e v e l apparently was s u f f i c i e n t but not the n i t r o g e n l e v e l when compared to the r e p o r t s of B l a i r e_t _ a l . (1969a) . In the present experiment, the growth r a t e d i f f e r e n c e occured only from 20 to 45 Kg body weight between the c o n t r o l d i e t and the 0.10% threonine supplemented d i e t . Chung and Beames (1974) showed s i g n i f i c a n t growth improvement w i t h g i l t s but not w i t h barrows by i n c r e a s i n g the l y s i n e l e v e l from 0.75% to 0.9% of the a l l b a r l e y d i e t c o n t a i n i n g about 10% crude p r o t e i n . However, i t i s d i f f i c u l t to e x p l a i n the s i g n i f i c a n t Increase i n d a i l y weight gain obtained by adding 0.05% threonine to the b a r l e y d i e t c o n t a i n i n g 0.75% - 64 -l y s i n e when fed to g i l t s ; a s i g n i f i c a n t i n c r e a s e could a l s o be obtained by f u r t h e r i n c r e a s i n g the l y s i n e l e v e l to 0.90%. The response to other l i m i t i n g amino a c i d s obtained when the f i r s t l i m i t i n g amino a c i d i s not at an optimal l e v e l i s contrary to the general theory of a stepwise response, as i l l u s t r a t e d by the contour maps of Rosenberg et a l . (1959); although where s e v e r a l amino acids are e q u a l l y l i m i t i n g , a response i s obtained only when a l l are s u p p l i e d together ( F i s h e r , 1965). I t i s t h e r e f o r e apparent that 0.75% l y s i n e i n the d i e t i s adequate f o r the growing and f i n i s h i n g of the p i g . The i n c r e a s e d growth response and feed e f f i c i e n c y obtained by Chung and Beames (1974) i n d i c a t e d that threonine was a l i m i t i n g amino a c i d i n these a l l b a r l e y d i e t s . The l i m i t i n g f a c t o r of threonine may have been one of the main f a c t o r s which r e s u l t e d i n a f a i l u r e to show the response of i n c r e a s i n g l y s i n e from 0.75% to 0.90% of the d i e t . The evidence observed by M o r r i s o n e t a l . (1961) and Braude et a l . (1972) i n d i c a t e d t h a t l y s i n e supplementation r e s u l t s i n a p r o g r e s s i v e decrease i n plasma threonine c o n c e n t r a t i o n . The high l e v e l of l y s i n e could be more imbalanced i n the e a r l y stage than i n l a t e r stages of growth. I t i s t h e r e f o r e suggested that perhap l e v e l s higher than 0.75% l y s i n e w i t h p r o g r e s s i v e l y i n c r e a -s i n g threonine l e v e l s commencing at 0.47% i n a l l barley-amino a c i d d i e t s should be f u r t h e r i n v e s t i g a t e d to see i f a d d i t i o n a l improvements i n p i g performance could be achieved. The evidence based on p i g growth and feed e f f i c i e n c y i n the p r e -sent experiment suggested that threonine i s adequate f o r optimal growth a t 0.47% of the l o w - p r o t e i n barley-amino a c i d d i e t c o n t a i n i n g 0.75% l y s i n e f o r the g r o w i n g - f i n i s h i n g p i g . - 65 -Dressing percentage d i d not d i f f e r s i g n i f i c a n t l y between t r e a t -ments and sex. Jurger (1967), Meade et al_. (1969) and P i e r c e and Bowland (1972) obtained no e f f e c t on d r e s s i n g percentage from v a r y i n g d i e t a r y p r o t e i n l e v e l s , thus supporting the r e s u l t s of the present experiment showing the h i g h p r o t e i n c o n t r o l d i e t (about 17.6% crude p r o t e i n ) to produce a d r e s s i n g percentage s i m i l a r to that obtained w i t h the low p r o t e i n treatment (about 11% crude p r o t e i n ) . Many re p o r t s i n the l i t e r a t u r e have shown l y s i n e supplementation of low p r o t e i n c e r e a l to markedly improve carcass l e a n content (Brooks e t a l . , 1959; Bowland, 1962; C a h i l l y et a l . , 1963; N i e l s e n e t a l . , 1963; Braude et a l . , 1972). However, very l i t t l e i n f o r m a t i o n has been presented on the e f f e c t of threonine a d d i t i o n to a l o w - p r o t e i n c e r e a l w i t h adequate l y s i n e supplementation on carcass lean content. The data i n the present study i n d i c a t e d a gradual decrease (not s i g n i f i c a n t at P < 0.05)" i n " b a c k f a t thickness corresponding to increased l e v e l of threonine supplement. However, the thickness of b a c k f a t 40 mm from m i d - l i n e was s i g n i f i c a n t l y CP < 0.05) reduced- only at 0.15% l e v e l of threonine supplementation. This suggests that to achieve a s i g n i f i c a n t improvement i n carcass q u a l i t y , higher l e v e l of threonine supplementation may be r e q u i r e d . Chung and Beames (1974) were a l s o unable to show any d i f f e r e n c e from the a d d i t i o n of 0.05% threonine to a b a r l e y - l y s i n e d i e t (0.75% l y s i n e ) . Higher threonine a d d i t i o n s i n Treatments 5 and 6 gave ba c k f a t f i g u r e s not s i g n i f i c a n t l y d i f f e r e n t from values obtained on the barley-soybean c o n t r o l d i e t . S i m i l a r l y , Treatments 4 and 5 - 66 -were s t a t i s t i c a l l y as good as the c o n t r o l d i e t i n respect to b a c k f a t 40 mm from the m i d - l i n e . In gene r a l , the r e s u l t s i n d i c a t e that the a d d i t i o n of threonine to the barley-amino a c i d (0.75% l y s i n e ) d i e t tends to decrease the b a c k f a t and improve carcass q u a l i t y . V a r i a t i o n i n feed i n t a k e can also i n f l u e n c e carcass f a t n e s s . However, as there were not s i g n i f i c a n t d i f f e r e n c e s between treatments i n mean d a i l y feed i n t a k e t h i s would not have been a c o n t r i b u t o r y f a c t o r . Eye muscle index and eye muscle area were not a f f e c t e d s i g n i f i -c a n t l y over the 0.05% to 0.15% range of threonine a d d i t i o n although the l e v e l of threonine supplementation was the only l e v e l on which the eye muscle index d i d not d i f f e r s i g n i f i c a n t l y from that obtained cn the b a r l e y -soybean meal c o n t r o l d i e t . The o v e r a l l r e s u l t s of the present experiment showed that the lowest back f a t t h i c k n e s s , l a r g e s t eye muscle area and eye muscle index was obtained from the barley-soybean c o n t r o l d i e t , although some of the threo-nine supplemented d i e t s gave values which were not s t a t i s t i c a l l y lower than the c o n t r o l . Many r e p o r t s have i n d i c a t e d that lowering the crude p r o t e i n l e v e l below about 16 - 17%, at l e a s t i n the e a r l y stages of the growing p e r i o d , w i l l produce adverse e f f e c t s on carcass q u a l i t y (Ashton e t a l . , 1955; Bowland et a l . , 1959 and A.R.C., 1967; Tjong-A-Hung et a l . , 1972). The i n f e r i o r i t y of carcass q u a l i t y on the present barley-amino a c i d - 67 -d i e t s (about 11% crude p r o t e i n ) compared w i t h the carcasses from pigs on the barley-soybean c o n t r o l (17.5 crude p r o t e i n ) d i e t may be due to inadequate n i t r o g e n contents i n the former d i e t s f o r optimal body p r o t e i n s y n t h e s i s . C o n f l i c t i n g r e p o r t s by Clawson.(1967), Meade (1966b) and P i e r c e and Bowland (1972) i n d i c a t e no i n f l u e n c e of d i e t a r y p r o t e i n on l o i n area and back f a t .thickness. However, i t appeared that the p r o t e i n l e v e l i n d i e t s used by above authors were over 14% crude p r o t e i n and t h e r e f o r e not designed to c r i t i c a l l y t e s t the e f f e c t of p r o t e i n l e v e l s . Higher mean d a i l y gains were observed i n the barrow than the g i l t which was i n agreement w i t h the r e s u l t s of Tjong-A-Hung e_t a l . (1972) and Chung and Beames (1974). However i t i s i n disagreement w i t h the f i n d i n g s o f Newell and Bowland (1972) and P i e r c e and Bowland (1972). G i l t s were s u p e r i o r to barrows i n a l l carcass measurements. This r e s u l t i s supported by many previous i n v e s t i g a t o r s (e.g. Wong e t a l . , 1968; Young et a l . , 1968; Newell and Bowland, 1972 ; Tjong-A-Hung et a l . , 1972; Lodge e_t a l . , 1972b; Chung and Beames, 1974). There was no i n t e r a c t i o n between d i e t and sex i n the present experiment. Chung and Beames (1974) i n a s i m i l a r experiment observed i n t e r a c t i o n between d i e t and sex. I t appears that d i e t and sex i n t e r a c t i o n has been i n c o n s i s t e n t l y found i n s e v e r a l previous i n v e s t i g a t i o n s . - 68 -D. CONCLUSION I t may be concluded that threonine was the second l i m i t i n g amino a c i d i n b a r l e y as used i n t h i s experiment f o r the f i n i s h i n g p i g and that b a r l e y can be used as the s o l e p r o t e i n source f o r f i n i s h i n g p i g s p r o v i d i n g t h a t the proper l e v e l s of l y s i n e and threonine are added. With 0.75% l y s i n e i n the a l l b a r l e y d i e t , a 0.10% L-threonine a d d i t i o n to the d i e t (0.47% t o t a l threonine) produced the best mean d a i l y gain and feed conversion e f f i c i e n c y . F u r t h e r a d d i t i o n of 0.05% L-threonine and 0.05% threonine plus 0.10% DL-methionine d i d not give any a d d i t i o n a l improvement i n p i g performance. The b a r l e y - l y s i n e d i e t c o n t a i n i n g 0.75% t o t a l l y s i n e gave the poorest performance i n both r a t e of growth and feed conversion e f f i c i e n c y . The 0.10%, 0.15% threonine and 0.15% threonine p l u s 0.10% methionine improved the mean d a i l y gain by 22.1%, 13.0% and 14.7% r e s p e c t i v e l y and feed conversion e f f i c i e n c y by 15.5%, 12.9% and 12.8% r e s p e c t i v e l y when they were added to a b a r l e y - l y s i n e d i e t c o n t a i n i n g 0.75% t o t a l l y s i n e . D i f f e r e n c e s i n the r a t e of growth on the b a r l e y -soybean d i e t (17.6% crude p r o t e i n ) and b a r l e y amino a c i d d i e t s c o n t a i n i n g 0.10% or more added threonine (about 11% crude p r o t e i n ) occured only at the 20 to 45 Kg body weight p e r i o d . The e s s e n t i a l amino a c i d content of the h i g h e r b a r l e y - l y s i n e d i e t s c o n t a i n i n g 0.10% or more threonine are s u f f i c i e n t to meet the requirements of the growing p i g according to the N.A.S,-N,R,C, (1968) feeding standards. However the t o t a l n i t r o g e n content of the amino a c i d supplemented d i e t s may have been i n s u f f i c i e n t during the - 6 9 -growing (prior to 45 Kg body weight) period of growth. It i s possible that growth rate could have been improved by increasing the nitrogen level of diet during this period. The barley-soybean meal diet gave the best carcass quality. The addition of graded levels of L-threonine to the barley-lysine diet improved carcass quality. Gilts grew more slowly and produced leaner carcasses than barrows. - 70 -IV. PIG EXPERIMENT I I A. EXPERIMENTAL PROCEDURE a. General In feeding experiments, growth, performance as assessed by growth r a t e and feed e f f i c i e n c y gives no i n d i c a t i o n s of the v a r i a t i o n i n n i t r o g e n r e t e n t i o n i n the t i s s u e s , p a r t i c u l a r l y when the animals are fed ad l i b i t u m . Although carcass measurements are reasonably w e l l c o r r e l a t e d w i t h l e a n content and t o t a l carcass n i t r o g e n these give no i n d i c a t i o n of the causes of v a r i a t i o n s between treatments i n n i t r o g e n storage. I t i s g e n e r a l l y accepted t h a t n i t r o g e n balance techniques are much more v a l u a b l e i n assess-i n g p r o t e i n q u a l i t y . Therefore, the n i t r o g e n metabolism experiment was designed to determine the causes of v a r i a t i o n s i n n i t r o g e n r e t e n t i o n i n t i s s u e s of pigs r e c e i v i n g the d i e t s used i n Experiment I . b. Animals A t o t a l of 18 male c a s t r a t e Yorkshire-Landrace crossbred pigs weighing between 39 and 51 Kg were assigned to three groups. Each group c o n s i s t e d of s i x animals which were placed i n s i x metabolism c r a t e s f o r a three-week p e r i o d . W i t h i n groups s i x experimental d i e t s were assigned randomly. The f i r s t week was an a c c l i m a t i z a t i o n p e r i o d . The second and t h i r d weeks were d i v i d e d i n t o two one-week c o l l e c t i o n p e r i o d s . - 71 -c. D i e t s D i e t s were the same as those used i n P i g Experiment I . Each d i e t was prepared by passing the b a r l e y through the f i n e screen (7 mm diameter) of a hammer m i l l . Feed samples were taken f o r moisture and n i t r o g e n determination at the s t a r t of the t r i a l . d. Management ( i ) Housing The metabolism room was maintained a t a temperature of 21° C w i t h t h e r m o s t a t i c a l l y - c o n t r o l l e d space h e a t e r s . The metabolism crates were a m o d i f i c a t i o n of the S h i n f i e l d design (Frape e_t a l . , 1968) . The f i b r e g l a s s u r i n e t r a y was i n c l i n e d from f r o n t to r e a r at an angle of approximately 30° from the h o r i z o n t a l . A f i n e w i r e screen was pla c e d on the u r i n e t r a y i n order to r e t a i n the s m a l l amount of feces f a l l i n g beyond the feces t r a y which was l o c a t e d at the r e a r of the f l o o r as shown i n F i g u r e 1 . Each p i g had attached a canvas b e l t to which was attached a pi e c e of rubber t u b i n g . The canvas b e l t was stuck w i t h cement around the abdomen of the p i g w i t h the rubber tube hanging j u s t i n f r o n t of the p e n i s . The purpose of t h i s design was to l e a d the u r i n e down to the u r i n e t r a y and to minimize p o s s i b l e u r i n e l o s s from s p r a y i n g during u r i n a t i o n ( F i g u r e 2 ) . ( i i ) Feeding method P i g s were fed twice d a i l y ad l i b i t u m at 9:00 a.m. and 1:00 p.m. - 72 -Figure 2. P i g i n the modified a d j u s t a b l e cage, h e l d w i t h canvas b e l t and rubber tubing to minimize u r i n e l o s s - 73 -f o r 40 minutes per feed. The d i e t was mixed w i t h equal amounts of water by weight j u s t b e f o r e f e e d i n g . Water was provided ad l i b i t u m f o r a 20 minute p e r i o d a f t e r each feeding. P r i o r to the p r o v i s i o n of water, feed residues were removed, placed i n p l a s t i c bags and s t o r e d at 3° C. At the end of each one-week c o l l e c t i o n p e r i o d , the residues f o r each p i g were pooled and d r i e d at 60° C to constant weight (approximately 72 h o u r s ) . e. Feces and Urine C o l l e c t i o n A l l feces were c o l l e c t e d d a i l y i n p l a s t i c bags and s t o r e d at 3° C u n t i l completion of the seven-day t r i a l . The t o t a l f e c a l output of each p i g was then pooled and weighed and an a c c u r a t e l y weighed a l i q u o t of appro-xi m a t e l y 500 g was d r i e d at 60° C f o r 72 hours (Saben and Bowland, 1971) . Urine was c o l l e c t e d i n a p l a s t i c j a r to which was .added 50 ml of... d i l u t e d s u l p h u r i c a c i d (50% V/V) each day to avoid l o s s of n i t r o g e n . Urine volume was measured each day and a 10% a l i q u o t c o l l e c t e d i n t o a p l a s t i c j a r and s t o r e d at 3° C f o r p o o l i n g at the end of each one-week c o l l e c t i o n p e r i o d . The sample of pooled u r i n e was used subsequently f o r n i t r o g e n d e t e r m i n a t i o n . f. S t a t i s t i c a l A n a l y s i s of Data The analyses were done as f o r a one-way c l a s s i f i c a t i o n a n a l y s i s of v a r i a n c e ( d i e t as treatment) w i t h or without a c o v a r i a b l e . The data f o r n i t r o g e n balance, apparent n i t r o g e n d i g e s t i b i l i t y , n i t r o g e n absorbed as a percentage of n i t r o g e n i n t a k e , n i t r o g e n r e t a i n e d as a percentage of n i t r o g e n absorbed and dry matter d i g e s t i b i l i t y were subjected to an - 74 -a n a l y s i s of vari a n c e (UBC-MFAV, Halm and Le, 1974) and the means from comparison showing s i g n i f i c a n t F values were t e s t e d according to Tukey's Test (1953). Nitrogen balance data were a l s o analyzed by covariance a n a l y s i s UBC-MFAV (Halm and Le, 1974) w i t h feed i n t a k e and n i t r o g e n i n t a k e as c o v a r i a b l e s . - 75 -B. RESULTS . The summarized r e s u l t s of the p i g metabolism s t u d i e s are shown i n Table 7 . With equal l e v e l s of l y s i n e (0.75%) i n the d i e t , the supple-mentation of L-threonine to the barley-amino a c i d d i e t improved the n i t r o g e n balance s i g n i f i c a n t l y . The n i t r o g e n balance improved p r o g r e s s i v e l y but not s i g n i f i c a n t l y from 0.05 to 0.15% threonine a d d i t i o n . No f u r t h e r response was obtained from methionine supplementation of the h i g h e s t t h r e o -nine d i e t . The n i t r o g e n balance f i g u r e s of the barley-amino a c i d d i e t s were s i g n i f i c a n t l y i n f e r i o r to those of the barley-soybean c o n t r o l d i e t except f o r the 0.15% threonine supplemented d i e t which d i d not d i f f e r s i g n i f i -c a n t l y from the c o n t r o l . When n i t r o g e n balance was adjusted f o r feed i n t a k e , comparative r e s u l t s were s i m i l a r to those without feed i n t a k e adjusted except that the non-threonine supplemented d i e t became i n s i g n i f i -c a n t l y d i f f e r e n t from Treatments 3, 4 and 6. However, when n i t r o g e n balance was adjusted f o r n i t r o g e n i n t a k e the 0.15% threonine added d i e t became the b e s t although i t was i n s i g n i f i c a n t l y d i f f e r e n t from a l l other threonine supplemented and c o n t r o l d i e t . However, i t was s i g n i f i c a n t l y b e t t e r than the non-threonine supplemented d i e t . Moreover, the d i e t without threonine added d i d not d i f f e r s i g n i f i c a n t l y from Treatments 1, 3, 4 and 6. N i t r o g e n absorbed as a percentage of n i t r o g e n i n t a k e showed no s i g n i f i c a n t d i f f e r e n c e s between amino a c i d supplemented d i e t s but the l y s i n e only and l y s i n e p l u s 0.05% threonine supplemented d i e t s gave s i g n i f i c a n t l y lower values than that of c o n t r o l . Hie c o n t r o l d i e t gave a s i g n i f i c a n t l y lower n i t r o g e n r e t e n t i o n as a percentage of n i t r o g e n i n t a k e than a l l b a r l e y -- 76 -amino acid diets. The diets with added threonine did not diff e r s i g n i f i -cantly from each other but tended to improve from the low to the higher levels of threonine addition. The picture for nitrogen retained as a per-centage of nitrogen absorbed was similar to that obtained when nitrogen retention was expressed as a percentage of nitrogen intake. Dry matter d i g e s t i b i l i t y showed no significant differences between diets. However, nitrogen intake and nitrogen d i g e s t i b i l i t y were s i g n i f i -cantly higher i n the barley-soybean control treatment but there were no significant differences among the barley-amino acid supplemented diets. In summary, the results showed that the barley diet with 0.444% lysine-HCl addition only was s t i l l limiting i n threonine values for nitro-gen balance. Nitrogen absorbed as percentage of nitrogen intake, nitrogen retained as percentage of nitrogen intake and nitrogen retained as percen-tage of nitrogen absorbed were generally improved by threonine supplementa-tion. - 77 -Table 7 . Summary of the e f f e c t s of supplementation of b a r l e y w i t h amino acid s on apparent dry matter d i g e s t i b i l i t y , n i t r o g e n balance, apparent n i t r o g e n d i g e s t i b i l i t y and on va r i o u s n i t r o g e n r e t e n t i o n i n d i c e s 1 2 3 4 5 6 Result Treatment S o y _ b a r l e y 0.75%lys B a s a l + Ba s a l + B a s a l + B a s a l + of s i g n i -( c o n t r o l ) (Basal) 0.05%thr 0.10%thr 0.15%thr 0.15%thr+ f i c a n t 0.10%met. t e s t * * T o t a l feed i n t a k e ( g ) T o t a l N intake(g) D.M. D i g e s t i -b i l i t y ( % ) N digested N balance(g) N balance^ adjusted by feed i n t a k e ( g ) N balance § adjusted by N intake(g) N absorbed N i n t a k e W o ) N r e t a i n e d N i n t a k e N r e t a i n e d N absorbed 13424 11833 12701 12538 12567 12244 ±595 135462 (%) 337.60 78.43 289.80 133.27 128.53 104.14 76.78 35.44 46.24 208.36 230.72 227.88 227.66 223.49 ±11.78 134562 77.76 143.66 87.78 69.11 42.07 76.26 152.40 103.65 77.95 159.37 107.63 77.61 158.09 114.48 77.14 ±1.11 142563 154.73 ±7.39 145632 102.12 ±6.23 154362 91.67 102.84 107.70 114.40 103.78 ±5.42 154632 97.07 107.83 112.50 119.40 107.98 ±5.72 546312 66.15 44.84 69.91 47.32 69.54 50.52 69.77 ±2.41 146523 45.99 ±2.12 546321 61.18 67.91 67.70 72.26 66.16 ±3.37 534621 ** Standard e r r o r of treatment means. Treatment numbers not underscored by the same l i n e are s i g n i f i c a n t l y d i f f e r e n t at the 5% l e v e l of p r o b a b i l i t y (Tukey, 1953). Co v a r i a b l e feed i n t a k e i s s i g n i f i c a n t (P < 0.05). Co v a r i a b l e N i n t a k e i s h i g h l y s i g n i f i c a n t (P < 0.01). - 78 -C. DISCUSSION The r e s u l t s of the experiment showed that the a d d i t i o n of threonine to the b a r l e y - l y s i n e d i e t p r o g r e s s i v e l y improved the d i e t a r y p r o t e i n q u a l i t y . There were s l i g h t v a r i a t i o n s f o r n i t r o g e n balance, n i t r o g e n absorbed/nitrogen i n t a k e , n i t r o g e n r e t a i n e d / n i t r o g e n i n t a k e and n i t r o g e n r e t a i n e d / n i t r o g e n absorbed f o r a l l the threonine supplemented d i e t s . The 0.15% threonine supplemented d i e t gave the best r e s u l t s but not s i g n i f i c a n t l y so except f o r n i t r o g e n absorbed/nitrogen i n t a k e . The 0.15% threonine supplemented d i e t a l s o appeared to be as good as (p < 0.05) the barley-soybean c o n t r o l d i e t i n terms of n i t r o g e n balance. An improvement i n n i t r o g e n balance of animals by the a d d i t i o n of L - l y s i n e - H C l to g r a i n has been reported by many i n v e s t i g a t o r s (e.g. E r i c s o n e_t a l . , 1962; S o l d e v i l a and Meade, 1964; Bowland and Grimson, 1969; P i c k and Meade,1971; Braude et al.,1972). Braude et a l . (1972) reported that n i t r o g e n r e t a i n e d as percentage of i n t a k e was s i g n i f i c a n t l y h i g h e r i n the l y s i n e supplemented h i g h b a r l e y d i e t (0.57% l y s i n e ) than n o n - l y s i n e supplemented d i e t (0.45% l y s i n e ) f o r the growing p i g . Chung and Beames (1974) showed no response to adding f u r t h e r l y s i n e to a b a r l e y p l u s l y s i n e d i e t c o n t a i n i n g 0.75% l y s i n e , i n d i c a t i n g some other amino a c i d to be l i m i t i n g at t h i s l e v e l . P i c k and Meade (1971) reported that r a t s fed d i e t s c o n t a i n i n g 0.73% l y s i n e has s i g n i f i c a n t l y (p < 0.01) g r e a t e r gain/feed r a t i o and r e t a i n e d more n i t r o g e n than those fed 0.54% l y s i n e d i e t s . However, i n c r e a s i n g the - 79 -d i e t a r y l y s i n e to 0.92% d i d not e f f e c t f u r t h e r improvements i n any of the response c r i t e r i a . Improvement of n i t r o g e n balance by s y n t h e t i c threonine supplemen-t a t i o n to a low p r o t e i n d i e t has been s t u d i e d by some researchers (Evans, 1963; M l i l l e r and Rozman, 1968; B r e s s a n i , 1971; Chung and Beames, 1974) . Evans (1963) reported that maximum n i t r o g e n r e t e n t i o n was obtained when the d i e t was s u p p l i e d w i t h c r y s t a l l i n e amino acid s w i t h the threo-nine content up to 0.43% of the d i e t . F u r t h e r a d d i t i o n of threonine up to 0.58% of the d i e t d i d not improve n i t r o g e n r e t e n t i o n , r a t e of growth or feed conversion e f f i c i e n c y under the metabolism cage c o n d i t i o n s . Both d i e t s contained approximately 0.70% l y s i n e and 0.64% methionine. The present experiment showed that w i t h 0.75% t o t a l l y s i n e i n the b a s a l d i e t , n i t r o g e n r e t e n t i o n improved p r o g r e s s i v e l y up to 0.10% threonine a d d i t i o n (0.47% of the d i e t ) . The h i g h e r l e v e l a d d i t i o n of 0.15% threonine (0.52% of the d i e t ) d i d not show a s i g n i f i c a n t improvement w i t h respect to n i t r o g e n r e t e n t i o n . This c l o s e l y supported the r e s u l t s of Evan (1963). B r e s s a n i (1971) reported that l y s i n e and threonine supplements to r o l l e d oats improved n i t r o g e n balance i n c h i l d r e n above that obtained w i t h l y s i n e alone. No f u r t h e r n i t r o g e n balance improvement was obtained by methionine a d d i t i o n . Chung and Beames (1974) obtained the s i m i l a r r e s u l t s w i t h b a r l e y d i e t s f o r growing p i g s . They demonstrated that n i t r o g e n r e t e n -t i o n was improved by threonine supplementation of a b a r l e y - l y s i n e d i e t . The marked response f o r improvement i n n i t r o g e n r e t e n t i o n by threonine a d d i t i o n confirmed Chung and Beames's r e s u l t . - 80 -M i i l l e r and Rozman (1968) showed that e i t h e r l y s i n e , threonine or tryptophan, and methionine supplementation of b a r l e y f o r g r o w i n g - f i n i s h i n g pigs improved the n i t r o g e n r e t a i n e d / n i t r o g e n d i g e s t e d r a t i o as w e l l as d a i l y gain and feed e f f i c i e n c y . Chung and Beames (1974) a l s o i n d i c a t e d that l y s i n e , threonine and methionine supplementation of b a r l e y gave an improvement i n n i t r o g e n balance and n i t r o g e n r e t a i n e d / n i t r o g e n i n t a k e . The present experiment gave r e s u l t s which were i n agreement w i t h those of M i i l l e r and Rozman (1968) and Chung and Beames (1974) . However, the a d d i -t i o n of methionine to the b a r l e y - l y s i n e - t h r e o n i n e d i e t d i d not improve the n i t r o g e n r e t e n t i o n , n i t r o g e n r e t a i n e d / n i t r o g e n i n t a k e and n i t r o g e n r e t a i n e d / n i t r o g e n absorbed but r a t h e r s l i g h t l y depressed the maximum values f o r the above c r i t e r i a obtained f o r l y s i n e and threonine a d d i t i o n s only. This was i n agreement w i t h the r e s u l t s reported by some other i n v e s t i g a t o r s ( S o l d e v i l a and Meade, 1964; Bowland and Grimson, 1969). The reason f o r methionine a d d i t i o n depressing n i t r o g e n balance i s probably that the methionine content i n d i e t may have been adequate f o r the growing-pig and t h a t the a d d i t i o n of methionine may have created a s l i g h t imbalance. Bowland and Grimson (1969) i n d i c a t e d that a b e t t e r n i t r o g e n r e t a i n e d / n i t r o g e n absorbed value was obtained w i t h a l y s i n e - m e t h i o n i n e supplemented low p r o t e i n d i e t (14% crude p r o t e i n ) than w i t h a h i g h e r pro-t e i n or an amino a c i d unsupplemented low p r o t e i n d i e t f o r e a r l y weaned p i g s . D a i l y n i t r o g e n r e t e n t i o n was i n c r e a s e d when only L - l y s i n e , i n s t e a d of both L - l y s i n e and DL—methionine, was added to d i e t s c o n t a i n i n g three percent urea. The o b s e r v a t i o n suggested that the methionine was not the second l i m i t i n g amino a c i d a f t e r l y s i n e . This was a l s o i n agreement w i t h - 81 -the present experiment which showed no improvement i n p i g performance w i t h methionine a d d i t i o n to the b a r l e y amino a c i d d i e t . On the other hand, the great response to threonine i n the a l l b a r l e y d i e t s t r o n g l y suggested that threonine i s the second l i m i t i n g amino a c i d a f t e r l y s i n e f o r the growing-f i n i s h i n g p i g . Moreover, Chung and Beames (1974) demonstrated that i s o l e u -c ine added to a b a r l e y + lysine. + threonine + methionine d i e t d i d not give f u r t h e r improvement i n n i t r o g e n - r e t e n t i o n i n d i c a t i n g that i s o l e u c i n e was not l i m i t i n g at t h i s l e v e l s of i n c l u s i o n of the other amino acids i n these d i e t s f o r growing p i g s . The dry matter d i g e s t i b i l i t y of barley-soybean and barley-amino a c i d d i e t s were not s i g n i f i c a n t l y d i f f e r e n t . The barley-soybean c o n t r o l d i e t gave the lowest f i g u r e f o r n i t r o g e n r e t a i n e d / n i t r o g e n i n t a k e and n i t r o g e n r e t a i n e d / n i t r o g e n absorbed, which would be understandable as t h i s d i e t contained a h i g h l e v e l of n o n - e s s e n t i a l amino acid s and thus a h i g h e r t o t a l n i t r o g e n l e v e l than that r e q u i r e d f o r maximum performance. The r e s u l t s of Chung and Beames (1974) w i t h p i g s and Bowland and Grimson (1969) w i t h r a t s support t h i s f i n d i n g . According to Metta and M i t c h e l l (1956) and Rippon (1959), the b i o l o g i c a l value of d i f f e r e n t p r o t e i n s decreased l i n e a r l y as the p r o t e i n c o n c e n t r a t i o n or i n t a k e i s i n c r e a s e d , (e.g. Becker e t a l . , 1963, A.R.C., 1967) . I t i s suggested that the lower apparent b i o l o g i c a l value of the soybean-barley c o n t r o l d i e t was due to h i g h e r p r o t e i n (17.6% of the d i e t ) i n t a k e . This i s above the need of the growing p i g . The p r o t e i n r a t i o of barley-soybean and barley-amino a c i d d i e t was about 1.5 to 1 i n the - 82 -present experiment. I t was shown i n t h i s experiment that 0.15% threonine added to the b a r l e y - l y s i n e d i e t gave a n i t r o g e n r e t e n t i o n not s i g n i f i c a n t l y l e s s than that obtained w i t h the barley-soybean c o n t r o l but s i g n i f i c a n t l y higher i n n i t r o g e n r e t a i n e d / n i t r o g e n i n t a k e and n i t r o g e n r e t a i n e d / n i t r o g e n absorbed. The data suggested that a 0.15% threonine supplemented b a r l e y -l y s i n e d i e t may be able to r e p l a c e p r o t e i n c o n c e n t r a t i o n i n order to support the equal n i t r o g e n r e t e n t i o n . I n n i t r o g e n metabolism experiments, there i s the p o s s i b i l i t y of n i t r o g e n l o s s i n feces and u r i n e i f one does not take adequate p r e c a u t i o n s . M a r t i n (1966) i n d i c a t e d that NH^ l o s s from feces was n e g l i g i b l e and l o s s e s from u r i n e depended on the. temperature and pH at which i t was c o l l e c t e d . The average l o s s of n i t r o g e n on c o l l e c t i o n of u r i n e at a pH value below 2.0 was . 1.33% when the ambient temperature was between 25 and 28° C and 0.97% at the lower temperature of 15 to 18° C. The l o s s e s of n i t r o g e n from feces and u r i n e should have been n e g l i g i b l e i n t h i s experiment as the u r i n e was c o l l e c t e d i n strong s u l p h u r i c a c i d w i t h a pH below 2 "and s t o r e d at 3° C u n t i l the end of the one-week c o l l e c t i o n p e r i o d . The samples were analysed immediately a f t e r the end of each one-week c o l l e c t i o n p e r i o d . - 83 -D. CONCLUSION I t may be concluded, from the metabolism experiment that threonine i s the second l i m i t i n g amino a c i d i n b a r l e y f o r growing p i g s , thereby supporting the r e s u l t s of the growth experiment. The a d d i t i o n of graded l e v e l s of L-threonine of the b a r l e y - l y s i n e d i e t p r o g r e s s i v e l y improved the n i t r o g e n balance, n i t r o g e n balance adjusted f o r feed i n t a k e and n i t r o g e n i n t a k e , n i t r o g e n r e t a i n e d / n i t r o g e n i n t a k e and n i t r o g e n r e t a i n e d / n i t r o g e n absorbed. Although not a l l r e s u l t s from the graded .L-threonine a d d i t i o n s d i f f e r e d s i g n i f i c a n t l y from each other, the h i g h e s t threonine (0.15%) supplemented l e v e l was s i g n i f i c a n t l y b e t t e r than the b a s a l b a r l e y - l y s i n e d i e t . At t h i s l e v e l of threonine a d d i t i o n (0.52% of the d i e t ) , the n i t r o g e n balance and n i t r o g e n balance adjusted f o r feed i n t a k e were s t a t i s t i c a l l y n o n - s i g n i f i c a n t l y d i f f e r e n t from the b a r l e y -soybean c o n t r o l d i e t . A l l threonine supplemented d i e t s were s i g n i f i c a n t l y b e t t e r than barley-soybean c o n t r o l d i e t i n terms of n i t r o g e n r e t a i n e d / n i t r o g e n i n t a k e and n i t r o g e n r e t a i n e d / n i t r o g e n absorbed. This phenomenon occurred because the barley-soybean d i e t contained 17.5% crude p r o t e i n , and i n excess of the requirements of growing p i g s . The excess n i t r o g e n i n t a k e was e x c r e t e d i n the u r i n e . Therefore i t s b i o l o g i c a l value was reduced. The methionine supplement d i d not f u r t h e r improve n i t r o g e n r e t e n -t i o n i n d i c a t i n g that methionine was mot l i m i t i n g i n the d i e t . There was no s i g n i f i c a n t d i f f e r e n c e i n dry matter d i g e s t i b i l i t y between d i e t s . - 84 -V. RAT EXPERIMENT I . A. EXPERIMENTAL PROCEDURE a. General The r e s u l t s obtained from P i g Experiment I i n d i c a t e d t h a t the performance of the pigs r e c e i v i n g d i e t s w i t h graded l e v e l s of threonine added to the b a r l e y - l y s i n e b a s a l d i e t , was s t i l l i n f e r i o r to th a t obtained w i t h the barley-soybean c o n t r o l d i e t , although performance on one of the graded threonine supplemented d i e t was not s t a t i s t i c a l l y i n f e r i o r to the performance on the c o n t r o l d i e t on the b a s i s of Tukey's t e s t (1953). Conse-quently i t was considered d e s i r a b l e to i n v e s t i g a t e the e f f e c t of modifying the l e v e l s of amino a c i d supplementation on the complete removal of t h i s gap-Owing to the time and expense i n v o l v e d w i t h c a r r y i n g out t h i s i n v e s t i g a t i o n w i t h any degree of thoroughness w i t h p i g s , i t was decided to use r a t s i n t h i s i n v e s t i g a t i o n . . I t i s g e n e r a l l y known that the l a b o r a t o r y r a t has been accepted as a s a t i s f a c t o r y p i l o t animal i n swine n u t r i t i o n research as the p a t t e r n of amino a c i d requirements f o r both species are q u i t e s i m i l a r (N.A.S.-N.R.C. 1968, 1972). This experiment was designed as a p r e l i m i n a r y i n v e s t i g a t i o n to compare the response i n r a t s given d i e t s s i m i l a r to those r e c e i v e d by the pigs i n P i g Experiment I . I f s i m i l a r growth patt e r n s were obtained, i t was intended to do f u r t h e r experiments as f i r s t experiments f o r p o s s i b l e - 85 -subsequent work w i t h pigs i n an attempt to formulate the " i d e a l " b a r l e y -amino a c i d d i e t . This experiment c o n s i s t e d of s i x treatments w i t h 6 r a t s i n d i -v i d u a l l y housed per treatment. Three e x t r a r a t s were needed as the c o n t r o l f o r i n i t i a l body composition a n a l y s i s . The experiment was conducted f o r 26 days. b. Animals A t o t a l of 39 a l b i n o male r a t s (Woodlyn/Wistar s t r a i n ) aged 30±1 days w i t h average body weight approximately 80 g were employed. T h i r t y -s i x of these were, randomly a l l o c a t e d to i n d i v i d u a l w i r e cages (25.4 x 17.8 2, cm ). D i e t s were assigned randomly to the r a t s . Three r a t s were k i l l e d by d i e t h y l e t h e r o v e r - a n e s t h e t i z a t i o n and used as c o n t r o l s f o r body composition a n a l y s i s . c. D i e t s The s i x t e s t d i e t s were of a s i m i l a r f o r m u l a t i o n to those used i n P i g Experiments I and I I as shown i n Table 2 . However, a l l the i n g r e -d i e n t s f o r r a t d i e t s were f i n e l y ground through 30 mesh screen by C & N l a b o r a t o r y m i l l * b e f o r e mixing i n a Hobart p l a n e t a r y feed mixer. d. Management ( i ) Housing Si z e 8 inches l a b o r a t o r y m i l l , C h r i s t y & N o r r i s L t d . , Chelmsford, England. - 86 -The experiment was conducted i n the r a t l a b o r a t o r y of the Depart-ment of Animal Science, U.B.C. The temperature of the room was maintained between 24 to 25° C throughout the experiment. ( i i ) Feeding methods Rats were fed ad_ l i b i t u m . Feed was added d a i l y . Fresh water was provided d a i l y and was a v a i l a b l e c ontinuously from automatic d r i n k e r s ; e. Records A l l r a t s were weighted i n i t i a l l y and t h e r e a f t e r at 24 hours i n t e r v a l s throughout the experiment. D a i l y feed consumption was recorded. S p i l l a g e of fe e d , when i t occured, was c o l l e c t e d d a i l y and sub t r a c t e d from gross i n t a k e . f. Chemical a n a l y s i s ( i ) Feed A l l d i e t s were sampled and analysed s i m i l a r to P i g Experiment I . ( i i ) Carcass C o n t r o l r a t s were k i l l e d by d i e t h y l e t h e r o v e r - a n a e s t h e t i z a t i o n at the beginning of the experiment. The gut of each r a t was cleaned by f l u s h i n g w i t h tap water b e f o r e s t o r i n g at -10° C f o r l a t e r a n a l y s i s . S i m i l a r l y a l l the experimented r a t s were k i l l e d at the end of the feeding p e r i o d by the same method, cleaned and st o r e d at -10° C . - 87 -For a n a l y s i s , a l l r a t s were d r i e d at 95° C f o r 72 hours and weighed. Each d r i e d r a t carcass was e x t r a c t e d i n a so x h l e t apparatus using s i z e 43 x 123 mm thimbles. The carcass was then d r i e d and weighed and ground i n a C & N Laboratory m i l l (30 mesh screen) and then b a l l m i l l e d f o r 24 hours. The powder from the carcass sample was then used to determine p r o t e i n and ash content. Analyses were done according to A.O.A.C. (1965). g. C a l c u l a t i o n s Average d a i l y gain, feed conversion e f f i c i e n c y and average d a i l y feed i n t a k e of each r a t was c a l c u l a t e d f o r each week* and the over a l l p e r i o d . P r o t e i n u l t i l i z a t i o n r a t i o f o r each r a t was a l s o c a l c u l a t e d . The o v e r a l l average d a i l y gain was used as a c o v a r i a b l e f o r a n a l y s i s of a l l the carcass data. Means of f a c t o r s w i t h s i g n i f i c a n t F values were t e s t e d by Tukey's t e s t (1953). Standard e r r o r s of treatment means were c a l c u l a t e d from a n a l y s i s of va r i a n c e t a b l e s . Carcass analyses were used to c a l c u l a t e t o t a l carcass p r o t e i n , p r o t e i n r e t e n t i o n during the whole experiment p e r i o d , p r o t e i n r e t a i n e d / p r o t e i n i n t a k e (%), p r o t e i n % i n dry car c a s s , p r o t e i n % i n f a t f r e e c a r c a s s , t o t a l carcass f a t , f a t % i n dry carcass, t o t a l carcass ash, ash % i n dry carcass and ash % i n f a t f r e e c a r c a s s . The f o u r t h "week" was only of four days d u r a t i o n . - 88 -h . S t a t i s t i c a l A n a l y s i s A l l t h e d a t a w e r e a n a l y s e d by a n a l y s i s o f c o v a r i a n c e u s i n g compu te r p r o g r a m , UBC-MFAV (Halm and L e , 1974) f o r a one way c l a s s i f i c a t i o n . A v e r a g e d a i l y g a i n and f e e d c o n v e r s i o n e f f i c i e n c y f o r e a c h week and f o r t h e o v e r a l l p e r i o d were a n a l y s e d w i t h w e e k l y and o v e r a l l a v e r a g e d r y m a t t e r f e e d i n t a k e r e s p e c t i v e l y and w e i g h t a t t h e b e g i n n i n g o f t he e x p e r i m e n t as c o v a r i a b l e s . C a r c a s s measurement d a t a w e r e a n a l y s e d w i t h a v e r a g e d a i l y g a i n as a c o v a r i a b l e . - 89 -B. RESULTS AND DISCUSSION The r e s u l t s obtained w i t h r a t s i n average d a i l y gain (ADG) on a weekly and o v e r a l l b a s i s , feed conversion e f f i c i e n c y (FCE) and o v e r a l l p r o t e i n e f f i c i e n c y r a t i o are summarized i n Table 8 . The a d d i t i o n of 0.444% L - l y s i n e - H C l alone to the a l l b a r l e y d i e t ( b a s a l d i e t ) gave the lowest ADG and poorest FCE on both weekly and o v e r a l l f i g u r e s w h i l e the a d d i t i o n of graded l e v e l s of L-threonine p r o g r e s s i v e l y improved ADG and FCE. The r a t s appeared to respond b e t t e r to 0.15% L-threonine a d d i t i o n than to 0.10% a d d i t i o n , but the improve-ment was not s i g n i f i c a n t . The a d d i t i o n of 0.10% methionine to 0.15% added L-threonine improved the response s l i g h t l y but not s i g n i f i c a n t l y . The 0.15% threonine and 0.15% threonine + 0.10% methionine added d i e t s were s i g n i f i c a n t b e t t e r then the b a s a l d i e t . The o v e r a l l ADG and FCE of r a t s r e c e i v i n g the b a r l e y - l y s i n e d i e t s supplemented w i t h 0, 0.05, 0.10, 0.15% threonine and 0.15% threonine p l u s 0.10% DL-methionine were 3.98, 3.71; 4.18, 3.51; 4.38, 3.34; 4.55, 3.21; 4.73, 3.09, r e s p e c t i v e l y . The trend obtained was s i m i l a r to that obtained w i t h the p i g s which r e c e i v e d the same d i e t s i n P i g Experiment I . The r a t growth r e s u l t s i n d i c a t e d t h a t threonine i s a l s o l i m i t i n g i n b a r l e y f o r the growing r a t s . This i s i n agreement w i t h the r e s u l t s reported by Sure (1955). No f u r t h e r s i g n i f i c a n t response to the a d d i t i o n 0.10% methionine suggests t h a t methionine i s adequate i n b a r l e y f o r the growing r a t . This i n t u r n i n d i c a t e d t h a t threonine, but not methionine, i s the second l i m i t i n g Table 8 . E f f e c t of amino acid supplementation of low protein barley on average d a i l y gain, feed conversion e f f i c i e n c y and protein e f f i c i e n c y r a t i o of rats 1 1 3 4 5 6 Result of Barley-soy l y s 3 + B + B + B + SE* s i S n i f i ~ Week cont r o l (Basal) 0.05%Thr 0.10%Thr 0.15%Thr 0.15%Thr+ O.lOZMet. cance t e s t * * Average Daily Gain(g) 1 5.35 5.58 5.25 5.47 o v e r a l l period Feed Conver-sion E f f i -ciency 5.28 2.30 2.49 Protein E f f i c i e n c y Ratio 2.03 3.36 3.78 3.68 4.07 4.22 4.26 4.24 4.42 4.02 4.45 4.50 4.60 3.98 4.18 4.38 3.77 3.30 . 3.08 3.96 3.49 3.23 4.27 4.47 4.71 4.81 4.55 3.02 3.20 2.36 2.48 2.63 2.69 4.72 ±0.15 1 6 5 4 3 2 4.58 4.83 5.11 4.73 2.67 tO.20 1 6 5 4 3 2 ±0.21 ±0.26 ±0.10 N.S. N.S. 1 6 5 4 3 2 ±0.13 2 3 4 5 6 1 3.11 ±0.16 2 3 4 5 6 1 g feed 3 3.15 3.74 3.95 3.65 3.43 3.31 ±0.23 8 gain ^ 3.07 3.90 3.90 3.61 3.41 3.27 ±0.22 o v e r a l l 2.79 3.71 3.51 3.34 3.21 3.09 ±0.07 period I n i t i a l 84 78 weight 78 86 77 79 ±2.81 N.S. N.S. S i g n i f i c a n t covariables I n i t i a l weight(-) Average feed intake I n i t i a l weight(-) Average feed intake Average feed intake I n i t i a l weight(-) Average feed intake I n i t i a l weight(-) Average feed intake I n i t i a l weight Average feed intake I n i t i a l weight N.S. N.S. 2_3 4 5 6 1 I n i t i a l weight N.S. 2.80 ±0.06 6 5 4 3 2 1 N.S. Computed from 5 days data. Standard error of treatment means. Treatment means not underscored by the same l i n e are s i g n i f i c a n t l y d i f f e r e n t at the 5% l e v e l of p r o b a o i l i t y (Tukey, 1953). ** - 91 -amino a c i d i n b a r l e y . The o v e r a l l ADG on the barley-soybean c o n t r o l d i e t was s i g n i f i c a n t l y b e t t e r than the gain on any amino acid-supplemented b a r l e y d i e t which tends to show that at these l e v e l s of amino a c i d a d d i t i o n to b a r l e y , p r o t e i n requirements s t i l l were not adequately met. However, the 0.15% threonine p l u s 0.10% methionine a d d i t i o n to the b a s a l d i e t t produced a FCE which d i d not d i f f e r s i g n i f i c a n t l y from that of the c o n t r o l d i e t . In t h i s case, methionine d i d show an e f f e c t by improving "FCE of the growing r a t s . The weekly ADG showed that the p r o t e i n and amino a c i d r e q u i r e -ments decreased as the age of r a t incr e a s e d . The r a t s had responded to the higher l e v e l s of threonine supplementation during the f i r s t two weeks on t r i a l . There was no s i g n i f i c a n t respond to the higher l e v e l s i n the l a t t e r two weeks. This r e d u c t i o n i n p r o t e i n s and amino a c i d requirements w i t h age confirms the work of Forbes and Rao (1959) and Hartsook and M i t c h e l l (1956). The weekly FCE r e s u l t s i n d i c a t e d that more u n i t s of feed were re q u i r e d per u n i t of gain as age i n c r e a s e d . This g e n e r a l l y appeared i n a l l the treatments. The 0.15% threonine p l u s 0.10% methionine supple-mented d i e t and the c o n t r o l d i e t showed the b e s t FCE, and d i d not d i f f e r s i g n i f i c a n t l y from each other. The former d i e t a l s o d i d not d i f f e r from the 0.10% and 0.15% threonine a d d i t i o n d i e t s . The b a s a l d i e t r e q u i r e d more feed per u n i t gain than a l l other treatments. The f a c t that threonine and methionine added to the b a s a l d i e t produced a FCE s i m i l a r to the c o n t r o l i n d i c a t e d that a low p r o t e i n d i e t (about 11% crude p r o t e i n ) w i t h a - 92 -proper amino a c i d balance can perform as w e l l as a higher p r o t e i n d i e t (about 17.5% crude p r o t e i n ) c o n t a i n i n g a conventional p r o t e i n supplement. The p r o t e i n e f f i c i e n c y r a t i o (PER) was improved p r o g r e s s i v e l y w i t h an i n c r e a s e i n the l e v e l of threonine supplementation. D i e t s w i t h 0.10% or over threonine a d d i t i o n gave s i g n i f i c a n t l y b e t t e r PER values than the b a s a l d i e t i n d i c a t i n g threonine to be l i m i t i n g i n the b a s a l d i e t . This agrees w i t h the ADG and FCE r e s u l t s and i n d i c a t e s t h a t threonine i s the second l i m i t i n g amino a c i d i n b a r l e y f o r growing r a t s . The barley-soybean c o n t r o l d i e t produced the poorest PER although the ADG and FCE were the best based on t h i s d i e t . This i s probably because the c o n t r o l d i e t contained a h i g h e r crude p r o t e i n l e v e l than r e q u i r e d per se. The r e s u l t s agree w i t h those of Metta and M i t c h e l l (1956) who i n d i c a t e d that the b i o l o g i c a l value of d i f f e r e n t p r o t e i n decreased l i n e a r l y as the p r o t e i n c o n c e n t r a t i o n i n the d i e t i n c r e a s e d . Covariance a n a l y s i s showed that there was a s i g n i f i c a n t negative r e l a t i o n s h i p between ADG and i n i t i a l weight and between FCE and feed Intake. The r a t carcass a n a l y s i s data are summarized i n Table 9 . No s i g n i f i c a n t d i f f e r e n c e s i n t o t a l p r o t e i n r e t e n t i o n were obtained among the c o n t r o l , 0.10% threonine and 0.15% threonine plus 0.10% methionine added d i e t s . L e v e l s of 0.10% threonine or h i g h e r produced s i g n i f i c a n t l y b e t t e r p r o t e i n r e t e n t i o n than the b a s a l d i e t . No s i g n i f i c a n t d i f f e r e n c e s Table 9 . Effect of amino acid supplementation of low protein barley on carcass characteristics of rats. Result of s i g n i f i -Barley-soy lys B+ B + B+ B+ SE* control (Basal) 0.05%thr 0.10%thr 0.15%thr 0.15%thr+ ^ " r * * 0.10%met 1 Significant covariable Total carcass protein(g) Protein reten-tion during 26 days on t r i a l (g) Protein retained Protein intake Protein % in dry carcass Protein % in fat free carcass Total carcass fat (g) Fat % in dry carcass Total carcass ash (g) Ash % in dry carcass Ash % in fat free carcass 38.31 29.96 31.82 35.07 33.26 34.25 +1.17 14 5 6 3 2 28.07 19.72 21.58 24.82 23.03 24.01 ± 1.17 14 6 5 3 2 45.29 63.02 81.46 13.21 22.37 6.48 10.78 44.51 47.73 48.62 53.18 80.26 80.21 23.07 20.63 38.37 33.85 5.42 5.72 8.94 9.57 Protein+fat+ash Dry carcass wt % 13.88 14.51 96.39 96.75 14.47 96.46 54.47 53.60 81.66 22.79 34.30 6.06 9.30 14.13 97;12 52.07 53.21 80.54 21.55 33.81 6.03 9.68 14.62 96.77 55.93 + 2.21 6 4 5 3 12 60.42+1.51 16 4 5 3 2 81.00 + 0.53 N.S. 14.29 ± 1.47 2 4 5 3 6 1 25.32 + 1.60 2 4 3 5 6 1 5.99 + 0.17 N.S. 10.61 + 0.26 1 6 5 3-4 2 14.21 ± 0.16 N.S. 96.43 + 0.39 N.S. N.S. N.S. N.S. Average dally gain(-) N.S. Average daily gain Average daily gain N.S. Average daily gain(-) N.S. N.S. Standard error of treatment means. Treatment means not underscored by the same line are significantly different at the 5% level of probability (Tukey, 1953) - 94 -were obtained among the threonine supplemented d i e t s . Hie r e s u l t s i n d i c a t e t h a t the b a r l e y - l y s i n e d i e t was l i m i t i n g i n threonine. The p r o t e i n r e t a i n e d as percentage of p r o t e i n i n t a k e was p r o g r e s s i v e l y improved by threonine a d d i t i o n , s i m i l a r i n manner to improvements i n PER. The methionine supplemen-ted d i e t appeared to produce a higher p r o t e i n r e t a i n e d / p r o t e i n i n t a k e value but the d i f f e r e n c e was not s i g n i f i c a n t l y d i f f e r e n t as measured by Tukey's (1953) t e s t . The barley-soybean c o n t r o l d i e t r e s u l t e d the poorest value i n d i c a t i n g an excess p r o t e i n i n t a k e . Carcass f a t was lowest i n both the c o n t r o l group and the methionine supplemented group. The a d d i t i o n of threonine had no i n f l u e n c e on the h i g h carcass f a t content obtained on the b a r l e y - l y s i n e d i e t . T o t a l ash showed no v a r i a t i o n w i t h treatment. The threonine p l u s methionine supplemented d i e t r e s u l t e d i n s i g n i f i c a n t l y lower carcass f a t . This i n d i c a t e d that methionine a d d i t i o n e f f e c t i v e l y reduced t o t a l carcass f a t i n growing r a t s . However, t o t a l b a c k f a t t h i c k n e s s was not reduced i n the previous p i g n u t r i t i o n a l t r i a l w i t h the same l e v e l of methionine supplementation. This evidence suggests th a t the growing r a t can probably u l t i l i z e t h i s l e v e l of methionine more e f f e c t i v e l y then the g r o w i n g - f i n i s h i n g p i g by enhancing n i t r o g e n r e t e n t i o n r e s u l t i n g i n l e s s f a t d e p o s i t i o n . Thus, the growing r a t s do not c o r r e l a t e w e l l w i t h the g r o w i n g - f i n i s h i n g pigs i n t h i s r e s p e c t . C. CONCLUSION The r e s u l t s of the r a t experiments i n d i c a t e a r a t response s i m i l a r to that given by pigs i n P i g Experiment I . The a d d i t i o n to the b a r l e y - l y s i n e d i e t of graded threonine l e v e l s improved the ADG, FCE and PER of the r a t . The f u r t h e r a d d i t i o n of methionine d i d not improve the above c r i t e r i a s i g n i f i c a n t l y . The carcass p r o t e i n r e t e n t i o n , expressed e i t h e r i n absolute terms or as a percentage of p r o t e i n i n t a k e a l s o gave r e s u l t s i n favor of threonine supplementation. The data measured gave strong evidence that threonine i s the second l i m i t i n g amino a c i d i n b a r l e y f o r growing r a t s . The r e s u l t i s i n agreement w i t h the p i g experiments. Carcass f a t was lowest i n the c o n t r o l group and the methionine supplemented group. However, the r e s u l t s obtained from the methionine supplemented group of r a t s , of low carcass f a t and h i g h p r o t e i n r e t e n t i o n , d i d not occur i n the p i g s . Fat content i n the r a t was not reduced by threonine supplementa-t i o n of the b a r l e y - l y s i n e d i e t . T o t a l ash d i d not vary w i t h treatment. - 96 -VI. RAT EXPERIMENT I I A. EXPERIMENTAL PROCEDURE a. General Results of Rat Experiment I i n d i c a t e d t h a t the response i n r a t s to amino a c i d supplementation of b a r l e y was somewhat s i m i l a r to that i n p i g s . However there were q u a n t i t a t i v e d i f f e r e n c e s i n that the r a t s appeared to be responding to the higher l e v e l of threonine and, at l e a s t i n the f i r s t week, growth and carcass composition were responding to the a d d i t i o n of methionine. However, i t was obvious that even 0.15% threonine plus 0.10% methionine a d d i t i o n d i d not give optimal performance. This experiment was designed to i n v e s t i g a t e the e f f e c t of (1) an improved c o n t r o l d i e t (higher p r o t e i n ) (2) A d d i t i o n a l l y s i n e (3) A d d i t i o n a l threonine and (4) The p r o v i s i o n of an amino acids mixture to the b a r l e y d i e t on r a t growth and carcass composition. The experimental design c o n s i s t e d of 48 a l b i n o male r a t s which were fed f o r 4 weeks. Four e x t r a r a t s were slaughted a t the beginning of the experiment as an i n i t i a l c o n t r o l group f o r the assessment of body n u t r i e n t storage. b. Animals A t o t a l of 52 a l b i n o male r a t s (Woodlyn/Wistar s t r a i n ) - 9 7 -average weight 86 g and age 30 ± 1 days were used. Method of allocation of rats was similar to that used in Rat Experiment I. c. Diets The diet formulation is shown in Table 10. The amino acid mixture was formulated to provide a slight margin of each of the essential amino acids over requirement .levels lis.ted i n "Nutrient Requirements of Laboratory Animals." N.A.S.-N.R.C (1972). The li s t e d requirements for non-essential amino acids were not adhered to as these had been merely calculated from levels included in "successful" diets, and thus were not shown to be essential. Total level of added amino acid mixture (air dry basis) was 1.75%. The composition of the added amino acid mixture and treatments were given below. The amino acid content of each diet i s shown in Table 11. Amino acid mixture L-arginine 0.20% L-tryptophan 0.05% L-histidine 0.20% L-leucine 0.20% L-isoleucine 0.20% L-phenylalanine 0.30% DL-methionine 0.20% L-valine 0.10% L-Glutamic acid 0.30% - 98 -Treatments 1. B a r l e y only to supply 0.41% t o t a l l y s i n e . 2. B a r l e y + soybean meal to supply 0.75% t o t a l l y s i n e . 3. B a r l e y + soybean meal to supply 0.90% t o t a l l y s i n e . 4. B a r l e y + 0.637% L - l y s i n e - H C l to supply 0.90% t o t a l l y s i n e . 5. B a r l e y + 0.444% L - l y s i n e - H C l to supply 0.75% t o t a l l y s i n e + 0.10% L-threonine. 6. D i e t 5 + 0 . 1 0 % L-threonine ( i . e . 0.20% added L-threonine) 7. D i e t 4 + 0.10% L-threonine. 8. D i e t 4 + 0.20% L-threonine. 9. D i e t 5 + amino a c i d mixture. 10. D i e t 6 + amino a c i d mixture. 11. D i e t 7 + amino a c i d mixture. 12. D i e t 8 + amino a c i d mixture. d. Management Same as Rat Experiment I . e. Records Same as Rat Experiment 1. f. Chemical A n a l y s i s Same as Rat Experiment I . _ 99 -g. C a l c u l a t i o n s Same as Rat Experiment I . h. S t a t i s t i c a l A n a l y s i s Same as Rat Experiment I . Table 10. Percentage composition of d i e t s used i n Rat Experiment I I ( A i r dry b a s i s ) I n g r e d i e n t s 1 2 3 4 5 D i e t No. 6 7 8 9 10 11 12 B a r l e y 96.82 81.34 75.16 96.19 96.23 96.18 96.04 95.99 94.63 94.43 94.34 94.24 Soybean meal - 15.60 21.80 - - - - - - - - -L - l y s i n e HC1* - - - 0.637 0.444 0.444 0.637 0.637 0.444 0.444 0.637 0.637 L-thr e o n i n e * - - - 0.10 0.20 0.10 0.20 6.10 0.20 0.10 0.20 L - A r g i n i n e * - - - - - - - - 0.20 0.20 0.20 0.20 L-tryptophan* - - - - - - - - 0.05 0.05 0.05 0.05 L - h i s t i d i n e * - - - - - - - - 0.20 0.20 0.20 0.20 L - l e u c i n e * - - - - - - - - 6.20 0.20 0.20 0.20 L - i s o l e u c i n e * - - - - - - - - 0.20 0.20 0.20 0.20 L-phenylalanine* - - - - - - - - 0.30 0.30 0.30 0.30 DL-me t h i o n i n e * - - - - - - - - 0.20 0.20 0.20 0.20 L - v a l i n e * - - - - - - - - 6.10 0.10 0.10 0.10 L-glutamic a c i d * - - - - - - - - 0.30 0.30 0.30 0.30 Trace m i n e r a l and v i t a m i n promix 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 D e f l u o r i n a t e d rock phosphate 1.64 1.49 1.42 1.64 1.64 1.64 1.64 1.64 1.64 1.64 1.64 1.64 Limes tone 0.54 0.57 0.62 0.54 0.54 0.54 0.54 0.54 0.54 0.54 0.54 0.54 Io d i z e d s a l t 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 The composition of t r a c e m i n e r a l and v i t a m i n premix i s same as that used i n P i g Experiments I , I I and Rat Experiment I . The L - l y s i n e HC1 was fyed grade and was 98% pure, c o n t a i n i n g 78% L - l y s i n e . The threonine was pure and DL-methionine was 98% pure. A l l were produced by the Ajinomoto Co., Japan. Table 11. Content of e s s e n t i a l amino acids of each d i e t f o r Rat Experiment I I (g amino acid/lOOg mixed feed) on a i r dry b a s i s . Amino A c i d 1 2 3 4 5 Diet 6 No 7 8 9 i 10 11 12 A r g i n i n e 0 .45 0. .82 0. .96 0 .45 0 .45 0 .45 0 .45 0 .45 0. 64 0 .64 0 .64 0, .64 H i s t i d i n e 0 .21 0. .40 0. .48 0 .21 0 .21 0 .21 0 .21 0 .21 0. 40 0 .40 0 .40 0. ,40 I s o l e u c i n e 0 .40 0, .58 0, .66 0 .40 0 .40 0 .40 0 .40 0 .40 0. 59 0 .59 0 .59 0, .59 Leucine 0 .73 1. .18 1, .36 0 .73 0 .73 0 .73 0 .73 0 .73 0. 91 0 .91 0 .91 0, .91 Lysine 0 .41 0 .75 0, .90 0 .90 0 .75 0 .75 0 .90 0 .90 0. 75 0 .75 0 .90 0, .90 Methionine 0 .17 0. .24 0, .27 0 .17 0 .17 0 .17 0 .17 0 .17 0. 36 0 .36 0 .36 0, .36 ( C y s t i n e ) * 0 .20 0, .27 0, .29 0 .20 0 .20 0 .20 0 .20 0 .20 0. 20 0 .20 0 .20 0, .20 Phenylalanine 0 .53 0. ,80 0, .90 0 .53 0 .53 0 .53 0 .53 0 .53 0. 82 0 .82 0 .82 0, .82 ( T y r o s i n e ) * 0 .28 0. .48 0. .55 0 .28 0 .28 0 .28 0 .28 0 .28 0. 28 0 .28 0 .28 0, .28 Threonine 0 .37 0. .56 0, .63 0 .37 0 .47 0 .57 0 .47 0 .57 0. 47 0 .57 0 .47 0, .57 Tryptophan 0 .11 0. .19 0, .23 0 .11 0 .11 0 .11 0 .11 0 .11 0. 16 0 .16 0 .16 0, .16 V a l i n e 0 .68 0. .86 0, .93 0 .68 0 .68 0 .68 0 .68 0 .68 0. 76 0 .76 0 .76 0, .76 Glutamic a c i d * * 2 .36 2. ,97 3, .21 2 .36 2 .36 2 .36 2 .36 2 .36 2. 60 2 .60 2 .60 2, .60 Dry matter 89 .93 90. ,26 90, .34 89 .76 89 .75 90 .22 90 .22 89 .74 90. 47 89 .91 90 .21 90, .13 content % Crude p r o t e n t % (N x 6.25) 10 .93 17. .91 19, .35 11 .54 11 .81 11 .38 11 .39 11 .66 12. 58 12 .58 12 .99 12, .93 S e m i - e s s e n t i a l amino a c i d N o n - e s s e n t i a l amino a c i d - 102 -B. RESULTS AND DISCUSSION The r e s u l t s of the experiment f o r ADG, FCE and PER are summarized i n Table 12 Rats fed a s o l e b a r l e y d i e t gave the poorest ADG, FCE and PER. The above c r i t e r i a were improved by a d d i t i o n of l y s i n e alone. Further improvements were obtained by v a r i ous l e v e l s of graded l y s i n e , threonine and other amino a c i d mixtures. With the 0.444% l y s i n e - H C l added d i e t (0.75% of the d i e t ) , 0.10% and 0.20% threonine ( d i e t s 5 and 6 r e s p e c t i v e l y ) supplementa-t i o n gave s i m i l a r ADG and FCE, i . e . no b e n e f i c i a l e f f e c t was obtained by i n c r e a s i n g the threonine l e v e l from 0.10% to 0.20%. In a d d i t i o n , no improvement was obtained based on these two l e v e l s of threonine by i n c r e a s i n g l y s i n e l e v e l s of the d i e t s from 0.75% to 0.90%. The r e s u l t s suggests that 0.75% l y s i n e was adequate f o r growing r a t s , although i n the f i r s t week d a i l y growth r a t e was g r e a t e r on the 0.20% added threonine d i e t when the l y s i n e l e v e l was 0.90% than when i t was 0.75%. These r e s u l t s are i n agreement w i t h the f i n d i n g s of P i c k and Meade (1970) who reported t h a t the l y s i n e requirement of growing r a t s was 0.70% of the d i e t although Rama Rao et a l . (1959) and N.A.S.-N.R.C. (1972) i n d i c a t e a h i g h er requirement (0.90% of the d i e t ) . The supplementation of amino a c i d mixtures to d i e t s 5 and 6 ( i . e . d i e t s 9 and 11 r e s p e c t i v e l y ) d i d not improve ADG and FCE Table 12. Effect of amino acid ftupplcMntaelon of low protein barley on average dolly gain, feed conversion efficiency end protein efficiency ratio ot rara. 1 2 3 4 i 6 7 8 10 11 12 Barley Barley-soy Barley-aoy B + » + B + B + B + B + B + B + B + Week only (0.75Jlya> (0.90Uya) lye(0.90Z) lye(0.75J)+ lys(0.73Z)+ lys(0.901)+ lys(0.90I) lys(0.75I)+ lys(0.75X)+ lya(0.90Z)+ lys(0.90Z)+ SE* Result of Significant (Basal) 0.10Z thr. 0.20X thr. 0.10Z thr. 0.20X thr. 0.10X thr + 0.201 thr + 0.10X thr + 0.20X thr + Blgnlfleance teat** covariable. amino acid amino acid amioo a d d aalDo acid mixture mixture mixture mixture Average Daily Cain (g) 2.83 3.76 5.90 3.21 4.06 J.59 1.40 5.85 3.37 3.60 2.70 6.03 5.62 3.48 3.7* 4.78 5.38 10.15 3 2 12 10 8 11 9 5 7 6 4 1 I n i t i a l velght(-) Average feed intake 6.12 10.22 12 2 10 3 5 8 11 6 7 9 4 1 Avareg. feed Intake 5.51 10.22 2 10 3 12 9 11 8 6 7 5 4 1 I n i t i a l velght(-) Average feed intake 2.67 3.38 4.94 10.25 3 2 10 12 9 11 3 6 8 4 7 1 I n i t i a l velght(-) Average feed intake Overall period 3.32 5.41 3.15 10.09 2 3 10 12 9 11 8 3 6 7 4 1 I n i t i a l velght(-) Average feed Intake reed Conversion 2 Efficiency g gain Ore r a i l period I n i t i a l weight 4.36 2.23 2.17 4.61 4.40 2.55 2.93 5.39 2.43 3.79 2.81 4.31 2.73 2.69 4.30 4.01 4.91 4.17 3.45 3.48 4.37 2.92 2.88 2.40 10.23 4 1 6 7 3 9 11 8 10 12 2 3 Average feed ' intake(-) 2.46 10.17 1 4 9 7 6 11 8 3 3 10 2 12 U.S. 2.81 10.19 1_4 5 7 6 11 9 8 12 10 3 2 I n i t i a l velght — A v e r a g e feed lntake(-) 3.14 D0.31 1 8 4 7 6 5 11 9 12 10 2 3 I n i t i a l velght — H Z Z Z m ^ Z ^ » ^ „ . Average feed lntake(-) 2.92 10.06 1, 4 7 6 5 6 11 9 12 10 3 2 I n l t l e l velght — Averege feed lnteke(-) 87 12.20 Protein Efficiency 2.03 Ratio 2.23 2.43 2.32 2.66 10.03 10 12 8 6 9 5 7 11 4 1 2 3 I n i t i a l velght — — A v e r e g e feed lotake(-) Standard error of tr.atment means. Treatment means not underscored by the same line a n slgnlficantly different at the 31 l e v e l of probebility (Tukey; 1953). - 104 -s i g n i f i c a n t l y . However, the a d d i t i o n of amino a c i d mixtures to d i e t s 6 and 8 ( i . e . d i e t s 10 and 12 r e s p e c t i v e l y ) gave r e s u l t s which were s i g n i f i -c a n t l y b e t t e r than those obtained w i t h a l l other barley-amino a c i d d i e t s . Moreover, the r e s u l t s obtained w i t h these two d i e t s were e q u i v a l e n t to those r e s u l t i n g from the barley-soybean d i e t s ( d i e t s 2 and 3 ) . When comparing a l l the d i e t s c o n t a i n i n g 0.75% l y s i n e ( i . e . d i e t s 5, 6, 9 and 10), d i e t s 5 and 6 c o n t a i n i n g 0.10% and 0.20% threonine r e s p e c t i v e l y has s i m i l a r ADG and FCE. However, the marked improvement of d i e t 6 a f t e r a d d i t i o n of the amino a c i d mixture and r e l a -t i v e l y no improvement i n d i e t 5 w i t h s i m i l a r amino a c i d mixture a d d i t i o n . tend to suggest that threonine became l i m i t i n g when the amino a c i d mixture was added to the d i e t c o n t a i n i n g 0.10% added threonine. I n c r e a s i n g the supplemented threonine l e v e l to 0.20% brought the d i e t i n t o proper balance, thereby improving the ADG and FCE d r a m a t i c a l l y . Higher l y s i n e a d d i t i o n w i t h amino a c i d mixture d i d not f u r t h e r improve the ADG and FCE as i n d i e t s 11 and 12. D i e t 12 contained 0.90% l y s i n e and 0.20% threonine plus amino a c i d mixtures gave the r e s u l t s i m i l a r to d i e t 10 w i t h 0.75% l y s i n e . The evidence f u r t h e r comfirmed that 0.75% l y s i n e was s u f f i c i e n t i n t h i s b a r l e y d i e t f o r the growing r a t s . Again 0.10% threonine added to the b a r l e y d i e t c o n t a i n i n g 0.90% l y s i n e p l u s amino a c i d mixture ( d i e t 11) gave i n f e r i o r r e s u l t to those w i t h 0.20% added threonine p l u s the amino a c i d mixture. The o b s e r v a t i o n suggested that w i t h the amino a c i d mixture, a d d i t i o n of 0.20% threonine must be r e q u i r e d i n t h i s b a r l e y d i e t (or 0.57% t o t a l threonine) i n order to a t t a i n the proper balance. However, t h i s balance could not be a t t a i n e d without the amino a c i d mixture. - 105 -supplementation. The r e s u l t s obtained w i t h d i e t s 10 and 12 were s i m i l a r to the r e s u l t s obtained w i t h the barley-soybean d i e t , i n d i c a t i n g that optimum ADG and FCE can be achieved by amino acids supplementation of the b a r l e y d i e t . The adequacy of the 0.52% threonine i n the d i e t f o r optimal growth was i n agreement w i t h the re p o r t s by N.A.S.-N.R.C. (1972) and Rama Rao et a l . (1959) but lower than l e v e l s suggested by P i c k and Meade (1971). Rosenberg j2t a l . (1959) s t u d i e d the e f f e c t of supplementation of r i c e d i e t w i t h l y s i n e and threonine and i n d i c a t e d that the r a t i o of t o t a l l y s i n e to t o t a l threonine i n the d i e t f o r optimum response was 1.4 to 1 by weight. This r a t i o was c o i n c i d e n t l y i n agreement w i t h d i e t s 2, 3, 6 and 10 i n the present experiment. However, t h i s d i f f e r e d from the r a t i o s of 1.66 : 1 or 2.01 : 1 suggested by Rose (1937) and Rose et a l . (1949) f o r the growing r a t s . The barley-soybean d i e t (2) c o n t a i n i n g 17.9% crude p r o t e i n produced the maximum growth. Further i n c r e a s i n g the p r o t e i n l e v e l ( d i e t 3) to 19.4% crude p r o t e i n d i d not improve the ADG and FCE. The l y s i n e and threonine of d i e t 2 were 0.75%, 0.56% r e s p e c t i v e l y and of d i e t 3 were 0.90%, 0.63% r e s p e c t i v e l y . The- o v e r a l l r e s u l t s f o r ADG and FCE of d i e t s 2, 3, 10 and 12 were s i g n i f i c a n t l y b e t t e r than those of any other barley-amino a c i d d i e t s . D i e t s 10 and 12 gave the best PER but values were not - 106 -s i g n i f i c a n t l y b e t t e r than those of d i e t s 6 and 8. The l a t t e r d i e t s were not s i g n i f i c a n t l y b e t t e r than the other threonine and l y s i n e supplemented d i e t s but were s i g n i f i c a n t l y b e t t e r than values obtained w i t h the 0.90% l y s i n e supplemented d i e t and the s o l e b a r l e y d i e t . The barley-soybean d i e t s produced the poorest PER's as i n Rat Experiment I due to the h i g h i n t a k e s of n o n - e s s e n t i a l n i t r o g e n . The r e s u l t s i n d i c a t e d that the p r o t e i n of d i e t s 10 and 12 was b e s t u t i l i z e d followed by d i e t s 8 and 6. The r a t s carcass a n a l y s i s r e s u l t s are presented i n Table 13 Barley-soybean meal d i e t s gave the h i g h e s t carcass p r o t e i n r e t e n t i o n but gave the lowest f i g u r e s (except f o r the s o l e b a r l e y d i e t ) i n terms of p r o t e i n r e t a i n e d as a percentage of p r o t e i n i n t a k e . As w i t h the PER f i g u r e s , the u n n e c e s s a r i l y h i g h i n t a k e of p r o t e i n produced s m a l l e r values f o r p r o t e i n r e t a i n e d as percentage of p r o t e i n i n t a k e . D i e t s 10 and 12 had the h i g h e s t p r o t e i n r e t e n t i o n and p r o t e i n r e t a i n e d as percentage of p r o t e i n i n t a k e when compared w i t h a l l other barley-amino a c i d d i e t s . The s o l e b a r l e y d i e t gave lowest p r o t e i n r e t e n t i o n and p r o t e i n r e t a i n e d / p r o t e i n i n t a k e . S i m i l a r to the p i c t u r e produced by the PER f i g u r e s , r e s u l t s obtained w i t h the b a r l e y amino a c i d d i e t s tended to i n d i c a t e t h a t a d d i t i o n of the f i r s t l i m i t i n g amino a c i d l y s i n e to b a r l e y improved the above c r i t e r i a . A d d i t i o n of graded l e v e l s of threonine f u r t h e r improved the performance. I n c r e a s i n g d i e t a r y l y s i n e l e v e l from 0.75% to 0.90% d i d not f u r t h e r improve performance. This again suggested t h a t 0.75% l y s i n e i s s u f f i c i e n t f o r the growing r a t . Carcass f a t content was n e g a t i v e l y c o r r e l a t e d w i t h p r o t e i n Table 1). Effect of n t n o acta" e u p p l c M n t a t l o n of low protein barley on carcase characterUtlcs of r a t e 1 2 3 4 5 6 7 8 9 10 11 12 Barley only (Basal) Barley-aoy (0.752lya) Barlcy-aoy (0.90Ztyo) B + lys(0.901) B + lys (0.751) B • lys(0.751) B + lyo(0.90Z) B + lys(0.90Z) B + lyo(0.73Z) B + lya(0.752) B + lys(0.90Z) 8 + lra(0.90Z) SE* Result of significance test** Significant covariable •tO.lOXthr +0.20Zthr +0.10Zthr 4O.20Zthr +0.10Zthr+ amino acid mixture 4O.20Zthr+ emlno acid mixture +0.10Zthr+ amino acid mixture +0.202 th r+ offline acid mixture Total carcaaa proteln(g) Protein retention during 28 days oo t r i a l (g) Protein retained j Protein intake Protein Z i n dry carcase Protein Z In fat -free carcaaa 32.25 . 41.31 40.44 34.29 35.66 37.63 28.64 22.49 41.81 77.90 Total carcass fat(g) 31.73 Pat Z In dry carcase 46.09 83.19 9.65 17.81 38.81 41.49 63.79 48.37 31.57 57.14 83.09 79.93 12.04 26.18 20.81 39.12 80.37 24.87 38.63 29.21 48.96 51.14 80.73 80.37 80.82 19.52 21.26 16.41 31.73 26.06 51.63 38.83 80.18 13.05 24.03 82.98. 13.56 81.82 19.22 22.26 29.34 39.92 z 0.89 2 10 3 12 9 6 8 11 7 3 4 1 Average daily • gain 28.15 t 0.89 2 10 3 12 9 11 8 3 6 7 4 1 Averege daily — gain 55.90 1 1.34 10 12 9 6 8 7 11 5 2 4 3 1 Average daily gain 61.78 i 1.72 2 3 10 9 12 8 11 6 7 5 4 1 Averege daily 8aln(-) 81.61 ± 0.66 U.S. 15.53 x 1.63 1 4 3 7 6 11 B 12 9 10 3 2 Averege dally — gain 24.33 * 1.78 1 4 5 7 11 6 8 12 9 10 3 2 Average delly gain O Total carcass ash(g) Aah Z In dry carcass Ash Z in fat free cercasa Proteln+fat+ash , Dry carcaaa wt. 6.22 8.36 15.52 6.55 10.98 13.98 6.72 .11.02 13.93 6.08. 8.68 6.20 8.99 14.30 14.00 6.69 10.19 14.37 6.30 9.67 14.18 6.69 10.71 14.48 97.61 96.39 6.44 6.49 6.13 6.47 ± 0.13 3 6 8 2 7 10 12 9 1 3 11 4 Average daily gain 10.36 10.41 9.50 10.10 ± 0.30 3 2 8 10 9 6 12 7 11 5 4 1 Average dally —- gain(-) 13.65 13.38 13.44 13.32 ±0.14 1 8 6 4 7 3 3 9 11 10 2 12 Average dally F galn(-). 96.83 97.16 96.67 96.19 ±0.43 U.S. M.S. Standard error of treatment meens. Treatment means not uodaraeored by the line are significantly different at the 52 level of probability (Tukey,1953). 108 -r e t e n t i o n . I t appeared that the b e t t e r balanced d i e t such as d i e t s 2, 3, 10 and 12 produce minimum f a t and maximum p r o t e i n r e t e n t i o n . The s o l e b a r l e y d i e t which was l i m i t i n g i n both l y s i n e and threonine produced maximum f a t and minimum p r o t e i n r e t e n t i o n i n the carcass. D i e t had a r a t h e r s m a l l s i g n i f i c a n t e f f e c t on carcass t o t a l ash content. However, these d i f f e r e n c e s could not be detected by Tukey fs Test (1953). The response of r a t s to l y s i n e and threonine a d d i t i o n appeared to be s i m i l a r to that obtained w i t h the p i g . The i n c r e a s e o f added th r e o -nine over 0.10% to the 0.75% l y s i n e b a r l e y d i e t d i d not f u r t h e r improve the growth of r a t s . Rats were observed to improve growth by a d d i t i o n of a complete amino a c i d mixture. However, i t i s d i f f i c u l t to p r e d i c t whether the a d d i t i o n of some amino acids to the p i g d i e t would f u r t h e r improve the growth as shown by the r a t s , a s the b a r l e y d i e t has a c t u a l l y s a t i s f i e d the other e s s e n t i a l amino a c i d f o r the g r o w i n g - f i n i s h i n g pigs except f o r l y s i n e and threonine as i n d i c a t e d by comparing a n a l y s i s f i g u r e s w i t h the N.A.S.-N.R.C. (1972) feeding standards. - 109 -C. CONCLUSION I t may be concluded that d i e t s based on b a r l e y alone w i t h adequate mineral and v i t a m i n supplementation do not support optimal growth and feed conversion e f f i c i e n c y i n r a t s . The PER, p r o t e i n r e t e n t i o n and p r o t e i n r e t a i n e d as percentage of p r o t e i n i n t a k e i n d i c a t e d t h a t such a d i e t i s of poor p r o t e i n q u a l i t y . Using the above c r i t e r i a , performance was improved by l y s i n e supplementation w i t h a f u r t h e r improvement produced when graded l e v e l s of threonine were added to the d i e t . Threonine was probably the second l i m i t i n g amino a c i d i n b a r l e y f o r the growing r a t . A 0.75% d i e t a r y l y s i n e was observed to be adequate f o r normal growth i n c o n j u n c t i o n w i t h 0.10% threonine a d d i t i o n (0.47% t o t a l threo-nine i n the d i e t ) , as supplementation of 0.90% d i e t a r y l y s i n e d i d not f u r t h e r improve performance of the r a t s . The r e s u l t s a l s o i n d i c a t e d that 0.20% threonine supplementation was r e q u i r e d i n t h i s b a r l e y - l y s i n e d i e t ( e i t h e r 0.75 or 0.90% d i e t a r y l y s i n e ) to maximize n i t r o g e n balance when an amino acid s mixture was added to the d i e t . The evidence o b v i o u s l y showed that a low q u a l i t y b a r l e y d i e t w i t h proper supplementation of l i m i t -i n g amino acid s can r e s u l t i n performance equal to that obtained w i t h a barley-soybean d i e t . The barley-soybean d i e t s produced poor values f o r PER and p r o t e i n r e t a i n e d as percentage of p r o t e i n i n t a k e because of excess p r o t e i n i n t a k e . - 110 -V I I . RAT EXPERIMENT I I I A. EXPERIMENTAL PROCEDURE a. General The evidence of Rat Experiment I I i n d i c a t e d that a 0.75% l y s i n e b a r l e y d i e t w i t h 0.20% threonine (0.57% t o t a l threonine i n the d i e t ) and an amino a c i d mixture provided o p t i m a l growth e q u i v a l e n t to that obtained w i t h a barley-soybean c o n t r o l d i e t . A requirement f o r a t t a i n i n g o ptimal growth appeared to be the a d d i t i o n of the e s s e n t i a l amino a c i d mixture. However, i t i s s t i l l p o s s i b l e that the improvement may have been a t t a i n e d by a d d i t i o n of o t h e r n i t r o g e n sources i n s t e a d of e s s e n t i a l amino a c i d s . Such an a d d i t i o n of n o n - s p e c i f i c n i t r o g e n would be economically more d e s i r a b l e than the a d d i t i o n of amino acid s e i t h e r i n pure form or as p r o t e i n s i n p r a c t i c a l p i g f e e d i n g . Therefore the f o l l o w i n g experiment was designed to i n v e s t i g a t e the e f f e c t of supplementation of g l y c i n e as a n i t r o g e n source on growth performance, feed conversion e f f i c i e n c y , p r o t e i n u t i l i z a t i o n and r e t e n t i o n of r a t s and to compare g l y c i n e a d d i t i o n w i t h a d d i t i o n of an amino a c i d mixture. The experiment c o n s i s t e d of 10 treatments w i t h 4 r a t s per treatment. Duration was 4 weeks. b. Animals A t o t a l of 44 a l b i n o male r a t s (Woodlyn/Wistar s t r a i n ) w i t h average weight of 88 g and 30 ± 1 days of age were employed. Four of - I l l -these were k i l l e d i n i t i a l l y to o b t a i n a c o n t r o l measure of body composition. A l l other procedures were e x a c t l y the same as i n the previous experiment. c. D i e t s Hie composition of the d i e t i s shown i n Table 14.Th e p r e p a r a t i o n of these d i e t were the same as f o r Rat Experiment I . The experiment c o n s i s t e d of 10 treatments as f o l l o w s : 1. B a r l e y only to supply 0.41% t o t a l l y s i n e . 2. B a r l e y + 0.444% L - l y s i n e - H C l to supply 0.75% t o t a l l y s i n e . 3. B a r l e y + 0.637% L - l y s i n e - H C l to supply 0.90% t o t a l l y s i n e . f 4. D i e t 2 + 0.10% threonine. 5. D i e t 4 + amino a c i d mixture. 6. D i e t 4 + g l y c i n e . 7. D i e t 2 + 0.20% threonine. 8. D i e t 7 + amino a c i d mixture. 9. D i e t 7 + g l y c i n e . 10. Barley-soybean to supply 0.75% l y s i n e . The composition of., the amino a c i d mixture was the same as that i n Rat Experiment I I . Management Same as Rat Experiment I Records Same as Rat Experiment I Chemical A n a l y s i s Same as Rat Experiment I C a l c u l a t i o n s Same as Rat Experiment I S t a t i s t i c a l A n a l y s i s Same as Rat Experiment I - 113 -Table 14. Percentage composition of d i e t s used i n Rat Experiment I I I ( a i r dry b a s i s ) D i e t No. Ingre d i e n t s 1 2 3 4 5 6 7 8 9 10 B a r l e y 96.82 96.38 96.19 96.28 94.53 94.93 96.18 94.43 94.83 81.34 Soybean meal - - - - - - - - - 15.60 L - l y s i n e HC1* - 0.444 0.637 0.444 0.444 0.444 0.444 0.444 0.444 -L-threonine* - - - 0.10 0.10 0.10 0.20 0.20 0.20 -L - A r g i n i n e * - - • - - 0.20 - - 0.20 - -L-tryptophan* - - - - 0.05 - - 0.05 - -L - h i s t i d i n e * - - - - 0.20 - 0.20 - -L - l e u c i n e * - - - - 0.20 - - 0.20 - -L - i s o l e u c i n e * - - - - 0.20 - - 0.20 - -L-phenylalanine* - - - - 0.30 - - 0.30 - -DL-methionine* - - - - 0.20 - - 0.20 - -L - v a l i n e * - - - - 0.10 - - 0.10 - -L-Glutamic a c i d * - - - 0.30 - - 0.30 - -L - G l y c i n e * - - - - - 1.355 - - 1.355 -Trace m i n e r a l and vi t a m i n premix 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 D e f l u o r i n a t e d rock phosphate 1.64 1.64 1.64 1.64 1.64 1.64 1.64 1.64 1.64 1.49 Limestone 0.54 0.54 0.54 0.54 0.54 0.54 0.54 0.54 0.54 0.57 Iod i z e d s a l t 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 The composition of t r a c e m i n e r a l and v i t a m i n premix was the same as that of used i n P i g Experiments I and I I . * The L - l y s i n e HC1 was feed grade and was 98% pure, c o n t a i n i n g 78% L - l y s i n e . Hie L-threonine was pure and DL-methionine was 98% pure. A l l were produced by the Ajinomoto Co., Japan. - 114 -Table 15. Content of e s s e n t i a l amino acid s of each d i e t f o r Rat Experiment I I I (g amino acid/lOOg mixed feed) on a i r dry b a s i s . Amino A c i d 1 2 3 4 Di e t No. 5 6 7 8 9 10 A r g i n i n e 0 .45 0, .45 0, .45 0. .45 0 .64 0. ,45 0 .45 0 .64. 0 .45 0 .82 H i s t i d i n e 0 .21 0. .21 0, .21 0, .21 0 .40 0. ,21 0 .21 0 .40 0 .21 0 .40 I s o l e u c i n e 0 .40 0, .40 0, .40 0. .40 0 .59 0, .40 0 .40 0 .59 0 .40 0 .58 Leucine 0 .73 0. .73 0, .73 0, .73 0 .91 0, .73 0 .73 0 .91 0 .73 1 .18 Lysine 0 .41 0, .75 0, .90 0. .75 0 .75 0. .75 0 .75 0 .75 0 .75 0 .75 Methionine 0 .17 0. .17 0, .17 0, .17 0 .36 0, .17 0 .17 0 .36 0 .17 0 .24 ( C y s t i n e ) * 0 .20 0. .20 0, .20 0, .20 0 .20 0. .20 0 .20 0 .20 0 .20 0 .27 Phenylalanine 0 .53 0, .53 0. .53 0, .53 0 .82 0, .53 0 .53 0 .82 0 .53 0 .80 ( T y r o s i n e ) * 0 .28 0, .28 0, .28 0, .28 0 .28 0. .28 0 .28 0 .28 0 .28 0 .48 Threonine 0 .37 0, .37 0, .37 0, .47 0 .47 0. .47 0 .57 0 .'57 0 .57 0 .57 Tryptophan 0 .11 0 .11 0 .11 0, .11 0 .16 0 .11 0 .11 0 .16 0 .11 0 .19 V a l i n e 0 .68 0. .68 0. .68 0. ,68 0 .76 0. .68 0 .68 0 .76 0 .68 0 .86 Glutamic a c i d * * 2 .36 2. ,36 2. .36 2. .36 2 .60 2, ,36 2 .36 2 .60 2 .36 2 .97 G l y c i n e * * 0 .42 0, ,42 0. .42 0. ,42 0 .41 1. .77 0 .42 0 .41 1 .77 0 .56 Dry matter content % 90 .14 89. .13 89. .69 90. .12 90 .00 89. ,93 89 .94 89 .57 89 .85 90 .45 Crude p r o t e i n % (N x 6.25) 10 .58 11. ,17 11. .39 11. .15 12 .74 12. .86 11 .24 12 .89 12 .91 17 .40 * S e m i - e s s e n t i a l amino a c i d ** N o n - e s s e n t i a l amino a c i d - 115 -B. RESULTS AND DISCUSSION The r e s u l t s of ADG, FCE and PER are presented i n Table 16 w h i l e the carcass a n a l y s i s data are summarized i n Table 17 The r a t s fed w i t h d i e t s the same as those i n Rat Experiment I I obtained b a s i c a l l y s i m i l a r r e s u l t s i n terms of ADG, FCE, PER and carcass p r o t e i n a n a l y s i s v a l u e s . This f u r t h e r confirmed the importance of threonine a d d i t i o n to b a r l e y - l y s i n e to improve the p r o t e i n q u a l i t y . The evidence a l s o confirmed threonine as the second l i m i t i n g amino a c i d i n b a r l e y . The r a t s ' performance on the 0.75% and 0.90% l y s i n e d i e t s d i d not d i f f e r s i g n i f i c a n t l y , f u r t h e r confirming the previous experimental r e s u l t s that 0.75% l y s i n e i s adequate f o r the growing r a t . With the same l e v e l of l y s i n e i n the d i e t s , a d d i t i o n of 0.10% threonine was s l i g h t l y but non-s i g n i f i c a n t l y b e t t e r than 0.20 % threonine a d d i t i o n . However, the r e s u l t s were reversed and s i g n i f i c a n t l y i n favor of the 0.20% threonine added d i e t when f u r t h e r amino a c i d mixtures were added. This observation was i n agreement w i t h Rat Experiment I I . Replacement of the amino a c i d mixture w i t h g l y c i n e on an i s o n i t r o g e -nous b a s i s f a i l e d to show the same performance i n a l l measured c r i t e r i a i n d i -c a t i n g that the improved performance i n d i e t 8 was not due to n i t r o g e n l e v e l per se but r a t h e r due to the proper balance of e s s e n t i a l amino acid s i n the d i e t . As a matter of f a c t , g l y c i n e a d d i t i o n depressed the r a t performance. This suggests t h a t the a d d i t i o n of g l y c i n e a t these l e v e l s c r e a t s an imbalance. Rama Rao e t a l . (1960) reported that g l y c i n e might depress Table 16. Effect of emlno acid aupplenentatton of low protein barley on ayerat* dally gain, feed ' eonveralon efficiency and protein efficiency ratio of rata. 1 2 3 4 5 6 7 e 9 10 Veek Barley • only (Basal) B + B + B +' ly.(0.75W lys(0.90I) lys(0.75J> +0.10*thr B + lye (0.732) +O.10Zthr+ amino acid mixture . B + lya (0.751) +0.10Ithr+ glycine B + lys(0.751) +0.201thr B + lys(0.752) +0.202thr+ 'amino acid mixture B + lys(0.751)+ 0.20Ithr + glycine Barley-soy (control) SE* Result o.t significance test** • Significant eovsrlablea Daily 2.77 3.21 3.28 3.93 3.54 4.35 4.82 4.66 5.22 4.11 3.93 i 0.17 10 8 5 6 9 7 4 3 2 1 I n i t i a l velght(-) " Average feed intake 3.73 ± 0.23 10 8 5 9 4 7 6 5 2 1 Average feed Intake Caln(g) 3.04 3.68 4.69 4.70 5.68 t 0.21 8 10 9 5 4 6 7 3 2 1 I n i t i a l velght(-) • Average feed Intake 2.59 3.49 3.47 4.30 4.93 4.23 4.71 t 0.30 8 3 10 9 4 6 7 2 3 1 I n i t i a l velght(-) •—^Z^^ZZI^I™1__ Average feed intake Overall 3.28 period 3.93 4.42 4.81 4.41 3.19 * 0.11 10 8 5 9 4 6 7 3 2 1 I n i t i a l uelght(-) — Average feed lnteke Conver-alon E f f i -ciency 3 ( gale Overall period I n i t i a l velght 4.47 4.06 4.62 3.89 5.01 4.40 3.74 4.58 Protein Efficiency 2.04 Ratio 4.61 3.97 2.22 3.47 4.05 4.63 3.78 3.48 3.73 3.37 3.43 J.18 3.12 3.32 3.77 3.33 3.68 4.41 3.47 2.47 2.68 2.63 2.88 2.97 3.56 3.22 2.37 Standard error of treatment means. Treatment means not underscored by the seme line are significantly different at the 5X level of probability (Tukey, 1953). 2.23 x 0.16 1_2 3 4 7 5 9 6 8 10 I n i t i a l velght — — Average feed lntakef,-) 2.78 t 0.18 1 2 6 3 4 7 9 3 10 8 8.S. ON 2.79 t 0.17 1 _ 2 3 7 6 4 3 9 10 8 I n i t i a l weight ZZZ^^L Average feed intake(-) 3.60 i 0.36 . N.S. I n i t i a l weight Averege feed intake(-) 2.94 1 0.08 1 2 J 7 * ( ! ! 10 8 I n i t i a l velght ~ — Averege feed lotake(-) 92 x 2.33 2.02 10.06 8 4 7 5 9 3 6 2 1 10 Table 17. Effect of amino and uuppU'tauntntlon of low protein bnrley on carcaaa char net 4 r la t lce of rata • 1 2 3 4 5 6 7 8 9 10 Barley only (Basal) B + lys(0.75Z) B 4-lys(0.90Z) 0 + lye(0.75Z) +0.10Zthr B + lys(0.75Z) +0.10Zthr+ anlno scld mixture B + lys(0.75Z) +0.102thr+ glycine 8 + ly.i(0.75Z> +0.20Ztlir B + lys(0.75Z) *O.20Zthr+ amino acid mixture B T lys(0.75Z) •K).20Zthr+ glycine Barley-soy (control) SE« Result of significance test Slcntftcant covariable Toc.1 c l r e u i protein (*,) 31.19 33.91 34.33 37.38 37.76 35.90 36.80 39.33 36.39 42.81 • t 0.68 10 8 5 4 7 9 6 3 2 1 Average dally gain Protein retention during 29 daye on t r i a l (g)-18.39 21.12 21.55 24.60 25.00 23.12 24.03 26.58 23.61 30.06 t 0.68 10 8 5 4 7 9 6 3 2 1 Average daily gain Protein retalnedj Protelo Intake 33.84 41.63 42.29 32.90 49.01 44.23 50.76 55.45 45.97 42.71 t 1.23 8 4 7 5 9 6 3 10 2 1 Average delly gain Protein Z lo dry carcase 40.39 47.58 49.65 60.34 59.04 53.27 38.53 63.06 59.47 71.88 t 1.67 10 8 4 9 5 7 6 3 2 1 Averege dally galn(-) Protein Z In fat free carcase 78.13 79.88 79.48 80.09 80.63 80.88 81.45 81.78 81.08 82.39 t 0.78 N.S. N.S. Total carcaas fat (g) 34.33 28.83 26.42 13.28 16.68 20.41 17.62 13.46 16.58 3.40 t 1.38 1 2 3 6 7 5 9 4 8 10 Average dally gain Fat Z l a dry carcess 49.86 41.63 38.23 24.43 25.76 31.26 27.94 21.93 26.81 10.77 i 1.69 1 1 3 6 7 9 3 4 8 10 Average dally gain Total carcaaa ash (g) 5.84 6.02 8.23 6.81 6.45 6.39 6.37 6.63 6.34 7.18 0.15 10 4 8 5 6 7 9 3 2 1 Average delly Ash Z In dry carcaaa Aeh Z In fat free carcaaa 7.49 14.95 8.JO 14.28 8.95 14.49 11.03 14.60 10.31 13.84 9.99 14.31 10.18 14.12 10.87 15.89 10.33 14.12 12.31 14.07 + 0.28 0.23 10 4 8 9 5 7 6 3 2 1 R.S. gein Average daily gain N.S. Proteln+fat+Ash Dry carcass wt. 96.18 96.42 96.19 96.01 93.96 97.20 96.80 96.66 96.47 96.73 + 0.33 N.S. N.S. Standard error of treatment aean. »t underacored by the same l ine are algnlf lcantly different at the 51 l eve l of probability (Tukey, 1933). - 118 -growth when given i n l a r g e amounts. Since g l y c i n e has been reported to be l e s s e f f e c t i v e when used as a n i t r o g e n source than ammonium s a l t s and L-glutamic a c i d (Rose e_t a l . , 1949), i t i s s t i l l p o s s i b l e that r a t growth coulb be improved by using e i t h e r ammonium s a l t s or glutamic a c i d to i n c r e a s e the d i e t a r y n i t r o g e n l e v e l . Carcass p r o t e i n r e t e n t i o n showed the 0.75% l y s i n e b a r l e y d i e t w i t h 0.20% added threonine, and an amino a c i d mixture not to be s i g n i f i -c a n t l y d i f f e r e n t from the barley-soybean c o n t r o l . This was s i m i l a r to the r e s u l t s obtained i n Rat Experiment I I . The former d i e t had the best value f o r p r o t e i n r e t a i n e d as a percentage of p r o t e i n i n t a k e . The a d d i t i o n of g l y c i n e d i d not improved the carcass p r o t e i n r e t e n t i o n and the value f o r p r o t e i n r e t a i n e d as a percentage of p r o t e i n i n t a k e . I t a l s o appeared to depress the values of the above c r i t e r i a when compared w i t h values obtained w i t h s i m i l a r d i e t s w ithout g l y c i n e . The carcass data f u r t h e r confirmed that g l y c i n e n i t r o g e n cannot rep l a c e the n i t r o g e n of an amino a c i d mixture f o r optimal r a t growth. The t o t a l carcass f a t content produced on the glycine-added d i e t s was higher than that produced on the corresponding amino a c i d supplemented d i e t s , supporting the f a c t that imbalanced d i e t s tend to deposite more f a t than balanced d i e t s . - 119 -C. CONCLUSION Experiment I I I f u r t h e r i n d i c a t e d that 0.75% l y s i n e was adequate f o r the growing r a t . The b a r l e y - l y s i n e d i e t c o n t a i n i n g 0.75% l y s i n e showed an improvement i n average d a i l y g ain, feed conversion e f f i c i e n c y and n i t r o g e n r e t e n t i o n and n i t r o g e n r e t a i n e d / n i t r o g e n i n t a k e when graded l e v e l s of threonine were added. "The supplementation w i t h amino a c i d mixtures to the 0.20% threonine-added b a r l e y - l y s i n e d i e t s gave r a t performance e q u i v a l e n t to that obtained w i t h the barley-soybean c o n t r o l d i e t . This confirmed the r e s u l t of Rat Experiment I I . When g l y c i n e was used as a n i t r o g e n source, i t f a i l e d to s a t i s -f a c t o r i l y r e p l a c e the amino a c i d mixture as a supplement to the b a r l e y -l y s i n e - threonine d i e t . The depression of r a t growth and n i t r o g e n r e t e n t i o n i n d i c a t e d t h a t g l y c i n e at t h i s l e v e l cannot be u l t i l i z e d e f f e c t i v e l y as a n i t r o g e n source f o r the growing r a t . Amino a c i d 'imbalance' might have been created when t h i s l e v e l of g l y c i n e was added. I t i s p o s s i b l e that e i t h e r supplementation of one or more other e s s e n t i a l amino acids i n s t e a d of t h i s t o t a l amino a c i d s mixture or r a i s i n g the t o t a l n i t r o g e n l e v e l w i t h another source of n i t r o g e n could give b e t t e r performance. T h i s , however, was not i n v e s t i g a t e d i n t h i s experiment. - 120 -V I I I . GENERAL CONCLUSIONS These experiments i n the p i g s and the r a t s have shown threonine to be the second l i m i t i n g amino a c i d i n b a r l e y , w i t h l y s i n e b e i n g the f i r s t . The t r e n d showed th a t the a d d i t i o n of graded l e v e l s of L-threonine to the b a r l e y - l y s i n e d i e t (0.75% t o t a l l y s i n e ) p r o g r e s s i v e l y improved the r a t e of growth, feed conversion . e f f i c i e n c y , carcass q u a l i t y and n i t r o g e n r e t e n t i o n f o r the g r o w i n g - f i n s i h i n g pigs to a l e v e l of 0.10% threonine. Threonine a d d i t i o n at a l e v e l of 0.10% to the d i e t (0.47% t o t a l threonine) produced the best mean d a l l y g a i n , feed conversion e f f i c i e n c y , eye muscle index and eye muscle area. Further a d d i t i o n of threonine and threonine p l u s methionine d i d not. improve the above c r i t e r i a . However, a l l amino a c i d supplemented d i e t s gave growth r a t e s which were i n f e r i o r to that obtained w i t h the soybean c o n t r o l d i e t p r i o r 45 Kg l i v e w e i g h t but not s i g n i f i c a n t l y d i f f e r e n t over 45 Kg l i v e w e i g h t . The n i t r o g e n balance study showed th a t 0.15% threonine a d d i t i o n gave n i t r o g e n r e t e n t i o n i n s i g n i f i c a n t l y d i f f e r e n t from other threonine added d i e t s and from the c o n t r o l d i e t although the c o n t r o l s t i l l gave greater n i t r o g e n r e t e n t i o n . The b a r l e y - l y s i n e d i e t w i t h no threonine a d d i t i o n gave s i g n i f i c a n t l y poorer performance i n growht r a t e , feed conver-s i o n e f f i c i e n c y , carcass q u a l i t y and n i t r o g e n r e t e n t i o n than the threonine supplemented d i e t s and the c o n t r o l d i e t . The o v e r a l l r e s u l t s of the p i g experiments suggested that 0.10% threonine added to an 0.75% l y s i n e - b a r l e y d i e t was comparable to the c o n t r o l d i e t a f t e r 45 Kg l i v e -weight. However, a s l i g h t i n c r e a s e i n the t o t a l l y s i n e and threonine l e v e l s beyond 0.75% and 0.47%, r e s p e c t i v e l y , or p r o v i s i o n of a d d i t i o n a l - 121 -n i t r o g e n may be able to reduce the d i f f e r e n c e between the barley-amino a c i d and barley-soybean meal d i e t s . More work i s needed to c l a r i f y t h i s p o i n t . The r a t experiments showed that 0.75% l y s i n e was adequate f o r the growing r a t s i n c e no s i g n i f i c a n t improvement was obtained by f u r t h e r a d d i t i o n of l y s i n e (to 0.90% t o t a l l y s i n e ) . S i m i l a r to the r e s u l t s of the p i g experiment, the mean d a i l y g a i n , feed conversion e f f i c i e n c y , p r o t e i n e f f i c i e n c y r a t i o and n i t r o g e n r e t e n t i o n improved p r o g r e s s i v e l y w i t h the a d d i t i o n of graded threonine l e v e l s . The 0.10% added threonine (0.47% t o t a l threonine) apparently provided an adequately balanced d i e t f o r r a t growth. However, 0.20% threonine had to be added to the d i e t (0.57% t o t a l threonine) when the d i e t was f u r t h e r supplemented w i t h a p u r i f i e d e s s e n t i a l amino a c i d mixture i n order to o b t a i n optimum r a t performance e q u i v a l e n t to that obtained on the barley-soybean d i e t . G l y c i n e could not be used as the n i t r o g e n source to r e p l a c e the amino a c i d mixture. The evidence of the present study s t r o n g l y i n d i c a t e s t h a t p u r i f i e d amino a c i d s are as e f f e c t i v e as n a t u r a l p r o t e i n concentrates when added to b a r l e y f o r the f i n i s h i n g p i g and the growing r a t . F i n e adjustment of the balance among amino a c i d s , e s p e c i a l l y between the f i r s t and second l i m i t i n g amino a c i d s i s needed to achieve o p t i m a l growth. - 122 -IX. LITERATURE CITED Abernathy, R.P., S e w e l l , R.F. and Tarpley, R.L. 1958. I n t e r r e l a t i o n s h i p s of p r o t e i n , l y s i n e and energy i n d i e t s f o r growing swine. J . Anim. S c i . 17: 635. A d i b i , S.A., Seymour, G.J. and Menden, E. 1967. The k i n e t i c s of amino a c i d a b s o r p t i o n and a l t e r a t i o n of plasma composition of f r e e amino acids a f t e r i n t e s t i n a l p e r f u s i o n of amino a c i d mixtures. Am. J . C l i n . Nutr. 20: 24. A.R.C. 1967. The N u t r i e n t Requirements of Farm L i v e s t o c k No. 3. P i g s . T e c h n i c a l Review and Summaries. A g r i c u l t u r a l Research C o u n c i l , London. Almquist, N.J. 1972. P r o t e i n s and Amino Acids i n Animal N u t r i t i o n . S.B. Penick & Co., A g r i b i s t i c s - N u t r i t i o n a u n i t of CPC I n t e r n a -t i o n a l Inc. New York. 5th E d i t i o n . A.O.A.C. 1965. O f f i c i a l Methods of A n a l y s i s (10th Ed.). A s s o c i a t i o n of O f f i c i a l A g r i c u l t u r a l Chemists. Washington, D.C. Anderson, G.H. and Bowland, J.P. 1967. L y s i n e and f a t supplementation of weanling p i g d i e t s . Can. J . of Anim. S c i . 47: 47 Ashton, G.C., K a s t e l i c , J . , A r c k e r , D.C, Jensen, A.H., Maddock, H.H., K l i n e , E.A. and C a r t r o n , D.V. 1955. D i f f e r e n t p r o t e i n l e v e l s w i t h and without a n t i b i o t i c s f o r g r o w i n g - f i n i s h i n g swine : e f f e c t on carcass leaness. J . Anim. S c i . 14: 82. B a l d i n i , J.T., Marvel, J.P. and Rosenberg, H.R. 1957. The e f f e c t of the p r o d u c t i v e energy l e v e l of the d i e t on the methionine requirement of the p o u l t . P o u l t r y S c i . 36: 1037. B a l d i n i , J.T. and Rosenberg H.R. 1955. The e f f e c t of p r o d u c t i v e energy l e v e l of the d i e t on the methionine requirement of the c h i c k . P o u l t r y S c i . 34: 1301. B a l d i n i , J.T. and Rosenberg, H.R. 1957. The e f f e c t of c a l o r i e source i n a c h i c k d i e t on growth, feed u t i l i z a t i o n , and body composition. P o u l t r y S c i . 36: 432. Bayley, H.S. and Summers, J.D. 1968. E f f e c t of p r o t e i n l e v e l and l y s i n e and methionine supplementation on the performance of growing p i g s : Response of d i f f e r e n t sexs and s t r a i n s of p i g s . Can. J . Anim. S c i . 48: 181. - 123 -Beames, R.M., D a n i e l s , L . J . and S e w e l l , J.O. 1968. The t o t a l replacement of p r o t e i n supplements by s y n t h e t i c l y s i n e i n r a t i o n s based on sorghum g r a i n f o r pigs over 45 Kg l i v e w e i g h t . Proc. Aust. Soc. Anim. Prod. 7: 391. Beames, R.M. and Pepper, P.M. 1969. The p a r t i a l replacement of soybean meal amino a c i d i n p i g r a t i o n s based on wheat and sorghum. Aust. J . Exp. Agr. Anim. Husb. 9: 400. Becker, D.E. 1958. The Feed-bag Red Book Buyer's Guide. Milwaukee, Wis., U.S.A., E d i t o r i a l S e r v i c e s Co. pp. 157. Becker, D.E. 1959. B a l a n c i n g Swine Rations. U n i v e r s i t y of I l l i n o i s . C o l l . of A g r i . C i r c . 811. Becker, D.E., Jensen, A.H. and Harmon, B.G. 1963. B a l a n c i n g Swine R a t i o n s . U n i v e r s i t y of I l l i n o i s . C o l l . of Agr. C i r . 866. Becker, D.E., Jensen, A.H. and Harmon, B.G. 1966. B a l a n c i n g Swine R a t i o n s . 111. Agr. Exp. S t a . C i r . 866. Becker, D.E., A.H. Jensen, S.W. T e r r i l l , l.D. Smith and H.W. Norton. 1957. The i s o l e u c i n e requirement of weanling swine fed two p r o t e i n l e v e l s . J . Anim. S c i . 16: 26. Becker, D.E., Adams, CR. , T e r r i l l , S.W. and Meade, K.J. 1955b. The methi o n i n e - c y s t i n e need of the young p i g . J . Anim. S c i . 14: 1086. Becker, D.E., L a s s i t e i , J.W., T e r r i l l , S.W. and Norton, H.W. 1954b. Levels of p r o t e i n i n p r a c t i c a l r a t i o n s f o r the p i g . J . Anim. S c i . 13: 611. Becker, D.E., N o t z o l t , R.A., Jesen, A.N., T e r r i l l , S.W. and Norton, H.W. 1955a. The tryptophan requirement of the young p i g . J . Anim. S c i . 14: 664. Becker, D.E., U l l r e y , D.E. and T e r r i l l , S.W. 1954a. P r o t e i n and amino a c i d i n t a k e s f o r optimum growth r a t e i n the young p i g . J . Anim. S c i . 13: 346. Becker, M. and Ha r n i s c h , S. 1958. Recent s t u d i e s on the s i g n i f i c a n c e and exactness of n i t r o g e n balances i n metabolism t r i a l s on l i v i n g animals. 1. Studies on the c o n t r o l of a n a l y t i c a l procedures, e s p e c i a l l y n i t r o g e n analyses. Arch. Tierernahrung 8: 309. Beeson, W.M., Jackson, H.D. and Mertz, E.T. 1953. Q u a n t i t a t i v e threonine requirement of the weanling p i g . J : Anim. S c i . 12: 870. - 124 -Beeson, W.M., Mertz, E.T. and Shelton, D.C. 1948. The amino a c i d r e q u i r e -ment of swine. 1. Tryptophan. Science 107: 599. Beeson, W.M., Mertz, E.T. and Shelton, D.C. 1949. E f f e c t of tryptophan d e f i c i e n c y i n the p i g . J . Anim. S c i . 8: 532. B e l l , J.M. 1965. N u t r i e n t requirement of Canadian Y o r k s h i r e swine. V. A study of the l y s i n e and p r o t e i n requirement of f i n i s h i n g p igs weighing from 100 to 200 pounds. Can. J . Anim. S c i . 45: 105. B e l l , J.M. and Voldeng, L.O. 1968. F u r t h e r observations on l y s i n e and p r o t e i n requirements of 23 to 89 Kg p i g s . Can. J . Anim. S c i . 48: 251. B e l l , J.M., W i l l i a m , H.H., L o o s l i , J.K. and Maynard, L.A. 1950. The e f f e c t of methionine supplementation of a soybean o i l meal p u r i f i e d r a t i o n f o r growing p i g s . J . N u t r i t i m . 40: 551. Benton, D.A., Harper, A.E., Spivey, H.E. and Elvehjem, C.A. 1956. Leucine, i s o l e u c i n e and v a l i n e r e l a t i o n s h i p i n the r a t . Arch Biochem. Biophys. 60: 147. Bergen, W.G. and P u r s e r , D.B. 1968. E f f e c t of feeding d i f f e r e n t p r o t e i n sources on plasma and gut amino a c i d s i n the growing r a t . J . Nutr. 95: 333. Blackburn, S. 1968. Amino A c i d Determination. Methods and Techniques. Ma r c e l Dekker, Inc., New York. B l a i r , R., Dent, J.B., E n g l i s h , P.R. and Raeburn, J.R. 1969a. P r o t e i n , l y s i n e and feed i n t a k e l e v e l e f f e c t s on p i g growth. 1. Main e f f e c t s . J . Agr. S c i . 72: 379. B l a i r , R., Dent, J.B., E n g l i s h , P.R. and Raeburn, J.R. 1969b. P r o t e i n , l y s i n e and feed i n t a k e e f f e c t s on p i g growth. 2. E f f e c t s on carcass composition and q u a l i t y . J . Arg. S c i . 73: 395. Bl o c h , R.O., F r o s e t h , J.A. and Kromann, P.R. 1972. Energy and feeding value of wheat and b a r l e y . J . Anim. S c i . 35: 211 ( A b s t r . ) . Boomgaard, J . and Baker, D.H. 1970. The l y s i n e requirement of the c h i c k at f i v e p r o t e i n l e v e l s . P o u l t r y S c i . 49: 1369. Boomgaard, J . and Baker, D.H. 1971. Tryptophan requirement of growing chi c k s as a f f e c t e d by d i e t a r y p r o t e i n l e v e l . J . Anim. S c i . 33: 595. Boomgaard, J . and Baker, D.H. 1973. Tryptophan requirement of growing p i g s at three l e v e l s of d i e t a r y p r o t e i n . J . Anim. S c i . 36: 303. - 125 -Booth, V.H. 1971. Problem i n the determination of FDNB-available l y s i n e . J . S c i . Fd. A g r i c . 22: 658. Bowland, J.P. 1962. A d d i t i o n of l y s i n e and/or a t r a n q u i l i z e r to low p r o t e i n , soybean meal supplemented r a t i o n f o r growing bacon p i g s . J . Anim. S c i . 21: 852. Bowland, J.P. 1964. The r e l a t i o n s h i p of energy and p r o t e i n i n the d i e t on the growth and energy e f f i c i e n c y of young p i g s . Seminaire I n t e r n a t i o n a l Organise" par L ' l n s t i t u t N a t i o n a l De La Recherche Agronomique. Bowland, J.P. and Berg, R.T. 1959. Inf l u e n c e of s t r a i n and sex on the r e l a t i o n s h i p of p r o t e i n to energy i n the r a t i o n s of growing and f i n i s h i n g bacon p i g s . Can. J . Anim. S c i . 39: 102. Bowland, J.P. and Grimson, R.E. 1969. Lysine and methionine supplementa-t i o n of swine and r a t d i e t s c o n t a i n i n g up to three percent urea. Wissen S c h a f t l i c h e Z e i t s c h r i f t der U n i v e r s i t a t Rostock T e i l I I . p. 213. Bragg,-D.B., Ivy, C.A. and Stephenson E.L. 1969. Methods f o r determining — a m i n o a c i d a v a i l a b i l i t y of feeds. P o u l t r y S c i . 48: 2135. Braude, R. and Esnaola, M.A. 1973. Methionine plus c y s t i n e requirement of growing p i g s performance, N r e t e n t i o n and carcass composition of growing pigs given s e m i - p u r i f i e d d i e t s supplemented w i t h graded l e v e l s of DL-methionine. Br. J . Nutr. 30: 437. Braude, R. and Lerman, P. 1970. P r o t e i n and l y s i n e l e v e l s i n p r a c t i c a l r a t i o n s . J . Agr. S c i . 74: 575. Braude, R., M i t c h e l l , K.G., Myres, A.W. and Newport, M.J. 1972. The replacement of p r o t e i n concentrates by s y n t h e t i c l y s i n e i n the d i e t of growing p i g s . Br. J . Nutr. 27: 169. Bravo, F.O., Meade, R.J., S t o c k l a n d , W.L. and Nordstrom, J.W. 1970. Reevaluation of the i s o l e u c i n e requirement of the growing p i g -plasma f r e e i s o l e u c i n e as a response c r i t e r i o n . J . Anim. S c i . 31: 1137. B r e e s a n i , R. 1971. Amino a c i d supplementation of c e r e a l g r a i n f l o u r s t e s t e d i n c h i l d r e n . In Amino A c i d F o r t i f i c a t i o n of P r o t e i n Foods. E d i t e d by N.S. Scrimshaw and A.M. A l t s c h u l . The MIT P r e s s . B r i n e g a r , M.J., L o o s l i , J.K., W i l l i a m s , H.H. and Maynard, L.A. 1949. L y s i n e requirement f o r growing p i g s . Fed. Proc. 8: 379. - 126 -Br i n e g a r , M.J., W i l l i a m s , H.H., F e r r i s , F.E., L o o s l i , J.K. and Maynard, L.A. 1950a. The l y s i n e requirement f o r the growth of swine. J . Nutr. 42: 129. Br i n e g a r , M.J., W i l l i a m s , H.H., F e r r i s , F.H., L o o s l i , J.K. and Maynard, L.A. 1950b. L y s i n e requirement f o r growing swine. Fed. Am. Soc. Expt. B i o l . 9: 353. Brooks, C.C. and Thomas, H.R. 1959. Supplements to peanut o i l meal p r o t e i n f o r growing f a t t e n i n g swine. " J . Anim. S c i . 18: 1119. Brown, H.W., Harmon, B.G,. .and Jensen, A..H. 1973a. The l y s i n e requirement of the f i n i s h i n g p i g f o r maximum r a t e of gain and feed e f f i c i e n c y . J . Anim. S c i . 37: 708. Brown, H.W., Harmon, B.G. and Jensen, A.H. 1973b. Lysine requirement of the f i n i s h i n g p i g f o r maximum carcass leanness. J . Anim. S c i . 37: 1159. Brown, H.W., Harmon, B.G. and Jensen, A.H. 1974. T o t a l s u l f u r c o n t a i n i n g amino acid s i s o l e u c i n e and tryptophan requirements of the f i n i s h i n g p i g f o r maximum n i t r o g e n r e t e n t i o n . J . Anim. S c i . 38: 59. Broxra, J.A. and C l i n e , T.R. 1974. Urea e x c r e t i o n i n the p i g : an i n d i c a t o r of p r o t e i n q u a l i t y and amino a c i d requirements. J . Nutr. 104: 542. Buck, S.F., H a r r i n g t o n , G. and Johnson, R.F. 1962. The p r e d i c t i o n of l e a n percentage of pigs of bacon weight from carcass measurements. Anim. Prod. 4: 25. Bujard, E., Handwerik, V. and Mauron, J . 1967. The d i f f e r e n t i a l determina-t i o n of l y s i n e i n heated m i l k . 1. I n v i t r o methods. J . S c i . Fd. A g r i c . 18: 52. C a h i l l y , Jr.,G.M., M i l l e r , R.F., K e l l y , R.F. and Brooks, C.C. 1963. E f f e c t of v a r i o u s l e v e l s of d i e t a r y l y s i n e on c e r t a i n blood phenomena, muscle development, and muscle-protein b i o l o g i c a l value of growing swine. J . Anim. S c i . 22: 726. Canada Depqrtment of A g r i c u l t u r e . 1968. Canada's new hog carcass v a l u a t i o n system. L i v e s t o c k D i v i s i o n , P r o d u c t i o n and Maketing Branch, Ottawa, Ontario. Cannon, P.R., S t e f f e e , C H . , F r a z i e r , L.E., Rawley, D.A. and Stepto, R.C 1947. The i n f l u e n c e of time of i n g e s t i o n of e s s e n t i a l amino acids upon u t i l i z a t i o n i n t i s s u e s y n t h e s i s . F e d e r a t i o n Proc. 6: 390. Calhoun, W.K., Hepburn, F.N. and Bradley, W.D. 1960. The a v a i l a b i l i t y of l y s i n e i n wheat, f l o u r , bread and g l u t e n . J . Nutr. 70: 337. - 127 -Carlson, K.H. and Bayley, H.S. 1970. Nitrogen and amino acids i n the feces of young pigs r e c e i v i n g a p r o t e i n - f r e e d i e t and d i e t s containing graded l e v e l s of soybean o i l meal or casein. J . Nutr. 100: 1353. Carpenter, K.J. 1960. The estimation of a v a i l a b l e l y s i n e i n animal p r o t e i n food. Biochem. J. 77: 604. Carpenter, K.J., McDonald, I. and M i l l e r , W.S. 1972. P r o t e i n q u a l i t y of feeding-stuffs. 5. C o l l a b o r a t i v e studies on the b i o l o g i c a l assay of a v a i l a b l e methionine using chicks. Br. J. Nutr. 27: 7. Change, R.E., Germann, A.F.O., Beeson, W.M. and Mertz, E.T. 1958. E f f e c t of p r o t e i n l e v e l on the l y s i n e requirement of weanling p i g s . J. Anim. S c i . 17: 1161. Chung, A.S. 1973. Amino a c i d supplementation of Peace River barley f or growing-finishing pigs. M.Sc. Thesis. U n i v e r s i t y of B r i t i s h Columbia. Chung, A.S. and Beames, R.M. 19 72. Amino acid supplementation of swine r a t i o n s . J . Anim. S c i . 35: 1103 (Abstr.). Chung, A.S. and Beames, R.M. 1974. Lysine, threonine, methionine and i s o l e u c i n e supplementation of Peace River barley f or growing pigs. Can. J . Anim. S c i . ( i n p r e s s ) . Clark, A.J., Hays, V.W., McCall, J.T. and Speer, V.C. 1963. Free amino acids i n the plasma and i n t e s t i n a l contents of baby pigs during d i g e s t i o n . J . Anim. S c i . 22: 1118 (Abstr.). Clausen, H. et. a l . 1959, 1960, 1961 and 1962. Experiments with p i g s . Reports at the annual meetings of the National P^esearch I n s t i t u t e of Animal Hunbandry, Copenhagen. Clawson, A.J. 1967. Influence of protein level,,amino acid r a t i o n and c a l o r i c density of the d i e t on feed intake and performance of pigs. J . Anim. S c i . 26: 328. Combs, G.E., Conness, R.G., Berry, T.H. and Wallace, H.D. 1967. E f f e c t of raw and heated soybeans on gain, n u t r i e n t d i g e s t i b i l i t y , plasma amino acids and other constituents of growing pigs. J . Anim. S c i . 26: 1067. Cooke, R., Lodge, G.A. and Lewis, D. 1972a. Influence of energy and p r o t e i n concentration i n the d i e t on the performance of growing pigs. 1. Response to p r o t e i n intake on high-energy d i e t . Anim. Prod. 14: 35. Cooke, R., Lodge, G.A. and Lewis, D. 1972b. Influence of energy and p r o t e i n concentration i n the d i e t on the performance of growing p i g s . 3. Response to d i f f e r e n c e s i n l e v e l s of both energy and prot e i n . Anim Prod. 14: 219. - 128 -Coulson, R.A. and Hernandez, T. 1970. P r o t e i n d i g e s t i o n and amino a c i d a b s o r p t i o n i n the cayman. J . Nutr. 100: 810. C u r t i n , L.V., Abraham, J.., W i l l i a m s , H.H. , L o o s l i , J.R. and Maynard, L.A. 1952b. The methionine requirement f o r the growth of swine. J . Nutr. 48: 499. C u r t i n , L.V., L o o s l i , J.K., W i l l i a m , J.P. and W i l l i a m s , H.H. 1952a. Methionine as a supplement to soybean o i l meal f o r weanling p i g s . J . Anim. S c i . 11: 459. Davidson, R.M., Young, L.G. and Thomas, 0.0. 1962. E f f e c t of l y s i n e a d d i t i o n s to a b a r l e y , b a r l e y soybean meal r a t i o n f o r growing and f a t t e n i n g swine. J . Anim. S c i . 21: 670 ( A b s t r . ) . D e v i l a t , J . , Pond, W.G. and Walker, E.F., J r . 1970. D i e t a r y amino a c i d balance i n g r o w i n g - f i n i s h i n g pigs : e f f e c t on plasma amino a c i d c o n c e n t r a t i o n . Nutr. Report I n t e r n a t i o n a l 2: 257. Delhumeau, G., V e l e z P r a t t , G. and G i t l e r , C. 1962. The a b s o r p t i o n of amino a c i d mixtures from s m a l l i n t e s t i n e of the r a t . I . Equimolar mixtures and those s i m u l a t i n g egg albumin, c a s e i n and z e i n . J . Nutr. 77: 52. D'Mello, J.P.F. 1973. Amino a c i d i n t e r a c t i o n s i n p o u l t r y n u t r i t i o n . 4th Europ. P o u l t . Conf., London, p.331. D'Mello, J.P.F. 1974. Plasma concentrations and d i e t a r y requirements of l e u c i n e , i s o l e u c i n e and v a l i n e : Studies w i t h the young c h i c k . J . S c i . Fd. A g r i c . 25: 187. D'Mello, J.P.F. and Lewis, D. 1970a. Amino a c i d i n t e r a c t i o n i n c h i c k n u t r i t i o n . 1. The i n t e r r e l a t i o n s h i p between l y s i n e and a r g i n i n e . Br. P o u l t . S c i . 11: 299. D'Mello, J.P.F. and Lewis, D. 1970b. Amino a c i d i n t e r a c t i o n i n c h i c k n u t r i t i o n . 2. The i n t e r r e l a t i o n s h i p between l e u c i n e , i s o l e u c i n e and v a l i n e . Br. P o u l t . S c i . 11: 313. D'Mello, J.P.F. and Lewis, D. 1970c. Amino a c i d i n t e r a c t i o n i n c h i c k n u t r i t i o n . 3. The interdependence i n amino a c i d requirements. • Br. P o u l t . S c i . 11: 367. E r i c s o n , L.E. 1961. A c r i t i c i s m of the carcass a n a l y s i s procedure f o r the determination of amino a c i d requirements. A c t a . P h y s i o l . Scand. 52: 90. E r i c s o n , L.E. , Larsson, S. and Ostholm, CO. 1962. P r e l i m i n a r y s t u d i e s on the p r o t e i n value of c e r e a l s f o r swine by l y s i n e supplementation. A c t a . Agr. Scand. 12: 157. - 129 -Evans, R.E. 1958. N u t r i t i o n of bacon p i g . XIX. The requirement of the bacon p i g f o r c e r t a i n e s s e n t i a l amino a c i d s . J . A g r i c . S c i . Camb. 50: 230. Evans, R.E. 1959. The minimum amount of w h i t e - f i s h meal r e q u i r e d to supplement the p r o t e i n s i n some vegetable p r o t e i n concentrates. J . A g r i c . S c i . Camb. 53: 230. Evans, R.E. 1960. The e f f e c t of adding l y s i n e and methionine to the d i e t of p i g s kept on lo w - p r o t e i n vegetable foods. J . A g r i c . S c i . Camb. 54: 266. Evans, R.E. 1962. The i s o l e u c i n e requirement of the weanling p i g s . J . A g r i c . S c i . Camb. 58: 413. Evans, R.E. 1963. The threonine requirement of the weanling p i g s . J . A g r i c . S c i . Camb. 60: 259. F i s h e r , H. 1965. Fu r t h e r s t u d i e s on the l i m i t i n g amino acid s i n d i f f e r e n t l y processed groundnut meal. J . S c i . Fd. A g r i c . 16: 390. F i n o t , P.A. 1973. Non-Enzymic Browing. I n 'Proteins i n human n u t r i t i o n ' . E d i t e d by P o r t e r , J.W.G. and R o l l s , B.A. Academic Press, London and New York. p. 501. Firnney, D.H. 1964. S t a t i s t i c a l Method i n B i o l o g i c a l Assay. 2nd e d i t i o n by Charles G r i f f i n and Co. L t d . London. F l o r e n t i n o , R.F. and Peason, W.N. 1962. E f f e c t of threonine induced amino a c i d imbalance on the e x c r e t i o n of t r y p t o p l a n metabolites by the r a t . J . Nutr. 78: 101. Forbes, R.M. and Rao, T. 1959. The e f f e c t of age on the net requirements of n i t r o g e n , l y s i n e and tryptophan by w e l l - f e d r a t . Arch. Biochem. Biophys. 82: 348. Ford, J.E. 1962. A m i c r o b i o l o g i c a l method f o r a s s e s s i n g the n u t r i t i o n a l value of p r o t e i n s . 2. The measurement of ' a v a i l a b l e ' methionine, : l e u c i n e , i s o l e u c i n e , a r g i n i n e , h i s t i d i n e , tryptophan and v a l i n e . B r . J . Nutr. 16: 409. Ford, J.E. 1960. A m i c r o b i o l o g i c a l method f o r a s s e s s i n g the n u t r i t i o n a l v a l u e of p r o t e i n s . B r . J . Nutr. 14: 485. Frape, D.L., Wolf, K.L., W i l k i n s o n , J . and Cubb, L.G. 1968. M o d i f i c a t i o n to s h i n f i e l d metabolism c r a t e . J . I n s t . Anim. Tech. 19: 61. F u l l e r , M.F. and Boyne, A.W. 1971. The e f f e c t s of environmental tempera-tur e on growth and metabolism of pigs given d i f f e r e n t amounts of food. 1. Nitrogen metabolism, growth and body composition. B r i t . J . Nutr. 25: 259. - 130 -G a l l o , J.T. and Pond, VI.G. 1968. Amino a c i d supplementation to a l l - c o r n d i e t s f o r p i g s . J . Anim. S c i . 27: 73. Germann, A.F.O., Mertz, E.T. and Beeson, W.M. 1958. Reevaluation of the L - l y s i n e requirement of weanling p i g . J . Anim. S c i . 17: 52. G i o v a n e t t i , P.M., S t o t h e r s , S.C. and Parker, R.J. 1970. Coprophagy p r e v e n t i o n and a v a i l a b i l i t y of amino acids i n wheat f o r the growing r a t . Can. J . Anim. S c i . 50: 269. Grau, C.R. and C a r r o l l , R.W. 1958. E v a l u a t i o n of P r o t e i n Q u a l i t y i n Processed P l a n t P r o t e i n F o o d s t u f f s , p. 153-189. A.M. A l t s c h u l ( E d i t o r ) . Academic P r e s s , New York. Halm, J . and Le. CD. 1974. UBC MFAV. A n a l y s i s of v a r i a n c e / c o v a r i a n c e . Computing Centre. The U n i v e r s i t y of B r i t i s h Columbia, Canada. Harper, A.E. 1959. Advances i n our knowledge of p r o t e i n and amino a c i d requirements. Feb. Proc. Supplement. No. 3, P a r t I I . 104. Harper, A.E. 1961. Amino a c i d imbalance, chemical score and e f f i c i e n c y of p r o t e i n u t i l i z a t i o n . Nat. Acad. Sci.-Nat. Res. Counc. Washington, D.C. P u b l . No. 843. p. 443-450. Harper, A.E. 1964. Amino A c i d T o x i c i t i e s and Imbalances. In Mammalian P r o t e i n Metabolism. Academic Press, New York. N.Y. Harper, A.E., Beaton, D.A., Wineje, M.E. and Elvehjem, C.A. 1954. Leucine-i s o l e u c i n e antagonism i n the r a t . Arch. Biochem. Biophys. 51: 523. Hartsook, E.W. and M i t c h e l l , H.H. 1956. The e f f e c t of age on the p r o t e i n and methionine requirements of the r a t . J . Nutr. 60: 173. Henry, Y. and R£rat, A. 1970. E s t i m a t i o n du b e s o i n en threonine chez l e pore femalle entre 20 e t 50 kg de poids v i f . Journees de l a Recherche Porcine en France. P a r i s . INRA, p. 73. Henry, Y., R£rat, A. and Tomassone, B. 1971. L y s i n e requirement of the g r o w i n g - f i n i s h i n g p i g . A p p l i c a t i o n of the m u l t i v a r i a t e s t a t i s t i -c a l a n a l y s i s . Annales de Zootechnie 20: 521. Hetzer, H.O., Harvey, W.R. and P e t e r s , W.H. 1963. G e n e t i c s . Proceedings of the 11th I n t e r n a t i o n a l Congress, The Hagire, The Nethelands. 1: 268. H i n t z , H.F. and lleitman, H. J r . 1967. Amino a c i d and v i t a m i n supplementa-t i o n to barley-cottonseed meal d i e t s f o r g r o w i n g - f i n i s h i n g swine. J . Anim. S c i . 26: 474. - 131 -H u g l i , T.E. and Moore, S . 1972. Determination of the tryptophan content of p r o t e i n s by i o n exchange chromatography of a l k a l i n e h y d r o l y -s a t e s . J . B i o l . Chem. 247: 2828. Jensen, A.H., Becker, D.E. and Harmon, B.G. 1965. N u t r i t i o n a l adequacy of mil o f o r the f i n i s h i n g p i g . J . Anim. S c i . 24: 398. Jones, A.S., Cadenhead, A. and L i v i n g s t o n e , R.M. 1968. V a r i a t i o n i n the composition of b a r l e y and i t s e f f e c t on the performance of p i g s . J . S c i . Fd. A g r i c . 19: 446. Jones, A.S., Hepburn, W.R., Cadenhead, A. and Boyne, A.W. 1962. The e f f e c t of v a r i a t i o n of p r o t e i n q u a l i t y and p r o t e i n l e v e l i n d i e t s on the performance of young p i g s . Anim. Prod. 4: 185. Jones, J.D. 1964 L y s i n e - a r g i n i n e antagonism i n the c h i c k . J . Nutr. 84: 313. Jurgens, M.H., Hudman, D.B., Adams, C H . and Peo, E.R., J r . 1967. In f l u e n c e of d i e t a r y supplement of l y s i n e fed at two l e v e l s of p r o t e i n on growth, feed e f f i c i e n c y and carcass c h a r a c t e r i s t i c s of swine. J . Anim. S c i . 26: 323. K e i t h , M.O., Ch r i s t e n s e n , D.A. and Owen, B.D. 1972. Determination of the methionine requirement of growing p i g s u s i n g serum f r e e amino a c i d s . Can. J . Anim. S c i . 52: 163. Koh l e r , G.O. and P a l t e r , R. 1967. Studies on methods f o r amino a c i d a n a l y s i s of wheat products. Cereal Chem. 44: 512. K r a t z e r , F.H. 1944. Amino a c i d a b s o r p t i o n and u t i l i z a t i o n i n the c h i c k . J . B i o l . Chem. 153: 237. Kroening, G.H., Pond, W.G. and L o o s l i , J.K. 1962. Methionine requirement of the s u c k l i n g age p i g . Summary of Research Swine D i v i s i o n . Department of Animal Husbandry, C o r n e l l U n i v e r s i t y . Kroening, G.H., Pond, W.G. and L o o s l i , J.K. 1965. D i e t a r y methionine-c y s t i n e requirement of the baby p i g as a f f e c t e d by threonine and p r o t e i n l e v e l s . J . Anim. S c i . 24: 519. Laxv-rence, T.L.J. 1971. H i g h - l e v e l c e r e a l d i e t s f o r the g r o w i n g / f i n i s h i n g p i g . V. A comparison of f i n i s h e r d i e t s c o n t a i n i n g h i g h l e v e l of maize on b a r l e y w i t h wide or narrow c a l o r i e / p r o t e i n / l y s i n e r a t i o n s when fed to give two d i f f e r e n t c a l o r i e i n t a k e s . J . Agr. S c i . Camb. 73: 433. Lee, C , McBee, J.L., J r . and Horvath, D.T. 1967. D i e t a r y p r o t e i n l e v e l and some carcass t r a i t s . J . Anim. S c i . 26: 490. - 132 -Lerner, J.T. 1968, C o n t r i b u t i o n a l'etude du b e s o i n en l y s i n e , en r e l a t i o n avec l e taux energetique chez l e r a t et l e pore. T h e s i s . Univ. P a r i s . France. Lodge, G.A., Cundy, M.E., Cooke, R. and Lewis, D. 1972a. I n f l u e n c e of energy and p r o t e i n c o n c e n t r a t i o n i n the d i e t on the performance of growing p i g s . 2. D i f f e r i n g n u t r i e n t d e n s i t y at a constant energy: p r o t e i n r a t i o . Anim. Prod. 14: 47. Lodge, G.A., Hardy, B. and Lewis, D. 1972b. I n f l u e n c e of energy and p r o t e i n c o n c e n t r a t i o n i n the d i e t on the performance of growing p i g s . 4. E f f e c t s of sex on response to d i e t a r y p r o t e i n l e v e l . Anim. Prod. 14: 229. Lougnon, J . and B r e t t e , A. 1971. C o n t r i b u t i o n to the e s t i m a t i o n of the threonine requirement of growing p i g s . Ann. Zootech. 20: 398. Manner, M.J. and McCrea, M.R. 1963. Changes i n the chemical composition of sow-reared p i g l e t s d u r i n g the 1st month of l i f e . B r i t . J . Nutr. 17: 495. Mauron, J . 1961. The concept of amino a c i d a v a i l a b i l i t y and i t s b e a r i n g on p r o t e i n e v a l u a t i o n . I n "Progress i n Meeting P r o t e i n Needs of I n f a n t s and Preschool C h i l d r e n " . Proc. Symp. 1960. (L. V o r i s , ed.) p. 425-442. Pubis. Natn. Res. Coun., Wash. No. 843. Mauron, J . , Mottu, F., Bu j a r d , E. and E g l i , R.H. 1955. The a v a i l a b i l i t y of l y s i n e , methionine and tryptophan i n condensed m i l k and m i l k powder. I n v i t r o , d i g e s t i o n s t u d i e s . Archs. Biochem. Biophys. 59: 433. McWard, G.W., Becker, D.E., Norton, H.W., T e r r i l l , S.W. and Jensen, A.H. 1959. The l y s i n e requirement of weanling swine a t two l e v e l s of d i e t a r y p r o t e i n . J . Anim. S c i . 18: 1059. Meade, R.J. 1956a. The i n f l u e n c e of methionine supplementation of 12, 14 and 16 percent p r o t e i n corn-soybean o i l meal d i e t s upon n i t r o g e n balance of growing swine. J . Nutr. 60: 599... Meade, R.J. 1956b. The i n f l u e n c e of tryptophan, methionine and l y s i n e supplementation of. a corn-soybean o i l meal d i e t on n i t r o g e n balance of growing swine. J . Anim. S c i . 15: 288. Meade, R.J., Dukelow, W.R. and Grant, R.S. 1966a. I n f l u e n c e of percent oats i n the d i e t , l y s i n e and methionine supplementation, and of p e l l e t i n g on r a t e and e f f i c i e n c y of gain of growing p i g s and on carcass c h a r a c t e r i s t i c s . J . Anim. S c i . 25: 58. - 133 -Meade, R.J., Dukelow, W.R. and Grand, R.S. 1966b. L y s i n e and methionine a d d i t i o n to corn-soybean meal d i e t s f o r growing swine : E f f e c t s on r a t e and e f f i c i e n c y of gain and carcass c h a r a c t e r i s t i c s . J . Anim. Soc. 25: 78. Meade, R.J., Vermedahl, L.D., Rusk, J.W. andWass, D.F. 1969. E f f e c t s of p r o t e i n content of the d i e t of the young p i g on r a t e and e f f i -ciency of gain during e a r l y development and subsequent to 23.5 kg and carcass composition of le a n t i s s u e . J . Anim. S c i . 28: 473. Mertz, E.T., Beeson, W.M. and Jackson, II.D. 1952. C l a s s i f i c a t i o n of e s s e n t i a l amino acids f o r the weanling p i g . Arch. Biochem. 38: 121. Mertz, E.T., Delong, D.C. and Beeson, W.M. 1955. Histidine and leucine reauirements of the weanling pig. J . Anim. Sci. 14: 1217. Mertz, E.T., Shelton, D.C. and Beeson, W.M. 1949. The amino acid requirement of swine : lysine. J . Anim. Sci. 8: 524. Metta, V.C. and Mitchell, H.H. 1956. A comparison of the biological values of dietary protein incorporated i n high-fat and low-fat diets. J . Nutr. 59: 501. Miner, J . J . , Clower, W.B. and Noland, P.R. 1955. Amino a c i d supplementa-t i o n of a corn-cottonseed meal d i e t f o r growing-fattening swine. J . Anim. S c i . 14: 24. M i t c h e l l , J.R., J r . , Becker,. D.E., Jensen, A.H., Norton, H.W. and Harmon, B.G. 1965a. Ly s i n e need of swine at two stages of development. J . Anim. S c i . 24: 409. M i t c h e l l , J.R., J r . , Becker, D.E., Jensen, A.H., Norton, H.W. and Harmon, B.G. 1965b. C a l o r i c d e n s i t y of the d i e t and the l y s i n e need of growing swine. J . Anim. S c i . 24: 977. M i t c h e l l , J.R., J r . , Becker, D.E., Norton, H.W., Harmon, B.G. and Jensen, A.H. 1968b. Determination of amino a c i d needs of the young p i g by n i t r o g e n balance and plasma f r e e amino a c i d s . J . Anim. S c i . 27: 1327. M i t c h e l l , J.R., J r . , Becker, D.E., Harmon, B.G., Norton, H.W. and Jensen, A.H. 1968a. Some amino a c i d needs of the young p i g fed a s e m i s y n t h e t i c d i e t . J . Anim. S c i . 27: 1322. Moore, S. 1963. On the determination of c y s t i n e as c y s t e i c a c i d . J . B i o l . Chem. 238: 235. - 134 -Mor r i s o n , A.E., Mi d d l e t o n , E.J. and McLaughlan, J.M. 1961. Blood amino a c i d s t u d i e s . I I . E f f e c t s of d i e t a r y l y s i n e c o n c e n t r a t i o n , sex and growth r a t e on plasma f r e e l y s i n e and threonine l e v e l s i n the r a t . Can. J . Biochem. P h y s i o l . 39: 1675. Mo r r i s o n , M.A. and Harper, A.E. 1960. Amino a c i d balance and imbalance. IV. S p e c i f i t y of threonine i n producing an imbalance i n d i e t s d e f i c i e n t i n n i a c i n and tryptophan. J . Nutr. 71: 296. M i i l l e r , Z., K o z e l , V., Bauer, B., Strunc, M. and Moravec, J . 1967c. Enrichment of c e r e a l d i e t s f o r f a t t e n i n g pigs from 35 Kg l i v e -weight w i t h l y s i n e , threonine, tryptophan and methionine. B i o l . Chem. Vyzivy Z v i r a t . 3: 399. M i i l l e r , Z., K o z e l , V., H e j z l a r , Z., Baver, B. and Moravec, J . 1967a. L y s i n e i n combination w i t h another l i m i t i n g amino a c i d i n the n u t r i t i o n of weaned p i g l e t s . B i o l . Chem. V y z i v y Z v i r a t 3: 411. M i i l l e r , Z.and Malek, I . 1967a. Monodiet based on wheat f o r weaned p i g l e t s . B i o l . Chem. Vyzivy Z v i r a t 3: 535. M i i l l e r , Z. and Malek, I . 1967b. Monodiet based on b a r l e y f o r weaned p i g l e t s . B i o l . Chem. Vyzivy Z v i r a t 3: 543. M i i l l e r , Z. and Malek, I . 1967c. Monodiet based on maize f o r weaned p i g l e t s . B i o l . Chem. Vyzivy Z v i r a t 3: 551. M i i l l e r , Z., Palkoska, J . , Drevjany, L. and Moravec, J . 1967b. C e r e a l d i e t s e n r i c h e d w i t h l y s i n e , threonine, tryptophan and methionine i n the n u t r i t i o n of weaned p i g l e t s . B i o l . Chem. Vyzi v y Z v i r a t 3: 423. M i i l l e r , Z. and Rozman, J . 1968. Monocereal d i e t s f o r t i f i e d w i t h l y s i n e , t h r e o n i n e , tryptophan and methionine i n p i g f a t t e n i n g . B i o l . Chem. Vyzi v y Z v i r a t 4: 433. N.A.S.-N.R.C. 1968. N u t r i e n t Requirement of Domestic Animals. 2. N u t r i e n t Requirement of Swine. 6th ed. P u b l . 1599, Washington,.D.C. N.A.S.-N.R.C. 1972. N u t r i e n t Requirements of Domestic Animals. 10. N u t r i e n t Requirements of Laboratory Animals. 2nd E d i t i o n . N a t i o n a l Academy of Sciences, Washington, D.C. Newell, J.A. and Bowland, J.P. 1972. Performance, carcass composition, and f a t composition of boars, g i l t s and barrows fed two l e v e l s of p r o t e i n . Can. J . Anim. S c i . 52: 543. N i e l s e n , A.J. 1971. The d i g e s t i b i l i t y of amino a c i d d i f f e r e n t balanced feed r a t i o n s as r e l a t e d to d i g e s t i b i l i t y of n i t r o g e n i n growing p i g s . A c t a . Agr. Scand. 21: 189. - 135 -N i e l s e n , H.E., Hays, V.W., Speer, V.C. and Catron, D.V. 1963. L y s i n e supplementation of corn- and barley-base d i e t s f o r growing-f i n i s h i n g swine. J . Anim. S c i . 22: 454. Nixon, S.E. and Mawer, G.E. 1970a. The d i g e s t i o n and absorption of p r o t e i n i n man. 1. The s i t e of a b s o r p t i o n . B r i t . J . Nutr. 24: 227. Nixon, S.E. and Mawer, G.E. 1970b. The d i g e s t i o n and abs o r p t i o n of p r o t e i n i n man. 2. The form i n which d i g e s t e d p r o t e i n i s absorbed. B r i t . J . Nutr. 24: 241. O'Dell, B.L., L a e r d a l , O.A., J e f f a y , A.M. and Savage, J.E. 1958. A r g i n i n e metabolism i n the growing c h i c k . P o u l t r y S c i . 37: 817. Oestemer, G.A., Hanson, L.E. and Meade, R.J. 1973. L e u c i n e - i s o l e u c i n e i n t e r r e l a t i o n s h i p i n the young p i g . J . Anim. S c i . 36: 674. Oestemer, G.A., Meade, R.J., Stockland, W.L. and Hanson, L.E. 1970. Methionine supplementation of opaque-2 corn f o r growing swine. J . Anim. S c i . 31: 1133. Oh, S., Summers, J.D. and Wood, A.S. 1972. A v a i l a b i l i t y of methionine i n v a r i o u s p r o t e i n supplements as determined by c h i c k b i o a s s a y . Can. J . Anim. S c i . 52: 171. Olsen, E.M., Summers, J.D. and S l i n g e r , S.J. 1968. B i o l o g i c a l a v a i l a b i l i t y of amino aci d s i n wheat by-products. Can. J . Anim. S c i . 48: 221. Orten, A.V. 1963. Research approaches to amino a c i d n u t r i t i o n . I n t e s t i n a l phase of amino a c i d n u t r i t i o n . Fed. Proc. 22: 1103. Ostrowski, H. 1969. The e f f e c t s of d i e t a r y supplementation w i t h l y s i n e and methionine on body and t i s s u e composition i n p i g . Anim. Prod. 11: 521. Oslage, H.J. 1962. Body composition and a c c r e t i o n of matter by growing f a t t e n i n g pigs and the i n f l u e n c e of n u t r i t i o n . 1. Statement of the problem and methods of study. Z e i t s c h r i f t f u r T i e r p h y s i o l o g i e , Tierernahrung und F u t t e r m i t t e l k u n d e 17: 350. Oslage, H.J., F l i e g e l , H., F a r r i e s , F.E. and R i c h t e r , K. 1966. A c c r e t i o n of N, f a t and energy by growing f a t t e n i n g p i g s . Z e i t h s c h r i f t f u r T i e r p h y s i o l o g i e , Tierernahrung und F u t t e r m i t t e l k u n d e 21: 50. P e l l e t t , P.L. (Ed.) 1963. E v a l u a t i o n of P r o t e i n Q u a l i t y . Pubis. Natn. Res. Coun., Wash. No. 1100. . P i c k , R.T. and Meade, R.J. 1971. Amino a c i d supplementation of opaque-2 corn d i e t s f o r growing r a t s . J . Nutr. 101: 1241. - 136 -P i e r c e , A.B. and Bowland, J.P. 1972. P r o t e i n amino a c i d l e v e l s and sequence i n swine d i e t s : E f f e c t s on gain feed conversion and carcass c h a r a c t e r i s t i c s . Can. J . Anim. S c i . 52: 531. Pond, W.G., H i l l i e r , J.C. and Benton, D.A. 1958. The amino a c i d adequacy of mi l o f o r the growth of r a t s . J . Nutr. 65: 493. Pond, W.G. and Jones, J.R. 1964. Amino a c i d supplementation to corn d i e t s f o r f i n i s h i n g p i g s . C o r n e l l Swine Mimeo. 64-1. Rama Rao, P.B., Metta, V.C. and Johnson, B.C. 1959. The amino a c i d compo-s i t i o n and n u t r i t i v e value of p r o t e i n s . I . E s s e n t i a l amino a c i d requirements of the growing r a t . J . Nutr. 69: 387. Rama Rao, P.B., Metta, V . C, Norton, H.W. and Johnson, B.C. 1960. The amino a c i d composition and n u t r i t i v e value of p r o t e i n s . I I I . The t o t a l p r o t e i n and the n o n e s s e n t i a l amino a c i d requirement. J . Nutr. 71: 361. R6rat, A. 1961. A l i m e n t a t i o n azotee des animaux domestiques. Colloque S c i e n t i f i q u e , P a r i s , France. A s s o c i a t i o n F r a n c a i s e de Zootechnie. Rerat, A. 1972. P r o t e i n n u t r i t i o n and metabolism i n the growing p i g . Nutr. A b s t r . and Rev. 42: 13. R£rat, A. and Henry, Y. 1963. Besoin en l y s i n e du r a t en cr q i s s a n c e : p r i n c i p e d'une methodologie et r e s u l t a t s experimentaux. C.R.. Acad. S c i . , P a r i s . 257: 3045. Rerat, A. and Henry, Y. 1964. The p r o t e i n requirements of the growing p i g 1. The u t i l i z a t i o n of the f i s h meal at three d i f f e r e n t l e v e l s . Ann. Zootech. 13: 5. R£rat, A. and Henry, Y. 1969. Supplementation des c£r£ales par l e s acides amines chez l e pore pendant l a periode de d e f i n i t i o n . Journees de l a Recherche Porcine en France. I.N.R.A., P a r i s , 143. Rerat, A. and Henry, Y. 1970. Sulfur-amino a c i d s need of the growing p i g . Annales Zootech. 19: 486. R£rat, A., Henry, Y. and Desmoulin, B. 1971 The i n f l u e n c e of energy r e s t r i c t i o n on the p r o t e i n need of the female growing p i g . Journees de Recherche P o r c i n e en France, P a r i s . I.N.R.A. p. 65. R£rat, A. and Lougnon, J . 1968. Amino a c i d requirements of growing p i g s . World Review of Animal Production.. 4: 84. R£rat, A. and Lougnon, J . 1965. Supplementation pur l a L - l y s i n e d'un regime v e g e t a l s i m p l i f i e chez l e pore en c r o i s s a n c e . Ann. Zootech. 14: 247. - 137 -Rerat, A., Lougnon, J . and P i o n , R. 1962. Supplementation by DL-methionine of a complex d i e t f o r growing f i n i s h i n g p i g . Ann. Zootech. 11: 159. Rdrat, A., Lerner, J . , Henry, Y. and Bourdon, D. 1970. Etude du beso i n de l y s i n e du pore en cro i s s a n c e en r e l a t i o n avec l e taux energetique du regime. Jourees de l a Recherche Porcine en France. P a r i s . I.N.R.A. p. 79. Rippon, W.P. 1959. Some e r r o r s i n the determination of n i t r o g e n r e t e n t i o n of sheep by n i t r o g e n balance s t u d i e s . B r i t . J . Nutr. 13: 243. Robinson, D.W. 1964. The plane of n u t r i t i o n and compensatory growth i n p i g s . Anim. Prod. 6: 229. Robinson, D.W. 1965 . The p r o t e i n and energy n u t r i t i o n of the p i g . 1. The e f f e c t of v a r y i n g the p r o t e i n and energy l e v e l s i n the ' f i n i s h i n g d i e t ' of heavy p i g s . J . A g r i c . S c i . 65: 405. Robinson, D.W. and Lewis, D. 1963. Amino a c i d supplementation of a b a r l e y r a t i o n f o r the p i g . J . S c i . Fd. Agr. 14: 806. Robinson, D.W. and Lewis, D. 1964. P r o t e i n and energy n u t r i t i o n of the bacon p i g . I I . The e f f e c t of v a r y i n g the p r o t e i n and energy l e v e l s i n d i e t s of ' f i n i s h i n g ' p i g s . J . A g r i c . S c i . 63: 185. Robinson, D.W., Morgan, J.T. and Lewis, D. 1964. P r o t e i n and energy n u t r i t i o n of the bacon p i g . 1. The e f f e c t of v a r y i n g p r o t e i n and energy l e v e l s i n the d i e t s of 'growing' p i g s . J . A g r i c . S c i . 62: 369. Rogers, Q.R., Tannous, R.T. and Harper, A.E. 1967. E f f e c t s of excess l e u c i n e on growth and food s e l e c t i o n . J . Nutr. 91: 561. Rose, W.C. 1938. The n u t r i t i v e s i g n i f i c a n c e of the amino a c i d s . P h y s i o l . Review. 18: 109. Rose, W.C, Smith, L . C , Womack, M. and Shane, M. 1949. The u t i l i z a t i o n of the n i t r o g e n of ammonium s a l t s , urea, and c e r t a i n other compounds i n the sy n t h e s i s of n o n e s s e n t i a l amino acid s i n v i v o . J . B i o l . Chem. 181: 307. Rosenberg, H.R. 1959. Amino a c i d supplementation of foods and feeds. I n P r o t e i n and Amino A c i d N u t r i t i o n . E d i t . A.A. Albanese, Academic P r e s s , New York. p. 381. Rosenberg, H.R., B a l d i n i , J.T., Sunde, M.L., B i r d , H.R. and Runnels, T.D. 1955. The concomitant of f a t and methionine i n b r o i l e r d i e t s . P o u l t r y S c i . 34: 1308. - 138 -Rosenberg, H.R. and C u l i k , R. 1955. Lysine requirement of the growing r a t as a f u n c t i o n of the pr o d u c t i v e energy l e v e l of the d i e t . J . Anim. S c i . 14: 1221. Rosenberg, H.R., C u l i k , R. and E c k e r t , R.E. 1959. Lys i n e and threonine supplementation of r i c e . J . Nutr. 69: 217. Rozman, I . , M u l l e r , Z., Plocek, F. and S i l e r , R. 1968. The i n f l u e n c e of l y s i n e , t h r e o n i n e , tryptophan and methionine supplemented to pi g s fed c e r e a l d i e t without p r o t e i n feeds. B i o l . Chem. Vyzi v y Z v i r a t . 4: 441. Saben, H.S. and Bowland, J.P. 1971. Comparative e v a l u a t i o n of some techniques used i n determination of n i t r o g e n and energy content of feces from p i g s . Can. J . Anim. S c i . 51: 793. Salmon, W.D. 1954. The tryptophan requirement of the r a t as a f f e c t e d by n i a c i n and l e v e l of d i e t a r y n i t r o g e n . Arch. Biochem. Biophys. 51: 30. Sauer, W.C. 1972. A v a i l a b i l i t y of amino aci d s from b a r l e y , wheat, t r i t i -c a l e and soybean meal f o r growing p i g s . M.Sc. Thesis, U n i v e r s i t y of Manitoba, Winnipeg, Manitoba. Scrimshaw, N.S. and A l t s c h u l , A.M. 1971. Amino A c i d F o r t i f i c a t i o n of P r o t e i n Foods. Ed. by Scrimshaw and A l t s c h u l , MIT P r e s s . S e w e l l , R.F., Abernathy, R.P. and Tarpley, R.L. 1956. I n t e r r e l a t i o n s h i p of p r o t e i n , l y s i n e and energy i n d i e t s f o r growing swine. J . Anim. S c i . 15: 1233. Se w e l l , R.F., L o o s l i , J.K., Maynard, L.A., W i l l i a m s , H.H. and Sheffy, B.E. 1952. The q u a n t i t a t i v e threonine requirement of s u c k l i n g p i g . J . Nutr. 49: 435. Shelton, D.C, Beeson, W.M. and Mertz, E.T. 1950. Growth of weanling p i g s on a d i e t c o n t a i n i n g ten p u r i f i e d amino a c i d s . Arch. Biochem. Biophys. 29: 446. Shelton, D.C, Beeson, W.M. and Mertz, E.T. 1951a. The e f f e c t of methionine and c y s t i n e on the growth of weanling p i g s . J . Anim. S c i . 10: 57. Shelton, D.C, Beeson, W.M. and Mertz, E.T. 1951b. Q u a n t i t a t i v e DL-trypto-phan requirement of the weanling p i g . J . Anim. S c i . 10: 73. Shimada, A. and C l i n e , T.R. 1974. Limiting,amino a c i d s of t r i t i c a l e f o r the growing r a t and p i g . J . Anim. S c i . 38: 941. - 139 -Smith, R.C. 1972. A c e t y l a t i o n of methionine s u l f o x i d e and methionine s u l f o n e by the r a t . Biochim. Biophys. Acta. 261: 304. Smith, J . , J r . , Clawson, A.J. and B a r r i c k , E.R. 1967. E f f e c t of r a t i o of p r o t e i n from corn and soybean meal i n d i e t s of v a r y i n g t o t a l p r o t e i n on performance, carcass d e s i r a b i l i t y and d i e t d i g e s t i b i -l i t y i n swine. J . Anim. S c i . 26: 752. Smith, G.H. and Lewis, D. 1966. A r g i n i n e i n p o u l t r y n u t r i t i o n . 3. Agent and t a r g e t i n amino a c i d i n t e r a c t i o n . B r i t . J . Nutr. 20: 621. Snedecor, G.W. and Cochran, W.G. 1967. S t a t i s t i c a l Methods. 6th E d i t i o n . The Iowa St a t e U n i v e r s i t y P r e s s , Ames Iowa, U.S.A. S o l d e v i l a , M. and Meade, R.J. 1964. B a r l e y r a t i o n s f o r swine. I I . The i n f l u e n c e of L - l y s i n e and DL-methionine supplementation of b a r l e y -soybean meal d i e t s upon r a t e and e f f i c i e n c y of gain and upon n i t r o g e n r e t e n t i o n of growing swine. J . Anim. S c i . 23: 394. Sowers, J.E. and Meade, R.J. 1972a. Threonine requirement of the growing p i g . J . Anim. S c i . 35: 224 ( A b s t r . ) . Sowers, J.E. and Meade, R.J. 1972b. E f f e c t of p r o t e i n l e v e l on the th r e o -nine requirement of the p i g . J . Anim. S c i . 35: 224 ( A b s t r . ) . S t andish, J.F. and Bowland, J.P. 1967. E f f e c t s of v a r y i n g d i e t a r y energy, p r o t e i n and amino a c i d l e v e l s on growth, n u t r i e n t d i g e s t i b i l i t y , and serum p r o t e i n l e v e l s of e a r l y weaned p i g s . Can. J . Anim. S c i . 47: 77. Stockland, W.L., L a i , Y.F., Meade, R.J., Sowers, J.E. and Oestemer, G.A. 1971. L-phenylalanine and L - t y r o s i n e requirements of the growing r a t . J . Nutr. 101: 177. Stockland, W.L., Meade, R.J. and M e l l i e r e , A.L. 1970. Lys i n e requirement of the growing r a t : plasma f r e e l y s i n e as a response c r i t e r i o n . J . Nutr. 100: 925. Stuc k i , W.P. and Harper, A.E. 1962. E f f e c t s of a l t e r i n g the r a t i o of i n d i s p e n s a b l e to dispensable amino a c i d s i n d i e t s f o r r a t s . J . Nutr. 78: 278. Sure, B. 1954. P r o t e i n supplementation r e l a t i v e n u t r i t i v e values of p r o t e i n i n whole wheat and whole rye and e f f e c t of amino a c i d supplements. J . Agr. Food Chem. 2: 1008. Sure, B. 1955. R e l a t i v e n u t r i t i v e value of p r o t e i n i n food and supplemen-t a r y value of amino aci d s i n pe a r l e d b a r l e y and peanut f l o u r . J . Agr. Food. Chem. 3: 789. - 140 -Thorbek, G. 1969. Nitrogen metabolism i n growing p i g s . Commission d'Etude de l a Fe d e r a t i o n Europ£ene de Zootechnie, H e l s i n k i , F i n l a n d . Tjong-A-Hung, Hans on, L.E., Rust, J.W. and Meade, R.J. 1972. E f f e c t s of p r o t e i n l e v e l sequence and sex on r a t e and e f f i c i e n c y of gain of growing swine, and on carcass c h a r a c t e r i s t i c s , i n c l u d i n g composition of lean t i s s u e . J . Anim. S c i . 35: 760. Tukey, J.W. 1953. The Problem of M u l t i p l e Comparisons. D i t t o P r i n c e t o n U n i v e r s i t y , P r i n c e t o n , N.J. Welch, J . , Cordts, R.H. and Vander Noot, G.W. 1966. E f f e c t of l y s i n e , methionine and tryptophan supplementation upon n i t r o g e n r e t e n t i o n of barrows. J . Anim. S c i . 25: 806. W i l l i a m s , H.H., C u r t i n , L.V., Abraham, J . , L o o s l i , J.K. and Maynard, L.A. 1954. E s t i m a t i o n of growth requirements f o r amino acid s by assay of the c a r c a s s . J . B i o l . Chem. 141: 375. W i l l i a m s , M.A. and Grau, C.R. 1956. Food i n t a k e and u t i l i z a t i o n of l y s i n e -d e f i c i e n t p r o t e i n by chick i n r e l a t i o n to the d i g e s t i b l e energy c o n c e n t r a t i o n of the d i e t . J . Nutr. 59: 243. Wong, W.C., Boylan, W.J. and S t o t h e r s , S.C. 1968. E f f e c t s of d i e t a r y p r o t e i n l e v e l and sex on swine performance and carcass t r a i t s . Can. J . Anim. S c i . 48: 383. Wood, A.J. and Groves, T.D.D. 1965. Body composition s t u d i e s on the s u c k l i n g p i g . 1. M o i s t u r e , chemical f a t , t o t a l p r o t e i n , and t o t a l ash i n r e l a t i o n to age and body weight. Can. J . Anim. S c i . 45: 8. W r e t l i n d , K.A.J. 1949. The e f f e c t of s y n t h e t i c amino acids e s s e n t i a l f o r growth on the body weight of growing r a t s , and the s y n t h e s i s of the amino acids used. Acta p h y s i o l . Scand. m. Suppl. 59. Young, L.G., Ashton,.G.C., Forshaw, R.P. and Ingram, R.H. 1968. R e l a t i o n -s h i p of d i e t a r y p r o t e i n l e v e l s to performance and carcass m e r i t of market swine. Can. J . Anim. S c i . 48: 71. Van Soest,P.J.1963.Use of detergents i n the a n a l y s i s of f i b r o u s feed. I I . A r a p i d method f o r the determination of f i b r e and l i g n i n . J.A.O.A.C. 46: 829. Veum, W.H., Pfander, W.H. and Bellamy, C.G. 1973. Opaque-2 and normal corn supplemented w i t h soybean-meal and/or amino a c i d f o r growing r a t s . J . Anim. S c i . 37: 63. - 141 -Zimmerman, R.A. and S c o t t , H.M. 1965. I n t e r r e l a t i o n s h i p s of plasma amino a c i d l e v e l s and weight gain i n the c h i c k as i n f l u e n c e d by suboptimal and superoptimal d i e t a r y concentrations of s i n g l e amino a c i d s . J . Nutr. 87: 13. - 142 -X. APPENDIX Table Page 1A Average feed intake (Kg) per pig per day for each diet on dry matter basi§ in Pig Experiment I 145 IB Analysis of variance of average daily feed intake in Pig Experiment I 145 2A Mean daily gain (Kg) from start to 45 Kg body weight of 108 pigs in Pig Experiment I 146 2B Analysis of variance of main daily gain from start to 45 Kg body weight of 108 pigs with starting weight as covariable in Pig Experiment I 147 3A Mean daily gain (Kg) from 45 Kg body weight to finish of 108 pigs in Pig Experiment I 148 3B Analysis of variance of main daily gain from 45 Kg body weight to finish of 108 pigs with no covariable in Pig Experiment I 149 4A Mean daily gain (Kg) from start to finish of 108 pigs in Pig Experiment I 150 4B Analysis of varaince of mean daily gain from start to finish of 108 pigs with starting weight as covariable in Pig Experiment I 151 5A Average feed conversion efficiency ratio per pig (D.M. feed intake (Kg)/weight gain (Kg)) in Pig Experiment I 152 5B Analysis of variance of feed conversion efficiency ratio in Pig Experiment I 152 6A Total back fat (mm) of 107 pigs in Pig Experiment I 153 6B Analysis of variance of total back fat thickness with fi n a l body weight, average body weight gain and body length as covariables in Pig Experiment I 154 - 143 -Table Page 7A Back f a t 40 mm from m i d - l i n e (mm) of 107 pigs i n P i g Experiment I 155 7B A n a l y s i s of v a r i a n c e of back f a t 40 mm from mid l i n e w i t h f i n a l body weight, average d a i l y gain and body length as c o v a r i a b l e s i n P i g Experiment I 156 2 8A Eye muscle area (mm ) of 107 pigs i n P i g Experiment I 157 8B A n a l y s i s of var i a n c e of eye muscle area w i t h f i n a l body weight, average d a i l y gain and body le n g t h as c o v a r i a b l e s i n P i g Experiment I 158 2 9A Eye muscle index ( l e n g t h 'A' x width 'B') (mm ) of the 107 p i g s i n P i g Experiment I 159 9B A n a l y s i s of var i a n c e of eye muscle index ( l e n g t h x width) w i t h f i n a l body weight, average d a i l y g ain and body le n g t h as c o v a r i a b l e s i n P i g Experiment I 160 10A Days on t r i a l s of 108 pigs i n P i g Experiment I 161 10B A n a l y s i s of v a r i a n c e of days on t r i a l s w i t h i n i t i a l body weight as c o v a r i a b l e i n P i g Experiment I 162 11A D.M. feed i n t a k e (g)/wk. i n P i g Experiment I I 163 11B A n a l y s i s of v a r i a n c e of D.M. feed i n t a k e (g)/wk. i n P i g Experiment I I 163 12 A n a l y s i s of v a r i a n c e of N i n t a k e (g)/wk. i n P i g Experiment I I 164 13A Percentage D.M. d i g e s t i b i l i t y i n P i g Experiment I I 165 13B A n a l y s i s of v a r i a n c e of dry matter d i g e s t i b i l i t y i n percent i n P i g Experiment I I 165 14A N i t r o g e n r e t e n t i o n (g)/wk. i n P i g Experiment I I 166 14B A n a l y s i s of va r i a n c e f o r n i t r o g e n r e t e n t i o n i n P i g Experiment I I 166 - 144 -Table Page 14C A n a l y s i s of co-variance of n i t r o g e n r e t e n t i o n w i t h feed i n t a k e as c o v a r i a b l e i n P i g Experiment I I 167 14D A n a l y s i s of co-variance f o r n i t r o g e n r e t e n t i o n w i t h n i t r o g e n i n t a k e as c o v a r i a b l e i n P i g Experiment I I 167 15A Ni t r o g e n absorbed as a percentage of n i t r o g e n i n t a k e i n P i g Experiment I I 168 15B A n a l y s i s of v a r i a n c e f o r n i t r o g e n absorbed as a percentage of n i t r o g e n i n t a k e i n P i g Experiment I I 168 16A Ni t r o g e n r e t a i n e d as a percentage of n i t r o g e n i n t a k e i n P i g Experiment I I 169 16B A n a l y s i s of var i a n c e f o r n i t r o g e n r e t a i n e d as a percentage of n i t r o g e n i n t a k e i n P i g Experiment I I 169 17A N i t r o g e n r e t a i n e d as a percentage of n i t r o g e n absorbed i n P i g Experiment I I 170 17B A n a l y s i s of var i a n c e of n i t r o g e n r e t a i n e d as a percentage of n i t r o g e n absorbed i n P i g Experiment I I 170 18 A n a l y s i s of co-variance f o r average d a i l y g ain and feed conversion e f f i c i e n c y w i t h i n i t i a l weight and d a i l y feed i n t a k e as c o v a r i a b l e s i n Rat Experiments I , I I and I I I 171 19 A n a l y s i s of co-variance f o r PER w i t h i n i t i a l weight as a c o v a r i a b l e i n Rat Experiments I , I I and I I I 172 20 A n a l y s i s of co-variance f o r carcass c h a r a c t e r i s t i c s of r a t s i n Rat Experiments I , I I and I I I 173 - 1 4 5 -Table 1A. Average feed i n t a k e (kg) per p i g per day f o r each d i e t on dry matter b a s i s i n P i g Experiment I . Treatment Rep 1 Rep 2 Rep 3 1 1.986 1.854 1.988 2 1.815 1.768 1.880 3 1.863 1.754 1.899 4 1.886 1.822 2.024 5 1.824 1.755 1.889 6 1.765 1.910 1.894 Table IB. A n a l y s i s of variance of average d a i l y feed i n t a k e i n P i g Experiment I . Source D.F. S.S. M.S. F-value Variance r a t i o F(nec.) P=0.05 P=0.01 T o t a l Treatment Covariable s t a r t i n g weight Resid u a l 17 0.1103 5 0.0389 0.0078 1.8616 1 0.0264 0.0264 6.3247* 11 0.0460 0.0042 3.20 4.82 5.32 9.65 *P < 0.05 - 146 -Table 2A. Mean d a i l y gain (Kg) from s t a r t to 45 Kg body weight of 108 pigs i n P i g Experiment I . Treatment Rep 1 Rep 2 Rep 3 Male Female Male Female Male Female 0.7500 0.6760 0.5561 0.6122 0.3036 0.4658 0.6714 0.5136 0.4680 0.6755 0.5561 0.6071 0.5060 0.5536 0.5442 0.5782 0.5405 0.6490 0.6071 0.6653 0.6403 0.4949 0.4821 0.4506 0.4949 0.4609 0.4797 0.5383 0.5357 0.5012 0.5893 0.5765 0.5578 0.5013 0.5000 0.6327 0.7194 0.5833 0.5893 0.4216 0.3857 0.5867 0.6551 0.5274 0.4913 0.4879 0.5643 0.7296 0.5833 0.4727 0.6276 0.5867 0.5167 0.5910 0.7898 0.6735 0.6505 0.4595 0.4216 0.5214 0.5632 0.5500 0.3866 0.5434 0.5286 0.5459 0.5262 0.5405 0.3583 0.5052 0.5119 0.6020 0.7143 0.6449 0.5310 0.4738 0.5281 0.4390 0.5714 0.5485 0.5095 0.6148 0.6490 0.5655 0.5017 0.5689 0.4524 0.6857 0.4900 0.5281 0.6684 0.5748 0.6378 0.4274 0.4770 0.4512 0.4645 0.5561 0.5918 0.5289 0.5578 0.5714 0.5689 0.4869 0.5408 0.5740 0.5493 0.5918 - 147 -Table 2B. A n a l y s i s of va r i a n c e of mean d a i l y gain from s t a r t to 45 Kg body weight of 108 pigs w i t h s t a r t i n g weight as c o v a r i a b l e i n P i g Experiment I Source D.F. S.S. M.S. F-value Variance (F nec P=0.05 r a t i o ..) P=0.01 T o t a l 107 0.7442 Treatment (a) 5 0.3222 0.0644 19.96** 2.35 3.29 Sex (b) 1 0.0043 0.0043 1.32 3.98 7.01 Block (c) 2 0.0117 0.0058 1.81 3.13 4.92 a x b 5 0.0136 0.0027 0.84 2.35 3.29 a x e 10 0.0334 0.0033 1.04 1.97 2.59 b x c 2 0.0020 0.0010 0.31 3.13 4.92 a x b x c 10 0.0257 0.0026 0.80 1.97 2.59 S t a r t i n g weight 1 0.1023 0.1023 31.60** 3.98 7.01 Residue 71 0.2292 0.0032 ** P < 0.01 - 148 -Table 3A. Mean d a i l y gain (Kg) from 45 Kg body v;eight to f i n i s h of 108 pi g s i n P i g Experiment I . Rep 1 Rep 2 Rep 3 Treatment Male Female Male Female Male Female 0.8002 0.8257 0.7619 0.6372 0.5434 0.7083 0.8129 0.6797 0.6750 0.7993 0.8112 0.8197 0.8112 0.7631 0.7369 0.8070 0.6048 0.7039 0.7917 0.7333 0.7214 0.6247 0.6299 0.5710 0.5263 0.7476 0.6074 0.7107 0.7524 0.6301 0.6905 0.5539 0.5658 0.5560 0.7452 0.6821 0.6976 0.6186 0.6173 0.4645 0.6344 0.5071 0.5939 0.6881 0.5100 0.8112 0.7058 0.7296 0.7857 0.7279 0.5558 0.6881 0.7107 0.7131 0.6345 0.6964 0.7738 0.5125 0.4283 0.6368 0.5656 0.5015 0.4710 0.8248 0.6632 0.6883 0.6494 0.6463 0.4408 0.6395 0.6845 0.7131 0.7310 0.7071 0.8648 0.6952 0.7262 0.7310 0.8206 0.7345 0.5020 0.8078 0.6976 0.7942 0.8291 0.6909 0.8172 0.6502 0.7645 0.6455 0.7071 0.6917 0.7440 0.6359 0.5169 0.6567 0.5701 0.5474 0.5900 0.6024 0.7440 0.8019 0.7655 0.6709 0.6571 0.7152 0.6385 0.6848 - 149 -Table 3B. A n a l y s i s of v a r i a n c e of mean d a i l y gain from 45 Kg body weight to f i n i s h of 108 pigs w i t h no c o v a r i a b l e i n P i g Experiment I . Variance r a t i o (F nec.) Source D.F. S.S. M.S. F-value P=0.05 P=0.i T o t a l 107 1.0421 Treatment (a) 5 0.2904 0.0581 10.07** 2.35 3.29 Sex (b) 1 0.1021 0.1021 17.69** 3.98 7.01 Block (c) 2 0.0909 0.0455 7.88** 3.13 4.92 a x b 5 0.0381 0.0076 1.32 2.35 3.29 a x e 10 0.0701 0.0070 1.22 1.97 2.59 b x c 2 0.0117 0.0058 1.01 3.13 4.92 a x b x c 10 0.0235 0.0024 0.41 1.97 2.59 Residue 72 0.4153 0.0058 * P < 0.01 - 150 -Table 4A. Mean d a i l y gain (Kg) from s t a r t to f i n i s h of 108 pi g s i n P i g Experiment I . Treatment Rep 1 Rep 2 Rep 3 Male Female Male Female Male Female 0.7812 0.7333 0.7026 0.7028 0.7276 0.6957 0.7736 0.7173 0.6194 0.6702 0.6933 0.6411 0.6837 0.7079 0.6446 0.7536 0.6988 0.6995 0.6508 0.5882 0.4897 0.5337 0.5778 0.5548 0.3747 0.5830 0.5344 0.4587 0.6322 0.4993 0.5994 0.5185 0.5627 0.6318 0.5899 0.5774 0.7687 0.4942 0.5903 0.5791 0.7146 0.5521 0.6315 0.6162 0.6409 0.5226 0.6747 0.5739 0.5805 0.5774 0.5197 0.4116 0.5203 0.6088 0.7724 0.6577 0.6775 0.6928 0.7581 0.6077 0.7235 0.6679 0.6607 0.6267 0.7080 0.6850 0.7301 0.5518 0.5795 0.6210 0.7190 0.7203 0.6768 0.6528 0.7141 0.6177 0.6859 0.6852 0.6681 0.5860 0.6098 0.6098 0.6544 0.5603 0.6615 0.5797 0.6038 0.3983 0.6342 0.5995 0.6962 0.5505 0.6533 0.6122 0.6626 0.6412 0.5949 0.6134 0.6231 0.6465 0.6183 0.5973 0.6151 0.6781 0.6796 0.7036 0.5749 0.6381 Table 4B. Analysis of variance of mean d a i l y gain from s t a r t to f i n i s h of 108 pigs with s t a r t i n g weight as covariable i n Pig Experiment I Source D.F. S.S. M.S. F-value Variance F(nec. P=0.05 Ratio ) P=0.01 Tota l 107 0.6734 Treatment (a) 5 0.2772 00.0554 17.549** 2.35 3.29 Sex (b) 1 0.0306 0.0306 9.688** 3.98 7.01 Block (c) 2 0.0019 0.0010 0.315 3.13 4.92 a x b 5 0.0299 0.0597 1.891 2.35 3.29 a x e 10 0.0256 0.0026 0.811 1.97 2.59 b x c 2 0.0036 0.0018 0.577 3.13 4.92 a x b x c 10 0.0173 0.0017 0.548 1.97 2.59 Covariable 1 0.0347 0.0347 10.995** 3.98 7.01 Residual 71 0.2243 0.0032 - 152 -Table 5A. Average feed conversion e f f i c i e n c y r a t i o per p i g (D.M. feed i n t a k e (kg)/weight gain (kg)) i n P i g Experiment I . Treatment Rep 1 Rep 2 Rep 3 1 2.762 2.787 2.943 2 3.414 3.466 3.341 3 3.222 3.289 3.341 4 2.890 2.924 3.040 5 2.971 3.049 3.035 6 2.919 3.048 3.093 Table 5B. A n a l y s i s of v a r i a n c e of feed conversion e f f i c i e n c y r a t i o i n P i g Experiment I . Source D.F. S.S. M.S. F-value Variance F(nec. P=0.05 r a t i o .) P=0.01 T o t a l 17 0.7699 Treatment 5 0.7014 0.1403 23.4865** 3.20 5.32 Cov a r i a b l e s t a r t i n g 1 0.0007 0.0007 0.1128 4.82 9.65 weight R e s i d u a l 11 0.0657 0.0060 - 153 -Table 6A. T o t a l back f a t (mm) of 107 p i g s i n P i g Exper iment I . Treatment Rep . Male 1 Female Rep Male 2 Female Rep Male 3 Female 1 91 80 90 82 84 62 96 88 83 81 103 76 87 78 83 84 90 77 2 104 95 70 93 91* 101 79 82 100 78 100 95 103 96 104 97 120 108 3 85 81 97 89 100 109 106 86 120 95 104 87 92 84 109 75 105 92 4 98 91 104 82 106 . 79 95 94 96 99 102 79 85 66 90 95 99 102 5 81 82 101 64 100 86 • o 87 88 103 75 85 79 97 100 115 55 97 91 6 86 89 92 86 103 98 81 100 86 95 91 68 94 87 105 83 84 96 C a l c u l a t e d m i s s i n g v a l u e (Snedecor and Cochran, 1 9 6 7 ) . Table 6B. A n a l y s i s of v a r i a n c e of t o t a l back f a t thickness w i t h f i n a l body weight, average body weight gain and body length as c o v a r i a b l e s i n P i g Experiment I . Source D.F. S.S. M.S. F-value Variance F(nec. P=0.05 Ratio • ) P=0.01 T o t a l 106 10860.683 Treatment (a) 5 1157.6 231.53 3.207** 2.35 3.29 Sex (b) 1 656.35 656.35 9.090** 3.98 7.01 Block (c) 2 40.553 20.276 0.281 3.13 4.92 a x b 5 358.44 71.687 0.993 2.35 3.29 a x e 10 838.37 83.837 1.161 1.97 2.59 b x c 2 153.20 76.599 1.061 3.13 4.92 a x b x c 10 1190.0 119.00 1.648 1.97 2.59 F i n a l body wt. 1 947.86 974.86 13.501** 3.98 7.01 A.D.G. 1 25.24 25.24 0.350 3.98 7.01 Body le n g t h 1 556.17 556.17 7.703** 3.98 7.01 R e s i d u a l 68 4909.9 72.204 P < 0.01 - 155 -Table 7A. Back f a t 40 mm from mid l i n e (mm) of 107 pigs i n P i g Experiment I . Treatment Rep Male 1 Female Rep Male 2 Female Rep Male 3 Female 1 21 14 16 15 19 12 22 17 16 9 17 12 17 14 20 12 20 9 2 20 22 12 16 17* 17 13 21 23 11 24 19 20 18 23 21 25 22 3 18 11 20 16 20 19 23 21 21 20 21 14 14 15 23 12 25 21 4 16 16 18 14 24 14 23 17 18 17 22 13 20 12 17 17 22 20 5 15 13 17 13 22 18 15 15 17 15 15 17 15 17 28 6 17 19 6 17 16 19 14 19 17 13 22 22 21 16 8 22 21 22 16 13 21 C a l c u l a t e d m i s s i n g value (Scedecor and Cochran, 1967). Table 7B. Analysis of variance of back f a t 40 mm from mid l i n e with f i n a l body weight, average d a i l y weight gain and body length as covariables i n Pig Experiment I . Source D.F. S.S. M.S. F-value Variance F(nec.) P=0.05 Ratio P=0.01 Total 106 1348.190 Treatment (a) 5 154.03 30.807 3.698** 2.35 3.29 Sex (b) 1 48.793 48.793 5.858** 3.98 7.01 Block (c) 2 7.407 3.7035 0.445 . 3.13 4.92 a x b 5 55.658 11.132 1.336 2.35 3.29 a x e 10 150.61 15.061 1.808 1.97 2.59 b x c 2 30.150 15.075 1.810 3.13 4.92 a x b x c 10 49.043 4.9043 0.589 1.97 2.59 F i n a l body wt. 1 138.575 138.575 16.636** 3.98 7.01 A.D.G. 1 21.131 21.131 2.537 3.98 7.01 Body length 1 126.343 126.343 15.167** 3.98 7.01 Residual 68 566.45 8.330 * p < 0.05 ** P < 0.01 - 157 -Table 8A. Eye muscle area (mm ) of 107 pi g s i n P i g Experiment I . Treatment Rep 1 Male Female Rep 2 Male Female Rep 3 Male Female 2430 2510 2550 2190 2080 2390 2410 2880 2620 2900 2410 2540 2760 3120 2480 2760 2850 2320 2900 2830 3060 2680 2600 2580 3030 2810 2740 2910 2880 2450 3010 3000 2520 2690 2470 2440 2700 2960 2690 2560 2500 2420 2540 2520 2660 2280 2810 2880 2900 2770 2320 2550 2440 2400 2650 3200 3220 2820 2910 2580 2780 2400 2910 2390 3320 2690 2880 2750 2560 2880 2600 2710 2750 2750 3080 2333* 2250 2170 2250 2660 2360 2890 2500 2590 2490 2580 2470 2330 2630 2710 3080 3660 3440 2540 2800 2670 2890 3290 2540 2770 2750 2680 2740 1980 2420 3330 3010 2730 * C a l c u l a t e d m i s s i n g value (Snedecor and Cochran, 1967). Table 8B. Analysis of variance of eye muscle area with f i n a l body weight, average d a i l y gain and body length as covariables i n Pig Experiment I . Variance Ratio Source D.F. S ;.S M.S. F-value F(nec. P=0.05 • ) P=0.01 Tota l 106 0.78515 X i o 7 Treatment (a) 5 0.14274 X 10 7 0.28547 x i o 6 5.558** 2.35 3.29 Sex (b) 1 0.76233 X i o 6 0.76233 x 10 6 14.842** 3.98 7.01 Block (c) 2 36818.0 18409 0.358 3.13 4.92 a x b 5 0.46021 X i o 6 92043 1.792 2.35 3.29 a x e 10 0.93779 X i o 6 93779 1.826 1.97 2.59 b x c 2 70083 35042 0.682 3.13 4.92 a x b x c 10 0.26036 X i o 6 26036 0.507 1.97 2.59 F i n a l body wt. 1 0.11576 X i o 6 0.11576 x i o 6 2.254 3.98 7.01 A.D.G. 1 0.22464 X i o 6 0.22464 x i o 6 4.374* 3.98 7.01 Body length 1 63524 63524 1.237 3.98 7.01 Residual 68 0.34926 X i o 7 51361.8 tick P < 0.05 P < 0.01 - 159 -Table 9 A . Eye muscle index (length 'A' x widt h 'B') (mm ) of the 107 pigs i n P i g Experiment I . Treatment Rep Male 1 Female . Rep Male 2 Female Rep Male 3 Female 1 3195 3927 3696 3840 3840 4717 3750 3840 4345 4592 4056 5187 3476 4312 3854 4536 4480 5162 2 2982 3871 3483 4050 3254* 3724 3280 3619 3465 3864 3634 3876 3600 3476 3300 3822 3358 3713 3 3234 4361 3600 4100 3212 4160 4088 3850 3572 3577 3700 4346 3854 3850 3555 3773 3168 3619 4 4250 4345 3337 3431 4100 3984 3408 4081 4067 4644 3969 4080 3564 3690 4080 3950 3750 3920 5 3672 4346 4182 4539 3528 3960 4582 4592 3978 4018 4067 3139 3724 3577 3225 3696 3650 3572 6 3927 . 4018 3431 4104 3360 4720 3822 3672 3588 3936 3569 4316 3192 3384 3397 3772 3840 3634 C a l c u l a t e d m i s s i n g value (Snedecor and Cochran, 1967). Table 9B. A n a l y s i s of va r i a n c e of eye muscle index (length x width) w i t h f i n a l body weight, average d a i l y gain and body length as c o v a r i a b l e s i n P i g Experiment I . Source D.F. S. .S. M.S. F-value Variance F(nec. P=0.05 Ratio ) P=0.01 T o t a l 106 0.17293 X i o 8 Treatment (a) 5 0.31403 X i o 7 0.62807 X i o 6 5.718** 2.35 3.29 Sex (b) 1 0.17253 X i o 7 0.17253 X i o 7 15.707** 3.98 7.01 Block (c) 2 0.29667 X i o 6 0.14834 X i o 6 1.351 3.13 4.92 a x b 5 0.61179 X i o 6 0.12236 X 10 6 1.114 2.35 3.29 a x e 10 0.22437 X i o 7 0.22437 X i o 6 2.043* 1.97 2.59 b x c 2 94287 47144 0.429 3.13 4.92 a x b x c 10 0.56122 X i o 6 56122 0.511 1.97 2.59 F i n a l body wt. 1 0.33189 X i o 6 0.33189 X i o 6 3.022 3.98 7.01 A.D.G. 1 0.57957 X i o 6 0.57957 X i o 6 5.276* 3.98 7.01 Body le n g t h 1 0.23945 X i o 6 0.23945 X i o 6 2.180 3.98 7.01 R e s i d u a l 68 0.74690 i o 7 0.10984 X i o 6 * ** P < 0.05 P < 0.01 - 161 -Table 10A. Days on t r i a l s of 1 0 8 pigs i n P i g Experiment I . Treatment Rep 1 Rep 2 Rep 3 Male Female Male Female Male Female 70 91 98 91 84 98 91 91 119 98 91 105 98 98 119 98 105 98 98 112 140 113 119 119 113 105 140 140 105 119 119 133 119 119 119 119 77 126 98 112 91 133 112 105 112 133 98 119 119 126 140 140 133 105 84 98 112 91 91 105 91 98 105 119 91 105 98 98 110 112 105 91 105 105 105 119 91 98 98 112 112 119 105 105 105 112 119 119 105 105 98 119 98 112 91 105 119 105 112 112 112 112 105 98 105 105 112 105 Table 10B. Analysis of variance of days on t r i a l s with i n i t i a l body weight as covariable i n Pig Experiment I . Variance Ratio F(nec.) Source D.F. S.S. M.S. F-value P=0.05 P=0.01 Tota l 107 21601 .74 Treatment (a) 5 8072 .284 1614. 457 26 .6750** 2 .35 3 .29 Sex (b) 1 268. 3129 268. 3129 4 .4332* 3 .98 7 .01 Block (c) 2 134. 3030 67. 1515 1 .1095 3 .13 4 .92 a x b 5 605. 5736 121. 1147 2 .0011 2 .35 3 .29 a x e 10 588. 8364 58. 8836 0 .9729 1 .97 2 .59 b x c 2 95.29601 47. 6480 0 .7873 3 .13 4 .92 a x b x c 10' 274. 5964 27. 4596 0 .4537 1 .97 2 .59 Covariable 1 4620 .856 4620. 856 76 .3485** 3 .98 7 .01 Residual 71 4297 .144 60. 5232 *P < 0.05 **P < 0.01 - 163 -Table 11A. D.M. feed i n t a k e (g)/wk. i n P i g Experiment I I . Treatment Rep 1 Rep 2 Rep 3 Rep 4 Rep 5 Rep 6 1 12580 .375 14140.000 13166. 950 14990 .225 12767 .850 12900.675 2 11212 .620 13109.890 11204. 960 12840 .660 11165 .510 11465.850 3 11478 .130 123821230 11865. 790 13845 .210 12656 .450 13978.210 4 11870 .400 12939.050 12250. 950 14297 .700 11515 .750 12355.900 5 . 10855 .400 12309.730 12093. 620 13764 .010 12466 .520 13911.380 6 12779 .450 14392.600 11454. 800 11752 .350 11735 .550 11350.850 Table 11B. A n a l y s i s of v a r i a n c e of D.M. feed i n t a k e (g)/wk. i n P i g Experiment I I . Source D.F. S.S. M.S. F-value Variance R a t i o F(nec.) P=0.05 P=0.01 T o t a l 35 4.021 x 10 Treatment 5 8.369 x 10 6 1.674 x 10 6 1.577 N S 2.53 3.70 Residu a l 30 3.185 x 10 7 1.0615 x 10 6 NS : n o n - s i g n i f i c a n c e - 164 -Table 12 . A n a l y s i s of va r i a n c e of N i n t a k e (g)/wk. i n P i g Experiment I I . Variance R a t i o Source D.F. S.S. M.S. F-value F(nec.) P=0.05 P=0.01 T o t a l 35 13290.6 Treatment 5 120407.6 24081.51 57.81** 2.53 3.70 Residua l 30 12496.99 416.57 P < 0.01 - 165 -Table 13A. Percentage D.M. d i g e s t i b i l i t y i n P i g Experiment I I . Treatment Rep 1 Rep 2 Rep 3 Rep 4 Rep 5 Rep 6 1 79.26 78.91 77.59 76.58 78.41 79.85 2 78.98 75.19 78.41 75.58 80.51 77.86 3 78.07 75.93 78.34 76.04 74.57 74.59 4 79.36 78.96 78.20 77.06 78.22 75.92 5 78.90 77.89 77.65 75.64 77.55 78.08 6 72.70 73.20 79.02 78.96 80.58 78.40 Table 13B, A n a l y s i s of vari a n c e of dry matter d i g e s t i b i l i t y i n percent i n P i g Experiment I I . Variance R a t i o Source D.F. S.S. M.S. F-value F(nec.) P=0.05 P=0.01 T o t a l 35 Treatment 5 R e s i d u a l 30 127.679 16.943 3.389 0.9180 N S 2.53 3.70 110.74 3.691 NS : N o n - s i g n i f i c a n c e - 166 -Table 14A. N i t r o g e n r e t e n t i o n (g)/wk. i n P i g Experiment I I . Treatment Rep 1 Rep 2 Rep 3 Rep 4 Rep 5 Rep 6 1 129 .70 143 .52 123 .82 115 .01 146 .59 140 .97 2 99 .89 102 .48 79 .86 95 .33 72 .09 77 .00 3 87 .40 91 .74 106 .73 121 .46 103 .43 111 .17 4 109 .65 107 .84 98 .75 120 .17 103 .97 105 .40 5 104 .80 121 .54 103 .81 118 .90 109 .56 128 .29 6 101 .48 115 .68 98 .40 99 .99 105 .82 91 .36 Table 14B. A n a l y s i s of var i a n c e f o r n i t r o g e n r e t e n t i o n i n P i g Experiment I I . Variance R a t i o Source D.F. S.S. M.S. F-value F(nec.) P=0.05 P=0.01 T o t a l 35 10351.80 Treatment 5 6857.99 1371.60 11.7773** 2.53 3.70 R e s i d u a l 30 3493.81 116.46 P < 0.01 - 167 -Table 14C. A n a l y s i s of co-variance of n i t r o g e n r e t e n t i o n w i t h feed i n t a k e as c o v a r i a b l e i n P i g Experiment I I . Source D.F. S.S. M.S. F-value Variance R a t i o F(nec.) P=0.05 P=0.01 T o t a l Treatment C o v a r i a b l e feed i n t a k e R e s i d u a l 35 10351.800 5 3818.843 763.769 8.668** 1 938.654 938.654 10.653** 29 2555.155 88.109 2.55 4.18 3.73 7.60 P < 0.01 Table 14D. A n a l y s i s of co-variance f o r n i t r o g e n r e t e n t i o n w i t h n i t r o g e n i n t a k e as c o v a r i a b l e i n P i g Experiment I I . Source D.F. S .S. M.S, F-value Variance R a t i o F(nec.) P=0.05 P=0.01 T o t a l Treatment C o v a r i a b l e n i t r o g e n i n t a k e R e s i d u a l 35 10351.800 5 1665.369 333.074 3.3897* 1 644.268 644.268 6.5568** 29 2849.541 98.260 2.55 4.18 3.73 7.60 P < 0.05 P < 0.01 - 168 -Table 15A- Nit r o g e n absorbed as a percentage of n i t r o g e n i n t a k e i n P i g Experiment I I . Treatment Rep 1 Rep 2 Rep 3 Rep 4 Rep 5 Rep 6 1 78.07 78.24 75.99 75.15 75.59 77.61 2 68.89 64.00 68.45 67.92 74.49 70.91 3 69.45 66.20 69.84 68.33 62.22 60.84 4 71.37 71.14 69.87 70.44 68.96 67.67 5 72.74 70.84 69.29 66.76 67.01 70.60 6 59.05 59.91 75.66 74.73 78 08 71.19 Table 15B. A n a l y s i s of v a r i a n c e f o r n i t r o g e n absorbed as a percentage of n i t r o g e n i n t a k e i n P i g Experiment I I . Variance R a t i o Source D.F. S.S. M.S. F-value F(nec.) P=0.05 P=0.01 T o t a l 35 891.128 Treatment 5 369.320 73.864 4.247** 2.53 3.70 R e s i d u a l 30 521.808 17.394 ** P < 0.01 - 169 -Table 16A. Nitrogen retained as a percentage of nitrogen intake i n P i g Experiment I I . Treatment Rep 1 Rep 2 Rep 3 Rep 4 Rep 5 Rep 6 1 36.65 36.09 33.09 27.16 40.82 38.85 2 50.60 44.39 40.48 42.16 36.67 38.14 3 41.88 40.75 49.47 48.25 44.95 43.74 4 50.83 45.86 44.35 46.25 49.68 46.93 5 53.29 54.50 47.38 47.69 48.51 50.91 6 43.51 44.03 47.06 46.61 49.40 ' 44.10 Table 16B. Analysis of variance f o r nitrogen retained as a percentage of nitrogen intake i n Pig Experiment I I . Variance Ratio Source D.F. S.S. M.S. F-value F ( n e c ) P=0.05 P=0.01 To t a l 35 1195.433 Treatment 5 791.771 158.354 11.769** 2.53 3.70 Residual 30 403.663 13.455 * P < 0.01 - 170 -Table 17A. Ni t r o g e n r e t a i n e d as a percentage of• n i t r o g e n absorbed i n P i g Experiment I I . Treatment Rep 1 Rep 2 Rep 3 Rep 4 Rep 5 Rep 6 1 46.97 46.12 44.00 36.30 54.00 50.06 2 73.45 69.37 59.13 62.08 49.23 53.79 3 60.30 61.55 70.84 70.61 72.24 71.90 4 71.22 64.46 63.48 65.65 72.04 69.35 5 73.27 76.94 68.39 71.42 72.40 71.11 6 73.67 73.51 62.21 62.37 63.27 61.95 Table 17B. A n a l y s i s of var i a n c e of n i t r o g e n r e t a i n e d as a percentage of n i t r o g e n absorbed i n P i g Experiment I I Variance R a t i o Source D.F. S.S. M.S. F-value F(nec, .) P=0.05 P=0.01 T o t a l 35 3565.118 Treatment 5 2544.151 508.802 14.951** 2.53 3.70 Re s i d u a l 30 1020.967 34.032 ** P < 0.01 Table 18 Analysis of covariance for average daily gain and feed conversion efficiency with i n i t i a l weight and daily feed intake as covariables i n Rat Experiments I, II and I I I . Experi- _ r Source ment DF M e a n Average da i l y gain S q u a r e Feed efficiency week 1 2 3 4 overall 1 2 3 4 overall Treatments 5 2.8962** 1.4948** 0.4414 0.4165 1.1954** 1.4702** 0.8876** 0.3294 0.2658 0.6117** I n i t i a l weight 1 1.6993** 1.6769* 0.0009 1.5153 0.4853** 0.8956** 0.7978* 0.0453 0.7881 1.7953* I Daily feed intake 1 11.9174** 5.'2256** 3.6885** 4.4858* 5.3272**. 1.1758** 0.2282 0.5174 0.3543 1.7057* Residual 28 0.1396 0.2499 0.2720 0.4196 0.0630 0.0978 0.1605 0.3204 0.2975 0.0298 Treatments 11 3.0793** 1.0924** 1.0618** 1.1032** 1.4758** 1.8036** 0.6671** 0.8411** 1.5654** 0.9539** I n i t i a l weight i i Daily feed intake 1 0.7948** 0.2835 1.9857** 2.4942** 0.3330** 0.8460 0.1672 1.5175** 3.7148** 0.1896** 1 13.0126** 3.1398** 9.0803** 13.2613** 4.0303** 2.8256** 0.0046 0.9732* 7.2121** 0.0990* • Residual 34 0.0960 0.1932 0.1894 0.2534 0.0293 0.2482 0.1093 0.1395 0.3732 0.0159 Treatments 9 2.8886** 1.0166** 1.0979** 0.9896* 1.2320** 1.9398** 0.6131** 0.8235** 1.1636 0.9381** I n i t i a l weight III 1 1.0608** 0.2580 1.5302** 2.4655* 0.2132* 2.0862** 0.1295 0.9242** 3.5582*. 0.1446* Daily feed intake 1 9.8727** 6.2404** 8.2364** 6.6900** 3.9260** 2.9883** 0.4623 1.2065** 3.8612* 0.1169* Residual 28 0.1115 0.2091 0.1812 0.3553 . 0.04755 0.1020 0.1360 0.1101 0.5217 0.02324 * P < 0.05 P < 0.01 Table 19 A n a l y s i s of Covariance f o r PER w i t h i n i t i a l weight as a c o v a r i a b l e i n Rat Experiments I , I I and I I I . Source Treatment I n i t i a l weight R e s i d u a l Rat Experiment I PER ( 5 ) * 0.4564** ( 1 ) * 0.0450 (29) * 0.0199 Mean Square Rat Experiment I I PER (11)* 0.3021** (1 ) * 0.0807** (35)* 0.0092 Rat Experiment I I I PER ( 9 ) * 0.2419** ( 1 ) * 0.0142 (29)* 0.0479 Degree of freedom P < 0.01 . Table 20. Analysis of eovarlanee for carcass c h a r a c t e r i s e s of rats In Rat Experlrcents 1, It and l i t . Experi- Source cent DP T o t a l carcass P r o t e i n Protein retained P r o t e i n X i n Protein X l n prote i n r e t e n t i o n Protein i n t a k t dry carcass fat free carcass Treatments Average d a l l y gains Residual Treatments Average d a l l y gains Residual Treatments Average d a i l y gains Residual 29 11 3J 29 26.670* 17.2980 8.17B7 7.1153* 84.275** 3.1826 9.1631** 62.279** 1.8261 26.658* 17.3055 8.1784 7.1153* 64.2752** 3.1826 9.2307** 61.609** 1.8490 130.78** 31.0380 29.392 173.80** C.6210 7.1922-111.21** 0.0575 6.0226 105.00** 81.935* 13.665 63,485** 41.167 11.350 80.424** 128.11** 11.418 1.62M 0.1379 1.7171 2.8013 0,0000 1.7418 2.3840 0.0227 2.4095 S Q U A R E Total Pat X l n dry carcass f a t carcass T o t a l ash Ash X i n dry Ash X i n f a t Prqtein»faf.+ash carcass free carcans Dry carcass ut. 85.464** 163.53** 12.974 46.360** 135.41** 10.894 76.464** 399.38** 10.002 144.61** 146.31 15.382 88.043** 61.029* 12.680 116.67** 289.25** 11.492 0.3846 2.1107** 0.2765 5.039 »« 0.1729 0.4064 0.1493* 1.2674** • 1.2933" 3.3327** 0.0716 0.3530 0.1926* 2.2518** 0.7570** 10.8075** 0.0849 0.3168 0.3526 0.5558 0.1615 0.7986** 1.0066** 0.0821 . 0.2757 1.4870* 0.2045 0.4640 0.0448 0.8599 0.4577 0.1453 0.8086 0.5970 0.0813 1.1074 P < 0.05 P < 0.01 

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