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Nutritional studies on high lysine barley lines from the Alberta agriculture breeding program, 1977 harvest Imbeah, Maleena 1980

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NUTRITIONAL STUDIES ON HIGH LYSINE BARLEY LINES FROM THE ALBERTA AGRICULTURE BREEDING PROGRAM - 197 7 HARVEST by MALEENA IMBEAH B . S c . Hons. ( A g r i c . ) The U n i v e r s i t y o f Ghana, L e g o n , 1976 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES (Department o f A n i m a l S c i e n c e ) We a c c e p t t h i s t h e s i s as c o n f o r m i n g to the r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA May 1980 Maleena Imbeah, 1980 I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r a n a d v a n c e d d e g r e e a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e a n d s t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by t h e Head o f my D e p a r t m e n t o r by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . D e p a r t m e n t n f /fM /m fl U ScigA*C£ The U n i v e r s i t y o f B r i t i s h C o l u m b i a 2075 W e s b r o o k P l a c e V a n c o u v e r , C a n a d a V6T 1W5 n at . P Tu»«. >^ HIP ABSTRACT Recent years have brought a g r e a t e r awareness of the need f o r more p l e n t i f u l as w e l l as more n u t r i t i o u s foods. D i s c o v e r i e s of s t r a i n s of maize, b a r l e y and other c e r e a l s having h i g h e r l e v e l s of e s s e n t i a l amino ac i d s have shown that n u t r i t i o n a l q u a l i t y of c e r e a l s can be improved through p l a n t b r e e d i n g . In the experiments r e p o r t e d here, the n u t r i t i o n a l value of three c u l t i v a r s from the A l b e r t a A g r i c u l t u r e h i g h l y s i n e breeding program ( C u l t i v a r s 1, 2 and 3), h i p r o l y , and a normal commercial v a r i e t y was s t u d i e d . Amino a c i d a n a l y s i s was done on each b a r l e y l i n e to compare the a b i l i t y of each to supply e s s e n t i a l amino a c i d s . The amino a c i d composition (g/16 g N) i n d i c a t e d v i r t u a l l y no d i f f e r e n c e s except f o r c u l t i v a r 3 which had a much lower l e v e l o f t o t a l e s s e n t i a l amino a c i d s than the r e s t . B a r l e y p r o t e i n s were separated by s o l u b i l i t y t e s t s to study the p r o p o r t i o n s i n which albumins and g l o b u l i n s , g l u t e l i n s and hordeins occur and to r e l a t e the l e v e l s o f these p r o t e i n f r a c t i o n s to the q u a l i t y of the t o t a l p r o t e i n . H i p r o l y and c u l t i v a r s 1 and 2 were found to c o n t a i n approximately the same l e v e l s of albumin plus g l o b u l i n w h i le c u l t i v a r 3 and G a i t had lower p r o p o r t i o n s of these. The l a t t e r a l s o had the h i g h e s t p r o p o r t i o n of h o r d e i n , an i n d i c a t i o n t h a t i t s p r o t e i n q u a l i t y i s low. Chemical d e t e r m i n a t i o n o f a v a i l a b l e l y s i n e by dye b i n d i n g d i f f e r e n c e gave i n c o n c l u s i v e r e s u l t s . However, r a t growth assa y s i n d i c a t e d H i p r o l y and c u l t i v a r 2 t o be s i g n i f i c a n t l y s u p e r i o r i n n u t r i t i o n a l q u a l i t y to G a i t and c u l t i v a r s 1 and 3. N i t r o g e n b a l a n c e t e s t s 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 any o f the f i v e b a r l e y s . When t h e b a r l e y s were d i l u t e d t o a s t a n d a r d p r o t e i n c o n t e n t (12%) , the s u p p l e m e n t a t i o n w i t h soybean meal o r l y s i n e p l u s o t h e r e s s e n t i a l amino a c i d s made s i g n i f i c a n t improvements i n r a t p e r f o r m a n c e , but l y s i n e a l o n e had no b e n e f i c i a l e f f e c t . T h i s i s an i n d i c a t i o n t h a t the p r o t e i n o f the s e h i g h l y s i n e b a r l e y s i s d e f i c i e n t i n e s s e n t i a l amino a c i d s o t h e r than l y s i n e . A pparent a v a i l a b i l i t y o f l y s i n e by f a e c a l a n a l y s i s gave r e s u l t s s u p p o r t i n g the f a c t t h a t h i g h l y s i n e b a r l e y s a re s u p e r i o r i n q u a l i t y t o the normal b a r l e y . TABLE OF CONTENTS Page ABSTRACT i i LIST OF TABLES v i i LIST OF FIGURES x i LIST OF APPENDICES X 1 1 ACKNOWLEDGEMENTS x i i i INTRODUCTION 1 LITERATURE REVIEW 10 1. P r o t e i n Q u a l i t y 10 Factor s A f f e c t i n g P r o t e i n Q u a l i t y 10 2. Methods of Measuring A v a i l a b l e Lysine .... 18 3. Supplementation of C e r e a l s with E s s e n t i a l Amino Acids 28 4. P r o t e i n : C a l o r i e R a t i o 34 5. B i o l o g i c a l Methods of Measuring P r o t e i n Q u a l i t y 36 i v Page EXPERIMENTAL 4 5 CHEMICAL EVALUATION OF PROTEIN QUALITY 45 Proximate Composition 45 Sep a r a t i o n of B a r l e y P r o t e i n s 5u Amino A c i d A n a l y s i s 61 Measurement of A v a i l a b l e Lysine 72 BIOLOGICAL EVALUATION OF PROTEIN QUALITY 83 Rat T r i a l I 83 Rat Growth Assay and the Measurement of True N i t r o g e n D i g e s t i b i l i t y , True B i o l o g i c a l Value and Net P r o t e i n U t i l i -z a t i o n 83 I n t r o d u c t i o n 83 M a t e r i a l s and Methods 84 Re s u l t s and D i s c u s s i o n 90 Rat T r i a l II 95 R e l a t i v e P r o t e i n Value as a B i o l o g i c a l Measure of P r o t e i n Q u a l i t y 95 I n t r o d u c t i o n 95 v: Page M a t e r i a l s and Methods ^5 R e s u l t s 100 D i s c u s s i o n 101 Rat T r i a l I I I 103 E f f e c t of Supplementing B a r l e y with Soy-Bean Meal or E s s e n t i a l Amino Acids on the Growth of Weanling Rats 103 I n t r o d u c t i o n 103 M a t e r i a l s and Methods 104 R e s u l t s and Discussion.' 105 GENERAL DISCUSSION AND CONCLUSIONS 126 LITERATURE CITED 136 APPENDICES 150 v i LIST OF TABLES Table Page 1 Proximate composition of b a r l e y s 48 2 N i t r o g e n content of b a r l e y e x t r a c t s as a percentage of t o t a l n i t r o g e n 55 3 Amino a c i d n i t r o g e n as a percentage of p r o t e i n n i t r o g e n 60 4 P r o t e i n content and t o t a l amino a c i d composition of f i v e b a r l e y s 64 5 Amino a c i d contents of fo u r b a r l e y s (g/lOOg DM) r e l a t i v e to amino a c i d composition of H i p r o l y 67 6 Amino a c i d contents of four b a r l e y s (g/16 g N) r e l a t i v e to amino a c i d composition of H i p r o l y 68 7 Proximate a n a l y s i s and t o t a l amino a c i d composition of f i v e b a r l e y s from A l b e r t a A g r i c u l t u r e Program - 1975 Harvest 71 v i i Table: Page 8 Standard curve f o r dye c o n c e n t r a t i o n ... 77 9 A n a l y s i s of b a r l e y g r a i n samples f o r dye b i n d i n g d e t e r m i n a t i o n of a v a i l a b l e l y s i n e 78 10 P r o t e i n content (g/100 g DM), l y s i n e and b a s i c amino a c i d content (g/16 g N) and dye b i n d i n g c a p a c i t y (mmol/16 g N) of f i v e b a r l e y samples . 79 11 D i e t composition, t r i a l I 86 12 Vi t a m i n mix composition, t r i a l s I, I I , III 87 13 M i n e r a l mix composition, t r i a l s I, I I , I I I 88 14 Average d a i l y body weight g a i n , food consumption, growth r a t e and f e e d e f f i c i e n c y of r a t s , t r i a l I 91 15 N i t r o g e n balance t e s t , t r i a l I 93 v i i i Table Page 16 Composition of RPV d i e t s , t r i a l II 97 17 R e s u l t s of a 14-day RPV assay, t r i a l II .. 100 18 Estimates of RPV of f i v e b a r l e y s , t r i a l II 102 19 Composition of b a s a l and supplemented d i e t s , t r i a l III 106 20 B a r l e y content and c a l c u l a t e d e s s e n t i a l amino a c i d content of b a s a l d i e t s , t r i a l I I I 107 21 Dry matter and crude p r o t e i n a n a l y s i s of d i e t s , t r i a l I I I 108 22 Feed consumption and growth data, t r i a l I I I n o 23 Standard curve f o r l y s i n e d e t e r m i n a t i o n by n i n h y d r i n r e a c t i o n . 122 24 F a e c a l measurements f o r l y s i n e d e t e r -mination 123 i x Table Page 25 Apparent a v a i l a b i l i t y of l y s i n e by f a e c a l a n a l y s i s 125 26 Summary of experimental r e s u l t s 127 0 X LIST OF FIGURES Fi g u r e Page 1 Standard curve f o r dye c o n c e n t r a t i o n and absorbance 76 2 R e l a t i v e p r o t e i n value d e t e r m i n a t i o n .... 99 3 Rat growth r a t e s on b a s a l and supple-mented b a r l e y d i e t s 109 4 Standard curve f o r l y s i n e d e t e r m i n a t i o n . 121 x i LIST OF APPENDICES Appendix Page 1 E s s e n t i a l amino a c i d requirements of growing c h i c k s ( l a y e r replacements) 150 2 E s s e n t i a l amino a c i d requirements of the growing r a t 151 3 E s s e n t i a l amino a c i d requirements of the growing p i g . 152 x i i ACKNOWLEDGEMENT I wish to express my s i n c e r e g r a t i t u d e to Dr. R.M. Beames f o r h i s c o u n s e l , guidance and p a t i e n c e with me throughout the course of these s t u d i e s . I wish to thank Anne M c C l e l l a n d f o r her s t a t i s t i c a l help; and my f a t h e r , S.O. Imbeah, who supported me with unceasing prayer throughout my stay i n Canada. I a l s o wish to thank Johnny Asafu-Adjaye f o r being my emotional support and the g r e a t e s t i n c e n t i v e . I a l s o thank Twum Ampofo Ansah f o r a l l that he has done f o r me throughout the course of my s t u d i e s here i n Canada. 1. INTRODUCTION Ce r e a l s were once c o n s i d e r e d to be sources of o n l y poor q u a l i t y p r o t e i n , d e f i c i e n t i n e s s e n t i a l amino a c i d s (EAA), e s p e c i a l l y l y s i n e ( P a u l i s and Wall, 1974). The growing awareness of the r o l e of p r o t e i n i n world n u t r i t i o n i n the 1960's, s t i m u l a t e d by i n t e n s e r e s e a r c h i n the f i e l d of n u t r i t i o n , l e d to the develop-ment of h i g h l y s i n e v a r i e t i e s of maize and other c e r e a l s (Munk, 1976). Work on other h i g h l y s i n e c e r e a l s s t a r t e d a f t e r the d i s c o v e r y of the high l y s i n e maize c u l t i v a r s opaque-2 and f l o u r y - 2 i n 1965 ( P a u l i s and Wall, 1974). Studies on p r o t e i n q u a l i t y and q u a n t i t y of r i c e s t a r t e d i n 196:8, wheat and b a r l e y i n 1969 and Sorghum i n 1972 (Munk, 1972). Rice and b a r l e y g r a i n s have b e t t e r amino a c i d balance than maize and Sorghum (Eggum, 1977b). L y s i n e content (g/16 g N) of c e r e a l p r o t e i n has been shown to be n e g a t i v e l y c o r r e l a t e d with crude p r o t e i n content (g/100 g DM) (Eggum, 1977a). Since the d i s c o v e r y of a s s o r t e d high l y s i n e genes i n maize,there has been much r e s e a r c h to over-come the d e p r e s s i o n i n y i e l d a s s o c i a t e d with the high l y s i n e c h a r a c t e r and a l s o to develop high y i e l d i n g 2. s t r a i n s of other g r a i n s that w i l l have improved l e v e l s of e s s e n t i a l amino a c i d s (Helm, 1972; P a u l i s and Wall, 1974). B a r l e y i s a major component of the d i e t of pi g s i n Canada. When used as the so l e g r a i n i n the d i e t , which i s commonly the case i n Canada, i t pr o v i d e s most of the energy and c o n t r i b u t e s more than h a l f of the t o t a l p r o t e i n . As a p r o t e i n source to support p i g growth i t i s poor i n q u a l i t y . L i k e other c e r e a l s , b a r l e y i s s e r i o u s l y d e f i c i e n t i n l y s i n e and to a l e s s e r extent i n other e s s e n t i a l amino a c i d s (EAA) ( F u l l e r , L i v i n g s t o n e , B a i r d and A t k i n s o n , 1979; F u l l e r , Mennie, and C r o f t s , 1979). The a d d i t i o n of s u i t a b l e p r o t e i n supplements to b a r l e y i n the f o r m u l a t i o n of p i g d i e t s not only i n c r e a s e s the d i e t a r y p r o t e i n but a l s o c o r r e c t s imbalances o f amino a c i d s . S e v e r a l sources of high l y s i n e , h i g h p r o t e i n b a r l e y have been developed (Munk, 1969). P r e s e n t l y , there are two major breeding l i n e s of high l y s i n e b a r l e y . These two are H i p r o l y from S v a l o f , Sweden (Munk and W e t t s t e i n , 1974) and 1508 from Ri s o , Denmark (Ingversen and Koie, 1973; D o l l , Koie and Eggum, 1974). H i p r o l y was d i s c o v e r e d i n the world b a r l e y c o l l e c t i o n i n 1968 at the Swedish Seed A s s o c i a t i o n 3. Research L a b o r a t o r i e s , S v a l o f , (Munk, K a r l s s o n and Hagberg, 1971). I t was s e l e c t e d on the b a s i s of i t s high content of b a s i c amino a c i d s as measured by the dye-binding c a p a c i t y (DBC) technique, r e l a t i v e to i t p r o t e i n content by K j e l d a h l a n a l y s i s (Munk and W e t t s t e i n , 1974). Mutant 1508 was d i s c o v e r e d by Ingversen, Koie and D o l l i n 1970 i n the Danish two-rowed v a r i e t y Bomi t r e a t e d with ethyleneimine at Riso (Eggum, 1977a). Both H i p r o l y and mutant 1508 have a h i g h content of e s s e n t i a l amino a c i d s (g/16 g N) e s p e c i a l l y l y s i n e , methionine and t h r e o n i n e (Munk and W e t t s t e i n , 1974). Mutant 1508 has 451 more- lysine than the parent v a r i e t y Bomi, while H i p r o l y has about 301 more l y s i n e than normal b a r l e y (Eggum, 1977a). High l y s i n e b a r l e y s are low i n prolamins (hordeins) ( D o l l , 1977) due to the r e d u c t i o n i n t h e i r s y n t h e s i s . I t has been shown that i n maize, sorghum and b a r l e y the genes c o n t r o l l i n g the s y n t h e s i s of prolamins are i n a c t i v a t e d i n the high l y s i n e genotypes ( D o l l , 1977). Mutant 1508 c o n t a i n s o n l y one t h i r d of the normal prolamin content, while the l y s i n e content (g/16 g N) i s i n c r e a s e d to 1501 of the parent v a r i e t y . The r e d u c t i o n i n y i e l d i n high l y s i n e genotypes i s a s s o c i a t e d w i t h the reduced prolamin s y n t h e s i s ( D o l l , 4. 1977). There i s an i n d i c a t i o n t h a t r e d u c t i o n i n prolamin s y n t h e s i s impairs the accumulation of carbohydrates i n the endosperm ( D o l l , 197 7). Increased p r o t e i n content i n h i g h l y s i n e genotypes i s due to reduced s t a r c h content of the endosperm (Koie and K r e i s , 1977). I f improving l y s i n e content (g/16 g N) leads to reduced s t a r c h syn-t h e s i s and hence reduced g r a i n y i e l d , one should c o n s i -der s e r i o u s l y whether p r o t e i n q u a l i t y should be improved at the expense of energy y i e l d , b e a r i n g i n mind that c e r e a l s are i n c l u d e d i n l i v e s t o c k d i e t s mainly f o r energy. Reduced g r a i n y i e l d i s demonstrated as reduced s i n g l e seed weight but the t o t a l number of seeds/unit area i s u n a f f e c t e d ; so i s the t o t a l p r o t e i n y i e l d / u n i t area. Breeding c e r e a l s f o r improved p r o t e i n q u a l i t y and q u a n t i t y r e q u i r e s r a p i d s c r e e n i n g methods ( T a l l b e r g , 1979). Chemical s c r e e n i n g methods are necessary to i d e n t i f y h i g h l y s i n e mutants i n b a r l e y , but simple v i s u a l s e l e c t i o n f o r opacity can be used f o r high l y s i n e maize and sorghum ( T a l l b e r g , 1979). Dye-binding c a p a c i t y (DBC) has been used e x t e n s i v e l y as a chemical method f o r s c r e e n i n g high l y s i n e b a r l e y (Munk, 1972; D o l l et a l . , 1974; Munk, 1976; and Eggum, 1977a). Most normal b a r l e y v a r i e t i e s show a common negative c o r r e l a t i o n 5. between the p r o t e i n l e v e l i n the seed and the l y s i n e content (g/16 g N) of the p r o t e i n (Munk, K a r l s s o n and Hagberg, 1971; D o l l et a l . , 1974 and Eggum, 1977a). However, i f l y s i n e content i s expressed on a dry matter (DM) b a s i s the l e v e l of p r o t e i n and the l e v e l of l y s i n e are p o s i t i v e l y c o r r e l a t e d f o r normal b a r l e y ( D o l l et a l . , 1974). Within high l y s i n e v a r i e t i e s , the same r e l a t i o n s h i p holds but at a higher l e v e l (Doll et al.,' 1974). T a l l b e r g (1979) has demonstrated these r e l a t i o n s h i p s i n normal b a r l e y s and high l y s i n e v a r i e t i e s . She obtained two r e g r e s s i o n l i n e s , each with a p o s i t i v e g r a d i e n t , the higher r e g r e s s i o n l i n e f o r high l y s i n e samples and the lower l i n e f o r normal samples. Research f o r the improvement of the n u t r i t i o n a l q u a l i t y of c e r e a l s i s j u s t i f i e d because c e r e a l s are the most important s t a p l e food i n the world but are d e f i c i e n t i n e s s e n t i a l amino a c i d s (EAA). An improve-ment i n amino a c i d balance w i l l supply the EAA at higher l e v e l s i n the p r o t e i n without r e q u i r i n g an i n c r e a s e i n p r o t e i n content (g/100 g DM) i n the g r a i n . C e r e a l s c o u l d then approach and p o s s i b l y become a 6. complete balanced food f o r the growing animal, thereby e l i m i n a t i n g the need f o r the a d d i t i o n o f p r o t e i n supple-ments. The ext e n s i v e use of high l y s i n e c e r e a l s would thus reduce the s e n s i t i v i t y of the c o s t of f e e d i n g to f l u c t u a t i o n s i n the p r i c e of p r o t e i n concentrates (Munk, 1976). Improved p r o t e i n q u a l i t y i s determined by chemical e v a l u a t i o n of e a r l y breeding l i n e s and by both chemical and b i o l o g i c a l e v a l u a t i o n on more advanced l i n e s (PAG, 1975). S e l e c t i o n f o r c h a r a c t e r s that are of d i r e c t economic importance f o r the g r a i n farmer such as di s e a s e r e s i s t a n c e , seed q u a l i t y and y i e l d are e a s i e r to j u s t i f y i n a p l a n t breeding program than q u a l i t y parameters, e.g., l y s i n e content, which exer t s i t s e f f e c t and importance i n the l a t t e r stages of the pro-d u c t i o n c h a i n (Munk, 1972). There i s a need f o r the p l a n t breeder to work together w i t h the n u t r i t i o n i s t and biochemist who can a s s i s t with s c r e e n i n g analyses and q u a l i t y e v a l u a t i o n . Methods that are normally used i n the 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 can be d i v i d e d i n t o ( i ) chemical, ( i i ) b i o l o g i c a l , ( i i i ) m i c r o b i o l o g i c a l , and (i v ) enzymatic. Microorganisms and enzymes have been wi d e l y used f o r d i g e s t i b i l i t y s t u d i e s and a l s o i n s t u d i e s on 7. the a v a i l a b i l i t y of amino a c i d s (Ford, 1960, 1962, 1964, 1966; S t o t t and Smith, 1966; Boyne, P r i c e , Rosen and S t o t t , 1967; Hsu, Vavak, S a t t e r l e e and M i l l e r , 1977; Buchmann, 1979a, 1979b and Ford and Hewitt, 1979). There are a v a r i e t y of chemical methods used i n e v a l u a t i n g p r o t e i n q u a l i t y . These i n c l u d e amino a c i d a n a l y s i s by i o n exchange chromatography a f t e r h y d r o l y s i s , and dye b i n d i n g techniques. Chemical methods have been used f o r measuring a v a i l a b l e l y s i n e and the a v a i l a b i l i t y of other EAA. The FDNB method of Carpenter (1960) i s widely used f o r measuring a v a i l a b l e l y s i n e i n animal products. In p l a n t m a t e r i a l s , there i s an i n t e r f e r e n c e by s t a r c h which makes t h i s method u n r e l i a b l e and u n s u i t a b l e . B i o l o g i c a l e v a l u a t i o n of n u t r i t i o n a l q u a l i t y may be done e i t h e r with l a b o r a t o r y animals or the a c t u a l animal f o r which that p r o t e i n i s to be fed e v e n t u a l l y . L aboratory animals most commonly used are r a t s and mice. Amino a c i d composition has been used as one of the c r i t e r i a f o r a s s e s s i n g p r o t e i n q u a l i t y . Since l y s i n e i s the most l i m i t i n g amino a c i d i n c e r e a l s , i n c l u d i n g b a r l e y , l y s i n e l e v e l has been used as a measure of n u t r i t i o n a l v a l u e . Improving the l y s i n e l e v e l has been shown to improve n u t r i t i o n a l q u a l i t y (Eggum, 1977a) p r o v i d i n g the p r o t e i n i s not heat 8. damaged before f e e d i n g . However, one cannot, on the b a s i s of amino a c i d composition alone, make a c o n c l u s i o n about the q u a l i t y of a p r o t e i n . T h i s i s because p r o t e i n q u a l i t y i s a f f e c t e d both by the d i g e s t i b i l i t y of the p r o t e i n and the a v a i l a b i l i t y of i t s amino a c i d s . I t i s a l s o a f f e c t e d by a n t i n u t r i t i o n a l f a c t o r s such as protease i n h i b i t o r s , saponins and t a n n i n s . B a r l e y g r a i n has no saponins or protease i n h i b i t o r s such as are found i n legumes but i t can c o n t a i n low l e v e l s of t a n n i n s . Tannins are known to impair p r o t e i n d i g e s t i b i l i t y and the a v a i l a b i l i t y of amino a c i d s and thereby reduce pro-t e i n q u a l i t y (Ford and Hewitt, 1979). Tannins exert t h e i r e f f e c t by b i n d i n g with the p r o t e i n s i n the d i g e s t a such that they are p r e c i p i t a t e d out, or they b i n d the d i g e s t i v e enzymes so t h a t they are i n c a p a b l e of hydro-l y s i n g the d i e t a r y p r o t e i n s (Eggum and C h r i s t e n s e n , 1975). These authors found t a n n i n content of t h e i r b a r l e y samples to range from 0.55 to 1.23%. They observed a s i g n i f i c a n t n e gative c o r r e l a t i o n between the t a n n i n l e v e l of a b a r l e y and i t s p r o t e i n d i g e s t i -b i l i t y . They a l s o suggested that t h i s c o r r e l a t i o n may p a r t l y e x p l a i n the r e l a t i v e l y low d i g e s t i b i l i t y of b a r l e y p r o t e i n when compared with other c e r e a l p r o t e i n s . 9. The q u a l i t y of a p r o t e i n a l s o depends on how much albumin and g l o b u l i n i t c o n t a i n s . Albumins and g l o b u l i n s are high q u a l i t y p r o t e i n s r e a d i l y s o l u b l e i n water, and hig h i n l y s i n e when compared with prolamins, the storage p r o t e i n s (Folkes and Yemm, 1956 and Ingversen and Koie, 1973). G l u t e l i n s are onl y i n t e r m e d i a t e i n q u a l i t y when compared with albumins and g l o b u l i n s . I t has been shown that albumins and globulins together c o n t r i b u t e 44% of the t o t a l l y s i n e (Ingversen and Koie, 1973) i n Emir b a r l e y , a normal v a r i e t y . The present study i n v o l v e s f i v e l i n e s of b a r l e y from the same p l o t i n Southern A l b e r t a . The f i v e l i n e s were H i p r o l y , G a i t (a wi d e l y grown commercial v a r i e t y ) and three l i n e s c o n t a i n i n g the high l y s i n e gene. 10. LITERATURE REVIEW P r o t e i n Q u a l i t y The q u a l i t y of a p r o t e i n may be d e f i n e d as i t s a b i l i t y to supply e s s e n t i a l amino a c i d s (EAA) i n the amounts and p r o p o r t i o n s r e q u i r e d f o r a given p h y s i o l o -g i c a l f u n c t i o n , e.g., growth. T h i s a b i l i t y to supply e s s e n t i a l amino a c i d s i s a f f e c t e d both by amino a c i d composition and by amino a c i d a v a i l a b i l i t y . T o t a l amino a c i d s present i n a p r o t e i n are u s u a l l y measured by chemical methods f o l l o w i n g h y d r o l y -s i s . Because amino a c i d s are g e n e r a l l y o n l y p a r t i a l l y a v a i l a b l e , t o t a l q u a n t i t y g i v e s o n l y an i n a c c u r a t e assessment o f the amount absorbable from the gastro -i n t e s t i n a l t r a c t . However, there are s e v e r a l a d d i t i o n a l f a c t o r s t hat a f f e c t p r o t e i n q u a l i t y apart from a v a i l -a b i l i t y o f amino a c i d s . F a c t o r s A f f e c t i n g P r o t e i n Q u a l i t y  1. Amino A c i d Composition Amino a c i d composition i s a b a s i c measure of p r o t e i n q u a l i t y . I t has been used f o r computing the 11. chemical score by comparing with the amino a c i d composi-t i o n o f a standard p r o t e i n ( M i t c h e l l and Block, 1946). C e r e a l s are known to be d e f i c i e n t i n l y s i n e , t hreonine and tryptophan i n v a r y i n g combinations f o r d i f f e r e n t c e r e a l s , f o r the monogastric animal. L y s i n e , however, i s the f i r s t l i m i t i n g amino a c i d i n most c e r e a l s (FAO n u t r i t i o n a l s t u d i e s no. 16, 1957; D o l l , Koie and Eggum, 1973; P a u l i s and Wall, 1974; Sauer, G i o v a n n e t t i and S t o t h e r s , 1974 ; Eggum, 1977a and Batterham, Murri s o n and Lewis, 1978). L y s i n e , apart from being the f i r s t l i m i t i n g amino a c i d i n c e r e a l p r o t e i n s , i s a l s o the l e a s t a v a i l a b l e amino a c i d i n c e r e a l p r o t e i n s (Eggum, 1977a). In p r a c t i c e , due to t h e i r d e f i c i e n c y i n essen-t i a l amino a c i d s (EAA), c e r e a l - b a s e d d i e t s f o r animals are supplemented with p r o t e i n concentrates such as f i s h m e a l , meatmeal and soybean meal (SBM), each of which has an amino a c i d p a t t e r n s a t i s f a c t o r y f o r complement-ing t h a t of c e r e a l s . S y n t h e t i c amino a c i d s are a l s o used as supplements f o r c e r e a l based d i e t s . L y s i n e supplementation of g r a i n g e n e r a l l y improves animal performance. A d d i t i o n of other EAA, e s p e c i a l l y t h r e o n i n e , has a l s o proved beneficial (Pond, H i l l i e r and Benton, 1958 and Chung and Beames, 1974). 12. In a study with Peace R i v e r B a r l e y f o r growing p i g s , Chung and Beames (1974) observed t h a t supplementation of b a r l e y with l y s i n e to p r o v i d e 0.75% improved the per-formance of growing p i g s . F u r t h e r improvement was observed with the a d d i t i o n of th r e o n i n e . Bayley and Summers (1968) observed that the response to supplementation of a corn-SBM r a t i o n w i t h l y s i n e and methionine depended on the a d d i t i o n o f t h r e o n i n e , i n d i c a t i n g t h i s amino a c i d to be d e f i c i e n t i n low p r o t e i n corn-SBM d i e t s . Rosenberg, C u l i k and Eck e r t (1959) have found t h a t l y s i n e and threonine supplementation o f r i c e d i e t s caused substan-t i a l growth response i n weanling r a t s . A d d i t i o n o f other EAA r e s u l t e d i n f u r t h e r improvement i n performance. Waggle, P a r r i s h and Deyoe (1966) a l s o observed improved performance i n r a t s when f e d corn or sorghum supplemented with l y s i n e , h i s t i d i n e and a r g i n i n e . B a r l e y i s an important g r a i n i n the animal feed i n d u s t r y . Normal b a r l e y i s d e f i c i e n t i n l y s i n e , as are other c e r e a l g r a i n s . A number of hig h l y s i n e b a r l e y l i n e s have been developed through p l a n t breed-in g ( D o l l et a l . , 1974; P a u l i s and Wall, 1974 and Munk et a l . , 1969). Among the high l y s i n e l i n e s o f b a r l e y , H i p r o l y and mutant 1508 have been much s t u d i e d . H i p r o l y was i s o l a t e d from the world b a r l e y c o l l e c t i o n 13. i n Sweden while mutant 1508 was c h e m i c a l l y induced i n a breeding program at Ri s o , Denmark. Pomeranz, Robbins, Wessenberg, Hockett and G i l b e r t s o n (1973) have shown that the high l y s i n e content (g/16 g N) of H i p r o l y i s a s s o c i a t e d with high a s p a r t i c a c i d content (g/16 g N). I t i s known that a s p a r t i c a c i d i s a key i n t e r m e d i a r y i n the b i o s y n t h e s i s of l y s i n e i n higher p l a n t s , b a c t e r i a and algae (Pomeranz et a l . , 1973). There i s a n e g a t i v e c o r r e l a t i o n between crude p r o t e i n l e v e l i n g r a i n and l y s i n e content i n p r o t e i n i n most c e r e a l s except oats (Munk, 1971). The n e g a t i v e c o r r e l a t i o n i s due to a g r e a t e r r e l a t i v e s y n t h e s i s of storage p r o t e i n s low i n l y s i n e and other e s s e n t i a l amino a c i d s as the t o t a l p r o t e i n content i n c r e a s e s (Munk, 1972). In normal 6-row b a r l e y and 2-row b a r l e y , the amino a c i d composition depends on the p r o t e i n content. In H i p r o l y , the genes c o n t r o l l i n g p r o t e i n l e v e l and l y s i n e concentra-t i o n are not g e n e t i c a l l y l i n k e d so that they segregate independently (Munk, K a r l s s o n , Hagberg and Eggum, 1970 and Pomeranz et a l . , 1973). Amino a c i d composition by i t s e l f i s not a good measure of p r o t e i n q u a l i t y . In p r o t e i n s that are damaged during p r o c e s s i n g , the amino a c i d composition 14. remains unchanged but b i o l o g i c a l value i s d r a s t i c a l l y reduced as i n heat damaged f i s h meal (Eggum, 1973). 2. P r o c e s s i n g and Storage Prolonged storage and v a r i o u s procedures used i n food p r o c e s s i n g a f f e c t p r o t e i n q u a l i t y i n s e v e r a l ways (Eggum, 1973). The p r i n c i p a l f a c t o r s i n v o l v e d are temperature l e v e l , d u r a t i o n of heat treatment and the presence of moisture and reducing substances. P r o t e i n q u a l i t y may be reduced when a food i s exposed to high temperatures d u r i n g p r o c e s s i n g . Heat damage of p r o t e i n s may occur through l o s s e s of amino n i t r o g e n by t r a n s -f o r m a t i o n to n o n - p r o t e i n n i t r o g e n (NPN) (Eggum, 1973). There may be formation of e s t e r s w i t h i n the p r o t e i n molecule which decreases the s o l u b i l i t y and d i g e s t i b i -l i t y o f the p r o t e i n and a l s o cause reduced a v a i l a b i l i t y o f the amino a c i d s i n v o l v e d i n the e s t e r formation (Bjarnason and Carpenter, 1970). Peptides with such l i n k a g e s are r e s i s t a n t to enzyme a c t i o n ; hence the reduced d i g e s t i b i l i t y and a v a i l a b i l i t y . In heat damaged p r o t e i n s there c o u l d be r a c i m i s a t i o n , i . e . , the L form of amino a c i d s may be converted to the D form which either cannot be used by 15. the animal or used o n l y p a r t i a l l y . A major process that occurs i n heat damage of p r o t e i n s i s the M a i l l a r d reac-t i o n , which i s the r e a c t i o n between l y s i n e and reducing sugars. In t h i s r e a c t i o n the e-amino group of l y s i n e forms an enzyme r e s i s t a n t bond with simple sugars and thus becomes n u t r i t i o n a l l y u n a v a i l a b l e . L y s i n e i s the most heat s e n s i t i v e amino a c i d (Carpenter, Morgan, Lea and P a r r , 1962) . Temperature and d u r a t i o n o f h e a t i n g are important i n determining the extent of damage, e.g., baking a r a t d i e t at 200°C f o r 15 to 20 minutes reduced PER from 3.5 to 2.4.. A f u r t h e r h e a t i n g by t o a s t i n g at 130°C f o r 40 to 60 minutes reduced PER to 0.8. An a d d i t i o n of l y s i n e c o r r e c t e d the PER to the o r i g i n a l value of 3.5, thus i n d i c a t i n g t h a t l y s i n e was destroyed by the heat treatments (Eggum, 1973). The extent of heat damage i s a l s o a f f e c t e d by the moisture content of the food m a t e r i a l (Carpenter et a l . , 1962). These authors have shown that the l o s s of a v a i l a b l e l y s i n e was g r e a t e s t at 5 to 14% moisture. T h i s means that d r y i n g of food m a t e r i a l s should not go below 14% moisture. Eggum (1973) r e p o r t e d that s t e r i l i z a t i o n of dry and soaked r a t d i e t s (11.2% and 71.6% moisture) caused more damage i n the dry feed than i n the soaked feed. 16. 3. A v a i l a b i l i t y of Amino A c i d s A v a i l a b l e amino a c i d s are those t h a t are a c c e s s i b l e to the organism f o r a n a b o l i c p h y s i o l o g i c a l f u n c t i o n s such as p r o t e i n s y n t h e s i s . A l l amino a c i d s that are absorbed and u t i l i z e d are termed a v a i l a b l e (Bodwell, 1976 and Erb e s d o b l e r , 1976). The a v a i l a b i l i t y of amino a c i d s may be impaired by incomplete d i g e s t i o n o f the p r o t e i n and incomplete a b s o r p t i o n r e s u l t i n g from i n d i g e s t i b l e c e l l w a l l s , a bulky p r o t e i n s t r u c t u r e or many c r o s s - l i n k a g e s i n the p r o t e i n molecule ( M i l l e r , Carpenter and M i l n e r , 1965 and Erbesdobler, 1976). A v a i l a b i l i t y o f amino a c i d s may be impaired by the presence of enzyme i n h i b i t o r s , i n h i b i t i o n o f enzyme b i n d i n g s i t e s i n the p r o t e i n and a l s o the i n h i b i t i o n o f amino a c i d a b s o r p t i o n by pe p t i d e s and p e p t i d e - l i k e compounds (Sauer et al.., 1974; and Erbesdobler, 1976). E x c e s s i v e heat treatment d u r i n g p r o c e s s i n g tends to reduce amino a c i d a v a i l a b i l i t y by causing i n t e r a c t i o n s between p r o t e i n s i d e groups and red u c i n g sugars (Erbesdobler, 1976). The s t u r c t u r e of l y s i n e i n the f r e e form has a f r e e e-amino group, a f r e e a-amino group and a f r e e : c a r b o x y l group. In p r o t e i n s , l y s i n e i s l i n k e d to other amino a c i d s through pep t i d e l i n k a g e s 17. i n v o l v i n g the a-amino and a - c a r b o x y l groups. Only the e p s i l o n amino group i s f r e e to r e a c t w i t h v a r i o u s s u b s t a n c e s such as sugars. I f the e-amino group i s bound th r o u g h a c h e m i c a l r e a c t i o n the a f f e c t e d l y s i n e becomes n u t r i t i o n a l l y u n a v a i l a b l e ( C a r p e n t e r and B j a r n a s o n , 1969; 1970; V a l l e - R i e s t r a and Barnes, 1970; B o d w e l l , 1976 and E r b e s d o b l e r , 1976). L y s i n e may a l s o form c r o s s l i n k a g e s w i t h o t h e r amino a c i d s such as g l u t a m i c a c i d and a s p a r t i c a c i d , thus making them u n a v a i l a b l e i n heat damaged p r o t e i n s ( M i l l e r et a l . , 1965 and E r b e s d o b l e r , 1976). C r o s s - 1 i n k a g e s h i n d e r the enzyme a t t a c k and r e l e a s e o f even those amino a c i d s t h a t are not d i r e c t l y i n v o l v e d i n the r e a c t i o n ( M i l l e r e t a l . , 1965). 4. D i g e s t i b i l i t y o f P r o t e i n s D i g e s t i b i l i t y o f p r o t e i n s , i . e . , the r e d u c t i o n to d i p e p t i d e s and amino a c i d s c a p a b l e o f a b s o r p t i o n from the g a s t r o - i n t e s t i n a l t r a c t i s a p r i m a r y f a c t o r a f f e c t i n g p r o t e i n q u a l i t y (Sauer, 1974 and E r b e s d o b l e r , 1976). The d i g e s t i b i l i t y o f a p r o t e i n may be reduced by heat damage as was o b s e r v e d by M i l l e r e t a l . (1965) w i t h h eated cod muscle. Such f a c t o r s as enzyme i n h i b i t o r s and i n a c e s s i b i l i t y o f p r o t e i n to enzyme 18. a c t i o n due to a bulky s t r u c t u r e c o u l d reduce p r o t e i n d i g e s t i b i l i t y (Erbesdobler, 1976). Some p r o t e i n mole-c u l e s are bulky due to t h e i r t e r t i a r y s t r u c t u r e . In such cases i t i s d i f f i c u l t f o r enzymes to c l e a v e the p e p t i d e bonds on the i n s i d e o f t h i s molecule and r e l e a s e the amino a c i d s . Apart from heat damage and protease i n h i b i t o r s i n legumes, the presence of t a n n i n s , quinones and saponins c o u l d reduce the d i g e s t i b i l i t y of p r o t e i n s (Cheeke and Myer, 1975). Such a n t i n u t r i t i o n a l f a c t o r s , do not occur i n b a r l e y except f o r tannins which occur i n small amounts of 0.55% to 1.23% (Eggum and C h r i s t e n s e n , 1975). Tannins are not a s e r i o u s problem i n b a r l e y g r a i n s . Methods of Measuring A v a i l a b l e Lysine Chemical Determination o f A v a i l a b l e Lysine A number of methods have been proposed, the m a j o r i t y of them depending on the r e a c t i o n of p r o t e i n w i t h l - F l u o r o - 2 , 4 - D i n i t r o b e n z e n e (Milner and Westgarth, 1973). Of the chemical methods a v a i l a b l e , the Carpenter (1960) and- S i l c o c k (see Roach et a l . , 1967) methods are most f r e q u e n t l y used. Although the Carpenter method i s w e l l e s t a b l i s h e d f o r measuring a v a i l a b l e l y s i n e i n animal products, i t has shortcomings when a p p l i e d to c e r e a l and o i l seed meals (Roach et a l . , 1967). T h i s i s because i t gives v a r i a b l e r e s u l t s with these feed-s t u f f s and poor r e c o v e r i e s o f the l y s i n e d e r i v a t i v e e-DNP-lysine due to their 'carbohydrates d u r i n g h y d r o l y s i s . A l s o there i s the formation o f other y e l l o w products which are not e a s i l y separated from the d e r i v a t i v e (Roach et a l . , 1967). The a l t e r n a t i v e method suggested by these authors i s the S i l c o c k method. While the Carpenter method measures a v a i l a b l e l y s i n e by measuring the amount of FDNB that has r e a c t e d with e-amino groups to form the l y s i n e d e r i v a t i v e , the S i l c o c k method measures a v a i l a b l e l y s i n e by d i f f e r e n c e , i . e . , the d i f f e r e n c e between t o t a l l y s i n e before the r e a c t i o n with FDNB and the r e s i d u a l l y s i n e a f t e r the r e a c t i o n . The r e s i d u a l l y s i n e i s u n a v a i l a b l e l y s i n e because i t does not r e a c t with the FDNB. Th i s d i f f e r e n c e method has no problem with c o l o u r i n t e r f e r e n c e s because i t does not measure c o l o u r as i n the Carpenter method but measures l y s i n e i n the h y d r o l y s a t e s by ion-exchange chromatography (Roach et a l . , 1967). However, i t does have the shortcoming i n r e q u i r i n g an a n a l y s i s f o r t o t a l l y s i n e i n the o r i g i n a l m a t e r i a l and again i n the m a t e r i a l 20. a f t e r the r e a c t i o n . T h i s i s more time consuming and expensive than a measurement of the c o l o u r e d r e a c t i o n products by spectrophotometry as used i n the d i r e c t method. G e n e r a l l y , chemical methods f o r e s t i m a t i n g a v a i l a b l e l y s i n e are e i t h e r d i r e c t or i n d i r e c t . Bodwell (1976) d e s c r i b e d a number of d i r e c t methods using v a r i o u s chemicals i n the formation of v a r i o u s d e r i v a t i v e s . Examples o f the d i r e c t methods which he mentions i n c l u d e Carpenter's FDNB method and the method of Kakade and L i e n e r (1969) u s i n g t r i n i t r o b e n z e n e s u l f o n i c a c i d (TNBS), forming TNP-lysine as a d e r i v a t i v e . The value of the d i r e c t chemical methods i s reduced, due to the f a c t t h a t the d e r i v a t i v e s are p a r t i a l l y h y d r o l y s e d d u r i n g a c i d h y d r o l y s i s (Bodwell, 1976). The i n d i r e c t methods can be done wi t h the same chemicals as used f o r the d i r e c t e s t i m a t i o n . Apart from the S i l c o c k i n d i r e c t method, dye b i n d i n g d i f f e r e n c e i s wi d e l y used f o r e s t i m a t i n g 21. (a) FDNB Direct-Method (Carpenter Method) A schematic r e p r e s e n t a t i o n (Bodwell, 1976) P r o t e i n R eaction with FDNB A c i d H y d r o l y s i s F i l t r a t i o n E x t r a c t i o n s and treatments to remove  i n t e r f e r i n g substances I Spectrophotometric e s t i m a t i o n o f DNP-Lysine - AVAILABLE LYSINE (b) I n d i r e c t FDNB Method A schematic r e p r e s e n t a t i o n (Bodwell, 1976) P r o t e i n A c i d H y d r o l y s i s 4 Column chromatography I T o t a l L y s i n e Reaction with FDNB 4 A c i d H y d r o l y s i s I Column chromatography 4/ Unreacted Lysine UNAVAILABLE LYSINE A v a i l a b l e l y s i n e = T o t a l l y s i n e - unreacted l y s i n e 22. a v a i l a b l e l y s i n e ( H u f r e l and Carpenter, 1975, 1976 and Walker, 1979). Dye b i n d i n g , u s i n g a c i d dyes at alow pH i n v o l v e s the formation of i o n i c l i n k a g e s with the b a s i c amino a c i d s l y s i n e , h i s t i d i n e and a r g i n i n e present i n the p r o t e i n (Walker, 1979). The dye b i n d i n g method of e s t i m a t i n g a v a i l a b l e l y s i n e i n v o l v e s the b l o c k i n g of the e-amino group of l y s i n e with reagents such as e t h y l c h loroformate (Sandler and Warren, 1974) and p r o p i o n i c anhydride (Hurrel and Carpenter, 1975 and Walker, 1979). The dye b i n d i n g d i f f e r e n c e (DBD) was c a l c u l a t e d as the d i f f e r e n c e between the dye b i n d i n g c a p a c i t y of the pro-t e i n b efore and a f t e r b l o c k i n g the e-amino group of l y s i n e . The DBD i s then used to c a l c u l a t e the percent a v a i l a b l e l y s i n e (Walker, 1979). Values of r e a c t i v e l y s i n e measured by DBD compare f a v o u r a b l y with v a l u e s f o r d i r e c t FDNB r e a c t i v e l y s i n e . T h i s f a c t i s supported by r e s u l t s presented below, obtained by H u r r e l and Carpenter (1976). Walker (1979) has observed a c o r r e -l a t i o n c o e f f i c i e n t o f 0.90 between DBD and FDNB reac-t i v e l y s i n e . 23. R e l a t i v e l y s i n e c o n c e n t r a t i o n s (mmol l y s i n e / k g crude p r o t e i n ) i n vegetable and pure p r o t e i n s determined by dye b i n d i n g d i f f e r e n c e and by r e a c t i o n with FDNB. DBD-reactive FDNB-reactive l y s i n e l y s i n e Wheat g l u t e n 110 100 Whole wheat 190 180 Rice 200 200 Sweet corn 220 220 Groundnut f l o u r 230 220 Broad bean 330 340 Soya bean 370 390 Chick pea 440 410 Wing bean 500 470 Bovine plasma albumin 810 830 B i o l o g i c a l Determination of A v a i l a b l e L y s i n e This may be done by the use of l a b o r a t o r y animals or farm animals. Lysine i s normally the f i r s t l i m i t i n g amino a c i d i n c e r e a l - b a s e d d i e t s f o r growing p i g s . T h i s l i m i t a t i o n i s u s u a l l y overcome by 24. supplementing with p r o t e i n c o n c e n t r a t e s . A v a i l a b i l i t y of l y s i n e i n most p r o t e i n c o n c e n t r a t e s that have not been heat damaged i s h i g h , e.g., f i s h meal and soybean-meal have h i g h l y s i n e l e v e l s of 7.60 g/16 g N and 5.98 g/16 g N r e s p e c t i v e l y w i t h a v a i l a b i l i t i e s of 96.8% and 91.6% as determined by r a t experiments (Eggum, 1973). Other p r o t e i n c o n c entrates such as groundnut meal and sunflower seed meal that are used as supplements have l y s i n e a v a i l a b i l i t i e s of 86.9% and 88.0% r e s p e c t i v e l y (Eggum, 1973) as determined i n r a t experiments. However, M i l n e r and Westgarth (1973) u s i n g S i l c o c k d i f f e r e n c e method ob t a i n e d l y s i n e a v a i l a b i l i t i e s of 94.6%, 92.7%, 96.7% and 94.7% f o r f i s h meal, groundnut meal, soybean meal and sunflower seed meal. Batterham, Murison and Lewis (1978) u s i n g wheat-based d i e t s supplemented with v a r i o u s p r o t e i n c o n c entrates ( f i s h meal, meat meal skim m i l k , soybean meal, etc.) and l y s i n e , and a con-t r o l d i e t w i t h no supplement, showed the d i f f e r e n c e s i n growth response to be due to d i f f e r e n c e s i n a v a i l -a ble l y s i n e . The a v a i l a b l e l y s i n e contents of the d i f f e r e n t p r o t e i n c o n c entrates were measured by r a t bioassay, and these were s a i d to correspond to the v a l u e s estimated by the S i l c o c k d i f f e r e n c e method, although the authors showed no f i g u r e s f o r t h i s comparison. B i o l o g i c a l a v a i l a b i l i t y of amino a c i d s has been determined from the d i f f e r e n c e between the amino a c i d content of the feed consumed and the amino a c i d content of the faeces (Ousterhout, Grau and Lundholm, 1959; DeMuelenaere and Feldman, 1960 and Eggum, 1973) Th i s method has two disadvantages, which are: (a) The a c t i o n o f i n t e s t i n a l m i c r o f l o r a on food (b) The f a c t t h at the d i e t can i n f l u e n c e the s e c r e t i o n of p r o t e i n r i c h m a t e r i a l i n t o the g a s t r o - i n t e s t i n a l t r a c t . (Lymman, 1957 and Sauer, Stothers and.Parker, 1977). These f a c t o r s a f f e c t the magnitude of the metabolic f a e c a l and endogenous u r i n a r y n i t r o g e n so t h a t true a v a i l a b i l i t y of amino a c i d s obtained by f a e c a l ana-l y s i s i s i n a c c u r a t e . Another b i o l o g i c a l method that has been used i n v o l v e s the comparison between growth r a t e s obtained on d i e t s low i n EAA and growth r a t e s obtained on s i m i l a r d i e t s to which known amounts of t e s t p r o t e i n have been added. By supplementing the ba s a l d i e t with s e v e r a l l e v e l s of the l i m i t i n g amino 26. a c i d s a growth response curve can be obtained and the amount of the amino a c i d i n the t e s t p r o t e i n determined (Ousterhout et a l . , 1959). Researchers u s i n g t h i s method o f t e n end up wit h d i f f e r e n t r e s u l t s f o r the same amino a c i d (Schweigert and Guthneck, 1953; Guthneck, Bennet and Schweigert, 1953, and Gupta, Dakrouy, Harper and Elvehjem, 1958). Sauer et a l . (1974) f e d v a r i o u s c e r e a l s ( b a r l e y , wheat, t r i t i c a l e ) and soybean meal to barrows and determined a v a i l a b l e l y s i n e by f a e c a l analy-s i s . The metabolic and endogenous amino a c i d s were determined with n i t r o g e n f r e e d i e t s . In t h i s study, they observed t h a t l y s i n e a v a i l a b i l i t y was 77% i n normal b a r l e y , 80.8% i n wheat, 94.2% i n t r i t i c a l e and 94.9% i n soybean meal with 10 kg p i g s . The same d i e t f e d to 30 kg p i g s produced a v a i l a b i l i t i e s of 65%, 67%, 77.5% and 91.2% i n b a r l e y , wheat, t r i t i c a l e and soybean meal r e s p e c t i v e l y . No e x p l a n a t i o n was given f a r the decreased a v a i l a b i l i t y i n . t h e o l d e r animals. 27. M i c r o b i o l o g i c a l E s t i m a t i o n of A v a i l a b l e Lysine A number of microorganisms have been used to estimate p r o t e i n q u a l i t y i n feed m a t e r i a l s . Tetrahymena  p y r i f o r m i s , W; Streptococcus zymogenes and Streptococcus  f a e c a l i s have been used i n e s t i m a t i n g gross p r o t e i n v a l u e , net p r o t e i n u t i l i z a t i o n and net p r o t e i n r e t e n t i o n . Use-f u l c o r r e l a t i o n s have been found between p r o t e i n q u a l i t y f o r r a t s and c h i c k s and the r e s u l t s from assays with T. p y r i f o r m i s , S. zymogenes and S. f a e c a l i s (Boyne, P r i c e , Rosen and S t o t t , 1967). T. p y r i f o r m i s i s s i m i l a r to higher animals i n the response to p r o t e i n q u a l i t y , e s p e c i a l l y a v a i l a b l e l y s i n e (Boyne et a l . , 1967). Several m i c r o b i o l o g i c a l methods have been recommended f o r use i n the study of p r o t e i n q u a l i t y (Ford, 1960). Indic e s of p r o t e i n v a l u e found with m i c r o b i o l o g i c a l methods agree with the f i n d i n g s from r a t growth t e s t s ; T. p y r i f o r m i s simulates more c l o s e l y the circumstances of b i o l o g i c a l t e s t s (Ford, 1960). I t has the same amino a c i d requirements.-similar to the- girowirtg-.rat and:.is able to use i n t a c t p r o t e i n . Ford (I960)- found a good p o s i -t i v e c o r r e l a t i o n between the r e l a t i v e n u t r i t i o n a l v a l u e s f o r S. zymogenes on a number of p r o t e i n s and t h e i r net p r o t e i n u t i l i z a t i o n (NPU) as determined with 28. r a t s . S t o t t and Smith (1966) have observed that T e t r a -hymena estimates of a v a i l a b l e l y s i n e are s i m i l a r to those obtained by the FDNB method of Carpenter f o r samples high i n a v a i l a b l e l y s i n e . For samples of lower a v a i l -able l y s i n e content Tetrahymena estimates were higher than those with FDNB. The authors attempted to e x p l a i n t h i s d i f f e r e n c e by saying that f a c t o r s other than b i n d i n g of the amino group--appear to--have i n f l u e n c e d the T e t r a -hymena assays. Supplementation of C e r e a l s with E s s e n t i a l Amino  A c i d s Supplementation of p r o t e i n with the f i r s t l i m i t i n g EAA i s h i g h l y b e n e f i c i a l i f i t b r i n g s the t o t a l amount of t h i s amino a c i d present i n the p r o t e i n and a v a i l a b l e to the organism i n t o balance with the second l i m i t i n g amino a c i d (Rosenberg, C u l i k and E c k e r t , 1959). When the proper balance has been achieved between the f i r s t two l i m i t i n g amino a c i d s , f u r t h e r improvement can be achieved i f both are added s i m u l t a n e o u s l y i n the p r o p o r t i o n s of the body's requirement (Rosenberg et a l . , 1959). There are.a number of r e p o r t s i n the l i t e r a t u r e t h a t combination of l y s i n e and threonine 29. causes s u b s t a n t i a l growth responses i n weanling r a t s f e d c e r e a l - b a s e d d i e t s . Rosenberg et a l . (1959) have observed that a d d i t i o n of other EAA to l y s i n e - t h r e o n i n e supplemented d i e t s caused f u r t h e r improvement i n r a t performance. Chung and Beames (1974) observed improve-ment i n the performance of.growing p i g s when f e d b a r l e y supplemented to a l e v e l of 0.75% l y s i n e . An a d d i t i o n a l improvement was observed with the a d d i t i o n of threonine. Other workers have made s i m i l a r o b s e r v a t i o n s w i t h other c e r e a l s (Pond., H i l l i e r and Benton, 1958 and Bayley and Summers, 1968). Margruder, Sherman and Reynolds (1961) observed a s i g n i f i c a n t i n c r e a s e i n the r a t e of g a i n of baby p i g s by adding 0.1% l y s i n e to a 14% crude p r o t e i n d i e t c o n t a i n i n g 0.74% l y s i n e . The r a t e of g a i n with supplementation was equal to that on a 16% p r o t e i n s t a r t e r , not supplemented with l y s i n e . No s i g n i f i c a n t i n c r e a s e i n feed e f f i c i e n c y was observed with l y s i n e supplementation. They a l s o observed a tendency toward improvement of some c a r c a s s c h a r a c t e r i s t i c s i n l y s i n e f e d p i g s . Bayley and Summers (1968) observed no b e n e f i c i a l e f f e c t on growth r a t e and feed e f f i c i e n c y when p r a c t i c a l corn-SBM r a t i o n s were supplemented with 0.1% l y s i n e or 0.05% methionine. However, when both were 30. given s i m u l t a n e o u s l y there was a p o s i t i v e e f f e c t on ga i n . I t i s known that corn i s d e f i c i e n t i n l y s i n e and soybean meal i s d e f i c i e n t i n methionine. Berry,. Combs, Wallace and Robbins (1966) observed that with corn-soybean d i e t s , the response to supplementation w i t h l y s i n e and methion-ine depended on the a d d i t i o n of th r e o n i n e . T h i s c o u l d be because threonine i s the second l i m i t i n g amino a c i d i n the d i e t . I t i s necessary to o b t a i n a balance between the f i r s t and second l i m i t i n g amino a c i d s to see the b e n e f i c i a l e f f e c t o f supplementation. F u l l e r et a l . (1979) have observed that growth r a t e o f p i g s i n c r e a s e d from .36 kg/day to a maximum of 0.65 kg/day with the a d d i t i o n o f 3.8 g l y s i n e / k g and 1.8 g threonine/kg to t h e i r b arley-based d i e t . Carcass q u a l i t y was a l s o found to be improved with a d d i t i o n of l y s i n e ( F u l l e r et a l . , 1979). They a l s o observed t h a t back f a t t h i c k n e s s decreased p r o g r e s s i v e l y with i n c r e a s i n g a d d i t i o n o f l y s i n e and thre o n i n e . I t has been e s t a b l i s h e d 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 (Chung and Beames, 1974). F u l l e r et a l . (1979) have proved that much improvement i n the u t i l i z a t i o n o f c e r e a l p r o t e i n can be achieved by the simultaneous a d d i t i o n of j u s t l y s i n e and threonine. 31. Supplementation o f a d i e t w i t h amino a c i d s f o r animals r e q u i r e a knowledge of the amino a c i d r e q u i r e -ments of the p a r t i c u l a r animal. Amino a c i d s u t i l i z e d by animals may be e s s e n t i a l or n o n - e s s e n t i a l . E s s e n t i a l amino a c i d s (EAA) are those that e i t h e r cannot be syn-t h e s i z e d by the animal or are not s y n t h e s i z e d i n ade-quate q u a n t i t i e s to meet requirements. A r g i n i n e , h i s t i -d ine, i s o l e u c o n e , l e u c i n e , l y s i n e , methionine, t r y p t o -phan and v a l i n e are the e s s e n t i a l amino a c i d s f o r the growth of r a t s , p i g s and man. N o n - e s s e n t i a l amino a c i d s can be s y n t h e s i z e d by animals, however, they need an adequate n i t r o g e n supply to achieve t h i s . Some amino a c i d s that may be e s s e n t i a l f o r the young animal may not be e s s e n t i a l f o r the a d u l t animal. Amino a c i d requirements are a f f e c t e d by g e n e t i c f a c t o r s , age and p h y s i o l o g y , food intake and d i e t a r y p r o t e i n and energy l e v e l . D i f f e r e n t s p e c i e s of animals of the same p h y s i o l o g i c a l age r e q u i r e d i f f e r e n t amounts of EAA, e.g., growing c h i c k s 0-6 weeks o l d r e q u i r e 1.25% l y s i n e i n t h e i r d i e t while growing p i g s 5-10 kg l i v e w e i g h t r e q u i r e 0.96% l y s i n e (NRC, 1971, 1973). D i f f e r e n t s p e c i e s have d i f f e r e n t n u t r i e n t requirements 32. and even d i f f e r e n t s t r a i n s o f the same breed of animals have d i f f e r e n t n u t r i e n t requirements. There i s a s i g n i f i c a n t i n t e r a c t i o n between age and amino a c i d l e v e l s i n the d i e t (Hays, Speer, Hartman and Catron, 1959). G e n e r a l l y , amino a c i d requirements decrease as the animal gets o l d e r . The r a t e of d e c l i n e of amino a c i d requirement with age v a r i e s f o r d i f f e r e n t amino a c i d s and f o r d i f f e r e n t s p e c i e s f o r the same amino a c i d s (Homb, 1976). In the growing p i g , l y s i n e r e q u i r e -ment d e c l i n e s f a s t e r than methionine p l u s c y s t i n e requirements (Homb, 1976). Amino a c i d requirements a l s o vary f o r d i f f e r e n t p h y s i o l o g i c a l s t a t e s or f u n c t i o n of animal. P r o t e i n d e p l e t e d animals have higher amino a c i d requirements than undepleted animals due to i n c r e a s e d p r o t e i n r e q u i r e -ments ( F i s h e r , Griminger and L e v e i l l e , 1959). Growing animals have higher requirements than mature animals because o f t h e i r need to form new t i s s u e s . I t has been demonstrated by Kielanowski (1972) u s i n g n i t r o g e n balance s t u d i e s , that boars, c a s t r a t e s and g i l t s have d i f f e r e n t amino a c i d requirements because they vary i n t h e i r r a t e o f " p r o t e i n - d e p o s i t i o n . I t i s general knowledge that amino a c i d 33. requirements i n c r e a s e d u r i n g pregnancy, l a c t a t i o n and e g g l a y i n g . S t r e s s e s such as i n j u r y or i n f e c t i o n cause an i n c r e a s e i n amino a c i d requirements ( L e v e i l l e et a l . , 1959). Amino a c i d requirement i n c r e a s e s with l e v e l o f i n t a k e i f requirement i s expressed on per day b a s i s . Thus, i t i s a f f e c t e d by a l l the f a c t o r s that g e n e r a l l y a f f e c t food i n t a k e (March and B i e l y , 1972). F a c t o r s such as age, h e a l t h and f u n c t i o n a f f e c t the in t a k e of an animal. A l s o sex a f f e c t s l e v e l of i n t a k e . There i s evidence to support the f a c t that the requirement f o r the most l i m i t i n g amino a c i d i n a d i e t r i s e s as the d i e t a r y p r o t e i n l e v e l i s increased. (Munaver and Harper, 1959). These workers r e p o r t e d that i n c r e a s i n g the l e v e l of a p r o t e i n d e f i c i e n t i n a c e r t a i n amino a c i d i n c r e a s e s the requirement f o r that amino a c i d . L y s i n e requirement of r a t s f o r maximum growth i n c r e a s e d from 0.9% of the d i e t c o n t a i n i n g 30% wheat g l u t e n to 1.2% of a d i e t con-t a i n i n g 60% wheat g l u t e n (Munaver and Harper, 1959). This they e x p l a i n e d as being due to the poor amino a c i d balance of wheat gl u t e n . . Imbalance i s due to excess l e v e l s o f other amino a c i d s . . In an attempt to pr o v i d e an adequate amount of the most l i m i t i n g amino 34. a c i d by i n c r e a s i n g p r o t e i n content, the requirement f o r t h i s amino a c i d i n c r e a s e s because of the high l e v e l s of the other EAA. The growth r e t a r d i n g e f f e c t s o f excess amino a c i d s can be c o r r e c t e d by adding the l i m i t i n g amino a c i d i n s t e a d o f i n c r e a s i n g the p r o t e i n l e v e l . This i s the b a s i s of amino a c i d supplementation of poor q u a l i t y p r o t e i n s . March and B i e l y (19 72) have shown t h a t energy content of the d i e t a f f e c t s the requirement of amino a c i d by c h i c k s . Animals on h i g h energy d i e t s tend to have a h i g h e r amino a c i d requirement (g/100 g DM d i e t ) due to the f a c t that food consumption g e n e r a l l y decreases as energy content i n c r e a s e s , i . e . , there i s a need to i n c r e a s e the amino a c i d c o n c e n t r a t i o n i n h i g h energy d i e t s . In supplementing any d i e t with amino a c i d s , one has to c o n s i d e r the p r o t e i n l e v e l , energy l e v e l , the age and p h y s i o l o g i c a l s t a t e and f u n c t i o n of the animal f o r which such a d i e t i s going to be given. P r o t e i n : C a l o r i e Ratio The importance of d i e t a r y p r o t e i n : c a l o r i e r a t i o has long been r e c o g n i z e d f o r a c h i e v i n g maximum 35. growth r a t e and e f f i c i e n c y of feed c o n v e r s i o n with animals (March and B i e l y , 1972). The e f f i c i e n c y of feed u t i l i z a -t i o n i n c r e a s e s as the energy content i s i n c r e a s e d (Rosenberg, 1957). Animals eat to s a t i s f y t h e i r energy requirement so that the higher the energy content the lower the amount consumed per u n i t weight g a i n . But t h i s lower amount must a l s o c o n t a i n a l l the n u t r i e n t r e q u i r e -ment o f the animal. Energy intake has been shown to be one of the f a c t o r s c o n t r o l l i n g amino a c i d requirement (Rosenberg, 1957). In e v a l u a t i n g p r o t e i n q u a l i t y i t i s necessary to p r o v i d e adequate energy i n the d i e t i n the N-free f r a c t i o n . T h i s ensures that p r o t e i n w i l l not be used as a source of energy. Forbes and Yohe (1954) i n v e s t i -gated the e f f e c t of energy consumption on BV u s i n g r a t s . They showed that at. a low feed i n t a k e , BV was low because not enough energy i s consumed and p r o t e i n i s used as energy source. Eggum (1973) c i t e s a number of authors who have suggested some e f f e c t s of energy i n t a k e on p r o t e i n u t i l i z a t i o n . However, Eggum h i m s e l f (1973) observed no b e n e f i c i a l e f f e c t of high energy content of d i e t on p r o t e i n u t i l i z a t i o n . When the energy content of d i e t 36. i s adequate such t h a t no p r o t e i n i s used for.energy pur-p o s e s , e x t r a energy w i l l not have any e f f e c t on p r o t e i n u t i l i z a t i o n . However, i f energy i s low and p r o t e i n i s b e i n g deaminated, added energy w i l l improve BV. B i o l o g i c a l Methods o f M e a s u r i n g P r o t e i n Q u a l i t y N u t r i t i o n a l q u a l i t y o f p r o t e i n s has been measured by c h e m i c a l , b i o l o g i c a l , e nzymatic and m i c r o -b i o l o g i c a l methods. B i o l o g i c a l methods o f a s s e s s i n g p r o t e i n q u a l i t y , d i r e c t l y o r i n d i r e c t l y , measure body p r o t e i n changes i n response to the t e s t p r o t e i n . D i r e c t measurements o f body p r o t e i n , i s c o m p l i c a t e d but changes i n body w e i g h t are easy to measure. Changes i n body weight a r e w e l l c o r r e l a t e d w i t h changes i n body p r o t e i n but a re not an a c c u r a t e measure o f body p r o t e i n changes. Methods t h a t have been used i n b i o l o g i c a l e s t i m a t i o n o f p r o t e i n q u a l i t y i n c l u d e p r o t e i n e f f i c i e n c y r a t i o (PER), net p r o t e i n u t i l i z a t i o n (NPU), net p r o t e i n r e t e n t i o n (NPR) and b i o l o g i c a l v a l u e (BV). P E R = Weight g a i n (g) P r o t e i n i n t a k e (g) 37. Weight g a i n of t e s t p r o t e i n group (g) - Weight l o s s N P R = o f p r o t e i n f r e e group (g) P r o t e i n i n t a k e (g) TD = True d i g e s t i b i l i t y of n i t r o g e n N i t r o g e n i n t a k e - ( f a e c a l n i t r o g e n - metabolic f a e c a l n i t r o g e n ) - ( u r i n a r y n i t r o g e n - endogenous _ u r i n a r y n i t r o g e n )  N i t r o g e n i n t a k e - ( f a e c a l n i t r o g e n - metabolic f a e c a l n i t r o g e n ) NPU = T D * B V 100 These d e f i n i t i o n s of the b i o l o g i c a l measures are as given by Eggum (1973). Ac c o r d i n g to McLaughlan and K e i t h (1974), PER i s not a good measure of p r o t e i n q u a l i t y . I t i s h i g h l y c o r r e l a t e d with weight gain but i s not c h a r a c t e r i s t i c of the p r o t e i n (Hegsted and Chang, 1965). I t i s not a t r u e measure of p r o t e i n e f f i c i e n c y s i n c e not a l l the p r o t e i n goes i n t o weight gain (Hegsted et a l . , 1965). PER v a r i e s with the sex o f the animal (Block and M i t c h e l l , 38. 1946). NPU, on the other hand, i s a f f e c t e d by amino a c i d composition, p r o t e i n : c a l o r i e r a t i o , adequacy of c a l o r i e i n t ake and v i t a m i n and m i n e r a l balance ( M i l l e r and Payne, 1961). A c c o r d i n g to P i a t t and M i l l e r (1958), NPU i s not d i r e c t l y r e l a t e d to amino a c i d l e v e l s i n the d i e t but to the amino a c i d balance. NPU i s a f u n c t i o n of d i g e s t i -b i l i t y and b i o l o g i c a l value so t h at a l l f a c t o r s a f f e c t i n g TD and BV w i l l a f f e c t NPU. B i o l o g i c a l t e s t s f o r p r o t e i n q u a l i t y can be put i n t o three c a t e g o r i e s : (1) T e s t s i n which the t e s t m a t e r i a l serves as the s o l e source of p r o t e i n . The l e v e l of t h i s p r o t e i n i s u s u a l l y f i x e d at 10%. Tests u s i n g t h i s c a t e g o r y i n c l u d e BV, NPU and PER. NPU and BV are best deter-mined on r e s t r i c t e d f e e d i n g while PER i s done on ad l i b i t u m f e e d i n g . (2) Tests which measure the supplementary e f f e c t of p r o t e i n when used i n combina-t i o n with a t e s t d i e t which i s d e f i c i e n t i n a p a r t i c u l a r amino a c i d , u s u a l l y l y s i n e . 39. (3). T ests using the supplement at p r a c t i c a l l e v e l s such as those used i n animal husbandry ( O l l e y and Payne, 1967). Hegsted and Yet-Oy Chang (1965) examined the a p p l i c a t i o n of standard b i o a s s a y procedures f o r the e v a l u a t i o n of n u t r i t i v e v alue o f p r o t e i n f o r r a t s . The slope r a t i o technique or r e l a t i v e p r o t e i n value appeared to them to be the most s a t i s f a c t o r y . T h i s method uses g a i n as the response and n i t r o g e n i n t a k e as a measure of dose. In t h i s slope r a t i o method, d i e t s of v a r y i n g p r o t e i n l e v e l s are used. At low l e v e l s o f p r o t e i n such as 2% or 5% one should c r i t i c a l l y ponder how meaningful the changes i n body weight are i n r e l a t i o n to n i t r o g e n i n t a k e . At such l e v e l s , f a r below the requirement of the animal, the animal w i l l be i n a poor p h y s i o l o g i c a l s t a t e and weight changes may not be the d i r e c t response to n i t r o g e n intake but to other p h y s i o l o g i c a l f a c t o r s . For some time BV and PER had been the two b i o l o g i c a l procedures f o r e v a l u a t i n g the n u t r i t i v e v a lue o f p r o t e i n s (Hegsted and Yet-Oy Chang, 1965). A c c o r d i n g to these authors, BV can be determined o n l y when the animal i s i n negative n i t r o g e n balance or 40. near balance; so, low p r o t e i n d i e t s are fed f o r t h i s d e t e r m i n a t i o n . The p r o t e i n e f f i c i e n c y r a t i o was o r i g i n a l l y proposed by Osborne and coworkers i n 1919. In t h i s method, the gain per gram of p r o t e i n eaten by young r a t s on low p r o t e i n d i e t s (9-101) i s c a l c u l a t e d (Hegsted and Yet-OY Chang, 1965). T h i s method has been c r i t i c i z e d because PER i s not c h a r a c t e r i s t i c o f the p r o t e i n but of the r a t e o f gain of the animals consuming t h e ' d i e t . It i s a l s o not a measure of e f f i c i e n c y s i n c e not a l l the p r o t e i n i s used f o r growth (Hegsted and Yet-OY Chang, 1965). NPU as a measure of p r o t e i n q u a l i t y i s a r e l a t i v e l y recent procedure when compared to BV and PER. I t has long been r e c o g n i z e d t h a t the BV appears to over-estimate the n u t r i t i v e value of poor q u a l i t y p r o t e i n s . M i t c h e l l et a l . (1952) and Dobbins, K r i d e r , Hamilton and T e r r i l l (1950) r e p o r t e d that the BV of corn p r o t e i n decreased as the p r o t e i n content i n c r e a s e d . I t has been e s t a b l i s h e d that BV i s independent of food i n t a k e but decreases as the p r o t e i n content of the d i e t i n c r e a s e s above a c e r t a i n l e v e l ( u s u a l l y 101) (Eggum, 1973). 41. NPU can be determined by c a r c a s s a n a l y s i s or by n i t r o g e n balance techniques (Chalupa and F i s h e r , 1963) NPU d e t e r m i n a t i o n was developed by M i l l e r and Bender (1955) who used t o t a l c a r c a s s a n a l y s i s . They fed d i e t s with s t a n d a r d i z e d amounts of p r o t e i n , f a t , minerals and v i t a m i n s . In t h e i r study, i t was suggested that a p r o t e i n - f r e e d i e t or a d i e t c o n t a i n i n g 4 to 4.5% egg p r o t e i n be f e d to the c o n t r o l group from which endogenous and m e t a b o l i c n i t r o g e n was determined. Eggum (1973) recommends the use of a 4% egg p r o t e i n and not a n i t r o -gen-free d i e t . Egg p r o t e i n i s completely d i g e s t e d and u t i l i z e d (Eggum, 1973) so that i t makes no c o n t r i b u t i o n to metabolic or endogenous n i t r o g e n , and the animal i s i n a b e t t e r p h y s i o l o g i c a l s t a t e than i t would be on a n i t r o g e n - f r e e d i e t . A m o d i f i e d form of NPU r e f e r r e d to as net p r o t e i n value (NPV) has been used by some workers who have observed a c l o s e c o r r e l a t i o n between s u l f u r content of foods and t h e i r NPV (Morrison, Sabry and Campbell (1962). L y s i n e l e v e l of d i e t s have been found to a f f e c t NPU v a l u e s . M o r r i s o n et a l . (1962) observed that when l y s i n e c o n c e n t r a t i o n i n r a t d i e t s was r a i s e d from 0.3.21 to 0.72% NPU i n c r e a s e d from 21 to 48 and they observed a h i g h c o r r e l a t i o n (r = 0.85) between NPU and l y s i n e l e v e l of the d i e t , i n d i c a t i n g t h at NPU i s 42. h i g h l y dependent on l y s i n e . Other measures of p r o t e i n q u a l i t y such as PER and net p r o t e i n r e t e n t i o n (NPR) have a l s o been found to be w e l l c o r r e l a t e d with l y s i n e l e v e l i n d i e t . M o r r i s o n et a l . (1962) have observed that NPU measured by c a r c a s s a n a l y s i s i s s e n s i t i v e to d i e t a r y l y s i n e l e v e l . Chalupa and F i s h e r (1963) observed that NPU determined by c a r c a s s a n a l y s i s showed much v a r i a t i o n when animals were fed ad l i b i t u m . However, the n i t r o g e n balance method gave c o n s i s t e n t r e s u l t s . Based on t h i s o b s e r v a t i o n these authors suggested the use of a n i t r o -gen balance method f o r measuring NPU when animals are on ad l i b i t u m f e e d i n g . Eggum (1973) determined NPU u s i n g both c a r c a s s a n a l y s i s and a n i t r o g e n balance method w i t h r e s t r i c t e d f e e d i n g , so t h a t the e f f e c t o f v a r i a b l e feed i n t a k e i s removed. It has been r e p o r t e d that NPU and BV estimated at r e l a t i v e l y low l e v e l s of p r o t e i n may be expected to be e r r o n e o u s l y high f o r foods i n which l y s i n e i s the l i m i t i n g amino a c i d (Eggum, 1973). I t i s known that BV overestimates the n u t r i t i v e value o f poor q u a l i t y p r o t e i n s , thus the l y s i n e d e f i c i e n t p r o t e i n w i l l be overestimated i n n u t r i t i o n a l v a l u e . A l s o , at low 43. l e v e l s of p r o t e i n one tends to get h i g h BV. These f a c t o r s have n e c e s s i t a t e d the need f o r standard c o n d i -t i o n s being used i n such d e t e r m i n a t i o n s . However, BV and NPU are g e n e r a l l y regarded as s p e c i f i c c h a r a c t e r i s -t i c s of food p r o t e i n s . A c c o r d i n g to Eggum (1973) n i t r o g e n r e t e n t i o n may be measured by the n i t r o g e n balance t e c h n i -que or by t o t a l body a n a l y s i s . The two methods have giv e n r e s u l t s t h a t show c l o s e agreement by some workers but others have r e p o r t e d the c a r c a s s a n a l y s i s method to be more ac c u r a t e . These d i f f e r e n c e s i n o p i n i o n c o u l d be due to d i f f e r e n c e s i n accuracy of the d i f f e r e n t r e s e a r c h e r s . In n i t r o g e n balance methods, metabolic and endogenous n i t r o g e n are important f a c t o r s . I t i s noted that h e a v i e r r a t s excrete.more n i t r o g e n i n t h e i r faeces than l i g h t e r ones (Eggum, 1973). T h i s i s o n l y important when l a r g e d i f f e r e n c e s i n body weight are i n v o l v e d . In b i o l o g i c a l 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 , animals of uniform weights are used. This i s u s u a l l y achieved by u s i n g animals of the same age. In the i n v e s t i g a t i o n s on H i p r o l y , the e a r l y experiments with r a t s and mice have shown that animals on t h i s h i g h l y s i n e b a r l e y show b e t t e r n i t r o g e n r e t e n -t i o n and net body n i t r o g e n gain than those on normal 44. b a r l e y s with high p r o t e i n (Munk, 1972). The a v a i l a b i l i t y of l y s i n e i n H i p r o l y was higher than i n normal b a r l e y with an i n c r e a s e i n n i t r o g e n content (Munk, 1972). An improved a v a i l a b i l i t y p a t t e r n r e f l e c t s the enhancement o f water s o l u b l e , l y s i n e r i c h p r o t e i n s that are e a s i l y d i g e s t i b l e . Since a v a i l a b i l i t y depends on the p r o t e i n d i g e s t i b i l i t y , the i n c r e a s e d a v a i l a b i l i t y i n H i p r o l y i s due to the f a c t t h a t h i g h l y s i n e b a r l e y s c o n t a i n higher l e v e l s of albumins and g l o b u l i n s than normal b a r l e y and have a reduced prolamin l e v e l ( D o l l , 1977; Anderson, 1977 and Koie and K r e i s , 1977). Albumins and g l o b u l i n s are r e a d i l y d i g e s t i b l e and high i n l y s i n e , so i t i s no wonder that the i n c r e a s e d l e v e l s of these p r o t e i n s i n b a r l e y g r a i n causes an i n c r e a s e i n a v a i l a b l e l y s i n e . Eggum (1969) has demonstrated that the stage of r i p e n e s s at h a r v e s i n g a f f e c t s the p r o t e i n q u a l i t y . He has observed that l y s i n e content decreases when b a r l e y i s harvested o v e r r i p e , with the BV and NPU a l s o reduced. U n f o r t u n a t e l y , he does not t e l l us what stage of r i p e n e s s i s o v e r r i p e . 45. EXPERIMENTAL This p r o j e c t was designed to evaluate high l y s i n e l i n e s o f b a r l e y from the A l b e r t a A g r i c u l t u r e breeding program us i n g chemical and b i o l o g i c a l methods. The chemical procedures used i n t h i s r e s e a r c h i n c l u d e proximate a n a l y s i s , s e p a r a t i o n and q u a n t i t a t i o n of b a r l e y p r o t e i n s , amino a c i d a n a l y s i s and a v a i l a b l e l y s i n e e s t i m a t i o n by the dye-binding technique. The b i o l o g i c a l d e terminations i n v o l v e d growth assays, measurements of true n i t r o g e n d i g e s t i b i l i t y (TD) , true b i o l o g i c a l value (BV), net p r o t e i n u t i l i z a -t i o n (NPU) and r e l a t i v e p r o t e i n value (RPV). CHEMICAL. EVALUATION. OF PROTEIN'QUALITY Proximate Composition  I n t r o d u c t i o n Proximate a n a l y s i s of f e e d s t u f f s i s the most b a s i c chemical method a p p l i e d i n n u t r i t i o n a l s t u d i e s . I t g i v e s a crude estimate of the composition o f a f e e d s t u f f . 46. However, i t has marked shortcomings i n that i t gives o n l y estimates of n u t r i e n t content. I t s value i s i n the f a c t that i t i s a simple e a s i l y r e p r o d u c i b l e system f o r c a t e g o r i z i n g a food. Moisture d e t e r m i n a t i o n allows an assessment to be made on s u i t a b i l i t y f o r storage; a l s o the:dry matter content sets an upper l i m i t on n u t r i e n t l e v e l . Crude p r o t e i n , although measuring o n l y t o t a l n i t r o g e n content, permits assumptions to be made on amino a c i d content, i n a c c u r a t e as they may be, from standard t a b l e s such as those p u b l i s h e d by the N a t i o n a l Research C o u n c i l (NAS-NRC-1971, 1973). For monogastric animals, energy content would be i n c r e a s e d with i n c r e a s i n g l e v e l s o f ether e x t r a c t ; decreased with i n c r e a s i n g l e v e l s of ash and f i b r e . Consequently, i n terms of bro a d l y d e f i n i n g a food, the proximate a n a l y s i s s t i l l serves a u s e f u l purpose. M a t e r i a l s and Methods F i v e b a r l e y samples, H i p r o l y , G a i t and three c u l t i v a r s , H69019109000, H69024038000 and H690240004191 0 were obtained from the 1977 harvest of the A l b e r t a A g r i c u l t u r e breeding program. 47. H i p r o l y , a mutant d i s c o v e r e d i n Sweden (Munk, Kar l s o n and Hagberg, 1971) with h i g h p r o t e i n , high l y s i n e content, i s low y i e l d i n g and d e f i c i e n t i n s e v e r a l agrono-mic t r a i t s ( f l i n t y , s h r i v i l l e d seed, small s p i k e s and uneven t i l l e r i n g p a t t e r n ) . G a i t i s a wi d e l y used commer-c i a l v a r i e t y . C u l t i v a r H69019109000 ( C u l t i v a r 1) i s a s e l e c t i o n from the F ^ Q g e n e r a t i o n o f a H i p r o l y x Orange Lemma c r o s s . The Orange Lemma c a r r i e s a marker f o r lemma c o l o u r p l u s a f a c t o r f o r hig h a-amylase a c t i v i t y . I t i s a 6-row b a r l e y with covered seed, high y i e l d and high number of seeds per culm. Both c u l t i v a r s H69024038000 ( c u l t i v a r 2) and H690240004191 ( c u l t i v a r 3) are s e l e c t i o n s from the F ^ Q g e n e r a t i o n of a H i p r o l y x c u l t i v a r 0R61-2141-9. This l a t t e r c u l t i v a r was s e l e c t e d from the Oregon breeding program as a p o t e n t i a l high-y i e l d i n g 2-row m a l t i n g b a r l e y . I t has good agronomic c h a r a c t e r i s t i c s and y i e l d i n g a b i l i t y . F u r t h e r informa-t i o n on the parent c u l t i v a r s has been presented by Helm (1972). A l l g r a i n was combine harvested when l e s s than 20% moisture content and d r i e d i n a dryer o p e r a t i n g at 32 to 38°C. Moisture content, crude p r o t e i n , ether e x t r a c t and ash were determined a c c o r d i n g to AOAC (1970). A c i d 48. detergent f i b r e (ADF) was determined a c c o r d i n g to the method of Waldern (1971). R e s u l t s Table 1. Proximate composition of barleys - % DM basis Sample Dry Matter Crude Protein N x 6.25 Ether Extract Ash Acid Detergent Fibre NFE* Hiproly 92.62 18.57 1.94 2.04 3.06 74.39 Gait 92.69 15.16 1.78 2.14 4.66 76.26 Cultivar 1 91.92 17.45 1.65 2.46 5.42 73.02 Cultivar 2 92.55 18.10 2.13 2.28 4.34 73.15 Cultivar 3 92.64 16.81 1.69 2.47 4.38 74.65 C u l t i v a r 1 = H69019109000 C u l t i v a r 2 = H69024038000 C u l t i v a r 3 = H69024004191 NFE* = M o d i f i e d NFE due to the use of ADF. M o d i f i e d NFE has been used by Morgan, Cole and Lewis (1975). 49. D i s c u s s i o n From Table 1 one can see the hig h p r o t e i n con-t e n t s o f the f i v e b a r l e y s being s t u d i e d . Canadian commercial b a r l e y g e n e r a l l y c o n t a i n 10 to 14% crude p r o t e i n (Coates, S l i n g e r , Summers and Bayley, 1977). A l l the b a r l e y s i n c l u d i n g G a i t , the commercial c o n t r o l , have a high e r p r o t e i n content than t h i s range of 10 to 14%. I t i s p o s s i b l e that the environment f o r c u l t i v a -t i o n c o n t r i b u t e d to t h i s r e l a t i v e l y h i g h crude p r o t e i n content i n G a i t . H i p r o l y c o n t a i n s the hi g h e s t l e v e l o f p r o t e i n and the lowest l e v e l of f i b r e and ash, the former being a s s o c i a t e d with the f a c t t h a t H i p r o l y i s h u l l - l e s s . In normal b a r l e y i n Canada, the ether e x t r a c t i s low, rang-ing from 1.96 to 3.16% (Coates et a l . , 1977) with an average of 2.43%. The b a r l e y c u l t i v a r s being s t u d i e d i n t h i s work have ether e x t r a c t s ranging from 1.65 to 2.13%. Coates et a l . (1977) r e p o r t e d ADF values f o r 16 normal b a r l e y samples from a number of coun-t r i e s . The values ranged from 5.64 to 8.81%. The ADF values of the 5 b a r l e y s under i n v e s t i g a t i o n range from 3.06 to 5.42%. Ash i n t h i s study ranged from 50. 2.04 to 2.47% while values f o r the 16 b a r l e y s of Coates et a l . (1977) ranged from 2.25 to 3.82%. Se p a r a t i o n of B a r l e y P r o t e i n s by S o l u b i l i t y  I n t r o d u c t i o n There are four d i f f e r e n t types of p l a n t pro-t e i n s . These have c h a r a c t e r i s t i c s o l u b i l i t i e s and, based on these s o l u b i l i t i e s , p r o t e i n s are c l a s s i f i e d i n t o four c a t e g o r i e s namely: Albumins G l o b u l i n s G l u t e l i n s Prolamins Albumins are s o l u b l e i n water, g l o b u l i n s i n s a l t s o l u t i o n , prolamins i n a l c o h o l and g l u t e l i n s i n d i l u t e a c i d or a l k a l i . This, method of s e p a r a t i o n was f i r s t used by Osborne i n 1895 f o r b a r l e y p r o t e i n s . S t r u c t u r e of the B a r l e y G r a i n In most c u l t i v a t e d b a r l e y s , the husk adheres to the g r a i n (e.g., Bomi) but some v a r i e t i e s are naked 51. ( K r e i s , 1979). The embryo occupies only a small p a r t of the k e r n n e l , i . e . , about 2 to 5% of the dry weight, and i t c o n t r o l s the m o b i l i z a t i o n of s t o r e d n u t r i e n t s . E i g h t y percent o f the g r a i n p r o t e i n and most of the carbohydrates are l o c a t e d i n the endosperm ( K r e i s , 1979). In normal b a r l e y albumins plus g l o b u l i n s form 18% of the crude p r o t e i n , non-protein, n i t r o g e n forms 12%, prolamins form 42%, and g l u t e l i n 23% ( B a l a r a v i et a l . , 1976; Koie and N i e l s o n , 1977; M i l f l i n and Shewry, 1978; and K r e i s , 1979). M a t e r i a l s and Methods Sample P r e p a r a t i o n The f i v e samples of b a r l e y g r a i n from the A l b e r t a A g r i c u l t u r e 1977 harvest were ground with a l a b o r a t o r y hammer m i l l ( C h r i s t y and N o r r i s L t d . , Chelmsford, England) u s i n g an 0.5 mm screen. Reagents S o l u t i o n s were prepared by methods d e s c r i b e d by Koie and N i e l s o n (1977). The s a l t s o l u t i o n c o n s i s t e d o f 29.2 g NaCl and. 0.02 g NaEDTA (sodium e t h y l d i a m i n e t e t r a a c e t i c a c i d ) d i s s o l v e d i n water and made up to 1 l i t r e 52. with water. The a l c o h o l s o l u t i o n was made of 500 ml i s o - p r o p a n o l , 6 ml 2-mercaptoethanol and 500 ml water. The a l k a l i n e s o l u t i o n c o n t a i n e d 4.8 g Na2B^O^ • lOI-^O (sodium t e t r a b o r a t e ) , 1.7 g NaOH and 5.0 g SDS (sodium dodecyl sulphate) per l i t r e . Procedure A ground sample of 1.5 g was weighed i n t o a 60 ml p l a s t i c c e n t r i f u g e tube and e x t r a c t e d three times with 10 ml of s o l v e n t . E x t r a c t i o n was done by mechanical shaking. The mechanical shaker was improvised from a water bath. Cut g l a s s rods 3 cm x 0.5 cm diameter, rounded at both ends were p l a c e d i n the c e n t r i f u g e tubes and each'tube f i t t e d with a no. 4 rubber stopper. Each e x t r a c t i o n was c a r r i e d out f o r 30 minutes at room temperature which ranged from 23 to 25°C. Samples were c e n t r i f u g e d i n a S o r v r a l l superspeed RC2-B r e f r i g e r a t e d c e n t r i f u g e at 12000 rpm f o r 20 minutes. The supernatants were p i p e t t e d i n t o 50 ml v o l u m e t r i c f l a s k s . A f t e r the t h i r d e x t r a c t i o n a l l e x t r a c t s were made up to 50 ml with more s o l v e n t . Samples were f i r s t e x t r a c t e d with s a l t s o l u t i o n f o r albumins a n d , g l o b u l i n s as w e l l as NPN, then with a l c o h o l f o r hordeins (the prolamins of b a r l e y ) and f i n a l l y w ith the a l k a l i n e s o l u t i o n f o r the 53. g l u t e l i n s . A f t e r a l l the e x t r a c t i o n s , the f i n a l r e s i d u e s were completely t r a n s f e r r e d i n t o aluminium weighing dishes by washing with d i s t i l l e d water. Residues were d r i e d i n an oven at 85°C o v e r n i g h t . They were weighed a f t e r c o o l i n g i n a d e s s i c a t o r . Ten m i l l i l i t r e s of each e x t r a c t was used f o r K j e l d a h l n i t r o g e n d e t e r m i n a t i o n . The 10 ml of each a l c o h o l e x t r a c t was f i r s t evaporated on a water bath to about 3 ml before being d i g e s t e d . T h i s ensured that there was no foaming which c o u l d l e a d to l o s s o f s a m p l e s d u r i n g d i g e s t i o n . The n i t r o g e n content of the f i n a l r e s i d u e s was a l s o determined by the K j e l d a h l method. The n i t r o g e n content of the f r a c t i o n s were expressed as a percent of the t o t a l n i t r o g e n which was determined i n a 1.5 g sample. The percentages of n i t r o g e n were summed up f o r each sample to determine the t o t a l percentage recov e r y of n i t r o g e n . 54. E x t r a c t i o n Procedure f o r Solu b l e P r o t e i n F r a c t i o n s B a r l e y Grain 4 M i l l i n g 4 B a r l e y f l o u r I Three e x t r a c t i o n s of 1.5 g with 10 ml s a l t s o l u t i o n f o r 30 minutes a t room temperature. C e n t r i f u g a t i o n at 1200 rpm f o r 20 minutes at room temperature. E x t r a c t made up to 50 ml with s a l t s o l u t i o n . S a l _ t _ s o l u b l e _ f r a c t ion Residue Three e x t r a c t i o n s as before but wit h a l c o h o l s o l u t i o n Hordeins Residue Three e x t r a c t i o n s as before but with a l k a l i n e s o l u t i o n I n s o l u b l e r e s i d u e G l u t e i i n s R e s u l t s and D i s c u s s i o n Table 2. N i t r o g e n content o f b a r l e y e x t r a c t s as a per centage of t o t a l n i t r o g e n (a, b, d u p l i c a t e s ) a , Salt Alcohol Alkaline n ., M n Sample Residue N-Recovery Hiproly a 20.38 38.11 30.49 8.04 97.02 b 20.52 35.94 31.00 7.15 94.61 Gait a 18.50 46.41 21.59 9.05 95.55 b 19.27 47.10 21.88 7.85 96.07 Cultivar 1 a 22.57 41.23 22.53 8.85 95.18 b 22.70 38.62 27.57 7.90 96.78 Cultivar 2 a 21.88 37.94 27.55 6.88 94.25 b 21.87 39.40 28.86 6.32 96.46 Cultivar 3 a 19.20 44.78 20.64 8.67 93.29 b 19.47 43.44 22.46 7.57 92.94 Cultivar 1 = H6901910900, Cultivar 2 = H69024038000, Cultivar 3 = H690240004191 56. Table 2 shows the p r o p o r t i o n s i n which the p r o t e i n f r a c t i o n s occur i n these f i v e b a r l e y s . The s a l t s o l u b l e f r a c t i o n which c o n t a i n s the albumins and g l o b u l i n s forms about 20% of the t o t a l p r o t e i n , with a range of 18.5 to 22.7%. The a l c o h o l f r a c t i o n which c o n t a i n s the hor-deins forms 37.0% i n H i p r o l y p r o t e i n , 46.5% G a i t , 40% i n c u l t i v a r 1, 38.5% i n c u l t i v a r 2 and 44% i n c u l t i v a r 3. In H i p r o l y , g l u t e l i n s form 30.5% of the p r o t e i n while i n G a i t i t forms 21.7%, c u l t i v a r 1, 25.0%, c u l t i v a r 2, 28.0%, and c u l t i v a r 3, 21.5%. Ingversen and Koie (1973) found t h a t the s a l t s o l u b l e f r a c t i o n of normal Emir b a r l e y accounted f o r 24% of the t o t a l p r o t e i n , and 44% o f the t o t a l l y s i n e . The d i f f e r e n c e between Ingversen and Koie's p a r t i t i o n i n g of the s o l u b l e f r a c t i o n and t h a t i n t h i s study c o u l d be due to incomplete e x t r a c -t i o n i n the l a t t e r . I t c o u l d a l s o be c h a r a c t e r i s t i c of the type of b a r l e y being used. B a l a r a v i , Bansal, Eggum and Bhaskaran (1976) i n t h e i r study of the c h a r a c t e r i s t i c s of induced high p r o t e i n and high l y s i n e 57. mutants i n b a r l e y compared H i p r o l y , notch 1 and notch 2 and t h e i r normal parent f o r the p r o p o r t i o n s of the d i f -f e r e n t f r a c t i o n s and f o r t h e i r n u t r i t i v e value by animal experiments. T h e i r r e s u l t s f o r s o l u b l e f r a c t i o n s are presented below. S a l t A l c o h o l A l k a l i n e % S o l u b l e S o l u b l e S o l u b l e Recovery Normal Parent 20% 29% ' 45% 94 Notch 1 29% 22% 49% 99 Notch 2 28% 25% 41% 94 H i p r o l y 25% 22% 46% 92 I f the s a l t s o l u b l e f r a c t i o n c o n t a i n s the best q u a l i t y p r o t e i n s , the higher the p r o p o r t i o n of t h i s f r a c t i o n the b e t t e r . They observed that notch 1 and notch 2 had more albumins and g l o b u l i n s than H i p r o l y . In t h i s p resent study c u l t i v a r s 1 and 2 a l s o appear to have more s a l t s o l u b l e f r a c t i o n than H i p r o l y . Helm (1972) found the s a l t s o l u b l e f r a c t i o n of H i p r o l y to be 21.12%, which i s very c l o s e to the value obtained i n t h i s study (20.38%). Helm (1972) observed that the a l c o h o l s o l u b l e f r a c t i o n of H i p r o l y was o n l y 19.26% of the 58. t o t a l p r o t e i n and the a l k a l i n e f r a c t i o n was 59.63%. The a l c o h o l f r a c t i o n seems r a t h e r low and the a l k a l i n e f r a c -t i o n too high when compared to the values obtained i n the present study and -those observed by B a l a r a v i et a l . (1976) f o r H i p r o l y , and values were obtained by Koie and N i e l s o n (1977), M i l f l i n and Shewry (1978) and K r e i s (1979) f o r normal b a r l e y s . I t appears that Helm's a l c o h o l s o l u b l e f r a c t i o n was not completely e x t r a c t e d and i t got e x t r a c t e d i n t o the a l k a l i n e s o l u b l e f r a c t i o n . T h i s may account f o r the low value and very high value obtained by Helm (1972) f o r h i s a l c o h o l and a l k a l i n e s o l u b l e f r a c t i o n s . A c c o r d i n g to M i l f l i n and Shewry, the a l c o h o l s o l u b l e f r a c t i o n which c o n t a i n s the hordeins has low l y s i n e and other b a s i c amino a c i d s but i s hig h i n glutamic a c i d and p r o l i n e . They a l s o suggest that c e r e a l prolamins i n c r e a s e at a g r e a t e r r a t e than other p r o t e i n c l a s s e s i n response to added n i t r o g e n . Thus, one can say that the percentage of hordein i n b a r l e y i s d i r e c t l y r e l a t e d to the n i t r o g e n s t a t u s of the s o i l on which i t was grown. Folkes and Yemm (1956) separated b a r l e y p r o t e i n s and determined the amino a c i d compositions of the f r a c t i o n s . They found that over 60% of the hordein n i t r o g e n i s made 59. up of glutamic a c i d , amides and p r o l i n e . T h i s i s i n agreement with that observed w i t h normal Emir b a r l e y by M i l f l i n and Shewry (1977). The amino a c i d composi-t i o n of the b a r l e y p r o t e i n f r a c t i o n s of Folkes and Yemm (1956) are quoted f o r comparison (Table 3). From t h i s t a b l e i t i s c l e a r t h a t albumins and g l o b u l i n s have a h i g h b a s i c amino a c i d content, e s p e c i a l l y a r g i n i n e and l y s i n e . G l u t e l i n has a moderate l e v e l of l y s i n e , an a r g i n i n e and a h i s t i d i n e l e v e l which are s i m i l a r to those of albumin and g l o b u l i n . T h e g l u t e -l i n f r a c t i o n has a higher l e v e l of t h r e o n i n e , l e u c i n e , and i s o l e u c i n e and other e s s e n t i a l amino a c i d s (EAA) than g l o b u l i n . In the hordein f r a c t i o n , l y s i n e l e v e l i s extremely low; h i s t i d i n e and a r g i n i n e l e v e l s are a l s o v e ry low. G e n e r a l l y the l e v e l s of EAA i n the hordein f r a c t i o n are much lower than the l e v e l s found i n the other f r a c t i o n s . Animal performance tends to be a f f e c t e d to a g r e a t e r extent by the t o t a l percentage l y s i n e i n the seed than by the l y s i n e content of the separate Osborne f r a c t i o n s (Eggum, 1977a). E x t r a c t i o n experiments wi t h normal and high l y s i n e b a r l e y s have i n d i c a t e d that the s o l u b i l i t y of the d i f f e r e n t p r o t e i n s w i t h the Osborne 60. Table 3. Amino a c i d n i t r o g e n as a percentage of p r o t e i n n i t r o g e n (N. x.6.25) (Folkes and Yemm, 1956). Albumin G l o b u l i n Hordein G l u t e l i n s [Water [ S a l t [ A l c o h o l [ A l k a l i n e s o l u b l e ] s o l u b l e ] s o l u b l e ] s o l u b l e ] Amide 5.9 5.1 23.0 10.3 A s p a r t i c A c i d 8.0- 5.6 1.2 4.3 Glutamic A c i d 8.7 6 . 8 23.0 11.6 P r o l i n e 4.2 2, 1 15.3 6.6 G l y c i n e 6 . 7 10 . 7 1.7 5.2 Al a n i n e 7 . 2 0.65 2 . 2 6.6 V a l i n e 5 . 8 4.1 3.5 4.9 Leucine 5 . 7 4.5 4.6 5.8 I s o l e u c i n e 4.1 2.2 3.6 3.5 Phen y l a l a n i n e 3.0 2.1 3.6 2 . 7 Tyr o s i n e 2.7 1.5 1.6 1.9 Tryptophan 1.3 0.65 0 . 7 1.1 Serine 4.1 3.9 3.2 4.2 Threonine 3.4 2.4 1.9 3.1 Cy s t i n e + C y s t e i n 1.5 2.6 1.5 0.9 Lysi n e 7.9 6 . 3 0 . 80 4.8 Methionine 1.4 0.9 0.75 1.1 A r g i n i n e 13.0 22.0 6.0 12.0 H i s t i d i n e 4.3 3.1 0 .22 4.3 61. technique i s h i g h l y dependent on the r i p e n e s s of the seed and the c h a r a c t e r of the l y s gene (Munk, 1977). I t i s b e l i e v e d that the l y s gene which gives the high l y s i n e content a l s o changes the e x t r a c t a b i l i t y o f s p e c i f i c p r o t e i n s (Munk, 1977). Eggum (1977a) has observed that f r a c t i o n a t i o n of Bomi and mutant 1508 v a r i e d w i t h l o c a t i o n s of c u l t i v a t i o n , so that the d i f f e r e n c e s between the r e s u l t s of t h i s work and that of other r e s e a r c h e r s c o u l d be due to d i f f e r e n c e s i n l o c a t i o n . Amino A c i d A n a l y s i s I n t r o d u c t i o n Measurements of t o t a l amino a c i d content are very important i n the study of the q u a l i t y of any. p r o t e i n . I t gives i n f o r m a t i o n on the t o t a l amino a c i d s p r e s e n t , l i m i t i n g amino a c i d s and the p r o p o r t i o n s i n which the amino ac i d s occur. Amino a c i d balance i s a good i n d i c a t o r of p r o t e i n q u a l i t y as i n the measurement of amino a c i d score. The accuracy of amino a c i d measure-ments, however, depends on the e f f i c i e n c y of the hyd r o l y -s i s procedure and the accuracy of the ion-exchange 62. chromatographic method. H y d r o c h l o r i c a c i d i s widely used f o r p r o t e i n h y d r o l y s i s and i t i s known to give higher r e c o v e r i e s of most amino a c i d s than any other reagent (Mason, Bech-Andersen and Rudemo, 1979). Due to the above f a c t , d i f f e r e n t reagents are used to hydro-l y s e p r o t e i n s to analyse f o r d i f f e r e n t amino a c i d s , thus we have a c i d h y d r o l y s i s or a l k a l i n e h y d r o l y s i s . To reduce l o s s e s of s u l p h u r - c o n t a i n i n g amino a c i d s i n HCl h y d r o l y s i s , samples have to be o x i d i z e d with p e r f o r m i c a c i d b e f o r e being h y d r o l y s e d . D i f f e r e n t o x i d i z i n g agents have been used and sometimes there may be reac-t i o n s w i t h some amino a c i d s , thus reducing t h e i r recov-e r i e s . C e r t a i n c o n s t i t u e n t s o f feeds such as carbo-hydrates, l i p i d s and n u c l e i c a c i d s are known to cause l o s s e s i n c e r t a i n amino a c i d s d u r i n g a c i d h y d r o l y s i s (Bech-Andersen, Rudemo and Mason, 1979). Thus the accur-acy of the amino a c i d composition of a feed m a t e r i a l depends on i t s c o n s t i t u e n t s and method of h y d r o l y s i s . M a t e r i a l s and Methods Amino a c i d composition (except f o r c y s t i n e , methionine and tryptophan) of the f i v e b a r l e y samples was determined on h y d r o l y s a t e s prepared by the method of Kohler and P a l t e r (1967). C y s t i n e was determined as c y s t e i c a c i d and methionine as methionine sulphone a f t e r o x i d a t i v e h y d r o l y s i s w i t h p e r f o r m i c a c i d (Moore, 1963). The samples were analysed u s i n g an amino a c i d a n a l y s e r (Durrum Model D500; Durrum, Palo A l t o , C a l i -f o r n i a ) . Tryptophan was determined a f t e r a l k a l i n e hydro l y s i s ( H u g l i and Moore, 1972) using a Beckman-Spinco A n a l y s e r (Model 120B; Beckman Instruments Inc., Palo A l t o , C a l i f o r n i a ) . R e s u l t s The amino a c i d composition as p r e s e n t e d i n Table 4 gives a g e n e r a l impression on the p r o t e i n s i n these f i v e b a r l e y s . When expressed as g/100 g DM H i p r o l y had the h i g h e s t l e v e l of most of the e s s e n t i a l amino a c i d s (EAA), except a r g i n i n e and l y s i n e where c u l t i v a r 2 had the h i g h e s t l e v e l s and c y s t i n e where c u l t i v a r 3 had the h i g h e s t l e v e l . For t o t a l l y s i n e supply per u n i t of dry matter, c u l t i v a r 2 was the best, C u l t i v a r 2 was mostly second to H i p r o l y i n the l e v e l s of EAA c o n t a i n e d on a DM b a s i s , f o l l o w e d by c u l t i v a r 1 Table 4. Protein content and amino acid composition (total) of f i v e barleys. HIPROLY g/lOOg DM g/16g N g/lOOg DM g/lbg N CULTIVAR 1 g/lOOg DM g/16g N g/lOOg DM g/16g N g/lOOg DM g/16g N Protein (N x 6.25) Alanine Arginine Aspartic Acid Cystine Glutamic Acid Glycine H i s t i d i n e Isoleucine Leucine Lysine Methionine Phenylalamine Proline Serine Threonine Tryptophan Tyrosine Valine Total EAA 18.57 0.67 0.71 1.04 0.30 3.88 0.57 0.37 0.65 1.26 0.58 0.30 0.99 1.79 0.65 0.54 0.50 0.51 0.81 7.52 3.61 3.82 5.60 1.62 20.89 3.07 1.48 3.50 6.79 3.12 1.62 5.33 9.64 3.50 2.91 2.69 2.75 4.36 35.58 15.16 0.50 0.54 0.74 0.31 3.26 0.41 0.30 0.49 1.00 0.46 0.20 0.73 1.45 0.50 0.40 0.40 0.41 0.57 5.81 30 56 88 04 21.50 2.70 1.98 3.23 6.60 3.03 1.32 6.53 9.56 3.30 2.64 2.64 2.70 3.76 35.29 17.45 0.68 0.66 1.01 0.27 3.67 0.53 0.34 0.52 1.20 0.56 0.27 0.91 1.62 0.61 0.51 0.45 0.48 0.74 6.91 3.90 3.78 5.79 1.55 21.03 3.04 1.95 2.98 6.88 3.21 1.55 5.21 9.28 3.50 2.92 2.58 2.75 4.24 35.30 18.10 0.65 0.78 0.92 0.33 3.67 0.58 0.34 0.60 1.17 0.62 0.27 0.89 1.62 0.69 0.52 0.45 0.51 0.72 7.18 3.59 4.31 5.08 1.82 20.28 3.20 1.88 3.31 6.46 3.43 1.49 4.92 8.95 3.81 2.87 2.49 2.82 3.98 35.14 16.81 0.56 0.69 0.81 0.37 3.80 0.55 0.32 0.54 1.09 0.47 0.22 0.77 1.71 0.60 0.47 0.45 0.46 0.65 6.50 3.30 4.10 4.82 2.20 22.61 3.27 1.90 3.21 6.48 2.80 1.31 4.58 10.17 3.57 2.80 2.68 2.74 3.87 33.73 Cu l t i v a r 1 = H69019109000 Amino acid analysis was done by AAA Laboratories C u l t i v a r 2 = H69024038000 Cu l t i v a r 3 = H690240004191 65. which - had the second lowest p r o t e i n content of 16.81% on DM b a s i s . Based on amino a c i d composition measured as g/100 g DM, the f i v e b a r l e y s may be arranged i n order of q u a l i t y as: H i p r o l y , c u l t i v a r 2, c u l t i v a r 1, c u l t i v a r 3 and G a i t i n order of d e c r e a s i n g q u a l i t y . The high l e v e l of a s p a r t i c a c i d i n H i p r o l y , c u l t i v a r 2 and c u l t i v a r 1 compared with G a i t and c u l t i v a r 3 i s worth n o t i n g . Pomeranz, Robbins, Wesenberg, Hockett and G i l b e r t s o n (1973) have shown that the h i g h l y s i n e content of H i p r o l y i s a s s o c i a t e d with the h i g h l e v e l o f a s p a r t i c a c i d . I t i s known t h a t a s p a r t i c a c i d i s a key i n t e r m e d i a t e i n the b i o s y n t h e s i s of l y s i n e i n b a c t e r i a , algae and higher p l a n t s (Pomeranz et a l . , 1973). The r e l a t i o n s h i p between l y s i n e and a s p a r t i c a c i d pre-sumably holds f o r other high l y s i n e v a r i e t i e s . The amino a c i d composition of notch-1 and notch-2 and t h e i r normal parent given by B a l a r a v i et a l . (1976) showed that, the high l y s i n e mutants have higher a s p a r t i c a c i d con-c e n t r a t i o n than the normal parent. In r e g u l a r 2-row and 6-row rbarleys,•the p r o t e i n content determines the amino a c i d composition, i . e . , the higher the l e v e l of p r o t e i n , the higher the l e v e l s of the amino a c i d s (Pomeranz et a l . , 1973). In H i p r o l y 66. the genes c o n t r o l l i n g p r o t e i n content, l y s i n e concentra-t i o n i n the p r o t e i n and k e r n e l development are not g e n e t i c a l l y l i n k e d , so that they segregate independently (Pomeranz et a l . , 1973; Munk, K a r l s s o n , Hagberg and Eggum, 1970). I f the l y s i n e content (g/100 g DM) i n H i p r o l y i s given a value of 100 then: G a i t - 79.13 C u l t i v a r 1 - 96.55 C u l t i v a r 2 - 106.90 C u l t i v a r 3 - 81.08 I f the same i s done f o r the other EAA, the values i n Table 5 are obtained. From Table 5 with H i p r o l y as the standard, c u l t i v a r 2 i s the next best source of EAA, f o l l o w e d by c u l t i v a r 1. I t i s c l e a r from t h i s t a b l e t h a t c u l t i v a r 3 i s not a very good source of EAA when compared wi t h the other high l y s i n e types. G a i t being "a normal commercial b a r l e y , i s poor i n the supply of EAA. For s t u d i e s of p r o t e i n q u a l i t y , however, i t i s more meaningful to make comparisons i n terms of g/16 g N. Table 6 shows such a comparison. In terms of g/100 g N, c u l t i v a r 2 was s u p e r i o r to H i p r o l y i n the Table 5. Amino a c i d content of four b a r l e y s (g/100 g DM) r e l a t i v e to amino a c i d composition of H i p r o l y . Barley Arg. Lysine Three: Hist. ^Isoleu. Leu. Meth. Tryp. Tyro Val Hiproly 100 100 100 ," 100 "100 100 100 100 100 100 Gait 76.06 79.31 74.07: P 81.08 75.38 79.36 66.70 80.00 83.97 70.37 Cultivar 1 97.96 96.55 94.44 91.87 .,80.00 95.24 90.00 90.00 94.12 91.36 Cultivar 2 109.86 106.90 96.30, 91.89- . 92.31 92.87 90.00 90.00 100.00 88.89 Cultivar 3 97.18 81.08 87,0,4, • 86.49 : 83.08 86.51 73.33 90.00 90.20 80.25 Table 6. Amino a c i d content of four b a r l e y s (g/16 g N) r e l a t i v e to amino a c i d composition of H i p r o l y . Barley Arg. Lysine Threo. Hist. Isoleu. Leu. Meth. Tryp. Typo. Val. Hiproly 100 100 100 100 100 100 100 100 100 100 Gait 93.19 97.12 90.72 133.78 92.29 97.20 81.48 98.14 98.18 86.24 Cultivar 1 98.95 102.88 100.34 131.76 85.14 101.33 95.68 95.91 100.00 97.25 Cultivar 2 112.83 109.94 98.63 127.03 94.57 95.14 91.98 92.56 102.55 91.28 Cultivar 3 107.33 89.74 96.22 128.38 91.71 95.43 80.86 99.63 99.64 88.76 69. supply of b a s i c amino a c i d s . C u l t i v a r 1 was .superior to H i p r o l y i n the supply of l y s i n e and h i s t i d i n e . G a i t had the h i g h e s t l e v e l of h i s t i d i n e among the f i v e b a r l e y s . C u l t i v a r 3 was s u p e r i o r to H i p r o l y but not c u l t i v a r 2 i n the supply o f a r g i n i n e and h i s t i d i n e . The e s s e n t i a l amino a c i d l e v e l s o f . t h e b a r l e y s used i n t h i s study seem r a t h e r low. T h i s i s demonstrated by the f a c t t h a t H i p r o l y had 3.12 g l y s i n e / 16 g N while B a l a r a v i et a l . (1976) obtained 4.08 g l y s i n e / 1 6 g N. B a l a r a v i ' s amino a c i d composition of H i p r o l y i s quoted here f o r comparison; a l l expressed as g/16 g N. H i p r o l y ( B a l a r a v i et a l . , 1976) H i p r o l y (Imbeah, 1980) A r g i n i n e 4.4 3.82 H i s t i d i n e 2.1 1.48 I s o l e u c i n e 3.9 3.50 Leucine 7.0. 6. 79 Ly s i n e 4.0 3.12 Methionine 2.0 1.62 Threonine 3.5 2. 91 Tryptophan 1.2 2.69 Ty r o s i n e 2.9 2.75 V a l i n e 5.3 4.36 70. I t appears t h a t the l y s i n e content and e s s e n t i a l amino a c i d content o f some normal b a r l e y s from the world c o l l e c -t i o n (Pomeranz et a l . , 1976) are higher than that obtained f o r H i p r o l y i n t h i s study. Climate, s o i l and c u l t i v a t i o n p r a c t i c e s i n f l u e n c e amino a c i d composition. A l s o the a v a i l a b l e n i t r o g e n l e v e l i n the s o i l i s important. A hig h n i t r o g e n l e v e l decreases l y s i n e c o n c e n t r a t i o n (g/ 16 g N) as was observed with normal Bomi and mutant 1508 (Eggum, 1977a), by i n c r e a s i n g the p r o p o r t i o n of storage p r o t e i n (hordein) i n the t o t a l p r o t e i n . Eggum (1977a) has r e p o r t e d v a r i a t i o n i n amino a c i d composition due to l o c a t i o n . B a l a r a v i ' s H i p r o l y and other h i g h l y s i n e b a r l e y s were grown i n Denmark while the b a r l e y s f o r t h i s study were grown i n A l b e r t a , Canada. The same b a r l e y c u l t i v a r s from the 1975 ha r v e s t of A l b e r t a A g r i c u l t u r e program had the amino a c i d composition i n Table 7. The amino a c i d composition of the same f i v e b a r l e y s harvested i n 1975 were h i g h e r than those obtained f o r the 1977 h a r v e s t . Since they were grown i n the same geo g r a p h i c a l l o c a t i o n , the d i f f e r e n c e i n amino a c i d composition may be a r e s u l t of c l i m a t e . The year 1977 was wet compared with 1975. In a wet year, the r a i n s bring.about i n c r e a s e d y i e l d . Table 7. Proximate a n a l y s i s and t o t a l amino a c i d composition of f i v e b a r l e y s from A l b e r t a A g r i c u l t u r e program - 1975 harvest*. H i p r o l y Gait C u l t i v a r 1 C u l t i v a r 2 C u l t i v a r 3 Dry matter 92. , 2 91. ,4 92 . ,0 92. ,0 91. , 80 Components (DM bas i s ) 14. 18. 18. A P r o t e i n (N x 6. .25) 19. ,5. ,4 , 4 , 6 17. , 4 Ether e x t r a c t 2. ,03 1. ,46 1. ,67 1. , 80 1. , 56 Acid-detergent 2 . ,50 6. .92 4. .80 4. , 78 5 . , 35 f i b r e .21 Ash 2. ,22 2. .63 2. , 26 2 . , 35 2  g/100 g DM 0. ,82 0 . ,67 Alanine 0 . 90 0 . 59 0. .87 A r g i n i n e 0. , 88 0 , .65 0, .93 0. , 90 0 , . 77 A s p a r t i c a c i d 1. .31 0. . 83 1. ,30 1. .08 0 . 96 Cys t i n e 0. .27 0. .27 0 , . 34 0 . 33 0 , .33 Glutamic a c i d 4. .60 3. . 91 5. .07 4. .57 5. .08 Gl y c i n e 0. .74 0. . 53 0 . 74 0. .69 0 , .64 H i s t i d i n e 0. .43 0 , .32 0 , .43 0 . 41 0 , . 38 I s o l e u c i n e 0. . 74 0, .55 0 , .78 0. .71 0 , .67 Leucine 1, .35 1, .04 1, .42 1, .28 1, .25 Lysine 0, .79 0 , .47 0 , .78 0, .72 0 , .35 Methionine 0. .36 0 , .22 0 , .37 0. . 32 0, .27 Phenylalanine 1. .10 0 , . 80 1, .20 1, .04 1, .08 P r o l i n e 2 , .15 1, . 81 2, . 33 2. . 13 2. . 36 Serine 0 , .86 0, .69 0, .89 0, .84 0 , . 80 Threonine 0. . 70 0, . 52 0 , . 72 0 , .64 0 , .60 Tryptophan 0, .38 0, .24 0, .43 0 , .30 0, .24 Tyr o s i n e 0, .60 0, .46 0 , .66 0. . 59 0 , .60 V a l i n e 0, .97 0, . 74 1, .05 0. . 95 0, . 88 C u l t i v a r 1 = H69019109000, C u l t i v a r 2 = H69024038000, C u l t i v a r 3 = H690240004191 * Beames, unpublished. 72. Increased y i e l d s are a s s o c i a t e d with h i g h e r p r o p o r t i o n s of n o n - p r o t e i n n i t r o g e n (NPN). Since NPN does not c o n t r i b u t e any amino acids i t causes low amino a c i d composition (g/16g N). This c o u l d be the reason f o r the d i f f e r e n c e between the amino a c i d compositions o f the 1977 and 1975 h a r v e s t . Determination o f A v a i l a b l e L y s i n e by Dye-Binding Procedure I n t r o d u c t i o n Methods f o r measuring r e a c t i v e or a v a i l a b l e l y s i n e i n food p r o t e i n s u s i n g FDNB or TNBS ( t r i n i t r o -benzene s u l p h o n i c a c i d ) f o l l o w e d by a c i d h y d r o l y s i s o f the p r o t e i n are r e l a t i v e l y complicated ( H u r r e l and Carpenter, 1976). Measurements of DBC are simpler and f a s t e r . I t has been found by H u r r e l and Carpenter (1975) t h a t f o r many food m a t e r i a l s the DBC with a c i d orange 12 was e q u i v a l e n t to the sum of t o t a l h i s t i d i n e , a r g i n i n e and r e a c t i v e l y s i n e . The d i f f e r e n c e i n DBC before and a f t e r b i n d i n g the r e a c t i v e l y s i n e has been used as a measure of a v a i l a b l e l y s i n e specifically (Hurrel and Carpenter, 1976). Masking or binding of reactive lysine may be achieved by reacting with propionic 73. anhydride, e t h y l c h l o r o f o r m a t e or TNBS (Hurrel and Carpenter, 1976). Sandler and Warren (1974) used e t h y l -c h l o r o f ormate f o r b i n d i n g the r e a c t i v e l y s i n e when they measured a v a i l a b l e l y s i n e i n fis h m e a l s u s i n g the dye b i n d i n g procedure. H u r r e l , Lerman and Carpenter (1979) used the dye-binding method to measure a v a i l a b l e l y s i n e i n a number of f e e d s t u f f s i n c l u d i n g b a r l e y . M a t e r i a l s and Methods F i v e b a r l e y s from the 1977 harvest of A l b e r t a A g r i c u l t u r e breeding program were used. Dye reagent was obtained from the Udy Analyzer Co., Boulder, Colorado. I t c o n t a i n e d 3.89 mmo l / l i t r e of a c i d orange 12 (MW 350.3), 200 g o x a l i c a c i d d i h y d r a t e , 34 g potassium dihydrogen phosphate and 600 ml g l a c i a l a c e t i c a c i d with water added to make 10 l i t r e s . P r o p i o n i c anhydride, sodium a c e t a t e , g l a s s beads and Watman g l a s s i f b r e f i l t e r paper (GF/A) were ob t a i n e d from F i s h e r Chemical Company. Pol y e t h y l e n e b o t t l e s were p r o v i d e d by the Udy Company with the dye reagent. For dye b i n d i n g d e t e r m i n a t i o n of a v a i l a b l e l y s i n e there i s a need to make two measurements, Dye 74. Bi n d i n g C a p a c i t y (DBC) and Dye Binding A f t e r Propiony-l a t i o n (DBAP). DBC 1.5 g b a r l e y , hammer^milled through a 0.5 mm screen, weighed i n d u p l i c a t e s i n t o p o l y e t h y l e n e b o t t l e s and ten g l a s s beads (3 mm] were added to each. Four m i l l i l i t r e s of 2.2 M sodium a c e t a t e were added and mixed w e l l (Hurrel et a l . , 1979 suggested that when the sample i s more than 0.8 g 4 ml of 2.2 M sodium a c e t a t e should be used). F o r t y m i l l i l i t r e s o f dye reagent were, added and the contents of the b o t t l e were shaken on a mechanical shaker o v e r n i g h t at room temperature. The r e a c t i o n mixture was f i l t e r e d through Watman GF/A f i l t e r s . An a l i q u o t of the f i l t r a t e was d i l u t e d 50 f o l d (Carpenter, p e r s o n a l communication) and the absorbance measured at 475 nm. A reagent blank c o n t a i n i n g 40 ml dye reagent and 4 ml sodium a c e t a t e and no sample was d i l u t e d to b r i n g i t i n the same absorbance range as the sample f i l t r a t e . T h i s was used as the c o n c e n t r a t i o n of dye before r e a c t -ing with the sample. G i l f o r d Spectrophotometer Model Stasar II.was used f o r a l l the measurements. 75. A standard curve was obtained by d i l u t i n g the reagent dye to d i f f e r e n t c o n c e n t r a t i o n s and the absor-bance measured. A r e g r e s s i o n of absorbance (Y) and c o n c e n t r a t i o n (X) [mmol/1] was obtained from the stand-ard curve and t h i s r e g r e s s i o n equation was used to pre-d i c t the c o n c e n t r a t i o n of dye i n the r e a c t i o n f i l t r a t e s from t h e i r absorbance. The equation being Y = 20.99 X; r = 0.99. DBAP Four m i l l i l i t r e s o f 2.2 sodium a c e t a t e s o l u t i o n and 0.4 ml p r o p i o n i c anhydride were added to 1.5 g ground samples and l e f t to stand o v e r n i g h t . F o r t y m i l l i l i t r e s of dye reagent were added and then .the pro-cedure was as f o r DBC. A f t e r measuring the dye con-c e n t r a t i o n s , the t o t a l dye content l e f t a f t e r the reac-t i o n was c a l c u l a t e d by m u l t i p l y i n g the c o r r e c t e d concen-t r a t i o n by the volume of r e a c t i o n mixture. DBC and DBAP were c a l c u l a t e d as the d i f f e r e n c e between reagent blanks and sample f i l t r a t e s . A v a i l a b l e l y s i n e was c a l c u l a t e d as the dye b i n d i n g l y s i n e (DBL) which i s the d i f f e r e n c e between DBC and DBAP i n mmol/16 g N. These values were then expressed as percentages of t o t a l l y s i n e i n the v a r i o u s b a r l e y s . Oym oonosneratlon lmma(/LJ F i g . I S t a n d a r d c u r v e f o r d y e c o n c e n t r a t i o n a n d a b s o r b a n c e ON R e s u l t s Table 8. Standard curve f o r dye c o n c e n t r a t i o n . D i l u t i o n C o n c e n t r a t i o n X Absorbance Y 1/100 0.0389 0. 799 1/125 0.0311 0.646 1/150 0.0259 0.496 1/175 0.0222 0.471 1/200 0.0195 0. 396 1/225 0.0173 0. 366 1/250 0.0153 0.323 1/275 0. 0141 0. 304 1/300 0.0130 0. 210 D i s c u s s i o n Dye b i n d i n g measurements on the b a r l e y s are shown i n Tables 9 and 10. I t i s b e l i e v e d that dye b i n d i n g c a p a c i t y i s p o s i t i v e l y c o r r e l a t e d with t o t a l b a s i c amino a c i d content and with t o t a l l y s i n e content Table 9. A n a l y s i s of ba r l e y g r a i n samples f o r dye b i n d i n g d e t e r m i n a t i o n of a v a i l a b l e l y s i n e . B a r l e y Hiproly : Gait. H69019109000 H69024038000 H690240004191 DBC 61.76 67.10 60.33 68.11 60.50 (mmol/16 g N) DBAP 43.89 50.95 47.10 50.97 47.57 (mmol/16 g N) DBD (DBC-DBAP) 17.87 16.15 13.28 17.14 12.93 •i L I ~i • r mmol % available lysine (^ ^) Total lysine from .. 21.33 20.71 21.94 23.44 19.14 i f mmol ion exchange ( 1 6 N) % avai l a b i l i t y of 83.78 77.98 60.53 73.12 67.55 lysine DBD/total lysine Table 10. P r o t e i n content (g/100 DM), l y s i n e and b a s i c amino a c i d content (g/16 g N) and dye b i n d i n g c a p a c i t y (mmol/16 g N) of f i v e b a r l e y samples. Ba r l e y Hiproly Gait H69019109000 H69024038000 H690240004191 Protein (N x 6.25) 18.57 15.16 17.45 18.10 16.81 Lysine (g/16 g N) 3.12 3.03 3.21 3.43 2.80 from ion exchange Total basic amino acids 8.42 8.57 8.94 8.64 8.80 (g/16 g N) from ion exchange DBC (mmol/16 g N)_ 61.71 67.10 60.38 68.11 60.50 80. Cg/16 g N) (Munk, 1972; T a l l b e r g , 1979; Walker, 1979). T h i s f a c t i s r e p o r t e d throughout the l i t e r a t u r e . In the pr e s e n t study, the dye b i n d i n g c a p a c i t i e s (DBC) were r a t h e r low and the c o r r e l a t i o n between DBC and t o t a l b a s i c amino a c i d s i s too low (r = 0.502). H u r r e l et a l . (1979) have measured DBC i n b a r l e y s and used t h i s method to estimate l y s i n e a v a i l a b i l i t i e s . DBC of 93.0, 88.5, 97.5 and 101.1 have been obtained f o r mutant 1508, b a r l e y h y b r i d s 2188, 2187 and 2186 r e s p e c t i v e l y (Hurrel et a l . , 1979). Ly s i n e a v a i l a b i l i t i e s observed were 91.19%, 102.84%, 94.99% and 102.87% f o r mutant 1508, hy b r i d s 2188, 2187 and 2186 r e s p e c t i v e l y . With a v a i l a b i l i t i e s of more than 100%, one ought to be f a i r l y c a u t i o u s about the dye b i n d i n g method f o r measuring a v a i l a b l e l y s i n e . B i o l o g i c a l measurements of a v a i l a b l e l y s i n e elsewhere i n t h i s t h e s i s have given values of 70.78%, 55.98%, 52.64%, 67.12% and 57.96% f o r H i p r o l y , G a i t , c u l t i v a r s 1, 2 and 3 r e s p e c t i v e l y . In t h i s study, H i p r o l y gave the h i g h e s t a v a i l a b i l i t y of 83.78% by dye b i n d i n g d i f f e r e n c e . However, H i p r o l y gave onl y 70.78% by b i o l o g i c a l d e t e r -m i n a t i o n ( i . e . , f a e c a l a n a l y s i s ) . G a i t which had 55.98% 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 by f a e c a l a n a l y s i s 81. gave 77.98% a v a i l a b i l i t y by dye b i n d i n g d i f f e r e n c e . C u l t i v a r 1 had the lowest l y s i n e a v a i l a b i l i t y by f a e c a l a n a l y s i s (52.641) and a l s o by dye b i n d i n g d i f f e r e n c e (60.53). I f one were to judge by l y s i n e a v a i l a b i l i t y measured by dye b i n d i n g d i f f e r e n c e i n t h i s study, Ga i t would be co n s i d e r e d s u p e r i o r i n p r o t e i n q u a l i t y to the three h i g h l y s i n e c u l t i v a r s . With the low c o r r e -l a t i o n c o e f f i c i e n t o b t a i n e d between dye b i n d i n g c a p a c i t y and t o t a l b a s i c amino a c i d s one ought to be c r i t i c a l about making c o n c l u s i o n s about p r o t e i n q u a l i t y based on dye b i n d i n g d i f f e r e n c e . In the dye b i n d i n g procedure d e s c r i b e d by H u r r e l et a l . (1979) and Walker (1979) they c a l l f o r the use of p o l y e t h y l e n e b o t t l e s f o r c a r r y i n g out the reac-t i o n . T h i s m a t e r i a l absorbed the dye and c o u l d be a source o f e r r o r i n t h i s procedure. However, H u r r e l et a l . (1979) and Walker (1979) made no r e f e r e n c e to the p o s s i b i l i t y of the b o t t l e s absorbing the dye. Udy A n a l y s z e r Co. s u p p l i e s these p o l y e t h y l e n e b o t t l e s with the dye. I would recommend that c o r r e c t i o n s be made f o r the dye absorbed by the b o t t l e s , a l t e r n a t i v e l y g l a s s b o t t l e s should be used. In papers d e s c r i b i n g dye b i n d i n g c a p a c i t i e s (Hurrel et a l . (1979) and 82. Walker , (1979) had c o r r e c t i o n f a c t o r s f o r machine d r i f t . In the present study, no machine d r i f t was observed. 83. BIOLOGICAL EVALUATION OF PROTEIN QUALITY Rat T r i a l I Rat Growth Assay and the Measurement of True N i t r o g e n  D i g e s t i b i l i t y , True B i o l o g i c a l Value and Net P r o t e i n  U t i l i z a t i o n I n t r o d u c t i o n The use of l a b o r a t o r y animals f o r n u t r i t i o n a l s t u d i e s i s a w e l l e s t a b l i s h e d p r a c t i c e . Change i n body weight i s a good estimate of changes i n body p r o t e i n i n response to the t e s t p r o t e i n , under w e l l c o n t r o l l e d con-d i t i o n s such as r e s t r i c t e d f e e d i n g , constant e n v i r o n -mental c o n d i t i o n s and young growing animals. Parameters that are commonly used i n a s s e s s i n g p r o t e i n q u a l i t y i n c l u d e True d i g e s t i b i l i t y of n i t r o g e n (TD) Net p r o t e i n u t i l i z a t i o n (NPU) 84. B i o l o g i c a l value (BV) P r o t e i n e f f i c i e n c y r a t i o (PER) R e l a t i v e p r o t e i n value (RPV) (Munk, 1972; Eggum, 1973; P.A.G. G u i d e l i n e s - FAO No. 16) Measurements of BV, PER and NPU are u s u a l l y done with approximately 101 crude p r o t e i n i n the d i e t , f e d to male r a t s . (Eggum, 1973; Chalupa and F i s h e r , 1963 and Hegsted and Chang, 1965). I t i s b e l i e v e d t h a t sex a f f e c t s the valu e s of these parameters. BV and NPU are sometimes determined with r e s t r i c t e d f e e d i n g . M a t e r i a l s and Methods Design o f Experiment A completely randomized design was used. There were s i x d i e t a r y treatments with 4 r a t s per treatment per p e r i o d and two p e r i o d s . Rats were housed i n d i v i -d u a l l y i n cages at a room temperature of 25°C. Animals and Cages Male r a t s (Woodlyn/Wistar S t r a i n , Woodlyn L a b o r a t o r i e s , Guelph, Ontario) 28 days of age at the beginning of the 14-day f e e d i n g p e r i o d were used. The s t a i n l e s s s t e e l cages had wire screen f l o o r s and were 85. f i t t e d w i t h t r a y s f o r c o l l e c t i n g faeces and u r i n e . Trays were l i n e d with paper towels which were changed d a i l y . During the c o l l e c t i o n p e r i o d , funnels w i t h 0.5 mm wire mesh l i n i n g were f i t t e d under the cages i n s t e a d o f the t r a y s . The wire mesh separated the faeces from the u r i n e . D i e t s D i e t s (Table 11) were based on b a r l e y g r a i n ground through 0.5 mm screen on a l a b o r a t o r y hammer m i l l ( C h r i s t y and N o r r i s L t d ; Chelmsford, England). There were f i v e t e s t d i e t s , one f o r each b a r l e y type, made to pro-v i d e approximately 101 crude p r o t e i n (DM b a s i s ) . The s i x t h d i e t was a 41 egg p r o t e i n p u r i f i e d d i e t . T h i s was used to estimate metabolic f a e c a l n i t r o g e n (MFN) and endogenous u r i n a r y n i t r o g e n (EUN) as suggested by Eggum (1973) and expressed as f o l l o w s EUN = mean N m g / r a t / c o l l e c t i o n p e r i o d MFN = N. mg/DM intake (g) Table 11. Diet Composition {% DM basis). Ingredients Hiproly Gait C u l t i v a r 1 C u l t i v a r 2 E R R Protein C u l t i v a r 3 c * n c e n t r a t e Barley 53.85 65.96 57.31 55.25 59.49 -" C o m starch 39.15 27.04 35.69 37.75 33.51 88.63 Corn o i l 2.00 2.00 2.00 2.00 2.00 2.00 •Mineral mix 4.00 4.00 4.00 4.00 4.00 4.00 •Vitamin mix 1.00 1.00 1.00 1.00 1.00 1.00 +Egg protein cone. _ 4.87 ~ Dry matter 94.20 93.83 94.27 93.91 94.03 97.38 Crude protein 10.01 10.08 10.01 10.27 10.36 4.26 4.50 Cellulose - -— GE(cal/g) . 4318 4342.32 4313.81 4321.57 4288.91 ND + Egg p r o t e i n c o n c e n t r a t e was o b t a i n e d from U n i t e d S t a t e s B i o c h e m i c a l C o r p o r a t i o n . I t i s f a t f r e e and 96% o f i t c h o l e s t e r o l has been removed. Co m p o s i t i o n s are shown i n T a b l e s 12 and 13. Corn s t a r c h was moistened and a u t o c l a v e d f o r 3 h o u r s , d r i e d o v e r n i g h t i n an oven at 95°C and ground t h r o u g h an 0.5 mm s c r e e n . ND = not determined. oo 87. Table 12. V i t a m i n mix composition (g) (Eggum, 1973) Vi t a m i n A 325000 IU 0.3125 Vi t a m i n D 3 200000 IU 0.0375 Thiamine HCl 0.0225 Nicotinamide 0.2000 R i b o f l a v i n 0.0500 Calcium pantothenate 0.1087 P y r i d o x a l HCl 0.0060 ct-tocopherol phosphoric a c i d disodium s a l t 0.1285 Potato s t a r c h to make up to 500 g 88. Table 13. M i n e r a l mix composition (g/kg) (Eggum, 1973) Calcium carbonate (CaCO^) 68.6 Calcium C i t r a t e ( C a ^ ^ Q 0 1 4 • 4 H 2 0 ) 308,3 Calcium hydrogen phosphate (CaHP0 4•2H 20) 112.8 Dipotassium hydrogen phosphate (K-^HPO^) 218.8 Potassium c h l o r i d e (KC1) 124.7 Sodium c h l o r i d e (NaCl) 77.1 Magnesium sulphate (MgSO^) 38.3 Magnesium carbonate (MgCO^) 35.3 Manganese sulphate (MnSO^'H^^O) 0.201 Ammonium f e r r i c c i t r a t e (Brown) 15.3 Copper sulphate (CuS0 4•5H 20) 0.078 Potassium Iodide (KI) 0.041 Sodium f l u o r i d e (NaF) 0.507 Aluminium ammonium sulphate (AINH 4(S0 4) 2-12H 20) 0.090 89. Feeding Procedure Food and water were p r o v i d e d ad l i b i t u m . Water was renewed every other day. Body weight and food con-sumption were recorded d a i l y . Faeces and u r i n e were s e p a r a t e l y c o l l e c t e d i n 5% ^SO^ durin g the l a s t f o u r days of the t r i a l . Each day's c o l l e c t i o n was t r a n s -f e r r e d i n t o a 24 oz mason j a r and s t o r e d at 0°C u n t i l ready f o r a n a l y s i s . . . T h i s procedure has been used by Eggum (1973). A n a l y t i c a l Methods Proximate a n a l y s i s was done on each b a r l e y sample. A f t e r the d i e t s were mixed, they were analyzed f o r crude p r o t e i n by the K j e l d a h l procedure (AOAC, 1970). Faeces and u r i n e were thawed. Faeces samples were d i s s o l v e d , each i n c o n c e n t r a t e d s u l p h u r i c a c i d with 25 ml at a time. I t was done four times; a f t e r each a d d i t i o n the mixture was s t i r r e d w e l l and allowed to c o o l before f u r t h e r a d d i t i o n o f a c i d . A f t e r the 4th time, the r e s u l t a n t s o l u t i o n was homogeneous. I t was then t r a n s f e r r e d into a 500 ml v o l u m e t r i c f l a s k and made up to volume with d i s t i l l e d water. 25 ml was used f o r each d u p l i c a t e n i t r o g e n d e t e r m i n a t i o n . 90. The u r i n e was t r a n s f e r r e d i n t o 250 ml volume-t r i c f l a s k s . C o l l e c t i n g b o t t l e s and f u n n els were washed three times w i t h d i s t i l l e d water i n t o the f l a s k s , and more d i s t i l l e d water was added to b r i n g to volume. A volume of 25 ml was used f o r d u p l i c a t e n i t r o g e n d eter-m i n a t i o n f o r a l l samples. Energy content of the d i e t s were determined u s i n g a Gallenkamp a d i a b a t i c bomb c a l o r i m e t e r (Model CB110). Faeces were a l s o analyzed f o r energy f o r use i n c a l c u l a t i n g d i g e s t i b l e energy (DE). R e s u l t s and D i s c u s s i o n Table 14 summarizes the feed i n t a k e and growth data. Those r a t s on the H i p r o l y d i e t gained s i g n i f i -c a n t l y b e t t e r than those on G a i t and c u l t i v a r 3. H i p r o l y , c u l t i v a r 1 and c u l t i v a r 2 were not s i g n i f i c a n t l y d i f f e r -ent from each other i n terms of weight g a i n of r a t s . However, n e i t h e r were c u l t i v a r s 1 and 2 s i g n i f i c a n t l y d i f f e r e n t from G a i t and c u l t i v a r 3. Feed e f f i c i e n c i e s a l s o showed s i g n i f i c a n t d i f f e r e n c e s . H i p r o l y and c u l t i v a r 2 gave s i g n i f i c a n t l y higher feed e f f i c i e n c i e s than G a i t , c u l t i v a r 1 and c u l t i v a r 3, 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 each other. H i p r o l y and c u l t i v a r 2 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 each other. Table 14. Average d a i l y body weight gain, food consumption, growth r a t e and feed e f f i c i e n c y per r a t s . Diets 1 2 3 4 5 Hiproly. Gait Cultivar Cultivar: Cultivar SE F-test Newman-Keul's 1 2. 3 I n i t i a l body 85 .37 83.00 84.25 88 .62 88.75 - - -weigh .(g) Food consumption (g/14 days) 211 .00 193.00 189.00 210 .20 199.50 9.90 NS 3,2,5,4,1 DM intake 198 .20 179.80 176.70 196 .10 186.40 9.55 NS 3,2,5,4,1 (g/14 days) Total gain (g) . 37 .81 25.56 31.13 33 .63 27.06 4.96 * 2,5,3,4,1 Growth rate (g/day) 2 .60 1.80 2.20 2 .30 1.80 0.34 * 2,5,3,4,1 Feed efficiency 0 .20 0.14 0.16 0 .19 0.15 0.026 * 2,5,3,4,1 (g gain/g DM) DE Kcal/kg 3809 3628 3565 3684 3708 Cultivar 1 = H69019109000, Cultivar 2 = H69024038000, Cultivar 3 = H690240004191 92. In the n i t r o g e n balance p a r t of t h i s t r i a l , t here were no s i g n i f i c a n t d i f f e r e n c e s between the d i e t s f o r n i t r o g e n i n t a k e , true n i t r o g e n d i g e s t i b i l i t y , b i o -l o g i c a l value and net p r o t e i n u t i l i z a t i o n (Table 15). B a l a r a v i et a l . (1976) observed true n i t r o g e n d i g e s t i -b i l i t y (TD) of 78.9% f o r notch-1 and 83.6% f o r notch-2. T h e i r normal parent had a TD of 86.6% which was higher than that of e i t h e r notch-1 or notch-2. They obtained BV of 75.8%, 86.4% and 87.6% f o r the normal parent, notch-1 and notch-2 r e s p e c t i v e l y . The same authors observed NPU v a l u e s of 68.2 and 73.3 f o r notch-1 and notch-2. The f i n d i n g s of the present study agree f a i r l y w e l l with the r e s u l t s of B a l a r a v i et a l . (1976). G e n e r a l l y , normal b a r l e y has a b i o l o g i c a l value of 72.7% (Munk, 1972) and t h i s i s not changed s i g n i f i c a n t l y with amino a c i d supplements ( i t goes up to 73.2%). Ac c o r d i n g to Munk (1972) true d i g e s t i b i l i t y of n i t r o g e n i n b a r l e y g r a i n i n c r e a s e s as the crude p r o t e i n content (g/100 g DM) i n c r e a s e s . However, the l y s i n e percen-tage and BV decrease s i m u l t a n e o u s l y as the p r o t e i n content o f the g r a i n i n c r e a s e s . T h i s i s because there i s an i n c r e a s e d c o n t r i b u t i o n by prolamins to the t o t a l crude p r o t e i n and prolamins are poor q u a l i t y p r o t e i n s with low Table 15. Nitrogen balance t e s t (4 days c o l l e c t i o n p e r i o d ) . Barley Hiproly Gait Cultivar 1 Cultivar 2 Cultivar 3 S.E. F-Test N intake (g) .1.00 0.90 0.93 1.05 0.95 0.056 NS True dige s t i b i l i t y 0.88 0.85 0.86 0.86 0.86 0.010 NS of nitrogen True BV 0.83 0.86 0.84 0;.85 0.84 0.011 NS NPU 73.09 73.00 72.09 74.19 72.62 0.77 NS 94. l y s i n e content and a g e n e r a l l y poor amino a c i d p a t t e r n . It i s s u r p r i s i n g then t h a t H i p r o l y and c u l t i v a r 2 which are both high i n l y s i n e should tend towards reduced BV, (the d i f f e r e n c e s were not s i g n i f i c a n t ) . Munk (1972) observed t h a t at the same l e v e l of p r o t e i n i n t a k e , H i p r o l y had a higher TD and BV than normal h i g h p r o t e i n b a r l e y . In t h i s present study, although, the d i e t s c o n t a i n e d approximately equal l e v e l s of p r o t e i n , t o t a l p r o t e i n i n t a k e by r a t s were d i f f e r e n t because they were fed ad. l i b i t u m and so feed intake and hence p r o t e i n i n t a k e v a r i e d . The TD and BV values obtained were higher than those obtained by Eggum (1977a) who observed TD of 85.2% and BV of 76.0% f o r H i p r o l y , compared to valu e s o f 88% and 83% obtained i n t h i s study. However, Eggum (1977a) used r e s t r i c t e d f e e d i n g , a l l o w i n g 10 g DM and 150 mg N per r a t per day and t h i s c o u l d have made the d i f f e r e n c e . NPU of about 70% has been observed f o r high, l y s i n e l i n e s while normal b a r l e y s had about 60% NPU (Eggum, 1977a). I t has been r e p o r t e d by Hegsted and Chang (1965) that BV values, tend to overestimate the n u t r i t i v e value o f poor q u a l i t y p r o t e i n s . In t h i s study the BV of G a i t was the h i g h e s t . One would 95. expect the high l y s i n e c u l t i v a r s to have higher b i o l o -g i c a l v a l u e s . However, the d i f f e r e n c e s were not s i g n i -f i c a n t . Rat T r i a l II R e l a t i v e P r o t e i n Value as.a Measure of P r o t e i n  Q u a l i t y I n t r o d u c t i o n The use of R e l a t i v e P r o t e i n Value (RPV) as a measure of p r o t e i n q u a l i t y assumes that the body weight response i s l i n e a r over the range of t e s t p r o t e i n i n t a k e s s t u d i e d (PAG, 1975; Eggum and Campbell, 1979). The accuracy, however, i s dependent on the number of animals used, the v a r i a t i o n around the r e g r e s s i o n l i n e s and the range o f p r o t e i n i n t a k e s i n c l u d e d i n the t e s t (PAG, 1975). M a t e r i a l s and Methods  Design of Experiment A completely randomized design was used with 4 r a t s per treatment.per p e r i o d . There were s i x d i e t s 96. wi t h three l e v e l s of p r o t e i n per d i e t , thus g i v i n g 18 d i f f e r e n t treatments. The experiment was done i n two d i f f e r e n t p e r i o d s of 14 days each. Animals and Cages Male r a t s (Woodlyn/Wistar s t r a i n , Woodlyn L a b o r a t o r i e s , Guelph, O n t a r i o ) 23 days of age at the beginning of the experiment were used. Cages were the same as used f o r t r i a l I . The room temperature was 25°C on the average d u r i n g the t r i a l p e r i o d . D i e t s There were s i x d i f f e r e n t d i e t s , each w i t h three l e v e l s of p r o t e i n . High n i t r o g e n c a s e i n ( S h e f f i e l d Chemical Co., Norwich, N.Y. 13815) was used as the r e f e r e n c e p r o t e i n . The other p r o t e i n sources were the f i v e b a r l e y s being evaluated f o r p r o t e i n q u a l i t y . Each p r o t e i n source was f e d at three l e v e l s , v i z . 2, 5 and 8 g crude p r o t e i n per 100 g DM. The composition of the d i e t s i s shown i n Table 16. Food and water were p r o v i d e d ad l i b i t u m . Water was renewed every other day and the bottom t r a y Table 16. Co m p o s i t i o n of RPV d i e t s 1 2 3 Ingredients Hiproly Gait H69019109C 2% Crude Protein Barley 10.77 13.19 11.46 Corn starch 82.23 79.81 81.64 Corn o i l 2.0 2.0 2.0 Mineral mix 4.0 4.0 4.0 Vitamin mix 1.0 1.0 1.0 Casein - - -Cellulose powder - - -Dry matter 95.68 96.41 97.18 Crude protein 2.18 2.14 2.08 51 Crude Protein Barley 26.93 32.98 28.65 Corn starch 66.07 60.02 64.35 Corn o i l 2.0 2.0 2.0 Mineral mix 4.0 4.0 4.0 Vitamin mix 1.0 1.0 1.0 Casein - - -Cellulose powder - - -Dry matter 94.10 95.63 95.52 Crude protein 4.84 4.47 4.67 81 Crude Protein Barley 43.08 52.77 45.85 Corn starch 49.92 40.23 47.15 Corn o i l 2.0 2.0 2.0 Mineral mix 4.0 4.0 4.0 Vitamin mix 1.0 1.0 1.0 Casein - - -Cellulose powder - - 94.88 Dry matter 94.50 95.00 Crude protein 7.58 7.32 7.98 % DM b a s i s 4 5 6 H69024038000 H690240004191 Casein 11.05 11.20 81.95 81.10 85.90 2.0 2.0 2.0 4.0 4.0 4.0 1.0 1.0 1.0 2.10 5.00 96.55 95.64 96.32 2.06 2.16 2.07 27.62 29.74 65.38 63.26 82.80 2.0 2.0 2.0 4.0 4.0 4.0 1.0 1.0 1.0 5.26 5.00 95.86 95.89 96.06 4.85 4.33 5.15 44.20 47.59 48.80 45.41 79.68 2.0 2.0 2.0 4.0 4.0 4.0 1.0 1.0 1.0 8.40 5.00 95.43 95.53 96.25 7.97 6.69 8.05 was c l e a n e d d a i l y . The m i n e r a l and v i t a m i n supplements used i n t h i s experiment had the same c o m p o s i t i o n s as those used i n t r i a l 1. The body w e i g h t s and fo o d consumption were measured d a i l y . F i g . 8 R e l a t i v e p r o t e i n v a l u e d e t e r m i n a t i o n Table 17. Results of a 14-day RPV assay. Dietary Regime Protein Content g/100 g DM Average Feed Intake Cg) Average DM Intake (g) Average Nitrogen Intake (g) I n i t i a l Body Weight Cg) Average Body Weight Change g/14 days Casein 2.07 72.50 69.83 0.23 :47.75 -4.875 5.15 104.50 100.38 0.83 46.00 14.127 8.05 112.19 107.98 1.37 40.90 27.437 Hiproly 2.18 69.50 66.50 0.235 55.0 -9.75 4.84 97.25 91.52 0.71 53.4 2.50 7.58 125.50 118.66 1.44 51.0 19.003 Gait 2.14 68.75 66.23 0.23 53.25 -10.19 4.47 83.00 79.37 0.57 52.94 -3.325 7.32 119.875 113.88 1.335 48.63 14.50 Cutlivar 1 2.08 61.375 51.68 0.20 50.63 -9.57 4.67 89.19 85.20 0.635 54.63 -1.415 7.46 122.065 115.88 1.385 47.63 12.625 Cultivar 2 2.06 57.90 55.90 0.185 51.50 -9.312 4.85 92.25 88.43 0.69 46.63 1.362 7.97 116.07 110.76 1.36 47.50 14.94 Cultivar 3 2.16 58.56 56.01 0.19 51.44 -10.437 4.33 83.44 80.01 0.555 46.00 1.937 6.69 102.13 97.56 0.045 44.50 9.625 101. D i s c u s s i o n From Table 18, H i p r o l y appears to have the h i g h e s t RPV among the b a r l e y s , i n d i c a t i n g a s u p e r i o r i t y i n q u a l i t y . H i p r o l y i n t h i s study had 87% of the q u a l i t y of c a s e i n . C u l t i v a r 3, a c c o r d i n g to t h i s t e s t , was next to H i p r o l y i n q u a l i t y f o l l o w e d by c u l t i v a r 1. T h i s t e s t showed»cultivar 2 to be o f the lowest q u a l i t y . There does not seem to be much l i t e r a t u r e on the use of RPV as a measure of p r o t e i n q u a l i t y . Most r e s e a r c h e r s doing b i o l o g i c a l e v a l u a t i o n s use TD, BV and NPU as measures of p r o t e i n q u a l i t y . Eggum and Campbell (1979) have r e p o r t e d a number of p o i n t s to be c o n s i d e r e d before a c c e p t i n g RPV as a standard method f o r p r o t e i n e v a l u a t i o n . It i s b e l i e v e d that the two low p r o t e i n l e v e l s (2 and 5%) cause low and v a r i a b l e food i n t a k e , l e a d i n g to small and v a r y i n g weight ga i n s , e s p e c i a l l y when the p r o t e i n i s of poor q u a l i t y (Eggum and Campbell, 1979). T h i s low and v a r i a b l e consumption was observed i n t h i s present study. It i s b e l i e v e d that l y s i n e d e f i c i e n t p r o t e i n s may not y i e l d a v a l i d slope r a t i o (Eggum and Campbell, 1979). 102'. Table 18. Estimates of RPV of f i v e b a r l e y s P r o t e i n Source I n t e r c e p t Slope RPV Casein -10.61 27.85 1.00 H i p r o l y -15.53 24.24 0.87 G a i t -14.33 21.52 0.77 C u l t i v a r 1 -14.96 23.19 0.83 C u l t i v a r 2 -12.34 19.84 0.71 C u l t i v a r 3 -13.73 23.36 0.84 103. Rat T r i a l I I I E f f e c t of Supplementing B a r l e y with Soybean Meal  (SBM) or E s s e n t i a l Amino A c i d s (EAA) on the Growth  of Weanling Rats I n t r o d u c t i o n C e r e a l s p l a y an important r o l e as a p r o t e i n source i n the d i e t of non-ruminant animals. I t i s known t h a t c e r e a l p r o t e i n s are d e f i c i e n t i n c e r t a i n e s s e n t i a l amino a c i d s , e s p e c i a l l y l y s i n e . In c e r e a l -based d i e t s such as barley-based d i e t s f o r p i g s , the c e r e a l p o r t i o n s u p p l i e s about 60% of the amino a c i d requirements (Thomke, 1974). In order to meet a l l the amino a c i d requirements, such d i e t s need to be supple-mented with other sources of p r o t e i n . P r o t e i n concen-t r a t e s such as f i s h meal, meat meal and soybean meal can be used to improve p r o t e i n s t a t u s and the amino a c i d supply. P u r i f i e d amino a c i d s have been used as supple-ments f o r c e r e a l based d i e t s . Such supplements have been known to cause improved performance i n animals. In the use of p r o t e i n c o n c e n t r a t e s , i t i s b e l i e v e d that t h e i r amino a c i d s complement those of the c e r e a l p r o t e i n . 104 . M a t e r i a l s and Methods Design "o£ Experiment. The experiment was a 5 x 4 f a c t o r i a l w i t h a completely randomized design. The two f a c t o r s being s t u d i e d were b a r l e y types and supplements. The b a r l e y s were H i p r o l y , G a i t , c u l t i v a r 1, c u l t i v a r 2 and c u l t i -var 3 and the supplements were none, soybean meal, l y s i n e and l y s i n e p l u s other e s s e n t i a l amino a c i d s . There were 20 d i f f e r e n t d i e t s (treatments) w i t h four r a t s per treatment per p e r i o d and two p e r i o d s . The r a t s were housed i n d i v i d u a l l y i n cages at a room temperature of 25°C. Animal and Cages Male r a t s (Woodlyn/Wistar S t r a i n , Woodlyn L a b o r a t o r i e s , Guelph, Ontario) 28 days of age at the s t a r t o f the experiment were used. The cages were the same as those used i n t r i a l s I and I I . D i e t s The d i e t s were formulated' to p r o v i d e a p p r o x i -mately 12% CP (DM b a s i s ) , the NRC net p r o t e i n requirement 105 . f o r growing r a t s (NRC, 1972). This was achieved by d i l u t i n g the b a r l e y s with s t a r c h . In the barley-SBM d i e t , SBM was added to supply 50% of the dietary p r o t e i n and to m a i n t a i n the p r o t e i n l e v e l at approximately 12%. Lysine and the other e s s e n t i a l amino a c i d s were added to r a i s e t h e i r l e v e l s i n the d i e t s to NRC (1972) recom-mended l e v e l s f o r growing r a t s , s i n c e the b a s a l d i e t s s u p p l i e d these amino a c i d s at l e v e l s lower than those r e q u i r e d by growing r a t s . The compositions of the d i e t s are shown i n Table 19 and the c a l c u l a t e d amino a c i d compositions of the b a s a l d i e t s are shown i n Table 20. R e s u l t s and D i s c u s s i o n The r e s u l t of t r i a l I I I can be seen i n Table 22. The e f f e c t of supplementation can be seen c l e a r l y i n t h i s t a b l e . A f t e r s t a r c h d i l u t i o n of each b a r l e y to produce a standard p r o t e i n (N x 6.25) content of 12%, supplementation with l y s i n e alone had no b e n e f i c i a l e f f e c t on r a t performance. T h i s i n d i c a t e s that l y s i n e i s not the o n l y l i m i t i n g amino a c i d i n b a r l e y p r o t e i n artd thus c o u l d not c o r r e c t the imbalance. The f a c t t h a t a d d i t i o n o f other EAA made a s i g n i f i c a n t improvement Table 19. Composition of b a s a l and supplemented d i e t s f o r t r i a l III (DM b a s i s to make 1 kg). Basal Diet Barley Corn starch Corn o i l *Mineral mix *Vitamin mix Hiproly 646.3 283.7 20.0 40.0 10.0 Gait 791.6 138.4 20.0 40.0 10.0 Cultivar 1 Cultivar 2 Cultivar 3 687.7 242.3 20.0 40.0 10.0 663.0 267.0 20.0 40.0 10.0 713.9 216.1 20.0 40.0 10.0 Basal + Soybean meal Barley Corn starch Corn o i l *Mineral mix *Vitamin mix ** Soybean meal Basal + L-Lysine Barley Corn starch Corn o i l *Mineral mix *Vitamin mix L-Lysine Basal + EM Barley Corn starch Corn o i l *Mineral mix *Vitamin mix Arginine Histidine Isoleucine Leucine Methionine + cystine Lysine Threonine Valine 323.15 395.8 343.85 331.5 486.85 414.2 466.15 478.5 20.0 20.0 20.0 20.0 40.0 40.0 40.0 40.0 10.0 10.0 10.0 10.0 120.0 120.0 120.0 120.0 646.3 791.6 687.7 663.0 283.7 138.4 242.3 267.0 20.0 20.0 20.0 20.0 40.0 40.0 40.0 40.0 10.0 10.0 10.0 10.0 6.3 6.4 6.1 5.9 646.3 791.6 687.7 663.0 283.7 138.4 242.3 267.0 20.0 20.0 20.0 20.0 40.0 40.0 40.0 40.0 10.0 10.0 10.0 10.0 1.8 2.4 2.2 1.5 0.9 0.9 1.0 1.0 1.9 2.2 2.5 2.1 0.2 0.4 0.0 0.5 2.8 2.7 3.0 2.7 6.3 6.4 6.1 5.9 2.1 2.4 2.5 2.6 1.5 2.2 1.6 1.9 356.95 453.05 20.0 40.0 10.0 120.0 713.9 216.1 20.0 40.0 10.0 6.6 713.9 216.1 20.0 40.0 10.0 1.8 1.0 2.2 0.5 2.5 6.6 2.4 2.1 o ON * mineral and vitamin mix compositions are shown i n Tables 7 and 8. ** soybean meal added to provide 50% of the p r o t e i n . Table 20. Barley content and c a l c u l a t e d EAA content of b a s a l d i e t s {% DM b a s i s ) . Component H i p r o l y Gait C u l t i v a r 1 C u l t i v a r 2 C u l t i v a r 3 B a r l e y 64. .63 79. .16 68, . 77 66, .30 71, .39 A r g i n i n e 0 , .49 0. .43 0 , .45 0, .52 0, .49 H i s t i d i n e 0, .24 0, .24 0. . 23 0, .23 0 , .23 I s o l e u c i n e 0. .42 0. .39 0. .36 0, .40 0 , . 39 Leucine 0, .81 0 . 79 0, .83 0, .78 0 , .78 L ysine 0 , .37 0 , .36 0 . 39 0 , .41 0 , . 34 Methionine + Cystine 0 , .39 0. .40 0 . 37 0 , .40 0 . 42 P h e y l a l a n i n e + Tyrosine 0 . ,97 0. .90 0. .96 0. . 93 0. . 89 Threonine 0 . , 35 0. .32 0. .35 0 . 34 0. .34 Tryptophan 0. .32 0 . 32 0 . 31 0. .30 0 . , 32 V a l i n e 0. .52 0. .45 0. .51 0. .48 0 . .46 Table 21. DM and crude p r o t e i n a n a l y s i s of d i e t s Hiproly Gait Cultivar 1 Cultivar 2 Cultivar 3 Basal Dry matter I 93.45 94.71 93.31 93.79 93.22 Protein (N x 6.25) (DM basis) 12.06 12.22 12.03 11.92 12.03 GE (Kcal/kg) 4328 4323 4314 4326 4298 Basal + SBM Dry matter % 94.05 94.92 93.87 93.98 93.61 Protein (N x 6.25) (DM basis) 12.56 12.73 12.55 12.43 12.63 Basal + Lysine Dry matter % 93.90 94.85 93.76 93.75 93.93 Protein (N x 6.25) (DM basis) 12.34 12.43 12.28 12.27 12.20 Basal + EAA Dry Matter % 93.98 94.80 93.56 94.03 93.80 Protein (N x 6.25) (DM basis) 12.75 12.80 12.29 12.46 12.91 Results Table 22 Feed consumption and growth data, T r i a l III Parameter Supplement Barley Hiproly Gait C u l t i v a r 1 I n i t i a l body None 80.50 84.38 81.13 weight (g) SBM 80.88 84.00 84.25 Lysine 77.75 82.50 79.88 Lys ine+EAA 82.25 82.25 80.38 j. • Mean 80.35 83.28 81.41 Food intake (g) None 180.10 188.50 189.00 SBM 213.50 214.60 224.40 Lysine 200.14 186.13 178.63 Lysine+EAA 202.00 220.38 197.00 / _ — — Mean 199.8a 202.4 a 197.3 a DM intake (g) None 168.50 178.50 176.80 SBM 200.60 198.90 210.60 Lysine 194.66 176.54 167.48 Lysine+EAA 189.83 208.92 184.31 Mean 189.4a 191.9 a 184.8a Total gain (g) None 36.62 30.62 39.12 SBM 69.37 58.62 62.12 Lysine 46.43 35.50 35.38 Lysine+EAA 58.38 60.75 54.13 Mean 52.90 3 46.37 b 47.69 b Growth rate None 2.62 2.18 2.80 (g/day) SBM 4.96 4.19 4.44 Lysine 3.32 2.53 2.50 Lysine+EAA 4.17 4.33 3.87 Mean 3.779a 3.309b 3.400b Food E f f i c i e n c y None 0.22 0.17 0.22 (g gain/g DM SBM 0.35 0.29 0.30 in take) Lysine 0.25 0.20 0.21 Lysine+EAA 0.31 0.29 0.28 j -Mean 0.2823 0.237c 0.250 b c C u l t i v a r 2 C u l t i v a r 3 Mean 82.00 83.13 82.23 84.38 83.75 83.45 83.88 81.75 81.15 82.75 81.88 81.90 83.25 82.63 191.90 187.00 187.3 b 225.50 210.90 217.8 a 200.63 183.13 189.5 b 195.13 201.25 203.I 3 203.3 a 195.6 a 180.00 174.30 175.6 b 211.90 197.40 204.9 a 188.09 170.95 180.4b 183.48 188.93 191.l a 190.9 a 182.9 a 39.13 31.75 35.45 b 68.00 63.25 64.27 a 39.38 36.25 38.38 57.38 56.75 57.47 a 50.97 a b 47.00 b 2.80 2.33 2.544b 4.86 4.52 4.591 3 2.81 2.59 2.736° 4.10 4.05 4.103 3 3.640 a b 3.372b 0.22 0.18 0.201 b 0.32 0.32 0.313 0.21 0.21 0.214b 0.31 0.30 0.298 3 0.264 a b 0.252 b c 111. goes to support the f a c t t h a t other amino a c i d s c o u l d be l i m i t i n g i n these b a r l e y p r o t e i n s . A f t e r d i l u t i n g each b a r l e y to a standard p r o t e i n l e v e l o f 12%, SBM was j u s t as good as EAA i n improving r a t performance. On the whole, improvements w i t h SBM were hi g h e r than with EAA but the d i f f e r e n c e s between the two supplements were not s i g n i f i c a n t . H i p r o l y was s i g n i f i c a n t l y s u p e r i o r i n i t s response to supplementation, to the other b a r l e y s but not c u l t i v a r 2. A l l the other b a r l e y s i n c l u d i n g c u l t i v a r 2, were not s i g n i f i c a n t l y d i f f e r e n t i n t h e i r response to supplementation i n terms of t o t a l g a i n and growth r a t e . In terms of feed e f f i c i e n c y , H i p r o l y and c u l t i v a r 2 were not s i g n i f i c a n t l y d i f f e r e n t i n t h e i r response to supplementation but H i p r o l y was s i g n i f i c a n t l y b e t t e r than G a i t , c u l t i v a r s 1 and 3 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 each other i n t h e i r response to supplementation. G e n e r a l l y l y s i n e supplementation o f g r a i n has been known to improve performance i n animals and the a d d i t i o n of other e s s e n t i a l amino ac i d s e s p e c i a l l y t h r eonine has proved b e n e f i c i a l (Pond et a l . , 1958). 112. Chung and Beames (1974) observed t h a t supplementation of b a r l e y w i t h l y s i n e to p r o v i d e 0.75 g/100 g DM improved the performance of growing p i g s , and. observed f u r t h e r improvement w i t h the a d d i t i o n of t h r e o n i n e . T h i s i n d i c a t e s t h a t t h r e o n i n e c o u l d be the second l i m i t i n g amino a c i d i n b a r l e y g r a i n . Chung and Beames (1974) have c i t e d a number of papers r e p o r t i n g improvements i n growth response w i t h g r a i n d i e t supplemented with l y s i n e alone and a l s o w i t h other EAA. Bayley and Summers (1968) observed t h a t supplementation of corn-SBM r a t i o n w i t h 0.1% l y s i n e and 0.05% methionine f o r growing p i g s had no b e n e f i c i a l e f f e c t of growth r a t e on feed e f f i c i e n c y . However, when both were given together, there was a p o s i t i v e e f f e c t on g a i n . They a l s o observed that r e s -ponse to l y s i n e and methionine supplements depends on the a d d i t i o n of t h r e o n i n e , i n d i c a t i n g that t h i s amino a c i d i s a l s o d e f i c i e n t i n low p r o t e i n corn-SBM d i e t s . In t h i s t r i a l , the responses to supplementation might have been g r e a t e r without the s t a r c h d i l u t i o n . In d i l u t i n g the p r o t e i n down to the recommended l e v e l of 12%, the amino a c i d s are a l s o d i l u t e d . For s t u d i e s on p r o t e i n q u a l i t y t h i s d i l u t i o n i s necessary. However, i f b a r l e y had- been used as a complete d i e t there would be no 113. d i l u t i o n with s t a r c h . Beames (unpublished) f e d the same types of b a r l e y (but from the 1975 h a r v e s t ) t t o r a t s as a complete d i e t . He observed much g r e a t e r responses with supplementation. 114. Determination o f Apparent A v a i l a b i l i t y o f Lysine i n  Bar l e y Using the F a e c a l A n a l y s i s Method I n t r o d u c t i o n A v a i l a b i l i t y o f amino a c i d s may be d e f i n e d as the p r o p o r t i o n of the p a r t i c u l a r amino a c i d s t h a t are a c c e s s i b l e to the organism. This assumes t h a t a l l amino a c i d s that are absorbed from the gastro -i n t e s t i n a l t r a c t are a v a i l a b l e to the animal. Through-out the l i t e r a t u r e one encounters a term l i k e d i g e s t i b l e amino a c i d s which I c o n s i d e r as a misnomer. This i s because amino a c i d s are not d i g e s t e d ; they are the s m a l l e s t u n i t s o f the p r o t e i n molecule. They are absorbed as such and used as such. Amino a c i d s may be deaminated but a f t e r t h a t they are of no use to the animal f o r p r o t e i n s y n t h e s i s . I b e l i e v e t h a t when the p r o t e i n i s d i g e s t e d , the amino a c i d s do not change form be f o r e a b s o r p t i o n . Therefore amino a c i d s are absorbed a f t e r p r o t e i n d i g e s t i o n without any f u r t h e r break down. A v a i l a b i l i t y i s t h e r e f o r e a b e t t e r word to use with amino a c i d s than d i g e s t i b i l i t y . A v a i l a b i l i t y o f amino a c i d s may be determined c h e m i c a l l y , b i o l o g i c a l l y , e n z y m a t i c a l l y or m i c r o b i o l o -g i c a l l y . In b i o l o g i c a l d e t e r m i n a t i o n , l a b o r a t o r y 115. animals such as r a t s and mice have been used or some-times farm animals such as p i g s are used (Batterham et a l . , 1978). In b i o l o g i c a l e v a l u a t i o n one -can measure true or apparent a v a i l a b i l i t y of amino a c i d s u s i n g i l e a l of f a e c a l a n a l y s i s methods. Sauer, Stoth e r s and Parker (1977) determined apparent a v a i l a b i l i t i e s o f amino a c i d s u s i n g i l e a l and f a e c a l a n a l y s i s f o r wheats and m i l l i n g by-products f o r growing p i g s . They showed that the use of a c o r r e c t i o n f o r metabolic f a e c a l amino a c i d l e v e l s ( i . e . , amino a c i d e x c r e t i o n on zero n i t r o g e n intake) f o r c a l c u l a t i n g true a v a i l a b i l i t y was not accur-ate. They observed t h a t metabolic amino a c i d l e v e l s i n the faeces were g r e a t e r f o r some amino a c i d s and l e s s f o r others than the l e v e l s found at the t e r m i n a l ileum. T h i s they e x p l a i n e d as being due to net e f f e c t of micro-b i a l s y n t h e s i s and degr a d a t i o n between the t e r m i n a l ileum and the fae c e s . For example, a r g i n i n e at the te r m i n a l ileum was 0.49 g/kg DM intake but was 0.22 g/kg DM intake i n f a e c e s ; h i s t i d i n e was 0.21 g/kg DM in t a k e at the t e r m i n a l ileum but 0.27 g/kg DM intake i n the faeces; l y s i n e was 0.27 g/kg DM in t a k e at the t e r m i n a l ileum but 0.37 g/kg DM intake i n the fae c e s . In a l l the d i e t s , the c a l c u l a t e d true f a e c a l a v a i l a b i l i t i e s 116. of some amino a c i d s were markedly i n excess of 100% and the r e s u l t s were not i n c l u d e d i n the p u b l i c a t i o n (Sauer et a l . , 1977). Fa e c a l a n a l y s i s method may be f a r from accurate f o r the d e t e r m i n a t i o n of true a v a i l a -b i l i t y of amino a c i d s (Sauer et a l . , 1977). T h i s i s because these authors have found d i f f e r e n c e s of 9.1% and 17.3% between i l e a l and f a e c a l r e c o v e r i e s of l y s i n e and threonine r e s p e c t i v e l y . One would expect i l e a l and f a e c a l r e c o v e r i e s to be the same i f there i s n e i t h e r a b s o r p t i o n nor s y n t h e s i s i n the lower gut. However, the m i c r o b i a l p o p u l a t i o n i n the c o l o n and caecum must a f f e c t the f i n a l l e v e l of amino a c i d s i n the e x c r e t a . True amino a c i d a v a i l a b i l i t y determined by the f a e c a l a n a l y s i s method can be found throughout the l i t e r a t u r e on b i o l o g i c a l a v a i l a b i l i t y of amino a c i d s (Eggum, 1973, de Muelenare and Feldman, 1960). In the l i t e r a t u r e authors of such p u b l i c a t i o n s do not t e l l us anything about the p o s s i b l e i n t e r f e r e n c e by micro-b i a l a c t i o n . Due to the modifying a c t i o n of micro-f l o r a i n the l a r g e i n t e s t i n e s , i t should be more a p p r o p r i a t e to measure j u s t apparent a v a i l a b i l i t i e s by f a e c a l a n a l y s i s and to use i l e a l a n a l y s i s i f true a v a i l a b i l i t y i s to be measured. M e t a b o l i c f a e c a l amino 117. a c i d s determined from faeces of animals on N-free d i e t s or 4% egg p r o t e i n d i e t s i s not accurate s i n c e m i c r o b i a l e f f e c t s are v a r i a b l e . For a t r u e measure of t r u e a v a i -l a b i l i t y o f amino a c i d s , chemical or enzymatical methods may be b e t t e r than f a e c a l or even i l e a l a n a l y s i s . T h i s experiment was done to compare r e s u l t s with v a l u e s obtained i n the chemical d e t e r m i n a t i o n of a v a i l a b l e l y s i n e u s i n g a dye b i n d i n g method. Procedure Rats on the unsupplemented d i e t s of t r i a l I I I were used f o r t h i s d e t e r m i n a t i o n . Rats were f e d ad l i b i t u m f o r 14 days. Faeces were c o l l e c t e d from day 11 to day 14 f o r l y s i n e d e t e r m i n a t i o n . L y s i n e i n t a k e was c a l c u l a t e d from DM intake and l y s i n e c o n c e n t r a t i o n i n the d i e t s . Faecal l y s i n e was measured by the n i n h y d r i n method d e s c r i b e d by Beckwith, P a u l i s and Wall- (1975). P r i n c i p l e s of the N i n h y d r i n Method f o r L y s i n e  Determination The c o l o u r e d d e r i v a t i v e produced by the reac-t i o n o f n i n h y d r i n w i t h alpha and e p s i l o n amino groups 118 . on amino a c i d s , p e p t i d e s and p r o t e i n s i s the b a s i s f o r the r a p i d spectrophotometric e s t i m a t i o n of l y s i n e i n corn meal (Beckwith et a l . , 1975). I f the meal i s f i r s t e x t r a c t e d with T r i c h l o r o a c e t i c a c i d (TCA) to remove f r e e amino a c i d s , the amount of c o l o u r e d d e r i v a t i v e produced i n a dimethyl s u l f o x i d e s o l v e n t system can be used f o r a q u a n t i t a t i v e d e t e r m i n a t i o n of l y s i n e con-te n t of g r a i n to w i t h i n the accuracy found with amino a c i d analyses f o r l y s i n e (Beckwith et a l . , 1975). The amount of f r e e l y s i n e i n the g r a i n i s r e l a t i v e l y small compared to the t o t a l l y s i n e , hence i t s c o n t r i b u t i o n of amino groups i s very s m a l l . When f r e e l y s i n e i s removed together with other f r e e amino a c i d s by p r e c i p i t a t i o n with TCA, the l o s s of amino groups from f r e e l y s i n e i s n e g l i -g i b l e . In measuring l y s i n e i n faeces by the n i n h y d r i n method the removal of f r e e amino a c i d s (of endogenous o r i g i n ) w i t h TCA leaves the undigested p r o t e i n , thus l y s i n e may be measured f a i r l y a c c u r a t e l y i n f a e c e s . Several chemical methods have been used f o r l y s i n e d e t e r m i n a t i o n but they a l l l a c k s i m p l i c i t y or accuracy (Beckwith et a l . , 1975). The r e a c t i o n of alpha and e p s i l o n amino groups i s f a c i l i t a t e d by the use of a s p e c i a l s o l v e n t system. In developing the ninhy-d r i n reagent f o r use i n amino a c i d a n a l y s e s , i t was 119. found t h a t the reduced form, h y d r i n d a n t i n , i s necessary f o r c o l o u r formation but i t i s i n s o l u b l e i n water. Dimethyl s u l f o x i d e (DMSO) was used as the s o l v e n t i n t h i s method because i t has b e t t e r s o l v e n t a c t i o n than other o r g a n i c s o l v e n t s (Beckwith et a l . , 1975). Determination of Lys i n e i n Faeces Twenty m i l l i g r a m s (.20 mg) ground faeces were p l a c e d i n pyrex screw cap tubes and 2 ml of 10% (W/V) TCA was added. This was shaken v i g o r o u s l y on an e l e c t r i c shaker (Canlab) f o r one minute to remove f r e e amino a c i d . Samples were c e n t r i f u g e d at 1000 g f o r 10 minutes and the supernatant was decanted. Sodium m e t a b i s u l f i t e (0.2 ml of 0.125 M) was added to the r e s i d u e f o l l o w e d by 2.3 ml of DMSO-Water (4:1 V/V) and 1 ml of n i n h y d r i n reagent. The b i s u l f i t e was added to break d i s u l f i d e bonds i n g l u t e l i n to y i e l d s m a l l e r p r o t e i n p a r t i c l e s , and to remove c o l o u r s due to DMSO-soluble pigments i n the sample. The r e a c t i o n mixture was heated f o r 30 minutes on a b o i l i n g water bath. F i v e m i l l i l i t r e s of DMSO-water was added and the mixture was c e n t r i f u g e d at 1000 g 120. f o r 10 minutes. The supernatant was decanted i n t o a c l e a n tube, d i l u t e d with 5 ml DMSO and the absorbance measured at 580 nm with the necessary d i l u t i o n s to make re a d i n g p o s s i b l e . Known weights of H i p r o l y were used to o b t a i n a standard curve. Beckwith et a l . (1975) used maize with known l y s i n e content to o b t a i n t h e i r standard curve. A r e g r e s s i o n of absorbance on l y s i n e content was done. The r e g r e s s i o n equation obtained was used to p r e d i c t the l y s i n e content i n the f a e c e s . The r e g r e s s -i o n equation was Y = 0.83 X, r = 0.9968 where y = absorbance and x = l y s i n e content (mg). R e s u l t s and D i s c u s s i o n The r e s u l t s on apparent a v a i l a b i l i t y of l y s i n e i n d i c a t e t h a t H i p r o l y has the h i g h e s t a v a i l a b i l i t y of l y s i n e , t h i s value .being 70.31%. I t i s an i n d i c a t i o n of s u p e r i o r , q u a l i t y to the other b a r l e y s . C u l t i v a r 2 has 93.57% of the l y s i n e a v a i l a b i l i t y of H i p r o l y ; c u l t i v a r 3 has 83%, c u l t i v a r 1 has 75.68% and G a i t has 78.3% of the q u a l i t y of H i p r o l y based on apparent a v a i l a b i l i t y o f l y s i n e determined by f a e c a l a n a l y s i s . o - a r L.ysln« oontant (mg] F i g * 4 S t a n d a r d c u r v e f o r l y s i n e d e t e r m i n a t i o n 122. Table 23. Standard curve f o r l y s i n e d e t e r m i n a t i o n by n i n h y d r i n r e a c t i o n . Weight of H i p r o l y T o t a l L y s i n e Absorbance (g) (g) .{10% concentration) 0.0108 0.58 0.034 0.0270 0.145 0.105 0.0540 0.290 0.234 0.1080 0.580 0.524 0.1620 0.870 0.768 0.2700 1.45 1.163 Table 24. Faecal measurements f o r l y s i n e d e t e r m i n a t i o n . Cultivar 1 Cultivar 2 Cultivar 3 Hiproly Gait (H69019109000) (H69024038000) (H690240004191) Dry weight of faeces used (g) 0.0133 0.01325 0.0121 0.0123 0.0120 Absorbance (1/10 cone) 0.085 0.095 0.093 0.085 0.090 Estimated lysine content in faeces used (mg) 0.1243 0.1359 0.136 0.1243 0.1301 Estimated lysine cone in faeces (mg/g Dry faeces) 9.346 10.2566 11.0413 10.1057 10.8417 Table 25. Apparent a v a i l a b i l i t y of l y s i n e by f a e c a l a n a l y s i s . H i p r o l y G a i t C u l t i v a r 1 C u l t i v a r 2 C u l t i v a r 3 DM intake (g) 55.50 49.30 54.94 .56.82 51.16 *Lysine intake (mg) 205.40 177.50 214.30 233.00 173.94 Dry weight of faeces 26.10 31.05 36.30 31.55 26.70 '(g) Lysine content of 60.98 79.74 100.20 79.71 72.37 dry faeces (mg) Apparent % l y s i n e 70.31 55.08 53.21 65.79 58.39 a v a i l a b i l i t y C a l c u l a t e d from amino a c i d composition of b a r l e y by ion exchange chromatography, and dry matter i n t a k e . 125. Sauer et a l . (1977) observed apparent l y s i n e a v a i l a b i -l i t y of 77.5% f o r normal b a r l e y (Herta) u s i n g barrows as experimental animals. They observed v a l u e s of 8 3.0% and 80.7% f o r commercial corn and hard wheat. The v a l u e s obtained i n t h i s study are markedly lower than those obtained by Sauer et a l . (1977). Eggum (1973) observed true d i g e s t i b i l i t y of l y s i n e ( i . e . , t r u e a v a i l a b i l i t y ) i n normal b a r l e y to be 76% u s i n g r a t s and 72.3% u s i n g p i g s . The r e s u l t s of the apparent a v a i l a b i l i t y t e s t seem to f o l l o w the same tr e n d as the other measures of p r o t e i n q u a l i t y except f o r the r e l a -t i v e p r o t e i n value r e s u l t s . The chemical a v a i l a b i l i t y of l y s i n e measured by dye b i n d i n g d i f f e r e n c e d i d not f o l l o w the same trend as the b i o l o g i c a l a v a i l a b i l i t y f i g u r e s . However, because the c o r r e l a t i o n c o e f f i c i e n t f o r dye b i n d i n g c a p a c i t y and t o t a l b a s i c amino a c i d s i n t h i s experiment was low (0.502), the i n t e r p r e t a t i o n of the r e s u l t s must be done with c a u t i o n . 126. GENERAL DISCUSSIONS AND CONCLUSIONS T h i s p r o j e c t was designed to evaluate the pro-t e i n q u a l i t y o f f i v e b a r l e y c u l t i v a r s from the 1977 harv e s t of the A l b e r t a A g r i c u l t u r e breeding program. T h i s was done by: proximate a n a l y s i s measurement of p r o t e i n f r a c t i o n s amino a c i d composition a v a i l a b i l i t y of l y s i n e n i t r o g e n balance s t u d i e s r e l a t i v e p r o t e i n value growth performance and feed u t i l i z a t i o n o f r a t s the e f f e c t of supplementation on growth per-formance and feed c o n v e r s i o n . A summary o f a l l the experimental r e s u l t s can be seen i n Table 26. Based on the proximate a n a l y s i s v a l u e s , H i p r o l y and the other h i g h - l y s i n e c u l t i v a r s supply more p r o t e i n (N x 6.25) than G a i t , the commercial v a r i e t y . On t h i s c r i t e r i o n alone, these high l y s i n e c u l t i v a r s would appear to be a b e t t e r source of p r o t e i n f o r p i g s Table 26. Summary of experimental r e s u l t s . Experiment Parameter Hiproly Gait Cultivar 1 Cultivar 2 Cultivar 3 Proximate analysis Dry matter % (DM) 92.62 92.69 91. .92 92.55 92.64 Crude protein (°s DM basis) (N x 6.25) 18.57 15.94 17. .45 18.10 16.81 Solubility test Albumin + globulin (°s of total protein) 20.45 18.89 22 .63 21.88 19.34 Hordein 37.02 46.75 39. .93 38.67 44.11 (°a of total protein) Amino acid Total EAA 7.52 5.81 6. ,91 7.18 6.50 composition (g/100 g DM) Total EAA 35.58 35.29 35 .30 35.14 33.73 (g/16 g N) Total basic amino 8.43 8.59 8, .94 8.64 8.80 acids (g/16 g N) Lysine (g/16 g N) 3.12 3.03 3 .21 3.43 2.80 Lysine (g/100 g DM) 0.58 0.46 0 .56 0.62 0.47 Availability of *Chemical % 83.78 77.98 60. .53 73.12 67.55 lysine availability "Biological % availability 70.31 55.08 53. .21 65.79 58.39 Trial I Growth rates (g/gain/day) 2.60 1.80 2, .20 2.30 1.80 Feed efficiency 0.20 0.14 0. .16 0.19 0.15 (g gain/g DM intake) True N digestibility 0.88 0.85 0, .86 0.86 0.86 Net protein 73.09 73.00 , 72, .09 74.19 72.62 utilization Trial II Relative protein value 0.87 0.77 0, .83 0.71 0.84 Trial III Growth rates (g gain/day) Basal 2.62 2.18 2, .80 2.80 2.33 Basal + SBM 4.96 4.19 4. .44 4.86 4.50 Basal + lysine 3.32 2.53 2 .50 2.81 2.59 Basal + EM 4.17 4.33 3, .89 4.10 4.05 Feed efficiencey (g gain/g DM intake) Basal 0.22 0.17 0, .22 0.22 0.18 Basal + SBM 0.35 0.29 0. .30 0.32 0.32 Basal + lysine 0.25 0.20 0. 21 0.21 0.21 Basal + EAA 0.31 0.29 0. 28 0.31 0.30 * measured by dye b i n d i n g d i f f e r e n c e * measured by f a e c a l a n a l y s i s 128.. and would r e q u i r e l e s s p r o t e i n supplement than G a i t . C u l t i v a r 3 does not have as h i g h a p r o t e i n l e v e l as the other high l y s i n e c u l t i v a r s (Table 26). However, p r o t e i n l e v e l alone i s not enough f o r judging the pro-t e i n c o n t r i b u t i o n of these b a r l e y s ; the q u a l i t y of the p r o t e i n i s more important than the q u a n t i t y . A c c o r d i n g to Ingrersen and Koie (1973), the q u a l i t y of a p r o t e i n i s r e l a t e d to the p r o p o r t i o n s of albumins, g l o b u l i n s , g l u t e l i n s and prolamins i t c o n t a i n s . Albumins and g l o b u l i n s are h i g h q u a l i t y p r o t e i n s so the higher the p r o p o r t i o n s of these i n a p r o t e i n the b e t t e r the q u a l i t y of that p r o t e i n . - On-this b a s i s and. on • " the r e s u l t s of the p r o t e i n s e p a r a t i o n experiment, i t can be s a i d that c u l t i v a r 1, c u l t i v a r 2 and H i p r o l y are s u p e r i o r to c u l t i v a r 3 and G a i t i n q u a l i t y . The amino a c i d composition o f the d i f f e r e n t p r o t e i n f r a c -t i o n s of b a r l e y (Folkes and Yemm, 1956; see Table 3) support the f a c t that prolamins are poor q u a l i t y pro-t e i n s w i t h low l e v e l s of e s s e n t i a l amino a c i d s , espec-i a l l y l y s i n e . C u l t i v a r 3 and G a i t which have low l e v e l s of albumin and g l o b u l i n have high l e v e l s of p rolamin and t h i s would c o n t r i b u t e to t h e i r poor q u a l i t y . T h i s i s not r e f l e c t e d i n the amino a c i d composition (g/16 g N) 129. of the b a r l e y s . One would expect that H i p r o l y and c u l t i -v a r s 1 and 2 would have higher t o t a l e s s e n t i a l amino a c i d s , e s p e c i a l l y l y s i n e because they had higher l e v e l s of albumins and globulins in t h e i r p r o t e i n , but t h i s was not observed. The t o t a l e s s e n t i a l amino a c i d (EAA) l e v e l s i n the p r o t e i n s were approximately the same f o r G a i t and the hi g h l y s i n e c u l t i v a r s except i n c u l t i v a r 3 which had a s l i g h t l y lower t o t a l EAA than the r e s t . The amino a c i d composition (g/16 g 'N)'. (Table : 4) i n d i c a t e very l i t t l e or no d i f f e r e n c e s between the b a r l e y s . I f amino a c i d composition were to be used as the s o l e c r i t e r i o n f o r judging p r o t e i n q u a l i t y one c o u l d say t h a t the; f i v e b a r l e y s have the same p r o t e i n q u a l i t y . However, i f amino a c i d composition i s expressed as g/100 g DM (Table 4) H i p r o l y and the other high l y s i n e c u l t i v a r s are s u p e r i o r to the normal v a r i e t y i n t h e i r a b i l i t y to supply EAA. For p r o t e i n q u a l i t y s t u d i e s amino a c i d composition expressed as g/16 g N i s more meaningful. High l y s i n e b a r l e y s are bred f o r the l y s i n e content expressed as g/100 gm DM. For p r a c t i c a l f e e d i n g purposes t h i s : - i s a b e t t e r e x p r e s s i o n than g/16 g N, s i n c e . t h e amino a c i d composition of the d i e t o f p i g s and other farm animals i s expressed on a DM b a s i s . A l s o an animal's requirement of amino a c i d s i s expressed as a percentage of the DM of the d i e t . 130. Lysine a v a i l a b i l i t y measured by dye b i n d i n g d i f f e r e n c e s gave values that do not f o l l o w any p a r t i c u l a r t r e n d though H i p r o l y was s t i l l p o r t r a y e d as being super-i o r to the o t h e r b a r l e y s as other t e s t s i n t h i s study have shown. Based on the r e s u l t s of the dye b i n d i n g t e s t i n t h i s study i t i s d i f f i c u l t to make c o n c l u s i o n s about the p r o t e i n q u a l i t y . On the other hand, measure-ments of apparent a v a i l a b i l i t y of l y s i n e done by f a e c a l a n a l y s i s show that H i p r o l y and c u l t i v a r 2 are s u p e r i o r i n q u a l i t y to the other b a r l e y s because of t h e i r r e l a t i v e l y h i g h l y s i n e a v a i l a b i l i t y (70.31% and 65.79% r e s p e c t i v e l y ) . C u l t i v a r 3 which has shown low values f o r q u a l i t y parameters thus f a r has a b e t t e r a v a i l a b i l i t y of l y s i n e than c u l t i v a r 1. This does not seem very l i k e l y , c o n s i d e r i n g the prolamin contents of the two. There appears to be no obvious e x p l a n a t i o n f o r t h i s i n c o n s i s t e n c y . The n i t r o g e n balance t e s t s 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 the f i v e b a r l e y s . The t e s t s used (TD, BV, NPU) c a l l e d f o r 10% CP and t h i s was achieved by d i l u t i n g the b a r l e y s with s t a r c h as suggested by Eggum (1973). The results obtained for the nitrogen balance tests in this study (Table 4) are comparable to results obtained by Eggum •131. (1977b) who worked on some hig h l y s i n e b a r l e y s . For example, he obtained a TD o f 85%, true BV of 83% and NPU o f 71%: f o r O i l y . 71/669; Mutant 1508 had JD of 78%, true, BV o f 90% and NPU of 70; H i p r o l y had TD. of 85.2%,"" true BV of 76% and NPU of 64%. For n i t r o g e n balance t e s t s , r e s t r i c t e d f e e d i n g i s recommended (Eggum, 1973). Animals are fed 10 g DM per day with 150 mg p r o t e i n per day. The r a t s used i n t h i s study ( i . e . , t r i a l I) even though f e e d i n g was ad l i b i t u m , consumed on the average 11 g DM per day, which i s c l o s e to the recom-mended 10 g DM per day. R e l a t i v e p r o t e i n value (RPV) as a measure of p r o t e i n q u a l i t y gave a d i f f e r e n t p i c t u r e a l t o g e t h e r ( t r i a l I I ) . H i p r o l y s t i l l maintained i t s s u p e r i o r i t y (87%) but c u l t i v a r 3 became second to H i p r o l y (84%) while c u l t i v a r 2 was the poorest by t h i s t e s t . T h i s does not seem to f i t i n very w e l l with the q u a l i t y trend set by the other methods. There was so much v a r i a t i o n •-. i n food consumption and body weight changes (Table 17) that i t i s not c e r t a i n how much weight should be a t t a c h e d to the RPV r e s u l t s . Eggum and Campbell (1979) have suggested that l y s i n e d e f i c i e n t p r o t e i n s may not give v a l i d slope r a t i o s . For high l y s i n e b a r l e y s 132, one would imagine t h a t the RPV would be a s u i t a b l e t e s t f o r p r o t e i n q u a l i t y . However, wit h the amino a c i d com-p o s i t i o n expressed as g/16 g N, these high l y s i n e b a r l e y s are not d i f f e r e n t from the normal v a r i e t i e s and t h e i r p r o t e i n can s t i l l be d e s c r i b e d as l y s i n e d e f i c i e n t . Eggum and Campbell (1979) b e l i e v e that the 1% and 5% CP l e v e l s are too low and are r e s p o n s i b l e f o r the v a r i a t i o n " i n food i n t a k e and body weight changes. Rat growth assays ( t r i a l s I and II) i n d i c a t e t h a t H i p r o l y has a s i g n i f i c a n t l y b e t t e r ( P «0.05) a b i l i t y to support growth than the other b a r l e y s being s t u d i e d except c u l t i v a r 2. T h i s i s demonstrated i n the growth r a t e s and feed e f f i c i e n c i e s of r a t s fed these b a r l e y s as the s o l e source of p r o t e i n . In terms of the a b i l i t y to support growth c u l t i v a r 3 was as poor as G a i t which i s an i n d i c a t i o n of the q u a l i t y of i t s p r o t e i n . Work done on high l y s i n e b a r l e y s has been c e n t r e d more or l e s s on n i t r o g e n balance t e s t s and amino a c i d analyses^ There appears to be no i n f o r m a t i o n on growth s t u d i e s w i t h high l y s i n e b a r l e y s ; however;^Beames (unpublished) has worked on the same v a r i e t i e s of b a r l e y as the ones being d i s c u s s e d i n t h i s r e p o r t , but from a 1975 h a r v e s t . He fed the b a r l e y u n d i l u t e d with s t a r c h and obtained growth 133. r a t e s of 6.3, 4.6, 4.8, 5.9 and 4.6 g/day f o r H i p r o l y , G a i t , c u l t i v a r 1, c u l t i v a r 2 and c u l t i v a r 3 r e s p e c t i v e l y , compared with r e s p e c t i v e values o f 2.6, 1.8, 2.2, 2.3 and 1.8 g/day i n t r i a l I and 2.62, 2.18, 2.8, 2.8 and 2.33 g/ day i n T r i a l I I I . In the present study the p r o t e i n l e v e l i n the b a r l e y s were brought down to 12% crude p r o t e i n (DM b a s i s ) but NRC (1972) recommends 12% i d e a l p r o t e i n (DM b a s i s ) f o r growing r a t s . I t i s p o s s i b l e that i f the 1977 b a r l e y s were u n d i l u t e d , the r a t s would have done j u s t as w e l l as those on the 19 75 h a r v e s t . With the d i l u t i o n of b a r l e y , the amino a c i d c o n c e n t r a t i o n was reduced and t h i s l e d to the low growth r a t e s and feed e f f i c i e n c i e s when compared wi t h the r e s u l t s of Beames (unpublished). The NRC recommended 12% net p r o t e i n a p p a r e n t l y a p p l i e d to good q u a l i t y p r o t e i n s and t h a t with very h i g h q u a l i t y p r o t e i n s 9% CP i n the d i e t i s adequate to support r a t growth. In t h i s study a l e v e l o f 12% CP i n the d i e t was s e l e c t e d because the aim o f the i n v e s t i g a t i o n was to eva l u a t e the q u a l i t y o f the b a r l e y p r o t e i n s . Without knowing the q u a l i t y of the p r o t e i n s i n advance allowance c o u l d not have been made f o r poor q u a l i t y by f e e d i n g above the recommended l e v e l . 134. The f a c t t h a t supplementation o f these b a r l e y s w i t h l y s i n e alone made no s i g n i f i c a n t improvement i n r a t performance and th a t a d d i t i o n of other e s s e n t i a l amino a c i d s made a s i g n i f i c a n t improvement (P < 0.05) i n the performance of a l l r a t s i s an i n d i c a t i o n t h a t there may be some other e s s e n t i a l amino a c i d s l i m i t i n g i n these b a r l e y s . I t seems that because other amino a c i d s are l i m i t i n g , l y s i n e supplementation c o u l d not c o r r e c t the imbalance. A l s o that soybean meal made a s i g n i f i c a n t (P < 0.05) improvement i n r a t performance shows that SBM p r o t e i n has an amino a c i d p a t t e r n that complements that e x i s t i n g i n these b a r l e y s . To conclude I would say that c u l t i v a r 2 i s as good as H i p r o l y i n q u a l i t y s i n c e r o 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 between them f o r almost a l l the t e s t s except i n one case where there was an i n c o n s i s t e n c y i n the r e s u l t s . C u l t i v a r 1 appears to have i n t e r m e d i a t e q u a l i t y while c u l t i v a r 3 has a q u a l i t y as low as that o f G a i t , the commercial v a r i e t y . A l s o t h a t r e l a t i v e p r o t e i n value may not be a good t e s t f o r p r o t e i n q u a l i t y i n b a r l e y . Eggum and Campbell (1979) have suggested that l y s i n e d e f i c -i e n t p r o t e i n s may not y i e l d v a l i d slope r a t i o s . High 135 . l y s i n e b a r l e y s , although they c o n t a i n higher l e v e l s of l y s i n e i n the DM than normal b a r l e y , the l y s i n e concen-t r a t i o n (g/16 g N) i n t h e i r p r o t e i n i s not much d i f f e r e n t from those of normal b a r l e y s . In t h i s case, I would s t i l l c o n s i d e r high l y s i n e b a r l e y s as a source of l y s i n e d e f i c i e n t p r o t e i n . P o s s i b l y t h i s w i l l e x p l a i n the type of r e s u l t s obtained with the RPV t e s t . However, s i n c e animal d i e t s are formulated to p r o v i d e amino a c i d s on a DM b a s i s , the high l y s i n e b a r l e y s on t h i s b a s i s had a b e t t e r n u t r i t i o n a l q u a l i t y or n u t r i t i o n a l v a l u e than the normal v a r i e t y G a i t used i n t h i s study. 136. REFERENCES A.O.A.C. 1970. 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 n a l y t i c a l Chemists. 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Determination of p r o t e i n and r e a c t i v e l y s i n e i n l e a f - p r o t e i n c o n c e ntrates by dye-binding. Br. J . Nutr. 42: 44 5. 1979c. A comparison of dye-binding and f l u o r o d i n i t r o b e n z e n e methods f o r determining r e a c t i v e l y s i n e i n l e a f - p r o t e i n c o n c e n t r a t e s . Br. J . Nutr. 42: 455. 150. APPENDIX I E s s e n t i a l amino a c i d requirements of growing (0-6 wks) c h i c k s ( l a y e r replacements) (NRC, 1971) A r g i n i n e 1. 20 G l y c i n e 1. 00 H i s t i d i n e 0. 40 I s o l e u c i n e 0. 75 Leucine 1. 40 Lysine 1. 10 Methionine + c y s t i n e 0. 7 5 P h e n y l a l a n i n e + t y r o s i n e 1. 30 Threonine 0. 70 Tryptophan 0. 20 V a l i n e 0. 85 APPENDIX II E s s e n t i a l amino a c i d requirements (% DM d i e t ) of the grow ing r a t . 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 Methionine + c y s t i n e P h e n y l a l a n i n e + t y r o s i n e Threonine Tryptophan V a l i n e NRC Rama Rao et a l . (1972) (1979) 0.67 0.33 0.21 0.61 0.55 0.83 0.69 1.00 0.90 0.67 0.49 0.89 0.72 0.56 0.51 0.17 0.11 0.67 0.56 152. APPENDIX I I I E s s e n t i a l amino a c i d r e q u i r e m e n t s (% DM d i e t ) o f the growing p i g (NRC, 1973) L i v e weight (kg) Amino a c i d 5-•10 10-•20 20-•35 35-•60 60--100 A r g i n i n e 0. , 28 0. , 23 0. , 20 0. 18 0. ,16 H i s t i d i n e 0. ,25 0. , 20 0. ,18 0. ,16 0. .15 I s o l e u c i n e 0. .69 0. , 56 0. , 50 0. ,44 0. .41 L e u c i n e 0. ,83 0. , 68 0. , 60 0. ,52 0. ,48 L y s i n e 0. , 96 0. , 79 0. , 70 0. ,61 0. .57 M e t h i o n i n e + 0. , 69 0. , 56 0. , 50 0. ,44 0. .41 c y s t i n e P h e n y l a l a n i n e + 0. .69 0. , 56 0. , 50 0. .44 0. .41 t y r o s i n e T h r e o n i n e 0. . 62 0. . 51 0. .45 0. .39 0, . 37 Tryptophan 0. .18 0. .15 0. , 13 0. .11 0. .11 V a l i n e 0. .69 0. . 56 0. . 50 0, .44 0, .41 

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