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Qualitative and quantitative aspects of the protein nutrition of juvenile chinook salmon (Oncorhynchus.. 1985

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QUALITATIVE AND QUANTITATIVE ASPECTS OF THE PROTEIN NUTRITION OF JUVENILE CHINOOK SALMON (Oncorhynchus ts hawytscha) by IAN McCALLUM B. Sc . ( A g r . ) . M c G i l l U n i v e r s i t y , 1973 M. S c . , U n i v e r s i t y of Saskatchewan, 1978 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES Department of P o u l t r y Sc i ence We accept t h i s t h e s i s as conforming t o _ t i i e _ r e q u i r e d s tandard THE UN/IVERSI^KOF BRITISH COLUMBIA J u l y 1985 Ian McCal lum, 1985 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. I t i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of 9 o u L T f i . Y ^ ( H E M c g The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 DE-6 (3/81) ABSTRACT Two experiments were conducted to study the e f f e c t of p r o t e i n source and l e v e l i n the d i e t of j u v e n i l e ch inook salmon (Oncorhynchus tshawytscha) r e a r e d i n f r e s h water t a n k s . The p r o t e i n sources compared were a f r e e z e - d r i e d p o l l o c k muscle and euphausid mix ( 9 : 1 ) ( F P E ) , three whole h e r r i n g meals processed d i f f e r e n t l y from the same l o t of raw h e r r i n g and a c a s e i n - g e l a t i n mix supplemented with a r g i n i n e and DL-meth ion ine ( C S ) . The p r o t e i n sources were t e s t e d at three l e v e l s of d i e t a r y p r o t e i n i n i s o c a l o r i c d i e t s fed to s a t i a t i o n to d u p l i c a t e groups of f i s h for a 42-day p e r i o d . P r o t e i n was r e p l a c e d by d e x t r i n and g lucose on an es t imated m e t a b o l i z a b l e energy b a s i s . The v a r i o u s methods employed to e v a l u a t e p r o t e i n q u a l i t y y i e l d e d d i f f e r e n t va lues r e l a t i v e to F P E . In terms of growth r a t e and assays based on body p r o t e i n g a i n , FPE was found to be the best p r o t e i n s o u r c e . Low temperature ( 7 5 ° C ) d r y i n g of h e r r i n g meal caused a s l i g h t r e d u c t i o n i n p r o t e i n q u a l i t y compared to f r e e z e - d r y i n g . High temperature ( 1 5 0 ° C ) d r i e d h e r r i n g meal was found to be an extremely poor q u a l i t y p r o t e i n s o u r c e . Al though h igh e s t imates of p r o t e i n q u a l i t y were obta ined for CS, lower food i n t a k e depressed growth i n f i s h fed CS d i e t s . The d e t e r m i n a t i o n of the endogenous l o s s of n i t r o g e n from f i s h enabled the p a r t i t i o n i n g of p r o t e i n i n t a k e i n t o the amounts used for growth, maintenance and exogenous e x c r e t i o n f o r each p r o t e i n s o u r c e . - 11 _ In Experiment 2, two s e r i e s of i s o c a l o r i c d i e t s were t e s t e d c o n t a i n i n g 17 to 47% p r o t e i n , in increments of 10%, p r o v i d e d by FPE at two l e v e l s of d i e t a r y energy . The e q u a t i o n y = -0 .50699 + 0.25398x - 8.37872x ,(where y = s p e c i f i c growth r a t e , and x = p r o t e i n e n e r g y : t o t a l energy ( P E : T E ) ) was d e r i v e d to q u a n t i f y the d i e t a r y p r o t e i n requirement for j u v e n i l e ch inook salmon over a 105-day p e r i o d . Maximum growth was ach ieved at a P E : T E r a t i o of 0 .55 . However, f or p r a c t i c a l purposes the P E : T E r a t i o r e q u i r e d was found to l i e i n the range between 0.35 and 0 .55 . The range permits the f i s h c u l t u r i s t to c o n s i d e r economic e f f i c i e n c y i n d i e t f o r m u l a t i o n . T a b l e of Contents S e c t i o n Page CHAPTER 1 1.0 INTRODUCTION 1 CHAPTER 2 2.0 REVIEW OF LITERATURE 3 2.1 The u t i l i z a t i o n of d i e t a r y p r o t e i n 3 2.2 G e n e r a l a spec t s of p r o t e i n metabol ism 5 2.3 N i t r o g e n ba lance 8 2.4 Endogenous n i t r o g e n e x c r e t i o n 12 2.5 Methods used to determine p r o t e i n q u a l i t y 15 2.6 The p r o t e i n requ irements of sa lmonids w i th r e s p e c t to d i e t a r y energy 22 CHAPTER 3 3.0 EXPERIMENT 1. P r o t e i n u t i l i z a t i o n and the measurement of p r o t e i n q u a l i t y i n d i e t s for chinook salmon f r y 28 3.1 I n t r o d u c t i o n 28 3.2 M a t e r i a l s and methods 32 3 . 2 . Tes t p r o t e i n sources 32 3 . 2 . 2 D i e t s 37 3 . 2 . 3 Aquarium f a c i l i t y 42 3 .2 .4 P r o t o c o l 42 3 . 2 . 5 Measurement of growth 42 3 . 2 .'6 Chemica l a n a l y s i s of f i s h and d i e t s 43 3 . 2 . 7 A v a i l a b l e l y s i n e 43 3 . 2 . 8 Data a n a l y s i s 43 3.3 R e s u l t s .: 46 3 .3 .1 The e f f e c t of d i e t a r y p r o t e i n source and l e v e l on •body weight ga in 46 3 .3 .2 The e f f e c t of d i e t a r y p r o t e i n source and l e v e l on food i n t a k e and gross food c o n v e r s i o n e f f i c i e n c y 52 3 . 3 . 3 The e f f e c t of d i e t a r y p r o t e i n source and l e v e l on energy i n t a k e and gross energy u t i l i z a t i o n 57 3 . 3 . 4 The e s t i m a t i o n of endogenous n i t r o g e n l o s s by j u v e n i l e ch inook salmon 59 3 . 3 . 5 The r e l a t i o n s h i p between p r o t e i n i n t a k e and p r o t e i n u t i l i z a t i o n 65 — i v — T a b l e of Contents ( c o n t ' d ) 3 . 3 . 6 The measurement of p r o t e i n q u a l i t y by p r o t e i n e f f i c i e n c y r a t i o and net p r o t e i n r a t i o 70 3 . 3 . 7 The measurement of p r o t e i n q u a l i t y by p r o t e i n p r o d u c t i v e va lue and net p r o t e i n u t i l i z a t i o n . . . . 80 3 . 3 . 8 The measurement of p r o t e i n q u a l i t y by s l o p e r a t i o . . . . 8 8 3 . 3 . 9 A v a i l a b l e l y s i n e content of the p r o t e i n sources 93 3.4 D i s c u s s i o n 95 3 .4 .1 The e f f e c t of d i e t a r y p r o t e i n source and l e v e l on body weight ga in 95 3 . 4 . 2 The e f f e c t of d i e t a r y p r o t e i n source and l e v e l on food i n t a k e and gross food c o n v e r s i o n e f f i c i e n c y 97 3 . 4 . 3 The e f f e c t of d i e t a r y p r o t e i n source and l e v e l on energy i n t a k e and gross energy u t i l i z a t i o n . . . 98 3 .4 .4 The e s t i m a t i o n of maintenance requirements f o r p r o t e i n 100 3 . 4 . 5 The e f f e c t of d i e t a r y p r o t e i n source on p r o t e i n u t i l i z a t i o n 105 3 . 4 . 6 The d e t e r m i n a t i o n of p r o t e i n q u a l i t y by b ioas say . . . . 112 3.5 Summary of Experiment 1 127 CHAPTER 4 4.0 EXPERIMENT 2. P r o t e i n requirements of j u v e n i l e chinook salmon i n r e l a t i o n to d i e t a r y energy content 129 4.1 I n t r o d u c t i o n 129 4.2 M a t e r i a l s and methods 130 4 .2 .1 P r o t o c o l 130 4 .2 .2 D i e t s 130 4 . 2 . 3 Data a n a l y s i s 13,1 4.3 R e s u l t s 135 4 .3 .1 E s t i m a t i o n of the p r o t e i n requirement of j u v e n i l e chinook salmon from growth data 135 4 . 3 . 2 The e f f e c t of d i e t a r y p r o t e i n and energy l e v e l on food c o n v e r s i o n and gross energy u t i l i z a t i o n 147 4 . 3 . 3 The e f f e c t of d i e t a r y p r o t e i n and energy l e v e l on p r o t e i n u t i l i z a t i o n 148 4 . 3 . 4 The e f f e c t of d i e t a r y p r o t e i n and energy l e v e l on the proximate body c o m p o s i t i o n of f i s h 150 v T a b l e of Contents ( c o n t ' d ) 4.4 D i s c u s s i o n 1 5 5 A . 4 . 1 The d i e t a r y p r o t e i n requirement of j u v e n i l e chinook salmon 155 A . 4 . 2 The e f f e c t of d i e t a r y energy on the e f f i c i e n c y of p r o t e i n u t i l i z a t i o n 162 4 . 4 . 3 The e f f e c t of the source and l e v e l of d i e t a r y energy on the proximate body c o m p o s i t i o n of f i s h 165 4.5 Summary of Experiment 2 168 CHAPTER 5 5.0 C o n c l u s i o n s 170 6.0 B i b l i o g r a p h y 174 7.0 Appendices 189 vi L i s t of T a b l e s Table Page 1. Amino a c i d requirements for chinook salmon, c o m p o s i t i o n of eggs and the ana lyzed e s s e n t i a l amino a c i d c o m p o s i t i o n of f r e e z e - d r i e d p o l l o c k musc le , f r e e z e - d r i e d whole euphausids and v i t a m i n - f r e e c a s e i n 30 2. Compos i t ion of p r o t e i n sources 34 3. Compos i t ion of d i e t s 35-36 4. Proximate c o m p o s i t i o n of d i e t s 38-39 5. Summary of d i e t a r y treatments and codes 41 6. F i n a l mean wet body weights and s p e c i f i c growth r a t e s of f i s h fed the v a r i o u s p r o t e i n sources at each d i e t a r y p r o t e i n c o n c e n t r a t i o n 50 7. S lopes of body weight a g a i n s t p r o t e i n i n t a k e for the v a r i o u s p r o t e i n sources 53 8. T o t a l dry food i n t a k e , mean d a i l y food i n t a k e and gross food c o n v e r s i o n of the v a r i o u s d i e t s 56 9. Gross food c o n v e r s i o n ( G F C ) , r e l a t i v e GFC and r a n k i n g of d i f f e r e n t p r o t e i n sources at d i f f e r e n t d i e t a r y c o n c e n t r a t i o n 58 10. Mean d a i l y gross energy i n t a k e , mean d a i l y m e t a b o l i z a b l e energy i n t a k e and gross energy u t i l i z a t i o n of f i s h fed the v a r i o u s d i e t s 60 11. Gross energy u t i l i z a t i o n (GEU) , r e l a t i v e GEU and r a n k i n g of d i f f e r e n t p r o t e i n sources at d i f f e r e n t d i e t a r y c o n c e n t r a t i o n s 61 12. Wet body weight and percent of i n i t i a l body weight of f i s h fed p r o t e i n - f r e e and a maintenance d i e t 63 13. E s t i m a t i o n of mean d a i l y endogenous n i t r o g e n l o s s by c a r c a s s a n a l y s i s of f i s h fed a p r o t e i n - f r e e and a low p r o t e i n d i e t 63 14. The u t i l i z a t i o n of d i e t a r y p r o t e i n on a d a i l y b a s i s by f i s h fed the v a r i o u s e x p e r i m e n t a l d i e t s d u r i n g the 42 day p e r i o d 66 VII L i s t of T a b l e s ( c o n t ' d ) 15. P r o t e i n e f f i c i e n c y r a t i o c a l c u l a t e d on a dry body weight b a s i s (PER) , r e l a t i v e PER and r a n k i n g of d i f f e r e n t p r o t e i n sources at d i f f e r e n t d i e t a r y c o n c e n t r a t i o n s 72 16. Net p r o t e i n r a t i o c a l c u l a t e d on a dry body weight b a s i s (NPR), r e l a t i v e NPR and r a n k i n g of d i f f e r e n t p r o t e i n sources at d i f f e r e n t d i e t a r y c o n c e n t r a t i o n s . . . . 75 17. P r o t e i n e f f i c i e n c y r a t i o ( P E R ) , r e l a t i v e PER, 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 NPU f o r v a r i o u s p r o t e i n s when fed as the so l e source of p r o t e i n to d i f f e r e n t f i s h s p e c i e s he ld under d i s s i m i l a r c o n d i t i o n s 76-79 18. P r o t e i n p r o d u c t i v e va lue (PPV) , r e l a t i v e PPV and r a n k i n g of d i f f e r e n t p r o t e i n sources at d i f f e r e n t d i e t a r y c o n c e n t r a t i o n s 81 19. Net p r o t e i n u t i l i z a t i o n c a l c u l a t e d by the method of Bender and M i l l e r (1953) (NPU-1) , r e l a t i v e NPU-1 and r a n k i n g of d i f f e r e n t p r o t e i n sources at d i f f e r e n t d i e t a r y c o n c e n t r a t i o n s 82 20. Net p r o t e i n u t i l i z a t i o n c a l c u l a t e d by the method of Ogino et a l . (1980) (NPU-2) , r e l a t i v e NPU-2 and r a n k i n g of d i f f e r e n t p r o t e i n sources at d i f f e r e n t d i e t a r y c o n c e n t r a t i o n s 83 21. The r e l a t i o n s h i p between p r o t e i n i n t a k e and net p r o t e i n u t i l i z a t i o n c a l c u l a t e d by the Bender and M i l l e r ( 1953) formula 84 22. The r e l a t i o n s h i p between p r o t e i n i n t a k e and net p r o t e i n u t i l i z a t i o n c a l c u l a t e d by the method of Ogino et a l . ( 1980) 85 23. Slope r a t i o s of dry body weight gains on p r o t e i n i n t a k e for f i s h fed the v a r i o u s p r o t e i n sources 89 24. Slope r a t i o s of body p r o t e i n ga ins on p r o t e i n i n t a k e for f i s h fed the v a r i o u s p r o t e i n sources 90 25. A v a i l a b l e l y s i n e , l y s i n e requirements for chinook salmon and percentage of requirements s u p p l i e d by each p r o t e i n source 94 26. E s t i m a t e d absorbed n i t r o g e n requirement for maintenance of f i s h , determined by f e e d i n g and c a r c a s s a n a l y s i s exper iments 104 — vm - L i s t of T a b l e s ( c o n t ' d ) 27. Summary of the r e l a t i v e e s t imates of the n u t r i t i v e va lue of the t e s t p r o t e i n sources employed i n t h i s study determined by d i f f e r e n t methods 119 28. C o r r e l a t i o n c o e f f i c i e n t s and l e v e l of s i g n i f i c a n c e between the d i f f e r e n t parameters used to e s t imate the n u t r i t i v e va lue of d i e t s c o n t a i n i n g the v a r i o u s p r o t e i n sources 119 29. Compos i t ion of d i e t s c o n t a i n i n g v a r i o u s l e v e l s of p r o t e i n and energy (Exper iment 2) 132 30. Proximate c o m p o s i t i o n and c a l c u l a t e d energy content s of d i e t s employed i n Experiment 2 133 31. Wet f i s h body weight at day 43 and day 105, and s p e c i f i c growth r a t e s of f i s h fed d i e t s c o n t a i n i n g d i f f e r e n t l e v e l s of p r o t e i n and energy , and OMP 138 32. Gross food c o n v e r s i o n e f f i c i e n c y and gross energy u t i l i z a t i o n of d i e t s c o n t a i n i n g d i f f e r e n t l e v e l s of p r o t e i n and energy 149 33. P r o t e i n e f f i c i e n c y r a t i o , p r o t e i n p r o d u c t i v e va lue and net p r o t e i n u t i l i z a t i o n of d i e t s c o n t a i n i n g d i f f e r e n t l e v e l s of p r o t e i n and energy 151-152 34. Whole body proximate c o m p o s i t i o n at day 42 of f i s h fed the d i e t s c o n t a i n i n g d i f f e r e n t l e v e l s of p r o t e i n and energy 153 35. Whole body proximate c o m p o s i t i o n at day 105 of f i s h fed the d i e t s c o n t a i n i n g d i f f e r e n t l e v e l s of p r o t e i n and energy 154 ix L i s t of F i g u r e s F i g u r e Page 1. The u t i l i z a t i o n of food n i t r o g e n i n growing j u v e n i l e f i s h 6 2. . S t y l i z e d r e p r e s e n t a t i o n of n i t r o g e n ba lance 10 3A. Growth of chinook salmon fed the v a r i o u s p r o t e i n sources i n d i e t s c o n t a i n i n g a p p r o x i m a t e l y 17% p r o t e i n . . 4 7 3B. Growth of ch inook salmon fed the v a r i o u s p r o t e i n sources i n d i e t s c o n t a i n i n g approx imate ly 27% p r o t e i n . . 4 8 3C. Growth of ch inook salmon fed the v a r i o u s p r o t e i n sources i n d i e t s c o n t a i n i n g a p p r o x i m a t e l y 37% p r o t e i n . . 4 9 4. S p e c i f i c growth r a t e s of ch inook salmon fed v a r i o u s sources of p r o t e i n at d i f f e r e n t d i e t a r y c o n c e n t r a t i o n s . 5 2 5. S lopes of weight ga in a g a i n s t p r o t e i n i n t a k e of ch inook salmon fed d i e t s c o n t a i n i n g the t e s t p r o t e i n sources 55 6A-E Percent u t i l i z a t i o n of p r o t e i n fed f o r maintenance and growth, and percent e x c r e t e d by f i s h fed the t e s t p r o t e i n sources 68-69 7. E f f e c t of d i e t a r y p r o t e i n l e v e l on p r o t e i n e f f i c i e n c y r a t i o c a l c u l a t e d on a wet body weight b a s i s for ch inook salmon f r y fed f r e e z e - d r i e d p o l l o c k and e u p h a u s i d s , f o r p l a i c e fed f r e e z e - d r i e d cod muscle and f o r r a t s fed c a s e i n c o n t a i n i n g d i e t s 71 8. The r e l a t i o n s h i p between p r o t e i n i n t a k e and p r o t e i n e f f i c i e n c y r a t i o and net p r o t e i n r a t i o of f i s h fed d i e t s c o n t a i n i n g f r e e z e - d r i e d p o l l o c k and e u p h a u s i d s . . . 7 3 9. The r e l a t i o n s h i p between p r o t e i n i n t a k e and net p r o t e i n u t i l i z a t i o n of the t e s t p r o t e i n sources 86 10. S lopes of dry body weight ga in on p r o t e i n i n t a k e of chinook salmon fed d i e t s c o n t a i n i n g the t e s t p r o t e i n sources 91 11. S lopes of body p r o t e i n ga in on p r o t e i n i n t a k e of chinook salmon fed d i e t s c o n t a i n i n g the t e s t p r o t e i n s o u r c e s . . . 9 2 12. Weight ga in of chinook salmon from the f r y - to s m o l t - stage fed d i e t s c o n t a i n i n g v a r i o u s l e v e l s of p r o t e i n and energy , and fed OMP 137 13. S p e c i f i c growth r a t e of j u v e n i l e chinook salmon fed d i e t s c o n t a i n i n g d i f f e r e n t l e v e l s of p r o t e i n and energy 139 x L i s t of F i g u r e s ( c o n t ' d ) 14. S p e c i f i c growth r a t e of j u v e n i l e chinook salmon fed d i e t s c o n t a i n i n g d i f f e r e n t p r o t e i n e n e r g y r t o t a l energy r a t i o s 142 15. The second order p o l y n o m i a l curve f i t t e d to s p e c i f i c growth r a t e of j u v e n i l e chinook salmon fed d i e t s wi th d i f f e r e n t p r o t e i n e n e r g y : t o t a l energy r a t i o s 145 xi L i s t of Appendices T a b l e s T a b l e Page 1. A n a l y s i s of v a r i a n c e of day 42 body weights i n Experiment 1 191 2. A n a l y s i s of v a r i a n c e and c o v a r i a n c e for s lope (GR%) t e s t of l og body weights i n Experiment 1 192 3. A n a l y s i s of c o v a r i a n c e of body weight ga in a g a i n s t p r o t e i n i n t a k e (Exper iment 1) 193 4. A n a l y s i s of v a r i a n c e of gross food c o n v e r s i o n and gross energy u t i l i z a t i o n of d i e t s fed i n Experiment 1.193 5. A n a l y s i s of v a r i a n c e of p r o t e i n e f f i c i e n c y r a t i o and net p r o t e i n r a t i o of d i e t s fed i n Experiment 1 194 6. A n a l y s i s of v a r i a n c e of p r o t e i n p r o d u c t i v e va lue of d i e t s fed i n Experiment 1 194 7. A n a l y s i s of v a r i a n c e of net p r o t e i n u t i l i z a t i o n -1 and net p r o t e i n u t i l i z a t i o n -2 of d i e t s fed i n Exp . 1 195 8. Summary of s t a t i s t i c a l a n a l y s i s f o r the s l opes of dry body weight and body p r o t e i n ga in (Exper iment 1 ) . . 1 9 5 9. A n a l y s i s of v a r i a n c e of day 42 body weights i n Experiment 2 196 10. A n a l y s i s of v a r i a n c e of day 105 body weights i n Experiment 2 196 11. A n a l y s i s of c o v a r i a n c e for body weights i n Exp . 2 197 12. S t a t i s t i c a l a n a l y s i s f o r the second order p o l y n o m i a l model to e s t imate p r o t e i n requirements i n Exp . 2 198 13. S t a t i s t i c a l a n a l y s i s f or the second order p o l y n o m i a l model to e s t imate the p r o t e i n e n e r g y : t o t a l energy i n Experiment 2 . .198 14. A n a l y s i s of v a r i a n c e of gross food c o n v e r s i o n and gross energy u t i l i z a t i o n i n Experiment 2 199 15. A n a l y s i s of v a r i a n c e of p r o t e i n e f f i c i e n c y r a t i o and p r o t e i n p r o d u c t i v e va lue i n Experiment 2 199 16. A n a l y s i s of v a r i a n c e of net p r o t e i n u t i l i z a t i o n -2 i n Experiment 2 200 _ xii — L i s t of Appendices T a b l e s ( c o n t ' d ) 17. T a b l e of mean squares f o r f i s h body m o i s t u r e , a s h , l i p i d and p r o t e i n at day 42 (Exper iment 2) 201 18. T a b l e of mean squares for f i s h body m o i s t u r e , a s h , l i p i d and p r o t e i n at day 105 (Experiment 2) 201 xiii _ ACKNOWLEDGEMENTS The a s s i s t a n c e of many people was i n s t r u m e n t a l i n the comple t ion of t h i s s t u d y . In p a r t i c u l ar the author i s very much indebted to D r . David Higgs for h i s p a t i e n c e , encouragement and d e d i c a t e d gu idance . The author i s a l s o g r e a t l y indebted to P r o f e s s o r B e r y l March for her keen i n t e r e s t i n f i s h s t u d i e s and s k i l l e d s u p e r v i s i o n . S p e c i a l thanks are due to D r . Bruce Owen who was i n s t r u m e n t a l i n i n i t i a t i n g t h i s s tudy . The author g r a t e f u l l y acknowledges D r . Edward Donaldson of the F i s h e r i e s Research Branch (Department of F i s h e r i e s and Oceans, Canada) for h i s support i n making the f a c i l i t i e s of the West Vancouver L a b o r a t o r y a v a i l a b l e for s tudent use . Being exposed to the r e s e a r c h a c t i v i t i e s of the F i s h C u l t u r e S e c t i o n has been most r e w a r d i n g . The author would l i k e to thank the s t a f f of the West Vancouver L a b o r a t o r y for t h e i r s i n c e r e a s s i s t a n c e , good humor and f r i e n d s h i p . In p a r t i c u l a r Nancy R i c h a r d s o n , Bakhshish Dosanjh , Dianne P l o t n i k o f f , Jack M a r k e r t , Helen Dye, Andy Lamb and P h i l l E d g e l l a l l p r o v i d e d t e c h n i c a l a s s i s t a n c e . The a s s i s t a n c e p r o v i d e d by Helen Crepeau with the s t a t i s t i c a l ana lyse s i s acknowledged. D r . Bob McKay p r o v i d e d v a l u a b l e a d v i c e on s t a t i s t i c a l i n f e r e n c e . The author would l i k e to thank Debra Wadsworth for p r o c e s s i n g the m a n u s c r i p t . Very s p e c i a l thanks are due to the a u t h o r ' s w i f e , L e s l e y , for assuming the burden of e x t r a work on the f a m i l y farm whi le the author completed t h i s s t u d y . — x i v — CHAPTER 1 1.0 INTRODUCTION The r e l a t i o n s h i p between food and growth i s one of the most important aspects of f i s h p r o d u c t i o n . F u r t h e r m o r e , the a b i l i t y of h a t c h e r y - r a i s e d f i s h to s u r v i v e i n the w i l d may be a f u n c t i o n of t h e i r n u t r i t i o n a l h i s t o r y (Burrows, 1969; P l o t n i k o f f et a l . , 1984). Compared to most other c u l t u r e d animal s p e c i e s , sa lmonids have h igh d i e t a r y p r o t e i n r e q u i r e m e n t s . P r o t e i n i s predominant ly f u r n i s h e d by f i s h meal , and i s the most expens ive component of f i s h d i e t s . S u i t a b l e h igh q u a l i t y p r o t e i n sources d e r i v e d from the B r i t i s h Columbia f i s h e r y are becoming i n c r e a s i n g l y s carce on a year round b a s i s . T o t a l replacement of f i s h m e a l in p r a c t i c a l d i e t s for chinook salmon (Oncorhynchus t shawytscha) does not , however, appear to be a r e a d i l y a t t a i n a b l e goa l (Westgate , 1979; Fowler , 1980, 1981a,b; Higgs et a l . , 1982, 1983). C o n s e q u e n t l y , an important o b j e c t i v e among f i s h n u t r i t i o n i s t s i s to maximize the u t i l i z a t i o n of d i e t a r y p r o t e i n s . T h i s cou ld be ach ieved by o b t a i n i n g a c o r r e c t balance of a l l n u t r i e n t s i n the d i e t . In p a r t i c u l a r , the q u a l i t y of the p r o t e i n source i n terms of meeting the e s s e n t i a l amino a c i d requirements of the f i s h and the r a t i o of p r o t e i n energy to t o t a l energy i n the d i e t would be expected to play a major r o l e (Cowey, 1979). F u r t h e r , there i s a need to develop the c r i t e r i a for the b i o l o g i c a l measurement of the n u t r i t i v e va lue of p r o t e i n in f i s h . 1 The o v e r a l l o b j e c t i v e s of t h i s study on chinook salmon f r y were t w o f o l d . F i r s t , to e v a l u a t e the e f f e c t of d i e t a r y p r o t e i n source and l e v e l on p r o t e i n u t i l i z a t i o n (Experiment 1 ) . Second, to determine the e f f e c t of d i e t a r y energy l e v e l on the requirements and u t i l i z a t i o n of p r o t e i n for growth (Experiment 2 ) . The f o l l o w i n g review of l i t e r a t u r e i s in tended to p r o v i d e the reader wi th a background i n p r o t e i n n u t r i t i o n to f a c i l i t a t e unders tand ing of the e x p e r i m e n t a l goals and procedures employed. 2 CHAPTER 2 2.0 REVIEW OF LITERATURE 2.1 The u t i l i z a t i o n of d i e t a r y p r o t e i n The u t i l i z a t i o n of d i e t a r y p r o t e i n i s mainly dependent upon the a b i l i t y of the p r o t e i n source to f u l f i l l the t o t a l p r o t e i n and i n d i v i d u a l amino a c i d requirements of the a n i m a l . The energy content of the d i e t , source of d i e t a r y energy , the g e n e t i c p r e d i s p o s i t i o n , the e n v i r o n m e n t a l c o n d i t i o n s and the p h y s i o l o g i c a l s t a t e of the animal a l s o p lay a major r o l e . Most f i s h s p e c i e s of concern to f i s h c u l t u r i s t s are c a r n i v o r o u s and have a h igh p r o t e i n requirement (Cowey, 1979). A l l f i s h s p e c i e s s t u d i e d r e q u i r e the same ten e s s e n t i a l d i e t a r y amino a c i d s that are needed by most other a n i m a l s . These have been reviewed for over a dozen f i s h spec i e s ( K e t o l a , 1982). C a r n i v o r o u s f i s h u t i l i z e c a r b o h y d r a t e s p o o r l y ( P h i l l i p s , 1969; Shimeno et a l . , 1979; H i l t o n and A t k i n s o n , 1982). Compared to most animal s p e c i e s r a i s e d for f o o d , sa lmonids metabo l i ze a g r e a t e r p r o p o r t i o n of d i e t a r y p r o t e i n for energy (Walton and Cowey, 1982). However, the energy requ irements of f i s h are lower than for mammals which r e q u i r e a c o n s i d e r a b l e amount of energy to m a i n t a i n body t emperature . F i s h are p o i k i l o t h e r m i c . A l s o , most of the n i t r o g e n excre ted by f i s h i s i n the form of ammonia, whi le b i r d s and mammals r e q u i r e energy to s y n t h e s i z e u r i c a c i d and urea r e s p e c t i v e l y for e x c r e t i o n (Smith et a l . , 1978) . 3 U n l i k e c a r b o h y d r a t e s and l i p i d s , p r o t e i n s , or the amino a c i d s d e r i v e d from them, cannot be s t o r e d as such by the animal i f fed i n excess of r e q u i r e m e n t s . T h i s means that no c o n s i d e r a b l e i n e r t d e p o s i t s of p r o t e i n or amino a c i d s are found i n the animal body comparable to g lycogen granu le s or f a t g l o b u l e s . For t h i s reason the e f f i c i e n c y of d i e t a r y p r o t e i n u t i l i z a t i o n i s dependent upon the q u a n t i t i e s and p r o p o r t i o n s of amino a c i d s absorbed with each meal (Albanese and O r t o , 1959). P r o t e i n sources vary i n t h e i r d i g e s t i b i l i t y and m e t a b o l i c u t i l i z a t i o n by v a r i o u s animal s p e c i e s . The q u a l i t y or e f f i c i e n c y of u t i l i z a t i o n of d i e t a r y p r o t e i n i s s u b j e c t to three g e n e r a l f a c t o r s : i n t a k e , d i g e s t i b i l i t y i n the g a s t r o i n t e s t i n a l t r a c t , and the metabo l i c u t i l i z a t i o n of the d i g e s t i o n p r o d u c t s . A genera l r e p r e s e n t a t i o n of the u t i l i z a t i o n of food n i t r o g e n as i t a p p l i e s to the growing f i s h i s d e p i c t e d in F i g . 1 . Once a b s o r b e d , amino a c i d s are u t i l i z e d through a n a b o l i c and c a t a b o l i c enzymatic processes which g ive r i s e to n i t r o g e n l o s s e s v i a the g i l l s and k i d n e y . Another f r a c t i o n of n i t r o g e n i n t a k e i s r e t a i n e d and used for growth and maintenance . The endogenous n i t r o g e n f r a c t i o n s ( F i g . l ) are of i n t e r e s t p r i m a r i l y because they play a r o l e i n the c a l c u l a t i o n of some e s t i m a t o r s of p r o t e i n q u a l i t y . The f e a t u r e of the endogenous n i t r o g e n l o s s e s i s that a l though they are i n d i s t i n g u i s h a b l e from exogenous n i t r o g e n wastes , they r e p r e s e n t f r a c t i o n s which have a c t u a l l y been u t i l i z e d by the a n i m a l . These and other aspec t s of p r o t e i n 4 u t i l i z a t i o n and e v a l u a t i o n w i l l based on the scheme d e p i c t e d i n be d i s c u s s e d F i g . l . i n t h i s t h e s i s '2 .2 G e n e r a l a spec t s of p r o t e i n metabol ism In f i s h , a s s i m i l a t e d amino a c i d s enter the same pathways and are presumed to undergo the same complex b i o c h e m c i a l r e a c t i o n s as i n other animals (Walton and Cowey, 1982). These are p r o t e i n s y n t h e s i s , deaminat ion f o l l o w e d by o x i d a t i o n and p o s s i b l y c o n v e r s i o n to l i p i d and g l u c o s e , and s y n t h e s i s of v a r i o u s p o l y p e p t i d e s , p u r i n e s and n u c l e i c a c i d s . The r a t e s of p r o t e i n s y n t h e s i s and breakdown are extremely s e n s i t i v e to d i e t a r y p r o t e i n i n t a k e and are under hormonal c o n t r o l . Body p r o t e i n s are i n a c o n t i n u a l s t a t e of t u r n o v e r , be ing broken down and r e s y n t h e s i z e d . Recent s t u d i e s have conf irmed t h a t , not only does each t i s s u e and each p r o t e i n have a d i f f e r e n t ra t e of t u r n o v e r , but a l s o the mechanisms for r e g u l a t i n g the mass of each t i s s u e p r o t e i n may d i f f e r ( G a r l i c k , 1980). For example, changes i n the r a t e s of p r o t e i n s y n t h e s i s are p r i m a r i l y r e s p o n s i b l e for the r e g u l a t i o n of muscle p r o t e i n mass. In the l i v e r , on the other hand, p r o t e i n mass i s thought to be r e g u l a t e d by the r a t e of p r o t e i n breakdown. In a d u l t s , non-growing muscle maintenance r e s u l t s from a balance between the r a t e of s y n t h e s i s and breakdown ( M i l l w a r d et a l . , 1978). In the growing animal p r o t e i n growth r e s u l t s from the f a c t that p r o t e i n s y n t h e s i s exceeds p r o t e i n breakdown. In s t u d i e s concerned with whole body p r o t e i n metabol ism two pools of amino a c i d s are c o n c e p t u a l i z e d ( G a r l i c k , 1980). One 5 FECAL NITROGEN (FN) (Undigested protein) BRANCHIAL ( B N ) - » and URINARY (UN) NITROGEN ( Protein utilized for energy) PROTEIN INTAKE DIGESTION ABSORBED PROTEIN METABOLISM (Anabolic-catabolic processes) PROTEIN RETAINED (NET PROTEIN RATIO) MAINTENANCE ENDOGENOUS PROTEIN LOSS ENDOGENOUS: BRANCHIAL (EBN) and URINARY (EUN) NITROGEN GROWTH PROTEIN PRODUCTIVE VALUE (PPV) METABOLIC FECAL NITROGEN (MFN) INSENSIBLE NITROGEN LOSS (SN) (Mucus, scales, etc.) F i g . 1. S i m p l i f i e d scheme f o r the u t i l i z a t i o n of food p r o t e i n i n j u v e n i l e g rowing f i s h . 6 - r e p r e s e n t s the t o t a l f r e e amino a c i d s of the body known as the "metabol i c p o o l " and the other the whole body p r o t e i n p o o l . These e x i s t i n dynamic e q u i l i b r i u m . The i n p u t s i n t o the m e t a b o l i c pool are from food i n t a k e and body p r o t e i n breakdown. The outputs from the m e t a b o l i c poo l are from p r o t e i n s y n t h e s i s and amino a c i d o x i d a t i o n with subsequent n i t r o g e n e x c r e t i o n . C h a r a c t e r i s t i c n i t r o g e n e x c r e t i o n curves from s t a r v e d f i s h have served to demonstrate the e x i s t e n c e of the above compartments i n t e l e o s t s ( Iwata , 1970; Smith and T h o r p e , 1976). The r a t e of d e p l e t i o n of body p r o t e i n s t o r e s i n f i s h have been shown to be dependent on water temperature ( S a v i t z , 1971) and s a l i n i t y (Smith and T h o r p e , 1976). When p r o t e i n i s p r o v i d e d a f t e r a p e r i o d of s t a r v a t i o n , r e t e n t i o n of the d i e t a r y p r o t e i n w i l l be h igh i n order to r e p l e n i s h the body p r o t e i n s t o r e s . Dur ing r e g e n e r a t i o n , the t i s s u e s that were dep le t ed l a s t are probably r e f i l l e d f i r s t . T h i s may i n d i c a t e that the t i s s u e d e p l e t e d l a s t was more e s s e n t i a l . The dynamic nature of p r o t e i n metabol ism has l e d to the concept of a m e t a b o l i c pool of amino a c i d s . The poo l i s not c o n s i d e r e d to be a d i s o r g a n i z e d f l u i d c o n t a i n e r of the body, but r a t h e r as an i n t e g r a t e d mechanism for the t r a n s f e r of amino a c i d s from one t i s s u e to another ( S h a p i r o and F i s h e r , 1962). The above concepts of the a n a b o l i c / c a t a b o l i c i n t e r r e l a t i o n s h i p s p r o v i d e the background for n i t r o g e n balance s t u d i e s of a n i m a l s . 7 2.3 N i t r o g e n balance The p r i n c i p l e of n i t r o g e n balance i n n u t r i t i o n a l s t u d i e s i s i d e n t i c a l to input versus output i n any other s c i e n c e , s i n c e i t s purpose i s to measure net ga in or l o s s o c c u r r i n g wi th d i e t a r y p r o t e i n u t i l i z a t i o n . N i t r o g e n balance can be d e f i n e d as the q u a n t i t y of n i t r o g e n i n t a k e that has been r e t a i n e d by the body. Many e x p e r i m e n t a l procedures based on n i t r o g e n balance have been dev i sed for the measurement of the n u t r i t i v e va lue of foods and n u t r i e n t requirements of a n i m a l s . The dynamic a n a b o l i c - c a t a b o l i c s t a t e of p r o t e i n metabol ism i s s imply d e s c r i b e d by the n i t r o g e n balance e q u a t i o n ( A l l i s o n , 1951) as f o l l o w s : NB = NI - (UN + FN) where NB = n i t r o g e n b a l a n c e , NI = n i t r o g e n i n t a k e , UN = u r i n a r y n i t r o g e n , and FN = f e c a l n i t r o g e n . I f n i t r o g e n ba lance i s p o s i t i v e , the animal i s g a i n i n g p r o t e i n , e i t h e r through the growth of new t i s s u e s or the r e p l e t i o n of d e p l e t e d s t o r e s . A l l growing animals a r e , under normal c o n d i t i o n s , i n p o s i t i v e n i t r o g e n b a l a n c e . A d u l t a n i m a l s , under normal c o n d i t i o n s , are i n n i t r o g e n e q u i l i b r i u m . M a i n t a i n i n g n i t r o g e n e q u i l i b r i u m does not mean that a l l t i s s u e s are i n t h i s s t a t e because some t i s s u e s may be mainta ined at the expense of o t h e r s (Young and Scrimshaw, 1977). The a forement ioned n i t r o g e n balance formula of A l l i s o n (1951) has not been found to be t o t a l l y c o r r e c t . S t u d i e s with human s u b j e c t s have demonstrated that s i g n i f i c a n t amounts of n i t r o g e n are e l i m i n a t e d i n p e r s p i r a t i o n . A l s o , l o s s e s from s k i n - 8 - and h a i r must - be accounted f o r i n long term n i t r o g e n balance s t u d i e s . These l o s s e s have been termed i n s e n s i b l e n i t r o g e n l o s s e s (SN) ( B r e s s a n i , 1977) . They are accounted for when the f a c t o r i a l procedure i s employed to c a l c u l a t e p r o t e i n requirements f o r maintenance i n farm animals (Maynard and L o o s l i , 1969). S i m i l a r l y , the a p p l i c a t i o n of n i t r o g e n balance to f i s h must c o n s i d e r a l l sources of n i t r o g e n l o s s . B r a n c h i a l e x c r e t i o n (BN) i s the major mechanism for n i t r o g e n l o s s i n f i s h . A l s o f i s h are covered i n mucus which f u n c t i o n s to p r o t e c t (Ingram, 1980) and a i d i n swimming and osmoregu la t ion (Cameron and Endean, 1973). E p i t h e l i a l mucus c o n t a i n s both p r o t e i n and c a r b o y d r a t e (Ingram, 1980). S t r e s s , as a r e s u l t of i n f e c t i o n or h a n d l i n g , i s known to cause an i n c r e a s e d mucus p r o d u c t i o n i n f i s h ( P i c k e r i n g and Macey, 1977). S a v i t z (1969) r e c o g n i z e d that c o n s i d e r a t i o n f o r the unknown amount and r a t e of mucus n i t r o g e n s e c r e t i o n would b i a s e s t imates of p r o t e i n metabo l i sm. I n s e n s i b l e n i t r o g e n l o s s may be of s i g n i f i c a n c e i n a q u a c u l t u r e where f i s h are crowded and f r e q u e n t l y h a n d l e d . T h e r e f o r e , an a p p r o p r i a t e n i t r o g e n ba lance r e l a t i o n s h i p for f i s h would be: NB = NI - (FN + UN + BN + SN) When f i s h are fed a s e r i e s of d i e t s which supply equa l amounts of m e t a b o l i z a b l e energy , t h e i r n i t r o g e n ba lances can be expected to form a curve of the type shown i n F i g . 2 (McDonald et a l . , 1976). As i n t a k e i n c r e a s e s from zero there i s a gradual r e d u c t i o n in the n e g a t i v e balance u n t i l the po in t of exact e q u i l i b r i u m i s r e a c h e d . The extent to which f u r t h e r n i t r o g e n - 9 Fig. (2). Stylized representation of nitrogen balance. Taken from McDonald et al.(1976). - 10 - F i g u r e 2 i n t a k e promotes t i s s u e growth depends on the growth p o t e n t i a l of the a n i m a l , the q u a l i t y of the p r o t e i n and the supply of other n u t r i e n t s . The curve becomes h o r i z o n t a l when f u r t h e r n i t r o g e n i n t a k e f a i l s to promote a d d i t i o n a l n i t r o g e n r e t e n t i o n . The i n f o r m a t i o n d e r i v e d from n i t r o g e n balance s t u d i e s has been used s i n c e 1909 (Thomas, c i t e d by M i t c h e l l , 1962) i n the c a l c u l a t i o n of the B i o l o g i c a l Value of p r o t e i n s . B i o l o g i c a l Value (BV) i s de f i ne d as the percentage of absorbed n i t r o g e n which i s r e t a i n e d by the organism ( M i t c h e l l , 1924): BV = Reta ined food N x ioo Absorbed food-N where: Absorbed food N = Food N - f e c a l food N F e c a l food N = F e c a l N - M e t a b o l i c N of feces Reta ined food N = Absorbed N - E x c r e t e d food N E x c r e t e d food N = U r i n e N - Endogenous u r i n a r y N With r e s p e c t to f i s h n u t r i t i o n r e s e a r c h , the formula for b i o l o g i c a l va lue r e p o r t e d by C a s t e l l and Tiews (1979) was: BV = NI - (FN - MFN) - (UN - EUN) - (BN - EBN) x 100 NI - (FN - MFN) where: MFN = M e t a b o l i c F e c a l N i t r o g e n EUN = Endogenous U r i n a r y N i t r o g e n EBN = Endogenous B r a n c h i a l N i t r o g e n MFN, EUN, and EBN are n i t r o g e n f r a c t i o n s whose e x c r e t i o n i s independent of food n i t r o g e n ( F i g . l ) . T h e i r e x i s t e n c e i s demonstrated by the n i t r o g e n e x c r e t e d when an animal i s g iven a p r o t e i n - f r e e d i e t . They r e p r e s e n t f r a c t i o n s which have a c t u a l l y 11 been u t i l i z e d - by the animal even though they appear as e x c r e t i o n s . The numerator i n the above e q u a t i o n r e p r e s e n t s t o t a l n i t r o g e n u t i l i z e d for both maintenance and t i s s u e growth. I t i s noteworthy that C a s t e l l and Tiews (1979) have not accounted for mucus n i t r o g e n l o s s . 2 .4 Endogenous n i t r o g e n e x c r e t i o n Endogenous n i t r o g e n e x c r e t i o n o r i g i n a t i n g from amino a c i d c a t a b o l i s m r e p r e s e n t s the maintenance f r a c t i o n of n i t r o g e n metabol ism and i s independent of d i e t a r y p r o t e i n i n t a k e . In mammals the p r i n c i p a l c o n s t i t u e n t of endogenous n i t r o g e n e x c r e t i o n i s u r e a , which a r i s e s from the breakdown of p r o t e i n and other n i t r o g e n o u s compounds which are c a t a b o l i z e d to y i e l d ammonia. The major end product of n i t r o g e n metabol ism i n t e l e o s t f i s h i s ammonia, though u r e a , u r i c a c i d , t r i m e t h y l a m i n e o x i d e , amino a c i d s , c r e a t i n i n e and c r e a t i n e are a l s o e x c r e t e d (Watts and Watts , 1974). The o r i g i n of urea and u r i c a c i d i s from p u r i n e metabol ism and c r e a t i n i n e i s formed from the breakdown of c r e a t i n e i n muscle a c t i o n ( F o r s t e r and G o l d s t e i n , 1969) . Endogenous u r i n a r y n i t r o g e n e x c r e t i o n v a r i e s i n homeothermous animals and i s r e l a t e d to body weight i n a manner s i m i l a r to c a l o r i c metabol ism (Brody , 1945). B a s a l metabol ism and endogenous n i t r o g e n e x c r e t i o n are r e l a t e d to one another and both are a f u n c t i o n of m e t a b o l i c body s i z e . In mammals the m e t a b o l i c r a t e i s p r o p o r t i o n a l to the t h r e e - f o u r t h s power of 0.75 body weight (W ) (Maynard and L o o s l i , 1 9 6 9 , ) . 12 G e r k i n g (1955) r e p o r t e d that endogenous n i t r o g e n e x c r e t i o n i n the b l u e g i l l s u n f i s h (Lepomis m a c r o c h i r u s ) decreased with 0.54 i n c r e a s i n g s i z e by W . S ince then , the exponent a p p l i c a b l e to f i s h has been r e p o r t e d to vary from 0.34 to 1.0 ( B r e t t and Groves , 1979). Smith et a l . ( 1 9 7 8 ) employed a method of d i r e c t c a l o r i m e t r y to study the e f f e c t of f i s h s i z e on m e t a b o l i c r a t e i n four s p e c i e s of s a l m o n i d s . T h e i r r e s u l t s i n d i c a t e d that 1.0 to 4.0g f i s h have a m e t a b o l i c r a t e p r o p o r t i o n a l to W . F i s h from 4.0g to 50.Og i n weight have a m e t a b o l i c r a t e p r o p o r t i o n a l 0.63 to W McDonald et a l . ( 1 9 7 6 ) s t a t e a g e n e r a l l y accepted va lue of 2 mg of endogenous u r i n a r y n i t r o g e n e x c r e t e d per k c a l at b a s a l metabol ism for warm blooded a n i m a l s . G e r k i n g (1955) obta ined a va lue of 7 mg of endogenous n i t r o g e n e x c r e t e d per k c a l i n the b l u e g i l l s u n f i s h . T h i s agrees wi th a va lue of 7.2 mg/kca l ob ta ined by Bonnet (1933) wi th f r o g s . The f o r e g o i n g r e s u l t s suggest that f i s h and amphibians e x c r e t e three and a h a l f t imes more endogenous n i t r o g e n per b a s a l k c a l than do warm blooded a n i m a l s . T h i s e s t a b l i s h e d that a h igh r a t e of n i t r o g e n e x c r e t i o n i s c h a r a c t e r i s t i c of p o i k i l o t h e r m o u s a n i m a l s . Hence, a l a r g e p o r t i o n of the energy requirements of f i s h are d e r i v e d from p r o t e i n , whereas homeotherms can use l a r g e p r o p o r t i o n s of c a r b o h y d r a t e and f a t f or t h i s purpose . The d a i l y r a t e s of endogenous n i t r o g e n e x c r e t i o n have been shown to i n c r e a s e wi th r i s i n g e n v i r o n m e n t a l temperature i n b l u e g i l l s u n f i s h ( S a v i t z , 1971) and carp (Ogino et a l . , 1973). At low temperatures n i t r o g e n e x c r e t i o n r a t e s were found not to 13 f l u c t u a t e , s u g g e s t i n g that s u n f i s h have the a b i l i t y for c a p a c i t y a d a p t a t i o n i n c o l d c l i m a t e s ( S a v i t z , 1971). Smith and Thorpe (1971) employed n i t r o g e n ba lance to study the a s s o c i a t i o n of p r o t e i n metabol ism with the s m o l t i f i c a t i o n process i n t r o u t . Endogenous n i t r o g e n e x c r e t i o n was found to i n c r e a s e wi th the onset of s m o l t i f i c a t i o n and c o n t i n u e i n sea water . By c o n t r a s t , post smolts decreased t h e i r r a t e of n i t r o g e n e x c r e t i o n when r e t a i n e d i n f r e s h water . These r e s u l t s i n d i c a t e that the p r o t e i n requirements for maintenance of t r o u t may be g r e a t e r i n sea water than i n f r e s h water ( Z e i t o u n , 1973). Another component of endogenous n i t r o g e n l o s s i s m e t a b o l i c f e c a l n i t r o g e n (MFN) or that p r o p o r t i o n of f e c a l n i t r o g e n that does not o r i g i n a t e from undiges ted food p r o t e i n ( F i g . l ) . I t a r i s e s from enzyme and c e l l r e s i d u e s s loughed o f f the d i g e s t i v e t r a c t . MFN output i n c r e a s e s wi th bulk food i n t a k e because the h igher the i n t a k e , the g r e a t e r the s e c r e t i o n of d i g e s t i v e j u i c e s and a b r a s i o n of the i n t e s t i n a l e p i t h e l i a l c e l l s . M i t c h e l l and Bert (1954) determined MFN with a n i t r o g e n - f r e e r a t i o n or with r a t i o n s c o n t a i n i n g low l e v e l s of almost t o t a l l y d i g e s t i b l e p r o t e i n . They e s t imated that MFN i s a p p r o x i m a t e l y O . l g / l O O g dry matter consumed for r a t s , p igs and man. T i t u s (1927) p l o t t e d t o t a l n i t r o g e n i n t a k e a g a i n s t t o t a l n i t r o g e n e x c r e t i o n of s t e e r s fed at a f i x e d r a t i o n l e v e l wi th d i e t s of v a r y i n g p r o t e i n c o n t e n t . He a r r i v e d at the e s t imated MFN e x c r e t e d at any l e v e l of food i n t a k e by s t r a i g h t l i n e e x t r a p o l a t i o n to the p o i n t of zero p r o t e i n i n t a k e . M i t c h e l l and 14 - Bert (1954) o b t a i n e d good agreement with the T i t u s (1927) method by d i r e c t d e t e r m i n a t i o n for s e v e r a l s p e c i e s . Ogino et a l . ( 1 9 7 3 ) f o l l o w e d a s i m i l a r approach to the above to determine MFN e x c r e t i o n i n c a r p . From the r e l a t i o n s h i p between p r o t e i n content of the d i e t and the amount of n i t r o g e n e x c r e t e d i n the f e c e s , they obta ined MFN e x c r e t i o n va lues at d i f f e r e n t water t e m p e r a t u r e s . These va lues were w i t h i n the range of va lues obta ined when a n o n - p r o t e i n d i e t was employed. Ogino et a l . ( 1 9 7 3 ) showed that MFN e x c r e t i o n i n carp i n c r e a s e s as water temperature i n c r e a s e s . Skrede et a l . ( 1 9 8 0 ) o b t a i n e d a va lue for MFN i n rainbow t r o u t of 180 mg/lOOg of d i e t a r y dry matter when a p r o t e i n - f r e e d i e t was f e d . 2.5 Methods used to determine p r o t e i n q u a l i t y The purpose of measuring p r o t e i n q u a l i t y i s to e v a l u a t e the e f f i c a c y of d i f f e r e n t p r o t e i n sources to support r a p i d growth. Then the economic r e t u r n of a p r o t e i n can be e s t i m a t e d . The q u a l i t y of a p r o t e i n r e f e r s to how w e l l the a v a i l a b l e c o n c e n t r a t i o n and balance of i t s c o n s t i t u e n t e s s e n t i a l amino a c i d s correspond to the needs of a g iven s p e c i e s . As mentioned e a r l i e r , there are major q u a n t i t a t i v e d i f f e r e n c e s i n the amino a c i d requirements of f i s h s p e c i e s . The d e t e r m i n a t i o n of amino a c i d s i n a f e e d s t u f f i s an expens ive procedure which i s o f t en s u b j e c t to c o n s i d e r a b l e i n t e r - l a b o r a t o r y v a r i a t i o n . A l t e r n a t i v e l y , the r e l i a n c e on t a b u l a t e d amino a c i d c o m p o s i t i o n t a b l e s (NRC, 1973,1981) o f ten l eads to erroneous e s t imates (Payne, 1972) . F u r t h e r m o r e , amino a c i d c o m p o s i t i o n data g ive no 15 i n d i c a t i o n of amino a c i d a v a i l a b i l i t y and hence u s e f u l n e s s to the animal when i n c l u d e d i n the f i n a l d i e t . Methods commonly used to assess p r o t e i n q u a l i t y may be d i v i d e d i n t o c h e m i c a l methods, m i c r o b i o l o g i c a l assay systems, animal f e e d i n g experiments and procedures based on m e t a b o l i c i n d i c e s of p r o t e i n metabo l i sm. These methods have been c r i t i c a l l y reviewed by Bodwel l ( 1977), S a t t e r l e e et a l . (1977) , Evans and W i t t y (1978) and Von der Decken (1983) . S t u d i e s wi th f i s h have u s u a l l y employed growth t r i a l s coupled with c a r c a s s a n a l y s i s to e v a l u a t e the p r o t e i n q u a l i t y of f e e d s t u f f s . The use of m e t a b o l i c i n d i c e s was attempted by Cowey et a l . ( 1 9 8 1 ) who found no c o n s i s t e n t r e l a t i o n s h i p between the presumed e q u i l i b r i u m p o i n t of opposing h e p a t i c enzyme a c t i v i t i e s and maximum weight ga in by t r o u t fed d i e t s c o n t a i n i n g f i s h m e a l , c a s e i n or corn g l u t e n . Nose (1972) and Kaushik and Luquet (1979) demonstrated a correspondence between the p r o p o r t i o n s of e s s e n t i a l amino a c i d s i n the d i e t and the plasma f r e e amino a c i d p a t t e r n . However, these t echn iques r e q u i r e f u r t h e r re f inement before they can be employed s u c c e s s f u l l y . A common es t imate of p r o t e i n q u a l i t y i s p r o t e i n e f f i c i e n c y r a t i o ( P E R ) . T h i s i s weight ga in of an animal per gram of p r o t e i n i n t a k e . Osborne et a l . ( 1 9 1 9 ) c a r r i e d out PER d e t e r m i n a t i o n s at s e v e r a l p r o t e i n l e v e l s and accepted the h i g h e s t va lue as the PER for that p r o t e i n . PER e s t imates do not a l l ow for maintenance needs and assume that a l l food i s used for growth. Net p r o t e i n r a t i o (NPR) (Bender and D o e l l , 1957) i s s i m i l a r i n concept to PER except f o r the i n c l u s i o n of a group 16 fed a d i e t devo id of p r o t e i n . NPR i s d e f i n e d by the f o l l o w i n g e q u a t i o n : ga in i n weight weight l o s s of group NPR = of t e s t group + fed a n o n - p r o t e i n d i e t p r o t e i n i n t a k e I n v e s t i g a t o r s wi th f i s h r o u t i n e l y r e p o r t PER (Cowey and S a r g e n t , 1972; S t e f f e n s , 1981; P f e f f e r , 1982), but not NPR because of the problems a s s o c i a t e d wi th m a i n t a i n i n g f i s h on a p r o t e i n f ree d i e t ( Z e i t o u n et a l . , 1973). U s u a l l y PER i s e s t imated only at one d i e t a r y p r o t e i n l e v e l u s u a l l y at the recommended d i e t a r y p r o t e i n l e v e l f or maximum growth of a g iven s p e c i e s at a p a r t i c u l a r stage of l i f e h i s t o r y . The use of weight ga in as a c r i t e r i o n for e s t a b i l i s h i n g p r o t e i n q u a l i t y has been c r i t i c i z e d (Maynard and L o o s l i , 1969) because i t may not r e f l e c t t r u e growth, s i n c e ga in i n weight may r e s u l t from d e p o s i t i o n of f a t r a t h e r than e l a b o r a t i o n of new t i s s u e . The a d d i t i o n of p r o t e i n sources to animal d i e t s a l t e r s the o v e r a l l m i n e r a l , v i t a m i n and f a t t y a c i d p r o f i l e s of these d i e t s , and these e f f e c t s cannot be d i s c o u n t e d (Evans and W i t t y , 1978). D i e t a c c e p t a b i l i t y and the presence of a n t i - n u t r i t i o n a l f a c t o r s i n p r o t e i n s o u r c e s , p a r t i c u l a r l y those of p l a n t o r i g i n , w i l l a l s o a l t e r growth r a t e (Higgs et a l . , 1979). F i s h f e e d i n g t r i a l s may be more b e n e f i c i a l to the assessment of p r o t e i n q u a l i t y when an attempt i s made to measure the f a t e of i n g e s t e d p r o t e i n . Techniques u t i l i z i n g n i t r o g e n balance d e p i c t the causes for v a r i a t i o n i n p r o t e i n s o u r c e s , f or example impa ired d i g e s t i o n and a b s o r p t i o n or a l t e r e d r e t e n t i o n (Evans and W i t t y , 1978). 17 Some i n v e s t i g a t o r s have m o d i f i e d the s i m p l e r M i t c h e l l formula for BV f o r f i s h s t u d i e s and c a l l e d i t the gross e f f i c i e n c y of n i t r o g e n u t i l i z a t i o n ( B i r k e t t , 1969; Iwata, 1970;Bret t and Z a l a , 1975; Smith and Thorpe , 1976). Endogenous n i t r o g e n l o s s has been determined from the n i t r o g e n e x c r e t i o n ra te of s t a r v e d f i s h . Iwata (1970) and B r e t t and Groves (1979) c i t e S t o r e r (1967) as ev idence that the r a t e of n i t r o g e n e x c r e t i o n of s t a r v e d f i s h p r o v i d e s a measure of endogenous n i t r o g e n e x c r e t i o n . Stover (1967) r e p o r t e d that the r a t e of p r o t e i n c a t a b o l i s m d i d not i n c r e a s e with the onset of s t a r v a t i o n . However, S a v i t z (1969,1971) found that the r a t e of n i t r o g e n e x c r e t i o n i n s t a r v e d b l u e g i l l s u n f i s h was h igher than i n c o n t r o l s fed g lucose a l o n e . Walton and Cowey (1982) present c o n s i d e r a b l e ev idence that s t a r v a t i o n i n f i s h , as i t does i n mammals, l eads to i n c r e a s e s i n the l e v e l s of amino a c i d degrading enzymes. Under normal c o n d i t i o n s a major p o r t i o n of the c a l o r i c requirement of f i s h e s i s d e r i v e d from p r o t e i n ( G e r k i n g 1955). S a v i t z (1971) showed by a n a l y s i s of body c o m p o s i t i o n , that t h i s s i t u a t i o n changes i n s t a r v a t i o n when f a t i s r e l i e d upon h e a v i l y to s a t i s f y energy r e q u i r e m e n t s . N u t r i e n t balance s t u d i e s are used e x t e n s i v e l y i n e v a l u a t i n g f e e d s t u f f s for domest ic animals (Maynard and L o o s l i , 1969). Q u a n t i t a t i v e c o l l e c t i o n of feces and e x c r e t o r y product s are f r a u g h t wi th problems i n f i s h s t u d i e s because they must be s epara ted from l a r g e volumes of water . I n d i r e c t measurement us ing an i n d i g e s t i b l e marker such as chromic oxide has been used e x t e n s i v e l y to determine the d i g e s t i b i l i t y of f e e d s t u f f s for 18 f i s h (Nose, 1960; Smith and L o v e l l , 1973; Cho and S l i n g e r , 1979; W i n d e l l et a l . , 1978; Wi l son et a l . , 1981; P f e f f e r , 1982). By d e t e r m i n i n g the r a t i o of the c o n c e n t r a t i o n of the marker to that of a n u t r i e n t i n the food and the same r a t i o i n the feces r e s u l t i n g from the f o o d , the d i g e s t i b i l i t y of the n u t r i e n t can be es t imated wi thout having to measure food i n t a k e or feces o u t p u t . U r i n a r y and g i l l e x c r e t i o n s are not c o l l e c t e d and only d i g e s t i b i l i t y can be c a l c u l a t e d . D i f f i c u l t i e s i n o b t a i n i n g a r e p r e s e n t a t i v e sample of feces and i n a v o i d i n g n u t r i e n t l e a c h i n g ( W i n d e l l et a l . , 1978; Choubert et a l . , 1979) are the major problems of t h i s method. T o t a l c o l l e c t i o n of feces and n i t r o g e n o u s e x c r e t i o n s f a c i l i t a t e s the d e t e r m i n a t i o n of d i g e s t i b i l i t y , n i t r o g e n ba lance and m e t a b o l i z a b l e energy . These methods vary from s imply scooping or s i p h o n i n g feces from the f i s h tank and a n a l y s i n g the water for n i t r o g e n ( G e r k i n g , 1955; B i r k e t t , 1969; Iwata, 1970; Rychly and Spanhof f , 1979), to the development of automated metabol ism chambers for f i s h (Cho et a l . , 1975). With these methods the assumption i s made that a l l i n s o l u b l e matter i s f e c a l i n o r i g i n and that s o l u b l e m a t e r i a l i s e x c r e t e d . Ogino (1973) developed an apparatus that f i l t e r s e f f l u e n t tank water to p r e c i p i t a t e , s eparate and preserve feces wi th c u p r i c hydrox ide and c h l o r o f o r m . N i t r o g e n e x c r e t i o n s are r e c o v e r e d from the e f f l u e n t water with an i o n exchange column. Smith (1971,1976) employed an apparatus that p e r m i t t e d the separate c o l l e c t i o n of f e c e s , u r i n a r y and g i l l e x c r e t i o n s . However, the f i s h were r e s t r a i n e d i n such a way that the d e t e r m i n a t i o n of the 19 n u t r i t i v e va lue of f e e d s t u f f s was made when the f i s h were under c o n s i d e r a b l e s t r e s s and i n a s t a t e of n e g a t i v e n i t r o g e n b a l a n c e . Data on the d i g e s t i b i l i t y of m e t a b o l i z a b l e energy contents of f o o d s t u f f s determined by these methods are t a b u l a t e d i n t a b l e s of n u t r i e n t requirements for f i s h e s (NRC,1981) . The u t i l i z a t i o n of a p r o t e i n by an animal w i l l depend on i t s d i g e s t i b i l i t y as w e l l as i t s b i o l o g i c a l v a l u e . Net p r o t e i n u t i l i z a t i o n (NPU) i s the product of these two va lues : NPU = BV x (% d i g e s t i b i l i t y ) M i l l e r and Bender (1953) dev i s ed NPU to assess the e f f i c i e n c y of n i t r o g e n u t i l i z a t i o n of a t e s t p r o t e i n . T h e i r method i s l e s s cumbersome f o r the d e t e r m i n a t i o n of NPU i n s m a l l animals than i s the procedure i n v o l v i n g the c o l l e c t i o n of e x c r e t o r y p r o d u c t s . I t i s based on a comparison of the body n i t r o g e n content r e s u l t i n g from a t e s t p r o t e i n source wi th that r e s u l t i n g over the same p e r i o d of time from a n i t r o g e n - f r e e d i e t : body N of _ body N of group fed NPU = t e s t group a n o n - p r o t e i n d i e t x 100 N consumed by t e s t group Ogino et a l (1980) d e s c r i b e d a method that i s p a r t i c u l a r l y s u i t e d to f i s h s t u d i e s . The endogenous l o s s of n i t r o g e n was determined by c a r c a s s a n a l y s i s a f t e r f e e d i n g a p r o t e i n - f r e e d i e t d u r i n g the e x p e r i m e n t a l p e r i o d . They found that the va lue for the endogenous l o s s of n i t r o g e n i n rainbow t r o u t was 9.5 mg/lOOg body we ight /day and changed i n d i r e c t p r o p o r t i o n to body weight . 20 A c c o r d i n g l y , NPU for rainbow t r o u t was c a l c u l a t e d as f o l l o w s : Body N ga in of W W -3 t e s t group (g) + 1 + 2 (g) x 9.5 x 10 xd NPU = 2 100 x 100 N i t r o g e n i n t a k e of t e s t group(g) Where: = i n i t i a l b o d y w e i g h t ^2 = f i n a l body weight d = days of f e e d i n g By t h i s method the p r i n c i p l e s of n i t r o g e n balance can be r e a d i l y adapted to s t u d i e s wi th f i s h by c a r c a s s a n a l y s i s . The d i f f i c u l t task of r e c o v e r i n g n i t r o g e n o u s wastes from the water can be e n t i r e l y a v o i d e d . F u r t h e r m o r e , the d e t e r m i n a t i o n of i n i t i a l and f i n a l body n i t r o g e n would be more a c c u r a t e because i t would account for a l l n i t r o g e n l o s s e s i n c l u d i n g the d i f f i c u l t task of measuring i n s e n s i b l e l o s s e s . S e v e r a l workers i n f i s h n u t r i t i o n have e s t imated the e f f i c i e n c y of p r o t e i n u t i l i z a t i o n by the r a t i o of body p r o t e i n gained to p r o t e i n fed ( G e r k i n g , 1971; Z e i t o u n et a l . , 1973; Rumsey and K e t o l a , 1975; De l a Higuera et a l . , 1977; Higgs et a l . , 1979; P f e f f e r , 1982). T h i s has been termed apparent net p r o t e i n u t i l i z a t i o n or p r o t e i n p r o d u c t i v e va lue (PPV): (%)PPV = c a r c a s s p r o t e i n gained x 100 p r o t e i n fed T h i s method has the advantage of being s imple and s u f f i c i e n t l y r e p r o d u c i b l e to p r o v i d e r e l a t i v e r a t i n g s to v a r i o u s p r o t e i n s when conducted under s t a n d a r d i z e d e x p e r i m e n t a l c o n d i t i o n s (Cowey and S a r g e n t , 1979). However, PPV does not make a l lowances for maintenance requirements and assumes that 21 a l l food p r o t e i n i s used f o r growth. As both maintenance and growth demand p r o t e i n , maintenance r e c e i v e s f i r s t p r i o r i t y . A l l i s o n (1959) proposed that the s lope of the l i n e measuring the r a t e of change of n i t r o g e n balance wi th p r o t e i n i n t a k e be used to compare p r o t e i n s . Hegsted and Chang (1965a) d e s c r i b e d an assay with r a t s u s i n g the s lope of the r e g r e s s i o n of weight ga in on n i t r o g e n i n t a k e as a percentage of the s lope obta ined with r a t s fed l a c t a l b u m i n . T h i s method was a l s o undertaken with n i t r o g e n ga in r e p l a c i n g weight ga in (Hegsted and Chang 1965b). These methods are s l o p e - r a t i o assays and have been c r i t i c a l l y reviewed by McLaughlan (1979) wi th r e s p e c t to l i n e a r i t y and o r i g i n of the i n t e r c e p t s of the dose -response c u r v e s . S l o p e - r a t i o assay p r o t o c o l s have been proposed both wi th and wi thout use of a zero p r o t e i n fed group (Hegsted and Chang, 1965a; Samonds and Hegs ted , 1977; McLaughlan and K e i t h , 1977). 2.6 The p r o t e i n requirements of sa lmonids wi th r e s p e c t to d i e t a r y energy . The gross p r o t e i n requirement has been the s u b j e c t of many s t u d i e s i n f i s h n u t r i t i o n . As mentioned e a r l i e r , the va lue of a p r o t e i n i s c h i e f l y determined by i t s a b i l i t y to s a t i s f y the amino a c i d requirements of the an imal under c o n s i d e r a t i o n . Perhaps of equal importance i n the des ign of economic f eed ing programs are the d i e t a r y c o n c e n t r a t i o n s of p r o t e i n and energy . About 70% of the energy i n n a t u r a l foods of salmon i s p r o v i d e d i n the form of p r o t e i n and most of the remainder i s s u p p l i e d by l i p i d (Gulbrandsen and Utne , 1977). Most commercia l salmon 22 d i e t s , however, c o n t a i n f a r l e s s of the t o t a l energy as p r o t e i n and l i p i d . There i s c o n s i d e r a b l e r e l i a n c e on c a r b o h y d r a t e as an energy s o u r c e . The o b j e c t i v e of p r o t e i n requirement s t u d i e s i s to f i n d the minimum amount of p r o t e i n r e q u i r e d to produce maximal growth. Delong et a l . ( 1 9 5 8 ) conducted one of the f i r s t of these s t u d i e s w i th chinook salmon. The f i s h were fed a d i e t i n which the p r o t e i n was s u p p l i e d by a mixture of c a s e i n , g e l a t i n , and c r y s t a l l i n e amino a c i d s wi th an o v e r a l l e s s e n t i a l amino a c i d c o m p o s i t i o n s i m u l a t i n g that of whole c h i c k e n egg p r o t e i n . T h i s c o m p o s i t i o n was thought to c o n t a i n an excess of i n d i s p e n s a b l e amino a c i d s . A s e r i e s of d i e t s c o n t a i n i n g d i f f e r e n t l e v e l s of p r o t e i n was formula ted by s u b s t i t u t i n g d i g e s t i b l e c a r b o h y d r a t e ( d e x t r i n ) for p r o t e i n i n an attempt to m a i n t a i n the d i e t s i s o c a l o r i c on a gross energy b a s i s . A f t e r a ten-week f e e d i n g p e r i o d Delong et a l . ( 1 9 5 8 ) found that weight gains of ch inook salmon were h i g h e s t when the d i e t c o n t a i n e d p r o t e i n at l e v e l s of 40 and 55% when water temperature was 8.3 and 1 4 . 5 ° C r e s p e c t i v e l y . Employing s i m i l a r d i e t s , Z e i t o u n et a l . ( 1 9 7 4 ) found that the minimum p r o t e i n requirement of coho salmon mainta ined at e i t h e r 10 or 20 ppt s a l i n i t y was a p p r o x i m a t e l y 40%. By c o n t r a s t , the p r o t e i n requirement of j u v e n i l e rainbow t r o u t was found to be d i r e c t l y r e l a t e d to water s a l i n i t y and i n c r e a s e d from 40% to 45% as s a l i n i t y was r a i s e d from 10 to 20 ppt ( Z e i t o u n et a l . , 1973). S a t i a (1974) conducted a study to determine the p r o t e i n requ irements of a p a r t i c u l a r g e n e t i c s t r a i n of rainbow t r o u t . 23 T h i s s t r a i n i s noted f o r i t s f a s t growth r a t e (Donaldson and O l s o n , 1957). These f i s h were r e a r e d at h igh temperatures of 16 to 2 7 ° C and they were fed d i e t s rang ing i n p r o t e i n content from 30% to 50%. The d i e t s were formula ted to be i s o c a l o r i c by a d j u s t i n g d e x t r i n i n r e l a t i o n to p r o t e i n from f i s h m e a l on a m e t a b o l i z a b l e energy b a s i s ( P h i l l i p s , 1969). The f i s h were fed at a r a t e of A.5% of body weight per day. Based on food *• c o n v e r s i o n d a t a , S a t i a (197A) showed that the p r o t e i n requirements of the t r o u t dropped from 50% to A0% as the f i s h grew over 20g i n we ight . The major u n c e r t a i n t y of a l l of the f o r e g o i n g s t u d i e s r e l a t e s to the c a l o r i c va lues a s s igned by i n d i v i d u a l i n v e s t i g a t o r s to the f e e d s t u f f s . The m e t a b o l i z a b l e energy va lues of p r o t e i n , l i p i d and c a r b o h y d r a t e are u s u a l l y e s t imated from the gross energy content of each component which i s then c o r r e c t e d for d i g e s t i b i l i t y and i n the case of p r o t e i n , f o r energy l o s t i n n i t r o g e n o u s e x c r e t o r y product s ( P h i l l i p s , 1969; B r e t t and G r o v e s , 1979). U n f o r t u n a t e l y , there i s a l a c k of s t a n d a r d i z a t i o n i n these procedures and the va lues employed d i f f e r between i n v e s t i g a t o r s . For example, Smith (1971) has shown that A . 5 k c a l / g i s a more a p p r o p r i a t e m e t a b o l i z a b l e energy va lue f o r p r o t e i n than the more f r e q u e n t l y used va lue of 3.9 k c a l / g of P h i l l i p s (1969) . T h i s i s because f i s h p r i m a r i l y e x c r e t e ammonia. Thus , the n o n - m e t a b o l i z a b l e energy f r a c t i o n of i n g e s t e d p r o t e i n should be accounted f o r by the heat of combustion va lue f o r ammonia r a t h e r than that of u r e a . A l s o , wi th r e s p e c t to c a r b o h y d r a t e the p i c t u r e i s c o m p l i c a t e d f u r t h e r - 2A - because for example, i n c r e a s e d amounts of s t a r c h or d e x t r i n i n the d i e t r e s u l t i n lowered d i g e s t i b i l i t y (S ingh and Nose, 1967). Hence, the m e t a b o l i z a b l e energy va lues of the v a r i o u s d i e t a r y components can be expected to vary wi th the l e v e l of d i e t a r y c a r b o h y d r a t e . J o b l i n g (1983) po in ted out that the e s t imated m e t a b o l i z a b l e energy content of a d i e t w i l l depend not only upon d i e t c o m p o s i t i o n , but a l s o upon r a t i o n l e v e l . The minimum d i e t a r y p r o t e i n l e v e l f or optimum weight ga in or feed e f f i c i e n c y has g e n e r a l l y been s t a t e d as a percentage of the dry d i e t ( D e l o n g , 1958; L u q u e t , 1971; S a t i a , 1974; Cowey, 1975; Z e i t o u n et a l . , 1976). P h i l l i p s (1969) , R ingrose (1971) , Lee and Putman (1973) , Takeda et a l . ( 1 9 7 5 ) and Cowey et a l . ( 1 9 7 5 ) have r e p o r t e d that the n o n - p r o t e i n energy components of the d i e t can promote the u t i l i z a t i o n of d i e t a r y p r o t e i n and have a p r o t e i n s p a r i n g e f f e c t . P r o t e i n ac t s both as a source of energy and of amino a c i d s f o r t i s s u e s y n t h e s i s . F i s h have been shown to eat to s a t i s f y t h e i r energy requirements (Lee and Putman, 1973; B r e t t and Groves , 1979). R e c e n t l y , Cowey and Sargent (1979) have s t a t e d that s i n c e the u l t i m a t e o b j e c t i v e of p r o t e i n requirement s t u d i e s i s to e s t imate the d i e t a r y p r o t e i n l e v e l i n r e l a t i o n to that of energy , the p r o t e i n content of the d i e t can best be expressed i n terms of the p r o p o r t i o n of energy that i t c o n t r i b u t e s . P r e v i o u s work with chinook salmon has i n d i c a t e d that the best r a t i o of p r o t e i n energy to t o t a l energy ( P E : T E ) i n the d i e t i s 0.50 (Combs et a l . , 1962; Fowler et a l . , 1964). Gulbrandsen and Utne (1977) r e p o r t e d that d i e t a r y P E : T E r a t i o s 25 v a r y i n g between 0.37 and 0.41 were o p t i m a l for maximal growth of rainbow t r o u t . They f u r t h e r noted that p r a c t i c a l Norwegian dry feeds for t r o u t had e x c e s s i v e l y h igh P E : T E r a t i o s . The l i p i d content of the d i e t employed by Gulbrandsen and Utne (1977) was a p p r o x i m a t e l y 20%. The l i p i d component, s u p p l i e d by c a p e l i n o i l , accounted for 40% to 42% of the t o t a l d i e t a r y energy c o n t e n t . Gulbrandsen and Utne (1977) found that l i p i d l e v e l s h igher than t h i s caused a d e p r e s s i o n i n growth. U n l i k e most other s t u d i e s on f i s h , the l e v e l of energy f u r n i s h e d by d i g e s t i b l e c a r b o h y d r a t e ( d e x t r i n ) was kept at 20% of the t o t a l energy i n the d i e t and consequent ly the energy s u p p l i e d by i p r o t e i n was ba lanced by v a r y i n g d i e t a r y l e v e l s of f i s h o i l . Whether p r o t e i n energy i s r e p l a c e d by energy from l i p i d or from c a r b o h y d r a t e cou ld be of great s i g n i f i c a n c e to the outcome of a p r o t e i n requirement exper iment . Recent ev idence (Shimeno et a l , 1979; H i l t o n and A t k i n s o n , 1982) support s the statement of P h i l l i p s (1969) that c a r n i v o r o u s f i s h are not ab le to t o l e r a t e d i g e s t i b l e c a r b o h y d r a t e l e v e l s above 14% of the d i e t . D e s p i t e the r e p o r t s i n d i c a t i n g that c a r n i v o r o u s f i s h are i n f e r i o r to omnivorous f i s h i n t h e i r a b i l i t y to u t i l i z e c a r b o h y d r a t e e f f e c t i v e l y , some experiments have shown that the former do adapt to h igh d i e t a r y l e v e l s of c a r b o h y d r a t e (Shimeno et a l . , 1979). For example, B u h l e r and H a l v e r (1961) fed j u v e n i l e ch inook salmon a s e r i e s of d i e t s i n which the p r o t e i n l e v e l was decreased from 71% to 40% and l e v e l s of d e x t r i n were i n c r e a s e d to 43%. They found that p r o t e i n e f f i c i e n c y r a t i o s were i n c r e a s e d by s u b s t i t u t i n g d e x t r i n f o r p r o t e i n whereas 26 growth r a t e remained s i m i l a r among f i s h r e c e i v i n g the d i f f e r e n t d i e t a r y t r e a t m e n t s . S i m i l a r l y , Bergot (1979a) found i n t r o u t that e l e v a t i n g the d i e t a r y l e v e l of g lucose from 15% to 30% whi le m a i n t a i n i n g e q u i v a l e n t p r o t e i n i n t a k e r e s u l t e d i n improved growth and p r o t e i n u t i l i z a t i o n . T h e r e f o r e , these exper iments have demonstrated that c a r b o h y d r a t e may have a p r o t e i n s p a r i n g a c t i o n i n f i s h , perhaps through r e d u c i n g the extent of g l u c o n e o g e n e s i s . A d d i t i o n a l s t u d i e s d i r e c t e d to determine the o p t i m a l p r o t e i n / e n e r g y r a t i o (Lee and Putman, 1973; R i n g r o s e , 1971; G a r l i n g and W i l s o n , 1977; and Cowey et a l . , 1953 i n d i e t s of f i s h a l s o support the concept that to some extent d i e t a r y c a r b o h y d r a t e spares p r o t e i n . In summary, proper d e f i n i t i o n of the c o r r e c t l e v e l of p r o t e i n i n r e l a t i o n to the energy content of a sa lmonid d i e t i s d i f f i c u l t owing to the d i f f e r e n t n u t r i e n t i n t e r a c t i o n s which can o c c u r . T h e r e f o r e , the p r o t e i n requirements of f i s h may have to be c o n t i n u o u s l y r e - e v a l u a t e d as more fundamental knowledge i s gained i n f i s h n u t r i t i o n . 27 CHAPTER 3 EXPERIMENT 1 3.0 P r o t e i n u t i l i z a t i o n and the measurement of p r o t e i n q u a l i t y i n d i e t s for chinook salmon f r y . 3.1 I n t r o d u c t i o n Recent ev idence suggests that the s u r v i v a l of h a t c h e r y - r e a r e d chinook salmon i n the ocean i s d i r e c t l y r e l a t e d to t h e i r s i z e at the time of seawater en try (Fowler et a l . , 1980; B i l t o n et a l . , 1982). T h e r e f o r e i t i s e s s e n t i a l that j u v e n i l e chinook salmon r e a l i z e t h e i r f u l l growth p o t e n t i a l d u r i n g the f reshwater stage of t h e i r l i f e h i s t o r y through the best p o s s i b l e n u t r i t i o n . A l s o , i t i s w e l l r e c o g n i z e d that the s u c c e s s f u l development of salmon farming i n B r i t i s h Columbia i s p a r t l y dependent on the supply of p r o f i t a b l e f i s h f e e d s . In p a r t i c u l a r , the q u a l i t y of p r o t e i n s c o m p r i s i n g sa lmonid foods i s c r i t i c a l to f i s h performance and cos t e f f e c t i v e n e s s of salmon c u l t u r e . Hence, i n t h i s s t u d y , the f i r s t o b j e c t i v e was an u n d e r s t a n d i n g of which p r o t e i n sources have the h i g h e s t n u t r i t i v e va lue for chinook salmon and how p r o c e s s i n g c o n d i t i o n s e i t h e r enhance or d i m i n i s h p r o t e i n q u a l i t y . F i shmea l i s the p r i n c i p a l source of p r o t e i n i n commercial sa lmonid d i e t s , and not only p r o v i d e s p r o t e i n to supply e s s e n t i a l amino a c i d s , but a l s o c o n t r i b u t e s l i p i d s , v i t a m i n s , and m i n e r a l s . The p r o t e i n q u a l i t y of f i s h m e a l p r e s e n t l y 28 a v a i l a b l e i n B r i t i s h Columbia i s known to vary c o n s i d e r a b l y . T h i s v a r i a b i l i t y i s a f u n c t i o n of the nature of the raw m a t e r i a l ( o r i g i n , s p e c i e s , s eason , whole f i s h or f i s h s c r a p s , s torage c o n d i t i o n s ) , p r o c e s s i n g methods ( c o o k i n g , d r y i n g , g r i n d i n g , a n t i o x i d a n t s t a b i l i z a t i o n ) and s torage c o n d i t i o n s of the f i n a l p r o d u c t . S e v e r a l f i s h s p e c i e s are commonly processed i n t o f i s h m e a l and of these h e r r i n g and p o l l o c k were s e l e c t e d for a comparison of n u t r i t i v e va lue i n t h i s s t u d y . Both h e r r i n g and p o l l o c k muscle were f r e e z e - d r i e d to m a i n t a i n amino a c i d a v a i l a b i l i t y . Moreover , the p o l l o c k was blended with a s m a l l p o r t i o n of e u p h a u s i d s , a s p e c i e s of marine z o o p l a n k t o n , i n an attempt to promote a p p e t i t e and improve amino a c i d b a l a n c e . In t h i s regard i t was observed that the amino a c i d p r o f i l e of f r e e z e - d r i e d p o l l o c k muscle and euphausids compared f a v o u r a b l y to that of s t a t e d e s s e n t i a l amino a c i d requirements of chinook salmon ( T a b l e 1 ) . A l s o , i t i s noteworthy , that Cowey et a l .( 1971 , 1972) o b t a i n e d e x c e l l e n t r e s u l t s i n s t u d i e s wi th p l a i c e us ing f r e e z e - d r i e d cod muscle as a p r o t e i n s o u r c e . To e v a l u a t e the e f f e c t s of d i f f e r e n t p r o c e s s i n g methods a l o n e , on p r o t e i n q u a l i t y , a common batch of raw h e r r i n g was cooked and then d r i e d under d i f f e r e n t c o n d i t i o n s . A t e n t a t i v e assumption was made that damage due to heat d u r i n g d r y i n g would be mani fes ted by l o s s e s i n the a v a i l a b i l i t y of c e r t a i n amino a c i d s , e s p e c i a l l y l y s i n e , and t h i s would c o n t r i b u t e to decreased p r o t e i n q u a l i t y . The q u a l i t y of a l l of the f o r e g o i n g p r o t e i n sources was compared to that of a mix of c a s e i n and g e l a t i n f o r t i f i e d with 29 T a b l e 1. A m i n o a c i d r e q u i r e m e n t s f o r c h i n o o k s a l m o n ( N R C . 1 9 8 1 ) , c o m p o s i t i o n o f e g g s and t h e a n a l y z e d e s s e n t i a l a m i n o a c i d c o m p o s i t i o n o f f r e e z e - d r i e d p o l l o c k m u s c l e , f r e e z e - d r i e d w h o l e e u p h a u s i d s a n d v i t a m i n - f r e e c a s e i n . R a m i n o a c i d / 16g n i t r o g e n " 1 3 3 A m i n o a c i d R e q u i r e m e n t s F r e e z e - d r i e d F r e e z e - d r i e d F r e e z e - d r i e d V i t a m i n - f r e e (NRC, 1 9 8 1 ) e y e d c h i n o o k e g g s p o l l o c k m u s c l e w h o l e e u p h a u s i d s c a s e i n A r g i n i n e 6 .0 6 .39 9, .30 5 .09 3 .83 H i s t i d i n e 1. .8 2, .88 2. .15 2 , .00 2 .95 I s o l e u c i n e 2, .2 5. .94 4 . 34 3. .65 4 .10 L e u c i n e 3, ,9 9. .74 8. .61 5, .81 7 .60 L y s i n e 5, .0 8, .65 12 . ,92 6, .17 7, .39 M e t h i o n i n e 2, .51 4, .10 4. .27 3, .01 C y s t i n e 2 4, .0 1 , .56 1 . ,68 0, .99 0, .74 P h e n y l a l a n i n e 5. .39 4. ,39 3, .74 4, .91 T y r o s i n e 2 5. . 1 4, .62 4. ,13 3. .60 5, .59 T h r e o n i n e 2 .2 5, .02 5. ,13 3. ,55 3, .83 T r y p t o p h a n 0 .5 1 .42 1 . 22 1 , .00 1 .23 V a l i n e 3 .2 7 .32 4, .26 3, .75 5 .39 1. D e t e r m i n e d by A.A.A. L a b o r a t o r y , S e a t t l e , Wash. 2. C y s t i n e a n d t r y o s i n e a r e d i s p e n s a b l e , b u t s p a r e r e q u i r e m e n t s f o r m e t h i o n i n e a n d p h e n y l a l a n i n e r e s p e c t i v e l y . 3. D e t e r m i n e d a t t h e D e p a r t m e n t o f A n i m a l a nd P o u l t r y S c i e n c e , U n i v e r s i t y o f S a s k a t c h e w a n , S a s k a t o o n , C a n a d a . a r g i n i n e and methionine to meet the e s s e n t i a l amino a c i d requirements of chinook salmon (NRC, 1981). T h i s was done because a b lend of c a s e i n and g e l a t i n has been c o n s i d e r e d to be an e x c e l l e n t source of p r o t e i n for j u v e n i l e salmon and other f i s h s p e c i e s i n p r e v i o u s n u t r i t i o n s t u d i e s , and i n d e e d , d i e t s based on c a s e i n and g e l a t i n have been used e x t e n s i v e l y to study the n u t r i t i o n a l requirements of f i s h (Cowey, 1976; H a l v e r , 1982). A l s o , the case f o r a s tandard r e f e r e n c e d i e t c o n t a i n i n g c a s e i n and g e l a t i n as a p r o t e i n source has been proposed to permit d i r e c t comparison of r e s u l t s among l a b o r a t o r i e s ( C a s t e l l and T iews , 1980) . Owing to the p a u c i t y of s t a n d a r d i z e d t echn iques f o r the e v a l u a t i o n of food p r o t e i n s i n f i s h (Cowey and S a r g e n t , 1979), the second goa l of t h i s study was to assess the m e r i t s versus demer i t s of s e v e r a l p r o t e i n q u a l i t y b ioassay procedures used i n assessment of the n u t r i t i v e va lue of p r o t e i n i n f i s h d i e t s . T h i s n e c e s s i t a t e d 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 at s e v e r a l c o n c e n t r a t i o n s of d i e t a r y p r o t e i n . As par t of t h i s g o a l , e s t imates were made of endogenous n i t r o g e n l o s s by the d i f f e r e n t groups of f i s h d u r i n g the e x p e r i m e n t a l p e r i o d to permit measurement of p r o t e i n q u a l i t y by methods which c o n s i d e r use of d i e t a r y p r o t e i n for maintenance as w e l l as growth. T h i s approach enabled d e t e r m i n a t i o n of the amounts of p r o t e i n u t i l i z e d for maintenance , growth and that l o s t from exogenous e x c r e t i o n s by f i s h i n g e s t i n g p r o t e i n s of v a r y i n g q u a l i t y . 31 3 . 2 M a t e r i a l s and methods 3 .2 .1 Test p r o t e i n s o u r c e s . Frozen p o l l o c k ( T h e r a g r a chalcogramma) f i l l e t s were p a r t i a l l y thawed, comminuted through a meat g r i n d e r (6 .35 mm g r i n d e r p l a t e ) , l o o s e l y spread over t r a y s , r e f r o z e n and f r e e z e - d r i e d . The f r e e z e - d r i e d p o l l o c k muscle was ground ( F i t z m i l l , model JT) so that p a r t i c l e s would pass 100% through a s i z e U . S . 20 s i e v e . E t h o x y q u i n (,025%)was added to prevent o x i d a t i v e r a n c i d i t y . Whole f r o z e n euphausids ( E u p h a s i a p a c i f i c a ) were prepared i n an i d e n t i c a l manner. The proximate compos i t i ons of f r e e z e - d r i e d p o l l o c k and euphausids were r e s p e c t i v e l y : m o i s t u r e , 1.80% and 3.40%; ash , 4.97% and 12.10%; l i p i d , 2.01% and 16.45%; and crude p r o t e i n , 89.74% and 60.01%. Nine p a r t s of of the former were combined with one part of the l a t t e r on a dry weight b a s i s to produce f r e e z e - d r i e d p o l l o c k muscle and whole euphausid meal ( F P E ) . The h e r r i n g meals were prepared from a s i n g l e batch of h e r r i n g (Clupea harangus p a l l a s i ) caught on January 8, 1981, o f f L a d y s m i t h , B . C . The h e r r i n g was t r a n s p o r t e d on i c e and kept deep f r o z e n ( - 2 0 ° C ) i n a i r t i g h t p l a s t i c bags for not more than a month whi l e be ing p r o c e s s e d . F r e e z e - d r i e d whole raw h e r r i n g (FRH) was prepared i n a s i m i l a r manner to the p o l l o c k muscle except that e thoxyquin was added to the mixed h e r r i n g at a l e v e l of 0.005% before f r e e z e - d r y i n g . The h e a t - d r i e d h e r r i n g meals were prepared i n a c o n t i n o u s p i l o t f i s h meal manufac tur ing machine (Chemica l Research O r g a n i z a t i o n , E s b j e r g , Denmark). The machine c o n s i s t s of a 32 s t eam- jacke ted c o o k e r , screw press and s t eam- jacke ted r o t a r y d r y e r . The temperature of the cooker and dryer can be c o n t r o l l e d . M i n c e d , whole h e r r i n g was cooked at 7 5 ° C . A f t e r p r e s s i n g , the o i l i n the press l i q u i d was decanted and the aqueous f r a c t i o n was condensed to approx imate ly 30% s o l i d s i n a s t e a m - j a c k e t e d bowl c o o k e r . The condensed s o l u b l e s were added to the presscake i n the r o t a r y d r y e r . The d r y e r was set to operate at 7 5 ° C to produce low temperature d r i e d h e r r i n g meal ( L T H ) . The h igh temperature d r i e d h e r r i n g meal (HTH) was mainly d r i e d i n the r o t a r y dryer at 1 2 0 ° C and f u r t h e r o v e n - d r i e d at 1 5 0 ° C for one hour . Because both the LTH and HTH product s c o n t a i n e d an excess of l i p i d , i t was necessary to p a r t i a l l y e x t r a c t l i p i d from the meals wi th hexane (5:1 v/w) to permit g r i n d i n g and d i e t f o r m u l a t i o n . A f t e r mixing and a l l o w i n g to stand for 30 minutes , the s o l v e n t and meal s l u r r y was f i l t e r e d through a Buchner f u n n e l over Whatman N o . l f i l t e r paper . T h i s procedure was repeated three t i m e s . The f i n a l h e r r i n g meal product s were p laced i n wire mesh t r a y s over a c u r r e n t of ambient a i r for f i v e hours to remove any t r a c e s of r e s i d u a l hexane. E t h o x y q u i n (0.025%) was added to the hexane e x t r a c t e d products which were then ground through a s i z e U . S . 20 screen with a hammer m i l l . T a b l e 2 shows the c o m p o s i t i o n s of the f i s h p r o t e i n s o u r c e s . The c a s e i n - g e l a t i n mix supplemented with 1.4% L - a r g i n i n e and 0.57% DL-meth ion ine (CS) was a m o d i f i e d v e r s i o n of the Oregon Tes t D i e t o u t l i n e d by NRC (1973)(Swarok and H i g g s , 1981). 33 T a b l e 2. C o m p o s i t i o n o f p r o t e i n s o u r c e s ( v a l u e s i n p a r e n t h e s i s show p e r c e n t a g e s f o r e a c h p r o x i m a t e c o n s t i t u e n t a nd l y s i n e e x p r e s s e d on a d r y m a t t e r b a s i s ) . A v a i l a b l e l y s i n e % A v a i l a b l e (% o f c r u d e C o d e % M o i s t u r e % P r o t e i n % L i p i d % A s h l y s i n e p r o t e i n ) F r e e z e - d r i e d p o l l o c k m u s c l e & f r e e z e - d r i e d e u p h a u s i d s F P E 1 .96 86 ( 8 8 .72 .45) 3, ( 3 , .43 .50) 5.69 ( 5 . 8 0 ) 7 ( 7 .42 .57) 8 .56 F r e e z e - d r i e d raw h e r r i n g ( h e x a n e e x t r a c t e d ) FRH 8 .11 70 ( 7 6 , .56 .79) 10, ( 1 1 . .48 .40) 11 .49 ( 1 2 . 5 0 ) 3 ( 4 .86 .20) 5, .47 Low t e m p e r a t u r e d r i e d h e r r i n g m e a l ( h e x a n e e x t r a c t e d ) LTH 7 .77 63, ( 6 9 , .95 .34) 16. ( 1 8 . .98 4 1 ) 10.21 ( 1 1 . 0 7 ) 3 ( 3 , .45 .74) 5, .39 H i g h t e m p e r a t u r e d r i e d h e r r i n g m e a l ( h e x a n e e x t r a c t e d ) HTH 3, .83 72. ( 7 5 . .13 ,00) 13. ( 1 3 . ,12 ,64) 1 1 . 1 5 ( 1 1 .59) 3, ( 3 , .28 .41) 4. .55 2 C a s e i n m i x CS 2. ,61 9 0 . (93.. ,82 25) 6. ( 6 . ,69 ,87) 7 . ,37 T. 90T" f r e e z e - d r i e d p o l l o c k an d 10% f r e e z e - d r i e d e u p h a u s i d s on a d r y w e i g h t b a s i s . 2. C o n t a i n s 8 8 % c a s e i n , 10% g e l a t i n , 1.4% L - a r g i n i n e , 0.57% D L - m e t h i o n i n e ( I . C . N . , S t . L o u i s , HO.) T a b l e 3. Co m p o s i t i o n of d i e t s (g/kg d i e t on a dry matter b a s i s ) . D i e t Code ( P r o t e i n s o u r c e and % d i e t a r y p r o t e i n ) I n g r e d i e n t s PF FPE FPE FRH LTH HTH CS FPE FRH 0 7 17 17 17 17 17 27 27 F r e e z e - d r i e d p o l l o c k - e u p h a u s i d 79. 14 192. 20 - 305 . 26 F r e e z e - d r i e d raw h e r r i n g 221 . 38 - 351 .61 Low temp d r i e d h e r r i n g meal 245 . 17 High temp d r i e d h e r r i n g meal - 226 .67 C a s e i n - g e l a t i n , a r g i n i n e , and m e t h i o n i n e mix - 182 . 31 H e r r i n g o i l ^ 130 . 0 127 . 22 123 . 22 104, .76 84 .1 86 99 .08 130 . 0 119 , 32 89, .92 M i n e r a l mix^ 83. 7 79 . 6 71 . 1 50, .4 50 .4 50 .4 83 . 7 62 , 7 28 , . 1 Ground c e l l u l o s e 85 . 5 69 . 24 85 . 5 91 . 66 87 . 77 92 . 05 105 . ,49 127 . 52 110 .57 Dex t r i n 346 . 0 307 . 0 250 . 5 250 .5 250 .5 250 . 5 250 . 5 194 , .5 194 .5 G l u c o s e 346 . 0 307 . 0 250. 5 250, .5 250 . 5 250 . 5 250 . 5 194 , 5 194 , .5 Car boxy-me thy 1 c e l l u l o s e 20 20 20 20 20 20 20 20 20 ... . 3 V i t a m i n mix 7 . 3 7 . 3 7 . 3 7 .3 7 .3 7 . 3 7 . 3 7 , .3 7 . 3 C h o l i n e c h l o r i d e 3. 5 3 . 5 3. 5 3 .5 3 .5 3 .5 3 . 5 3 , .5 3, .5 ( 50%) 1. S t a b l i z e d w i t h BHA-BHT (1:1) 0.33%. 2. M i n e r a l mix f o r m u l a t e d so t h a t each d i e t c o n t a i n e d (g/kg dry d i e t ) : Ca 12.6, P 9.0, Mg 1.8, Fe 0.2, Na 2.4, A l 0.05, Cu 0.01, Mn 0.03, Co 0.01, Zn 0.08, K 8.3, I 0.004, Na:K 0.29, Ca:Mg 7, P:Mg 5, Ca:P 1.4. C a l c i u m l e v e l s i n FRH, LTH and HTH c o n t a i n i n g d i e t s were c a l c u l a t e d to be 13.9 and 19.6 g/kg f o r d i e t s c o n t a i n i n g 27 and 37% p r o t e i n . S i m i l a r l y , phosphorus l e v e l s were 10.0 and 14.0 r e s p e c t i v e l y to m a i n t a i n e q u i v a l e n t Ca:P i n a l l d i e t s . S o u r c e s of m i n e r a l s were p o t a s s i u m phosphate monobasic, p o t a s s i u m phosphate d i b a s i c , sodium phosphate monobasic, c a l c i u m phosphate d i b a s i c , c a l c i u m c a r b o n a t e , magnesium s u l p h a t e , f e r r i c o x i d e , z i n c o x i d e , sodium c h l o r i d e , manganese s u l p h a t e , c o b a l t c h l o r i d e , c u p rous c h l o r i d e , p o t a s s i u m i o d i d e and aluminum c h l o r h y d r a t e . 3. V i t a m i n mix to s u p p l y (mg/kg dry d i e t u n l e s s o t h e r w i s e i n d i c a t e d ) : a s c o r b i c a c i d 1200, i n o s i t o l 400, n i a c i n 300, C a - p a n t o t h e n a t e 150, r i b o f l a v i n 60, menadione 80, p y r i d o x i n e HCL 30, t h i a m i n HCL 30, f o l i c a c i d 20, b i o t i n 3, v i t a m i n A 10,000 IU, v i t a m i n D3 1,000 IU, v i t a m i n E 600 IU. 35 T a b l e 3. co n t ' d D i e t Code ( P r o t e i n s o u r c e and % d i e t a r y p r o t e i n ) I n g r e d i e n t s LTH HTH CS FPE FRH LTH HTH CS FPE 27 27 27 37 37 37 37 37 47 F r e e z e - d r i e d p o l l o c k - e u p h a u s i d 418 . 32 — 531.37 F r e e z e - d r i e d raw h e r r i n g - 481 . 83 - - Low temp d r i e d h e r r i n g meal 389 .39 - 533 . 60 - - High temp d r i e d h e r r i n g meal 360.0 493 . 33 - C a s e i n - g e l a t i n , a r g i n i n e , and m e t h i o n i n e mix - 289 . 54 396 . 78 - H e r r i n g o i l ' 58 .31 80.90 130 115. 36 75 . 07 31 . 76 • . 71 130 I l l . 4 0 M i n e r a l mix^ 28 . 1 28 . 1 83, . 7 56 . 00 17 . 04 17, .4 17 . 4 83 . 7 47.4 Ground c e l l u l o s e 104 .40 11 .20 132 , .56 142 . 12 118. 90 110 , .44 119 . 76 149 . 02 115.43 D e x t r i n 194 . 5 194 .5 194 . 5 138 138 138 138 138 82 G l u c o s e 194 .5 194 . 5 194 , .5 138 138 138 1 38 138 82 Car b o x y - m e t h y l c e l l u l o s e 20 20 20 20 20 20 20 20 20 3 V i t a m i n mix 7 , .3 7.3 7 . 3 7 . 3 7 . 3 7 . 3 7 . 3 7 . 3 7.3 C h o l i n e c h l o r i d e 3, .5 3.5 3 . 5 3 . 5 3. 5 3 . ,5 3 .5. 3 . 5 3.5 ( 50%) 1. S t a b l i z e d w i t h BHA-BHT (1:1) 0.33%. 2. M i n e r a l mix f o r m u l a t e d so t h a t each d i e t c o n t a i n e d (g/kg dry d i e t ) : Ca 12.6, P 9.0, Mg 1.8, Fe 0.2, Na 2.4, A l 0.05, Cu 0.01, Mn 0.03, Co 0.01, Zn 0.08, K 8.3, I 0.004, Na:K 0.29, Ca:Mg 7, P:Mg 5, Ca:P 1.4. C a l c i u m l e v e l s i n FRH, LTH and HTH c o n t a i n i n g d i e t s were c a l c u l a t e d to be 13.9 and 19.6 g/kg f o r d i e t s c o n t a i n i n g 27 and 37% p r o t e i n . S i m i l a r l y , phosphorus l e v e l s were 10.0 and 14.0 r e s p e c t i v e l y to m a i n t a i n e q u i v a l e n t Ca:P i n a l l d i e t s . S o u r c e s of m i n e r a l s were p o t a s s i u m phosphate monobasic t p o t a s s i u m phosphate d i b a s i c , sodium phosphate monobasic, c a l c i u m phosphate d i b a s i c , c a l c i u m c a r b o n a t e , magnesium s u l p h a t e , f e r r i c o x i d e , z i n c o x i d e , sodium c h l o r i d e , manganese s u l p h a t e , c o b a l t c h l o r i d e , c u p rous c h l o r i d e , p o t a s s i u m i o d i d e and aluminum c h l o r h y d r a t e . 3. V i t a m i n mix to s u p p l y (mg/kg dry d i e t u n l e s s o t h e r w i s e i n d i c a t e d ) : a s c o r b i c a c i d 1200, i n o s i t o l 400, n i a c i n 300, C a - p a n t o t h e n a t e 150, r i b o f l a v i n 60, menadione 80, p y r i d o x i n e HCL 30, t h i a m i n HCL 30, f o l i c a c i d 20, b i o t i n 3, v i t a m i n A 10,000 IU, v i t a m i n D3 1,000 IU, v i t a m i n E 600 IU. 36 3 . 2 . 2 D i e t s The formulae of the e x p e r i m e n t a l d i e t s are shown i n T a b l e 3. Each p r o t e i n source was i n c l u d e d i n the d i e t s as the s o l e source of p r o t e i n . D i e t s were formula ted to c o n t a i n 17%, 27%, and 37% of dry matter as crude p r o t e i n (N x 6 . 2 5 ) . An a d d i t i o n a l two d i e t s were prepared c o n t a i n i n g FPE at l e v e l s of 7% and 47% p r o t e i n . In order to e s t imate endogenous and m e t a b o l i c p r o t e i n l o s s a n o n - p r o t e i n d i e t was o f f e r e d to two groups of f i s h . B a s a l i n g r e d i e n t s were p r o p o r t i o n a t e l y common to a l l d i e t s . M i n e r a l supplements were prepared to e q u a l i z e the m i n e r a l c o m p o s i t i o n of a l l d i e t s ( T a b l e 3 ) . A l l e x p e r i m e n t a l d i e t s were formula ted to c o n t a i n 4000 k c a l / kg by a s c r i b i n g m e t a b o l i z a b l e energy (ME) va lues of 4.5 k c a l / g crude p r o t e i n , 9.5 k c a l / g l i p i d and 4.0 k c a l / g f o r d e x t r i n and g l u c o s e . The h igh va lues employed for f i s h o i l , d e x t r i n and g lucose are c o n s i d e r e d v a l i d because each has been found to be h i g h l y d i g e s t i b l e i n rainbow t r o u t d i e t s (Cho and S l i n g e r , 1979). The va lue for p r o t e i n was d e r i v e d by d e d u c t i n g the heat of combustion of e x c r e t e d ammonia n i t r o g e n (0 .95 k c a l / g of p r o t e i n ) from the gross energy of p r o t e i n (5 .66 k c a l / g ) ( B r e t t and G r o v e s , 1979) and a p p l y i n g a d i g e s t i b i l i t y c o e f f i c i e n t for p r o t e i n of 95% for c a s e i n - g e l a t i n ( S m i t h , 1971) and F P E . The c a l c u l a t e d energy va lues of the d i e t s based on a n a l y s i s are shown i n T a b l e 4. L a s t l y , two groups of f i s h r e c e i v e d a commercia l d i e t , Oregon Moi s t P e l l e t s (0MP) which i s the s tandard hatchery d i e t for chinook and coho salmon i n B r i t i s h C o l u m b i a . Values of 4.2 37 Table k. Proximate c o m p o s i t i o n of d i e t s (% d r y - m a t t e r b a s i s ) and c a l c u l a t e d a v a i l a b l e l y s i n e and energy v a l u e s based on the a n a l y s e s . PF FPE FPE FRH LTH HTH CS FPE FRH A n a l y s i s 0 7 17 17 17 17 17 27 27 Crude p r o t e i n (Z N x 6.25) 1 . 07 6. . 73 16 .94 18 .01 16, .78 17 .00 18 , .55 27 .24 27 . 59 A v a i l a b l e l y s i n e 0 .58 1 .45 0. .99 0 .90 0, . 77 1 , .37 2 .33 1 , . 51 T o t a l crude l i p i d 14 .08 13. .22 12 , .40 12 .46 13 , .33 11 , .95 11 , .61 12. .31 12 . 81 L i p i d c o n t r i b u t e d by f i s h meal (• .28) (. .67) (2. .52) (4, .51) (3, .09) (1, .07) (4, .01: Ash 7 .46 7. .38 7 . 15 6 .67 7 , .13 7 , .06 7 , .73 7, .37 7 , .34 D i g e s t i b l e c a r b o h y d r a t e 69. .2 61 . 4 50, . 1 50, . 1 50 , . 1 50 , . 1 50 . 1 38, .9 38. ,9 M o i s t u r e 9 .17 10. .44 7, .89 9 .20 8, .12 6, .10 8 , .88 7, .72 10, .95 Energy ( k c a l / k g ) P r o t e i n : ME (A.5 k c a l / g ) 48 303 762 811 755 765 835 1126 1242 GE (5.7 k c a l / g ) 61 384 966 1027 956 969 1057 1553 1573 L i p i d : ME S GE (9.5 k c a l / g ) 1338 1256 1178 1184 1266 1135 1103 1169 1217 C a r b o h y d r a t e : ME & GE (4.0 k c a l / g ) 2768 2456 2004 2004 2004 2004 2004 1556 1556 T o t a l (ME) 4154 4015 3495 3998 4026 3904 3942 3951 4015 T o t a l (GE) 4167 4096 4148 4215 4226 4108 4164 4278 4346 mg p r o t e i n / k c a l (ME) 2 .58 16, ,8 42, .9 45, .0 41 , 7 43, .5 47 . , 1 68, .9 68. . 7 P r o t e i n e n e r g y : t o t a l energy (ME) 0. .01 0. .08 0 , .19 0 , .20 0. .19 0. ,20 0 . ,21 0, .28 0. ,31 T a b l e 4. c o n t ' d . A n a l y s i s LTH HTH CS FPE FRH LTH HTH CS FPE OMP 27 27 27 37 37 37 37 37 47 Crude P r o t e i n (% N x 6.25) 28, .45 27 , .38 26 .40 35 .96 39, .58 38, .42 39 .33 35, .99 46 .15 49. 40 A v a i l a b l e l y s i n e 1 .53 1 . 25 1 .95 3 .08 2 .17 2 .07 1 .79 2 .65 3 .95 T o t a l crude l i p i d 11 , .43 12 , .60 12 .60 11 .79 12, .20 11 , .81 11 .29 11 , .52 12 .95 14 . 53 L i p i d c o n t r i b u t e d by f i s h meal (7, .17) (4, .91) (1 .46) (5, .49) (9 .82) (6 •73) (1 .86) Ash 7 , . 78 7 , .11 7, .21 7, .61 8, .07 7 , .50 7, .84 7 , .30 8 .30 11 . 92 D i g e s t i b l e c a r b o h y d r a t e 38, .9 38 .9 38 .9 27 .6 27 , .6 27 .6 27 .6 27 , .6 16 .4 16. 4 M o i s t u r e 8, .36 6 . 28 8 .32 6 . 72 9 .12 8 . 19 6 .73 8 . 59 6 .03 24. 62 Energy ( k c a l / k g ) P r o t e i n : ME (4.5 k c a l / g ) 1281 1232 1188 1618 1781 1729 1797 1619 2077 2075 1 GE (5.7 k c a l / g ) 1622 1561 1595 2050 2256 2190 2276 2051 2631 2816 L i p i d : ME S GE (9.5 k c a l / g ) 1086 1197 1146 1120 1159 1122 1073 1094 1230 2380 C a r b o h y d r a t e : ME & GE (4.0 k c a l / g ) 1556 1556 1556 1104 1104 1104 1104 1104 656 2 2662 T o t a l (ME) 3922 3985 3890 4044 3955 3974 3818 3818 3963 3721 T o t a l (GE) 4264 4314 4207 4274 4519 4416 4453 4249 4517 4462 mg p r o t e i n / c a l (ME) 72. 5 68. 7 67 . 9 93. 6 97 . 9 97. 1 100 . 5 94. 3 116. .5 132 . 8 P r o t e i n e n e r g y : t o t a l energy (ME) 0. 33 0. 31 0. 31 0. 42 0. 44 0. 44 0. 45 0- 42 0. ,52 0. 56 1. Based on 4.2 kcal/g protein. 2. Based on 1.6 Xcal/g carbohydrate. k c a l / g of p r o t e i n and 1.6 k c a l / g of n i t r o g e n - f r e e e x t r a c t were employed to e s t imate the ME content of OMP because they more adequate ly r e f l e c t the d i g e s t i b i l i t y of p r o t e i n and raw s t a r c h i n t h i s d i e t (Tab le 4 ) . The e x p e r i m e n t a l feeds were prepared by mixing the i n g r e d i e n t s with a p o r t i o n of the o i l f or 20 minutes i n a Hobart m i x e r . The mix was then c o l d p e l l e t e d i n a C a l i f o r n i a model C L - t y p e 2 l a b o r a t o r y p e l l e t m i l l wi th 1.59 mm d i e . S u b s e q u e n t l y , the p e l l e t s were crumbled with a r o l l i n g p i n and hand screened to o b t a i n the a p p r o p r i a t e s i z e d c r u m b l e s . The remainder of the h e r r i n g o i l was s p r a y e d , by means of a hand he ld s y r i n g e (needle s i z e 18) , i n t o the crumbles . The d i e t s were mixed thorough ly and s t o r e d r e f r i g e r a t e d i n a i r t i g h t c o n t a i n e r s d u r i n g the s t u d y . The d i e t a r y treatment codes , r e s p e c t i v e p r o t e i n sources and p r o t e i n c o n c e n t r a t i o n s are summarized i n Tab le 5. T h i s code may be used to i n t e r p r e t the r e s u l t s of t h i s s t u d y . 3 . 2 . 3 Aquarium f a c i l i t y The e x p e r i m e n t a l aquarium f a c i l i t y c o n t a i n e d two rows of f i b e r g l a s s t a n k s . Each tank c o n t a i n e d 150 l i t r e s of a e r a t e d 1 0 . 5 ° C w e l l - w a t e r . Flow r a t e was 4 to 6 l i t r e s / m i n u t e / t a n k . A n a t u r a l p h o t o p e r i o d was p r o v i d e d by a s e r i e s of f l u o r e s c e n t l i g h t s ( V i t a l i t e , Durotes t 40w) c o n t r o l l e d by an a s t r o n o m i c a l time c l o c k . 40 Table 5. Summary of d i e t a r y treatments and codes . D i e t a r y p r o t e i n source Formulated D i e t a r y p r o t e i n treatment c o n c e n t r a t i o n code P r o t e i n - f r e e 0 PF F r e e z e - d r i e d p o l l o c k 7 FPE -7 muscle & f r e e z e - d r i e d 17 FPE--17 euphaus ids 27 FPE -27 37 FPE -37 47 FPE -47 F r e e z e - d r i e d raw whole 17 FRH -17 h e r r i n g ( h e x a n e - e x t r a c t e d ) 27 FRH -27 37 FRH -37 Low temperature d r i e d 17 LTH -17 whole h e r r i n g meal 27 LTH -27 ( h e x a n e - e x t r a c t e d ) 37 LTH -37 High temperature d r i e d 17 HTH -17 whole h e r r i n g meal 27 HTH -27 ( h e x a n e - e x t r a c t e d ) 37 HTH -37 C a s e i n - g e l a t i n mix, p lus 17 CS- 17 L - a r g i n i n e & DL-meth ion ine 27 CS- 27 37 CS- 37 Oregon moist p e l l e t s ( # 2 ) OMP (complete d i e t ) 41 3 .2 .4 P r o t o c o l Chinook salmon f r y were o b t a i n e d i n March, 1981 from Qual icum H a t c h e r y , B . C . A p o p u l a t i o n of 1900 f i s h was s e l e c t e d for un i form s i z e with a mean weight of 1.03g + 0 .24 . F i s h from the s e l e c t e d p o p u l a t i o n were randomly d i s t r i b u t e d i n t o groups of 150 i n each of 38 t a n k s . Each row of tanks c o n s t i t u t e d a b lock i n the e x p e r i m e n t a l d e s i g n . The 19 treatments were a l l o t e d at random to the tanks w i t h i n each row. Dur ing a 14-day a c c l i m a t i o n p e r i o d the f i s h were fed to excess with a d i e t s i m i l a r i n c o m p o s i t i o n to FPE-47 (Tab le 3) except that p r o t e i n comprised 50% and l i p i d 10% of the d i e t dry m a t t e r . Mean weight of f i s h at the s t a r t of t e s t f e e d i n g was 1.56 +_ 0 .024g(SEM). Dur ing the e x p e r i m e n t a l p e r i o d f i s h were fed by hand three times per day u n t i l s a t i a t e d and a d a i l y r e c o r d of feed i n t a k e was mainta ined for each group . The p o i n t of s a t i a t i o n was determined when " a c t i v e " f eed ing ceased over a f e e d i n g p e r i o d of one hour . The i n t e r v a l between the f i r s t and second, and second and t h i r d f eed ings was two and one h a l f hours i n each c a s e . Food p a r t i c l e s i z e was a d j u s t e d to s u i t f i s h s i z e a c c o r d i n g to Fowler and Burrows (1971) . 3 . 2 . 5 Measurement of growth On day 0, 21 and 42 of the f e e d i n g t r i a l , f o l l o w i n g 16 hrs of food d e p r i v a t i o n , 60 f i s h were randomly removed from each tank, a n a e s t h e s i z e d i n 0.5 ml 2 - p h e n o x y e t h a n o l / l and p laced on an absorbent t o w e l . I n d i v i d u a l f i s h weights ( to 0.01 g) were r e c o r d e d . 42 3 . 2 . 6 Chemica l a n a l y s i s of f i s h and d i e t s Four samples c o n t a i n i n g approx imate ly 25g of f i s h each , common to a l l treatment groups , were taken at day 0. At the end of the t r i a l , f o l l o w i n g 48 hrs of s t a r v a t i o n , samples of 12 f i s h were taken from each group. The f i s h were k i l l e d i n 2 ml of 2 - p h e n o x y e t h a n o l / l , b l o t t e d dry on a towel and s t o r e d for a n a l y s i s at - 2 0 ° C i n h e a t - s e a l e d bags. P r i o r to chemica l a n a l y s i s p a r t i a l l y thawed f i s h samples were homogenized i n a b l e n d e r . A l i q u o t s of the homogenat'e were used to determine mois ture and ash (AOAC, 1975), l i p i d ( B l i g h and Dyer , 1959) and t o t a l n i t r o g e n (Techn icon Instrument Co. L t d . i n d u s t r i a l methods 369-75 A/A and 334-74 W/B) . Percent n i t r o g e n was m u l t i p l i e d by 6.25 to e s t imate p r o t e i n c o n t e n t . D i e t s were ana lyzed employing s i m i l a r p r o c e d u r e s . 3 . 2 . 7 A v a i l a b l e l y s i n e A v a i l a b l e l y s i n e i n the p r o t e i n sources was determined by C a r p e n t e r ' s (1960) method as m o d i f i e d by Booth (1971) . The va lues were r e p o r t e d i n Tab le 2 as a percentage of the meal and as a percentage of crude p r o t e i n , i . e . g/16g N. 3 . 2 . 8 Data a n a l y s i s The body weight data were s u b j e c t e d to a n a l y s i s of v a r i a n c e (ANOVA). The a n a l y s i s of the randomized b lock tank means was conducted as a mixed model two-way ANOVA without r e p l i c a t i o n . The d i e t a r y treatments were not r e p l i c a t e d w i t h i n rows. P r o t e i n source and d i e t a r y p r o t e i n c o n c e n t r a t i o n were assumed to be f i x e d e f f e c t s and row ( b l o c k ) e f f e c t s were random. The 43 a p p r o p r i a t e i n t e r a c t i o n mean square was used to t e s t f o r s i g n i f i c a n t d i f f e r e n c e s due to the f i x e d e f f e c t s . A c c o r d i n g to Zar (1974) there i s no c o r r e c t term a v a i l a b l e to t e s t for b lock e f f e c t s i n a n o n - r e p l i c a t e d model . N e v e r t h e l e s s , the r e s i d u a l mean squares d e r i v e d from the v a r i a n c e between i n d i v i d u a l f i s h i s i n d u e d i n the ANOVA. However, no major i n f e r e n c e was drawn. From a s t r i c t v i ewpoint the i n d i v i d u a l f i s h body weights w i t h i n tanks were not r e p l i c a t e s . The approach taken i n t h i s study was c o n s e r v a t i v e to assure that the p r o b a b i l i t y of a Type 1 e r r o r i s r e d u c e d . The treatment means were then s u b j e c t e d to Duncan's (1955) New M u l t i p l e Range Test (DMR)(P = 0 . 0 5 ) . The data were trans formed to l og body weight and s u b j e c t e d to an a n a l y s i s of c o v a r i a n c e wi th day as the c o v a r i a t e . S p e c i f i c growth r a t e (GR%)(Higgs et a l . , 1979) was d e r i v e d from the c o v a r i a t e s l o p e ; GR% = ( e s l o p e - i ) x 100. GR% expresses percent i n c r e a s e i n body weight per day ( B r e t t , 1979). The c o v a r i a t e s lopes were s u b j e c t e d to S c h e f f e ' s (1959) t e s t (P = 0.05) to de tec t s i g n i f i c a n t d i f f e r e n c e s . These ana lyse s were performed by computer us ing a g e n e r a l l e a s t squares a n a l y s i s of v a r i a n c e program ( U . B . C . GENLIN) . R e g r e s s i o n equat ions of body weight ga in a g a i n s t p r o t e i n i n t a k e were computed by means of a g e n e r a l l i n e a r models procedure (SAS, 1982). The s lopes were compared by DMR t e s t (P = 0 . 0 5 ) . Food i n t a k e data were used to c a l c u l a t e gross food c o n v e r s i o n ( G F C ) , ( d r y weight ga in (g ) x 100 -f- dry food i n t a k e (g)) and gross energy u t i l i z a t i o n ( G E U ) , ( g r o s s energy ga in 44 ( k c a l ) x 100 -r gross energy i n t a k e ( k c a l ) ) . P r o t e i n e f f i c i e n c y r a t i o ( P E R ) , net p r o t e i n r a t i o (NPR), and s lope r a t i o ( S R ) , ( f o r weight ga in) were c a l c u l a t e d on a dry body weight b a s i s (Higgs et a l . , 1979) a c c o r d i n g to the formulas d e s c r i b e d i n the p r e v i o u s c h a p t e r . From f i s h c a r c a s s c o m p o s i t i o n , p r o t e i n p r o d u c t i v e va lue ( P P V ) , net p r o t e i n u t i l i z a t i o n and SR ( f o r p r o t e i n ga in) were c a l c u l a t e d . Net p r o t e i n u t i l i z a t i o n was c a l c u l a t e d both by the Bender and M i l l e r (1953) method, des igna ted (NPU-1) , and by the method of Ogino et a l . ( 1980)(NPU-2) . The procedure for these methods i s d e s c r i b e d i n the p r e v i o u s c h a p t e r . The above i n d i c e s were s u b j e c t e d to ANOVA. As d e s c r i b e d above a two-way randomized b lock model wi thout r e p l i c a t i o n was assumed. The ana lyse s are t a b u l a t e d i n the a p p e n d i c e s . As an e s t imate of sample v a r i a n c e the s tandard e r r o r of the mean (SE) was d e r i v e d from the r e s p e c t i v e e r r o r mean square of the ANOVA. These are shown on each t a b l e with the f i r s t va lue they d e s c r i b e . S u p e r s c r i p t s were used to des ignate s i g n i f i c a n t d i f f e r e n c e s d e r i v e d from m u l t i p l e comparison t e s t s . These are shown with each t a b l e . 45 - 3.3 RESULTS 3 .3 .1 The e f f e c t of d i e t a r y p r o t e i n source and l e v e l on body weight ga in The changes i n body weight of f i s h fed the v a r i o u s d i e t s are shown i n F i g s . 3A, 3B, and 3C. The growth response over the 42 day p e r i o d was g e n e r a l l y l i n e a r for f i s h fed d i e t s c o n t a i n i n g 27% and 37% p r o t e i n . The e x c e p t i o n s were h igh temperature d r i e d h e r r i n g meals . F i s h fed these d i e t s , and those c o n t a i n i n g 7 and 17% p r o t e i n showed a d e c l i n e i n the ra t e of growth from day 21 to 42 compared to day 0 to 21 ( F i g . 3 A ) . P r e d i c t a b l y , f i s h fed the PF d i e t l o s t body weight d u r i n g the e n t i r e p e r i o d . The growth response of the e x p e r i m e n t a l f i s h to the v a r i o u s d i e t a r y treatments as d e p i c t e d i n F i g s . 3A, 3B, and 3C are i n the c l a s s i c a l n u t r i t i o n a l form showing body weight ga ins over t ime . Tab le 6 and F i g . 4 on the other hand summarize the responses to the d i e t a r y treatments i n terms of the s p e c i f i c growth r a t e s e l i c i t e d i n response to d i e t a r y c o n c e n t r a t i o n of p r o t e i n from the d i f f e r e n t s o u r c e s . A l though the d i e t s were formula ted to c o n t a i n exact l e v e l s of n u t r i e n t s , proximate a n a l y s i s showed that the compos i t i ons of the d i e t s were not a l l e q u i v a l e n t at each p r e s c r i b e d p r o t e i n l e v e l (Tab le 4 ) . However, over the range of p r o t e i n l e v e l s fed to groups of f i s h wi th FPE as the so l e source of p r o t e i n the i n c r e a s e i n growth r a t e d i m i n i s h e d with each increment of d i e t a r y p r o t e i n c o n c e n t r a t i o n ( F i g . 4 ) . The p a t t e r n of d i m i n i s h i n g r e t u r n s was not as c l e a r for the other p r o t e i n 46 D A Y S F i g . 3A. Growth of chinook salmon fed the v a r i o u s p r o t e i n sources i n d i e t s c o n t a i n i n g a p p r o x i m a t e l y 17% p r o t e i n . Growth of f i s h fed FPE-7 and p r o t e i n f r e e (PF) d i e t s are a l s o shown. Va lues p l o t t e d are mean wet f i s h w e i g h t s , +_ 2 s tandard e r r o r s ( S E ) . - 47 DAYS F i g . 3B. Growth of chinook salmon fed the v a r i o u s p r o t e i n sources i n d i e t s c o n t a i n i n g a p p r o x i m a t e l y 27% p r o t e i n . Growth of f i s h fed OMP and p r o t e i n f r e e (PF) d i e t s are a l s o shown. Va lues p l o t t e d are mean wet f i s h w e i g h t s , +_ 2 s tandard e r r o r s ( S E ) . - 48 - D A Y S F i g . 3C. Growth of chinook salmon fed the various protein sources in diets containing approximately 37% prote in . Growth of f i sh fed FPE-47, OMP, and prote in free (PF) diets are also shown. Values plotted are mean wet f i sh weights, + 2 standard errors (SE) . - 49 - T a b l e 6. F i n a l (day 42) mean wet body w e i g h t s (g) and s p e c i f i c growth r a t e s (G.R.I; p e r c e n t body weight per day) o f f i s h f e d the v a r i o u s p r o t e i n s o u r c e s a t each d i e t a r y p r o t e i n c o n c e n t r a t i o n . P r o t e i n Source 17 Z P r o t e i n i n D i e t 27 37 Mean F P E FRH LTH HTH CS F i n a l wt. (+SE) G.R. ( I ) (+SE) F i n a l wt. G.R. ( Z ) F i n a l wt. G.R. (Z) F i n a l wt. G.R. (Z) F i n a l wt. G.R. (Z) c l 2.33 +0.06 C l 1.04 +0.06 2.43 1.04 be 2.16 BC 0.74 1.59 0.06 2.16 be BC 3.45 DE 1.86 de 3.02 1.52 2.99 1.52 ab 1.85 0.70 0.39 3.06 1.52 AB 3.94 i EF 2.26 4.25 2.38 3.90 EF 2.20 2.34 0.89 de 3.34 1.76 3.24 +0.03 Y 1.72 +0.03 3.23 1.64 M 3.02 X 1 .48 v 1.92 V 0.44 v 2.85 h 1.33 Mean F i n a l wt. G.R. (Z) 2.13 +0.02 P 0.71 +0.03 2.87 1.36 3.55 1.90 OMP F i n a l wt. G.R.(Z) 3.59 2.08 1. V a l u e s w i t h the same s u p e r s c r i p t f o r each parameter w i t h r e s p e c t t o the s o u r c e x l e v e l (a - f ) t p r o t e i n s o u r c e (v - y ) , and p r o t e i n l e v e l (p - r ) e f f e c t s do not d i f f e r s i g n i f i c a n t l y . S u p e r s c r i p t s f o r G.R.(I) a r e c a p i t a l i z e d (DMR t e s t f o r body wei g h t , P • 0.05); S c h e f f e ' a t e s t f o r G.R. ( Z ) , P - 0.05). 2. OMP d a t a waa not a n a l y z e d s t a t i s t i c a l l y . - 50 - sources as was noted f o r f i s h fed F P E . C l e a r l y , HTH was an inadequate p r o t e i n s o u r c e , demonstrat ing the e f f e c t of severe d r y i n g temperature on h e r r i n g meal . The performance of f i s h fed OMP was found to be comparable to that of f i s h fed F P E , FRH and LTH at the 37% l e v e l of d i e t a r y p r o t e i n . OMP i s a popular commercial salmon feed c o n t a i n i n g 49% p r o t e i n . The performance of f i s h fed the t e s t d i e t s c o n t a i n i n g the v a r i o u s p r o t e i n sources at three p r o t e i n c o n c e n t r a t i o n s was compared by a two-way randomized b lock f a c t o r i a l a n a l y s e s of v a r i a n c e . At day 42 marked d i f f e r e n c e s i n body weight due to p r o t e i n source (P < 0.001) and p r o t e i n l e v e l (P < 0.01) were found (Tab le 6 ) . S i m i l a r l y , a f a c t o r i a l a n a l y s i s of c o v a r i a n c e of l o g g body weights a l s o i n d i c a t e d a s i g n i f i c a n t e f f e c t due to p r o t e i n source (P < 0.01) and d i e t a r y p r o t e i n l e v e l (P < 0 . 0 5 ) . The s p e c i f i c growth r a t e s ( d e r i v e d from the s l o p e s ) of groups of f i s h fed each p r o t e i n source at a l l l e v e l s were compared (Tab le 6 ) . Both the mean f i n a l body weights and s p e c i f i c growth r a t e s showed that FPE and FRH promoted the f a s t e s t growth, f o l l o w e d by L T H , CS and l a s t l y , HTH. The above r e s u l t s showed that d i f f e r e n c e s i n performance of f i s h fed p r o t e i n s of v a r y i n g q u a l i t y were mani fes ted at w ide ly d i f f e r e n t d i e t a r y c o n c e n t r a t i o n s . S ince p r o t e i n i n t a k e may have been r e s t r i c t e d due to poor p a l a t i b i l i t y of p a r t i c u l a r d i e t s , weight ga in was r e g r e s s e d a g a i n s t p r o t e i n i n t a k e for each p r o t e i n source ( T a b l e 7 ) . The s lopes of "the r e g r e s s i o n equat ions i n d i c a t e the r a t e of weight ga in r e l a t i v e to p r o t e i n i n t a k e . These are d e p i c t e d g r a p h i c a l l y 51 * 0 5 10 15 20 25 30 35 40 45 50 PERCENT PROTEIN IN DIET F i g . 4. S p e c i f i c growth r a t e s of chinook salmon fed v a r i o u s sources of p r o t e i n at d i f f e r e n t d i e t a r y c o n c e n t r a t i o n . Values p l o t t e d are s p e c i f i c growth r a t e (% wet body w e i g h t s / d a y ) , + 2 s tandard e r r o r s ( S E ) . 52 T a b l e 7. S lopes of body weight gain a g a i n s t the v a r i o u s p r o t e i n s o u r c e s . p r o t e i n i n t a k e for P r o t e i n E q u a t i o n SE of s lope source a FPE y = -0 .263 + 3.33x 0.28 (n=8, r=0.9969, P<0.01) a FRH y = -0 .2483 + 3.23x 0.28 (n=8, r=0.9957, P<0.01) b LTH y = -0 .1522 + 2.56x 0.27 (n=8, r=0.9843, P<0.01) c HTH y = -0 .3842 + 1.78x 0.33 (n=8, r=0.9966, P<0.01) a CS y = -0.4271 + 3.52x 0.24 (n=8, r=0.9848, P<0.01) 1. S lopes wi th the same s u p e r s c r i p t do not d i f f e r s i g n i f i c a n t l y (DMR t e s t , P  0 . 0 5 ) - 53 - ( F i g . 5 ) . The a n a l y s i s i n d i c a t e d that the e f f e c t s of the p r o t e i n source and the c o v a r i a t e ( p r o t e i n i n t a k e ) were s i g n i f i c a n t (P < 0 .0 0 1 ) . The i n t e r c e p t s d i d not d i f f e r s i g n i f i c a n t l y (P > 0.05) s i n c e the r e l a t i o n s h i p s between weight ga in and p r o t e i n i n t a k e were l i n e a r (P < 0.001) and the data for the p r o t e i n - f r e e d i e t fed groups was common for a l l p r o t e i n s o u r c e s . R e l a t i v e to p r o t e i n i n t a k e the c a s e i n - b a s e d d i e t s (CS) supported the h i g h e s t r a t e of weight g a i n . T h i s c o n t r a s t s the growth r e s u l t s above where i n t a k e was not c o n s i d e r e d . T h i s suggests that food i n t a k e was r e s t r i c t e d i n f i s h fed the CS d i e t s . S i m i l a r s l opes were obta ined with the f r e e z e - d r i e d meals . The s lope o b t a i n e d with the low temperature d r i e d meal (LTH) was lower than that obta ined wi th the f r e e z e - d r i e d meals . The lowest s lope was o b t a i n e d with the h igh temperature d r i e d meal (HTH). 3 . 3 . 2 The e f f e c t of d i e t a r y p r o t e i n source and l e v e l on food i n t a k e and gross food c o n v e r s i o n e f f i c i e n c y Food i n t a k e of the groups of f i s h fed by hand to s a t i e t y at each f e e d i n g are shown both on a per 100 f i s h b a s i s ( T F I ) , and on a dry body weight b a s i s ( D F I ) ( H i g g s et a l . 1979, 1982) (Tab le 8 ) . The l a t t e r may be c o n s i d e r e d more meaningfu l s i n c e i t c o r r e c t s for f i s h s i z e and mois ture c o n t e n t . G e n e r a l l y , DFI decreased with i n c r e a s i n g body weight and d i e t a r y p r o t e i n c o n c e n t r a t i o n . The t rend was most ev ident for f i s h fed d i e t s c o n t a i n i n g FPE over a wide range of d i e t a r y p r o t e i n l e v e l s . 54 025 0.50 0.76 LOO PROTEN NTAKE (g/FRH ) F i g . 5 . S lopes of weight ga in a g a i n s t p r o t e i n i n t a k e of chinook salmon fed d i e t s c o n t a i n i n g the t e s t p r o t e i n s o u r c e s . - 55 - T a b l e 8. T o t a l d r y f o o d i n t a k e ( T F l ) ( g / 1 0 0 f i s h ) , mean d a i l y f o o d i n t a k e ( D F I ) ( g / 1 0 0 g mean d r y bod y w e i g h t / d a y ) a nd g r o s s f o o d c o n v e r s i o n ( G F C ) ( P e r c e n t ) o f t h e v a r i o u s d i e t s d u r i n g t h e e x p e r i m e n t a l p e r i o d . P r o t e i n S o u r c e % C r u d e P r o t e i n i n D i e t s 7 17 27 37 47 On O F P E T F I DFI 123 11 .00 162 12 . ,13 188 10, .91 234 9. .89 233 8, .86 247 8, .21 GFC ( + S E ) 0. ,44 +0.74 11 , .50 2 1 . .15 27 .11 29, .86 FRH T F I DFI 196 11 . ,01 180 8. ,49 233 8. .04 GFC 12 , ,68 20. ,69 31 , .12 LTH T F I DFI GFC 173 10 9, . 18 .79 187 8 19, .85 .84 271 10 22 .48 .67 HTH T F I DFI 133 9 , .83 140 9. .54 156 8. .88 GFC 1 , .26 4 , .99 12 , .60 CS T F I DFI 178 10 .98 186 8 .95 176 7 .77 GFC 7 . 19 19 .67 24 .72 OMP T F I DFI 203 8.70 GFC 25.2 8 Gross food c o n v e r s i o n e f f i c i e n c y (GFC) was c a l c u l a t e d as percent c o n v e r s i o n to dry body weight ( B r e t t , 1971; Higgs et a l . , 1982). A r a p i d i n c r e a s e i n GFC was found as d i e t a r y p r o t e i n l e v e l was i n c r e a s e d from 7% to 37% (Tab le 8) . The i n c r e a s e i n GFC between f i s h groups fed FPE i n d i e t s c o n t a i n i n g 37% and 47% p r o t e i n was r e l a t i v e l y s m a l l . The GFC data for f i s h fed the t e s t d i e t s c o n t a i n i n g p r o t e i n sources at l e v e l s of 17%, 27% and 37% were ana lyzed as a two way randomized b lock f a c t o r i a l exper iment . The e f f e c t s of p r o t e i n source and p r o t e i n l e v e l were s i g n i f i c a n t (P < 0.001 and P < 0 . 0 5 ) . The average GFC va lues o b t a i n e d for each s o u r c e , a c r o s s a l l l e v e l s of p r o t e i n , were used as a means of e v a l u a t i n g p r o t e i n q u a l i t y . In t h i s r e g a r d , r e l a t i v e GFC and the r a n k i n g of the p r o t e i n s i s shown i n Tab le ( 9 ) . Values f o r f i s h fed d i e t s with FRH and FPE were h i g h e s t , f o l l o w e d by those for f i s h r e c e i v i n g LTH and CS (Tab le 9 ) . The GFC of f i s h i n g e s t i n g d i e t s wi th HTH r e l a t i v e to tha t of the other p r o t e i n s shows that the HTH d i e t s were extremely p o o r l y u t i l i z e d . 3 . 3 . 3 The e f f e c t of d i e t a r y p r o t e i n source and l e v e l on energy i n t a k e and gross energy u t i l i z a t i o n Food i n t a k e on a d i e t a r y energy b a s i s by the v a r i o u s groups of f i s h was compared on both a gross (GEI) and m e t a b o l i z a b l e (MEI) b a s i s ( T a b l e 10) . Food energy i n t a k e was r e p o r t e d on a dry f i s h body weight b a s i s i n an attempt to c o r r e c t for f i s h s i z e and mois ture c o n t e n t . Food energy i n t a k e decreased as the d i e t a r y p r o t e i n l e v e l i n c r e a s e d . T h i s t rend was noted to be 57 T a b l e 9. G r o s s f o o d c o n v e r s i o n r a n k i n g o f d i f f e r e n t c a l c u l a t e d on p r o t e i n s o u r c e s a d r y b o d y w e i g h t b a s i s ( G F C ) ; r e l a t i v e a t d i f f e r e n t d i e t a r y c o n c e n t r a t i o n . GFC ( i n p a r e n t h e s i s ) and P r o t e i n S o u r c e 17 % C r u d e P r o t e i n i n D i e t s 27 37 Mean FP E GFC ( + S E ) R e l a t i v e GFC R a n k i n g 1 c d 11.50 +1. ( 1 0 0 ) 2 .55 e f 21 . 15 ( 1 0 0 ) 1 h 2 7 . 1 1 ( 1 0 0 ) 2 wx 19. 9 2 +0.89 ( 1 0 0 ) 2 FRH GFC R e l a t i v e GFC R a n k i n g d 12.68 ( 1 1 0 ) 1 e f 2 0 . 6 9 ( 9 8 ) 2 j 31 .12 ( 1 1 5 ) 1 X 21 .50 ( 1 0 8 ) 1 LTH GFC R e l a t i v e GFC R a n k i n g c 9.79 ( 8 5 ) 3 e 19.84 ( 9 4 ) 3 22 .67 ( 8 4 ) 4 w 17 .43 ( 8 8 ) 3 HTH GFC R e l a t i v e GFC R a n k i n g a 1 .26 ( 1 1 ) 5 b 4.99 ( 2 4 ) 5 d 1 2 . 6 0 ( 4 6 ) 5 V 6.28 ( 3 2 ) 5 CS GFC R e l a t i v e GFC R a n k i n g b 7.19 ( 6 3 ) 4 e 19.67 ( 9 3 ) 4 gh 2 4 . 7 2 ( 9 1 ) 3 w 17 . 1 9 ( 8 6 ) 4 Mean GFC P 8.48 +0. .70 q 17 .27 r 2 3 . 6 4 1. V a l u e s w i t h t h e same s u p e r s c r i p t w i t h r e s p e c t t o s o u r c e x l e v e l ( a - i ) a n d p r o t e i n s o u r c e ( v - x ) e f f e c t s d i d n o t d i f f e r s i g n i f i c a n t l y (DMR t e s t P = 0 . 0 5 ) . lower for GEI than for MEI . Energy i n t a k e was s i m i l a r between f i s h fed OMP and f i s h fed FPE-47 which c o n t a i n e d a s i m i l a r p r o t e i n and gross energy c o n c e n t r a t i o n (Tab le 10) . However, OMP supported a lower gross energy u t i l i z a t i o n (GEU) than F P E - 4 7 . Gross energy u t i l i z a t i o n of the d i e t s was noted to be d i r e c t l y r e l a t e d to d i e t a r y p r o t e i n l e v e l (Tab le 10) . At d i e t a r y p r o t e i n l e v e l s above 37% no f u r t h e r i n c r e a s e i n energy u t i l i z a t i o n was noted for f i s h fed d i e t s c o n t a i n i n g F P E . GEU was a l s o employed to compare the q u a l i t y of the p r o t e i n s o u r c e s . The t rend i n GEU of f i s h fed the v a r i o u s p r o t e i n s i s shown i n T a b l e ( 1 1 ) . F i s h fed d i e t s c o n t a i n i n g the f r e e z e - d r i e d meals ( F P E , FRH) and the low-temperature d r i e d meal (LTH) u t i l i z e d d i e t a r y energy most e f f e c t i v e l y . The c a s e i n based d i e t (CS) produced s i m i l a r GEU to L T H . The GEU of the HTH d i e t s was extremely poor . 3 . 3 . 4 The e s t i m a t i o n of endogenous n i t r o g e n l o s s (maintenance r e q u i r e m e n t s ) by j u v e n i l e chinook salmon Two d i e t s were employed to measure endogenous n i t r o g e n l o s s by chinook salmon f r y . One was a p r o t e i n - f r e e d i e t ( fed to s a t i a t i o n ) i n which a l l of the d i e t a r y energy that cou ld p o s s i b l y be used o r i g i n a t e d from c a r b o h y d r a t e and l i p i d . T h e r e f o r e , t i s s u e p r o t e i n would be the only source of n i t r o g e n a v a i l a b l e f o r maintenance purposes . The second d i e t was des igned to p r o v i d e s u f f i c i e n t m e t a b o l i z a b l e energy to s a t i s f y maintenance requirements under more normal p h y s i o l o g i c a l c o n d i t i o n s than a l lowed by the p r o t e i n - f r e e d i e t . E x a c t l y 59 T a b l e 10. Mean d a i l y g r o s s e n e r g y i n t a k e ( G E I ) ( k c a l / l O O g mean d r y b o d y w e i g h t / d a y ) , mean d a i l y m e t a b o l i z a b l e e n e r g y i n t a k e (ME1) ( k c a l / l O O g mean d r y body w e i g h t / d a y ) a n d g r o s s e n e r g y u t i l i z a t i o n ( G E U ) o f t h e v a r i o u s d i e t s d u r i n g t h e e x p e r i m e n t a l p e r i o d . P r o t e i n % C r u d e P r o t e i n i n D i e t s S o u r c e 0 7 17 27 37 47 O o FPE GEI 45, .87 49. .73 45. , 28 42, .33 37. .83 37. .11 MEI 45, .65 48. ,76 4 3 . 09 39. .56 35. .71 32 . 51 GEU ( + SE) - 0 . .29 +1.37 16. ,41 31 , .86 39 , .66 41 . .71 FRH G E I 46, .46 39 .93 36 .34 MEI 44, .04 34 .13 35 .79 GEU 17. .08 29, .43 4 0 , .93 LTH GEI 43 . .06 37 , .70 46. ,32 MEI 41 , .03 34, .69 41 , .50 GEU 14, .42 29 .11 32 . 14 HTH G E I 40, .40 41 .12 39, .52 MEI 38, .34 38 .06 35, .25 GEU 0, .96 6 .38 15, .54 CS G E I 45, .68 37 .68 33, .02 MEI 43, .26 34, .82 29, .60 GEU 8. .20 26, .09 3 3 , .49 OMP GEI 38, .80 MEI 32, .36 GEU 3 3 , .88 T a b l e 1 1 . G r o s s e n e r g y u t i l i z a t i o n ( G E U ) , r e l a t i v e GEU ( i n p a r e n t h e s i s ) a n d r a n k i n g o f d i f f e r e n t p r o t e i n s o u r c e s a t d i f f e r e n t d i e t a r y c o n c e n t r a t i o n s . P r o t e i n S o u r c e 17 % C r u d e P r o t e i n i n D i e t s 27 37 Mean F P E FRH LTH HTH CS GEU ( + S E ) R e l a t i v e GEU R a n k i n g GEU R e l a t i v e GEU R a n k i n g GEU R e l a t i v e GEU R a n k i n g GEU R e l a t i v e GEU R a n k i n g GEU R e l a t i v e GEU R a n k i n g 1 c 16.41 +1.48 ( 1 0 0 ) 2 c 17.08 ( 1 0 4 ) 1 c 14.42 ( 8 8 ) 3 a 0.96 ( 6 ) 5 b 8. 20 ( 5 0 ) 4 31 .86 ( 1 0 0 ) 1 2 9 . 4 3 ( 9 2 ) 2 3 9 . 1 1 ( 9 1 ) 3 h 6.38 ( 2 0 ) 5 d 2 6 . 0 9 ( 8 2 ) 4 de de 3 9 . 6 6 ( 1 0 0 ) 2 f 4 0 . 9 3 ( 1 0 3 ) 1 e 32 .14 ( 8 1 ) 4 c 15.54 ( 3 9 ) 5 e 3 3 . 4 9 ( 8 4 ) 3 29.31 ±0.85 ( 1 0 0 ) 1 x 2 9 . 1 5 ( 9 9 ) 2 wx 2 5 . 2 2 ( 8 6 ) 7.62 ( 2 6 ) 2 2 . 5 9 ( 7 7 ) 4 Mean GEU 11.41 +0.66 24.57 3 2 . 3 5 1. V a l u e s w i t h t h e same s u p e r s c r i p t w i t h r e s p e c t t o s o u r c e x l e v e l ( a - f ) , p r o t e i n s o u r c e ( v - x ) , and p r o t e i n l e v e l ( p - r ) e f f e c t s d i d n o t d i f f e r s i g n i f i c a n t l y (DMR t e s t P = 0 . 0 5 ) . s u f f i c i e n t p r o t e i n energy was p r o v i d e d so that when the f i s h were fed to s a t i a t i o n , over an extended p e r i o d of time (more than 42 d a y s ) , the f i s h would m a i n t a i n t h e i r body we ight . In p r e l i m i n a r y s t u d i e s FPE was shown to be h i g h l y d i g e s t i b l e (97%) by chinook salmon fed d i e t s v a r y i n g i n l e v e l s of p r o t e i n and energy . T h e r e f o r e , i t may be reasonably assumed that the p r o t e i n i n d i e t FPE-7 was almost t o t a l l y absorbed by the f i s h . I t was p o s t u l a t e d that the p r o t e i n i n t a k e r e q u i r e d to support zero growth and exact n i t r o g e n balance r e p r e s e n t e d the maintenance r e q u i r e m e n t . As mentioned p r e v i o u s l y , t h i s corresponds to the endogenous p r o t e i n - n i t r o g e n l o s s of a group of f i s h mainta ined on a p r o t e i n - f r e e d i e t d u r i n g the e x p e r i m e n t a l f eed ing p e r i o d . Body weight data of f i s h fed the p r o t e i n - f r e e and FPE-7 d i e t s for 63 days (Tab le 12) show the e f f e c t s of severe p r o t e i n d e p r i v a t i o n . From day 0 to 42 the f i s h o f f e r e d the p r o t e i n - f r e e d i e t l o s t an average of 22.5% of t h e i r i n i t i a l body we ight . From day 42 to 63 there was no f u r t h e r r e d u c t i o n i n body we ight . F i s h fed FPE-7 c o n t a i n i n g 7% p r o t e i n showed a s l i g h t i n c r e a s e i n body weight from day 0 to day 21. By day 42, however, there was no f u r t h e r ga in i n weight and by day 63 the groups were observed to have mainta ined t h e i r i n i t i a l we ight . I t i s noteworthy that f i s h fed the p r o t e i n - f r e e d i e t and the 7% p r o t e i n d i e t mainta ined t h e i r r e s p e c t i v e day 42 body weights to day 63 on r a t i o n s of s i m i l a r m e t a b o l i z a b l e energy c o n t e n t . The data employed and the e s t imates observed for endogenous n i t r o g e n l o s s are shown i n Tab le 13. Endogenous n i t r o g e n l o s s 62 T a b l e 12. Wet body weight (BW) and percent of i n i t i a l body weight (%BW) of f i s h fed p r o t e i n f r e e (PF) and a maintenance d i e t ( F P E - 7 ) . D i e t PF BW g / f i s h 1.56 (+SE) (+0.04) % BW FPE-7 BW g / f i s h 1.56 (+SE) (+0.04) % BW 21 42 _Day_ 63 1 .44 1.21 1.21 91.93 77.51 77.80 1.67 1 .55 1 .52 07.04 99.33 97 .09 T a b l e 13. E s t i m a t i o n of mean d a i l y endogenouse n i t r o g e n l o s s by c a r c a s s a n a l y s i s of f i s h fed a p r o t e i n - f r e e (PF) and a low p r o t e i n (FPE-7 ) d i e t f or the 42 day e x p e r i m e n t a l p e r i o d . N i t r o g e n N i t r o g e n Endogenous (SE) D i e t ga in i n t a k e n i t r o g e n l o s s ( C a l c u l a t e d on a wet body weight b a s i s (mg/lOOgBW/day)) PF -17 .09 3.63 20.73 (2 .77) FPE-7 -1 .15 26.72 25.52 (0 .18) ( C a l c u l a t e d on a dry body weight b a s i s (mg/1OOgDBW/day)) PF -88 .48 18.80 107.28 (15.60) FPE-7 6.16 130.64 124.96 (0 .16) 63 - was c a l c u l a t e d by adding n i t r o g e n i n t a k e to n i t r o g e n l o s s . The low n i t r o g e n content of the p r o t e i n - f r e e d i e t was assumed to be d e r i v e d from p r o t e i n and was t h e r e f o r e i n c l u d e d i n the d e t e r m i n a t i o n . Endogenous n i t r o g e n l o s s was c a l c u l a t e d to be 20.73 mg n i t r o g e n (N)/100g body weight (BW)/day (SE = 2.77) from the f i s h fed the p r o t e i n - f r e e d i e t and 25.52 mg N/lOOg BW/day (SE = 0.18) from the f i s h fed the maintenance p r o t e i n l e v e l d i e t ( F P E - 7 ) . Because f i s h fed the p r o t e i n - f r e e d i e t were found to have an e l e v a t e d mois ture c o n t e n t , endogenous n i t r o g e n l o s s was a l s o c a l c u l a t e d on a dry body weight b a s i s . In t h i s case , endogenous n i t r o g e n l o s s was es t imated to be 107.28 mg N/lOOg dry BW/day (SE = 15.60) from the groups fed the zero p r o t e i n d i e t and 124.96 mg N/lOOg dry BW/day (SE = 0.16) for the FPE-7 d i e t group. The above va lues show that the p r o t e i n i n t a k e r e q u i r e d f o r maintenance approximates the p r o t e i n l o s s tha t o c c u r r e d when f i s h were fed a n o n - p r o t e i n d i e t . The va lues obta ined from f i s h fed the 7% p r o t e i n d i e t (Tab le 13) can be employed to c o r r e c t p r o t e i n q u a l i t y e s t imates for endogenous n i t r o g e n l o s s (ENL) or p r o t e i n l o s s (EPL)(ENL x 6 . 2 5 ) . S i n c e , endogenous n i t r o g e n l o s s changes i n d i r e c t p r o p o r t i o n to f i s h body weight ( s i z e ) ( B r e t t and Groves , 1979) the method of Ogino (1980) was adapted as f o l l o w s : ENL (g) = W W - 5 1 + 2 x 25.52 x 10 x d 2 EPL (g) = W 1 + W 2 x 159.5 x 10 - 5 x d 2 64 DW DW _ 5 ENL (g) = 1 + 2 x 124.96 x 10 x d 2 DW DW _ 5 EPL (g) 1 • + 2 x 781.0 x 10 x d 2 ENL = endogenous n i t r o g e n l o s s EPL = endogenous p r o t e i n l o s s W l = i n i t i a l body weight (g) W2 = f i n a l body weight (g) D w l = i n i t i a l dry body weight (g) D W 2 = f i n a l dry body weight (g) d = days of f e e d i n g 3 . 3 . 5 The r e l a t i o n s h i p between p r o t e i n i n t a k e and p r o t e i n u t i l i z a t i o n To i l l u s t r a t e the r e l a t i o n s h i p of n i t r o g e n balance and p r o t e i n q u a l i t y , the way i n which the groups of f i s h u t i l i z e d the v a r i o u s d i e t a r y p r o t e i n sources was i n v e s t i g a t e d . P r o t e i n i n t a k e was p a r t i t i o n e d i n t o the amounts of p r o t e i n u t i l i z e d for maintenance and growth of body t i s s u e s and the amount l o s t through exogenous f e c a l and metabo l i c e x c r e t i o n (Tab le 14) . In t h i s d i s c u s s i o n , exogenous e x c r e t i o n s r e f e r to the f r a c t i o n of p r o t e i n i n t a k e that was not d i g e s t e d p lus the amount used for energy ( F i g . l ) . T h i s was o b t a i n e d by the d i f f e r e n c e between p r o t e i n i n t a k e and , endogenous p r o t e i n l o s s (equal to maintenance , F i g . 1) p lus body p r o t e i n g a i n . The q u a n t i t i e s were expressed i n mg per lOOg wet body weight per day 65 Table 14. The u t i l i z a t i o n of dietary protein on a d a i l y basis by f i s h fed the various experimental diets during the 42 day period. Amount of Protein U t i l i z e d by lOOg f i s h for: Percent of Protein Intake used for: Diet Protein Intake (mg/lOOgBW/d) Maintenance Growth (mg/lOOgBW/d) Excretion Maintenance Growth Excretion FPE-7 167.0 160 7.1 0 96.0 4.3 0 FPE-17 395.3 160 146.6 88.7 40.5 37.0 22.5 FPE-27 604.1 160 298.2 145.9 26.6 49.4 24.1 FPE-37 728.9 160 351.4 217.5 22.0 48.2 29.9 FPE-47 898.6 160 378.8 359.8 17.6 42.2 40.3 OMP 941.4 160 312.7 468.7 17.0 32.3 49.8 FRH-17 422.8 160 154.0 108.9 38.3 40.5 21.3 FRH-27 511.8 160 227.5 111.7 31.3 44.5 24.2 FRH-37 749.9 160 321.4 268.6 21.3 41.8 36.9 LTH-17 366.4 160 131.0 75.5 43.7 35.8 20.5 LTH-27 554.5 160 232.6 161.9 ° 28.9 42.0 29.1 UTH-27 914.3 160 340.5 413.8 17.5 37.3 45.2 HIH-17 341.7 160 -16.2 197.8 46.9 -4.1 57.2 HIH-27 534.8 160 53.3 321.6 30.0 10.0 60.1 HIH-37 754.8 160 121.9 473.0 21.2 16.2 62.6 CS-17 417.7 160 88.6 169.2 38.4 21.4 40.2 CS-27 500.9 160 238.6 102.5 32.0 43.1 25.0 CS-37 609.6 160 257.6 191.9 26.3 42.3 31.5 - 66 - (mg/lOOg BW/day) . Next , the u t i l i z a t i o n of p r o t e i n for maintenance , growth and the q u a n t i t y e x c r e t e d , was expressed as a percentage of the p r o t e i n fed (Tab le 14) and has been d e p i c t e d g r a p h i c a l l y ( F i g s . 6A - 6 E ) . In regard to f i s h i n g e s t i n g F P E , the l e v e l of p r o t e i n i n t a k e for maintenance was 160mg/100g BW/day. At t h i s l e v e l p r a c t i c a l l y a l l of the p r o t e i n was absorbed and u t i l i z e d for maintenance and none was wasted or a v a i l a b l e f o r growth ( F i g . 6 A ) . Between maintenance and o p t i m a l p r o t e i n u t i l i z a t i o n (Op) , the percentage of p r o t e i n i n t a k e used for growth rose s h a r p l y , whereas the p r o p o r t i o n employed for maintenance dropped r a p i d l y ( F i g . 6 A ) . A d d i t i o n a l p r o t e i n i n t a k e above the optimum r e s u l t e d i n a decreased r a t e of u t i l i z a t i o n for growth because of an i n c r e a s e i n the percentage of p r o t e i n l o s t i n e x c r e t a . FPE p r o t e i n was most e f f i c i e n t l y u t i l i z e d for growth at a p r o t e i n i n t a k e of approx imate ly 600mg/100g BW/day. In c o n t r a s t to f i s h r e c e i v i n g d i e t s c o n t a i n i n g F P E , the f i s h fed OMP u t i l i z e d p r o t e i n l e s s e f f e c t i v e l y for growth l a r g e l y because of i n c r e a s e d e x c r e t o r y l o s s e s ( F i g . 6 A ) . The method of h e r r i n g meal p r o c e s s i n g markedly i n f l u e n c e d the way p r o t e i n was u t i l i z e d ( F i g . 6B, 6C, 6D) . In comparison to a l l o ther groups , chinook fed d i e t s c o n t a i n i n g HTH u t i l i z e d p r o t e i n p o o r l y . Only 16.5% of the p r o t e i n fed was u t i l i z e d for growth i n d i e t s wi th a p r o t e i n c o n c e n t r a t i o n of 37% (Tab le 14) . A h igher p r o p o r t i o n of p r o t e i n i n t a k e was r e q u i r e d by the f i s h fed d i e t s wi th HTH r e l a t i v e to those fed d i e t s c o n t a i n i n g FRH or LTH to meet t h e i r maintenance 67 F i g . 6A - E . Percent u t i l i z a t i o n of p r o t e i n fed for maintenance (O) and growth ( A ) , and percent e x c r e t e d (•) when f i s h fed the t e s t p r o t e i n sources at v a r i o u s l e v e l s of p r o t e i n i n t a k e . (Op) stands for the p r o t e i n i n t a k e that promotes o p t i m a l u t i l i z a t i o n of p r o t e i n f o r growth. I t i s noteworthy that percent u t i l i z a t i o n for growth (A) i s the same as p r o t e i n p r o d u c t i v e va lue (PPV) (see F i g . 1 ) . The p r o t e i n u t i l i z a t i o n of f i s h fed OMP i s shown i n dark symbols f or comparison with each of the t e s t p r o t e i n s . The dashed p o r t i o n s of the curves were e x t r a p o l a t e d . - 68 PROTEIN UTILIZATION I X Of PROTEIN P(0| PROTEIN UTILIZATION I Z OP PROTEIN FED ) 8 3 8 8 PROTEIN UTILIZATION (X OP PROTEIN o o o o o S o o PED| 2* 2 M « 8 I § m\ \ \ • M T l » FPE. ? V ! • FPE. PROTEIN UTILIZATION (Z OP PROTEIN requirements for p r o t e i n . F u r t h e r , at the h i g h e s t l e v e l of p r o t e i n i n t a k e over 60% of the p r o t e i n fed was e x c r e t e d ( F i g . 6D) . Because of an i n c o n s i s t e n c y i n the performance of f i s h fed CS at low l e v e l s , and a p a l a t a b i l i t y problem noted i n f i s h i n g e s t i n g h igh d i e t a r y l e v e l s of CS, the p a t t e r n of p r o t e i n u t i l i z a t i o n was d i f f i c u l t to c o n s t r u c t and i n t e r p r e t f o r the CS groups ( F i g . 6 E ) . The data i n d i c a t e d that CS was a good source of p r o t e i n for meeting the p r o t e i n requirements of chinook salmon i n terms of p r o t e i n u t i l i z a t i o n . 3 . 3 . 6 The measurement of p r o t e i n q u a l i t y by p r o t e i n e f f i c i e n c y r a t i o and net p r o t e i n r a t i o P r o t e i n e f f i c i e n c y r a t i o (PER)(Osborne et a l . , 1919) and net p r o t e i n r a t i o (NPR)(Bender and D o e l l , 1957) are commonly used to assess p r o t e i n q u a l i t y . F i g u r e 7 shows the r e l a t i o n s h i p between d i e t a r y p r o t e i n c o n c e n t r a t i o n and PER i n f i s h fed FPE d i e t s . P r e d i c t a b l y i t f o l l o w e d a c l a s s i c a l t rend s i m i l a r to tha t r e p o r t e d for r a t s and p l a i c e by other i n v e s t i g a t o r s . The noteworthy d i f f e r e n c e s among s p e c i e s are the p r o t e i n c o n c e n t r a t i o n s at which maximum PER o c c u r r e d . F i g u r e 8 shows the extent to which PER was dependent on p r o t e i n i n t a k e . The e f f e c t of low p r o t e i n i n t a k e was p a r t i a l l y overcome once an a l lowance was made f o r maintenance by the NPR method. The d i f f e r e n c e between the PER and NPR curve at d i f f e r e n t p r o t e i n 70 -0-5 1 • • • 1 • 1 • 1 1 ' ' ' 0 5 10 15 20 25 30 35 40 45 50 55 60 P E R C E N T P R O T E I N I N D I E T F i g . 7. E f f e c t of d i e t a r y p r o t e i n l e v e l on p r o t e i n e f f i c i e n c y r a t i o (PER) c a l c u l a t e d on a wet body weight b a s i s for chinook salmon f r y fed F P E , for p l a i c e fed f r e e z e - d r i e d cod muscle (from Cowey et a l . , 1972) and for r a t s fed c a s e i n (from Hegsted and Chang, 1965) c o n t a i n i n g d i e t s . 71 T a b l e 1 5 . P r o t e i n e f f i c i e n c y r a t i o c a l c u l a t e d on a d r y body w e i g h t b a s i s ( P E R ) , r e l a t i v e PER ( i n p a r e n t h e s i s ) and r a n k i n g o f d i f f e r e n t p r o t e i n s o u r c e s a t d i f f e r e n t d i e t a r y c o n c e n t r a t i o n s . VI P r o t e i n S o u r c e 17 % C r u d e 27 P r o t e i n i n D i e t s 37 Mean FP E PER (+SE) R e l a t i v e R a n k i n g PER 1 d e f 0 .679 +0.010 ( 1 0 0 ) 2 f 0.776 ( 1 0 0 ) 1 f 0.754 ( 1 0 0 ) 2 X 0.736 +0.06 ( 1 0 0 ) 2 FRH PER R e l a t i v e R a n k i n g PER f 0.704 ( 1 0 3 ) 1 f 0.750 ( 9 6 ) 2 f 0.786 ( 1 0 5 ) 1 X 0.747 ( 1 0 1 ) 1 LTH PER R e l a t i v e R a n k i n g PER d 0.583 ( 8 5 ) 3 e f 0.697 ( 9 0 ) 4 de 0 . 590 ( 7 9 ) 4 w 0.623 ( 8 4 ) 3 HTH PER R e l a t i v e R a n k i n g PER a 0.074 ( 1 0 ) 5 b 0.182 ( 2 3 ) 5 c 0.315 ( 4 3 ) 5 v 0.191 ( 2 6 ) 5 CS PER R e l a t i v e R a n k i n g PER c 0.387 ( 5 7 ) 4 f 0.745 ( 9 6 ) 3 d e f 0.687 ( 9 2 ) 3 w 0.606 ( 8 2 ) 4 Mean PER 0.486 +0.055 0.630 0.626 1. V a l u e s w i t h t h e same s u p e r s c r i p t w i t h r e s p e c t t o s o u r c e x l e v e l ( a - f ) and p r o t e i n s o u r c e ( v - x ) e f f e c t s d i d n o t d i f f e r s i g n i f i c a n t l y (DMR t e s t P = 0 . 0 5 ) . 100 200 300 400 500 600 700 800 900 PROTEIN INTAKE (mg/IOOg BV^doy) F i g . 8. The r e l a t i o n s h i p between p r o t e i n i n t a k e and p r o t e i n e f f i c i e n c y r a t i o (PER)(0) and net p r o t e i n r a t i o n (NPR)(A) of f i s h fed d i e t s c o n t a i n i n g F P E . Values for OMP are shown i n dark symbols for c o m p a r i s o n . 73 i n t a k e s i s due to the a l lowance made f o r maintenance by the l a t t e r method ( F i g . 8 ) . Those d i e t s f ormula ted to have a p r o t e i n c o n c e n t r a t i o n of 17, 27, and 37% were employed to e v a l u a t e p r o t e i n q u a l i t y . The e f f e c t of d i e t a r y p r o t e i n source on PER and NPR was s i g n i f i c a n t (P < 0 . 0 0 5 ) . Desp i t e the d i f f e r e n c e s i n PER and NPR both methods d i s t i n g u i s h e d between the d i f f e r e n t p r o t e i n sources i n an almost i d e n t i c a l manner ( T a b l e s 15 and 16) . I n c o n s i s t a n t d i f f e r e n c e s i n r e l a t i v e PER and NPR va lues were noted for the p r o t e i n s at d i f f e r e n t d i e t a r y c o n c e n t r a t i o n s . E x c l u d i n g the CS d i e t s , i t was found that r e l a t i v e PER was lower for th~e poor q u a l i t y p r o t e i n (HTH) at low compared to h igh d i e t a r y p r o t e i n c o n c e n t r a t i o n s . T h i s e f f e c t was not noted for the f r e e z e - d r i e d meals ( F P E , FRH) . Low-temperature d r i e d meal (LTH) show maximum PER at the i n t e r m e d i a t e p r o t e i n c o n c e n t r a t i o n . The above o b s e r v a t i o n s may suggest that PER underes t imates the p r o t e i n q u a l i t y of poor p r o t e i n s when t e s t e d at low d i e t a r y l e v e l s . NPR on the other hand prov ided s i m i l a r va lues at d i f f e r e n t d i e t a r y p r o t e i n c o n c e n t r a t i o n s . However, an i d e n t i c a l r a n k i n g of the p r o t e i n s a c c o r d i n g to mean r e l a t i v e (FPE = 100) PER and NPR va lues was o b t a i n e d . Comparisons with r e p o r t s by other i n v e s t i g a t o r s (Tab le 17) were made by t a b u l a t i n g PER r e l a t i v e to CS ( i . e . PER of CS = 100). The r e s u l t s i n d i c a t e d that HTH was an extremely poor q u a l i t y p r o t e i n whereas the other f i s h m e a l s were comparable to those r e p o r t e d i n the l i t e r a t u r e . 74 T a b l e 16. Net p r o t e i n r a t i o c a l c u l a t e d on a d r y bo d y v e i g h t b a s i s ( N P R ) , r e l a t i v e NPR ( i n p a r e n t h e s i s ) a n d r a n k i n g o f d i f f e r e n t p r o t e i n s o u r c e s a t d i f f e r e n t d i e t a r y c o n c e n t r a t i o n s . P r o t e i n S o u r c e 17 % C r u d e P r o t e i n i n D i e t s 27 37 Mean FPE FRH NPR (+SE) R e l a t i v e NPR R a n k i n g NPR R e l a t i v e NPR R a n k i n g 1 e f 0.974 +0.009 ( 1 0 0 ) 2 0.993 ( 1 0 2 ) 1 c d e f 0.921 ( 1 0 0 ) 3 d e f 0.934 ( 1 0 1 ) 1 c d 0.864 ( 1 0 0 ) 2 c d e 0.884 ( 1 0 2 ) 1 0 . 9 20 +0.005 ( 1 0 0 ) 2 0.937 ( 1 0 2 ) 1 xy LTH NPR R e l a t i v e NPR R a n k i n g c d e f 0.901 ( 9 3 ) 3 c d 0.872 ( 9 5 ) 4 0.679 ( 7 9 ) 4 0.817 ( 8 9 ) 3 HTH CS NPR R e l a t i v e NPR R a n k i n g NPR R e l a t i v e NPR R a n k i n g a 0.481 ( 4 9 ) 5 0.667 ( 6 9 ) 4 0.422 ( 4 6 ) 5 d e f 0.934 ( 1 0 1 ) 1 0.464 ( 5 3 ) 5 0.833 ( 9 7 ) 3 0.456 ( 5 0 ) 5 0.811 ( 8 8 ) 4 Mean NPR 0.803 +0.004 0.816 0 . 7 4 5 1. V a l u e s w i t h t h e same s u p e r s c r i p t w i t h r e s p e c t t o s o u r c e x l e v e l ( a - f ) a n d p r o t e i n s o u r c e ( v - y ) e f f e c t s d i d n o t d i f f e r s i g n i f i c a n t l y (DMR t e s t P = 0 . 0 5 ) . Table 17. Protein e f f i c i e n c y r a t i o (PER), r e l a t i v e PER, net protein u t i l i z a t i o n (NPU), and r e l a t i v e NPU for various proteins, when fed as the sole source of protein to d i f f e r e n t f i s h species held under d i s s i m i l a r experimental conditions. Protein Source of Diet Species Size (g) Temperature degree C Ration Protein l e v e l % of dry d i e t PER wet body weight basis Relative PER (casein=100) NPU 1' 2 Relative NPU (casein=100) Reference Freeze dried cod muscle Plaice (Pleuronectes platessa 13 15 s a t i a t i o n 50 1.78 42 Cowey et a l . (1974) Single c e l l protein plus methionine 14 15 it 50 1.23 38 White f i s h meal M 15 15 50 1.29 35 Soy protein plus methionine M 14 15 " 50 0.89 34 F i s h protein concentrate " 13 15 50 0.60 23 L i p i d extracted herring muscle II 13 15 50 0.84 30 L i p i d extracted sprats 14 15 50 0.98 33 Leaf protein concentrate Carp (cyprinos carpio) 2.5 25 not stated 41 1.78 68 34 74 Ogino et a l . (1978) Casein 2.5 25 43 2.60 100 46 100 Leaf protein concentrate 2.5 25 30 2.09 59 38 72 Casein 2.5 25 27 3.55 100 53 100 cont'd.. ./2 Table 17. cont'd..(2) Protein Source of Diet Species Size (g) Temperature degree C Ration Protein l e v e l % of dry d i e t PER wet body weight basis Relative PER (casein=100) NPU 1 ' 2 Relative NPU (casein=>100) Reference Leaf protein concentrate Rainbow trout (salmo gairdneri) 3.5 19 sat i a t i o n 41 2.03 70 38 69 Ogino et a l . (1978) Casein ii 3.5 19 39 2.91 100 55 100 Herring meal Rainbow trout (salmo gairdneri) 30 12 •• 30 1.91 97 38 95 Atadc and Matty (1978) htethanophilic bacteria 30 12 II 30 1.62 82 37 93 S p i r u l i n a algae 30 12 30 1.33 68 32 80 Petroyeast n 30 12 « 30 2.01 102 42 105 Soybean » 30 12 30 18 45 Brewers yeast 30 12 II 30 1.17 59 30 75 Casein " 30 12 30 1.97 100 40 100 Herring meal Carp (cyprinus carpio) 30 25 4% of body weight 25 2.82 114 64 131 Atadc et a l . (1979) Methanophilic bacteria " 30 25 " 35 2.54 102 49 100 Spiru l i n a algae 30 25 30 1.15 42 36 73 Petroyeast " 30 25 31 2.08 84 47 97 Soybean 30 25 29 1.35 54 42 86 cont'd.../3 Table 17. cont'd..(3) Protein Source Species Size Temperature Ration Protein PER Relative 1 2 NPU ' Relative Reference of Diet (g) degree C l e v e l % wet body PER NPU of dry weight (casein=100) (casein=100) d i e t basis Casein Carp (cyprinus carpioT 30 25 4% of body 30 weight 2.48 100 49 100 Atack et a l (1979) White f i s h meal Rainbow trout 34 (saliro gairdneri) Peruvian anchovy " 34 meal Sardine and pollock " 34 scrap meal Greenling meal " 34 Casein " 34 10.7 not stated 31 10.7 " 31 10.7 10.7 10.7 32 30 33 3.2 3.7 3.2 3.6 3.6 89 103 89 100 100 63 61 63 66 66 95 92 95 100 100 Watanabe et a l . (1983) Egg yolk Whole egg Egg albumin Trout muscle protein Squid muscle protein Rainbow trout 2.0 (salmo gairdneri) 2.0 2.0 2.0 2.0 2.0 17 17 17 17 17 17 not stated 30 30 30 30 30 30 3.8 3.8 3.9 3.8 3.9 3.5 109 109 111 109 111 100 60 61 62 62 62 56 107 109 111 111 IU 100 Ogino and Nanri (1980) cont'd.../4 Table 17. cont'd..(4) -o Protein Source of Diet Species Size (g) Temperature degree C Ration Protein l e v e l % of dry d i e t PER wet body weight basis Relative PER (casein=100) NPU 1 , 2 Relative NPU (casein=100) Reference Casein & squid muscle (1:1) Rainbow trout (salrtD gairdneri) 4.0 17 not stated 30 3.9 108 61 105 Ogino and Nanri (1980) Casein & whole (1:1) egg 4.0 17 30 3.8 106 63 109 Casein 4.0 17 30 3.6 100 58 100 FPE Chinock salmon (oncorhynchus tshawytscha) 1.50 11 s a t i a t i o n 27 2.94 98 79 103 Present study FRH 1.50 11 28 2.87 96 78 101 LTH 1.50 11 " 28 2.65 88 73 95 HTH II 1.50 11 » 27 0.76 25 40 52 CS » 1.50 11 26 3.00 100 77 100 (casein-gelatin, + arginine & inetJuonine) T. Determined by method of Bender and M i l l e r (1953). 2. rjetermined by method o f Ogino et al.(1980). 3 . 3 . 7 The measurement of p r o t e i n q u a l i t y by p r o t e i n p r o d u c t i v e va lue and net p r o t e i n u t i l i z a t i o n P r o t e i n p r o d u c t i v e va lue (PPV) and net p r o t e i n u t i l i z a t i o n (NPU) ( M i l l e r and Bender , 1955; Ogino et a l . , 1980) r e l a t e p r o t e i n r e t e n t i o n to p r o t e i n i n t a k e . PPV and NPU are analogous to PER and NPR r e s p e c t i v e l y . The l a t t e r methods, however, cannot d i f f e r e n t i a t e between p r o t e i n s that promote s i m i l a r ga ins i n body weight but d i f f e r e n t ga ins i n body p r o t e i n . T h e r e f o r e , PPV and NPU would best measure the e f f e c t i v e n e s s of a p r o t e i n to p r o v i d e for muscle p r o t e i n s y n t h e s i s . The va lues obta ined for PPV are shown i n Tab le (18) and are d e p i c t e d g r a p h i c a l l y for the v a r i o u s p r o t e i n sources i n F i g . ( 6 A - 6 E ) . PPV i s i d e n t i c a l to the es t imate of the percentage of p r o t e i n fed that was r e t a i n e d . The r e l a t i o n s h i p between t h i s parameter and p r o t e i n i n t a k e was p r e v i o u s l y d e s c r i b e d wi th r e s p e c t to the mode of p r o t e i n u t i l i z a t i o n for growth ( s e c t i o n 3 . 3 . 5 ) . A p r o g r e s s i v e decrease i n mean PPV was observed as the p r o t e i n source i n chinook salmon d i e t s changed i n the f o l l o w i n g o r d e r : FPE > FRH > LTH > CS > HTH (Tab le 18) . As s t a t e d p r e v i o u s l y the q u a l i t y of the f r e e z e - d r i e d meals was h i g h e s t . Low temperature d r i e d meal was found to have a lower (not s t a t i s t i c a l l y s i g n i f i c a n t P > 0.05) q u a l i t y than f r e e z e - d r i e d h e r r i n g meal (FRH) . The c a s e i n based p r o t e i n source (CS) was of s i m i l a r q u a l i t y to L T H . An i d e n t i c a l r a n k i n g (of mean v a l u e s ) for the p r o t e i n sources was o b t a i n e d by PPV and NPU ( T a b l e s 18, 19, 20) . A l though NPU was c a l c u l a t e d by two methods, the 80 T a b l e 18. P r o t e i n p r o d u c t i v e v a l u e s ( P P V ) , r e l a t i v e PPV ( i n p a r e n t h e s i s ) a n d r a n k i n g o f d i f f e r e n t p r o t e i n s o u r c e s a t d i f f e r e n t d i e t a r y c o n c e n t r a t i o n s . P r o t e i n S o u r c e 17 % C r u d e P r o t e i n i n 27 D i e t s 37 Mean FPE PPV (+SE) R e l a t i v e PPV R a n k i n g 1 d 3 6 . 8 7 +3.23 ( 1 0 0 ) 2 f 4 9 . 5 5 ( 1 0 0 ) 1 e f 4 8 . 1 1 ( 1 0 0 ) 1 X 4 4 . 8 4 +1.87 ( 1 0 0 ) 1 FRH PPV R e l a t i v e PPV R a n k i n g d e f 4 0 . 4 5 ( 1 1 0 ) 1 d e f 4 4 . 5 6 ( 9 0 ) 2 d e f 41 .78 ( 8 7 ) 3 wx 4 2 . 2 6 ( 9 4 ) 2 LTH PPV R e l a t i v e PPV R a n k i n g d 34 .23 ( 9 3 ) 3 d e f 4 2 . 0 8 ( 8 5 ) 4 de 3 7 . 5 3 ( 7 8 ) 4 wx 3 7 . 9 5 ( 8 5 ) 3 HTH PPV R e l a t i v e PPV R a n k i n g a - 4 . 8 1 (-13) 5 b 9.81 ( 2 0 ) 5 be 1 6 . 2 3 ( 3 4 ) 5 v 7.07 ( 1 6 ) 5 CS PPV R e l a t i v e PPV R a n k i n g c 2 1 . 5 0 ( 5 8 ) 4 d e f 4 3 . 0 6 ( 8 7 ) 3 d e f 4 2 . 2 3 ( 8 8 ) 2 w 3 5 . 5 9 ( 7 9 ) 4 Mean PPV 25.65 +1.45 37.81 3 7 . 18 1. d i d V a l u e s w i t h t h e same s u p e r s c r i p t w i t h r e s p e c t n o t d i f f e r s i g n i f i c a n t l y (DMR t e s t P - 0 . 0 5 ) . t o s o u r c e x l e v e l ( a • - f ) and p r o t e i n s o u r c e ( v - x ) e f f e c t s T a b l e 1 9 . Net p r o t e i n u t i l i z a t i o n c a l c u l a t e d by t h e m e t h o d o f B e n d e r a nd M i l l e r ( 1 9 5 3 ) ( N P U - 1 ) , r e l a t i v e NPU-1 ( i n p a r e n t h e s i s ) a n d r a n k i n g o f d i f f e r e n t p r o t e i n s o u r c e s a t d i f f e r e n t d i e t a r y c o n c e n t r a t i o n s . P r o t e i n S o u r c e 17 % C r u d e P r o t e i n 27 i n D i e t s 37 Mean F P E NPU-1 ( + S E ) R e l a t i v e NPU R a n k i n g 1 de 5 6 . 8 8 +0.90 ( 1 0 0 ) 2 e 59.31 ( 1 0 0 ) 1 de 5 5 . 6 5 ( 1 0 0 ) 1 w 5 7 . 3 2 +0.52 ( 1 0 0 ) 1 FRH NPU-1 R e l a t i v e NPU R a n k i n g e 60.02 ( 1 0 5 ) 1 de 57 .00 ( 9 6 ) 2 bed 4 8 . 4 2 ( 8 7 ) 3 V 5 5 . 1 4 ( 9 6 ) 2 LTH NPU-1 R e l a t i v e NPU R a n k i n g de 55.74 ( 9 8 ) 3 de 5 3 . 8 6 ( 9 1 ) 4 be 4 3 . 5 ( 7 8 ) 4 w 5 1 . 1 0 ( 8 9 ) 3 HTH NPU-1 R e l a t i v e NPU R a n k i n g a 22 .69 ( 4 0 ) 5 a 26.12 ( 4 4 ) 5 a 2 6 . 3 2 ( 4 7 ) 5 V 2 5 . 0 4 ( 4 4 ) 5 CS NPU-1 R e l a t i v e NPU R a n k i n g b 40.41 ( 7 1 ) 4 de 5 5 . 7 8 ( 9 4 ) 3 c d e 5 2 . 2 7 ( 9 4 ) 2 w 4 9 . 5 1 ( 8 6 ) 4 Mean NPU-1 47.11 +0.40 50.41 4 5 . 2 3 1. V a l u e s w i t h t h e same s u p e r s c r i p t w i t h r e s p e c t t o s o u r c e x l e v e l ( a - e ) a n d p r o t e i n s o u r c e ( v - x ) e f f e c t s d i d n o t d i f f e r s i g n i f i c a n t l y (DMR t e s t P = 0 . 0 5 ) . T a b l e 2 0 . Net p r o t e i n u t i l i z a t i o n c a l c u l a t e d by t h e m e t h o d o f O g i n o e t a l . ( 1 9 8 0 ) ( N P U - 2 ) , r e l a t i v e N P U - 2 ( i n p a r e n t h e s i s ) and r a n k i n g o f d i f f e r e n t p r o t e i n s o u r c e s a t d i f f e r e n t d i e t a r y c o n c e n t r a t i o n s . P r o t e i n Sou r c e 17 % C r u d e P r o t e i n i n D i e t s 27 37 Mean 00 CO FPE FRH LTH NPU-2 ( + S E ) R e l a t i v e NPU-2 Rank i n g NPU-2 R e l a t i v e NPU-2 R a n k i n g NPU-2 R e l a t i v e NPU-2 R a n k i n g 1 e f 7 9 . 0 6 +3.46 ( 1 0 0 ) 3 f 8 4 . 0 5 ( 1 0 6 ) 1 e f 7 9.57 ( 1 0 1 ) 2 e f d e f 7 8 . 6 9 ( 1 0 0 ) 1 7 7 . 6 9 ( 9 9 ) 2 73.07 ( 9 3 ) 4 e f d e f 7 2 . 5 6 ( 1 0 0 ) 1 6 5 . 7 8 ( 9 1 ) 3 b 5 6 . 9 9 ( 7 9 ) 4 bed 7 6 . 7 4 +2.00 ( 1 0 0 ) 1 wx 75.84 ( 9 9 ) 2 wx 6 9 . 8 8 ( 9 1 ) HTH CS NPU-2 R e l a t i v e NPU-2 R a n k i n g NPU-2 R e l a t i v e NPU-2 R a n k i n g 41 .85 ( 5 3 ) 5 59 .82 ( 7 6 ) 4 be 3 9 . 7 0 ( 5 1 ) 5 7 6 . 5 6 ( 9 7 ) 3 d e f 3 8.24 ( 5 3 ) 5 7 0 . 1 0 ( 9 7 ) 2 c d e 39 .93 ( 5 2 ) 6 8 . 8 9 ( 9 0 ) 4 Mean NPU-2 68.87 +1.55 6 9 . 1 6 6 0 . 7 4 1. V a l u e s w i t h t h e same s u p e r s c r i p t w i t h r e s p e c t t o s o u r c e x l e v e l ( a - f ) and p r o t e i n s o u r c e ( v - x ) e f f e c t s d i d n o t d i f f e r s i g n i f i c a n t l y (DMR t e s t P = 0 . 0 5 ) . T a b l e 21. The r e l a t i o n s h i p between p r o t e i n i n t a k e (x) and net p r o t e i n u t i l i z a t i o n (NPU)(y) c a l c u l a t e d by the Bender and M i l l e r (1953) f o r m u l a . Chinook Salmon (present s tudy) P r o t e i n Source E q u a t i o n FPE y = = 65 .01 -- 0 .0161x (n=10, r = -0 .5611 , NS) FRH y = = 73 .81 -- 0 .0333x (n=6, r = - 0 . 8 8 4 8 , P<0 .05) LTH y = = 65 .28 -- 0 .0233x (n = 6, r = - 0 . 8 3 5 8 , P<0.05) HTH y = = 20 .38 -- 0 .0085x (n=6, r = 0 .3732, NS) CS y = = 22 .17 -- 0 .0536x (M=6, r = 0 .6036, NS) - 84 - T a b l e 22. The r e l a t i o n s h i p between p r o t e i n i n t a k e (x) and net p r o t e i n u t i l i z a t i o n (NPU)(y) c a l c u l a t e d by the method of Ogino et a l . (1980) Chinook Salmon (present s tudy) P r o t e i n Source E q u a t i o n FPE y = 104.76 - 0.0476x (n=10, r= - 0 . 9 1 2 0 , P<0.01) FRH y = 106.45 - 0.0545x (n=6, r= - 0 . 9 5 9 4 , P<0.01) LTH y = 95.69 - 0.0422x (n=6, r= - 0 . 8 5 9 5 , P<0.05) HTH y = 45.15 - 0.0096x (N=6, r= - 0 . 6 5 9 9 , NS) CS y = 47.93 + 0.0412x (n=6, r= 0 .4076, NS) Rainbow T r o u t ( c a l c u l a t e d from Ogino et a l . , 1980) Egg yo lk y = 97.9 - 0.0458x (n=5, r= -0 .9664 , P<0.01) P l a i c e ( c a l c u l a t e d from Cowey et a l . , 1972) F r e e z e - d r i e d cod muscle y = 75.8 - 0.0579x (n=6, r= - 0 . 9 8 2 3 , P<0.01) 85 - I U Z 10 . 0 0 100 200 300 400 500 600 700 800 900 1000 PROTEIN INTAKE (mg/100g BW/day) F i g . 9. The r e l a t i o n s h i p between p r o t e i n i n t a k e and net p r o t e i n u t i l i z a t i o n (NPU) of the t e s t p r o t e i n sources determined by the method of Ogino et a l . (1980) . S lopes for rainbow t r o u t fed egg yo lk ( taken from Ogino et a l . , 1980) and p l a i c e fed cod muscle ( taken from Cowey et a l . , 1972) c o n t a i n i n g d i e t s are a l s o shown f o r compar i son . The NPU va lue for f i s h fed OMP i s a l s o shown. 86 Bender and M i l l e r (1953) (NPU-1) and Ogino et a l . ( 1 9 8 0 ) (NPU-2) r e s p e c t i v e l y , very c l o s e r e l a t i v e NPU (FPE = 100) va lues were obta ined by both p r o c e d u r e s . As o u t l i n e d p r e v i o u s l y , NPU-2 a l l o w s for maintenance on a body weight b a s i s . R e l a t i v e NPU (CS = 100) va lues obta ined i n the present study are t a b u l a t e d below those r e p o r t e d by other i n v e s t i g a t o r s (Tab le 17) for compar i son . The r e l a t i o n s h i p between p r o t e i n i n t a k e and NPU was d e s c r i b e d by r e g r e s s i o n equat ions ( T a b l e s 21, 22) . When the method of Ogino et a l . ( 1 9 8 0 ) was employed a s i g n i f i c a n t i n v e r s e dependence of NPU on p r o t e i n i n t a k e was e v i d e n t f o r FPE (P < 0.01) FRH (P < 0 . 0 1 ) , and LTH (P < 0 . 0 5 ) . However, when the method of Bender and M i l l e r (1953) was used , the c o r r e l a t i o n between NPU and p r o t e i n i n t a k e was c o n s i d e r a b l y l e s s (Tab le 21) . Co wey and Sargent (1972) s t a t e that i n the absence of s t a n d a r d i z e d d i e t a r y c o n c e n t r a t i o n s for d e t e r m i n i n g the n u t r i t i v e va lue of p r o t e i n s o u r c e s , r e g r e s s i o n equat ions may p r o v i d e the best r e l a t i v e measure of t h e i r q u a l i t y f o r f i s h s p e c i e s . These equat ions are d e p i c t e d ( F i g . 9) f or NPU determined by the method of Ogino et a l . ( 1 9 8 0 ) . The l i n e f o r CS was omit ted because the p o s i t i v e s lope o b t a i n e d cannot be adequate ly e x p l a i n e d . A g r e a t e r i n v e r s e r e l a t i o n s h i p of NPU on p r o t e i n i n t a k e would have been expected f o r f i s h fed HTH than was found i n t h i s a s say . The r e g r e s s i o n l i n e s o b t a i n e d i n t h i s study f o r F P E , FRH, and LTH compare w e l l wi th one another and that o b t a i n e d with rainbow t r o u t fed egg y o l k (Ogino et a l . , 1 9 8 0 ) ( F i g . 9 ) . 87 3 . 3 . 8 The measurement of p r o t e i n q u a l i t y by s lope r a t i o The s lope r a t i o (SR) assay i s a m u l t i - d o s e procedure which i n c l u d e s a n o n - p r o t e i n d i e t and three or more d i e t a r y l e v e l s of p r o t e i n (Hegsted and Chang, 1965). A l t e r n a t i v e l y , i n some r a t b i o a s s a y s , the zero p r o t e i n fed group has been omit ted when c a l c u l a t i n g the s lope (McLaughlan , 1974; Samonds and Hegs ted , 1977). The assay i s based on the response ( s l o p e ) of body weight ga in or body p r o t e i n ga in on p r o t e i n i n t a k e . In the i d e a l SR as say , the s lopes of the r e f e r e n c e p r o t e i n (FPE) and the t e s t p r o t e i n s are l i n e a r and meet at a common i n t e r c e p t . The s lopes for the v a r i o u s p r o t e i n s fed to chinook f r y were compared by a n a l y s i s of c o v a r i a n c e of dry body weight ga in (Tab le 23) and body p r o t e i n ga in (Tab le 24) wi th p r o t e i n i n t a k e as the c o v a r i a t e . The s l o p e s for data i n c l u d i n g the zero p r o t e i n fed groups are d e p i c t e d i n F i g s . 10, 11. The s tandard e r r o r s of the s lopes were c a l c u l a t e d as percentages (Hegsted and Chang, 1965) (Tables 23 and 24) and may be c o n s i d e r e d as e s t imates of p r e c i s i o n of the a s s a y . These were reduced c o n s i d e r a b l y when the data for the p r o t e i n f r e e d i e t fed groups were i n c l u d e d i n the a s s a y . The c o r r e l a t i o n c o e f f i c i e n t s obta ined for each s lope i n d i c a t e d that under the c o n d i t i o n s of the present s t u d y , the r e l a t i o n s h i p between body weight or p r o t e i n ga in and p r o t e i n i n t a k e s a t i s f i e d the requirements for l i n e a r i t y i n a s lope assay (Hegsted and Chang, 1965; McLaughlan , 1979). When the s lopes of body p r o t e i n ( i n c l u d i n g the p r o t e i n free d i e t fed groups) were compared, the assay d i s t i n g u i s h e d F P E , LTH and HTH from one another (Tab le 24) . With the 88 Table 23. Slope r a t i o s of dry body weight gains on protein intake for f i s h fed the various protein sources. Calculated both by including and excluding the protein free d i e t (PF) fed groups. Protein Slope r a t i o s of dry body weight gains Source excluding PF groups including PF groups Slope 1 , z 0 . 8 0 4 8 m 0.8817  8 0 SE of slope +0.0623 +0.0432 SE of slope as % ~7.74 4.90 Intercept -0.0329 -0.0852 Correlation c o e f f i c i e n t 0.9937 0.9957 Relative slope (100) (100) Ranking 3 3 Slope 0.8382 b 0.8940 C SE of slope +0.0517 +0.0405 SE of slops as % 6.17 4.53 Intercept -0.0445 -0.0840 Correlation c o e f f i c i e n t 0.9973 0.9970 Relative slope (104) (101) Ranking 2 1 Slope 0.5752* 0.6769™ SE of slope +0.0431 +0.0361 SE of slope as % 7.49 5.33 Intercept 0.0275 -0.0520 Correlation c o e f f i c i e n t 0.9858 0.9827 Relative slope (71) (77) Ranking 4 4 Slope 0.4618 3 0.4675 A SE of slope +0.0826 +0.0611 SE of slope as % T7.89 T3.07 Intercept -0.0943 -0.0972 Correlation c o e f f i c i e n t 0.9873 0.9928 Relative slope (57) (53) Ranking 5 5 Slope 1.0202 b 0.8875 8 0 SE of slope +0.1093 +0.0591 SE of slope as % TO. 71 6.66 Intercept -0.1859 -0.1168 Correlation c o e f f i c i e n t 0.9566 0.9835 Relative slope (127) (101) Ranking 1 2 ! • A n analysis of oovariance of dry body weights with the oovariate (protein intake) having d i f f e r e n t slopes fo r d i f f e r e n t protein sources indicated P > 0.001, when calculated both by including and excluding PF groups. 2. Slopes with the same superscript do not d i f f e r s i g n i f i c a n t l y (Scheffes' t e s t P = 0.05). 89 Table 24. Slope r a t i o s of body protein gains on protein intake for f i s h fed the various protein sources. Calculated both by including and excluding the protein free (PF) fed groups. Protein Source Slope r a t i o s of body protein gains excluding PF groups including PF groups FPE Slope SE of slope SE of slope as % Intercept Correlation c o e f f i c i e n t Relative slope Ranking FRH Slope SE of slope SE of slope as % Intercept Correlation c o e f f i c i e n t Relative slope Ranking LTH Slope SE of slope SE of slope as % Intercept Correlation c o e f f i c i e n t Relative slope Ranking HTH Slope SE of slope SE of slope as % Intercept Correlation c o e f f i c i e n t Relative slope Ranking CS Slope SE of slope SE of slope as % Intercept Correlation c o e f f i c i e n t Relative slope Ranking 0.5588"- +0.0422 7.55 -0.0541 0.9849 (100) 2 0.4255" +0.0350 8.23 0.0000 0.9986 (76) 3 0.3785 +0.0292 7.71 -0.0029 0.9818 (71) 4 0.28073 +0.0560 19.95 -O.0715 0.9807 (57) 5 0.6489 1 +0.0740 Tl.40 . -0.1327 0.9661 (116) 1 ab ab 0.5756 c +0.0286 4.97 -0.0652 0.9943 (100) 1 0.4896 +O.0268 5.47 -0.0454 0.9887 (85) 3 0.4345' +0.0238 5.48 -0.0405 0.9839 (76) 4 0.2711* +0.0404 14.90 -0.0671 0.9881 (47) 5 BC •B 0.5483 +0.0390 7.11 -0.0790 0.9838 (95) 2 BC T~. An analysis of covariance of body protein gains with the covariate (protein intake) having d i f f e r e n t slopes for d i f f e r e n t protein sources indicated P > 0.001, when calculated both by including and excluding PF groups. 2. Slopes with the same superscript do not d i f f e r s i g n i f i c a n t l y (Scheffes' t e s t P = 0.05). - 90 -0-20 0 0 10 0 20 0 30 0-40 0-50 0-60 070 0-80 0-90 100 MO PROTEIN INTAKE (g/ FISH) F i g . 10. S lopes of dry body weight ga in on p r o t e i n i n t a k e of chinook salmon fed d i e t s c o n t a i n i n g the t e s t p r o t e i n s o u r c e s . The s lopes were c a l c u l a t e d i n c l u d i n g p o i n t s ob ta ined from groups of f i s h fed the p r o t e i n f r e e d i e t s (see Tab le 23) . 91 -0-10 1 . . . . . . . . . . . 0 0 10 0-20 0-30 0 40 0-50 0-60 070 0-80 0-90 1 00 1 10 PROTEIN INTAKE (g/ FISH) F i g . 11. S lopes of body p r o t e i n ga in on p r o t e i n i n t a k e of ch inook salmon fed d i e t s c o n t a i n i n g the t e s t p r o t e i n s o u r c e s . The s lopes were c a l c u l a t e d i n c l u d i n g p o i n t s o b t a i n e d from groups of f i s h fed the p r o t e i n f r e e d i e t s (see Tab le 24) . - 92 e x c e p t i o n of FRH s i m i l a r r e l a t i v e s lope va lues were obta ined by e i t h e r body weight or body p r o t e i n g a i n . 3 . 3 . 9 A v a i l a b l e l y s i n e content of the p r o t e i n sources The a v a i l a b l e l y s i n e content of the p r o t e i n sources was determined i n an attempt to e v a l u a t e the extent of p r o t e i n damage i n r e l a t i o n to the p r o c e s s i n g method used . The l y s i n e va lues are shown i n T a b l e ( 2 ) . They were used to c a l c u l a t e the a v a i l a b l e l y s i n e content of the d i e t s and are compared to the known l y s i n e requirements of sa lmonids ( T a b l e 25) . H a l v e r et a l . ( 1 9 5 8 ) s t a t e d that the l y s i n e requirement for chinook salmon i s 5% of the d i e t a r y p r o t e i n or 2% of the d i e t c o n t a i n i n g 40% p r o t e i n . Ogino (1980) and K e t o l a (1982) showed that the minimum requirement of rainbow t r o u t for l y s i n e was 5.3% and 6.1% of d i e t a r y p r o t e i n r e s p e c t i v e l y . Employing the l y s i n e requirement va lue of 5% of d i e t a r y p r o t e i n ( H a l v e r et a l . , 1958) only HTH f a i l e d to meet the requirement (Tab le 25) . However, i f the l y s i n e requirement i s based on f i s h egg c o m p o s i t i o n , only FPE c o n t a i n e d s u f f i c i e n t s u r p l u s a v a i l a b l e l y s i n e r e l a t i v e to the l y s i n e requ irements s t a t e d by K e t o l a (1982) (Tab le 25) . LTH and FRH c o n t a i n e d almost s u f f i c i e n t a v a i l a b l e l y s i n e to meet requirements ( K e t o l a , 1982). Based on the a v a i l a b l e l y s i n e content o b t a i n e d for HTH coupled wi th the performance of f i s h fed HTH, the r e s u l t s may suggest that a c e r t a i n amount of l y s i n e was rendered u n a v a i l a b l e i n t h i s p r o t e i n source due to the severe h e a t i n g d u r i n g the d r y i n g of the meal . 93 Table 25. Available l y s i n e , lysine requirements for chinook salmon and percentages of requirements supplied by each protein source ( i n parenthesis). % Crude Protein i n Diets 7 17 27 37 47 % of % of % of % of % of Req Req Req Req Req FPE Available lysine % of dry diet 0.59 1.45 2.33 3.08 3.95 Requirement: Halver et al.(1958)5% of protein 0.34 (171) 0.85 (171) 1.36 (171) 1.80 (171) 2.31 (171) Ketola (1982) 8.8% protein 0.59 (97) 1.49 (97) 2.40 (97) 3.16 (97) 4.06 (97) FRH Available lysine % of dry diet 0.99 1.51 2.17 Requirement: Halver et al.(1958)52 of protein 0.90 (109) 1.38 (109) 1.98 (109) Ketola (1982) 8.8% protein 1.58 (62) 2.43 (62) 3.48 (62) LTH Available lysine % of dry diet 0.90 1.53 2.07 Requirement: Halver et al.(1958)5% of protein 0.84 (108) 1.42 (108) 1.92 (108) Ketola (1982) 8.8% protein 1.48 (61) 2.50 (61) 3.38 (61) HTH Available lysine % of dry diet 0.77 1.25 1.79 Requirement: Halver et al.(1958)5% of protein 0.85 (91) 1.27 (91) 2.00 (91) Ketola (1982) 8.8% protein 1.50 (52) 2.41 (52) 3.51 (52) CS Available lysine % of dry diet 1.37 1.95 2.65 Requirement: Halver et al.(1958)5% of protein 0.93 (148) 1.32 (148) 1.80 (148) Ketola (1982) 8.8% protein 1.63 (84) 2.32 (84) 3.17 (84) 3.A DISCUSSION 3 . 4 . 1 The e f f e c t of d i e t a r y p r o t e i n source and l e v e l on body weight ga in An inadequate supply of p r o t e i n would be expected to have a profound i n f l u e n c e on growth. V a r i o u s i n v e s t i g a t o r s i n f i s h growth s t u d i e s c a l c u l a t e growth r a t e by d i f f e r e n t methods. Z e i t o u n (1973) compared average d a i l y g a i n , percent ga in of i n i t i a l weight and s p e c i f i c growth r a t e (% i n c r e a s e i n body weight per d a y ) . He and other i n v e s t i g a t o r s ( B r e t t et a l . , 1969) conc luded that the l a t t e r p r o v i d e d the most u s e f u l way to compare growth of d i f f e r e n t s i z e f i s h . The use of average d a i l y ga in i n animal exper iments has been c r i t i z e d because i t does not a c c u r a t e l y d e s c r i b e weight change at any s p e c i f i c age (Crampton and L l o y d , 1959). However, i n the present s t u d y , s i m i l a r i n f e r e n c e s could be made by e x p r e s s i n g growth by both f i n a l body weight and s p e c i f i c growth r a t e (Tab le 6 ) . The r e s u l t s of the growth study demonstrated the e f f e c t that p r o t e i n source and l e v e l had on the performance of chinook salmon. With the e x c e p t i o n of f i s h fed HTH body weight i n c r e a s e was a p p r o x i m a t e l y l i n e a r for f i s h fed d i e t a r y p r o t e i n l e v e l s of 27% and h i g h e r . The d e c l i n e i n growth r a t e from day 21 to 42 compared to day 0 to 21 for f i s h fed the low p r o t e i n d i e t s or HTH ( F i g s . 3A, 3B, 3C) i s l i k e l y due to the i n f l u e n c e of body n u t r i e n t r e s e r v e s accumulated d u r i n g the a c c l i m a t i o n p e r i o d . D a i l y growth r a t e s do not a d j u s t i n s t a n t l y to d i e t a r y change. The e x i s t e n c e of a l a g e f f e c t i n the metabo l i c a d a p t a t i o n to d i e t s of d i f f e r i n g n u t r i e n t c o m p o s i t i o n has been w e l l 95 - e s t a b l i s h e d (Maynard and L o o s l i , 1969). T h i s was r e c o g n i z e d i n f i s h n u t r i t i o n by S t e f f e n s (1981) who s t a t e d that a r e l i a b l e i n d i c a t i o n of p r o t e i n u t i l i z a t i o n by means of growth r a t e t e s t s would be reached only a f t e r a 1 to 2 week p e r i o d of a d a p t a t i o n . The e f f e c t of the a c c l i m a t i o n p e r i o d d i e t , which was s i m i l a r i n c o m p o s i t i o n to F P E - 4 7 , i s the most l i k e l y reason that f i s h weight ga in was not noted to i n c r e a s e e x p o n e n t i a l l y for most of the d i e t a r y t r e a t m e n t s . From the r e s u l t s of f e e d i n g t r i a l s with j u v e n i l e chinook salmon, Higgs et a l . ( 1 9 8 2 , 1983) r e p o r t e d that weight ga in over time was n e a r l y e x p o n e n t i a l . In c o n t r a s t to the present s t u d y , the growth r a t e s r e p o r t e d by Higgs et a l . (1982, 1983) were measured d u r i n g a longer e x p e r i m e n t a l p e r i o d , which perhaps had the e f f e c t of masking the i n f l u e n c e of the a c c l i m a t i o n d i e t d u r i n g the t r i a l p e r i o d . N e v e r t h e l e s s , f o l l o w i n g a 42-day e x p e r i m e n t a l p e r i o d the growth response to i n c r e a s i n g l e v e l s of p r o t e i n s u p p l i e d by FPE fo l l owed a smooth curve wi th a s t e a d i l y d e c r e a s i n g r a t e of i n c r e a s e ( F i g . 4 ) . A s i m i l a r o b s e r v a t i o n was r e p o r t e d f o r p l a i c e fed f r e e z e - d r i e d cod muscle (Cowey et a l . , 1972). A smooth curve was not o b t a i n e d with the other p r o t e i n sources t e s t e d . S ince t h i s was c o n s i d e r e d to be due to d i f f e r i n g food i n t a k e s , the performance of the f i s h fed the v a r i o u s p r o t e i n sources was compared by the s lopes of weight ga in to p r o t e i n i n t a k e r a t h e r than to d i e t a r y p r o t e i n c o n c e n t r a t i o n . Thus , a l though the c a s e i n based d i e t s (CS) supported r a p i d growth per u n i t of p r o t e i n i n t a k e , a c t u a l weight ga in was not r e a l i z e d . I t was c o n j e c t u r e d that t h i s was due to a p a l a t a b i l i t y problem with 96 the CS d i e t s . The n u t r i t i o n a l q u a l i t y of the v a r i o u s p r o t e i n sources w i l l be d i s c u s s e d l a t e r . The growth r a t e s of chinook fed OMP were s i m i l a r to those of f i s h fed d i e t s c o n t a i n i n g 37% p r o t e i n s u p p l i e d by f r e e z e - d r i e d or low temperature d r i e d f i s h meals . OMP i s the d i e t most used to r e a r j u v e n i l e chinook salmon at l o c a l h a t c h e r i e s . The f i n d i n g s of t h i s study suggest that the p r o t e i n content of OMP c o u l d be reduced from the present 50%. However, OMP c o n t a i n s a v a r i e t y of p r o t e i n sources i n c l u d i n g some of p l a n t o r i g i n , and l o w e r i n g the p r o t e i n l e v e l of OMP would perhaps impa ir the growth r a t e of f i s h and consequent ly ocean s u r v i v a l . 3 .4 .2 The e f f e c t of d i e t a r y p r o t e i n source and l e v e l on food i n t a k e and gross food c o n v e r s i o n e f f i c i e n c y Food i n t a k e i n f i s h i s r e g u l a t e d by the c a l o r i c content of the d i e t (Lee and Putman, 1973), and f i s h s i z e ( B r e t t , 1971); assuming that a l l o ther b i o t i c and a b i o t i c f a c t o r s ( B r e t t , 1979) have been s t a n d a r d i z e d . The r e s u l t s of t h i s study agree wi th a r e p o r t by B r e t t (1971) who showed that food i n t a k e of sockeye salmon was i n f l u e n c e d by f i s h we ight , the s m a l l e r f i s h consuming the l a r g e s t r e l a t i v e amount. The i n f l u e n c e of f i s h s i z e on l e v e l of d i e t a r y i n t a k e i s accounted for i n hatchery f e e d i n g t a b l e s (Fowler and Burrows, 1971). Food c o n v e r s i o n i s a parameter of p a r t i c u l a r i n t e r e s t to f i s h p r o d u c e r s . The r a t i o of weight ga in to food i n t a k e r e p r e s e n t s a major i n f l u e n c e on the p r o f i t margin i n an animal - 97 p r o d u c t i o n e n t e r p r i s e . The t rend of a s t e a d i l y d e c r e a s i n g r a t e of i n c r e a s e i n GFC to increments i n d i e t a r y p r o t e i n c o n c e n t r a t i o n are c o n s i s t e n t with the r e s u l t s of other i n v e s t i g a t o r s who have s t u d i e d the p r o t e i n requirements of s a l m o n i d s . For example, Z e i t o u n (1973) , S a t i a (1974) , Gulbrandsen and Utne (1977) and K o s h i i s h i (1980) o b t a i n e d maximum GFC when the p r o t e i n l e v e l of the d i e t s lay between 35% and 45%. More p r e c i s e comparisons wi th these r e p o r t s cannot adequate ly be made because of wide ly d i f f e r i n g e x p e r i m e n t a l c o n d i t i o n s . Comparison with an experiment by Higgs et a l . ( 1 9 8 2 ) i s more v a l i d because i t was conducted on the same s p e c i e s and under s i m i l a r e x p e r i m e n t a l c o n d i t i o n s as the present s t u d y . They o b t a i n e d GFC va lues rang ing between 20% and 25% for a s e r i e s of t e s t p r a c t i c a l d i e t s c o n t a i n i n g a p p r o x i m a t e l y 50% p r o t e i n . Higher va lues were o b t a i n e d i n t h i s study with d i e t s c o n t a i n i n g s u b s t a n t i a l l y l e s s p r o t e i n . T h i s suggests that the p r o t e i n l e v e l i n p r a c t i c a l chinook salmon d i e t s cou ld be reduced wi thout s u p p r e s s i n g GFC, p r o v i d e d that the q u a l i t y of d i e t a r y p r o t e i n i s h i g h . The p r o t e i n sources judged to be of h i g h e s t va lue by GFC i n the present study were the f r e e z e - d r i e d f i s h meals . 3 . 4 . 3 The e f f e c t of d i e t a r y p r o t e i n source and l e v e l on energy i n t a k e and gross energy u t i l i z a t i o n As mentioned p r e v i o u s l y , f i s h eat to s a t i s f y t h e i r energy needs (Lee and Putman, 1973). In t h i s study an attempt was made 98 to formula te the d i e t s (except OMP) to be i s o c a l o r i c on a m e t a b o l i z a b l e energy b a s i s . The i n a c c u r a c y and i n c o n s i s t e n c y of t h i s p r a c t i c e i n f i s h growth and n u t r i t i o n s t u d i e s has r e c e n t l y been reviewed and h a r s h l y c r i t i c i z e d by J o b l i n g (1983) . His main o b j e c t i o n s p e r t a i n to the u n r e l i a b i l i t y of methods used to measure m e t a b o l i z a b l e energy , the dependency of e s t imates of m e t a b o l i z a b l e energy on l e v e l of f e e d i n g , and the use of i n a c c u r a t e c a l o r i c c o n v e r s i o n c o e f f i c i e n t s . In t h i s study d i e t a r y l i p i d was kept cons tant whi l e p r o t e i n was r e p l a c e d by c a r b o h y d r a t e on a m e t a b o l i z a b l e energy b a s i s ( p r o t e i n = 4.5 k c a l / g ; c a r b o h y d r a t e = 4 k c a l / g ) . S ince the f i s h were fed to s a t i e t y one may expect m e t a b o l i z a b l e energy i n t a k e s (MEI) to be s i m i l a r at each p r o t e i n l e v e l . The g e n e r a l t rend n o t e d , however, was a decreased energy i n t a k e by f i s h consuming the d i e t s c o n t a i n i n g more p r o t e i n and l e s s c a r b o h y d r a t e . T h i s suggests that e i t h e r the energy va lue a s s igned to p r o t e i n was too low or the va lue for c a r b o h y d r a t e was too h i g h , c a u s i n g an o v e r e s t i m a t i o n of MEI i n f i s h r e c e i v i n g the h igh c a r b o h y d r a t e d i e t s . A c t u a l l y , the m e t a b o l i z a b l e energy of p o l y s a c c h a r i d e s i s dependent on t h e i r c o n c e n t r a t i o n i n the d i e t s i n c e t h e i r d i g e s t i b i l i t y i n t r o u t was shown to decrease p r o g r e s s i v e l y as t h e i r d i e t a r y c o n c e n t r a t i o n was i n c r e a s e d (S ingh and Nose, 1967). However, the a b s o r p t i o n of monosaccharides was not found to depend on d i e t a r y l e v e l . In the present study equal p o r t i o n s of g lucose monohydrate and p a r t i a l l y h y d r o l y z e d d e x t r i n were employed. Thus an e r r o r i n the present study may have been i n t r o d u c e d by not c o r r e c t i n g the ME va lue a s c r i b e d to 99 c a r b o h y d r a t e with c o n s i d e r a t i o n for i t s l e v e l i n the d i e t . Higgs et a l . ( 1 9 8 3 ) encountered a s i m i l a r dilemma which they a s c r i b e d to i m p a i r e d d i g e s t i b i l i t y of p r o t e i n and energy due to the h igh f i b e r and d e x t r i n content s of the low p r o t e i n d i e t s . They a l s o suggested that d i e t a r y f i b e r ( c e l l u l o s e i n the present s tudy) depresses m i n e r a l b i o a v a i l a b i l i t y and i n c r e a s e s the r a t e of t r a n s i t of the d i g e s t a . I t i s i n t e r e s t i n g to note that the f i s h fed the p r o t e i n - f r e e d i e t a l s o appeared to eat to s a t i s f y t h e i r energy r e q u i r e m e n t s . Lee and Putnam (1973) showed that d i e t a r y energy u t i l i z a t i o n was i n f l u e n c e d by the p r o t e i n : c a l o r i e r a t i o of the d i e t . S i m i l a r l y , i n the present s t u d y , d i e t a r y p r o t e i n l e v e l and source had an e f f e c t on GEU, s u g g e s t i n g that t h i s parameter may be used as a c r i t e r i a for e v a l u a t i n g the n u t r i t i v e va lue of p r o t e i n s for f i s h d i e t s . P i e p e r and P f e f f e r (1980) found GEU to be u s e f u l i n comparing the r e l a t i v e u t i l i z a t i o n of v a r i o u s supplements of c a r b o h y d r a t e s , f a t s , and p r o t e i n s to a b a s a l d i e t . The GEU of f i s h r e c e i v i n g HTH was extremely poor i n comparison to va lues recorded for f i s h fed d i e t s c o n t a i n i n g the other p r o t e i n s o u r c e s . Perhaps t h i s was due not only to the inadequacy of HTH as a source of a v a i l a b l e energy from p r o t e i n but a l s o from l i p i d . 3 .4 .4 The e s t i m a t i o n of maintenance requirements for p r o t e i n The r e l a t i o n s h i p between n i t r o g e n (N) i n t a k e and n i t r o g e n r e t e n t i o n s t a t e s that when n i t r o g e n i n t a k e i s z e r o , n i t r o g e n 100 r e t e n t i o n i s , and must be, equal to the t o t a l endogenous n i t r o g e n e x c r e t i o n ( B r e s s a n i , 1977). C o n v e r s e l y , when n i t r o g e n r e t e n t i o n i s z e r o , n i t r o g e n i n t a k e should equa l endogenous n i t r o g e n l o s s . S e v e r a l s t u d i e s d i s c u s s e d e a r l i e r ( G e r k i n g , 1955a; B i r k e t t , 1969; S a v i t z , 1971; B r e t t and G r o v e s , 1979) c o n s i d e r endogenous n i t r o g e n l o s s to r e p r e s e n t the p r o t e i n - n i t r o g e n requirement f o r maintenance i n f i s h (see F i g . 1 ) . The p r o t e i n requirement f o r maintenance of ch inook salmon f r y was es t imated to be 0.160g/100g BW/day f o r f i s h fed d i e t s c o n t a i n i n g a p p r o x i m a t e l y 3950 k c a l / k g dry f o o d . The assumption was made that FPE was t o t a l l y a s s i m i l a t e d by the f i s h . T h i s assumption i s v a l i d s i n c e a p r e l i m i n a r y study found that FPE was 97% d i g e s t e d and i s i n agreement wi th r e p o r t s on d i g e s t i b i l i t y of raw cod by p l a i c e (Cowey et a l . , 1974) and rainbow t r o u t (Skrede et a l . , 1980; Opstvedt et a l . , 1984). The maintenance requirement i n the present study may be c o n t r a s t e d wi th e s t imates of d a i l y endogenous n i t r o g e n e x c r e t i o n i n f i s h e s ( B r e t t and Groves , 1979). The p r o t e i n requirement above corresponds to an endogenous n i t r o g e n e x c r e t i o n r a t e of 25.5mg/100g BW/day. T h i s va lue i s i n c l o s e agreement wi th that r e p o r t e d by B r e t t and Z a l a (1975) for sockeye salmon, namely . 22.1mg N/ lOOg BW/day, a l though t h e i r e s t imate was o b t a i n e d us ing l a r g e r f i s h and at a h i g h e r water temperature than i n the present s t u d y . G e r k i n g (1955a,b) was the f i r s t to study maintenance p r o t e i n requirements of f i s h . B l u e g i l l s u n f i s h were s t a r v e d for three days and then they were fed d a i l y an amount of g lucose v i a 101 stomach tube e q u i v a l e n t to the m e t a b o l i c r a t e of the f i s h . G e r k i n g (1955a) found that a 29.7g s u n f i s h e x c r e t e d 5.84mg of n i t r o g e n d a i l y (19.7mg N/lOOg BW) . However, i t i s u n l i k e l y that t h i s i n i t i a l e s t imate based upon a three day f a s t e d f i s h p r o v i d e d a good measure of endogenous n i t r o g e n e x c r e t i o n s i n c e the f i s h were probably not f u l l y d e p l e t e d of p r o t e i n r e s e r v e s . When f i s h are p laced on a p r o t e i n - f r e e d i e t there i s an a d a p t i v e d e c l i n e i n the r a t e of n i t r o g e n e x c r e t i o n over a p e r i o d of time u n t i l a new e q u i l i b r i u m i s e s t a b l i s h e d . Dur ing t h i s p e r i o d the p r o t e i n r e s e r v e i s dep le t ed and u r i n a r y n i t r o g e n e x c r e t i o n ensues at a minimal cons tant l e v e l . The h i g h e r the l e v e l of p r e v i o u s n u t r i t i o n , the longer the p e r i o d of time necessary to e s t a b l i s h the minimum l e v e l of n i t r o g e n e x c r e t i o n . M o r g u l i s (1918) determined n i t r o g e n balance i n brook t r o u t and found no s t a b i l i z a t i o n of n i t r o g e n e x c r e t i o n f o l l o w i n g a four week f a s t . Body weight data i n t h i s study ( T a b l e 12) suggest that s t a b i l i z a t i o n had o c c u r r e d by day 42 i n f i s h fed the p r o t e i n - f r e e d i e t . G e r k i n g (1955b) regarded endogenous n i t r o g e n e x c r e t i o n as be ing e q u i v a l e n t to the minimum amount of p r o t e i n r e q u i r e d to m a i n t a i n the f i s h i n n i t r o g e n e q u i l i b r i u m . T h i s was determined from the d i f f e r e n c e i n body n i t r o g e n content of f i s h sampled at the b e g i n n i n g and the end of a 30 day f e e d i n g p e r i o d . The f i s h were fed mealworms at d i f f e r e n t r a t e s of i n t a k e . The s u n f i s h were shown to have absorbed 98% of the p r o t e i n i n the mealworms. A c c o r d i n g l y , G e r k i n g (1955b) es t imated that 7.2mg N/day was r e q u i r e d to m a i n t a i n a 29.7g s u n f i s h i n n i t r o g e n 102 e q u i l i b r i u m at 2 5 ° C . Thus , the maintenance n i t r o g e n requirement amounted to 24.24mg N/lOOg BW/day. Gerk ing (1955b) regarded t h i s va lue to be i n c l o s e agreement with that obta ined from n i t r o g e n d e t e r m i n a t i o n of aquarium water f o l l o w i n g o r a l g lucose a d m i n i s t r a t i o n . T h i s va lue agrees with the e s t imate r e p o r t e d i n t h i s study for chinook salmon f r y . Ogino et a l . ( 1 9 8 0 ) ana lysed rainbow t r o u t c a r c a s s e s and e x c r e t a a f t e r f e e d i n g them a p r o t e i n - f r e e d i e t f or p e r i o d s of 14 to 19 days . They es t imated n i t r o g e n l o s t endogenously by c a r c a s s a n a l y s i s and by t o t a l n i t r o g e n e x c r e t e d to be 8.89 and 9.96mg/100g BW/day, r e s p e c t i v e l y . These va lues are c o n s i d e r a b l y lower than the one o b t a i n e d for chinook salmon i n t h i s s t u d y . Other e s t imates of maintenance p r o t e i n requirements i n f i s h e s , ob ta ined from experiments where f i s h were fed e i t h e r maintenance p r o t e i n d i e t s , or e s t imated from feed ing a s e r i e s of low p r o t e i n d i e t s and c o n d u c t i n g c a r c a s s a n a l y s i s , are shown i n T a b l e 26. In c o n t r a s t to the present e s t imate for chinook salmon fed a d i e t c o n t a i n i n g 7% p r o t e i n from F P E , Z e i t o u n et a l . ( 1973,1974) found that the maintenance d i e t a r y p r o t e i n requirement for coho salmon and rainbow t r o u t , fed c a s e i n - g e l a t i n based d i e t s was 23.5% and 15% r e s p e c t i v e l y . D i s c o u n t i n g the e f f e c t s of low s a l i n i t y of the water i n t h e i r s t u d y , i t would appear that e i t h e r c a s e i n - g e l a t i n was p o o r l y a s s i m i l a t e d , or more l i k e l y , t h e i r method of e x t r a p o l a t i o n of percentage d i e t a r y p r o t e i n at zero growth was i n a c c u r a t e . In a study with young r a t s , Henry (1965) found that the maintenance p r o t e i n l e v e l was about 2% f o r egg a lbumin and 103 Table 26. Estimated absorbed nitrogen requirement for maintenance of f i s h , determined by feeding and carcass analysis experiments. Species Size (grams) Water Temperature (degrees C) Diet Requirement (mg/lOOgBW/d) Reference B l u e g i l l sunfish 30 (Lepomls macrochlrus) Yearling plaice 11 - 34 (Pleuronectes platessa) Yearling sole 0.2 - 1.8 (Solea vulgaris) Yearling sole 5 - 5 3 (Solea vulgaris) Perch 100 - 150 (Perca f l u v l a l i s ) Carp 2.5 - 4.5 (Carassius auratus) Rainbow trout 0.9 - 26 (Salmo gairdneri) Carp 1.5 - 12 (Carassius auratus) Chinook salmon 1.56 (Oncorhynchus tshawytscha) Chinook salmon 1.56 (Oncorhynchus t&hawytscha) 25 17 17 17 17 20 12 - 19 19 - 25 11 lugwonns whiteworms lugwonns earthworms chironomid larvae protein-free protein-free protein-free FPE-7 24.24 20.3 24.4 38.1 17.3 22 13.9 20.7 Gerking (1955b) Bi r k e t t (1969) Birkett (1969) Birkett (1969) Birkett (1969) Iwata (1970) Ogino (1980) Ogino (1980) Present study Present study - 10A - h e r r i n g meal and between 2% and 3% for c a s e i n . The requirement i n c r e a s e d when poor q u a l i t y p r o t e i n s such as g l u t e n and z e i n were f e d . S i m i l a r l y , i t was observed that the d i e t a r y p r o t e i n l e v e l f or maintenance v a r i e d wi th p r o t e i n s o u r c e . Whereas a d i e t a r y p r o t e i n i n t a k e of a p p r o x i m a t e l y 180mg/100g BW/day was r e q u i r e d for f i s h fed d i e t s c o n t a i n i n g FRH and L T H , double the amount of p r o t e i n was r e q u i r e d when HTH was employed ( F i g . 6 ) . A f i n a l note should be added wi th r e s p e c t to the i n t e r p r e t a t i o n of maintenance requirements for p r o t e i n i n growing f i s h . These va lues (Tab le 26) are based on t o t a l body n i t r o g e n t u r n o v e r by f i s h . Maintenance n i t r o g e n ba lance does not n e c e s s a r i l y r e f l e c t maintenance of a steady s t a t e l e v e l of i n d i v i d u a l t i s s u e p r o t e i n metabol ism or the n u t r i t i o n a l s t a t u s of a p a r t i c u l a r organ w i t h i n the body. The p r a c t i c a l a p p l i c a t i o n of maintenance p r o t e i n requirements to f i s h c u l t u r e i s dangerous s i n c e they do not c o n s i d e r the p h y s i o l o g i c a l requirements of the growing a n i m a l . Rather they serve to a i d i n the p a r t i t i o n i n g of p r o t e i n i n t a k e a c c o r d i n g to the scheme d e s c r i b e d by F i g . 1. 3 . 4 . 5 The e f f e c t of d i e t a r y p r o t e i n source on p r o t e i n u t i l i z a t i o n The r e s u l t s of t h i s study demonstrated the magnitude of the e f f e c t that p r o t e i n s of d i f f e r e n t n u t r i t i v e va lue had on the u t i l i z a t i o n of d i e t a r y p r o t e i n i n growing salmon. S i n c e , p r o t e i n u t i l i z a t i o n i s a l s o a f u n c t i o n of p r o t e i n i n t a k e , the 105 p r o t e i n sources were compared at d i f f e r e n t d i e t a r y c o n c e n t r a t i o n s . The r e l a t i o n s h i p between p r o t e i n i n t a k e and the u t i l i z a t i o n of p r o t e i n f o r maintenance and growth and amount excre ted d e s c r i b e d i n s e c t i o n ( 3 . 3 . 5 ) f o l l o w e d a s i m i l a r p a t t e r n to that d e s c r i b e d by Ogino et a l . (1973) for c a r p . As the amount of p r o t e i n consumed by carp i n c r e a s e d above that needed for maximum e f f i c i e n c y , the p r o t e i n used for growth decreased g r a d u a l l y as d i d the p r o p o r t i o n used for maintenance . Exogenous l o s s , however, c o n c o m i t a n t l y i n c r e a s e d . Apart from r e p r e s e n t i n g an economic l o s s , n i t r o g e n o u s waste a c c u m u l a t i o n i n the form of ammonia and decomposing feces pose a hazard to c o n f i n e d f i s h . The u t i l i z a t i o n of d i e t a r y p r o t e i n was found to vary c o n s i d e r a b l y between p r o t e i n sources of e x c e l l e n t and poor n u t r i t i o n a l v a l u e . That FPE supported e x c e l l e n t growth and the most d e s i r e d mode of p r o t e i n u t i l i z a t i o n i s not s u r p r i s i n g s i n c e i t i s e n t i r e l y made up of t e l e o s t e a n s k e l e t a l muscle p r o t e i n s . The amino a c i d c o m p o s i t i o n of f r e e z e - d r i e d p o l l o c k muscle compares f a v o u r a b l y with the s t a t e d amino a c i d requirements for chinook salmon and the c o m p o s i t i o n of f i s h eggs (Table 1 ) . I t has been amply demonstrated that supplementa l amino a c i d s pa t t erned a f t e r the c o m p o s i t i o n of f i s h eggs or whole f r y produced the h i g h e s t ga in i n f i s h fed a b a s a l d i e t ( A r a i , 1981; K e t o l a , 1982). FPE was processed by f r e e z e - d r y i n g which i s not known to cause any d e t r i m e n t a l e f f e c t s on amino a c i d a v a i l a b i l i t y . Exper iments conducted by Cowey et a l . ( 1 9 7 1 ) wi th p l a i c e and M i l l e r and Bender (1953) with r a t s have shown 106 e x c e l l e n t p r o t e i n u t i l i z a t i o n with vacuum-dr ied cod muscle . Cowey et a l . ( 1 9 7 2 ) r e p o r t e d that f r e e z e - d r i e d cod muscle p r o t e i n was comple te ly d i g e s t e d by p l a i c e at lower d i e t a r y l e v e l s and over 90% d i g e s t e d at the h i g h e s t l e v e l of i n c l u s i o n i n the d i e t . FRH was u t i l i z e d almost as e f f i c i e n t l y as F P E . Al though both meals were f r e e z e - d r i e d , FRH was prepared from whole f i s h and some of the in tegumenta l p r o t e i n s may not have been comple te ly a s s i m i l a t e d . R e c e n t l y , Watanabe et a l . (1983) compared three k i n d s of brown meals made from g r e e n l i n g , s a r d i n e and anchovy to white f i s h meal i n rainbow t r o u t d i e t s . They r e p o r t e d comparable p r o t e i n u t i l i z a t i o n i n t r o u t r e g a r d l e s s of the meal s o u r c e . The r e s u l t s o b t a i n e d with FPE and FRH, a white f i s h m e a l and a brown meal r e s p e c t i v e l y , support the o b s e r v a t i o n s of Watanabe et a l . ( 1 9 8 3 ) . Cowey et a l . ( 1 9 7 1 ) observed that a l though f r e e z e - d r i e d and low temperature d r i e d ( 3 0 ° C ) cod meal were almost i n d i s t i n g u i s h a b l e i n t h e i r e s s e n t i a l amino a c i d c o n t e n t , they supported d i f f e r e n t growth r a t e s . They presumed that the d i f f e r e n t d r y i n g c o n d i t i o n s f o r the cod meal had to some extent a f f e c t e d the b i o l o g i c a l a v a i l a b i l i t y of c e r t a i n e s s e n t i a l amino a c i d s . The f i n d i n g s i n t h i s study would c o r r o b o r a t e the same presumption a l though the e f f e c t of a mi ld d r y i n g temperature was m i n i m a l . LTH was processed i n a l a b o r a t o r y f a c i l i t y to s i m u l a t e an i d e a l commercia l h e r r i n g meal . C o n s i d e r i n g past and present d e p l e t i o n of the h e r r i n g s t o c k s i n B r i t i s h C o l u m b i a , every attempt should be made towards improv ing the u t i l i z a t i o n of the 107 rendered p r o d u c t . The performance of f i s h fed LTH demonstrates that whole h e r r i n g can be cooked, pressed and d r i e d to produce a product s u i t a b l e for chinook salmon f r y d i e t s wi th l i t t l e adverse e f f e c t on p r o t e i n q u a l i t y . I t i s noteworthy that the h e r r i n g meal i n c l u d e d i n OMP t e s t e d i n t h i s study appears l i k e l y to have been of s i m i l a r q u a l i t y to L T H . The s l i g h t l y lower u t i l i z a t i o n of p r o t e i n i n OMP compared to p r o t e i n from LTH i s probably due to the other i n g r e d i e n t s present i n t h i s d i e t . I t i s w e l l e s t a b l i s h e d tha t e x c e s s i v e heat d u r i n g d r y i n g can s e v e r e l y i m p a i r p r o t e i n d i g e s t i b i l i t y and b i o l o g i c a l va lue ( T a r r and B i e l y , 1972). T h i s was amply demonstrated by the extremely low ra te of u t i l i z a t i o n of HTH for growth and h igh d i g e s t i v e and m e t a b o l i c l o s s of t h i s p r o t e i n i n the present s t u d y . T a r r et a l . (1954) observed a r e d u 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 va lue of menhaden meal s u b j e c t e d to flame d r y i n g as compared to steam d r i e d meal . Dur ing p r o c e s s i n g the damage to HTH may have r e s u l t e d from the d e s t r u c t i o n of amino a c i d s by o x i d a t i o n , m o d i f i c a t i o n of some of the l i n k a g e s between the amino a c i d s so that t h e i r r e l e a s e was de layed d u r i n g d i g e s t i o n , and, the f o r m a t i o n of l i n k a g e s that are not h y d r o l y z e d d u r i n g d i g e s t i o n (Bender , 1972). C e r t a i n l y , as ev idenced by odor and dark c o l o r , some c h a r r i n g of HTH had o c c u r r e d . Al though a r e l a t i v e l y minor d e s t r u c t i o n of a v a i l a b l e l y s i n e due to temperature ( T a b l e 1) was o b s e r v e d , o ther amino a c i d s may have s u f f e r e d more damage. For example, i n heated h e r r i n g meal C a r p e n t e r et a l . (1962) found that t r y p t o p h a n , a r g i n i n e , meth ionine and l y s i n e were not t o t a l l y des troyed but rendered somewhat l e s s a v a i l a b l e . Opstvedt 108 et a l . (1984) r e p o r t e d a d e p r e s s i o n i n d i g e s t i b i l i t y i n t r o u t of p o l l o c k muscle s u b j e c t e d to v a r i o u s heat t r e a t m e n t s . T h e s e i n v e s t i g a t o r s noted that the d e p r e s s i o n i n d i g e s t i b i l i t y due to heat (above 9 5 ° C ) c o i n c i d e d with the f o r m a t i o n of d i s u l f i d e bonds. C r o s s - l i n k a g e s v i a l y s i n e r e s i d u e s were not found to a f f e c t p r o t e i n d i g e s t i b i l i t y i n t h e i r s t u d y . S e v e r a l i n v e s t i g a t o r s have attempted to r e l a t e p r o t e i n q u a l i t y measured b i o l o g i c a l l y to the chemica l 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 . Hence, both the l y s i n e requirement of the animal and the a v a i l a b l e l y s i n e content of the d i e t have to be c o n s i d e r e d . K e t o l a (1982) p u b l i s h e d the amino a c i d c o m p o s i t i o n of chinook salmon eggs and proposed that the f i s h egg amino a c i d p a t t e r n should be used as a g u i d e l i n e for f o r m u l a t i n g f i s h foods . He r e p o r t e d that the l y s i n e content of chinook eggs i s 8.8% of p r o t e i n which agrees wi th the va lue of 8.65% o b t a i n e d from the a n a l y s i s of eyed chinook eggs i n t h i s study ( T a b l e 1 ) . Based on a f e e d i n g t r i a l , H a l v e r et a l . ( 1958) s t a t e d that the l y s i n e requirement was 5% of p r o t e i n . Tab le 25 shows that FPE and CS c o n t a i n e d l y s i n e i n excess of most s t a t e d r e q u i r e m e n t s . FRH and LTH appeared to be p r o t e i n sources that would not warrant l y s i n e supp lementa t ion i n p r a c t i c a l d i e t s for j u v e n i l e chinook salmon a c c o r d i n g to the l y s i n e requirement proposed by H a l v e r et a l . ( 1958) . March et a l . ( 1 9 6 6 ) r e p o r t e d that a l though a v a i l a b l e l y s i n e va lues were not found to be s i g n i f i c a n t l y c o r r e l a t e d with the b i o l o g i c a l a s s a y s , supp lementa t ion of the f i s h meals with l y s i n e i n combinat ion wi th a r g i n i n e and methionine s i g n i f i c a n t l y 109 i n c r e a s e d c h i c k weight g a i n s . Cowey et a l . ( 1 9 7 1 ) observed that the t o t a l l y s i n e content i n f r e e z e - d r i e d cod muscle and low temperature d r i e d cod were almost i n d i s t i n g u i s h a b l e . B u t , the a v a i l a b l e l y s i n e content of the l a t t e r was lower than i n the f o r m e r . The low temperature a i r - d r i e d product a l s o supported a somewhat ( s i c ) reduced growth r a t e of p l a i c e . However, both product s c o n t a i n e d a v a i l a b l e l y s i n e i n excess of the requirements for p l a i c e (Cowey et a l . , 1971). March et a l . ( 1 9 6 1 ) conc luded that e s t imates of p r o t e i n q u a l i t y of f i s h meals based upon l a b o r a t o r y ana lyse s for the a v a i l a b i l i t y of i n d i v i d u a l amino a c i d s were u s e f u l i n d e t e c t i n g meals of d i s t i n c t l y i n f e r i o r q u a l i t y . However, the e s t imates f a i l e d to d i f f e r e n t i a t e among meals of average or improved q u a l i t y for p o u l t r y . S i m i l a r l y , i n t h i s s t u d y , i t may be conc luded that the 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 i n the v a r i o u s p r o t e i n sources cannot be r e l i e d upon to p r e d i c t the n u t r i t i v e va lue of p r o t e i n s f o r i n c l u s i o n i n d i e t s for j u v e n i l e chinook salmon. The u t i l i z a t i o n of p r o t e i n by f i s h fed d i e t s c o n t a i n i n g HTH may a l s o have been i m p a i r e d by damage done to other n u t r i e n t s i n h e r r i n g meal . T a r r et a l . ( 1 9 5 4 ) conducted a number of f e e d i n g t r i a l s to determine the e f f e c t s of heat on the n u t r i t i v e va lue of h e r r i n g meal i n c h i c k d i e t s . They compared presscake d r i e d at 4 3 ° C to p o r t i o n s of t h i s meal heated for 1, 2, or 3 hours at 1 4 9 ° C i n a r o t a t i n g drum. They noted that h e a t - l a b i l e v i t a m i n s were d e s t r o y e d a f t e r h e a t i n g for 1 hour , whereas the impairment i n n u t r i t i v e va lue a t t r i b u t e d to a v a i l a b i l i t y of e s s e n t i a l amino a c i d s o c c u r r e d a f t e r pro longed h e a t i n g ( B i s s e t and T a r r , 1954). 110 - By comparing h e x a n e - e x t r a c t e d and u n e x t r a c t e d meals s u b j e c t e d to heat damage, B i e l y et a l . ( 1 9 5 5 ) found no d i f f e r e n c e s i n c h i c k growth that cou ld be a t t r i b u t e d to l i p i d q u a l i t y except when h e a t i n g was e x c e s s i v e . C h i c k performance was improved by hexane e x t r a c t i o n of meals heated for 2 hours at 1 4 9 ° C . However, pro longed h e a t i n g s e v e r e l y i m p a i r e d the q u a l i t y of both e x t r a c t e d and complete meals . T h i s was not improved by the a d d i t i o n of f r e s h h e r r i n g o i l , s u g g e s t i n g that severe damage to p r o t e i n had o c c u r r e d . S i m i l a r l y , i n t h i s study an attempt was made to e x t r a c t the l i p i d s i n the t e s t h e r r i n g meals with hexane. However, e x t r a c t i o n was not complete and at h igher l e v e l s of HTH i n the d i e t s some impairment of growth and p r o t e i n u t i l i z a t i o n due to l i p i d damage may have o c c u r r e d . C a s e i n has been used e x t e n s i v e l y as a p r o t e i n source i n t e s t d i e t s for f i s h . However, c a s e i n a lone has been shown to be d e f i c i e n t i n c e r t a i n amino a c i d s (Ogino and N a n r i , 1980; Rumsey and K e t o l a , 1975). In the t e s t d i e t f or s a l m o n i d s , Ha lver et a l . ( 1 9 5 8 ) employed g e l a t i n to c o r r e c t for an a r g i n i n e d e f i c i e n c y i n c a s e i n . The c a s e i n - g e l a t i n mix employed i n t h i s study was supplemented with a r g i n i n e and m e t h i o n i n e . The r e s u l t s i n d i c a t e d that a l though CS supported an e f f i c i e n t ra t e of p r o t e i n s y n t h e s i s i n f i s h t i s s u e s , food i n t a k e was depressed at a h igher l e v e l of CS i n the d i e t . The major problem encountered with CS i n t h i s study was i t s apparent low p a l a t a b i l i t y for chinook salmon r e l a t i v e to h igh q u a l i t y f i s h meal p r o t e i n . Consequent ly i t d i d not support a growth r a t e comparable to F P E . These f i n d i n g s concur wi th r e p o r t s by other i n v e s t i g a t o r s 111 who conc luded that p r o t e i n sources of marine o r i g i n were p r e f e r a b l e to c a s e i n to meet the g e n e t i c p o t e n t i a l f or growth i n f i s h , and to determine the d i e t a r y p r o t e i n requirement of v a r i o u s f i s h s p e c i e s (Cowey et a l . , 1970,1972; P f e f f e r et a l . , 1980; Winfree and S t i c k n e y , 1981; Watanabe et a l . , 1983). 3 . 4 . 6 The d e t e r m i n a t i o n of p r o t e i n q u a l i t y by b ioas say The methodology for p r o t e i n q u a l i t y e v a l u a t i o n has been d i s c u s s e d f r e q u e n t l y wi th r e s p e c t to human, animal and b i r d a p p l i c a t i o n s ( A l l i s o n , 1959; Samonds and Hegs ted , 1977; McLaughlan , 1979). Al though i t has been g e n e r a l l y agreed that there i s an urgent need for adequate measures of p r o t e i n q u a l i t y so that d i e t s or i n g r e d i e n t s which vary i n p r o t e i n q u a l i t y as w e l l as q u a n t i t y can be compared, there has been no consensus on the most a p p r o p r i a t e method to use . The same problem e x i s t s i n f i s h n u t r i t i o n (Cowey and S a r g e n t , 1972, 1979) and very few i n v e s t i g a t i o n s with f i s h have attempted to e v a l u a t e p r o t e i n q u a l i t y by more than one method. There were three main p o i n t s to c o n s i d e r i n the a p p l i c a t i o n of the p r o t e i n q u a l i t y e s t imates determined i n t h i s s t u d y . F i r s t , the pr imary f u n c t i o n of d i e t a r y p r o t e i n i s to p r o v i d e a mixture of e s s e n t i a l amino a c i d s i n the r i g h t p r o p o r t i o n s f o r the s y n t h e s i s and maintenance of t i s s u e p r o t e i n s . In t h i s r e s p e c t f i s h are no d i f f e r e n t than other a n i m a l s . In sa lmonids and other f i s h s p e c i e s , p r o t e i n s are a l s o major sources of energy (Walton and Cowey, 1982). T h i s c o n t r a s t s with omnivorous 112 mammals where p r o t e i n c a t a b o l i s m i s of l e s s s i g n i f i c a n c e i n s u p p l y i n g energy . The q u a l i t y of a p r o t e i n , as determined by a measurable b i o l o g i c a l re sponse , i s dependent on the p r o t e i n s a t i s f y i n g the r e q u i r e d l e v e l s and ba lance of e s s e n t i a l amino a c i d s , f o r growth and maintenance , as w e l l as s u p p l y i n g an unknown q u a n t i t y of p r o t e i n f o r energy . Rat s t u d i e s have shown that the amino a c i d needs for maintenance d i f f e r both a q u a n t i t a t i v e l y and q u a l i t a t i v e l y from those for r a p i d growth (Maynard and L o o s l i , 1969). T h e r e f o r e , i t would be expected that p r o t e i n sources would d i f f e r i n t h e i r a b i l i t y to support growth and p r o v i d e f o r maintenance . Methods which determine p r o t e i n q u a l i t y at d i f f e r e n t l e v e l s of p r o t e i n i n t a k e have the advantage of g i v i n g a more complete p i c t u r e of p r o t e i n u t i l i z a t i o n than would be o b t a i n e d at s i n g l e l e v e l s of p r o t e i n i n t a k e (Samonds and Hegs ted , 1977; McLaughlan , 1979). On the o ther hand, as p r o t e i n i s r e p l a c e d by c a r b o h y d r a t e on an es t imated m e t a b o l i z a b l e energy b a s i s , the e f f e c t of c a r b o h y d r a t e i n t a k e i n excess of that b e l i e v e d to be t o l e r a b l e by sa lmonids ( P h i l l i p s , 1969; H i l t o n et a l . , 1982) on p r o t e i n q u a l i t y e s t imates i s not f u l l y u n d e r s t o o d . The second aspect concerns the purpose of the p r o t e i n q u a l i t y assay i n f i s h s t u d i e s . T h i s was to c l a s s i f y b i o l o g i c a l l y the s u i t a b i l i t y of the e s s e n t i a l amino a c i d balance of a p r o t e i n as the so l e source of p r o t e i n i n a d i e t . I t i s most d e s i r a b l e to make t h i s c l a s s i f i c a t i o n i n r e l a t i o n to a w e l l i d e n t i f i e d s t a n d a r d . In t h i s study p r o t e i n sources were c l a s s i f i e d r e l a t i v e to FPE (Tab le 27) . U l t i m a t e l y , i t was 113 T a b l e 27. Summary of r e l a t i v e (FPE = 100) e s t i m a t e s of the n u t r i t i v e v a l u e of the t e s t p r o t e i n s o u r c e s employed i n t h i s s t u d y d e t e r m i n e d by d i f f e r e n t methods. P r o t e i n s o u r c e GR GFC GEU PER NPR PPV NPU-1 NPU-2 S l o p e ( w e i g h t g a i n ) S l o p e ( p r o t e i n g a i n ) FPE 100 100 100 100 100 100 100 100 100 100 FRH 95 108 99 101 102 94 96 99 101 85 LTH 86 88 86 84 89 85 89 91 77 76 HTH 26 32 26 26 50 16 44 52 53 47 CS 77 86 77 82 88 79 86 90 101 95 1. NPU-1 and NPU-2 r e f e r s to the methods of Bender and M i l l e r (1953) and Ogino e t a l . (1980) r e s p e c t i v e l y employed to c a l c u l a t e t h i s p a r a m e t e r . d e s i r e d to i d e n t i f y the p r o t e i n s as good, i n t e r m e d i a t e or poor i n q u a l i t y , j u s t as the chemica l d e t e r m i n a t i o n of p r o t e i n content i n d i c a t e s the amount of the i n g r e d i e n t that i s to be i n c l u d e d i n the d i e t . In t h i s sense , the r e s u l t s of a b ioassay served to c o n f i r m the s u i t a b i l i t y of the e s s e n t i a l amino a c i d content of the p r o t e i n for growth of f i s h . For example, Hegsted (1972) s t a t e s that what i s r e q u i r e d by human d i e t i c i a n s i s the measure of some f a c t o r ( f ) , such that the p r o t e i n content of the f e e d s t u f f x f = p r o t e i n a v a i l a b l e to the a n i m a l . Such a f a c t o r should vary from 0 to 1.0 and be p r o p o r t i o n a l to the t rue q u a l i t y . T h e r e f o r e , t h i s scheme would enable the f i s h n u t r i t i o n i s t to formula te d i e t s based on the b i o l o g i c a l va lue of p r o t e i n a v a i l a b l e to the f i s h r a t h e r than on p r o t e i n content a l o n e . Thus , the 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 i n sa lmonids has s e v e r a l p o t e n t i a l a p p l i c a t i o n s which are as f o l l o w s : i ) The measurement of the e f f e c t i v e n e s s of a p r o t e i n source to meet s p e c i f i c performance requirements of c u l t u r e d f i s h (Cowey and S a r g e n t , 1972). i i ) The m o n i t o r i n g of p r o c e s s i n g methods that are used i n the manufacture of f i s h food i n g r e d i e n t s ( i . e . f i s h m e a l ) and f i s h f eed . i i i ) The d e t e r m i n a t i o n of the minimum requirements for amino a c i d s and p r o t e i n s ( Z e i t o u n , 1973). i v ) The comparison of r e s u l t s o b t a i n e d with nove l p r o t e i n sources by other i n v e s t i g a t o r s when expressed on a r e l a t i v e b a s i s . 115 - v) The p r e d i c t i o n of the n u t r i t i v e va lue of d i e t a r y p r o t e i n s f o r the o ther a n i m a l s , p a r t i c u l a r l y c a r n i v o r o u s s p e c i e s . The t h i r d aspect that should be r e c o g n i z e d i s that a l l the p r o t e i n q u a l i t y parameters determined i n t h i s study are i n t e r r e l a t e d and are a f f e c t e d by the same f a c t o r s . For example, the l e v e l of p r o t e i n i n t a k e a f f e c t s i n the same manner PER, NPR, PPV, NPU and s lope a s s a y . Feeding t r i a l methods wi th r a t s are u s u a l l y s u b d i v i d e d i n t o those based on weight ga in and those based upon body n i t r o g e n r e t e n t i o n (McLaughlan , 1979). In the present s t u d y , p r o t e i n ga in was h i g h l y c o r r e l a t e d with weight ga in ( c o r r e l a t i o n c o e f f i c i e n t = 0.9888) and the former c o u l d be p r e d i c t e d from the l a t t e r by the f o l l o w i n g e q u a t i o n : P r o t e i n ga in = 0.6172 x dry weight ga in - 0 .0096. Z e i t o u n et a l . (1976) a l s o r e p o r t e d that percent weight ga in and p r o t e i n r e t e n t i o n were h i g h l y c o r r e l a t e d . The important p o i n t i s whether the e x t r a work i n d e t e r m i n i n g the p r o t e i n content of the c a r c a s s i s worth the e f f o r t . C l e a r l y i t was f o r the s lope a s s a y . Under the e x p e r i m e n t a l c o n d i t i o n s of the present study disagreement was found i n the r e l a t i v e va lues and r a n k i n g of the good p r o t e i n sources t e s t e d at d i f f e r e n t l e v e l s . T h i s shortcoming i n terms of s e n s i t i v i t y and p r e c i s i o n suggests that m o d i f i c a t i o n s are necessary to the e x p e r i m e n t a l p r o t o c o l . The mean va lues for PER, NPR, PPV, and NPU r e s p e c t i v e l y (Table 27) were taken from the va lues o b t a i n e d at a l l d i e t a r y p r o t e i n c o n c e n t r a t i o n s for each p r o t e i n s o u r c e . In the s lope assays t h i s problem was p a r t l y overcome s i n c e i n the a n a l y s i s of l i n e a r r e g r e s s i o n a l l va lues were c o n s i d e r e d . A l though there i s 116 disagreement wi th r e s p e c t to the use of a p r o t e i n - f r e e d i e t fed group i n p r o t e i n q u a l i t y assays (Samonds and Hegs ted , 1977) i t was noted to be advantageous i n the present s t u d y . In s p i t e of the shortcomings i n e s t i m a t i n g p r o t e i n q u a l i t y i t was found to be c l e a r that p r o t e i n s were measurably d i f f e r e n t i n t h e i r a b i l i t i e s to support growth and meet maintenance needs. The i n f e r e n c e i s i n that a poor q u a l i t y p r o t e i n such as HTH cou ld s u f f i c e i f fed at a s u f f i c i e n t l y h igh l e v e l . I f HTH has a n u t r i t i v e va lue 50% of the best q u a l i t y p r o t e i n (FPE) based on NPR, NPU, and s lope e s t imates (Tab le 27) , then double the amount of that p r o t e i n would be needed i n f i s h d i e t s . The va lue w i l l be quadrupled i f the r e l a t i v e va lue of HTH l i e s i n the r e g i o n of 25% of FPE as e s t imated by PER, and PPV. In f a c t s i m i l a r performances i n j u v e n i l e chinook salmon i n terms of growth, feed c o n v e r s i o n and d i e t a r y energy u t i l i z a t i o n were o b t a i n e d wi th the good q u a l i t y p r o t e i n sources ( F P E , FRH, LTH and CS) and a poor q u a l i t y p r o t e i n (HTH) when the p r o t e i n content of the d i e t wi th the l a t t e r source was a p p r o x i m a t e l y doubled (Tab le 5, 7, 9 ) . U l t i m a t e l y , the u s e f u l n e s s of a p r o t e i n source l i e s i n i t s cos t e f f e c t i v e n e s s i n the d i e t as a whole for growth and feed e f f i c i e n c y . In the case of farmed f i s h r a i s e d for f o o d , one should c o n s i d e r the q u a l i t y and y i e l d of the f i n a l p r o d u c t . For h a t c h e r y - r e a r e d f i s h c o n s i d e r a t i o n should be g iven to the e f f e c t i v e n e s s of the d i e t to promote ocean s u r v i v a l . I f the above s tatements are a c c e p t e d , then i t i s d i f f i c u l t to s t a t e c a t e g o r i c a l l y which p r o t e i n q u a l i t y assay method should be recommended f o r r o u t i n e f e e d i n g t r i a l s wi th s a l m o n i d s . When 117 the r e l a t i v e s cores f o r the v a r i o u s p r o t e i n s were compared ( T a b l e 27) , there was a g e n e r a l agreement between the v a r i o u s b i o l o g i c a l assays f o r e v a l u a t i n g p r o t e i n q u a l i t y and growth r a t e , gross food c o n v e r s i o n e f f i c i e n c y and gross energy u t i l i z a t i o n . Henry and T o o t h i l l (1962) , Johnston and Coon (1979) and McLaughlan (1979) found that PER va lues i n r a t s and c h i c k s were c o r r e l a t e d with those f o r NPU and NPR for a v a r i e t y of p r o t e i n s . Henry (1965) o b t a i n e d n u m e r i c a l l y s i m i l a r r e l a t i v e va lues ( c a s e i n = 100) f o r h igh q u a l i t y p r o t e i n s by PER, NPU and NPR. For poor q u a l i t y p r o t e i n s , PER y i e l d s much lower va lues than the o ther methods. The r e s u l t s of t h i s study c o n f i r m both these f i n d i n g s . S ince PER and PPV on one hand, and NPR and NPU on the o t h e r , are e s s e n t i a l l y the same assay ( i . e . weight ga in and p r o t e i n ga in are r e l a t e d ) , agreement between these two methods was e x c e l l e n t (Tab le 28) . The va lues f o r PER were c a l c u l a t e d on a dry f i s h weight ba s i s (Higgs et a l . , 1979) to f a c i l i t a t e comparison wi th v a l u e s o b t a i n e d by Higgs et a l . ( 1 9 8 2 ) and P l o t n i k o f f et a l . ( 1 9 8 3 ) wi th chinook salmon f r y . Comparisons between the r e s u l t s r e p o r t e d h e r e i n (Tab le 15) and those of other i n v e s t i g a t o r s should be i n t e r p r e t e d c a u t i o u s l y because of d i f f e r i n g e x p e r i m e n t a l c o n d i t i o n s . M o r r i s o n and Campbel l (1960) s t a t e d that PER v a r i e d not only wi th sex , age, g e n e t i c s , the q u a n t i t y and q u a l i t y of the p r o t e i n , and other d i e t a r y components, but a l s o wi th the d u r a t i o n of the t e s t . The PER va lues for a commercial dry d i e t (Abernathy) and v a r i o u s m o d i f i c a t i o n s of t h i s d i e t w i th p r o t e i n from rapeseed byproducts (Higgs et a l . , 1982) were determined 118 - Table 28. C o r r e l a t i o n c o e f f i c i e n t s and l e v e l of s i gn i f i cance between the d i f f e r e n t parameters used to estimate the n u t r i t i v e value of d iets containing the various p r o t e i n sources. GFC 0.9668 P - 0.000 GEU 0.9658 P = 0.000 0.9936 P = 0.000 PER 0.7172 P = 0.000 0.4635 P = 0.002 0.8096 P = 0.000 NPR 0.2360 P = 0.083 0.2790 P = 0.50 0.3126 P = 0.032 0.6765 P = 0.000 PPV 0.7391 P = 0.000 0.8056 P = 0.000 0.8308 P = 0.000 0.9765 P = 0.000 0.6519 P = 0.000 NPU-1 0.2525 P = 0.069 0.2863 P = 0.045 0.3271 P = 0.026 0.6319 P = 0.000 0.9620 P = 0.000 0.6669 P = 0.000 NPU-2 0.0213 P = 0.451 0.0396 P = 0.409 0.0727 P = 0.337 0.4037 P = 0.007 0.8896 P = 0.000 0.4374 P = 0.004 0.9266 P = 0.000 LYS 0.8503 P = 0.000 0.8410 P = 0.000 0.8379 P = 0.000 0.5349 P = 0.001 0.0497 P = 0.390 0.5776 P = 0.000 0.0955 P = 0.295 -0.1220 P = 0.246 GR GFC GEU PER NPR PPV NPU-1 NPU-2 under s i m i l a r e x p e r i m e n t a l c o n d i t i o n s to those of the present study except that a 69 day t r i a l p e r i o d was employed. The va lue obta ined f o r a commercia l d i e t (OMP) ( T a b l e 15) i s of the same order as the PER r e p o r t e d by Higgs et a l . ( 1 9 8 2 ) f o r v a r i o u s p r a c t i c a l d i e t s . However, i n the present study a h igher PER was o b t a i n e d with FPE at a s i m i l a r p r o t e i n l e v e l to a l l of the above d i e t s . I t may be suggested t h e r e f o r e that i f a premium p r o t e i n source such as FPE i s employed as a c o n t r o l i n p r a c t i c a l d i e t e v a l u a t i o n , a more meaningfu l comparison may be made with a group of f i s h per forming c l o s e r to t h e i r g e n e t i c p o t e n t i a l f or growth. Higgs et a l . ( 1 9 8 2 ) and P l o t n i k o f f et a l . ( 1 9 8 3 ) a l s o e v a l u a t e d p r a c t i c a l d i e t s , i n c l u d i n g OMP under ha tchery management c o n d i t i o n s i n a d i f f e r e n t l o c a l i t y (Robertson Creek Hatchery ) to the present s t u d y . They r e p o r t e d PER va lues c o n s i d e r a b l y h i g h e r than those determined at the West Vancouver l a b o r a t o r y . T h i s i s probably due to d i f f e r e n c e s between the chinook s t o c k s i n g e n e t i c c o n s t i t u t i o n and to e l e v a t e d water temperatures and d i f f e r e n t f e e d i n g s t r a t e g y at the h a t c h e r y . I t may be p o s t u l a t e d that i f FPE were used as a s o l e p r o t e i n s o u r c e , or only premium q u a l i t y h e r r i n g meal were employed i n d i e t s fed to Robertson Creek chinook salmon, o u t s t a n d i n g PER va lues may be o b t a i n e d . Higgs et a l . ( 1 9 8 3 ) showed that d i e t a r y p r o t e i n content s i g n i f i c a n t l y i n f l u e n c e d p r o t e i n u t i l i z a t i o n as determined by PER with chinook f r y . The p r o t e i n l e v e l s employed i n the present study f o r d i e t s c o n t a i n i n g FPE covered a wider range 120 than those of Higgs et a l . (1983) . The downward t rend i n PER i n d i e t s c o n t a i n i n g more than 27% p r o t e i n ( T a b l e 15, F i g . 7) i s c o n s i s t e n t wi th that found wi th chinook salmon (Higgs et a l . , 1983), c a r p . ( O g i n o and S a i t o , 1970), j u v e n i l e rainbow t r o u t ( T a k e u c h i et a l , 1978), j u v e n i l e chum salmon ( K o s h i i s h i , 1980) and grass carp f r y (Dabrowski and Kosak, 1979). Over a wider range of p r o t e i n l e v e l s the r e l a t i o n s h i p wi th PER f o l l o w e d a s i m i l a r p a t t e r n to that observed i n p l a i c e fed d i e t s c o n t a i n i n g f r e e z e - d r i e d cod muscle (Cowey et a l . , 1972) and r a t s fed c a s e i n d i e t s (Hegsted and Chang, 1965) ( F i g . 7 ) . The noteworthy d i f f e r e n c e between these curves i s the l e v e l of d i e t a r y p r o t e i n at which maximum PER was o b t a i n e d . Maximum PER with the r e s p e c t i v e p r o t e i n s o c c u r r e d at d i e t a r y p r o t e i n l e v e l s of 16% i n r a t s , 27% i n c h i n o o k , and 40% i n p l a i c e . D i f f e r e n c e s between the d i e t a r y p r o t e i n l e v e l f or maximum PER i n t h i s study (27%) and other s t u d i e s may be due to s p e c i e s d i f f e r e n c e s , a l though confounded by the p r e v i o u s l y mentioned f a c t o r s a f f e c t i n g the d e t e r m i n a t i o n of PER. In the U n i t e d S t a t e s and Canada, PER i s used to measure p r o t e i n q u a l i t y , p a r t i c u l a r l y f o r r e g u l a t o r y purposes , and i s an o f f i c i a l method of the 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 C h e m i s t s . T e s t s are performed with r a t s . I t i s customary to feed c a s e i n as a c o n t r o l p r o t e i n . C a s e i n i s used because i t i s a r e a d i l y o b t a i n a b l e pure p r o t e i n . PER es t imates r e l a t i v e to that determined f o r c a s e i n are r e p o r t e d i n an attempt to f a c i l i t a t e comparisons i n PER measurements among i n v e s t i g a t o r s . For example, when the PER va lue for c a s e i n was set at 100, f or 121 each as say , the r e l a t i v e PER f o r f i s h meal was 109, d e f a t t e d beef 88, soy 78, pea p r o t e i n 23 and wheat g l u t e n 6 (McLaughlan , 1979). S i m i l a r l y , the va lues obta ined i n t h i s study can be c a u t i o u s l y compared with those of o ther i n v e s t i g a t o r s ; b e a r i n g i n mind that c a s e i n was supplemented with g e l a t i n , a r g i n i n e and methionine i n the present study (Tab le 17) . G e n e r a l l y , r e l a t i v e PER va lues o b t a i n e d wi th f i s h fed animal p r o t e i n s ( f i s h meals , egg p r o t e i n s ) by other i n v e s t i g a t o r s were of a s i m i l a r order to those determined f o r F P E , FRH, and LTH i n t h i s s t u d y . The PER va lues for p r o t e i n s of p l a n t o r i g i n are c o n s i s t e n t l y lower than those of animal o r i g i n , probably because p l a n t p r o t e i n s have lower d i g e s t i b i l i t y and a poorer amino a c i d ba lance r e l a t i v e to animal p r o t e i n s . A l s o , each p l a n t p r o t e i n source c o n t a i n s one or more a n t i - n u t r i t i o n a l f a c t o r s (Higgs et a l . , 1983). S i n g l e c e l l p r o t e i n sources such as p e t r o y e a s t and m e t h a n o p h i l i c b a c t e r i a look p r o m i s i n g f o r i n c l u s i o n i n t o chinook salmon d i e t s whi l e a l g a l p r o t e i n s do not (Tab le 17) . S e v e r a l s t u d i e s i n f i s h r e p o r t the e f f i c i e n c y of p r o t e i n d e p o s i t i o n to measure the n u t r i t i v e va lue of a f e e d s t u f f (Ogino and S a i t o , 1970; Z e i t o u n et a l . , 1973; De l a Higuera et a l . , 1977; Higgs et a l . , 1979; P f e f f e r , 1982; C l a r k e et a l . , 1982). In the present d i s c u s s i o n t h i s i s termed p r o t e i n p r o d u c t i v e va lue ( P P V ) . S ince PPV and PER are e s s e n t i a l l y the same assay (the former r e l a t e s body p r o t e i n ga in and the l a t t e r body weight ga in to p r o t e i n i n t a k e ) the r e s u l t s obta ined by the two methods were h i g h l y c o r r e l a t e d ( T a b l e 28) . 122 Under s i m i l a r e x p e r i m e n t a l c o n d i t i o n s to the present s t u d y , Higgs et a l . ( 1 9 8 2 ) r e p o r t e d an e q u i v a l e n t PPV (32%) f o r another commercial d i e t ( A b e r n a t h y ) . At h i g h e r water temperatures and with a d i f f e r e n t s tock of chinook f r y (Robertson Creek) Higgs et al.(1982) and P l o t n i k o f f et a l . ( 1 9 8 3 ) r e p o r t e d a va lue f o r PPV of approx imate ly 40% with f i s h fed OMP and v a r i o u s t e s t p r a c t i c a l dry d i e t s . Higher e s t imates for PPV have been r e p o r t e d f o r chinook salmon i n the above i n v e s t i g a t i o n s and i n the present study compared to those f o r coho salmon ( Z e i t o u n et a l . , 1973; Higgs et a l . , 1979; C l a r k e et a l . , 1982) and rainbow t r o u t ( Z e i t o u n et a l . , 1973; P f e f f e r , 1982). T h i s may suggest that chinook salmon can u t i l i z e d i e t a r y p r o t e i n more e f f e c t i v e l y than coho salmon for body p r o t e i n s y n t h e s i s and t h e r e f o r e the former s p e c i e s may be more p r e f e r a b l e than the l a t t e r f o r f i s h f a r m i n g . In f i s h n u t r i t i o n s t u d i e s two methods have been adopted f o r measuring NPU ( T a b l e 17) . A l though NPU was c a l c u l a t e d by the Bender and M i l l e r (1953) and Ogino et a l . (1980) methods, very c l o s e r e l a t i v e NPU (FPE = 100) va lues were obta ined by both- p r o c e d u r e s . The main d i f f e r e n c e between the two methods i s that the l a t t e r c o r r e c t s for maintenance requirements on a body weight b a s i s . T h e o r e t i c a l l y t h i s i s more a c c u r a t e s i n c e i t a v o i d s o v e r e s t i m a t i n g the q u a l i t y of p r o t e i n sources measured i n f i s h a c h i e v i n g a h i g h e r mean body we ight . With the method of Ogino et a l . ( 1 9 8 0 ) a NPU of 100% was obta ined wi th a d i e t a r y p r o t e i n i n t a k e that e x a c t l y met maintenance requirements ( F i g . 9 ) . 123 In t h i s study NPU was determined at s e v e r a l l e v e l s of d i e t a r y p r o t e i n . T h i s avo ided the problem encountered by Atack et a l . ( 1 9 7 9 ) who had d i f f i c u l t y comparing NPU va lues o b t a i n e d with carp f o r m e t h a n o p h i l i c b a c t e r i a and h e r r i n g meal because t h e i r d i e t s c o n t a i n e d 35% and 25% p r o t e i n , r e s p e c t i v e l y ( T a b l e 17) . The l e v e l of d i e t a r y p r o t e i n for e v a l u a t i n g p r o t e i n sources for f i s h has not been s t a n d a r d i z e d at one a r b i t r a r y l e v e l as i t has been for methods employing r a t s . However, s i n c e 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 i s dose dependent , an e s t imate at one l e v e l of i n t a k e may not be p r o p o r t i o n a l to t r u e p r o t e i n q u a l i t y (Samonds and Hegs ted , 1977). As mentioned p r e v i o u s l y , s t a n d a r d i z a t i o n at one l e v e l of d i e t a r y p r o t e i n p e n a l i z e d both h igh q u a l i t y and low q u a l i t y p r o t e i n s (McLaughlan , 1979). A dependence of NPU on p r o t e i n i n t a k e has a l s o been r e p o r t e d for carp fed c a s e i n (Ogino and S a i t o , 1970), t r o u t fed c a s e i n (Watanabe et a l . , 1978) and p l a i c e fed f r e e z e - d r i e d cod muscle (Cowey et a l . , 1 9 7 2 ) ( F i g . 9 ) . The range of NPU va lues r e p o r t e d for j u v e n i l e chinook i n t h i s study are comparable to those obta ined with rainbow t r o u t for s i m i l a r types of p r o t e i n by a s i m i l a r method ( T a k e u c h i et a l . , 1978; O g i n o , 1980; Ogino and N a n r i , 1980; Watanabe et a l . , 1983). The d i f f e r e n c e s i n va lues between the s t u d i e s ( T a b l e 17) are most l i k e l y due to d i f f e r e n c e s i n s p e c i e s , s t r a i n , f i s h s i z e , t e m p e r a t u r e , f e e d i n g regimen and other i n g r e d i e n t s i n the d i e t s . Comparison of r e s u l t s o b t a i n e d by other i n v e s t i g a t o r s can only be meaningfu l i f r e l a t i v e NPU ( c a s e i n = 100) va lues are used as they were for PER (Tab le 17) . However, s i n c e c a s e i n was 124 not used as the s o l e source of p r o t e i n i n the present s t u d y , a meaningful comparison with other s t u d i e s i s h i g h l y s u b j e c t i v e . Because few i n v e s t i g a t o r s have attempted to measure p r o t e i n q u a l i t y at more than one l e v e l of p r o t e i n i n t a k e , the s lope r a t i o method (Hegsted and Chang, 1965a) has not been adopted per se to e v a l u a t e p r o t e i n q u a l i t y i n f i s h . S e v e r a l s t u d i e s were d i s c u s s e d e a r l i e r where, i n r e p o r t i n g o b s e r v a t i o n s on n i t r o g e n b a l a n c e , body p r o t e i n ga in was r e g r e s s e d a g a i n s t p r o t e i n i n t a k e ( Iwata , 1970; G e r k i n g , 1971; Nose, 1971; R y c h l y , 1980). Up to the l e v e l of p r o t e i n i n t a k e where the minimum requirement of p r o t e i n for growth was met (see Chap. 4) a l i n e a r r e l a t i o n s h i p between i n t a k e and response (weight ga in or p r o t e i n ga in) was assumed (De Long et a l . , 1958; Hegsted and Chang, 1965; G e r k i n g , 1971). The q u e s t i o n of l i n e a r i t y i n the s lope assay has o f t e n been r a i s e d with r e s p e c t to the use of a p r o t e i n - f r e e d i e t fed group of animals (McLaughlan , 1977). O b j e c t i o n s to the employment of a z e r o - p r o t e i n fed group i n r a t assays i s due to r e p o r t s of e x c e s s i v e downward c u r v a t u r e of the s l opes at very low i n t a k e s , which i s encountered with some p r o t e i n s . S p e c i f i c a l l y , l y s i n e - d e f i c i e n t p r o t e i n s such as wheat g l u t e n have caused t h i s problem (Yanez and McLaughlan , 1979). The r a t e of c a t a b o l i s m of l y s i n e i n r a t s i s dependent upon the a v a i l a b i l i t y of that amino a c i d ( B o d w e l l , 1977). At low l y s i n e i n t a k e s , c a t a b o l i s m of l y s i n e i s r e t a r d e d , r e s u l t i n g i n the c o n s e r v a t i o n and r e - u t i l i z a t i o n of t h i s amino a c i d . Threon ine d e f i c i e n t p r o t e i n s , on the other hand, show upward c u r v a t u r e at low i n t a k e s as t h i s amino a c i d appears to have no c o n s e r v a t i o n 125 mechanism (McLaughlan and K e i t h , 1977). Hegsted et a l . ( 1 9 6 8 ) and McLaughlan (1979) conc luded that the s lope r a t i o assay which i n c l u d e s the p r o t e i n - f r e e d i e t fed group was a more s u i t a b l e method than the one e x c l u d i n g t h i s group . T h i s i s because the former s i t u a t i o n tended to reduce p a r a l l e l i s m between l i n e s which l ed to erroneous v a l u e s . In t h i s study the i n t e r c e p t s for the dose response l i n e s were brought c l o s e r toge ther when the data for the p r o t e i n - f r e e d i e t fed groups was i n c l u d e d . At the same time the c o r r e l a t i o n c o e f f i c i e n t s were h i g h . Hegsted et a l . ( 1 9 6 8 ) found that the p r e c i s i o n of the s lope r a t i o assay depended p r i m a r i l y on the number of r a t s used . When the number of animals fed each d i e t was reduced from s i x to t h r e e , the s tandard e r r o r of the s l opes i n c r e a s e d by a p p r o x i m a t e l y 30%. In the present s t u d y , d u p l i c a t e groups were employed and undoubtedly both p r e c i s i o n and s e n s i t i v i t y c o u l d have been improved i f a d d i t i o n a l groups of f i s h had been fed each d i e t . When the s lopes for body p r o t e i n ga in i n c l u d i n g the zero p r o t e i n d i e t fed groups were ana lyzed ( T a b l e 24) , meaningful comparisons were found. By t h i s method the assay was ab le to d i s t i n g u i s h between the t e s t p r o t e i n s o u r c e s . 126 3.5 Summary of Experiment 1 In t h i s experiment the r e l a t i o n s h i p between p r o t e i n source i n the d i e t of chinook salmon f r y , and performance i n terms of growth, food c o n v e r s i o n and p r o t e i n u t i l i z a t i o n was i n v e s t i g a t e d . The f i s h were fed the e x p e r i m e n t a l d i e t s to measured s a t i a t i o n for 42 days . The r e s u l t s showed that the p r o c e s s i n g c o n d i t i o n s of f i s h meal had a pronounced e f f e c t on growth and the u t i l i z a t i o n of p r o t e i n as measured by v a r i o u s parameters . Rapid growth and best p r o t e i n u t i l i z a t i o n was ach ieved by f r e e z e - d r y i n g the raw m a t e r i a l . Cooking f o l l o w e d by low temperature ( 7 5 ° C ) d r y i n g of h e r r i n g meal r e s u l t e d i n s l i g h t l y i n f e r i o r performance . High temperature (150"C) d r y i n g had a severe adverse e f f e c t on the p r o t e i n q u a l i t y of h e r r i n g meal . In a d d i t i o n a c a s e i n - g e l a t i n based d i e t , a l though a p p a r e n t l y adequate in terms of p r o t e i n q u a l i t y , gave i n c o n s i s t e n t r e s u l t s because of poor p a l a b i l i t y . Comparisons between f i s h fed the t e s t d i e t s and a commercial d i e t (OMP) r e v e a l e d that the p r o t e i n l e v e l of the l a t t e r cou ld probably be lowered . An attempt was made to compare v a r i o u s b i o l o g i c a l assay techniques for e v a l u a t i n g p r o t e i n q u a l i t y . The r e s u l t s were g e n e r a l l y found to be more r e l i a b l e when p r o t e i n q u a l i t y was measured at more than one l e v e l of d i e t a r y p r o t e i n . In t h i s re spec t s lope r a t i o s may have p r o v i d e d the best means of d i r e c t comparison between the p r o t e i n s . A l s o , the measurement of p r o t e i n ga in r a t h e r than weight ga in was c o n s i d e r e d to be b i o l o g i c a l l y more v a l i d . The maintenance requirements for 127 p r o t e i n were e s t imated by f e e d i n g f i s h a p r o t e i n - f r e e and a low p r o t e i n d i e t . T h i s enabled the e s t i m a t i o n of p r o t e i n q u a l i t y by methods which c o r r e c t f o r the endogenous l o s s of n i t r o g e n from the body. P r o t e i n i n t a k e was p a r t i t i o n e d i n t o the amounts u t i l i z e d for maintenance and growth, and the amount of exogenous e x c r e t i o n . T h i s p r o v i d e d a c l e a r d e p i c t i o n of how the v a r i o u s p r o t e i n sources compared as the l e v e l of p r o t e i n i n t a k e i n c r e a s e d . Thus the l e v e l of p r o t e i n i n t a k e for maxmimum e f f i c i e n c y wi th a p a r t i c u l a r p r o t e i n source cou ld be i n t e r p r e t e d . L a s t l y , the n u t r i t i v e va lue of the t e s t p r o t e i n s was e v a l u a t e d i n terms of t h e i r a v a i l a b l e l y s i n e c o n t e n t . A l though the p r o c e s s i n g c o n d i t i o n s of the meals a f f e c t e d p r o t e i n q u a l i t y as assessed by b i o a s s a y , the e f f e c t cou ld not have been adequate ly p r e d i c t e d by the chemica l 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 . In c o n c l u s i o n , c e r t a i n procedures were judged to be p r e f e r a b l e to o t h e r s , and s u b s t a n t i a l improvements i n e x p e r i m e n t a l p r o t o c o l may p r o v i d e a more s e n s i t i v e and p r e c i s e method to measure the n u t r i t i v e va lue of p r o t e i n s i n d i e t s f o r f i s h . 128 Chapter 4 EXPERIMENT 2 4.0 P r o t e i n requirements of j u v e n i l e chinook salmon i n r e l a t i o n to d i e t a r y energy content 4 .1 I n t r o d u c t i o n The p r e v i o u s experiment showed that a b lend of f r e e z e - d r i e d p o l l o c k muscle wi th f r e e z e - d r i e d whole euphausids (FPE) p r o v i d e d a more p a l a t a b l e and h igher q u a l i t y p r o t e i n source than a combinat ion of c a s e i n and g e l a t i n , supplemented with a r g i n i n e and m e t h i o n i n e , f or i n c l u s i o n i n j u v e n i l e chinook salmon d i e t s . The gross p r o t e i n requirements of j u v e n i l e chinook salmon were e s t a b l i s h e d (NRC 1973,1981) us ing c a s e i n - g e l a t i n based d i e t s (De Long et a l . , 1958). The requirements of j u v e n i l e chinook salmon for p r o t e i n have n o t , however been determined with p r o t e i n d e r i v e d from f i s h e r y product s wi th a demonstrated h igh b i o l o g i c a l v a l u e . Moreover , the b a s a l d i e t employed by De Long et a l . , ( 1 9 5 8 ) had a h igh content of omega-6 type f a t t y a c i d s from corn o i l wh ich , i n l i g h t of more recent i n f o r m a t i o n (Yu and S i n n h u b e r , 1979), i s known to depress growth r a t e of sa lmonids when fed at d i e t a r y c o n c e n t r a t i o n s i n excess of 1% . I t t h e r e f o r e seemed worthwhi le to i n v e s t i g a t e the p r o t e i n requ irements of j u v e n i l e chinook salmon us ing FPE as the so l e source of p r o t e i n i n d i e t s c o n t a i n i n g two l e v e l s of d i e t a r y l i p i d s u p p l i e d by marine o i l . T h i s approach a l s o enabled d e t e r m i n a t i o n of the o p t i m a l r a t i o of p r o t e i n to energy i n d i e t s for j u v e n i l e chinook salmon. - 129 - In regard to the l a t t e r a im, Lee and Putnam (1973) conducted one of the few experiments on sa lmonids i n which d i e t a r y l e v e l s of both p r o t e i n and energy were v a r i e d . T h e i r methodology p r o v i d e d e s t i m a t i o n of an a c c e p t a b l e r a t i o of p r o t e i n e n e r g y : t o t a l energy ( P E : T E ) i n p r a c t i c a l d i e t s f or rainbow t r o u t . S i m i l a r e s t imates have n o t , however, been o b t a i n e d for P a c i f i c salmon d i e t s . 4 . 2 M a t e r i a l s and methods 4 .2 .1 P r o t o c o l Whereas Experiment 1 was conducted for 43 days , Experiment 2 was c o n t i n u e d for 105 days . T h i s p e r i o d approximates the freshwater r e s i d e n c y p e r i o d of j u v e n i l e chinook salmon. The same s tock of chinook f r y were employed i n Experiment 2 that was used i n Experiment 1. The f i s h were d i s t r i b u t e d at the same time and the same manner as d e s c r i b e d i n the p r e v i o u s experiment . The experiment was des igned as a 4 x 2 randomized b lock f a c t o r i a l wi thout r e p l i c a t i o n ( Z a r , 1974). Four c o n c e n t r a t i o n s of d i e t a r y p r o t e i n and two l e v e l s of d i e t a r y energy were the f a c t o r s t e s t e d . Each of the e i g h t d i e t a r y treatments was randomly a s s i g n e d i n each of the two rows. As i n Experiment 1, each row of tanks c o n s t i t u t e d a b l o c k . 4 .2 .2 D i e t s The d i e t s were formula ted to c o n t a i n 17%, 27%, 37% or 47% of dry matter as crude p r o t e i n from FPE at each of two l e v e l s of - 130 - a v a i l a b l e d i e t a r y energy , 3150 and 3950 k c a l / k g dry d i e t ( T a b l e 29) . At each l e v e l of m e t a b o l i z a b l e energy , l i p i d l e v e l s were kept cons tant at 6% or 13% r e s p e c t i v e l y . C a r b o h y d r a t e , i n the form of equal p r o p o r t i o n s of d e x t r i n and g l u c o s e , was s u b s t i t u t e d for p r o t e i n on an equal m e t a b o l i z a b l e energy b a s i s as d e s c r i b e d i n the p r e v i o u s exper iment . The proximate a n a l y s i s of the d i e t s i s shown i n T a b l e 30, toge ther with the c a l c u l a t e d m e t a b o l i z a b l e and gross energy c o n c e n t r a t i o n of the d i e t s . 4 . 2 . 3 Data a n a l y s i s The data for f i s h body weight , feed consumpt ion , feed a n a l y s i s and f i s h c a r c a s s a n a l y s i s were t r e a t e d as d e s c r i b e d i n Experiment 1. The parameters were s u b j e c t e d to a two-way randomized f a c t o r i a l ANOVA. A mixed e f f e c t s model was employed s i m i l a r to that d e s c r i b e d for Experiment 1. D i e t a r y p r o t e i n and energy c o n c e n t r a t i o n were assumed to be f i x e d e f f e c t s and row a random e f f e c t . Treatment means were then s u b j e c t e d to Duncan's New M u l t i p l e Range Tes t (1955)(DMR) (P = 0 . 0 5 ) . Data on f i s h fed OMP was not i n c l u d e d i n the s t a t i s t i c a l a n a l y s i s but are i n c l u d e d i n the t a b l e s and f i g u r e s for compar i son . An a n a l y s i s of c o v a r i a n c e of l o g g body weights with day as the c o v a r i a t e was a l s o conducted as d e s c r i b e d i n Experiment 1. S p e c i f i c growth r a t e was s e l e c t e d as the p r i n c i p a l response c r i t e r i o n on which p r o t e i n requ irements were based . L i n e a r and po lynomina l r e g r e s s i o n equat ions were d e r i v e d ( Z a r , 1974) from growth r a t e data and i n t e r p r e t e d a c c o r d i n g to Cowey et a l . 131 Table 29. Composition of diets containing various levels of protein and energy (Experiment 2). % protein in diet 17 27 37 47 Metabolizable energy (kcal/kg) 3150 3950 3150 3950 3150 3950 315 3950 Freeze-dried pollock and euphausid 192.20 192.20 305.26 305.26 418.32 418.32 531.37 531.37 Herring o i l 54.1 173.27 50.1 119.32 46.2 115.36 42.2 111.40 Dextrin 225.0 250.5 169.0 194.5 112.5 138.0 56.5 82.0 Glucose 225.0 250.5 169.0 194.5 112.5 138.0 56.5 82.0 Ground cellulose 224.5 85.5 235.0 127.52 248.15 142.15 259.95 116.73 Carboxy methyl cellulose 20.0 20.0 20.0 20.0 20.0 20.0 20.0 20.0 Mineral mix 71.1 71.1 62.7 62.7 54.3 54.3 45.7 45.7 Vitamin mix 7.3 7.3 7.3 7.3 7.3 7.3 7.3 7.3 Choline Chloride (50%) 3.5 3.5 3.5 3.5 3.5 3.5 3.5 3.5 1,2,3. See Table (3). Table 30. Proximate composition and calculated energy contents of the diets employed i n Experiment 2 . Z protein i n diet 17 27 37 47 Metabolizable energy (kcal/kg) 3150 3950 3150 3950 3150 3950 3150 3950 OMP Crude protein (N x 6.25) 18.05 16.94 27.36 28.24 36.68 35.96 45.81 46.15 49.40 Total crude l i p i d 6.09 12.40 5.70 12.31 6.32 11.79 6.49 12.95 14.53 Ash 7.47 7.15 7.19 7.37 7.34 7.61 7.39 8.30 11.92 Digestible carbohydrate 45.0 50.1 33.8 38.9 22.5 27.6 11.3 16.4 16.60 Moisture 8.31 7.89 6.95 7.72 8.37 6.72 5.80 6.03 24.62 Energy (kcal/kg) Protein ME (4.5 kcal/g) 812 762 1231 1126 1651 1618 2062 2077 2075 GE (5.7 kcal/g) 1029 966 1560 1553 2091 2050 2611 2631 2816 L i p i d ME & GE (9.5kcal/g) 579 1179 542 1169 600 1120 617 1230 1280 Carbohydrate ME & GE (4.0kcal/g) 1800 2004 1352 1556 800 1104 452 655 266 Total ME 3191 3945 3125 3951 3151 3842 3130 3963 3721 Total GE 3408 4145 3453 4278 3591 4274 3680 4517 4462 mg protein/kcal ME 56.6 42.9 87.6 68.9 116.4 93.6 146.4 116.5 132.8 Protein energy (ME): t o t a l energy (ME) 0.25 0.19 0.38 0.28 0.52 0.42 0.66 0.52 0.56 1 . - 6 . See Table (4). (1972) and Z e i t o u n et a l . (1973,1976) to e s t imate p r o t e i n requirements as d i s c u s s e d i n the t e x t . S t a t i s t i c a l ana lyses were performed by computer through a g e n e r a l l e a s t squares a n a l y s i s of v a r i a n c e program (GENLIN). A g e n e r a l l i n e a r model (GLM)(SAS, 1982) procedure was used to f i t a r e g r e s s i o n model growth r a t e . 134 4.3 R e s u l t s A . 3 . 1 E s t i m a t i o n of the p r o t e i n requirement of j u v e n i l e chinook salmon from growth data The mean body weights of f i s h fed the v a r i o u s e x p e r i m e n t a l d i e t s and OMP i n c r e a s e d e x p o n e n t i a l l y d u r i n g the e x p e r i m e n t a l p e r i o d ( F i g . 12) . D i f f e r e n c e s i n wet f i s h body weight due to the d i f f e r e n t d i e t a r y c o n c e n t r a t i o n s of p r o t e i n and energy were apparent e a r l y i n the exper iment . An a n a l y s i s of v a r i a n c e i n d i c a t e d that by day A2 there were s i g n i f i c a n t d i f f e r e n c e s (P < 0.001) i n body weight a s s o c i a t e d with d i e t a r y p r o t e i n c o n c e n t r a t i o n (Table 31) . The mean d i f f e r e n c e s of f i s h body weights were s i g n i f i c a n t (Duncan's new m u l t i p l e range t e s t , P = 0.05) wi th each increment i n d i e t a r y p r o t e i n l e v e l . At day 105, s i g n i f i c a n t d i f f e r e n c e s (P < 0.001) due to d i e t a r y p r o t e i n l e v e l were s t i l l e v i d e n t . However, i n c o n t r a s t to day A2, at day 105 the d i f f e r e n c e s i n f i n a l weights between groups r e c e i v i n g d i e t s c o n t a i n i n g 37% and 47% p r o t e i n r e s p e c t i v e l y were no longer s i g n i f i c a n t (P > 0 . 0 5 ) ( T a b l e 31 ) . T h i s suggests that the p r o t e i n requirement for chinook salmon f r y i s h igher d u r i n g the f i r s t h a l f of t h e i r growing p e r i o d i n f r e s h water than d u r i n g the second. A three-way a n a l y s i s of c o v a r i a n c e of l o g e wet weights wi th time (day) as the c o v a r i a t e i n d i c a t e d a s i g n i f i c a n t d i f f e r e n c e (P < 0.001) i n s lope ( s p e c i f i c growth r a t e ) due to d i e t a r y p r o t e i n c o n c e n t r a t i o n . However, the s l o p e s obta ined with f i s h fed d i e t s c o n t a i n i n g 27% p r o t e i n were s i m i l a r to those with 135 f i s h fed d i e t s c o n t a i n i n g 37 and 47% p r o t e i n ( T a b l e 31) . The lowest l e v e l of p r o t e i n at which f i s h a t t a i n maximum weight ga in i s commonly c o n s i d e r e d to be the minimum requirement l e v e l f or growth . The q u a n t i t a t i v e p r o t e i n requirements for f i s h have been determined from "broken l i n e " type p l o t s (De Long et a l . , 1 9 5 8 ; S a t i a , 1974; Z e i t o u n et a l . , 1973, 1974; Ogata et a l . , 1983; Anderson et a l . , 1981). A s i m i l a r method was a p p l i e d to the data i n the present s t u d y . F o l l o w i n g the procedure of Z e i t o u n et a l . , ( 1 9 7 3 ) for the growing f i s h , a r e g r e s s i o n l i n e was f i t t e d to the ascending p o r t i o n of the response l i n e of s p e c i f i c growth r a t e to d i e t a r y p r o t e i n l e v e l . By a v e r a g i n g the h ighes t observed means which d i d not d i f f e r by more than two s tandard e r r o r s of the mean w i t h i n t r e a t m e n t s , a h o r i z o n t a l l i n e e s t a b l i s h i n g maximum growth ra t e was drawn ( F i g . 13) . The i n t e r s e c t i o n p o i n t of these two l i n e s was taken as the minimum p r o t e i n l e v e l , as a percent of dry d i e t , r e q u i r e d to support maximum growth i n j u v e n i l e ch inook salmon. Employing the above method, the p r o t e i n requirement for growth i n t h i s study was found to be a p p r o x i m a t e l y 35%, r e g a r d l e s s of the d i e t a r y energy l e v e l t e s t ed ( F i g . 13) . Other i n v e s t i g a t o r s s t u d y i n g the n u t r i e n t requirements of f i s h (Cowey et a l . , 1972; Z e i t o u n et a l . , 1976; Dabrowski , 1977; Murai et a l . , 1979) have employed n o n - l i n e a r growth curves to es t imate r e q u i r e m e n t s . T h e r e f o r e , the growth r a t e data were f i t t e d to a second degree po lynomina l by the procedure of Cowey et a l . (1972) . The f i t t e d e q u a t i o n for f i s h fed d i e t s 136 DIET Z protein - kcol/lOOg 47-315 47 -395 37-315 37-395 27-315 OMP 27-395 17-315 17-395 105 DAY F i g . 12. Weight ga in (+ 2 SE) of chinook salmon from the f r y - to s m o l t - s t a g e fed d i e t s c o n t a i n i n g v a r i o u s l e v e l s of p r o t e i n and energy , and fed OMP. 137 - T a b l e 31. Wet f i s h body weight (mean + SE) a t day 43 and day 105, and s p e c i f i c growth r a t e s of d i e t s c o n t a i n i n g d i f f e r e n t l e v e l s of p r o t e i n and energy and OMP. M e t a b o l i z a b l e energy P e r c e n t P r o t e i n i n D i e t c o n t e n t of d i e t ( k c a l / k g ) 17 27 37 47 Mean Mean body weight (+ SE) Day 42 3150 2.71 3.77 4.10 4.57 3.79 + 0.068 + 0.034 3950 2.33 3.45 3.94 4.48 3.55 Mean 2.52 P 3 . 6 i q 4.02' 4.52* + 0.048 OMP 3.59 + 0.025 Day 105 3150 5.66 10.47 13.25 13.49 10.72 + 0.23 + 0.12 3950 4.23 9.55 12.13 13.45 9.84 1 Mean 4.94 P 1 0 . 0 i q 12.69 r 13.47' + 0.16 OMP 10.06 + 0.46 S p e c i f i c Growth Rate (Z wet wt/day) 3150 1.17 1.77 2.00 1.99 1.73 + 0.024 + 0.012 3950 0.89 1.68 1.89 2.00 1.61 2 Mean 1.03 P 1.73' 1 . 9 s ' 2.00' + 0.017 OMP 1.74 + 0.06 1. V a l u e s w i t h the same s u p e r s c r i p t f o r each parameter w i t h r e s p e c t t o p r o t e i n l e v e l (p - r ) do not d i f f e r s i g n i f i c a n t l y (DMR t e a t P - 0.05). 2. S l o p e s w i t h the same s u p e r s c r i p t w i t h r e s p e c t to p r o t e i n l e v e l (p - r ) e f f e c t s do not d i f f e r s i g n i f i c a n t l y ( S c h e f f ^ ' s t e s t P - 0.05). - 138 - F i g . 13. S p e c i f i c growth r a t e ( p e r c e n t wet body weight per day) (+_ 2 SE) of j u v e n i l e chinook salmon fed d i e t s c o n t a i n i n g d i f f e r e n t l e v e l s of p r o t e i n and energy . A r e g r e s s i o n l i n e was f i t t e d to the data over the ascending p o r t i o n of the growth response to p r o t e i n c o n c e n t r a t i o n at both l e v e l s of d i e t a r y energy . A s t r a i g h t l i n e p a r a l l e l to the a b s c i s s a was drawn by a v e r a g i n g the h ighes t observed means which d i d not d i f f e r from each other by more than two s tandard e r r o r s of the means. The va lue o b t a i n e d f o r OMP i s i n c l u d e d for compar i son . 139 DIET ME O 3150 kcal/kg A 3950 " REQUIREMENT 0 10 20 30 40 50 PERCENT PROTEIN IN DIET O O Z 0-50 u L U a. 140 c o n t a i n i n g 3150 k c a l / k g was: y = -0 .81225 + 0.14212x - 0 . 00177x 2 , (n = 4, r = 0.9991) and 3950 k c a l / k g was: y = -1 .07943 + 0.14671x - 0 . 00174x 2 , (n = 4, r = 0.9956) where y = s p e c i f i c growth r a t e , x = percent p r o t e i n i n the d i e t . The data f i t the above equat ions wi th p r o b a b i l i t y l e v e l s of P < 0.05 and P < 0.10 for d i e t s c o n t a i n i n g 3150 and 3950 k c a l / k g r e s p e c t i v e l y . The maximum of the response curves o c c u r r e d at a p r o t e i n l e v e l of 40% and 42% for d i e t s c o n t a i n i n g 3150 and 3950 k c a l s / k g r e s p e c t i v e l y . A c c o r d i n g to the c r i t e r i a of Cowey et a l . ( 1972) these va lues would be the p r o t e i n requ irements of chinook salmon for maximum growth. The ana lyse s of v a r i a n c e conducted on f i s h body weights i n d i c a t e d that the m e t a b o l i z a b l e energy c o n c e n t r a t i o n of the d i e t s was not a f a c t o r a f f e c t i n g growth of j u v e n i l e ch inook salmon ( T a b l e 31) . D i e t a r y energy was p r o v i d e d by p r o t e i n and n o n - p r o t e i n sources (Tab le 30) . S ince p r o t e i n i s u t i l i z e d both for t i s s u e s y n t h e s i s and as an energy s o u r c e , i t i s the r e l a t i o n s h i p between p r o t e i n energy and n o n - p r o t e i n energy i n the d i e t that i s important i n the n i t r o g e n ba lance scheme d e s c r i b e d i n F i g . 1. The o b s e r v a t i o n s made above support the case made by Cowey and Sargent (1979) for s t a t i n g the p r o t e i n requirement of a f i s h d i e t i n terms of the p r o p o r t i o n of energy i t c o n t r i b u t e s , r e l a t i v e to t o t a l d i e t a r y energy . When the 141 F i g . 14. S p e c i f i c growth r a t e (percent wet body weight per day) (+_ 2 SE) of j u v e n i l e chinook salmon fed d i e t s c o n t a i n i n g d i f f e r e n t p r o t e i n e n e r g y : t o t a l energy r a t i o s ( P E : T E ) . The p r o t e i n requirement was o b t a i n e d by a s i m i l a r method to that d e s c r i b e d i n F ( 1 3 ) . The va lue obta ined for OMP i s i n c l u d e d for compar i son . 142 CO C N +1 < < U LU a. CO 200 1-50 £ 100 O O 0-50 y=i 97 4 — i O M P " DIET ME O 3150 kcal/kg A 3950 » REQUIREMENT 010 0-20 0-30 0-40 0-50 0-60 0-70 PE:TE 143 p r o t e i n requirement was es t imated r e l a t i v e to that of t o t a l d i e t a r y e n e r g y , - t h e minimum P E : T E r a t i o for growth of chinook salmon was found to be 0.41 by the broken l i n e method ( F i g . 14) . The p r o t e i n requirement expressed as P E : T E was a l s o q u a n t i f i e d by a p p l y i n g a second order p o l y n o m i a l e q u a t i o n . The q u a d r a t i c equat ion d e r i v e d was: y = - 0.50699 + 9.25398x - 8 .37872x 2 , (n = 8, r = 0 .9647) , where y = s p e c i f i c growth r a t e , x = P E : T E r a t i o • The curve o b t a i n e d ( F i g . 15) f i t the data adequate ly (P < 0 . 0 0 5 ) , both l i n e a r and q u a d r a t i c terms be ing s i g n i f i c a n t (P < 0 . 0 5 ) . Cowey et a l . (1972) d e f i n e d the d i e t a r y p r o t e i n requirement as the l e v e l that produced the h i g h e s t p o i n t on the c u r v e . In the present s t u d y , maximum growth of the f i s h was achieved at a P E : T E r a t i o of 0.55 (x max. , F i g . 15) . T h i s va lue corresponds to the P E : T E r a t i o i n OMP, a popular commercia l salmon feed (Tab le 30) . However, Z e i t o u n et a l . (1976) p o i n t e d out that x max on the q u a d r a t i c curve r e l a t i n g growth to dose "does not r e f l e c t the p r a c t i c a l l y i n s i g n i f i c a n t d i f f e r e n c e s i n percentage ga in below and beyond the maximum p o i n t , nor does i t c o n s i d e r the a b i l i t y of the animal to adapt to a range of d i e t a r y p r o t e i n l e v e l s . " They a p p l i e d a s t a t i s t i c a l approach to determine the n u t r i e n t l e v e l which cou ld p r o v i d e a response that l a y w i t h i n a c e r t a i n conf idence range of the maximum response . In the present s t u d y , upper and lower conf idence l i m i t s of 95% of the means were p l o t t e d , and a s t r a i g h t l i n e was drawn 144 F i g . 15. The second order p o l y n o m i a l curve ( s o l i d curved l i n e ) f i t t e d to s p e c i f i c growth r a t e (+_ 95% conf idence l i m i t s of the e s t imated means) of j u v e n i l e chinook salmon fed d i e t s wi th d i f f e r e n t P E : T E r a t i o s . Dashed l i n e s r e p r e s e n t the 95% c o n f i d e n c e l i m i t s of the r e l a t i o n s h i p . x max r e p r e s e n t s the P E : T E r a t i o for maximum growth. xO and x l , r e p r e s e n t the range of d i e t a r y P E : T E that would p r e d i c t a b l y r e s u l t i n r e l a t i v e l y minor decreases from maximum growth r a t e (y max). The concomitant r e d u c t i o n i n the d i e t a r y P E : T E r a t i o would be r e l a t i v e l y l a r g e . The va lue o b t a i n e d with f i s h fed OMP i s i n c l u d e d for c o m p a r i s o n . 145 010 0-20 0-30 0-40 0-50 0-60 0-70 PE :TE - 146 - p a r a l l e l to the a b s s i s s a and pass ing through the maximum l e v e l of the lower conf idence l i m i t ( F i g . . 15) . T h i s l i n e cros sed the p o l y n o m i a l curve at x l and i n t e r s e c t s the upper c o n f i d e n c e l i m i t at xO. S t a t i s t i c a l l y , the conf idence l i m i t s of the response expected with a d i e t hav ing a P E : T E r a t i o of 0.55 (x max) i n c l u d e the mean response o b t a i n e d with a d i e t having a P E : T E r a t i o of 0.41 ( x l ) and the upper l i m i t response wi th a P E : T E r a t i o of 0.35 ( x O ) . A c c o r d i n g to the c r i t e r i o n of Cowey et a l . (1982) the P E : T E requirement of chinook salmon was 0 . 5 5 . Other i n v e s t i g a t o r s (Robbins et a l . , 1979) would i n t e r p r e t the data d i f f e r e n t l y and s e l e c t the minimum P E : T E r a t i o that corresponds to the lower l i m i t of the maximum response ( x l , F i g . 15) . The P E : T E requirement by t h i s method was found to be 0.41 i n the present exper iment . Other i n t e r p r e t a t i o n s of the curve w i l l be d i s c u s s e d l a t e r . 4 .3 .2 The e f f e c t of d i e t a r y p r o t e i n and energy l e v e l on food c o n v e r s i o n and gross energy u t i l i z a t i o n . Gross food c o n v e r s i o n e f f i c i e n c y (GFC) was noted to i n c r e a s e as both the p r o t e i n l e v e l and the energy l e v e l of the d i e t s were r a i s e d ( T a b l e 32) . D i f f e r e n c e s i n GFC between f i s h fed 3150 and 3950 k c a l / k g were not s i g n i f i c a n t (P > 0.05) i n f i s h fed d i e t s c o n t a i n i n g low l e v e l s of d i e t a r y p r o t e i n . At h igh d i e t a r y p r o t e i n c o n c e n t r a t i o n food c o n v e r s i o n i n c r e a s e d as the energy l e v e l of the d i e t was i n c r e a s e d . 147 Gross energy u t i l i z a t i o n (GEU) was i n f l u e n c e d by both the l e v e l of d i e t a r y p r o t e i n and the m e t a b o l i z a b l e energy content of the d i e t (Tab le 32) . GEU was found to i n c r e a s e as the d i e t a r y p r o t e i n l e v e l i n c r e a s e d u n t i l a p p r o x i m a t e l y the p o i n t at which the p r o t e i n requirement of the f i s h was r e a c h e d . The e f f i c i e n c y of energy u t i l i z a t i o n i n f i s h as i n d i c a t e d by GEU f o l l o w s the laws of d i m i n i s h i n g r e t u r n s . At a l l l e v e l s of p r o t e i n , i n c r e a s i n g the energy content of the d i e t by 800 k c a l / k g caused a r e d u c t i o n of GEU i n the same order of magnitude. T h i s o b s e r v a t i o n suggests tha t the t o t a l energy content of d i e t s employed i n t h i s study was not l i m i t i n g . T h i s was c e r t a i n l y the case for the d i e t s c o n t a i n i n g 3950 k c a l / k g (Tab le 32) . 4 . 3 . 3 . The e f f e c t of d i e t a r y p r o t e i n and energy l e v e l on p r o t e i n u t i l i z a t i o n The e f f i c i e n c y of p r o t e i n u t i l i z a t i o n , as i n d i c a t e d by PER and PPV, g ive s an i n d i c a t i o n of the extent to which p r o t e i n was used for growth. The . r e l a t i o n s h i p between the p r o t e i n c o n c e n t r a t i o n of the d i e t and the u t i l i z a t i o n of d i e t a r y p r o t e i n f o l l o w e d a s i m i l a r p a t t e r n to that d e s c r i b e d i n the p r e v i o u s c h a p t e r . As the d i e t a r y p r o t e i n c o n c e n t r a t i o n was i n c r e a s e d , e f f i c i e n c y i n c r e a s e d , and reached a maximum at a lower d i e t a r y c o n c e n t r a t i o n than the minimum requirement l e v e l f or growth (Tab le 33) . At h igher p r o t e i n l e v e l s PER and PPV d e c r e a s e d . Because p r o t e i n u t i l i z a t i o n f o r maintenance i s accounted f o r , NPU-2 va lues are not lower i n f i s h fed d i e t s c o n t a i n i n g p r o t e i n 148 - T a b l e 32. Gross food c o n v e r s i o n e f f i c i e n c y (GFC)and g r o s s energy u t i l i z a t i o n (GEU) of d i e t s c o n t a i n i n g d i f f e r e n t l e v e l s o f p r o t e i n and energy. M e t a b o l i z a b l e energy Z Crude P r o t e i n i n D i e t c o n t e n t of d i e t ( k c a l / k g ) 17 27 37 47 Mean GFC ,1 3150 11.56 19.31 23.48 24.70 19.76 + 0.89 + 0.45 3950 11.26° 21.58 b 2 7 . 5 3 d e 30.06* 22.61* Mean 11.41 P 20.44 q 2 5 . 5 l r 27.38' + 0.63 OMP 24.40 + 0.20 GEU 3150 20.49° 3 6 . 7 1 D C 44.01 d 4 5 . 7 7 d 36.74* + 1.59 + 0.79 o b c d c d v 3950 16.41 31.25 39.86 41.20 32.18 Mean 18.45° 33.98 q 41.94' 43.48' + 1.12 OMP 32.03 + 1.54 1. V a l u e s w i t h the same s u p e r s c r i p t f o r each parameter w i t h r e s p e c t to p r o t e i n l e v e l x energy l e v e l ( a - c ) , p r o t e i n l e v e l (p - s ) and energy l e v e l (v,w) e f f e c t s do not d i f f e r s i g n i f i c a n t l y (DMR t e s t , P " 0.05). 149 l e v e l s below minimum r e q u i r e m e n t s . P r o t e i n u t i l i z a t i o n by a l l three i n d i c a t o r s was improved when the energy content of d i e t s was i n c r e a s e d from 3150 to 3950 k c a l / k g . The h igher energy content of the d i e t had a s p a r i n g e f f e c t on p r o t e i n , a l l o w i n g more d i e t a r y p r o t e i n to be u t i l i z e d for s y n t h e s i s of t i s s u e p r o t e i n . 4 . 3 . 4 The e f f e c t of d i e t a r y p r o t e i n and energy l e v e l on the proximate body c o m p o s i t i o n of f i s h A pooled sample of whole f i s h taken at day 0 c o n t a i n e d 79.88% m o i s t u r e ; and on a dry matter b a s i s 9.99% a s h , 16.6% l i p i d and 76.54% p r o t e i n . The gross c o m p o s i t i o n of ch inook salmon sampled at day 42 and 105 of the experiment v a r i e d wi th d i e t a r y energy c o n c e n t r a t i o n (Tab le 34 and 35) . O v e r a l l , the mois ture c o n c e n t r a t i o n of f i s h decreased s l i g h t l y from samples taken at day 0, 42, and 105 r e s p e c t i v e l y . The mois ture c o n c e n t r a t i o n of the f i s h at day 42 and 105 was found to decrease as the d i e t a r y energy l e v e l was r a i s e d . A trend of d e c r e a s i n g moi s ture as d i e t a r y p r o t e i n c o n c e n t r a t i o n i n c r e a s e d was a l s o n o t e d , which may have been due to concomi t tan t i n c r e a s e s i n f i s h s i z e . As one would expec t , i n c r e a s i n g the d i e t a r y energy l e v e l r e s u l t e d i n an i n c r e a s e i n body l i p i d . An i n v e r s e r e l a t i o n s h i p between d i e t a r y energy l e v e l and body p r o t e i n was o b s e r v e d . D i e t a r y p r o t e i n c o n c e n t r a t i o n was not found to have an e f f e c t on body a s h , l i p i d or p r o t e i n at day 42. At day 105 d i f f e r e n c e s in 150 Table 33. Protein e f f i c i e n c y ration (PER), protein productive value (PPV), and net protein u t i l i z a t i o n (NPU-2) of diets containing d i f f e r e n t levels of protein and energy. Metabolizable energy X Crude Protein l a Diet content of diet (kcal/kg) 17 27 37 47 Mean 1 PER 2 ab _ bed , ab _ a 3150 0.64 0.71 0.64 0.54 0.63 + 0.025 + 0.013 be d cd ob v 3950 0.66 0.79 0.77 0.65 0.72 Mean 0.65 P q 0.75f 0.72q 0.60 P + 0.18 OMP 0.49 + 0.02 1 PPV abc abc abc a w 3150 42.79 44.73 43.27 38.37 42.29 + 1.69 + 0.86 abc c be ab v 3950 42.77 48.87 46.48 41.20 44.83 Mean 42.78 P q 46.80 q f 44.87q 39.78 P + 1.20 OMP 31.42 + 1.05 Table 33. cont'd...(2) Metabolizable energy X Crude Protein i n Diet content of die t (kcal/kg) 17 27 37 47 Mean NPU-2 be be abc a v> 3150 60.60 59.60 56.08 49.24 56.38 + 2.20 + 1.10 be c be ab i — 1 Mean + 1.55 K> — OMP 54.80 + 1.35 v 3950 63.63 65.91 61.89 54.10 61.38 62.1i q 62.75q 58.99q 51.67 P 1. A two-way randomized ANOVA indicated s i g n i f i c a n t differences i n PER, PPV and NPU-2 due to protein l e v e l (P > 0.005) and energy l e v e l (P > 0.05). Differences due to protein l e v e l x energy l e v e l and block effects were not s i g n i f i c a n t (P < 0.05). 2. Values with the same superscript for each parameter with respect to protein l e v e l x energy l e v e l (a - d), protein l e v e l (p - r) and energy l e v e l (v,w) effects do not d i f f e r s i g n i f i c a n t l y (DMR t e s t , P - 0.05). T a b l e 34. Whole body proximate c o m p o s i t i o n a t day 42 of the d i e t s c o n t a i n i n g d i f f e r e n t l e v e l s of p r o t e i n and energy, and OMP. M e t a b o l i z a b l e energy P e r c e n t P r o t e i n i n D i e t c o n t e n t of d i e t ( k c a l / k g ) 17 27 37 47 Mean Z M o i s t u r e (+ SE) 1 ab abc a w 3150 78.09 77.54 72.09 77.80 77.68 + 0.27 + 0.14 3950 77.80° 7 6 . 7 6 . 1 1 C 76.18 c 76.65 V Mean 77.94 P 77.04 q 76.60° 76.99 q + 0.19 OMP 77.38 + 0.27 Z Ash (Dry B a s i s ) a ab ab ab w 3150 10.41 9.78 9.74 9.55 9.87 + 0.26 + 0.13 ab b b b v 3950 9.59 9.04 9.38 8.91 9.23 Mean 10.00 P 9.41°°- 9.56 P q 9.23 q + 0.18 OMP 9.86 + 0.07 Z L i p i d (Dry B a s i s ) (+ SE) c c be c w 3150 17.89 17.18 19.75 17.15 17.99 + 1.12 + 0.56 3950 22.05°° 24.52° 23.94° 24.81° 23.83 V Mean 19.97 20.85 21.85 20.98 + 0.79 OMP 20.96 + 0.78 Z P r o t e i n (Dry B a s i s ) (+ SE) 3150 72.98 73.02 67.47 71.02 71.12*' + 2.27 + 1.14 3950 65.57 67.71 67.06 68.69 67.25* Mean 69.27 70.36 67.26 69.85 + 1.61 OMP 68.67 + 1.94 1. Va l u e s w i t h the Bane s u p e r s c r i p t f o r each proximate component w i t h r e s p e c t t o p r o t e i n l e v e l x energy l e v e l (a - c ) , p r o t e i n l e v e l (p,q) and energy l e v e l (v,w) e f f e c t s do not d i f f e r s i g n i f i c a t n l y (DMR t e s t , P - 0.05). Table 35. Whole body proximate composition at day 105 of the diets containing different levels of protein and energy, and OMP. Metabolizable energy Percent Protein In Diet content of diet (kcal/kg) 17 27 37 47 Mean Mean Z Moisture (+ SE) 1 „„ , be b 3150 78.60 76.12 76.73 77.52 77.24 + 0.27 + 0.13 b d d d v 3950 77.38 75.43 75.23 75.40 75.86 77.99P 75.77r 75.98q 76.460, + 0.19 OMP 76.05 + 0.15 Z Ash (Dry Basis) a ob ab ab 3150 11.70 10.53 9.52 9.97 10.43 + 0.72 + 0.36 ab ab ab b 3950 10.18 9.65 10.06 9.57 9.57 Mean 10.94 10.09 9.79 9.19 + 0.51 OMP 8.89 + 0.43 Z Lipid (Dry Basis) (+ SE) 3150 18.70de 23.42DC 21.08cd 18.17* 20.34* + 0.85 + 0.42 3950 22.38C 26.59° 27.48° 25.21°° 25.41V Mean 20.54°- 25.01° 24.28° 21.69°" + 0.60 OMP 23.23 + 0.68 Z Protein (Dry Basis) (+ SE) 3150 66.61 C 62.43° 6 68.20° 74.91° 68.04* + 1.39 + 0.70 3950 60.12 58.58 58.32 64.23 60.31 Mean 63.37°" 60.50q 63.26° 69.57° + 0.98 OMP 63.58 + 0.41 1. Values with the same superscript for each proximate component with respect to protein level x energy level (a - c), protein level (p.q) and energy level (v,w) effects do not differ slgnificatnly (DMR teat, P - 0.05). - 154 body l i p i d and p r o t e i n due to d i e t a r y p r o t e i n c o n c e n t r a t i o n were found but no c l e a r t rends were noted ( T a b l e 35) . A . A D i s c u s s i o n A . A . I The d i e t a r y p r o t e i n requirement of j u v e n i l e chinook salmon The r e s u l t s of the growth t r i a l suggested that the chinook salmon may r e q u i r e d i e t s c o n t a i n i n g a h igher c o n c e n t r a t i o n of p r o t e i n d u r i n g the f r y stage than d u r i n g the l a t t e r stage of f reshwater r e a r i n g . T h i s i s i n agreement wi th a r e p o r t by S a t i a (197A) who found that the p r o t e i n requirement for rainbow t r o u t dropped down from 50 to A0% as the f i s h grew from the f r y to f i n g e r l i n g s tage . There i s a g e n e r a l t rend of lower p r o t e i n r e q u i r e m e n t s , as a percent of d i e t , i n o l d e r and l a r g e r f i s h of s e v e r a l s p e c i e s (Ogino and S a i t o , 1970; Nose and A r a i , 1972; Page and Andrews, 1971). Gulbrandsen and Utne (1977) on the other hand, found no change i n the p r o t e i n requ irements of rainbow t r o u t rang ing i n s i z e from 5 to 70g. In t h i s s tudy , the e x p e r i m e n t a l p e r i o d covered almost the e n t i r e p e r i o d of f r e s h water r e s i d e n c y f o r j u v e n i l e chinook salmon. I t i s l i k e l y that the h igher p r o t e i n requirement of sa lmonids i s i n d i c a t e d for d u r i n g the f r y stage only ( F o w l e r , 1980). Dur ing t h i s p e r i o d i t may be recommended to feed d i e t s with a p r o t e i n c o n c e n t r a t i o n to support maximum growth (x max, F i g . 15) . D i e t s f o r f r y should c o n t a i n a minimum P E : T E r a t i o of 0 .55 . 15.5 The e m p i r i c a l requirement for a n u t r i e n t depends, not only on the p h y s i o l o g i c a l and env ironmenta l c o n d i t i o n s of the exper iment , but a l s o on the method used to e s t imate the requirement (Robbins et a l . , 1979). When expressed as a percentage of dry d i e t the minimum d i e t a r y p r o t e i n c o n c e n t r a t i o n for maximum growth was found to be 35% of the d i e t by the "broken l i n e " method. T h i s va lue i s lower than that s t a t e d for chinook sa lmon, o b t a i n e d by a s i m i l a r method, r e a r e d i n 7 ° C water (40%) and i n 1 5 ° C water (50%)(Delong et a l . , 1958). In the present study the f i s h were r e a r e d at 10 .5 c C . A l t e r n a t i v e l y , o ther i n v e s t i g a t o r s have used cont inuous growth curves to a r r i v e at the p r o t e i n requirements for p l a i c e (Cowey et a l . , 1972), rainbow t r o u t ( Z e i t o u n et a l . , 1976), grass carp (Dab r o w s k i , 1977) and shad (Mura i et a 1 . , 1979). They s t a t e d that the r e l a t i o n s h i p of growth to dose d id not e x h i b i t the abrupt change from l i n e a r i t y i m p l i e d by the "broken l i n e " method. A l s o , the r i g h t - h a n d s e c t i o n of the response l i n e was assumed to be h o r i z o n t a l and r e p r e s e n t a t i v e of maximum average growth a f t e r the minimum p r o t e i n requirement l e v e l i s r e a c h e d . Cowey et a l . (1972) and Z e i t o u n et a l . (1976) found that very h igh d i e t a r y p r o t e i n l e v e l s depressed growth i n p l a i c e and rainbow t r o u t r e s p e c t i v e l y . These authors found that t h e i r data was best d e s c r i b e d by a second degree p o l y n o m i n a l . In the present s t u d y , d i e t a r y c o n c e n t r a t i o n s of p r o t e i n t e s t ed d i d not exceed 47%. However, Delong et a l . (1958) r e p o r t e d a d e c l i n e i n growth of chinook salmon with d i e t s c o n t a i n i n g more than 60% p r o t e i n . The d i e t a r y p r o t e i n 156 c o n c e n t r a t i o n p r o v i d i n g for maximum growth r a t e from a second degree p o l y n o m i a l e q u a t i o n was found to be 40 and 42% f o r d i e t s c o n t a i n i n g 3150 and 3950 k c a l s / k g r e s p e c t i v e l y . These requirements are approx imate ly 10% lower than the requirement a r r i v e d at by a s i m i l a r method f o r rainbow t r o u t u s i n g d i e t s c o n t a i n i n g c a s e i n and g e l a t i n as a p r o t e i n source ( Z e i t o u n et a l . , 1976). At t h i s p o i n t i n the d i s c u s s i o n , i t must be emphasized that i n t h i s study the f i s h were fed to s a t i a t i o n . Feed i n t a k e by f i s h , as by other a n i m a l s , i s governed by the energy content of the d i e t (Lee and Putnam, 1973; Page and Andrews, 1973). The p r o t e i n r e q u i r e m e n t , based on growth r a t e , s t a t e d above was found to be the same at both l e v e l s of d i e t a r y energy . T h i s i m p l i e s that p r o t e i n i n t a k e a lone was r e s p o n s i b l e f o r the d i f f e r e n c e s i n the growth r a t e . The e f f e c t s due to d i e t a r y energy l e v e l and the i n t e r a c t i o n of p r o t e i n l e v e l and energy l e v e l were m i n i m a l . The m e t a b o l i z a b l e energy content of the d i e t s was not a f a c t o r l i m i t i n g the growth of the f i s h . In f i s h fed d i e t s c o n t a i n i n g low p r o t e i n l e v e l s , i n c r e a s i n g the d i e t a r y energy l e v e l had the e f f e c t of d e p r e s s i n g growth r a t e due to a r e d u c t i o n i n p r o t e i n i n t a k e . Growth r a t e was not depressed by i n c r e a s i n g the d i e t a r y energy l e v e l i n d i e t s c o n t a i n i n g 47% p r o t e i n . P r o t e i n i n t a k e by these groups of f i s h was not r e s t r i c t e d by the energy content of t h e i r d i e t . I t i s i n t e r e s t i n g to note that growth r a t e s of f i s h were s i m i l a r when the d i e t s fed c o n t a i n e d the same p r o p o r t i o n of t o t a l energy as p r o t e i n energy even though the c o m p o s i t i o n of the d i e t s d i f f e r e d 157 ( i . e . , d i e t s wi th 37% p r o t e i n and 3150 k c a l / k g and 47% p r o t e i n and 3950 k c a l / k g r e s p e c t i v e l y ) . Ogino et a l . (1976) found that the d i e t a r y p r o t e i n c o n c e n t r a t i o n r e q u i r e d f o r maximum growth i n t r o u t was 30 to 35% when n o n - p r o t e i n d i e t a r y energy was p r o v i d e d by l i p i d ( 1 : 1 , soybean and cod l i v e r o i l ) but the requirement s h i f t e d to approx imate ly 40% with c a r b o h y d r a t e ( s t a r c h and d e x t r i n ) . In c o n t r a s t , i n the present study the source of n o n - p r o t e i n energy was not a major f a c t o r a f f e c t i n g p r o t e i n requirements f o r growth. T h i s aspect w i l l be d i s c u s s e d i n more d e t a i l l a t e r . F u r t h e r m o r e , the d i e t s a l s o d i f f e r e d markedly i n t h e i r content of ground c e l l u l o s e (Tab le 29) . Buh ler and H a l v e r " (1961) r e p o r t e d depressed growth r a t e s of ch inook salmon fed d i e t s with i n c r e a s i n g l e v e l s of c e l l u l o s e . H i l t o n et a l . (1983) conc luded that d i e t a r y f i b e r l e v e l s for rainbow t r o u t should be l e s s than 10% of the d i e t because t h e i r n a t u r a l d i e t c o n t a i n s l i t t l e f i b e r . Higgs et a l . (1983) suggested that f i b e r may depress m i n e r a l uptake , a d v e r s e l y a f f e c t t r a n s i t time of d i g e s t a * and decrease the d i g e s t i b i l i t y of n u t r i e n t s . In the present s t u d y , however, i t would seem u n l i k e l y that c e l l u l o s e i n t e r a c t e d with other n u t r i e n t s . R a t h e r , f i s h fed the low energy d i e t s were ab le to a t t a i n a s i m i l a r growth r a t e to those fed h igh energy d i e t s by i n c r e a s i n g feed i n t a k e , and hence, as noted above, p r o t e i n i n t a k e by a l t e r a t i o n of g a s t r i c e v a c u a t i o n and stomach d i s t e n t i o n ( H i l t o n et a l . , 1983). T h i s i s i n agreement wi th the c o n c l u s i o n s made by Dupree and Sneed (1966) and Lee and Putnam (1973) i n channel c a t f i s h and rainbow t r o u t r e s p e c t i v e l y . 158 - D i s c r e p a n c i e s wi th the other s t u d i e s may be due to e f f e c t s of temperature ( B r e t t and H i g g s , 1970), f e e d i n g frequency (Adron et a l . , 1973) and other e n v i r o n m e n t a l f a c t o r s ( B r e t t , 1979). The o b s e r v a t i o n s made above support the case made by Cowey and Sargent (1979) for s t a t i n g p r o t e i n requirement i n terms of the p r o p o r t i o n of energy c o n t r i b u t e d . By the broken l i n e method ( F i g . 14) the P E : T E r a t i o f o r maximum growth r a t e was found to be 0 .41 . T h i s va lue i s lower than that p r e v i o u s l y r e p o r t e d for chinook salmon, P E : T E of 0.50 (Combes et a l . , 1962; Fowler et a l . , 1964). D i s c r e p a n c i e s between the va lues l i k e l y r e s u l t e d from s e v e r a l sources i n c l u d i n g d i e t a r y i n g r e d i e n t s , d i f f e r e n t f i s h s t o c k s , water temperature and c a l o r i c va lues a s s i g n e d to the n u t r i e n t s . R e c e n t l y Fowler (1980) found that the best P E : T E r a t i o i n the d i e t f o r newly hatched chinook f r y was 0.51 to 0 .55 . However, the p r o t e i n was p r o v i d e d by s e v e r a l i n g r e d i e n t s , some of which may not have been d i g e s t e d by the very s m a l l f i s h . The P E : T E of 0.41 found i n t h i s study agrees wi th that r e p o r t e d for rainbow t r o u t , 0.37 - 0.41 (Gulbrandsen and Utne , 1977) and a r c t i c c h a r , 0.35 - 0.45 ( J o b l i n g and Wandsvik, 1983). Based on the f i n d i n g that the growth response to the p r o t e i n c o n c e n t r a t i o n of the d i e t s was not i n f l u e n c e d by changes i n the p r o p o r t i o n of m e t a b o l i z a b l e energy s u p p l i e d by c a r b o h y d r a t e and l i p i d , a s i n g l e second order p o l y n o m i a l curve was f i t t e d to the data ( F i g . 15) . With a p o l y n o m i a l curve such as the one d e s c r i b e d above, one i s c on f ron t ed with the task of recommending a P E : T E r e q u i r e m e n t . Whi le the P E : T E va lue of 0.55 (x max) may be p h y s i o l o g i c a l l y c o r r e c t , t h i s e s t imate i s u n s a t i s f a c t o r y for 159 p r a c t i c a l use because i t i g n o r e s economic c o n s i d e r a t i o n s . An i d e n t i c a l dilemma e x i s t s i n the e s t i m a t i o n of n u t r i e n t requ irements from growth data wi th other l i v e s t s o c k . For example, when Robbins et a l . (1979) compared n o n - l i n e a r models to l i n e a r models to r e - e v a l u a t e the amino a c i d and v i t a m i n requirements of the growing c h i c k , they a r b i t r a r i l y chose the dose at which the response reached 95% of the t o t a l r e sponse . D e s p i t e the s u b j e c t i v i t y i n d e f i n i n g the "requirement" these authors conc luded that the n o n - l i n e a r models were p r e f e r a b l e , a l though the e s t imated requirements by both methods were n e a r l y the same. An i d e n t i c a l c o n c l u s i o n may be drawn from the r e s u l t s i n the present study as the P E : T E r a t i o at x l i s i d e n t i c a l to that determined by the b r o k e n - l i n e model , namely P E : T E = 0.41 ( F i g s . 1 4 , 1 5 ) . From a s t r i c t s t a t i s t i c a l p o i n t of view one cannot e s t a b l i s h the p r o t e i n requirement at xO or x l on the b a s i s that the growth response at these l e v e l s i s not s t a t i s t i c a l l y s i g n i f i c a n t l y d i f f e r e n t f rom that at x max ( Z e i t o u n et a l , 1976). A l s o , one must bear i n mind that i n c r e a s i n g the number of o b s e r v a t i o n s i n the r e g r e s s i o n a n a l y s i s may have had the e f f e c t of narrowing the c o n f i d e n c e l i m i t s and hence s h i f t xO and x l to the r i g h t . Z e i t o u n et a l . (1976) very s u c c i n c t l y p o i n t out that " s t a t i s t i c a l s i g n i f i c a n c e i s somewhat i r r e l e v a n t f o r r e g r e s s i o n s wi th s i g n i f i c a n t parameters . What i s r e l e v a n t i s a d i f f e r e n c e which has a p r a c t i c a l i m p o r t a n c e . " E c o n o m i c a l l y , the d i f f e r e n c e i n P E : T E of the d i e t s cou ld be important s i n c e p r o t e i n i s one of the most expens ive components of a f i s h d i e t . The i m p l i c a t i o n 160 i s that the p r o t e i n requirement cou ld be based on the cos t of each d i e t a r y p r o t e i n increment r e l a t i v e to the c o r r e s p o n d i n g monetary va lue a s s igned to the growth r a t e of the f i s h . Thus the curve shown i n F i g . 15 cou ld be adapted to cos t b e n e f i t a n a l y s i s i n a f i s h p r o d u c t i o n e n t e r p r i s e when i n f o r m a t i o n on the cos t of f e e d , growth of the f i s h and s e l l i n g p r i c e of the product i s a v a i l a b l e . Feed c o n v e r s i o n was used by S a t i a (1974) as the p r i n c i p a l c r i t e r i a for d e t e r m i n i n g p r o t e i n requirements of rainbow t r o u t . In t h i s s t u d y , GFC cou ld be used as an i n d i c a t o r of p r o t e i n requirements on ly when i s o c a l o r i c d i e t s are c o n s i d e r e d . Hence, maximum food c o n v e r s i o n was o b t a i n e d at a d i e t a r y p r o t e i n l e v e l of a p p r o x i m a t e l y 3 7% at both l e v e l s of t o t a l d i e t a r y energy (Tab le 32) . P r e d i c t a b l y , the e f f i c i e n c y of d i e t a r y energy u t i l i z a t i o n i n c r e a s e d i n a s i m i l a r manner to GFC as d i e t a r y p r o t e i n c o n c e n t r a t i o n was r a i s e d to 37% of the d i e t . The t rend noted of i n c r e a s e d GEU as the P E : T E r a t i o of the d i e t s was i n c r e a s e d may support the p o i n t t h a t , for f i s h , p r o t e i n i s the p r e f e r r e d source of energy (Walton and Cowey, 1982). The h igher GEU by f i s h fed d i e t s c o n t a i n i n g a lower energy d e n s i t y may be due to i n c r e a s e d l i p i d d i g e s t i b i l i t y . T a k e u c h i et a l . (1978) on the other hand, r e p o r t e d that l i p i d d i g e s t i b i l i t y i n t r o u t was not a f f e c t e d by d i e t a r y l i p i d c o n c e n t r a t i o n . The r e s u l t s obta ined for GFC and GEU served to support the p r o t e i n requirement e s t imate made p r e v i o u s l y . 161 A . 4 . 2 The e f f e c t of d i e t a r y energy on the e f f i c i e n c y of p r o t e i n u t i l i z a t i o n The replacement of p r o t e i n energy with n o n - p r o t e i n energy to f u r n i s h the m e t a b o l i z a b l e energy r e q u i r e m e n t s , i s a p o t e n t i a l means for r e d u c i n g the cos t of r e a r i n g f i s h . T h i s p r o t e i n s p a r i n g e f f e c t has been observed by s e v e r a l i n v e s t i g a t o r s (Lee and Putnam, 1973; Page and Andrews, 1973; Takeda et a l . , 1975; Adron et a l . , 1976; G a r l i n g and W i l s o n , 1976; Gulbrandsen and Utne , 1977; Takeuch i et a l . , 1978; Shimeno et a l . , 1980). For example, Takeuch i et a l . (1978) fed t r o u t d i e t s c o n t a i n i n g l e v e l s of p r o t e i n r a n g i n g from 16 to 48% and l e v e l s of l i p i d rang ing from 5 to 25%. They found that best weight ga ins were o b t a i n e d when d i e t s c o n t a i n e d 18% l i p i d . At t h i s l e v e l of d i e t a r y l i p i d , the p r o t e i n content of the d i e t c o u l d be reduced from 48 to 35% with no l o s s i n weight g a i n . Buhler and H a l v e r (1961) fed chinook salmon a s e r i e s of d i e t s i n which the p r o t e i n l e v e l decreased from 71 to 40% by i n c r e a s i n g l e v e l s of d e x t r i n up to 43% of the d i e t . They showed that p r o t e i n e f f i c i e n c y r a t i o s were i n c r e a s e d by s u b s t i t u t i n g d e x t r i n for p r o t e i n a l though growth r a t e remained s i m i l a r among d i e t a r y t r e a t m e n t s . In the present s t u d y , p r o t e i n i n the d i e t s was r e p l a c e d by both c a r b o h y d r a t e and l i p i d on a m e t a b o l i z a b l e energy b a s i s . The h i g h e s t va lues of p r o t e i n u t i l i z a t i o n for growth as measured by PER and PPV o c c u r r e d when P E : T E r a t i o ranged from 0.28 to 0 .42 . P r o t e i n u t i l i z a t i o n was s i g n i f i c a n t l y (P < 0.05) lower when the P E : T E i n c r e a s e d from 0.42 to 0.66 (Tab le 33) . T h e r e f o r e i n c r e a s i n g the P E : T E r a t i o of the d i e t beyond the minimum 162 p r o t e i n requirement l e v e l ( i . e : P E : T E = 0.41) has the e f f e c t of r e d u c i n g the e f f i c i e n c y of p r o t e i n u t i l i z a t i o n wi thout promoting any i n c r e a s e i n the growth r a t e . In f a c t , the growth r a t e of f i s h fed a d i e t with a P E : T E r a t i o of 0.42 was s l i g h t l y lower , a l though not s i g n i f i c a n t l y lower (P > 0 . 0 5 ) , than that of f i s h fed d i e t s with a P E : T E r a t i o of 0.52 (Tab le 31 ) . S e v e r a l s t u d i e s (reviewed i n Chapter 2) have demonstrated the s p a r i n g of d i e t a r y p r o t e i n by l i p i d and c a r b o h y d r a t e . Increased use of l i p i d s i n f i s h d i e t s can be d i sadvantageous because of t h e i r h igh cos t and s u s c e p t i b i l i t y to o x i d a t i v e r a n c i d i t y . On the other hand, the use of c a r b o h y d r a t e s i s l i m i t e d by the d i g e s t i b i l i t y of s t a r c h and the amount of g lucose c a r n i v o u r o u s f i s h can t o l e r a t e . In the present s t u d y , i d e n t i c a l h igh growth r a t e s were ach ieved by f i s h fed e i t h e r of two d i e t s wi th a P E : T E r a t i o of 0.52 (Tab le 31) . One c o n t a i n e d 22.5% and 6.32%, and the other 16.4% and 12.95% d i g e s t i b l e c a r b o h y d r a t e and l i p i d r e s p e c t i v e l y . With both d i e t s d i e t a r y p r o t e i n was spared e q u a l l y ( T a b l e 33) . D i e t a r y p r o t e i n was f u r t h e r spared by f i s h fed the d i e t wi th a P E : T E r a t i o of 0.42 that c o n t a i n e d 27.6% d i g e s t i b l e c a r b o h y d r a t e and 11.79% l i p i d . S t i l l f u r t h e r sav ings may be ach ieved by f e e d i n g f i s h the d i e t wi th a P E : T E r a t i o of 0 .38 . ' T h i s d i e t promoted growth at only a s l i g h t l y lower r a t e than d i d the d i e t wi th a P E : T E r a t i o of 0.42 and s i m i l a r to that of f i s h fed OMP (Tab le 33) . However, i t c o n t a i n e d 33.8% d i g e s t i b l e c a r b o h y d r a t e , and .only 5.70% l i p i d and 27.4% p r o t e i n , which c o n t r a s t s markedly wi th the commercial d i e t (OMP, T a b l e 30) . 163 The above o b s e r v a t i o n s support the f i n d i n g s of Buh ler and Ha lver (1961) who found that the o p t i m a l l e v e l of d i e t a r y c a r b o h y d r a t e was a p p r o x i m a t e l y 20% a l though chinook salmon cou ld t o l e r a t e d i e t s c o n t a i n i n g h i g h e r l e v e l s . However, t h i s i s at v a r i a n c e wi th r e p o r t s by P h i l l i p s (1969) and H i l t o n and A t k i n s o n (1982) who c l a i m that d i g e s t i b l e c a r b o h y d r a t e l e v e l s i n t r o u t d i e t s should not exceed 12 to 14%. I t would seem d o u b t f u l that major d i f f e r e n c e s e x i s t between r e l a t e d s p e c i e s and c o n s i d e r i n g that rainbow t r o u t are a more domest ic s p e c i e s of sa lmonid than were the chinook salmon employed i n the present s t u d y . S u b s e q u e n t l y , H i l t o n et a l . , (1982) suggested that the optimum l e v e l s of i n c l u s i o n of d i g e s t i b l e c a r b o h y d r a t e may w e l l be v a r i a b l e depending on the o v e r a l l ba lance of p r o t e i n , f a t and c a r b o h y d r a t e as w e l l as the t o t a l energy content of the d i e t . These au thors a l s o r e p o r t e d that the d i g e s t i b i l i t y of g lucose was u n i f o r m l y h igh (96 to 99%) and was not a f f e c t e d by the l e v e l of i n c l u s i o n i n the d i e t . The d i g e s t i b i l i t y of raw s t a r c h and to a l e s s e r e x t e n t , cooked s t a r c h , and d e x t r i n decreases as the l e v e l i n the d i e t of rainbow t r o u t i s i n c r e a s e d (S ingh and Nose, 1967). T h e r e f o r e , s i n c e the r e s u l t s of present study i n d i c a t e that j u v e n i l e chinook salmon are ab le to u t i l i z e absorbed c a r b o h y d r a t e to a g r e a t e r extent than p r e v i o u s l y thought ( P h i l l i p s , 1969), i t may be recommended that s t a r c h - c o n t a i n i n g i n g r e d i e n t s be processed p r i o r to i n c l u s i o n i n p r a c t i c a l d i e t s for t h i s s p e c i e s . T h i s may l e s s e n the r e l i a n c e on h igh l i p i d l e v e l s as a source of energy i n f i s h d i e t s . T h i s p o i n t has been amply demonstrated ( S m i t h , 1976; P i e p e r and P f e f f e r , 1980; 164 H i l t o n et a l . , 1981) yet does not appear to have been wide ly adopted by f i s h feed m a n u f a c t u r e r s . A l though h igh c a r b o h y d r a t e d i e t s may be advantageous when a p p l i e d to f i s h f a r m i n g , f i s h fed these d i e t s develop e n l a r g e d l i v e r s and i n c r e a s e d l i v e r g lycogen l e v e l s ( P h i l l i p s , 1969; Lee and Putnam, 1973). T h i s cou ld be hazardous to the h e a l t h of f i s h d e s t i n e d for r e l e a s e s i n c e i t has been shown that abnormal l i v e r s i z e and l i v e r g lycogen content i n t r o u t reduce the t o l e r a n c e of these f i s h to waterborne t o x i c a n t s and impa ir l i v e r f u n c t i o n ( H i l t o n , 1982). 4 . 4 . 3 The e f f e c t of the source and l e v e l of d i e t a r y energy on the proximate body c o m p o s i t i o n of f i s h The r e s u l t s of t h i s study are i n agreement wi th those of Groves (1970) who observed that the ra t e of i n c r e a s e i n body p r o t e i n r e l a t i v e to body water i s r a p i d d u r i n g e a r l y growth but by the time the sockeye salmon reached smolt s i z e f u r t h e r change was not n o t e d . However, throughout the l i f e of sa lmonids gross c o m p o s i t i o n a l changes do occur as a r e s u l t of v a r i a t i o n s i n body l i p i d r e l a t i v e to the n o n - l i p i d f r a c t i o n of the f i s h (Buckley and G r o v e s , 1979). T h i s was a l s o observed i n the present study with chinook salmon and i n a recent study with s t e e l h e a d t r o u t ( F a g e r l u n d et a l . , 1984) where both e x p e r i m e n t a l and commercial d i e t s were employed. Imposed on the above o b s e r v a t i o n s i s the e f f e c t of d i e t c o m p o s i t i o n . I n c r e a s i n g the energy content of the d i e t wi th 165 h e r r i n g o i l had the e f f e c t of i n c r e a s i n g body l i p i d . T h i s has a l s o been noted by other i n v e s t i g a t o r s (Cowey and S a r g e n t , 1979; Watanabe, 1982). The concomitant decrease i n body m o i s t u r e , p r o t e i n and ash i s e s s e n t i a l l y independent of d i e t a r y l i p i d l e v e l when a l lowances are made for changes i n body l i p i d . Higgs et a l . , (1983) c i t e s e v e r a l s t u d i e s that r e p o r t an i n v e r s e r e l a t i o n s h i p between p r o t e i n l e v e l , or p r o t e i n to c a l o r i c r a t i o of the d i e t , and body l i p i d c o n t e n t . Lee and Putnam (1973) on the other hand observed that h igher p r o t e i n to c a l o r i e r a t i o s were p o s i t i v e l y c o r r e l a t e d with percentage body f a t . Over the range of p r o t e i n l e v e l s t e s t e d i n the present study body l i p i d content tended to r i s e and subsequent ly f a l l r e g a r d l e s s of age of the f i s h or t o t a l energy content of the d i e t s ( T a b l e 32, 35) . Comparison of r e s u l t s between s t u d i e s i s c o m p l i c a t e d because of d i f f e r e n c e s i n s p e c i e s , d i e t a r y i n g r e d i e n t s and e n v i r o n m e n t a l f a c t o r s . Comparison of proximate c o n s t i t u e n t s i n the present study between f i s h fed d i e t s wi th i d e n t i c a l P E : T E r a t i o s but d i f f e r i n g d i e t c o m p o s i t i o n r e v e a l e d d i f f e r e n c e s i n body c o m p o s i t i o n . C l e a r l y , the moi s ture content and the amount of l i p i d and p r o t e i n d e p o s i t e d i n f i s h i s a l s o a f u n c t i o n of the source of d i e t a r y n o n - p r o t e i n energy . These changes i n c o m p o s i t i o n would have a d i r e c t b e a r i n g on the q u a l i t y c h a r a c t e r i s t i c s of the f l e s h . C u r r e n t human d i e t a r y t rends suggest that l ean f l e s h i s more d e s i r a b l e to consumers. On the other hand, h igh l i p i d r e t e n t i o n may be advantageous to the p r o d u c t i o n of h a t c h e r y - r e a r e d f i s h where s u r v i v a l i n the w i l d environment may depend on l i p i d r e s e r v e s (Buckley and G r o v e s , 166 1979). The r e s u l t s of the present study demonstrate that the energy sources as w e l l as energy c o n c e n t r a t i o n of the sa lmonid d i e t may be manipulated to a l t e r the body c o m p o s i t i o n of f i s h with r e s p e c t to p r o t e i n , l i p i d and g l y c o g e n . As mentioned p r e v i o u s l y , h igh l i v e r g lycogen r e s e r v e s i n f i s h d e s t i n e d f o r r e l e a s e cou ld be a f a c t o r that a d v e r s e l y a f f e c t s ocean s u r v i v a l . - 167 - 4.5 Summary of Experiment 2 Determin ing the gross p r o t e i n requirement f o r j u v e n i l e chinook salmon present s a compromise between the o b j e c t i v e s of maximum growth r a t e and p r o t e i n u t i l i z a t i o n e f f i c i e n c y . A s i m i l a r dilemma was d i s c u s s e d by Z e i t o u n et a l . , (1976) with r e f e r e n c e to rainbow t r o u t who suggested the concept of an "economic p r o t e i n r e q u i r e m e n t . " T h e i r method i n v o l v e d the a p p l i c a t i o n of a second order p o l y n o m i a l e q u a t i o n . I t was m o d i f i e d and s u c c e s s f u l l y a p p l i e d to the r e s u l t s of the present s t u d y . The m o d i f i c a t i o n i n v o l v e d e x p r e s s i n g p r o t e i n requirements i n terms of m e t a b o l i z a b l e energy r a t h e r than as a percentage of the dry d i e t . Depending on the o b j e c t i v e s of the f i s h c u l t u r i s t , the P E : T E r a t i o of f i s h d i e t s may be a d j u s t e d to s u i t a p a r t i c u l a r need. Thus produc ing chinook salmon smolts for r e l e a s e with an enhanced a d a p t a b i l i t y to a w i l d environment may r e q u i r e a d i e t which meets the p h y s i o l o g i c a l requirement for p r o t e i n . A l t h o u g h , a s u c c e s s f u l commercial d i e t (OMP) c o n t a i n e d a P E : T E r a t i o s i m i l a r to that r e q u i r e d for maximum growth ( P E : T E = 0 .55 ) ; r e s u l t s on growth, d i e t a r y energy r e t e n t i o n and the e f f i c i e n c y of p r o t e i n u t i l i z a t i o n i n d i c a t e that major improvements can be made i n the s e l e c t i o n of i n g r e d i e n t s and manufacture of commercia l f i s h f e e d s . The f i s h farmer produc ing t a b l e f i s h may s e l e c t a P E : T E r a t i o that maximizes r e t u r n s over input c o s t s . Where t h i s i n f o r m a t i o n i s not a v a i l a b l e i t may be safe to s e l e c t an o p t i m a l P E : T E r a t i o of 0 . 4 1 . T h i s va lue l i e s j u s t w i t h i n the range of o p t i m a l p r o t e i n u t i l i z a t i o n e f f i c i e n c y . The above P E : T E r e l a t i o n s h i p was not found to be a f f e c t e d by 168 - the source of n o n - p r o t e i n energy i n the d i e t . When the p r o p o r t i o n s of c a r b o h y d r a t e ( s u p p l i e d by g lucose and h y d r o l y z e d d e x t r i n ) and l i p i d ( h e r r i n g o i l ) were v a r i e d i n d i e t s of s i m i l a r P E : T E r a t i o s , growth and the e f f i c i e n c y of d i e t a r y energy and I p r o t e i n u t i l i z a t i o n were s i m i l a r . However, body c o m p o s i t i o n of the f i s h r e f l e c t e d t h e i r d i e t , the h igher l i p i d d i e t fed f i s h having h igher content of body l i p i d . Experiment 2 showed that by m a n i p u l a t i n g the P E : T E r a t i o and the p r o p o r t i o n s of c a r b o h y d r a t e and l i p i d i n the d i e t of j u v e n i l e chinook salmon, f i s h can be r e a r e d to a p r e s c r i b e d performance and body c o m p o s i t i o n . 169 CHAPTER 5 5 .0 C o n c l u s i o n s The s u b j e c t of p r o t e i n n u t r i t i o n i s very wide i n scope and i n v o l v e s s e v e r a l f a c t o r s . An attempt was made i n t h i s t h e s i s to draw a t t e n t i o n to the two most r e l e v a n t a spec t s c o n c e r n i n g the p r o t e i n content of s u c c e s s f u l f i s h d i e t s . These are the e f f e c t s of q u a l i t y and q u a n t i t y of d i e t a r y p r o t e i n on growth and e f f i c i e n c y of p r o t e i n u t i l i z a t i o n . Along wi th the f i n d i n g s of other i n v e s t i g a t o r s , the c o n c l u s i o n s drawn from t h i s study may serve to a i d i n the f o r m u l a t i o n of d i e t s for the r e a r i n g of j u v e n i l e chinook salmon. The present p r o t e i n requirements for sa lmonids have been c a l c u l a t e d l a r g e l y from experiments i n which d e f i c i e n t d i e t s have been supplemented with pure p r o t e i n s and amino a c i d s to determine the l e v e l of n u t r i e n t g i v i n g maximum growth. Experiment 1 demonstrated the extent to which growth, feed c o n v e r s i o n and p r o t e i n u t i l i z a t i o n e f f i c i e n c y may d i f f e r i n response to both source and c o n c e n t r a t i o n of d i e t a r y p r o t e i n . The p r o t e i n s t e s t e d i n c l u d e d a pure p r o t e i n source ( i . e . a mixture of v i t a m i n - f r e e c a s e i n and g e l a t i n supplemented with amino a c i d s to s a t i s f y the known amino a c i d requirements for chinook salmon) and p r o t e i n sources d e r i v e d from the commercia l f i s h e r y . The responses o b t a i n e d with the l a t t e r were dependent on the nature of the raw m a t e r i a l and p r o c e s s i n g c o n d i t i o n s employed. In Experiment 2 the p r o t e i n requirements for j u v e n i l e chinook salmon were r e - e v a l u a t e d - 170 - employing a f r e e z e - d r i e d p o l l o c k and euphausid mix ( 9 : 1 ) , the p r o t e i n source found to have the h ighes t q u a l i t y i n Experiment 1 . In both experiments comparisons were made between groups fed the t e s t d i e t s and a popular commercial sa lmonid d i e t (OMP). The r e s u l t s showed that there i s ample scope for improvement i n commercial f i s h f eeds . The r e s u l t s a l s o i n d i c a t e d that the c a s e i n - g e l a t i n based p r o t e i n source may not be s a t i s f a c t o r y as a r e f e r e n c e s tandard for p r o t e i n q u a l i t y e v a l u a t i o n . The best responses i n terms of growth and p r o t e i n u t i l i z a t i o n were obta ined by groups fed a mix (9:1) of f r e e z e - d r i e d p o l l o c k and e u p h a u s i d . T h e r e f o r e i t may be conc luded that the p a t t e r n of a v a i l a b l e amino a c i d s present i n t h i s mix (FPE) i s o p t i m a l for chinook salmon and p r e f e r r e d over that present i n the p u r i f i e d d i e t . T h i s c o n c l u s i o n would only be v a l i d i f the assumption were made that no other growth enhancing f a c t o r s were present i n F P E . The r e s u l t s of the b i o a s s a y s for p r o t e i n q u a l i t y showed that the a v a i l a b i l i t y to the f i s h of amino a c i d s or l o s s of some amino a c i d s from h e r r i n g meal was d r a s t i c a l l y i m p a i r e d by h igh d r y i n g temperatures ( 1 5 0 ° C ) . M i l d d r y i n g temperature (75 C C) was found to cause a s l i g h t r e d u c t i o n i n p r o t e i n q u a l i t y compared to that of a f r e e z e - d r i e d meal made from the same l o t of raw m a t e r i a l . T h i s product may be c o n s i d e r e d to be s i m i l a r to that which can be manufactured by l o c a l p l a n t s equipped with steam jac ke te d f i s h meal d r y e r s . I t would seem d o u b t f u l that p r o d u c t i o n of a f r e e z e - d r i e d meal would be commerc ia l l y v i a b l e . 171 D e f i n i t i v e c o n c l u s i o n s r e g a r d i n g the best methodology f o r p r o t e i n q u a l i t y b i o a s s a y s can not be drawn from the r e s u l t s of t h i s s t u d y . N e v e r t h e l e s s , methods i n v o l v i n g r e g r e s s i o n of p r o t e i n ga in on p r o t e i n i n t a k e are c o n s i d e r e d to be more meaningfu l than e s t i m a t i o n s made at a s i n g l e l e v e l of p r o t e i n i n t a k e . When c o n s i d e r e d a long with body weight ga in or body p r o t e i n ga in the v a r i o u s p r o t e i n sources may be adequate ly compared. I t i s a l s o conc luded that p a r t i t i o n i n g p r o t e i n i n t a k e i n t o that used f o r maintenance , growth and the amount l o s t through exogenous e x c r e t i o n s p r o v i d e s an i n t e r e s t i n g means of d e p i c t i n g p r o t e i n u t i l i z a t i o n at d i f f e r e n t i n t a k e s of the t e s t p r o t e i n . The 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 was not found to be an a c c u r a t e measure of heat damage nor a r e l i a b l e p r e d i c t o r of the n u t r i t i v e va lue of f i s h m e a l . In c o n c l u s i o n , the use of a b ioassay to d e f i n e the presence of a l i m i t i n g amino a c i d i n a f i s h d i e t r e q u i r e s improvements i n methodology. The procedures d e s c r i b e d i n t h i s t h e s i s for b i o l o g i c a l t e s t i n g of p r o t e i n q u a l i t y were a l l n o n - s p e c i f i c i n that the p r o p o r t i o n s and a v a i l a b l e q u a n t i t i e s of a l l e s s e n t i a l amino a c i d s were t e s t e d for s i m u l t a n e o u s l y . The r e s u l t s of Experiment 2 p r o v i d e d c o n v i n c i n g ev idence that the p r o t e i n requirements for f i s h should be s t a t e d i n terms of the p r o p o r t i o n of d i e t a r y energy s u p p l i e d by p r o t e i n . S ince the f i s h i n t h i s study were fed to s a t i s f y t h e i r t o t a l energy needs, p r o t e i n i n t a k e was governed by the p r o t e i n energy to t o t a l energy r a t i o of the d i e t . A l though the v a r y i n g p r o p o r t i o n s of c a r b o h y d r a t e and l i p i d energy i n the d i e t s cou ld 172 have confounded the above o b s e r v a t i o n s , no i n d i c a t i o n of t h i s e f f e c t was de tec ted under the e x p e r i m e n t a l c o n d i t i o n s of t h i s s t u d y . T h e r e f o r e , i t may be conc luded that j u v e n i l e ch inook salmon are ab le to adapt to e i t h e r c a r b o h y d r a t e or l i p i d as a source of m e t a b o l i z a b l e energy, w i t h i n the l i m i t s of p r a c t i c a l d i e t f o r m a t i o n . T h i s c o n c l u s i o n would advance the s u i t a b i l i t y of chinook salmon as a s p e c i e s f o r d o m e s t i c a t i o n . I t i s a l s o conc luded that the e m p i r i c a l gross p r o t e i n requirements of chinook salmon depend, not only on the p h y s i o l o g i c a l requirement of the f i s h , but a l s o on the o b j e c t i v e s and economic c o n s i d e r a t i o n s of the f i s h c u l t u r i s t . T h i s study d e s c r i b e d the use of a p o l y n o m i a l curve r e l a t i n g a growth response to d i e t a r y p r o t e i n energy l e v e l , which , wi th the e s t a b l i s h m e n t of s u i t a b l e conf idence l i m i t s , would p r o v i d e the f i s h n u t r i t i o n i s t wi th a bas i s for f o r m u l a t i n g f i s h f e e d s . The range for the q u a n t i t a t i v e p r o t e i n requirements f o r j u v e n i l e chinook salmon found i n t h i s study agrees wi th those s t a t e d by other i n v e s t i g a t o r s for c a r n i v o r o u s f i s h s p e c i e s . 173 6.0 B i b l i o g r a p h y A l b a n e s e , A . A . and O r t o , L . A . (1953) . P r o t e i n s and Amino A c i d s . I n : Newer methods of n u t r i t i o n a l b i o c h e m i s t r y . E d . A l b a n e s e , A . A . , Academic P r e s s . New Y o r k . pp .1 -104 . A l l i s o n , J . B . (1951) . I n t e r p r e t a t i o n of n i t r o g e n ba lance d a t a . F e d . P r o c . 10 ,676-683. A l l i s o n , J . B . (1964) . The n u t r i t i v e va lue of d i e t a r y p r o t e i n s , p . 41-86. In H . N . Munro and J . B . A l l i s o n [ e d . ] Mammalian p r o t e i n metabo l i sm. V o l . 2. Academic Pres s I n c . , New Y o r k , N . Y . A l m q u i s t , H . J . (1970) . E v a l u a t i o n of n u t r i e n t r e q u i r e m e n t s . I n : A . A . Albanese [ e d . ] Newer methods of n u t r i t i o n a l b i o c h e m i s t r y . V o l . 4. Academic Press I n c . , New Y o r k , N . Y . pp. 1-36. Anderson , R . J . ; K i e n h o l z , E .W. and F l i c k i n g e r , S . A . (1981) . P r o t e i n requirements of smallmouth bass and largermouth b a s s . J . N u t r . I l l , 1085-1097. A0AC (1975) . O f f i c i a l methods of a n a l y s i s of the 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 C h e m i s t s . W i l l i a m Horwith [ e d . ] . A s s o c . of O f f i c i a l A n a l y t i c a l Chem. , Wash . , D . C . , 1094 p . A r a i , S. (1981) . A p u r i f i e d t e s t d i e t f or coho sa lmon, Oncorhynchus k i s u t c h , f r y . B u l l . J a p . Soc . S c i . F i s h . 47, 547-550. A t a c k , T . H . , Jauncey , K. & M a t t y , A . J . (1979) . The u t i l i z a t i o n of some s i n g l e c e l l p r o t e i n s by f i n g e r l i n g m i r r o r c a r p . ( C y p r i n u s c a r p i o ) . A q u a c u l t u r e 18, 337-348. A t a c k , T . H . and M a t t y , A . J . (1978) . The e v a l u a t i o n of some s i n g l e c e l l p r o t e i n s i n the d i e t of rainbow t r o u t . I I . The d e t e r m i n a t i o n of net p r o t e i n u t i l i z a t i o n , b i o l o g i c a l va lues and t rue d i g e s t i b i l i t y . I n : F i n f i s h n u t r i t i o n and f i s h feed t e c h n o l o g y , V o l . I . ( e d . H a l v e r , J . E . and Tiews , K . ) . B e r l i n , pp 261-273. A u s t r e n g , E . 1978. D i g e s t i b i l i t y d e t e r m i n a t i o n i n f i s h us ing chromic ox ide marking and a n a l y s i s of contents from d i f f e r e n t segments of the g a s t r o i n t e s t i n a l t r a c t . A q u a c u l t u r e 13, 265-272. Bender , A . E . (1972) . P r o c e s s i n g damage to p r o t e i n f o o d : A r e v i ew . J . F d . T e c h n o l . 7 ,239-250. Bender , A . E . and M i l l e r , D . S . (1953) . A new b r i e f method of e s t i m a t i n g net p r o t e i n v a l u e . Biochem. J . 53, V I I . 174 Bender , A . E . and D o e l l , B . H . (1957) . B i o l o g i c a l e v a l u a t i o n s of p r o t e i n s : a new a s p e c t . B r . J . N u t r . 11,140-148. B e r g o t , F . (1979) . Carbohydrate i n rainbow t r o u t d i e t s : E f f e c t s of the l e v e l and source of c a r b o h y d r a t e and the number of meals on growth and body c o m p o s i t i o n . A q u a c u l t u r e 18, 157-167. B i e l y , J . , March , B . E . and T a r r , H . L . A . (1955) . The n u t r i t i v e va lue of h e r r i n g meals , I I I . The e f f e c t s of heat treatment and s torage temperature as r e l a t e d to o i l c o n t e n t . P o u l t r y S c i . 34, 1274-1279. B i r k e t t , L . (1969) . The n i t r o g e n ba lance i n p l a i c e , s o l e and p e r c h . J . Exp . B i o l . 50, 375-386. B i s s e t t , H . M . and T a r r , H . L . A . (1954) . The n u t r i t i v e va lue of h e r r i n g meals . I I . E f f e c t of heat on the a v a i l a b i l i t y of e s s e n t i a l amino a c i d s . P o u l t r y S c i . 33, 250. B l i g h , E . G . and Dyer , W . J . (1959) . A r a p i d method of t o t a l l i p i d e x t r a t i o n s . a n d p u r i f i c a t i o n . Can . J . Biochem. P h y s i o l . 37, 911-947. B o d w e l l , C . E . (1977) . Problems i n the development and a p p l i c a t i o n of r a p i d methods of a s s e s s i n g p r o t e i n q u a l i t y . F d . T e c h n o l . 31, 73-77. Booth , V . H . (1971) . Problems i n d e t e r m i n a t i o n of FDNB - a v a i l a b l e l y s i n e . J . S c i . F d . A g r i c . 22, 658-666. B r e s s a n i , R. (1977) . Humans assays and a p p l i c a t i o n s . I n : E v a l u a t i o n of p r o t e i n s for humans. B o d w e l l , C . E . ( e d ) . A r i P u b l i s h i n g Company, I n c . , Wes tpor t , C t . pp 81-118. B r e t t , J . R . (1971) . Growth responses of young sockeye salmon (Oncorhynchus n e r k a ) t o d i f f e r e n t d i e t s and p lanes of n u t r i t i o n . J . F i s h Rs . Board Can . 28, 1635-1643. B r e t t , J . R . (1979) . E n v i r o n m e n t a l f a c t o r s and growth. I n : Hoar , W . S . , R a n d a l l , D . J . and B r e t t , J . R . ( e d s . ) , F i s h P h y s i o l o g y , V o l . 8. Academic P r e s s , New Y o r k . pp 599-675. B r e t t , J . R . and H i g g s , D . A . (1970) . E f f e c t of temperature on the r a t e of g a s t r i c d i g e s t i o n i n f i n g e r l i n g sockeye . J . F i s h Res . Board Can . 32,2103-2110. B r e t t , J . R . , S h e l b o u r n , J . E . and Shoop, C T . (1969) . Growth r a t e and body c o m p o s i t i o n of f i n g e r l i n g sockeye salmon, Oncorhynchus n e r k a , i n r e l a t i o n to temperature and r a t i o n s i z e . J . F i s h . Res . Board C a n . , 26, 2362-2394. - 175 Brody , S. (1945) . B i o e n e r g e t i c s and growth. R e i n h o l d , New York . B u c k l e y , J . T . and G r o v e s , T . D . (1979) . I n f l u e n c e of feed on the body c o m p o s i t i o n of f i n f i s h . I n : F i n f i s h n u t r i t i o n and f i s h feed t e c h n o l o g y . V o l . 2. E d . H a l v e r , J . E . And T iews , K . , Heenemann, B e r l i n . Buhl e r , D . R . and H a l v e r , J . E . (1961) . N u t r i t i o n of sa lmonid f i s h e s . IX . Carbohydrate requirement of chinook salmon. J . N u t r . 74, 307-318. Burrows, R . E . (1969) . The i n f l u e n c e of f i n g e r l i n g q u a l i t y on a d u l t salmon s u r v i v a l s . T r a n s . Am. F i s h . Soc . 98, 777-784. Cameron, A . M . and Endean, R. (1973) . E p i d e r m a l s e c r e t i o n s and the e v o l u t i o n of venom glands in f i s h e s . T o x i c o n 11, 401-410. C a r p e n t e r , K . J . (1960) . E s t i m a t i o n of a v a i l a b l e l y s i n e i n f o o d s . Biochem. J . 77, 604-610. C a r p e n t e r , K . J . , March , B . E . , M i l n e r , C . K . and C a m p b e l 1 , R . C . (1963) . A growth assay with c h i c k s for the l y s i n e content of p r o t e i n c o n c e n t r a t e s . B r i t . J . N u t r . 17, 309-323. C a s t e l l , J . D . and Tiews , K. (1979) . Report of the E I F A C , IUNS and ICES working group on the s t a n d a r d i z a t i o n of methodology i n f i s h n u t r i t i o n r e s e a r c h . EIFAC T e c h . Pap. 36.24 pp Cho, C . V . and S l i n g e r , S . J . (1979) . Apparent d i g e s t i b i l i t y measurement i n f e e d s t u f f s for rainbow t r o u t . I n : F i n f i s h N u t r i t i o n and F i s h f e e d T e c h n o l o g y , V o l . I I . ( E d i t e d by H a l v e r , J . E . and T i e w s , K . ) . pp 239-247. B e r l i n . Cho, C . Y . , B a y l e y , H . S . , and S l i n g e r , S . J . (1975) . An automated f i s h r e s p i r o m e t e r for n u t r i t i o n s t u d i e s . P r o c . 28 th . Ann. Meet ing of Can . Conf . for f i s h . R e s . , Vancouver , B . C . C h o u b e r t , G . , Noue, J . and Luquet , P. (1979) . Cont inuous q u a n t i t a t i v e automat ic c o l l e c t o r for f i s h f e c e s . P r o g . F i s h C u l t . 41, 64-67. C l a r k e , W . C . , H i g g s , D . A . , M a r k e r t , J . R . , S h e l b o u r n , J . E . and C a s t l e d i n e , A . J . (1982) . E f f e c t of v a r y i n g d i e t a r y p r o t e i n : l i p i d r a t i o s on growth and body c o m p o s i t i o n of coho salmon f r y (Oncorhynchus k i s u t c h ) reared at d i f f e r e n t t e m p e r a t u r e s . Can. Data Rep. F i s h . A q u a t i c S c i . No. 373. pp 18. 176 Crampton, E .W. and L l o y d , L . E . (1959) . Fundamentals of N u t r i t i o n , Freeman, San F r a n c i s c o . Combs, B . D . , Heinemann, W.W., Burrows, R . E . , Thomas, A . E . and F o w l e r , L . G . (1962) . P r o t e i n and c a l o r i e l e v e l s of meat meal , v i t a m i n supplemented salmon d i e t s . U . S . F i s h W i l d l i f e S e r v . , Spec . S c i . Rept . , F i s h e r i e s , 432, pp 1-24. Cowey, C . B . (1975) . Aspect s of p r o t e i n u t i l i z a t i o n by f i s h . P r o c . N u t r . Soc . 34, pp 57-63. Cowey, C . B . (1979) . P r o t e i n and amino a c i d requ irements of f i n f i s h . I n : F i n f i s h n u t r i t i o n and f i s h feed t e c h n o l o g y , V o l . I . E d . H a l v e r , J . E . and Tiew, K . , Heenemann, B e r l i n , pp 3-16. Cowey, C . B . and S a r g e n t , J . R . (1972) . F i s h N u t r i t i o n . Adv. Mar. B i o l . 10, 383-492. Cowey, C . B . and S a r g e n t , J . R . (1979) . N u t r i t i o n . I n : Hoar , W . S . , R a n d a l l , D . J . and B r e t t , J . R . ( e d s . ) , F i s h P h y s i o l o g y , V o l . 8. Academic P r e s s , New Y o r k , pp 1-69. Cowey, C . B . , A d r o n , J . W . , B l a i r , A. and Pope, J . (1970) . The growth of 0. group p l a i c e on a r t i f i c i a l d i e t s c o n t a i n i n g d i f f e r e n t l e v e l s of p r o t e i n . He lgo . Wis s . Meere. 20, 602-609. Cowey, C . B . , Pope, J . A . , A d r o n , J . W . and B l a i r , A. (1971) . S t u d i e s on the n u t r i t i o n of marine f l a t f i s h . Growth of p l a i c e , P l e u r o n e c t e s p l a t e s s a , on d i e t s c o n t a i n i n g p r o t e i n s d e r i v e d from p l a n t s and other s o u r c e s . Mar. B i o l . 10, 145-153. Cowey, C . B . , Pope, J . A . , A d r o n , J . W . and B l a i r , A. (1972) . S t u d i e s on the n u t r i t i o n of marine f l a t f i s h . The p r o t e i n requirements of p l a i c e ( P l e u r o n e c t e s p l a t e s s a ) . B r . J . N u t r . 28, 447-456. Cowey, C . B . , A d r o n , J . W . , B l a i r , A. and Shankes, A . M . (1974) . S t u d i e s on the n u t r i t i o n of marine f l a t f i s h . U t i l i z a t i o n of v a r i o u s d i e t a r y p r o t e i n s by p l a i c e ( P l e u r o n e c t e s p l a t e s s a ) . B r . J . N u t r . 31, 297-306. Cowey, C . B . , Cooke, D . J . , M a t t y , A . J . and A d r o n , J . W . (1981) . E f f e c t of q u a n t i t y and q u a l i t y of d i e t a r y p r o t e i n on c e r t a i n enzyme a c t i v i t i e s i n rainbow t r o u t . J . N u t r . I l l , 336-345. Dabrowski , K. (1979) . P r o t e i n requirements of grass carp f r y . A q u a c u l t u r e . 12, 63-73. Dabrowski , K. and Kozak, B. (1979) . The use of f i s h meal and soybean meal as a p r o t e i n source i n the d i e t of grass carp f r y . A q u a c u l t u r e . 18, 107-114. 177 DeLong, D . C . , H a l v e r , J . E . , and M e r t z , E . T . (11958) . N u t r i t i o n of sa lmonid f i s h e s . V I . P r o t e i n requirements of chinook salmon at two water t e m p e r a t u r e s . J . N u t r . 65, 589-599. Dona ldson , L . R . and O l s o n , P . R . (1957) . Development of rainbow t r o u t broods tock by s e l e c t i v e b r e e d i n g . T r a n s . Amer. F i s h Soc . 85, 93-101. Duncan, D . B . (1955) . M u l t i p l e range and m u l t i p l e F t e s t s . B i o m e t r i c s . 11, 1-42. Dupree , H . K . and Sneed, K . E . (1966) . Response of channe l c a t f i s h f i n g e r l i n g s to d i f f e r e n t l e v e l s of major n u t r i e n t s i n p u r i f i e d d i e t s . T e c h . paper No. 9. Bureau Sport F i s h e r i e s and W i l d l i f e , U . S . Dept . I n t . pp 21. E v a n s , E . and W i t t y , R. (1978) . An assessment of methods used to determine p r o t e i n q u a l i t y . Wld . Rev. N u t r . D i e t . 32, 1-26. F a g e r l u n d , U . H . M . , McCal lum, I . , H i g g s , D . A . , M c B r i d e , J . R . , P l o t n i k o f f , M . D . , and Dosanjh , B . S . (1984) . D i e t c o m p o s i t i o n as a f a c t o r i n the a n a b o l i c e f f i c a c y of 3 , 5 , 3 - t r i i o d o - L - t h y r o n i n e a d m i n i s t e r e d o r a l l y to s t e e l h e a d t r o u t . (Salmo g a i r d n e r i ) . A q u a c u l t u r e , 36, 49-59 . F o r s t e r , R . P . and G o l d s t e i n , L . (1969) . Format ion of e x c r e t o r y p r o d u c t s . I n : F i s h P h y s i o l o g y . V o l 1. (W.S. Hoar and D . J . R a n d a l l , e d s . ) , pp 313-350. Academic P r e s s , New York . F o w l e r , L . G . (1980) . S u b s t i t u t i o n of soybean and co t tonseed product s f o r f i s h meal i n d i e t s fed to chinook and coho sa lmon. P r o g . F i s h . C u l t . , 42, 87-91. F o w l e r , L . G . (1981a) . T e s t s of vege tab le p r o t e i n rep lacements and a l t e r n a t e i n g r e d i e n t s i n the Abernathy d i e t . Abernathy Salmon C u l t u r a l Development C e n t e r . Technology T r a n s f e r S e r i e s No. 81 -1 . pp 12. F o w l e r , L . G . (1981b) . E f f e c t of l i p i d and amino a c i d supp lementa t ion i n chinook salmon d i e t s c o n t a i n i n g p o u l t r y b y - p r o d u c t meal . Abernathy Salmon C u l t u r a l Development C e n t e r . Technology T r a n s f e r S e r i e s No. 81-2 . pp 12. F o w l e r , L . G . and Burrows, R . E . (1971) . The Abernathy salmon d i e t . P r o g . F i s h C u l t . 33, 67-75. 178 F o w l e r , L . G . , McCormick, J . H . , and Thomas, A . E . (1964) . F u r t h e r s t u d i e s of p r o t e i n and c a l o r i c l e v e l s of meat meal , v i t a m i n supplemented salmon d i e t s . U . S . F i s h W i l d l i f e S e r v i c e , Spec . S c i . R e p t . , F i s h e r i e s 480, pp 1-14. F o w l e r , L . G . , Burrows, R . E . , Combs, B . D . and Banks , J . L . (1980) . The e f f e c t of s i z e and time of r e l e a s e of f a l l chinook salmon f i n g e r l i n g s on a d u l t s u r v i v a l . Abernathy Salmon C u l t u r a l Development C e n t e r . Technology T r a n s f e r S e r i e s , No. 80-3 , pp 18. G a r l i c k , P . J . (1980) . P r o t e i n turnover i n the whole animal and s p e c i f i c t i s s u e s . In Comprehensive B i o c h e m i s t r y V o l . 19b, E d . by F l o r k i n , M. and Newberger, A. E l s e v i e r S c i e n t i f i c P u b l i s h i n g Company, Amsterdam. pp. 77-145. G a r l i n g , D . L . and W i l s o n , R . P . (1977) . E f f e c t s of d i e t a r y c a r b o h y d r a t e to l i p i d r a t i o s on growth and body c o m p o s i t i o n of f i n g e r l i n g channel c a t f i s h . P r o g . F i s h C u l t . 39, 43-37. G e r k i n g , S . D . (1955a) . I n f l u e n c e of f e e d i n g r a t e on body c o m p o s i t i o n and p r o t e i n metabol ism of b l u e g i l l s u n f i s h . P h y s i o l . Z o o l . 28, 267-283. G e r k i n g , S . D . (1955b) . Endogenous n i t r o g e n e x c r e t i o n of b l u e g i l l s u n f i s h . P h y s i o l . Z o o l . 28, 283-289. G e r k i n g , S . D . (1971) . I n f l u e n c e of r a t e of f e e d i n g and body weight on p r o t e i n metabol ism of b l u e g i l l s u n f i s h . P h y s i o l . Z o o l . 44, 9-19. G r o v e s , T . D . (1980) . Body c o m p o s i t i o n changes d u r i n g growth i n young sockeye (Oncorhynchus nerka) i n f r e s h water . J . F i s h . Res . Board C a n . 27, 929-942. G u l b r a n d s e n , K . E . and Utne , F . (1977) . The p r o t e i n requirement on energy b a s i s for rainbow t r o u t (Salmo g a i r d n e r i ) . F i s h . S k r . S e r . E r n a e r i n g 1, 75-86. H a l v e r , J . E . (1982) . The v i t a m i n s r e q u i r e d f o r c u l t i v a t e d s a l m o n i d s . Comp. Biochem. P h y s i o l . 73B, 43-50. H a l v e r , J . E . , DeLong, D . C . , and M e r t z , E . T . (1958) . Threon ine and l y s i n e requ irements of chinook sa lmon. F e d . P r o c . 17, 1873. H e g s t e d , D . M . and Chang, Y . 0 . (1965a) . P r o t e i n u t i l i z a t i o n i n growing r a t s . I . R e l a t i v e growth index as an assay p r o c e d u r e . J . N u t r . 85, 159-168. Hegs ted , D . M . and Chang, Y . 0 . (1965b) . P r o t e i n u t i l i z a t i o n i n growing r a t s at d i f f e r e n t l e v e l s of i n t a k e . J . N u t r . 87, 19-25. 179 - Hegsted , D . M . , N e f f , R. and W o r c e s t e r , J . (1968) . D e t e r m i n a t i o n of the r e l a t i v e n u t r i t i v e va lue of p r o t e i n s . F a c t o r s a f f e c t i n g p r e c i s i o n and v a l i d i t y . J . Ag . Food Chem. 16, 190-197. Henry , K . M . (1965) . A comparison of b i o l o g i c a l methods with r a t s for d e t e r m i n i n g the n u t r i t i v e va lue of p r o t e i n s . J . N u t r . 19, 125-135. Henry , K . M . and T o o t h h i l l , J . (1962) . A comparison of the body-water and n i t r o g e n b a l a n c e - s h e e t methods for d e t e r m i n i n g the n u t r i t i v e va lues of p r o t e i n s . B r i t . J . N u t r . 16, 125-133. H i g g s , D . A . , F a g e r l u n d , U . H . M . , M c B r i d e , J . R . , P l o t n i k o f f , M . D . , M a r k e r t , J . R . and D a v i d s o n , J . (1983) . P r o t e i n q u a l i t y of A l t e x c a n o l a meal for j u v e n i l e ch inook salmon (Oncorhynchus tshawytscha) c o n s i d e r i n g d i e t a r y p r o t e i n and 3 , 5 , 3 - t r i i o d o - L - t h y r o n i n e c o n t e n t . A q u a c u l t u r e , 34, 213-238. H i g g s , D . A . , M a r k e r t , J . R . , M a c Q u a r r i e , D . W . , M c B r i d e , J . R . , Dosanjh , B . S . , N i c h o l s , C . and H o s k i n s , G. (1979) . Development of p r a c t i c a l dry d i e t s for coho salmon, Oncorhynchus k i s u t c h , us ing p o u l t r y - b y - p r o d u c t meal , f e a t h e r meal , soybean meal and rapeseed meal as major p r o t e i n s o u r c e s . I n : F i n f i s h n u t r i t i o n and f i s h feed t e c h n o l o g y , J . E . Ha lver and K. Tiews ( e d s ) , V o l . I I . Heenemann V e r l a g s g e s e l l s c h a f t M b H . , B e r l i n , pp 191-218. H i l t o n , J . W . (1982) . The e f f e c t of p r e - f a s t i n g d i e t and water temperature on l i v e r g lycogen and l i v e r weight i n rainbow t r o u t , Salmo g a i r d n e r i , d u r i n g f a s t i n g . J . F i s h B i o l . 20, 69-78. H i l t o n , J . W . and A t k i n s o n , J . L . (1982) . Response of rainbow t r o u t to i n c r e a s e d l e v e l s of a v a i l a b l e c a r b o h y d r a t e i n p r a c t i c a l t r o u t d i e t s . B r . J . N u t r . 47, 597-607. H i l t o n , J . W . , Cho, C . Y . and S l i n g e r , S . J . (1981) . E f f e c t of e x t r u s i o n p r o c e s s i n g and steam p e l l e t i n g d i e t s on p e l l e t d u r a b i l i t y , p e l l e t water a b s o r p t i o n and the p h y s i o l o g i c a l response of rainbow t r o u t (Salmo g a i r d n e r i ) . A q u a c u l t u r e , 25, 185-194. H i l t o n , J . W . , A t k i n s o n , J . L . and S l i n g e r , S . J . (1982) . Maximum t o l e r a b l e l e v e l , d i g e s t i o n , and metabol ism of D - g l u c o s e ( c e r e l o s e ) i n rainbow t r o u t (Salmo g a i r d n e r i ) r e a r e d on a p r a c t i c a l t r o u t d i e t . Can. J . F i s h . Aquat . S c i . 39, 1229-1234. 180 H i l t on, J . W . , A t k i n s o n , J . L . and S l i n g e r , S . J . (1983) . E f f e c t of i n c r e a s e d d i e t a r y f i b e r on the growth of rainbow t r o u t (Salmo g a i r d n e r i ) . Can . J . F i s h . Aquat . S c i . 40, 81-85. Ingram, G . A . (1980) . Substances i n v o l e d i n the n a t u r a l r e s i s t a n c e of f i s h to i n f e c t i o n - A rev iew. J . F i s h B i o l . 16, 23-60. J o b l i n g , M. (1983) . A s h o r t review and c r i t i q u e of methodology used i n f i s h growth and n u t r i t i o n s t u d i e s . J . F i s h B i o l . 23, 685-703. J o b l i n g , M. and Wandsvik, A. (1983) . An i n v e s t i g a t i o n of f a c t o r s c o n t r o l l i n g food i n t a k e i n A r c t i c c h a r , S a l v e l i n u s a l p i n u s . J . F i s h B i o l . 23, 397-404. J o h n s t o n , J . and Coon, C . N . (1979) . A comparison of s i x p r o t e i n q u a l i t y assays u s i n g c o m m e r c i a l l y a v a i l a b l e p r o t e i n meals . P o u l t r y S c i . 58, 919-927. Iwata, K. (1970) . R e l a t i o n s h i p between food and growth i n young c r u c i a n c a r p s , C a r a s s i u s aura tus c u r i e r i , as determined by the n i t r o g e n b a l a n c e . J p n . J . L i m n o l . 31, 129-151. K a u s h i k , S . J . and Luquet , P. (1979) . I n f l u e n c e of d i e t a r y amino a c i d p a t t e r n s on the f r e e amino a c i d content s of b lood and muscle of rainbow t r o u t . Comp. Biochem. P h y s i o l . 64, 175-180. K e t o l a , H . G . (1982) . Amino a c i d n u t r i t i o n of f i s h e s . Requirments and supp lementa t ion of d i e t s . Comp. Biochem. P h y s i o l . 73B, 17-24. K o s h i i s h i , Y . (1980) . Growth of j u v e n i l e chum salmon (Oncorhyndrus k e t a ) , r e a r e d i n tanks s u p p l i e d sea water i n r e l a t i o n to food consumpt ion , s a l i n i t y and d i e t a r y p r o t e i n c o n t e n t . B u l l . J a p . Sea Reg. F i s h Res. L a b . 31, 41-55 . L e a , C . H . , P a r r , L . J . and C a r p e n t e r , K . J . (1958) . Chemica l and n u t r i t i o n a l changes i n s t o r e d meal . B r i t . J . N u t r . 12, 297-312. Lee , D . J . and Putnam, G . B . (1973) . The response of rainbow t r o u t to v a r y i n g p r o t e i n / e n e r g y r a t i o s i n a t e s t d i e t . J . N u t r . 103, 916-922. Luquet , P. (1971) . E f f i c a c i t e des p r o t e i n e s en r e l a t i o n avec l e u r taux d ' i n c o r p o r a t i o n dans 1 ' a l i m e n t a t i o n de l a t r u i t e a r e - e n - c i e l . Ann. H y d r o b i o l . 2, 175-186. 181 March , B . E . , B i e l y , J . , G o u d i e , C . , C l a g g e t , F . G . and T a r r , H . L . (1961) . The e f f e c t of s torage temperature and a n t i o x i d a n t treatment on the chemica l and n u t r i t i v e c h a r a c t e r i s t i c s of h e r r i n g meal . J . Am. O i l Chem. Soc . 38, 80-84. M a r c h , B . E . , B i e l y , J . , Goud ie , C . , and T a r r , H . L . A . (1966) . P r o t e i n n u t r i t i v e q u a l i t y of commercia l f i s h meals as assessed by b i o l o g i c a l and chemica l methods. J . F i s h Res . Board Can. 23, 395-414. Maynard, L . A . and L o o s l i , J . K . (1969) . Animal N u t r i t i o n , 6th e d . , M c G r a w - H i l l , New Y o r k . 613 pp. McDonald , P . , Edwards, R . A . and G r e e n h a l g h , J . F . (1976) . Animal N u t r i t i o n . McLaughlan , J . M . (1979) . C r i t i q u e of methods for e v a l u a t i o n of p r o t e i n q u a l t i y . I n : W i l k e , H . L . , H o p k i n s , D . T . and Waggle, D . H . ( e d s ) . Soy p r o t e i n and human n u t r i t i o n . Academic P r e s s , New Y o r k . pp 281-297. M c L a u g h l i n , J . M . and K e i t h , M . O . (1977) . E f f e c t of t h r e o n i n e supp lementa t ion i n the s lope assay for p r o t e i n q u a l i t y . J . A . O . A . C . , 60, 1291-1295. M i l l w a r d , D . J . , B a t e s , P . C . , L a u r e n t , G . J . and L o , C . C . (1978) . F a c t o r s a f f e c t i n g p r o t e i n breakd own i n s k e l e t a l musc le . E d . by S e g a l , H . L . and D o y l e , D . J . . Acadmeic P r e s s , New Y o r k , pp. 619-646. M o r g u l i s , S. (1918) . S t u d i e s on the n u t r i t i o n of f i s h . Exper iments on brook t r o u t . J . B i o l . Chem. 36, 391-413. M o r r i s o n , A . B . and C a m p b e l l , J . A . (1960) . E v a l u a t i o n of food p r o t e i n s . V. F a c t o r s i n f l u e n c i n g the p r o t e i n e f f i c i e n c y r a t i o of f o o d s . J . N u t r . 70, 112-118. M i l l e r , D . S . and Bender , A . E . (1955) . The d e t e r m i n a t i o n of the net u t i l i z a t i o n of p r o t e i n s by a shortened method. B r i t . J . N u t r . 15, 11-19. M i l l e r , D . S . and Bender , A . E . (1955) . The d e t e r m i n a t i o n of the net u t i l i z a t i o n of p r o t e i n s by a shortened method. B r i t . J . N u t r . 9, 382-388. M i t c h e l l , H . H . (1924) . A method of d e t e r m i n i n g the b i o l o g i c a l va lue of p r o t e i n . J . B i o l . Chem. 58, 873-903. M i t c h e l l , H . H . (1962) . Comparat ive n u t r t i o n of man and domest ic a n i m a l s , V o l I . Academic P r e s s , New Y o r k . 182 M i t c h e l l , H . H . and B e r t , M . H . (1954) . The d e t e r m i n a t i o n of m e t a b o l i c f e c a l n i t r o g e n . J . N u t r . 52, 83-497. M u r a i , T , F l ee twood , M . A . and Andrews, J . W . (1979) . Optimum l e v e l s of crude p r o t e i n for f i n g e r l i n g American shad . The P r o g . F i s h C u l t . 41, 5-7 . N . R . C . ( N a t i o n a l Research C o u n c i l ) (1981) . N u t r i e n t requirements of c o l d water f i s h e s . N a t i o n a l Academy of S c i e n c e s , Washington, D . C . Nose, T . (1960) . On the d i g e s t i o n of food p r o t e i n by g o l d f i s h ( C a r a s s i u s auratus L . ) and rainbow t r o u t (Salmo i r i d e u s G ,~J~. B u l l . Freshwater Res. L a b . , Tokyo 10, 11-22 . Nose, T . and A r a i , S. (1972) . Optimum l e v e l of p r o t e i n i n p u r i f i e d d i e t f or e e l , A n g u i l l a j a p o n i c a . B u l l . Freshwater Res. L a b . , Tokyo 22, 145-154. Ogata , H . , A r a i , S . , and Nose, T . (1983) . Growth responses of c h e r r y salmon, Oncorhynchus masou, and amago salmon, 0. r h o d u r u s , f r y fed p u r i f i e d c a s e i n d i e t s supplemented wi th amino a c i d s . B u l l . J a p . Soc . S c i . F i s h 49, 1381-1385. Ogino , C . and S a i t o , K. (1970) . P r o t e i n n u t r i t i o n i n F i s h . I . The u t i l i z a t i o n of d i e t a r y p r o t e i n by young c a r p . B u l l . J a p . Soc . S c i . F i s h 36, 250-254. Og ino , C . and N a n r i , H. (1980) . R e l a t i o n s h i p between the n u t r i t i v e va lue of d i e t a r y p r o t e i n s for rainbow t r o u t and the e s s e n t i a l amino a c i d c o m p o s i t i o n s . B u l l . J a p . Soc . S c i . F i s h 46, 109-112. Ogino , C , K a k i n o , J . and Chen, M . S . (1973) . P r o t e i n n u t r i t i o n i n f i s h . I I . D e t e r m i n a t i o n of metabo l i c f e c a l n i t r o g e n and endogenous n i t r o g e n e x c r e t i o n of c a r p . B u l l . J a p . Soc . S c i . F i s h 39, 519-523. Og ino , C , C h i o u , J . Y . and T a k e u c h i , T . (1976) . P r o t e i n n u t r i t i o n i n f i s h . V I . E f f e c t s of d i e t a r y energy sources on the u t i l i z a t i o n of p r o t e i n by rainbow t r o u t and c a r p . B u l l . J a p . Soc . S c i . F i s h 42, 213-218. Ogino , C . , Cowey, C . B . and C h i o u , J . Y . (1978) . Leaf p r o t e i n c o n c e n t r a t e as a p r o t e i n source i n d i e t s f o r carp and rainbow t r o u t . B u l l . J a p . Soc . S c i . F i s h 44, 49-52. Ogino , C . , Kawasaki , H. and N a n r i , H. (1980) . Method for d e t e r m i n a t i o n of n i t r o g e n r e t a i n e d i n the f i s h body by c a r c a s s a n a l y s i s . B u l l . J a p . Soc . S c i . F i s h 46, 105-108. - 183 - O p s t v e d t , J . , M i l l e r , R . , Hardy , R.W. and S p i n e l l i , J . 1984). H e a t - i n d u c e d changes i n s u l f h y d r y l groups and d i s u l f i d e bonds i n f i s h p r o t e i n and t h e i r e f f e c t on p r o t e i n and amino a c i d d i g e s t i b i l i t y i n rainbow t r o u t . J . A g r i c . Food Chem. 32, 929-935. Osborne , T . B . , Mendel , L . B . and F e r r y , E . L . (1919) . A method of e x p r e s s i n g n u m e r i c a l l y the growth promoting va lue of p r o t e i n s . J . B i o l . Chem. 37, 223-229. Page, J . W . and Andrews, J . W . (1973) . I n t e r a c t i o n s of d i e t a r y l e v e l s of p r o t e i n and energy on channel c a t f i s h , I c t a l u r u s p u n c t a t u s . J . N u t r . 103, 1339-1346. Payne, P . R . (1972) . P r o t e i n q u a l i t y of d i e t s , c h e m i c a l s cores and amino a c i d i m b a l a n c e s . I n : Bigwood ( e d . ) I n t e r n a t i o n a l e n c y c l o p e d i a of food and n u t r i t i o n , V o l . I I , pp 259-310. Pergamon P r e s s , O x f o r d . P f e f f e r , E . (1982) . U t i l i z a t i o n of d i e t a r y p r o t e i n by sa lmonid f i s h . Comp . Biochem. P h y s i o l . 73B, 51-57. P h i l l i p s , A . M . (1969) . N u t r i t i o n , d i g e s t i o n and energy u t i l i z a t i o n . I n : F i s h P h y s i o l o g y , V o l . I . Hoar , W.S. and R a n d a l l , D . J . ( E d ) . Academia P r e s s , New Y o r k . pp. P i c k e r i n g , A . D . and Macey, D . J . (1977) . S t r u c t u r e , h i s t o c h e m i s t r y and the e f f e c t of h a n d l i n g s t r e s s on the mucous c e l l s of the e p i d e r m i s of the char ( S a l v e n i u s a l p i n u s ) . J . F i s h B i o l . 10, 505-512. P i e p e r , A. and P f e f f e r , E . (1980) . S t u d i e s on the e f f e c t of i n c r e a s i n g p r o p o r t i o n s of sucrose or g e l a t e n i z e d maize s t a r c h i n d i e t s for rainbow t r o u t (Salmo g a i r d n e r i ) on the u t i l i z a t i o n of d i e t a r y energy and p r o t e i n . A q u a c u l t u r e 20, 333-342. P l o t n i k o f f , M . D . , H i g g s , D . A . , M a r k e r t , J . R . , Dosanjh , B . S . , M c B r i d e , J . R . and B u c k l e y , J . T . (1983) . N u t r i t i o n and marine s u r v i v a l of chinook sa lmon. 1. P o t e n t i a l r o l e of smolt body c o m p o s i t i o n (Robert son Creek Hatchery 1979 b r o o d ) . Can . T e c h . Rep. of F i s h e r i e s and A q u a t i c S c i e n c e s No. 1206. P l o t n i k o f f , M . D . , H i g g s , D . A . , M a r k e r t , J . R . , Dosanjh , B . S . , M c B r i d e , J . R . and B u c k l e y , J . T . (1984) . N u t r i t i o n and marine s u r v i v a l of chinook sa lmon. 2. F u r t h e r i n v e s t i g a t i o n of the p o t e n t i a l r o l e of smolt body c o m p o s i t i o n (Robertson Creek Hatchery 1980 b r o o d ) . Can T e c h . Rep. of F i s h e r i e s and A q u a t i c S c i e n c e s No. 1235. 184 R i n g r o s e , R . C . (1971) . C a l o r i e to p r o t e i n r a t i o for brook t r o u t ( S a l v e n i u s f o n t i n a l i s ) . J . F i s h . Res. Board Can . 28, 1113-1117. Robb ins , K . R . , N o r t o n , H.W. and Baker , D . H . (1979) . E s t i m a t i o n of n u t r i e n t requirements from growth d a t a . J . N u t r . 109, 1710-1714. Rumsey, G . L . and K e t o l a , H . G . (1975) . Amino a c i d supp lementa t ion of c a s e i n i n d i e t s of A t l a n t i c salmon (Salmo s a l a r ) f r y and of soybean meal for rainbow t r o u t (Salmo g a i r d n e r i ) f i n g e r l i n g s . J . F i s h Res. Bd . Can. 32, 422-426. R y c h l y , J . and Spannhoff , L . (1979) . N i t r o g e n ba lance i n t r o u t . I . D i g e s t i b i l i t y of d i e t s c o n t a i n i n g v a r y i n g l e v e l s of p r o t e i n and c a r b o h y d r a t e . A q u a c u l t u r e 16, 39-46. Samonds, K.W. and Hegsted , D . M . (1977) . Animal B i o a s s a y s : A c r i t i c a l e v a l u a t i o n with s p e c i f i c r e f e r e n c e to a s s e s s i n g n u t r i t i v e va lue for the human. I n : E v a l u a t i o n of p r o t e i n s f o r humans. B o d w e l l , C . E . ( e d . ) . A v i P u b l i s h i n g Company, I n c . , Wes tpor t , C t . pp 68-80. SAS I n s t i t u t e , I n c . , (1982) . SAS Users gu ide : s t a t i s t i c s . SAS I n s t . , I n c . , C a r y , N . C . S a t i a , B . P . (1974) . Q u a n t i t a t i v e p r o t e i n requirements of rainbow t r o u t . P r o g . F i s h . C u l t . 36, 80-85. S a t t e r l e e , L . D . , K e n d r i c k , J . G . and M i l l e r , G . A . (1977) . Rapid ITI v i t r o assays for e s t i m a t i n g p r o t e i n q u a l i t y . F d . T e c h n o l . 31, 78-81. S a v i t z , J . (1969) . E f f e c t s of temperature and body weight on endogenous n i t r o g e n e x c r e t i o n i n the b l u e g i l l ' s u n f i s h (Lepomis m a c r o c h i r u s ) . J . F i s h . Res. Board Can . 26, 1813-1821. S a v i t z , J . (1971) . N i t r o g e n e x c r e t i o n and p r o t e i n consumption of the b l u e g i l l s u n f i s h (Lepomis m a c r o c h i r u s ) . J . F i s h . Res . Board Can. 28, 449-451. Seagran , H . L . , Morey, D . E . and Dassow, J . A . (1954) . The amino a c i d content of roe at d i f f e r e n t s tages of m a t u r i t y from f i v e s p e c i e s of P a c i f i c sa lmon. J . N u t r . 53, 139-149. S c h e f f e , H. (1959) . The a n a l y s i s of v a r i a n c e . John Wiley and Sons, I n c . , New Y o r k . 477 pp. S h a p i r o , R. and F i s h e r , H. (1967) . P r o t e i n r e s e r v e s : R e l a t i o n s h i p of d i e t a r y e s s e n t i a l and n o n e s s e n t i a l amino a c i d s to f o r m a t i o n and maintenance i n the f o w l . J . N u t r . 76, 106-112. 185 Shimeno, S . , Hosokawa, H. and Takeda , M. (1979) . The importance of c a r b o h y d r a t e i n the d i e t of a c a r n i v o r o u s f i s h . I n : F i n f i s h n u t r i t i o n and feed t e c h n o l o g y . V o l . 1 H a l v e r , J . E . and Tiews , K. ( e d s . ) Heenemann, B e r l i n . pp 127-143. S i n g h , R . P . and Nose, T . (1967) . D i g e s t i b i l i t y of c a r b o h y d r a t e i n young rainbow t r o u t . B u l l . F r e s h . F i s h Res . L a b . Tokyo 17, 21-25. Skrede , A . , K r o g d a h l , A . and A u s t r e n g , E . (1980) . D i g e s t i b i l i t y of amino a c i d s i n raw f i s h and meat and bone meal f o r the c h i c k e n , fox , mink and rainbow t r o u t . Z . T i e r p h y s i c a l . T i e r e r n a h r . F u t t e r m i t t e l k 43, 92-101. Smi th , B.W. and L o v e l l , R . T . (1973) . D e t e r m i n a t i o n of apparent p r o t e i n d i g e s t i b i l i t y i n feeds for channe l c a t f i s h . T r a n s . Am. F i s h . Soc . 102, 831-835. S m i t h , M . A . K . and T h o r p e , A. (1976) . N i t r o g e n metabol ism and t r o p h i c i n p u t i n r e l a t i o n to growth i n f re shwater and s a l t w a t e r Salmo g a i r d n e r i . B i o l . B u l l . (Woods H o l e , Mass . ) 150, 139-151. S m i t h , R . R . (1971) . A method of measuring d i g e s t i b i l i t y and m e t a b o l i z a b l e energy of f i s h f e e d s . P r o g . F i s h C u l t . 33, 132-134. Smi th , R . R . , Rumsey, G . L . and S c o t t , M . L . (1978) . Heat increment a s s o c i a t e d with d i e t a r y p r o t e i n , f a t , c a r b o h y d r a t e and complete d i e t s i n sa lmonids : comparat ive e n e r g e t i c e f f i c i e n c y . J . N u t r . 108, 1025-1032. S t e f f e n s , W. (1981) . P r o t e i n u t i l i z a t i o n by rainbow t r o u t (Salmo g a i r d n e r i ) and carp ( C y p r i n u s c a r p i o ) : A b r i e f rev i ew . A q u a c u l t u r e 23, 337-345. S t o v e r , J . H . (1967) . S t a r v a t i o n and the e f f e c t s of C o r t i s o l i n the g o l d f i s h ( C a r a s s i u s a u r a t u s L . ) . Comp. Biochem. P h y s i o l . 20, 939-948. Swarok, M. and H i g g s , D . A . (1981) . P e r s o n a l communicat ion . Takeda , M . , Shimeno, S . , Hosokawa, H . , K a j i y a m a , H. and K a i s y o , T . (1975) . The e f f e c t of d i e t a r y p r o t e i n to c a l o r i e r a t i o on the growth, feed c o n v e r s i o n and body c o m p o s i t i o n of young y e l l o w t a i l . B u l l . Japan Soc . S c i e n . F i s h 41, 443-447. T a k e u c h i , T . , Watanabe, T . and Og ino , C . (1978) . Optimum r a t i o of p r o t e i n to l i p i d i n d i e t s of rainbow t r o u t . B u l l . Japan Soc . S c i e n . F i s h 44, 729-732. 186 T a r r , H . L . A . and B i e l y , J . (1972) . E f f e c t of p r o c e s s i n g on the n u t r i t i o n a l va lue of f i s h meal and r e l a t e d p r o d u c t s . I n : E f f e c t of p r o c e s s i n g on the n u t r i t i o n a l va lue of f e e d s . Nat . A c a d , of S c i . , Washington , D . C . , p p 252-281. T a r r , H . L . A . , B i e l y , J . and March , B . E . (1954) . The n u t r i t i v e va lue of h e r r i n g meals . 1. E f f e c t of h e a t . P o u l t r y S c i . 33, 242-250. T i t u s , H.W. (1927) . The n i t r o g e n metabol ism of s t e e r s , on r a t i o n s c o n t a i n i n g a l f a l f a as the so l e source of the n i t r o g e n . J . A g r . R e s e a r c h . 39, 49-58. Von der Decken, A . (1983) . E x p e r i m e n t a l s t u d i e s on the q u a l i t y of food p r o t e i n s . Comp. Biochem. P h y s i o l . 74B, 213-220. W a l t o n , M . J . and Cowey, C . B . (1982) . Aspects of i n t e r m e d i a r y metabol ism i n sa lmonid f i s h . Comp. Biochem. P h y s i o l . 73B, 59-79. Watanabe, T . , N a n r i , H . , Sa tok , S . , T a k e u c h i , M. and N o s e , T . (1983) . N u t r i t i o n a l e v a l u a t i o n of brown meals as a p r o t e i n source i n d i e t s for rainbow ' t r o u t . B u l l . J a p . Soc . S c i . F i s h 49, 1083-1087. Watt s , R . L . and Watt s , D . C . (1974) . N i t r o g e n metabol ism i n f i s h e s . Chem. Z o o l . 8, 369-446. Westgate , J . W . (1979) . Hatchery b i o l o g y . Columbia R i v e r F i s h e r y Development Program. Annual progres s r e p o r t . Oregon Dept . of F i s h and W i l d l i f e . 28 pp. W i l s o n , R . P . , R o b i n s o n , E . H . and Poe, W . E . (1981) . Apparent and t r u e a v a i l a b i l i t y of amino a c i d s from common feed i n g r e d i e n t s for channe l c a t f i s h . J . N u t r . I l l , 923-929 . W i n d e l l , J . T . , F o l t z , J . W . and Sarokon , J . A . (1978) . Methods of f e c a l c o l l e c t i o n and n u t r i e n t l e a c h i n g i n d i g e s t i b i l i t y s t u d i e s . P r o g . F i s h C u l t . 40, 51-55. W i n f r e e , R . A . and S t i c k n e y , R . R . (1981) . E f f e c t s of d i e t a r y p r o t e i n and energy on growth, feed c o n v e r s i o n e f f i c i e n c y and body c o m p o s i t i o n of T i l a p i a a u r e a . J . N u t r . I l l , 1001-1012. Young, V . R . and Scrimshaw, N . S . (1977) . Human p r o t e i n and amino a c i d metabol ism and requirements i n r e l a t i o n to p r o t e i n q u a l i t y . I n : E v a l u a t i o n of p r o t e i n s f o r humans. B o d w e l l , C . E . ( e d . ) . A v i P u b l i s h i n g Company, I n c . , W e s t p o r t , C t . pp 11-54. 187 Y u , T . C . and S i n n h u b e r , R . O . (1979) . E f f e c t of d i e t a r y W3 and W6 f a t t y a c i d s on growth and feed c o n v e r s i o n e f f i c i e n c y of coho salmon (Oncorhynchus k i s u t c h ) . A q u a c u l t u r e 16, 31-38. Z a r , J . H . (1974) . B i o s t a t i s t i c a l a n a l y s e s . P r e n t i c e - H a l l , New J e r s e y . Z e i t o u n , I . H . (1973) . The i n f l u e n c e of s a l i n i t y on p r o t e i n requ irements and u t i l i z a t i o n by rainbow t r o u t (Salmo g a i r d n e r i ) and coho salmon (Oncorhynchus k i s u t c h ) . P h . D . T h e s i s , M i c h i g a n S t a t e U n i v e r s i t y . Eas t L a n s i n g , M i . 90 pp . Z e i t o u n , I . H . , H a l v e r , J . E . , U l l r e y , D . E . and T a c k , P . I . (1973) . I n f l u e n c e of s a l i n i t y on p r o t e i n requ irements of rainbow t r o u t (Salmo g a i r d n e r i ) f i n g e r l i n g s . J . F i s h Res. Bd . Can. 30, 1867-1873. Z e i t o u n , I . H . , U l l r e y , D . E - . , H a l v e r , J . E . , T a c k , P . I . and Magee, W.T . (1974) . I n f l u e n c e of s a l i n i t y on p r o t e i n requirements of coho salmon (Oncorhynchus k i s u t c h ) s m o l t s . J . F i s h Res . B d . Can . 31, 1145-1148. Z e i t o u n , I . H . , U l l r e y D . E . , Magee, W . T . , G i l l , J . L . and Bergen , W.G. (1976) . Q u a n t i f y i n g n u t r i e n t requirements of f i s h . J . F i s h . Res . B d . Can . 33, 167-172. Zombade, S . S . , L o d k i , G . N . and I c h h p o n a n i , J . S . (1980) . 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 of p o u l t r y f e e d s t u f f s . 2. F i s h meal . J . A g r i . S c i . Camb. 94, 111-116. 188 Table 1. ANOVA of day 42 body weights i n Experiment 1. Source df Mean squares Prob.>F Tes t term P r o t e i n (PS) 4 106. .33 0. .0002 PSxRow L e v e l (PL) 2 301 , .82 0, .005 PLxRow PSxPL 8 9, .16 0, .006 PSxPLxRow Row 1 0. .0914 0. .62 R e s i d u a l PSxRow 4 0, .774 0. .078 R e s i d u a l PLxRow 2 1. .730 0. .009 R e s i d u a l PSxPLxRow 8 1, .309 0, .0004 R e s i d u a l R e s i d u a l 1770 0. .3688 T o t a l 1799 - 189 - Table 2. A n a l y s i s of v a r i a n c e and c o v a r i a n c e for s lope (GR%) t e s t of l og body weights i n Experiment 1. Source df Mean squares Prob.>F Tes t term P r o t e i n (PS) 4 12.5495 0.001 PSxRow L e v e l (PL) 2 29.0717 0.01 PLxRow PSxPL 8 0.4654 0.1 PSxPLxRow Row 1 0.02686 0.5 R e s i d u a l PSxRow 4 0.2592 0.0000 R e s i d u a l PLxRow 2 0.4192 0.0000 R e s i d u a l PSxPLxRow 8 0.2122 0.0000 R e s i d u a l R e s i d u a l 5369 0.00517 DayxPS 4 8.3211 0.05 DayxPSxPLxRow DayxPL 2 18.2168 0.01 DayxPSxPLxRow DayxPSxPL 14 5.1357 0.01 DayxPSxPLxRow DayxRow 1 0.00140 0.8 R e s i d u a l DayxPSxPLxRo w 29 2.5032 0.00.00 R e s i d u a l R e s i d u a l 5349 0.03840 T o t a l 5399 - 190 - T a b l e 3. A n a l y s i s of c o v a r i a n c e of body weight gain a g a i n s t p r o t e i n i n t a k e (Exper iment 1 ) . Source d_f Mean squares Prob . >F P r o t e i n (PS) 4 1.3426 0.0001 Row 1 0.0053 0.63 PSxRow 4 0.006 0.89 P r o t . i n t a k e (PI) 1 31 .5088 0.0001 PIxPS 4 0.2593 0.0001 PIxRow 1 0.0006 0.88 E r r o r 24 0.0219 T o t a l 39 Model 15 2.5297 0.0001 E r r o r 24 0.0219 T o t a l 39 Tab le 4. ANOVA of GFC and GEU of d i e t s fed i n Experiment 1. Parameter GFC GEU Source df Mean s quares Prob.>F Mean squares Prob .>F P r o t e i n (PS) 4 213. 62 0.0001 478.34 0.0016 L e v e l (PL) 2 579. 18 0.014 1120.04 0.015 PSxPL 8 9. 26 0.0002 17.70 0.0076 Row 1 0. 584 0.25 0.66 0.63 PSxRow 4 2. 26 0.017 6.90 0.11 PLxRow 2 2. 73 0.017 6.25 0.15 PSxPLxRow 8 0. 388 2 .63 T o t a l 29 - 191 - T a b l e 5. ANOVA of PER and NPR of d i e t s fed i n Experiment 1. Parameter PER NPR Source df Mean squares Prob.>F Mean squares Prob.>F P r o t e i n (PS) 4 0.3097 0.0013 0.2267 0.0019 L e v e l (PL) 2 0.0679 0.077 0.0144 0.064 PSxPL 8 0.0129 0.0004 0.0162 0.0001 Row 1 0.00348 0.057 0.00864 0.0049 PSxRow 4 0.00364 0.025 0.00373 0.0133 PLxRow 2 0.00508 0.017 0.000836 0.2921 PSxPLxRow 8 0.000715 0.000579 T o t a l 29 Tab le 6. ANOVA of PPV of d i e t s fed i n Experiment 1 • Parameter PPV Source df Mean squares P: rob.>F P r o t e i n (PS) 4 1391 , .96 0 .002 L e v e l (PL) 2 468 , .87 0 .097 PSxPL 8 49. .61 0 .04 Row 1 97. .64 0 .03 PSxRow 4 22 , .91 0 .24 PLxRow 2 46. .76 0 .08 PSxPLxRow 8 13, .45 T o t a l 29 192 T a b l e 7. ANOVA of NPU-1 and NPU-2 of d i e t s fed i n Experiment 1. Parameter NPU- 1 NPU- 2 Source df Mean squares Prob.>F Mean squares Prob .>F P r o t e i n (PS) 4 1011. .84 0. 003 1372 .41 0 .002 L e v e l (PL) 2 69. .70 0. 17 228 .83 0 .25 PSxPL 8 58, .52 0. 02 98 .00 0 .003 Row 1 1, .03 0. 77 164 .39 0 .004 PSxRow 4 24, .54 0. 20 24 .04 0 .15 PLxRow 2 13, .81 0. 39 77 .75 0 .016 PSxPLxRow 8 12 .95 10 .56 T o t a l 29 ' T a b l e 8. Summary of s t a t i s t i c a l a n a l y s i s for the s lopes of dry body weight and body p r o t e i n g a i n . S lopes were ana lyzed both i n c l u d i n g and e x c l u d i n g data f o r the p r o t e i n - f r e e d i e t (PF) (Exper iment 1 ) . Treatment df E r r o r df Treatment MS E r r o r MS Prob .>F Dry weight Rain Slope (exc . PF) Slope ( i n c . PF) P r o t e i n £ a i n Slope ( exc . PF) Slope ( i n c . PF) 4 4 15 25 15 25 0.00967 0.01901 0.00362 0.00786 0.00109 0.0007 0.00149 0.0000 0.00050 0.0019 0.00082 0.0000 193 T a b l e 9. ANOVA of day 42 body weights i n Experiment 2. Source df. Mean : squares Prob.>F Tes t term P r o t e i n (PL) 3 174 .32 0 .0002 PLxRow Energy (EL) 1 13 .52 0 .32 ELxRow PLxEL 3 1 . 142 0 .80 PLxELxRow Row 1 0 .527 0 .33 R e s i d u a l PLxRow 3 0 .334 0 .62 R e s i d u a l ELxRow 1 4 .021 0 .0076 R e s i d u a l PLxELxRow 3 3 .317 0 .0005 R e s i d u a l R e s i d u a l 944 0 .561 T o t a l 959 T a b l e 10. ANOVA of day 105 body weights i n Experiment 2. Source df Mean : squares P: rob.>F Tes t term P r o t e i n (PL) 3 3561 .3 0 .0004 PLxRow Energy (EL) 1 184 .35 0 .12 ELxRow PLxEL 3 21 .59 0 .35 PLxELxRow Row 1 58 .44 0 .003 R e s i d u a l PLxRow 3 13 .83 0 .09 R e s i d u a l ELxRow 1 6 .286 0 .32 R e s i d u a l PLxELxRow 3 13 .07 0 .11 R e s i d u a l R e s i d u a l 944 6 .46 T o t a l 959 - 194 - T a b l e 11. A n a l y s i s of c o v a r i a n c e for body weights i n Exp . 2. Source df Mean s quares Prob.>F Tes t term PL 3 3252. 7 0.0005 PLxRow EL 1 154. 17 0.14 ELxRow PLxEL 3 23. 74 0.37 PLxELxRow Row 1 57. 14 0.0000 R e s i d u a l PLxRow 3 8. 534 0.014 R e s i d u a l ELxRow 1 7. 466 0.079 R e s i d u a l PLxELxRow 3 15. 59 0.0002 R e s i d u a l Day 1 47510. 0.021 DayxRow DayxPL 3 2336. 1 0.0005 DayxPLxRow DayxEL 1 107 . 29 0.106 DayxELxRow DayxPLxEL 3 17 . 1 0.33 DayxPLxELxRow DayxRow 1 50. 49 0.0000 R e s i d u a l Da yxPLxRow 3 10. 40 0.0048 R e s i d u a l DayxELxRow 1 3. 002 0.26 R e s i d u a l DayxPLxELxRow 3 9. 976 0.006 R e s i d u a l R e s i d u a l 5727 2 . 412 T o t a l 5758 - 195 - T a b l e 12. S t a t i s t i c a l a n a l y s i s f or the second order p o l y n o m i a l model to es t imate p r o t e i n requirements i n Exp . 2. D i e t a r y ener gy 3150 k c a l / k g 3950 k c a l / k g Source df Mean square Prob.>F Mean square Prob.>F Model 2 0.22743 0.042 0.37356 0.093 E r r o r 1 0.00081 0.00658 T o t a l 3 L i n e a r term 1 0.36332 0.030 0.63271 0.065 Q u a d r a t i c term 1 0.09155 0.059 0.11441 0.150 E r r o r 1 0.00081 T o t a l 3 T a b l e 13. S t a t i s t i c a l a n a l y s i s f or the second order p o l y n o m i a l model to es t imate the P E : T E requirements i n Experiment 2. Source df Mean square Prob.>F Model 2 0.57562 0.0013 E r r o r 5 0.01715 T o t a l 7 L i n e a r term 1 0.90859 0.0008 Q u a d r a t i c term 1 0.24265 0.013 E r r o r 5 0.01715 T o t a l 7 196 T a b l e 1A . ANOVA of GFC and GEU i n Experiment 2. Parameter GFC GEU Source df Mean squares df Mean squares P r o t e i n (PL) 3 20A.23** 3 525 .22** Energy (EL) 1 32.29 1 83.25 PLxEL 3 6.003 3 0.A0 Row 1 0.360 1 1 .438 PLxRow 3 0.708 3 2.A51 ELxRow 1 1 .183 1 2.A95 PLxELxRow 3 1 .596 3 5.033 T o t a l 15 15 L e v e l of s i g n i f i c a n c e * * = 0 . 0 1 . T a b l e 15. ANOVA of PER and PPV i n Experiment 2. Parameter PER PPV Source df Mean square df Mean square P r o t e i n (PL) 3 0.017A* 3 36.136* Energy (EL) 1 0.0302 1 25.702 PLxEL 3 0.00202 3 3.223 Row 1 0.000A05 1 A.852 PLxRow 3 0.000769 3 1 .925 ELxRow 1 0.00132 1 7.930 PLxELxRow 3 0.00127 3 5. 738 T o t a l 15 15 L e v e l of s i g n i f i c a n c e * = 0 .05 . - 197 - T a b l e 16. ANOVA of NPU-2 i n Experiment 2. Parameter NPU-2 Source df Mean square P r o t e i n (PL) 3 103.36 * * Energy (EL) 1 100.11 PLxEL 3 2 .089 Row 1 10.652 PLxRow 3 3.269 ELxRow 1 10.115 PLxELxRow 3 9.656 T o t a l 15 L e v e l of s i g n i f i c a n c e * * = 0 . 0 1 . 198 Table 17. Tab le of mean squares f o r f i s h body m o i s t u r e , a s h , l i p i d and p r o t e i n at day 42 (Exper iment 2) • Source df Mean squares M o i s t u r e Ash L i p i d P r o t e i n P r o t e i n (PL) 3 1.2915** 0.4367 2.355 7 .368 Energy (EL) 1 3.8025** 1.6448 136.422** 59.714* PLxEL 3 0.2934 0.0403 3.713 9.661 Row 1 0.4356 0.1278 0.483 50.730 PLxELxRow . 7 0.1473 0.1325 2.527 10.331 T o t a l 15 L e v e l of s i g n i f i c a n c e * = 0 .05 , * * = 0 . 0 1 . Tab le 18. T a b l e of mean squares for f i s h body m o i s t u r e , a s h , l i p i d and p r o t e i n at day 105 (Exper iment 2 ) . Source df Mean squares M o i s t u r e Ash L i p i d P r o t e i n P r o t e i n (PL) "3 4.0058** 2.1109 17 .8373** 58.782** Energy (EL) 1 7 .6591** 2.9241 102 .8703** 238 .780** PLxEL 3 0.3493 0.9528 3.7150 9.959 Row 1 0.6931 0.3080 0.1871 7 .798 PLxELxRow 7 0.1436 1.0499 1.4302 3 .868 T o t a l 15 L e v e l of s i g n i f i c a n c e * * = 0 . 0 1 . - 199 -

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