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Evaluation of feedstuff digestibility in post-juvenile chinook salmon (Oncorhynchus tshawytscha) in seawater Hajen, Walter Ernesto 1990

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EVALUATION OF FEEDSTUFF DIGESTIBILITY IN POST-JUVENILE CHINOOK SALMON (Oncorhynchus tshawytscha) IN SEAWATER by Walter E. Hajen B.Sc. (Agr.), The U n i v e r s i t y of B r i t i s h Columbia, 198! A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES Department of Animal Science We accept t h i s t h e s i s as conforming t o the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA November 1990 © Walter E. Hajen, 1990 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of ANIMAL SCIENCE The University of British Columbia Vancouver, Canada Date December 26, 1990 DE-6 (2/88) i i ABSTRACT Feed accounts f o r 40% to 60% of the o p e r a t i n g c o s t s of P a c i f i c salmon farms. P r e s e n t l y , commercial d i e t s f o r P a c i f i c salmon i n seawater are formulated a c c o r d i n g t o the n u t r i e n t requirements of j u v e n i l e chinook salmon and d i g e s t i b i l i t y i n f o r m a t i o n d e r i v e d from s t u d i e s on rainbow t r o u t i n f r e s h water. Information on f e e d s t u f f d i g e s t i b i l i t y by P a c i f i c salmon i s completely l a c k i n g , r e g a r d l e s s of l i f e h i s t o r y stage. Hence, t h i s t h e s i s was undertaken t o determine the apparent d i g e s t i b i l i t y o f o r g a n i c matter, crude p r o t e i n and energy i n c o n v e n t i o n a l and novel f e e d s t u f f s u s i n g p o s t - j u v e n i l e chinook salmon (Oncorhynchus tshawytscha) i n seawater. The v a l i d i t y of determining apparent n u t r i e n t d i g e s t i b i l i t y u s i n g the "Guelph system" of f e c a l c o l l e c t i o n and chromic oxide (C^C^) as the i n d i g e s t i b l e i n d i c a t o r i n the d i e t was assessed i n experiment I. In t h i s regard, f e c a l samples were c o l l e c t e d e i t h e r from a s e t t l i n g column a f f i x e d t o each n o v e l l y designed d i g e s t i b i l i t y tank at 6 and 18 hour i n t e r v a l s or d i r e c t l y from the t e r m i n a l s e c t i o n o f the i n t e s t i n a l t r a c t by s t r i p p i n g or i n t e s t i n a l d i s s e c t i o n . D i g e s t i b i l i t y c o e f f i c i e n t s were noted t o be s i g n i f i c a n t l y i n c r e a s e d (P<0.001) when the feces remained i n the water f o r 18 hours i n s t e a d o f 6 hours, owing t o n u t r i e n t l e a c h i n g . The c o l l e c t i o n o f feces d i r e c t l y from the f i s h r e s u l t e d i n lower d i g e s t i b i l i t y c o e f f i c i e n t s than those found when feces were obtained from the "Guelph system". T h i s was a t t r i b u t e d t o a flaw i n the design of the d i g e s t i b i l i t y tank d r a i n system, whereby the feces d i d not s e t t l e q u i c k l y i n t o the i i i c o l l e c t i o n column. The problem was r e c t i f i e d b e f o r e conducting the subsequent experiments d e s c r i b e d below. In t h r e e a d d i t i o n a l experiments on chinook salmon i n seawater, the o v e r a l l goal was t o a s c e r t a i n the o r g a n i c matter, crude p r o t e i n and energy d i g e s t i b i l i t y c o e f f i c i e n t s and the d i g e s t i b l e energy v a l u e s f o r commercial sources of f i s h meal ( h e r r i n g meal, anchovy meal, menhaden meal, Norwegian low temperature f i s h meal), p o u l t r y by-product meal (two s u p p l i e r s ) , f e a t h e r meal, b l o o d meal, d r i e d whey, canol a meal, soybean meal, soybean p r o t e i n i s o l a t e , extruded wheat and wheat m i d d l i n g s . A l s o , t h r e e n o vel sources of rapeseed p r o t e i n products (two types of g l u c o s i n o l a t e - f r e e c a n o l a meal and rapeseed p r o t e i n concentrate) were e v a l u a t e d i n t h i s regard. The i n i t i a l minimum s i z e of the chinook i n these s t u d i e s v a r i e d between 10.3 g and 40.5 g. The d i g e s t i b i l i t y tank complex c o n s i s t e d of 27 open-c i r c u i t 150 L f i b e r g l a s s d i g e s t i b i l i t y tanks, each s u p p l i e d with aerated, 8.0^C t o 12.5^C f i l t e r e d seawater at a r a t e of 6 L/min. The f i s h were f e d by hand twice d a i l y t o s a t i a t i o n e i t h e r a r e f e r e n c e d i e t or a t e s t d i e t (70% r e f e r e n c e : 30% t e s t i n g r e d i e n t ) . In some i n s t a n c e s i n v o l v i n g p l a n t p r o t e i n products, the t e s t i n g r e d i e n t was i n c l u d e d at two d i e t a r y l e v e l s (15% and 30%). W i t h i n each t e s t , each d i e t treatment was a s s i g n e d t o t h r e e groups of f i s h u s i n g a completely random or randomized complete b l o c k d e s i g n . Chromic oxide (0.5%) was i n c l u d e d i n a l l d i e t s as the i n d i g e s t i b l e marker. At the end of each experiment, f i s h were s a c r i f i c e d and t h e i r feces were removed e i t h e r by s t r i p p i n g or i n t e s t i n a l d i s s e c t i o n f o r comparisons of methodology. i v In general, the f i s h meals had higher a v a i l a b l e energy content f o r chinook salmon than the other animal and p l a n t p r o t e i n sources assessed. The importance of screening f e e d s t u f f s f o r p o t e n t i a l n u t r i t i v e value by d i g e s t i b i l i t y measurements was p a r t i c u l a r l y evident from an examination of the d i g e s t i b i l i t y c o e f f i c i e n t s obtained f o r menhaden meal versus the other f i s h meal sources, the two sources of p o u l t r y by-product meal, wheat products, canola products and blood meal i n t h i s study. The assessment of soybean products, regardless of d i e t a r y i n c l u s i o n l e v e l (15% or 30%), could not be a s c e r t a i n e d i n chinook salmon because of poor d i e t acceptance. Canola p r o t e i n sources appear h i g h l y promising as p a r t i a l or complete ( i n the case of rapeseed p r o t e i n concentrate) replacements of f i s h meal, based on d i g e s t i b i l i t y assessment. The use of the "Guelph system" f o r f e c a l c o l l e c t i o n r e s u l t e d i n organic matter d i g e s t i b i l i t y c o e f f i c i e n t s s i m i l a r t o those obtained by i n t e s t i n a l d i s s e c t i o n . Thus, n u t r i e n t l e a c h i n g must have been minimal w i t h the "Guelph system" and i t i s concluded t h a t t h i s i s a s a t i s f a c t o r y procedure f o r d i g e s t i b i l i t y assessment using chinook salmon i n seawater. TABLE OF CONTENTS S e c t i o n Page ABSTRACT i i LIST OF TABLES v i i i LIST OF FIGURES , x i ACKNOWLEDGEMENTS x i i CHAPTER 1 1.0 INTRODUCTION 1 CHAPTER 2 2.0 LITERATURE REVIEW 5 2.1 P r o t e i n r e q u i r e m e n t s o f f i s h 5 2.2 Amino a c i d r e q u i r e m e n t s o f f i s h 6 2.3 Energy b a l a n c e i n . f i s h 7 2.4 Sources o f energy f o r f i s h 9 2.4.1 P r o t e i n as a d i e t a r y energy s o u r c e 9 2.4.2 C a r b o h y d r a t e s as d i e t a r y energy s o u r c e s 9 2.4.3 L i p i d s as d i e t a r y energy s o u r c e s 12 2.5 S e l e c t i o n o f d i e t a r y i n g r e d i e n t s f o r s a l m o n i d d i e t s . . 14 2.5.1 F i s h e r y p r o d u c t s 14 2.5.2 A n i m a l b y - p r o d u c t s 15 2.5.3 P l a n t b y - p r o d u c t s . . . 16 2.6 E v a l u a t i n g t h e n u t r i e n t and energy v a l u e o f f e e d i n g r e d i e n t s 19 2.7 D i g e s t i o n and a b s o r p t i o n 22 2.8 G e n e r a l a l i m e n t a r y t r a c t anatomy and i t s a s s o c i a t e d enzymes 23 2.8.1 The midgut 23 2.8.2 The h i n d g u t 25 2.9 P r o t e i n d i g e s t i o n and a b s o r p t i o n 26 2.10 L i p i d d i g e s t i o n and a b s o r p t i o n 27 2.11 C a r b o h y d r a t e d i g e s t i o n and a b s o r p t i o n 28 2.12 Apparent and t r u e d i g e s t i b i l i t y 28 2.13 D i g e s t i b i l i t y measurements i n f i s h 29 2.14 E v a l u a t i o n o f f e e d s t u f f d i g e s t i b i l i t y 35 CHAPTER 3 3.0 EXPERIMENT I - D i g e s t i b i l i t y measurements i n j u v e n i l e c h i n o o k salmon (Oncorhynchus tshawytscha) i n s eawater: Comparison o f t h e d i r e c t ( t o t a l ) and i n d i r e c t (Cr 203) methods o f d e t e r m i n i n g d i g e s t i b i l i t y and r e l i a b i l i t y o f d i g e s t i o n c o e f f i c i e n t s i n r e l a t i o n t o t e c h n i q u e s used i n f e c a l c o l l e c t i o n 36 3.1 I n t r o d u c t i o n 36 3.2 M a t e r i a l s and methods 39 3.2.1 E x p e r i m e n t a l d e s i g n 39 3.2.2 D i g e s t i b i l i t y t a n k s 40 3.2.3 Aquarium f a c i l i t y 42 3.2.4 Water f i l t e r i n g system 42 3.2.5 D i e t p r e p a r a t i o n and c o m p o s i t i o n 43 3.2.6 E x p e r i m e n t a l p r o c e d u r e s and s a m p l i n g 46 v i TABLE OF CONTENTS c o n t . . . . S e c t i o n Page 3.2.6.1 F i s h h i s t o r y and d i s t r i b u t i o n 46 3.2.6.2 E x p e r i m e n t a l p r o t o c o l 47 3.2.6.3 F e c a l c o l l e c t i o n t e c h n i q u e s 48 3.2.6.3.1 "Guelph system" 4 8 3.2.6.3.2 S t r i p p i n g t e c h n i q u e 4 9 3.2.6.3.3 I n t e s t i n a l d i s s e c t i o n t e c h n i q u e 4 9 3.2.7 Che m i c a l a n a l y s e s o f f e e d and f e c e s 50 3.2.7.1 P r o x i m a t e c o m p o s i t i o n and g r o s s energy d e t e r m i n a t i o n 50 3.2.7.2 Chromic o x i d e d e t e r m i n a t i o n s 50 3.2.8 C a l c u l a t i o n s o f apparent d i g e s t i b i l i t y and f i s h p erformance 51 3.2.9 S t a t i s t i c a l a n a l y s i s 53 3.3 R e s u l t s 55 3.3.1 D i e t s 55 3.3.2 Chromic o x i d e a n a l y s i s 55 3.3.3 Comparison o f t h e d i r e c t and i n d i r e c t methods o f measuring d i g e s t i b i l i t y 58 3.3.4 I n g e s t e d chromic o x i d e r e c o v e r y 60 3.3.5 E f f e c t o f t i m e between f e c a l c o l l e c t i o n s on f e c a l n u t r i e n t l o s s due t o l e a c h i n g 63 3.3.6 E f f e c t o f f i s h management on apparent n u t r i e n t d i g e s t i b i l i t y c o e f f i c i e n t s / f e e d i n t a k e , growth r a t e and f i s h m o r t a l i t y 66 3.4 D i s c u s s i o n 69 3.4.1 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 s o f f i s h r a i s e d i n seawater 69 3.4.2 Chromic o x i d e a n a l y s i s 71 3.4.3 Comparison o f t h e d i r e c t and i n d i r e c t methods o f measuring d i g e s t i b i l i t y i n s a l t water f i s h 72 3.4.4 N u t r i e n t l e a c h i n g l o s s e s i n f e c a l samples c o l l e c t e d w i t h t h e "Guelph system" 75 3.4.5 E f f e c t o f f i s h management on apparent n u t r i e n t d i g e s t i b i l i t y , f e e d i n t a k e , growth r a t e and f i s h m o r t a l i t y 79 CHAPTER 4 4.0 EXPERIMENTS I I , I I I AND IV. - Apparent d i g e s t i b l e o r g a n i c m a t t e r , crude p r o t e i n and energy c o e f f i c i e n t s o f common and n o v e l f e e d i n g r e d i e n t s i n p o s t - j u v e n i l e c h i n o o k salmon (Oncorhynchus tshawytscha) i n seawater 82 4.1 I n t r o d u c t i o n 82 4.2 M a t e r i a l s and methods 84 4.2.1 E x p e r i m e n t a l d e s i g n 84 4.2.2 H i s t o r y o f e x p e r i m e n t a l f i s h 84 4.2.3 Aquarium f a c i l i t y 85 4.2.4 Water f i l t e r i n g system 87 4.2.5 D i e t s 87 v i i TABLE OF CONTENTS cont.... S e c t i o n Page 4.2.6 D i e t p r e p a r a t i o n 88 4.2.7 Experimental procedures and f i s h h a n d l i n g 95 4.2.7.1 D i e t a l l o c a t i o n 95 4.2.7.2 P r o t o c o l 95 4.2.8 F e c a l c o l l e c t i o n procedure 97 4.2.9 Chemical analyses of t e s t i n g r e d i e n t s , d i e t s and f e c a l samples 99 4.2.10 D i g e s t i b i l i t y and f i s h performance c a l c u l a t i o n s . . 100 4.2.11 S t a t i s t i c a l a n a l y s i s ' 102 4.3 R e s u l t s 104 4.3.1 Chemical composition of t e s t i n g r e d i e n t s 104 4.3.2 Proximate composition and m i n e r a l content of r e f e r e n c e and t e s t d i e t s 109 4.3.3 Apparent n u t r i e n t d i g e s t i b i l i t y c o e f f i c i e n t s of t e s t i n g r e d i e n t s 115 4.3.4 E f f e c t o f f e c a l c o l l e c t i o n technique on apparent o r g a n i c matter d i g e s t i b i l i t y 119 4.3.5 I n f l u e n c e of D i e t on f i s h performance 122 4.3.6 P r e d i c t i o n o f o r g a n i c matter, crude p r o t e i n and energy d i g e s t i b i l i t y of the r e f e r e n c e d i e t s used i n experiments I I , I I I and IV based on the apparent n u t r i e n t d i g e s t i b i l i t y v a l u e s of i n d i v i d u a l d i e t a r y i n g r e d i e n t s 128 4.4 D i s c u s s i o n 132 4.4.1 F e e d s t u f f e v a l u a t i o n a c c o r d i n g t o proximat composition and n u t r i e n t d i g e s t i b i l i t y 132 4.4.1.1 F i s h meals 132 4.4.1.2 Animal by-product meals 137 4.4.1.3 P l a n t by-product meals 142 4.4.1.3.1 G r a i n products 147 4.4.2 E f f e c t o f f e c a l c o l l e c t i o n technique on apparent o r g a n i c matter d i g e s t i b i l i t y . . . . . 150 4.4.3 I n f l u e n c e of d i e t on f i s h performance 152 4.4.4 A d d i t i v i t y of i n d i v i d u a l i n g r e d i e n t n u t r i e n t d i g e s t i b i l i t y c o e f f i c i e n t s f o r the p r e d i c t i o n of the n u t r i e n t d i g e s t i o n o f a complete d i e t 153 CHAPTER 5 5.0 CONCLUSIONS 154 BIBLIOGRAPHY 158 APPENDICES 177 v i i i LIST OF TABLES Table Page 1. Composition of c o n t r o l and p r o t e i n - f r e e d i e t s (g/kg dry matter) 45 2. Proximate composition, energy content and m i n e r a l content of the c o n t r o l and p r o t e i n - f r e e d i e t s of Experiment 1 56 3. Recovery of chromic oxide from a feed mash mixture u s i n g the wet a s h i n g c o l o r i m e t r i c method of Stevenson and De Langen (1960) 57 4. Comparison of the t h r e e week mean apparent d i g e s t i b i l i t y c o e f f i c i e n t v a l u e s determined by d i r e c t and i n d i r e c t measurement u s i n g the "Guelph system" of f e c a l c o l l e c t i o n , as w e l l as v a l u e s o b t a i n e d f o r feces c o l l e c t e d by i n t e s t i n a l d i s s e c t i o n and s t r i p p i n g techniques 61 5. Recovery r a t e of chromic oxide (0^03) from i n g e s t a and e x c r e t a a f t e r a three-week ( r e p l i c a t e s 1,2,3,4,7 and 8) and a two-week ( r e p l i c a t e s 5,6 and 9) c o l l e c t i o n p e r i o d 62 6. Three-week mean apparent d i g e s t i b i l i t y c o e f f i c i e n t s f o r o r g a n i c matter, crude p r o t e i n and gross energy by j u v e n i l e chinook salmon i n seawater f e d the c o n t r o l d i e t ( d i e t 1), i n r e l a t i o n t o f e c a l c o l l e c t i o n method and time between c o l l e c t i o n s 65 7. E f f e c t of f i s h management (feeding regime and f i s h handling) on mean apparent o r g a n i c matter, crude p r o t e i n and gross energy d i g e s t i b i l i t y c o e f f i c i e n t s c a l c u l a t e d u s i n g d i g e s t i b i l i t y data from f e c a l samples c o l l e c t e d i n the morning and i n the a f t e r n o o n 67 8. E f f e c t of f i s h management on feed i n t a k e , s p e c i f i c growth r a t e and m o r t a l i t y of j u v e n i l e chinook salmon r e a r e d i n s a l t water and f e d the c o n t r o l d i e t 68 9. Reference and t e s t d i e t p r e p a r a t i o n p r o t o c o l employed i n experiments II and I I I 91 10. Reference and t e s t d i e t p r e p a r a t i o n p r o t o c o l employed i n experiment IV 92 11. Formulation of b a s a l mix 1 used i n the p r e p a r a t i o n 1 of the r e f e r e n c e and t e s t d i e t s employed i n experiments II and I I I 93 i x LIST OF TABLES cont Table Page 12. Formulation of b a s a l mix 2 used i n the p r e p a r a t i o n of the r e f e r e n c e and t e s t d i e t s employed i n experiment IV 94 13. Proximate composition and gross energy content o f t e s t i n g r e d i e n t s used i n experiment II (values i n parentheses show percentages f o r each proximate c o n s t i t u e n t on a dry matter b a s i s ) 106 14. Proximate composition and gross energy content of t e s t i n g r e d i e n t s used i n experiment I I I (values i n parentheses show percentages f o r each proximate c o n s t i t u e n t on a dry matter b a s i s ) 107 15. Proximate composition and gross energy content of t e s t i n g r e d i e n t s used i n experiment IV (values i n parentheses show percentages f o r each proximate c o n s t i t u e n t on a dry matter b a s i s ) 109 16. Proximate composition and m i n e r a l content of the r e f e r e n c e and f i v e t e s t d i e t s f e d to j u v e n i l e chinook salmon i n experiment I I . (Refer to Table 13 f o r a d d i t i o n a l i n f o r m a t i o n . REF-a r e f e r s t o r e f e r e n c e d i e t a) 110 17. Proximate composition and m i n e r a l content of the r e f e r e n c e and e i g h t t e s t d i e t s f e d to j u v e n i l e chinook salmon i n experiment I I I . (Refer t o Table 14 f o r a d d i t i o n a l i n f o r m a t i o n . REF-a r e f e r s t o r e f e r e n c e d i e t a) I l l 18 Proximate composition and m i n e r a l content of the r e f e r e n c e and seven t e s t d i e t s f e d to j u v e n i l e chinook salmon i n experiment IV. (Refer t o Table 15 f o r a d d i t i o n a l i n f o r m a t i o n . REF-b r e f e r s t o r e f e r e n c e d i e t b) 113 19 Mean percent (± SEM) apparent o r g a n i c matter, crude p r o t e i n and gross energy d i g e s t i b i l i t y c o e f f i c i e n t s and d i g e s t i b l e energy v a l u e s of i n g r e d i e n t s measured w i t h p o s t - j u v e n i l e chinook salmon i n seawater 117 20 E f f e c t of f e c a l c o l l e c t i o n technique on the apparent o r g a n i c matter d i g e s t i b i l i t y c o e f f i c i e n t s of the r e f e r e n c e d i e t s and t e s t i n g r e d i e n t s by j u v e n i l e chinook salmon 120 X LIST OF TABLES cont Table Page 21 Weight gai n , s p e c i f i c growth r a t e (SGR), d a i l y grams of dry feed i n t a k e per f i s h (DFI), d a i l y dry feed i n t a k e as a percent of wet body weight (FI ) , feed e f f i c i e n c y (FE), p r o t e i n e f f i c i e n c y r a t i o (PER), d a i l y grams of o r g a n i c matter d i g e s t e d , d a i l y grams of crude p r o t e i n , d a i l y KJ of energy d i g e s t e d and m o r t a l i t y data o f f i s h f e d r e f e r e n c e and t e s t d i e t s i n experiment I I . (Refer t o Table 16 f o r a d d i t i o n a l i n f o r m a t i o n on d i e t s and to m a t e r i a l s and methods f o r measurement of each parameter) 125 22 Weight gain, s p e c i f i c growth r a t e (SGR) , d a i l y grams of dry feed i n t a k e per f i s h (DFI), d a i l y dry feed i n t a k e as a percent o f wet body weight ( F I ) , feed e f f i c i e n c y (FE), p r o t e i n e f f i c i e n c y r a t i o (PER), d a i l y grams of o r g a n i c matter d i g e s t e d , d a i l y grams of crude p r o t e i n , d a i l y KJ of energy d i g e s t e d and m o r t a l i t y data of f i s h f e d r e f e r e n c e and t e s t d i e t s i n experiment I I I . (Refer t o Table 17 f o r a d d i t i o n a l i n f o r m a t i o n on d i e t s and t o m a t e r i a l s and methods f o r measurement of each parameter) 12 6 23 Weight gai n , s p e c i f i c growth r a t e (SGR), d a i l y grams of dry feed i n t a k e per f i s h (DFI), d a i l y dry feed i n t a k e as a percent of body weight (FI) and m o r t a l i t y data of f i s h f e d r e f e r e n c e and t e s t d i e t s i n experiment IV. (Refer t o Table 18 f o r a d d i t i o n a l i n f o r m a t i o n on d i e t ) 127 24 Determined and c a l c u l a t e d apparent d i g e s t i b i l i t y c o e f f i c i e n t s f o r r e f e r e n c e d i e t - a (REF-a) which was f e d t o chinook salmon i n experiment II 129 25 Determined and c a l c u l a t e d apparent d i g e s t i b i l i t y c o e f f i c i e n t s f o r r e f e r e n c e d i e t - a (REF-a) which was f e d to chinook salmon i n experiment I I I 130 2 6 Determined and c a l c u l a t e d apparent d i g e s t i b i l i t y c o e f f i c i e n t s f o r r e f e r e n c e d i e t - b (REF-b) which was f e d t o chinook salmon i n experiment IV 131 x i LIST OF FIGURES Figure Page 1. Diagram showing the "Guelph system" of f e c a l c o l l e c t i o n used i n experiments I, I I , I I I and IV 41 x i i ACKNOWLEDGEMENT S I would l i k e t o express my si n c e r e g r a t i t u d e to a l l those who helped me during the course of my stu d i e s at the U n i v e r s i t y of B r i t i s h Columbia. In p a r t i c u l a r my research s u p e r v i s o r , Dr. Richard M. Beames, deserves s p e c i a l thanks, not only f o r h i s knowledge, encouragement and dedicated guidance, but al s o f o r h i s s i n c e r i t y , good humor and f r i e n d s h i p . I would a l s o l i k e t o e s p e c i a l l y thank Dr. David Higgs of F i s h e r i e s and Oceans, whose unceasing enthusiasm and w i l l i n g n e s s to share h i s e x p e r t i s e i n f i s h n u t r i t i o n was e s s e n t i a l t o the s u c c e s s f u l completion of my st u d i e s . Dr. B e r y l E. March and Dr. A. Kozak are g r a t e f u l l y acknowledged f o r t h e i r v a l u a b l e help w i t h s t a t i s t i c a l analyses. S p e c i a l thanks t o Dr. B e r y l E. March f o r s t i m u l a t i n g my i n t e r e s t i n n u t r i t i o n and f o r her continuous support and motivation throughout my years at UBC. My a p p r e c i a t i o n a l s o goes to the members of my supervisory committee f o r t h e i r c o n t r i b u t i o n to t h i s t h e s i s . Among those who provided t e c h n i c a l a s s i s t a n c e , Maureen Evans deserves s p e c i a l mention f o r her d e d i c a t i o n and d i l i g e n c e , as w e l l as Bakhshish Dosanjh and Jim E l l i o t t f o r t h e i r help at the West Vancouver Laboratory of F i s h e r i e s and Oceans. I a l s o want t o express my thanks t o my long time f r i e n d A l b e r t Nussbaum, f o r h i s help i n u n r a v e l l i n g the mysteries of the d i f f e r e n t computer programs. I used throughout my academic career, as w e l l as f o r the use of h i s computer f o r the pr e p a r a t i o n of t h i s t h e s i s . My parents have, more than I can say, supported me both emotionally and s p i r i t u a l l y throughout my s c h o l a s t i c endeavors and t o them I extend my wholehearted thankfulness. I dedicate t h i s t h e s i s to my l o v i n g wife E l i z a b e t h , who has helped me immeasurably w i t h her understanding, support and encouragement, and who p a t i e n t l y endured many bo r i n g weekends while I was consumed i n my s t u d i e s . For her I have the greatest admiration, and t o her I owe my greatest a p p r e c i a t i o n . F i n a l l y , I would l i k e t o acknowledge the N a t i o n a l Sciences and Engineering Research C o u n c i l of Canada, the Science Council of B r i t i s h Columbia and the Department of F i s h e r i e s and Oceans f o r f i n a n c i a l support and Dr. C.Y.Cho f o r p r o v i d i n g the prototype f e c a l c o l l e c t i o n column. 1 CHAPTER 1 1.0 INTRODUCTION Aquaculture i n B r i t i s h Columbia has become a dynamic and important developing i n d u s t r y and the amount of investment i n aquaculture i s r i s i n g d a i l y . Production of farmed salmon has d r a m a t i c a l l y increased from 120 tonnes marketed i n 1985, t o more than 12,300 tonnes i n 1989 w i t h a wholesale value of $85 m i l l i o n (Egan and Kenney, 1990). By the end of 1990, the commercially reared P a c i f i c salmon harvests are expected to reach 15,000 tonnes according to the B r i t i s h Columbia Salmon Farmers A s s o c i a t i o n (BCSFA) (Archibald, 1990; Egan and Kenney, 1990) . This p r o j e c t i o n i s 25% below the p r e v i o u s l y f o r e c a s t e d 20,000 tonnes by the BCSFA and was p r i m a r i l y due to a d e c l i n e i n the world market p r i c e of salmon causing v a r i o u s companies w i t h massive debt loads t o go i n t o r e c e i v e r s h i p . The general t r e n d i n the past year has t h e r e f o r e been a decrease i n the number of companies, although the more s u c c e s s f u l and w e l l e s t a b l i s h e d ones continue to expand t h e i r operations while at the same time they look, f o r ways to t r i m t h e i r production c o s t s . According to a study conducted i n 1988 by the B r i t i s h Columbia M i n i s t r y of A g r i c u l t u r e and F i s h e r i e s (BCMAF), food costs alone account f o r over 41% of the t o t a l operating expense of a r e l a t i v e l y e f f i c i e n t P a c i f i c salmon grow-out operation w i t h an annual production of 240 tonnes of dressed salmon. In small s c a l e farm operations, the feed costs account f o r an even l a r g e r p r o p o r t i o n of the t o t a l o perating c o s t s , w i t h estimates being as l a r g e as 55% t o 60% (Higgs, 1986) . I t i s t h e r e f o r e of c r i t i c a l importance t h a t f i s h feed costs be reduced so t h a t salmon farmers can r e a l i z e the most 2 p r o f i t f o r t h e i r l a b o r and investment. Current commercial d i e t s f o r P a c i f i c salmon i n seawater are formulated on the b a s i s of knowledge of t h e i r n u t r i e n t requirements d u r i n g the f r e s h water stages of t h e i r l i f e c y c l e , coupled with n u t r i t i o n a l i n f o r m a t i o n o b t a i n e d with A t l a n t i c salmon (Salmo salar) as w e l l as with f e e d s t u f f d i g e s t i b i l i t y i n f o r m a t i o n d e r i v e d from s t u d i e s on rainbow t r o u t (Oncorhynchus mykiss) i n f r e s h water. The work of numerous authors has i n d i c a t e d however, t h a t f e e d s t u f f d i g e s t i b i l i t y v a r i e s among s p e c i e s of f i s h ( B i r k e t t , 1969; J o b l i n g , 1983) as w e l l as w i t h water s a l i n i t y (Zeitoun et al., 1974; MacLeod, 1977; Pandey and Singh, 1980; L a l l et al., 1984; F e r r a r i s et al., 1986; Usher et al., 1990). As a r e s u l t , one can suspect t h a t the present d i e t s f o r s a l t - w a t e r P a c i f i c salmon are not of optimum q u a l i t y with r e s p e c t to support of f i s h growth and cost e f f e c t i v e n e s s (Higgs, 1986). Moreover, commercial d i e t s c o n t a i n 25% t o 65% f i s h meal (Tacon and Jackson, 1985). The v a r i a b l e supply and q u a l i t y of f i s h meal, and perhaps more im p o r t a n t l y the h i g h cost a s s o c i a t e d with i t , have been i d e n t i f i e d as major drawbacks i n the u t i l i z a t i o n of t h i s product i n f i s h feeds ( S p i n e l l i et al., 1979; P f e f f e r , 1982; McCallum and Higgs, 1989). Savings i n the c o s t of f i s h feed can be e f f e c t e d by r e p l a c i n g as much as p o s s i b l e of the f i s h meal with l e s s expensive f e e d s t u f f s . The f i r s t t ask i n seeking a more cost e f f i c i e n t d i e t f o r f i s h c u l t u r e i s to a s c e r t a i n the d i g e s t i b i l i t y of the major d i e t a r y components, p r i n c i p a l l y p r o t e i n and energy i n l o c a l l y a v a i l a b l e f e e d s t u f f s ( H i l t o n and S l i n g e r , 1981; Cho et al., 1982). To be of any value, these measurements should be conducted using the t a r g e t species i n t h e i r n a t u r a l environment ( H i l t o n and S l i n g e r , 1981) which i n B r i t i s h Columbia, would be P a c i f i c salmon i n seawater. In B r i t i s h Columbia, chinook salmon (Oncorhynchus tshawytscha) has, over the years been the dominant species farmed, p r e s e n t l y comprising an estimated 77% of the t o t a l production (Egan and Kenney, 1990) . Due t o poor growth and e a r l y maturation, coho salmon (Oncorhynchus kisutch) have decreased i n p o p u l a r i t y , w i t h production d e c l i n i n g s t e a d i l y from 76% i n 1986, to an expected 7% i n 1990 (Egan and Kenney, 1990). Therefore, an improvement i n the economics of chinook salmon production would have the greatest impact on the l o c a l aquaculture i n d u s t r y . This t h e s i s was designed to e s t a b l i s h d i g e s t i b i l i t y i n f o r m a t i o n f o r an array of conventional and hovel f e e d s t u f f s which could p o t e n t i a l l y p a r t i a l l y or wholly replace f i s h meal i n d i e t s f o r chinook salmon h e l d i n seawater. In a d d i t i o n , the d i g e s t i b i l i t y of v a r i o u s f i s h meals was determined t o f a c i l i t a t e the s u b s t i t u t i o n of one meal f o r another, depending on market trends and a v a i l a b i l i t y . This t h e s i s a l s o documents an i n v e s t i g a t i o n of the v a l i d i t y of determining n u t r i e n t d i g e s t i b i l i t y i n f i s h by the i n d i r e c t method (chromic oxide i n d i c a t o r ) . In a d d i t i o n , the r o u t i n e methodology of c o l l e c t i n g feces from the water, developed f o r rainbow t r o u t by Cho and S l i n g e r (1979), was compared wi t h the i n t e s t i n a l d i s s e c t i o n and the s t r i p p i n g techniques, which r e l y on c o l l e c t i n g the feces d i r e c t l y from the f i s h . 4 The f o l l o w i n g l i t e r a t u r e review i s designed t o provide background knowledge on the general n u t r i t i o n a l requirements of salmonids, the p h y s i o l o g i c a l processes i n v o l v e d i n the d i g e s t i o n and absorption of n u t r i e n t s and the fundamental problems a s s o c i a t e d w i t h the v a r i o u s systems commonly used i n the determination of d i g e s t i b i l i t y c o e f f i c i e n t s i n f i s h . 5 CHAPTER 2 2.0 LITERATURE REVIEW 2.1 P r o t e i n requirements of f i s h F i s h , as a group r e q u i r e a high percentage of d i e t a r y p r o t e i n compared to other - animals (Cowey and Sargent, 1979; Tacon and Cowey, 1985). A l s o , i t has been suggested t h a t f i s h are l e s s e f f i c i e n t than other animals i n p r o t e i n u t i l i z a t i o n (Rumsey, 1981). More r e c e n t l y , however, Bowen (1987) compared the p r o t e i n requirements of various f i s h e s w i t h the requirements of other v e r t e b r a t e s and found t h a t , although the two animal groups undoubtedly d i f f e r i n the concentration of p r o t e i n i n the d i e t , they are remarkably s i m i l a r i n the use of p r o t e i n f o r growth. This r e f l e c t s the homeotherms' great requirement f o r energy to maintain homeostasis. P r o t e i n requirements of f i s h are l a r g e l y a f f e c t e d by environmental c o n d i t i o n s . DeLong et al. (1958) obtained maximum gains i n 1.5 to 5.6 gram chinook salmon reared i n 6-8®C f r e s h water when the d i e t a r y p r o t e i n l e v e l was 39-40%. However, at a water temperature of 14.5°C, maximum gains were observed at 55-60% d i e t a r y p r o t e i n . S i m i l a r observations on the e f f e c t of water temperature on p r o t e i n requirements f o r maximum growth were noted by Hastings (1973) working w i t h channel c a t f i s h (Ictalurus punctatus). Zeitoun et al. (1973) on the other hand, found that water s a l i n i t y a l s o a f f e c t s the p r o t e i n requirements of young rainbow t r o u t . At a s a l i n i t y of 10 ppt, the optimal d i e t a r y p r o t e i n l e v e l was 40%, while at a s a l i n i t y of 20 ppt i t was 45%. More r e c e n t l y , Archdekin et al. (1988) determined that post-j u v e n i l e chinook salmon h e l d i n seawater r e q u i r e a minimum of 44% 6 p r o t e i n i n t h e i r d i e t to a t t a i n maximum growth. An increase i n d i e t a r y p r o t e i n requirement at higher s a l i n i t i e s may be due, at l e a s t i n p a r t , to a decrease i n i t s d i g e s t i b i l i t y . Work by MacLeod (1977), F e r r a r i s et al. (1986) and Usher et a l . (1990), would seem to support such a hypothesis although r e s u l t s obtained by L a l l et al. (1984) and L a l l (1988, c i t e d by Higgs et a l . , 1990) working w i t h A t l a n t i c salmon c o n t r a d i c t such t h e s i s . Other f a c t o r s a f f e c t i n g p r o t e i n requirements are r e l a t e d to the f i s h i t s e l f . Page and Andrews (1973) working w i t h channel c a t f i s h and S a t i a (1974) working w i t h rainbow t r o u t , concluded that d i e t a r y p r o t e i n requirements are i n v e r s e l y c o r r e l a t e d w i t h f i s h s i z e . 2.2 Amino a c i d requirements of f i s h F i s h , l i k e other animals, do not have a tr u e p r o t e i n requirement per se (Wilson, 1985) but r e q u i r e a w e l l balanced mixture of e s s e n t i a l amino acids and adequate l e v e l s of non-e s s e n t i a l amino a c i d s . F i s h r e q u i r e the same ten e s s e n t i a l amino acids i n t h e i r d i e t as t h e i r t e r r e s t r i a l counterparts, namely, threonine, v a l i n e , methionine, i s o l e u c i n e , l e u c i n e , phenylalanine, l y s i n e , h i s t i d i n e , a r g i n i n e and tryptophan. F i s h can r e a d i l y convert phenylalanine to t y r o s i n e and they u t i l i z e d i e t a r y t y r o s i n e t o meet t h e i r metabolic needs f o r t h i s amino a c i d . Cystine i s considered n o n e s s e n t i a l because i t can be synthesized by the f i s h from the e s s e n t i a l amino a c i d methionine. The presence of c y s t i n e i n the d i e t has t h e r e f o r e , a sparing e f f e c t on d i e t a r y methionine requirements. Improved growth and feed conversion have been reported by s e v e r a l workers when experimental d i e t s f o r salmonids were 7 supplemented w i t h e s s e n t i a l amino acids t o b r i n g t o t a l l e v e l s c l o s e to the l e v e l s found i n i s o l a t e d f i s h p r o t e i n or i n the egg or whole body t i s s u e of the species being s t u d i e d (Rumsey and K e t o l a , 1975; A r a i , 1981; K e t o l a , 1982; Ogata et al., 1983). Wilson and Cowey, (1985), analyzed the amino a c i d composition of rainbow t r o u t and A t l a n t i c salmon t i s s u e and found t h a t there was no s i g n i f i c a n t d i f f e r e n c e between the two species. They showed that the amino a c i d composition of these t i s s u e s was a l s o s i m i l a r to that of t i s s u e from coho salmon, cherry salmon (Oncorhynchus masou) and channel c a t f i s h . They suggested t h a t d i e t s f o r f i s h may be improved by formulating them to simulate the amino a c i d balance of the whole body t i s s u e of the f i s h being s t u d i e d . 2.3 Energy balance i n f i s h F i s h . a r e among the most e f f i c i e n t animals i n converting feed energy i n t o body p r o t e i n (Smith et al., 1978a) and s e v e r a l f a c t o r s c o n t r i b u t e t o t h e i r high e n e r g e t i c e f f i c i e n c y . F i r s t l y , i n c o n t r a s t t o warm-blooded animals, f i s h are aquatic ectotherms and consequently do not have to expend a l a r g e p r o p o r t i o n of energy i n maintaining body temperature. Secondly, f i s h are n e u t r a l l y buoyant and thus have no need f o r l a r g e a n t i g r a v i t y muscles i n t h e i r aquatic environment. F i n a l l y , f i s h have a very e f f i c i e n t mechanism f o r the excetion of waste n i t r o g e n . The primary waste product of p r o t e i n catabolism i n a l l animals i s ammonia which possesses no b i o l o g i c a l energy value but i s t o x i c even at very low concentrations and thus must be r a p i d l y excreted or converted to l e s s t o x i c compounds. T e r r e s t r i a l homeotherms, at considerable energy cost, convert ammonia to urea and u r i c 8 a c i d which, i n a d d i t i o n , must be concentrated and excreted by the kidneys. F i s h , on the other hand, excrete approximately 85% of the waste n i t r o g e n as ammonia (Wood, 1958; Randall and Wright, 1987) while the remainder i s excreted as urea, u r i c a c i d , trimethylamine oxide, c r e a t i n i n e and c r e a t i n (Watts and Watts, 1974). Ammonia i s excreted by passive d i f f u s i o n , from blood to the surrounding water, v i a the g i l l s , w i t h l i t t l e or no energy expenditure (Goldstein and F o r s t e r , 1970). The energy requirement per u n i t p r o t e i n gain i s , t h e r e f o r e , lower w i t h f i s h than w i t h land animals (Smith et al., 1978a; Cho and Kaushik, 1985; Smith, 1989). Most domesticated land animals are, however, herbivorous or omnivorous. By c o n t r a s t , f i s h farming i n c o l d c l imates has concentrated on r e a r i n g mostly carnivorous f i s h s pecies. Given t h a t food p r o t e i n i s expensive, t h i s has r e q u i r e d a focus on esteemed species of f i s h such as salmonids, which have a high market value. P h i l l i p s (1969), Ringrose (1971), Lee and Putnam (1973), Takeda et al. (1975) and Cowey et al. (1975) have reported that the non-protein "energy y i e l d i n g " n u t r i e n t s l i k e l i p i d s and carbohydrates can improve 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 means of a p r o t e i n sparing e f f e c t , s ince f i s h , i n common w i t h other animals, g e n e r a l l y eat to s a t i s f y t h e i r energy requirements. An optimal 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 o r f i s h i s thus d i c t a t e d by a d e l i c a t e balance of the d i e t a r y p r o t e i n to energy r a t i o . Excessive nonprotein energy intake r e s u l t i n g from high d i g e s t i b l e energy-to-dietary p r o t e i n r a t i o s , causes a p p e t i t e or demand to be s a t i s f i e d before a s u f f i c i e n t q u a n t i t y of p r o t e i n (and p o s s i b l y other n u t r i e n t s ) i s ingested t o s a t i s f y demand f o r 9 maximal ra t e s of growth (Page and Andrews, 1973). In a d d i t i o n , e x c e s s i v e l y high energy t o p r o t e i n r a t i o s can le a d t o d e p o s i t i o n of l a r g e amounts of body f a t . Conversely, when a d i e t i s d e f i c i e n t i n nonprotein energy, a l a r g e r f r a c t i o n of the p r o t e i n w i l l be used f o r en e r g e t i c purposes, thus r e s u l t i n g i n a decrease i n the p r o p o r t i o n a v a i l a b l e f o r p r o t e i n synthesis and growth (Cowey, 1979). 2.4 Sources of energy f o r f i s h As i n the case of warm-blooded animals, carbohydrates, l i p i d s and p r o t e i n s are the three primary energy sources f o r f i s h . 2.4.1 P r o t e i n as d i e t a r y energy source P r o t e i n s are r e a d i l y usable sources of energy f o r salmonids. P r o t e i n w i l l be used f o r energy i f : i n s u f f i c i e n t energy i s a v a i l a b l e from other sources (carbohydrates and l i p i d ) , p r o t e i n i s fed i n excess of minimal requirements, or the p r o t e i n i s of a poor q u a l i t y . 2.4.2 Carbohydrates as d i e t a r y energy sources In recent years, much work has been devoted t o the e v a l u a t i o n of the p r o t e i n - s p a r i n g e f f e c t s of non-protein energy-y i e l d i n g n u t r i e n t s i n f i s h d i e t s . While i t i s unanimously agreed that the a d d i t i o n of l i p i d s c o n t r i b u t e s t o p r o t e i n - s p a r i n g by i n c r e a s i n g the d i g e s t i b l e energy of the d i e t s (Watanabe et al., 1979; Cho and Kaushik, 1985), some controversy p e r s i s t s regarding the u t i l i z a t i o n of d i e t a r y carbohydrates by carnivorous f i s h such 10 as the salmonids. Carbohydrate u t i l i z a t i o n i s g e n e r a l l y poor and t h i s r e s u l t s from d e f i c i e n c i e s both i n d i g e s t i v e and metabolic c a p a c i t y . As carnivorous f i s h , salmonids are i l l - a d a p t e d to d i g e s t complex carbohydrates. D i g e s t i o n c o e f f i c i e n t s of l e s s than 40% f o r raw s t a r c h have been reported i n the l i t e r a t u r e (Inaba et al., 1963; Singh and Nose, 1967; Spannhof and Kiihne, 1977; Bergot and Breque, 1983). Furthermore, carbohydrate d i g e s t i b i l i t y v a r i e s i n v e r s e l y w i t h the s t a r c h content of the d i e t (Singh and Nose, 1967; Spannhof and Kiihne, 1977; Spannhof and Plantikow, 1983; Kirchgessner et al., 1986). One f a c t o r having a major e f f e c t on carbohydrate d i g e s t i b i l i t y i s the degree of s t a r c h g e l a t i n i z a t i o n . For t h i s reason, any p h y s i c a l treatment t h a t might decrease the complexity of the d i e t a r y s t a r c h (e.g. cooking, extrusion) d r a m a t i c a l l y improves i t s a v a i l a b i l i t y to these f i s h (Inaba et al., 1963; Smith, 1971; Luquet and Bergot, 1976; Bergot and Breque, 1983). The a b i l i t y of carnivorous f i s h t o metabolize glucose, the end product of s t a r c h d i g e s t i o n i s , nevetheless, l i m i t e d . S t i l l , Kaushik and O l i v a - T e l e s , (1985) have r e c e n t l y shown th a t i n c o r p o r a t i o n of g e l a t i n i z e d s t a r c h i n t o the d i e t of rainbow t r o u t can lead to an increase i n p r o t e i n and energy r e t e n t i o n e f f i c i e n c i e s by decreasing the nitrogenous metabolic l o s s e s a s s o c i a t e d w i t h p r o t e i n deamination f o r energy production. The t o t a l carbohydrate f r a c t i o n c o n s i s t s predominantly of s t a r c h i n very few vegetable feeds. The c e l l w a l l f r a c t i o n s i n c l u d i n g c e l l u l o s e , pentosane, h e m i c e l l u l o s e , and l i g n i n a l s o belong i n t h i s group. These f r a c t i o n s are mostly i n d i g e s t i b l e by f i s h , i n p a r t i c u l a r carnivorous f i s h . The small c a p a c i t y of the d i g e s t i v e system, the short food r e t e n t i o n time and the low body temperature of p o i k i l o t h e r m i c carnivorous f i s h , a l l o w l i t t l e o pportunity f o r the m i c r o b i a l enzymatic a c t i o n which i s necessary to u t i l i z e these non-starch carbohydrates. The i n c l u s i o n of f i b r e i n the d i e t s of monogastric animals has a l s o been shown to increase the sloughing of i n t e s t i n a l mucosal c e l l s (Bergner et al., 1975; Beames and Eggum, 1981) and to enhance mucus production (Schneeman et a l . , 1982), thereby l e a d i n g to increased losses of endogenous amino a c i d s . F i b r e i s a l s o capable of adsorbing amino acids and peptides and w i t h h o l d i n g these from absorption, the extent of which depends on the degree of l i g n i f i c a t i o n of the f i b r e (Bergner et al., 1975; M i t a r u et al., 1984). For these reasons, the f i b r e content f o r salmonid d i e t s should not exceed 4 percent, according t o the N a t i o n a l Research C o u n c i l (NRC, 1978). No adaptive r e g u l a t i o n of the s t r u c t u r e and f u n c t i o n of the d i g e s t i v e system seems to operate i n response t o the long term feeding of carbohydrate-rich d i e t s t o salmonids. In f a c t , enzymatic a c t i v i t y has been shown to decrease with time of exposure. The r e s u l t s of Buddington and H i l t o n (1987) and Kaushik et al. (1989) showed that both disaccharidase a c t i v i t y and glucose uptake r a t e were considerably reduced i n rainbow t r o u t a f t e r 30 weeks on a high l e v e l of d i e t a r y glucose. Various s t u d i e s have i n d i c a t e d that d i e t s c o n t a i n i n g d i g e s t i b l e carbohydrate l e v e l s ranging from 30% (Buhler and Halver, 1961) to as high as 38% (Kaushik et al., 1989), may be used e f f i c i e n t l y by salmonids. Other authors however, have reported t h a t d i g e s t i b l e carbohydrate l e v e l s should not exceed 14% of the d i e t ( H i l t o n and Atkinson, 1982; H i l t o n et a l . , 1982). The reason f o r such c o n f l i c t i n g r e s u l t s may be due to d i f f e r e n c e s i n the age and s i z e of f i s h and d i f f e r e n c e s i n water temperature and c u l t u r e c o n d i t i o n s , as w e l l as the form of carbohydrate used by the d i f f e r e n t authors. Absorbed glucose a l s o appears to be very p o o r l y u t i l i z e d by carnivorous f i s h as evidenced by prolonged hyperglycemia accompanied by enlargement of the l i v e r at high l e v e l s of absorption (Palmer and Ryman, 1972; Bergot, 1979; F u r u i c h i and Yone, 1981, 1982). H i l t o n and S l i n g e r (1981) s t a t e d t h a t excessive amounts of carbohydrate i n the salmonid d i e t depress growth r a t e , increase m o r t a l i t y and produce an abnormally high glycogen content of the l i v e r , e s p e c i a l l y at low temperatures. 2.4.3 L i p i d s as d i e t a r y energy sources L i p i d s p l a y an important r o l e as an energy source i n f i s h d i e t s , e s p e c i a l l y f o r carnivorous f i s h i n which d i e t a r y carbohydrate i s p o o r l y u t i l i z e d . L i p i d s c o n t ain over 1.5 times as much gross energy per u n i t weight r e l a t i v e t o p r o t e i n s and ne a r l y 2.5 times as much as carbohydrates (Lehninger, 1982). Several s t u d i e s have shown th a t the p r o v i s i o n of adequate l e v e l s of d i e t a r y l i p i d s can minimize the use of c o s t l y p r o t e i n as an energy source (Fowler et al., 1966; Ringrose, 1971; Watanabe,, 1977; Takeuchi et al., 1978; Cowey and Sargent, 1979; Watanabe, 1982). Although the q u a n t i t y of l i p i d i n salmon d i e t s i s important as a p r o t e i n - s p a r i n g energy source, the q u a l i t y of the l i p i d i s of even greater importance. Cold water f i s h e s have a l i m i t e d a b i l i t y t o synthesize f a t t y acids of the l i n o l e i c and l i n o l e n i c f a m i l i e s . Therefore, these must be s u p p l i e d i n the d i e t (Yu and Sinnhuber, 1972; Watanabe et a l . , 1974; Watanabe, 1982). Takeuchi et al. (1979), determined the d i g e s t i b i l i t y of beef t a l l o w and v a r i o u s hydrogenated f i s h o i l s w i t h v a r i o u s m e l t i n g p o i n t s w i t h common carp (Cyprinus carpio) and rainbow t r o u t and found an i n v e r s e r e l a t i o n s h i p between mel t i n g p o i n t (which i s a f u n c t i o n of the degree of s a t u r a t i o n or length of the carbon chain) and d i g e s t i b i l i t y . These authors a l s o concluded that i n general, a p o s i t i v e r e l a t i o n s h i p e x i s t s between l i p i d d i g e s t i b i l i t y and f i s h s i z e . D i e t a r y l i p i d i n c l u s i o n l e v e l s of 15% t o 20% of dry matter have y i e l d e d 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 and growth rates f o r a v a r i e t y of species (Stickney and Andrews, 1972; Lee and Putnam, 1973; H i l t o n and S l i n g e r , 1981; NRC, 1981; Watanabe, 1982). By c o n t r a s t , d i e t s c o n t a i n i n g higher l e v e l s of l i p i d than t h i s range have l e d t o remarkable increases i n v i s c e r a l f a t ( S a t i a , 1974; Ogino et al., 1976; Takeuchi et al., 1978; C a s t l e d i n e and Buckley, 1980; Boggio et al., 1985). The d i f f i c u l t y of d e f i n i n g an optimal balance of energy components stems from the f a c t that although d i e t a r y p r o t e i n l e v e l s can be reduced while d i e t a r y l i p i d l e v e l s are increased, without lowering the food conversion r a t e or growth r a t e , these changes w i l l be at the expense of an i n c r e a s i n g l y f a t t y carcass, which may a f f e c t market a c c e p t a b i l i t y . 14 2.5 S e l e c t i o n of d i e t a r y i n g r e d i e n t s f o r salmonid d i e t s The i n g r e d i e n t s u t i l i z e d i n the formulation of salmonid d i e t s can be ca t e g o r i z e d as f i s h e r y products (e.g. h e r r i n g meal, anchovy meal), by-products of the animal slaughter i n d u s t r y (e.g. meat and bone meal, p o u l t r y by-product meal, feather meal, blood meal) or by-products of the processing and m i l l i n g i n d u s t r i e s (e.g. soybean meal, canola meal, wheat m i d d l i n g s ) . When d i e t s are formulated, i n g r e d i e n t s w i t h s i m i l a r p r o p e r t i e s may be s u b s t i t u t e d f o r one another depending upon t h e i r market p r i c e , a v a i l a b i l i t y and n u t r i t i v e value. In making s u b s t i t u t i o n s , p a r t i c u l a r a t t e n t i o n must be p a i d t o the e s s e n t i a l n u t r i e n t content and balance of the f i n a l d i e t . P a l a t a b i l i t y or a c c e p t a b i l i t y of the d i e t i s a l s o dependent on the s e l e c t i o n of the i n g r e d i e n t s . Thus, i n c l u s i o n of a h i g h l y d i g e s t i b l e yet unpalatable i n g r e d i e n t can reduce feed i n t a k e and growth r a t e . Another c o n s i d e r a t i o n i n s e l e c t i n g feed i n g r e d i e n t s i s v a r i a b i l i t y w i t h i n the f e e d s t u f f s as a f f e c t e d by source, season of the year and processing methods. The lower the v a r i a b i l i t y of the product, the more r e l i a n c e can be placed on i t s q u a l i t y . In summary, s e l e c t i o n of i n g r e d i e n t s based on composition, n u t r i e n t d i g e s t i b i l i t y , a c c e p t a b i l i t y , a v a i l a b i l i t y and p r i c e are c r i t i c a l c o n s i d e r a t i o n s i n the manufacture of q u a l i t y feed f o r f i s h . 2.5.1 F i s h e r y products F i s h meal made from good q u a l i t y whole f i s h t h a t i s p r o p e r l y processed i s the highest q u a l i t y p r o t e i n source commonly a v a i l a b l e t o f i s h feed manufacturers. F i s h meals made from whole f i s h have high l e v e l s of p r o t e i n , a high energy value and are a l s o h i g h l y d i g e s t i b l e and p a l a t a b l e (Ogino and Chen, 1973/ Smith et a l . , 1980; P f e f f e r , 1982; M i l l e r and De Boer, 1988). Furthermore, the e s s e n t i a l amino a c i d content of commonly used f i s h meals c l o s e l y resembles the d i e t a r y requirement of f i s h such as rainbow t r o u t and carp (Higgs et al., 1990). I t i s t h e r e f o r e not s u r p r i s i n g t h a t at present, commercial d i e t s formulated f o r salmonid c u l t u r e c o n tain 25% to 65% f i s h meal (Tacon and Jackson, 1985), w i t h the higher l e v e l s being used f o r s t a r t e r and f i n g e r l i n g d i e t s (McCallum and Higgs, 1989) . The v a r i a b l e supply and q u a l i t y of f i s h meal, and perhaps more importantly the high cost, even of poorer q u a l i t y meals, have been i d e n t i f i e d as the major drawbacks of u t i l i z i n g t h i s product i n f i s h feeds ( S p i n e l l i et al., 1979; Higgs et al., 1982; P f e f f e r , 1982; H i l t o n and S l i n g e r , 1986; Higgs, 1986; McCallum and Higgs, 1989). Consequently, v a r i o u s attempts have been made to replace some, i f not a l l , of the f i s h meal w i t h other animal or p l a n t p r o t e i n sources. 2.5.2 Animal by-products The production of food f o r human consumption, whether of animal or of p l a n t o r i g i n , i n c r e a s i n g l y i n v o l v e s the processing of raw m a t e r i a l s , w i t h consequent production of by-products. The most common method of p r e s e r v a t i o n of the by-product i s dr y i n g , but the high i n i t i a l moisture content i n e v i t a b l y means that t h i s i s an expensive process. The m a j o r i t y of animal by-products have a higher content of d i g e s t i b l e p r o t e i n and energy than p l a n t by-products (NRC, 1981). Compared to f i s h meal however, the m a j o r i t y of a l t e r n a t e p r o t e i n sources, p l a n t and animal, are e i t h e r d e f i c i e n t i n a s p e c i f i c amino a c i d or s u f f e r from an imbalance of amino a c i d s . Blood meal f o r instance, i s low i n i s o l e u c i n e but r i c h i n l e u c i n e (NRC, 1983) and the a n t a g o n i s t i c e f f e c t of excess l e u c i n e on i s o l e u c i n e w i l l cause an i s o l e u c i n e d e f i c i e n c y i n animals fed high d i e t a r y l e v e l s of blood meal (Taylor et al., 1977). On the other hand, feather meal i s low i n l y s i n e (NRC, 1983), but a mixture of the two gives a r a t h e r w e l l balanced amino a c i d p r o f i l e . A d i e t a r y combination of p o u l t r y by-product meal and hydrolyzed feather meal has been employed wit h success to replace most of the f i s h meal p r o t e i n i n p r a c t i c a l d i e t s f o r j u v e n i l e rainbow t r o u t (Tiews et al., 1976; Gropp et al, 1979) and j u v e n i l e coho salmon (Higgs et al., 1979). In the s t u d i e s on t r o u t by Tiews et al. (197 6) and Gropp et al. (1979), a l l of the f i s h meal was replaced s u c c e s s f u l l y by a mixture of p o u l t r y by-product meal and feather meal supplemented wit h the e s s e n t i a l amino acids l y s i n e , methionine and tryptophan. 2.5.3 P l a n t by-products Fee d s t u f f s of p l a n t o r i g i n are g e n e r a l l y lower i n p r o t e i n content than those of animal o r i g i n . Although a n a l y t i c a l data may give an adequate amino a c i d p r o f i l e i n a given p l a n t p r o t e i n source, the amino acids may not be b i o l o g i c a l l y a v a i l a b l e because of processing losses or d e l e t e r i o u s f a c t o r s i n the p r o t e i n source that i n t e r f e r e w i t h p r o t e i n u t i l i z a t i o n . Most p l a n t p r o t e i n sources undergo considerable processing during production. Meals of d i f f e r e n t o i l - r i c h seeds, such as soybean, rapeseed, canola, e t c . , a f t e r the e x t r a c t i o n of the o i l , are good and r e l a t i v e l y inexpensive sources of p r o t e i n . The e x t r a c t i o n of o i l from these seeds e n t a i l s the production of considerable heat which can be detr i m e n t a l t o the n u t r i t i v e value of the p r o t e i n . Many pl a n t p r o t e i n s c o n t a i n reducing carbohydrates such as glucose which may react w i t h the free amino groups of a p r o t e i n , p a r t i c u l a r l y the r e a c t i v e groups of l y s i n e , a r g i n i n e , h i s t i d i n e and tryptophan (Potter, 1984). Such carbohydrate-amino a c i d r e a c t i o n s ( M a i l l a r d reaction) r e s u l t i n linkages t h a t are not hydrolyzed by d i g e s t i v e enzymes, although the amino acids may s t i l l be recovered from the p r o t e i n by a c i d h y d r o l y s i s . Such amino acids are b i o l o g i c a l l y u n a v a i l a b l e , even though they are chemically present. Nearly a l l p l a n t p r o t e i n sources, u n f o r t u n a t e l y , have compounds a s s o c i a t e d w i t h them that make some a d d i t i o n a l processing necessary to render them of maximum n u t r i t i v e value. These compounds, i f not i n a c t i v a t e d , can markedly i n f l u e n c e the d i g e s t i o n and u t i l i z a t i o n of many n u t r i e n t s . One of the most abundant o i l seed meals i s soybean meal. Soybean contains s e v e r a l a n t i - n u t r i t i o n a l f a c t o r s , the main ones being i n h i b i t o r s of both t r y p s i n and chymotrypsin. Cottonseed meal contains gossypol which i s t o x i c t o monogastrics. Rapeseed contains s i g n i f i c a n t amounts of g l u c o s i n o l a t e s and e r u c i c a c i d (Ackman, 1977). ' The g l u c o s i n o l a t e compounds are hydrolyzed i n t o g o i t r o g e n i c substances by the enzyme myrosinase which i s found i n the seed i t s e l f , as w e l l as by the microorganisms of the i n t e s t i n a l t r a c t (Tookey et al., 1980). These g o i t r o g e n i c substances have marked negative e f f e c t s on feed i n t a k e and growth and al s o cause enlargement of the t h y r o i d (Jones, 1979; Higgs et al., 1979). E r u c i c a c i d i s a 22-carbon monounsaturated f a t t y a c i d which has been shown t o be c a r d i o t o x i c (Hendricks and B a i l e y , 1989). As a r e s u l t of these 18 a n t i - n u t r i t i o n a l f a c t o r s , advances have been made i n p l a n t breeding programs as w e l l as i n processing procedures to e l i m i n a t e or at the very l e a s t , reduce these f a c t o r s t o l e v e l s which enable the feed manufacturer to u t i l i z e higher l e v e l s of o i l s e e d meals i n animal d i e t s . New v a r i e t i e s of rapeseed have been developed which are low i n both e r u c i c a c i d and g l u c o s i n o l a t e s ( B e l l , 1982). Studies on salmonids have i n d i c a t e d that canola products can s a t i s f a c t o r i l y supply some of the d i e t a r y p r o t e i n and p a r t i a l l y replace f i s h meal (Yurkowski et al., 1978; Hardy and S u l l i v a n , 1983; Higgs et al., 1982, 1983). Further processing methods have been implemented as w e l l , i n order t o reduce the g l u c o s i n o l a t e content and al s o to remove water s o l u b l e non-proteinaceous c o n s t i t u e n t s i n canola and rapeseed meals. In these methods, the dehulled seed f r a c t i o n i s subjected to a water b o i l i n g treatment, followed by a hexane e x t r a c t i o n a f t e r the water has been removed. The r e s u l t i n g canola/rapeseed concentrates are much lower i n carbohydrate and f i b r e and s i g n i f i c a n t l y higher i n p r o t e i n content, r e f l e c t i n g high p o t e n t i a l f o r use i n f i s h d i e t s (Higgs et al., 1990). From the g r a i n m i l l i n g i n d u s t r y , wheat bran and middlings are commonly used i n salmonid feeds because of t h e i r s u i t a b i l i t y as p e l l e t b i n d i n g agents. However, t h e i r n u t r i t i o n a l c o n t r i b u t i o n to the d i e t i s l i m i t e d because of t h e i r low l e v e l s of p r o t e i n , l i p i d and energy and high carbohydrate and f i b r e contents. 19 2.6 E v a l u a t i n g the n u t r i e n t and energy value of feed i n g r e d i e n t s The organic compounds i n the food r e l e a s e energy i n the body upon t h e i r biochemical o x i d a t i o n . For salmonids, most of the energy i s d e r i v e d from the p r o t e i n and l i p i d f r a c t i o n s . Although energy f u r n i s h e d by d i e t a r y p r o t e i n , l i p i d and, t o a l e s s e r extent carbohydrate, above the maintenance requirements of the f i s h u l t i m a t e l y r e s u l t i n anabolism and growth, an increased need fo r maintenance energy can preclude t h e i r i n c o r p o r a t i o n i n t o t i s s u e s and l e a d s o l e l y t o t h e i r catabolism. In f a c t , i f the energy provided i n the d i e t i s l e s s than t h a t needed f o r maintenance, t i s s u e components w i l l be c a t a b o l i z e d . The energy requirements of a l l animals are i n f l u e n c e d by environmental f a c t o r s . In f i s h , temperature i s of p a r t i c u l a r importance, owing to i t s d i r e c t e f f e c t on r a t e of metabolism. The p a r t i c u l a r l i f e stage of the animal a l s o determines the energy needs, w i t h the younger growing animals r e q u i r i n g more energy per u n i t body weight than more mature ones (Winberg, 1961; Smith et al., 1978b). Since f i s h r egulate t h e i r feed i n t a k e i n r e l a t i o n t o t h e i r energy needs, the c o n c e n t r a t i o n of the n u t r i e n t s which must be provided i n the d i e t to adequately meet the animal's requirements, t h e r e f o r e , depends upon the energy value of the d i e t t o the animal. A l a r g e number of p o t e n t i a l feed i n g r e d i e n t s are a v a i l a b l e f o r f i s h d i e t s and t h e i r n u t r i e n t content can r e a d i l y be determined by standard chemical analyses. A n a l y s i s of p r o t e i n , l i p i d , ash and determination of carbohydrate by d i f f e r e n c e and m u l t i p l i c a t i o n by the appropriate f a c t o r s , w i l l g ive an approximate value f o r the t o t a l or gross energy (GE) content. A more p r e c i s e method of determining gross energy i s t o 20 measure the t o t a l energy l i b e r a t e d by the f e e d s t u f f upon complete combustion i n a bomb c a l o r i m e t e r . Although the chemical makeup of the f e e d s t u f f has a d i r e c t i n f l u e n c e on i t s gross energy content, n e i t h e r measure y i e l d s i n f o r m a t i o n regarding the a v a i l a b i l i t y of the i n d i v i d u a l n u t r i e n t s and energy to the f i s h . A b i o l o g i c a l , r a t h e r than a chemical e v a l u a t i o n of the f e e d s t u f f i s the best method f o r measuring the u t i l i z a t i o n of a p a r t i c u l a r feed i n g r e d i e n t by the animal. B i o l o g i c a l e v a l u a t i o n of feed i n g r e d i e n t s i n v o l v e s the feeding of f i s h and the a n a l y s i s of some aspect of f i s h performance and/or d i e t d i g e s t i b i l i t y . Performance s t u d i e s , i n which growth, feed e f f i c i e n c y and carcass n u t r i e n t and energy r e t e n t i o n are measured can be used t o evaluate a d i e t . Such s t u d i e s , however, have t o be c a r r i e d out fo r a long p e r i o d of time before v a l i d comparisons between f e e d s t u f f s can be made w i t h respect t o n u t r i t i v e value. Studies i n which the var i o u s l o s s e s of ingested food v i a feces, u r i n e , and g i l l s are measured, y i e l d r e s u l t s sooner, are l e s s expensive to perform and they give q u a n t i t a t i v e f i g u r e s on the u t i l i z a t i o n of t h e . n u t r i e n t s and energy of the i n d i v i d u a l feed i n g r e d i e n t s . During the passage of food through the d i g e s t i v e t r a c t , not a l l of the food i s dig e s t e d and absorbed. The undigested p o r t i o n i s l o s t as feces and the energy which would have been l i b e r a t e d by t h i s f r a c t i o n to the f i s h i s r e f e r r e d to as the f e c a l energy (FE) l o s s . The d i g e s t i b l e energy (DE) i s the d i f f e r e n c e between the gross energy' of the food and the gross energy of the feces, and thus represents the part of the food energy which i s a c t u a l l y absorbed by the f i s h . Some of the diges t e d energy i s l o s t through the e x c r e t i o n of the combustible c a t a b o l i t e s urea i n the u r i n e (UE) and ammonia through the g i l l s (ZE). These losses are l a r g e l y the r e s u l t of catabolism of p r o t e i n s and t h e i r deamination when amino acids are u t i l i z e d f o r energy ra t h e r than f o r s ynthesis of new t i s s u e p r o t e i n (Walton and Cowey, 1982). The energy a s s i m i l a t e d i n the body i s the metabolizable energy (ME) and i t can be summarized as f o l l o w s : ME = GE - ( FE + UE + ZE ) Consequently, metabolizable energy, i n c o n t r a s t t o d i g e s t i b l e energy, gives i n f o r m a t i o n regarding the amount of energy which i s e f f e c t i v e l y u t i l i z a b l e f o r growth by the f i s h . Determination of ME however, i s much simpler i n t e r r e s t r i a l animals than f o r those l i v i n g i n an aquatic medium. B i r d s , f o r instance, v o i d u r i n a r y and f e c a l wastes together, so that values obtained from c o l l e c t i o n of s o l i d c l o a c a l excreta are d i r e c t estimates of ME. In mammals, measurement of ME r e q u i r e s the separate c o l l e c t i o n and a n a l y s i s of feces and u r i n e , and i n the case of ruminants the gaseous products of fermentation need to be considered a l s o . The separation and recovery of g i l l and u r i n a r y e x c r e t i o n s from the aquarium water i s the most t e c h n i c a l l y d i f f i c u l t p a r t of determining ME i n f i s h . Since f e c a l energy l o s s i s the major l o s s of the ingested gross energy (Cho et a l . , 1982), measurement of DE gives a good i n d i c a t i o n of the a v a i l a b i l i t y of the energy of d i e t a r y feed i n g r e d i e n t s . The formulation of s u c c e s s f u l p r a c t i c a l f i s h d i e t s i s based, t h e r e f o r e , on an understanding not only of the chemical and p h y s i c a l c h a r a c t e r i s t i c s of the i n d i v i d u a l f e e d s t u f f s to be used, but a l s o of t h e i r r e l a t i v e 22 d i g e s t i b i l i t y by t h e t a r g e t s p e c i e s under s p e c i f i c c u l t u r e c o n d i t i o n s . Feed i n g r e d i e n t s are n o r m a l l y c a t e g o r i z e d by l i s t i n g t h e i r c o m p o s i t i o n and c o n t e n t o f d i g e s t i b l e n u t r i e n t s (Smith e t al., 1980, NRC, 1981). The apparent d i g e s t i b i l i t y c o e f f i c i e n t s a r e m o s t l y r e p r e s e n t e d by a s i n g l e v a l u e , s u g g e s t i n g t h a t each c o e f f i c i e n t n ever v a r i e s . H a s t i n g s (1969), however enumerated f a c t o r s w h i c h may a f f e c t d i g e s t i b i l i t y such as s i z e and age o f f i s h , s t o c k i n g d e n s i t y , water t e m p e r a t u r e , q u a l i t y o f f e e d , d i e t n u t r i e n t d e n s i t y , l e v e l o f f e e d i n g , f r e q u e n c y o f f e e d i n g , p r e v i o u s l e v e l o f n u t r i t i o n , f o r c e d v e r s u s ad l i b i t u m f e e d i n g and p r e s e n c e o f t o x i n s . The work o f numerous a u t h o r s has added t o t h i s l i s t o t h e r f a c t o r s w h i c h a f f e c t d i g e s t i b i l i t y , such as f i s h s p e c i e s ( B i r k e t t , 1969; J o b l i n g , 1983) and water s a l i n i t y ( Z e i t o u n , 1974; MacLeod, 1977; Pandey and S i n g h , 1980; L a l l e t al., 1984; F e r r a r i s e t al., 1986; Usher et al., 1990). 2.7 D i g e s t i o n and a b s o r p t i o n Many o f t h e o r g a n i c components o f f o o d a r e i n t h e form o f l a r g e i n s o l u b l e m o l e c u l e s which have t o be b r o k e n down by enzymes i n t o s i m p l e r compounds b e f o r e t h e y can pass t h r o u g h t h e mucosa o f t h e d i g e s t i v e t r a c t i n t o t h e b l o o d and lymph. The breaking-down p r o c e s s i s termed " d i g e s t i o n " , t h e passage o f t h e d i g e s t e d n u t r i e n t s t h r o u g h t h e i n t e s t i n a l mucosa " a b s o r p t i o n " . F i s h e s a r e t h e dominant v e r t e b r a t e group as f a r as number o f s p e c i e s i s concerned, and i n t h e i r immense v a r i e t y have adopted many d i f f e r e n t n u t r i t i o n a l h a b i t s . Not o n l y a r e t h e r e l a r g e d i f f e r e n c e s i n n u t r i t i o n a l h a b i t s , but a l s o tremendous d i v e r s i t y e x i s t s i n the form and f u n c t i o n of the d i g e s t i v e t r a c t . The most pronounced d i s t i n c t i o n among f i s h e s w i t h d i f f e r e n t d i g e s t i v e processes i s that some have a stomach, while others have none. Some f i s h e s feed e x c l u s i v e l y on plankton; others are s t r i c t l y bottom feeders and many species feed only on prey animals. Due to t h i s l a r g e d i v e r s i t y , i t i s improper t o make a g e n e r a l i z a t i o n about the d i g e s t i v e system of a l l f i s h . Consequently, the emphasis i n the f o l l o w i n g d i s c u s s i o n w i l l be on salmonids. 2.8 General alimentary t r a c t anatomy and i t s a s s o c i a t e d enzymes The s e c r e t i o n s i n the stomach t y p i c a l l y i n c l u d e the mucus produced by goblet c e l l s , as w e l l as pepsinogen which i s a c t i v a t e d i n t o pepsin by h y d r o c h l o r i c a c i d (HC1). Some n o n p r o t e o l y t i c d i g e s t i v e enzymes such as esterase (Kitamikado and Tachino, 1960b) l i p a s e and c h i t i n a s e (Lindsay, 1984) have al s o been i d e n t i f i e d i n the stomach of salmonids. 2.8.1 The midgut The midgut begins j u s t p o s t e r i o r t o the p y l o r i c s p h i n c t e r where the p y l o r i c cecae are found and ends at the region where an i l e o c e c a l valve i s present, j u s t p r i o r to the hindgut (Ezeasor and Stokoe, 1980). The p y l o r i c cecae c l o s e l y resemble the i n t e s t i n e i n s t r u c t u r e , w i t h the inner e p i t h e l i u m c o n t a i n i n g goblet c e l l s . Although many of the enzymes i n v o l v e d i n d i g e s t i o n are found i n lar g e numbers i n the cecae, l i g h t and e l e c t r o n microscopic s t u d i e s i n d i c a t e they l a c k c e l l s capable of s e c r e t i n g d i g e s t i v e enzymes (Vagas-Velez, 1972, c i t e d by Fange and Grove, 1979). M a t e r i a l from the i n t e s t i n a l lumen moves i n t o and out of the p y l o r i c cecae by p e r i s t a l t i c c o n t r a c t i o n of cecael muscles (Smith, 1989). The f u n c t i o n of the cecae i s not yet f u l l y c l e a r and v a r i o u s f u n c t i o n s have been suggested. A d i g e s t i v e r o l e supplementing t h a t of the stomach, absorption of n u t r i e n t s , r e s o r p t i o n of water and i n o r g a n i c ions, synthesis of enzymes and v i t a m i n production, have a l l been proposed. Since these appendages are i n c l o s e p r o x i m i t y t o where the b i l e and p a n c r e a t i c s e c r e t i o n s are released, t h e i r f u n c t i o n as d i g e s t i v e compartments a c t i v e i n absorption of c e r t a i n n u t r i e n t s seems l o g i c a l . However, f u r t h e r s t u d i e s are needed to c l a r i f y the r o l e of the p y l o r i c cecae i n n u t r i e n t d i g e s t i o n . Enzymes found i n the i n t e s t i n a l lumen can p o t e n t i a l l y o r i g i n a t e from e i t h e r the pancreas or the sec r e t o r y c e l l s i n the gut w a l l . The proteases t r y p s i n , chymotrypsin, carboxypeptidase, collagenase and e l a s t a s e are known to be s t o r e d i n the p a n c r e a t i c c e l l s as i n a c t i v e zymogens. Complementing the p a n c r e a t i c enzymes, the i n t e s t i n e a l s o secretes enzymes th a t act on o l i g o p e p t i d e s , such as t r i - and dipeptidases (Ash, 1980, Boge et al., 1981) and aminopeptidases (Plantikow and Plantikow, 1985). Upon a r r i v a l i n the i n t e s t i n a l lumen, trypsinogen i s transformed i n t o t r y p s i n by enterokinase which i s secreted by the i n t e s t i n a l mucosa. Other p a n c r e a t i c zymogens are a c t i v a t e d by t r y p s i n (Fange and Grove, 197 9). The existence of s e v e r a l carbohydrases i n e x t r a c t s from the i n t e s t i n e s , pancreas, p y l o r i c cecae and l i v e r of rainbow t r o u t and P a c i f i c salmon has been demonstrated by v a r i o u s authors (Kitamikado and Tachino, 1960a; P h i l l i p s , 1969). Salmonids a l s o 25 produce amylases, maltases, sucrases, l a c t a s e s , and glucosidases f o r the d i g e s t i o n of carbohydrates ( P h i l l i p s et a l . , 1948). Although l i p o l y t i c a c t i v i t y has been found i n e x t r a c t s of the stomach, p y l o r i c cecae and i n t e s t i n e of many f i s h , i n c l u d i n g rainbow t r o u t (Kitamikado and Tachino, 1960b; Swarup and Goel, 1975), the pancreas i s b e l i e v e d to be the major source of l i p a s e s (Kapoor et al., 1975). Enzymes from the m i c r o f l o r a i n the i n t e s t i n e could have a s i g n i f i c a n t r o l e i n d i g e s t i o n , e s p e c i a l l y f o r substrates such as c e l l u l o s e , which few animals can d i g e s t . Mowlah et al. (1979), determined t h a t Enterobacter spp. and Vibrio spp. produce c h i t i n a s e as w e l l as showing a high a c t i v i t y i n the breakdown of g e l a t i n , c a s e i n , s t a r c h and l e c i t h i n . Although the number of b a c t e r i a l species i n the i n t e s t i n e s of salmonids has been found to be low (Ugayin, 1979), the a c t u a l concentration can be q u i t e high (Trust and Sparrow, 1974). 2.8.2 The hindgut P r o t e o l y t i c a c t i v i t y decreases from midgut to hindgut (Fange and Grove, 1979) and since p r o t e o l y t i c enzymes are considered t o be r e s i s t a n t to a u t o l y s i s w i t h i n the small i n t e s t i n e (Hofer, 1982), i t has been p o s t u l a t e d by the l a t t e r author that some of these enzymes are resorbed i n t a c t i n p o s t e r i o r s e c t i o n s of the g a s t r o i n t e s t i n a l t r a c t . Gauthier and Landis (1971) and more r e c e n t l y Georgopoulou et al. (1985), reported t h a t the enterocytes from the p o s t e r i o r i n t e s t i n e were i n v o l v e d i n macromolecule uptake by the process of p i n o c y t o s i s . The study of the absorption of macromolecules and peptides has been g r e a t l y f a c i l i t a t e d by the use of horseradish peroxidase, which can be r e a d i l y i d e n t i f i e d h i s t o c h e m i c a l l y , as a model p r o t e i n . Once the p r o t e i n i s enclosed w i t h i n the enterocyte, i n t r a c e l l u l a r (lysosomal) p r o t e o l y s i s can occur. 2.9 P r o t e i n d i g e s t i o n and absorption The extent of p r o t e o l y s i s w i t h i n the stomach depends upon a number of f a c t o r s , such as s e c r e t o r y r a t e of. a c i d and pepsin, s p e c i f i c a c t i v i t y of pepsin, r e t e n t i o n time, feed i n t a k e e t c . , many of these f a c t o r s being i n t e r a c t i v e . Although the primary r o l e of the stomach may be one of preparing s u s c e p t i b l e peptide lin k a g e s f o r subsequent h y d r o l y s i s w i t h i n the small i n t e s t i n e , Austreng (1978) and Dabrowski and Dabrowska (1981) i n d i c a t e d t h a t to some extent p r o t e i n d i g e s t i o n and even amino a c i d absorption occurs w i t h i n the stomach of rainbow t r o u t . Once i n the i n t e s t i n e , most polypeptides are f u r t h e r hydrolyzed, l e a d i n g to a mixture of low molecular peptides and f r e e amino a c i d s . Much work has been done on amino a c i d uptake, showing th a t each amino a c i d has i t s own c h a r a c t e r i s t i c s . As i n the case of higher v e r t e b r a t e s , f r e e amino a c i d absorption i s an a c t i v e , group-s p e c i f i c , sodium dependent, ca r r i e r - m e d i a t e d mechanism which i s separate and independent from the d i - and t r i - p e p t i d e t r a n s p o r t system (Smith, 1969; S i l k et a l . , 1982; Ash, 1985). Amino a c i d t r a n s p o r t i s competitive and the t r a n s p o r t of one amino a c i d may be i n h i b i t e d i n the presence of other amino acids ( K i t c h i n and M o r r i s , 1971). Thus Dabrowski (1983), s t a t e d t h a t the r a t e l i m i t i n g f a c t o r i n the u t i l i z a t i o n of amino acids by f i s h i s not d i g e s t i o n but r a t h e r absorption. 27 The absorbed amino acids enter the p o r t a l blood stream and presumably undergo the same types of r e a c t i o n s as i n other animals, namely p r o t e i n s y n t h e s i s ; deamination followed by o x i d a t i o n and i n some cases, conversion t o f a t and/or glucose; and to a l e s s e r extent f o r synthesis of hormones, purines and n u c l e i c acids (Walton and Cowey, 1982). 2.10 L i p i d d i g e s t i o n and absorption In t r o u t , l i p i d s are absorbed at the l e v e l of the p y l o r i c cecae and i n the a n t e r i o r midgut (Gauther and Landis, 1972). Lipase, i n the presence of b i l e s a l t s and a s p e c i a l p r o t e i n c a l l e d c o l i p a s e , binds to t r i g l y c e r i d e s and c a t a l y z e s the removal of one or both of the outer f a t t y a c i d residues t o y i e l d an emulsion mixture of fr e e f a t t y acids and 2-monoglycerides c a l l e d a m i c e l l e (Lehninger, 1982). The m i c e l l e s formed i n the g a s t r o i n t e s t i n a l t r a c t of f i s h are r e a d i l y absorbed by i n t e s t i n a l c e l l s i n much the same way as i n higher v e r t e b r a t e s , although i n f i s h , absorption i s slower and temperature dependent ( N o a i l l a c -Depeyre and Gas, 1976; S i r e et al., 1981) . , A f t e r passage across the i n t e s t i n a l c e l l w a l l , d i e t a r y l i p i d s are tra n s p o r t e d mainly by lymph, although t h e i r presence i n blood as chylomicrons has als o been reported (Leger, 1977). 2.11 Carbohydrate d i g e s t i o n and absorption Amylases are the enzymes p r i m a r i l y r e s p o n s i b l e f o r the h y d r o l y s i s of s t a r c h i n t o glucose. Absorption of glucose by the i n t e s t i n a l e p i t h e l i u m occurs by an a c t i v e mechanism and can take place against considerable concentration gradients (Fange and 28 Grove, 197 9). Glucose t r a n s p o r t r a t e s have been shown t o vary up t o 200 f o l d among d i f f e r e n t f i s h species, (Buddington e t j a l . , 1985/ c i t e d by Smith, 1989) being lowest i n t r o u t ( c a r n i v o r e ) , intermediate i n c a t f i s h (omnivore) and highest i n carp (herbivore). 2.12 Apparent and t r u e d i g e s t i b i l i t y A p r o p o r t i o n of the f e c a l n i t r o g e n does not o r i g i n a t e from d i e t a r y n i t r o g e n but r a t h e r from endogenous residues. These substances i n c l u d e d i g e s t i v e enzymes and mucoproteins secreted i n t o the d i g e s t i v e t r a c t , as w e l l as sloughed o f f c e l l s abraded from the alimentary c a n a l . Since these substances are composed p r i m a r i l y of p r o t e i n , they are r e f e r r e d t o as endogenous f e c a l p r o t e i n or endogenous f e c a l n i t r o g e n . The i n c l u s i o n of the endogenous p r o t e i n w i t h the d i e t a r y p r o t e i n i n the feces causes a bi a s of the d i g e s t i b i l i t y c o e f f i c i e n t toward lower values. Such c o e f f i c i e n t s , are t h e r e f o r e "apparent d i g e s t i b i l i t y c o e f f i c i e n t s " t o be d i s t i n g u i s h e d from "true or c o r r e c t e d d i g e s t i b i l i t y c o e f f i c i e n t s " f o r which c o r r e c t i o n s f o r endogenous p r o t e i n are made. To determine the tru e d i g e s t i b i l i t y , the amount of endogenous f e c a l p r o t e i n i s f i r s t determined and subtracted from the p r o t e i n i n the feces. This can be accomplished by measuring the amount of n i t r o g e n excreted i n the feces when the f i s h are fed a non-protein d i e t . Nose (1967; c i t e d i n NRC, 1981), fed such a d i e t t o rainbow t r o u t and determined the endogenous f e c a l n i t r o g e n output t o be 500 mg per 100 g of dry d i e t , whereas Skrede et al. (1980) c a l c u l a t e d the value t o be 180 mg per 100 g of d i e t a r y dry matter. Such d i s c r e p a n c i e s may be due to d i f f e r e n c e s i n water temperature or to d i s s i m i l a r d i e t a r y composition and t e x t u r e between the two experiments, as the f i n d i n g s o f F o l t z (1978; c i t e d i n NRC, 1981), Ogino and Chen (1973) and Beames and Eggum (1981) suggest. A l s o , as the d i e t a r y p r o t e i n l e v e l i n c r e a s e s , the d i f f e r e n c e between apparent and t r u e p r o t e i n d i g e s t i b i l i t y estimates d i m i n i s h e s (Ogino and Chen, 1973). 2.13 D i g e s t i b i l i t y measurements i n f i s h The e a r l i e s t method f o r determining d i g e s t i b i l i t y c o e f f i c i e n t s f o r domestic animals was the d i r e c t or t o t a l c o l l e c t i o n method which i n v o l v e s a q u a n t i t a t i v e r e c o r d of the • n u t r i e n t s consumed and those v o i d e d i n the f e c e s . The apparent d i g e s t i o n c o e f f i c i e n t s are c a l c u l a t e d f o r crude p r o t e i n , l i p i d , carbohydrate, energy, and t o t a l dry matter by performing a p p r o p r i a t e chemical analyses f o r the n u t r i e n t s i n the feed consumed and i n the f e c e s . The apparent d i g e s t i b i l i t y c o e f f i c i e n t o f a g i v e n n u t r i e n t based on the d i r e c t method (ADCd) i s thus determined as: ADCd (%) = 100 feces dry weight x % n u t r i e n t i n feces 1 d i e t f e d weight x % n u t r i e n t i n d i e t The d i r e c t method of determining d i g e s t i b i l i t y i s c o n s i d e r e d t o g i v e very a c c u r a t e r e s u l t s when p r o p e r l y c a r r i e d out. The i m p r a c t i c a l i t y of t h i s method comes, however, from the need t o q u a n t i t a t i v e l y c o l l e c t the feces f o r an extended p e r i o d of time. An i n d i r e c t method, which i n v o l v e s the use o f an i n d i g e s t i b l e 30 i n d i c a t o r i n the d i e t , has been used e x t e n s i v e l y i n t e r r e s t r i a l animal d i g e s t i b i l i t y t r i a l s s i n c e i t g r e a t l y s i m p l i f i e s the procedure and a l s o reduces the amount of labour and expense in v o l v e d i n the determination of d i g e s t i b i l i t y c o e f f i c i e n t s . The r a t i o of i n d i c a t o r to n u t r i e n t i s determined i n the d i e t and i n the feces and thus the apparent d i g e s t i b i l i t y c o e f f i c i e n t of a d i e t determined by the i n d i r e c t or i n d i c a t o r method (ADCi) i s c a l c u l a t e d as: ADCi (%)= 100 % i n d i c a t o r i n d i e t x % n u t r i e n t i n feces 1 % i n d i c a t o r i n feces x % n u t r i e n t i n d i e t With t h i s i n d i r e c t method of determining d i g e s t i b i l i t y , the t o t a l c o l l e c t i o n of feces i s unnecessary, since the d i g e s t i b i l i t y can be computed from r e p r e s e n t a t i v e p o r t i o n s of feces i f the n u t r i e n t and i n d i c a t o r are evenly d i s t r i b u t e d (Kane et . a l . , 1950; Maynard and L o o s l i , 1969). To be an e f f e c t i v e i n d i c a t o r , a substance must be i n d i g e s t i b l e and unabsorbable, remain homogeneously mixed 'with the d i g e s t a during passage through the gut, and have no e f f e c t on the d i g e s t i v e metabolism of the animal (Schneider and F l a t t 1975). Two c l a s s e s of i n d i c a t o r s can be d i s t i n g u i s h e d : those that are added to a d i e t and those th a t are i n h e r e n t l y present as n a t u r a l components of a d i e t . Edin (1918) f i r s t proposed the use of chromic oxide (C^C^) and most i n d i c a t o r t r i a l s i n t e r r e s t r i a l animals have been c a r r i e d out w i t h t h i s e x t e r n a l d i e t a r y marker. D i g e s t i o n t r i a l s are more d i f f i c u l t w i t h f i s h than w i t h land animals because f e c a l and other metabolic e x c r e t i o n s must be c o l l e c t e d from an aquatic medium (Smith et al., 1980). The o r i g i n a l a p p l i c a t i o n of the d i r e c t method t o determine d i g e s t i b i l i t i e s i n f i s h i n v o l v e d water f i l t r a t i o n techniques t h a t r e q u i r e d c o l l e c t i o n , measurement and a n a l y s i s of a l l egesta and ex c r e t i o n s (Tunison et al., 1942). Post et al. (1965), Smith (1971, 1976) and Smith et al. (1980), used a metabolism chamber which permits separation and q u a n t i t a t i v e c o l l e c t i o n of feces, u r i n e and g i l l e x c r e t i o n s . This method has the advantage of p e r m i t t i n g the determination of d i g e s t i b i l i t y c o e f f i c i e n t s and metabolizable energy, however, metabolism chambers have been c r i t i c i z e d because f i s h are confined, c a t h e t e r i z e d and force fed, and consequently are under considerable s t r e s s which probably reduces t h e i r a b i l i t y to d i g e s t and u t i l i z e feed. Determination of d i g e s t i b i l i t y by the i n d i r e c t method has been used w i t h s e v e r a l species of f i s h (Nose, 1960; Hastings, 1969: Smith and L o v e l l , 1971, 1973; Windell et al., 1978a). The major disadvantages of t h i s method are the v a r i e t y of techniques used to c o l l e c t the f e c a l samples without inherent n u t r i e n t l e a c h i n g e r r o r s and the i n a b i l i t y to compute r e l i a b l e metabolizable energy values (NRC, 1981). To avoid completely any lea c h i n g of n u t r i e n t s i n the water, s e v e r a l authors p r e f e r to c o l l e c t the feces d i r e c t l y from the p o s t e r i o r region of the i n t e s t i n e before i t i s n a t u r a l l y e x p e l l e d . This can be done by s a c r i f i c i n g the f i s h and performing an i n t e s t i n a l d i s s e c t i o n (Smith and L o v e l l , 1971, 1973; Austreng, 1978; Windell et al., 1978a; Henken et al., 1985). Since repeated sampling i s not p o s s i b l e w i t h t h i s technique, other authors have r e s o r t e d t o applying manual pressure to the abdomen of anesthetized f i s h and 32 s t r i p p i n g the feces from the d i s t a l p a r t of the l a r g e i n t e s t i n e (Nose, 1960; Inaba et al., 1962; Austreng, 1978; W i n d e l l et al., 1978a; Tacon et al., 1984; Vens-Cappell, 1985) or to a p p l y i n g anal s u c t i o n w i t h vacuum ( L o v e l l , 1977; W i n d e l l et al., 1978a; Brown and Strange, 1985). Although the c o l l e c t i o n of feces by i n t e s t i n a l d i s s e c t i o n , s t r i p p i n g and anal s u c t i o n avoids the l e a c h i n g problem, the assumption t h a t d i g e s t i o n and a b s o r p t i o n are complete when the m a t e r i a l has reached the d i s t a l end of the l a r g e i n t e s t i n e may l e a d t o an u n d e r e s t i m a t i o n of d i g e s t i b i l i t y (Smith and L o v e l l , 1971; Austreng, 1978; W i n d e l l et al., 1978a). An a d d i t i o n a l disadvantage of these methods i s t h a t f e c a l samples are e a s i l y contaminated with mucus, blood, u r i n e or sexual p r o d u c t s . The p h y s i c a l a c t i o n of s t r i p p i n g a l s o r e s u l t s i n the contamination of the f e c a l .material with sloughed o f f c e l l s of the i n t e s t i n a l e p i t h e l i u m . The need t o a n e s t h e t i z e the f i s h p r i o r t o h a n d l i n g a l s o appears to induce sudden d e f e c a t i o n and a c c e l e r a t i o n of i n t e s t i n a l t r a n s i t ( S p y r i d a k i s et al., 1989). Furthermore, samples are taken i n an i n t e s t i n a l segment e m p i r i c a l l y d e f i n e d by the i n v e s t i g a t o r and sometimes d i f f i c u l t t o reproduce from one i n d i v i d u a l t o another, p a r t i c u l a r l y f o r s t r i p p i n g or s u c t i o n . Another source of e r r o r may come from the d i s c o n t i n u i t y i n the time i n h e r e n t i n i n t e s t i n a l sampling. Indeed, the composition of feces has been shown to have s i g n i f i c a n t d i u r n a l v a r i a t i o n s (Inaba et al., 1962; De l a Noue et al., 1980; De S i l v a and Perera, 1983, 1984). F i n a l l y , i n t e s t i n a l sampling techniques are not a p p l i c a b l e t o s m a l l f i s h because only small amounts of sample can be c o l l e c t e d at any s i n g l e o c c a s i o n . Problems a s s o c i a t e d w i t h i n t e s t i n a l sampling could be avoided by c o l l e c t i n g the feces as soon as they are n a t u r a l l y r e l e a s e d i n t o the water. A d i f f e r e n t source of e r r o r i s introduced when feces are c o l l e c t e d from the water since s o l u b l e compounds can be l o s t and d i g e s t i b i l i t i e s overestimated (Inaba et al., 1962; Win d e l l et al., 1978a). With the aim of avoid i n g manipulation of f i s h as w e l l as minimizing l e a c h i n g l o s s e s , devices f o r the separation and recovery of feces have been proposed by numerous workers. Ogino et al. (1973), c o l l e c t e d feces by passing the e f f l u e n t water from the f i s h tanks through a f i l t r a t i o n column. In the "Guelph system" (Cho et al., 1975; Cho and S l i n g e r , 1979; Cho et al., 1982; M a r t i n e z - P a l a c i o s , 1988) the e f f l u e n t water from the tank passes through a s e t t l i n g column wit h an o u t l e t at the bottom so that s e t t l e d m a t e r i a l r e s t s i n undisturbed water before c o l l e c t i o n . Mechanical methods f o r c o l l e c t i o n of the feces range from a simple removal of the f e c a l p e l l e t s w i t h a f i n e mesh net (Windell et al., 1978a), to elaborate mechanically r o t a t i n g screen devices (Kaushik and Luquet, 1976; Choubert et al., 1979, 1982; De l a Noiie and Choubert, 1986) th a t f i l t e r out the f e c a l m a t e r i a l from the outflow water. D i s r u p t i o n of the f e c a l p e l l e t s i s an unfortunate drawback of the n e t t i n g and automatic f i l t e r i n g methods, as i s the high cost a s s o c i a t e d w i t h the l a t t e r . The obvious advantage of the methods mentioned above i s th a t f i s h are not handled and are allowed to feed undisturbed, although the aquarium tanks need t o be f l u s h e d c l e a r of uneaten food before feces are c o l l e c t e d . A f u r t h e r advantage i s that repeated samplings can be performed and growth r a t e s can be monitored during the d i g e s t i b i l i t y e v a l u a t i o n . .Concern s t i l l remains among n u t r i t i o n i s t s w i t h respect to the degree of n u t r i e n t l e a c h i n g which may occur w i t h the various methods i n which feces are c o l l e c t e d from the aquarium water. Data from Windell et al. (1978a), showed th a t most n u t r i e n t l e a c h i n g from feces occurs during the f i r s t hour a f t e r d e f e c a t i o n . Systems which remove the feces from the water s w i f t l y are claimed by some authors t o cause minimum l e a c h i n g (Choubert et al., 1979, 1982/ Schmitz et al., 1983), while others c l a i m t h a t d i s r u p t i o n and breakage of f e c a l p e l l e t s during c o l l e c t i o n are the major cause of n u t r i e n t l e a c h i n g (Cho and S l i n g e r , 1979/ Cho et al., 1982/ Vens-Cappell, 1985). Indeed, Cho and S l i n g e r (1979), Cho et al. (1982) and Talbot (1985) showed th a t r e s u l t s obtained by i n t e s t i n a l d i s s e c t i o n and by anal s u c t i o n were not s i g n i f i c a n t l y d i f f e r e n t from the r e s u l t s obtained by the "Guelph system" f o r the d i g e s t i b i l i t y of dry matter, crude p r o t e i n and l i p i d s . This would i n d i c a t e that n u t r i e n t l e a c h i n g was not an important source of e r r o r w i t h t h i s system. 35 2.14 E v a l u a t i o n of f e e d s t u f f d i g e s t i b i l i t y R e l a t i v e l y few d i e t a r y components can s a t i s f a c t o r i l y be provided as the sole d i e t . Consequently, i t i s impossible to determine the d i g e s t i b i l i t y of a m a j o r i t y of feed i n g r e d i e n t s unless they are combined wi t h other feed components. Determination of the d i g e s t i b i l i t y of a t e s t i n g r e d i e n t r e q u i r e s comparing the d i g e s t i b i l i t y of a reference d i e t and a t e s t d i e t ; the t e s t d i e t being a known mixture of the reference d i e t and t e s t i n g r e d i e n t . This procedure assumes th a t there i s no i n t e r a c t i o n between the components of the d i e t during d i g e s t i o n , while at the same time a l l o w i n g the pr e p a r a t i o n of an adequately balanced diet, i n which to t e s t the s u s c e p t i b i l i t y of the t e s t i n g r e d i e n t t o d i g e s t i o n . This procedure has been widely used i n t e r r e s t r i a l animals as w e l l as i n f i s h (Cho- and S l i n g e r , 1979; Cho et a l . , 1982; Wilson and Poe, 1985). 36 CHAPTER 3 3.0 EXPERIMENT I - D i g e s t i b i l i t y measurements i n j u v e n i l e chinook salmon (Oncorhynchus tshawytscha) i n seawater: Comparison of the d i r e c t ( t o t a l ) and i n d i r e c t (C^C^) methods of determining d i g e s t i b i l i t y and r e l i a b i l i t y of d i g e s t i o n c o e f f i c i e n t s i n r e l a t i o n to techniques used i n f e c a l c o l l e c t i o n . 3.1 INTRODUCTION Before any f e e d s t u f f can be p r o p e r l y evaluated i n terms of i t s n u t r i e n t d i g e s t i b i l i t y by a given animal, a dependable method r f o r measuring d i g e s t i b i l i t y c o e f f i c i e n t s must be i n p l a c e . C o l l e c t i o n of the t o t a l f e c a l matter produced a f t e r consumption of a known amount of feed or measurement of the concentration of an i n d i g e s t i b l e marker (e.g. chromic oxide) i n the feed and feces have both been e x t e n s i v e l y used. The d i r e c t or t o t a l c o l l e c t i o n method i s very tedious and labour i n t e n s i v e and r e q u i r e s great care i n feeding, f e c a l c o l l e c t i o n and measurement of s p i l l a g e , although proper experimental procedures w i l l give very r e l i a b l e r e s u l t s . The i n d i r e c t method using chromic oxide has been developed to obviate q u a n t i t a t i v e c o l l e c t i o n problems. The use of i n e r t markers i n the feed, however, has not been c o n s i s t e n t l y s a t i s f a c t o r y . Major l i m i t a t i o n s to the use of chromic oxide are the v a r i a t i o n of i t s concentration i n the feces and incomplete recovery of the i n d i c a t o r , r e s u l t i n g i n v a r i a b l e r e s u l t s and reduced d i g e s t i o n c o e f f i c i e n t s i n f i s h (Bowen, 1978; F o l t z , 1979) and other species (Knapka et al., 1967; Hattan and 37 Owen, 1970). Hence, i n t h i s study, the f i r s t o b j e c t i v e was to compare the d i g e s t i b i l i t y data obtained by both the d i r e c t and i n d i r e c t methods i n f i s h r a i s e d i n seawater. The greatest problem w i t h both methods i s the d i f f i c u l t y of ob t a i n i n g r e p r e s e n t a t i v e f e c a l samples from the aquatic medium. For example, the various techniques employed f o r recovery of the feces from the water have been c r i t i c i z e d because of n u t r i e n t l e a c h i n g . By c o n t r a s t , those i n v o l v i n g d i r e c t removal of f e c a l matter from the lower i n t e s t i n e are subject t o e r r o r because of contamination of the feces w i t h body f l u i d s and incomplete n u t r i e n t d i g e s t i o n and absorption. The d i g e s t i b i l i t y r e s u l t s obtained by Cho and S l i n g e r (197 9) using the "Guelph system", i n d i c a t e t h a t n u t r i e n t l e a c h i n g i s i n s i g n i f i c a n t when feces of rainbow t r o u t are c o l l e c t e d a f t e r s e t t l i n g overnight i n a column of undisturbed water. More r e c e n t l y , however, S p y r i d a k i s et al. (1989) working w i t h sea bass (Dicentrarchus labrax), showed t h a t , compared to other f e c a l c o l l e c t i o n techniques, the "Guelph system" gave the highest p r o t e i n d i g e s t i b i l i t y values. The same authors a t t r i b u t e d the le a c h i n g losses to a flaw i n t h e i r tank design which prevented the feces from being r a p i d l y c a r r i e d t o the s e t t l i n g column. The second goal of t h i s study was t h e r e f o r e to assess the v a l i d i t y of the "Guelph system" of f e c a l c o l l e c t i o n by comparing d i g e s t i b i l i t y values obtained from feces c o l l e c t e d w i t h t h i s system to those obtained from feces c o l l e c t e d by the s t r i p p i n g and i n t e s t i n a l d i s s e c t i o n techniques. As part of t h i s g oal, the e f f e c t of f e c a l water-exposure time on n u t r i e n t 38 l e a c h i n g was a l s o assessed. The f i n a l goal of t h i s experiment was to measure the amount of endogenous f e c a l p r o t e i n excreted by f i s h fed a p r o t e i n - f r e e d i e t . This approach would enable the r e s u l t s to be presented on a "t r u e " d i g e s t i b i l i t y b a s i s . 39 3.2 MATERIALS AND METHODS 3.2.1 Experimental design Twenty j u v e n i l e chinook salmon (mean weight of 55.3 ± 0.50 g (SEM)) were d i s t r i b u t e d randomly i n t o each of nine d i g e s t i b i l i t y tanks. The f i s h i n s i x of the groups were fed a c o n t r o l d i e t (containing 0.45% chromic oxide on a dry-matter basis) by hand twice d a i l y on a r e s t r i c t e d b a s i s whereas the remaining three tanks were o f f e r e d a p r o t e i n - f r e e d i e t . But the l a t t e r d i e t was not r e a d i l y consumed by any of the groups during the a c c l i m a t i o n p e r i o d and consequently t h i s treatment was d i s c o n t i n u e d . Instead, each of the three groups was fed the c o n t r o l d i e t to s a t i a t i o n to i n v e s t i g a t e the e f f e c t of feeding l e v e l on n u t r i e n t d i g e s t i b i l i t y . To p r o p e r l y acclimate these f i s h to t h e i r new (control) d i e t and feeding p r o t o c o l , t h e i r feces were c o l l e c t e d eight days a f t e r i n i t i a t i o n of f e c a l c o l l e c t i o n i n the s i x groups fed the r e s t r i c t e d r a t i o n . Feces were q u a n t i t a t i v e l y c o l l e c t e d from a l l nine tanks twice d a i l y (0830 hours and 1430 hours) over a p e r i o d of three weeks ( s i x groups) or two weeks (three groups) using the "Guelph system". Since the feces contained chromic oxide, comparisons were made between the d i r e c t and i n d i r e c t methods of determining d i g e s t i b i l i t y . Further, because the feces were c o l l e c t e d twice d a i l y , i t was p o s s i b l e to monitor the extent of n u t r i e n t l e a c h i n g between the c o l l e c t i o n times. In a d d i t i o n to the above methodology comparisons, i t was a l s o of i n t e r e s t to compare the d i g e s t i b i l i t y c o e f f i c i e n t s based on the "Guelph system" of f e c a l c o l l e c t i o n versus those based on removal of feces by- s t r i p p i n g . In t h i s regard, the feces from 40 f i s h i n three of the s i x groups fed on a r e s t r i c t e d b a s i s were randomly c o l l e c t e d by the "Guelph system" but al s o by the s t r i p p i n g technique r e f e r r e d t o p r e v i o u s l y . The l a t t e r procedure i n v o l v e s a n e s t h e t i z i n g and s t r i p p i n g a l l f i s h i n each of the three groups once a week f o r three weeks. F i n a l l y , at the end of the experiment, a l l f i s h not p r e v i o u s l y s t r i p p e d were euthenized and the contents of t h e i r lower i n t e s t i n a l t r a c t between the p e l v i c f i n s and the anus were d i s s e c t e d out, t o permit f u r t h e r methodology comparisons. 3.2.2 D i g e s t i b i l i t y tanks The o r i g i n a l "Guelph system" developed by Cho and S l i n g e r (1977) f o r d i g e s t i b i l i t y determinations i n t r o u t e n t a i l s the c o l l e c t i o n of feces from f i s h h e l d i n groups of three interconnected 7 7 - l i t e r tanks by means of a common s e t t l i n g tube f o r each set of three tanks l o c a t e d at the outflow. This design was modified i n the present study on chinook salmon since t h i s species i s more subject to s t r e s s of confinement than t r o u t . Therefore, 150-L f i b r e g l a s s tanks were s p e c i a l l y designed and f a b r i c a t e d t o permit r a p i d e x i t of f e c a l p e l l e t s . Moreover, a separate f e c a l c o l l e c t i o n column of the Guelph design was a f f i x e d onto each tank as shown i n Figure 1. Funnels f i t t e d w i t h a f i n e p o l y e s t e r f a b r i c mesh were placed at the overflow of each tank to ensure t h a t the smaller, l e s s dense f e c a l p e l l e t s which were c a r r i e d out of the system, were a l s o i n c l u d e d i n the c o l l e c t i o n , sample. 41 in f lowing f i l t e r e d seawater to drain c o l l e c t i o n column c o l l e c t i o n port t ank -co l l ec t i on column connection pipe •feces Figure 1. Diagram showing the "Guelph system" of feca l c o l l e c t i o n used in experiments I, I I , III and IV. 42 3.2.3 Aquarium f a c i l i t y The experiment was conducted at the U n i v e r s i t y / D.F.O. aquarium f a c i l i t y l o c a t e d adjacent t o the Department of F i s h e r i e s and Oceans (D.F.O.), West Vancouver Laboratory. Each of the d i g e s t i b i l i t y tanks was s u p p l i e d w i t h 6 t o 8 l i t e r s per minute of f i l t e r e d Burrard I n l e t seawater (26 to 31 ppt) i n a flow through system. The water flow r a t e was c a r e f u l l y balanced to not only ensure the r a p i d removal of f e c a l matter from each tank, but a l s o to ensure a gentle s e t t l i n g of the feces by g r a v i t y i n t o the c o l l e c t i o n column w i t h minimal l o s s v i a the overflow. Water temperature v a r i e d by approximately 1°C d i u r n a l l y and ranged between 5°C and 8°C throughout the experimental p e r i o d . A e r a t i o n stones i n each tank maintained the water oxygen l e v e l c l o s e to s a t u r a t i o n . Overhead 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 Durotest 40W) c o n t r o l l e d by a time c l o c k were used to simulate a n a t u r a l photoperiod. 3.2.4 Water f i l t e r i n g system F i l t e r s were constructed f o r each of the tanks i n order to prevent sand, s h e l l s , algae and l i v e organisms such as copepods which are commonly c a r r i e d i n w i t h the pumped seawater, from e n t e r i n g the tanks. The f i l t e r s were constructed from 7.62 cm (I.D.) p o l y v i n y l c h l o r i d e (PVC) p i p e l i n e cut i n t o 30 cm lengths. Removable screw-in l i d s were attached to both ends of the pipe t o allow c l e a n i n g or r e p l a c i n g of the f i l t e r i n g m a t e r i a l , which i n t h i s case was f i n e (3-5 mm) aquarium grade g r a v e l . Each l i d was d r i l l e d and threaded at the center to f a c i l i t a t e the attachment o f t h e f i l t e r t o t h e 1.91 cm (O.D.) i n c o m i n g water s u p p l y p i p e l i n e a t one end, and t h e f l o w c o n t r o l v a l v e a t t h e o u t f l o w end. To p r e v e n t l o s s o f g r a v e l from t h e f i l t e r t h r o u g h e i t h e r end, c i r c l e s (7.60 cm i n diameter) were c u t out o f a 2.38 mm p e r f o r a t e d PVC sheet and p l a c e d between t h e g r a v e l and t h e l i d s . A f t e r a p r e l i m i n a r y r u n , i t became apparent t h a t t h e g r a v e l f i l t e r was not v e r y e f f e c t i v e i n p r e v e n t i n g f i n e a l g a e p a r t i c l e s and s m a l l copepods from e n t e r i n g t h e t a n k s . S u b s e q u e n t l y , a f i n e V e x l a r s c r e e n (mesh opening < 500um) was sandwiched between two PVC p e r f o r a t e d c i r c l e s a t t h e o u t f l o w end o f t h e f i l t e r . I n a d d i t i o n , water l e a v i n g t h e f i l t e r was d i r e c t e d i n t o a p l a s t i c f u n n e l f i t t e d w i t h a v e r y f i n e p o l y e s t e r f a b r i c mesh (mesh opening a p p r o x i m a t e l y 250 um) h e l d i n p l a c e w i t h r u b b e r bands. The " f u n n e l f i l t e r " was v e r y e f f e c t i v e i n r e t a i n i n g f i n e s i l t . R i n s i n g t h e f a b r i c mesh t w i c e d a i l y e n s u r e d t h a t p r o p e r water f l o w was m a i n t a i n e d . The g r a v e l f i l t e r , on t h e o t h e r hand, had t o be r e p l a c e d e v e r y t h r e e days. 3.2.5 D i e t p r e p a r a t i o n and c o m p o s i t i o n The f o r m u l a e o f t h e c o n t r o l and p r o t e i n - f r e e d i e t s are shown i n T a b l e 1. The m i n e r a l supplement o f t h e p r o t e i n - f r e e d i e t was p r e p a r e d t o e q u a l t h e m i n e r a l c o m p o s i t i o n o f t h e c o n t r o l d i e t . M i n e r a l c o n t e n t s were d e t e r m i n e d by plasma e m i s s i o n s p e c t r o s c o p y (ICAP) by Quanta Trace L a b o r a t o r i e s I n c . ( B u r n a b y , B r i t i s h C o l u m b i a ) . The p r o x i m a t e c o m p o s i t i o n and major m i n e r a l c o n t e n t s o f t h e d i e t s are l i s t e d i n Table 2. M i n e r a l supplements were ground t o a f i n e powder i n a p o r t a b l e c o f f e e g r i n d e r p r i o r t o w e i g h i n g . They were t h e n i n d i v i d u a l l y weighed and mixed f o r 45 minutes i n a Twin S h e l l Dry Blender (P a t t e r s o n - K e l l e y Co., D i v i s i o n of Harsco Corp., Pennsylvania) using ©^-cellulose (ICN N u t r i t i o n a l Biochemicals, Cleveland, Ohio.) as the c a r r i e r . The same procedure was followed f o r the v i t a m i n supplements, although g r i n d i n g was not req u i r e d . A l l f e e d s t u f f s used were commercially obtained and, i f necessary, were ground i n a hammer m i l l ( F i t z m i l l , model JT; The F i t z p a t r i c k Company, Elmhurst I l l i n o i s , U.S.A.) equipped w i t h a s i z e 30 U.S. screen (595 um). Ingredients f o r each d i e t were blended (with a p o r t i o n of the o i l ) f o r 20 minutes i n a Hobart commercial mixer (Hobart Manufacturing Co., Troy, Ohio). Chromic oxide (Cr203) was combined w i t h a p o r t i o n of the i n g r e d i e n t s and ground i n a mortar and p e s t l e u n t i l a l l lumps had d i s i n t e g r a t e d . Vitamin and mineral pre-mixes were added to the r e s p e c t i v e d i e t s and combined i n the Hobart mixer f o r an a d d i t i o n a l 20 minutes. Each d i e t was then steam-pelleted i n a C a l i f o r n i a model CL-type 2 la b o r a t o r y p e l l e t m i l l ( C a l i f o r n i a P e l l e t M i l l Co., San Fra n c i s c o , C a l i f o r n i a ) w i t h a 3.18 mm d i e . Immediately f o l l o w i n g p e l l e t i n g , d i e t s were placed i n a v e r t i c a l c o o l e r . A f t e r c o o l i n g , f i n e s were, screened out and the remainder of the o i l was sprayed on t o the tumbling p e l l e t s i n s i d e a cement-mixer using an e l e c t r i c a l l y operated spray-gun. The d i e t s were kept i n dark p l a s t i c bags i n a r e f r i g e r a t o r (3°C-4°C) throughout the experiment. 45 TABLE 1. Composition of c o n t r o l and p r o t e i n - f r e e d i e t s (g/kg dry d i e t ) . Ingredients C o n t r o l d i e t P r o t e i n - f r e e Steam-dried h e r r i n g meal 550 . 00 -Wheat middlings 126. 80 -Dried whey 7 5 . 00 -Blood meal 5 0 . 00 -Herring oil-*- 115 . 00 178 .00 M i n e r a l supplement 2 2 0 . 00 133 .70 D e x t r i n - 313 .15 Glucose - 313 .15 Vitamin supplement^ 3 5 . 00 35 .00 Carboxy-methyl c e l l u l o s e 1 5 . 00 15 .00 Choline c h l o r i d e (60%) 5 . 00 5 .00 Chromic oxide 5 . 00 5 .00 A s c o r b i c a c i d 2 . 00 2 . 0 0 DL-methionine 1. 20 -S t a b i l i z e d w i t h 0.025% ethoxyquin. The mineral supplement provided the f o l l o w i n g amounts per kg of dry d i e t (values i n brackets are the values f o r the p r o t e i n - f r e e d i e t ) : magnesium as MgSC^^^O, 250 mg (1.735 g) ; i r o n as FeSC^^HpO, 75 mg (480.5 mg) ; z i n c as ZnS04»7H20, 60mg (192 mg) ; manganese as MnSC^'^O, 75 mg (111 mg); copper as CuS04»5H20, 6 mg (22.5 mg); f l u o r i n e as NaF, 4.5 mg (4.5 mg); i o d i n e as KIO3, 5 mg (5 mg); cobal t as CoCl2*6H 20 / 1 mg (2.75 mg); selenium as Na2SeC>3, 0.10 mg (0.10 mg) ; calcium as CaHP0 4, (14.268 g) and as CaCC>3 (5.481 g) ; sodium c h l o r i d e , (5.095 g) ; potassium as K2SO4, (3.865 g) and as KH2PO4, (3.865 g); aluminum as A1C1 3»6H 20, (5 mg). A t o t a l of 16.55 g/kg (7.92 g/kg f o r the p r o t e i n - f r e e d i e t ) of o c - c e l l u l o s e was used as the c a r r i e r . The v i t a m i n supplement provided the f o l l o w i n g (mg/kg of dry d i e t unless otherwise i n d i c a t e d ) : i n o s i t o l 400; n i a c i n 300; pantothenate as D-calcium pantothenate 166; menadione as hetrazeen 26.1; r i b o f l a v i n 60; py r i d o x i n e as pyridoxine-HCl 36.5; thiamine as thiamine mononitrate 36.3; f o l i c a c i d 20; vi t a m i n B-12 0.06; r e t i n o l acetate 10,000 IU; c h o l e c a l c i f e r o l 2,400 IU; dl-«=-tocopheryl acetate 600 IU. A t o t a l of 32.38 g/kg of ^ - c e l l u l o s e was used as the c a r r i e r . 46 3.2.6 Experimental procedures and sampling 3.2.6.1 F i s h h i s t o r y and d i s t r i b u t i o n Chinook salmon (Oncorhynchus tshawytscha) f r y were obtained i n March, 1988 from the Robertson Creek hatchery (Vancouver I s l a n d , B.C.) and t r a n s f e r r e d i n t o 3,600 l i t e r f i b e r g l a s s tanks h e l d outdoors. Well water was g r a d u a l l y replaced by Burrard I n l e t seawater i n mid J u l y . The f i s h were i n s a l t water (28 to 31 ppt) approximately 10 days l a t e r . F i s h were p e r i o d i c a l l y removed f o r other experiments and s t o c k i n g d e n s i t y was consequently maintained below approximately 7.5 kg/m^. The Oregon moist p e l l e t d i e t (OMP) was fed by hand t o s a t i a t i o n twice d a i l y . On January 16, 198 9 f i s h were s e l e c t e d randomly and anesthetized w i t h 2-phenoxyethanol (0.5 ml/L water), placed on an absorbent c l o t h t o remove excess moisture, and i n d i v i d u a l l y weighed to the nearest gram. Those f i s h weighing between 45 g and 65 g were separated from the r e s t and were randomly d i s t r i b u t e d i n t o one row of 9 d i g e s t i b i l i t y tanks u n t i l there were 25 f i s h per tank. Feeding was resumed by the next day and feces (chromic oxide-free) were c o l l e c t e d from each s e t t l i n g column f o r futu r e use i n the chromic oxide analyses. At the end of a two-week a c c l i m a t i o n p e r i o d , a l l f i s h were removed from the d i g e s t i b i l i t y tanks, anesthetized and reweighed. A po p u l a t i o n of 180 f i s h was s e l e c t e d f o r uniform s i z e w i t h a mean weight of 55.3g ± 0.50 (SEM). I n d i v i d u a l f i s h weights were recorded to the nearest O.lg. 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 groups of 2, t o provide 20 f i s h t o each of the 9 d i g e s t i b i l i t y tanks. 47 3.2.6.2 Experimental p r o t o c o l A l l f i s h i n the d i g e s t i b i l i t y tanks were weaned from the OMP d i e t to the c o n t r o l d i e t over a two day p e r i o d . The presence of chromic oxide i n the feces was f i r s t observed approximately 56 hours a f t e r i t s i n i t i a l i n g e s t i o n . To minimize feed wastage during the experiment, the amount of ingested feed which would s a t i s f y a p p e t i t e had t o be determined. For the next seven days, f i s h were fed by hand twice d a i l y u n t i l " a c t i v e " feeding ceased over a feeding 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 feeding was s i x hours. A p r e c i s e record of feed consumption at each feeding p e r i o d was maintained as described i n Appendix 2. Three days before q u a n t i t a t i v e f e c a l c o l l e c t i o n s began, the mean d a i l y feed consumption f o r f i s h i n each tank was c a l c u l a t e d and feed allowances were reduced by 10% to minimize feed wastage. At t h i s stage, f i s h i n three randomly s e l e c t e d tanks were switched from the c o n t r o l d i e t t o the p r o t e i n - f r e e d i e t . F o l l o w i n g the l a s t feeding on day 0, tanks were f l u s h e d c l e a r of feces and feed i n p r e p a r a t i o n f o r the s t a r t of f e c a l c o l l e c t i o n s the next morning. Four days i n t o the experiment, i t was decided to abort the use of the p r o t e i n - f r e e d i e t as the f i s h , although obviously hungry, refused t o consume any of the p e l l e t s . F i s h on the c o n t r o l d i e t , however, continued t o consume t h e i r d a i l y r a t i o n w e l l and they produced adequate q u a n t i t i e s of feces. The p r o t e i n - f r e e d i e t was subsequently replaced by the c o n t r o l d i e t . To evaluate the e f f e c t of feeding l e v e l on n u t r i e n t d i g e s t i b i l i t y , the feed i n t a k e of the f i s h formerly on 48 the p r o t e i n - f r e e d i e t was no longer r e s t r i c t e d , the c o n t r o l d i e t being fed to s a t i a t i o n twice d a i l y .throughout the study. F i s h fed e n t h u s i a s t i c a l l y and f e c a l c o l l e c t i o n s from these tanks were resumed four days l a t e r . 3.2.6.3 F e c a l c o l l e c t i o n techniques 3.2.6.3.1 "Guelph system" Immediately a f t e r the l a s t feeding on the day p r i o r to the s t a r t of f e c a l c o l l e c t i o n s , the drain p i p e and the s e t t l i n g column of a l l nine tanks were brushed out t o remove feed and f e c a l residues from the system. The tanks were g e n t l y brushed clean while o n e - t h i r d of the water was drained out t o ensure the system had been c l e a r e d of a l l p o s s i b l e contaminants. On the f i r s t day of c o l l e c t i o n and f o r the remainder of the study, feces were q u a n t i t a t i v e l y c o l l e c t e d at 0830 and 1430 hours d a i l y as described i n Appendix 1. Thus the feces c o l l e c t e d at 0830 hours remained i n the water f o r a maximum of 18 hours (depending on when the feces had been voided by the f i s h ) , w hile those c o l l e c t e d at 1430 hours remained i n the water f o r a maximum of 6 hours. Feeding was begun immediately a f t e r each c o l l e c t i o n and refused feed p e l l e t s were recovered and accounted f o r as described i n Appendix 2. The c o l l e c t e d feces were c e n t r i f u g e d at 10,000 x g f o r 30 minutes at approximately 5°C and the supernatant discarded. Each f e c a l c o l l e c t i o n sample was sto r e d i n a sealed c e n t r i f u g e b o t t l e at -40°C. 49 3.2.6.3.2 S t r i p p i n g technique F i s h i n three tanks were anesthetized once a week and t h e i r feces were removed by applying gentle pressure w i t h the f i n g e r s between the p e l v i c f i n s and the anus as described by Singh and Nose (1967) and Nose (1967). S t r i p p e d f e c a l matter was c o l l e c t e d onto a lar g e piece of absorbent f i l t e r paper t o remove as much of the u r i n e as p o s s i b l e . Given the small q u a n t i t y of feces c o l l e c t e d , a l l f e c a l samples c o l l e c t e d by t h i s technique were pooled and st o r e d i n sealed v i a l s at -40°C. Immediately a f t e r f e c a l removal, the f i s h were returned t o t h e i r r e s p e c t i v e tanks and q u a n t i t a t i v e f e c a l c o l l e c t i o n with the "Guelph system" was continued. The amount of feces c o l l e c t e d by the s t r i p p i n g method was l a t e r taken i n t o account when c a l c u l a t i o n s of n u t r i e n t d i g e s t i b i l i t y based on the t o t a l c o l l e c t i o n procedure were performed. 3.2.6.3.3 I n t e s t i n a l d i s s e c t i o n technique Upon culmination of the experiment on day 22, a l l f i s h were euthenized using 2 ml of 2-phenoxyethanol/L of water, b l o t t e d dry on a towel and weighed. A l l f i s h , e xcluding those p r e v i o u s l y s t r i p p e d , had the contents of t h e i r lower i n t e s t i n e between the p e l v i c f i n s and the anus removed by d i s s e c t i o n . Blood was c a r e f u l l y removed by absorbent towel from the abdominal c a v i t y p r i o r to and a f t e r severing the i n t e s t i n e t o minimize f e c a l contamination. A l l c o l l e c t i o n s obtained i n t h i s manner were pooled together and st o r e d at -40°C i n a sealed p l a s t i c v i a l . 50 3.2.7 Chemical analyses of feed and feces 3.2.7.1 Proximate composition and gross energy determination Frozen f e c a l samples were l y o p h i l i z e d and ground using a mortar and p e s t l e . F i s h s c a l e s contaminating the feces c o l l e c t e d using the "Guelph system" were removed p r i o r to weighing using a 250 um s i e v e and a l l necessary sample t r a n s f e r s were performed q u a n t i t a t i v e l y using a f i n e camel h a i r brush. The morning f e c a l c o l l e c t i o n s (0830 hours) f o r each i n d i v i d u a l tank were pooled on a weekly b a s i s , as were those f o r the afternoon c o l l e c t i o n s (1430 hours). Feed and f e c a l , samples were analyzed f o r moisture and ash (AOAC, 1975) and t o t a l n i t r o g e n (Technicon Instrumental Co. L t d . , i n d u s t r i a l methods 369-75A/A and 334-74W/B)... The percent n i t r o g e n was m u l t i p l i e d by 6.25 to estimate crude p r o t e i n content. Energy content was determined by the procedure of P h i l l i p s o n (1964) and i n v o l v e d the use of a microbomb c a l o r i m e t e r manufactured by Gentry Instruments Inc., Aiken, South C a r o l i n a , U.S.A.. Crude l i p i d content of the d i e t s was determined by the B l i g h and Dyer (1959) method. A l l determinations were done i n d u p l i c a t e . 3.2.7.2 Chromic oxide determinations F o l l o w i n g extensive t e s t i n g of methods a v a i l a b l e f o r the determination of chromic oxide, such as those by Fenton and Fenton (1979) and L i e d et al. (1982), the wet d i g e s t i o n c o l o r i m e t r i c method of Stevenson and De Langen (1960) was chosen as the p r e f e r r e d method. Recoveries of chromic oxide were determined a f t e r a d d i t i o n of known amounts of the marker to a dry 51 d i e t mash mixture. A stock mixture c o n t a i n i n g 5.0 mg C^C^ per gram of mash was prepared by adding 5.0 g of dry chromium sesquioxide (Fisher S c i e n t i f i c Co. Chemical Manufacturing D i v i s i o n , F a i r Lawn, New Jersey) to 995.OOg of a f i n e l y ground dry d i e t mash. A mortar and p e s t l e was used i n the mixing process to ensure that no lumps remained. A homogeneous mix was achieved by f u r t h e r blending i n a small Hobart mixer f o r an a d d i t i o n a l 90 minutes. Sub stocks of 3, 2 and 1 mg C^C^/g mash were prepared by t a k i n g a l i q u o t s of the stock mix and mixing w i t h mash d i e t as described above. 3.2.8 C a l c u l a t i o n s of apparent d i g e s t i b i l i t y and f i s h performance Data on feed and feces composition were used to c a l c u l a t e apparent d i g e s t i b i l i t y c o e f f i c i e n t s of organic matter, crude p r o t e i n and gross energy. Two c a l c u l a t i o n methods were used, each r e s u l t i n g i n one set of c o e f f i c i e n t s . The apparent d i g e s t i b i l i t y c o e f f i c i e n t s according to the d i r e c t method (ADCd) and the i n d i r e c t method (ADCi) were c a l c u l a t e d as f o l l o w s : ADCd (%) = 100 1-feces dry weight x % n u t r i e n t i n feces d i e t fed weight x % n u t r i e n t i n d i e t ADCi (%) = 100 % Cr203 i n d i e t x % n u t r i e n t i n feces % C^C^ i n feces x % n u t r i e n t i n d i e t 52 The above formulae were al s o used f o r the determination of apparent gross energy d i g e s t i b i l i t y c o e f f i c i e n t s , w i t h gross energy data (MJ/kg) r e p l a c i n g the n u t r i e n t term i n the formulae. To determine weekly apparent d i g e s t i b i l i t y c o e f f i c i e n t s based on the i n d i c a t o r method, the apparent n u t r i e n t d i g e s t i b i l i t y c o e f f i c i e n t s d e r i v e d from weekly pooled morning and weekly pooled afternoon samples had t o be combined. However, since the q u a n t i t y of f e c a l organic matter c o l l e c t e d i n the morning d i f f e r e d from that c o l l e c t e d i n the afternoon, the apparent d i g e s t i b i l i t y c o e f f i c i e n t s f o r the morning and afternoon samples were m u l t i p l i e d by t h e i r p r o p o r t i o n a t e c o n t r i b u t i o n to the weekly f e c a l organic matter output. S p e c i f i c growth r a t e , which describes the percent increase i n body weight per day (Bret t , 1979) was c a l c u l a t e d as: (logn t l - logn tO) x 100 ( t l - tO) Logn t l and logn tO are the n a t u r a l l o g wet weights at the s t a r t (tO) and the end ( t l ) of the p e r i o d . Using data f o r the mean d a i l y dry feed intake (DFI) per f i s h f o r the p e r i o d , feed in t a k e was expressed as a percentage of mean wet body weight according to Richardson et a l . (1985). The formula used was as f o l l o w s : DFI (logn t l + logn tO - x 100 2 ) e 53 3.2.9 S t a t i s t i c a l analyses The a n a l y s i s of the experiment was performed i n two p a r t s . Part one was the a n a l y s i s of the d i r e c t versus the i n d i r e c t method of determining apparent n u t r i e n t d i g e s t i b i l i t y c o e f f i c i e n t s . This p a r t was analyzed as a 2 x 3 x 3 f a c t o r i a l experiment. The eighteen treatments r e s u l t e d from a combination of two methods of determining d i g e s t i b i l i t y ( d i r e c t vs i n d i r e c t ) , three f i s h management techniques ( r e s t r i c t e d f e e d i n g - f i s h not handled vs r e s t r i c t e d f e e d i n g - f i s h handled weekly vs s a t i a t i o n f e e d i n g - f i s h not handled) and three time r e p l i c a t i o n s (week 1 vs week 2 vs week 3). Part two was the a n a l y s i s of the e f f e c t of time between f e c a l c o l l e c t i o n s on n u t r i e n t l e a c h i n g . Part two was al s o analyzed as a 2 x 3 x 3 f a c t o r i a l w i t h two f e c a l c o l l e c t i o n times (morning vs afternoon), three f i s h management techniques ( r e s t r i c t e d f e e d i n g - f i s h not handled vs r e s t r i c t e d f e e d i n g - f i s h handled weekly vs s a t i a t i o n f e e d i n g - f i s h not handled) and three time r e p l i c a t i o n s (week 1 vs week 2 vs week 3). The analyses are t a b u l a t e d i n Appendix 4. Before performing a n a l y s i s of variance 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 between treatments, i t was f i r s t necessary t o confirm t h a t the assumptions regarding e q u a l i t y of variance between the treatments were tru e and Ha r t l e y ' s t e s t was used f o r t h i s purpose as described by Lyman (1984). Where necessary, the data were transformed by a r c s i n square root (Steel and T o r r i e , 1960). Data i n each f a c t o r i a l were subjected to a n a l y s i s of variance and t o Newman-Keuls (Keuls, 1952) m u l t i p l e range t e s t , u sing the SAS (1985) computer program. Apparent d i g e s t i b i l i t y c o e f f i c i e n t s f o r organic matter, crude p r o t e i n and gross energy, based on the s t r i p p i n g and i n t e s t i n a l d i s s e c t i o n samples, were c a l c u l a t e d according to the i n d i r e c t method but could not be compared s t a t i s t i c a l l y due to lack of r e p l i c a t e s . S p e c i f i c growth r a t e s and feed i n t a k e data were a l s o subjected to a n a l y s i s of variance procedures and t o Newman-Keuls m u l t i p l e range t e s t using the SAS computer program. 55 3.3 RESULTS 3.3.1 D i e t s The r e s u l t s of proximate and chemical analyses of the d i e t s are given i n Table 2. Only small v a r i a t i o n s between the two d i e t s were found i n the l i p i d , ash, moisture and major mineral content. The t e x t u r e however, v a r i e d g r e a t l y between d i e t s . The p r o t e i n - f r e e d i e t p e l l e t s were extremely hard and the l i p i d sprayed on a f t e r p e l l e t i n g was not absorbed but i n s t e a d coated each p e l l e t w i t h a heavy l a y e r of o i l . Some 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 were observed to take a p e l l e t i n t o t h e i r mouth and to immediately e j e c t i t , w hile others only l o s t i n t e r e s t a f t e r repeated attempts t o masticate the p e l l e t s f a i l e d . 3.3.2 Chromic oxide a n a l y s i s The r e s u l t s of the chromic oxide r e c o v e r i e s obtained w i t h the wet ashing c o l o r i m e t r i c method of Stevenson and De Langen (1960) are provided i n Table 3. In general, more than 94% (mean of a l l concentrations = 96.5%) of the chromic oxide was recovered. Small l o s s e s of chromic oxide, regardless of the t e s t c o ncentration, may have been due to l o s s e s while mixing as w e l l as the l a c k of p u r i t y of the chromic oxide used. Nevertheless, best r e c o v e r i e s were a t t a i n e d when the chromic oxide content of the sample was between 3.2 and 4.0 mg. The s i z e of subsequent samples of feed and feces being analyzed f o r C^C^, was t h e r e f o r e adjusted to y i e l d a marker content which would f a l l approximately w i t h i n t h i s range. The chromic oxide concentration of the c o n t r o l d i e t was determined to be 4.49 mg/g dry d i e t ± 0.114 (SD) and the c o e f f i c i e n t of v a r i a t i o n was c a l c u l a t e d to be 2.45% ( n = l l ) . 57 TABLE 2. Proximate composition, energy content and mineral content of the c o n t r o l and p r o t e i n - f r e e d i e t s of Experiment 1. DIET A n a l y s i s C o n t r o l P r o t e i n - f r e e Proximate composition (% of dry matter) P r o t e i n (%N x 6.25) 52.12 0.0 Crude l i p i d (Bligh-Dyer) 17.25 17.30 Ash 10.58 11.33 Moisture (as fed) 7.30 8.55 Gross Energy (MJ/kg) 23.03 — M i n e r a l c o n t e n t x (g/kg dry di e t ) Calcium (Ca) 24.0 24.0 Phosphorus (P) 14.7 16.9 Sodium (Na) 5.51 7.13 Chromic oxide (C^C^) 2 4.49 4.87 Magnesium (Mg) 1.86 2.05 Copper (Cu) 0.014 0.024 Iron (Fe) 0.319 0.567 Manganese (Mn) 0.094 0.121 Zinc (Zn) 0.151 0.226 Cobalt (Co) <0.0005 0.002 Ca:P 1.63 1.42 Determined by plasma emission spectroscopy (Higgs et al., 1982) . Determined by the wet d i g e s t i o n c o l o r i m e t r i c method of Stevenson and De Langen (1960). 58 Table 3. Recovery of chromic oxide from a feed mash mixture using the wet ashing c o l o r i m e t r i c method of Stevenson and De Langen (1960). Mg C r 2 0 3 assayed Recoveries mg % 1.36 1.20 88.2 1.86 1.87 100.5 2.71 2.62 96.7 3.11 2.98 95.8 3.26 3.18 97.5 3.59 3.54 98.6 3.79 3.80 100.3 4.05 3.93 97.0 4.57 4.30 94.1 59 3.3.3 Comparison of the d i r e c t and i n d i r e c t methods of measuring d i g e s t i b i l i t y . The three week mean d i g e s t i b i l i t y c o e f f i c i e n t s f o r organic matter, crude p r o t e i n and gross energy measured by the d i r e c t ( t o t a l c o l l e c t i o n ) and i n d i r e c t (chromic oxide marker) method are shown i n Table 4. D i f f e r e n c e s between the d i r e c t and the i n d i r e c t methods of measuring apparent n u t r i e n t d i g e s t i b i l i t i e s were not s i g n i f i c a n t (P>0.05) although the values f o r a l l n u t r i e n t s were s l i g h t l y higher w i t h the d i r e c t method. S t a t i s t i c a l comparisons of d i g e s t i b i l i t y values determined from feces acquired by the i n t e s t i n a l d i s s e c t i o n and s t r i p p i n g procedure were not p o s s i b l e due t o a l a c k of r e p l i c a t i o n s , given the l i m i t e d sample s i z e of feces c o l l e c t e d w i t h these two techniques. Apparent d i g e s t i b i l i t y values obtained f o r feces c o l l e c t e d d i r e c t l y from the f i s h by s t r i p p i n g and d i s s e c t i o n were, however, c o n s i s t e n t l y lower than the d i r e c t and i n d i r e c t values c a l c u l a t e d f o r feces c o l l e c t e d from the water by the "Guelph system". The s t r i p p i n g technique tended to y i e l d the lowest d i g e s t i b i l i t y values, being 3.5 percentage u n i t s lower f o r organic matter 4.5 percentage u n i t s lower f o r p r o t e i n and 3.4 percentage u n i t s lower f o r gross energy d i g e s t i b i l i t y than values obtained w i t h the i n t e s t i n a l d i s s e c t i o n technique. D i f f e r e n c e s between apparent d i g e s t i b i l i t y c o e f f i c i e n t s f o r organic matter, crude p r o t e i n and gross energy between weeks 1, 2 and 3 were found not to be s i g n i f i c a n t (P>0.05), i n d i c a t i n g t h a t the minimum f e c a l c o l l e c t i o n time i s determined p r i m a r i l y by the s i z e of the sample r e q u i r e d f o r a n a l y s i s r a t h e r than by the need to minimize weekly d i g e s t i b i l i t y v a r i a t i o n s . 60 3.3.4 Ingested chromic oxide recovery Besides being indigestible, chromic oxide has not been shown to have any adverse effects on the normal physiological processes of digestion (Davignon et al., 1968). Moreover, the indicator is thought to closely follow the solid phase of the intestinal contents (Balch et al., 1957). To confirm this in chinook salmon, the recovery rate of chromic oxide was evaluated by measuring the balance between the amount of chromic oxide ingested in the food versus the amount recovered in the feces. The recovery rate was noted to be more than 90% (Table 5) except for replicate 5. The overall average chromic oxide for a l l replicate groups was 93.9%. The loss of fecal particles over the water outflow and past the fine mesh screening as well as some minor losses during sample grinding and transfer between containers, probably account for the lack of complete recovery. 61 Table 4. Comparison of the three week mean apparent d i g e s t i b i l i t y c o e f f i c i e n t values determined by d i r e c t and i n d i r e c t measurement using the "Guelph system" of f e c a l c o l l e c t i o n , as w e l l as values obtained f o r feces c o l l e c t e d by i n t e s t i n a l d i s s e c t i o n and s t r i p p i n g techniques. D i g e s t i b i l i t y measuring method C o l l e c t i o n system APPARENT DIGESTIBILITY (%) ± SEM Organic matter Crude p r o t e i n Gross energy -'-Direct "Guelph" 7 4 . 5 a 4 85 .3 a 79 .8 a ± 0 . 8 6 ± 0 . 7 2 ± 0 . 8 3 I n d i r e c t "Guelph" 73 .2 a 84. 6 a 78. 9 a ± 0 . 7 4 ± 0 . 7 5 ± 0 . 7 2 ^ I n d i r e c t I n t e s t i n a l d i s s e c t i o n 65.0 75.0 71.9 ^ I n d i r e c t S t r i p p i n g 61.5 70.5 68.5 A n a l y s i s of variance w i t h d i g e s t i b i l i t y measurement ( d i r e c t , i n d i r e c t ) as the f a c t o r , based on the "Guelph system" of f e c a l c o l l e c t i o n , i n d i c a t e d a lack of s i g n i f i c a n t d i f f e r e n c e (P>0.05). Values f o r d i r e c t and i n d i r e c t methods based on the "Guelph system" are means ± SEM (n=24) f o r a three week p e r i o d . Feces d i s s e c t e d out from h a l f way between the p e l v i c f i n s and the anus. F e c a l samples were c o l l e c t e d from f i s h not p r e v i o u s l y s t r i p p e d and were pooled p r i o r t o chemical analyses. S t a t i s t i c a l a n a l y s i s was not p o s s i b l e due t o lack of r e p l i c a t e s . S t r i p p i n g of feces was performed on the same f i s h once weekly, f o r three weeks. F e c a l samples were pooled p r i o r to chemical analyses. S t a t i s t i c a l a n a l y s i s was not p o s s i b l e due to lack of r e p l i c a t e s . Within a column, values w i t h a common s u p e r s c r i p t l e t t e r are not s i g n i f i c a n t l y d i f f e r e n t (P>0.05). 62 Table 5. Recovery r a t e of chromic oxide (C^C^) from ing e s t a and excreta a f t e r a three-week ( r e p l i c a t e s 1,2,3,4,7 and 8) and a two-week ( r e p l i c a t e s 5,6 and 9) c o l l e c t i o n p e r i o d . Chromic oxide (mg) Recovery (%) R e p l i c a t e Ingested Excreted 1 472.4 462.2 97.8 2 562.6 520.9 92.6 3 171.6 164.2 95.7 4 220.2 211.7 96.1 5 263.1 235.7 89.6 6 516.0 471.2 91.3 7 246.2 241.8 98.2 8 450.2 406.0 90.2 9 240.3 225. 9 94.0 63 3.3.5 E f f e c t of time between f e c a l c o l l e c t i o n s on f e c a l  n u t r i e n t l o s s due to l e a c h i n g The e f f e c t of n u t r i e n t l e a c h i n g l o s s e s from feces was evaluated by comparing the three week mean apparent d i g e s t i b i l i t y c o e f f i c i e n t s f o r f e c a l samples c o l l e c t e d from the water at 6 hour (afternoon c o l l e c t i o n ) and 18 hour (morning c o l l e c t i o n ) i n t e r v a l s . These values, together w i t h those from feces obtained d i r e c t l y from the f i s h are presented i n Table 6. D i g e s t i b i l i t y values f o r organic matter, crude p r o t e i n and gross energy determined on f e c a l samples c o l l e c t e d i n the afternoon were s i g n i f i c a n t l y (P<0.001) lower than those of samples c o l l e c t e d the next morning. The longer contact p e r i o d of the feces w i t h the water increased the opportunity f o r n u t r i e n t s t o leach out, thereby causing d i g e s t i b i l i t i e s t o be overestimated. Feces remaining i n water f o r the longer time p e r i o d i n c u r r e d a l o s s of 11.7% organic matter, 13.9% crude p r o t e i n and 9.9% gross energy, i n c r e a s i n g d i g e s t i b i l i t y c o e f f i c i e n t s above the values obtained f o r the s h o r t e r p e r i o d by 7.9, 10.7 and 7.3 percentage u n i t s r e s p e c t i v e l y . F e c a l samples c o l l e c t e d from the water i n the afternoon had an increased estimate of organic matter, crude p r o t e i n and gross energy d i g e s t i b i l i t y of 2.3, 1.7 and 1.6 percentage u n i t s r e s p e c t i v e l y compared to values f o r feces obtained by d i s s e c t i o n . A l l o w i n g feces to remain i n the water an a d d i t i o n a l 12 hours (morning c o l l e c t i o n ) thus increased the c o e f f i c i e n t s by 10.2, 12.4 and 8.9 percentage u n i t s f o r organic matter, p r o t e i n and energy r e s p e c t i v e l y . The c l o s e agreement between d i g e s t i b i l i t i e s determined f o r feces c o l l e c t e d i n the afternoon w i t h the "Guelph system" and those determined f o r feces 64 c o l l e c t e d by i n t e s t i n a l d i s s e c t i o n i n d i c a t e that l e a c h i n g of n u t r i e n t s from feces was minimal during the i n i t i a l 6 hours i n the water. During the experiment, i t was observed t h a t a l a r g e p o r t i o n of the feces was not c a r r i e d i n t o the c o l l e c t i o n column but i n s t e a d r e s t e d on the pipe connecting the tank bottom d r a i n to the c o l l e c t i o n column. The feces i n t h i s pipe were thus exposed to a constant water current which presumably r e s u l t e d i n n u t r i e n t l e a c h i n g losses and consequently i n the d i f f e r e n c e s i n d i g e s t i b i l i t y values observed between morning and afternoon f e c a l c o l l e c t i o n times. I t a l s o e x p l a i n s , to some extent, d i f f e r e n c e s observed between samples c o l l e c t e d i n the water and samples c o l l e c t e d d i r e c t l y from the f i s h . The lowest d i g e s t i b i l i t y values f o r organic matter, crude p r o t e i n and gross energy were obtained f o r f e c a l samples obtained by the s t r i p p i n g technique. 65 TABLE 6. Three-week mean apparent d i g e s t i b i l i t y c o e f f i c i e n t s f o r organic matter, crude p r o t e i n and gross energy by j u v e n i l e chinook salmon i n seawater fed the c o n t r o l d i e t (diet 1), i n r e l a t i o n t o f e c a l c o l l e c t i o n method and time between c o l l e c t i o n s . Feces c o l l e c t i o n system ^-APPARENT DIGESTIBILITY (% ± SEM) and time between c o l l e c t i o n s Organic Matter Crude P r o t e i n Gross Energy ^ S t r i p p i n g 61.5 70.5 68.5 - ^ I n t e s t i n a l d i s s e c t i o n 65.0 75.0 71.9 4"Guelph system" (6 hours) ^"Guelph system" (18 hours) **67.3 ±0.98 **75.2 ±0.57 **76.7 '±1.51 **87.4 ±0.44 * * 73.5 ±1.20 **80.8 ±0.51 Apparent d i g e s t i b i l i t y c o e f f i c i e n t c a l c u l a t i o n s based on the i n d i r e c t method. S t r i p p i n g of feces was performed on the same f i s h once weekly, f o r three weeks. Fec a l samples were pooled p r i o r t o chemical analyses. S t a t i s t i c a l a n a l y s i s was not p o s s i b l e due to lack of r e p l i c a t e s . Feces d i s s e c t e d out from h a l f way between the p e l v i c f i n s and the anus. F e c a l samples were c o l l e c t e d from f i s h not p r e v i o u s l y s t r i p p e d and were pooled p r i o r t o chemical analyses. S t a t i s t i c a l a n a l y s i s was not p o s s i b l e due t o la c k of r e p l i c a t e s . Feces accumulated f o r a maximum p e r i o d of 6 hours p r i o r t o being c o l l e c t e d by the "Guelph system" at 1430 hours i n each tank d a i l y . Feces accumulated f o r a p e r i o d of 18 hours p r i o r to being c o l l e c t e d by the "Guelph system" at 0830 hours i n each tank d a i l y . Mean values (n=24) i n the same column preceded by two a s t e r i s k s are s i g n i f i c a n t l y d i f f e r e n t from each other (P<0.001). 4 66 3 . 3 . 6 E f f e c t of f i s h management on apparent n u t r i e n t d i g e s t i b i l i t y c o e f f i c i e n t s , feed i n t a k e , growth ra t e  and f i s h m o r t a l i t y The three-week mean apparent d i g e s t i b i l i t y c o e f f i c i e n t s f o r organic matter, crude p r o t e i n and gross energy as determined by the i n d i r e c t method using weekly f e c a l c o l l e c t i o n s were s i g n i f i c a n t l y (P<0.001) lower f o r groups of f i s h subjected to weekly handling than f o r those not d i s t u r b e d (Table 7). The data i n Table 8 a l s o r e v e a l t h a t those groups of f i s h which had t h e i r feces removed by s t r i p p i n g once a week, had a s i g n i f i c a n t l y lower (P<0.05) d a i l y feed i n t a k e (expressed as a percent of wet body weight) than groups of f i s h not d i s t u r b e d . The s t r e s s r e s u l t i n g from f i s h handling l e d to a dramatic l o s s of ap p e t i t e f o r a pe r i o d of three to four days subsequent t o s t r i p p i n g . The handling s t r e s s and the lower feed i n t a k e a s s o c i a t e d w i t h i t , r e s u l t e d i n a s i g n i f i c a n t l y lower (P<0.001) s p e c i f i c growth ra t e and higher m o r t a l i t y r a t e f o r these f i s h (Table 8). No s i g n i f i c a n t d i f f e r e n c e s (P>0.05) were observed between undisturbed f i s h on a r e s t r i c t e d or s a t i a t i o n feeding p r o t o c o l f o r s p e c i f i c growth r a t e . S u r p r i s i n g l y , the feed intake of undisturbed f i s h fed a r e s t r i c t e d amount of feed, was found not to be s i g n i f i c a n t l y d i f f e r e n t (P>0.05) from those f i s h fed t o s a t i a t i o n (Table 8). 67 Table 7. E f f e c t of f i s h management (feeding regime and f i s h handling) on mean apparent organic matter, crude p r o t e i n and gross energy d i g e s t i b i l i t y c o e f f i c i e n t s c a l c u l a t e d using d i g e s t i b i l i t y data from f e c a l samples c o l l e c t e d i n the morning and i n the afternoon over a three week c o l l e c t i o n p e r i o d . F i s h management •'-Apparent d i g e s t i b i l i t y (%) ± SEM ^Feed int a k e (% BW/day) F i s h handling organic matter crude p r o t e i n gross energy 0.22 weekly 69.7 a 3 ±0.51 81. l a ±0.73 75.7 a ±0.47 0.48 no 76.0 b ±0.62 87. l b ±0.56 81.5 b ±0.72 0.50 no 74. 4 b ±1.12 8 6. 0 b ±1.06 79.6 b ±1.22 D i g e s t i b i l i t y c a l c u l a t i o n s based on the i n d i r e c t (0:^03) method. A n a l y s i s of variance 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 between f i s h management and apparent n u t r i e n t d i g e s t i b i l i t y . Values are three week means ± SEM wit h n=9 fo r f i s h fed 0.22 and 0.48 % BW/day and n=6 f o r f i s h fed 0.50 % BW/day r e s p e c t i v e l y . Feed int a k e expressed as a percentage of body weight was c a l c u l a t e d according t o the formula: DFI x 100 (logn t l + logn tO 2 ) e , where DFI i s the mean d a i l y dry feed in t a k e per f i s h and logn tO, logn t l are the average n a t u r a l l o g weights at the s t a r t (tO) and at the end ( t l ) of the p e r i o d . Within a column, values w i t h a common s u p e r s c r i p t l e t t e r are not s i g n i f i c a n t l y d i f f e r e n t (Newman-Keuls w i t h P=0.05) Table 8. E f f e c t of f i s h management on feed i n t a k e , s p e c i f i c growth r a t e and m o r t a l i t y of j u v e n i l e chinook salmon reared i n s a l t water and fed the c o n t r o l d i e t . F i s h management -'-Performance Feeding p r o t o c o l F i s h handling Feed intake (% BW/day) S p e c i f i c growth ra t e , M o r t a l i t y R e s t r i c t e d weekly 0 . 2 2 a 2 +0.01 0.027 a ± 0 . 0 0 6 20/60 R e s t r i c t e d no 0.48 b ± 0 . 0 2 0.192 b ± 0 . 0 1 3 3/60 S a t i a t i o n no 0.50 b ± 0 . 0 9 0.178 b ± 0 . 0 2 6 6/60 S i g n i f i c a n t d i f f e r e n c e s were noted i n feed i n t a k e (P<0.05) and s p e c i f i c growth ra t e (P<0.001) between the d i f f e r e n t f i s h management techniques. Feed int a k e expressed as a percentage of body weight was c a l c u l a t e d according t o the formula: DFI x 100 •+ logn t l + logn tO -s- 2 ) e , where DFI i s the mean d a i l y dry feed i n t a k e per f i s h and logn tO, logn t l are the average n a t u r a l l o g weights at the s t a r t (tO) and at the end ( t l ) of the p e r i o d . S p e c i f i c growth r a t e determined f o r the p e r i o d between i n i t i a l and f i n a l f i s h weighing. Within each column, values w i t h a common s u p e r s c r i p t l e t t e r are not s i g n i f i c a n t l y d i f f e r e n t (Newman-Keuls wit h P=0.05) 69 3.4 DISCUSSION 3.4.1 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 s of f i s h r a i s e d i n seawater 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 s r e q u i r e t h a t f e c a l samples be c o l l e c t e d e i t h e r q u a n t i t a t i v e l y or as a r e p r e s e n t a t i v e p o r t i o n , depending on whether the d i r e c t or the i n d i r e c t method of determining d i g e s t i b i l i t y i s used. I t i s important however, t h a t the n a t u r a l l y v o i d e d feces remain as unchanged as p o s s i b l e p r i o r t o c o l l e c t i o n and a n a l y s i s . D i g e s t i b i l i t y d e t erminations i n f i s h p resent problems not encountered w i t h t e r r e s t r i a l animals, one of them b e i n g t h a t n a t u r a l l y v o i d e d feces are suspended i n l a r g e q u a n t i t i e s of water. Concern among n u t r i t i o n i s t s remains r e g a r d i n g the l o s s of n u t r i e n t s from the feces through l e a c h i n g . Since the l o s t n u t r i e n t s are t r e a t e d as i f they were absorbed by the f i s h , any l e a c h i n g r e s u l t s i n the d i g e s t i b i l i t y c o e f f i c i e n t s being b i a s e d toward h i g h e r v a l u e s . When feces are c o l l e c t e d from seawater, another f a c t o r must be c o n s i d e r e d . Upon d r y i n g , a s u b s t a n t i a l amount of s a l t w i l l remain i n the sample which w i l l d i l u t e the c o n c e n t r a t i o n s of a l l f e c a l c o n s t i t u e n t s i n the sample. The r e l a t i v e p r o p o r t i o n between the o r g a n i c matter c o n s t i t u e n t s (e.g. p r o t e i n , l i p i d , carbohydrate) and the i n d i g e s t i b l e marker w i l l , however, remain constant r e g a r d l e s s of the q u a n t i t y o f • s a l t contaminating the f e c e s . Given t h a t 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 i s based on the r e l a t i v e p r o p o r t i o n of n u t r i e n t to marker or n u t r i e n t t o t o t a l dry weight f o r the i n d i r e c t and d i r e c t methods r e s p e c t i v e l y , the e f f e c t of s a l t contamination becomes a n n u l l e d (see equations below) as long as the " n u t r i e n t " i n the equation i s an o r g a n i c n u t r i e n t . 70 Apparent d i g e s t i b i l i t y c o e f f i c i e n t equations f o r the i n d i r e c t (ADCi) and d i r e c t (ADCd) methods ADCi (%) = 100 1-% Cr2C>3 i n d i e t x % n u t r i e n t i n feces %^Cr2C>3 i n feces x % n u t r i e n t i n d i e t ADCd (%) = 100 1-feces dry weight x % n u t r i e n t i n feces d i e t fed weight x % n u t r i e n t i n d i e t In the case of dry matter d i g e s t i b i l i t y determinations however, the e f f e c t of s a l t contamination does not cancel i t s e l f out. The dry matter f r a c t i o n i s an absolute r a t h e r than a r e l a t i v e value and i n c l u d e s both the organic and i n o r g a n i c c o n s t i t u e n t s (e.g. s a l t ) . The contamination of the feces w i t h s a l t w i l l erroneously underestimate dry matter d i g e s t i b i l i t y r e gardless of whether the determinations are made by the i n d i r e c t or the d i r e c t methods. In the i n d i r e c t method, s a l t contamination w i l l d i l u t e the c o n c e n t r a t i o n of the i n d i g e s t i b l e marker while the percent dry matter ("nutrient" i n formula above) remains constant and consequently dry matter d i g e s t i b i l i t y i s underestimated. In the d i r e c t method, the amount of t o t a l f e c a l dry matter w i l l increase when s a l t contaminates the sample and dry matter d i g e s t i b i l i t y i s consequently a l s o underestimated. Unless a c o r r e c t i o n i s made f o r the presence of the contaminant i n the feces, dry matter d i g e s t i b i l i t y values w i l l be underestimated. The i n t r o d u c t i o n of such an e r r o r has, however, been overlooked by some workers (Seidman and Lawrence, 1985; Lee and Lawrence, 1985), while others have r e s o r t e d to washing out the s a l t s adhering t o the feces by repeatedly r i n s i n g the sample w i t h f r e s h water (MacLeod, 1977/ De S i l v a and Perera, 1984), which w i l l undoubtedly cause n u t r i e n t l e a c h i n g l o s s e s . In order to avoid both the n u t r i e n t l e a c h i n g and the s a l t . c o n t a m i n a t i o n problems a s s o c i a t e d w i t h the c o l l e c t i o n of feces d i r e c t l y from the seawater, most researchers working w i t h marine f i s h have opted t o c o l l e c t the feces d i r e c t l y from the f i s h by performing an i n t e s t i n a l d i s s e c t i o n (Lied et al., 1982/ F e r r a r i s et al., 1986) or by s t r i p p i n g ( L a l l and Bishop, 1977, 1979/ Dabrowski et al., 1986). These techniques, as mentioned below, however, have t h e i r shortcomings as w e l l . Dry matter d i g e s t i b i l i t y c o e f f i c i e n t s were, t h e r e f o r e , not c a l c u l a t e d i n the present study and r e l i a n c e was placed on organic matter as w e l l as on crude p r o t e i n and gross energy d i g e s t i b i l i t y c o e f f i c i e n t s . Another important t e c h n i c a l aspect to consider when feces are c o l l e c t e d from the aquarium water i s the need to separate f i s h s c a l e s from the f e c a l samples. In t h i s experiment, f i s h s c a l e l o s s was r e l a t i v e l y high (up t o 8.9% of the t o t a l weekly " f e c a l " dry weight) due p r i m a r i l y , t o a tendency of f i s h t o swim i n and out of the narrow d r a i n s l o t at the bottom of the tank. F a i l u r e t o screen out the s c a l e s would have r e s u l t e d i n lower apparent d i g e s t i b i l i t y c o e f f i c i e n t s , w i t h crude p r o t e i n d i g e s t i b i l i t y being a f f e c t e d the most since scales are comprised of k e r a t i n . 3.4.2 Chromic oxide analysis' Numerous methods f o r the determination of chromic oxide (C^C^) are s c a t t e r e d throughout the l i t e r a t u r e . L i e d et a l . (1982) describe a r a p i d procedure whereby a l i q u o t s from the 72 di g e s t of the m i c r o - K j e l d a h l n i t r o g e n determination technique are analyzed f o r chromium (Cr) by atomic absorption spectrophotometry (AAS). The advantage of t h i s procedure i s th a t the contents of both n i t r o g e n and chromium can be determined on samples as small as 40 mg r a p i d l y and w i t h ease. Given the small amount of feces c o l l e c t e d i n t h i s study, e s p e c i a l l y from the d i s s e c t i o n and s t r i p p i n g . t e c h n i q u e s , the use of the AAS was t e s t e d on a few samples as described by L i e d et al. (1982). Chromium concentration values obtained by the AAS method f l u c t u a t e d considerably and d i f f e r e n c e s of over 10% were observed when the same sample was t e s t e d again only 15 minutes l a t e r . According t o Lee et al. (1986), a combination of f a c t o r s c o n t r i b u t e t o the larg e v a r i a b i l i t y of chromium values obtained by atomic absorption spectrophotometry. These i n c l u d e the flame stoi c h i o m e t r y , chromium o x i d a t i o n s t a t e , the presence of p o t e n t i a l i n t e r f e r e n c e s , and the i n a b i l i t y t o reproduce optimized instrumental operating c o n d i t i o n s e x a c t l y . In p a r t i c u l a r c o n s i s t e n t s e t t i n g s of gas flows and burner p o s i t i o n f o r c o n t r o l of flame s t o i c h i o m e t r y are important. The wet d i g e s t i o n method f o r determination of chromic oxide described by Stevenson and De Langen (1960) was chosen as the p r e f e r r e d method a f t e r acceptable r e c o v e r i e s and good r e p r o d u c i b i l i t i e s were a t t a i n e d w i t h t h i s method (Table 3). 3.4.3 Comparison of the d i r e c t and i n d i r e c t methods of measuring d i g e s t i b i l i t y i n s a l t water f i s h . The r e s u l t s of t h i s i n v e s t i g a t i o n i n d i c a t e that there were no s i g n i f i c a n t d i f f e r e n c e s between the d i g e s t i o n c o e f f i c i e n t s f o r organic matter, crude p r o t e i n and gross energy obtained by the d i r e c t method and those secured by the i n d i r e c t method using chromic oxide as the i n d i g e s t i b l e marker. De l a Noue and , Choubert (1986), using an automatic feces c o l l e c t o r w i t h rainbow t r o u t , a l s o v a l i d a t e d the use of chromic oxide as an i n d i g e s t i b l e marker f o r use i n f i s h a s s i m i l a t i o n s t u d i e s . Bowen (1978) on the other hand, s t a t e d that the use of chromic oxide i n f i s h d i g e s t i b i l i t y s t u d i e s was inadequate a f t e r observing an apparent s e l e c t i v e r e j e c t i o n of the marker by t i l a p i a (Sarotherodon mossambicus). These f i s h , u n l i k e salmonids, were observed by the same author t o work the food i n t o t h e i r mouth and r e j e c t p a r t of each b i t e thereby a l l o w i n g the more dense chromic oxide to separate. A l s o , the chromic oxide content i n the d i e t used'by Bowen (1978) was 6.35% which s i g n i f i c a n t l y exceeds the maximum 1.0% recommended by Tacon and Rodrigues (1984). The r e s u l t s of the present study f u r t h e r show that i t was p o s s i b l e t o recover over 90% (over 98% i n one instance) of the chromic oxide ingested by the f i s h over a three week p e r i o d (Table 5), which i n d i c a t e s there l i k e l y i s l i t t l e , i f any, l o s s during the process of d i g e s t i o n . Incomplete recovery of chromic oxide was l i k e l y due t o a l o s s of small f e c a l fragments which, a f t e r being swept over the tank overflow onto the f i n e screen mesh, could not be r e t r i e v e d completely. A d d i t i o n a l losses l i k e l y occurred when water was decanted a f t e r c e n t r i f u g i n g as w e l l as during sample g r i n d i n g , screening and t r a n s f e r r i n g between c o n t a i n e r s . Such losses a l s o e x p l a i n why the d i g e s t i b i l i t y c o e f f i c i e n t s obtained by the d i r e c t method were c o n s i s t e n t l y higher f o r organic matter, crude p r o t e i n and energy than those measured by the i n d i r e c t method. Another p o s s i b l e f a c t o r c o n t r i b u t i n g to an incomplete recovery of the chromic oxide i n g e s t e d over a t h r e e week p e r i o d i s t h a t the a n a l y s i s f o r the marker i n a given sample d i d not y i e l d 100% r e c o v e r y (Table 3). N e v e r t h e l e s s , the f a c t t h a t no s i g n i f i c a n t d i f f e r e n c e s were found between the d i r e c t method ( t o t a l c o l l e c t i o n ) and the i n d i r e c t method demonstrates t h a t the l a t t e r method can be used s a t i s f a c t o r i l y t o reduce the labour i n v o l v e d i n 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 c o e f f i c i e n t s of seawater f i s h . F u r t h e r , the l a c k of s i g n i f i c a n t v a r i a b i l i t y i n d i g e s t i b i l i t y c o e f f i c i e n t s between weeks 1,2 and 3 i n d i c a t e s t h a t a c o l l e c t i o n p e r i o d g r e a t e r than seven days i s not necessary u n l e s s the q u a n t i t y of f e c a l sample r e q u i r e d f o r analyses warrants a longer c o l l e c t i o n p e r i o d . C o l l e c t i o n of feces f o r at l e a s t one week w i l l a l s o negate the e f f e c t of any p o s s i b l e d a i l y d i g e s t i b i l i t y f l u c t u a t i o n s as those observed by Inaba et a l . (1962), De l a Noue et al. (1980) and De S i l v a and Perera (1983, 1984). Due to the s m a l l amounts of feces c o l l e c t e d , measurements of l i p i d and carbohydrate d i g e s t i b i l i t i e s were not p o s s i b l e . N e v e r t h e l e s s , some g e n e r a l o b s e r v a t i o n s w i t h r e s p e c t t o the p a r t i t i o n i n g of n u t r i e n t a s s i m i l a t i o n were p o s s i b l e u s i n g a v a i l a b l e r e s u l t s . The d i g e s t i b l e energy (D.E.) determined by the i n d i r e c t method (Table 4) was c a l c u l a t e d t o be 18.17 MJ/kg dry d i e t (23.03 MJ/kg d i e t x 78.9% d i g e s t i b i l i t y ) of which crude p r o t e i n would have c o n t r i b u t e d 10.52 MJ (521.2 g p r o t e i n / k g dry d i e t x 23.848 MJ/kg p r o t e i n x 84.6% d i g e s t i b i l i t y ) or 57.9% of D.E., w i t h the remainder being p r o v i d e d by l i p i d and, to a l e s s e r extent, carbohydrate. 75 3.4.4 N u t r i e n t l e a c h i n g l o s s e s i n f e c a l samples c o l l e c t e d w i t h the "Guelph system" The r e s u l t s of t h i s study concur w i t h those of various authors (Inaba et al., 1962; Windell et al., 1978a; Vens-Cappell, 1985; S p y r i d a k i s et al., 1989) that feces c o l l e c t e d from water give higher n u t r i e n t d i g e s t i b i l i t y values than those determined from feces obtained d i r e c t l y from the f i s h . This d i f f e r e n c e could r e s u l t from any one, or a l l of the f o l l o w i n g f a c t o r s : 1. Leaching of s o l u b l e m a t e r i a l from feces i n t o the surrounding water, whereby d i g e s t i b i l i t y values are overestimated. 2. Contamination of feces obtained by manual s t r i p p i n g w i t h u r i n e , mucus and sloughed o f f i n t e s t i n a l c e l l s . In samples obtained by i n t e s t i n a l d i s s e c t i o n , contamination by blood i s of most concern. Such contaminations would tend to cause d i g e s t i b i l i t y values t o be underestimated. 3. The f e c a l samples obtained d i r e c t l y from the f i s h c o n t ain m a t e r i a l which would have been absorbed before the feces were n a t u r a l l y voided by the f i s h , thus b i a s i n g d i g e s t i b i l i t y values downward. The apparent d i g e s t i b i l i t y values f o r organic matter, crude p r o t e i n and gross energy i n t h i s experiment increased w i t h an increase i n contact time between feces and water, i n d i c a t i n g that n u t r i e n t l e a c h i n g took p l a c e . Windell et al. (1978a) used f e c a l samples obtained by i n t e s t i n a l d i s s e c t i o n t o determine the e f f e c t of exposure to water on the l o s s of n u t r i e n t s . These authors found t h a t the major l o s s i s i n c u r r e d during the f i r s t hour of immersion. During t h i s time about 21% of the dry matter, 12% of the p r o t e i n and 4% of the l i p i d s were l o s t , i n c r e a s i n g the d i g e s t i b i l i t y c o e f f i c i e n t s by 11.5%, 10% and 3.7% r e s p e c t i v e l y . Within 16 h the lo s s e s of n u t r i e n t s from the feces reached 31%, 12% and 9.8%, r e s p e c t i v e l y , and the r e s p e c t i v e increase i n d i g e s t i b i l i t y c o e f f i c i e n t s were 17%, 10% and 8.2%, according t o the authors. In the present study, p r o t e i n l o s s e s due to lea c h i n g over the 18 hour p e r i o d were g e n e r a l l y s i m i l a r t o values obtained over a 16 hour p e r i o d by Windell et al. (1978a). However, u n l i k e t h e i r f i n d i n g s , most of the lo s s e s i n the present study occurred a f t e r feces had been exposed to water between 6 and 18 hours. In f a c t , the d i f f e r e n c e s i n organic matter, p r o t e i n and gross energy d i g e s t i b i l i t i e s between f e c a l samples obtained by d i s s e c t i o n , and those obtained from feces r e s t i n g i n water f o r a maximum of 6 hours were only 2.3, 1.7 and 1.6 percentage u n i t s , r e s p e c t i v e l y . On the other hand, the d i f f e r e n c e s i n the same c o e f f i c i e n t s had increased by 10.2, 12.4 and 8.9 percentage u n i t s i n the f e c a l samples allowed to remain i n water f o r a maximum of 18 hours. These r e s u l t s c o n t r a d i c t those of Cho and S l i n g e r (1979), who found s i m i l a r d i g e s t i b i l i t y c o e f f i c i e n t s between feces obtained by d i s s e c t i o n and those obtained from feces which had s e t t l e d i n undisturbed water overnight (15.5 hours). Cho et a l . (1982) s t r e s s e d t h a t l e a c h i n g i s minimized when feces remain i n undisturbed water and tha t most los s e s occur i f f e c a l p e l l e t s are broken up during the c o l l e c t i o n process. The m o d i f i c a t i o n s made i n the present study t o the Guelph f e c a l c o l l e c t i o n tank design used by Cho and S l i n g e r (1979) and Cho et al. (1982) may account f o r the disagreement between r e s u l t s . In the present study, f e c a l m a t e r i a l o f t e n s e t t l e d i n the connection pipe between the tank and the s e t t l i n g column, a problem a l s o observed by S p y r i d a k i s et al. (1989). When the water flow i n t o the tanks was increased to ensure feces were c a r r i e d at a proper speed between the tank and the s e t t l i n g column, most of the smaller fragments were unable t o s e t t l e i n the column by g r a v i t y and i n s t e a d were c a r r i e d up over the overflow. Cho and S l i n g e r (1979) and Cho et al. (1982) probably d i d not encounter s i g n i f i c a n t f e c a l l o sses by way of the overflow i n t h e i r experiments since they operated t h e i r system w i t h f r e s h water which, being l e s s dense than s a l t water, allowed feces t o more r e a d i l y f a l l i n t o the s e t t l i n g column by g r a v i t y . Since q u a n t i t a t i v e f e c a l c o l l e c t i o n was c r i t i c a l i n the present study, flow r a t e s were adjusted t o a l e v e l which prevented f e c a l l o sses over the outflow. Leaching, t h e r e f o r e , most l i k e l y occurred i n the connection pipe between the tank and the s e t t l i n g column since a p o r t i o n of the feces would have been exposed to a constant flow of water at t h i s l o c a t i o n . The design of the tank can be g r e a t l y improved by reducing the c r o s s - s e c t i o n of the connecting pipe t o ensure that the water v e l o c i t y i s s u f f i c i e n t t o s w i f t l y c a r r y feces t o the s e t t l i n g column. A l t e r n a t i v e l y , the cross s e c t i o n of the stand pipe above the s e t t l i n g column can be increased to permit higher water flow r a t e s i n t o the tanks without a concomitant increase i n v e l o c i t y i n the v e r t i c a l pipe and an increase i n f e c a l l o sses v i a the overflow. The occurrence of n u t r i e n t l e a c h i n g from feces observed i n t h i s experiment does not, however, i n v a l i d a t e the o b j e c t i o n s against the d i f f e r e n t techniques of feces c o l l e c t i o n from the 78 i n t e s t i n e . Two major problems wi t h the s t r i p p i n g technique were observed i n t h i s study. E r r o r may have a r i s e n by the a d d i t i o n of u r i n e and body mucus that a d u l t e r a t e d the f e c a l samples. A d d i t i o n a l e r r o r was l i k e l y caused by the i n a b i l i t y t o c o n t r o l the t o t a l amount of feces s t r i p p e d . This amount ranged from none, to one well-formed p e l l e t , t o a s t r i n g or ribbon much longer than a n t i c i p a t e d . Upon subsequent s t r i p p i n g s , a y e l l o w i s h white ribbon or f e c a l - c a s t was the product s t r i p p e d from f i s h which had obviously not fed f o r some time. The f e c a l - c a s t s lacked chromic oxide and were composed of endogenous wastes, which would f u r t h e r depress apparent d i g e s t i b i l i t y c o e f f i c i e n t s . Although the method of s t r i p p i n g feces avoids the problem of l e a c h i n g , according t o Austreng (1978) i t a l s o a c c e l e r a t e s passage of m a t e r i a l through the d i g e s t i v e system w i t h a p o s s i b l e r e d u c t i o n i n absorption. The methodological o b j e c t i o n s against the s t r i p p i n g technique h o l d t r u e a l s o f o r the i n t e s t i n a l d i s s e c t i o n technique, although the e r r o r a s s o c i a t e d w i t h the l a t t e r i s not as s e r i o u s , according t o v a r i o u s authors (Austreng, 1978; W i n d e l l et al., 1978a; Henken et a l . , 1985; S p y r i d a k i s et al., 1989). The a r t i f i c i a l removal of feces d i r e c t l y from the f i s h by any one of these techniques assumes that d i g e s t i o n and absorption are complete when the m a t e r i a l has reached the d i s t a l end of the l a r g e i n t e s t i n e . Several authors (Gauthier and Landis, 1972; Georgopoulou et a l . , 1985) however, have described the a b i l i t y of enterocytes from the p o s t e r i o r i n t e s t i n e to ingest i n t a c t p r o t e i n s by p i n o c y t o s i s . Furthermore, another source of e r r o r may come from the assumption th a t d i g e s t i b i l i t y i s a continuous and steady process, having no v a r i a b i l i t y w i t h time. The d a i l y f l u c t u a t i o n s i n d i g e s t i b i l i t y observed by other i n v e s t i g a t o r s (Inaba et a l . , 1962; De l a Noue et a l . , 1980; De S i l v a and Perera, 1983, 1984), suggest t h a t s t r i p p i n g and i n t e s t i n a l d i s s e c t i o n techniques a p p l i e d f o r one day w i l l not provide a r e p r e s e n t a t i v e sample. 3.4.5 E f f e c t of f i s h management on apparent n u t r i e n t d i g e s t i b i l i t y , feed i n t a k e , growth r a t e and f i s h  m o r t a l i t y There i s some disagreement as t o whether d i g e s t i b i l i t y i s a f f e c t e d by r a t i o n l e v e l (Hastings, 1969; Windell et al., 1978b; Henken et a l . , 1985; Hudon and De l a Noiae, 1985; Storebakken and Austreng, 1987). Those researchers who have concluded t h a t r a t i o n l e v e l has an e f f e c t on apparent d i g e s t i b i l i t y , found the c o r r e l a t i o n to be negative (Henken et al., 1985; Hudon and De l a Noue, 1985). In the present study the reverse occurred, with f i s h i n g e s t i n g the l e a s t amount of feed showing the lowest a s s i m i l a t i o n e f f i c i e n c y . In c o n t r a s t to the experiments r e f e r r e d to above, however, f i s h i n t h i s study manifested poor a p p e t i t e and consequently poor feed intake because of the handling s t r e s s caused by weekly s t r i p p i n g . Primary s t r e s s e f f e c t s are manifested by increased s e c r e t i o n r a t e s and plasma l e v e l s of the c o r t i c o s t e r o i d hormone C o r t i s o l (Donaldson, 1981) and of the catecholamine hormone a d r e n a l i n (Mazeaud and Mazeaud, 1981). Ohnesorge and Rauch (1968) demonstrated t h a t a d r e n a l i n i n h i b i t s p e r i s t a l s i s of the i n t e s t i n e and t h e r e f o r e a l s o has a d i r e c t e f f e c t on d i g e s t i o n - r e l a t e d f u n c t i o n s . The poor feed intake 80 observed f o r three to four days a f t e r s t r i p p i n g agrees w i t h observations made by P i c k e r i n g et al. (1982) working w i t h brown t r o u t {Salmo trutta). Wedemeyer (1976) on the other hand, r e f e r s to young coho salmon l o s i n g a p p e t i t e f o r 4 t o 7 days a f t e r handling, but rainbow t r o u t , under i d e n t i c a l c o n d i t i o n s , resumed feeding the next day. U n l i k e the more domesticated rainbow t r o u t s t r a i n s , w i l d chinook salmon become f r i g h t e n e d very e a s i l y . The r e l a t i v e l y low feed i n t a k e experienced by even the groups of undisturbed f i s h , was due p a r t l y t o the low water temperatures experienced during the t r i a l (5°C to 8°C), and p a r t l y t o the f i s h being alarmed when anyone approached the tanks. The very low feed i n t a k e by the s t r i p p e d f i s h a l s o had a no t i c e a b l e e f f e c t on the appearance of the feces. Much of the feces c o l l e c t e d from the water, as w e l l as through subsequent s t r i p p i n g s , lacked the d i s t i n c t i v e green c o l o r of chromic oxide. I t i s p o s t u l a t e d t h e r e f o r e , that the depressed apparent d i g e s t i b i l i t y of organic matter, crude p r o t e i n and gross energy observed i n d i s t u r b e d f i s h , was due not only to the p h y s i o l o g i c a l e f f e c t s of s t r e s s on the d i g e s t i v e f u n c t i o n s , but a l s o t o a r e l a t i v e l y higher p r o p o r t i o n of endogenous f e c a l wastes being c o l l e c t e d . The low s p e c i f i c growth ra t e and the extreme m o r t a l i t y r a t e observed i n s t r i p p e d f i s h were caused d i r e c t l y and i n d i r e c t l y by the handling s t r e s s as w e l l . The excessive l o s s of f i s h s c a l e s due t o repeated p h y s i c a l manipulation f o r instance, may have upset the osmotic balance beyond the animal's osmoregulatory c a p a b i l i t i e s , l e a d i n g t o dehydration and death. 81 In r e l a t i o n to the undisturbed f i s h , no s t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e was observed i n feed in t a k e between f i s h fed t o s a t i a t i o n and those maintained on a r e s t r i c t e d i n t a k e l e v e l . Feed intake l e v e l s even p r i o r t o the s t a r t of the experiment were q u i t e low due to the c o l d water temperatures (8°C). The l e v e l of food intake of the f i s h on the r e s t r i c t e d p r o t o c o l was reduced to 90% of the maximum r a t i o n noted at 8°C p r i o r to the beginning of the experiment and the undisturbed f i s h r e a d i l y consumed t h i s r a t i o n throughout the study. F i s h fed to s a t i a t i o n , however, d i d not consume much more feed than those fed the r e s t r i c t e d amount since water temperatures and consequently a p p e t i t e d e c l i n e d t o the poin t where the r a t i o n s of the two groups of undisturbed f i s h were almost i d e n t i c a l (water temperature reached 5°C). I t i s noteworthy t h a t the growth rates of the undisturbed f i s h on the r e s t r i c t e d r a t i o n were s l i g h t l y g reater than those of f i s h fed to s a t i a t i o n . This d i f f e r e n c e , though s m a l l , was probably due to the l o s s i n weight of the f i s h w h ile on the p r o t e i n - f r e e d i e t f o r four days. D i f f e r e n c e s i n the apparent d i g e s t i b i l i t y c o e f f i c i e n t s f o r organic matter, crude p r o t e i n and gross energy between the two feeding groups of undisturbed f i s h were not s i g n i f i c a n t , although m o r t a l i t y was twice as high (6 versus 3 f i s h out of 60) i n the higher intake group. 82 CHAPTER 4 4.0 EXPERIMENTS I I , I I I AND IV. - Apparent d i g e s t i b l e organic matter, crude p r o t e i n and energy c o e f f i c i e n t s of common and novel feed i n g r e d i e n t s i n p o s t - j u v e n i l e chinook salmon (Oncorhynchus tshawytscha) i n seawater 4.1 INTRODUCTION F i s h meal i s w e l l recognized as the best source of p r o t e i n f o r salmonids. The high cost of high q u a l i t y f i s h meal, however, poses r e a l problems f o r c o s t - e f f e c t i v e feed formulations. The common p r a c t i c e i n animal husbandry i s to p a r t i a l l y or e n t i r e l y replace the high-cost animal p r o t e i n s w i t h l e s s expensive p l a n t and/or animal by-product sources to maximize r e t u r n s , even i f t h i s may be a s s o c i a t e d w i t h a moderate reduction i n feed e f f i c i e n c y . A long term feeding t r i a l i s the most dependable method of measuring the n u t r i t i v e value of a feed, but t h i s i s slow and too expensive. In vitro chemical methods, while g i v i n g some i n d i c a t i o n of feed q u a l i t y , are not yet r e l i a b l e enough t o i n d i c a t e the a v a i l a b i l i t y of n u t r i e n t s i n a feed to a p a r t i c u l a r s pecies. D i g e s t i o n t r i a l s are used e x t e n s i v e l y i n studying the n u t r i t i o n of domestic and la b o r a t o r y animals as w e l l as that of commercially important f i s h s pecies. Concerning the l a t t e r , most • d i g e s t i b i l i t y s t u d i e s have been confined t o rainbow t r o u t reared i n f r e s h water while almost no st u d i e s have been d i r e c t e d to assessing f e e d s t u f f d i g e s t i b i l i t y by salmonids i n the marine environment. Owing to the p a u c i t y of such v i t a l i n f ormation, a s e r i e s of three experiments were undertaken to determine the apparent d i g e s t i b i l i t y of organic matter, crude p r o t e i n and 83 energy i n conventional and novel f e e d s t u f f s i n seawater-adapted p o s t - j u v e n i l e chinook salmon (Oncorhynchus tshawytscha) using the "Guelph system" of f e c a l c o l l e c t i o n . 84 4.2 MATERIALS AND METHODS 4.2.1 Experimental design In a s e r i e s of three experiments, chinook salmon w i t h a minimum s i z e of 10.3 g t o 40.5 g at the s t a r t of the various experiments, were fed by hand twice d a i l y to s a t i a t i o n e i t h e r a reference d i e t (REF-a experiments I I and I I I , REF-b experiment IV) or a t e s t d i e t (70% reference : 30% t e s t i n g r e d i e n t ) . In the case of some pl a n t p r o t e i n sources, the t e s t i n g r e d i e n t was inc l u d e d not only at 30% but a l s o at 15% to determine p o s s i b l e e f f e c t s of a n t i n u t r i t i o n a l f a c t o r s or carbohydrate on d i g e s t i b i l i t y c o e f f i c i e n t s . Chromic oxide (0.5%) was in c l u d e d i n a l l d i e t s as the i n d i g e s t i b l e marker. Within t e s t s , each d i e t was assigned t o three groups of f i s h using a completely random (experiment II) or randomized complete block design (experiments I I I and IV). Feces were c o l l e c t e d from each tank every morning f o r f i f t e e n days using the "Guelph system" of c o l l e c t i o n . Apparent d i g e s t i b i l i t y c o e f f i c i e n t s of organic matter, crude p r o t e i n and gross energy were determined f o r each t e s t i n g r e d i e n t . At the end of each experiment, feces were a l s o obtained by the s t r i p p i n g or the i n t e s t i n a l d i s s e c t i o n techniques, t o obt a i n data f o r comparisons w i t h organic matter d i g e s t i b i l i t y values obtained w i t h the "Guelph system". 4.2.2 H i s t o r y of experimental f i s h Approximately 7,000 chinook salmon f r y were obtained from the Robertson Creek hatchery (Vancouver I s l a n d , B.C.) i n May, 1989. The f i s h were d i s t r i b u t e d i n t o f i v e outdoor c i r c u l a r 3,600 l i t e r f i b e r g l a s s tanks s u p p l i e d w i t h 10°C t o 12°C aerated w e l l water. B i o D i e t (Bioproducts, Inc., Warrenton, Oregon, U.S.A.) 85 was f e d by hand t o s a t i a t i o n seven t i m e s d a i l y f o r t h e f i r s t 3 weeks and t h e f r e q u e n c y was t h e r e a f t e r g r a d u a l l y r e d u c e d t o t h r e e t i m e s d a i l y . On June 22, 1989, f i s h had r e a c h e d t h e minimum weight (4-6 grams) r e q u i r e d t o e l i c i t an immune response and were i n d i v i d u a l l y immunized v i a i n t e r - p e r i t o n e a l i n j e c t i o n (0.1 m l / f i s h ) w i t h Ermogen-Furogen-Vibrogen B a c t e r i n (AquaHealth Inc.) t o p r o t e c t a g a i n s t v i b r i o s i s (caused by V i b r i o anguillarum and V i b r i o o r d a l i i ) , e n t e r i c redmouth d i s e a s e (caused by t y p e 1 Yersinia r u c k e r i ) , and f u r u n c u l o s i s (caused by t y p i c a l i s o l a t e s o f Aeromonas salmonicida). On J u l y 14, 1989, w e l l water was g r a d u a l l y r e p l a c e d by B u r r a r d I n l e t seawater i n s t e p s (1/3, 1/2, 3/4, 4/4) over a 7-day p e r i o d . B e f o r e commencement o f any e x p e r i m e n t , t h e r e q u i r e d number o f f i s h was s e l e c t e d f o r u n i f o r m s i z e and t h e n t h e f i s h were d i s t r i b u t e d randomly i n t o t h e d i g e s t i b i l i t y t a n k s . S t o c k i n g d e n s i t i e s i n t h e 3,600 l i t e r t a n k s t h e r e f o r e , never exceeded 10.5 kg/m 3. 4.2.3 Aquarium f a c i l i t y The e x p e r i m e n t a l i n d o o r aquarium f a c i l i t y ' l o c a t e d a t t h e West Vancouver L a b o r a t o r y o f t h e Department o f F i s h e r i e s and Oceans (D.F.O.), c o n t a i n e d one row o f 27 d i g e s t i b i l i t y t a n k s . These t a n k s were s i m i l a r t o t h o s e used i n experiment I ; however, t h e o r i g i n a l 3.81 cm (I.D.) c o n n e c t i o n p i p e between t h e t a n k bottom and t h e s e t t l i n g column i n t h e t a n k s i n experiment I was r e p l a c e d by a 2 cm (I.D.) p i p e t o ensure t h a t f e c e s were c a r r i e d s w i f t l y from t h e t a n k t o t h e s e t t l i n g column f o r reasons o u t l i n e d 86 p r e v i o u s l y . In a d d i t i o n , to minimize f i s h s c a l e l o s s r e s u l t i n g from f i s h rubbing against the tank bottom d r a i n s l o t , each tank was f i t t e d w i t h a rect a n g u l a r P l e x i g l a s s p l a t e which f i t t e d f l a t over the d r a i n s l o t . The corners of the p l a t e s were extended s l i g h t l y outward t o allo w water and feces to flow f r e e l y between the p l a t e edge and the tank to the d r a i n s l o t , yet the water s u c t i o n e f f e c t around the edges was reduced enough to prevent f i s h from g e t t i n g stuck. Each tank was s u p p l i e d w i t h 6 L/min of f i l t e r e d Burrard I n l e t seawater. P r i o r t o flo w i n g i n t o each tank, the f i l t e r e d water was passed through a 60 cm long a e r a t i o n column f i l l e d w i t h Koch r i n g s to supplement the a e r a t i o n provided i n each tank by porous rubber tubing. Water temperature v a r i e d between experiments and ranged from 10.0°C t o 12.5°C i n experiment I I , 10.0°C to 11.0°C i n experiment I I I and 8.0°C to 9.5°C i n experiment IV. D i u r n a l water temperature f l u c t u a t i o n s d i d not exceed 1.0°C i n any of the experiments. Water s a l i n i t y was monitored twice d a i l y i n each experiment and values v a r i e d between 28 ppt and 30 ppt i n experiment I I , 29 ppt and 31 ppt i n experiment I I I , and 30 ppt and 31 ppt i n experiment IV. In a l l experiments, f l u c t u a t i o n s i n water s a l i n i t y between readings (6 hours) v a r i e d from 1 to 2 ppt on some days while remaining constant on other days. The d i s s o l v e d oxygen l e v e l s were monitored i n random tanks throughout each experiment and the l e v e l s were found t o be near s a t u r a t i o n i n a l l determinations. Some chemical parameters of the f i l t e r e d sea-water i n ppb were as f o l l o w s : Cd <1.3; Cr <0.3; Cu 0.059; B 1600.0; Fe 1100.0; N i 310.0; Pb 3.2; Zn 280.0. A n a t u r a l photoperiod was provided by a 87 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 , Durotest 40W) c o n t r o l l e d by a p h o t o c e l l . 4.2.4 Water f i l t e r i n g system The f i l t e r i n g system used at the University/D.F.O. f a c i l i t y i n experiment I was found to be adequate at that l o c a t i o n . However, the l a r g e r i n f l u x of sand, algae, copepods and other i n v e r t e b r a t e s i n the seawater s u p p l i e d to the West Vancouver Laboratory, occluded the f i l t e r s completely w i t h i n 1 t o 2 days of operation. Subsequently, the V e x l a r mesh sandwiched between the two p e r f o r a t e d PVC d i s c s i n each f i l t e r was replaced w i t h 7.6 cm t h i c k aquarium f i l t e r foam (Cycle guard Aquaclear 2000, d i s t r i b u t e d by R.C. Hagen Inc., Montreal, Quebec). This f i l t e r foam was very e f f e c t i v e i n preventing the passage of even the smallest i n v e r t e b r a t e s and d i d not plug up as suddenly or as of t e n as w i t h the other system. Nevertheless, f i l t e r s were replaced every t h i r d day throughout each experiment. The f i l t e r e d seawater was passed through a funnel c o n t a i n i n g a handful of aquarium p o l y - f i l t e r wool (R.C. Hagen Inc., Montreal, Quebec) before entry i n t o each tank. The wool was very e f f e c t i v e i n f i l t e r i n g the very f i n e sediment which passed through the g r a v e l and foam f i l t e r s . The wool f i l t e r i n each funnel was replaced every afternoon before f l u s h i n g the tanks t o remove uneaten food and f e c a l accumulation. 4 .2 .5 D i e t s A l l t e s t d i e t s i n experiments I I and I I I were composed of 30% of a t e s t i n g r e d i e n t and 70% of a reference d i e t ( a i r dry b a s i s ) / while i n experiment IV, some of the t e s t i n g r e d i e n t s were incorporated at the 15% l e v e l of i n c l u s i o n , with the reference d i e t comprising the balance. Chromic oxide (0.5% a i r dry basis) was i n c l u d e d i n t o the reference and t e s t d i e t s as the i n d i g e s t i b l e marker. The reference d i e t used i n experiments IV (REF-b) was 4% higher i n p r o t e i n content than the reference d i e t (REF-a) used i n experiments I I and I I I t o compensate f o r the lower p r o t e i n content of the t e s t i n g r e d i e n t s used i n experiment IV. The d i e t p r e p a r a t i o n p r o t o c o l s are shown i n Tables 9 and 10. The formulations f o r the b a s a l mixes used i n the reference d i e t p reparations are l i s t e d i n Tables 11 and 12. The t e s t i n g r e d i e n t s and t h e i r r e s p e c t i v e proximate composition are l i s t e d i n Tables 13, 14 and 15. Each t e s t i n g r e d i e n t was assigned a code and the same code was used to d i f f e r e n t i a t e the t e s t d i e t s from one another. Since some f e e d s t u f f s were t e s t e d at two l e v e l s of i n c l u s i o n , the number f o l l o w i n g the t e s t d i e t code represents the l e v e l of i n c o r p o r a t i o n (e.g. number "1" or "2" r e p r e s e n t i n g the 15% or 30% l e v e l of i n c l u s i o n of the t e s t i n g r e d i e n t i n the t e s t d i e t , r e s p e c t i v e l y ) . The d i e t codes, together w i t h t h e i r r e s p e c t i v e proximate composition and d i e t a r y mineral contents are shown i n Tables 16, 17 and 18. 4.2.6 Diet p r e p a r a t i o n D i e t s were prepared i n J u l y , 1989 (experiments I I and I I I ) and i n October, 198 9 (experiment IV) at the West Vancouver Laboratory (D.F.O.). The mineral and v i t a m i n supplements were prepared as described i n experiment I, although f i n e l y ground extruded wheat replaced the o c - c e l l u l o s e used as a c a r r i e r i n 89 experiment I. A l l f e e d s t u f f s were stored i n the dark at 15°C and 28% r e l a t i v e humidity and, i f necessary, were ground i n a hammer m i l l ( F i t z m i l l , model JT; The F i t z p a t r i c k Company, Elmhurst I l l i n o i s , U.S.A.) equipped w i t h a s i z e U.S. 30 screen (595 um) p r i o r to d i e t p r e p a r a t i o n . The i n g r e d i e n t s f o r the bas a l mix (excluding the o i l ) were thoroughly blended i n a Marion mixer (Rapids Machinery L t d . , Marion, Iowa U.S.A.) f o r 30 minutes. To increase the moisture content of both b a s a l mixtures t o approximately 9%, 17.53 g water/kg mash and 19.67 g water/kg mash were added t o b a s a l mix 1 and bas a l mix 2 r e s p e c t i v e l y . A t o t a l of one hundred kilograms of b a s a l mix 1 (experiments I I and I I I ) and f i f t y kilograms of bas a l mix 2 (experiment IV) was prepared. Approximately 10 kg of b a s a l mix 1 and 5 kg of bas a l mix 2 were 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 CL-type 2 l a b o r a t o r y p e l l e t m i l l ( C a l i f o r n i a P e l l e t M i l l Co., San Fr a n c i s c o , C a l i f o r n i a ) w i t h a 3.18 mm d i e . Thereafter, the p e l l e t s were placed i n t o a v e r t i c a l c o o l e r and then they were sprayed w i t h h e r r i n g o i l as described i n experiment I. The p e l l e t e d b a s a l d i e t s were used to wean the f i s h from the Bi o D i e t p r i o r t o the s t a r t of each of the experiments and a l s o t o generate the chromic oxide-free feces r e q u i r e d f o r subsequent chromic oxide analyses. A t o t a l of 5 kg of each d i e t was prepared. Water was added to each t e s t d i e t before p e l l e t i n g to e q u a l i z e the moisture content i n a l l d i e t s at 9%. Chromic oxide (C^C^) was mixed i n t o each d i e t using a mortar and p e s t l e as described i n experiment I. 9 The reference d i e t s and t e s t d i e t s were each mixed f o r 20 minutes i n a Hobart commercial mixer (Hobart Manufacturing Co., Troy, Ohio). Each d i e t was then c o l d p e l l e t e d w i t h a 3.18 mm 90 (experiments I I and I I I ) or a 3.97 mm (experiment IV) d i e , the s i z e of the d i e being adjusted to s u i t f i s h s i z e according to Fowler and Burrows (1971). A f t e r c o o l i n g , p e l l e t s were sprayed w i t h the h e r r i n g o i l as described i n experiment I. The d i e t s were st o r e d i n the dark i n t h i c k p l a s t i c bags at 15°C and 28% r e l a t i v e humidity u n t i l r e q u i r e d . D i e t s i n use were kept i n a i r t i g h t c ontainers and were r e f r i g e r a t e d n i g h t l y at 3°C t o 4°C. 91 Table 9. Reference and t e s t d i e t p r e p a r a t i o n p r o t o c o l employed i n experiments I I and I I I Reference d i e t Test d i e t (REF-a) ( l e v e l 2) Composition (g/Kg) 995.0 696.5 0 298.5 5.0 5.0 Test d i e t s w i t h a 30% l e v e l of i n c l u s i o n of t e s t i n g r e d i e n t ( l e v e l 2) and 70% i n c l u s i o n l e v e l of b a s a l mix ( a i r - d r y b a s i s ) . Refer to Table 11 f o r b a s a l mix 1 form u l a t i o n . Ingredients t e s t e d i n experiment I I in c l u d e d : rapeseed p r o t e i n concentrate(BRONOWSKI FRI -73-5 s t r a i n ) ; canola meal (commercially a v a i l a b l e source); g l u c o s i n o l a t e - f r e e canola meal ( s t r a i n BC86-18); h e r r i n g meal ( B r i t i s h Columbia source); anchovy meal (Chilean source). Ingredients t e s t e d i n experiment I I I i n c l u d e d : p o u l t r y by-product meal (Kansas source); p o u l t r y by-product meal ( B r i t i s h Columbia source); menhaden meal (Louisiana source); hydrolyzed feather meal ( B r i t i s h Columbia source); LT (low temperature) h e r r i n g / c a p e l i n meal (Norwegian source); soybean meal; blood meal ( B r i t i s h Columbia source); soybean p r o t e i n i s o l a t e . Basal mix l z Test i n g r e d i e n t Chromic oxide 92 Table 10. Reference and t e s t d i e t p r e p a r a t i o n p r o t o c o l employed i n experiment IV. Reference d i e t Test d i e t Test d i e t (REF-b) ( l e v e l 1) ( l e v e l 2) Composition (g/Kg) Basal mix 2 Z 995.0 845.7 696.5 Test i n g r e d i e n t 3 0 149.3 298.5 Chromic oxide 5.0 5.0 5.0 Test i n g r e d i e n t at l e v e l 1 i s equivalent t o a 15% l e v e l of i n c l u s i o n of the t e s t i n g r e d i e n t and 85% i n c l u s i o n l e v e l of the b a s a l mix. While t e s t i n g r e d i e n t at l e v e l 2 i s equivalent to 30% i n c l u s i o n l e v e l of the t e s t i n g r e d i e n t and 70% i n c l u s i o n l e v e l of the b a s a l mix ( a i r - d r y b a s i s ) . Refer to Table 12 f o r b a s a l mix 2 f o r m u l a t i o n . Ingredients t e s t e d at l e v e l 1 of i n c l u s i o n i n c l u d e d : d r i e d whey ( B r i t i s h Columbia source); extruded wheat ( B r i t i s h Columbia source); soybean meal; soybean p r o t e i n i s o l a t e . Ingredients t e s t e d at l e v e l 2 of i n c l u s i o n i n c l u d e d : extruded wheat ( B r i t i s h Columbia source); wheat middlings ( B r i t i s h Columbia source); s o l v e n t - e x t r a c t e d g l u c o s i n o l a t e - f r e e canola meal. 93 Table 11. Formulation of b a s a l mix 1 used i n the p r e p a r a t i o n of the reference and t e s t d i e t s employed i n experiments I I and I I I . Ingredients Concentration (g/kg dry d i e t ) Steam-dried h e r r i n g meal (73 .58% CP)* 550. 0 Extruded wheat (17.83% CP)* 109. 0 Dried whey (14.13% CP)* 75. 0 F r e e z e - d r i e d euphausids 1 (71 .48 CP)* 60. 0 Blood meal (95.37% CP) 50. 0 Herring o i l 84. 0 Soybean l e c i t h i n 10. 0 Vitamin supplement 3 20. 0 M i n e r a l supplement 4 20. 0 Permapell ( l i g n i n - s u l p h o n a t e binder) 15. 0 Choline c h l o r i d e (60%) 5. 0 A s c o r b i c a c i d 2. 0 Dry matter b a s i s . S t a b i l i z e d w i t h 0.02% santoquin. The o i l was s t a b i l i z e d w i t h 0.025% santoquin and was sprayed onto the p e l l e t e d d i e t s . The v i t a m i n supplement provided the f o l l o w i n g amounts per kg of dry d i e t : i n o s i t o l 400 mg; n i a c i n 300 mg; pantothenate (as D-calcium pantothenate) 166 mg; r i b o f l a v i n 60 mg; p y r i d o x i n e (as pyridoxine-HCl) 36.5 mg; thiamine (as thiamine mononitrate) 36.3 mg; menadione (as MSBC) 30 mg; f o l i c a c i d 20 mg; b i o t i n 3 mg; v i t a m i n B-12 0.09 mg; r e t i n o l acetate 10,000 IU; c h o l e c a l c i f e r o l 2,400 IU; DL-c<-tocopheryl acetate 600 IU. A t o t a l of 17.93 g of extruded wheat per kg of dry d i e t was used as the c a r r i e r . The mineral supplement provided the f o l l o w i n g (mg/kg of dry d i e t ) : sodium (as NaCl) 1,500; magnesium (as MgS0 4»7H 20) 300; manganese (as MnS0 4*HoO) 75; i r o n (as FeS0 4«7H 20) 75; z i n c (as ZnS0 4»7H 20) 50; i o d i n e (as KI0 3) 10; f l u o r i n e (as NaF) 10; copper (as CuS0 4 #5H 20) 5; c o b a l t (as CoCl 2«6H 20) 1; selenium (as Na 2Se0 3) 0.1. A t o t a l of 12.58 g of extruded wheat per kg of dry d i e t was used as the c a r r i e r . 94 Table 12. Formulation o f b a s a l mix 2 used i n the p r e p a r a t i o n of the r e f e r e n c e and t e s t d i e t s employed i n experiment IV. In g r e d i e n t s C o n c e n t r a t i o n (g/kg dry d i e t ) Steam-dried h e r r i n g meal (73 .58% CP)* 610. 9 Extruded wheat (17.83% CP)* 39. 9 D r i e d whey (14.13% CP)* 83. 3 F r e e z e - d r i e d e u p h a u s i d s 1 (71 .48 CP)* 66. 6 Blood meal (95.37% CP) 55. 5 H e r r i n g o i l 2 71. 7 Soybean l e c i t h i n 10. 0 Vi t a m i n supplement-; 20. 0 M i n e r a l supplement 4 20. 0 Permapell ( l i g n i n - s u l p h o n a t e binder) 15. 0 Ch o l i n e c h l o r i d e (60%) 5. 0 A s c o r b i c a c i d 2. 0 Dry matter b a s i s . S t a b i l i z e d with 0.02% santoquin. The o i l was s t a b i l i z e d with 0.025% santoquin and was sprayed on t o the p e l l e t e d d i e t s . The v i t a m i n supplement p r o v i d e d the f o l l o w i n g amounts per kg o f dry d i e t : i n o s i t o l 400 mg; n i a c i n 300 mg; pantothenate (as D-calcium pantothenate) 166 mg; r i b o f l a v i n 60 mg; p y r i d o x i n e (as pyridoxine-HCl) 36.5 mg; thiamine (as thiamine mononitrate) 36.3 mg; menadione (as MSBC) 30 mg; f o l i c a c i d 20 mg; b i o t i n 3 mg; v i t a m i n B-12 0.09 mg; r e t i n o l a c e t a t e 10,000 IU; c h o l e c a l c i f e r o l 2,400 IU; DL - o c-tocopheryl a c e t a t e 600 IU. A t o t a l of 17.93 g of extruded wheat per kg of dry d i e t was used as the c a r r i e r . The m i n e r a l supplement p r o v i d e d the f o l l o w i n g (mg/kg of dry d i e t ) : sodium (as NaCl) 1,500; magnesium (as MgS0 4*7H 20) 300; manganese (as MnSC^'HoO) 75; i r o n (as FeS0 4*7H 20) 75; z i n c (as ZnS0 4*7H 20) 50; i o d i n e (as K I0 3 ) 10; f l u o r i n e (as NaF) 10; copper (as CuS 0 4 « 5 H 2 0 ) 5; c o b a l t (as C o C l 2 « 6 H 2 0 ) 1; selenium (as Na 2Se0 3) 0.1. A t o t a l of 12.58 g o f extruded wheat per kg of dry d i e t was used as the c a r r i e r . 95 4.2.7 Experimental procedures and f i s h handling 4.2.7.1 Diet a l l o c a t i o n Each d i e t was assigned randomly t o t r i p l i c a t e groups of f i s h i n a s i n g l e row of d i g e s t i b i l i t y tanks i n experiment I I . In experiments I I I and IV, a randomized complete block design was employed. 4.2.7.2 P r o t o c o l F i s h from the h o l d i n g tanks were anesthetized w i t h 2-phenoxyethanol (0.5 ml/L water) and weighed t o the nearest gram before commencement of experiments I I and I I I . A t o t a l p o p u l a t i o n of 1080 f i s h (experiment II) and 837 f i s h (experiment I I I ) was s e l e c t e d f o r uniform s i z e . The s e l e c t e d f i s h were d i s t r i b u t e d randomly, f i v e f i s h at a time, i n t o 18 and 27 d i g e s t i b i l i t y tanks i n experiments I I and I I I , r e s p e c t i v e l y . F o l l o w i n g t r a n s f e r i n t o the d i g e s t i b i l i t y tanks, the f i s h were g r a d u a l l y weaned from a commercial d i e t (BioDiet) to ba s a l d i e t 1 over a two-day p e r i o d . F i s h were acclimated t o the b a s a l d i e t over a p e r i o d of ten days during which feces (chromic oxide-free) were c o l l e c t e d from each c o l l e c t i o n column f o r subsequent chromic oxide standard curve p r e p a r a t i o n s . F i s h were fed to s a t i a t i o n at 0900 and 1430 hours, seven days a week. A f t e r the a c c l i m a t i o n p e r i o d , a l l f i s h i n each group were anesthetized w i t h 2-phenoxyethanol (0.5 ml/L water), placed onto an absorbent c l o t h t o remove excess moisture, and i n d i v i d u a l l y weighed t o the nearest 0.1 gram. Removal of a l l f i s h from each tank a l s o allowed f o r thorough tank c l e a n i n g . Mean weight (± SEM) of f i s h on day 0 was 13.3 ± 0.11 g and 26.7 ± 0.18 g f o r experiments II and I I I r e s p e c t i v e l y . On day 1, the s i x d i e t a r y treatments i n experiment II were a l l o t t e d at random to the row of tanks (completely random d e s i g n ) . In experiment I I I , however, the row of tanks was d i v i d e d i n t o t h r e e b l o c k s and each of the nine d i e t a r y treatments was a l l o t t e d at random to the tanks w i t h i n each b l o c k (randomized complete b l o c k d e s i g n ) . The p r o t o c o l p r i o r t o commencement of experiment IV was d i f f e r e n t from t h a t used i n experiments II and I I I . To minimize the s t r e s s and s c a l e l o s s a s s o c i a t e d w i t h e x c e s s i v e f i s h h a n d l i n g , i t was dec i d e d t o weigh the f i s h i n experiment IV only once. In t h i s regard, a t o t a l of 567 f i s h was s e l e c t e d from the 3600 L stock tanks w i t h i n a narrow weight range (mean weight of 45.1 ± 0.32 g (SEM)). The f i s h were d i s t r i b u t e d randomly i n t o the 27 d i g e s t i b i l i t y tanks. Three days a f t e r t r a n s f e r t o the d i g e s t i b i l i t y tanks, the f i s h were weaned from the B i o D i e t t o b a s a l d i e t 2 over a two-day p e r i o d . B a s a l d i e t 2 was f e d f o r seven days, d u r i n g which chromic o x i d e - f r e e feces were c o l l e c t e d . A f t e r the d i e t a c c l i m a t i o n p e r i o d , the row of 27 tanks was d i v i d e d i n t o t h r e e b l o c k s and each of the nine d i e t a r y treatments was a l l o t t e d at random t o the tanks w i t h i n each b l o c k . F i s h were f e d the r e f e r e n c e and t e s t d i e t s i n a l l experiments t o s a t i a t i o n d a i l y at 0900 and 1430 hours, seven days a week. To minimize f i s h d i s t u r b a n c e w hile f e e d i n g , a spoon a t t a c h e d t o a 1 m long c l e a r P l e x i g l a s s r od was used t o d e l i v e r the feed t o each of the groups. The p o i n t o f s a t i a t i o n was determined when i n d i v i d u a l l y d e l i v e r e d feed p e l l e t s were ign o r e d by the f i s h f o r the t h i r d s u c c e s s i v e time. Dry food consumption was recorded d a i l y . 97 C o l l e c t i o n o f f e c e s w i t h t h e "Guelph system" began t h r e e days a f t e r t h e i n t r o d u c t i o n o f t h e v a r i o u s d i e t a r y t r e a t m e n t s i n ex p e r i m e n t s I I and I I I . F e c a l c o l l e c t i o n s i n experiment IV were d e l a y e d s e v e r a l days s i n c e t h e f i s h o f f e r e d d i e t a r y t r e a t m e n t DW2 (30% d r i e d whey : 70% r e f e r e n c e d i e t ) f a i l e d t o f e e d a d e q u a t e l y . The poor f e e d i n t a k e o f f i s h f e d d i e t DW2 was most l i k e l y due t o th e e x t r e m e l y h a r d p e l l e t c o n s i s t e n c y r e s u l t i n g from t h e h i g h e r l e v e l o f d r i e d whey i n t h i s d i e t . S u b s e q u e n t l y , t h e groups r e c e i v i n g d i e t DW2 were g i v e n d i e t DW1 (15% d r i e d whey : 85% r e f e r e n c e d i e t ) and f e c a l c o l l e c t i o n s from a l l t a n k s were s t a r t e d t h r e e days l a t e r . 4.2.8 F e c a l c o l l e c t i o n p r o c e d u r e Immediately a f t e r t h e l a s t f e e d i n g on t h e day b e f o r e t h e morning f e c a l c o l l e c t i o n s were i n i t i a l e d and f o r t h e next t h r e e weeks i n experiment I I ( e x c l u d i n g weekends) or 15 c o n s e c u t i v e days i n e x p e r i m e n t s I I I and IV, t h e t a n k s were t h o r o u g h l y c l e a r e d o f any f e c e s o r uneaten f e e d . The c o t t o n f i l t e r i n each water i n l e t f u n n e l was r e p l a c e d b e f o r e t a n k c l e a n i n g . U s i n g a c l e a r (1.90 cm ID) p l e x i g l a s s p i p e a t t a c h e d t o a f l e x i b l e hose, a l l f e e d and f e c a l sediments on t h e s i d e s o f each ta n k were syphoned o u t . The c l e a r p l e x i g l a s s p i p e p r o v e d t o be v e r y e f f i c i e n t f o r c l e a n i n g t h e t a n k s and t h e f i s h appeared t o be u n d i s t u r b e d . A f t e r a l l t a n k s had been syphoned c l e a n , t h e clamp a t t h e s a m p l i n g p o r t o f t h e c o l l e c t i o n column was opened and water was a l l o w e d t o f l u s h o u t . A l a r g e b r u s h a t t a c h e d t o a 1 m l o n g p i p e was i n s e r t e d down t h e v e r t i c a l s t a n d p i p e t o t h e bottom o f t h e c o l l e c t i o n column (see F i g u r e 1) and t h e br u s h e d d e p o s i t s were 98 swept out w i t h the out f l o w i n g water. The f l u s h i n g cap of each tank was opened and a brush w i t h a long handle was used t o clean the i n s i d e of the connecting pipe between the tank bottom s l o t and the c o l l e c t i o n column. When the water l e v e l i n the tank had dropped by approximately 1/3 of the t o t a l tank volume, the f l u s h i n g cap and the c o l l e c t i o n column clamp were replaced. Water flow r a t e s were monitored i n each tank to ensure that the feces produced overnight would be s w i f t l y c a r r i e d i n t o the c o l l e c t i o n column. At 0800 hours the f o l l o w i n g day, the s e t t l e d feces and surrounding water were very gently withdrawn from the base of the s e t t l i n g column i n t o a 250 ml c e n t r i f u g e b o t t l e f i t t e d w i t h a rubber stopper. Immediately a f t e r c o l l e c t i o n , the feces were c e n t r i f u g e d at 10,000 x g f o r 30 minutes at approximately 5°C and the supernatant discarded. The d a i l y f e c a l c o l l e c t i o n of each group was pooled over the c o l l e c t i o n p e r i o d and h e l d i n a sealed p l a s t i c container at -40°C. F i s h were euthenized by an overdose of 2-phenoxyethanol (2ml/L water) and weighed to the nearest 0.1 gram at the end of each experiment. Feces were c o l l e c t e d from a l l f i s h at t h i s time (except from f i s h maintained on d i e t s c o n t a i n i n g soybean products) by e i t h e r s t r i p p i n g or i n t e s t i n a l d i s s e c t i o n as described i n experiment I. Subsequently, the f e c a l samples were stored i n sealed p l a s t i c v i a l s at -40°C. 99 4.2.9 Chemical analyses of t e s t i n g r e d i e n t s / d i e t s and f e c a l samples The frozen f e c a l samples were l y o p h i l i z e d and ge n t l y ground i n a mortar and p e s t l e before chemical analyses. Moreover, the samples c o l l e c t e d w i t h the "Guelph system" were passed through a 250um screen to remove f i s h s c a l e s . The t e s t i n g r e d i e n t s , d i e t s and l y o p h i l i z e d feces were analyzed f o r moisture and ash (AOAC, 1975) and t o t a l n i t r o g e n (Technicon Instrumental Co., L t d . , i n d u s t r i a l methods 369-75A/A and 334-74 W/B). The percent n i t r o g e n was m u l t i p l i e d by 6.25 t o obtain percent crude p r o t e i n values. Gross energy was determined by combustion of the t e s t i n g r e d i e n t s , d i e t s and feces using an a d i a b a t i c oxygen bomb c a l o r i m e t e r (Gallenkamp and Co. L t d . , Loughborough, England) using benzoic a c i d as a standard. The chromic oxide concentrations i n the d i e t s and feces were determined by the wet d i g e s t i o n c o l o r i m e t r i c method of Stevenson and De Langen (1960). The crude l i p i d contents i n the i n g r e d i e n t s and d i e t s were measured by the B l i g h and Dyer (1959) technique. A l l determinations were done i n d u p l i c a t e . Owing to the small amounts of feces c o l l e c t e d by the i n t e s t i n a l d i s s e c t i o n and s t r i p p i n g techniques, a n a l y s i s of these samples was r e s t r i c t e d t o chromic oxide and organic matter. 100 4.2.10 D i g e s t i b i l i t y and f i s h performance c a l c u l a t i o n s The apparent d i g e s t i b i l i t y c o e f f i c i e n t s (ADC) f o r organic matter, crude p r o t e i n and energy f o r each d i e t were c a l c u l a t e d by the i n d i r e c t method using the f o l l o w i n g formula: ADC (%) '=. 100 1-% Cr2C>3 i n d i e t x % n u t r i e n t i n feces % Cr 203 i n feces x % n u t r i e n t i n d i e t The apparent d i g e s t i b i l i t y c o e f f i c i e n t of gross energy was c a l c u l a t e d using gross energy data (MJ/kg) i n s t e a d of n u t r i e n t data. The apparent d i g e s t i b i l i t y c o e f f i c i e n t f o r a given t e s t i n g r e d i e n t was c a l c u l a t e d from the apparent d i g e s t i b i l i t y c o e f f i c i e n t s f o r the reference and t e s t d i e t s on the b a s i s of the percent s u b s t i t u t i o n of the t e s t i n g r e d i e n t (X) f o r the reference d i e t (100-X) on a dry matter b a s i s according to the f o l l o w i n g equation: ADC of t e s t ingr.= 100 X 100-X ADC t e s t d i e t ADC r e f e r , d i e t 100 F i s h performance was al s o assessed i n each experiment. However, the r e s u l t s of growth and feed e f f i c i e n c y r a t i o s should be i n t e r p r e t e d w i t h caution since the d i e t s were fed f o r a r e l a t i v e l y short p e r i o d of time and they were n e i t h e r i s o c a l o r i c nor isonitrogenous. 101 S p e c i f i c growth r a t e s or percent increases i n wet body weight per day (Bret t , 1979) were c a l c u l a t e d as: (logn t l - logn tO) x 100 ( t l - tO) In t h i s equation, logn t l and logn tO are the n a t u r a l l o g wet weights at the s t a r t (tO) and the end ( t l ) of the p e r i o d . Using data on the mean d a i l y dry feed intake per f i s h (DFI; g / f i s h ) , feed i n t a k e was expressed as a percentage of mean wet body weight according t o the procedure of Richardson et al. (1985). The formula used was as f o l l o w s : DFI x 100 (logn t l + logn tO 2 ) e Dry feed i n t a k e data were a l s o used t o c a l c u l a t e feed e f f i c i e n c y (FE; wet weight gain (g) -5- DFI) and p r o t e i n e f f i c i e n c y r a t i o (PER; wet weight gain (g) p r o t e i n i n t a k e (g) ) . Data f o r DFI coupled, w i t h those f o r d i e t proximate composition and r e s p e c t i v e organic matter, crude p r o t e i n and gross energy d i g e s t i b i l i t y , were used t o c a l c u l a t e mean d a i l y organic matter, p r o t e i n and energy d i g e s t e d by each f i s h (g/fish) i n experiments I I and I I I . Since the i n i t i a l mean weights of the f i s h i n each group were not obtained p r i o r t o i n i t i a l t e s t d i e t feeding i n experiment IV, data on weight gain and growth were c a l c u l a t e d using the o v e r a l l i n i t i a l mean f i s h weight.. 102 4.2.11 S t a t i s t i c a l a n a l y s i s A l l s t a t i s t i c a l analyses were performed using a general l i n e a r model program (SAS, 1985) a f t e r ensuring that'assumptions regarding homogeneity of variance were s a t i s f i e d (using H a r t l e y ' s t e s t as described by Lyman, 1984). In t h i s regard, one-way analyses of variance were performed a f t e r t r ansformation of the apparent organic matter, crude p r o t e i n and gross energy d i g e s t i b i l i t y data i n t o a r c s i n square root data (Steel and T o r r i e , 1960) t o determine s i g n i f i c a n t d i f f e r e n c e s between t e s t i n g r e d i e n t s . No block e f f e c t s were found (P>0.05) i n experiments I I I and IV and hence the degrees of freedom f o r blocks were pooled i n t o the e r r o r term. D i f f e r e n c e s between treatment means were detected by Newman-Keuls' (Keuls, 1952) t e s t w i t h P=0.05. Di f f e r e n c e s i n mean apparent organic matter d i g e s t i b i l i t y c o e f f i c i e n t s as i n f l u e n c e d by the f e c a l c o l l e c t i o n procedure ("Guelph system" vs i n t e s t i n a l d i s s e c t i o n or "Guelph system" vs s t r i p p i n g ) were assessed by the student's " t " t e s t (Steel and T o r r i e , 1960). In cases where the i n g r e d i e n t s were t e s t e d at two l e v e l s of i n c l u s i o n ( i . e . 15% or 30%), the d i f f e r e n c e s i n organic matter, crude p r o t e i n and gross energy d i g e s t i b i l i t y c o e f f i c i e n t s were revealed by the p a i r e d data a n a l y s i s as described by Lyman (1984). 103 Data on weight gain, s p e c i f i c growth r a t e , dry feed i n t a k e , feed intake as a percent of body weight, feed e f f i c i e n c y , p r o t e i n e f f i c i e n c y r a t i o and q u a n t i t y of organic matter, p r o t e i n and energy dig e s t e d by f i s h on each d i e t a r y treatment i n a l l experiments were subjected to a n a l y s i s of variance t o detect d i f f e r e n c e s between d i e t treatments. The treatment means were subjected to Newman-Keuls1 (Keuls, 1952) t e s t w i t h P=0.05. The s t a t i s t i c a l analyses are t a b u l a t e d i n Appendix 4. 104 4.3 RESULTS 4.3.1 Chemical composition of t e s t i n g r e d i e n t s The r e s u l t s of proximate analyses of the i n g r e d i e n t s t e s t e d i n experiments I I , I I I and IV, together w i t h t h e i r a b b r e v i a t i o n codes, are given i n Tables 13, 14 and 15, r e s p e c t i v e l y . Of the f i s h meals, the menhaden meal (MM) contained the highest ash content and subsequently a l s o the lowest crude p r o t e i n content. The p o u l t r y by-product meals from d i f f e r e n t sources (PMK and PMB) d i f f e r e d only by one percentage p o i n t i n t h e i r ash content. However, d i f f e r e n c e s i n the moisture, crude p r o t e i n and l i p i d content of the two p o u l t r y by-product meals were s u b s t a n t i a l . The lower crude p r o t e i n concentration i n the l o c a l p o u l t r y by-product meal (PMB) was compensated f o r by a higher l i p i d content, and thus a s l i g h t l y higher gross energy value, r e l a t i v e to PMK. With respect to other animal by-product sources, hydrolyzed feather meal (FM) and blood meal (BM) i n p a r t i c u l a r , c o n s i s t e d almost e x c l u s i v e l y of p r o t e i n , while d r i e d whey (DW) contained mainly carbohydrate. The g l u c o s i n o l a t e - f r e e c u l t i v a r canola meal (GCMa) was s i m i l a r t o the commercial canola meal (CM) i n proximate composition. The p r o t e i n content of the canola meal which was g l u c o s i n o l a t e - f r e e as a r e s u l t of solvent e x t r a c t i o n (GCMb) on the other hand, was s u b s t a n t i a l l y higher than t h a t of the former canola meals, p o s s i b l y due to some lo s s e s of l i p i d and s o l u b l e carbohydrates during the a d d i t i o n a l e x t r a c t i o n s processes performed t o reduce the g l u c o s i n o l a t e content of GCMb. In regards to the high p r o t e i n content p l a n t sources, soybean p r o t e i n i s o l a t e (SI) had the highest l e v e l of p r o t e i n , matching 105 t h a t of the blood meal, while rapeseed p r o t e i n concentrate (RP) was r e l a t i v e l y high i n both p r o t e i n and l i p i d . D i f f e r e n c e s between the proximate components of wheat middlings (WM) and extruded wheat (EW) would not appear to be s u f f i c i e n t to warrant s u b s t i t u t i o n of one f o r the other i n d i e t formulations on the b a s i s of these c r i t e r i a . 106 Table 1 3 . Proximate composition and gross energy content of t e s t i n g r e d i e n t s used i n experiment II (values i n parentheses show percentages f o r each proximate c o n s t i t u e n t on a dry matter b a s i s ) . Proximate c o n s t i t u e n t Water P r o t e i n L i p i d Ash G.E, Test I n g r e d i e n t Code (percent) (MJ/kg) Rapeseed p r o t e i n c o ncentrate Canola meal (commercial) RP 6 .57 60 .03 8 .89 6.64 21 .17 (64.25) (9 .51) (7 .11) (22.66) CM 10 .38 3 4 . 0 6 4 .34 6.84 18 .09 (38.00) (4 .84) (7 .63) (20.18) G l u c o s i n o l a t e f r e e GCMa c u l t i v a r c a n o l a 9.62 32 .14 3 .85 7 .36 17 .75 (35.56) (4 .26) (8 .14) (19.64) H e r r i n g meal HM 7 .96 68 .15 10 .93 12 .96 2 0 . 2 9 (74.04) (11.87) (14.08) (22.04) Anchovy meal AM 8.60 66 .12 11 .20 14 .07 20 .21 (72.34) (12.25) (15 .39) (22.11) 107 Table 14. Proximate composition and gross energy content of t e s t i n g r e d i e n t s used i n experiment I I I (values i n parentheses show percentages f o r each proximate c o n s t i t u e n t on a dry matter b a s i s ) . Proximate c o n s t i t u e n t Water P r o t e i n L i p i d Ash G.E. Test Ingredient Code (percent) (MJ/kg) P o u l t r y by-product PMK 5.37 meal (Kansas) P o u l t r y by-product PMB '2.91 meal (B.C.) 69.55 13.52 10.79 21.47 (73.50) (14.29) (11.40) (22.69) 64.20 20.18 12.03 22.52 (66.12) (20.78) (12.39) (23.20) Menhaden MM 9.66 meal Hydrolyzed feather FM 4.97 meal 60.54 11.36 18.36 18.30 (67.01) (12.57) (20.32) (20.26) 84.93 8.09 4.32 22.61 (89.37) (8.51) (4.55) (23.79) Low temperature Norwegian LT h e r r i n g / c a p e l i n meal 6.93 70.46 10.41 11.89 20.52 (75.71) (11.18) (12.77) (22.04) Soybean SM 8.42 47.91 1.96 6.84 17.89 meal (52.31) (2.14) (7.47) (19.53) Blood BM 3.44 91.39 2.95 1.81 23.72 meal (94.64) (3.05) (1.87) (24.57) Soybean SI 4.83 87.26 4.82 4.11 21.53 i s o l a t e s (91.69) (5.06) (4.32) (22.62) 108 Table 15. Proximate composition and gross energy content of t e s t i n g r e d i e n t s used i n experiment IV (values i n parentheses show percentages f o r each proximate c o n s t i t u e n t on a dry matter b a s i s ) . Proximate c o n s t i t u e n t Water P r o t e i n L i p i d Ash G.E. Test :  Ingredient Code (percent) (MJ/kg) Dried whey DW 5 .53 10 (11 .98 .62) 0. 96 (1.02) 7.74 (8.19) 15.29 (16.19) Extruded wheat EW 9 .42 14 (15 .15 .62) 1.22 (1.35) 1.57 (1.73) 16.78 (18.52) Soybean meal SM 8 .42 47 (52 .91 .31) 1.96 (2.14) 6.84 (7.47) 17.89 (19.53) Wheat middlings WM 11 .03 14 (15 .12 .87) 3.34 (3.75) 2.11 (2.37) 16.66 (18.72) Soybean i s o l a t e s SI 4 .83 87 (91 .26 .69) 4.82 (5.06) 4.11 (4.32) 21.53 (22.62) G l u c o s i n o l a t e e x t r a c t e d canola meal GCMb 9 .23 37. (41 62 .44) 2.02 (2.22) 7.73 (8.52) 17.61 (19.40) 109 4.3.2 P r o x i m a t e c o m p o s i t i o n and m i n e r a l c o n t e n t o f r e f e r e n c e and t e s t d i e t s The p r o x i m a t e and m i n e r a l c o m p o s i t i o n s o f a l l t h e d i e t s used i n e x p e r i m e n t s I I , I I I and IV a r e shown i n T a b l e s 16, 17 and 18, r e s p e c t i v e l y . The p r o t e i n c o n t e n t o f b o t h r e f e r e n c e d i e t s (REF-a and REF-b) was a p p r o x i m a t e l y 2.5 p e r c e n t a g e p o i n t s h i g h e r t h a n would be e x p e c t e d from t h e d i e t f o r m u l a t i o n s . On t h e o t h e r hand, t h e m o i s t u r e c o n t e n t o f a l l d i e t s was v e r y c l o s e t o t h e t h e o r e t i c a l amount o f 9%. The m i n e r a l l e v e l s i n b o t h r e f e r e n c e d i e t s (REF-a and REF-b) were found t o be v e r y s i m i l a r . However, t h e r e were c o n s i d e r a b l e d i f f e r e n c e s i n t h e major m i n e r a l c o n t e n t s between some t e s t d i e t s . F o r i n s t a n c e , t h e i r o n c o n t e n t s o f d i e t s MM2 and BM2 were two t o t h r e e t i m e s g r e a t e r t h a n t h e l e v e l s found i n any o t h e r d i e t s . L i k e w i s e , t h e copper c o n t e n t o f d i e t PMK2 was f o u r t i m e s g r e a t e r t h a n t h e h i g h e s t amount found i n o t h e r d i e t . The magnesium c o n t e n t o f a l l c a n o l a / r a p e s e e d p r o d u c t s appeared t o be q u i t e h i g h , as r e f l e c t e d by t h e s u b s t a n t i a l i n c r e a s e i n t h e l e v e l o f t h i s m i n e r a l i n a l l d i e t s i n w h i c h c a n o l a / r a p e s e e d was i n c l u d e d as t h e t e s t i n g r e d i e n t (e.g. CM2, RP2, GCMa2, GCMb2). The t e x t u r e o f t h e p e l l e t s v a r i e d c o n s i d e r a b l y between d i e t s , b e i n g r e l a t i v e l y h a r d i n some cases (BM2, DW1, DW2, WM2, EW2), and f r a g i l e i n o t h e r s (SI2, GCMb2). 110 Table 16. Proximate composition and mineral content of the reference and f i v e t e s t d i e t s fed to j u v e n i l e chinook salmon i n experiment I I . (Refer to Table 13 f o r a d d i t i o n a l i n f o r m a t i o n . REF-a r e f e r s to reference d i e t a ) . Proximate Diet code Composition (test i n g r e d i e n t and i n c l u s i o n l e v e l ) (% of dry matter) REF-a RP2 CM2 GCMa2 HM2 AM2 Crude p r o t e i n (N x 6.25) 55.50 Crude l i p i d (Bligh-Dyer) 16.72 Ash 10.81 T o t a l carbohydrate 1 (by d i f f e r e n c e ) 16.97 Gross energy (MJ/kg) 22.63 Moisture (as fed) 8.54 59.13 14.86 9.12 16.89 22.56 8.63 50.06 13.08 9.84 27.02 21.58 8.40 50.13 12.68 9. 97 27.22 21.45 8.58 61.44 15.06 11.76 11.74 22.52 8.42 61.31 15.42 12.19 11.08 22.43 8.76 o M i n e r a l content^ (g/kg dry d i e t ) Calcium (Ca) 27. 3 20.7 20 .9 20. 0 31.6 30 .6 Phosphorous (P) 17. 1 17.5 15 .2 14. 8 19.4 19 .5 Sodium (Na) 7. 68 5.35 5 .22 4. 91 7.10 8 .15 Magnesium (Mg) 2. 02 3.65 3 .26 3. 26 1.97 2 .18 Iron (Fe) 0. 33 0.28 0 .29 0. 29 0.32 0 .36 Zinc (Zn) 0. 12 0.11 0 .11 0. 11 0.11 0 .11 Manganese (Mn) 0. 11 0.12 0 .09 0. 09 0.08 0 .08 Copper (Cu) 0. 01 0.01 0 .01 0. 01 0.01 0 .01 Cobalt (Co) l e s s than 0. 0002 i n a l l d i e t s Ca:P 1. 60 1.18 1 .38 1. 35 1.63 1 .57 Chromic o x i d e 3 5. 65 5.46 5 .75 5. 78 5.86 5 .53 C a l c u l a t e d as: 100 - ( p r o t e i n + l i p i d + ash). Di e t s were analyzed f o r mineral content by plasma spectroscopy (Higgs et al., 1982). Chromic oxide (Cr2C>3) a n a l y s i s performed by the wet d i g e s t i o n c o l o r i m e t r i c method of Stevenson and De Langen (1960). I l l Table 17. Proximate composition and mineral content of the reference and e i g h t t e s t d i e t s fed to j u v e n i l e chinook salmon i n experiment I I I . (Refer to Table 14 f o r a d d i t i o n a l i n f o r m a t i o n . REF-a r e f e r s t o reference d i e t a ) . Proximate Diet code Composition (test i n g r e d i e n t and i n c l u s i o n l e v e l ) (% of dry matter) REF-a PMK2 PMB2 MM2 FM2 Crude p r o t e i n (N x 6.25) 55. 62 61 .44 58 .63 60. 13 65 .19 Crude l i p i d (Bligh-Dyer) 16. 14 16 .48 17 .68 16. 03 14 .53 Ash 11. 10 11 .41 12 .73 14. 23 9 .24 T o t a l carbohydrate 1 (by d i f f e r e n c e ) 17. 14 10 .67 10 .96 9. 61 11 .04 Gross energy (MJ/kg) 22. 91 23 .01 22 .91 22. 43 23 .25 Moisture (as fed) 9. 78 9 .36 9 .86 9. 74 9 .14 M i n e r a l content (g/kg dry d i e t ) Calcium (Ca) 27. 9 28 .9 33 .8 33. 6 23 .2 Phosphorous (P) 16. 6 17 .2 19 .0 19. 6 13 .4 Sodium (Na) 8. 30 7 .17 7 .52 8. 35 6 .24 Magnesium (Mg) 2. 17 1 .87 1 .95 2. 22 1 .61 Iron (Fe) 0. 42 0 .47 0 .42 0. 70 0 .39 Zinc (Zn) 0. 10 0 .10 0 .10 0. 09 0 .10 Manganese (Mn) 0. 11 0 .07 0 .07 0. 08 0 .07 Copper (Cu) 0. 01 0 .04 0 .01 0. 01 0 .01 Cobalt (Co) 0. 0009 l e s s than 0.0007— Ca:P 1. 68 1 . 68 1 .78 1. 71 1 .73 Chromic o x i d e 3 5. 51 5 .00 6 .13 5. 43 5 .57 C a l c u l a t e d as: 100 - (pr o t e i n + l i p i d + ash). D i e t s were analyzed f o r mineral content by plasma spectroscopy (Higgs et al., 1982). Chromic oxide (C^C^) a n a l y s i s performed by the wet d i g e s t i o n c o l o r i m e t r i c method of Stevenson and De Langen (1960). 112 T a b l e 17. C o n t i n u e d . P r o x i m a t e C o m p o s i t i o n (% o f d r y m a t t e r ) D i e t code ( t e s t i n g r e d i e n t and i n c l u s i o n l e v e l ) LT2 SM2 BM2 SI2 C r u d e p r o t e i n (N x 6.25) Crude l i p i d ( B l i g h - D y e r ) A s h 1 T o t a l c a r b o h y d r a t e -(by d i f f e r e n c e ) G r o s s e n e r g y (MJ/kg) M o i s t u r e (as fed) 64.25 15.54 11.88 8.33 22.98 9. 92 55.44 12.86 10.20 21.50 22.22 9.63 67.63 12.68 8.35 11.34 9.34 67.06 13.63 9.18 10.13 23.87 23.24 9.93 M i n e r a l content'' (g/kg d r y d i e t ) C a l c i u m (Ca) 26.5 18.2 18.6 15. 7 P h o s p h o r o u s (P) 17.7 13.1 12.1 11. 6 Sodium (Na) 8.58 5.28 6.92 6. 99 Magnesium (Mg) 2.20 2.50 1.49 1. 27 I r o n (Fe) 0.38 0.32 1.01 0. 23 Z i n c (Zn) 0.10 0.08 0.08 0. 08 Manganese (Mn) 0.07 0.08 0.07 0. 06 C o p p e r (Cu) 0.01 0.01 0.01 0. 01 C o b a l t (Co) 0.0007 0.0008 0.0009 0. 0006 Ca:P 1.50 1.39 1.54 1. 35 C h r o m i c o x i d e 3 5.83 5.30 4.97 5. 42 C a l c u l a t e d a s : 100 - ( p r o t e i n + l i p i d + ash) . D i e t s were a n a l y z e d f o r m i n e r a l c o n t e n t by p l a s m a s p e c t r o s c o p y ( H i g g s e t a l . , 1982) . C h r o m i c o x i d e (Cr2C>3 ) a n a l y s i s p e r f o r m e d by t h e wet 1 2 d i g e s t i o n c o l o r i m e t r i c method o f S t e v e n s o n a nd De Langen (19 6 0 ) . 113 Ta b l e 18. P r o x i m a t e c o m p o s i t i o n and m i n e r a l c o n t e n t o f t h e r e f e r e n c e and seven t e s t d i e t s f e d t o j u v e n i l e c h i n o o k salmon i n experiment IV. ( R e f e r t o T a b l e 15 f o r a d d i t i o n a l i n f o r m a t i o n . REF-b r e f e r s t o r e f e r e n c e d i e t b ) . P r o x i m a t e D i e t code C o m p o s i t i o n ( t e s t i n g r e d i e n t and i n c l u s i o n l e v e l ) (% o f d r y matter) REF-b DW1 DW2 EW1 EW2 Crude p r o t e i n (N x 6.25) 59.50 52.69 46.00 53.44. 46.94 Crude l i p i d ( B l i g h - D y e r ) 16.10 14.76 11.58 14.61 11.92 Ash 12.15 11.58 10.98 10.49 8.91 T o t a l c a r b o h y d r a t e 1 (by d i f f e r e n c e ) 12.25 20.97 31.44 21.46 32.95 Gross energy (MJ/kg) 22.72 21.96 21.19 22.18 21.64 M o i s t u r e (as fed) 8.37 8.64 8.66 8.24 8.19 M i n e r a l c o n t e n t (g/kg d r y d i e t ) C a l c i u m (Ca) 30 .3 25. 8 N/A 25 .2 16.0 Phosphorous (P) 17 .8 15. 8 N/A 15 .6 12.1 Sodium (Na) 8 .31 8. 11 N/A 7 .14 4.99 Magnesium (Mg) 2 .04 1. 90 N/A 2 .02 1.66 I r o n (Fe) 0 .42 0. 35 N/A 0 .39 0.23 Z i n c (Zn) 0 .10 0. 08 N/A 0 .09 0.07 Manganese (Mn) 0 .10 0. 08 N/A 0 .09 0.07 Copper (Cu) 0 .01 0. 01 N/A 0 .01 0.01 C o b a l t (Co) 0 .0009 0. 0008 N/A 0 .001 0.0007 Ca:P 1 .70 1. 63 N/A 1 .62 1.32 Chromic o x i d e 4 5 .20 5. 54 N/A 5 .31 5.77 C a l c u l a t e d as: 100 - ( p r o t e i n + l i p i d + ash). D i e t s were analyzed f o r mineral content by plasma spectroscopy (Higgs et a l . , 1982). Use of d i e t DW2 aborted i n the experiment. No mineral a n a l y s i s performed. Chromic oxide (C^C^) a n a l y s i s performed by the wet d i g e s t i o n c o l o r i m e t r i c method of Stevenson and De Langen (1960). Table 18. Continued. 114 Proximate Composition (% of dry matter) Diet code (test i n g r e d i e n t and i n c l u s i o n l e v e l ) SMI WM2 SI1 GCMb2 Crude p r o t e i n (N x 6.25) Crude l i p i d (Bligh-Dyer) Ash T o t a l carbohydrate 1 (by d i f f e r e n c e ) Gross energy (MJ/kg) Moisture (as fed) 58 .25 14.80 11.45 15.50 47.50 13.13 9.25 30.12 63.06 15.67 10.79 10.48 53.50 12.96 11.10 22.44 22.55 21.66 23.11 21.88 8.73 8.77 8.42 8.52 M i n e r a l content (g/kg dry d i e t ) Calcium (Ca) 19. 9 17.3 21.8 23.3 Phosphorous (P) 14 . 1 13.1 16.0 18.0 Sodium (Na) 5. 99 5.22 7.90 6.16 Magnesium (Mg) 1. 98 1.62 1.65 3.72 Iron (Fe) 0. 33 0.32 0.36 0.38 Zinc (Zn) 0. 07 0.07 0.09 0.09 Manganese (Mn) 0. 07 0.07 0.08 0.09 Copper (Cu) 0. 01 0.01 0.01 0.01 Cobalt (Co) . 0. 0009 0.0008 0.0008 0.0009 Ca:P 1. 41 1.32 1.36 1.29 Chromic o x i d e J 5. 12 5.21 4.87 5.47 C a l c u l a t e d as: 100 - ( p r o t e i n + l i p i d + ash). D i e t s were analyzed f o r mineral content by plasma spectroscopy (Higgs et al., 1982). Chromic oxide (C^C^) a n a l y s i s performed by the wet d i g e s t i o n c o l o r i m e t r i c method of Stevenson and De Langen (1960). 115 4.3.3 Apparent n u t r i e n t d i g e s t i b i l i t y c o e f f i c i e n t s of t e s t  i n g r e d i e n t s The apparent d i g e s t i b i l i t y c o e f f i c i e n t s f o r organic matter, crude p r o t e i n and gross energy, as w e l l as the d i g e s t i b l e energy content of a l l i n g r e d i e n t s t e s t e d i n experiments I I , I I I and IV are l i s t e d i n Table 19. D i f f e r e n c e s i n n u t r i e n t d i g e s t i b i l i t i e s between f e e d s t u f f s were s i g n i f i c a n t (P<0.001). The d i g e s t i b i l i t y of organic matter and gross energy of the f i s h meals was c o n s i s t e n t l y higher than the d i g e s t i b i l i t y of these components i n the other f e e d s t u f f s , although d i f f e r e n c e s were not always s i g n i f i c a n t (P>0.05). Within the f i s h meals, menhaden meal had the lowest n u t r i e n t d i g e s t i b i l i t y c o e f f i c i e n t s . The highest apparent crude p r o t e i n d i g e s t i b i l i t y was obtained f o r the rapeseed p r o t e i n concentrate (RP) and i t s crude p r o t e i n d i g e s t i b i l i t y was s i g n i f i c a n t l y (P<0.05) greater than t h a t of most other f e e d s t u f f s . In r e l a t i o n to the animal by-product meals assessed, the Kansas source of p o u l t r y by-product meal (PMK) g e n e r a l l y had s u p e r i o r n u t r i e n t d i g e s t i b i l i t y c o e f f i c i e n t s , although s i g n i f i c a n t d i f f e r e n c e s could not always be demonstrated. By c o n t r a s t , blood meal (BM) was very poorly d i g e s t e d by the f i s h . The d i g e s t i b i l i t y of the crude p r o t e i n and the energy of BM was s i g n i f i c a n t l y (P<0.05) lower than the d i g e s t i b i l i t y of these components i n the other f e e d s t u f f s . Reduction of the g l u c o s i n o l a t e content i n the canola meal, GCMa, r e s u l t e d i n improved n u t r i e n t and energy d i g e s t i b i l i t i e s r e l a t i v e t o those observed f o r the commercial canola meal (CM), although the d i f f e r e n c e s were not s i g n i f i c a n t (P<0.05). A l t e r n a t i v e l y , reduction of the g l u c o s i n o l a t e content i n canola meal by solvent e x t r a c t i o n , r e s u l t e d i n s i g n i f i c a n t l y (P<0.05) lower organic matter d i g e s t i b i l i t y compared t o CM and GCMa. Extruded wheat was w e l l d i g e s t e d by chinook salmon. D i f f e r e n c e s i n d i g e s t i b i l i t y c o e f f i c i e n t s between the two l e v e l s of i n c l u s i o n of extruded wheat were not s i g n i f i c a n t (P>0.05) f o r organic matter (t=0.58; 4 d f ) , crude p r o t e i n (t=1.57; 4df) and gross energy (t=0.47; 4 d f ) . The d i g e s t i b i l i t y of the organic matter and gross energy i n the wheat middling was very poor, although the crude p r o t e i n i n t h i s f e e d s t u f f was h i g h l y d i g e s t i b l e . D i g e s t i b i l i t y c o e f f i c i e n t s obtained f o r the soybean products are not r e l i a b l e s ince the food intakes of f i s h fed d i e t s c o n t a i n i n g soybean products were extremely poor due to reduced d i e t p a l a t a b i l i t y . F eedstuff d i g e s t i b i l i t y was noted to be more v a r i a b l e when the t e s t i n g r e d i e n t s were i n c l u d e d at the 15% l e v e l r e l a t i v e to the 30% l e v e l . 117 Table 19. Mean percent (± SEM) apparent o r g a n i c matter, crude p r o t e i n and gross energy d i g e s t i b i l i t y c o e f f i c i e n t s and d i g e s t i b l e energy v a l u e s of i n g r e d i e n t s measured wi t h p o s t - j u v e n i l e chinook salmon i n seawater. Apparent d i g e s t i b i l i t y (%) D i g e s t i b l e energy (MJ/kg) Ingredient Code Organic matter Crude p r o t e i n Gross energy H e r r i n g meal HM 8 7 . 3 a l ±1.03 9 0 . 5 b c d ±0.51 92. 6 a ±0.48 20.4 a ±0.11 Anchovy meal AM 87. 6 a ±2.11 9 1 . 7 a b c ±1.50 91. 6 a ±1.62 20.2 a ±0.36 Menhaden meal MM 84.0 a ±0.99 83.1 e f9 ±1.45 8 4 . 1 a b ±1.80 1 7 . 0 a b c d ±0.36 LT h e r r i n g / c a p e l i n meal LT 87.8 a ±0.62 9 3 . 6 a b ±0.81 88.4 a ±1.26 1 9 . 5 a b ±0.28 P o u l t r y by-product meal (Kansas) PMK 7 5 . 9 a b ±2.41 8 4 . 9 d e f g ±1.16 7 2 . 4 b c d ±1.69 1 6 . 4 b c d e ±0.38 P o u l t r y by-product meal (B.C.) PMB 6 5 . 6 b c d ±1.82 74.4 h ±1.11 6 5 . 4 c d ±2.17 1 5 . 2 c d e f ±0.50 Feather meal FM 6 3 . 1 b c d ±0.89 70. 8 h ±0.84 5 7 . 4 d e ±1.48 1 3 . 6 d e f ±0.35 Blood meal BM 34. 8 e ±2.27 29.4 1 ±1.20 31. 9 f ±1.68 7.8 h ±0.41 D r i e d whey DW 7 6.7 a b ±3.65 72. 5 h ±2.18 7 1 . 5 b c d ±5.82 11.6 f9 • ±0.60 1 Means i n each column f o l l o w e d by a common s u p e r s c r i p t are not s i g n i f i c a n t l y d i f f e r e n t (Newman-Keuls t e s t with P=0.05). Table 19. Continued. Apparent d i g e s t i b i l i t y (%) D i g e s t i b l e 118 Ingredient Code Organic Crude Gross energy matter p r o t e i n energy (MJ/kg) Canola CM 53 . 5 d l 84 > 5 d e f g 64 . 5 c d 13 . 0 e f meal ±2 .33 ±1 .65 ±1 .59 ±0 .32 G l u c o s i n o l a t e -free c u l t i v a r GCMa 58 . 7 c d 87 m gcde 71 . o b c d 13 > 9 d e f canola meal ±2 .36 ±1 .15 ±2 .34 ±0 .46 G l u c o s i n o l a t e e x t r a c t e d GCMb 37 . 5 e 78 > 6 f g h 51 . 2 d e 9 .93^ h canola meal ±1 .48 ±1 .23 ±1 .71 ±0 .33 Rapeseed p r o t e i n RP 69 . 8 b c 95 . 6 a 80 . 5 a b c 18 . 2 a b c concentrate ±1 .48 ±0 .61 ±2 .82 ±0 .64 Soybean meal SM 58 . 5 c d 74 . 6 h 66 . o b c d 12 . 9 e f ( l e v e l l ) 2 ±7 .19 ±3 .76 ±9 .32 ±1 .82 Soybean meal ( l e v e l 2 ) 3 SM 61 > 7 b c d 77 . 09 h 69 . 8 b c d 13 . 6 d e f ±6 . 67 ±4 .25 ±8 !98 ±1 .75 Soybean i s o l a t e s SI 58 . 9 c d 83 . 5 d e f g 62 . o c d e 14 . 0 d e f ( l e v e l 1) ±3 .07 ±4 .54 ±4 .11 ±0 .27 Soybean i s o l a t e s SI 68 ^ 4 b c d 86 # 3Cdef 59 . l d e 13 > 4 d e f ( l e v e l 2) ±1 .83 ±1 .08 ±2 .10 ±0 .47 Extruded wheat EW 75 > 7 a b 87 ^ 4cde 67 . 5 b c d 12 ( l e v e l 1) ±6 .80 ±0 .85 ±7 .85 ±1 .45 Extruded wheat EW 71 . 6 b c 84 > 3 d e f g 71 . 3 b c d 13 > 2 d e f ( l e v e l 2) ±1 . 64 ±0 .95 ±2 .82 ±0 .52 Wheat WM 31 . 4 e 85 , 7 d e f 45 . 3 e 8 . 5 h middlings ±2 . 98 ±1 .37 ±1 .95 ±0 .36 1 Means i n each column followed by a common s u p e r s c r i p t are not s i g n i f i c a n t l y d i f f e r e n t (Newman-Keuls t e s t w i t h P=0.05). 2 L e v e l 1 i s equivalent t o an i n c l u s i o n l e v e l of 15% t e s t i n g r e d i e n t and 85% reference d i e t ( a i r dry b a s i s ) . 3 L e v e l 2 i s equivalent to an i n c l u s i o n l e v e l of 30% t e s t i n g r e d i e n t and 70% reference d i e t ( a i r dry b a s i s ) . 119 4.3.4 E f f e c t of f e c a l c o l l e c t i o n technique on apparent organic  matter d i g e s t i b i l i t y . Organic matter (OM) d i g e s t i b i l i t y c o e f f i c i e n t s based on f e c a l samples c o l l e c t e d from the water using the "Guelph system" were, w i t h one exception, higher than values obtained when feces were c o l l e c t e d d i r e c t l y from the f i s h by the i n t e s t i n a l d i s s e c t i o n or the s t r i p p i n g techniques (Table 20). Nevertheless, d i f f e r e n c e s were not s i g n i f i c a n t (P>0.05) between the OM d i g e s t i b i l i t y values obtained c a l c u l a t e d from f e c a l samples c o l l e c t e d w i t h the "Guelph system" and those c a l c u l a t e d from samples obtained by the i n t e s t i n a l d i s s e c t i o n procedure. This was not always the case when OM values based on the "Guelph system" were compared to those based on the s t r i p p i n g procedure. For instance, the OM d i g e s t i b i l i t y of d i e t REF-a determined from f e c a l samples c o l l e c t e d by s t r i p p i n g was s i g n i f i c a n t l y lower i n experiments I I (t=6.56; P<0.05; 2df) and experiment I I I (t=4.43; P<0.05; 2df) than values determined from feces c o l l e c t e d by the "Guelph system". D i f f e r e n c e s i n O.M. d i g e s t i b i l i t y due t o the f e c a l c o l l e c t i o n technique employed ("Guelph system" versus s t r i p p i n g procedure) were a l s o s i g n i f i c a n t f o r the f o l l o w i n g t e s t i n g r e d i e n t s : h e r r i n g meal HM (t=20.22; P<0.01; 2 d f ) , menhaden meal MM (t=7 . 8 8 ; P<0.05; 2 d f ) , B r i t i s h Columbia p o u l t r y by-product meal PMB ( t=6.17; P<0.05; 2 d f ) , feather meal FM (t=5.93; P<0.05; 2 d f ) , blood meal BM (t=4.72; P<0.05; 2df) and dry whey DW (t=3.70; P<0.05; 5 d f ) . Attempts t o s t r i p the feces from f i s h which were fed d i e t s c o n t a i n i n g soybean products were abandoned owing t o the very poor feed i n t a k e s of these d i e t s . 120 Table 20. E f f e c t of f e c a l c o l l e c t i o n technique on the apparent organic matter d i g e s t i b i l i t y c o e f f i c i e n t s of the reference d i e t s and t e s t i n g r e d i e n t s by j u v e n i l e chinook salmon. Percent apparent organic matter d i g e s t i b i l i t y (± SEM) Diet or F e c a l c o l l e c t i o n technique Test Ingredient (N obs.) "Guelph" D i s s e c t i o n S t r i p p i n g REF-a (Experiment (3) I D *84.5 ±0. 60 - *77.2 ±0.57 REF-a (Experiment (3) I I I ) *80.3 ±0.18 - *76.2 ±1.10 REF-b (Experiment (3) IV) 81.2 ±0.16 77 ±1 .0 .02 -HM (3) **87.3 ±1.03 - **83.4 ±1.22 AM (3) 87.6 ±2.11 - 82.7 ±1.41 MM (3) *84.0 ±0.99 - *78.5 ±0.29 LT (3) 87.8 ±0.62 - 83.6 ±0.73 PMK (3) 75. 9 ±2.41 - 67.0 ±2.32 PMB (3) *65. 6 ±1.82 - *57.9 ±2.55 FM (3) *63.1 ±0.89 - *58.5 ±1.11 BM (3) *34.8 ±2.27 - *28.6 ±0.96 DW (6) *76.7 ±3. 65 - *66.7 ±1.91 CM (3) 53.5 ±2.33 - 51.9 ±1.78 1 Within each row, a s t e r i s k s denote s i g n i f i c a n t d i f f e r e n c e between p a i r e d means ( t - t e s t ; P<0.05* or P<0.01**). Table 20. Continued. 121 Percent apparent organic matter d i g e s t i b i l i t y (± SEM) Test Ingredient (N obs. Fec a l c o l l e c t i o n technique ) "Guelph" D i s s e c t i o n S t r i p p i n g GCMa (3) 58.7 — 56.3 ±2.36 ±0.38 GCMb (3) 37.5 36.8 — ±1.48 ±1.36 RP (3) 69.8 — 66.8 ±1.48 ±1.17 SM2 ( l e v e l 1) (3) 58.5 — — ±7.19 SM 3 ( l e v e l 2) (3) 61.7 — — ±6. 67 S I 2 ( l e v e l 1) (2) 58.9 - — ±3.07 S I 3 ( l e v e l 2) (3) 68.4 — — ±1.83 EW2 ( l e v e l 1) (3) 75.7 — 67.3 ±6.80 ±1.67 EW3 ( l e v e l 2) (3) 71.6 72.4 — ±1.64 ±0.7 6 WM (3) 31.4 30.1 — ±2.98 ±1.25 Within each row, a s t e r i s k s denote s i g n i f i c a n t d i f f e r e n c e between p a i r e d means ( t - t e s t ; P<0.05* or P<0.01**). Level 1 i s equivalent t o an i n c l u s i o n l e v e l of 15% t e s t i n g r e d i e n t and 85% reference d i e t ( a i r dry b a s i s ) . L evel 2 i s equivalent to an i n c l u s i o n l e v e l of 30% t e s t i n g r e d i e n t and 70% reference d i e t ( a i r dry b a s i s ) . 122 4.3.5 I n f l u e n c e of D i e t on f i s h performance Data i n Tables 21, 22 and 23, show t h a t the d a i l y dry feed i n t a k e s (DFI) of the f i s h f e d a l l d i e t s i n the th r e e experiments were, i n g e n e r a l , s i m i l a r . However, when the d a i l y dry feed i n t a k e s per f i s h were expressed as a percentage of wet body weight, the f i s h i n experiment II were observed t o i n g e s t a g r e a t e r amount of feed than f i s h i n experiments I I I and IV. In a d d i t i o n , the feed i n t a k e s o f f i s h f e d the d i f f e r e n t d i e t s i n experiment II (Table 21) d i d not vary s i g n i f i c a n t l y (P>0.05), while the feed i n t a k e s o f f i s h i n g e s t i n g the d i e t s i n experiments I I I (Table 22) and IV (Table 23), d i f f e r e d s i g n i f i c a n t l y (P<0.001) w i t h i n experiments. F i s h f e d the t e s t d i e t s c o n t a i n i n g the soybean products, e s p e c i a l l y at the h i g h e r l e v e l , e x h i b i t e d the poorest a p p e t i t e i n experiments I I I and IV. Indeed, the feed i n t a k e of one group of f i s h maintained on d i e t SI1 i n experiment IV was so poor t h a t the f e c a l c o l l e c t i o n s were d i s c a r d e d . Although the a p p e t i t e s o f f i s h i n experiment II were not s i g n i f i c a n t l y d i f f e r e n t (P>0.05) r e g a r d l e s s of treatments, t h e r e 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) noted between groups f o r growth r a t e , FE and PER. The poorest performances were found f o r those groups which had the lowest d a i l y amounts of o r g a n i c matter, crude p r o t e i n and gross energy d i g e s t e d . Data on f i s h growth, FE and PER should be i n t e r p r e t e d with c a u t i o n as these parameters are not intended t o be used t o eva l u a t e the f e e d s t u f f u t i l i z a t i o n f o r t i s s u e growth i n t h i s t h e s i s . The shor t d u r a t i o n of the f e e d i n g t r i a l s , coupled w i t h the d i f f e r e n c e s i n the n u t r i e n t composition between most d i e t s , do not permit v a l i d comparisons t o be drawn between the v a r i o u s f e e d s t u f f s based on 123 f i s h growth performance. The data on f i s h growth performance i n t h i s t h e s i s i s t h e r e f o r e intended to be used mainly as an i n d i c a t i o n that a p o s i t i v e n i t r o g e n balance was maintained i n a l l f i s h fed the t e s t d i e t s throughout the experiments. In cases where the composition of the d i e t s was s i m i l a r , such as i n the d i e t s c o n t a i n i n g f i s h meal as the t e s t i n g r e d i e n t and d i e t s CM2 and GCMa2, r e s u l t s f o r growth r a t e , FE, and PER can be used as i n d i c a t o r s of the comparative short-term u t i l i z a t i o n of these i n g r e d i e n t s f o r growth. Nevertheless, proper long-term feeding t r i a l s should be performed t o reach more d e f i n i t e and v a l i d conclusions regarding the p o t e n t i a l of any f e e d s t u f f as a p a r t i a l or a complete f i s h meal s u b s t i t u t e . Information on f i s h growth i n experiment IV (Table 23), r e f e r s to the p e r i o d when the f i s h were f i r s t t r a n s f e r r e d to the d i g e s t i b i l i t y tanks and the end of the experiment (34 days l a t e r ) . During t h a t time, f i s h were g r a d u a l l y changed from the commercial d i e t to the b a s a l d i e t and t h e r e a f t e r they were switched t o the r e s p e c t i v e t e s t d i e t s as part of the d i e t a c c l i m a t i o n program. Although the growth data appear to be of l i t t l e use, they do i n d i c a t e , however, that f i s h maintained on the d i e t s c o n t a i n i n g soybean meal or soybean p r o t e i n i s o l a t e grew very slowly during the e n t i r e 34-day p e r i o d . Since the bas a l d i e t s were a v i d l y consumed by the f i s h before commencement of any of the experiments, i t can be p o s t u l a t e d that f i s h fed the d i e t s c o n t a i n i n g the soybean products i n experiments I I I and IV a c t u a l l y l o s t weight while on the t e s t d i e t s . M o r t a l i t y ranged from a low of 3.3% f o r f i s h on d i e t RP2, t o a high of 16.6% f o r f i s h on d i e t SI1. The need to handle the f i s h before the experimental p e r i o d and the i n e v i t a b l e disturbances to the f i s h w hile changing water f i l t e r s , f l u s h i n g the tanks, c o l l e c t i n g the feces as w e l l as during feeding, undoubtedly caused excessive s t r e s s and increased s u s c e p t i b i l i t y to diseases. The l a t t e r were p r i m a r i l y caused by the o p p o r t u n i s t i c pathogens Myxobacterium sporophytophygia and Vijbrio ordalii. 125 Table 21. Weight gain , s p e c i f i c growth r a t e (SGR), d a i l y grams of dry feed i n t a k e per f i s h (DFI), d a i l y dry feed i n t a k e as a percent o f wet body weight (FI), feed e f f i c i e n c y (FE), p r o t e i n e f f i c i e n c y r a t i o (PER), d a i l y grams of o r g a n i c matter d i g e s t e d , d a i l y grams of crude p r o t e i n d i g e s t e d , d a i l y KJ of energy d i g e s t e d and m o r t a l i t y data of f i s h f e d re f e r e n c e and t e s t d i e t s i n experiment I I . (Refer t o Table 16 f o r a d d i t i o n a l i n f o r m a t i o n on d i e t s and t o M a t e r i a l s and Methods f o r measurement of each parameter). Parameter D i e t REF-a RP2 CM2 GCMa 2 HM2 AM2 Weight g a i n ( g / f i s h i n 22 3.30 a l days) 3.17 a 0.71 c 1.31 b 3.18 a 3.63 a SGR (%/day) 1.09 a 1.02 a 0.37 c 0.55 b l . l l a 1.19 a DFI ( g / f i s h ) 0.25 a 0.23 a 0.21 a 0.23 a 0.24 a 0.24 a FI (% Of B.W.) 1.67 a 1.53 a 1.51 a 1.62 a 1.59 a 1.57 a F.E. 0.60 a 0.61 a 0.16 c 0.25 b 0.62 a 0.70 a P.E.R. 1.08 a 1.04 a 0.32° 0.50 b 1.00 a 1.14 a D i g e s t i b l e o r g a n i c matter ( g / f i s h ) 0.19 a 0.16 a b 0.14 b 0.16 a b 0.18 a 0.18 a D i g e s t i b l e p r o t e i n ( g / f i s h ) 0.12 a b 0.12 a b 0.09° 0.10 b c 0.13 a 0.13 a D i g e s t i b l e energy (KJ / f i s h ) M o r t a l i t y 4.86 a 9/180 4 2 9 a b 6/180 3.59 b 20/180 4 . 0 1 a b 20/180 4.68 a 14/180 4.64 a 11/180 Means i n each row with a common s u p e r s c r i p t l e t t e r , are not s i g n i f i c a n t l y d i f f e r e n t (Newman-Keuls t e s t P=0.05). 126 T a b l e 22 . W e i g h t g a i n , s p e c i f i c g r o w t h r a t e (SGR) , d a i l y d r y f e e d i n t a k e p e r f i s h ( D F I ) , d a i l y d r y f e e d ' i n t a k e as a p e r c e n t o f wet body w e i g h t ( F I ) , f e e d e f f i c i e n c y ( F E ) , 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 ) , d a i l y grams o f o r g a n i c m a t t e r d i g e s t e d , d a i l y grams o f c r u d e p r o t e i n d i g e s t e d , d a i l y K J o f e n e r g y d i g e s t e d and m o r t a l i t y d a t a o f f i s h f e d r e f e r e n c e and t e s t d i e t s i n e x p e r i m e n t I I I . ( R e f e r t o T a b l e 17 f o r a d d i t i o n a l i n f o r m a t i o n on d i e t s and t o M a t e r i a l s and Methods f o r measurement o f e a c h p a r a m e t e r ) . P a r a m e t e r D i e t R E F - a PMK2 PMB2 MM2 FM2 LT2 SM2 BM2 SI2 W e i g h t 2 . 4 9 a b l 2 . 0 4 b 1 . 8 2 b c 3 . 1 9 a 1 . 4 4 b c 2 . 1 4 a b 0 . 2 0 d 0 . 8 2 c d 0 . 3 l d g a i n ( g / f i s h i n 17 days) SGR 0 . 5 3 a b 0 . 4 5 a b 0 . 4 1 b c 0 . 6 7 a 0 . 3 7 b c 0 . 4 7 a b 0 . 0 5 d 0 . 1 9 c d 0 . 0 6 d (%/day) DFI 0 . 3 0 a b 0 . 2 8 a b c 0 . 2 7 b c 0 . 3 6 a 0 . 2 5 b c 0 . 2 8 a b c 0 . 1 4 d e 0 . 2 2 c d 0 . 1 2 e ( g / f i s h ) F I 1 . 1 4 b 1.06 b c 1 . 0 2 b c 1 . 3 6 a 0 . 9 5 b c 1 . 0 5 b c 0 . 5 7 d 0 . 8 5 c 0 . 4 8 d (% o f B . W . ) F . E . 0 . 4 8 a b 0 . 4 3 a b c 0 . 3 9 b c 0 . 5 3 a 0 . 3 4 c d 0 . 4 5 a b c 0 . 0 9 f 0 . 2 2 d e 0.15 e f P . E . R . 0 . 8 5 a b 0 . 7 0 a b c 0 . 6 6 b c 0 . 8 7 a 0 . 5 2 c d 0 . 7 1 a b c 0 . 1 5 e 0 . 3 3 d e 0 . 2 2 e D i g e s t i b l e , . , ' ' • , O . M . 0 . 2 2 a b 0 . 2 0 a b 0 . 1 8 b c 0 . 2 5 a 0 . 1 7 b c 0 . 2 0 a b 0 . 1 0 d 0 . 1 3 c d 0 . 0 9 d ( g / f i s h ) D i g e s t i b l e , , , , , p r o t e i n 0 . 1 5 a b 0 . 1 5 a b 0 . 1 3 b c 0 . 1 8 a 0 . 1 3 b c 0 . 1 5 a b 0 . 0 7 d 0 . 1 0 c d 0 . 0 7 d ( g / f i s h ) D i g e s t i b l e i . • w . , ^ ' j e n e r g y 5 . 6 6 a b 5 . 0 9 b 4 . 7 7 b c 6 . 6 2 a 4 . 2 9 b c 5 . 3 1 a b 2 . 5 0 d 3 . 4 8 c d 2.16 d ( K J / f i s h ) M o r t a l i t y 4 /93 7 /93 5 /93 4 /93 11 /93 6/93 6/93 4/93 5 /93 . ^ Means i n e a c h row w i t h a common s u p e r s c r i p t l e t t e r a r e n o t s i g n i f i c a n t l y d i f f e r e n t (Newman-Keuls t e s t P = 0 . 0 5 ) . 127 Table 23. Weight gain, s p e c i f i c growth ra t e (SGR), d a i l y dry feed intake per f i s h (DFI), d a i l y dry feed i n t a k e as a percent of body weight (FI) and m o r t a l i t y data of f i s h fed reference and t e s t d i e t s i n experiment IV. (Refer t o Table 18 f o r a d d i t i o n a l information on d i e t s and to M a t e r i a l s and Methods f o r measurement of each parameter). Parameter Diet REF-b DW1 EW1 EW2 SMI WM2 SI1 GCMb2 Weight 1 4.16 a 2 3.00 a b 3.96 a 2.67 a b 0.35 c 2.04 b 0.33 c 3.43 a gain ( g / f i s h i n 34 days) SGR1 0.29 a 0.23 a b 0.27 a b 0.19 a b 0.04° 0.15 b c0.08 c 0.23 a b (%/day) DFI 3 0.31 a 0.27 a 0.35 a 0.32 a 0.17 b 0.34 a 0.16 b 0.32 a (g/fish) F I 3 0.78 a 0.68 a 0.90 a 0.81 a 0.45 b 0.89 a 0.41 b 0.82 a (% of B.W.) M o r t a l i t y 6/63 16/126 4/63 3/63 4/63 3/63 7/42 3/63 1 Weight gain and s p e c i f i c growth ra t e was c a l c u l a t e d f o r the p e r i o d between i n i t i a l t r a n s f e r of f i s h to the d i g e s t i b i l i t y tanks and the end of the experiment. F i s h were f i r s t maintained on BioDiet followed by feeding of the b a s a l d i e t and f i n a l l y f i s h were fed the t e s t d i e t s during t h i s p e r i o d . 2 Means i n each row wit h the same s u p e r s c r i p t are not s i g n i f i c a n t l y d i f f e r e n t (Newman-Keuls t e s t P=0.05). 3 Dry feed intake expressed as g / f i s h and as % of wet body weight was c a l c u l a t e d only f o r the p e r i o d i n which the f i s h were o f f e r e d the t e s t d i e t s . 128 4.3.6 P r e d i c t i o n of organic matter, crude p r o t e i n and energy d i g e s t i b i l i t y of the reference d i e t s used i n experiments  I I , I I I and IV based on the apparent n u t r i e n t d i g e s t i b i l i t y  values of i n d i v i d u a l d i e t a r y i n g r e d i e n t s An attempt was made to i n v e s t i g a t e the r e l i a b i l i t y of p r e d i c t i n g the d i g e s t i b i l i t y of the i n d i v i d u a l n u t r i e n t s of a complete d i e t based on the d i g e s t i b i l i t y values of the i n d i v i d u a l i n g r e d i e n t s obtained i n t h i s study. Observations of the p r e d i c t i v e accuracy of the d i g e s t i b i l i t y data were made by comparing the observed values f o r organic matter, crude p r o t e i n and gross energy of the reference d i e t s REF-a (experiments I I and I I I ) and REF-b (experiment IV), w i t h c a l c u l a t e d values based on the i n d i v i d u a l i n g r e d i e n t c o n t r i b u t i o n s to the d i e t s . Unfortunately, the i n g r e d i e n t s t e s t e d f o r t h e i r n u t r i e n t d i g e s t i b i l i t y d i d not belong t o the same batch as those used f o r the p r e p a r a t i o n of the reference d i e t s . . Nevertheless, c l o s e agreement was found between the determined and the expected d i g e s t i b l e organic matter, crude p r o t e i n and energy values f o r the reference d i e t s i n a l l experiments (Tables 24, 25 and 26) i n d i c a t i n g that the d i g e s t i b i l i t y of the v a r i o u s i n g r e d i e n t s d i d not vary e x c e s s i v e l y between batches. 129 Table 24. Determined and c a l c u l a t e d apparent d i g e s t i b i l i t y c o e f f i c i e n t s f o r reference d i e t - a (REF-a) which was fed t o chinook salmon i n experiment I I . Organic Crude Energy matter p r o t e i n (%) (%) (MJ/kg) Experiment I I Reference Diet REF-a (Mean ± SEM) Proximate a n a l y s i s 89.19 ± 0 . 0 7 55.50 ± 0 . 6 2 22.63 ± 0 . 0 1 D i g e s t i b i l i t y c o e f f i c i e n t s 84.48 ± 0 . 6 0 84.59 ± 0 . 3 2 86.01 ± 0 . 7 3 Determined D i g e s t i b l e n u t r i e n t 75.35 46.95 19.46 Diet f o r m u l a t i o n 1 ( D i g e s t i b l e N u t r i e n t ) 2 Steam-dried h e r r i n g meal (86.13% O.M.,73.58% CP. , 2 2 .18 41.36 MJ/kg D.M. 36.61 basis) 11.30 Extruded wheat (98.20% O.M.,17.83% CP. , 1 8 .46 8.11 MJ/kg D.M. 1.70 basis) 1.36 D r i e d whey (90.60% O.M.,14.13% CP. , 1 6 .37 5.21 MJ/kg D.M. 0.77 basis) 0.88 F r e e z e - d r i e d euphausids 3 (86.36% O.M.,71.48% CP. , 1 8 .46 4.41 MJ/kg D.M. 4.08 basis) 1.19 Blood meal (98.36% O.M., 95.69% CP. , 2 4 .31 1.71 MJ/kg D.M. 1.41 basis) 0.39 Vitamin & Mi n e r a l c a r r i e r (see extruded wheat above) 2.02 0.42 0.34 H e r r i n g o i l 4 8.40 0.0 3.02 Supplements ~* 1.70 0.0 0.40 Expected T o t a l 72.92 44.99 18.88 Determined Expected (%) 103 104 103 Refer t o Table 11 f o r d i e t f o r m u l a t i o n . Refer t o Table 19 f o r d i g e s t i b i l i t y of i n g r e d i e n t s No p u b l i s h e d data on d i g e s t i b i l i t y a v a i l a b l e . Estimate 85% O.M. d i g e s t i b i l i t y , 95% C P . d i g e s t i b i l i t y and 90% energy d i g e s t i b i l i t y . Based on d i g e s t i b l e energy used by Cho et al. (1982). Includes soybean l e c i t h i n , c h o l i n e c h l o r i d e and as c o r b i c a c i d . D i g e s t i b l e energy assuming 95% d i g e s t i b i l i t y of soybean l e c i t h i n supplement. 130 Table 25. Determined and c a l c u l a t e d apparent d i g e s t i b i l i t y c o e f f i c i e n t s f o r reference d i e t - b (REF-a) which was fed to chinook salmon i n experiment I I I . Organic Crude Energy matter p r o t e i n (%) (%) (MJ/kg) Experiment I I I Reference Diet REF-a (Mean ± SEM) Proximate a n a l y s i s 88 ±0 .90 .08 55.62 ±0.21 22 ±0, .90 .01 D i g e s t i b i l i t y c o e f f i c i e n t s 80 ±0 .33 .18 84.84 ±0.25 81 ±0, .97 .32 Determined D i g e s t i b l e n u t r i e n t 71 .41 47.19 18 .77 Diet f o r m u l a t i o n 1 ( D i g e s t i b l e N u t r i e n t ) 2 Steam-dried h e r r i n g meal (86.1.3% O.M.,73.58% CP.,22 .18 . 41 MJ/kg .36 D.M. 36.61 basis) 11 .30 Extruded wheat (98.20% O.M.,17.83% CP.,18 .46 8.11 MJ/kg D.M. 1.70 basis) 1 .36 Dr i e d whey (90.60% CM.,14.13% CP.,16 .37 5 MJ/kg .21 D.M. 0.77 basis) 0 .88 Freeze - d r i e d euphausids 3 (86.36% CM.,71.48% CP.,18 .46 4 MJ/kg .41 D.M. 4.08 basis) 1 .19 Blood meal (98.36% O.M.,95.69% C P . ,24 .31 1 MJ/kg .71 D.M. 1.41 basis) 0 .39 Vitamin & M i n e r a l c a r r i e r (see extruded wheat above) 2 .02 0.42 0 .34 Herring o i l 4 8 .40 0.0 3 .02 Supplements^ 1 .70 0.0 0 .40 Expected T o t a l 72 .92 44.99 18 .88 Determined -s- Expected (%) 98 105 99 Refer t o Table 11 f o r d i e t f o r m u l a t i o n . Refer to Table 19 f o r d i g e s t i b i l i t y of i n g r e d i e n t s No p u b l i s h e d data on d i g e s t i b i l i t y a v a i l a b l e . Estimate 85% O.M. d i g e s t i b i l i t y , 95% C P . d i g e s t i b i l i t y and 90% energy d i g e s t i b i l i t y . Based on d i g e s t i b l e energy used by Cho et al. (1982). Includes soybean l e c i t h i n , c h o l i n e c h l o r i d e and a s c o r b i c a c i d . D i g e s t i b l e energy assuming 95% d i g e s t i b i l i t y of soybean l e c i t h i n supplement. 131 Table 26. Determined and c a l c u l a t e d apparent d i g e s t i b i l i t y c o e f f i c i e n t s f o r reference d i e t - b (REF-b) which was fed to chinook salmon i n experiment IV. Organic Crude Energy matter p r o t e i n (%) (%) (MJ/kg) Experiment IV Reference Diet REF-b (Mean ± SEM) Proximate a n a l y s i s 87.85 ±0.06 59.50 ±0.44 22.72 ±0.01 D i g e s t i b i l i t y c o e f f i c i e n t s 81.25 ±0.16 83.63 ±0.28 83.59 ±0.38 Determined D i g e s t i b l e n u t r i e n t 71.38 49.76 18.99 Diet formulation- 1- ( D i g e s t i b l e N u t r i e n t ) 2 Steam-dried h e r r i n g meal (86.13% O.M.,73.58% CP.,22 .18 45.93 MJ/kg, D.M, 40.68 . basis) 12.55 Extruded wheat (98.20% O.M.,17.83% CP.,18 .46 2.97 MJ/kg D.M. 0.62 basis) 0.50 Drie d whey (90.60% O.M.,14.13% CP.,16 .37 5.79 MJ/kg D.M. 0.85 basis) 0.97 Free z e - d r i e d euphausids J (86.36% O.M.,71.48% CP.,18 .46 4.89 MJ/kg D.M. 4.53 basis) 1.30 Blood meal (98.36% O.M., 95.69% CP.,24 .31 1.90 MJ/kg D.M. 1.56 basis) 0.43 Vitamin & Mi n e r a l c a r r i e r (see extruded wheat above) 2.02 0.42 0.34 Herring o i l 4 7.18 0.0 2.58 Supplements^ 1.70 0.0 0.40 Expected T o t a l 72.38 48.66 19.07 Determined -s- Expected (%) 99 102 100 Refer t o Table 12 f o r d i e t f o r m u l a t i o n . Refer to Table 19 f o r d i g e s t i b i l i t y of i n g r e d i e n t s No pub l i s h e d data on d i g e s t i b i l i t y a v a i l a b l e . Estimate 85% O.M. d i g e s t i b i l i t y , 95% C P . d i g e s t i b i l i t y and 90% energy d i g e s t i b i l i t y . Based on d i g e s t i b l e energy used by Cho et al. (1982). Includes soybean l e c i t h i n , c h o l i n e c h l o r i d e and a s c o r b i c a c i d . D i g e s t i b l e energy assuming 95% d i g e s t i b i l i t y of soybean l e c i t h i n supplement. 132 4.4 DISCUSSION The o b j e c t i v e of feed formulation i s to supply the c o r r e c t a v a i l a b l e n u t r i e n t d e n s i t y f o r optimal animal production. Numerical c o e f f i c i e n t s are used to enable the s e l e c t i o n of i n g r e d i e n t s on the b a s i s of t h e i r chemical p r o p e r t i e s and t h e i r n u t r i t i o n a l value f o r the o p t i m i z a t i o n of f i s h production at low feed c o s t s . Salmonid d i e t s r e l y h e a v i l y on the use of high q u a l i t y expensive f i s h meals and any attempt t o reduce feed costs n e c e s s i t a t e s the replacement of some, i f not a l l , of the f i s h meal w i t h l e s s expensive animal or p l a n t p r o t e i n sources. In t h i s t h e s i s , a v a r i e t y of i n g r e d i e n t s of animal and p l a n t by-product o r i g i n were evaluated on the b a s i s of t h e i r n u t r i e n t d i g e s t i b i l i t y , as p o t e n t i a l f i s h meal s u b s t i t u t e s . In a d d i t i o n , d i f f e r e n t f i s h meals were a l s o evaluated t o f a c i l i t a t e replacement of one f i s h meal w i t h another according t o f i s h meal p r i c e trends,and a v a i l a b i l i t y . 4.4.1 Feedstuff e v a l u a t i o n according t o proximate composition and n u t r i e n t d i g e s t i b i l i t y 4.4.1.1 F i s h meals On the b a s i s of proximate a n a l y s i s , the high p r o t e i n and organic matter content of the low temperature Norwegian h e r r i n g / c a p e l i n meal (LT) rank t h i s as the best of the f i s h meals of t h i s study. The p r o t e i n content of B r i t i s h Columbia h e r r i n g meal (HM) was lower than expected and t h i s was probably due to i n c l u s i o n of ground f i s h (approx. 64% crude p r o t e i n ) i n our sample (Brian G i l l e y , B r i t i s h Columbia Packers, personal 133 communication). Nevertheless, based on proximate composition, HM c l o s e l y r i v a l s LT as a good source of both p r o t e i n and energy. Although anchovy meal (AM) was lower i n crude p r o t e i n than the other f i s h meals evaluated, the l i p i d and gross energy content of AM surpassed that of a l l other f i s h meals t e s t e d i n t h i s study. Therefore, based on chemical a n a l y s i s , anchovy meal appears t o be a good s u b s t i t u t e f o r other f i s h meals. Menhaden meal (MM) on the other hand, contains a very high amount of ash and i t s use as a complete s u b s t i t u t e f o r more expensive f i s h meals would need c a r e f u l adjustments to be made i n the mineral supplementation of the complete d i e t since increased l e v e l s of d i e t a r y calcium and phosphorus reduce z i n c b i o a v a i l a b i l i t y i n f i s h (Hardy and Shearer, 1984), e s p e c i a l l y i n the presence of p h y t i c a c i d (Richardson et a l . , 1985). R e l a t i v e l y high d i g e s t i b i l i t y values, ranging from 83.1% t o 93.6% f o r apparent p r o t e i n d i g e s t i b i l i t y of f i s h meals, were found i n j u v e n i l e chinook salmon i n seawater. Among the f i s h meals, there was l i t t l e d i f f e r e n c e i n the d i g e s t i b i l i t y c o e f f i c i e n t s f o r LT, HM and AM, but a l l had considerably higher values than MM. I t was a l s o evident t h a t apparent organic matter and p r o t e i n d i g e s t i b i l i t y c o e f f i c i e n t s v a r i e d i n v e r s e l y w i t h the ash content of the f e e d s t u f f . S i m i l a r findings, on the r e l a t i o n s h i p between f i s h meal ash content and apparent p r o t e i n d i g e s t i b i l i t y have been reported f o r rainbow t r o u t (Nose and Mamiya, 1963; G u l l e y , 1980). The high ash content of the menhaden meal based d i e t may have a l s o increased the endogenous f e c a l n i t r o g e n of the f i s h due t o enhanced sloughing o f f of i n t e s t i n a l mucosa c e l l s r e l a t i v e t o the s i t u a t i o n i n f i s h fed 134 d i e t s c o n t a i n i n g the other f i s h meals. I f t h i s d i d indeed occur, the t r u e p r o t e i n d i g e s t i b i l i t y c o e f f i c i e n t of the menhaden meal would have been underestimated the most. Crude p r o t e i n and gross energy d i g e s t i b i l i t y c o e f f i c i e n t s found f o r the f i s h meals t e s t e d i n t h i s study are g e n e r a l l y higher than the f i g u r e s reported f o r rainbow t r o u t i n freshwater by numerous authors (see NRC, 1981; Cho et al., 1982), and thus support the f i n d i n g s of L a l l et al. (1984) and L a l l (1988, c i t e d by Higgs, et al., 1990) f o r A t l a n t i c salmon. In t h i s regard, the f e e d s t u f f d i g e s t i b i l i t y values obtained f o r A t l a n t i c salmon h e l d i n seawater were noted to be s i g n i f i c a n t l y higher than those f o r salmon i n freshwater. Of the f i s h meals t e s t e d i n the present study, the apparent crude p r o t e i n d i g e s t i b i l i t y of anchovy meal (91.6%) by chinook salmon i n seawater most resembled the value determined f o r anchovy meal using rainbow t r o u t i n freshwater (89.3%; Watanabe et al., 1983). The gross energy d i g e s t i b i l i t y of anchovy meal by rainbow t r o u t i n freshwater (91.3%) determined by Smith (1976) and Smith et al. (1980) a l s o approaches the value determined here (91.6%). The apparent crude p r o t e i n d i g e s t i b i l i t y of h e r r i n g meal (HM) by j u v e n i l e chinook salmon i n seawater i n the present study (90.5%, Table 19) i s s u b s t a n t i a l l y higher than values reported f o r A t l a n t i c salmon i n seawater (79.1% and 76.3%) by L a l l and Bishop (1977), or values reported f o r rainbow t r o u t i n freshwater (86.7%) by Smith et a l . (1980). Cho and S l i n g e r (1979) and Cho et al. (1982), a l s o working w i t h rainbow t r o u t i n freshwater, obtained higher apparent crude p r o t e i n d i g e s t i b i l i t y values f o r h e r r i n g meal than those obtained by Smith et al. (1980). Major causes of the d i s c r e p a n c i e s i n r e s u l t s between st u d i e s may 135 i n c l u d e d i f f e r e n c e s i n the o r i g i n of f i s h meal, processing methods of the raw products, d i e t f o rmulation, r a t i o n l e v e l and feeding frequency, f i s h age and s i z e , environmental c o n d i t i o n s and the d i s s i m i l a r methods of f e c a l c o l l e c t i o n employed by the d i f f e r e n t researchers. Consequently, the present r e s u l t s do not provide c o n c l u s i v e evidence f o r a d i f f e r e n c e between species i n d i g e s t i v e e f f i c i e n c y or an e f f e c t of s a l i n i t y on d i g e s t i b i l i t y . But they do h i g h l i g h t the problems i n e x t r a p o l a t i n g r e s u l t s across species and environmental c o n d i t i o n s and they suggest that r e s u l t s obtained w i t h one batch of a feed may not m i r r o r those f o r another when d i f f e r e n t sources of i n g r e d i e n t s are employed. Excessive heat during processing of f i s h meals has been shown t o severely impair 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 value (Tarr and B i e l y , 1973). Opstvedt et al. (1984) c o r r e l a t e d the reduced p r o t e i n d i g e s t i b i l i t y of overcooked (95°C) p o l l o c k f i s h meal by rainbow t r o u t , to the formation of d i s u l f i d e bonds (S-S) from -SH groups. Loss of l y s i n e can a l s o occur from m i l d heat treatment i n the presence of reducing sugars (Carpenter et al., 1963). Under severe heating, i n the presence of e i t h e r sugars or o x i d i z e d l i p i d s , the a v a i l a b i l i t y of not only l y s i n e , but a l s o tryptophan, a r g i n i n e and methionine can become reduced (Carpenter et al., 1963). From the informat i o n obtained from the s u p p l i e r s of the f i s h meals used i n the present experiments (Appendix 3), i t i s evident that the processing temperatures used i n the production of LT and HM d i d not appear t o d i f f e r s i g n i f i c a n t l y . Therefore, minor d i f f e r e n c e s i n the n u t r i e n t d i g e s t i b i l i t y c o e f f i c i e n t s between the two meals may have r e s u l t e d from the i n c l u s i o n of the ground f i s h or the c a p e l i n 136 i n t o the HM and LT meals, r e s p e c t i v e l y . Results on the p r o t e i n d i g e s t i b i l i t y of both c a p e l i n (89.4%) (Storebakken, 1985) and ground f i s h (79.0%) (Nose and Mamiya, 1963) by rainbow t r o u t i n freshwater, support the n o t i o n that mixing ground f i s h w i t h h e r r i n g meal, as was the case w i t h the HM source used i n t h i s study, w i l l depress the n u t r i e n t d i g e s t i b i l i t y c o e f f i c i e n t s of the meal. A l t e r n a t i v e l y , a blend of c a p e l i n meal wi t h the h e r r i n g meal would account f o r the higher n u t r i e n t d i g e s t i b i l i t y c o e f f i c i e n t s of the LT meal. Further, i n an experiment designed to evaluate the u t i l i z a t i o n of v a r i o u s kinds of f i s h meals, Ke t o l a (1980) found that growth rates of A t l a n t i c salmon f r y which had been fed d i e t s c o n t a i n i n g c a p e l i n , anchovy and Canadian h e r r i n g meal were 110%, 70% and 70% r e s p e c t i v e l y of that obtained w i t h a d i e t based on Norwegian whole h e r r i n g meal. This again suggests t h a t the use of c a p e l i n meal as a component of the LT meal i n the present study, was a l a r g e f a c t o r i n the enhanced u t i l i z a t i o n of t h i s meal. However, u n l i k e the experiment by K e t o l a (1980), v a l i d comparisons of the performance of f i s h on the v a r i o u s t e s t d i e t s was not p o s s i b l e since the d i e t s i n the present experiment were n e i t h e r isonitrogenous nor i s o c a l o r i c . A l s o , the p e r i o d over which these d i e t s were fed was r e l a t i v e l y short. The i n f o r m a t i o n on feed i n t a k e , on the other hand, provided v a l u a b l e i n f o r m a t i o n regarding the poor p a l a t a b i l i t y of d i e t s c o n t a i n i n g i n g r e d i e n t s such as soybean meal and soybean p r o t e i n i s o l a t e s . 137 4.4.1.2 Animal by-product meals P o u l t r y by-product meal i s being i n c r e a s i n g l y used i n animal feeds and i t i s becoming more r e a d i l y a v a i l a b l e as time progresses. Although the q u a l i t y of p o u l t r y by-product meal p r o t e i n i s reasonable (NRC, 1983), i t i s c h a r a c t e r i z e d by a high ash content which can cause mineral imbalances as mentioned p r e v i o u s l y f o r menhaden meal. With respect t o the two sources of p o u l t r y by-product meals t e s t e d , the crude p r o t e i n content of the meal from Kansas (PMK) was s u b s t a n t i a l l y higher than t h a t of the l o c a l source (PMB) even though the organic matter content of the former was only one percentage u n i t higher than the l a t t e r . Nevertheless, due to the higher l i p i d content of the PMB meal compared to tha t of the PMK meal, l i t t l e d i f f e r e n c e was found i n the gross energy content of these two p o u l t r y by-product meals. When c o n s i d e r i n g the other animal by-products, blood meal and feather meal stand out wi t h respect t o t h e i r high l e v e l s of crude p r o t e i n and energy and, on t h i s b a s i s alone, they are p o t e n t i a l l y u s e f u l as replacements f o r f i s h meal. The low crude p r o t e i n and high carbohydrate (lactose) contents of d r i e d whey on the other hand, make t h i s product more s u i t a b l e as a b i n d i n g agent r a t h e r than a p r o t e i n supplement. The p r o t e i n , being p r i m a r i l y l a c t a l b u m i n (Potter, 1984), would, however, be expected t o be h i g h l y d i g e s t i b i l e when processed at normal temperatures. The v a r i a t i o n i n apparent n u t r i e n t d i g e s t i b i l i t y of the d i f f e r e n t animal by-product meals t e s t e d i n t h i s study was greater than t h a t observed f o r the f i s h - meals. D i g e s t i b i l i t y of organic matter, crude p r o t e i n and gross energy of the imported p o u l t r y by-product meal (PMK) was 7 t o 10 percentage u n i t s higher 138 than r e s p e c t i v e values determined f o r the l o c a l meal (PMB). Apparent crude p r o t e i n d i g e s t i b i l i t y values f o r both p o u l t r y by-product meals were higher than those reported f o r rainbow t r o u t i n freshwater (NRC, 1981; Cho et al., 1982) and channel c a t f i s h (Brown and Strange, 1985). But the gross energy d i g e s t i b i l i t y of the PMB meal i n chinook salmon was lower than t h a t found i n rainbow t r o u t . Although the feed intake of the f i s h fed the d i e t s c o n t a i n i n g the two d i f f e r e n t p o u l t r y by-product meals as t e s t i n g r e d i e n 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), f i s h maintained on the PMK2 d i e t had s l i g h t l y improved growth r a t e s , feed e f f i c i e n c i e s and p r o t e i n e f f i c i e n c y r a t i o s than those maintained on the PMB2 d i e t . This may have been due t o the higher p r o t e i n and energy contents i n the PMK2 d i e t r e l a t i v e t o the PMB2 d i e t . Higgs et a l . (197 9) found th a t s u b s t i t u t i n g d e f a t t e d p o u l t r y by-product meal f o r a l l of the h e r r i n g meal of a c o n t r o l d i e t on an isonitrogenous b a s i s r e s u l t e d i n only a s l i g h t , yet s i g n i f i c a n t decrease i n the growth ra t e of coho salmon f r y maintained i n freshwater. The same authors found that growth r a t e , feed conversion e f f i c i e n c y and p r o t e i n and gross energy u t i l i z a t i o n of j u v e n i l e coho salmon i n seawater were not a f f e c t e d when 75% of the h e r r i n g meal i n the c o n t r o l d i e t was replaced by p r o t e i n from mixtures of d e f a t t e d or f u l l - f a t p o u l t r y by-product meal and feather meal. S i m i l a r f i n d i n g s have been reported f o r j u v e n i l e rainbow t r o u t by Tiews et al. (197 6) and Gropp et al. (197 9). These st u d i e s demonstrated that f i s h p r o t e i n could be e n t i r e l y replaced by a mixture of p o u l t r y by-product meal and feather meal on an isonitrogenous b a s i s , 139 provided t h a t the d i e t was supplemented wi t h the e s s e n t i a l amino acids l y s i n e , methionine and tryptophan. D i f f e r e n c e s i n the n u t r i e n t d i g e s t i b i l i t y of the two p o u l t r y by-product meals used i n the present study may have r e s u l t e d from d i f f e r e n c e s i n the raw m a t e r i a l s i n c l u d e d i n the meal (e.g. feathers, g i z z a r d , i n t e s t i n e s and organs) and/or d i s s i m i l a r p rocessing procedures. Information on the processing method employed i n the production of the l o c a l l y - p r o d u c e d p o u l t r y by-product meal revealed that cooking temperatures (135°C) were much higher than those employed during f i s h meal production (<100°C). D e t a i l s of the processing method i n v o l v e d i n the p r e p a r a t i o n of the imported p o u l t r y by-product meal (PMK) was, u n f o r t u n a t e l y , not made a v a i l a b l e . A reduction i n the ash content of the PMK meal may have been achieved through s e l e c t i v e removal of bone f o l l o w i n g the d r y i n g of the meal or e x c l u s i o n of high ash c o n t a i n i n g body p a r t s (e.g., head, f e e t , neck) from the v i s c e r a , before cooking and d r y i n g . I t i s noteworthy that bone p r o t e i n i s n e a r l y a l l c o l l a g e n (Eastoe and Eastoe, 1954) which i s of poor n u t r i t i o n a l value. Hence, the PMK meal not only contained more p r o t e i n than PMB, but a l s o the p r o t e i n i n PMK was l i k e l y of improved q u a l i t y due t o i t s lower c o l l a g e n content. The feather meal (FM) p r o t e i n was reasonably w e l l d i g e s t e d by chinook salmon i n t h i s study. However, the gross energy d i g e s t i b i l i t y c o e f f i c i e n t of t h i s product was lower than the values of 73.7% and 70.0% reported f o r rainbow t r o u t i n freshwater by Cho and S l i n g e r (1979) and Cho et al. (1982), r e s p e c t i v e l y . The t e s t d i e t c o n t a i n i n g 30% feather meal was l i k e l y imbalanced w i t h respect t o e s s e n t i a l amino a c i d content and t h i s may have depressed chinook growth r a t e , a p p e t i t e , food u t i l i z a t i o n and p r o t e i n u t i l i z a t i o n and s u r v i v a l r e l a t i v e to f i s h fed the reference d i e t . The apparent d i g e s t i b i l i t y of crude p r o t e i n and gross energy i n the blood meal was very low i n chinook salmon. The poor a s s i m i l a t i o n of t h i s f e e d s t u f f was probably due to excessive heating during processing since t h i s product was dark purple i n c o l o r and charred m a t e r i a l was present. A p p e t i t e was a l s o s i g n i f i c a n t l y (P<0.05) lower i n f i s h r e c e i v i n g the t e s t d i e t c o n t a i n i n g the blood meal than i n f i s h r e c e i v i n g the reference d i e t . I t was of i n t e r e s t to note t h a t feed p e l l e t s c o n t a i n i n g the blood meal as the t e s t f e e d s t u f f had a tendency to f l o a t on the surface f o r up t o 30 seconds before s i n k i n g to the tank bottom, a phenomenon not observed w i t h the other d i e t s . R e sults on the apparent d i g e s t i b i l i t y of crude p r o t e i n and gross energy of blood meal by rainbow t r o u t and A t l a n t i c salmon are extremely v a r i a b l e . Apparent p r o t e i n d i g e s t i b i l i t y values f o r rainbow t r o u t i n freshwater have been reported to be as low as 16% and as high as 99% (Cho et a l . , 1982) w i t h numerous intermediate values being reported by other authors (Cho and S l i n g e r , 1979; Smith et a l . , 1980). L a l l et al. (1984) a l s o reported that the crude p r o t e i n of blood meal was poorly d i g e s t e d by A t l a n t i c salmon i n both freshwater and seawater. By c o n t r a s t , Asgard and Austreng (1986) noted t h a t the apparent d i g e s t i b i l i t y of blood p r o t e i n by A t l a n t i c salmon i n seawater exceeded 97%. The blood used by Asgard and Austreng (1986) was not subjected t o heat, being preserved w i t h formic a c i d or by f r e e z i n g . Such f i n d i n g s i n d i c a t e t h a t blood from slaughterhouses can be a v a l u a b l e source of p r o t e i n and a p o t e n t i a l l y u s e f u l i n g r e d i e n t i n f i s h d i e t s provided t h a t processing c o n d i t i o n s are optimal and i n g r e d i e n t s such as feather meal or meat and bone meal are a l s o incorporated i n t o the d i e t to complement the amino a c i d imbalances of blood meal. The high l e u c i n e and low i s o l e u c i n e content i n blood meal (NRC, 1983) must be taken i n t o account i n the choice of complementary p r o t e i n sources i n f i s h d i e t s . An excess of d i e t a r y l e u c i n e can have an a n t a g o n i s t i c e f f e c t on i s o l e u c i n e absorption and can thereby increase the requirement f o r i s o l e u c i n e i n the d i e t . This has been demonstrated i n r a t s (Harper et al., 1955), pi g s (Oestemer et al., 1973) and chinook salmon (Chance et al., 1964). Dr i e d whey, despite i t s low p r o t e i n content, i s a good source of r e a d i l y a v a i l a b l e B vitamins (NRC, 1981). The f a c t that these vitamins are i n conjugate form makes them l e s s l i k e l y t o be leached i n t o the water during feeding. The p r o t e i n of whey, being mostly lac t a l b u m i n , i s r e p o r t e d l y of higher q u a l i t y than casein (Morrison, 1949) and the whey acts as a good p e l l e t binder. In t h i s study, a 30% i n c l u s i o n of d r i e d whey i n t o the d i e t r e s u l t e d i n very hard p e l l e t s which were r e j e c t e d by the f i s h . The d i e t w i t h a lower i n c l u s i o n l e v e l (15%) of whey, however, proved to be somewhat s o f t e r i n t e x t u r e and was accepted more r e a d i l y by the f i s h . D i g e s t i b i l i t y values r e v e a l that the whey p r o t e i n i s moderately dig e s t e d by chinook salmon i n seawater. The r e l a t i v e l y high d i g e s t i b l e energy content of d r i e d whey i n d i c a t e s that l a c t o s e i s a l s o r e l a t i v e l y w e l l d i g e s t e d by chinook salmon. Cho and S l i n g e r (1979) and Cho et al. (1982) found t h a t rainbow t r o u t i n freshwater were able t o almost completely d i g e s t a l l of the dry matter of t h i s f e e d s t u f f , a con c l u s i o n a l s o reached by L a l l et al. (1984) f o r A t l a n t i c salmon i n both freshwater and seawater. The lower d i g e s t i b i l i t y values obtained f o r a l l n u t r i e n t s i n the d r i e d whey i n the present experiment may have been caused by the r e l a t i v e l y poor feed int a k e of f i s h fed t h i s d i e t . The low feed in t a k e would tend to increase the p r o p o r t i o n of endogenous f e c a l n i t r o g e n waste i n the feces and thereby r e s u l t i n underestimation of organic matter, crude p r o t e i n and energy d i g e s t i b i l i t y . 4.4.1.3 P l a n t by-product meals In regard to the p l a n t f e e d s t u f f s t e s t e d i n t h i s experiment, only rapeseed p r o t e i n concentrate (RP), soybean meal (SM) and soybean p r o t e i n i s o l a t e (SI) contain s u f f i c i e n t l e v e l s of p r o t e i n to p o t e n t i a l l y completely replace f i s h meal i n salmonid d i e t s . The proximate compositions of the commercial canola meal (CM)(variety not s p e c i f i e d by manufacturer) and the g l u c o s i n o l a t e - f r e e canola meal deri v e d from the Brassica campestris c u l t i v a r Tobin v a r i e t y (GCMa) were s i m i l a r . By co n t r a s t , the solvent e x t r a c t i o n method employed to reduce the g l u c o s i n o l a t e content of canola meal (GCMb) tended t o elevate the p r o t e i n content of the meal because of removal of some carbohydrate and l i p i d during the e x t r a c t i o n process (Naczk et a l . , 1986). In general, the organic matter d i g e s t i b i l i t y of the p l a n t by-product meals was lower than that of the animal by-products and f i s h meals. However, apparent crude p r o t e i n and gross energy d i g e s t i b i l i t y of the p l a n t f e e d s t u f f s tended to be higher than 143 values obtained f o r animal by-products (excluding f i s h meals). Rapeseed p r o t e i n concentrate (RP) was extremely w e l l d i g e s t e d and i t s apparent p r o t e i n d i g e s t i b i l i t y c o e f f i c i e n t exceeded those of a l l other f e e d s t u f f s t e s t e d i n t h i s study. The energy d i g e s t i b i l i t y of RP was al s o the highest among a l l n o n - f i s h meal f e e d s t u f f s . Despite a lower p r o t e i n and gross energy content i n the d i e t RP2 compared to d i e t s HM2 and AM2, there were only small d i f f e r e n c e s among groups fed the three d i e t s f o r growth r a t e , feed e f f i c i e n c y and p r o t e i n e f f i c i e n c y r a t i o . In a d d i t i o n , the s u r v i v a l of f i s h at the end of experiment I I , was highest f o r the RP2 d i e t . Higgs et a l . ( 1 9 8 2 ) , a l s o working with rapeseed p r o t e i n concentrate, concluded t h a t rapeseed p r o t e i n concentrate could replace at l e a s t 25% of the p r o t e i n i n a modified Abernathy dry d i e t without compromising growth r a t e and feed u t i l i z a t i o n of j u v e n i l e chinook salmon. Yurkowski et al. (1978) found s i m i l a r r e s u l t s w i t h rainbow t r o u t when a l l of the soybean meal of t h e i r b a s a l d i e t (approximately 24% of the d i e t a r y p rotein) was replaced by rapeseed p r o t e i n concentrate. The same authors found, however, that complete replacement of the f i s h meal w i t h rapeseed p r o t e i n concentrate l e d t o poor growth, feed i n t a k e , and feed u t i l i z a t i o n . The d e l e t e r i o u s e f f e c t s caused by high d i e t a r y l e v e l s of rapeseed p r o t e i n concentrate observed by Yurkowski et al. (1978) p o s s i b l y can be e l i m i n a t e d by reducing the p h y t i c a c i d content i n rapeseed p r o t e i n concentrate and a l s o by supplementing the d i e t c o n t a i n i n g RP with z i n c (Higgs et a l . , 1 9 8 2 ) . The d i g e s t i b i l i t y c o e f f i c i e n t s f o r organic matter, crude p r o t e i n and gross energy i n the canola meals were lower than noted f o r the rapeseed p r o t e i n concentrate This may be due to the higher contents of carbohydrate and f i b r e i n the canola meals r e l a t i v e to RP (Jones, 1979; NRC, 1983). The solvent treatment of canola t o reduce the g l u c o s i n o l a t e content of the r e s u l t a n t meal (GCMb), d i d not improve n u t r i e n t d i g e s t i b i l i t y . Indeed, the apparent organic matter, crude p r o t e i n and gross energy d i g e s t i b i l i t y c o e f f i c i e n t s of GCMb were, r e s p e c t i v e l y 15.8, 5.9 and 13.3 percentage u n i t s lower than values determined f o r the commercial canola meal product (CM). Losses of the more r e a d i l y s o l u b l e carbohydrates and p r o t e i n s during the process may have r e s u l t e d i n a greater p r o p o r t i o n of more i n d i g e s t i b l e complex carbohydrates and g l o b u l i n molecules being l e f t i n the meal. Moreover, i t i s not c l e a r whether whole seed or commercial canola meal was solvent e x t r a c t e d and t h e r e f o r e whether the canola product had been subjected to heat treatment. Proper heat treatment would l i k e l y be necessary to improve p r o t e i n and energy d i g e s t i b i l i t y . A high f i b r e content i n the d i e t can a l s o have a negative e f f e c t on d i g e s t i b i l i t y . For instance, a high f i b r e content i n r a t d i e t s has been shown to reduce the d i g e s t a gut t r a n s i t time and consequently lower the d i g e s t i b i l i t y and absorption of n u t r i e n t s across the i n t e s t i n a l mucosa (Fleming and Lee, 1983). Bergner et al. (1975) concluded t h a t f i b r e i s capable of adsorbing amino acids and peptides thereby impending t h e i r absorption. F i b r e a l s o increases the endogenous f e c a l n i t r o g e n l o s s due t o an increased sloughing of i n t e s t i n a l mucosal c e l l s (Bergner et al., 1975) and concomitant enhancement of mucus production (Schneeman et a l . , 1982). This leads t o an underestimation of organic matter, crude p r o t e i n and energy d i g e s t i b i l i t y . 145 Although the apparent d i g e s t i b i l i t y c o e f f i c i e n t s f o r organic matter, crude p r o t e i n and gross energy were higher f o r the g l u c o s i n o l a t e - f r e e canola meal c u l t i v a r v a r i e t y (GCMa) than f o r the conventional canola meal (CM), the d i f f e r e n c e e were not s i g n i f i c a n t . Since both of the d i e t s c o n t a i n i n g the GCMa and CM as the t e s t i n g r e d i e n t s contained equal amounts of p r o t e i n and gross energy, some general comparisons on f i s h performance can be made between the two d i e t a r y treatments. The data i n d i c a t e t h a t , while the d i g e s t i b l e energy and p r o t e i n contents of the CM2 and GCMa2 d i e t s d i d not d i f f e r s i g n i f i c a n t l y (P>0.05), f i s h maintained on the l a t t e r d i e t had s i g n i f i c a n t l y (P<0.05) improved growth and food and p r o t e i n u t i l i z a t i o n . These r e s u l t s suggest th a t the g l u c o s i n o l a t e content i n d i e t s based on commercial canola meal (CM) can a f f e c t f i s h performance when the l e v e l of i n c l u s i o n of the meal i n t o the d i e t i s 30% or more. The apparent crude p r o t e i n d i g e s t i b i l i t y values f o r canola meal (84.5% f o r CM and 87.9% f o r GCMa) obtained w i t h chinook salmon i n seawater i n the present study, c l o s e l y agree w i t h data obtained by H i l t o n and S l i n g e r (1986) with rainbow t r o u t i n freshwater (83.2% to 87.1% depending on the method of f e c a l c o l l e c t i o n used) as w e l l as w i t h r e s u l t s obtained by L a l l , (1988, c i t e d by Higgs et al., 1990) wi t h A t l a n t i c salmon i n freshwater (80.8%) and seawater (86.3%). The d i g e s t i b l e energy c o e f f i c i e n t of the CM i n the present study was 8.2 percentage u n i t s lower than that determined f o r A t l a n t i c salmon i n seawater ( L a l l , 1988 c i t e d by Higgs et al., 1990) while the d i g e s t i b l e energy c o e f f i c i e n t of GCMa d i f f e r e d by only 1.7 percentage u n i t s . 146 Soybean meal (SM) and soybean p r o t e i n i s o l a t e (SI) were both t e s t e d at two l e v e l s of i n c l u s i o n i n the d i e t (15% and 30% on an a i r dry b a s i s ) . The feed intakes of f i s h fed d i e t s c o n t a i n i n g each of the soybean products were poor. Soybean meal i s reported to be unpalatable f o r chinook salmon (Fowler and Banks, 1967; Fowler, 1980), coho salmon (Fowler, 1980) and some other f i s h species (Akiyama et al., 1984). Recent work by Murai et al. (1989), has shown that treatment of soybeans w i t h methanol improves i t s u t i l i z a t i o n i n j u v e n i l e rainbow t r o u t . The same authors concluded that u t i l i z a t i o n of soybean meal improves as the s i z e otf the f i s h i n c r e a s e s . In t h i s regard, the l a r g e r s i z e d f i n g e r l i n g s i n t h e i r study fed a d i e t i n which 77% of the f i s h meal was replaced by methanol e x t r a c t e d soybean f l o u r supplemented w i t h e s s e n t i a l a c i d s , achieved 87% of the feed e f f i c i e n c y of a c o n t r o l group fed a f i s h meal based d i e t . Tacon et al. (1984) reported s i m i l a r f i n d i n g s f o r rainbow t r o u t weighing over 30 g. M o r t a l i t y of chinnok fed d i e t s c o n t a i n i n g soybean products as the t e s t i n g r e d i e n t was high i n the present study. The use of commercially prepared hexane-extracted soybean meals i s not recommended f o r chinook salmon on the b a s i s of t h i s and previous s t u d i e s c i t e d above. Apparent crude p r o t e i n and gross energy d i g e s t i b i l i t y c o e f f i c i e n t s f o r soybean meal i n t h i s study were lower, regardless of the d i e t a r y l e v e l of i n c l u s i o n , than c o e f f i c i e n t s determined f o r rainbow t r o u t i n freshwater (Cho and S l i n g e r , 1979, Cho et al., 1982) and those f o r A t l a n t i c salmon i n freshwater ( L a l l , 1988; c i t e d by Higgs et a l . , 1990) and seawater ( L a l l and Bishop, 1977; L a l l , 1988; c i t e d by Higgs et al., 1990). 147 Owing to the extremely poor feed intakes of f i s h fed the d i e t s c o n t a i n i n g soybean products as t e s t i n g r e d i e n t s , the high p r o p o r t i o n of endogenous f e c a l losses very l i k e l y caused an underestimation of organic matter, crude p r o t e i n and gross energy d i g e s t i b i l i t y . The n u t r i e n t d i g e s t i b i l i t y c o e f f i c i e n t s f o r soybean meal and soybean p r o t e i n i s o l a t e determined i n the present study were a l s o very i n c o n s i s t e n t , e s p e c i a l l y when the soybean products were t e s t e d at the lower d i e t a r y l e v e l . Due t o the probable underestimation of the n u t r i e n t d i g e s t i b i l i t y of soybean products and the h i g h l y v a r i a b l e d i g e s t i o n values obtained i n the present study, the use of these c o e f f i c i e n t s i s questionable f o r formulation of salmonid d i e t s . The p o s s i b i l i t y of using s p e c i a l l y processed soybean products as p a r t i a l or complete s u b s t i t u t e s f o r f i s h meals remains, however, a v i a b l e option. 4.4.1.3.1 Grain products Various by-products of the g r a i n processing i n d u s t r y are used i n animal feeds. Wheat m i l l i n g , which produces f l o u r as i t s main product, a l s o y i e l d s wheat sh o r t s , wheat m i l l - r u n , and wheat middlings, and a l l of these products have been used i n f i s h feed formulations. Wheat middlings (WM) have been used t r a d i t i o n a l l y i n t r o u t d i e t s at a l e v e l of 30% or more ( H i l t o n and S l i n g e r , 1983) p r i m a r i l y because they are a good b i n d i n g agent i n p e l l e t manufacturing. This i n g r e d i e n t has a p r o t e i n content of approximately 16%, which f o r rainbow t r o u t i n freshwater, has a r e l a t i v e l y high order of d i g e s t i b i l i t y (95.3%, Cho and S l i n g e r , 1979; 92.0%, Cho et al., 1982). The d i g e s t i b i l i t y of the 148 remaining dry matter i n t r o u t i s , on the other hand, q u i t e low (Smith et al., 1980; Cho et al., 1982). Thus the use.of WM as a p e l l e t b i n d i n g agent r a t h e r than l i g n i n sulphonate or bentonite can only be j u s t i f i e d on the b a s i s of supply and cost although, u n l i k e the p e l l e t b inders, WM does enhance the d i e t a r y n u t r i e n t content. The apparent p r o t e i n d i g e s t i b i l i t y of WM by chinook salmon i n the present study was approximately 10 percentage u n i t s lower than f i g u r e s reported f o r rainbow t r o u t i n freshwater by the authors mentioned above. In a d d i t i o n , the d i g e s t i b l e energy content of WM was l e s s than h a l f i t s gross energy value, i n d i c a t i n g very poor d i g e s t i b i l i t y of the carbohydrate f r a c t i o n of t h i s product by chinook salmon i n seawater. S i m i l a r f i n d i n g s have been reported f o r rainbow t r o u t i n freshwater - (Cho end S l i n g e r , 1979; Smith et al., 1980; Cho et al., 1982) and f o r A t l a n t i c salmon i n both freshwater and seawater ( L a l l et a l . , 1984). The d i g e s t i b i l i t y of the organic matter, and consequently of the energy y i e l d i n g n u t r i e n t s , i n the extruded wheat (EW) by chinook salmon i n the present experiment was f a r s u p e r i o r t o that of the WM. The apparent d i g e s t i b i l i t y of the p r o t e i n i n EW and WM was s i m i l a r , however, the gross energy d i g e s t i b i l i t y of the EW was over 50% greater than t h a t of the WM. Extruded wheat, which i s made from whole hard wheat, i s h i g h l y d i g e s t i b l e by chinook salmon because the high temperatures and pressures i n v o l v e d during the e x t r u s i o n process f a c i l i t a t e s t a r c h g e l a t i n i z a t i o n . A l s o , the ^-amylase i n h i b i t o r normally found i n raw wheat (Hofer and Sturmbauer, 1985) i s i n a c t i v a t e d by the high processing temperatures i n v o l v e d . Numerous authors (Inaba et al., 1963; Smith, 1971; Luquet and Bergot, 1976; Bergot and Breque, 1983; Kaushik and O l i v a - T e l e s , 1985) have demonstrated t h a t g e l a t i n i z a t i o n of s t a r c h g r e a t l y improves i t s d i g e s t i b i l i t y by salmonids. Work by Spannhof and Plantikow (1983), has f u r t h e r demonstrated t h a t amylases becomes adsorbed onto crude s t a r c h , thereby r e d u c i n g s t a r c h h y d r o l y s i s . The same authors a l s o i n d i c a t e d t h a t crude s t a r c h moves through the g a s t r o i n t e s t i n a l t r a c t f a s t e r than does g e l a t i n i z e d s t a r c h . A decrease i n the r e t e n t i o n time of feed i n the d i g e s t i v e system can, t h e r e f o r e , be expected to reduce the amount of h y d r o l y s i s and a b s o r p t i o n of n u t r i e n t s across the i n t e s t i n a l mucosa. THe h i g h e r d i g e s t i b i l i t y of the carbohydrate i n the EW r e l a t i v e t o t h a t i n the WM, i s a l s o l i k e l y due t o the lower crude f i b r e content i n the hard wheat which i s processed to produce EW ( i . e . 2.5 % C F . on an a i r dry b a s i s i n hard wheat) versus f o r the WM ( i . e . 7.3% C F . on an a i r dry b a s i s ; NRC, 1981) . Hence, the f i n d i n g s of t h i s study suggest t h a t EW i s p r e f e r a b l e t o WM as a b i n d i n g agent and energy source f o r the i n c l u s i o n i n chinook salmon d i e t s . A decrease i n the apparent p r o t e i n d i g e s t i b i l i t y c o e f f i c i e n t of EW was observed when i t s i n c l u s i o n l e v e l i n the d i e t was i n c r e a s e d . Other workers have a l s o shown t h a t apparent p r o t e i n d i g e s t i b i l i t y decreases i n salmonids f e d d i e t s c o n t a i n i n g h i g h carbohydrate and low p r o t e i n content (Inaba et a l . , 1963; Singh and Nose, 1967; Page and Andrews, 1973; Rychly and Spannhof, 1979; Bergot and Breque,.1983). T h i s may be a t t r i b u t e d t o enhancement of m e t a b o l i c f e c a l n i t r o g e n p r o d u c t i o n (Ogino and Chen, 1973). Under these c o n d i t i o n s , the h i g h e r l e v e l of carbohydrate i n the d i e t may have a l s o reduced p r o t e i n d i g e s t i b i l i t y due t o n u t r i e n t i n t e r a c t i o n . For i n s t a n c e , 150 Watanabe et al. (197 9) showed that e l e v a t i o n of d i e t a r y l i p i d from 5% t o 23% increased the d i g e s t i o n of t o t a l energy by rainbow t r o u t . This was due t o an increase i n the d i g e s t i b i l i t y of both carbohydrates (from 50.7% to 58.5%) and l i p i d s (from 74.7% to 87.5%), w i t h p r o t e i n d i g e s t i b i l i t y remaining the same (about 98.5%) 4.4.2 E f f e c t of f e c a l c o l l e c t i o n technique on apparent organic matter d i g e s t i b i l i t y The c l o s e agreement between organic matter d i g e s t i b i l i t y c o e f f i c i e n t s based on feces c o l l e c t e d d i r e c t l y from the f i s h by i n t e s t i n a l d i s s e c t i o n and those based on feces c o l l e c t e d by the "Guelph system" suggests t h a t the re d u c t i o n i n the cross s e c t i o n of the pipe connecting the tank w i t h the c o l l e c t i o n column g r e a t l y reduced the amount of n u t r i e n t l e a c h i n g from the feces to the surrounding water medium. In experiment I, a d i f f e r e n c e of 10.2 percentage u n i t s was found between organic matter d i g e s t i b i l i t y c a l c u l a t e d using feces c o l l e c t e d by i n t e s t i n a l d i s s e c t i o n and t h a t c a l c u l a t e d using feces c o l l e c t e d overnight by the "Guelph system". In experiments I I - IV, d i f f e r e n c e s i n organic matter d i g e s t i b i l i t y c o e f f i c i e n t s due t o the method of f e c a l c o l l e c t i o n ("Guelph system" vs i n t e s t i n a l d i s s e c t i o n ) were small and they ranged from 0.8 to 4.2 percentage u n i t s . S u r p r i s i n g l y , the mean organic matter d i g e s t i b i l i t y c o e f f i c i e n t of extruded wheat which was determined using feces c o l l e c t e d by i n t e s t i n a l d i s s e c t i o n was higher than t h a t determined using feces c o l l e c t e d d i r e c t l y from the water w i t h the "Guelph system". As discussed i n experiment I, d i g e s t i b i l i t y c o e f f i c i e n t s c a l c u l a t e d from f e c a l samples c o l l e c t e d by i n t e s t i n a l d i s s e c t i o n are u s u a l l y lower than those determined from samples c o l l e c t e d from the water f o r two main reasons. F i r s t , feces c o l l e c t e d by i n t e s t i n a l d i s s e c t i o n underestimate a s s i m i l a t i o n e f f i c i e n c y because of contamination of feces w i t h body f l u i d s and because d i g e s t i o n and absorption may not be f u l l y complete at the time of sampling. Second, feces c o l l e c t e d from the water medium i n v a r i a b l y undergo some degree of n u t r i e n t l e a c h i n g and t h i s subsequently r e s u l t s i n overestimation of d i g e s t i b i l i t y . D i g e s t i b i l i t y determinations based on the c o l l e c t i o n of feces by s t r i p p i n g and i n t e s t i n a l d i s s e c t i o n assume that d i g e s t i o n i s a continuous process without d i u r n a l v a r i a t i o n . This has not been shown to be t r u e by various authors (Inaba et al., 1962/ De l a Noue et al., 1980; De S i l v a and Perera 1983, 1984) who have observed d i u r n a l v a r i a t i o n s i n both t o t a l d i e t and p r o t e i n d i g e s t i b i l i t y . Because of such v a r i a t i o n , a f e c a l sample obtained by i n t e s t i n a l d i s s e c t i o n does not n e c e s s a r i l y provide a r e p r e s e n t a t i v e sample. I t can th e r e f o r e be p o s t u l a t e d t h a t , as the l o s s of n u t r i e n t s due to le a c h i n g i s minimized, the d i f f e r e n c e i n d i g e s t i b i l i t y between samples c o l l e c t e d from the water and those c o l l e c t e d d i r e c t l y from the f i s h become i n c r e a s i n g l y more i n f l u e n c e d by the r h y t h m i c i t y i n d i g e s t i b i l i t y than by any n u t r i e n t l e a c h i n g e r r o r s of the "Guelph system" or by the f e c a l contamination e r r o r s of the d i s s e c t i o n technique. D i f f e r e n c e s between organic matter d i g e s t i b i l i t y c o e f f i c i e n t s determined using f e c a l samples c o l l e c t e d by s t r i p p i n g and those determined using f e c a l samples c o l l e c t e d d i r e c t l y from the water w i t h the "Guelph system" were found to be 152 s i g n i f i c a n t (P<0.05) f o r the reference d i e t s as w e l l as f o r some t e s t i n g r e d i e n t s (P<0.01 f o r HM). D i f f e r e n c e s i n d i g e s t i b i l i t y between the two methods of c o l l e c t i o n ranged from 1.6 to 10.0 percentage u n i t s ; the c o e f f i c i e n t being c o n s i s t e n t l y lower f o r feces c o l l e c t e d by s t r i p p i n g . I t i s g e n e r a l l y agreed t h a t the use of feces obtained by s t r i p p i n g underestimates d i g e s t i b i l i t y c o e f f i c i e n t s more than the use of feces c o l l e c t e d by i n t e s t i n a l d i s s e c t i o n . This i s due t o the a d d i t i o n a l contamination of the feces obtained by the former method wi t h u r i n e and body mucus, as w e l l as the i n a b i l i t y to c o n s i s t e n t l y c o n t r o l the s e c t i o n from the lower i n t e s t i n e from which the feces are obtained. Therefore, because the organic matter d i g e s t i b i l i t y c o e f f i c i e n t s based on the "Guelph system" and i n t e s t i n a l d i s s e c t i o n procedure agree c l o s e l y , whereas those f o r s t r i p p i n g d i d not, i t i s concluded t h a t the s t r i p p i n g procedure i s the l e a s t r e l i a b l e method f o r determining f e e d s t u f f d i g e s t i b i l i t y i n chinook salmon. 4.4.3 Influence of d i e t on f i s h performance Due t o the short d u r a t i o n of each experiment, r e s u l t s on f i s h performance i n the present study were only intended to determine whether the f i s h were i n p o s i t i v e n i t r o g e n balance while f e c a l c o l l e c t i o n s were conducted. However, information on a p p e t i t e i s of great importance i n the present experiments s i n c e l e v e l of d i e t a r y intake determines the i n f l u e n c e t h a t metabolic f e c a l n i t r o g e n exerts on the estimates f o r the apparent d i g e s t i b i l i t y c o e f f i c i e n t s of f e e d s t u f f s (Ogino and Chen, 1973). A l s o , data on 153 a p p e t i t e provides information regarding the u n a c c e p t a b i l i t y of c e r t a i n i n g r e d i e n t s due to t a s t e (e.g. soybean products) or te x t u r e (e.g. d r i e d whey) which need to be taken i n t o c o n s i d e r a t i o n i n subsequent s t u d i e s . 4.4.4 A d d i t i v i t y of i n d i v i d u a l i n g r e d i e n t n u t r i e n t d i g e s t i b i l i t y c o e f f i c i e n t s f o r the p r e d i c t i o n of the n u t r i e n t d i g e s t i o n of a complete d i e t Very c l o s e agreement was found between the determined and the c a l c u l a t e d d i g e s t i b l e organic matter, crude p r o t e i n and energy c o e f f i c i e n t s f o r the reference d i e t used i n experiments I I and I I I (REF-a) and f o r the reference d i e t used i n experiment IV (REF-b). A shortcoming of using t h i s i n f o r m a t i o n i n an attempt to confirm t h a t n u t r i e n t d i g e s t i b i l i t y values determined f o r the i n d i v i d u a l feed i n g r e d i e n t s i n a d i e t are indeed a d d i t i v e , i s that i n t h i s study, the i n g r e d i e n t s which were t e s t e d f o r t h e i r d i g e s t i b i l i t y were not from the same batches as those i n g r e d i e n t s used to prepare the reference d i e t s . The c l o s e agreement between the expected and the determined d i g e s t i b i l i t i e s of organic matter, crude p r o t e i n and gross energy of the reference d i e t s does, however, r e i n f o r c e the not i o n that the i n d i v i d u a l i n g r e d i e n t d i g e s t i b i l i t y c o e f f i c i e n t s can be used s u c c e s s f u l l y t o estimate the d i g e s t i b l e p r o t e i n and energy contents of formulated d i e t s f o r chinook salmon i n seawater. 154 CHAPTER 5 5.0 CONCLUSIONS This t h e s i s was undertaken w i t h the o b j e c t i v e of e s t a b l i s h i n g d i g e s t i b i l i t y i n f o r m a t i o n f o r an array of conventional and novel f e e d s t u f f s which are c u r r e n t l y being used or are of p o t e n t i a l use i n the d i e t s of chinook salmon reared i n seawater. The r e s u l t s suggest t h a t f e e d s t u f f chemical analyses coupled w i t h d i g e s t i b i l i t y determinations may a l l o w a reasonable p r e d i c t i o n of the p o t e n t i a l n u t r i t i o n a l value of a p a r t i c u l a r f e e d s t u f f i n a complete d i e t f o r chinook salmon i n seawater. In experiment I, the a p p l i c a b i l i t y of using chromic oxide i n the d i e t as a r e l i a b l e i n d i c a t o r f o r f i s h d i g e s t i b i l i t y s t u d i e s i n seawater was e s t a b l i s h e d . As p a r t of t h i s experiment, concerns r a i s e d p r e v i o u s l y by f i s h n u t r i t i o n i s t s regarding the p o s s i b l e overestimation of n u t r i e n t d i g e s t i b i l i t y c o e f f i c i e n t s d e r i v e d from the use of f e c a l samples c o l l e c t e d a f t e r being n a t u r a l l y voided i n the water were addressed. The d i s c r e p a n c i e s i n organic matter, crude p r o t e i n and energy d i g e s t i b i l i t y c o e f f i c i e n t s using f e c a l samples c o l l e c t e d d i r e c t l y from the f i s h by the i n t e s t i n a l d i s s e c t i o n and s t r i p p i n g techniques and those determined w i t h f e c a l samples c o l l e c t e d from the water by the "Guelph system" were due t o a combination of e r r o r s inherent i n each c o l l e c t i o n technique. Methods which r e l y on the c o l l e c t i o n of feces d i r e c t l y from the f i s h were deemed i m p r a c t i c a l due to the l a r g e number of f i s h r e q u i r e d , the small amount of f e c a l sample c o l l e c t e d , the i n a b i l i t y t o o b t a i n a r e p r e s e n t a t i v e sample and perhaps most importantly, due t o the p h y s i o l o g i c a l changes a s s o c i a t e d w i t h s t r e s s induced by the repeated handling of the 155 f i s h . With regard t o the l a s t p o i n t , t h i s t h e s i s a l s o demonstrated t h a t w i l d chinook salmon, i n p a r t i c u l a r , are very e a s i l y s t r e s s e d . Every e f f o r t should t h e r e f o r e be taken t o minimize handling and disturbance during the d i g e s t i b i l i t y study. In p a r t i c u l a r , care should be e x e r c i s e d t o avoid s c a l e l o s s and to c a r r y a l l c u l t u r e operations such as feeding changing water f i l t e r s , c o l l e c t i n g feces and f l u s h i n g the tank system without e l i c i t i n g f r e i g h t responses i n the f i s h . The n u t r i e n t l e a c h i n g losses a s s o c i a t e d w i t h feces c o l l e c t e d w i t h the "Guelph system" i n experiment I were caused mainly by a d e f i c i e n c y i n the design of the e x i t pipe from the d i g e s t i b i l i t y tank which prevented feces from being r a p i d l y c a r r i e d to the area of undisturbed water i n the c o l l e c t i o n column. I t was p o s t u l a t e d at the end of experiment I and subsequently confirmed i n experiments I I - IV, t h a t m o d i f i c a t i o n s t o the tank f l u s h i n g hydrodynamics would enable l e a c h i n g l o s s e s to be minimized and hence the generation of r e l i a b l e i n f o r m a t i o n on f e e d s t u f f d i g e s t i b i l i t y . Consequently, the "Guelph system" was found to be the p r e f e r r e d technique f o r c o l l e c t i n g feces f o r the determination of f e e d s t u f f d i g e s t i b i l i t y i n chinook salmon h e l d i n seawater. Further, methodology f i n d i n g s , h i t h e r t o ignored i n p r e v i o u s l y p u b l i s h e d research on f e e d s t u f f d i g e s t i b i l i t y i n f i s h suggest the need to remove f i s h s c a l e s from f e c a l samples c o l l e c t e d salmon h e l d i n seawater since these would cause d i g e s t i b i l i t y c o e f f i c i e n t s , e s p e c i a l l y t h a t of crude p r o t e i n , t o be underestimated. 156 In experiments I I , I I I and IV, the apparent d i g e s t i b i l i t y of organic matter, crude p r o t e i n and gross energy, as w e l l as the d i g e s t i b l e energy values of various f i s h meals (he r r i n g meal, anchovy meal, menhaden meal, Norwegian LT f i s h meal), p o u l t r y by-product meals (two sources), feather meal, blood meal, d r i e d whey, canola p r o t e i n products (commercial canola meal, g l u c o s i n o l a t e - f r e e canola meals, rapeseed p r o t e i n concentrate), soybean meal, soybean p r o t e i n i s o l a t e , extruded wheat and wheat middlings were determined w i t h chinook salmon i n seawater as the t a r g e t species. In general, i t was apparent t h a t chinook salmon were able to d i g e s t the energy of the f i s h meals more e f f i c i e n t l y than t h a t of the animal and p l a n t by-product f e e d s t u f f s . This i s thought t o occur because the energy content of the f i s h meals r e s i d e s l a r g e l y i n the p r o t e i n and o i l f r a c t i o n s , both of which are h i g h l y d i g e s t i b l e by chinook salmon, while much of the energy content of the p l a n t by-products stems from complex carbohydrates, which are e i t h e r i n d i g e s t i b l e or po o r l y d i g e s t e d by chinook salmon. Likewise, the crude p r o t e i n d i g e s t i b i l i t y of the animal by-products, e s p e c i a l l y t h a t of blood meal, i s mainly determined by the t e c h n i c a l p rocessing procedure employed. The source of blood meal used i n t h i s study was so overheated during processing, t h a t i t s crude p r o t e i n was only 2 9.4% dige s t e d by chinook salmon. On the other hand, based on d i g e s t i b i l i t y assessment, novel f e e d s t u f f s such as rapeseed p r o t e i n concentrate appear t o be h i g h l y promising as p a r t i a l or even complete replacements of f i s h meal i n salmonid d i e t s . Even though there are problems a s s o c i a t e d w i t h the use of c e r t a i n plant-based p r o t e i n sources, such as the u n p a l a t a b i l i t y of the soybean 157 products i n the present study, these problems can l i k e l y be overcome through i n n o v a t i v e research e f f o r t s . F i n a l l y , the r e s u l t s of t h i s t h e s i s support the view that wheat middlings are not an adequate source of energy f o r chinook salmon. Due t o the low d i g e s t i b i l i t y of the carbohydrate p o r t i o n of t h i s product, most of t h i s i n g r e d i e n t i s wasted and i s a cause of p o l l u t i o n i n f i s h farming. By c o n t r a s t , the s t a r c h component of extruded wheat became a valuable source of energy f o r chinook salmon owing to enhanced s t a r c h g e l a t i n i z a t i o n . I t i s t h e r e f o r e recommended t h a t wheat middlings, which are c u r r e n t l y i n c o r p o r a t e d i n t o salmonid d i e t s as a b i n d i n g agent, be completely replaced by more e f f i c i e n t l y d i g e s t e d binders such as extruded wheat or d r i e d whey. 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Using a f i n e s p a t u l a , c a r e f u l l y scrape any feces which may have been c a r r i e d over the tank overflow onto the funnel screen mesh and place i t i n t o the appropriate c e n t r i f u g e b o t t l e . 2. Increase the water flow i n t o the two tanks by f u l l y opening the flow valves (10-12 l i t e r s / m i n u t e ) . 3. As the water f l o w i n g i n t o the tanks increases, feces r e s t i n g on the h o r i z o n t a l pipe connecting the tank w i t h the c o l l e c t i o n column w i l l be dislod g e d and w i l l enter the v e r t i c a l stand pipe (see tank Figure 1). C l o s e l y monitor the movement of the f e c a l matter as i t i s c a r r i e d by the upwel l i n g water. 4. Using a 3.81 cm rubber stopper, plug the overflow d r a i n before any f e c a l p a r t i c l e s are c a r r i e d over the s i d e . 5. Allow the water l e v e l i n the tanks t o r i s e an a d d i t i o n a l 3 t o 5 cm and the shut o f f the water completely. I n t e r r u p t i o n of the u p w e l l i n g water movement i n the stand pipe w i l l a llow suspended f e c a l matter t o s e t t l e i n t o the c o l l e c t i o n column by g r a v i t y . 6. While w a i t i n g f o r f e c a l matter to s e t t l e i n t o the c o l l e c t i o n column, steps 1 to 4 are repeated on the next p a i r of tanks u n t i l a l l tanks have been f l u s h e d c l e a r of feces from the connecting pipe. At t h i s stage, a l l tank d r a i n s should be plugged and water flow i n t o the tanks i n t e r r u p t e d . 7. Remove the d r a i n plug from the f i r s t p a i r of tanks. The higher "head" i n the tank w i l l cause a surge of water flow t o car r y any remaining f e c a l p a r t i c l e s r e s t i n g on the connecting pipe t o the v e r t i c a l stand pipe area (see Figure 1). 178 APPENDIX 1. (cont'd) 8. Once again, plug the overflow d r a i n before any p a r t i c l e s reach t h i s l e v e l and repeat the procedure on the remaining tanks. 9. Once the water l e v e l i n the tanks has reached the l e v e l of the overflow d r a i n , plug the d r a i n as before and open the water valve i n the f i r s t tank u n t i l a flow r a t e of approximately 2 l i t r e s / m i n u t e has been a t t a i n e d . 10. When a 3 to 5 cm tank head has been achieved, i n s e r t the 0.64 cm s e m i - r i g i d hose i n t o the tank bottom d r a i n s l o t and open the small clamp which i s connected t o the incoming water l i n e . This w i l l create a high v e l o c i t y j e t of water to come out of the f a r end of the 0.64 cm hose. 11. At the same time, remove the plug from the tank overflow and c a r e f u l l y s l i d e the s e m i - r i g i d hose as f a r as i t w i l l go i n t o the connecting pipe which connects the tank bottom d r a i n w i t h the c o l l e c t i o n column. The sudden increase i n water flow caused by the drop i n water l e v e l from the tank, coupled w i t h the high v e l o c i t y water j e t out of the 0.64 cm s e m i - r i g i d hose w i l l ensure a l l f e c a l deposits are d i s l o d g e d from the connecting pipe and are c a r r i e d to the c o l l e c t i o n column. 12. When s a t i s f i e d t h a t a l l feces has s e t t l e d i n the c o l l e c t i o n column and none remains i n the connecting pipe, the 0.64 cm s e m i - r i g i d hose i s p u l l e d out of the tank and the water i s once again turned o f f . 13. P r i o r to c o l l e c t i n g the feces i n t o the c e n t r i f u g e b o t t l e s , a 1 meter long aluminum rod (approx. 2 mm i n diameter) i s i n s e r t e d down i n s i d e the v e r t i c a l stand pipe and the t i p i s used to dislodge f e c a l p a r t i c l e s which may have become stuck i n the pipe j o i n t s . 14. The f i r s t c e n t r i f u g e b o t t l e i s capped w i t h a s p e c i a l l y made rubber plug w i t h a spout i n the middle. The f l e x i b l e t u b i n g at the most d i s t a l end of the tank c o l l e c t i n g column i s i n s e r t e d i n t o the plug spout. 15. Gently squeeze the f l e x i b l e t u b i n g above the clamp a few times to a l l o w feces i n the column to s l i d e i n t o the t u b i n g . 16. While f i r m l y squeezing the f l e x i b l e t u b i n g , remove the clamp and a l l o w feces to g e n t l y s l i d e i n t o the c e n t r i f u g e b o t t l e . I t i s of utmost importance t o ensure f e c a l p e l l e t s do not d i s i n t e g r a t e during t h i s procedure as t h i s w i l l cause seri o u s l e a c h i n g l o s s e s . 179 APPENDIX 1. (cont'd) 17. Once a l l f e c a l matter has been c o l l e c t e d i n t o the c e n t r i f u g e b o t t l e , replace the clamp and squeeze the tu b i n g t i g h t l y to prevent water from l e a k i n g out. 18. Open the water valve and adjust the water flow i n t o the tank. 19. Repeat steps 9 to 18 f o r a l l other tanks. 20. C a r e f u l l y take c e n t r i f u g e b o t t l e s to l a b o r a t o r y and c e n t r i f u g e samples f o r 30 minutes at 10,000 x g. 21. The same procedure i s repeated at 1430 hours d a i l y f o r three weeks. 180 APPENDIX 2. Procedures followed for the quantitative feed consumption determination by chinook salmon i n Experiment I . A) Pre-feeding preparations to be ca r r i e d out i n the laboratory. 1. Sieve feed p e l l e t s to remove p e l l e t s smaller than 3 mm i n length. 2. Weigh empty pre-labelled feed v i a l s (Wl). 3. Add feed ration to each corresponding feed v i a l and record weight (W2). Thus, d a i l y feed ration ( i f f u l l y consumed) i s W2 - Wl. B) Quantitative feeding procedure to be followed immediately afte r f e c a l c o l l e c t i o n s . 1. Increase the water flow into each tank to approximately 10 l i t e r s per minute to ensure that refused feed p e l l e t s are quickly flushed out of the tank. 2. Insert s p e c i a l l y made perforated cup into the c o l l e c t i o n column v i a the v e r t i c a l stand pipe. The cup should rest below the l e v e l of the pipe connecting the tank bottom drain with the c o l l e c t i o n column to ensure that refused feed p e l l e t s drop into the cup after being flushed out of the tank. 3. Using a spoon, take some p e l l e t s out of the f i r s t feed v i a l and feed t h i s to the appropriate f i s h , one p e l l e t at a time. When f i s h lose interest i n the feed, move to the next tank and feed those f i s h with t h e i r feed ration and return to the f i r s t tank once these f i s h lose t h e i r feeding enthusiasm. Feed only two tanks at a time. 4. Attempt to feed at least one half the d a i l y ration to each tank i n the morning and save the remainder for the afternoon feeding. 5. Once feeding the f i r s t two tanks has been completed, increase the water flow into these two tanks to maximum flow (approx. 12 l i t e r s / m i n ) . 6. Insert t h i n 0.64 cm semi-rigid tube into the tank bottom s l o t and open the clamp to allow water under pressure to flow through the tube. 181 APPENDIX 2. (cont'd) 7. S l i d e the s e m i - r i g i d tube as f a r as i t w i l l t r a v e l i n s i d e the pipe connecting the tank bottom s l o t and the c o l l e c t i o n column. The water j e t at the end of the s e m i - r i g i d tube, coupled w i t h the increased flow r a t e i n t o the tank w i l l ensure that a l l feed p e l l e t s are f l u s h e d out of the tank and drop i n t o the cup s i t t i n g at the bottom of the c o l l e c t i o n column. 8. Close r i g i d tube hose clamp and p u l l out the tube from the tank. 9. Adjust water flow i n t o the tank and then b r i n g up the cup from the c o l l e c t i o n column. 10. Quickly count and record the number of feed p e l l e t s c o l l e c t e d i n the cup before they d i s i n t e g r a t e . 11. The same procedure i s repeated f o r every tank. 12. Feed the remainder of p e l l e t s i n the afternoon as described above and i f p e l l e t s remain i n the feed v i a l s , reweigh each v i a l again (W3). 13. C a l c u l a t e the amount of dry feed consumed f o r the day as f o l l o w s : % dry matter x [ (W2 - WI) - (W3 - WI) - (W4)] where W4 = number of feed p e l l e t s recovered from cup x mean feed p e l l e t weight (0.0465 grams). 182 APPENDIX 3 . Information on the p r o c e s s i n g methods o f v a r i o u s f e e d s t u f f s t e s t e d i n t h i s t h e s i s as d e s c r i b e d by the s u p p l i e r s . (Note: a b b r e v i a t i o n i n p a r e n t h e s i s i s the code g i v e n t o the f e e d s t u f f i n t h i s t h e s i s ) . 1. B r i t i s h Columbia h e r r i n g meal (HM) s u p p l i e d by Mr. B r i a n D. G i l l e y , B r i t i s h Columbia Packers L t d . a) The meal was prepared u s i n g whole male and female (minus the roe) h e r r i n g (Clupea harengus pallasi) c a r c a s s e s from the 1988 h e r r i n g roe season. b) Once caught, the h e r r i n g were t r a n s p o r t e d on i c e b e f o r e being deep f r o z e n (-20°C) fou r days l a t e r . c) The f i s h were thawed at 4 to 7°C p r i o r t o roe e x t r a c t i o n . d) Carcasses were processed w i t h i n two t o th r e e days of roe e x t r a c t i o n . e) The minced h e r r i n g was cooked by i n d i r e c t steam at 95°C, and was then passed through a s i n g l e screw auger t o remove s t i c k water. f) The temperature i n the steam-jacket r o t a r y d r y e r was set at 77°C. g) The f i n a l h e r r i n g meal was ground i n a hammer m i l l b e f o r e b e i n g s t a b i l i z e d with a n t i o x i d a n t . Anchovy meal (AM) from C h i l e s u p p l i e d by Mr. Jack Markert, Moore-Clark Co. (Canada) Inc. No f u r t h e r p r o c e s s i n g i n f o r m a t i o n a v a i l a b l e . 3 . Menhaden meal (MM) of a s p e c i a l s e l e c t grade from L o u i s i a n a s u p p l i e s by Mr. Kelsey Short, Zapata Haynie Corp. a) Meal prepared u s i n g whole, f r e s h menhaden with a TVN of l e s s than 0.05%. b) Temperature o f meal at time o f e x i t from the d r y e r was l e s s than 54°C. 4. Low temperature Norwegian h e r r i n g meal (LT) s u p p l i e d by Mr. Jason Mann, EWOS Canada L t d . a) Used whole f r e s h h e r r i n g and c a p e l i n from southern Norway with a TVN of l e s s than 0.05%. b) P r o c e s s i n g temperatures never reach 100°C. c) 200-400 ppm ethoxyquin a n t i o x i d a n t added a f t e r d r y i n g d) F i s h meal c o n t a i n e d 0.0 9% TVN. 183 APPENDIX 3. (cont'd) 5. P o u l t r y by-product meal from Kansas (PMK) manufactured by N a t i o n a l By-products Inc. (Kansas C i t y ) and sample s u p p l i e d f o r t h i s t h e s i s by Dr. Ron W. Hardy, Northwest F i s h e r i e s Center, S e a t t l e , Washington. a) Meal was d r i e d by i n d i r e c t methods and the dry meal was then passed through an a i r c l a r i f i e r which removes l a r g e p a r t i c l e s of bone.. No f u r t h e r processing d e t a i l s were made a v a i l a b l e . 6. P o u l t r y by-product meal from B r i t i s h Columbia (PMB) manufactured by West Coast Reduction L t d . , Vancouver, B r i t i s h Columbia. a) P o u l t r y by-products are processed continuously by means of a 2 stage vacuum evaporation system. b) Maximum temperature a t t a i n e d i s 135°C f o r 20 minutes. 7. Hydrolyzed feather meal (FM) manufactured by West Coast Reduction L t d . , Vancouver, B r i t i s h Columbia. a) Raw feathers are batch hydrolyzed at 40 p s i and 142°C f o r 4 0 minutes. b) The feathers are then d r i e d i n a steam tube dryer. c) The maximum temperature i n the dryer i s approximately 100°C wit h the contact temperature being 170°C. 8. Blood meal (BM) manufactured by West Coast Reduction L t d . , Vancouver, B r i t i s h Columbia. a) Raw blood processing c o n s i s t s of continuous co a g u l a t i o n at approximately 98°C by means of l i v e steam. b) Separation i s accomplished by means of a c e n t r i f u g e , and d r y i n g t o the f i n a l moisture content i s performed i n a continuous d i s k dryer. c) The maximum product temperature i n the dryer i s approximately 100°C wit h the contact temperature being 143°C. 184 APPENDIX 3. (cont'd) 9. Dr i e d whey powder (DW) was obtained from Ms. Nancy Richardson, White Crest M i l l s . a) L i q u i d whey, separated from cheese curds, i s f i r s t evaporated to b r i n g s o l i d s down to 32%. b) This l i q u i d i s then pumped under pressure through spray nozzles i n t o a dryer u n i t . c) When the mist h i t s the hot a i r i n the dryer, the s o l i d s drop t o the bottom of the dryer and are augered out. 10. Commercial canola meal (CM) obtained through R i t c h i e - S m i t h Feeds, Inc., Abbotsford B.C. and processed by Canbra Foods Ltd . , Lethbridge, A l b e r t a . This canola meal i s a blend of both types of seed ( P o l i s h and Argentinian) to a r r i v e at a p r o t e i n l e v e l of 36%. The clean seed i s f l a k e d i n r o l l e r m i l l s as the f i r s t step i n breaking the seed s t r u c t u r e t o allo w separation of o i l and meal. Many o i l bearing c e l l s are made permeable to o i l by cooking which a l s o coalesces the o i l i n t o l a r g e r d r o p l e t s . Cooking i s done (temperature not given) f o r 20 t o 30 minutes i n stack cookers heated i n d i r e c t l y by steam. The method of o i l e x t r a c t i o n i s performed through an e x p e l l e r / s o l v e n t or mechanical/chemical operation using hexane as the so l v e n t . The solvent i s s t r i p p e d from the meal using l i v e steam. The meal i s d r i e d and i s then t o a s t e d f o r complete solvent e x t r a c t i o n (temperatures not g i v e n ) . 11. G l u c o s i n o l a t e f r e e canola meal (GCMa) obtained through Dr. J.M. B e l l , Dept. of Animal Science, U n i v e r s i t y of Saskatchewan, Saskatoon. a) This s p e c i a l g l u c o s i n o i a t e - f r e e s t r a i n was developed by Dr. David Hutcheson and i s a s e l e c t i o n d e r i v e d from the Brassica campestris c u l t i v a r Tobin. This g e n e t i c a l l y s e l e c t e d canola i s r e f e r r e d to as BC86-18 and i s not a l i c e n s e d v a r i e t y as yet. b) The seed was grown on very clean land and was fre e of w i l d mustard and other weed seed contaminants. c) The seed was crushed i n the POS P i l o t P l a n t Corp. f a c i l i t i e s u s ing a pre-press solvent (hexane) e x t r a c t i o n method designed t o simulate t y p i c a l commercial procedures i n use i n Canada, th a t i s cl e a n i n g , tempering, f l a k i n g , a) b) c) d) e) f) g) 185 APPENDIX 3. (cont'd) c o o k i n g / c o n d i t i o n i n g , e x p e l l i n g i n a screw pr e s s , s o l v e n t e x t r a c t i o n , d e s o l v e n t i z a t i o n and g r i n d i n g . d) The maximum temperature i n the c o o k i n g / c o n d i t i o n i n g phase are 90 t o 100°C. e) Steam i s used i n the d e s o l v e n t i z e r . 12. G l u c o s i n o l a t e e x t r a c t e d c a n o l a meal (GCMb) p r o v i d e d by Dr. Rubin Diosady and sample was e x t r a c t e d w i t h a combination of methanol/ammonia t o reduce the g l u c o s i n o l a t e content of the meal. F u r t h e r e x t r a c t i o n d e t a i l s were not made a v a i l a b l e s i n c e t h i s i s a patented p r o c e s s . 13. Rapeseed p r o t e i n concentrate (RP) s u p p l i e d by Dr. John D. Jones, Food Research Centre, A g r i c u l t u r e Canada, Ottawa. a) Rapeseed p r o t e i n concentrate shipped t o the Department of F i s h e r i e s and Oceans i n West Vancouver i n August 1987, and s t o r e d at -20°C at t h i s f a c i l i t y . b) Lot number o f product t e s t e d was 343 which i d e n t i f i e s both the seed and the method of p r e p a r a t i o n [BRONOWSKI FRI-73-5(343)]. c) Prepared u s i n g d e h u l l e d , s o l v e n t and water e x t r a c t e d rapeseed f l o u r . d) D e h u l l e d seed i s t r e a t e d twice by e x t r a c t i n g with b o i l i n g water f o r 2 minutes. 14. Soybean meal (SM) obt a i n e d through R i t c h i e Smith Feeds, Inc., Ab b o t s f o r d B.C. -No d e t a i l e d p r o c e s s i n g i n f o r m a t i o n p r o v i d e d . 15. Soybean p r o t e i n i s o l a t e (SI) obtained from Van Waters and Rogers L t d . A b b o t s f o r d B.C. -No d e t a i l e d p r o c e s s i n g i n f o r m a t i o n p r o v i d e d . 16. Extrude wheat (EW) obtained through Ms. Nancy Richardson, White C r e s t M i l l s , Campbell R i v e r . a) Used hard wheat of p r a i r i e o r i g i n . b) Moisture i s added i n the form of steam t o hard wheat. c) The wheat i s cooked i n an extruder at 14 9 t o 193°C and i s then f o r c e d through a dye. 17. Wheat mid d l i n g s (WM) obt a i n e d from Van Waters and Rogers, Abbotsford, B r i t i s h Columbia. -No d e t a i l e d p r o c e s s i n g i n f o r m a t i o n p r o v i d e d . 186 APPENDIX 4. S t a t i s t i c a l analyses. ANOVA f o r organic matter d i g e s t i b i l i t y based on the d i r e c t or i n d i r e c t method. (Experiment I ) . Source DF Mean Square F Value Pr >F WEEK 2 8.1305833 1.31 0.2848 METHOD 1 15.8802083 2.55 0.1141 MANAGEMENT 2 218.2812500 35.07 0.0001 WEEK*METHOD 2 2.3355833 0.38 0.6901 WEEK*MANAGEMENT 3 10.6902315 1.72 0.1830 METHOD*MANAGEMENT 2 2.5112500 0.40 0.6713 WEEK*METHOD*MANAGEMENT 3 2.2446759 0.36 0.7818 E r r o r 32 6.2233333 Corrected T o t a l 47 ANOVA f o r crude p r o t e i n . d i g e s t i b i l i t y based on the d i r e c t or i n d i r e c t method. (Experiment I ) . Source DF Mean Square F Value Pr > F WEEK 2 10.6989167 2.03 0.1485 METHOD 1 16.6352083 3.14 0.0828 MANAGEMENT 2 173.3805833 32. 65 0.0001 WEEK*METHOD 2 0.6935833 0.13 0.8774 WEEK*MANAGEMENT 3 4.3375926 0.82 0.4919 METHOD ^MANAGEMENT 2 1.1072500 0.21 0.8120 WEEK*METHOD*MANAGEMENT 3 0.5070370 0.10 0.9617 E r r o r 32 5.2829167 Corrected T o t a l 47 ANOVA f o r gross energy d i g e s t i b i l i t y based on the d i r e c t or i n d i r e c t method. (Experiment I ) . Source DF Mean Square F Value Pr > WEEK 2 8.7622500 1.34 0.2766 METHOD 1 11.8133333 1.80 0.1966 MANAGEMENT 2 197.7112500 30.19 0.0001 WEEK*METHOD 2 4.7605833 0.73 0.4911 WEEK*MANAGEMENT 3 4.7706019 0.73 0.5425 METHOD *MANAGEMENT 2 7.5179167 1.15 0.3299 WEEK*METHOD*MANAGEMENT 3 4.0868981 0.62 0.6046 E r r o r 32 6.5475000 Corrected T o t a l 47 187 APPENDIX 4. Continued. ANOVA f o r organic matter d i g e s t i b i l i t y based on the morning and afternoon f e c a l c o l l e c t i o n s . (Experiment I ) . Source DF Mean Square F Value Pr > WEEK 2 5.815139 1.30 0 .2857 METHOD 1 769.600833 173.37 0 .0001 MANAGEMENT 2 228.405069 51.45 0 . 0001 WEEK*METHOD 2 11.451389 2.58 0 .0915 WEEK*MANAGEMENT 3 8.258750 1.86 0 .1562 METHOD*MANAGEMENT 2 35.970625 8.10 0 .0014 WEEK*METHOD*MANAGEMENT 3 4.006157 0.90 0 .4507 E r r o r 32 4.43896 Corrected T o t a l 47 ANOVA f o r crude p r o t e i n d i g e s t i b i l i t y based on the morning and afternoon f e c a l c o l l e c t i o n s . (Experiment I ) . Source DF Mean Square F Value Pr > F WEEK 2 13. .29503 1. .64 0, .2113 METHOD 1 1406. ,23521 173. ,80 0. .0001 MANAGEMENT 2 318. ,11951 . 39. ,32 0. .0001 WEEK*METHOD 2 23. .96837 2. .96 0, .0796 WEEK*MANAGEMENT 3 14. ,58912 1. ,80 0, .1665 METHOD ^MANAGEMENT 2 120, .98951 14, .95 0, .0001 WEEK*METHOD*MANAGEMENT 3 18, . 90245 2, .34 0, .0923 E r r o r 32 8, .09125 Corrected T o t a l 47 ANOVA f o r gross energy d i g e s t i b i l i t y based on the morning and afternoon f e c a l c o l l e c t i o n s . (Experiment I ) . Source DF Mean Square F Value Pr > F WEEK 2 19. ,123090 2, .85 0. .0867 METHOD 1 642. ,210208 95, .56 0, .0001 MANAGEMENT 2 248. . 685625 37, .01 0. .0001 WEEK*METHOD 2 16. , 607535 2, .47 0, .1033 WEEK * MANAGEMENT 3 8, . 172454 1, .22 0, .3197 METHOD *MANAGEMENT 2 58, .967292 8, .78 0, .0009 WEEK*METHOD*MANAGEMENT 3 5, .586157 0, .83 0, .4865 E r r o r 32 6, .71937 Corrected T o t a l 47 188 APPENDIX 4. Continued A n a l y s i s of variance of feed intake data from experiment I. Source DF Mean Sq. (10 - 5) F Value Pr > F MANAGEMENT 2 49.398 9.88 0.012 6 E r r o r 6 4.998 To t a l 8 A n a l y s i s of variance of s p e c i f i c growth r a t e of chinook salmon i n experiment I. Source DF Mean Sq. (10~ 5) F Value Pr > F Model 2 72.218 48.01 0.0002 E r r o r 6 1.504 To t a l 8 A n a l y s i s of variance f o r organic matter d i g e s t i b i l i t y of the f e e d s t u f f s t e s t e d i n experiments I I , I I I and IV. Mean Source DF Square F Value Pr > F Feedstuff 19 869.1843 27.11 0.0001 E r r o r 42 32.0596 T o t a l 61 A n a l y s i s of variance f o r crude p r o t e i n d i g e s t i b i l i t y of the f e e d s t u f f s t e s t e d i n experiments I I , I I I and IV. Mean Source DF Square F Value Pr > F Feedstuff 19 595.0453 60.29 0.0001 E r r o r 42 9.8698 To t a l 61 A n a l y s i s of variance f o r gross energy d i g e s t i b i l i t y of the f e e d s t u f f s t e s t e d i n experiments I I , I I I and IV. Mean Source DF Square F Value Pr > F Feedstuff E r r o r T o t a l 19 42 61 690.1523 13.69 0.0001 50.4111 189 APPENDIX 4. Continued A n a l y s i s of variance f o r d i g e s t i b l e energy of the f e e d s t u f f s t e s t e d i n experiments I I , T i l and IV. Mean Source DF Square F Value Pr > F Feedstuff 19 0.00822056 19.82 0.0001 E r r o r 42 0.00041478 T o t a l 61 A n a l y s i s of variance of weight gain (g/fish) f o r Experiment I I . Mean Source DF Square F Value Pr > F DIET 5 4.4512667 33.22 0.0001 E r r o r 12 0.1340056 T o t a l 17 A n a l y s i s of variance of s p e c i f i c growth r a t e (SGR) f o r Experiment I I . Mean Source DF Square F Value Pr > F DIET 5 0.35170343 31.65 0.0001 E r r o r 12 0.01111144 T o t a l 17 A n a l y s i s of variance of d a i l y dry feed i n t a k e (DFI) f o r Experiment I I . Mean Source DF Square F Value Pr > F DIET 5 0.00053960 1.58 0.2392 E r r o r 12 0.00034183 T o t a l 17 A n a l y s i s of variance of d a i l y dry feed i n t a k e as a percent of B.W. (FI) f o r Experiment I I . Mean Source DF, Square F Value Pr > F DIET E r r o r T o t a l 5 12 17 0.01094222 0.01535556 0.71 0.6257 190 APPENDIX 4. C o n t i n u e d A n a l y s i s o f v a r i a n c e o f f e e d e f f i c i e n c y r a t i o (FE) f o r Experiment I I . Mean Source DF Square F V a l u e P r > F DIET 5 0.15511222 24.58 0.0001 E r r o r 12 0.00631111 T o t a l 17 A n a l y s i s o f v a r i a n c e o f p r o t e i n e f f i c i e n c y r a t i o (PER) f o r Experiment I I . Mean Source DF Square F V a l u e P r > F DIET 5 0.36289889 20.14 0.0001 E r r o r 12 0.01802222 T o t a l 17 A n a l y s i s o f v a r i a n c e o f d i g e s t i b l e o r g a n i c m a t t e r p e r f i s h f o r Experiment I I . Mean Source DF Square F V a l u e P r > F DIET 5 0.00083289 5.02 0.0100 E r r o r 12 0.00016578 T o t a l 17 A n a l y s i s o f v a r i a n c e o f d i g e s t i b l e p r o t e i n p e r f i s h f o r Experiment I I . Mean Source DF Square F V a l u e P r > F DIET 5 0.00068413 8.93 0.0010 E r r o r 12 0.00007661 T o t a l 17 A n a l y s i s o f v a r i a n c e o f d i g e s t i b l e energy p e r f i s h f o r Experiment I I . Mean Source DF Square F V a l u e P r > F DIET 5 0.68997423 6.40 0.0040 E r r o r 12 0.10778794 T o t a l 17 191 APPENDIX 4. Continued A n a l y s i s of variance of f i s h weight gain f o r Experiment I I I . Mean Source DF Square F Value Pr > F DIET 8 3.0449204 22.15 0.0001 E r r o r 18 0.1374444 T o t a l 26 A n a l y s i s of variance of s p e c i f i c growth r a t e (SGR) f o r Experiment I I I . Mean Source DF Square F Value Pr > F DIET 8 0.13321233 19.47 0.0001 E r r o r 18 0.00684156 T o t a l 26 A n a l y s i s of variance of d a i l y dry feed intake (DFI) per f i s h f o r Experiment I I I . Mean Source DF Square F Value Pr > F DIET 8 0.01656420 19.85 0.0001 E r r o r 18 0.00083444 T o t a l 26 A n a l y s i s of variance of d a i l y dry feed i n t a k e (FI) as a percent of body weight f o r Experiment I I I . Mean Source DF Square F Value Pr > F DIET 8 0.22575648 21.05 0.0001 E r r o r 18 0.01072593 T o t a l 26 A n a l y s i s of variance of feed e f f i c i e n c y r a t i o (FE) f o r Experiment I I I . Mean Source DF Square F Value Pr > F DIET E r r o r T o t a l 8 0.07175833 39.38 0.0001 18 0.00182222 26 192 APPENDIX 4 . Continued 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 (PER) f o r Experiment I I I . Mean Source DF Square F Value Pr > F DIET 8 0.2109.8333 4 3 . 1 6 0 .0001 E r r o r 18 0 .00488889 T o t a l 26 A n a l y s i s o f v a r i a n c e of d i g e s t i b l e o r g a n i c matter per f i s h f o r Experiment I I I . Mean Source DF Square F Value Pr > F DIET 8 0 .00897970 24 .00 0 .0001 E r r o r 18 0 .00037411 T o t a l 26 A n a l y s i s o f v a r i a n c e o f d i g e s t i b l e p r o t e i n per f i s h f o r Experiment I I I . Mean Source DF Square F Value Pr > F DIET 8 0 .00449975 2 3 . 0 5 0 .0001 E r r o r 18 0 .00019526 T o t a l 26 A n a l y s i s o f v a r i a n c e o f d i g e s t i b l e energy per f i s h f o r Experiment I I I . Mean Source DF Square F Value Pr > F DIET 8 6 .5478872 ,24 .26 0 .0001 E r r o r 18 0 .2699349 T o t a l 26 A n a l y s i s o f v a r i a n c e of weight g a i n f o r Experiment IV. Mean Source DF Square F Value Pr > F DIET E r r o r T o t a l 7 18 25 6.0356907 16 .70 0 .0001 0 .3614556 193 APPENDIX- 4 . Continued A n a l y s i s of variance of s p e c i f i c growth r a t e (SGR) f o r Experiment IV. Mean Source DF Square F Value Pr > F DIET 7 0 .02285464 8.28 0 .0001 E r r o r 18 0 .00276025 T o t a l 25 A n a l y s i s of variance f o r d a i l y dry feed i n t a k e (DFI) f o r Experiment IV. Mean Source DF Square F Value Pr > F DIET 7 0 .01459376 6 .59 0 .0006 E r r o r 18 0 .00221345 T o t a l 25 A n a l y s i s of variance of d a i l y dry feed i n t a l e as a percent of body weight (FI) f o r experiment IV. Mean Source DF Square F,Value Pr > F DIET 7 0 .09586190 6.60 0 .0006 E r r o r 18 0 .01453426 T o t a l 25 T-test between the organic matter d i g e s t i b i l i t y c o e f f i c i e n t s determined from feces c o l l e c t e d w i t h the "Guelph system" and from feces c o l l e c t e d by s t r i p p i n g i n experiment I I . REF-a DIET N Obs Mean Std E r r o r T Prob>|T 3 7 .3066667 1.1134980 6.5619038 0.0224 T-test between the organic matter d i g e s t i b i l i t y c o e f f i c i e n t s determined from feces c o l l e c t e d w i t h the "Guelph system" and from feces c o l l e c t e d by s t r i p p i n g i n experiment I I I . 194 APPENDIX 4. Continued REF-a DIET N Obs Mean Std E r r o r 3 4.1166667 0.9249617 T Prob>|T| 4.4332249 0.0376 T-test between the organic matter d i g e s t i b i l i t y c o e f f i c i e n t s determined from feces c o l l e c t e d w i t h the "Guelph system" and from feces c o l l e c t e d by i n t e s t i n a l d i s s e c t i o n i n experiment IV. REF-b DIET N Obs Mean Std E r r o r T Prob>|T| 3 4.2933333 1.1743130 3.6560382 0.0673 T-test between the organic matter d i g e s t i b i l i t y c o e f f i c i e n t s of the t e s t i n g r e d i e n t s i n experiments I I , I I I and IV, determined from feces c o l l e c t e d w i t h the "Guelph system" and from feces c o l l e c t e d d i r e c t l y from the f i s h . INGR=RP N Obs Mean Std E r r o r T Prob>|T| 3 3.0600000 2.2670024 1.3498001 0.3096 INGR=CM-N Obs Mean Std E r r o r T Prob>|T 3 1 .5833333 4. 0896835 0.3871530 0.7360 N Obs Mean Std E r r o r T Prob>|T| 3 2 .3800000 2. 2908805 1.0389019 0.4080 195 APPENDIX 4. Continued INGR=HM N Obs Mean Std E r r o r T Prob>|T| 3 3. 9866667 0.1971745 20 .2189784 0.0024 N Obs Mean Std E r r o r T Prob>|T| 3 4. 9633333 3.3772589 1 .4696336 0.2794 N Obs Mean Std E r r o r T Prob>|T| 3 8.9400000 4.7207203 1.8937788 0.1988 N Obs INGR Mean =PMB Std E r r o r T Prob>IT| 3 7 .7633333 1.2581777 6 .1702996 0.0253 N Obs Mean Std E r r o r T Prob>|T| 3 5. 5633333 0.7056990 7. 8834364 0.0157 N Obs Mean Std E r r o r T Prob>IT| 3 4 .6433333 0.7822262 5 .9360496 0.0272 N Obs Mean Std E r r o r T Prob>|T| 3 4 .2066667 1.3520396 3 .1113487 0.0896 N Obs Mean Std E r r o r T Prob>|T| 3 6.2033333 1.3131937 4.7238524 0.0316 196 APPENDIX 4. Continued. N Obs Mean Std E r r o r T Prob>|T| 6 10 .0166667 2.7072676 3 .6999175 0.0140 TVI/'-T-> I C Q . TPT.T N Obs Mean Std E r r o r T Prob>IT| 3 8 .4400000 6.5796454 1 .2827439 0.3282 N Obs Mean -L IN J U T ) Hi »V Std E r r o r T Prob>IT| 3 -0 .7266667 1.2939646 -0 .5615816 0.6309 N Obs Mean Std E r r o r . T Prob>|T| 3 1 .3233333 3.9183854 0 .3377241 0.7677 N Obs Mean Std E r r o r T Prob>|T| 3 0.6966667 2.5172097 0.2767615 0.8079 

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