Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

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

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-UBC_1991_A6_7 H34.pdf [ 10.31MB ]
Metadata
JSON: 831-1.0098489.json
JSON-LD: 831-1.0098489-ld.json
RDF/XML (Pretty): 831-1.0098489-rdf.xml
RDF/JSON: 831-1.0098489-rdf.json
Turtle: 831-1.0098489-turtle.txt
N-Triples: 831-1.0098489-rdf-ntriples.txt
Original Record: 831-1.0098489-source.json
Full Text
831-1.0098489-fulltext.txt
Citation
831-1.0098489.ris

Full Text

EVALUATION POST-JUVENILE  OF FEEDSTUFF D I G E S T I B I L I T Y  CHINOOK SALMON IN  (Oncorhynchus  IN  tshawytscha)  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  C o l u m b i a , 198!  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE  i n  THE FACULTY  OF GRADUATE  Department We a c c e p t t h i s  o f Animal thesis  to the required  THE UNIVERSITY  Science  as c o n f o r m i n g standard  OF B R I T I S H COLUMBIA  November ©  STUDIES  1990  W a l t e r E . H a j e n , 1990  In  presenting this  degree at the  thesis  in  University of  partial  fulfilment  of  of  department  this thesis for or  by  his  or  requirements  British Columbia, I agree that the  freely available for reference and study. I further copying  the  representatives.  an advanced  Library shall make it  agree that permission for extensive  scholarly purposes may be her  for  It  is  granted  by the  understood  that  head of copying  my 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  DE-6 (2/88)  December 26, 1990  ii ABSTRACT F e e d a c c o u n t s f o r 40% t o 60% o f t h e o p e r a t i n g c o s t s o f Pacific salmon  salmon  farms.  i n seawater  Presently,  Information completely this  c h i n o o k salmon  lacking,  from s t u d i e s  on r a i n b o w t r o u t  i n fresh  regardless  of l i f e  history  salmon  o f organic matter, crude p r o t e i n  salmon i s  stage.  Hence,  (Oncorhynchus  system"  of fecal  indigestible In t h i s  tshawytscha)  collection  indicator  regard,  settling  fecal  i n seawater.  digestibility  i n the diet samples  of the i n t e s t i n a l  dissection.  Digestibility  significantly  increased  tract  The v a l i d i t y o f  (C^C^)  as t h e  was a s s e s s e d i n e x p e r i m e n t I .  were c o l l e c t e d  intervals  chinook  u s i n g t h e "Guelph  and chromic o x i d e  column a f f i x e d t o e a c h n o v e l l y  t a n k a t 6 a n d 18 h o u r section  and e n e r g y i n  and n o v e l 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  determining apparent n u t r i e n t  either  designed  or d i r e c t l y by s t r i p p i n g  from a  digestibility  from t h e t e r m i n a l or i n t e s t i n a l  c o e f f i c i e n t s were n o t e d t o be  (P<0.001) when t h e f e c e s  remained  i n the  f o r 18 h o u r s i n s t e a d o f 6 h o u r s , owing t o n u t r i e n t  leaching. resulted  The c o l l e c t i o n  of feces d i r e c t l y  i n lower d i g e s t i b i l i t y  coefficients  f e c e s were o b t a i n e d f r o m t h e " G u e l p h  from t h e f i s h than those found  system".  This  a t t r i b u t e d t o a flaw i n the design o f the d i g e s t i b i l i t y drain  water.  t h e s i s was u n d e r t a k e n t o d e t e r m i n e t h e a p p a r e n t  conventional  when  for Pacific  and d i g e s t i b i l i t y  on f e e d s t u f f d i g e s t i b i l i t y b y P a c i f i c  digestibility  water  diets  are formulated according to the nutrient  requirements of j u v e n i l e information derived  commercial  system,  whereby t h e f e c e s d i d n o t s e t t l e  quickly  was tank  into the  iii collection the  column.  subsequent In  the o v e r a l l  crude p r o t e i n digestible  to ascertain  meal,  b l o o d meal,  protein  isolate,  and  menhaden m e a l ,  Norwegian (two  d r i e d whey, c a n o l a m e a l , e x t r u d e d wheat and wheat  concentrate)  meal  low suppliers),  soybean  (two t y p e s  c a n o l a m e a l and r a p e s e e d p r o t e i n  were e v a l u a t e d  in this  regard.  The  initial  size  o f t h e c h i n o o k i n t h e s e s t u d i e s v a r i e d between 10.3  40.5  g.  The  circuit  150  aerated, The  digestibility L fiberglass  8.0^C  ingredient). the  test  30%).  t o 12.5^C f i l t e r e d  or a t e s t  was  W i t h i n each t e s t , of f i s h  stripping  methodology.  : 30%  Chromic  dietary  open-  (0.5%)  was  o f 6 L/min.  either  (15% and  assigned to randomized included  A t t h e end o f e a c h  f o r comparisons  of  i na l l  experiment,  f e c e s were removed e i t h e r  dissection  a  test  levels  t r e a t m e n t was  oxide  and  protein products,  a c o m p l e t e l y random o r  and t h e i r  or i n t e s t i n a l  to satiation  involving plant  as t h e i n d i g e s t i b l e m a r k e r .  f i s h were s a c r i f i c e d  at a rate  (70% r e f e r e n c e  each d i e t  g  tanks, each s u p p l i e d w i t h  i n c l u d e d a t two  using  complete b l o c k d e s i g n . diets  diet  minimum  c o n s i s t e d o f 27  seawater  twice daily  I n some i n s t a n c e s  ingredient  t h r e e groups  t a n k complex  digestibility  f i s h were f e d by hand  reference diet  meal,  middlings.  three novel sources of rapeseed p r o t e i n products  glucosinolate-free  the  sources of f i s h  f i s h m e a l ) , p o u l t r y b y - p r o d u c t meal  f e a t h e r meal,  in  the organic matter,  coefficients  energy v a l u e s f o r commercial  temperature  of  e x p e r i m e n t s on c h i n o o k salmon  g o a l was  anchovy  before conducting  below.  and e n e r g y d i g e s t i b i l i t y  ( h e r r i n g meal,  soybean  rectified  experiments d e s c r i b e d  three additional  seawater,  Also,  The p r o b l e m was  by  iv In g e n e r a l , t h e f i s h meals had h i g h e r a v a i l a b l e energy c o n t e n t f o r chinook salmon t h a n t h e o t h e r a n i m a l and p l a n t p r o t e i n sources assessed.  The importance o f s c r e e n i n g 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 v a l u e 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 e v i d e n t from an e x a m i n a t i o n o f t h 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 o b t a i n e d f o r menhaden meal v e r s u s t h e o t h e r f i s h meal s o u r c e s , t h e two s o u r c e s o f p o u l t r y b y - p r o d u c t meal, wheat p r o d u c t s , c a n o l a p r o d u c t s and b l o o d meal i n t h i s s t u d y . assessment level  o f soybean p r o d u c t s , r e g a r d l e s s o f d i e t a r y  inclusion  (15% or 3 0 % ) , c o u l d not be a s c e r t a i n e d i n chinook  because o f poor d i e t a c c e p t a n c e . h i g h l y p r o m i s i n g as p a r t i a l  The  salmon  Canola p r o t e i n s o u r c e s appear  o r complete  ( i n t h e case o f rapeseed  p r o t e i n c o n c e n t r a t e ) replacements o f f i s h meal, based on d i g e s t i b i l i t y assessment.  The use o f t h e "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  digestibility  c o e f f i c i e n t s s i m i l a r t o t h o s e o b t a i n e d by i n t e s t i n a l  dissection.  Thus, n u t r i e n t l e a c h i n g must have been m i n i m a l w i t h t h e  "Guelph  system" and i t i s c o n c l u d e d 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  u s i n g chinook salmon i n seawater.  TABLE OF CONTENTS  Section ABSTRACT L I S T OF TABLES L I S T OF FIGURES ACKNOWLEDGEMENTS  Page  ,  i i viii xi x i i  CHAPTER 1 1.0  INTRODUCTION  1 CHAPTER 2  2.0 2.1 2.2 2.3 2.4 2.4.1 2.4.2 2.4.3 2.5 2.5.1 2.5.2 2.5.3 2.6 2.7 2.8 2.8.1 2.8.2 2.9 2.10 2.11 2.12 2.13 2.14  LITERATURE REVIEW P r o t e i n requirements of f i s h Amino a c i d r e q u i r e m e n t s o f f i s h Energy balance i n . f i s h Sources o f energy f o r f i s h P r o t e i n as a d i e t a r y energy s o u r c e 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 L i p i d s as d i e t a r y energy s o u r c e s 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 . . Fishery products Animal by-products Plant by-products... 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 ingredients D i g e s t i o n and a b s o r p t i o n G e n e r a l a l i m e n t a r y t r a c t anatomy a n d i t s a s s o c i a t e d enzymes The m i d g u t The h i n d g u t 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 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 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 Apparent and t r u e 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 measurements i n f i s h Evaluation of feedstuff d i g e s t i b i l i t y  5 5 6 7 9 9 9 12 14 14 15 16 19 22 23 23 25 26 27 28 28 29 35  CHAPTER 3 3.0  EXPERIMENT I - D i g e s t i b i l i t y m e a s u r e m e n t s i n j u v e n i l e chinook salmon (Oncorhynchus tshawytscha) i n seawater: 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 ( C r 0 3 ) 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 techniques used in fecal collection  36  Introduction M a t e r i a l s and methods Experimental design D i g e s t i b i l i t y tanks Aquarium f a c i l i t y Water f i l t e r i n g system 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 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  36 39 39 40 42 42 43 46  2  3.1 3.2 3.2.1 3.2.2 3.2.3 3.2.4 3.2.5 3.2.6  vi TABLE OF CONTENTS c o n t . . . . Section  Page  3.2.6.1 3.2.6.2 3.2.6.3 3.2.6.3.1 3.2.6.3.2 3.2.6.3.3 3.2.7 3.2.7.1 3.2.7.2 3.2.8 3.2.9 3.3 3.3.1 3.3.2 3.3.3 3.3.4 3.3.5 3.3.6  3.4 3.4.1 3.4.2 3.4.3 3.4.4 3.4.5  F i s h h i s t o r y and d i s t r i b u t i o n Experimental protocol Fecal c o l l e c t i o n techniques "Guelph system" Stripping technique I n t e s t i n a l d i s s e c t i o n technique Chemical a n a l y s e s o f f e e d and f e c e s Proximate c o m p o s i t i o n and gross energy determination Chromic oxide d e t e r m i n a t i o n s 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 performance Statistical analysis  46 47 48 48 49 49 50 50 50 51 53  Results Diets Chromic oxide a n a l y s i s 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 Ingested chromic oxide recovery E f f e c t o f t i m e b e t w e e n 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 E f f e c t o f f i s h management on a p p a r 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 / 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  55 55 55  Discussion D i g e s t i b i l i t y determinations of f i s h raised i n seawater Chromic oxide a n a l y s i s 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 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" E f f e c t o f f i s h management on a p p a r e n t 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 mortality  69  58 60 63 66  69 71 72 75 79  CHAPTER 4 4.0  EXPERIMENTS I I , I I I AND I V . - A p p a r e n t d i g e s t i b l e o r g a n i c matter, 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 a n d 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 4.2 4.2.1 4.2.2 4.2.3 4.2.4 4.2.5  Introduction M a t e r i a l s and methods Experimental design History of experimental f i s h Aquarium f a c i l i t y Water f i l t e r i n g system Diets  82 84 84 84 85 87 87  vii TABLE OF CONTENTS c o n t . . . . Section 4.2.6 4.2.7 4.2.7.1 4.2.7.2 4.2.8 4.2.9 4.2.10 4.2.11  Page Diet preparation E x p e r i m e n t a l p r o c e d u r e s and f i s h h a n d l i n g Diet allocation Protocol F e c a l c o l l e c t i o n procedure Chemical analyses o f 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 s a m p l e s 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 . . Statistical analysis '  4.3 4.3.1 4.3.2  Results Chemical composition o f t e s t i n g r e d i e n t s 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 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 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 t e c h n i q u e on a p p a r e n t organic matter d i g e s t i b i l i t y 4.3.5 I n f l u e n c e o f D i e t on f i s h p e r f o r m a n c e 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 e n e r g y d i g e s t i b i l i t y o f t h e r e f e r e n c e d i e t s u s e d i n e x p e r i m e n t s I I , I I I and IV b a s e d on t h e a p p a r e n t 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 o f individual dietary ingredients 4.4 Discussion 4.4.1 F e e d s t u f f e v a l u a t i o n according t o proximat c o m p o s i t i o n 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 4.4.1.2 Animal b y - p r o d u c t meals 4.4.1.3 P l a n t by-product meals 4.4.1.3.1 Grain products 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 t e c h n i q u e on apparent o r g a n i c matter d i g e s t i b i l i t y . . . . . 4.4.3 I n f l u e n c e o f d i e t on f i s h p e r f o r m a n c e 4.4.4 A d d i t i v i t y of individual ingredient nutrient 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 prediction of t h e 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  88 95 95 95 97 99 100 102 104 104 109 115 119 122  128 132 132 132 137 142 147 150 152 153  CHAPTER 5 5.0  CONCLUSIONS  BIBLIOGRAPHY APPENDICES  154 158 177  viii L I S T OF  TABLES  Table  Page  1.  Composition of c o n t r o l (g/kg d r y m a t t e r )  2.  3.  4.  and p r o t e i n - f r e e  diets 45  P r o x i m a t e c o m p o s i t i o n , e n e r g y c o n t e n t and m i n e r a l c o n t e n t 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 o f Experiment 1  56  R e c o v e r y o f c h r o m i c o x i d e f r o m a f e e d mash m i x t u r e u s i n g t h e wet a s h i n g c o l o r i m e t r i c method o f S t e v e n s o n and De L a n g e n (1960)  57  C o m p a r i s o n o f t h e t h r e e week mean a p p a r 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 v a l u e s d e t e r m i n e d by d i r e c t and i n d i r e c t measurement u s i n g t h e " G u e l p h s y s t e m " o f 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 f e c e s 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 t e c h n i q u e s  5.  6.  61  R e c o v e r y r a t e o f c h r o m i c o x i d e ( 0 ^ 0 3 ) from i n g e s t a and e x c r e t a a f t e r a t h r e e - w e e k (replicates 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  Three-week mean a p p a r 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 o r g a n i c m a t t e r , c r u d e p r o t e i n and g r o s s e n e r g y by j u v e n i l e c h i n o o k salmon i n s e a w a t e r f e d t h e 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 t i m e between c o l l e c t i o n s  65  7.  E f f e c t o f f i s h management ( f e e d i n g r e g i m e and f i s h h a n d l i n g ) on mean a p p a r e n t o r g a n i c m a t t e r , c r u d e p r o t e i n and g r o s s e n e r g y 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 d a t a f r o m f e c a l samples c o l l e c t e d i n t h e m o r n i n g and i n t h e a f t e r n o o n 67  8.  E f f e c t o f f i s h management on f e e d i n t a k e , s p e c i f i c g r o w t h r a t e and m o r t a l i t y o f j u v e n i l e c h i n o o k salmon r e a r e d i n s a l t w a t e r and f e d the c o n t r o l d i e t  68  R e f e r e n c e and t e s t d i e t p r e p a r a t i o n e m p l o y e d i n e x p e r i m e n t s I I and I I I  protocol 91  R e f e r e n c e and t e s t d i e t p r e p a r a t i o n employed i n e x p e r i m e n t IV  protocol  9. 10. 11. 1  F o r m u l a t i o n o f b a s a l mix 1 u s e d i n t h e p r e p a r a t i o n o f t h e r e f e r e n c e and t e s t d i e t s employed i n e x p e r i m e n t s I I and I I I  92  93  ix LIST  OF  TABLES c o n t  Table 12.  13.  14.  15.  16.  17.  18  19  20  Page F o r m u l a t i o n o f b a s a l mix 2 u s e d i n t h e p r e p a r a t i o n o f t h e r e f e r e n c e and t e s t d i e t s e m p l o y e d i n e x p e r i m e n t IV  94  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 e n e r g y c o n t e n t o f t e s t i n g r e d i e n t s used i n experiment II ( v a l u e s i n p a r e n t h e s e s show p e r c e n t a g e s f o r e a c h p r o x i m a t e c o n s t i t u e n t on a d r y m a t t e r b a s i s )  106  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 e n e r g y c o n t e n t of t e s t i n g r e d i e n t s used i n experiment III ( v a l u e s i n p a r e n t h e s e s show p e r c e n t a g e s f o r e a c h p r o x i m a t e c o n s t i t u e n t on a d r y m a t t e r b a s i s )  107  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 e n e r g y c o n t e n t o f t e s t i n g r e d i e n t s u s e d i n e x p e r i m e n t IV ( v a l u e s i n p a r e n t h e s e s show p e r c e n t a g e s f o r e a c h p r o x i m a t e c o n s t i t u e n t on a d r y m a t t e r b a s i s )  109  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 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 c h i n o o k salmon i n experiment I I . 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 e n c e d i e t a)  content of the to juvenile (Refer to Table refers to  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 e i g h t 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 I I I . (Refer t o T a b l e 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) 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 s e v e n 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 s a l m o n i n e x p e r i m e n t IV. ( R e f e r t o T a b l e 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)  13 110  14 I l l  15 113  Mean p e r c e n t (± SEM) a p p a r e n t o r g a n i c m a t t e r , c r u d e p r o t e i n and g r o s s e n e r g y 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 e n e r g y v a l u e s o f i n g r e d i e n t s m e a s u r e d w i t h p o s t - j u v e n i l e c h i n o o k salmon i n seawater  117  E f f e c t o f f e c a l c o l l e c t i o n t e c h n i q u e on t h e a p p a r e n t 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 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 c h i n o o k salmon  120  X  L I S T OF  TABLES c o n t  Table 21  Page Weight 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 grams o f 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 (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 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 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 . ( R e f e r t o T a b l e 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 o f e a c h parameter)  125  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 grams o f 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 (PER), 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 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 ) 12 6  23  Weight 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 grams o f 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 i n t a k e as a p e r c e n t o f body w e i g h t (FI) 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 IV. ( R e f e r t o T a b l e 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 )  24  25  26  feed  127  D e t e r m i n e d and c a l c u l a t e d a p p a r 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 r e f e r e n c e d i e t - a (REF-a) w h i c h was f e d t o c h i n o o k salmon i n e x p e r i m e n t I I  129  D e t e r m i n e d and c a l c u l a t e d a p p a r 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 r e f e r e n c e d i e t - a (REF-a) w h i c h was f e d t o c h i n o o k salmon i n e x p e r i m e n t I I I  130  D e t e r m i n e d and c a l c u l a t e d a p p a r 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 r e f e r e n c e d i e t - b (REF-b) w h i c h was f e d t o c h i n o o k salmon i n e x p e r i m e n t IV  131  xi LIST OF FIGURES Figure 1.  Page Diagram showing t h e "Guelph system" o f f e c a l c o l l e c t i o n used i n e x p e r i m e n t s I , I I , I I I and IV  41  xii ACKNOWLEDGEMENT S I would l i k e t o express my s i n c e r e g r a t i t u d e t o a l l those who h e l p e d me d u r i n g t h e course o f my s t u d i e s a t t h e U n i v e r s i t y of B r i t i s h Columbia. I n p a r t i c u l a r my r e s e a r c h s u p e r v i s o r , Dr. R i c h a r d M. Beames, d e s e r v e s s p e c i a l t h a n k s , n o t o n l y f o r h i s knowledge, encouragement and d e d i c a t e d guidance, b u t a l 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. D a v i d Higgs o f F i s h e r i e s and Oceans, whose u n c e a s i n g e n t h u s i a s m and w i l l i n g n e s s t o 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 t h e s u c c e s s f u l c o m p l e t i o n o f my s t u d i e s . Dr. B e r y l E. March and Dr. A. Kozak a r e 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 h e l p w i t h s t a t i s t i c a l a n a l y s e s . S p e c i a l t h a n k s 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 h e r c o n t i n u o u s support and m o t i v a t i o n throughout my years a t UBC. My a p p r e c i a t i o n a l s o goes t o t h e members o f my s u p e r v i s o r y committee f o r t h e i r c o n t r i b u t i o n t o this thesis. Among t h o s e who p r o v i d e d t e c h n i c a l a s s i s t a n c e , Maureen Evans deserves s p e c i a l mention f o r h e r 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 B a k h s h i s h Dosanjh and J i m E l l i o t t f o r t h e i r h e l p a t t h e West Vancouver L a b o r a t o r y o f F i s h e r i e s and Oceans. I a l s o want t o express my t h a n k s t o my l o n g t i m e f r i e n d A l b e r t Nussbaum, f o r h i s h e l p i n u n r a v e l l i n g t h e m y s t e r i e s o f t h e d i f f e r e n t computer programs. I used t h r o u g h o u t my academic c a r e e r , as w e l l as f o r t h e use o f h i s computer f o r t h e p r e p a r a t i o n o f this thesis. My p a r e n t s have, more t h a n I can say, s u p p o r t e d me b o t h e m o t i o n a l l y and s p i r i t u a l l y t h r o u g h o u t my s c h o l a s t i c endeavors and t o them I e x t e n d my w h o l e h e a r t e d t h a n k f u l n e s s . I d e d i c a t e t h i s t h e s i s t o my l o v i n g w i f e E l i z a b e t h , who has h e l p e d me immeasurably w i t h h e r u n d e r s t a n d i n g , support and encouragement, and who p a t i e n t l y endured many b o r i n g weekends w h i l e I was consumed i n my s t u d i e s . F o r h e r I have t h e g r e a t e s t a d m i r a t i o n , and t o h e r I owe my g r e a t e s t 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 t h e N a t i o n a l S c i e n c e s and E n g i n e e r i n g Research C o u n c i l o f Canada, t h e S c i e n c e C o u n c i l o f B r i t i s h Columbia and t h e Department o f 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 t h e p r o t o t y p e 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  i m p o r t a n t d e v e l o p i n g i n d u s t r y and the amount o f investment aquaculture i s r i s i n g d a i l y .  in  P r o d u c t i o n o f farmed salmon has  d r a m a t i c a l l y i n c r e a s e d from 120 tonnes marketed i n 1985,  t o more  than 12,300 tonnes i n 1989 w i t h a w h o l e s a l e v a l u e o f $85  million  (Egan and Kenney, 1990).  By the end o f 1990,  the  commercially  r e a r e d P a c i f i c salmon h a r v e s t s are e x p e c t e d t o r e a c h 15,000 tonnes a c c o r d i n g t o the B r i t i s h Columbia Salmon Farmers Association  (BCSFA) ( A r c h i b a l d , 1990;  Egan and Kenney, 1990) .  T h i s p r o j e c t i o n i s 25% below t h e p r e v i o u s l y f o r e c a s t e d 20,000 tonnes by t h e BCSFA and was p r i m a r i l y due t o a d e c l i n e i n the w o r l d market p r i c e o f salmon c a u s i n g v a r i o u s companies w i t h massive debt l o a d s t o go i n t o r e c e i v e r s h i p . The g e n e r a l t r e n d i n the p a s t year has t h e r e f o r e been a decrease  i n t h e number o f  companies, a l t h o u g h t h e 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 c o n t i n u e t o expand t h e i r o p e r a t i o n s w h i l e at t h e same time look, f o r ways t o t r i m t h e i r p r o d u c t i o n c o s t s . study conducted  i n 1988  they  According to a  by the B r i t i s h Columbia M i n i s t r y o f  A g r i c u l t u r e and F i s h e r i e s  (BCMAF), f o o d c o s t s a l o n e account  over 41% o f t h e t o t a l o p e r a t i n g expense o f a r e l a t i v e l y  for  efficient  P a c i f i c salmon grow-out o p e r a t i o n w i t h an annual p r o d u c t i o n o f 240 tonnes o f d r e s s e d salmon. the f e e d c o s t s account  In s m a l l s c a l e farm o p e r a t i o n s ,  f o r an even l a r g e r p r o p o r t i o n o f t h e t o t a l  o p e r a t i n g c o s t s , w i t h e s t i m a t e s b e i n g as l a r g e as 55% t o (Higgs, 1986) .  I t i s t h e r e f o r e o f c r i t i c a l importance  f e e d c o s t s be reduced  60%  that f i s h  so t h a t salmon farmers can r e a l i z e t h e most  2 profit for  for t h e i r labor  and  investment.  P a c i f i c salmon i n seawater are  Current  commercial  f o r m u l a t e d on  the  knowledge o f t h e i r n u t r i e n t  requirements during  stages of t h e i r l i f e  coupled with n u t r i t i o n a l  cycle,  o b t a i n e d w i t h A t l a n t i c salmon feedstuff  digestibility  rainbow t r o u t  (Salmo  information  (Oncorhynchus  indicated  digestibility  among s p e c i e s  Jobling, 1974;  1983)  suspect of  cost  et  the  25%  to  supply  importantly identified fish  as  1989).  The  high cost  Savings as  much as  As  Moreover,  1979; cost  possible  of  The  work  first  task  i s to  i n seeking ascertain  et  of  et  al.,  al.,  1984;  fish  growth  commercial  Jackson,  available To  feedstuffs be  of  any  ( H i l t o n and  fish  value,  and  diets  1985).  The  feed  of t h i s product  1982;  McCallum  can  be  f i s h meal w i t h  efficient  digestibility and  Slinger,  are  p e r h a p s more  Pfeffer,  components, p r i n c i p a l l y p r o t e i n  can  i t , have b e e n  a more c o s t  the  of  1969;  a r e s u l t , one  utilization  of the  on  P a c i f i c salmon  f i s h m e a l , and  al.,  i n the  Lall  support  with  with  (Zeitoun  for salt-water  associated  et  as  (Birkett,  1990).  (Tacon and  q u a l i t y of  information  feedstuff  1980;  to  water  less  energy  1981;  diet  of the  Cho  in et  t h e s e measurements s h o u l d  and  effected  feedstuffs.  culture  1982).  al.,  1986).  f i s h meal  (Spinelli  fish  Singh,  major drawbacks i n the  feeds  expensive  dietary  diets  (Higgs,  and  the  replacing  fish  65%  well  w i t h water s a l i n i t y  Usher et  present  of  of  fresh  from s t u d i e s  however, t h a t  Pandey and  1986;  effectiveness  Higgs,  as  basis  i n f r e s h water.  optimum q u a l i t y w i t h r e s p e c t  variable  by  well  1977;  al.,  that  contain  in  as  MacLeod,  Ferraris  not  varies  as  derived  mykiss)  numerous a u t h o r s has  salar)  the  diets  for  major locally al., be  conducted  u s i n g the t a r g e t s p e c i e s 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. (Oncorhynchus  tshawytscha)  I n B r i t i s h Columbia, chinook  salmon  has, over the y e a r s been t h e dominant  s p e c i e s farmed, p r e s e n t l y c o m p r i s i n g an e s t i m a t e d 77% o f the t o t a l production  (Egan and Kenney, 1990) .  e a r l y m a t u r a t i o n , coho salmon (Oncorhynchus decreased  Due  t o poor growth and  kisutch)  have  i n p o p u l a r i t y , w i t h p r o d u c t i o n d e c l i n i n g s t e a d i l y from  76% i n 1986,  t o an e x p e c t e d 7% i n 1990  (Egan and Kenney, 1990).  T h e r e f o r e , an improvement i n the economics o f chinook p r o d u c t i o n would have the g r e a t e s t impact on t h e  salmon  local  aquaculture i n d u s t r y . T h i s t h e s i s was  designed to e s t a b l i s h  digestibility  i n f o r m a t i o n f o r an a r r a y o f c o n v e n t i o n a l and h o v e l f e e d s t u f f s which c o u l d p o t e n t i a l l y p a r t i a l l y  or w h o l l y r e p l a c e f i s h meal i n  d i e t s f o r chinook salmon h e l d i n seawater. d i g e s t i b i l i t y o f v a r i o u s f i s h meals was  In a d d i t i o n ,  determined  the  to f a c i l i t a t e  the s u b s t i t u t i o n o f one meal f o r another, depending on market t r e n d s and a v a i l a b i l i t y .  T h i s 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 digestibility indicator).  of determining n u t r i e n t  i n f i s h by the i n d i r e c t method (chromic  In a d d i t i o n , the r o u t i n e methodology o f c o l l e c t i n g  f e c e s from t h e water, developed Slinger  oxide  (1979), was  f o r rainbow t r o u t by Cho  and  compared w i t h t h e i n t e s t i n a l d i s s e c t i o n  the s t r i p p i n g t e c h n i q u e s , which r e l y on c o l l e c t i n g the f e c e s d i r e c t l y from t h e  fish.  and  4  The f o l l o w i n g l i t e r a t u r e r e v i e w i s d e s i g n e d t o p r o v i d e background  knowledge on t h e g e n e r a l n u t r i t i o n a l r e q u i r e m e n t s o f  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 a b s o r p t i o n o f n u t r i e n t s and t h e fundamental  problems  a s s o c i a t e d w i t h t h e v a r i o u s systems commonly used i n t h e 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 h i g h p e r c e n t a g e o f d i e t a r y  p r o t e i n compared t o o t h e r - a n i m a l s and Cowey, 1985). efficient 1981).  (Cowey and Sargent, 1979;  Tacon  A l s o , i t has been suggested t h a t f i s h are l e s s  than other animals i n p r o t e i n u t i l i z a t i o n  (Rumsey,  More r e c e n t l y , however, Bowen (1987) compared t h e p r o t e i n  requirements of v a r i o u s 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 , a l t h o u g h t h e two a n i m a l groups undoubtedly d i f f e r i n t h e c o n c e n t r a t i o n o f p r o t e i n i n t h e d i e t , they are remarkably s i m i l a r i n t h e use o f p r o t e i n f o r growth. T h i s r e f l e c t s t h e homeotherms' g r e a t requirement f o r energy t o maintain homeostasis. 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 are l a r g e l y a f f e c t e d by environmental conditions.  DeLong et al.  (1958) o b t a i n e d maximum  g a i n s i n 1.5 t o 5.6 gram chinook salmon r e a r e d i n 6-8®C f r e s h water when t h e 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 o f 14.5°C, maximum g a i n s were observed at 5560% d i e t a r y p r o t e i n .  S i m i l a r o b s e r v a t i o n s on t h e e f f e c t o f water  temperature on p r o t e i n r e q u i r e m e n t s f o r maximum growth were n o t e d by H a s t i n g s (1973) w o r k i n g w i t h channel c a t f i s h punctatus).  Z e i t o u n e t al.  (Ictalurus  (1973) on t h e o t h e r hand, found t h a t  water s a l i n i t y a l s o a f f e c t s t h e p r o t e i n r e q u i r e m e n t s o f young rainbow t r o u t .  At a s a l i n i t y o f 10 p p t , t h e o p t i m a l d i e t a r y  p r o t e i n l e v e l was  40%, w h i l e at a s a l i n i t y o f 20 ppt i t was  More r e c e n t l y , A r c h d e k i n e t al.  45%.  (1988) d e t e r m i n e d t h a t p o s t -  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 o f 44%  6 p r o t e i n i n t h e i r d i e t t o a t t a i n maximum growth.  An i n c r e a s e i n  d i e t a r y p r o t e i n requirement a t h i g h e r 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 , t o a decrease i n i t s d i g e s t i b i l i t y . MacLeod (1977), F e r r a r i s et al.  Work by  (1986) and Usher e t a l . (1990),  would seem t o support such a h y p o t h e s i s a l t h o u g h r e s u l t s o b t a i n e d by L a l l et al. 1990)  (1984) and L a l l  (1988, c i t e d by Higgs et a l . ,  w o r k i n g 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 r e q u i r e m e n t s are r e l a t e d t o  the f i s h i t s e l f .  Page and Andrews (1973) w o r k i n g w i t h c h a n n e l  c a t f i s h and S a t i a  (1974) w o r k i n g w i t h rainbow t r o u t ,  concluded  t h a t d i e t a r y p r o t e i n r e q u i r e m e n t s are i n v e r s e l y c o r r e l a t e d w i t h fish  size.  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 F i s h , l i k e o t h e r a n i m a l s , do not have a t r u e p r o t e i n  requirement per  se  ( W i l s o n , 1985) but r e q u i r e a w e l l b a l a n c e d  m i x t u r e o f e s s e n t i a l amino a c i d s and adequate e s s e n t i a l amino a c i d s .  l e v e l s o f non-  F i s h r e q u i r e t h e same t e n e s s e n t i a l amino  a c i d s 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  c o u n t e r p a r t s , namely,  threonine, v a l i n e , methionine, i s o l e u c i n e ,  leucine,  p h e n y l a l a n i n e , l y s i n e , h i s t i d i n e , a r g i n i n e and t r y p t o p h a n .  Fish  can r e a d i l y c o n v e r t p h e n y l a l a n i n e t o t y r o s i n e and t h e y 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 m e t a b o l i c needs f o r t h i s amino acid.  C y s t i n e i s c o n s i d e r e d n o n e s s e n t i a l because i t can be  s y n t h e s i z e d by t h e f i s h from t h e e s s e n t i a l amino a c i d m e t h i o n i n e . The p r e s e n c e o f c y s t i n e i n t h e d i e t has t h e r e f o r e , a s p a r i n g e f f e c t on d i e t a r y m e t h i o n i n e r e q u i r e m e n t s . Improved growth and f e e d c o n v e r s i o n have been r e p o r t e d by s e v e r a l workers when e x p e r i m e n t a l 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 a c i d s t o b r i n g t o t a l l e v e l s c l o s e t o t h e 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 or whole body t i s s u e of the s p e c i e s b e i n g s t u d i e d K e t o l a , 1975; Wilson  A r a i , 1981;  and Cowey, (1985),  K e t o l a , 1982;  1983).  a n a l y z e d t h e amino a c i d c o m p o s i t i o n  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 t h a t t h e amino a c i d c o m p o s i t i o n  masou) and c h a n n e l c a t f i s h .  of  found t h a t t h e r e  species.  of t h e s e t i s s u e s was  t o t h a t of t i s s u e from coho salmon, c h e r r y salmon  was  They showed also similar (Oncorhynchus  They suggested t h a t d i e t s f o r f i s h  be improved by f o r m u l a t i n g them t o s i m u l a t e t h e amino a c i d  b a l a n c e of t h e whole body t i s s u e of t h e f i s h b e i n g  2.3  (Rumsey and  Ogata et al.,  rainbow t r o u t and A t l a n t i c salmon t i s s u e and  may  egg  studied.  Energy b a l a n c e i n f i s h F i s h . a r e among t h e most e f f i c i e n t a n i m a l s i n c o n v e r t i n g  energy i n t o body p r o t e i n (Smith et al.,  1978a) and  several  factors contribute to t h e i r high energetic e f f i c i e n c y . i n c o n t r a s t t o warm-blooded a n i m a l s , and c o n s e q u e n t l y do not  feed  Firstly,  f i s h are a q u a t i c ectotherms  have t o expend a l a r g e p r o p o r t i o n  energy i n m a i n t a i n i n g body t e m p e r a t u r e .  Secondly, f i s h  of  are  n e u t r a l l y buoyant and t h u s 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 a q u a t i c environment.  F i n a l l y , f i s h have a v e r y  e f f i c i e n t mechanism f o r t h e e x c e t i o n o f waste n i t r o g e n .  The  p r i m a r y waste p r o d u c t of p r o t e i n c a t a b o l i s m i n a l l a n i m a l s i s ammonia which p o s s e s s e s no b i o l o g i c a l energy v a l u e but i s t o x i c even at v e r y low c o n c e n t r a t i o n s or c o n v e r t e d  and t h u s must be r a p i d l y e x c r e t e d  t o l e s s t o x i c compounds.  at c o n s i d e r a b l e energy c o s t , c o n v e r t  T e r r e s t r i a l homeotherms, ammonia t o u r e a and  uric  8 acid  which,  i n a d d i t i o n , must be c o n c e n t r a t e d and e x c r e t e d by  the kidneys.  F i s h , on t h e o t h e r hand, e x c r e t e a p p r o x i m a t e l y 85%  o f t h e waste n i t r o g e n as ammonia (Wood, 1958; R a n d a l l and Wright, 1987)  w h i l e t h e remainder i s e x c r e t e d as u r e a , u r i c  t r i m e t h y l a m i n e o x i d e , c r e a t i n i n e and c r e a t i n 1974).  acid,  (Watts and Watts,  Ammonia i s e x c r e t e d by p a s s i v e d i f f u s i o n , from b l o o d t o  the s u r r o u n d i n g water, v i a t h e g i l l s , w i t h l i t t l e o r no energy expenditure  ( G o l d s t e i n and F o r s t e r , 1970).  requirement  p e r u n i t p r o t e i n g a i n i s , t h e r e f o r e , lower w i t h  than w i t h l a n d animals 1985;  Smith, 1989).  The energy  (Smith e t al., 1978a; Cho and Kaushik,  Most d o m e s t i c a t e d  h e r b i v o r o u s o r omnivorous.  l a n d animals a r e , however,  By c o n t r a s t , f i s h f a r m i n g i n c o l d  c l i m a t e s has c o n c e n t r a t e d on r e a r i n g m o s t l y c a r n i v o r o u s species.  fish  fish  Given t h a t food p r o t e i n i s e x p e n s i v e , t h i s has r e q u i r e d  a f o c u s on esteemed s p e c i e s o f f i s h such as s a l m o n i d s , which have a h i g h market v a l u e . Phillips  (1969), R i n g r o s e  (1971), Lee and Putnam  (1973),  Takeda e t al. (1975) and Cowey e t al. (1975) have r e p o r t e d  that  the n o n - p r o t e i n "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 t h e u t i l i z a t i o n o f d i e t a r y p r o t e i n by  means o f a p r o t e i n s p a r i n g e f f e c t ,  s i n c e f i s h , i n common w i t h  o t h e r a n i m a l s , g e n e r a l l y e a t t o s a t i s f y t h e i r energy requirements.  An o p t i m a l d i e t a r y p r o t e i n c o n c e n t r a t i o n f o r f i s h  i s t h u s d i c t a t e d by a d e l i c a t e b a l a n c e o f t h e d i e t a r y p r o t e i n t o energy r a t i o .  E x c e s s i v e n o n p r o t e i n energy i n t a k e r e s u l t i n g from  h i g h d i g e s t i b l e e n e r g y - t o - d i e t a r y p r o t e i n r a t i o s , causes a p p e t i t e or demand t o be s a t i s f i e d b e f o r e a s u f f i c i e n t q u a n t i t y o f p r o t e i n (and p o s s i b l y o t h e r n u t r i e n t s ) i s i n g e s t e d t o s a t i s f y demand f o r  9 maximal r a t e s o f growth  (Page and Andrews, 1973).  In addition,  e x c e s s i v e l y h i g h energy t o p r o t e i n r a t i o s can l e a d t o d e p o s i t i o n of l a r g e amounts o f body f a t . C o n v e r s e l y , when a d i e t i s d e f i c i e n t i n n o n p r o t e i n energy, a l a r g e r f r a c t i o n o f t h e p r o t e i n w i l l be used f o r e n e r g e t i c p u r p o s e s , thus r e s u l t i n g i n a d e c r e a s e i n t h e 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 s y n t h e s i s and growth (Cowey, 1979).  2.4  Sources o f energy f o r f i s h As i n t h e case o f warm-blooded a n i m a l s , c a r b o h y d r a t e s ,  l i p i d s and p r o t e i n s a r e t h e t h r e e p r i m a r y energy s o u r c e s f o r fish.  2.4.1  P r o t e i n as d i e t a r y energy s o u r c e P r o t e i n s a r e r e a d i l y u s a b l e s o u r c e s o f energy f o r s a l m o n i d s .  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 o t h e r s o u r c e s ( c a r b o h y d r a t e s and l i p i d ) ,  protein  i s f e d 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  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 In r e c e n t y e a r s , much work has been d e v o t e d t o t h e  e v a l u a t i o n o f t h e p r o t e i n - s p a r i n g e f f e c t s o f n o n - p r o t e i n energyyielding nutrients i n fish diets.  W h i l e i t i s unanimously agreed  t h a t t h e a d d i t i o n o f 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 t h e d i g e s t i b l e energy o f t h e d i e t s  (Watanabe e t al.,  1979; Cho and K a u s h i k , 1985), some c o n t r o v e r s y p e r s i s t s r e g a r d i n g the  u t i l i z a t i o n o f d i e t a r y c a r b o h y d r a t e s by c a r n i v o r o u s f i s h such  10 as t h e s a l m o n i d s .  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 b o t h i n d i g e s t i v e and m e t a b o l i c capacity. As c a r n i v o r o u s f i s h , salmonids are i l l - a d a p t e d t o d i g e s t complex c a r b o h y d r a t e s .  D i g e s t i o n c o e f f i c i e n t s o f l e s s than  f o r raw s t a r c h have been r e p o r t e d i n t h e l i t e r a t u r e al.,  1963;  S i n g h and Nose, 1967;  and Breque, 1983).  (Inaba et  Spannhof and Kiihne, 1977;  Furthermore,  carbohydrate  Spannhof and Kiihne, 1977;  1983; K i r c h g e s s n e r et al.,  1986).  gelatinization.  f a c t o r h a v i n g a major  i s t h e degree o f s t a r c h  F o r t h i s reason, any p h y s i c a l t r e a t m e n t t h a t  might decrease t h e c o m p l e x i t y o f t h e d i e t a r y s t a r c h c o o k i n g , e x t r u s i o n ) d r a m a t i c a l l y improves these f i s h  (Singh and  Spannhof and P l a n t i k o w ,  One  e f f e c t on c a r b o h y d r a t e d i g e s t i b i l i t y  Bergot  digestibility  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 Nose, 1967;  40%  (Inaba et al.,  1963;  1976; Bergot and Breque, 1983).  (e.g.  i t s availability  to  Smith, 1971; Luquet and B e r g o t , The a b i l i t y o f c a r n i v o r o u s f i s h  t o m e t a b o l i z e g l u c o s e , t h e end p r o d u c t o f 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 .  Still,  Kaushik and O l i v a - T e l e s ,  (1985)  have r e c e n t l y shown t h a t i n c o r p o r a t i o n o f 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 o f rainbow t r o u t can l e a d t o an i n c r e a s e 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 d e c r e a s i n g t h e n i t r o g e n o u s m e t a b o l i c 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 d e a m i n a t i o n f o r energy production. The t o t a l c a r b o h y d r a t e f r a c t i o n c o n s i s t s p r e d o m i n a n t l y o f s t a r c h i n v e r y few v e g e t a b l e f e e d s . i n c l u d i n g c e l l u l o s e , pentosane, b e l o n g i n t h i s group.  The c e l l w a l l  fractions  h e m i c e l l u l o s e , and l i g n i n  also  These f r a c t i o n s are m o s t l y 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 s m a l l c a p a c i t y o f t h e  d i g e s t i v e system, t h e s h o r t food r e t e n t i o n time and t h e low body temperature  of p o i k i l o t h e r m i c carnivorous f i s h , allow l i t t l e  o p p o r t u n i t y f o r t h e m i c r o b i a l enzymatic a c t i o n which i s n e c e s s a r y to u t i l i z e these non-starch carbohydrates.  The i n c l u s i o n o f  f i b r e i n t h e d i e t s o f m o n o g a s t r i c animals has a l s o been shown t o i n c r e a s e t h e s l o u g h i n g o f i n t e s t i n a l mucosal c e l l s al.,  1975; Beames and Eggum, 1981)  production  (Schneeman e t a l . ,  (Bergner e t  and t o enhance mucus  1982), t h e r e b y l e a d i n g t o i n c r e a s e d  l o s s e s o f endogenous amino a c i d s .  F i b r e i s a l s o capable of  a d s o r b i n g amino a c i d s and p e p t i d e s and w i t h h o l d i n g t h e s e  from  a b s o r p t i o n , t h e e x t e n t o f which depends on t h e degree o f l i g n i f i c a t i o n of the f i b r e 1984).  (Bergner et al.,  1975; M i t a r u et  al.,  For these reasons, the f i b r e content f o r salmonid d i e t s  s h o u l d not exceed 4 p e r c e n t , a c c o r d i n g t o t h e N a t i o n a l Research Council  (NRC,  1978).  No a d a p t i v e r e g u l a t i o n o f t h e s t r u c t u r e and f u n c t i o n o f t h e d i g e s t i v e system seems t o o p e r a t e i n response t o t h e l o n g term feeding of c a r b o h y d r a t e - r i c h 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 t o decrease w i t h t i m e o f exposure. The r e s u l t s o f Buddington et al.  and H i l t o n  (1987) and  (1989) showed t h a t b o t h d i s a c c h a r i d a s e a c t i v i t y  Kaushik  and  g l u c o s e uptake r a t e were c o n s i d e r a b l y reduced i n rainbow  trout  a f t e r 30 weeks on a h i g h l e v e l o f d i e t a r y g l u c o s e . V a r i o u s s t u d i e s have i n d i c a t e d t h a t 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 c a r b o h y d r a t e l e v e l s r a n g i n g from 30% H a l v e r , 1961)  t o as h i g h as 38%  (Kaushik et al.,  ( B u h l e r and 1989), may  used e f f i c i e n t l y by s a l m o n i d s . Other a u t h o r s however, have  be  r e p o r t e d t h a t d i g e s t i b l e c a r b o h y d r a t e l e v e l s s h o u l d not 14% o f t h e d i e t  exceed  ( H i l t o n and A t k i n s o n , 1982; H i l t o n e t 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 t o d i f f e r e n c e s  i n t h e age and s i z e o f 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 t h e form o f c a r b o h y d r a t e used by t h e d i f f e r e n t a u t h o r s . Absorbed g l u c o s e a l s o appears t o be v e r y p o o r l y u t i l i z e d  by  c a r n i v o r o u s f i s h as e v i d e n c e d by p r o l o n g e d h y p e r g l y c e m i a accompanied by enlargement absorption  o f t h e l i v e r at h i g h l e v e l s o f  (Palmer and Ryman, 1972; B e r g o t , 1979; F u r u i c h i  Yone, 1981,  1982).  H i l t o n and S l i n g e r  and  (1981) s t a t e d t h a t  e x c e s s i v e amounts o f c a r b o h y d r a t e i n t h e s a l m o n i d d i e t  depress  growth r a t e , i n c r e a s e m o r t a l i t y and produce an a b n o r m a l l y h i g h g l y c o g e n c o n t e n t o f t h e l i v e r , e s p e c i a l l y at low t e m p e r a t u r e s .  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 i m p o r t a n t r o l e as an energy s o u r c e i n f i s h d i e t s , e s p e c i a l l y f o r c a r n i v o r o u s f i s h i n which carbohydrate i s p o o r l y u t i l i z e d .  dietary  L i p i d s c o n t a i n over 1.5  as much g r o s s energy p e r u n i t weight r e l a t i v e t o p r o t e i n s  times and  n e a r l y 2.5 t i m e s as much as c a r b o h y d r a t e s ( L e h n i n g e r , 1982). S e v e r a l s t u d i e s have shown t h a t t h e p r o v i s i o n o f adequate  levels  of d i e t a r y l i p i d s can m i n i m i z e t h e use o f c o s t l y p r o t e i n as an energy source  (Fowler et al.,  1977; Takeuchi et al., 1982).  1966; R i n g r o s e , 1971; Watanabe,,  1978; Cowey and Sargent, 1979; Watanabe,  A l t h o u g h t h e q u a n t i t y o f l i p i d i n salmon d i e t s i s  i m p o r t a n t as a p r o t e i n - s p a r i n g energy s o u r c e , t h e q u a l i t y o f t h e l i p i d i s o f even g r e a t e r i m p o r t a n c e .  C o l d 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 s y n t h e s i z e f a t t y a c i d s o f the l i n o l e i c linolenic families. diet  Therefore,  (Yu and Sinnhuber, 1972;  and  t h e s e must be s u p p l i e d i n the  Watanabe et a l . , 1974;  Watanabe,  1982). T a k e u c h i et al.  (1979),  d e t e r m i n e d the d i g e s t i b i l i t y of b e e f  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 p o i n t s w i t h common c a r p  (Cyprinus  carpio)  melting  and rainbow t r o u t  found an i n v e r s e r e l a t i o n s h i p between m e l t i n g p o i n t  and  (which i s a  f u n c t i o n o f t h e degree o f s a t u r a t i o n or l e n g t h of t h e carbon chain)  and d i g e s t i b i l i t y .  These a u t h o r s a l s o c o n c l u d e d t h a t i n  g e n e r a l , 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 d i g e s t i b i l i t y and  fish  lipid  size.  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 o f 15% t o 20% o f dry m a t t e r 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 r a t e s f o r a v a r i e t y of species 1973;  ( S t i c k n e y and Andrews, 1972;  H i l t o n and S l i n g e r , 1981;  NRC,  1981;  Lee and Putnam,  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 h i g h e r l e v e l s o f l i p i d t h a n t h i s range have l e d t o remarkable i n c r e a s e s i n v i s c e r a l f a t ( S a t i a , Ogino et al., Buckley,  1980;  1976;  Takeuchi et al.,  Boggio et al.,  1978;  1985).  The  Castledine  1974;  and  d i f f i c u l t y of d e f i n i n g  an o p t i m a l b a l a n c e of energy components stems from t h e f a c t t h a t although  d i e t a r y p r o t e i n l e v e l s can be reduced w h i l e d i e t a r y  l i p i d l e v e l s are i n c r e a s e d , w i t h o u t  l o w e r i n g the food  conversion  r a t e or growth r a t e , t h e s e changes w i l l be at t h e expense of i n c r e a s i n g l y f a t t y c a r c a s s , which may acceptability.  a f f e c t market  an  14  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 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 o f salmonid  d i e t s can be c a t e g o r i z e d as f i s h e r y p r o d u c t s  (e.g. h e r r i n g meal,  anchovy m e a l ) , b y - p r o d u c t s o f t h e a n i m a l s l a u g h t e r i n d u s t r y (e.g. meat and bone meal, p o u l t r y b y - p r o d u c t meal, f e a t h e r meal, meal) o r b y - p r o d u c t s o f t h e p r o c e s s i n g  and m i l l i n g i n d u s t r i e s  (e.g. soybean meal, c a n o l a meal, wheat m i d d l i n g s ) . are f o r m u l a t e d ,  blood  When d i e t s  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 a n o t h e r depending upon t h e i r market p r i c e , availability  and n u t r i t i v e v a l u e .  I n 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 t h e e s s e n t i a l n u t r i e n t c o n t e n t and b a l a n c e o f t h e 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 o f t h e d i e t i s a l s o dependent on t h e s e l e c t i o n o f the i n g r e d i e n t s . unpalatable  Thus, i n c l u s i o n o f a  highly d i g e s t i b l e yet  i n g r e d i e n t can reduce f e e d 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 t h e f e e d s t u f f s as a f f e c t e d by s o u r c e , season of t h e y e a r and p r o c e s s i n g methods.  The lower t h e v a r i a b i l i t y o f  the p r o d u c t , t h e more r e l i a n c e can be p l a c e d on i t s q u a l i t y . I n summary, s e l e c t i o n o f i n g r e d i e n t s based on c o m p o s i t i o n ,  nutrient  digestibility,  acceptability, availability  considerations  i n t h e manufacture o f q u a l i t y f e e d f o r f i s h .  2.5.1  and p r i c e a r e c r i t i c a l  Fishery 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  p r o c e s s e d i s t h e h i g h e s t 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 h i g h l e v e l s o f p r o t e i n , a h i g h energy v a l u e 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 e t a l . , 1980; P f e f f e r , 1982; M i l l e r and De Boer, Furthermore,  1988).  t h e e s s e n t i a l amino a c i d c o n t e n t o f commonly used  f i s h meals c l o s e l y resembles t h e d i e t a r y requirement o f f i s h  such  as rainbow t r o u t and c a r p (Higgs e t 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 a t p r e s e n t , commercial  d i e t s formulated f o r  s a l m o n i d c u l t u r e c o n t a i n 25% t o 65% f i s h meal (Tacon and J a c k s o n , 1985), w i t h t h e h i g h e r l e v e l s b e i n g used f o r s t a r t e r and fingerling diets  (McCallum and Higgs, 1989) .  The v a r i a b l e s u p p l y  and q u a l i t y o f f i s h meal, and perhaps more i m p o r t a n t l y t h e h i g h c o s t , even o f p o o r e r q u a l i t y meals, have been i d e n t i f i e d as t h e major drawbacks o f u t i l i z i n g t h i s p r o d u c t i n f i s h feeds  (Spinelli  et al., 1979; Higgs e t 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 t o r e p l a c e some, i f not a l l , o f t h e f i s h meal w i t h o t h e r a n i m a l o r p l a n t p r o t e i n sources.  2.5.2  Animal  by-products  The p r o d u c t i o n o f food f o r human consumption,  whether o f  animal or o f 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 p r o c e s s i n g of raw m a t e r i a l s , w i t h consequent  production of by-products.  most common method o f p r e s e r v a t i o n o f t h e by-product  The  i s drying,  but t h e h i g h i n i t i a l m o i s t u r e c o n t e n t i n e v i t a b l y means t h a t t h i s i s an e x p e n s i v e p r o c e s s .  The m a j o r i t y o f a n i m a l b y - p r o d u c t s have  a h i g h e r c o n t e n t o f d i g e s t i b l e p r o t e i n and energy t h a n p l a n t byproducts  (NRC, 1981).  Compared t o f i s h meal however, t h e  m a j o r i t y o f a l t e r n a t e p r o t e i n s o u r c e s , p l a n t and a n i m a l , a r e  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 o f amino a c i d s .  B l o o d meal f o r i n s t a n c e , 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 t h e  antagonistic  e f f e c t o f 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 a n i m a l s f e d h i g h d i e t a r y l e v e l s of b l o o d meal ( T a y l o r et al., lysine  (NRC,  1977).  On the o t h e r hand, f e a t h e r meal i s low i n  1983), but a m i x t u r e of the two  b a l a n c e d amino a c i d p r o f i l e .  gives a rather w e l l  A d i e t a r y c o m b i n a t i o n of p o u l t r y  b y - p r o d u c t meal and h y d r o l y z e d  f e a t h e r meal has been employed  w i t h s u c c e s s t o r e p l a c e most o f 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 Gropp et al,  1979)  and  (Tiews et al.,  j u v e n i l e coho salmon (Higgs et al.,  In the s t u d i e s on t r o u t by Tiews et al. (1979), a l l o f t h e f i s h meal was  al.  a  f e a t h e r meal supplemented  w i t h t h e e s s e n t i a l amino a c i d s l y s i n e , m e t h i o n i n e and  tryptophan.  Plant by-products Feedstuffs  o f 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  c o n t e n t t h a n t h o s e of a n i m a l o r i g i n . may  1979).  (197 6) and Gropp et  r e p l a c e d s u c c e s s f u l l y by  m i x t u r e of p o u l t r y b y - p r o d u c t meal and  2.5.3  1976;  Although a n a l y t i c a l data  g i v e an adequate amino a c i d p r o f i l e i n a g i v e n p l a n t p r o t e i n  s o u r c e , t h e amino a c i d s may of processing  not be b i o l o g i c a l l y a v a i l a b l e because  l o s s e s 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 with protein u t i l i z a t i o n . s o u r c e s undergo c o n s i d e r a b l e  processing  Most p l a n t p r o t e i n during production.  o f d i f f e r e n t o i l - r i c h seeds, such as soybean, rapeseed, e t c . , a f t e r t h e e x t r a c t i o n of t h e o i l , inexpensive  s o u r c e s of p r o t e i n .  The  are good and  Meals  canola,  relatively  e x t r a c t i o n of o i l from t h e s e  seeds e n t a i l s t h e p r o d u c t i o n o f c o n s i d e r a b l e heat which can be detrimental t o the n u t r i t i v e value of the protein.  Many p l a n t  p r o t e i n s c o n t a i n r e d u c i n g c a r b o h y d r a t e s such as g l u c o s e which may r e a c t w i t h t h e f r e e amino groups o f a p r o t e i n , p a r t i c u l a r l y t h e r e a c t i v e groups o f l y s i n e , a r g i n i n e , h i s t i d i n e and t r y p t o p h a n ( P o t t e r , 1984).  Such carbohydrate-amino a c i d r e a c t i o n s  (Maillard  r e a c t i o n ) r e s u l t i n l i n k a g e s t h a t a r e not h y d r o l y z e d by d i g e s t i v e enzymes, a l t h o u g h t h e amino a c i d s may s t i l l be r e c o v e r e d from t h e p r o t e i n by a c i d h y d r o l y s i s .  Such amino a c i d s a r e 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 t h e y a r e c h e m i c a l l y p r e s e n t .  Nearly a l l  p l a n t p r o t e i n s o u r c e s , 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 t h a t make some a d d i t i o n a l p r o c e s s i n g n e c e s s a r y t o r e n d e r them o f maximum n u t r i t i v e v a l u e .  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 t h e d i g e s t i o n and u t i l i z a t i o n o f many n u t r i e n t s . soybean meal.  One o f t h e most abundant  Soybean c o n t a i n s s e v e r a l  o i l seed meals i s  anti-nutritional  f a c t o r s , t h e main ones b e i n g i n h i b i t o r s o f b o t h t r y p s i n and chymotrypsin.  C o t t o n s e e d meal c o n t a i n s g o s s y p o l which i s t o x i c  to monogastrics.  Rapeseed c o n t a i n s s i g n i f i c a n t amounts o f  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 a r e h y d r o l y z e d i n t o g o i t r o g e n i c s u b s t a n c e s by t h e enzyme myrosinase which i s found i n t h e seed i t s e l f , as w e l l as by t h e m i c r o o r g a n i s m s o f t h e i n t e s t i n a l t r a c t 1980).  (Tookey e t al.,  These g o i t r o g e n i c s u b s t a n c e s have marked n e g a t i v e e f f e c t s  on f e e d i n t a k e and growth and a l s o cause enlargement o f t h e thyroid  (Jones, 1979; Higgs e t al., 1979).  carbon monounsaturated cardiotoxic  E r u c i c a c i d i s a 22-  f a t t y a c i d which has been shown t o be  ( H e n d r i c k s and B a i l e y , 1989).  As a r e s u l t o f t h e s e  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 b r e e d i n g programs as w e l l as i n p r o c e s s i n g p r o c e d u r e s t o e l i m i n a t e o r a t t h e v e r y l e a s t , reduce t h e s e f a c t o r s t o l e v e l s which enable t h e f e e d manufacturer t o u t i l i z e h i g h e r l e v e l s o f 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 o f rapeseed have  been developed which a r e low i n b o t h e r u c i c a c i d and glucosinolates  ( B e l l , 1982).  S t u d i e s on salmonids have i n d i c a t e d  t h a t c a n o l a p r o d u c t s can s a t i s f a c t o r i l y s u p p l y some o f t h e d i e t a r y p r o t e i n and p a r t i a l l y r e p l a c e f i s h meal (Yurkowski e t al.,  1978; Hardy and S u l l i v a n , 1983; Higgs e t al., 1982, 1983).  F u r t h e r p r o c e s s i n g methods have been implemented as w e l l , i n o r d e r t o reduce t h e g l u c o s i n o l a t e c o n t e n t and a l s o t o remove water s o l u b l e n o n - p r o t e i n a c e o u s rapeseed meals.  c o n s t i t u e n t s i n c a n o l a and  I n t h e s e methods, t h e d e h u l l e d seed f r a c t i o n i s  s u b j e c t e d t o a water b o i l i n g t r e a t m e n t , f o l l o w e d by a hexane e x t r a c t i o n a f t e r t h e water has been removed.  The r e s u l t i n g  c a n o l a / r a p e s e e d c o n c e n t r a t e s a r e much lower i n c a r b o h y d r a t e and f i b r e and s i g n i f i c a n t l y h i g h e r i n p r o t e i n c o n t e n t , r e f l e c t i n g h i g h 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 e t al., 1990).  From t h e g r a i n m i l l i n g i n d u s t r y , wheat b r a n and m i d d l i n g s are commonly used i n s a l m o n i d feeds because o f t h e i r as p e l l e t b i n d i n g agents.  However, t h e i r  suitability  nutritional  c o n t r i b u t i o n t o t h e d i e t i s l i m i t e d because o f t h e i r low l e v e l s o f p r o t e i n , l i p i d and energy and h i g h c a r b o h y d r a t e 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 v a l u e o f f e e d i n g r e d i e n t s The o r g a n i c compounds i n the food r e l e a s e energy i n t h e body  upon t h e i r b i o c h e m i c a l o x i d a t i o n .  For s a l m o n i d s , most o f 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 r e q u i r e m e n t s  of the  f i s h u l t i m a t e l y r e s u l t i n a n a b o l i s m and growth, an i n c r e a s e d need f o r maintenance energy can p r e c l u d e 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 c a t a b o l i s m .  In f a c t , i f the  energy p r o v i d e d 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 r e q u i r e m e n t s environmental importance,  factors.  o f a l l animals are i n f l u e n c e d by  In f i s h , temperature  i s of p a r t i c u l a r  owing t o i t s d i r e c t e f f e c t on r a t e o f  metabolism.  The p a r t i c u l a r l i f e stage o f the animal a l s o determines energy needs, w i t h the younger growing  the  animals r e q u i r i n g more  energy p e r u n i t body weight than more mature ones (Winberg, Smith e t al.,  1978b).  1961;  Since f i s h r e g u l a t e 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, t h e c o n c e n t r a t i o n o f the n u t r i e n t s which must be p r o v i d e d i n t h e d i e t t o a d e q u a t e l y meet the a n i m a l ' s r e q u i r e m e n t s ,  t h e r e f o r e , depends upon t h e energy  v a l u e o f t h e d i e t t o the a n i m a l .  A l a r g e number o f p o t e n t i a l  f e e d 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 c o n t e n t can  r e a d i l y be determined  A n a l y s i s of p r o t e i n , l i p i d ,  by s t a n d a r d c h e m i c a l a n a l y s e s .  ash and d e t e r m i n a t i o n o f  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 a p p r o p r i a t e f a c t o r s , g i v e an approximate v a l u e f o r t h e t o t a l o r g r o s s energy content.  will  (GE)  A more p r e c i s e method o f d e t e r m i n i n g g r o s s energy i s t o  20  measure t h e t o t a l energy l i b e r a t e d by t h e 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 . of t h e f e e d s t u f f  A l t h o u g h t h e c h e m i c a l makeup  has a d i r e c t i n f l u e n c e on i t s g r o s s energy  c o n t e n t , 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 r e g a r d i n g t h e availability  o f t h e i n d i v i d u a l n u t r i e n t s and energy t o t h e 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 t h e f e e d s t u f f i s t h e b e s t method f o r measuring t h e u t i l i z a t i o n o f a p a r t i c u l a r f e e d i n g r e d i e n t by t h e a n i m a l .  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 t h e f e e d i n g o f f i s h and t h e a n a l y s i s o f some aspect o f f i s h performance and/or d i e t  digestibility.  Performance s t u d i e s , i n which growth, f e e d e f f i c i e n c y and c a r c a s s n u t r i e n t and energy r e t e n t i o n a r e 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  f o r a l o n g p e r i o d o f t i m e b e f o r e v a l i d comparisons between f e e d s t u f f s can be made w i t h r e s p e c t t o n u t r i t i v e v a l u e .  Studies  i n which t h e v a r i o u s l o s s e s o f i n g e s t e d f o o d v i a f e c e s , u r i n e , and g i l l s a r e measured, y i e l d r e s u l t s sooner, a r e l e s s e x p e n s i v e t o p e r f o r m and t h e y g i v e q u a n t i t a t i v e f i g u r e s on t h e 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 o f t h e i n d i v i d u a l f e e d i n g r e d i e n t s . D u r i n g t h e passage o f f o o d t h r o u g h t h e d i g e s t i v e t r a c t , n o t a l l o f t h e f o o d i s d i g e s t e d and absorbed.  The u n d i g e s t e d p o r t i o n  i s l o s t as f e c e s and t h e 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 t o t h e f i s h i s r e f e r r e d t o as t h e f e c a l energy (FE) l o s s . the  The d i g e s t i b l e energy  (DE) i s t h e d i f f e r e n c e between  g r o s s energy' o f t h e f o o d and t h e g r o s s energy o f t h e f e c e s ,  and t h u s r e p r e s e n t s t h e p a r t o f t h e f o o d energy which i s a c t u a l l y absorbed by t h e f i s h .  Some o f t h e d i g e s 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  urine  (UE)  and ammonia t h r o u g h the g i l l s  (ZE).  These l o s s e s  l a r g e l y t h e r e s u l t o f c a t a b o l i s m o f p r o t e i n s and  are  their  d e a m i n a t i o n when amino a c i d s are u t i l i z e d f o r energy r a t h e r t h a n f o r s y n t h e s i s o f new The (ME)  t i s s u e p r o t e i n (Walton and Cowey, 1982).  energy a s s i m i l a t e d i n the body i s t h e m e t a b o l i z a b l e  energy  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, g i v e s i n f o r m a t i o n r e g a r d i n g the amount of energy which i s effectively utilizable  f o r growth by the f i s h .  ME however, i s much s i m p l e r i n t e r r e s t r i a l l i v i n g i n an a q u a t i c medium. and  f e c a l wastes t o g e t h e r ,  Determination  animals than f o r those  Birds, for instance, void urinary  so t h a t v a l u e s o b t a i n e d  from  c o l l e c t i o n of s o l i d c l o a c a l e x c r e t a are d i r e c t e s t i m a t e s In mammals, measurement o f ME  of  r e q u i r e s the s e p a r a t e  of  ME.  collection  and a n a l y s i s o f f e c e s and u r i n e , and i n t h e case of ruminants t h e gaseous p r o d u c t s o f f e r m e n t a t i o n s e p a r a t i o n and r e c o v e r y  need t o be c o n s i d e r e d  ME  in fish.  of  S i n c e f e c a l energy l o s s i s the major  l o s s o f t h e i n g e s t e d g r o s s energy  (Cho et a l . , 1982), measurement  of DE g i v e s a good i n d i c a t i o n of t h e a v a i l a b i l i t y of d i e t a r y feed i n g r e d i e n t s .  The  of t h e energy  f o r m u l a t i o n 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 u n d e r s t a n d i n g only of the chemical  The  o f 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 determining  also.  and p h y s i c a l c h a r a c t e r i s t i c s o f t h e  i n d i v i d u a l f e e d s t u f f s t o be used, but a l s o of t h e i r  relative  not  22 digestibility  by t h e t a r g e t s p e c i e s u n d e r s p e c i f i c c u l t u r e  conditions. Feed i n g r e d i e n t s are normally composition 1980,  NRC,  mostly  and  content  1981).  The  represented  by  fish,  apparent d i g e s t i b i l i t y a single value, Hastings  (1969),  their  et  al.,  coefficients  suggesting  affect digestibility  that  are  each  however enumerated  s u c h as s i z e and  age  s t o c k i n g d e n s i t y , water temperature, q u a l i t y of feed,  n u t r i e n t density, l e v e l of feeding, previous  level  of n u t r i t i o n ,  presence of t o x i n s . this  listing  o f d i g e s t i b l e n u t r i e n t s (Smith  c o e f f i c i e n t never v a r i e s . f a c t o r s w h i c h may  c a t e g o r i z e d by  list  species  other  1969;  ad  J o b l i n g , 1983)  1984;  Ferraris  2.7  D i g e s t i o n and  e t al.,  and  P a n d e y and  1986;  libitum feeding has  water  added t o  s u c h as  1980;  Lall  et  form  of  1990).  components o f f o o d a r e  i n the  l a r g e i n s o l u b l e m o l e c u l e s w h i c h h a v e t o be b r o k e n down by i n t o s i m p l e r compounds b e f o r e  process  i n t o the blood  n u t r i e n t s through the  species  they  and  i n t e s t i n a l mucosa  lymph.  The  in their  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 .  breaking-down digested  "absorption".  a r e t h e d o m i n a n t v e r t e b r a t e g r o u p as  i s c o n c e r n e d , and  enzymes  c a n p a s s t h r o u g h t h e mucosa o f  i s termed " d i g e s t i o n " , the passage of the  Fishes  fish  salinity  Singh,  U s h e r e t al.,  and  absorption  organic  the d i g e s t i v e t r a c t  diet  feeding,  work o f numerous a u t h o r s  M a c L e o d , 1977;  al.,  Many o f t h e  forced versus  of  f a c t o r s which a f f e c t d i g e s t i b i l i t y ,  (Birkett,  ( Z e i t o u n , 1974;  The  frequency  of  f a r as number o f  immense v a r i e t y h a v e a d o p t e d Not  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  only are there a l s o tremendous  large diversity  e x i s t s i n t h e form and f u n c t i o n o f t h e 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 p r o c e s s e s i s t h a t some have a stomach, w h i l e o t h e r s have none. Some f i s h e s f e e d e x c l u s i v e l y on p l a n k t o n ; bottom f e e d e r s and many s p e c i e s  others are s t r i c t l y  feed o n l y on p r e y a n i m a l s .  Due  t o 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 t h e d i g e s t i v e system o f a l l f i s h .  Consequently, the  emphasis i n t h e 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 s a l m o n i d s .  2.8  General alimentary The  t r a c t anatomy and i t s a s s o c i a t e d  enzymes  s e c r e t i o n s i n t h e stomach t y p i c a l l y i n c l u d e t h e mucus  produced by g o b l e t  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 p e p s i n by h y d r o c h l o r i c a c i d (HC1). nonproteolytic  d i g e s t i v e enzymes such as e s t e r a s e  Tachino, 1960b) l i p a s e and c h i t i n a s e (Lindsay,  Some (Kitamikado and  1984) have a l s o  been i d e n t i f i e d i n t h e stomach o f s a l m o n i d s .  2.8.1 The  The midgut midgut b e g i n s j u s t p o s t e r i o r t o t h e 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 a r e found and ends a t t h e r e g i o n where an i l e o c e c a l valve i s present, j u s t p r i o r t o the hindgut and  (Ezeasor  Stokoe, 1980). The  p y l o r i c cecae c l o s e l y resemble t h e i n t e s t i n e i n  structure, with the inner epithelium containing goblet  cells.  A l t h o u g h many o f t h e enzymes i n v o l v e d i n d i g e s t i o n a r e found i n l a r g e numbers i n t h e 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 o f 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 t h e i n t e s t i n a l lumen moves i n t o and out o f t h e 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 o f c e c a e l muscles (Smith,  1989).  The f u n c t i o n o f t h e cecae i s not y e t 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 s u p p l e m e n t i n g t h a t o f t h e stomach, a b s o r p t i o n o f n u t r i e n t s , r e s o r p t i o n o f water and i n o r g a n i c i o n s , s y n t h e s i s o f enzymes and v i t a m i n p r o d u c t i o n , have a l l been proposed.  Since these  appendages a r e i n c l o s e p r o x i m i t y t o where t h e 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 r e l e a s e d , 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 a b s o r p t i o n o f c e r t a i n n u t r i e n t s seems logical.  However, f u r t h e r s t u d i e s a r e needed t o c l a r i f y t h e r o l e  of t h e 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 t h e 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 t h e pancreas or t h e s e c r e t o r y c e l l s i n t h e gut w a l l .  The p r o t e a s e s t r y p s i n , c h y m o t r y p s i n , c a r b o x y p e p t i d a s e ,  collagenase  and e l a s t a s e a r e known t o be s t o r e d i n t h e 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 pancreatic  enzymes, t h e i n t e s t i n e a l s o s e c r e t e s enzymes t h a t a c t on o l i g o p e p t i d e s , such as t r i al.,  1981)  and d i p e p t i d a s e s  (Ash, 1980, Boge et  and a m i n o p e p t i d a s e s ( P l a n t i k o w and P l a n t i k o w ,  Upon a r r i v a l i n t h e i n t e s t i n a l lumen, t r y p s i n o g e n i n t o t r y p s i n by e n t e r o k i n a s e mucosa.  1985).  i s transformed  which i s s e c r e t e d by t h e i n t e s t i n a l  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 e x i s t e n c e o f s e v e r a l c a r b o h y d r a s e s i n e x t r a c t s from t h e i n t e s t i n e s , p a n c r e a s , p y l o r i c cecae and l i v e r o f 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 a u t h o r s (Kitamikado and Tachino, 1960a; P h i l l i p s , 1969).  Salmonids a l s o  25 produce amylases, m a l t a s e s , s u c r a s e s , l a c t a s e s , and g l u c o s i d a s e s 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).  A l t h o u g h 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 o f t h e stomach, p y l o r i c cecae and i n t e s t i n e o f many f i s h , rainbow t r o u t  including  (Kitamikado and Tachino, 1960b; Swarup and G o e l ,  1975), the pancreas i s b e l i e v e d t o be t h e major s o u r c e o f l i p a s e s (Kapoor et al.,  1975).  Enzymes from t h e m i c r o f l o r a i n t h e i n t e s t i n e c o u l d 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 s u b s t r a t e s such c e l l u l o s e , which few a n i m a l s can d i g e s t . d e t e r m i n e d t h a t Enterobacter  Mowlah et al.  spp. and Vibrio  as  (1979),  spp. produce  c h i t i n a s e as w e l l as showing a h i g h a c t i v i t y i n t h e breakdown o f 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 .  A l t h o u g h t h e number o f  b a c t e r i a l s p e c i e s i n t h e i n t e s t i n e s o f s a l m o n i d s has been found t o be low (Ugayin, 1979), t h e a c t u a l c o n c e n t r a t i o n can be q u i t e high  2.8.2  (Trust and Sparrow,  1974).  The h i n d g u t P r o t e o l y t i c a c t i v i t y d e c r e a s e s from midgut t o h i n d g u t  and Grove, 1979)  (Fange  and s i n c e p r o t e o l y t i c enzymes a r e c o n s i d e r e d t o  be r e s i s t a n t t o a u t o l y s i s w i t h i n the s m a l l 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 t h e l a t t e r a u t h o r t h a t some o f t h e s e enzymes a r e r e s o r b e d 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 o f t h e gastrointestinal tract.  G a u t h i e r and L a n d i s (1971) and more  r e c e n t l y Georgopoulou et al.  (1985), r e p o r t e d t h a t t h e  e n t e r o c y t e s from t h e 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 t h e p r o c e s s o f p i n o c y t o s i s .  The s t u d y o f  t h e a b s o r p t i o n o f macromolecules and p e p t i d e s has been g r e a t l y  f a c i l i t a t e d by the use  of h o r s e r a d i s h  readily identified histochemically,  p e r o x i d a s e , which can  as a model p r o t e i n .  p r o t e i n i s e n c l o s e d w i t h i n the e n t e r o c y t e , (lysosomal) p r o t e o l y s i s can  occur.  2.9  absorption  P r o t e i n d i g e s t i o n and The  be  Once the  intracellular  e x t e n t of p r o t e o l y s i s w i t h i n the stomach depends upon a  number o f 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 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 intake  many o f t h e s e f a c t o r s b e i n g i n t e r a c t i v e . r o l e of the stomach may  pepsin,  be one  etc.,  A l t h o u g h the p r i m a r y  of preparing  susceptible peptide  l i n 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 s m a l l 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  t o some e x t e n t p r o t e i n d i g e s t i o n and even amino a c i d o c c u r s w i t h i n the stomach of rainbow t r o u t . i n t e s t i n e , most p o l y p e p t i d e s  Once i n the  are f u r t h e r h y d r o l y z e d ,  m i x t u r e of low m o l e c u l a r p e p t i d e s and  absorption  leading to a  f r e e amino a c i d s .  Much  work has been done on amino a c i d uptake, showing t h a t each amino a c i d has  i t s own  vertebrates,  c h a r a c t e r i s t i c s . As i n the case of  f r e e amino a c i d a b s o r p t i o n  i s an a c t i v e , group-  s p e c i f i c , sodium dependent, c a r r i e r - m e d i a t e d s e p a r a t e and  mechanism which i s  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; transport  S i l k et a l . , 1982;  i s competitive  Ash,  and the t r a n s p o r t  1985).  of one  be i n h i b i t e d i n the p r e s e n c e of o t h e r amino a c i d s Morris,  higher  1971).  Amino a c i d  amino a c i d  may  ( K i t c h i n and  Thus Dabrowski (1983), s t a t e d t h a t the  rate  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 a c i d s by f i s h i s not d i g e s t i o n but  rather  absorption.  27 The absorbed  amino a c i d s e n t e r t h e p o r t a l b l o o d stream and  presumably undergo t h e same t y p e s o f r e a c t i o n s as i n o t h e r a n i m a l s , namely p r o t e i n s y n t h e s i s ; d e a m i n a t i o n  f o l l o w e d by  o x i d a t i o n and i n some c a s e s , c o n v e r s i o n t o f a t and/or g l u c o s e ; and t o a l e s s e r e x t e n t f o r s y n t h e s i s o f  hormones, p u r i n e s and  n u c l e i c a c i d s (Walton and Cowey, 1982).  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 In t r o u t , l i p i d s a r e absorbed  at the l e v e l of the p y l o r i c  cecae and i n t h e a n t e r i o r midgut (Gauther and L a n d i s , 1972). L i p a s e , i n t h e presence  o f 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 , b i n d s t o t r i g l y c e r i d e s and c a t a l y z e s t h e removal o f one o r b o t h o f t h e o u t e r f a t t y a c i d r e s i d u e s t o y i e l d an emulsion m i x t u r e o f f r e e f a t t y a c i d s 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 t h e  g a s t r o i n t e s t i n a l t r a c t o f f i s h a r e 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 t h e same way as i n h i g h e r v e r t e b r a t e s , a l t h o u g h i n f i s h , a b s o r p t i o n i s s l o w e r and temperature  dependent  (Noaillac-  Depeyre and Gas, 1976; S i r e e t al., 1981) . , A f t e r passage a c r o s s 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 t r a n s p o r t e d mainly by lymph, a l t h o u g h t h e i r presence  i n b l o o d as c h y l o m i c r o n s has  a l s o been r e p o r t e d (Leger, 1977).  2.11  Carbohydrate  d i g e s t i o n and a b s o r p t i o n  Amylases a r e t h e enzymes p r i m a r i l y r e s p o n s i b l e f o r t h e hydrolysis of starch into glucose.  A b s o r p t i o n o f g l u c o s e by t h e  i n t e s t i n a l e p i t h e l i u m o c c u r s by an a c t i v e mechanism and can t a k e p l a c e a g a i n s t c o n s i d e r a b l e c o n c e n t r a t i o n g r a d i e n t s (Fange and  28 Grove, 197 9).  Glucose t r a n s p o r t r a t e s have been  up t o 200 f o l d among d i f f e r e n t f i s h s p e c i e s , 1985/  c i t e d by Smith, 1989) b e i n g lowest  intermediate  in catfish  shown t o v a r y  (Buddington e t j a l . ,  i n trout (carnivore),  (omnivore) and h i g h e s t i n c a r p  (herbivore).  2.12 Apparent and t r u e  digestibility  A p r o p o r t i o n o f t h e 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 r e s i d u e s .  These  s u b s t a n c e s i n c l u d e d i g e s t i v e enzymes and m u c o p r o t e i n s s e c r e t e d i n t o t h e d i g e s t i v e t r a c t , as w e l l as s l o u g h e d o f f c e l l s abraded from t h e a l i m e n t a r y  c a n a l . S i n c e t h e s e s u b s t a n c e s a r e composed  p r i m a r i l y o f p r o t e i n , t h e y a r e r e f e r r e d t o as endogenous f e c a l p r o t e i n o r endogenous f e c a l n i t r o g e n .  The i n c l u s i o n o f t h e  endogenous p r o t e i n w i t h t h e d i e t a r y p r o t e i n i n t h e f e c e s causes a b i a s o f t h 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 toward lower v a l u e s . c o e f f i c i e n t s , a r e t h e r e f o r e "apparent d i g e s t i b i l i t y t o be d i s t i n g u i s h e d from " t r u e o r c o r r e c t e d  Such  coefficients"  digestibility  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 a r e made.  To determine t h e t r u e d i g e s t i b i l i t y , t h e amount o f  endogenous f e c a l p r o t e i n i s f i r s t d e t e r m i n e d and s u b t r a c t e d the p r o t e i n i n t h e f e c e s .  from  T h i s can be a c c o m p l i s h e d by measuring  the amount o f n i t r o g e n e x c r e t e d i n t h e f e c e s when t h e f i s h a r e fed a non-protein  diet.  Nose  (1967; c i t e d i n NRC,  1981), f e d  such a d i e t t o rainbow t r o u t and d e t e r m i n e d t h e endogenous f e c a l n i t r o g e n output t o be 500 mg p e r 100 g o f d r y d i e t , whereas Skrede e t al. (1980) c a l c u l a t e d t h e v a l u e t o be 180 mg p e r 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 t o  differences  i n water  composition  and t e x t u r e between t h e two e x p e r i m e n t s ,  findings  of Foltz  temperature  (1978;  cited  (1973) and Beames and Eggum protein  level  increases,  protein  digestibility  or t o d i s s i m i l a r  i n NRC,  1981),  (1981) s u g g e s t .  dietary  Ogino Also,  as t h e and Chen  as t h e d i e t a r y  t h e d i f f e r e n c e between a p p a r e n t  and t r u e  e s t i m a t e s 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 The e a r l i e s t coefficients collection  measurements i n f i s h method f o r d e t e r m i n i n g d i g e s t i b i l i t y  f o r d o m e s t i c a n i m a l s was  method w h i c h  involves  the d i r e c t  a quantitative  or t o t a l record of the •  nutrients  consumed and t h o s e v o i d e d i n t h e f e c e s .  digestion  coefficients  carbohydrate, appropriate  energy,  are c a l c u l a t e d and t o t a l  coefficient is  f o r crude p r o t e i n ,  The a p p a r e n t  of a given nutrient  lipid,  d r y m a t t e r by p e r f o r m i n g  chemical analyses f o r the nutrients  consumed and i n t h e f e c e s .  The a p p a r e n t  i n the feed  digestibility  b a s e d on t h e d i r e c t  method  (ADCd)  thus determined as:  ADCd  (%) =  100  1  f e c e s dry weight diet  The d i r e c t  f e d weight  x % nutrient  i n feces  x % nutrient  i n diet  method 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  g i v e v e r y a c c u r a t e r e s u l t s when p r o p e r l y impracticality  of t h i s  method comes,  quantitatively  collect  the feces  An i n d i r e c t  method, w h i c h  i s considered to  carried  however,  out.  The  from t h e need t o  f o r an e x t e n d e d p e r i o d  i n v o l v e s t h e u s e o f an  o f time.  indigestible  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  animal d i g e s t i b i l i t y t r i a l s since p r o c e d u r e and involved  a l s o reduces the  f e c e s and  i t greatly simplifies  amount o f l a b o u r and  the  expense  calculated  The  i s d e t e r m i n e d i n the d i e t and  in  t h u 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 of a  d i e t d e t e r m i n e d by the  ADCi (%)=  terrestrial  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 .  r a t i o of i n d i c a t o r to n u t r i e n t the  in  i n d i r e c t or i n d i c a t o r method (ADCi) i s  as:  % indicator in diet x % nutrient  100 1  i n feces  % i n d i c a t o r i n feces x % n u t r i e n t  in diet  W i t h t h i s i n d i r e c t method of d e t e r m i n i n g d i g e s t i b i l i t y , the  total  c o l l e c t i o n o f f e c e s i s unnecessary, s i n c e 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  portions  and  i n d i c a t o r are e v e n l y d i s t r i b u t e d  and  L o o s l i , 1969).  must be  To be  i n d i g e s t i b l e and  'with the d i g e s t a  o f f e c e s i f the  nutrient  (Kane e t . a l . , 1950;  Maynard  an e f f e c t i v e i n d i c a t o r , a substance u n a b s o r b a b l e , remain homogeneously mixed  d u r i n g passage t h r o u g h the gut,  and  e f f e c t on the d i g e s t i v e m e t a b o l i s m of the a n i m a l F l a t t 1975).  Two  classes  of i n d i c a t o r s can be  t h o s e t h a t are added t o a d i e t and p r e s e n t as n a t u r a l proposed the use  external  dietary  Digestion  Edin  of chromic o x i d e ( C ^ C ^ ) and  trials in terrestrial  (Schneider  and  distinguished:  t h o s e t h a t are  components of a d i e t .  have no  inherently  (1918) f i r s t most i n d i c a t o r  a n i m a l s have been c a r r i e d out w i t h t h i s  marker.  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 t h a n w i t h  a n i m a l s because f e c a l and  o t h e r m e t a b o l i c e x c r e t i o n s must be  land  c o l l e c t e d from an a q u a t i c medium (Smith e t al., 1980).  The  o r i g i n a l a p p l i c a t i o n o f t h e 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 t e c h n i q u e s 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 o f a l l e g e s t a and excretions  (Tunison e t al., 1942).  Post e t al. (1965),  (1971, 1976) and Smith e t al. (1980), used a metabolism  Smith chamber  which p e r m i t s s e p a r a t i o n and q u a n t i t a t i v e c o l l e c t i o n o f f e c e s , u r i n e and g i l l e x c r e t i o n s .  T h i s method has t h e advantage o f  p e r m i t t i n g t h e d e t e r m i n a t i o n o f 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 m e t a b o l i z a b l e energy, however, metabolism  chambers have been  c r i t i c i z e d because f i s h a r e c o n f i n e d , c a t h e t e r i z e d and f o r c e f e d , and c o n s e q u e n t l y  a r e under c o n s i d e r a b l e s t r e s s which p r o b a b l y  reduces t h e i r a b i l i t y t o d i g e s t and u t i l i z e f e e d . D e t e r m i n a t i o n o f d i g e s t i b i l i t y by t h e i n d i r e c t method has been used w i t h s e v e r a l s p e c i e s o f f i s h 1969:  (Nose, 1960; H a s t i n g s ,  Smith and L o v e l l , 1971, 1973; W i n d e l l e t al., 1978a).  major d i s a d v a n t a g e s  The  o f t h i s method a r e t h e v a r i e t y o f t e c h n i q u e s  used t o c o l l e c t t h e f e c a l samples w i t h o u t i n h e r e n t n u t r i e n t l e a c h i n g e r r o r s and t h e i n a b i l i t y t o compute r e l i a b l e m e t a b o l i z a b l e energy v a l u e s  (NRC, 1981).  l e a c h i n g o f n u t r i e n t s i n t h e water,  To a v o i d c o m p l e t e l y any  s e v e r a l authors p r e f e r t o  c o l l e c t t h e f e c e s d i r e c t l y from t h e p o s t e r i o r r e g i o n o f t h e i n t e s t i n e before i t i s n a t u r a l l y expelled.  T h i s can be done by  s a c r i f i c i n g t h e f i s h and p e r f o r m i n g an i n t e s t i n a l (Smith and L o v e l l , 1971, 1973; A u s t r e n g , 1978a; Henken e t al., 1985).  dissection  1978; W i n d e l l e t al.,  S i n c e r e p e a t e d s a m p l i n g i s not  p o s s i b l e w i t h t h i s t e c h n i q u e , o t h e r a u t h o r s have r e s o r t e d t o a p p l y i n g manual p r e s s u r e t o t h e abdomen o f a n e s t h e t i z e d f i s h and  32  stripping (Nose,  the  1960;  f e c e s from Inaba  e t al.,  1978a; T a c o n e t al., anal  Strange,  intestinal  l e a c h i n g problem, the  large  (Smith and  Austreng,  (Lovell,  1977;  Although  stripping assumption  Lovell,  additional  large  1978;  1985)  that  W i n d e l l e t al.,  anal suction digestion  reached  Austreng,  disadvantage  1978;  avoids  and  o f t h e s e methods i s t h a t  products.  The  action  the  intestinal  prior  f e c a l .material with  epithelium.  to handling  also  The  appears  acceleration  of i n t e s t i n a l  Furthermore,  samples a r e t a k e n  empirically to  d e f i n e d by  reproduce  stripping  from  al.,  1980;  sampling small  i n the time  the composition  significant  Another  Silva  techniques  and  o f f e c e s has  Perera,  are not  off cells  fish  1989).  difficult  particularly  o f e r r o r may  for  come from  the  sampling.  b e e n shown t o have  (Inaba  e t al.,  1962;  De  l a Noue e t  1983,  1984).  Finally,  intestinal  a p p l i c a b l e t o s m a l l f i s h because  amounts o f sample can be  of  segment  sometimes  inherent i n i n t e s t i n a l  diurnal variations De  and  t o another,  source  i n the  sudden d e f e c a t i o n and  intestinal  investigator  samples  sexual  ( S p y r i d a k i s e t al.,  i n an  individual  or s u c t i o n .  discontinuity Indeed,  one  fecal  results  sloughed  to induce  the  1978a).  need t o a n e s t h e t i z e t h e  transit  the  also  of  digestibility  w i t h mucus, b l o o d , u r i n e o r  of the  end  W i n d e l l e t al.,  of s t r i p p i n g  the  absorption  the d i s t a l  contaminated  contamination  1978a;  o f f e c e s by  are e a s i l y  physical  al.,  or t o a p p l y i n g  l e a d t o an u n d e r e s t i m a t i o n o f 1971;  intestine  Windell et  the c o l l e c t i o n  and  when t h e m a t e r i a l has  i n t e s t i n e may  p a r t of the  Vens-Cappell,  1985).  dissection,  are complete  1962;  1984;  s u c t i o n w i t h vacuum  Brown and  An  the d i s t a l  collected  a t any  single  only  occasion.  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 s a m p l i n g c o u l d be a v o i d e d by c o l l e c t i n g t h e f e c e s as soon as they a r e n a t u r a l l y r e l e a s e d i n t o t h e water.  A d i f f e r e n t source o f e r r o r i s  i n t r o d u c e d when f e c e s a r e c o l l e c t e d from t h e water s i n c e 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 o v e r e s t i m a t e d al.,  1962; W i n d e l l e t al., 1978a).  (Inaba e t  With t h e aim o f a v o i d i n g  m a n i p u l a t i o n o f f i s h as w e l l as m i n i m i z i n g l e a c h i n g l o s s e s , d e v i c e s f o r t h e s e p a r a t i o n and r e c o v e r y o f f e c e s have been proposed by numerous workers.  Ogino e t al. (1973),  collected  f e c e s by p a s s i n g t h e e f f l u e n t water from t h e f i s h t a n k s t h r o u g h a f i l t r a t i o n column.  I n t h e "Guelph system" (Cho e t al., 1975; Cho  and S l i n g e r , 1979; Cho e t al., 1982; M a r t i n e z - P a l a c i o s , 1988) t h e e f f l u e n t water from t h e tank passes t h r o u g h a s e t t l i n g column w i t h an o u t l e t a t t h e bottom so t h a t s e t t l e d m a t e r i a l r e s t s i n u n d i s t u r b e d water b e f o r e c o l l e c t i o n .  M e c h a n i c a l methods f o r  c o l l e c t i o n o f t h e f e c e s range from a s i m p l e removal o f t h e f e c a l p e l l e t s w i t h a f i n e mesh n e t ( W i n d e l l e t al., 1978a), t o elaborate mechanically r o t a t i n g screen devices  (Kaushik and  Luquet, 1976; Choubert e t al., 1979, 1982; De l a Noiie and Choubert, 1986) t h a t f i l t e r out t h e f e c a l m a t e r i a l from t h e o u t f l o w water.  D i s r u p t i o n o f t h e f e c a l p e l l e t s i s an u n f o r t u n a t e  drawback o f t h e n e t t i n g and a u t o m a t i c  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 o f t h e methods mentioned above i s t h a t f i s h a r e n o t h a n d l e d and a r e a l l o w e d t o f e e d u n d i s t u r b e d , a l t h o u g h t h e aquarium t a n k s need t o be f l u s h e d c l e a r o f 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 t h a t  r e p e a t e d 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 .Concern s t i l l  evaluation.  remains among n u t r i t i o n i s t s w i t h r e s p e c t t o  t h e degree o f n u t r i e n t l e a c h i n g which may o c c u r w i t h t h e v a r i o u s methods i n which f e c e s a r e c o l l e c t e d from t h e aquarium w a t e r . Data from W i n d e l l e t al. (1978a), showed t h a t most n u t r i e n t l e a c h i n g from f e c e s o c c u r s d u r i n g t h e f i r s t hour a f t e r defecation.  Systems which remove t h e f e c e s from t h e water  s w i f t l y a r e c l a i m e d by some a u t h o r s t o cause minimum  leaching  (Choubert e t al., 1979, 1982/ Schmitz e t al., 1983), w h i l e o t h e r s c l a i m t h a t d i s r u p t i o n and breakage o f f e c a l p e l l e t s d u r i n g c o l l e c t i o n a r e t h e major cause o f 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 e t al., 1982/ V e n s - C a p p e l l , 1985). Cho and S l i n g e r  Indeed,  (1979), Cho e t al. (1982) and T a l b o t (1985)  showed t h a t r e s u l t s o b t a i n e d by i n t e s t i n a l d i s s e c t i o n and by a n a l 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 t h e r e s u l t s o b t a i n e d by t h e "Guelph system" f o r t h e d i g e s t i b i l i t y o f d r y m a t t e r , crude p r o t e i n and l i p i d s .  T h i s would 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 was not an i m p o r t a n t s o u r c e o f 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 o f 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 p r o v i d e d as t h e s o l e d i e t .  Consequently,  i t i s impossible to  determine t h e 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  u n l e s s t h e y a r e combined w i t h o t h e r f e e d components. Determination  of the d i g e s t i b i l i t y of a t e s t ingredient requires  comparing t h e d i g e s t i b i l i t y o f a r e f e r e n c e d i e t and a t e s t  diet;  the t e s t d i e t b e i n g a known m i x t u r e o f t h e r e f e r e n c e d i e t and test ingredient.  T h i s p r o c e d u r e assumes t h a t t h e r e i s no  i n t e r a c t i o n between t h e components o f t h e d i e t d u r i n g d i g e s t i o n , w h i l e a t t h e same time a l l o w i n g t h e p r e p a r a t i o n o f an a d e q u a t e l y b a l a n c e d d i e t , i n which t o t e s t t h e s u s c e p t i b i l i t y o f t h e t e s t ingredient to digestion.  T h i s p r o c e d u r e has been w i d e l y 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 e t a l . , 1982; W i l s o n 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)  Comparison o f t h e d i r e c t  in  ( t o t a l ) and i n d i r e c t  seawater: (C^C^)  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 digestion  of  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  fecal collection.  3.1  INTRODUCTION B e f o r e any f e e d s t u f f  i t s nutrient  can be p r o p e r l y e v a l u a t e d i n terms o f  d i g e s t i b i l i t y by a g i v e n a n i m a l , 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 o f the t o t a l f e c a l matter produced a f t e r consumption o f a known amount o f f e e d or measurement o f t h e c o n c e n t r a t i o n o f an i n d i g e s t i b l e marker (e.g. chromic oxide) i n t h e f e e d and have b o t h been e x t e n s i v e l y  used.  feces  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 v e r y t e d i o u s and l a b o u r i n t e n s i v e  and r e q u i r e s g r e a t  c a r e i n f e e d i n g , f e c a l c o l l e c t i o n and measurement o f s p i l l a g e , a l t h o u g h p r o p e r e x p e r i m e n t a l procedures results.  The  developed  to obviate quantitative  w i l l give very r e l i a b l e  i n d i r e c t method u s i n g chromic  o x i d e has been  c o l l e c t i o n problems.  o f i n e r t markers i n t h e f e e d , however, has not been satisfactory.  The  use  consistently  Major l i m i t a t i o n s t o the use o f chromic o x i d e are  the v a r i a t i o n o f i t s c o n c e n t r a t i o n i n the f e c e s and recovery of the i n d i c a t o r , reduced d i g e s t i o n  resulting i n variable  coefficients in fish  and o t h e r s p e c i e s (Knapka e t al.,  1967;  results  (Bowen, 1978; Hattan  incomplete  and  and  Foltz,  1979)  37 Owen, 1970).  Hence, i n t h i s study, t h e f i r s t o b j e c t i v e was t o  compare t h e d i g e s t i b i l i t y d a t a o b t a i n e d by b o t h t h e 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 g r e a t e s t problem w i t h b o t h methods i s t h e d i f f i c u l t y o f o b 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 t h e a q u a t i c medium. F o r example, t h e v a r i o u s t e c h n i q u e s employed f o r r e c o v e r y o f t h e f e c e s from t h e water have been c r i t i c i z e d because o f n u t r i e n t leaching. matter  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 o f f e c a l  from t h e lower i n t e s t i n e a r e s u b j e c t t o e r r o r because o f  c o n t a m i n a t i o n o f t h e f e c e s w i t h body f l u i d s and i n c o m p l e t e n u t r i e n t d i g e s t i o n and a b s o r p t i o n .  The d i g e s t i b i l i t y  results  o b t a i n e d by Cho and S l i n g e r (197 9) u s i n g t h e "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 f e c e s o f rainbow t r o u t a r e c o l l e c t e d a f t e r s e t t l i n g o v e r n i g h t i n a column o f u n d i s t u r b e d water.  More r e c e n t l y , however, S p y r i d a k i s e t al.  (1989) w o r k i n g w i t h sea bass (Dicentrarchus  labrax),  showed t h a t ,  compared t o o t h e r 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 , t h e "Guelph system" gave t h e h i g h e s t p r o t e i n d i g e s t i b i l i t y v a l u e s .  The same  authors a t t r i b u t e d the l e a c h i n g l o s s e s t o a flaw i n t h e i r  tank  d e s i g n which p r e v e n t e d t h e f e c e s from b e i n g 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 g o a l o f t h i s study was t h e r e f o r e  t o a s s e s s t h e v a l i d i t y o f t h e "Guelph system" o f f e c a l  collection  by comparing d i g e s t i b i l i t y v a l u e s o b t a i n e d from f e c e s c o l l e c t e d w i t h t h i s system t o t h o s e o b t a i n e d from f e c e s c o l l e c t e d by t h e 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 t e c h n i q u e s .  As p a r t o f t h i s  g o a l , t h e e f f e c t o f 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 a s s e s s e d .  The f i n a l g o a l o f t h i s experiment  was t o measure t h e amount o f endogenous f e c a l p r o t e i n e x c r e t e d fish fed a protein-free diet.  T h i s approach would e n a b l e t h e  r e s u l t s t o be p r e s e n t e d on a " t r u e " d i g e s t i b i l i t y  basis.  by  39 3.2  MATERIALS AND  3.2.1  METHODS  Experimental  design  Twenty j u v e n i l e chinook  salmon (mean weight o f 55.3  (SEM)) were d i s t r i b u t e d randomly i n t o each o f n i n e tanks.  The  ± 0.50  g  digestibility  f i s h i n s i x o f t h e groups were f e d a c o n t r o l d i e t  ( c o n t a i n i n g 0.45%  chromic o x i d e on a d r y - m a t t e r  b a s i s ) by hand  t w i c e d a i l y on a r e s t r i c t e d b a s i s whereas t h e r e m a i n i n g t a n k s were o f f e r e d a p r o t e i n - f r e e d i e t .  three  But the l a t t e r d i e t  was  not r e a d i l y consumed by any o f the groups d u r i n g the a c c l i m a t i o n p e r i o d and c o n s e q u e n t l y  t h i s treatment  I n s t e a d , each o f the t h r e e groups was  was  discontinued.  fed the c o n t r o l d i e t to  s a t i a t i o n t o i n v e s t i g a t e the e f f e c t o f f e e d i n g l e v e l on n u t r i e n t digestibility.  To p r o p e r l y a c c l i m a t e t h e s e f i s h t o t h e i r  new  ( c o n t r o l ) d i e t and f e e d i n g p r o t o c o l , t h e i r f e c e s were c o l l e c t e d e i g h t days a f t e r i n i t i a t i o n o f f e c a l c o l l e c t i o n i n t h e s i x groups fed the r e s t r i c t e d  ration.  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 n i n e twice d a i l y  (0830 hours and 1430  tanks  hours) over a p e r i o d o f t h r e e  weeks ( s i x groups) or two weeks (three groups) u s i n g the "Guelph system".  S i n c e the f e c e s c o n t a i n e d chromic o x i d e , comparisons  were made between t h e d i r e c t and i n d i r e c t methods o f digestibility. d a i l y , i t was  determining  F u r t h e r , because the f e c e s were c o l l e c t e d t w i c e p o s s i b l e t o monitor the e x t e n t o f n u t r i e n t l e a c h i n g  between t h e c o l l e c t i o n t i m e s . In a d d i t i o n t o t h e above methodology comparisons, i t was a l s o o f i n t e r e s t t o 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 t h e "Guelph system" o f f e c a l c o l l e c t i o n v e r s u s those based on removal o f f e c e s by- s t r i p p i n g .  In t h i s r e g a r d , the f e c e s from  40 f i s h i n t h r e e o f t h e s i x groups f e d 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 t h e "Guelph system" but a l s o by t h e s t r i p p i n g technique r e f e r r e d t o previously.  The l a t t e r p r o c e d u r e  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 o f t h e t h r e e groups once a week f o r t h r e e weeks. the  F i n a l l y , a t t h e end o f  experiment, a l l f i s h n o t p r e v i o u s l y s t r i p p e d were e u t h e n i z e d  and t h e c o n t e n t s o f t h e i r lower i n t e s t i n a l t r a c t between t h e p e l v i c f i n s and t h e anus were d i s s e c t e d out, t o p e r m i t f u r t h e r methodology  3.2.2  comparisons.  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" d e v e l o p e d 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 d e t e r m i n a t i o n s i n t r o u t e n t a i l s t h e c o l l e c t i o n o f f e c e s from f i s h h e l d i n groups o f t h r e e i n t e r c o n n e c t e d 7 7 - l i t e r t a n k s by means o f a common s e t t l i n g tube f o r each s e t o f t h r e e t a n k s l o c a t e d a t t h e o u t f l o w .  This design  was m o d i f i e d i n t h e p r e s e n t s t u d y on chinook salmon s i n c e t h i s s p e c i e s i s more s u b j e c t t o s t r e s s o f confinement t h a n T h e r e f o r e , 150-L f i b r e g l a s s  trout.  t a n k s were s p e c i a l l y d e s i g n e d 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  s e p a r a t e f e c a l c o l l e c t i o n column o f t h e Guelph d e s i g n was a f f i x e d onto each tank as shown i n F i g u r e 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 p l a c e d a t t h e o v e r f l o w o f each tank t o ensure t h a t t h e s m a l l e r , 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 o f t h e system, were a l s o i n c l u d e d i n t h e c o l l e c t i o n , sample.  41  inflowing filtered seawater  to drain  t a n k - c o l l e c t i o n column connection pipe collection column  •feces  collection port  Figure 1.  Diagram showing the "Guelph system" of f e c a l used in experiments I,  II,  III  and IV.  collection  42 3.2.3  Aquarium  facility  The experiment was conducted a t t h e 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 a d j a c e n t t o t h e Department and Oceans (D.F.O.),  West Vancouver L a b o r a t o r y .  of F i s h e r i e s  Each o f t h e  d i g e s t i b i l i t y t a n k s was s u p p l i e d w i t h 6 t o 8 l i t e r s p e r minute o f f i l t e r e d B u r r a r d I n l e t seawater system.  (26 t o 31 ppt) i n a f l o w t h r o u g h  The water f l o w r a t e was c a r e f u l l y b a l a n c e d t o not o n l y  ensure t h e r a p i d removal o f f e c a l m a t t e r from each t a n k , but a l s o t o ensure a g e n t l e s e t t l i n g o f t h e f e c e s by g r a v i t y i n t o t h e c o l l e c t i o n column w i t h m i n i m a l l o s s v i a t h e o v e r f l o w .  Water  t e m p e r a t u r e v a r i e d by a p p r o x i m a t e l y 1°C d i u r n a l l y and ranged between 5°C and 8°C t h r o u g h o u t t h e e x p e r i m e n t a l p e r i o d .  Aeration  s t o n e s i n each tank m a i n t a i n e d t h e water oxygen l e v e l c l o s e t o saturation. Overhead  fluorescent 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 t i m e c l o c k were used t o s i m u l a t e 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 c o n s t r u c t e d f o r each o f t h e t a n k s i n o r d e r t o  p r e v e n t sand, s h e l l s , a l g a e and l i v e organisms such as  copepods  which a r e commonly c a r r i e d i n w i t h t h e pumped seawater, from e n t e r i n g the tanks.  The f i l t e r s were c o n s t r u c t e d from 7.62  (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 c u t i n t o 30 cm  cm  lengths.  Removable s c r e w - i n l i d s were a t t a c h e d t o b o t h ends o f t h e p i p e t o a l l o w c l e a n i n g or r e p l a c i n g o f t h e f i l t e r i n g m a t e r i a l , w h i c h 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 t h r e a d e d a t t h e c e n t e r t o f a c i l i t a t e t h e attachment  of  the f i l t e r  t o t h e 1.91  p i p e l i n e a t one end.  end,  To p r e v e n t  end,  circles  (O.D.) i n c o m i n g w a t e r  cm  i n diameter)  and  was  w e r e c u t o u t o f a 2.38  (mesh o p e n i n g  < 500um) was  a d d i t i o n , water  l e a v i n g the f i l t e r  was  that the gravel  "funnel f i l t e r "  250  was  um)  directed into a  in retaining  to  be  maintained.  r e p l a c e d every three  3.2.5  gravel f i l t e r ,  Table  formulae 1.  The  plastic  (mesh bands.  fine  that proper  silt. water  on t h e o t h e r h a n d ,  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 a r e  shown  m i n e r a l supplement of the p r o t e i n - f r e e d i e t  was  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 p l a s m a e m i s s i o n  (ICAP) by Q u a n t a T r a c e L a b o r a t o r i e s I n c . ( B u r n a b y ,  of  The  had  composition  prepared t o equal the mineral composition of the c o n t r o l  Columbia).  two  days.  D i e t p r e p a r a t i o n and The  in  The  fine  In  h e l d i n p l a c e w i t h rubber  very e f f e c t i v e  a  of the f i l t e r .  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 f l o w was  particles  Subsequently,  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 approximately  lids.  sandwiched between  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  The  mm  s h e e t and p l a c e d b e t w e e n t h e g r a v e l and t h e  s m a l l copepods from e n t e r i n g t h e t a n k s .  opening  either  not very 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 algae  Vexlar screen PVC  outflow  through  A f t e r a p r e l i m i n a r y r u n , i t became a p p a r e n t filter  supply  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  l o s s o f g r a v e l from t h e f i l t e r  (7.60  p e r f o r a t e d PVC  cm  proximate  the d i e t s are l i s t e d  spectroscopy  British  c o m p o s i t i o n and m a j o r m i n e r a l  i n Table  diet.  contents  2.  M i n e r a l s u p p l e m e n t s w e r e g r o u n d t o a f i n e powder i n a portable coffee grinder p r i o r to weighing.  They w e r e  then  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 B l e n d e r ( P a t t e r s o n - K e l l e y Co., D i v i s i o n o f Harsco Corp., P e n n s y l v a n i a ) u s i n g ©^-cellulose (ICN N u t r i t i o n a l B i o c h e m i c a l s , C l e v e l a n d , Ohio.) as t h e c a r r i e r .  The same p r o c e d u r e was  f o l l o w e d f o r t h e v i t a m i n supplements, a l t h o u g h g r i n d i n g was n o t required.  A l l f e e d s t u f f s used were c o m m e r c i a l l y o b t a i n e d and, i f  n e c e s s a r y , 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, E l m h u r s t I l l i n o i s , U.S.A.) equipped w i t h a s i z e 30 U.S. s c r e e n (595 um).  I n g r e d i e n t s f o r each d i e t were  b l e n d e d ( w i t h a p o r t i o n o f t h e o i l ) f o r 20 minutes i n a Hobart commercial m i x e r (Hobart M a n u f a c t u r i n g Co., Troy, O h i o ) . oxide  Chromic  (Cr203) was combined w i t h a p o r t i o n o f t h e i n g r e d i e n t s and  ground i n a m o r t a r 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 . V i t a m i n and m i n e r a l pre-mixes were added t o t h e r e s p e c t i v e d i e t s and combined i n t h e Hobart m i x e r f o r an a d d i t i o n a l 20 m i n u t e s . Each d i e t was t h e n s t e a m - 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 laboratory 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  F r 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 .  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 p l a c e d i n a v e r t i c a l c o o l e r .  After  c o o l i n g , f i n e s were, s c r e e n e d out and t h e remainder o f t h e o i l was s p r a y e d on t o t h e t u m b l i n g p e l l e t s i n s i d e a cement-mixer u s i n g an e l e c t r i c a l l y o p e r a t e d spray-gun. p l a s t i c bags i n a r e f r i g e r a t o r experiment.  The d i e t s were kept i n dark  (3°C-4°C) t h r o u g h o u t t h e  45 TABLE 1. C o m p o s i t i o n o f c o n t r o l and p r o t e i n - f r e e diet). Ingredients  Control  S t e a m - d r i e d h e r r i n g meal Wheat m i d d l i n g s D r i e d whey B l o o d meal H e r r i n g oil-*Mineral supplement Dextrin Glucose V i t a m i n supplement^ Carboxy-methyl c e l l u l o s e C h o l i n e c h l o r i d e (60%) Chromic o x i d e Ascorbic acid DL-methionine 2  550. 126. 75. 50. 115. 20.  diet  35. 15. 5. 5. 2. 1.  (g/kg d r y  Protein-free -  00 80 00 00 00 00  -  diets  -  178.00 133.70 313.15 313.15 35.00 15.00 5.00 5.00 2.00 -  00 00 00 00 00 20  S t a b i l i z e d w i t h 0.025% e t h o x y q u i n . The m i n e r a l supplement p r o v i d e d t h e f o l l o w i n g amounts p e r kg o f d r y d i e t ( v a l u e s i n b r a c k e t s a r e t h e v a l u e s f o r t h e p r o t e i n - f r e e d i e t ) : magnesium as M g S C ^ ^ ^ 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»5H 0, 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); c o b a l t as C o C l 2 * 6 H 0 1 mg (2.75 mg); s e l e n i u m as Na SeC>3, 0.10 mg (0.10 mg) ; c a l c i u m as CaHP0 , (14.268 g) and as CaCC>3 (5.481 g) ; sodium c h l o r i d e , (5.095 g) ; p o t a s s i u m as K2SO4, (3.865 g) and as KH2PO4, (3.865 g ) ; aluminum as A1C1 »6H 0, (5 mg). A t o t a l o f 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 ) o f o c - c e l l u l o s e was used as t h e carrier. The v i t a m i n supplement p r o v i d e d t h e f o l l o w i n g (mg/kg o f dry d i e t u n l e s s o t h e r w i s e i n d i c a t e d ) : i n o s i t o l 400; n i a c i n 300; p a n t o t h e n a t e as D - c a l c i u m p a n t o t h e n a t e 166; menadione as h e t r a z e e n 26.1; r i b o f l a v i n 60; p y r i d o x i n e as p y r i d o x i n e - H C l 36.5; t h i a m i n e as t h i a m i n e m o n o n i t r a t e 36.3; f o l i c a c i d 20; v i t a m i n B-12 0.06; r e t i n o l a c e t a t e 10,000 I U ; c h o l e c a l c i f e r o l 2,400 I U ; dl-«=-tocopheryl a c e t a t e 600 I U . A t o t a l o f 32.38 g/kg o f ^ - c e l l u l o s e was used as t h e c a r r i e r . 2  2  /  2  4  3  2  46 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  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 o b t a i n e d  i n March, 1988 from t h e R o b e r t s o n Creek h a t c h e r y  (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 t a n k s held outdoors.  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 mid J u l y .  The f i s h were i n s a l t water  31 ppt) a p p r o x i m a t e l y 10 days l a t e r .  (28 t o  F i s h were p e r i o d i c a l l y  removed f o r o t h e r experiments and s t o c k i n g d e n s i t y was c o n s e q u e n t l y m a i n t a i n e d below a p p r o x i m a t e l y 7.5 kg/m^. Oregon moist p e l l e t d i e t  The  (OMP) was f e d by hand t o s a t i a t i o n t w i c e  daily. On January 16, 198 9 f i s h were s e l e c t e d randomly and a n e s t h e t i z e d w i t h 2-phenoxyethanol  (0.5 ml/L w a t e r ) , p l a c e d on an  absorbent c l o t h t o remove excess m o i s t u r e , and i n d i v i d u a l l y weighed t o t h e n e a r e s t gram.  Those f i s h w e i g h i n g between 45 g  and 65 g were s e p a r a t e d from t h e r e s t and were randomly d i s t r i b u t e d i n t o one row o f 9 d i g e s t i b i l i t y t a n k s u n t i l t h e r e were 25 f i s h p e r t a n k . feces  F e e d i n g was resumed by t h e next day and  (chromic o x i d e - f r e e ) were c o l l e c t e d from each  settling  column f o r f u t u r e use i n t h e chromic o x i d e a n a l y s e s . A t t h e end o f 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 t h e d i g e s t i b i l i t y t a n k s , a n e s t h e t i z e d and reweighed. A p o p u l a t i o n o f 180 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 w i t h a mean weight o f 55.3g ± 0.50 (SEM). r e c o r d e d t o t h e n e a r e s t O.lg.  I n d i v i d u a l f i s h w e i g h t s were  F i s h from t h e 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 o f 2, t o p r o v i d e 20 f i s h t o each o f t h e 9 d i g e s t i b i l i t y t a n k s .  47  3.2.6.2 All  Experimental p r o t o c o l f i s h i n the d i g e s t i b i l i t y t a n k s were weaned from the  d i e t t o t h e c o n t r o l d i e t over a two day p e r i o d . chromic o x i d e i n t h e f e c e s was  f i r s t observed  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 . d u r i n g t h e experiment,  The presence o f 56  approximately  To m i n i m i z e  OMP  f e e d wastage  the amount o f i n g e s t e d f e e d which would  s a t i s f y a p p e t i t e had t o be determined.  F o r the next seven days,  f i s h were f e d by hand t w i c e d a i l y u n t i l " a c t i v e " f e e d i n g ceased over a f e e d i n g p e r i o d o f one hour. f i r s t and second f e e d i n g was  The  s i x hours.  consumption at each f e e d i n g p e r i o d was Appendix  i n t e r v a l between the A p r e c i s e r e c o r d of feed maintained  as d e s c r i b e d i n  2.  Three days b e f o r e 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 f e e d consumption f o r f i s h i n each tank was c a l c u l a t e d and f e e d a l l o w a n c e s  were reduced by 1 0 % t o m i n i m i z e  f e e d wastage.  At t h i s s t a g e , f i s h i n t h r e e randomly s e l e c t e d tanks were s w i t c h e d 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 f e e d i n g on day  0, t a n k s were f l u s h e d  c l e a r o f f e c e s and f e e d i n p r e p a r a t i o n f o r the s t a r t o f f e c a l c o l l e c t i o n s t h e next morning. was  Four days i n t o the experiment,  it  d e c i d e d t o a b o r t the use o f the p r o t e i n - f r e e d i e t as the  f i s h , a l t h o u g h o b v i o u s l y hungry, r e f u s e d t o consume any o f t h e pellets.  F i s h on the c o n t r o l d i e t , however, c o n t i n u e d 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 o f feces.  The p r o t e i n - f r e e d i e t was  control diet.  subsequently  r e p l a c e d by  To e v a l u a t e the e f f e c t o f f e e d i n g l e v e l  the  on  n u t r i e n t d i g e s t i b i l i t y , t h e f e e d i n t a k e o f t h e f i s h f o r m e r l y on  48 the p r o t e i n - f r e e d i e t was no l o n g e r r e s t r i c t e d , t h e c o n t r o l b e i n g f e d t o s a t i a t i o n t w i c e d a i l y .throughout t h e s t u d y . 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  diet Fish  from t h e s e t a n k s were  resumed f o u r days l a t e r .  3.2.6.3  Fecal c o l l e c t i o n techniques  3.2.6.3.1 "Guelph system" 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 p r i o r t o t h e s t a r t o f f e c a l c o l l e c t i o n s , t h e d r a i n p i p e and t h e s e t t l i n g column of a l l n i n e t a n k s were b r u s h e d out t o remove f e e d and f e c a l r e s i d u e s from t h e system.  The t a n k s were g e n t l y brushed c l e a n  w h i l e o n e - t h i r d o f t h e water was d r a i n e d out t o ensure t h e system had been c l e a r e d o f a l l p o s s i b l e c o n t a m i n a n t s .  On t h e f i r s t day  of c o l l e c t i o n and f o r t h e remainder o f t h e s t u d y , f e c e s were quantitatively  collected  d e s c r i b e d i n Appendix 1.  a t 0830 and 1430 hours d a i l y as Thus t h e f e c e s c o l l e c t e d  a t 0830 hours  remained i n t h e water f o r a maximum o f 18 hours (depending on when t h e f e c e s had been v o i d e d by t h e f i s h ) , w h i l e t h o s e collected hours.  a t 1430 hours remained i n t h e water f o r a maximum o f 6  F e e d i n g was begun i m m e d i a t e l y a f t e r each c o l l e c t i o n and  r e f u s e d f e e d p e l l e t s were r e c o v e r e d and a c c o u n t e d f o r as d e s c r i b e d i n Appendix 2. The c o l l e c t e d  f e c e s were c e n t r i f u g e d a t 10,000 x g f o r 30  minutes a t a p p r o x i m a t e l y 5°C and t h e s u p e r n a t a n t d i s c a r d e d . f e c a l c o l l e c t i o n sample was s t o r e d i n a s e a l e d c e n t r i f u g e at -40°C.  Each bottle  49 3.2.6.3.2 S t r i p p i n g  technique  F i s h i n t h r e e t a n k s were a n e s t h e t i z e d once a week and t h e i r f e c e s were removed by a p p l y i n g g e n t l e p r e s s u r e w i t h t h e f i n g e r s between t h e p e l v i c f i n s and t h e anus as d e s c r i b e d by Singh and Nose (1967) and Nose (1967).  S t r i p p e d f e c a l m a t t e r was  collected  onto a l a r g e p i e c e o f absorbent f i l t e r paper t o remove as much o f the u r i n e as p o s s i b l e . collected,  Given t h e s m a l l q u a n t i t y o f f e c e s  a l l f e c a l samples c o l l e c t e d by t h i s t e c h n i q u e  p o o l e d and s t o r e d i n s e a l e d v i a l s a t -40°C.  Immediately  were after  f e c a l removal, t h e f i s h were r e t u r n e d t o t h e i r r e s p e c t i v e t a n k s and q u a n t i t a t i v e continued.  f e c a l c o l l e c t i o n w i t h t h e "Guelph system" was  The amount o f f e c e s c o l l e c t e d by t h e s t r i p p i n g method  was l a t e r t a k e n i n t o account when c a l c u l a t i o n s  of  nutrient  d i g e s t i b i l i t y based on t h e t o t a l c o l l e c t i o n p r o c e d u r e 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 c u l m i n a t i o n o f t h e experiment on day 22, a l l f i s h were e u t h e n i z e d u s i n g 2 ml o f 2-phenoxyethanol/L o f water, b l o t t e d d r y on a t o w e l and weighed. stripped,  A l l f i s h , e x c l u d i n g those p r e v i o u s l y  had t h e c o n t e n t s  o f t h e i r lower i n t e s t i n e between t h e  p e l v i c f i n s and t h e anus removed by d i s s e c t i o n .  B l o o d was  c a r e f u l l y removed by absorbent t o w e l from t h e abdominal p r i o r t o and a f t e r s e v e r i n g t h e i n t e s t i n e t o m i n i m i z e contamination.  A l l collections  cavity  fecal  o b t a i n e d i n t h i s manner were  p o o l e d t o g e t h e r and s t o r e d a t -40°C i n a s e a l e d p l a s t i c v i a l .  50 3.2.7  Chemical a n a l y s e s o f f e e d and  feces  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  F r o z e n f e c a l samples were l y o p h i l i z e d and ground u s i n g 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  u s i n g t h e "Guelph system" were removed p r i o r t o w e i g h i n g u s i n g a 250 um s i e v e and a l l n e c e s s a r y sample t r a n s f e r s were q u a n t i t a t i v e l y u s i n g a f i n e camel h a i r b r u s h . collections  (0830 hours)  performed  The morning f e c a l  f o r each i n d i v i d u a l tank were p o o l e d  a weekly b a s i s , as were t h o s e f o r t h e a f t e r n o o n c o l l e c t i o n s hours). ash  Feed and f e c a l , samples were a n a l y z e d f o r m o i s t u r e  (AOAC, 1975)  n i t r o g e n was m u l t i p l i e d by 6.25 Energy c o n t e n t was  Phillipson  and  t o e s t i m a t e crude  The  percent  protein  determined by t h e procedure  of  (1964) and i n v o l v e d t h e use o f a microbomb c a l o r i m e t e r  manufactured U.S.A..  (1430  and t o t a l n i t r o g e n (Technicon I n s t r u m e n t a l Co.  L t d . , i n d u s t r i a l methods 369-75A/A and 334-74W/B)...  content.  on  by Gentry Instruments  I n c . , A i k e n , South  Crude l i p i d c o n t e n t o f t h e d i e t s was  B l i g h and Dyer (1959) method.  Carolina,  determined by t h e  A l l d e t e r m i n a t i o n s were done i n  duplicate.  3.2.7.2  Chromic o x i d e d e t e r m i n a t i o n s  F o l l o w i n g e x t e n s i v e t e s t i n g o f methods a v a i l a b l e f o r t h e d e t e r m i n a t i o n o f chromic o x i d e , such as t h o s e by Fenton Fenton  (1979) and L i e d et al. (1982), t h e wet  c o l o r i m e t r i c method o f Stevenson as t h e p r e f e r r e d method. determined  and  digestion  and De Langen (1960) was  chosen  R e c o v e r i e s o f chromic o x i d e were  a f t e r a d d i t i o n o f known amounts o f t h e marker t o a dry  51 d i e t mash m i x t u r e .  A s t o c k m i x t u r e c o n t a i n i n g 5.0 mg C ^ C ^ p e r  gram o f mash was p r e p a r e d by adding 5.0 g o f d r y 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 J e r s e y ) t o 995.OOg o f a f i n e l y dry d i e t mash.  ground  A mortar and p e s t l e was used i n t h e m i x i n g  p r o c e s s t o ensure t h a t no lumps remained. A homogeneous mix was a c h i e v e d by f u r t h e r b l e n d i n g i n a s m a l l Hobart mixer f o r an a d d i t i o n a l 90 m i n u t e s .  Sub s t o c k s o f 3, 2 and 1 mg C ^ C ^ / g mash  were p r e p a r e d by t a k i n g a l i q u o t s o f t h e s t o c k mix and m i x i n g w i t h mash d i e t as d e s c r i b e d above.  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 performance Data on f e e d and f e c e s c o m p o s i t i o n were used t o 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 o f o r g a n i c m a t t e r , p r o t e i n and g r o s s energy.  crude  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 s e t o f 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 a c c o r d i n g t o t h e d i r e c t method and t h e 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  ADCi (%) = 100  f e c e s d r y weight x % n u t r i e n t i n f e c e s 1-  d i e t f e d weight x % n u t r i e n t i n d i e t  % Cr203 i n d i e t x % n u t r i e n t i n f e c e s % C^C^  i n feces x % n u t r i e n t i n d i e t  (ADCd)  52 The above formulae were a l s o used f o r t h e d e t e r m i n a t i o n o f apparent g r o s s 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 g r o s s energy d a t a (MJ/kg) r e p l a c i n g t h e n u t r i e n t term i n t h e f o r m u l a e . To d e t e r m i n e weekly apparent d i g e s t i b i l i t y  coefficients  based on t h e i n d i c a t o r method, t h e 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 p o o l e d morning and weekly p o o l e d a f t e r n o o n samples had t o be combined.  However,  since t h e q u a n t i t y o f f e c a l organic matter c o l l e c t e d i n t h e morning d i f f e r e d from t h a t c o l l e c t e d i n t h e a f t e r n o o n , t h 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 f o r t h e morning and a f t e r n o o n 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 t o the  weekly f e c a l o r g a n i c m a t t e r o u t p u t . S p e c i f i c growth r a t e , which d e s c r i b e s t h e p e r c e n t i n c r e a s e  i n body weight p e r day ( B r e t t , 1979) was c a l c u l a t e d a s :  (logn t l - l o g n tO) ( t l - tO)  x 100  Logn t l and l o g n tO a r e t h e n a t u r a l l o g wet w e i g h t s a t t h e s t a r t (tO) and t h e end ( t l ) o f t h e p e r i o d . d a i l y dry feed intake  U s i n g d a t a f o r t h e mean  (DFI) p e r f i s h f o r t h e p e r i o d , f e e d i n t a k e  was e x p r e s s e d as a p e r c e n t a g e o f mean wet body weight a c c o r d i n g t o R i c h a r d s o n e t a l . (1985).  The f o r m u l a used was as f o l l o w s :  DFI (logn t l + l o g n tO  - x 100 2) e  53 3.2.9  Statistical  analyses  The a n a l y s i s o f t h e experiment  was performed  i n two p a r t s .  P a r t one was t h e a n a l y s i s o f t h e d i r e c t v e r s u s t h e i n d i r e c t method o f d e t e r m i n i n g apparent n u t r i e n t coefficients. experiment.  digestibility  T h i s p a r t was a n a l y z e d as a 2 x 3 x 3  factorial  The e i g h t e e n t r e a t m e n t s r e s u l t e d from a c o m b i n a t i o n  of two 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 t h r e e f i s h management t e c h n i q u e s  (direct vs i n d i r e c t ) ,  ( r e s t r i c t e d f e e d i n g - f i s h not  h a n d l e d v s r e s t r i c t e d f e e d i n g - f i s h h a n d l e d weekly v s s a t i a t i o n f e e d i n g - f i s h n o t handled) and t h r e e time r e p l i c a t i o n s  (week 1 v s  week 2 v s week 3 ) . P a r t two was t h e a n a l y s i s o f t h e e f f e c t o f 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 . was a l s o a n a l y z e d as a 2 x 3 x 3 f a c t o r i a l c o l l e c t i o n times techniques  P a r t two  w i t h two f e c a l  (morning v s a f t e r n o o n ) , t h r e e f i s h management  ( r e s t r i c t e d f e e d i n g - f i s h not h a n d l e d v s r e s t r i c t e d  f e e d i n g - f i s h h a n d l e d weekly v s s a t i a t i o n f e e d i n g - f i s h not handled) and t h r e e time r e p l i c a t i o n s 3).  (week 1 vs week 2 v s week  The a n a l y s e s a r e 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 t o 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 t r e a t m e n t s , i t was f i r s t n e c e s s a r y t o c o n f i r m t h a t t h e assumptions  regarding equality of  v a r i a n c e between t h e t r e a t m e n t s were t r u e and H a r t l e y ' s t e s t was used f o r t h i s purpose as d e s c r i b e d by Lyman (1984).  Where  n e c e s s a r y , t h e d a t a were t r a n s f o r m e d by a r c s i n square r o o t and T o r r i e , 1960).  (Steel  Data i n each f a c t o r i a l were s u b j e c t e d t o  a n a l y s i s o f v a r i a n c e and t o Newman-Keuls ( K e u l s , 1952) m u l t i p l e range t e s t , u s i n g t h e 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 g r o s s 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 a c c o r d i n g t o the i n d i r e c t method but c o u l d 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 f e e d i n t a k e d a t a were a l s o s u b j e c t e d t o a n a l y s i s o f v a r i a n c e p r o c e d u r e s and t o Newman-Keuls m u l t i p l e range t e s t u s i n g the SAS  computer program.  55 3.3  RESULTS  3.3.1  Diets The r e s u l t s o f p r o x i m a t e and c h e m i c a l a n a l y s e s o f t h e d i e t s  are g i v e n i n Table 2.  Only s m a l l v a r i a t i o n s between t h e two  d i e t s were found i n t h e l i p i d ,  ash, m o i s t u r e and major m i n e r a l  c o n t e n t . 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 e x t r e m e l y h a r d and t h e 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 c o a t e d each p e l l e t w i t h a heavy l a y e r o f o i l .  Some f i s h o f f e r e d t h e  p r o t e i n - f r e e d i e t were observed t o t a k e a p e l l e t i n t o t h e i r mouth and t o i m m e d i a t e l y e j e c t i t , w h i l e o t h e r s o n l y l o s t a f t e r r e p e a t e d attempts t o m a s t i c a t e t h e p e l l e t s  3.3.2  interest  failed.  Chromic o x i d e a n a l y s i s The r e s u l t s o f t h e chromic o x i d e r e c o v e r i e s o b t a i n e d w i t h  t h e wet a s h i n g c o l o r i m e t r i c method o f Stevenson (1960) a r e p r o v i d e d i n Table 3. of  and De Langen  I n g e n e r a l , more than 94%  (mean  a l l c o n c e n t r a t i o n s = 96.5%) o f t h e chromic o x i d e was  recovered.  S m a l l l o s s e s o f chromic o x i d e , r e g a r d l e s s o f t h e t e s t  c o n c e n t r a t i o n , may have been due t o l o s s e s w h i l e m i x i n g as w e l l as t h e l a c k o f p u r i t y o f t h e chromic o x i d e used.  Nevertheless,  b e s t r e c o v e r i e s were a t t a i n e d when t h e chromic o x i d e c o n t e n t o f t h e sample was between 3.2 and 4.0 mg.  The s i z e o f subsequent  samples o f f e e d and f e c e s b e i n g a n a l y z e d f o r C^C^,  was t h e r e f o r e  a d j u s t e d t o y i e l d a marker c o n t e n t which would f a l l  approximately  w i t h i n t h i s range. c o n t r o l d i e t was  The chromic o x i d e c o n c e n t r a t i o n o f t h e  determined t o be 4.49  and t h e c o e f f i c i e n t o f v a r i a t i o n was (n=ll).  mg/g  dry d i e t ± 0.114  c a l c u l a t e d t o be  2.45%  (SD)  57 TABLE 2. P r o x i m a t e c o m p o s i t i o n , energy c o n t e n t and m i n e r a l c o n t e n t 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 o f Experiment 1. DIET Analysis Proximate composition 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 ) Ash M o i s t u r e (as fed) Gross Energy (MJ/kg) Mineral content C a l c i u m (Ca) Phosphorus (P) Sodium (Na) Chromic o x i d e ( C ^ C ^ ) Magnesium (Mg) Copper (Cu) I r o n (Fe) Manganese (Mn) Z i n c (Zn) C o b a l t (Co) Ca:P x  2  Control Protein-free (% o f d r y matter) 52.12 0.0 17.25 17.30 10.58 11.33 7.30 8.55 23.03 —  (g/kg d r y d i e t ) 24.0 24.0 14.7 16.9 5.51 7.13 4.49 4.87 1.86 2.05 0.014 0.024 0.319 0.567 0.094 0.121 0.151 0.226 <0.0005 0.002 1.63 1.42  Determined by plasma e m i s s i o n s p e c t r o s c o p y (Higgs et al., 1982) . Determined by t h e wet d i g e s t i o n c o l o r i m e t r i c method o f Stevenson and De Langen (1960).  58  Table 3. Recovery o f chromic o x i d e from a f e e d mash m i x t u r e u s i n g t h e wet a s h i n g c o l o r i m e t r i c method o f Stevenson and De Langen (1960).  Mg C r 0 assayed 2  3  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 measuring  o f t h e d i r e c t and i n d i r e c t methods o f digestibility.  The t h r e e 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 o r g a n i c m a t t e r , crude p r o t e i n  and g r o s s energy measured by t h e d i r e c t  (total collection)  and i n d i r e c t  (chromic o x i d e marker) method are  shown i n Table 4.  D i f f e r e n c e s between t h e d i r e c t and t h e  i n d i r e c t methods o f measuring apparent n u t r i e n t were not s i g n i f i c a n t nutrients  digestibilities  (P>0.05) a l t h o u g h t h e v a l u e s f o r a l l  were s l i g h t l y h i g h e r w i t h t h e d i r e c t method.  S t a t i s t i c a l comparisons o f d i g e s t i b i l i t y v a l u e s d e t e r m i n e d from f e c e s a c q u i r e d by t h e i n t e s t i n a l d i s s e c t i o n p r o c e d u r e were not p o s s i b l e the  and  stripping  due t o a l a c k o f r e p l i c a t i o n s , g i v e n  l i m i t e d sample s i z e o f f e c e s c o l l e c t e d w i t h t h e s e two  techniques.  Apparent d i g e s t i b i l i t y v a l u e s o b t a i n e d f o r f e c e s  c o l l e c t e d d i r e c t l y from t h e f i s h by s t r i p p i n g and were, however, c o n s i s t e n t l y  dissection  lower t h a n t h e d i r e c t and  indirect  values calculated  f o r f e c e s c o l l e c t e d from t h e water by t h e  "Guelph system".  The s t r i p p i n g t e c h n i q u e tended t o y i e l d t h e  l o w e s t d i g e s t i b i l i t y v a l u e s , b e i n g 3.5 p e r c e n t a g e u n i t s o r g a n i c m a t t e r 4.5 p e r c e n t a g e u n i t s percentage u n i t s  lower f o r p r o t e i n  lower f o r  and  3.4  lower f o r g r o s s energy d i g e s t i b i l i t y t h a n v a l u e s  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 o r g a n i c m a t t e r , crude p r o t e i n  and g r o s s energy between weeks 1, 2  and 3 were found not t o be s i g n i f i c a n t  (P>0.05),  indicating that  the minimum f e c a l c o l l e c t i o n t i m e i s d e t e r m i n e d p r i m a r i l y by t h e s i z e o f t h e sample r e q u i r e d f o r a n a l y s i s t o m i n i m i z e weekly d i g e s t i b i l i t y  r a t h e r t h a n by t h e need  variations.  60  3.3.4  Ingested chromic oxide recovery Besides being i n d i g e s t i b l e , 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 i s  thought to closely follow the s o l i d phase of the i n t e s t i n a l contents  (Balch et al., 1957).  To confirm t h i s i n chinook  salmon, the recovery rate of chromic oxide was evaluated by measuring the balance between the amount of chromic oxide ingested i n the food versus the amount recovered i n the feces. The recovery rate was noted to be more than 90% for r e p l i c a t e 5.  (Table 5) except  The overall average chromic oxide for a l l  r e p l i c a t e groups was  93.9%.  The loss of fecal p a r t i c l e s 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  T a b l e 4. Comparison o f t h e 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 d e t e r m i n e d by d i r e c t and i n d i r e c t measurement u s i n g t h e "Guelph system" o f 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 f e c e s 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 t e c h n i q u e s . Digestibility measuring method  Collection system  -'-Direct  "Guelph"  74.5 ±0.86  85.3 ±0.72  "Guelph"  73.2 ±0.74  84. 6 ±0.75  Indirect  APPARENT DIGESTIBILITY Organic Crude matter protein a 4  a  a  a  (%) ± SEM Gross energy 79.8 ±0.83  a  78. 9 ±0.72  ^Indirect  Intestinal dissection  65.0  75.0  71.9  ^Indirect  Stripping  61.5  70.5  68.5  a  A n a l y s i s o f v a r i a n c e 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 t h e f a c t o r , based on t h e "Guelph system" o f 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 l a c k o f s i g n i f i c a n t d i f f e r e n c e (P>0.05). V a l u e s f o r d i r e c t and i n d i r e c t methods based on t h e "Guelph system" a r e means ± SEM (n=24) f o r a t h r e e 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 t h e p e l v i c f i n s and t h e anus. F e c a l samples were c o l l e c t e d from f i s h n o t p r e v i o u s l y s t r i p p e d and were p o o l e d p r i o r t o c h e m i c a l a n a l y s e s . 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 o f f e c e s was performed on t h e same f i s h once weekly, f o r t h r e e weeks. F e c a l samples were p o o l e d p r i o r t o c h e m i c a l a n a l y s e s . S t a t i s t i c a l a n a l y s i s was n o t p o s s i b l e due t o l a c k o f r e p l i c a t e s . W i t h i n a column, v a l u e s w i t h a common s u p e r s c r i p t l e t t e r are 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 (P>0.05).  62 Table 5. Recovery r a t e o f chromic o x i d e ( C ^ C ^ ) 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 .  Chromic o x i d e  (mg)  Recovery  Replicate  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 o f time between f e c a l c o l l e c t i o n s on  fecal  n u t r i e n t l o s s due t o l e a c h i n g The e f f e c t o f n u t r i e n t l e a c h i n g l o s s e s from f e c e s e v a l u a t e d by comparing t h e t h r e e week mean apparent  was  digestibility  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 t h e water at 6 hour ( a f t e r n o o n c o l l e c t i o n ) and 18 hour (morning  collection)  i n t e r v a l s . These v a l u e s , t o g e t h e r w i t h t h o s e from f e c e s o b t a i n e d d i r e c t l y from the f i s h are p r e s e n t e d i n Table 6.  Digestibility  v a l u e s f o r o r g a n i c m a t t e r , crude p r o t e i n and g r o s s energy determined  on f e c a l samples c o l l e c t e d i n the a f t e r n o o n were  significantly next morning.  (P<0.001) lower than those o f samples c o l l e c t e d the The l o n g e r c o n t a c t p e r i o d o f the f e c e s w i t h t h e  water i n c r e a s e d the o p p o r t u n i t y f o r n u t r i e n t s t o l e a c h out, t h e r e b y c a u s i n g d i g e s t i b i l i t i e s t o be o v e r e s t i m a t e d .  Feces  r e m a i n i n g i n water f o r t h e l o n g e r time p e r i o d i n c u r r e d a l o s s o f 11.7%  organic matter,  13.9%  crude p r o t e i n and 9.9%  g r o s s 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 v a l u e s o b t a i n e d f o r t h e s h o r t e r p e r i o d by 7.9, respectively.  10.7  and 7.3 p e r c e n t a g e  units  F e c a l samples c o l l e c t e d from t h e water i n the  a f t e r n o o n had an i n c r e a s e d e s t i m a t e o f o r g a n i c m a t t e r ,  crude  p r o t e i n and g r o s s energy d i g e s t i b i l i t y o f 2.3,  1.6  percentage  1.7  and  u n i t s r e s p e c t i v e l y compared t o v a l u e s f o r f e c e s  o b t a i n e d by d i s s e c t i o n .  A l l o w i n g f e c e s t o remain i n t h e water an  a d d i t i o n a l 12 hours (morning c o e f f i c i e n t s by 10.2,  12.4  c o l l e c t i o n ) t h u s i n c r e a s e d the  and 8.9 percentage  m a t t e r , p r o t e i n and energy r e s p e c t i v e l y . between d i g e s t i b i l i t i e s determined  u n i t s f o r organic  The c l o s e agreement  f o r f e c e s c o l l e c t e d i n the  a f t e r n o o n w i t h t h e "Guelph system" and t h o s e 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 t h a t l e a c h i n g o f n u t r i e n t s from f e c e s was minimal d u r i n g t h e i n i t i a l the water.  D u r i n g the experiment,  i t was  6 hours i n  observed t h a t a l a r g e  p o r t i o n o f the f e c e s was not c a r r i e d i n t o t h e 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 p i p e c o n n e c t i n g t h e tank bottom d r a i n t o the c o l l e c t i o n column.  The  f e c e s i n t h i s p i p e were thus  exposed t o a c o n s t a n t water c u r r e n t 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 l o s s e s and c o n s e q u e n t l y  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 v a l u e s observed between morning and a f t e r n o o n c o l l e c t i o n times.  fecal  I t a l s o e x p l a i n s , t o some e x t e n t , 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 The  fish.  lowest d i g e s t i b i l i t y v a l u e s f o r o r g a n i c m a t t e r ,  crude  p r o t e i n and g r o s s energy were o b t a i n e d f o r f e c a l samples o b t a i n e d by t h e s t r i p p i n g t e c h n i q u e .  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 o r g a n i c m a t t e r , crude p r o t e i n and g r o s s energy by j u v e n i l e c h i n o o k salmon i n seawater f e d t h e 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 t i m e between c o l l e c t i o n s . Feces c o l l e c t i o n system and t i m e between collections ^Stripping -^Intestinal 4  dissection  " G u e l p h system" (6 hours)  ^"Guelph system" (18 hours)  ^-APPARENT DIGESTIBILITY Organic Matter  Crude Protein  (% ± SEM) Gross Energy  61.5  70.5  68.5  65.0  75.0  71.9  **67.3 ±0.98  **76.7 '±1.51  **75.2 ±0.57  **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 t h e i n d i r e c t method. S t r i p p i n g o f f e c e s was p e r f o r m e d on t h e same f i s h once weekly, f o r t h r e e weeks. F e c a l samples were p o o l e d p r i o r t o c h e m i c a l a n a l y s e s . S t a t i s t i c a l a n a l y s i s was n o t p o s s i b l e due t o l a c k o f replicates. Feces d i s s e c t e d out from h a l f way between t h e 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 p o o l e d p r i o r t o c h e m i c a l a n a l y s e s . S t a t i s t i c a l a n a l y s i s was n o t p o s s i b l e due t o l a c k o f replicates. Feces accumulated f o r a maximum p e r i o d o f 6 hours p r i o r t o b e i n g c o l l e c t e d by t h e "Guelph system" a t 1430 hours i n each tank d a i l y . Feces accumulated f o r a p e r i o d o f 18 hours p r i o r t o b e i n g c o l l e c t e d by t h e "Guelph system" a t 0830 hours i n each tank daily. Mean v a l u e s (n=24) i n t h e same column p r e c e d e d 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 o t h e r (P<0.001).  4  66  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 The  fish mortality  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 m a t t e r , crude p r o t e i n and g r o s s energy as d e t e r m i n e d by the i n d i r e c t method u s i n g weekly f e c a l c o l l e c t i o n s were significantly  (P<0.001)  weekly h a n d l i n g  lower f o r groups o f f i s h s u b j e c t e d t o  than f o r those not d i s t u r b e d  (Table 7).  The d a t a  i n T a b l e 8 a l s o r e v e a l t h a t t h o s e groups o f f i s h w h i c h had t h e i r f e c e s 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 (P<0.05) d a i l y f e e d i n t a k e  (expressed  as a p e r c e n t o f wet body  weight) t h a n groups o f f i s h n o t d i s t u r b e d . from f i s h h a n d l i n g  lower  The s t r e s s r e s u l t i n g  l e d t o a dramatic l o s s o f appetite f o r a  p e r i o d o f t h r e e t o f o u r days subsequent t o s t r i p p i n g . The handling  s t r e s s and the lower f e e d 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 r a t e  and h i g h e r m o r t a l i t y r a t e f o r t h e s e f i s h significant differences undisturbed  8).  No  (P>0.05) were o b s e r v e d between  f i s h on a r e s t r i c t e d o r s a t i a t i o n f e e d i n g  f o r s p e c i f i c growth r a t e . undisturbed  (Table  protocol  S u r p r i s i n g l y , t h e feed i n t a k e o f  f i s h f e d a r e s t r i c t e d amount o f f e e d , was found n o t  t o 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 t h o s e f i s h f e d t o satiation  (Table  8).  67 Table 7. E f f e c t o f f i s h management ( f e e d i n g regime and f i s h h a n d l i n g ) on mean apparent o r g a n i c m a t t e r , crude p r o t e i n and g r o s s 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 d a t a from f e c a l samples c o l l e c t e d i n t h e morning and i n t h e a f t e r n o o n over a t h r e e week c o l l e c t i o n period.  F i s h management  •'-Apparent d i g e s t i b i l i t y (%) ± SEM  ^Feed i n t a k e (% BW/day)  Fish handling  organic matter  crude protein  gross energy  0.22  weekly  69.7 ±0.51  l ±0.73  75.7 ±0.47  0.48  no  76.0 ±0.62 b  87. l ±0.56  b  81.5 ±0.72  0.50  no  74. 4 ±1.12  8 6. 0 ±1.06  79.6 ±1.22  a 3  b  81.  a  b  a  b  b  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 t h e i n d i r e c t (0:^03) method. A n a l y s i s o f v a r i a n c e i n d i c a t e d a s i g n i f i c a n t d i f f e r e n c e 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 . V a l u e s a r e t h r e e week means ± SEM w i t h n=9 f o r f i s h f e d 0.22 and 0.48 % BW/day and n=6 f o r f i s h f e d 0.50 % BW/day r e s p e c t i v e l y . Feed i n t a k e e x p r e s s e d as a p e r c e n t a g e o f body weight was c a l c u l a t e d a c c o r d i n g t o t h e f o r m u l a : DFI x 100 (logn t l + l o g n tO 2 ) , where DFI i s t h e mean 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 and l o g n tO, l o g n t l a r e t h e average n a t u r a l l o g w e i g h t s a t t h e s t a r t (tO) and a t t h e end ( t l ) of the p e r i o d . W i t h i n a column, v a l u e s w i t h a common s u p e r s c r i p t l e t t e r are 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 w i t h P=0.05) e  Table 8. E f f e c t o f 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 o f 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 t h e c o n t r o l d i e t . -'-Performance  F i s h management Feeding protocol  Fish handling  Feed i n t a k e S p e c i f i c , Mortality (% BW/day) growth r a t e 0.027 ±0.006  a  20/60  b  0.192 ±0.013  b  3/60  b  0.178 ±0.026  b  6/60  Restricted  weekly  0.22 +0.01  Restricted  no  0.48 ±0.02  Satiation  no  0.50 ±0.0 9  a 2  S i g n i f i c a n t d i f f e r e n c e s were n o t e d i n f e e d i n t a k e (P<0.05) and s p e c i f i c growth r a t e (P<0.001) between t h e d i f f e r e n t f i s h management t e c h n i q u e s . Feed i n t a k e e x p r e s s e d as a p e r c e n t a g e o f body weight was c a l c u l a t e d a c c o r d i n g t o t h e f o r m u l a : DFI x 100 •+ l o g n t l + l o g n tO -s- 2 ) , where DFI i s t h e mean 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 and l o g n tO, l o g n t l a r e t h e average n a t u r a l l o g w e i g h t s a t t h e s t a r t (tO) and a t t h e end ( t l ) o f t h e p e r i o d . S p e c i f i c growth r a t e d e t e r m i n e d f o r t h e 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. W i t h i n each column, v a l u e s 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 w i t h P=0.05) e  69 3.4  DISCUSSION  3.4.1  Digestibility Digestibility  collected  determinations of  determinations  e i t h e r q u a n t i t a t i v e l y or  d e p e n d i n g on  whether the  to  n a t u r a l l y voided collection  present  and  them b e i n g  that  quantities  of water.  regarding  the  Since  lost  the  the  fish,  being  any  biased  seawater,  dilute sample.  the  feces  constituents  leaching  quantity  determination proportion f o r the salt as  feces  treated  f a c t o r must be salt  will  method  and  that  prior in  animals,  fish  one  of  large  remains  through  leaching.  i f t h e y were a b s o r b e d  by  digestibility coefficients are  c o l l e c t e d from  Upon d r y i n g ,  a  remain i n the  sample w h i c h  will  constituents  between t h e  lipid,  and  however, r e m a i n c o n s t a n t feces.  i s b a s e d on  in  organic  carbohydrate)  contaminating the  the  equation  (see  i s an  the  matter the  regardless  Given that  the  dry  weight  effect  e q u a t i o n s below) as organic  of  relative  d i r e c t methods r e s p e c t i v e l y , t h e  " n u t r i e n t " i n the  of  however,  of n u t r i e n t t o marker or n u t r i e n t t o t o t a l  indirect  portion,  suspended i n  feces  be  considered.  proportion  of d i g e s t i b i l i t y  samples  determinations  When f e c e s  c o n t a m i n a t i o n becomes a n n u l l e d  the  as  of a l l f e c a l  protein,  of•salt  are  from the  values.  seawater  u n c h a n g e d as p o s s i b l e  r e s u l t s i n the  i n d i g e s t i b l e marker w i l l , the  are  toward higher  (e.g.  indirect  C o n c e r n among n u t r i t i o n i s t s  nutrients  relative  fecal  encountered with t e r r e s t r i a l  concentrations  The  that  a representative  Digestibility  loss of nutrients  amount o f  raised in  It i s important  r e m a i n as  n a t u r a l l y voided  another  substantial  i s used.  analysis.  problems not  as  d i r e c t or the  determining d i g e s t i b i l i t y the  require  fish  nutrient.  of long  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 e q u a t i o n s f o r t h e (ADCi) and d i r e c t (ADCd) methods  ADCi (%) =  100  1-  ADCd (%) =  100  1-  indirect  % Cr2C>3 i n d i e t x % n u t r i e n t i n f e c e s %^Cr2C>3 i n f e c e s x % n u t r i e n t i n d i e t  f e c e s dry weight x % n u t r i e n t i n f e c e s d i e t f e d weight x % n u t r i e n t i n d i e t  In t h e case o f dry m a t t e r d i g e s t i b i l i t y  determinations  however, t h e e f f e c t o f s a l t c o n t a m i n a t i o n does not c a n c e l out.  itself  The d r y m a t t e r f r a c t i o n i s an a b s o l u t e r a t h e r t h a n a  r e l a t i v e v a l u e and i n c l u d e s b o t h t h e o r g a n i c and i n o r g a n i c constituents  (e.g. s a l t ) .  The c o n t a m i n a t i o n o f t h e f e c e s w i t h  s a l t w i l l erroneously underestimate dry matter  digestibility  r e g a r d l e s s o f whether t h e d e t e r m i n a t i o n s are made by t h e or t h e d i r e c t methods.  In t h e i n d i r e c t method,  salt  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 marker w h i l e t h e p e r c e n t d r y m a t t e r  indirect  indigestible  ( " n u t r i e n t " i n f o r m u l a above)  remains c o n s t a n t and c o n s e q u e n t l y dry m a t t e r d i g e s t i b i l i t y i s underestimated.  In t h e d i r e c t method, t h e amount o f t o t a l  dry m a t t e r w i l l i n c r e a s e when s a l t contaminates dry matter d i g e s t i b i l i t y  t h e sample and  i s consequently a l s o underestimated.  U n l e s s a c o r r e c t i o n i s made f o r t h e presence  of the  i n the feces, dry matter d i g e s t i b i l i t y values w i l l underestimated.  fecal  contaminant be  The i n t r o d u c t i o n o f such an e r r o r has, however,  been o v e r l o o k e d by some workers  (Seidman and Lawrence, 1985;  Lee  and Lawrence, 1985), w h i l e o t h e r s have r e s o r t e d t o washing out t h e s a l t s a d h e r i n g t o t h e f e c e s by r e p e a t e d l y r i n s i n g t h e sample  w i t h f r e s h water  (MacLeod,  1977/ De S i l v a and P e r e r a , 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 o r d e r  t o a v o i d b o t h t h e n u t r i e n t l e a c h i n g and t h e 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 t h e c o l l e c t i o n o f f e c e s d i r e c t l y  from  t h e seawater, most r e s e a r c h e r s w o r k i n g w i t h marine f i s h have opted t o c o l l e c t t h e f e c e s d i r e c t l y from t h e f i s h by p e r f o r m i n g an i n t e s t i n a l d i s s e c t i o n 1986) o r by s t r i p p i n g al.,  1986).  ( L i e d e t al.,  1982/ F e r r a r i s e t al.,  ( L a l l and B i s h o p , 1977, 1979/ Dabrowski et  These t e c h n i q u e s , as mentioned below, however, have  t h e i r shortcomings as w e l l .  Dry m a t t e 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 were, t h e r e f o r e , not c a l c u l a t e d i n t h e p r e s e n t study and r e l i a n c e was p l a c e d on o r g a n i c m a t t e r as w e l l as on crude p r o t e i n and g r o s s energy d i g e s t i b i l i t y  coefficients.  Another i m p o r t a n t t e c h n i c a l a s p e c t t o c o n s i d e r when f e c e s are c o l l e c t e d from t h e aquarium water i s t h e need t o s e p a r a t e f i s h s c a l e s from t h e f e c a l samples.  I n 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 h i g h (up t o 8.9% o f t h e t o t a l weekly " f e c a l " d r y weight) due p r i m a r i l y , t o a tendency o f f i s h t o swim i n and out o f t h e narrow d r a i n s l o t at t h e bottom o f t h e t a n k . F a i l u r e t o s c r e e n out t h e 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 b e i n g a f f e c t e d t h e most s i n c e s c a l e s a r e comprised of k e r a t i n .  3.4.2  Chromic o x i d e a n a l y s i s ' Numerous methods f o r t h e d e t e r m i n a t i o n o f chromic o x i d e  (C^C^) are s c a t t e r e d throughout the l i t e r a t u r e . (1982)  L i e d et a l .  d e s c r i b e a r a p i d p r o c e d u r e whereby a l i q u o t s from t h e  72 digest of the micro-Kjeldahl analyzed (AAS).  f o r chromium  nitrogen determination  (Cr) by atomic a b s o r p t i o n  technique are  spectrophotometry  The advantage o f t h i s p r o c e d u r e i s t h a t t h e c o n t e n t s o f  b o t h n i t r o g e n and chromium can be d e t e r m i n e d on samples as s m a l l as 40 mg r a p i d l y and w i t h ease.  Given t h e s m a l l amount o f f e c e s  c o l l e c t e d i n t h i s s t u d y , e s p e c i a l l y from t h e 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 , t h e use o f t h e AAS was t e s t e d on a few samples as d e s c r i b e d by L i e d e t al. (1982). concentration considerably  values  Chromium  o b t a i n e d by t h e AAS method f l u c t u a t e d  and d i f f e r e n c e s o f over 10% were o b s e r v e d when t h e  same sample was t e s t e d a g a i n o n l y 15 minutes l a t e r . Lee  e t al.  to  (1986), a c o m b i n a t i o n o f f a c t o r s c o n t r i b u t e t o t h e  l a r g e v a r i a b i l i t y o f chromium v a l u e s absorption  According  spectrophotometry.  stoichiometry,  o b t a i n e d by atomic  These i n c l u d e t h e flame  chromium o x i d a t i o n s t a t e , t h e p r e s e n c e o f  p o t e n t i a l i n t e r f e r e n c e s , and t h e i n a b i l i t y t o reproduce instrumental  operating conditions exactly.  optimized  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 o f gas f l o w s and b u r n e r p o s i t i o n f o r c o n t r o l o f 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 d e t e r m i n a t i o n  o f chromic  oxide  d e s c r i b e d by Stevenson and De Langen (1960) was chosen as t h e p r e f e r r e d method a f t e r a c c e p t a b l e  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  3.4.3  (Table 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  fish.  The r e s u l t s o f 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 t h a t t h e r e  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 t h e d i g e s t i o n c o e f f i c i e n t s f o r  o r g a n i c m a t t e r , crude p r o t e i n and g r o s s energy o b t a i n e d by the d i r e c t method and t h o s e s e c u r e d by the i n d i r e c t method u s i n g chromic o x i d e as t h e i n d i g e s t i b l e marker. Choubert (1986), u s i n g an a u t o m a t i c  De l a Noue and ,  f e c e s 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 o f chromic o x i d e 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 t h e  o t h e r hand, s t a t e d t h a t t h e use o f chromic o x i d e 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 o b s e r v i n g an apparent  s e l e c t i v e r e j e c t i o n o f the marker by t i l a p i a mossambicus).  (Sarotherodon  These f i s h , u n l i k e salmonids, were observed by the  same a u t h o r t o work t h e food i n t o t h e i r mouth and r e j e c t p a r t o f each b i t e t h e r e b y a l l o w i n g t h e more dense chromic o x i d e t o separate.  A l s o , t h e chromic  o x i d e c o n t e n t 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 o f t h e p r e s e n t study f u r t h e r show t h a t i t was p o s s i b l e t o r e c o v e r over 90% (over 98% i n one i n s t a n c e ) o f the chromic  o x i d e i n g e s t e d by the f i s h over a t h r e e week p e r i o d  (Table 5), which i n d i c a t e s t h e r e l i k e l y i s l i t t l e , during the process o f d i g e s t i o n .  Incomplete  i f any,  loss  r e c o v e r y o f chromic  o x i d e was l i k e l y due t o a l o s s o f s m a l l f e c a l fragments which, a f t e r b e i n g swept over the tank o v e r f l o w onto the f i n e mesh, c o u l d not be r e t r i e v e d c o m p l e t e l y . l i k e l y o c c u r r e d when water was decanted  Additional losses a f t e r c e n t r i f u g i n g as  w e l l as d u r i n g sample g r i n d i n g , s c r e e n i n g and between c o n t a i n e r s .  screen  transferring  Such l o s s e s a l s o e x p l a i n why t h 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 o b t a i n e d by the d i r e c t method were c o n s i s t e n t l y h i g h e r f o r o r g a n i c m a t t e r , crude p r o t e i n and energy  than  those  factor oxide  m e a s u r e d by  c o n t r i b u t i n g t o an i n g e s t e d over  Nevertheless,  found  between t h e  indirect  g r e a t e r than  al. Due  and  to the  and  partitioning  o f any  (total  D.E.,  and  necessary  fish.  f o r at l e a s t  Further,  possible daily  observed Silva  and  by  some g e n e r a l  quantity  of  collection  week w i l l  also  digestibility  (1983,  digestibilities  the  period  I n a b a e t a l . (1962),  Perera  used  coefficients  a longer  one  be  determination  unless the  warrants  the  method c a n  in digestibility  De  l a Noue  1984). measurements  of  were n o t p o s s i b l e .  observations with  respect to  the  o f 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  method  The  digestible  ( T a b l e 4)  (23.03 MJ/kg d i e t  was  energy  with the  x 78.9%  digestibility) MJ  18.17  of which  digestibility) lipid  o r 57.9%  and,  by  MJ/kg  (521.2 g p r o t e i n / k g  r e m a i n d e r b e i n g p r o v i d e d by  carbohydrate.  (D.E.) d e t e r m i n e d  c a l c u l a t e d t o be  x 23.848 MJ/kg p r o t e i n x 84.6%  extent,  collection) latter  (Table  d i f f e r e n c e s were  i n v o l v e d i n the  p r o t e i n w o u l d have c o n t r i b u t e d 10.52 diet  recovery  s m a l l amounts o f f e c e s c o l l e c t e d ,  results.  indirect  dry d i e t  of feces  De  100%  chromic analysis for  3 indicates that a c o l l e c t i o n  carbohydrate  Nevertheless,  available  yield  of seawater  variability  as t h o s e  (1980) and  of the  significant  labour  seven days i s not  effect  fluctuations  the  method  coefficients  Collection  negate the  lipid  t h a t no  sample r e q u i r e d f o r a n a l y s e s  period.  et  fact  direct  between weeks 1,2  Another p o s s i b l e  recovery  sample d i d n o t  the  of s i g n i f i c a n t  fecal  incomplete  to reduce the  digestibility  lack  method.  method d e m o n s t r a t e s t h a t t h e  satisfactorily of  indirect  a t h r e e week p e r i o d i s t h a t t h e  the marker i n a g i v e n 3).  the  to a  crude dry of lesser  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 t h e "Guelph system" The r e s u l t s o f t h i s study concur w i t h those o f v a r i o u s  authors 1985;  (Inaba et al.,  1962;  S p y r i d a k i s e t al.,  W i n d e l l e t al.,  1989)  1978a; V e n s - C a p p e l l ,  t h a t f e c e s c o l l e c t e d from water  g i v e h i g h e r 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 t h a n those from f e c e s o b t a i n e d d i r e c t l y from the f i s h . c o u l d r e s u l t from any one,  determined  This d i f f e r e n c e  or a l l o f the f o l l o w i n g  factors:  1. L e a c h i n g o f s o l u b l e m a t e r i a l from f e c e s i n t o the water, whereby d i g e s t i b i l i t y v a l u e s are 2. C o n t a m i n a t i o n  surrounding  overestimated.  o f f e c e s o b t a i n e d 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  o b t a i n e d by i n t e s t i n a l d i s s e c t i o n , c o n t a m i n a t i o n by b l o o d i s o f most c o n c e r n .  Such c o n t a m i n a t i o n s would t e n d t o cause  d i g e s t i b i l i t y v a l u e s t o be 3. The  underestimated.  f e c a l samples o b t a i n e d d i r e c t l y from t h e f i s h c o n t a i n  m a t e r i a l which would have been absorbed  b e f o r e the f e c e s were  n a t u r a l l y v o i d e d by t h e f i s h , t h u s b i a s i n g d i g e s t i b i l i t y v a l u e s downward. The apparent  d i g e s t i b i l i t y values f o r organic matter,  p r o t e i n and g r o s s energy i n t h i s experiment  i n c r e a s e d w i t h an  i n c r e a s e i n c o n t a c t time between f e c e s and water, n u t r i e n t l e a c h i n g took p l a c e .  crude  W i n d e l l e t al.  indicating that  (1978a) used f e c a l  samples o b t a i n e d by i n t e s t i n a l d i s s e c t i o n t o determine o f exposure t o water on the l o s s o f n u t r i e n t s .  the e f f e c t  These a u t h o r s  found t h a t the major l o s s i s i n c u r r e d d u r i n g t h e f i r s t hour o f immersion.  D u r i n g t h i s time about 21% o f t h e d r y m a t t e r , 12% of  t h e p r o t e i n and 4% o f t h e l i p i d s were l o s t , i n c r e a s i n g t h 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 by 11.5%, 10% and 3.7% r e s p e c t i v e l y . W i t h i n 16 h t h e l o s s e s o f n u t r i e n t s from t h e f e c e s reached 31%, 12% and 9.8%, r e s p e c t i v e l y , and t h e r e s p e c t i v e i n c r e a s 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 were 17%, 10% and 8.2%, a c c o r d i n g t o the a u t h o r s .  I n t h e p r e s e n t s t u d y , p r o t e i n l o s s e s due t o  l e a c h i n g over t h e 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 v a l u e s o b t a i n e d over a 16 hour p e r i o d by W i n d e l l e t al.  (1978a).  However, u n l i k e t h e i r f i n d i n g s , most o f t h e l o s s e s i n t h e p r e s e n t study o c c u r r e d a f t e r f e c e s had been exposed t o water between 6 and 18 h o u r s .  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 g r o s s energy d i g e s t i b i l i t i e s between f e c a l samples o b t a i n e d by d i s s e c t i o n , and t h o s e o b t a i n e d from f e c e s r e s t i n g i n water f o r a maximum o f 6 hours were o n l y 2.3, 1.7 and percentage  units, respectively.  1.6  On t h e o t h e r hand, t h e  d i f f e r e n c e s i n t h e same c o e f f i c i e n t s had i n c r e a s e d by 10.2, 12.4 and 8.9 p e r c e n t a g e  u n i t s i n t h e f e c a l samples a l l o w e d t o remain  i n water f o r a maximum o f 18 h o u r s .  These r e s u l t s c o n t r a d i c t  those o f Cho and S l i n g e r (1979), who found s i m i l a r  digestibility  c o e f f i c i e n t s between f e c e s o b t a i n e d by d i s s e c t i o n and t h o s e o b t a i n e d from f e c e s which had s e t t l e d i n u n d i s t u r b e d water overnight  (15.5 h o u r s ) .  Cho e t 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 m i n i m i z e d when f e c e s remain i n u n d i s t u r b e d water and t h a t most l o s s e s occur i f f e c a l p e l l e t s a r e broken up d u r i n g t h e 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 t h e p r e s e n t study t o t h e  Guelph f e c a l c o l l e c t i o n tank d e s i g n used by Cho and S l i n g e r (1979) and Cho e t al. (1982) may account between r e s u l t s .  f o r t h e disagreement  In the p r e s e n t 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 c o n n e c t i o n p i p e 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. water f l o w i n t o the t a n k s was  (1989).  When t h e  i n c r e a s e d t o ensure f e c e s were  c a r r i e d a t a p r o p e r speed between t h e tank and t h e  settling  column, most o f the s m a l l e r 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 t h e overflow.  Cho  and S l i n g e r (1979) and Cho  d i d not encounter  e t al.  (1982) p r o b a b l y  s i g n i f i c a n t f e c a l l o s s e s by way  i n t h e i r experiments  of the overflow  s i n c e they o p e r a t e d t h e i r system w i t h f r e s h  water which, b e i n g l e s s dense than s a l t water, a l l o w e d f e c e s 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 . 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  f l o w r a t e s were a d j u s t e d t o a l e v e l which p r e v e n t e d over t h e o u t f l o w .  Since study,  fecal losses  L e a c h i n g , t h e r e f o r e , most l i k e l y o c c u r r e d i n  the c o n n e c t i o n p i p e between the tank and the s e t t l i n g column s i n c e a p o r t i o n o f t h e f e c e s would have been exposed t o a c o n s t a n t f l o w o f water a t t h i s l o c a t i o n .  The d e s i g n o f the tank  can be g r e a t l y improved by r e d u c i n g t h e c r o s s - s e c t i o n o f the c o n n e c t i n g p i p e t o ensure t h a t 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 f e c e s t o the s e t t l i n g column.  Alternatively,  the c r o s s s e c t i o n o f the s t a n d p i p e above t h e s e t t l i n g column can be i n c r e a s e d t o p e r m i t h i g h e r water f l o w r a t e s i n t o the w i t h o u t a concomitant  tanks  i n c r e a s e i n v e l o c i t y i n the v e r t i c a l p i p e  and an i n c r e a s e i n f e c a l l o s s e s v i a t h e o v e r f l o w . The o c c u r r e n c e o f n u t r i e n t l e a c h i n g from f e c e s 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  a g a i n s t t h e d i f f e r e n t t e c h n i q u e s o f f e c e s c o l l e c t i o n from the  78 intestine.  Two  major problems w i t h the s t r i p p i n g t e c h n i q u e 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 t h a t 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 f e c e s s t r i p p e d . none, t o one  w e l l - f o r m e d p e l l e t , t o a s t r i n g or r i b b o n 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  w h i t e r i b b o n or f e c a l - c a s t was which had  T h i s amount ranged from  obviously  not  the p r o d u c t s t r i p p e d from f i s h  f e d f o r some t i m e .  The  fecal-casts  l a c k e d chromic o x i d e 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 . A l t h o u g h the method o f s t r i p p i n g f e c e s a v o i d s the problem o f leaching, according  to Austreng  (1978) i t a l s o  accelerates  passage o f m a t e r i a l t h r o u g h the d i g e s t i v e system w i t h a p o s s i b l e reduction  i n absorption.  The  methodological objections  the s t r i p p i n g t e c h n i q u e h o l d t r u e a l s o f o r the  against  intestinal  d i s s e c t i o n t e c h n i q u e , a l t h o u g h the e r r o r a s s o c i a t e d w i t h l a t t e r i s not as s e r i o u s , a c c o r d i n g 1978; al.,  Windell  1978a; Henken et a l . , 1985;  Spyridakis  one  a r t i f i c i a l removal o f f e c e s d i r e c t l y from the f i s h o f t h e s e t e c h n i q u e s assumes t h a t d i g e s t i o n and  are complete when the m a t e r i a l has large i n t e s t i n e .  Several  Georgopoulou et a l . , 1985) enterocytes  et  authors  absorption  reached the d i s t a l end (Gauthier  and L a n d i s ,  however, have d e s c r i b e d  by  of  the  1972;  the a b i l i t y  from the p o s t e r i o r i n t e s t i n e t o i n g e s t 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 . may  (Austreng,  1989). The  any  et al.,  to various authors  the  Furthermore, a n o t h e r source o f e r r o r  come from the assumption t h a t d i g e s t i b i l i t y  i s a continuous  of  and steady p r o c e s s , h a v i n g no v a r i a b i l i t y w i t h t i m e .  The  daily  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 o t h e r 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 . , P e r e r a , 1983,  1980; De S i l v a  1984), suggest t h a t s t r i p p i n g and  and  intestinal  d i s s e c t i o n t e c h n i q u e s a p p l i e d f o r one day w i l l not p r o v i d e 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 o f f i s h management on apparent digestibility,  nutrient  f e e d i n t a k e , growth r a t e and  fish  mortality There i s some disagreement a f f e c t e d by r a t i o n l e v e l Henken e t a l . ,  as t o whether d i g e s t i b i l i t y i s  ( H a s t i n g s , 1969; W i n d e l l et al.,  1985; Hudon and De l a Noiae, 1985;  A u s t r e n g , 1987).  Those r e s e a r c h e r s who  Storebakken  and  have c o n c l u d e d 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 , c o r r e l a t i o n t o be n e g a t i v e (Henken e t al., Noue, 1985).  1978b;  found t h e  1985; Hudon and De l a  In the present study the reverse occurred, w i t h  f i s h i n g e s t i n g t h e l e a s t amount o f f e e d showing t h e l o w e s t assimilation efficiency.  In c o n t r a s t t o t h e experiments  referred  t o above, however, f i s h i n t h i s study m a n i f e s t e d poor a p p e t i t e and c o n s e q u e n t l y poor f e e d i n t a k e because o f t h e h a n d l i n g s t r e s s caused by weekly s t r i p p i n g .  P r i m a r y s t r e s s e f f e c t s are  m a n i f e s t e d by i n c r e a s e d s e c r e t i o n r a t e s and plasma l e v e l s o f t h e corticosteroid  hormone C o r t i s o l  (Donaldson,  1981)  and o f t h e  c a t e c h o l a m i n e hormone a d r e n a l i n (Mazeaud and Mazeaud, Ohnesorge and Rauch (1968) demonstrated  1981).  that adrenalin i n h i b i t s  p e r i s t a l s i s o f t h e 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 f e e d i n t a k e  80 o b s e r v e d f o r t h r e e t o f o u r 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 e t al. (1982) w o r k i n g w i t h brown  trout  trutta).  {Salmo  Wedemeyer (1976) on t h e o t h e r hand, r e f e r s  t o 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,  b u t 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  f e e d i n g t h e next day.  U n l i k e t h e more d o m e s t i c a t e d 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 v e r y e a s i l y .  The  r e l a t i v e l y low f e e d i n t a k e e x p e r i e n c e d by even t h e groups o f undisturbed  f i s h , was due p a r t l y t o t h e low water t e m p e r a t u r e s  experienced during the t r i a l  (5°C t o 8°C), and p a r t l y t o t h e f i s h  b e i n g alarmed when anyone approached t h e t a n k s . The  v e r y low f e e d i n t a k e by t h e s t r i p p e d f i s h a l s o had a  n o t i c e a b l e e f f e c t on t h e appearance o f t h e f e c e s .  Much o f t h e  f e c e s c o l l e c t e d from t h e water, as w e l l as t h r o u g h subsequent s t r i p p i n g s , l a c k e d t h e d i s t i n c t i v e green c o l o r o f 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 , t h a t t h e d e p r e s s e d apparent d i g e s t i b i l i t y o f o r g a n i c m a t t e r , crude p r o t e i n and g r o s s energy o b s e r v e d i n d i s t u r b e d f i s h , was due n o t o n l y t o t h e p h y s i o l o g i c a l e f f e c t s o f s t r e s s on t h e 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 h i g h e r p r o p o r t i o n o f endogenous f e c a l wastes  being  collected. The  low s p e c i f i c growth r a t e and t h e extreme m o r t a l i t y r a t e  o b s e r v e d 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 due  s t r e s s as w e l l .  The e x c e s s i v e  t o repeated p h y s i c a l manipulation  loss of f i s h  scales  f o r i n s t a n c e , may have  upset t h e o s m o t i c b a l a n c e beyond t h e a n i m a l ' s o s m o r e g u l a t o r y c a p a b i l i t i e s , leading t o dehydration  and d e a t h .  81  In r e l a t i o n t o t h e u n d i s t u r b e d 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 f e e d i n t a k e between f i s h fed t o s a t i a t i o n and t h o s e m a i n t a i n e d on a r e s t r i c t e d level.  intake  Feed i n t a k e l e v e l s even p r i o r t o t h e s t a r t o f t h e  experiment were q u i t e low due t o t h e c o l d water t e m p e r a t u r e s (8°C).  The l e v e l o f f o o d i n t a k e o f t h e f i s h on t h e r e s t r i c t e d  p r o t o c o l was reduced t o 90% o f t h e maximum r a t i o n n o t e d a t 8°C p r i o r t o t h e b e g i n n i n g o f t h e experiment and t h e u n d i s t u r b e d f i s h r e a d i l y consumed t h i s r a t i o n throughout t h e s t u d y .  Fish fed to  s a t i a t i o n , however, d i d not consume much more f e e d t h a n t h o s e f e d the  r e s t r i c t e d amount s i n c e water t e m p e r a t u r e s and c o n s e q u e n t l y  a p p e t i t e d e c l i n e d t o t h e p o i n t where t h e r a t i o n s o f t h e two groups o f u n d i s t u r b e d f i s h were almost i d e n t i c a l t e m p e r a t u r e reached 5°C).  (water  I t i s noteworthy t h a t t h e growth  of t h e u n d i s t u r b e d f i s h on t h e r e s t r i c t e d r a t i o n were greater than those of f i s h f e d t o s a t i a t i o n .  rates  slightly  This difference,  though s m a l l , was p r o b a b l y due t o t h e l o s s i n weight o f t h e f i s h w h i l e on t h e p r o t e i n - f r e e d i e t f o r f o u r days.  Differences 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 o r g a n i c m a t t e r , crude p r o t e i n and g r o s s energy between t h e two f e e d i n g groups o f u n d i s t u r b e d f i s h were not s i g n i f i c a n t , a l t h o u g h m o r t a l i t y was t w i c e as h i g h (6 v e r s u s 3 f i s h out o f 60) i n t h e h i g h e r i n t a k e 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 matter,  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 chinook (Oncorhynchus  4.1  organic  tshawytscha)  salmon  i n seawater  INTRODUCTION F i s h meal i s w e l l r e c o g n i z e d as t h e b e s t source o f p r o t e i n  for salmonids.  The h i g h c o s t o f h i g h 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 f e e d f o r m u l a t i o n s . common p r a c t i c e i n animal husbandry i s t o p a r t i a l l y  The  or e n t i r e l y  r e p l a c e t h e h i g h - c o s t animal p r o t e i n s w i t h l e s s e x p e n s i v e  plant  and/or animal b y - p r o d u c t sources t o 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 r e d u c t i o n i n f e e d efficiency.  A l o n g term f e e d i n g t r i a l i s t h e most dependable  method o f measuring t h e n u t r i t i v e v a l u e o f a f e e d , b u t t h i s i s slow and t o o e x p e n s i v e .  In vitro  c h e m i c a l methods, w h i l e g i v i n g  some i n d i c a t i o n o f f e e d q u a l i t y , a r e n o t y e t r e l i a b l e enough t o indicate the a v a i l a b i l i t y species.  o f n u t r i e n t s i n a feed t o a p a r t i c u l a r  D i g e s t i o n t r i a l s a r e used e x t e n s i v e l y i n s t u d y i n g t h e  n u t r i t i o n o f d o m e s t i c and l a b o r a t o r y animals as w e l l as t h a t o f commercially  important  f i s h species.  Concerning  t h e 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 c o n f i n e d t o rainbow t r o u t r e a r e d i n f r e s h water w h i l e almost no s t u d i e s have been d i r e c t e d t o a s s e s s i n g f e e d s t u f f d i g e s t i b i l i t y by salmonids environment.  i n t h e marine  Owing t o t h e p a u c i t y o f such v i t a l i n f o r m a t i o n , a  s e r i e s o f t h r e e e x p e r i m e n t s were u n d e r t a k e n t o determine t h e apparent d i g e s t i b i l i t y o f o r g a n i c m a t t e r ,  crude p r o t e i n and  83  energy i n c o n v e n t i o n a l and n o v e l 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  "Guelph system" o f f e c a l  collection.  tshawytscha)  u s i n g the  84 4.2 MATERIALS AND METHODS 4.2.1 Experimental design In a s e r i e s o f t h r e e e x p e r i m e n t s , c h i n o o k salmon w i t h a minimum s i z e o f 10.3 g t o 40.5 g a t t h e s t a r t o f t h e v a r i o u s e x p e r i m e n t s , were f e d by hand t w i c e d a i l y t o s a t i a t i o n e i t h e r a reference diet  (REF-a e x p e r i m e n t s I I and I I I , REF-b experiment  IV) 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 ) .  case o f some p l a n t p r o t e i n s o u r c e s , t h e t e s t i n g r e d i e n t  In t h e  was  i n c l u d e d not o n l y a t 30% but a l s o a t 15% t o determine p o s s i b l e e f f e c t s o f a n t i n u t r i t i o n a l f a c t o r s or c a r b o h y d r a t e on digestibility coefficients.  Chromic o x i d e (0.5%) was i n c l u d e d i n  a l l d i e t s as t h e i n d i g e s t i b l e marker.  W i t h i n t e s t s , each d i e t  was a s s i g n e d t o t h r e e groups o f f i s h u s i n g a c o m p l e t e l y random (experiment I I ) o r randomized complete b l o c k d e s i g n (experiments I I I and I V ) . for  Feces were c o l l e c t e d from each tank e v e r y morning  f i f t e e n days u s i n g t h e "Guelph system" o f 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 o f o r g a n i c m a t t e r , crude p r o t e i n and g r o s s energy were d e t e r m i n e d f o r each t e s t ingredient.  A t t h e end o f each experiment, f e c e s were a l s o  o b t a i n e d by t h e s t r i p p i n g o r the i n t e s t i n a l  dissection  t e c h n i q u e s , t o o b t a i n d a t a f o r comparisons w i t h o r g a n i c m a t t e r d i g e s t i b i l i t y v a l u e s o b t a i n e d w i t h t h e "Guelph system".  4.2.2  History of experimental f i s h A p p r o x i m a t e l y 7,000 c h i n o o k salmon f r y were o b t a i n e d from  the  R o b e r t s o n Creek h a t c h e r y (Vancouver I s l a n d , B.C.)  1989.  i n May,  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 t a n k s s u p p l i e d w i t h 10°C t o 12°C a e r a t e d w e l l water.  BioDiet  ( B i o p r o d u c t s , I n c . , Warrenton, Oregon, U.S.A.)  85 was  f e d by h a n d t o s a t i a t i o n  weeks a n d t h e f r e q u e n c y was times  seven t i m e s d a i l y  J u n e 22,  1989,  t h e r e a f t e r g r a d u a l l y reduced t o three  f i s h h a d r e a c h e d t h e minimum w e i g h t  grams) r e q u i r e d t o e l i c i t individually  an immune r e s p o n s e  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  protect against v i b r i o s i s  Vibrio ordalii), Yersinia  On  July  and  (AquaHealth  V i b r i o anguillarum  Inc.) and  furunculosis  ( c a u s e d by t y p i c a l  isolates  salmonicida). 14,  1989,  B u r r a r d I n l e t seawater period.  (0.1  e n t e r i c r e d m o u t h d i s e a s e ( c a u s e d by t y p e 1  ruckeri),  Aeromonas  ( c a u s e d by  (4-6  and were  m l / f i s h ) w i t h Ermogen-Furogen-Vibrogen B a c t e r i n  of  3  daily. On  to  f o r the f i r s t  w e l l w a t e r was  3/4,  4/4)  B e f o r e commencement o f any e x p e r i m e n t ,  the  number o f f i s h was  i n steps  g r a d u a l l y r e p l a c e d by  ( 1 / 3 , 1/2,  o v e r a 7-day required  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  were d i s t r i b u t e d randomly  i n t o the d i g e s t i b i l i t y tanks.  fish Stocking  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 , n e v e r e x c e e d e d  10.5  kg/m . 3  4.2.3  Aquarium The  e x p e r i m e n t a l i n d o o r aquarium  West V a n c o u v e r Oceans  facility facility  'located at the  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  (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  These t a n k s were s i m i l a r t o t h o s e u s e d i n e x p e r i m e n t the o r i g i n a l bottom  3.81  (I.D.)  tanks.  I ; however,  c o n n e c t i o n p i p e between t h e  tank  and t h e s e t t l i n g c o l u m n i n t h e t a n k s i n e x p e r i m e n t  r e p l a c e d by a 2 cm swiftly  cm  and  (I.D.) p i p e t o e n s u r e t h a t f e c e s w e r e  from t h e tank t o t h e s e t t l i n g column f o r reasons  I  was  carried outlined  86  previously.  In a d d i t i o n , t o minimize f i s h scale l o s s  resulting  from f i s h r u b b i n g a g a i n s t t h e 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 r e c t 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 over t h e d r a i n s l o t .  flat  The c o r n e r s o f t h e p l a t e s were extended  s l i g h t l y outward t o a l l o w water and f e c e s t o f l o w f r e e l y between the  p l a t e edge and t h e t a n k t o t h e d r a i n s l o t , y e t t h e water  s u c t i o n e f f e c t around t h e edges was reduced enough t o p r e v e n t f i s h from g e t t i n g s t u c k .  Each tank was s u p p l i e d w i t h 6 L/min o f  f i l t e r e d B u r r a r d I n l e t seawater.  P r i o r t o f l o w i n g i n t o each  t a n k , t h e f i l t e r e d water was p a s s e d t h r o u g h a 60 cm l o n g 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 t o supplement t h e a e r a t i o n p r o v i d e d i n each tank by porous r u b b e r t u b i n g .  Water temperature v a r i e d  between e x p e r i m e n t s and ranged from 10.0°C t o 12.5°C i n experiment I I , 10.0°C t o 11.0°C i n experiment I I I and 8.0°C t o 9.5°C i n experiment IV. D i u r n a l water t e m p e r a t u r e f l u c t u a t i o n s d i d not exceed 1.0°C i n any o f t h e e x p e r i m e n t s .  Water s a l i n i t y  was m o n i t o r e d t w i c e d a i l y i n each experiment and v a l u e s v a r i e d between 28 p p t and 30 p p t i n experiment I I , 29 p p t and 31 p p t i n experiment I I I , and 30 p p t and 31 p p t i n experiment IV.  In a l l  e x p e r i m e n t s , f l u c t u a t i o n s i n water s a l i n i t y between r e a d i n g s (6 hours) v a r i e d from 1 t o 2 p p t on some days w h i l e r e m a i n i n g c o n s t a n t on o t h e r days.  The d i s s o l v e d oxygen l e v e l s were  m o n i t o r e d i n random t a n k s t h r o u g h o u t each experiment and t h e 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 d e t e r m i n a t i o n s . Some c h e m i c a l parameters o f t h e 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 p h o t o p e r i o d was p r o v i d e d 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  (Vitalite,  D u r o t e s t 40W)  controlled  by a p h o t o c e l l .  4.2.4  Water f i l t e r i n g The  system  f i l t e r i n g system used at the U n i v e r s i t y / D . F . O .  i n experiment  I was  facility  found t o be adequate at t h a t l o c a t i o n .  However, t h e l a r g e r i n f l u x o f sand, a l g a e , 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 t o the West Vancouver L a b o r a t o r y , o c c l u d e d t h e f i l t e r s c o m p l e t e l y w i t h i n 1 t o 2 days o f operation.  Subsequently,  two p e r f o r a t e d PVC  the V e x l a r mesh sandwiched between the  d i s c s i n each f i l t e r was  r e p l a c e d w i t h 7.6  t h i c k aquarium f i l t e r foam ( C y c l e guard A q u a c l e a r d i s t r i b u t e d by R.C.  cm  2000,  Hagen I n c . , M o n t r e a l , Quebec).  This f i l t e r  foam was 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 t h e passage o f even the s m a l l e s t i n v e r t e b r a t e s and d i d not p l u g up as suddenly o f t e n as w i t h t h e o t h e r system.  o r as  N e v e r t h e l e s s , f i l t e r s were  r e p l a c e d every t h i r d day throughout  each experiment.  The  f i l t e r e d seawater was passed t h r o u g h a f u n n e l c o n t a i n i n g a h a n d f u l o f aquarium p o l y - f i l t e r wool (R.C. Hagen I n c . , M o n t r e a l , Quebec) b e f o r e e n t r y i n t o each t a n k .  The wool was v e r y  effective  i n f i l t e r i n g the v e r y f i n e sediment which passed t h r o u g h  the  g r a v e l and foam f i l t e r s .  was  The wool f i l t e r i n each f u n n e l  r e p l a c e d every a f t e r n o o n b e f o r e f l u s h i n g the t a n k s t o remove uneaten f o o d and f e c a l  4.2.5  accumulation.  Diets A l l t e s t d i e t s i n experiments  30%  o f a t e s t i n g r e d i e n t and 70%  I I and I I I were composed o f  of a reference d i e t  ( a i r dry  b a s i s ) / w h i l e i n experiment IV, some o f t h e t e s t i n g r e d i e n t s were i n c o r p o r a t e d a t t h e 15% l e v e l o f i n c l u s i o n , w i t h t h e d i e t c o m p r i s i n g the b a l a n c e . was  Chromic o x i d e  (REF-b) was  The  4% h i g h e r i n p r o t e i n c o n t e n t  lower p r o t e i n c o n t e n t  The  The  the  r e f e r e n c e d i e t used i n e x p e r i m e n t s IV  (REF-a) used i n e x p e r i m e n t s I I and  IV.  (0.5% a i r dry b a s i s )  i n c l u d e d i n t o the r e f e r e n c e and t e s t d i e t s as  i n d i g e s t i b l e marker.  reference  than the reference  I I I t o compensate f o r the  o f the t e s t i n g r e d i e n t s used i n experiment  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  f o r m u l a t i o n s f o r the b a s a l mixes used i n the r e f e r e n c e  p r e p a r a t i o n s are l i s t e d i n Tables  11 and  12.  The  13,  14 and  15.  Each t e s t i n g r e d i e n t was  code and t h e same code was from one  another.  10.  diet  test  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 p r o x i m a t e c o m p o s i t i o n i n Tables  diet  are  listed  assigned  used t o d i f f e r e n t i a t e the t e s t  S i n c e some f e e d s t u f f s were t e s t e d a t  a  diets  two  l e v e l s o f 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 r e p r e s e n t s the l e v e l o f i n c o r p o r a t i o n (e.g. number "1" o r r e p r e s e n t i n g t h e 15% or 30% l e v e l o f i n c l u s i o n o f t h e 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  4.2.6  are shown i n Tables  16,  17 and  test  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 mineral contents  "2"  and  dietary  18.  Diet preparation D i e t s were p r e p a r e d  i n J u l y , 1989  (experiments  I I and I I I )  and i n October, 198 9 (experiment IV) a t t h e West Vancouver Laboratory prepared  (D.F.O.).  The m i n e r a l and v i t a m i n supplements were  as d e s c r i b e d i n experiment I , a l t h o u g h  f i n e l y ground  e x t r u d e d wheat r e p l a c e d t h e 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 s t o r e d i n t h e dark a t 15°C and  28% r e l a t i v e h u m i d i t y and, i f n e c e s s a r y , were ground i n a hammer mill  ( 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 s c r e e n (595 um) p r i o r t o 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 t h e b a s a l mix ( e x c l u d i n g t h e o i l ) were t h o r o u g h l y b l e n d e d i n a M a r i o n mixer (Rapids Machinery L t d . , M a r i o n , Iowa U.S.A.) f o r 30 m i n u t e s .  To  increase the moisture content of both b a s a l mixtures t o a p p r o x i m a t e l y 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 b a s a l mix 2 r e s p e c t i v e l y .  A total  o f one hundred k i l o g r a m s o f b a s a l mix 1 (experiments I I and I I I ) and f i f t y k i l o g r a m s o f b a s a l mix 2 (experiment IV) was p r e p a r e d . A p p r o x i m a t e l y 10 kg o f b a s a l mix 1 and 5 kg o f b a s 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 mill  ( C a l i f o r n i a P e l l e t M i l l Co., San F r 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 .  T h e r e a f t e r , t h e p e l l e t s were p l a c e d i n t o a  v e r t i c a l c o o l e r and t h e n t h e y were s p r a y e d w i t h h e r r i n g o i l as d e s c r i b e d 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 t o  wean t h e f i s h from t h e B i o D i e t p r i o r t o t h e s t a r t o f each o f t h e e x p e r i m e n t s and a l s o t o g e n e r a t e t h e chromic o x i d e - f r e e f e c e s r e q u i r e d f o r subsequent  chromic o x i d e a n a l y s e s .  A t o t a l o f 5 kg o f each d i e t was p r e p a r e d .  Water was added  t o each t e s t d i e t b e f o r e p e l l e t i n g t o e q u a l i z e t h e m o i s t u r e c o n t e n t i n a l l d i e t s a t 9%.  Chromic  o x i d e ( C ^ C ^ ) was mixed  into  each d i e t u s i n g a mortar and p e s t l e as d e s c r i b e d i n experiment I . 9 The r e f e r e n c e 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 Ohio).  (Hobart M a n u f a c t u r i n g Co., Troy,  Each d i e t was t h e n 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 ) o r a 3.97 mm  (experiment IV) d i e , t h e  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 F o w l e r and Burrows (1971).  A f t e r c o o l i n g , p e l l e t s were s p r a y e d  w i t h t h e h e r r i n g o i l as d e s c r i b e d i n experiment  I.  The d i e t s  were s t o r e d i n t h e dark i n t h i c k p l a s t i c bags a t 15°C and 28% r e l a t i v e humidity u n t i l required.  D i e t s i n use were kept i n  a i r t i g h t c o n t a i n e r s and were r e f r i g e r a t e d n i g h t l y a t 3°C t o 4°C.  91  Table 9. R e f e r e n c e 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 e x p e r i m e n t s I I and I I I  Reference d i e t (REF-a) Composition B a s a l mix l Test i n g r e d i e n t Chromic o x i d e z  995.0 0 5.0  Test d i e t ( l e v e l 2) (g/Kg) 696.5 298.5 5.0  Test d i e t s w i t h a 30% l e v e l o f i n c l u s i o n o f 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 o f b a s a l mix (air-dry basis). R e f e r t o Table 11 f o r b a s a l mix 1 f o r m u l a t i o n . I n g r e d i e n t s t e s t e d i n experiment I I i n c l u d e d : r a p e s e e d p r o t e i n concentrate(BRONOWSKI F R I - 7 3 - 5 s t r a i n ) ; c a n o l a meal ( c o m m e r c i a l l y a v a i l a b l e s o u r c e ) ; g l u c o s i n o l a t e - f r e e c a n o l a 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 s o u r c e ) ; anchovy meal ( C h i l e a n s o u r c e ) . I n g r e d i e n t s 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 byp r o d u c t meal (Kansas s o u r c e ) ; p o u l t r y b y - p r o d u c t meal ( B r i t i s h Columbia s o u r c e ) ; menhaden meal ( L o u i s i a n a s o u r c e ) ; h y d r o l y z e d f e a t h e r meal ( B r i t i s h Columbia s o u r c e ) ; LT (low temperature) h e r r i n g / c a p e l i n meal (Norwegian s o u r c e ) ; soybean meal; b l o o d meal ( B r i t i s h Columbia s o u r c e ) ; soybean p r o t e i n i s o l a t e .  92  Table 10. R e f e r e n c e 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 (REF-b)  Test d i e t ( l e v e l 1)  Composition B a s a l mix 2  995.0  Z  Test i n g r e d i e n t Chromic o x i d e  3  0 5.0  Test d i e t ( l e v e l 2)  (g/Kg)  845.7  149.3 5.0  696.5  298.5 5.0  Test i n g r e d i e n t at l e v e l 1 i s e q u i v a l e n t t o a 15% l e v e l o f i n c l u s i o n o f 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. W h i l e t e s t i n g r e d i e n t at l e v e l 2 i s e q u i v a l e n t t o 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 t h e b a s a l mix ( a i r - d r y b a s i s ) . R e f e r t o Table 12 f o r b a s a l mix 2 f o r m u l a t i o n . I n g r e d i e n t s t e s t e d a t 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 s o u r c e ) ; e x t r u d e d wheat ( B r i t i s h Columbia s o u r c e ) ; soybean meal; soybean p r o t e i n i s o l a t e . I n g r e d i e n t s 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 : e x t r u d e d wheat ( B r i t i s h Columbia s o u r c e ) ; wheat m i d d l i n g s ( B r i t i s h Columbia s o u r c e ) ; 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 c a n o l a meal.  93 Table 11. F o r m u l a t i o n o f b a s a l mix 1 used i n the p r e p a r a t i o n o f the r e f e r e n c e and t e s t d i e t s employed i n e x p e r i m e n t s I I and I I I . Ingredients  Concentration (g/kg d r y d i e t ) 550. 109. 75. 60. 50. 84. 10. 20. 20. 15. 5. 2.  S t e a m - d r i e d h e r r i n g meal (73 .58% CP)* E x t r u d e d wheat (17.83% CP)* D r i e d whey (14.13% CP)* Freeze-dried euphausids (71 .48 CP)* B l o o d meal (95.37% CP) Herring o i l Soybean l e c i t h i n Vitamin supplement M i n e r a l supplement Permapell (lignin-sulphonate binder) C h o l i n e c h l o r i d e (60%) Ascorbic acid 1  3  4  0 0 0 0 0 0 0 0 0 0 0 0  Dry m a t t e r 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% s a n t o q u i n and was s p r a y e d onto t h e 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 d r y d i e t : i n o s i t o l 400 mg; n i a c i n 300 mg; p a n t o t h e n a t e (as D - c a l c i u m p a n t o t h e n a t e ) 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 p y r i d o x i n e - H C l ) 36.5 mg; t h i a m i n e (as t h i a m i n e m o n o n i t r a t e ) 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-c<-tocopheryl a c e t a t e 600 IU. A t o t a l o f 17.93 g o f e x t r u d e d wheat per kg o f dry d i e t was used as the carrier. 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 o f d r y d i e t ) : sodium (as NaCl) 1,500; magnesium (as MgS0 »7H 0) 300; manganese (as M n S 0 * H o O ) 75; i r o n (as FeS0 «7H 0) 75; z i n c (as ZnS0 »7H 0) 50; i o d i n e (as K I 0 ) 10; f l u o r i n e (as NaF) 10; copper (as C u S 0 5 H 0 ) 5; c o b a l t (as CoCl «6H 0) 1; s e l e n i u m (as N a S e 0 ) 0.1. A t o t a l of 12.58 g o f e x t r u d e d wheat per kg o f d r y d i e t was used as the c a r r i e r . 4  2  4  2  4  4  2  3  #  4  2  2  2  3  2  94 T a b l e 12. F o r m u l a t i o n o f b a s a l mix 2 u s e d i n t h e p r e p a r a t i o n o f t h e r e f e r e n c e and t e s t d i e t s employed i n experiment IV.  Ingredients  Concentration (g/kg d r y d i e t )  S t e a m - d r i e d h e r r i n g meal (73 .58% C P ) * E x t r u d e d wheat (17.83% C P ) * D r i e d whey (14.13% C P ) * Freeze-dried euphausids (71 .48 C P ) * B l o o d m e a l (95.37% CP) Herring o i l Soybean l e c i t h i n V i t a m i n supplement-; Mineral supplement Permapell (lignin-sulphonate binder) C h o l i n e c h l o r i d e (60%) Ascorbic acid  610. 9 39. 9 83. 3 66. 6 55. 5 71. 7 10. 0 20. 0 20. 0 15. 0 5. 0 2. 0  1  2  4  Dry m a t t e r b a s i s . S t a b i l i z e d w i t h 0.02% s a n t o q u i n . The o i l was s t a b i l i z e d w i t h 0.025% s a n t o q u i n a n d was s p r a y e d on t o t h e p e l l e t e d d i e t s . The v i t a m i n s u p p l e m e n t p r o v i d e d t h e f o l l o w i n g amounts p e r kg o f d r y 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 - c a l c i u m p a n t o t h e n a t e ) 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 p y r i d o x i n e - H C l ) 36.5 mg; t h i a m i n e (as t h i a m i n e m o n o n i t r a t e ) 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; D L - o c - t o c o p h e r y l a c e t a t e 600 I U . A t o t a l o f 17.93 g o f e x t r u d e d wheat p e r k g o f d r y d i e t was u s e d as t h e carrier. The m i n e r a l s u p p l e m e n t p r o v i d e d t h e f o l l o w i n g (mg/kg o f d r y d i e t ) : s o d i u m (as N a C l ) 1,500; magnesium (as M g S 0 * 7 H 0 ) 300; manganese (as M n S C ^ ' H o O ) 75; i r o n (as F e S 0 * 7 H 0 ) 75; z i n c (as Z n S 0 * 7 H 0 ) 50; i o d i n e (as K I 0 ) 10; f l u o r i n e (as NaF) 10; c o p p e r (as C u S 0 « 5 H 0 ) 5; c o b a l t (as C o C l « 6 H 0 ) 1; s e l e n i u m (as N a S e 0 ) 0.1. A t o t a l o f 12.58 g o f e x t r u d e d wheat p e r k g o f d r y d i e t was u s e d a s the c a r r i e r . 4  2  4  2  4  3  2  4  2  2  2  3  2  95 4.2.7  E x p e r i m e n t a l p r o c e d u r e s and f i s h h a n d l i n g  4.2.7.1  Diet allocation  Each d i e t was a s s i g n e d randomly t o t r i p l i c a t e groups o f f i s h i n a s i n g l e row o f d i g e s t i b i l i t y t a n k s i n experiment experiments I I I and IV, a randomized  I I . In  complete b l o c k d e s i g n was  employed.  4.2.7.2  Protocol  F i s h from t h e h o l d i n g t a n k s were a n e s t h e t i z e d w i t h 2phenoxyethanol  (0.5 ml/L water) and weighed t o t h e n e a r e s t gram  b e f o r e commencement o f experiments I I and I I I . p o p u l a t i o n o f 1080 f i s h III)  (experiment I I ) and 837 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 .  d i s t r i b u t e d randomly,  A total (experiment  The s e l e c t e d f i s h were  f i v e f i s h a t a t i m e , i n t o 18 and 27  d i g e s t i b i l i t y t a n k s i n experiments I I and I I I ,  respectively.  Following 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 d i e t 1 over a two-day p e r i o d .  (BioDiet) t o b a s a l  F i s h were a c c l i m a t e d t o t h e b a s a l  d i e t over a p e r i o d o f t e n days d u r i n g which f e c e s (chromic o x i d e f r e e ) 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 o x i d e s t a n d a r d c u r v e p r e p a r a t i o n s . F i s h were f e d t o s a t i a t i o n a t 0900 and 1430 h o u r s , seven days a week. A f t e r t h e 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 a n e s t h e t i z e d w i t h 2-phenoxyethanol  (0.5 ml/L w a t e r ) , p l a c e d onto  an absorbent c l o t h t o remove excess m o i s t u r e , and i n d i v i d u a l l y weighed t o t h e n e a r e s t 0.1 gram.  Removal o f a l l f i s h from each  t a n k a l s o a l l o w e d f o r thorough t a n k c l e a n i n g .  Mean weight  (±  SEM) o f f i s h on day 0 was 13.3 ± 0.11 g and 26.7 ± 0.18 g f o r  experiments treatments tanks  I I and I I I r e s p e c t i v e l y . i n experiment  (completely  nine d i e t a r y within  divided  t r e a t m e n t s was  each b l o c k  the  from t h a t  stress  handling, once.  loss  regard,  g  (SEM)).  t h e 27 d i g e s t i b i l i t y digestibility diet  was  a t random t o t h e t a n k s  I I and I I I .  a total  a narrow weight  The f i s h tanks.  d u r i n g which  into three blocks  allotted  after  To m i n i m i z e  fish  the  d e t e r m i n e d when i n d i v i d u a l l y by t h e f i s h was  f o r the t h i r d  recorded daily.  diet  2 was  fedfor  f e c e s were  each diets  was treatments  in a l l  a  seven  days  spoon  r o d was u s e d t o d e l i v e r  The p o i n t  delivered  collected.  block.  disturbance while feeding,  feed t o each o f the groups.  into  t r a n s f e r to the  a t 0900 a n d 1430 h o u r s ,  attached t o a 1 m long c l e a r P l e x i g l a s s  from t h e  randomly  and e a c h o f t h e n i n e d i e t a r y  daily  IV o n l y  (mean w e i g h t o f  t h e row o f 27 t a n k s  F i s h were f e d t h e r e f e r e n c e a n d t e s t  a week.  fish  from t h e B i o D i e t t o  Basal  a t random t o t h e t a n k s w i t h i n  experiments t o s a t i a t i o n  range  chromic o x i d e - f r e e  acclimation period,  To m i n i m i z e  selected  were d i s t r i b u t e d  Three days  IV was  i n experiment  o f 567 f i s h was  t a n k s , t h e f i s h were weaned  the diet  design).  associated with excessive  2 o v e r a two-day p e r i o d .  seven days,  divided  allotted  i t was d e c i d e d t o w e i g h t h e f i s h  In t h i s  I I I , however,  i n t o t h r e e b l o c k s and each o f t h e  used i n experiments  and s c a l e  45.1 ± 0.32  After  In experiment  t o commencement o f e x p e r i m e n t  3600 L s t o c k t a n k s w i t h i n  basal  a t random t o t h e row o f  (randomized complete b l o c k  The p r o t o c o l p r i o r different  I I were a l l o t t e d  random d e s i g n ) .  t h e row o f t a n k s was  On day 1, t h e s i x d i e t a r y  of satiation  feed p e l l e t s  successive time.  Dry f o o d  were  was ignored  consumption  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 experiments delayed  I I and I I I .  Fecal collections  i n experiment  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  (30% d r i e d whey : 7 0 % 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 The  poor feed i n t a k e o f f i s h  the extremely  hard p e l l e t  consistency resulting Subsequently,  r e c e i v i n g d i e t DW2 w e r e g i v e n d i e t DW1 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 t h r e e days  DW2  adequately.  f e d d i e t DW2 was most l i k e l y  l e v e l o f d r i e d whey i n t h i s d i e t .  4.2.8  I V were  due t o  from t h e h i g h e r t h e groups  ( 1 5 % d r i e d whey : 8 5 % from a l l t a n k s were  started  later.  Fecal collection Immediately  procedure  after the last  f e e d i n g on t h e d a y b e f o r e t h e  m o r n i n g 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  three  weeks i n e x p e r i m e n t  I I ( e x c l u d i n g weekends) o r 15 c o n s e c u t i v e  days i n experiments  I I I and IV, t h e t a n k s were t h o r o u g h l y  o f any f e c e s o r u n e a t e n f e e d .  The c o t t o n f i l t e r  i n each water  inlet  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  (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 h o s e , a l l  f e e d and f e c a l out.  cleaning.  cleared  Using a clear  s e d i m e n t s on t h e s i d e s o f e a c h t a n k w e r e s y p h o n e d  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 a n d t h e f i s h a p p e a r e d 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 sampling  p o r t o f t h e c o l l e c t i o n c o l u m n was o p e n e d a n d w a t e r was  allowed t o f l u s h out. A large brush was i n s e r t e d down t h e v e r t i c a l c o l l e c t i o n column  attached t o a 1 m long  pipe  stand pipe t o t h e bottom o f t h e  ( s e e F i g u r e 1) a n d t h e b r u s h e d  d e p o s i t s were  98 swept out w i t h t h e o u t f l o w i n g water.  The f l u s h i n g  cap o f each  t a n k was opened and a b r u s h w i t h a l o n g h a n d l e was used t o c l e a n the  inside  o f t h e c o n n e c t i n g p i p e between t h e tank bottom s l o t  and t h e c o l l e c t i o n column.  When t h e water l e v e l i n t h e tank had  dropped by a p p r o x i m a t e l y 1/3 o f t h e t o t a l t a n k volume, t h e flushing  cap and the c o l l e c t i o n column clamp were r e p l a c e d .  Water f l o w r a t e s were m o n i t o r e d i n each tank t o ensure t h a t t h e f e c e s produced o v e r n i g h t would be s w i f t l y c a r r i e d i n t o t h e collection At  column.  0800 hours t h e f o l l o w i n g  day, t h e s e t t l e d f e c e s and  s u r r o u n d i n g water were v e r y g e n t l y withdrawn from t h e base o f t h e 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 rubber s t o p p e r .  f i t t e d with a  Immediately a f t e r c o l l e c t i o n , t h e f e c e s were  c e n t r i f u g e d a t 10,000 x g f o r 30 minutes a t a p p r o x i m a t e l y 5°C the  supernatant discarded.  and  The d a i l y f e c a l c o l l e c t i o n o f each  group was p o o l e d over t h e c o l l e c t i o n p e r i o d and h e l d i n a s e a l e d p l a s t i c c o n t a i n e r a t -40°C. F i s h were e u t h e n i z e d by an overdose o f 2-phenoxyethanol (2ml/L water) and weighed t o t h e n e a r e s t 0.1 gram a t t h e end o f each e x p e r i m e n t .  Feces were c o l l e c t e d  from a l l f i s h a t t h i s t i m e  (except from f i s h m a i n t a i n e d on d i e t s c o n t a i n i n g p r o d u c t s ) by e i t h e r  soybean  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 e s c r i b e d i n experiment I .  as  S u b s e q u e n t l y , t h e f e c a l samples were  s t o r e d i n s e a l e d p l a s t i c v i a l s a t -40°C.  99 4.2.9  Chemical  a n a l y s e s o f 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 f r o z e n f e c a l samples were l y o p h i l i z e d and g e n t l y ground i n a mortar and p e s t l e b e f o r e c h e m i c a l a n a l y s e s .  Moreover, t h e  samples c o l l e c t e d w i t h t h e "Guelph system" were passed t h r o u g h a 250um s c r e e n t o 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 f e c e s were a n a l y z e d f o r m o i s t u r e and ash (AOAC, 1975) and t o t a l n i t r o g e n (Technicon I n s t r u m e n t a l Co., L t d . , i n d u s t r i a l methods 369-75A/A and 334-74 W/B).  The p e r c e n t n i t r o g e n was m u l t i p l i e d by 6.25 t o  o b t a i n p e r c e n t crude p r o t e i n v a l u e s .  Gross energy was  determined  by combustion o f t h e t e s t i n g r e d i e n t s , d i e t s and f e c e s u s i n g 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) u s i n g b e n z o i c a c i d as a s t a n d a r d .  The  chromic o x i d e c o n c e n t r a t i o n s i n t h e d i e t s and f e c e s were determined  by t h e wet d i g e s t i o n c o l o r i m e t r i c method o f Stevenson  and De Langen (1960).  The crude l i p i d c o n t e n t s i n t h e  i n g r e d i e n t s and d i e t s were measured by t h e B l i g h and Dyer (1959) technique.  A l l d e t e r m i n a t i o n s were done i n d u p l i c a t e .  Owing t o t h e s m a l l amounts o f f e c e s c o l l e c t e d by t h e 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 t e c h n i q u e s , a n a l y s i s o f t h e s e samples was r e s t r i c t e d t o chromic  o x i d e and o r g a n i c m a t t e r .  100 4.2.10  Digestibility  and f i s h performance  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 m a t t e r , crude p r o t e i n  (ADC) f o r o r g a n i c  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 u s i n g t h e f o l l o w i n g  ADC  (%) '=. 100  formula:  % Cr C>3 i n d i e t x % n u t r i e n t 2  1-  calculations  % Cr 03 i n feces  i n feces  x % nutrient  2  i n diet  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 g r o s s energy was c a l c u l a t e d u s i n g g r o s s energy d a t a data.  (MJ/kg) i n s t e a d  of nutrient  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 g i v e n t e s t  ingredient  was c a l c u l a t e d  from t h e apparent  digestibility  c o e f f i c i e n t s f o r t h e r e f e r e n c e and t e s t d i e t s on t h e b a s i s percent s u b s t i t u t i o n of the t e s t ingredient diet  (100-X) on a d r y m a t t e r b a s i s  of the  (X) f o r t h e r e f e r e n c e  according t o the following  equation:  ADC o f t e s t  ingr.=  100 X  ADC t e s t d i e t  100-X 100  ADC r e f e r , d i e t  F i s h performance was a l s o a s s e s s e d i n each e x p e r i m e n t . However, t h e r e s u l t s o f growth and f e e d e f f i c i e n c y r a t i o s s h o u l d be i n t e r p r e t e d  w i t h c a u t i o n s i n c e t h e d i e t s were f e d f o r a  r e l a t i v e l y short period nor  isonitrogenous.  o f time and t h e y were n e i t h e r  isocaloric  101 S p e c i f i c growth r a t e s o r p e r c e n t  i n c r e a s e s i n wet body  weight p e r day ( B r e t t , 1979) were c a l c u l a t e d a s : (logn t l - l o g n tO) ( t l - tO)  x 100  In t h i s e q u a t i o n , l o g n t l and l o g n tO a r e t h e n a t u r a l l o g wet weights  at the s t a r t  (tO) and t h e end ( t l ) o f t h e p e r i o d .  d a t a on t h e mean 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 f e e d i n t a k e was e x p r e s s e d  Using  (DFI; g / f i s h ) ,  as a p e r c e n t a g e o f mean wet body weight  according t o the procedure of Richardson  e t al. (1985).  The  f o r m u l a used was as f o l l o w s : DFI (logn t l + l o g n tO  2)  e  x 100  Dry f e e d i n t a k e d a t a were a l s o used t o c a l c u l a t e f e e d e f f i c i e n c y (FE; wet weight g a i n (g) -5- DFI) and p r o t e i n e f f i c i e n c y (PER; wet weight g a i n  (g)  p r o t e i n i n t a k e (g) ) .  c o u p l e d , w i t h t h o s e f o r d i e t proximate organic matter,  ratio  Data f o r DFI  c o m p o s i t i o n and r e s p e c t i v e  crude p r o t e i n and g r o s s energy  digestibility,  were used t o c a l c u l a t e mean d a i l y o r g a n i c m a t t e r , p r o t e i n and energy d i g e s t e d by each f i s h S i n c e t h e i n i t i a l mean w e i g h t s  ( g / f i s h ) i n e x p e r i m e n t s I I and I I I . o f t h e f i s h i n each group were n o t  o b t a i n e d p r i o r t o i n i t i a l t e s t d i e t f e e d i n g i n experiment IV, d a t a on weight g a i n and growth were c a l c u l a t e d u s i n g t h e o v e r a l l i n i t i a l mean f i s h  weight..  102 4.2.11  Statistical analysis  A l l s t a t i s t i c a l a n a l y s e s were performed u s i n g a g e n e r a l l i n e a r model program (SAS, 1985) a f t e r e n s u r i n g  that'assumptions  r e g a r d i n g homogeneity o f v a r i a n c e were s a t i s f i e d  (using H a r t l e y ' s  t e s t as d e s c r i b e d by Lyman, 1984).  In t h i s regard,  one-way  a n a l y s e s o f v a r i a n c e were performed a f t e r t r a n s f o r m a t i o n o f t h e apparent o r g a n i c m a t t e r ,  crude p r o t e i n and g r o s s energy  d i g e s t i b i l i t y d a t a i n t o a r c s i n square r o o t d a t a  ( S t e e l 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 ingredients.  No b l o c k e f f e c t s were found (P>0.05) i n e x p e r i m e n t s  I I I and IV and hence t h e degrees o f freedom f o r b l o c k s were p o o l e d i n t o t h e e r r o r term.  D i f f e r e n c e s between treatment  were d e t e c t e d by Newman-Keuls'  means  ( K e u l s , 1952) t e s t w i t h P=0.05.  D i f f e r e n c e s i n mean apparent o r g a n i c m a t t e r  digestibility  c o e f f i c i e n t s as i n f l u e n c e d by t h e f e c a l c o l l e c t i o n p r o c e d u r e ("Guelph system" v s i n t e s t i n a l d i s s e c t i o n o r "Guelph system" vs s t r i p p i n g ) were a s s e s s e d by t h e s t u d e n t ' s T o r r i e , 1960).  ( S t e e l and  I n cases where t h e i n g r e d i e n t s were t e s t e d a t two  levels of i n c l u s i o n matter,  " t " test  ( i . e . 15% o r 3 0 % ) , t h e d i f f e r e n c e s i n o r g a n i c  crude p r o t e i n and g r o s s energy d i g e s t i b i l i t y  coefficients  were r e v e a l e d by t h e p a i r e d d a t a a n a l y s i s as d e s c r i b e d by Lyman (1984).  103 Data on weight g a i n , s p e c i f i c growth r a t e , d r y f e e d i n t a k e , f e e d i n t a k e as a p e r c e n t o f 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 o f o r g a n i c m a t t e r , p r o t e i n and energy d i g e s t e d by f i s h on each d i e t a r y treatment  in a l l  e x p e r i m e n t s were s u b j e c t e d t o a n a l y s i s o f v a r i a n c e t o d e t e c t d i f f e r e n c e s between d i e t t r e a t m e n t s . s u b j e c t e d t o Newman-Keuls  1  The t r e a t m e n t  means were  ( K e u l s , 1952) t e s t w i t h P=0.05.  s t a t i s t i c a l a n a l y s e s a r e t a b u l a t e d i n Appendix 4.  The  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 o f proximate i n experiments  analyses of the i n g r e d i e n t s t e s t e d  I I , I I I and IV, t o g e t h e r w i t h t h e i r a b b r e v i a t i o n  codes, a r e g i v e n i n Tables 13, 14 and 15, r e s p e c t i v e l y .  Of t h e  f i s h meals, t h e menhaden meal (MM) c o n t a i n e d t h e h i g h e s t ash c o n t e n t and s u b s e q u e n t l y  a l s o t h e lowest crude p r o t e i n c o n t e n t .  The p o u l t r y by-product  meals from d i f f e r e n t sources  PMB) d i f f e r e d o n l y by one percentage  (PMK and  p o i n t i n t h e i r ash c o n t e n t .  However, d i f f e r e n c e s i n t h e m o i s t u r e , crude p r o t e i n and l i p i d c o n t e n t o f t h e 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 c o n c e n t r a t i o n i n t h e l o c a l p o u l t r y byp r o d u c t meal (PMB) was compensated f o r by a h i g h e r l i p i d  content,  and thus a s l i g h t l y h i g h e r g r o s s energy v a l u e , r e l a t i v e t o PMK. With r e s p e c t t o o t h e r animal by-product  sources,  hydrolyzed  f e a t h e r meal (FM) and b l o o d 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 o f p r o t e i n , w h i l e d r i e d whey (DW) c o n t a i n e d 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 c a n o l a meal (GCMa) was s i m i l a r t o t h e commercial c a n o l a meal (CM) i n p r o x i m a t e composition.  The p r o t e i n c o n t e n t o f t h e c a n o l a 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 o f s o l v e n t e x t r a c t i o n  (GCMb) on  t h e o t h e r hand, was s u b s t a n t i a l l y h i g h e r t h a n t h a t o f t h e former c a n o l a meals, p o s s i b l y due t o some l o s s e s o f 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 t h e g l u c o s i n o l a t e c o n t e n t o f GCMb.  In  r e g a r d s t o t h e h i g h p r o t e i n c o n t e n t p l a n t s o u r c e s , soybean protein isolate  (SI) had t h e h i g h e s t l e v e l o f p r o t e i n , matching  105 t h a t o f t h e b l o o d meal, w h i l e rapeseed p r o t e i n c o n c e n t r a t e was  r e l a t i v e l y h i g h i n b o t h p r o t e i n and l i p i d .  between t h e p r o x i m a t e  (RP)  Differences  components o f wheat m i d d l i n g s  (WM)  and  e x t r u d e d wheat (EW)  would not appear t o be s u f f i c i e n t t o  s u b s t i t u t i o n o f one  f o r t h e o t h e r i n d i e t f o r m u l a t i o n s on the  b a s i s of these  criteria.  warrant  106 Table 13. Proximate c o m p o s i t i o n and g r o s s energy c o n t e n t o f t e s t i n g r e d i e n t s used i n experiment I I (values i n parentheses show p e r c e n t a g e s f o r e a c h p r o x i m a t e c o n s t i t u e n t on a d r y matter b a s i s ) . Proximate Water Test Ingredient  Code  Rapeseed protein concentrate  RP  Canola meal (commercial)  CM  Glucosinolate free GCMa c u l t i v a r canola  Protein  constituent Lipid  Ash  G.E, (MJ/kg)  (percent) 6.57  60.03 (64.25)  8.89 (9.51)  6.64 (7.11)  21.17 (22.66)  10.38  34.06 (38.00)  4.34 (4.84)  6.84 (7.63)  18.09 (20.18)  9.62  32.14 (35.56)  3.85 (4.26)  7.36 (8.14)  17.75 (19.64)  Herring meal  HM  7.96  68.15 (74.04)  10.93 (11.87)  12.96 (14.08)  20.29 (22.04)  Anchovy meal  AM  8.60  66.12 (72.34)  11.20 (12.25)  14.07 (15.39)  20.21 (22.11)  107 Table 14. 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 c o n t e n t o f 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 p a r e n t h e s e s show p e r c e n t a g e s f o r each p r o x i m a t e c o n s t i t u e n t on a d r y matter b a s i s ) . Proximate Test Ingredient  Water Code  Protein  constituent Lipid  Ash  G.E.  (MJ/kg)  (percent)  Poultry by-product meal (Kansas)  PMK  5.37  69.55 13.52 10.79 (73.50) (14.29) (11.40)  21.47 (22.69)  Poultry by-product meal (B.C.)  PMB  '2.91  64.20 20.18 12.03 (66.12) (20.78) (12.39)  22.52 (23.20)  MM  9.66  60.54 11.36 18.36 (67.01) (12.57) (20.32)  18.30 (20.26)  FM  4.97  84.93 (89.37)  Low t e m p e r a t u r e 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 (75.71) (11.18) (12.77)  Soybean meal  SM  8.42  47.91 (52.31)  1.96 (2.14)  6.84 17.89 (7.47) (19.53)  Blood meal  BM  3.44  91.39 (94.64)  2.95 (3.05)  1.81 23.72 (1.87) (24.57)  Soybean isolates  SI  4.83  87.26 (91.69)  4.82 (5.06)  4.11 21.53 (4.32) (22.62)  Menhaden meal Hydrolyzed feather meal  8.09 (8.51)  4.32 22.61 (4.55) (23.79) 20.52 (22.04)  108 Table 15. 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 c o n t e n t o f t e s t i n g r e d i e n t s used i n experiment IV (values i n p a r e n t h e s e s show p e r c e n t a g e s f o r each p r o x i m a t e c o n s t i t u e n t on a d r y matter b a s i s ) . Proximate Test Ingredient  Water  Protein  constituent Lipid  Ash  G.E.  :  Code  (percent)  (MJ/kg)  Dried whey  DW  5 .53  10 .98 (11 .62)  0. 96 (1.02)  7.74 15.29 (8.19) (16.19)  Extruded wheat  EW  9 .42  14 .15 (15 .62)  1.22 (1.35)  1.57 16.78 (1.73) (18.52)  Soybean meal  SM  8 .42  47 .91 (52 .31)  1.96 (2.14)  6.84 17.89 (7.47) (19.53)  Wheat middlings  WM  11 .03  14 .12 (15 .87)  3.34 (3.75)  2.11 16.66 (2.37) (18.72)  Soybean isolates  SI  4 .83  87 .26 (91 .69)  4.82 (5.06)  4.11 21.53 (4.32) (22.62)  9 .23  37. 62 (41 .44)  2.02 (2.22)  7.73 17.61 (8.52) (19.40)  Glucosinolate extracted GCMb c a n o l a meal  109 4.3.2  Proximate  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  r e f e r e n c e and t e s t The  proximate  diets  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  i n experiments  I I , I I I and  respectively.  The  and REF-b) was  a p p r o x i m a t e l y 2.5  w o u l d be e x p e c t e d  I V a r e shown i n T a b l e s  diets  16,  from t h e d i e t  percentage  amount o f 9%.  The  18,  (REF-a  p o i n t s higher than  formulations. was  used  17 and  p r o t e i n content of both reference diets  the moisture content of a l l d i e t s theoretical  of  On t h e o t h e r h a n d ,  very close to the  mineral levels  i n both reference  (REF-a and REF-b) w e r e f o u n d 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 b e t w e e n some t e s t  diets.  d i e t s MM2  w e r e two  and BM2  f o u n d i n any PMK2 was  For i n s t a n c e , the i r o n contents  other diets.  t o three times greater than the  levels  L i k e w i s e , the copper  diet  content of  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 f o u n d i n  other 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 a p p e a r e d t o be q u i t e h i g h , as r e f l e c t e d by substantial which CM2,  The  i n c l u d e d as t h e t e s t  and  i n g r e d i e n t (e.g.  GCMa2, GCMb2). 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 c a s e s EW2),  the  increase i n the l e v e l of t h i s mineral i n a l l d i e t s  c a n o l a / r a p e s e e d was RP2,  of  fragile  i n others  ( S I 2 , GCMb2).  (BM2,  DW1,  DW2,  WM2,  in  110 Table 16. 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 f i v e t e s t d i e t s f e d t o j u v e n i l e chinook salmon i n experiment I I . (Refer t o 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 ) . Proximate Composition (% o f dry matter)  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  level)  REF-a  RP2  CM2  GCMa2  HM2  55.50  59.13  50.06  50.13  61.44  61.31  16.72  14.86  13.08  12.68  15.06  15.42  10.81  9.12  9.84  9. 97  11.76  12.19  Total carbohydrate (by d i f f e r e n c e ) 16.97  16.89  27.02  27.22  11.74  Gross energy (MJ/kg)  22.56  21.58  21.45  22.52  22.43  8.40  8.58  8.42  8.76  Crude p r o t e i n (N x 6.25) Crude l i p i d (Bligh-Dyer) Ash  AM2  1  Moisture  22.63  (as fed)  8.54  8.63  11.08  o  Mineral content^ (g/kg dry d i e t ) C a l c i u m (Ca) Phosphorous (P) Sodium (Na) Magnesium (Mg) I r o n (Fe) Z i n c (Zn) Manganese (Mn) Copper (Cu) C o b a l t (Co) Ca:P Chromic o x i d e 3  20.7 27. 3 20. 0 31.6 20 .9 17. 1 17.5 15 .2 14. 8 19.4 7. 68 5.35 5 .22 4. 91 7.10 2. 02 3.65 3 .26 3. 26 1.97 0. 33 0.28 0 .29 0. 29 0.32 0. 12 0.11 0 .11 0. 11 0.11 0. 11 0.12 0 .09 0. 09 0.08 0. 01 0.01 0 .01 0. 01 0.01 l e s s than 0. 0002 i n a l l d i e t s 1. 60 1.18 1 .38 1. 35 1.63 5. 65 5.46 5 .75 5. 78 5.86  30 .6 19 .5 8 .15 2 .18 0 .36 0 .11 0 .08 0 .01 1 .57 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 + a s h ) . 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 plasma s p e c t r o s c o p y (Higgs e t al., 1982). Chromic o x i d e (Cr C>3) a n a l y s i s performed by t h e wet d i g e s t i o n c o l o r i m e t r i c method o f Stevenson and De Langen (1960). 2  Ill  Table 17. 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 e i g h t t e s t d i e t s f e d t o 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 ) . Proximate Composition (% o f d r y matter)  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  level)  REF-a  PMK2  PMB2  MM2  FM2  Crude p r o t e i n (N x 6.25) Crude l i p i d (Bligh-Dyer)  55. 62  61 .44  58 .63  60. 13  65 .19  16. 14  16 .48  17 .68  16. 03  14 .53  Ash  11. 10  11 .41  12 .73  14. 23  9 .24  17. 14  10 .67  10 .96  9.61  11 .04  22. 91  23 .01  22 .91  22. 43  23 .25  9.78  9 .36  9 .86  9.74  9 .14  Total carbohydrate (by d i f f e r e n c e )  1  Gross energy (MJ/kg) Moisture  (as fed)  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) Phosphorous (P) Sodium (Na) Magnesium (Mg) I r o n (Fe) Z i n c (Zn) Manganese (Mn) Copper (Cu) C o b a l t (Co) Ca:P Chromic o x i d e 3  27. 9 28 .9 33 .8 33. 6 16. 6 17 .2 19 .0 19. 6 8.30 7 .17 7 .52 8.35 2. 17 1 .87 1 .95 2. 22 0. 42 0 .47 0 .42 0. 70 0. 10 0 .10 0 .10 0. 09 0. 11 0 .07 0 .07 0. 08 0. 01 0 .04 0 .01 0. 01 0. 0009 l e s s than 0.0007— 1. 68 1 . 68 1 .78 1. 71 5 .00 5. 51 6 .13 5. 43  23 .2 13 .4 6 .24 1 .61 0 .39 0 .10 0 .07 0 .01 1 .73 5 .57  C a l c u l a t e d as: 100 - ( p r o t e i n + l i p i d + a s h ) . 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 plasma s p e c t r o s c o p y (Higgs e t al., 1982). Chromic o x i d e ( C ^ C ^ ) a n a l y s i s performed by t h e wet d i g e s t i o n c o l o r i m e t r i c method o f Stevenson and De Langen (1960).  112 Table  17. Continued.  Proximate Composition (% o f d r y m a t t e r )  (test  D i e t code i n g r e d i e n t and i n c l u s i o n  level)  LT2  SM2  BM2  SI2  Crude p r o t e i n (N x 6.25) Crude lipid (Bligh-Dyer)  64.25  55.44  67.63  67.06  15.54  12.86  12.68  13.63  Ash  11.88  10.20  8.35  9.18  8.33  21.50  11.34  10.13  22.98  22.22  23.87  23.24  9.63  9.34  1 Total carbohydrate(by d i f f e r e n c e ) Gross energy (MJ/kg) Moisture  (as  fed)  9. 92  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) P h o s p h o r o u s (P) S o d i u m (Na) M a g n e s i u m (Mg) I r o n (Fe) Z i n c (Zn) M a n g a n e s e (Mn) C o p p e r (Cu) C o b a l t (Co) Ca:P Chromic o x i d e 3  26.5 17.7 8.58 2.20 0.38 0.10 0.07 0.01 0.0007 1.50 5.83  18.2 13.1 5.28 2.50 0.32 0.08 0.08 0.01 0.0008 1.39 5.30  18.6 12.1 6.92 1.49 1.01 0.08 0.07 0.01 0.0009 1.54 4.97  15. 11. 6. 1. 0. 0. 0. 0. 0. 1. 5.  7 6 99 27 23 08 06 01 0006 35 42  1 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) . 2 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 spectroscopy ( H i g g s e t a l . , 1982) . Chromic oxide (Cr2C>3 ) a n a l y s i s p e r f o r m e d b y t h e w e t d i g e s t i o n c o l o r i m e t r i c m e t h o d o f S t e v e n s o n a n d De L a n g e n (1960).  113 Table 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 s e v e n 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 s a l m o n i n e x p e r i m e n t I V . ( 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 ) . Proximate Composition (% 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 REF-b  DW1  DW2  EW1  level) EW2  Crude p r o t e i n (N x 6.25) Crude l i p i d  59.50  52.69  46.00  53.44.  46.94  (Bligh-Dyer)  16.10  14.76  11.58  14.61  11.92  12.15  11.58  10.98  10.49  8.91  12.25  20.97  31.44  21.46  32.95  22.72  21.96  21.19  22.18  21.64  8.37  8.64  8.66  8.24  8.19  N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A  25 .2 15 .6 7 .14 2 .02 0 .39 0 .09 0 .09 0 .01 0 .001 1 .62 5 .31  Ash Total carbohydrate (by d i f f e r e n c e ) Gross energy (MJ/kg) M o i s t u r e (as f e d ) Mineral content (g/kg d r y d i e t ) C a l c i u m (Ca) P h o s p h o r o u s (P) Sodium (Na) M a g n e s i u m (Mg) I r o n (Fe) Z i n c (Zn) Manganese (Mn) C o p p e r (Cu) C o b a l t (Co) Ca:P Chromic o x i d e 4  1  25. 8 30 .3 17 .8 15. 8 8 .31 8. 11 2 .04 1. 90 0 .42 0. 35 0 .10 0. 08 0 .10 0. 08 0 .01 0. 01 0 .0009 0. 0008 1 .70 1. 63 5 .20 5. 54  16.0 12.1 4.99 1.66 0.23 0.07 0.07 0.01 0.0007 1.32 5.77  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 + a s h ) . 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 plasma s p e c t r o s c o p y (Higgs e t a l . , 1982). Use o f d i e t DW2 a b o r t e d i n t h e e x p e r i m e n t . No m i n e r a l a n a l y s i s performed. Chromic o x i d e ( C ^ C ^ ) a n a l y s i s p e r f o r m e d by t h e wet d i g e s t i o n c o l o r i m e t r i c method o f Stevenson and De Langen (1960).  114 Table  18.  Continued.  Proximate Composition (% o f dry matter)  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 SMI  WM2  level)  SI1  GCMb2  Crude p r o t e i n (N x 6.25) Crude l i p i d (Bligh-Dyer)  58 .25  47.50  63.06  53.50  14.80  13.13  15.67  12.96  Ash  11.45  9.25  10.79  11.10  15.50  30.12  10.48  22.44  22.55  21.66  23.11  21.88  8.77  8.42  8.52  Total carbohydrate (by d i f f e r e n c e ) Gross energy (MJ/kg) Moisture  (as fed)  1  8.73  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) Phosphorous (P) Sodium (Na) Magnesium (Mg) I r o n (Fe) Z i n c (Zn) Manganese (Mn) Copper (Cu) C o b a l t (Co) . Ca:P Chromic o x i d e J  19. 9 17.3 21.8 23.3 14 . 1 13.1 16.0 18.0 5. 99 5.22 7.90 6.16 1. 98 1.62 1.65 3.72 0. 33 0.32 0.36 0.38 0. 07 0.07 0.09 0.09 0. 07 0.07 0.08 0.09 0. 01 0.01 0.01 0.01 0. 0009 0.0008 0.0008 0.0009 1. 41 1.32 1.36 1.29 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 + a s h ) . 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 plasma s p e c t r o s c o p y (Higgs et al., 1982). Chromic o x i d e ( C ^ C ^ ) a n a l y s i s performed by t h e wet d i g e s t i o n c o l o r i m e t r i c method o f 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 o f t e s t ingredients 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 o r g a n i c  matter,  crude p r o t e i n and g r o s s energy, as w e l l as the d i g e s t i b l e energy c o n t e n t of a l l i n g r e d i e n t s t e s t e d i n e x p e r i m e n t s I I , I I I and are l i s t e d i n Table 19.  Differences  i n nutrient  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).  IV  digestibilities The  o f o r g a n i c m a t t e r and g r o s s energy of the f i s h meals  digestibility was  c o n s i s t e n t l y h i g h e r t h a n the d i g e s t i b i l i t y o f t h e s e components i n the o t h e r f e e d s t u f f s , a l t h o u g h d i f f e r e n c e s were not significant  (P>0.05).  W i t h i n the f i s h meals, menhaden meal  the l o w e s 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 . The apparent crude p r o t e i n d i g e s t i b i l i t y was rapeseed p r o t e i n c o n c e n t r a t e d i g e s t i b i l i t y was  always  (RP)  significantly  most o t h e r f e e d s t u f f s .  and  obtained  had  highest  f o r the  i t s crude p r o t e i n  (P<0.05) g r e a t e r t h a n t h a t  of  In r e l a t i o n t o the a n i m a l by-product  meals a s s e s s e d , the Kansas source of p o u l t r y b y - p r o d u c t meal (PMK)  g e n e r a l l y had  superior nutrient 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 l t h o u g h s i g n i f i c a n t d i f f e r e n c e s c o u l d not always be demonstrated.  By c o n t r a s t , b l o o d meal (BM)  d i g e s t e d by the f i s h . the energy of BM was  The  was  very  poorly  d i g e s t i b i l i t y of the crude p r o t e i n  significantly  and  (P<0.05) lower t h a n the  d i g e s t i b i l i t y of t h e s e components i n the o t h e r f e e d s t u f f s . R e d u c t i o n of the g l u c o s i n o l a t e c o n t e n t i n the c a n o l a GCMa, r e s u l t e d i n improved n u t r i e n t and energy  digestibilities  r e l a t i v e t o t h o s e o b s e r v e d f o r the commercial c a n o l a meal a l t h o u g h the d i f f e r e n c e s were not A l t e r n a t i v e l y , reduction  meal,  (CM),  s i g n i f i c a n t (P<0.05).  of the g l u c o s i n o l a t e c o n t e n t i n  canola  meal by s o l v e n t 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 o r g a n i c m a t t e r d i g e s t i b i l i t y compared t o CM and GCMa. E x t r u d e d 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 t h e two l e v e l s o f i n c l u s i o n o f e x t r u d e d wheat were not s i g n i f i c a n t organic matter g r o s s energy  (t=0.58; 4 d f ) , crude p r o t e i n  (P>0.05) f o r  (t=1.57; 4df) and  (t=0.47; 4 d f ) . The d i g e s t i b i l i t y o f t h e o r g a n i c  m a t t e r and g r o s s energy i n t h e wheat m i d d l i n g was v e r y poor, a l t h o u g h t h e 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 digestible.  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 o b t a i n e d f o r t h e soybean  p r o d u c t s a r e not r e l i a b l e s i n c e t h e f o o d i n t a k e s o f f i s h f e d d i e t s c o n t a i n i n g soybean p r o d u c t s were e x t r e m e l y poor due t o reduced d i e t p a l a t a b i l i t y .  F e e d s t u f f d i g e s t i b i l i t y was n o t e d t o  be more v a r i a b l e when t h e t e s t i n g r e d i e n t s were i n c l u d e d a t t h e 15% l e v e l r e l a t i v e t o t h e 30% l e v e l .  117 T a b l e 19. Mean p e r c e n t (± SEM) a p p a r e n t o r g a n i c m a t t e r , c r u d e p r o t e i n and g r o s s e n e r g y 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 o f 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. Apparent Ingredient  Code  digestibility  (%)  Organic matter  Crude protein  Gross energy  Digestible energy (MJ/kg)  Herring meal  HM  87.3 ±1.03  90.5 ±0.51  b c d  92. 6 ±0.48  20.4 ±0.11  Anchovy meal  AM  87. 6 ±2.11  91.7 ±1.50  a b c  91. 6 ±1.62  20.2 ±0.36  Menhaden meal  MM  84.0 ±0.99  83.1 9 ±1.45 e f  84.1 ±1.80  17.0 ±0.36  LT h e r r i n g / c a p e l i n meal  LT  87.8 ±0.62  93.6 ±0.81  a b  88.4 ±1.26  Poultry by-product m e a l (Kansas)  PMK  75.9 ±2.41  Poultry by-product m e a l (B.C.)  PMB  65.6 ±1.82  Feather meal  FM  63.1 ±0.89  Blood meal  BM  34. 8 ±2.27  Dried whey  DW  7 6.7 ±3.65  a l  a  a  a  a b  8 4  . defg 9  a  a  a  a  a b  19.5 ±0.28  a  7 2  a b  . bcd  16.4 ±0.38  b c d e  c d  15.2 ±0.50  c d e f  d e  4  ±1.16  ±1.69  b c d  74.4 ±1.11  h  65.4 ±2.17  b c d  70. 8 ±0.84  57.4 ±1.48  13.6 ±0.35  29.4 ±1.20  31. 9 ±1.68  7.8 ±0.41  72. 5 ±2.18  71.5 ±5.82  e  h  1  a b  h  a b c d  f  d e f  h  b c d  11.6 9 • ±0.60 f  Means i n e a c h column f o l l o w e d by a common s u p e r s c r i p t a r e 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). 1  Table  19. C o n t i n u e d . Apparent  digestibility  (%) Digestible  118  Ingredient  Code  Organic matter  Crude protein  Gross energy  energy (MJ/kg)  84 d e f g ±1 .65  64 . 5 ±1 .59  13 . 0 ±0 .32  87 gcde ±1 .15  71 . o ±2 .34  Canola meal  CM  53 . 5 ±2 .33  Glucosinolatefree c u l t i v a r c a n o l a meal  GCMa  58 . 7 ±2 .36  Glucosinolate extracted c a n o l a meal  GCMb  37 . 5 ±1 .48  78 f g h ±1 .23  51 . 2 ±1 .71  9 .93^ ±0 .33  Rapeseed protein concentrate  RP  69 . 8 ±1 .48  95 . 6 ±0 .61 a  80 . 5 ±2 .82  18 . 2 ±0 .64  Soybean meal (level l )  SM  58 . 5 ±7 .19  74 . 6 ±3 .76  66 . o ± 9 .32  SM  61 b c d ±6 . 67  77 . 0 9 ±4 .25  Soybean isolates ( l e v e l 1)  SI  58 . 9 ±3 .07  83 . d e f g ±4 .54  62 . o ±4 .11  Soybean isolates ( l e v e l 2)  SI  68 ^ b c d ±1 .83  86 3Cdef ±1 .08  59 . l ±2 .10  Extruded wheat ( l e v e l 1)  EW  75 a b ±6 .80  87 ^ c d e ±0 .85  67 . 5 ±7 .85  12 ±1 .45  Extruded wheat ( l e v e l 2)  EW  71 . 6 ±1 . 64  84 d e f g ±0 .95  71 . 3 ±2 .82  13 d e f ±0 .52  WM  31 . 4 ±2 . 98  85 , d e f ±1 .37  45 . 3 ±1 .95  8.5 ±0 .36  Soybean meal (level 2)  2  3  Wheat middlings 1  3  c d  e  b c  c d  > 7  c d  4  > 7  b c  e  > 5  m  > 6  c d  b  c  e f  d  d e  a b c  h  h  5  #  4  > 3  7  b  69 . 8 ±8 !98  c  b c d  c  d  d  d  e  e  b c d  b c d  e  13 d e f ±0 .46 > 9  h  a b c  12 . 9 ±1 .82  e f  13 . 6 ±1 .75  d e f  14 . 0 ±0 .27  d e f  13 d e f ±0 .47 > 4  > 2  h  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 a r e 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  d l  L e v e l 1 i s e q u i v a l e n t t o an i n g r e d i e n t and 85% r e f e r e n c e L e v e l 2 i s e q u i v a l e n t t o an i n g r e d i e n t and 70% r e f e r e n c e  inclusion diet (air inclusion diet (air  l e v e l o f 15% t e s t dry basis). l e v e l o f 30% t e s t dry basis).  119 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 t e c h n i q u e on apparent o r g a n i c matter  digestibility.  O r g a n i c m a t t e r (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 t h e water u s i n g t h e "Guelph system" were, w i t h one e x c e p t i o n , h i g h e r t h a n v a l u e s o b t a i n e d when f e c e s were c o l l e c t e d d i r e c t l y from t h e f i s h by t h e i n t e s t i n a l d i s s e c t i o n o r t h e s t r i p p i n g t e c h n i q u e s (Table 2 0 ) . N e v e r t h e l e s s , differences  were not s i g n i f i c a n t (P>0.05) between t h e OM  d i g e s t i b i l i t y values obtained calculated  from f e c a l  samples  c o l l e c t e d w i t h t h e "Guelph system" and t h o s e c a l c u l a t e d samples o b t a i n e d by t h e i n t e s t i n a l d i s s e c t i o n p r o c e d u r e .  from This  was not always t h e case when OM v a l u e s based on t h e "Guelph system" were compared t o t h o s e based on t h e s t r i p p i n g p r o c e d u r e . For  i n s t a n c e , t h e OM d i g e s t i b i l i t y o f d i e t REF-a d e t e r m i n e d 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 e x p e r i m e n t s I I (t=6.56; P<0.05; 2df) and experiment I I I (t=4.43; P<0.05; 2df) t h a n v a l u e s d e t e r m i n e d from f e c e s c o l l e c t e d by t h e "Guelph system".  D i f f e r e n c e s i n O.M.  f e c a l c o l l e c t i o n t e c h n i q u e employed  d i g e s t i b i l i t y due t o t h e ("Guelph system" v e r s u s  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 t h e f o l l o w i n g ingredients: meal MM  h e r r i n g meal HM  (t=20.22; P<0.01; 2 d f ) , menhaden  ( 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  p r o d u c t meal PMB  ( t=6.17; P<0.05; 2 d f ) , f e a t h e r meal FM  P<0.05; 2 d f ) , b l o o d meal BM  test  by(t=5.93;  (t=4.72; P<0.05; 2df) and d r y whey DW  (t=3.70; P<0.05; 5 d f ) . A t t e m p t s t o s t r i p t h e f e c e s from f i s h which were f e d d i e t s c o n t a i n i n g soybean p r o d u c t s were abandoned owing t o t h e v e r y 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 o f f e c a l c o l l e c t i o n t e c h n i q u e on t h e apparent o r g a n i c m a t t e 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 o f t h e 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. P e r c e n t apparent o r g a n i c m a t t e r d i g e s t i b i l i t y Diet or Test Ingredient  (± SEM)  Fecal c o l l e c t i o n technique (N obs.)  "Guelph"  Dissection  Stripping  REF-a (3) (Experiment I D  *84.5 ±0. 60  -  *77.2 ±0.57  REF-a (3) (Experiment I I I )  *80.3 ±0.18  -  *76.2 ±1.10  REF-b (3) (Experiment IV)  81.2 ±0.16  77 .0 ±1 .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  W i t h i n 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* o r P<0.01**). 1  121 Table 20.  Continued.  P e r c e n t apparent o r g a n i c m a t t e r d i g e s t i b i l i t y Test Ingredient  Fecal collection (N obs. )  "Guelph"  GCMa  (3)  58.7 ±2.36  GCMb  (3)  37.5 ±1.48  RP  (3)  69.8 ±1.48  SM  2  ( l e v e l 1)  (3)  58.5 ±7.19  SM  3  ( l e v e l 2)  (3)  61.7 ±6. 67  36.8 ±1.36 —  Stripping 56.3 ±0.38 —  66.8 ±1.17  —  —  —  —  -  SI  2  ( l e v e l 1)  (2)  58.9 ±3.07  SI  3  ( l e v e l 2)  (3)  68.4 ±1.83  EW  2  ( l e v e l 1)  (3)  75.7 ±6.80  EW  3  ( l e v e l 2)  (3)  71.6 ±1.64  72.4 ±0.7 6  (3)  31.4 ±2.98  30.1 ±1.25  WM  technique  Dissection —  (± SEM)  —  —  —  —  67.3 ±1.67 —  —  W i t h i n 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* o r P<0.01**). L e v e l 1 i s e q u i v a l e n t t o an i n c l u s i o n l e v e l o f 15% t e s t i n g r e d i e n t and 85% r e f e r e n c e d i e t ( a i r d r y b a s i s ) . L e v e l 2 i s e q u i v a l e n t t o an i n c l u s i o n l e v e l o f 30% t e s t i n g r e d i e n t and 70% r e f e r e n c e d i e t ( a i r d r y b a s i s ) .  122 4.3.5  Influence  of Diet  Data i n Tables intakes were,  fed a l l d i e t s i n the three  similar.  However, when t h e d a i l y  i n t a k e s p e r f i s h were e x p r e s s e d weight, t h e f i s h greater  amount  addition, experiment while III  i n experiment  of feed than  the feed  (Table  intakes  I I (Table  the feed  22) and IV  fish  the poorest  appetite  i n experiments  of fish  of fish  i n experiments  so p o o r t h a t t h e f e c a l  g r o w t h r a t e , F E a n d PER. those  of fish  utilization  nutrient  composition  exhibited  Indeed, t h e f e e d SI1 i n e x p e r i m e n t discarded. I I were n o t  of treatments,  there  p e r f o r m a n c e s were f o u n d f o r  daily  amounts o f o r g a n i c  energy d i g e s t e d .  be i n t e r p r e t e d w i t h  coupled  with  thesis.  D a t a on  caution  t o be u s e d t o e v a l u a t e  f o r t i s s u e growth i n t h i s  of the feeding t r i a l s ,  diets containing  i n experiment  The p o o r e s t  parameters are not intended  (P>0.05),  (P<0.001) n o t e d b e t w e e n g r o u p s f o r  c r u d e p r o t e i n and g r o s s  g r o w t h , F E and PER s h o u l d  were  regardless  groups which had t h e lowest  matter,  on d i e t  collections  (P>0.05)  differences  diets in  level,  I I I and IV.  IV was  different  In  significantly  at the higher  o f one g r o u p o f f i s h m a i n t a i n e d  were s i g n i f i c a n t  significantly  F i s h fed the test  intake  significantly  I I I and IV.  i n g e s t i n g the d i e t s i n experiments  especially  Although the appetites  dry feed  fed the d i f f e r e n t  (Table 23), d i f f e r e d  soybean p r o d u c t s ,  experiments  as a p e r c e n t a g e o f wet body  21) d i d n o t v a r y  intakes  dry feed  I I were o b s e r v e d t o i n g e s t a  (P<0.001) w i t h i n e x p e r i m e n t s . the  performance  21, 22 and 23, show t h a t t h e d a i l y  (DFI) o f t h e f i s h  i n general,  on f i s h  as  fish these  the feedstuff  The s h o r t  duration  the d i f f e r e n c e s i n the  b e t w e e n most d i e t s ,  do n o t p e r m i t  c o m p a r i s o n s t o be drawn b e t w e e n t h e v a r i o u s  valid  f e e d s t u f f s b a s e d on  123 f i s h growth performance.  The d a t a 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 i n t e n d e d t o be used m a i n l y as an i n d i c a t i o n t h a t a p o s i t i v e n i t r o g e n b a l a n c e was m a i n t a i n e d i n a l l f i s h f e d t h e t e s t d i e t s throughout t h e e x p e r i m e n t s .  I n cases  where t h e c o m p o s i t i o n o f t h e d i e t s was s i m i l a r , such as i n t h e d i e t s c o n t a i n i n g f i s h meal as t h e 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 o f t h e comparative s h o r t - t e r m u t i l i z a t i o n o f t h e s e 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 s h o u l d be performed t o r e a c h more d e f i n i t e and v a l i d c o n c l u s i o n s r e g a r d i n g t h e p o t e n t i a l o f 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 .  I n f o r m a t i o n on f i s h growth i n experiment  IV (Table 2 3 ) ,  r e f e r s t o t h e p e r i o d when t h e f i s h were f i r s t t r a n s f e r r e d t o t h e d i g e s t i b i l i t y t a n k s and t h e end o f t h e experiment later).  (34 days  D u r i n g t h a t t i m e , f i s h were g r a d u a l l y changed from t h e  commercial  d i e t t o t h e b a s a l d i e t and t h e r e a f t e r t h e y were  s w i t c h e d t o t h e r e s p e c t i v e t e s t d i e t s as p a r t o f t h e d i e t a c c l i m a t i o n program.  A l t h o u g h t h e growth d a t a appear t o be o f  l i t t l e use, t h e y do i n d i c a t e , however, t h a t f i s h m a i n t a i n e d on t h e d i e t s c o n t a i n i n g soybean meal o r soybean p r o t e i n i s o l a t e grew v e r y s l o w l y d u r i n g t h e e n t i r e 34-day p e r i o d .  Since the b a s a l  d i e t s were a v i d l y consumed by t h e f i s h b e f o r e commencement o f any of t h e e x p e r i m e n t s , i t can be p o s t u l a t e d t h a t f i s h f e d t h e d i e t s c o n t a i n i n g t h e soybean p r o d u c t s i n experiments a c t u a l l y l o s t weight w h i l e on t h e t e s t  diets.  I I I and IV  M o r t a l i t y ranged from a low o f 3.3% f o r f i s h on d i e t RP2, t o a h i g h o f 16.6% f o r f i s h on d i e t S I 1 .  The need t o handle t h e  f i s h b e f o r e t h e e x p e r i m e n t a l p e r i o d and t h e i n e v i t a b l e d i s t u r b a n c e s t o t h e f i s h w h i l e changing water f i l t e r s ,  flushing  the t a n k s , c o l l e c t i n g t h e f e c e s as w e l l as d u r i n g f e e d i n g , undoubtedly caused e x c e s s i v e s t r e s s and i n c r e a s e d 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 t h e  o p p o r t u n i s t i c pathogens Myxobacterium ordalii.  sporophytophygia  and V i j b r i o  125 T a b l e 21. 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 grams o f d r y f e e d i n t a k e p e r f i s h (DFI), 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 (PER), 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 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 a n d t o M a t e r i a l s a n d Methods f o r measurement o f e a c h p a r a m e t e r ) . Parameter  Diet RP2  REF-a Weight g a i n 3.30 ( g / f i s h i n 22 days)  a l  CM2  GCMa 2  HM2  3.17  a  0.71  c  1.31  b  3.18  AM2 a  3.63  a  1.19  a  SGR (%/day)  1.09  a  1.02  a  0.37  c  0.55  b  l . l l  DFI (g/fish)  0.25  a  0.23  a  0.21  a  0.23  a  0.24  a  0.24  a  FI  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  Digestible 0.19 organic matter (g/fish)  a  0.16  a b  0.14  0.16  a b  0.18  a  0.18  a  0.12  a b  0.09°  0.10  b c  0.13  a  0.13  a  3.59  4.01  a b  4.68  a  4.64  a  (% O f  a  B.W.)  Digestible protein (g/fish)  0.12  Digestible energy (KJ/fish) Mortality  4.86  a b  a  9/180  4  29  6/180  a b  b  b  20/180  20/180  14/180  11/180  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=0.05).  126  T a b l e 22. Weight g a i n , s p e c i f i c growth 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 a s a p e r c e n t o f wet b o d y w e i g h t ( F I ) , f e e d efficiency ( F E ) , 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 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 o n d i e t s a n d 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 ) . Parameter  Diet REF-a  Weight gain (g/fish  2.49 i n 17  a b l  PMK2  PMB2  2.04  b  MM2  FM2  LT2  SM2  BM2  SI2  1.82  b c  3.19  a  1.44  b c  2.14  a b  0.20  d  0.82  c d  0.3l  d  0.41  b c  0.67  a  0.37  b c  0.47  a b  0.05  d  0.19  c d  0.06  d  0.27  b c  0.36  a  0.25  b c  0.28  0.22  c d  0.12  e  1.02  b c  1.36  a  0.95  b c  1.05  0.48  d  days)  SGR (%/day)  0.53  a b  0.45  DFI (g/fish)  0.30  a b  0.28  FI 1.14 (% o f B . W . )  a b  a b c  1.06  b  b c  a b c  b c  0.14  d e  0.57  d  0.85  c  F.E.  0.48  a b  0.43  a b c  0.39  b c  0.53  a  0.34  c d  0.45  a b c  0.09  f  0.22  d e  P.E.R.  0.85  a b  0.70  a b c  0.66  b c  0.87  a  0.52  c d  0.71  a b c  0.15  e  0.33  d e  0.22  Digestible O.M. (g/fish)  0.22  a b  0.20  a b  b c  0.25  a  0.17  b c  0.10  d  0.13  c d  0.09  Digestible protein (g/fish)  0.15  a b  0.15  a b  b c  0.18  a  d  0.10  c d  Digestible energy (KJ/fish)  ^  5.66  a b  b c  6.62  a  ' 3.48  Mortality  4/93  ,  .  i .•  w 5.09  7/93  b  , 0.18  0.13  . 4.77  5/93  '  4/93  , 0.13  , 4.29  ' 0.20  • a b  ,  ,  0.15  a b  0.07  b c  5.31  a b  2.50  ^ 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 different (Newman-Keuls t e s t P = 0 . 0 5 ) .  e  ,  b c  11/93  0.15  d  6/93  6/93  letter  are not  ,  4/93  d  , 0.07  d  j c d  2.16  d  5/93 .  significantly  e f  127 Table 23. Weight g a i n , s p e c i f i c growth 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 (DFI), 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 body weight (FI) 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 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 s and t o M a t e r i a l s and Methods f o r measurement o f each p a r a m e t e r ) . Parameter  Diet DW1  REF-b  EW1  EW2  Weight 4.16 3.00 gain ( g / f i s h i n 34 days)  ab  3.96  SGR (%/day)  a b  0.27  1  1  a2  0.29  DFI 0.31 (g/fish) 3  FI 0.78 (% o f B.W.) 3  0.23  a  a  a  M o r t a l i t y 6/63 1  2  3  a  ab  SMI 0.35  0.33  c  0.19  ab  0.04° 0 . 1 5 0 . 0 8  c  0.35  a  0.32  0.68  a  0.90  a  0.81  4/63  GCMb2  a b  a  3/63  a  2.04  SI1  2.67  0.27  16/126  WM2 c  bc  3.43  a  0.23  0.17 0.34  a  0.16 0.32  a  0.45  a  0 . 4 1 0.82  a  b  a  b  4/63  b  0.89 3/63  b  b  7/42  3/63  Weight g a i n and s p e c i f i c growth r a 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 o f f i s h t o t h e d i g e s t i b i l i t y t a n k s and t h e end o f t h e experiment. F i s h were f i r s t m a i n t a i n e d on B i o D i e t f o l l o w e d by f e e d i n g o f the b a s a l d i e t and f i n a l l y f i s h were f e d t h e t e s t d i e t s during t h i s period. Means i n each row w i t h t h e same s u p e r s c r i p t 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=0.05). Dry feed i n t a k e e x p r e s s e d as g / f i s h and as % o f wet body weight was c a l c u l a t e d o n l y f o r t h e p e r i o d i n which t h e f i s h were o f f e r e d t h e t e s t d i e t s .  ab  128 4.3.6  P r e d i c t i o n o f o r g a n i c m a t t e r , crude p r o t e i n and energy d i g e s t i b i l i t y o f t h e 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 t h e apparent n u t r i e n t values of i n d i v i d u a l d i e t a r y  digestibility  ingredients  An attempt was made t o i n v e s t i g a t e t h e 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 t h e d i g e s t i b i l i t y v a l u e s o f t h e 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 a c c u r a c y o f t h e d i g e s t i b i l i t y d a t a were made by comparing t h e o b s e r v e d v a l u e s f o r o r g a n i c m a t t e r , crude p r o t e i n and g r o s s energy o f t h e r e f e r e n c e d i e t s REF-a (experiments I I and III) the  and REF-b (experiment I V ) , w i t h c a l c u l a t e d v a l u e s based on 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 t o 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 b e l o n g t o t h e same b a t c h as t h o s e 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 t h e d e t e r m i n e d and t h e e x p e c t e d  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 v a l u e s f o r the  r e f e r e n c e 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 v a r y e x c e s s i v e l y between b a t c h e s .  129 T a b l e 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 c h i n o o k salmon i n experiment I I . Organic matter (%)  Crude protein (%)  Energy (MJ/kg)  Experiment I I R e f e r e n c e D i e t REF-a (Mean ± SEM) 89.19 Proximate a n a l y s i s 55.50 22.63 ±0.07 ±0.62 ±0.01 Digestibility  coefficients  Determined D i g e s t i b l e n u t r i e n t Diet  formulation  84.48 ±0.60  84.59 ±0.32  86.01 ±0.73  75.35  46.95  19.46  (Digestible N u t r i e n t )  1  S t e a m - d r i e d h e r r i n g meal 41.36 36.61 (86.13% O.M.,73.58% C P . , 2 2 .18 MJ/kg D.M. b a s i s )  11.30  E x t r u d e d wheat 8.11 1.70 (98.20% O.M.,17.83% C P . , 1 8 .46 MJ/kg D.M. b a s i s )  1.36  5.21 0.77 D r i e d whey .37 MJ/kg D.M. b a s is) (90.60% O.M.,14.13% C P . , 1 6  0.88  4.41 Freeze-dried euphausids 4.08 (86.36% O.M.,71.48% C P . , 1 8 .46 MJ/kg D.M. b a s i s ) 3  1.19  1.71 1.41 B l o o d meal (98.36% O.M., 95.69% C P . , 2 4 .31 MJ/kg D.M. b a s i s )  0.39  Vitamin & Mineral c a r r i e r (see e x t r u d e d wheat above)  2.02  0.42  0.34  Herring  8.40  0.0  3.02  1.70  0.0  0.40  72.92  44.99  18.88  103  104  103  oil  4  Supplements ~* Expected T o t a l Determined  E x p e c t e d (%)  2  R e f e r t o T a b l e 11 f o r d i e t f o r m u l a t i o n . R e f e r t o T a b l e 19 f o r d i g e s t i b i l i t y o f i n g r e d i e n t s No p u b l i s h e d d a t a on d i g e s t i b i l i t y a v a i l a b l e . E s t i m a t e 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 e t al. (1982). I n c l u d e s 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 o f soybean l e c i t h i n supplement.  130 T a b l e 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 - b (REF-a) which was chinook salmon i n experiment I I I . Organic matter (%)  Crude protein (%)  fed to Energy (MJ/kg)  Experiment I I I R e f e r e n c e D i e t REF-a (Mean ± SEM) 88 .90 55.62 Proximate a n a l y s i s 22 .90 ±0.21 ±0 .08 ±0,.01 Digestibility  coefficients  Determined D i g e s t i b l e n u t r i e n t Diet  formulation  80 .33 ±0 .18  84.84 ±0.25  81 .97 ±0,.32  71 .41  47.19  18 .77  (Digestible N u t r i e n t )  1  Steam-dried h e r r i n g meal . 41.36 (86.1.3% O.M.,73.58% C P . , 2 2 .18 MJ/kg D.M.  36.61 basis)  11 .30  E x t r u d e d wheat 8.11 1.70 (98.20% O.M.,17.83% C P . , 1 8 .46 MJ/kg D.M. b a s i s )  1 .36  5 .21 D r i e d whey .37 MJ/kg D.M. (90.60% CM.,14.13% C P . , 1 6  0.77 basis)  0 .88  4 .41 Freeze-dried euphausids (86.36% CM.,71.48% C P . , 1 8 .46 MJ/kg D.M.  4.08 basis)  1 .19  1 .71 B l o o d meal (98.36% O.M.,95.69% C P . ,24 .31 MJ/kg D.M.  1.41 basis)  0 .39  3  Vitamin & Mineral c a r r i e r (see e x t r u d e d wheat above)  2 .02  0.42  0 .34  Herring  8 .40  0.0  3 .02  1 .70  0.0  0 .40  oil  4  Supplements^  72 .92  Expected T o t a l Determined -s- E x p e c t e d  (%)  98  44.99 105  2  18 .88 99  R e f e r t o Table 11 f o r d i e t f o r m u l a t i o n . R e f e r t o Table 19 f o r d i g e s t i b i l i t y o f i n g r e d i e n t s No p u b l i s h e d d a t a on d i g e s t i b i l i t y a v a i l a b l e . E s t i m a t e 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 e t al. (1982). I n c l u d e s 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 o f 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 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. Organic matter (%)  Crude protein (%)  Energy (MJ/kg)  Experiment IV R e f e r e n c e D i e t REF-b (Mean ± SEM) 87.85 59.50 22.72 Proximate a n a l y s i s ±0.44 ±0.06 ±0.01 Digestibility  coefficients  Determined D i g e s t i b l e n u t r i e n t D i e t formulation- -  81.25 ±0.16  83.63 ±0.28  83.59 ±0.38  71.38  49.76  18.99  (Digestible N u t r i e n t )  1  Steam-dried h e r r i n g meal 45.93 40.68 (86.13% O.M.,73.58% C P . , 2 2 .18 MJ/kg, D.M,. b a s i s )  12.55  2.97 0.62 E x t r u d e d wheat (98.20% O.M.,17.83% C P . , 1 8 .46 MJ/kg D.M. b a s i s )  0.50  0.85 D r i e d whey 5.79 .37 MJ/kg D.M. b a s is) (90.60% O.M.,14.13% C P . , 1 6  0.97  Freeze-dried euphausids 4.89 4.53 (86.36% O.M.,71.48% C P . , 1 8 .46 MJ/kg D.M. b a s i s )  1.30  1.90 1.56 B l o o d meal (98.36% O.M., 95.69% C P . , 2 4 .31 MJ/kg D.M. b a s i s )  0.43  Vitamin & Mineral c a r r i e r (see e x t r u d e d wheat above)  2.02  0.42  0.34  Herring  7.18  0.0  2.58  1.70  0.0  0.40  J  oil  4  Supplements^ Expected T o t a l Determined -s- E x p e c t e d  72.38 (%)  99  2  48.66  19.07  102  100  R e f e r t o Table 12 f o r d i e t f o r m u l a t i o n . R e f e r t o Table 19 f o r d i g e s t i b i l i t y o f i n g r e d i e n t s No p u b l i s h e d d a t a on d i g e s t i b i l i t y a v a i l a b l e . E s t i m a t e 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 e t al. (1982). I n c l u d e s 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 o f soybean l e c i t h i n supplement.  132 4.4  DISCUSSION The o b j e c t i v e o f f e e d f o r m u l a t i o n i s t o s u p p l y t h e 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 p r o d u c t i o n . N u m e r i c a l c o e f f i c i e n t s are used t o e n a b l e t h e s e l e c t i o n o f i n g r e d i e n t s on t h e b a s i s o f t h e i r c h e m i c a l 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 v a l u e f o r t h e o p t i m i z a t i o n o f f i s h p r o d u c t i o n 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 t h e use o f h i g h  q u a l i t y e x p e n s i v e f i s h meals and any attempt t o reduce f e e d c o s t s n e c e s s i t a t e s t h e replacement o f some, i f not a l l , o f t h e  fish  meal w i t h l e s s e x p e n s i v e a n i m a l o r p l a n t p r o t e i n s o u r c e s .  In  t h i s t h e s i s , a v a r i e t y o f i n g r e d i e n t s o f a n i m a l and p l a n t byp r o d u c t o r i g i n were e v a l u a t e d on t h e b a s i s o f t h e i r n u t r i e n t digestibility,  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 e v a l u a t e d t o f a c i l i t a t e replacement o f one f i s h meal w i t h another a c c o r d i n g t o f i s h meal p r i c e trends,and  4.4.1  availability.  F e e d s t u f f e v a l u a t i o n according t o proximate composition and n u t r i e n t  4.4.1.1  digestibility  F i s h meals  On t h e b a s i s o f p r o x i m a t e a n a l y s i s , t h e h i g h p r o t e i n  and  o r g a n i c m a t t e r c o n t e n t o f t h e low temperature Norwegian herring/capelin  meal (LT) rank t h i s as t h e b e s t o f t h e  fish  meals o f t h i s s t u d y .  The p r o t e i n c o n t e n t o f B r i t i s h  h e r r i n g meal (HM) was  lower t h a n e x p e c t e d and t h i s was p r o b a b l y  due t o i n c l u s i o n o f ground f i s h  Columbia  (approx. 64% crude p r o t e i n ) i n  our sample ( B r i a n G i l l e y , B r i t i s h Columbia P a c k e r s , p e r s o n a l  133  communication).  N e v e r t h e l e s s , based on p r o x i m a t e  c o m p o s i t i o n , HM  c l o s e l y r i v a l s LT as a good source o f b o t h p r o t e i n and energy. A l t h o u g h anchovy meal (AM) was lower i n crude p r o t e i n t h a n t h e o t h e r f i s h meals e v a l u a t e d , t h e l i p i d and g r o s s energy c o n t e n t o f AM s u r p a s s e d t h a t o f a l l o t h e r f i s h meals t e s t e d i n t h i s  study.  T h e r e f o r e , based on c h e m i c a l 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 o t h e r f i s h meals.  Menhaden meal (MM) on  t h e o t h e r hand, c o n t a i n s a v e r y h i g h amount o f a s h and i t s use as a complete s u b s t i t u t e f o r more e x p e n s i v e c a r e f u l adjustments  f i s h meals would need  t o be made i n t h e m i n e r a l s u p p l e m e n t a t i o n o f  t h e complete d i e t s i n c e i n c r e a s e d l e v e l s o f d i e t a r y c a l c i u m 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 Shearer,  1984), e s p e c i a l l y i n t h e presence  (Hardy and  of phytic acid  (Richardson e t a l . , 1985). R e l a t i v e l y h i g h d i g e s t i b i l i t y v a l u e s , r a n g i n g 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 o f f i s h meals, were found i n j u v e n i l e chinook  salmon i n seawater.  Among t h e f i s h  meals, t h e r e was l i t t l e d i f f e r e n c e i n t h 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 f o r LT, HM and AM, b u t a l l had c o n s i d e r a b l y h i g h e r v a l u e s t h a n MM.  I t was a l s o e v i d e n t t h a t apparent  o r g a n i c 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 t h e ash c o n t e n t o f t h e f e e d s t u f f .  S i m i l a r f i n d i n g s , on t h e  r e l a t i o n s h i p between f i s h meal a s h c o n t e n t and apparent p r o t e i n d i g e s t i b i l i t y have been r e p o r t e d f o r rainbow t r o u t (Nose and Mamiya, 1963; G u l l e y , 1980).  The h i g h a s h c o n t e n t o f t h e  menhaden meal based d i e t may have a l s o i n c r e a s e d t h e endogenous f e c a l n i t r o g e n o f t h e f i s h due t o enhanced s l o u g h i n g o f f o f 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 t h e s i t u a t i o n i n f i s h f e d  134 d i e t s c o n t a i n i n g t h e o t h e r f i s h meals. the  I f t h i s d i d indeed occur,  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 o f t h e menhaden meal  would have been u n d e r e s t i m a t e d t h e most.  Crude p r o t e i n and g r o s s  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 t h e 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 h i g h e r than the f i g u r e s r e p o r t e d f o r rainbow t r o u t i n f r e s h w a t e r by numerous a u t h o r s (see NRC, Cho e t al.,  1982), and t h u s s u p p o r t t h e f i n d i n g s o f L a l l et al.  (1984) and L a l l salmon.  1981;  (1988, c i t e d by H i g g s , e t al.,  1990)  for Atlantic  In t h i s r e g a r d , t h e f e e d s t u f f d i g e s t i b i l i t y v a l u e s  o b t a i n e d f o r A t l a n t i c salmon h e l d i n seawater were n o t e d t o be s i g n i f i c a n t l y h i g h e r t h a n t h o s e f o r salmon i n f r e s h w a t e r .  Of t h e  f i s h meals t e s t e d i n t h e p r e s e n t s t u d y , t h e apparent crude p r o t e i n d i g e s t i b i l i t y o f anchovy meal (91.6%) by chinook  salmon  i n seawater most resembled t h e v a l u e d e t e r m i n e d f o r anchovy  meal  u s i n g rainbow t r o u t i n f r e s h w a t e r (89.3%; Watanabe e t al.,  1983).  The g r o s s energy d i g e s t i b i l i t y o f anchovy meal by rainbow t r o u t i n f r e s h w a t e r (91.3%) d e t e r m i n e d by Smith  (1976) and Smith et al.  (1980) a l s o approaches t h e v a l u e d e t e r m i n e d 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 o f h e r r i n g meal (HM) j u v e n i l e chinook salmon i n seawater i n t h e p r e s e n t s t u d y  by (90.5%,  Table 19) i s s u b s t a n t i a l l y h i g h e r t h a n v a l u e s r e p o r t e d 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 B i s h o p  (1977), or v a l u e s r e p o r t e d f o r rainbow t r o u t i n f r e s h w a t e r (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 w o r k i n g w i t h rainbow t r o u t i n f r e s h w a t e r , o b t a i n e d h i g h e r apparent crude p r o t e i n d i g e s t i b i l i t y v a l u e s f o r h e r r i n g meal t h a n t h o s e o b t a i n e d by Smith et al.  (1980).  causes o f t h e d i s c r e p a n c i e s i n r e s u l t s between s t u d i e s  may  Major  135 i n c l u d e d i f f e r e n c e s i n t h e o r i g i n o f f i s h meal, p r o c e s s i n g methods o f t h e raw p r o d u c t s , d i e t f o r m u l a t i o n , r a t i o n l e v e l and f e e d i n g f r e q u e n c y , f i s h age and s i z e , e n v i r o n m e n t a l c o n d i t i o n s and t h e d i s s i m i l a r methods o f f e c a l c o l l e c t i o n employed by t h e d i f f e r e n t r e s e a r c h e r s . Consequently, t h e p r e s e n t r e s u l t s do not p r o v i d e c o n c l u s i v e e v i d e n c e f o r a d i f f e r e n c e between s p e c i e s i n d i g e s t i v e e f f i c i e n c y o r an e f f e c t o f s a l i n i t y on d i g e s t i b i l i t y . But t h e y do h i g h l i g h t t h e problems i n e x t r a p o l a t i n g r e s u l t s a c r o s s s p e c i e s and e n v i r o n m e n t a l c o n d i t i o n s and t h e y suggest t h a t r e s u l t s o b t a i n e d w i t h one b a t c h o f a f e e d may not m i r r o r t h o s e f o r a n o t h e r when d i f f e r e n t s o u r c e s o f i n g r e d i e n t s a r e employed. E x c e s s i v e heat d u r i n g p r o c e s s i n g o f f i s h meals has been shown t o s e v e r e l y i m p a i r p r o t e i n d i g e s t i b i l i t y and b i o l o g i c a l value the  (Tarr and B i e l y , 1973).  Opstvedt e t al. (1984)  reduced p r o t e i n d i g e s t i b i l i t y o f overcooked  correlated  (95°C) p o l l o c k  f i s h meal by rainbow t r o u t , t o t h e f o r m a t i o n o f d i s u l f i d e bonds (S-S) from -SH groups.  Loss o f l y s i n e can a l s o o c c u r from m i l d  heat t r e a t m e n t i n t h e presence o f r e d u c i n g sugars (Carpenter e t al.,  1963).  Under s e v e r e h e a t i n g , i n t h e presence o f e i t h e r  sugars o r o x i d i z e d l i p i d s , t h e 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 t r y p t o p h a n , a r g i n i n e and m e t h i o n i n e can become reduced (Carpenter e t al., 1963).  From t h e i n f o r m a t i o n o b t a i n e d from t h e  s u p p l i e r s o f t h e f i s h meals used i n t h e p r e s e n t experiments (Appendix 3 ) , i t i s e v i d e n t t h a t t h e p r o c e s s i n g t e m p e r a t u r e s used i n t h e p r o d u c t i o n o f LT and HM d i d n o t appear t o d i f f e r significantly.  T h e r e f o r e , minor d i f f e r e n c e s i n t h e 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 t h e two meals may have r e s u l t e d from t h e i n c l u s i o n o f t h e ground f i s h o r t h e 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 .  R e s u l t s on t h e p r o t e i n  d i g e s t i b i l i t y o f b o t h c a p e l i n (89.4%) (Storebakken, 1985) ground f i s h  (79.0%) (Nose and Mamiya, 1963)  freshwater,  s u p p o r t the n o t i o n t h a t m i x i n g ground f i s h  h e r r i n g meal, as was  t h e case w i t h t h e HM  and  by rainbow t r o u t i n with  source used i n t h i s  s t u d y , w i l l depress t h e 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 h e meal.  A l t e r n a t i v e l y , a b l e n d of c a p e l i n meal w i t h  h e r r i n g meal would account f o r the h i g h e r n u t r i e n t c o e f f i c i e n t s of t h e LT meal.  Further,  the  digestibility  i n an experiment d e s i g n e d  t o e v a l u a t e t h e u t i l i z a t i o n of v a r i o u s k i n d s o f f i s h meals, Ketola  (1980) found t h a t growth r a t e s o f A t l a n t i c salmon f r y  which had been f e d d i e t s c o n t a i n i n g c a p e l i n , anchovy and 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 t h a t  w i t h a d i e t based on Norwegian whole h e r r i n g meal.  This  Canadian obtained again  suggests t h a t the use o f c a p e l i n meal as a component of the meal i n the p r e s e n t study, was u t i l i z a t i o n o f t h i s meal. Ketola  a l a r g e f a c t o r i n t h e enhanced  However, u n l i k e the experiment by  (1980), v a l i d comparisons o f the performance o f f i s h  the v a r i o u s t e s t d i e t s was  nor  isocaloric.  A l s o , the p e r i o d over which t h e s e d i e t s were f e d was The  on  not p o s s i b l e s i n c e the d i e t s i n the  p r e s e n t experiment were n e i t h e r i s o n i t r o g e n o u s  short.  LT  relatively  i n f o r m a t i o n on f e e d i n t a k e , on the o t h e r hand,  p r o v i d e d v a l u a b l e i n f o r m a t i o n r e g a r d i n g t h e poor p a l a t a b i l i t y 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 protein  isolates.  soybean  of  137 4.4.1.2  A n i m a l b y - p r o d u c t meals  P o u l t r y b y - p r o d u c t meal i s b e i n g i n c r e a s i n g l y used i n a n i m a l 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 t i m e progresses.  A l t h o u g h t h e q u a l i t y o f p o u l t r y b y - p r o d u c t meal  p r o t e i n i s r e a s o n a b l e (NRC, 1983), i t i s c h a r a c t e r i z e d by a h i g h ash c o n t e n t which can cause m i n e r a l imbalances as mentioned p r e v i o u s l y f o r menhaden meal.  With r e s p e c t t o t h e two s o u r c e s o f  p o u l t r y b y - p r o d u c t meals t e s t e d , t h e crude p r o t e i n c o n t e n t o f t h e meal from Kansas (PMK) was s u b s t a n t i a l l y h i g h e r t h a n t h a t o f t h e l o c a l source (PMB) even though t h e o r g a n i c m a t t e r c o n t e n t o f t h e former was o n l y one p e r c e n t a g e u n i t h i g h e r t h a n t h e l a t t e r . N e v e r t h e l e s s , due t o t h e h i g h e r l i p i d c o n t e n t o f t h e PMB meal compared t o t h a t o f t h e PMK meal, l i t t l e d i f f e r e n c e was found i n t h e g r o s s energy c o n t e n t o f t h e s e two p o u l t r y b y - p r o d u c t meals. When c o n s i d e r i n g t h e o t h e r a n i m a l b y - p r o d u c t s , b l o o d meal and f e a t h e r meal s t a n d out w i t h r e s p e c t t o t h e i r h i g h l e v e l s o f crude p r o t e i n and energy and, on t h i s b a s i s a l o n e , t h e y a r e 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  h i g h c a r b o h y d r a t e ( l a c t o s e ) c o n t e n t s o f d r i e d whey on t h e o t h e r hand, make t h i s p r o d u c t more s u i t a b l e as a b i n d i n g agent than a p r o t e i n supplement. lactalbumin  rather  The p r o t e i n , b e i n g p r i m a r i l y  ( P o t t e r , 1984), would, however, be e x p e c t e d t o be  h i g h l y d i g e s t i b i l e when p r o c e s s e d a t normal t e m p e r a t u r e s . 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 o f t h e d i f f e r e n t a n i m a l b y - p r o d u c t meals t e s t e d i n t h i s s t u d y was g r e a t e r t h a n t h a t observed f o r t h e f i s h - meals.  D i g e s t i b i l i t y of  o r g a n i c m a t t e r , crude p r o t e i n and g r o s s energy o f t h e i m p o r t e d p o u l t r y b y - p r o d u c t meal (PMK) was 7 t o 10 p e r c e n t a g e u n i t s h i g h e r  138  t h a n r e s p e c t i v e v a l u e s d e t e r m i n e d f o r t h e 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 v a l u e s f o r b o t h p o u l t r y byp r o d u c t meals were h i g h e r t h a n t h o s e r e p o r t e d i n freshwater  f o r rainbow t r o u t  (NRC, 1981; Cho e t al., 1982) and c h a n n e l c a t f i s h  (Brown and S t r a n g e , 1985).  But t h e g r o s s energy  digestibility  of t h e PMB meal i n chinook salmon was lower t h a n t h a t found i n rainbow t r o u t .  A l t h o u g h t h e feed i n t a k e o f t h e f i s h f e d t h e  d i e t s c o n t a i n i n g t h e two d i f f e r e n t p o u l t r y b y - p r o d u c t meals as t e s t ingredients d i d not d i f f e r s i g n i f i c a n t l y m a i n t a i n e d on t h e PMK2 d i e t had s l i g h t l y  (P>0.05),  fish  improved growth r a t e s ,  f e e d 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 t h a n t h o s e m a i n t a i n e d on t h e PMB2 d i e t .  T h i s may have been due t o t h e  h i g h e r p r o t e i n and energy c o n t e n t s i n t h e PMK2 d i e t r e l a t i v e t o the PMB2 d i e t .  Higgs e t a l . (197 9) found t h 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 b y - p r o d u c t meal f o r a l l o f t h e h e r r i n g meal o f a c o n t r o l d i e t on an i s o n i t r o g e n o u s slight,  basis r e s u l t e d i n only a  y e t s i g n i f i c a n t d e c r e a s e i n t h e growth r a t e o f coho  salmon f r y m a i n t a i n e d i n f r e s h w a t e r . growth r a t e , feed c o n v e r s i o n  The same a u t h o r s found t h a t  e f f i c i e n c y and p r o t e i n and g r o s s  energy u t i l i z a t i o n o f j u v e n i l e coho salmon i n seawater were n o t a f f e c t e d when 75% o f t h e h e r r i n g meal i n t h e c o n t r o l d i e t was r e p l a c e d by p r o t e i n from m i x t u r e s o f d e f a t t e d o r f u l l - f a t b y - p r o d u c t meal and f e a t h e r meal. reported  poultry  S i m i l a r f i n d i n g s have been  f o r j u v e n i l e rainbow t r o u t by Tiews e t al. (197 6) and  Gropp et al. (197 9 ) .  These s t u d i e s demonstrated t h a t  fish  p r o t e i n c o u l d be e n t i r e l y r e p l a c e d by a m i x t u r e o f p o u l t r y byp r o d u c t meal and f e a t h e r meal on an i s o n i t r o g e n o u s  basis,  139 p r o v i d e d t h a t the d i e t was  supplemented w i t h t h e e s s e n t i a l amino  a c i d s l y s i n e , m e t h i o n i n e 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 b y - p r o d u c t meals used i n the p r e s e n t  study may  poultry  have r e s u l t e d from  d i f f e r e n c e s i n t h e raw m a t e r i a l s i n c l u d e d i n the meal  (e.g.  f e a t h e r s , 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 processing procedures.  Information  employed i n t h e p r o d u c t i o n  on t h e p r o c e s s i n g method  o f the l o c a l l y - p r o d u c e d p o u l t r y  by-  p r o d u c t meal r e v e a l e d t h a t c o o k i n g t e m p e r a t u r e s (135°C) were much h i g h e r t h a n t h o s e employed d u r i n g f i s h meal p r o d u c t i o n  (<100°C).  D e t a i l s o f the p r o c e s s i n g method i n v o l v e d i n the p r e p a r a t i o n the i m p o r t e d p o u l t r y by-product meal (PMK) was,  meal may  unfortunately,  A r e d u c t i o n i n t h e ash content  not made a v a i l a b l e .  have been a c h i e v e d  of t h e PMK  t h r o u g h s e l e c t i v e removal o f bone  f o l l o w i n g t h e d r y i n g of the meal or e x c l u s i o n of h i g h c o n t a i n i n g body p a r t s before cooking  of  ash  (e.g., head, f e e t , neck) from t h e v i s c e r a ,  and d r y i n g .  I t i s noteworthy t h a t bone p r o t e i n i s  nearly a l l collagen  (Eastoe and E a s t o e , 1954)  which i s o f poor  n u t r i t i o n a l value.  Hence, t h e PMK meal not o n l y c o n t a i n e d more  p r o t e i n t h a n PMB, but a l s o t h e p r o t e i n i n PMK was improved q u a l i t y due t o i t s lower c o l l a g e n The  f e a t h e r meal (FM)  p r o t e i n was  by c h i n o o k salmon i n t h i s study.  content.  reasonably  and 70.0%  f r e s h w a t e r by Cho respectively.  well digested  However, t h e gross  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 p r o d u c t was v a l u e s of 73.7%  l i k e l y of  energy  lower t h a n the  r e p o r t e d f o r rainbow t r o u t i n  and S l i n g e r (1979) and Cho  et al.  (1982),  The t e s t d i e t c o n t a i n i n g 30% f e a t h e r meal  l i k e l y imbalanced w i t h r e s p e c t t o e s s e n t i a l amino a c i d  was  content  and t h i s may  have depressed  chinook growth r a t e , a p p e t i t e , f o o d  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 t o  fish  f e d the r e f e r e n c e d i e t . The apparent  d i g e s t i b i l i t y o f crude p r o t e i n and g r o s s energy  i n the b l o o d meal was v e r y low i n chinook salmon.  The  poor  a s s i m i l a t i o n o f t h i s f e e d s t u f f was p r o b a b l y due t o e x c e s s i v e h e a t i n g d u r i n g p r o c e s s i n g s i n c e t h i s p r o d u c t was c o l o r and c h a r r e d m a t e r i a l was p r e s e n t . significantly  dark p u r p l e i n  A p p e t i t e was  also  (P<0.05) lower i n f i s h r e c e i v i n g t h e t e s t  diet  c o n t a i n i n g t h e b l o o d meal t h a n i n f i s h r e c e i v i n g t h e r e f e r e n c e diet.  I t was  o f i n t e r e s t t o note t h a t f e e d p e l l e t s c o n t a i n i n g  the b l o o d meal as the t e s t f e e d s t u f f had a tendency t o f l o a t  on  the s u r f a c e f o r up t o 30 seconds b e f o r e s i n k i n g t o the tank bottom, a phenomenon not observed w i t h the o t h e r d i e t s . on the apparent  d i g e s t i b i l i t y o f crude p r o t e i n and g r o s s energy  o f b l o o d meal by rainbow t r o u t and A t l a n t i c salmon are variable.  Results  extremely  Apparent p r o t e i n d i g e s t i b i l i t y v a l u e s f o r rainbow  t r o u t i n f r e s h w a t e r have been r e p o r t e d t o be as low as 16% and h i g h as 99%  (Cho et a l . , 1982)  b e i n g r e p o r t e d by o t h e r a u t h o r s al.,  1980).  L a l l e t al.  p r o t e i n o f b l o o d meal was  w i t h numerous i n t e r m e d i a t e v a l u e s (Cho and S l i n g e r , 1979;  (1984) a l s o r e p o r t e d t h a t t h e  Smith  et  crude  p o o r l y d i g e s t e d by A t l a n t i c salmon i n  b o t h f r e s h w a t e r and seawater. (1986) noted t h a t t h e apparent  By c o n t r a s t , Asgard and  (1986) was  Austreng  d i g e s t i b i l i t y o f b l o o d p r o t e i n by  A t l a n t i c salmon i n seawater exceeded 97%. Asgard and A u s t r e n g  as  The b l o o d used by  not s u b j e c t e d t o heat,  p r e s e r v e d w i t h f o r m i c a c i d o r by f r e e z i n g . i n d i c a t e t h a t b l o o d from s l a u g h t e r h o u s e s  being  Such f i n d i n g s  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  that processing  c o n d i t i o n s are o p t i m a l  and  ingredients  such as f e a t h e r meal or meat and bone meal are a l s o  incorporated  i n t o the d i e t t o complement the amino a c i d imbalances of meal.  The  (NRC,  1983)  h i g h l e u c i n e and  blood  low i s o l e u c i n e c o n t e n t i n b l o o d meal  must be t a k e n i n t o account i n t h e c h o i c e  complementary p r o t e i n s o u r c e s i n f i s h d i e t s .  of  An excess o f  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 t h e r e b y i n c r e a s e the requirement f o r  i s o l e u c i n e i n the d i e t . (Harper et al.,  T h i s has been demonstrated i n r a t s  1955), p i g s  salmon (Chance et al.,  (Oestemer et al.,  1973)  source of r e a d i l y a v a i l a b l e B v i t a m i n s  t o be l e a c h e d  binder.  (NRC,  i s a good  1981).  The  fact  are i n c o n j u g a t e form makes them l e s s l i k e l y  i n t o t h e water d u r i n g f e e d i n g .  whey, b e i n g m o s t l y l a c t a l b u m i n , than c a s e i n  chinook  1964).  D r i e d whey, d e s p i t e i t s low p r o t e i n c o n t e n t ,  that these vitamins  and  (Morrison,  1949)  The p r o t e i n of  i s r e p o r t e d l y of h i g h e r q u a l i t y  and the whey a c t s as a good p e l l e t  In t h i s s t u d y , a 30% i n c l u s i o n o f d r i e d whey i n t o t h e  d i e t r e s u l t e d i n v e r y h a r d p e l l e t s which were r e j e c t e d by fish.  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, p r o v e d t o be somewhat s o f t e r i n t e x t u r e and was 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  the  accepted  reveal that  the  whey p r o t e i n i s m o d e r a t e l y d i g e s t e d by chinook salmon i n seawater.  The  r e l a t i v e l y h i g h d i g e s t i b l e energy c o n t e n t o f d r i e d  whey i n d i c a t e s t h a t 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 chinook salmon.  Cho  and  S l i n g e r (1979) and Cho  found t h a t rainbow t r o u t i n f r e s h w a t e r  et al.  (1982)  were a b l e t o almost  by  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 c o n c l u s i o n a l s o reached by L a l l e t al. (1984) f o r A t l a n t i c i n b o t h f r e s h w a t e r and seawater.  salmon  The lower d i g e s t i b i l i t y v a l u e s  o b t a i n e d f o r a l l n u t r i e n t s i n t h e d r i e d whey i n t h e p r e s e n t experiment may have been caused by t h e r e l a t i v e l y poor f e e d intake of f i s h fed t h i s d i e t .  The low f e e d i n t a k e would t e n d t o  i n c r e a s e t h e p r o p o r t i o n o f endogenous f e c a l n i t r o g e n waste i n t h e f e c e s and t h e r e b y r e s u l t i n u n d e r e s t i m a t i o n o f o r g a n i c m a t t e r , crude p r o t e i n and energy  digestibility.  4.4.1.3 P l a n t b y - p r o d u c t meals In r e g a r d t o t h e 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, o n l y rapeseed p r o t e i n c o n c e n t r a t e (RP), soybean meal (SM) and soybean p r o t e i n i s o l a t e  (SI) c o n t a i n s u f f i c i e n t l e v e l s o f p r o t e i n  t o p o t e n t i a l l y c o m p l e t e l y r e p l a c e f i s h meal i n s a l m o n i d d i e t s . The p r o x i m a t e c o m p o s i t i o n s o f t h e commercial c a n o l a meal ( C M ) ( v a r i e t y not s p e c i f i e d by manufacturer) and t h e g l u c o s i n o l a t e - f r e e c a n o l a meal d e r i v e d from t h e campestris  c u l t i v a r Tobin v a r i e t y  Brassica  (GCMa) were s i m i l a r .  By  c o n t r a s t , t h e s o l v e n t e x t r a c t i o n method employed t o reduce t h e g l u c o s i n o l a t e c o n t e n t o f c a n o l a meal (GCMb) tended t o e l e v a t e t h e p r o t e i n c o n t e n t o f t h e meal because o f removal o f some c a r b o h y d r a t e and l i p i d d u r i n g t h e e x t r a c t i o n p r o c e s s (Naczk e t al.,  1986). In g e n e r a l , t h e o r g a n i c m a t t e r d i g e s t i b i l i t y o f t h e p l a n t  b y - p r o d u c t meals was lower t h a n t h a t o f t h e a n i m a l b y - p r o d u c t s and f i s h meals.  However, apparent crude p r o t e i n and g r o s s energy  d i g e s t i b i l i t y o f t h e p l a n t f e e d s t u f f s tended t o be h i g h e r t h a n  143  v a l u e s o b t a i n e d f o r animal b y - p r o d u c t s Rapeseed p r o t e i n c o n c e n t r a t e i t s apparent  (excluding f i s h  meals).  (RP) was e x t r e m e l y w e l l d i g e s t e d 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 exceeded those o f  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 o f RP was a l s o t h e h i g h e s t among a l l n o n - f i s h meal feedstuffs.  D e s p i t e a lower p r o t e i n and g r o s s energy c o n t e n t i n  t h e d i e t RP2 compared t o d i e t s HM2 and AM2, t h e r e were o n l y s m a l l d i f f e r e n c e s among groups f e d t h e t h r e e d i e t s f o r growth r a t e , f e e d 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 . s u r v i v a l o f f i s h a t t h e end o f experiment RP2  diet.  Higgs e t a l .  (1982),  In a d d i t i o n , the  I I , was h i g h e s t f o r t h e  a l s o working w i t h  rapeseed  p r o t e i n c o n c e n t r a t e , c o n c l u d e d t h a t rapeseed p r o t e i n c o n c e n t r a t e c o u l d r e p l a c e a t l e a s t 25% o f t h e p r o t e i n i n a m o d i f i e d Abernathy dry d i e t w i t h o u t compromising growth r a t e and f e e d u t i l i z a t i o n o f j u v e n i l e chinook salmon.  Yurkowski  e t al.  (1978)  found  similar  r e s u l t s w i t h rainbow t r o u t when a l l o f t h e soybean meal o f t h e i r basal diet  ( a p p r o x i m a t e l y 24% o f t h e d i e t a r y p r o t e i n ) was  r e p l a c e d by rapeseed p r o t e i n c o n c e n t r a t e . found, however, t h a t complete replacement  The same a u t h o r s o f t h e f i s h meal w i t h  rapeseed p r o t e i n c o n c e n t r a t e l e d t o poor growth, f e e d 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 h i g h d i e t a r y  l e v e l s o f rapeseed p r o t e i n c o n c e n t r a t e observed by Yurkowski e t al.  (1978)  p o s s i b l y can be e l i m i n a t e d by r e d u c i n g t h e p h y t i c a c i d  c o n t e n t i n rapeseed p r o t e i n c o n c e n t r a t e and a l s o by supplementing t h e d i e t c o n t a i n i n g RP w i t h z i n c  (Higgs e t a l . ,  1982).  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 o r g a n i c m a t t e r , p r o t e i n and g r o s s energy i n t h e c a n o l a meals were lower noted f o r t h e rapeseed p r o t e i n c o n c e n t r a t e  crude than  T h i s may be due t o  t h e h i g h e r c o n t e n t s o f c a r b o h y d r a t e and f i b r e i n t h e c a n o l a meals r e l a t i v e t o RP  (Jones, 1979; NRC,  1983).  The s o l v e n t t r e a t m e n t  o f c a n o l a t o reduce t h e g l u c o s i n o l a t e c o n t e n t o f t h e 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 . apparent o r g a n i c m a t t e r , crude p r o t e i n and g r o s s  Indeed,  the  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 o f GCMb were, r e s p e c t i v e l y 15.8,  5.9  and 13.3 p e r c e n t a g e u n i t s lower than v a l u e s determined f o r t h e commercial  c a n o l a meal p r o d u c t  (CM).  Losses o f t h e more r e a d i l y  s o l u b l e c a r b o h y d r a t e s and p r o t e i n s d u r i n g t h e p r o c e s s may  have  r e s u l t e d i n a g r e a t e r p r o p o r t i o n o f more i n d i g e s t i b l e complex c a r b o h y d r a t e s and g l o b u l i n m o l e c u l e s b e i n g l e f t i n t h e meal. Moreover, i t i s not c l e a r whether whole seed o r commercial meal was  canola  s o l v e n t e x t r a c t e d and t h e r e f o r e whether t h e c a n o l a  p r o d u c t had been s u b j e c t e d t o heat t r e a t m e n t .  P r o p e r heat  t r e a t m e n t would l i k e l y be n e c e s s a r y t o improve p r o t e i n and digestibility.  energy  A h i g h f i b r e c o n t e n t i n t h e d i e t can a l s o have a  n e g a t i v e e f f e c t on d i g e s t i b i l i t y .  For i n s t a n c e , a h i g h f i b r e  c o n t e n t i n r a t d i e t s has been shown t o reduce t h e d i g e s t a gut t r a n s i t t i m e and c o n s e q u e n t l y lower t h e d i g e s t i b i l i t y  and  a b s o r p t i o n o f n u t r i e n t s a c r o s s t h e i n t e s t i n a l mucosa (Fleming and Lee, 1983).  Bergner et al.  (1975) c o n c l u d e d t h a t f i b r e i s  c a p a b l e o f a d s o r b i n g amino a c i d s and p e p t i d e s t h e r e b y t h e i r absorption.  impending  F i b r e a l s o i n c r e a s e s t h e endogenous f e c a l  n i t r o g e n l o s s due t o an i n c r e a s e d s l o u g h i n g o f i n t e s t i n a l mucosal cells  (Bergner e t al.,  production  1975)  (Schneeman et a l . ,  and concomitant enhancement o f mucus 1982).  T h i s l e a d s t o an  u n d e r e s t i m a t i o n o f o r g a n i c m a t t e r , crude p r o t e i n and digestibility.  energy  145 A l t h o u g h t h 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 f o r o r g a n i c m a t t e r , crude p r o t e i n and g r o s s energy were h i g h e r f o r t h e g l u c o s i n o l a t e - f r e e c a n o l a meal c u l t i v a r v a r i e t y t h e c o n v e n t i o n a l c a n o l a meal significant.  (GCMa) t h a n f o r  (CM), t h e d i f f e r e n c e e were n o t  S i n c e b o t h o f t h e d i e t s c o n t a i n i n g t h e GCMa and CM  as t h e t e s t i n g r e d i e n t s c o n t a i n e d e q u a l amounts o f p r o t e i n and g r o s s energy, some g e n e r a l comparisons on f i s h performance can be made between t h e two d i e t a r y t r e a t m e n t s .  The d a t a i n d i c a t e t h a t ,  w h i l e t h e d i g e s t i b l e energy and p r o t e i n c o n t e n t s o f t h e CM2 and GCMa2 d i e t s d i d n o t d i f f e r s i g n i f i c a n t l y  (P>0.05),  m a i n t a i n e d on t h e l a t t e r d i e t had s i g n i f i c a n t l y growth and f o o d and p r o t e i n u t i l i z a t i o n .  fish  (P<0.05) improved  These r e s u l t s suggest  t h a t t h e g l u c o s i n o l a t e c o n t e n t i n d i e t s based on commercial c a n o l a meal  (CM) can a f f e c t f i s h performance when t h e l e v e l o f  i n c l u s i o n o f t h e meal i n t o t h e d i e t i s 30% o r more. crude p r o t e i n d i g e s t i b i l i t y v a l u e s f o r c a n o l a meal  The apparent (84.5% f o r CM  and 87.9% f o r GCMa) o b t a i n e d w i t h c h i n o o k salmon i n seawater i n t h e p r e s e n t s t u d y , c l o s e l y agree w i t h d a t a o b t a i n e d by H i l t o n and Slinger  (1986) w i t h rainbow t r o u t i n f r e s h w a t e r (83.2% t o 87.1%  depending on t h e method o f 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 o b t a i n e d by L a l l ,  (1988, c i t e d by Higgs e t al., 1990)  w i t h A t l a n t i c salmon i n f r e s h w a t e r (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 o f t h e CM i n t h e p r e s e n t study was 8.2 p e r c e n t a g e u n i t s lower t h a n t h a t d e t e r m i n e d 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 e t al., 1990) w h i l e t h e d i g e s t i b l e energy c o e f f i c i e n t o f GCMa d i f f e r e d by o n l y 1.7 p e r c e n t a g e 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  t e s t e d a t two l e v e l s o f i n c l u s i o n i n t h e d i e t a i r dry b a s i s ) .  (15% and 30% on an  The f e e d i n t a k e s o f f i s h f e d d i e t s c o n t a i n i n g  each o f t h e soybean p r o d u c t s were poor. t o be u n p a l a t a b l e f o r c h i n o o k salmon F o w l e r , 1980), coho salmon species  (SI) were b o t h  Soybean meal i s r e p o r t e d  (Fowler and Banks, 1967;  (Fowler, 1980) and some o t h e r f i s h  (Akiyama e t al., 1984).  Recent work by M u r a i e t al.  (1989), has shown t h a t t r e a t m e n t o f 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  a u t h o r s c o n c l u d e d t h a t u t i l i z a t i o n o f soybean meal improves as the  s i z e otf t h e 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  sized  f i n g e r l i n g s i n t h e i r study f e d a d i e t i n which 77% o f t h e f i s h meal was r e p l a c e d by methanol e x t r a c t e d soybean  flour  supplemented w i t h e s s e n t i a l a c i d s , a c h i e v e d 87% o f t h e f e e d e f f i c i e n c y o f a c o n t r o l group f e d a f i s h meal based d i e t .  Tacon  et al. (1984) r e p o r t e d s i m i l a r f i n d i n g s f o r rainbow t r o u t w e i g h i n g over 30 g.  M o r t a l i t y o f chinnok f e d d i e t s c o n t a i n i n g  soybean p r o d u c t s as t h e t e s t i n g r e d i e n t was h i g h i n t h e p r e s e n t study.  The use o f c o m m e r c i a l l y p r e p a r e d h e x a n e - e x t r a c t e d soybean  meals i s n o t recommended f o r c h i n o o k salmon on t h e b a s i s o f t h i s and p r e v i o u s s t u d i e s c i t e d  above.  Apparent crude p r o t e i n and g r o s s energy  digestibility  c o e f f i c i e n t s f o r soybean meal i n t h i s s t u d y were l o w e r , 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 d e t e r m i n e d f o r rainbow t r o u t i n f r e s h w a t e r (Cho and S l i n g e r , 1979, Cho e t al., 1982) and t h o s e 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 e t a l . , 1990) and seawater  ( L a l l and B i s h o p , 1977; L a l l ,  1988; c i t e d by Higgs e t al., 1990).  147 Owing t o t h e e x t r e m e l y poor f e e d i n t a k e s o f f i s h f e d t h e d i e t s containing  soybean p r o d u c t s as t e s t i n g r e d i e n t s , t h e h i g h  proportion  o f endogenous f e c a l l o s s e s v e r y l i k e l y caused an  u n d e r e s t i m a t i o n o f o r g a n i c m a t t e r , crude p r o t e i n and g r o s s energy digestibility. 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 t h e p r e s e n t study were a l s o  v e r y i n c o n s i s t e n t , e s p e c i a l l y when t h e soybean p r o d u c t s were t e s t e d a t t h e lower d i e t a r y l e v e l .  Due t o t h e p r o b a b l e  u n d e r e s t i m a t i o n o f t h e n u t r i e n t d i g e s t i b i l i t y o f soybean p r o d u c t s and t h e h i g h l y v a r i a b l e d i g e s t i o n v a l u e s o b t a i n e d i n t h e p r e s e n t s t u d y , t h e use o f t h e s e c o e f f i c i e n t s i s q u e s t i o n a b l e f o r formulation  of salmonid d i e t s .  The p o s s i b i l i t y o f u s i n g  s p e c i a l l y p r o c e s s e d soybean p r o d u c t s as p a r t i a l o r 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 G r a i n p r o d u c t s Various by-products used i n animal f e e d s .  of the g r a i n processing industry are  Wheat m i l l i n g , which produces f l o u r as i t s  main p r o d u c t , a l s o y i e l d s wheat s h o r t s , wheat m i l l - r u n , and wheat m i d d l i n g s , and a l l o f t h e s e p r o d u c t s have been used i n f i s h f e e d formulations.  Wheat m i d d l i n g s  (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 a t a l e v e l o f 30% o r 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 a r e a good b i n d i n g agent i n p e l l e t  manufacturing. approximately  T h i s i n g r e d i e n t has a p r o t e i n c o n t e n t o f 16%, which f o r rainbow t r o u t i n f r e s h w a t e r , 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 1979;  92.0%, Cho e t al., 1982).  (95.3%, Cho and S l i n g e r ,  The d i g e s t i b i l i t y o f t h e  148 r e m a i n i n g d r y m a t t e r i n t r o u t i s , on t h e o t h e r hand, q u i t e low (Smith et al.,  1980; Cho et al.,  1982).  Thus t h e u s e . o f WM  as a  p e l l e t b i n d i n g agent r a t h e r t h a n l i g n i n s u l p h o n a t e or b e n t o n i t e can o n l y be j u s t i f i e d on t h e b a s i s o f s u p p l y and c o s t a l t h o u g h , u n l i k e t h e p e l l e t b i n d e r s , WM does enhance t h e 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 o f WM by chinook  salmon i n t h e p r e s e n t study was  a p p r o x i m a t e l y 10 p e r c e n t a g e u n i t s  lower t h a n f i g u r e s r e p o r t e d f o r rainbow t r o u t i n f r e s h w a t e r by t h e a u t h o r s mentioned c o n t e n t o f WM was  above.  In a d d i t i o n , t h e d i g e s t i b l e  energy  l e s s t h a n h a l f i t s g r o s s energy v a l u e ,  i n d i c a t i n g v e r y poor d i g e s t i b i l i t y o f t h e c a r b o h y d r a t e f r a c t i o n of t h i s p r o d u c t by chinook salmon i n seawater.  Similar  have been r e p o r t e d f o r rainbow t r o u t i n f r e s h w a t e r - (Cho S l i n g e r , 1979;  Smith e t al.,  1980; Cho e t al.,  A t l a n t i c salmon i n b o t h f r e s h w a t e r and seawater 1984).  The d i g e s t i b i l i t y  1982)  findings end  and f o r  ( L a l l et a l . ,  o f t h e o r g a n i c m a t t e r , and c o n s e q u e n t l y  of t h e energy y i e l d i n g n u t r i e n t s , i n t h e e x t r u d e d wheat (EW) chinook salmon i n the p r e s e n t experiment was of t h e WM. WM was  by  f a r superior to that  The apparent d i g e s t i b i l i t y o f t h e p r o t e i n i n  EW  and  s i m i l a r , however, t h e g r o s s energy d i g e s t i b i l i t y o f t h e  was over 50% g r e a t e r t h a n t h a t o f t h e WM.  EW  E x t r u d e d wheat, which  i s made from whole h a r d wheat, i s h i g h l y d i g e s t i b l e by chinook salmon because t h e h i g h t e m p e r a t u r e s and p r e s s u r e s 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 and Sturmbauer,  i n h i b i t o r n o r m a l l y found i n raw wheat (Hofer  1985)  temperatures i n v o l v e d .  i s i n a c t i v a t e d by t h e h i g h p r o c e s s i n g Numerous a u t h o r s (Inaba et al.,  Smith, 1971; Luquet and B e r g o t , 1976; B e r g o t and Breque,  1963; 1983;  Kaushik  and  Oliva-Teles,  gelatinization salmonids.  thereby  that  S p a n n h o f and  expected  to reduce  across the  likely  due  t o the  i n the d i g e s t i v e  the  carbohydrate  intestinal i n t h e EW  lower  crude  i n h a r d wheat) v e r s u s  basis; EW  for the  NRC,  1981) .  i s preferable  was  observed  increased.  Other  digestibility carbohydrate and Nose, 1979;  i n the  1967;  Bergot  Chen,  1973).  carbohydrate digestibility  protein  content  Breque,.1983).  due  i n t h e WM,  on  an  CF.  i s also wheat a i r dry on  and  energy  an a i r suggest source  digestibility coefficient i n the d i e t  was  apparent  protein high  (Inaba  Singh  1973; This  conditions,  to nutrient  digestibility  of t h i s study  agent  be  of  fed diets containing  have a l s o  e t a l . , 1963;  Rychly  may  fecal nitrogen  i n t h e d i e t may  higher  shown t h a t  i n salmonids  Under t h e s e  therefore,  absorption  % CF.  findings  w o r k e r s have a l s o  enhancement o f m e t a b o l i c  can,  i n the  salmon d i e t s .  Page and Andrews,  and  also  i n the hard  when i t s i n c l u s i o n l e v e l  low p r o t e i n  starch,  ( i . e . 7.3%  as a b i n d i n g  apparent  decreases and  content  f o r t h e WM  i n c l u s i o n i n chinook  A decrease  THe  ( i . e . 2.5  Hence, t h e  t o WM  and  r e l a t i v e to that  EW  crude  A decrease  system  mucosa.  fibre  further  the g a s t r o i n t e s t i n a l  amount o f h y d r o l y s i s  which i s p r o c e s s e d t o produce  o f EW  onto  by  has  same a u t h o r s  s t a r c h moves t h r o u g h  of feed  of the  that  (1983),  a m y l a s e s becomes a d s o r b e d  crude  time  nutrients  dry  Plantikow  The  that  its digestibility  f a s t e r t h a n does g e l a t i n i z e d s t a r c h .  retention  basis  improves  reducing starch hydrolysis.  indicated that tract  have d e m o n s t r a t e d  of starch g r e a t l y  Work by  demonstrated  1985)  be  and  Spannhof,  attributed to  production  (Ogino  the higher l e v e l reduced  interaction.  For  of  protein instance,  and  150 Watanabe et al.  (197 9)  showed t h a t e l e v a t i o n o f d i e t a r y  lipid  from 5% t o 23% i n c r e a s e d t h e d i g e s t i o n o f t o t a l energy by rainbow trout.  T h i s was due t o an i n c r e a s e i n t h e d i g e s t i b i l i t y o f b o t h  carbohydrates  (from 50.7% t o 58.5%) and l i p i d s  (from 74.7% t o  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 r e m a i n i n g t h e same (about 98.5%)  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 t e c h n i q u e on apparent organic matter  digestibility  The c l o s e agreement between o r g a n i c m a t t e 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 based on f e c e s c o l l e c t e d d i r e c t l y from t h e 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 t h o s e based on f e c e s c o l l e c t e d by t h e "Guelph system" s u g g e s t s t h a t t h e r e d u c t i o n i n t h e c r o s s s e c t i o n of t h e p i p e c o n n e c t i n g t h e t a n k w i t h t h e c o l l e c t i o n column g r e a t l y reduced t h e amount o f n u t r i e n t l e a c h i n g from t h e f e c e s t o the  s u r r o u n d i n g water medium.  10.2 p e r c e n t a g e u n i t s was  In experiment I , a d i f f e r e n c e o f  found between o r g a n i c m a t t e r  d i g e s t i b i l i t y c a l c u l a t e d u s i n g f e c e s c o l l e c t e d by  intestinal  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 u s i n g f e c e s c o l l e c t e d o v e r n i g h t by the  "Guelph system".  I n e x p e r i m e n t s I I - IV, d i f f e r e n c e s i n  o r g a n i c m a t t e 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 due t o t h e method o f fecal collection  ("Guelph system" vs i n t e s t i n a l d i s s e c t i o n ) were  s m a l l and t h e y ranged from 0.8 t o 4.2 p e r c e n t a g e u n i t s . S u r p r i s i n g l y , t h e mean o r g a n i c m a t t e r d i g e s t i b i l i t y  coefficient  of e x t r u d e d wheat which was d e t e r m i n e d u s i n g f e c e s 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 h i g h e r t h a n t h a t d e t e r m i n e d u s i n g f e c e s c o l l e c t e d d i r e c t l y from t h e water w i t h t h e "Guelph system". d i s c u s s e d 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  As  calculated  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 a r e u s u a l l y lower t h a n t h o s e d e t e r m i n e d from samples c o l l e c t e d from t h e water f o r two main r e a s o n s .  First,  f e c e s 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 u n d e r e s t i m a t e a s s i m i l a t i o n e f f i c i e n c y because o f c o n t a m i n a t i o n o f f e c e s w i t h body f l u i d s and because d i g e s t i o n and a b s o r p t i o n may not be f u l l y complete a t t h e t i m e o f s a m p l i n g . Second, f e c e s c o l l e c t e d from t h e water medium i n v a r i a b l y  undergo  some degree o f n u t r i e n t l e a c h i n g and t h i s s u b s e q u e n t l y 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 t h e c o l l e c t i o n o f f e c e s 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 t h a t d i g e s t i o n i s a c o n t i n u o u s p r o c e s s w i t h o u t diurnal variation. authors  T h i s has not been shown t o be t r u e by v a r i o u s  (Inaba e t al., 1962/ De l a Noue e t al., 1980; De S i l v a  and P e r e r a 1983, 1984) who have o b s e r v e d d i u r n a l v a r i a t i o n s i n b o t h 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 o f such  v a r i a t i o n , a f e c a l sample o b t a i n e d 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 p r o v i d e a r e p r e s e n t a t i v e sample.  I t can  t h e r e f o r e be p o s t u l a t e d t h a t , as t h e l o s s o f n u t r i e n t s due t o l e a c h i n g i s m i n i m i z e d , t h e 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 t h e water and t h o s e c o l l e c t e d  directly  from t h e 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 t h e 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 t h a n by any n u t r i e n t l e a c h i n g e r r o r s o f t h e "Guelph system" o r by t h e f e c a l c o n t a m i n a t i o n e r r o r s o f the d i s s e c t i o n technique. D i f f e r e n c e s between o r g a n i c m a t t e 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 d e t e r m i n e d u s i n g 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 t h o s e d e t e r m i n e d u s i n g f e c a l samples  collected  d i r e c t l y from t h e water w i t h t h e "Guelph system" were found t o be  152 significant  (P<0.05) f o r t h e r e f e r e n c e d i e t s as w e l l as f o r some  test ingredients  (P<0.01 f o r HM).  Differences i n d i g e s t i b i l i t y  between t h e two methods o f c o l l e c t i o n ranged from 1.6 t o 10.0 percentage u n i t s ; the c o e f f i c i e n t b e i n g c o n s i s t e n t l y lower f o r f e c e s 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 t h e  use o f f e c e s o b t a i n e d by s t r i p p i n g u n d e r e s t i m a t e s 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 t h a n t h e use o f f e c e s c o l l e c t e d by i n t e s t i n a l dissection.  T h i s i s due t o t h e a d d i t i o n a l c o n t a m i n a t i o n o f t h e  f e c e s o b t a i n e d by t h e former method w i t h u r i n e and body mucus, as w e l l as t h e i n a b i l i t y t o c o n s i s t e n t l y c o n t r o l t h e s e c t i o n from the  lower i n t e s t i n e from which t h e f e c e s a r e o b t a i n e d .  T h e r e f o r e , because t h e o r g a n i c m a t t e r d i g e s t i b i l i t y  coefficients  based on t h e "Guelph system" and i n t e s t i n a l d i s s e c t i o n p r o c e d u r e agree c l o s e l y , whereas t h o s e f o r s t r i p p i n g d i d n o t , 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 method f o r d e t e r m i n i n g f e e d s t u f f d i g e s t i b i l i t y  4.4.3  I n f l u e n c e o f d i e t on f i s h  reliable  i n chinook salmon.  performance  Due t o t h e s h o r t d u r a t i o n o f each experiment, r e s u l t s on f i s h performance i n t h e p r e s e n t s t u d y were o n l y i n t e n d e d t o determine whether t h e f i s h were i n p o s i t i v e n i t r o g e n b a l a n c e w h i l e f e c a l c o l l e c t i o n s were conducted.  However, i n f o r m a t i o n on  a p p e t i t e i s o f g r e a t importance i n t h e p r e s e n t e x p e r i m e n t s s i n c e l e v e l of d i e t a r y i n t a k e 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 e x e r t s on t h e e s t i m a t e s f o r t h 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 feedstuffs A l s o , d a t a on  (Ogino and Chen, 1973).  153 appetite provides  information regarding the unacceptability of  c e r t a i n i n g r e d i e n t s due t o t a s t e texture  (e.g. soybean p r o d u c t s ) o r  (e.g. d r i e d whey) which need t o be t a k e n 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 ingredient nutrient 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 V e r y c l o s e agreement was found between t h e d e t e r m i n e d and  t h e c a l c u l a t e d 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 f o r t h e r e f e r e n c e  d i e t used i n e x p e r i m e n t s I I  and  d i e t used i n experiment IV  I I I (REF-a) and f o r t h e r e f e r e n c e  (REF-b).  A shortcoming o f using t h i s information  i n an attempt  t o 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 t h a t i n t h i s study, t h e 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 t h e same b a t c h e s as t h o s e used t o p r e p a r e t h e r e f e r e n c e  diets.  ingredients  The c l o s e agreement between  t h e e x p e c t e d and t h e d e t e r m i n e d d i g e s t i b i l i t i e s o f o r g a n i c m a t t e r , crude p r o t e i n and g r o s s energy o f t h e r e f e r e n c e  diets  does, however, r e i n f o r c e t h e n o t i o n t h a t t h e 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  t h e d i g e s t i b l e p r o t e i n and energy c o n t e n t s o f  d i e t s f o r c h i n o o k salmon i n seawater.  formulated  154 CHAPTER 5 5.0  CONCLUSIONS T h i s t h e s i s was  u n d e r t a k e n w i t h the o b j e c t i v e o f  establishing digestibility conventional  i n f o r m a t i o n f o r an a r r a y  of  and n o v e l f e e d s t u f f s which are c u r r e n t l y b e i n g  used  or are o f p o t e n t i a l use i n the d i e t s o f c h i n o o k salmon r e a r e d 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 c h e m i c a l  coupled w i t h d i g e s t i b i l i t y determinations  may  analyses  allow a  reasonable  p r e d i c t i o n o f the p o t e n t i a l n u t r i t i o n a l v a l u e 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 u s i n g chromic o x i d e i n t h e 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 i n seawater was  established.  studies  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 r e g a r d i n g possible overestimation  of n u t r i e n t d i g e s t i b i l i t y  the  coefficients  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 b e i n g n a t u r a l l y v o i d e d i n the water were a d d r e s s e d . i n o r g a n i c m a t t e r , crude p r o t e i n and energy  The  discrepancies  digestibility  c o e f f i c i e n t s u s i n g 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 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 t e c h n i q u e s  and  d e t e r m i n e d w i t h f e c a l samples c o l l e c t e d from the water by  fish  those the  "Guelph system" were due t o a c o m b i n a t i o n of e r r o r s i n h e r e n t i n each c o l l e c t i o n t e c h n i q u e .  Methods which r e l y on t h e  collection  of f e c e s 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 o f f i s h r e q u i r e d , the s m a l l amount o f f e c a l sample c o l l e c t e d , t h e 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 i m p o r t a n t l y , 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 i n d u c e d by t h e r e p e a t e d  h a n d l i n g of t h e  155 fish.  With r e g a r d t o t h e 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 , a r e v e r y easily stressed.  Every e f f o r t s h o u l d t h e r e f o r e be t a k e n t o  m i n i m i z e h a n d l i n g and d i s t u r b a n c e d u r i n g t h e d i g e s t i b i l i t y s t u d y . In p a r t i c u l a r , c a r e s h o u l d be e x e r c i s e d t o a v o i d s c a l e l o s s and t o c a r r y a l l c u l t u r e o p e r a t i o n s such as f e e d i n g changing water filters,  c o l l e c t i n g f e c e s and f l u s h i n g t h e tank system w i t h o u t  e l i c i t i n g f r e i g h t responses i n t h e f i s h . The n u t r i e n t l e a c h i n g l o s s e s a s s o c i a t e d w i t h f e c e s c o l l e c t e d w i t h t h e "Guelph system" i n experiment I were caused m a i n l y by a d e f i c i e n c y i n t h e d e s i g n o f t h e e x i t p i p e from t h e d i g e s t i b i l i t y tank which p r e v e n t e d f e c e s from b e i n g r a p i d l y c a r r i e d t o t h e a r e a of u n d i s t u r b e d water i n t h e c o l l e c t i o n column.  I t was  postulated  at t h e end o f experiment I and s u b s e q u e n t l y c o n f i r m e d i n e x p e r i m e n t s I I - IV, t h a t m o d i f i c a t i o n s t o t h e tank f l u s h i n g hydrodynamics would e n a b l e l e a c h i n g l o s s e s t o be m i n i m i z e d and hence t h e g e n e r a t i o n o f 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 digestibility.  C o n s e q u e n t l y , t h e "Guelph system" was found t o  be t h e p r e f e r r e d t e c h n i q u e f o r c o l l e c t i n g f e c e s f o r t h e d e t e r m i n 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 i n chinook salmon h e l d i n seawater.  F u r t h e r , methodology  findings, hitherto ignored i n  p r e v i o u s l y p u b l i s h e d r e s e a r c h on f e e d s t u f f d i g e s t i b i l i t y suggest t h e need t o remove f i s h s c a l e s from f e c a l  in fish  samples  c o l l e c t e d salmon h e l d i n seawater s i n c e t h e s e 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 o f crude p r o t e i n , t o be u n d e r e s t i m a t e d .  156 In experiments I I , I I I and IV, t h e apparent d i g e s t i b i l i t y o f o r g a n i c m a t t e r , crude p r o t e i n and g r o s s energy, as w e l l as t h e d i g e s t i b l e energy v a l u e s o f v a r i o u s f i s h meals ( h e r r i n g meal, anchovy meal, menhaden meal, Norwegian  LT f i s h m e a l ) , p o u l t r y by-  p r o d u c t meals (two s o u r c e s ) , f e a t h e r meal, b l o o d meal,  dried  whey, c a n o l a p r o t e i n p r o d u c t s (commercial c a n o l a meal, g l u c o s i n o l a t e - f r e e c a n o l a meals, rapeseed p r o t e i n c o n c e n t r a t e ) , soybean meal, soybean p r o t e i n i s o l a t e , e x t r u d e d wheat and wheat m i d d l i n g s were determined w i t h chinook salmon i n seawater as t h e target species.  I n g e n e r a l , i t was apparent t h a t chinook  salmon  were a b l e t o d i g e s t t h e energy o f t h e f i s h meals more e f f i c i e n t l y t h a n t h a t o f t h e a n i m a l and p l a n t b y - p r o d u c t f e e d s t u f f s .  This i s  thought t o o c c u r because t h e energy c o n t e n t o f t h e f i s h meals r e s i d e s l a r g e l y i n t h e p r o t e i n and o i l f r a c t i o n s , b o t h o f which are  h i g h l y d i g e s t i b l e by chinook salmon, w h i l e much o f t h e energy  c o n t e n t o f t h e p l a n t b y - p r o d u c t s stems from complex c a r b o h y d r a t e s , which a r e e i t h e r i n d i g e s t i b l e o r p o o r l y d i g e s t e d by chinook salmon. the  L i k e w i s e , t h e crude p r o t e i n d i g e s t i b i l i t y o f  a n i m a l b y - p r o d u c t s , e s p e c i a l l y t h a t o f b l o o d meal, i s m a i n l y  d e t e r m i n e d by t h e t e c h n i c a l p r o c e s s i n g p r o c e d u r e employed.  The  s o u r c e o f b l o o d meal used i n t h i s s t u d y was so o v e r h e a t e d d u r i n g p r o c e s s i n g , t h a t i t s crude p r o t e i n was o n l y 2 9.4% d i g e s t e d by c h i n o o k salmon.  On t h e o t h e r hand, based on d i g e s t i b i l i t y  assessment, n o v e l f e e d s t u f f s such as rapeseed p r o t e i n c o n c e n t r a t e appear t o be h i g h l y p r o m i s i n g as p a r t i a l o r even r e p l a c e m e n t s o f f i s h meal i n s a l m o n i d d i e t s . are  complete  Even though t h e r e  problems a s s o c i a t e d w i t h t h e use o f c e r t a i n p l a n t - b a s e d  p r o t e i n s o u r c e s , such as t h e u n p a l a t a b i l i t y o f t h e soybean  157 p r o d u c t s i n the p r e s e n t s t u d y , t h e s e problems can l i k e l y overcome t h r o u g h i n n o v a t i v e r e s e a r c h Finally,  Due  efforts.  the r e s u l t s o f t h i s t h e s i s support the view t h a t  wheat m i d d l i n g s salmon.  be  are not an adequate source of energy f o r chinook  t o t h e low d i g e s t i b i l i t y o f the c a r b o h y d r a t e p o r t i o n  o f t h i s p r o d u c t , most of t h i s i n g r e d i e n t i s wasted and of p o l l u t i o n i n f i s h farming.  i s a cause  By c o n t r a s t , the s t a r c h component  of e x t r u d e d wheat became a v a l u a b l e source o f energy f o r chinook salmon owing t o enhanced s t a r c h g e l a t i n i z a t i o n . recommended t h a t wheat m i d d l i n g s , incorporated completely  It i s therefore  which are c u r r e n t l y  i n t o s a l m o n i d d i e t s as a b i n d i n g agent, be  r e p l a c e d by more e f f i c i e n t l y  digested binders  such as  e x t r u d e d wheat or d r i e d whey. The  f i n a l assessment of t h e e f f e c t i v e n e s s of d i e t s  formulated  upon the b a s i s o f the d i g e s t i b i l i t y of the n u t r i e n t s  and energy of t h e 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 must be evaluated  by o b s e r v a t i o n  o f weight g a i n , f e e d e f f i c i e n c y and body  c o m p o s i t i o n o f f i s h r e c e i v i n g the d i e t s under s p e c i a l c u l t u r e conditions.  Only t h e r e s u l t s of t h e s e l o n g - t e r m f e e d i n g  trails  w i l l v e r i f y t h e adequacy of the d i e t a r y amino a c i d b a l a n c e the a v a i l a b i l i t y  and p o s t a b s o r p t i v e u t i l i z a t i o n o f the d i e t a r y  n u t r i e n t s f o r t i s s u e d e p o s i t i o n as w e l l as the energy/protein  and  r a t i o s f o r growth.  optimal  158 BIBLIOGRAPHY Ackman, R.G. (1977). Rapeseed o i l : c h e m i c a l and p h y s i c a l c h a r a c t e r i s t i c s . P r o c e e d i n g s o f symposium on rapeseed o i l , meal, and by-product u t i l i z a t i o n . Rapeseed A s s o c i a t i o n o f Canada No. .45: 12-37. Akiyama, T., M u r a i , T., Hirasawa, Y. and Nose, T. (1984). Supplementation o f v a r i o u s meals t o f i s h meal d i e t f o r chum salmon f r y . A q u a c u l t u r e , 37: 217-222. AOAC. (1975). O f f i c i a l methods o f a n a l y s i s . A s s o c i a t i o n o f O f f i c i a l A n a l y t i c a l Chemists (12th e d i t i o n ) , Washington, 1094 pp.  D.C.  A r a i , S. (1981). A p u r i f i e d t e s t d i e t f o r coho salmon, Oncorhynchus kisutch, f r y . B u l l . Jap. Soc. S c i . F i s h . 47: 550.  547-  A r c h d e k i n , C.G., H i g g s , D.A., McKeown B.A. and P l i s e t s k a y a , E. (1988) P r o t e i n r e q u i r e m e n t s o f p o s t - j u v e n i l e chinook salmon i n seawater. P r o c . A q u a c u l t u r e symposium, Vancouver, B r i t i s h Columbia. A q u a c u l t u r e A s s o c i a t i o n o f Canada B u l l e t i n , December e d i t i o n 88-4; 78-80. A r c h i b a l d , D. (1990). M a r k e t i n g B r i t i s h Columbia's farmed salmon. A q u a c u l t u r e A s s o c i a t i o n o f Canada B u l l e t i n , June e d i t i o n 90-2; 5-12. Asgard, T., and A u s t r e n g , E. (1986). B l o o d , e n s i l e d o r f r o z e n , as f e e d f o r s a l m o n i d s . A q u a c u l t u r e , 55: 263-284. Ash, R. (1980) . H y d r o l y t i c c a p a c i t y o f t r o u t (Salmo gairdneri) i n t e s t i n a l mucosa w i t h r e s p e c t t o t h r e e s p e c i f i c d i p e p t i d e s . Comp. Biochem. P h y s i o l . 65B: 173-176. Ash, R. (1985). 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 . I n : Cowey, C.B., Mackie, A.M. and B e l l , J.G. ( e d s . ) , N u t r i t i o n and F e e d i n g o f F i s h . Academic P r e s s , London. A u s t r e n g , E. (1978). D i g e s t i b i l i t y d e t e r m i n a t i o n i n f i s h u s i n g chromic o x i d e marking and a n a l y s i s o f c o n t e n t s from d i f f e r e n t s e c t i o n s o f t h e g a s t r o i n t e s t i n a l t r a c t . A q u a c u l t u r e 13, 265272. Balch, C C , R e i d , J.T. and S t r o u d , J.W. (1957). F a c t o r s i n f l u e n c i n g t h e r a t e o f e x c r e t i o n o f a d m i n i s t e r e d chromium s e s q u i o x i d e by s t e e r s . B r . J . N u t r . , 11: 184-197. Beames, R.M. and Eggum, B.O. (1981). The e f f e c t o f food t y p e l e v e l o f p r o t e i n , f i b r e and s t a r c h on n i t r o g e n e x c r e t i o n p a t t e r n s i n r a t s . B r . J . N u t r . , 46: 301-313.  and  B e l l , J.M. ( 1 9 8 2 ) . F r o m r a p e s e e d t o c a n o l a : A b r i e f h i s t o r y o f r e s e a r c h f o r s u p e r i o r meal a n d e d i b l e o i l . P o u l t r y S c i e n c e 61: 613-622. B e r g n e r , H., S i m o n , 0. a n d Z i m m e r , M. ( 1 9 7 5 ) . C r u d e f i b e r c o n t e n t o f t h e d i e t as a f f e c t i n g t h e p r o c e s s o f amino a c i d a b s o r p t i o n in rats. Arch. Tierernahr., 25: 95-104. Bergot, F. (1979). E f f e c t s o f d i e t a r y c a r b o h y d r a t e s and t h e i r mode o f d i s t r i b u t i o n o n g l y c e m i a i n r a i n b o w t r o u t (Salmo gairdneri R i c h a r d s o n ) Comp. B i o c h e m . P h y s i o l . , 64A: 5 4 3 - 5 4 7 . Bergot, F. and Breque, J . (1983). D i g e s t i b i l i t y o f s t a r c h rainbow t r o u t : e f f e c t s o f t h e p h y s i c a l s t a t e o f s t a r c h t h e i n t a k e l e v e l . A q u a c u l t u r e , 22:81-96. B i r k e t t , L. (1969). The n i t r o g e n b a l a n c e p e r c h . J . Exp. B i o l . , 50: 375-386.  i n plaice,  sole  by and o f  and  B l i g h , E . G . a n d D y e r , W.A. (1959). A r a p i d method o f t o t a l l i p i d e x t r a c t i o n and p u r i f i c a t i o n . Can. J . Biochem. P h y s i o l . 37: 911-917. B o g e , G., R i g a l , A . a n d P e r e s , G. ( 1 9 8 1 ) . R a t e s o f in vivo i n t e s t i n a l a b s o r p t i o n o f g l y c i n e and g l y c y l g l y c i n e by rainbow  trout  (Salmo  gairdneri  R . ) . Comp. B i o c h e m .  P h y s i o l . , 69A:  455-  459. B o g g i o , S . T . , H a r d y , R.W., Babbitt, (1985). The i n f l u e n c e o f d i e t a r y a c e t a t e l e v e l on p r o d u c t q u a l i t y 51: 13-24.  J.K. and Brannon, E . L . l i p i d source and " - t o c o p h e r o l o f rainbow t r o u t . Aquaculture  Bowen, S.H. ( 1 9 7 8 ) . C h r o m i c a c i d i n a s s i m i l a t i o n s t u d i e s caution. T r a n s . Am. F i s h . S o c , 1 0 7 ( 5 ) : 7 5 5 - 7 5 6 .  - a  B o w e n , S.H. ( 1 9 8 7 ) . D i e t a r y p r o t e i n r e q u i r e m e n t s o f f i s h e s r e a s s e s s m e n t . C a . J . F i s h . A q u a t . S c i . 44: 1995-2001.  - a  B r e t t , J.R. ( 1 9 7 9 ) . E n v i r o n m e n t a l f a c t o r s a n d g r o w t h . I n : Hoar, W.S., R a n d a l l , D . J . a n d B r e t t , J . R . ( e d s . ) , F i s h P h y s i o l o g y , V o l . 8. A c a d e m i c P r e s s , New Y o r k , p p 5 9 9 - 6 7 5 . B r o w n , P.B. a n d S t r a n g e , R . J . ( 1 9 8 5 ) . P r o t e i n digestibility c o e f f i c i e n t s f o r y e a r l i n g channel c a t f i s h f e d high protein f e e d s t u f f s . P r o g . F i s h C u l t . 47(2) 94:97. B u d d i n g t o n , R.K., C h e n , J . a n d D i a m o n d , R. A n n u . M e e t . , L o s A n g e l e s , 26-31 May.  (1985).  Abstr.  AAAS.  B u d d i n g t o n , R.K. a n d H i l t o n , J.W. (1987) . I n t e s t i n a l a d a p t a t i o n s o f r a i n b o w t r o u t t o c h a n g e s i n d i e t a r y c a r b o h y d r a t e . Am. J . P h y s i o l . , 253 ( G a s t r o i n t e s t . L i v e r P h y s i o l . , 1 6 ) : G485-G496.  160 B u h l e r , D.R. and H a l v e r , J.E. (1961). N u t r i t i o n o f s a l m o n i d f i s h e s . IX. Carbohydrate requirement o f chinook salmon. J . N u t r . 74: 307-318. C a r p e n t e r , K . J . , March, B.E., M i l n e r , C.K. and Campbell, R.C. (1963) . A growth assay w i t h c h i c k s f o r t h e l y s i n e c o n t e n t o f p r o t e i n c o n c e n t r a t e s . B r . J . N u t r . , 17: 309-323. C a s t l e d e n e , A . J . and B u c k l e y , J.T. (1980). D i s t r i b u t i o n and m o b i l i t y o f omega 3 f a t t y a c i d s i n rainbow t r o u t f e d v a r y i n g l e v e l s and t y p e s o f d i e t a r y l i p i d . J . N u t r . 110 ( 4 ) : 675-685. Chance, R.E., M e r t z , E.T. and H a l v e r , J.E,. (1964). N u t r i t i o n o f s a l m o n i d f i s h e s . X I I . I s o l e u c i n e , l e u c i n e , v a l i n e and p h e n y l a l a n i n e r e q u i r e m e n t s o f chinook salmon and i n t e r r e l a t i o n s between i s o l e u c i n e and l e u c i n e f o r growth. J . N u t r . , 83: 177-185. Cho, C.Y., B a y l e y , H.S. and S l i n g e r , S.J. (1975). An automated f i s h r e s p i r o m e t e r f o r n u t r i t i o n s t u d i e s . P r o c . 28th Ann. M e e t i n g o f Can. Conf. f o r F i s h . Res., Vancouver, B.C. Cho, C.Y. and Kaushik, S . J . (1985). E f f e c t s o f p r o t e i n i n t a k e on m e t a b o l i z a b l e and n e t energy v a l u e s o f f i s h d i e t s . I n : C B . Cowey, A.M. Mackie and J.G. B e l l ( e d s . ) , N u t r i t i o n and F e e d i n g o f F i s h . Academic P r e s s , London, pp. 95-117. Cho, C.Y. and S l i n g e r , S . J . (1979). Apparent d i g e s t i b i l i t y measurement i n f e e d s t u f f s f o r rainbow t r o u t . I n : F i n f i s h n u t r i t i o n and f i s h f e e d t e c h n o l o g y , J.E. H a l v e r and K. Tiews (Eds), V o l . I I . Heenemann V e r l a g s g e s e l l s c h a f t MbH. B e r l i n , pp 239-247. Cho, C.Y., S l i n g e r , S . J . and B a y l e y , H.S. (1982). B i o e n e r g e t i c s o f s a l m o n i d f i s h e s : Energy i n t a k e , e x p e n d i t u r e and p r o d u c t i v i t y . Comp. Biochem. P h y s i o l . , 73B: 25-41. Choubert, C , De l a Noue, J . and Luquet, P. (1979). Continuous q u a n t i t a t i v e automatic c o l l e c t o r f o r f i s h feces. Prog. F i s h C u l t . , 41: 64-67. Choubert, C , De l a Noiie, J . and Luquet, P. (1982). D i g e s t i b i l i t y i n f i s h : improved d e v i c e f o r t h e a u t o m a t i c c o l l e c t i o n o f f a e c e s . A q u a c u l t u r e , 29: 185-189. Cowey, C B . (1979). P r o t e i n and amino a c i d r e q u i r e m e n t s o f f i n f i s h . I n : F i n f i s h n u t r i t i o n and f i s h f e e d t e c h n o l o g y , J.E. H a l v e r and K. Tiews (Eds), V o l . I . Heenemann V e r l a g s g e s e l l s c h a f t MbH. B e r l i n , pp 3-16.  161 Cowey, C.B., Adron, J.W., Brown, D.A. and Shanks, A.M. (1975). S t u d i e s on t h e n u t r i t i o n o f marine f l a t f i s h . The metabolism o f g l u c o s e by p l a i c e ( P l e u r o n e c t e s platessa) and t h e e f f e c t o f d i e t a r y energy source on p r o t e i n u t i l i z a t i o n i n p l a i c e . B r . J . N u t r . , 33: 219-231. Cowey, C.B. and Sargent, J.R. (1979). N u t r i t i o n . I n : Hoar, W.S., R a n d a l l , D.J. and B r e t t , J.R. ( e d s . ) , F i s h P h y s i o l o g y , V o l V I I I . B i o e n e r g e t i c s and growth. Academic P r e s s , New York, NY. Dabrowski, K. (1983). Comparative a s p e c t s o f p r o t e i n d i g e s t i o n and amino a c i d a b s o r p t i o n i n f i s h and o t h e r a n i m a l s . Comp. Biochem. P h y s i o l . , 74A: 417-426. Dabrowski, K. and Dabrowska, H. (1981). D i g e s t i o n o f p r o t e i n by rainbow t r o u t (Salmo gairdneri Rich.) and a b s o r p t i o n o f amino a c i d s w i t h i n t h e a l i m e n t a r y t r a c t . Comp. Biochem. P h y s i o l . , 69A: 99-111. Dabrowski, K., Leray, C , Nonnotte, G. and C o l i n , D.A. (1986). P r o t e i n d i g e s t i o n and i o n c o n c e n t r a t i o n s i n rainbow t r o u t (Salmo gairdneri Rich.) d i g e s t i v e t r a c t i n sea- and f r e s h w a t e r . Comp. Biochem. P h y s i o l . 83A: 27-39. Davignon, J . , Simmonds, S . J . and Ahrens, E.H. (1968). U s e f u l n e s s o f chromic o x i d e as an i n t e s t i n a l s t a n d a r d f o r b a l a n c e s t u d i e s i n f o r m u l a f e d p a t i e n t s and f o r assessment o f c o l o n i c f u n c t i o n . J . C l i n . I n v e s t . 47: 127-138. De l a Noue, J . and Choubert, G. (1986) . D i g e s t i b i l i t y i n rainbow t r o u t : Comparison o f t h e d i r e c t and i n d i r e c t methods o f measurement. P r o g . F i s h . C u l t . 48: 190-195. De l a Noiie, J . , Choubert, G., P a g n i e z , B., B l a n c , J.M. and Luquet, P. (1980) . Mesure des c o e f f i c i e n t s d u t i l i z a t i o n d i g e s t i v e chez l a t r u i t e a r c - e n - c i e l . Etude des temps d ' a d a p t a c i o n a un nouveau regime, du temps de r e c o l t e des f e c e s e t du nombre des r e p e t i t i o n s . Can. J . F i s h . Aquat. S c i . , 37 (12) : 2218-2224. 1  DeLong, D.C, H a l v e r , J.E. and M e r t z , E.T. (1958). N u t r i t i o n o f s a l m o n i d f i s h e s . V I . P r o t e i n requirements o f chinook salmon a t two water t e m p e r a t u r e s . J . N u t r . , 65: 589-600. De S i l v a , S.S. and P e r e r a , M.K. (1983). D i g e s t i b i l i t y o f an a q u a t i c macrophyte by t h e c i c h l i d Etroplus suratensis with o b s e r v a t i o n s on t h e r e l a t i v e m e r i t s o f t h r e e i n d i g e n o u s components as markers and d a i l y changes i n p r o t e i n d i g e s t i b i l i t y . J . F i s h . B i o l . , 23: 675-684. De S i l v a , S.S. and P e r e r a , M.K. (1984). D i g e s t i b i l i t y i n Sarotherodon niloticus f r y : effect of dietary protein level and s a l i n i t y w i t h f u r t h e r o b s e r v a t i o n s on v a r i a b i l i t y i n d i e t a r y d i g e s t i b i l i t y . A q u a c u l t u r e , 38: 293-306.  162 Donaldson, E.M. (1981) . The p i t u i t a r y - i n t e r r e n a l a x i s as an i n d i c a t o r o f s t r e s s i n f i s h . I n : A.D. P i c k e r i n g (Ed.), S t r e s s and F i s h . Academic P r e s s , London, pp. 11-47. E a s t o e , J E . and E a s t o e , B. (1954) . The o r g a n i c c o n s t i t u e n t s o f mammalian compact bone. The b i o c h e m i c a l J o u r n a l 57: 453. E d i n , M. (1918). O r i e n t e r a n d e Forsok Over en pa L e d k o p p r i n c i p e n Grundad Metod a t t Bestamma en F o d e r b l a n d i n g s S m a l t b a r h e t . Cent. F o r s o k s v a s e n d e t j o r d b r u c k . Stockholm Medd., 165: 1-28. Egan, D. and Kenney, A. (1990) . Salmon f a r m i n g i n B r i t i s h Columbia. World A q u a c u l t u r e 21 ( 2 ) ; pp 6-11, 24-29. E z e a s o r , D.N. and Stokoe, W.M. (1980) . Scanning m i c r o s c o p i c study on t h e gut mucosa o f t h e rainbow t r o u t (Salmo gairdneri). J . F i s h B i o l . , 17: 619-634. Fange, R. and Grove, D. (1979). D i g e s t i o n . I n : Hoar, W.S., R a n d a l l , D.J. and B r e t t , J.R. ( e d s . ) , F i s h P h y s i o l o g y , V o l . V I I I . Academic P r e s s , New York, pp 162-241. Fenton, T.W. and Fenton, M. (1979) . An improved procedure f o r t h e d e t e r m i n a t i o n o f chromic o x i d e i n f e e d and f e c e s . Can. J . Anim. S c i . 59: 631-634. F e r r a r i s , R.P., C a t a c u t a n , M.R. and Adan, P.J. (1986). D i g e s t i b i l i t y i n m i l k f i s h , Chanos chanos ( F o r s s k a l ) : E f f e c t o f p r o t e i n s o u r c e , f i s h s i z e and s a l i n i t y . A q u a c u l t u r e , 59: 93105. F l e m i n g , S.E. and Lee, B. (1983). Growth performance and i n t e s t i n a l t r a n s i t t i m e o f r a t s f e d p u r i f i e d and n a t u r a l d i e t a r y f i b r e s . J . N u t r . , 113: 592-601. F o l t z , J.W. (1978). The e f f e c t o f meal s i z e and temperature on g a s t r o i n t e s t i n a l m o t i l i t y and a b s o r p t i o n i n rainbow t r o u t (Salmo gairdneri) and t i l a p i a (Sarotherodon mossambicus) Ph.D. T h e s i s . U n i v e r s i t y o f C o l o r a d o , B o u l d e r . 52 pp. F o l t z , J.W. (1979). Chromic o x i d e i n food a s s i m i l a t i o n Trans. Amer. F i s h . Soc. 108: 650-652.  studies.  F o w l e r , L.G. (1980). S u b s t i t u t i o n o f soybean and c o t t o n s e e d p r o d u c t s f o r f i s h meal i n d i e t s f e d t o chinook and coho salmon. P r o . F i s h C u l t . , 4 2 ( 2 ) : 87-91. F o w l e r , L.G. and Banks, J.L. (1967). T e s t s o f d i f f e r e n t components i n t h e Abernathy salmon d i e t . U.S. F i s h W i l d l . Serv., Tech. Pap. 13. 18 pp. F o w l e r , L.G. and Burrows, R.E. (1971). The Abernathy P r o g . F i s h . C u l t . , 33: 67-75.  salmon d i e t .  163 F o w l e r , L.G., McCormick, J.H. a n d Thomas, A . E . (1966). S t u d i e s o f c a l o r i c a n d v i t a m i n l e v e l s o f s a l m o n d i e t s . U.S. F i s h . W i l d l . S e r v . , T e c h . Pap. 6. 12pp. F u r u i c h i , M. a n d Yone, Y. (1981) . Change i n b l o o d s u g a r a n d plasma i n s u l i n l e v e l s o f f i s h e s i n glucose t o l e r a n c e t e s t s . B u l l . J p n . S o c . S c i . F i s h . , 47: 761-764. F u r u i c h i , M. a n d Yone, Y. (1982). E f f e c t o f i n s u l i n on b l o o d s u g a r l e v e l s o f f i s h e s . B u l l . J p n . S o c . S c i . F i s h . , 48: 12891291. G a u t h i e r , G.F. a n d L a n d i s , S.C. (1972). The r e l a t i o n s h i p o f u l t r a s t r u c t u r a l and c y t o c h e m i c a l f e a t u r e s t o a b s o r p t i v e a c t i v i t y i n t h e g o l d f i s h i n t e s t i n e . A n a t . R e c , 172: 657-702. G e o r g o p o u l o u , U., S i r e , M.F. a n d V e r n i e r , J.M. (1985). M a c r o m o l e c u l a r a b s o r p t i o n o f p r o t e i n s by e p i t h e l i a l c e l l s o f t h e p o s t e r i o r i n t e s t i n a l segment a n d t h e i r i n t r a c e l l u l a r d i g e s t i o n i n t h e rainbow t r o u t . U l t r a s t r u c t u r a l and B i o c h e m i c a l s t u d y . B i o l . C e l l . , 53: 269-282. G o l d s t e i n , L. a n d F o r s t e r , R.P. (1970). N i t r o g e n m e t a b o l i s m i n f i s h e s . I n : C a m p b e l l , J.W. ( e d . ) , C o m p a r a t i v e B i o c h e m i s t r y o f N i t r o g e n M e t a b o l i s m , V o l I I . The V e r t e b r a t e s . A c a d e m i c P r e s s , New Y o r k a n d L o n d o n . Gropp, J . , Koops, H., T i e w s , K. a n d Beck, E . (1979). R e p l a c e m e n t o f f i s h m e a l i n t r o u t f e e d s by o t h e r f e e d s t u f f s . I n : A d v a n c e s i n a q u a c u l t u r e , T.V.R. P i l l a y a n d W.A. D i l l ( E d s . ) , p p . 596601. Farnham, E n g l a n d : F i s h i n g News B o o k s . G u l l e y , D.D. (1980). E f f e c t o f m i n e r a l a s h c o n t e n t on A t l a n t i c menhaden m e a l (Brevoortia tyrannus) d i g e s t i b i l i t y by r a i n b o w t r o u t (Salmo gairdneri) . J . C o l . / Wyo. A c a d . S c i . 1 2 ( 1 ) : 17. Hardy, R.W. a n d S h e a r e r , K.D., (1984). E f f e c t o f d i e t a r y c a l c i u m p h o s p h a t e a n d z i n c s u p p l e m e n t a t i o n on whole body z i n c c o n c e n t r a t i o n o f r a i n b o w t r o u t (Salmo gairdneri). Can. J . F i s h . A q u a t . S c i . , 42: 181-184. Hardy, R.W. a n d S u l l i v a n , t r o u t (Salmo gairdneri) S c i . 40: 281-286.  C.V. (1983) . C a n o l a m e a l i n r a i n b o w p r o d u c t i o n d i e t s . Can. J . F i s h . Aquat.  H a r p e r , A.E., B e n t o n , D.A. a n d E l v e h j e m , C A . (1955). L - l e u c i n e , an i s o l e u c i n e a n t a g o n i s t i n t h e r a t . A r c h . Biochem., 57: 1-12. H a s t i n g s , W.H. (1969) . N u t r i t i o n a l s c o r e . I n : Neuhaus, O.W. a n d H a l v e r , J . E . ( e d s . ) , F i s h i n R e s e a r c h . A c a d e m i c P r e s s . New Y o r k , p p . 263-292. Hastings, Cult.,  W.H. (1973) . P h a s e f e e d i n g f o r c a t f i s h . 35: 195-196.  Prog.  Fish  164  H a t t a n , G.L. a n d Owen, F.G. ( 1 9 7 0 ) . E f f i c i e n c y o f t o t a l c o l l e c t i o n and chromic oxide techniques i n s h o r t - t e r m d i g e s t i o n t r i a l s . J . D a i r y S c i . 5 3 : 325-329. H e n d r i c k s , J.D. a n d B a i l e y , G.S. ( 1 9 8 9 ) . A d v e n t i t i o u s t o x i n s . I n : J . E . H a l v e r (ed.) F i s h N u t r i t i o n . S e c o n d e d i t i o n . A c a d e m i c P r e s s . L o n d o n , p p 606-645. Henken, A.M., K l e i n g e l d , D.W. a n d T i j s s e n , P.A.T. ( 1 9 8 5 ) . The e f f e c t o f f e e d i n g l e v e l on a p p a r e n t d i g e s t i b i l i t y o f d i e t a r y dry m a t t e r , crude p r o t e i n and g r o s s energy i n t h e A f r i c a n c a t f i s h ( C l a r i a s gariepinus B u r c h e l l , . 1822) . A q u a c u l t u r e , 5 1 : 1-11. H i g g s , D.A. ( 1 9 8 6 ) . F e e d i n g f i s h e f f i c i e n t l y . Canadian A q u a c u l t u r e m a g a z i n e , s p r i n g e d i t i o n 2 ( 2 ) ; 15, 1 7 , 1 8 , 2 6 . H i g g s , D.A.,' M a r k e r t , J.R., M a c Q u a r r i e , D.W., M c B r i d e , J.R., D o s a n j h , B.S., N i c h o l s , C. a n d H o s k i n s , G. ( 1 9 7 9 ) . D e v e l o p m e n t o f p r a c t i c a l d r y d i e t s f o r c o h o s a l m o n , Oncorhynchus kisutch, u s i n g p o u l t r y - b y - p r o d u c t meal, f e a t h e r meal, soybean meal and / r a p e s e e d meal as major p r o t e i n s o u r c e s . I n : F i n f i s h n u t r i t i o n and f i s h f e e d t e c h n o l o g y , J . E . H a l v e r a n d K. T i e w s ( E d s ) , V o l . I I . Heenemann V e r l a g s g e s e l l s c h a f t MbH. B e r l i n , p p 191-218. H i g g s , D.A., M c B r i d e , J.R., M a r k e r t , J.R., D o s a n j h , B.S., P l o t n i k o f f , M.D. a n d C l a r k e , W.C. ( 1 9 8 2 ) . E v a l u a t i o n o f Tower and C a n d l e r a p e s e e d ( c a n o l a ) m e a l a n d B r o n o w s k i r a p e s e e d p r o t e i n c o n c e n t r a t e as p r o t e i n supplements 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 c h i n o o k s a l m o n (Oncorhynchus tshawytscha) . A q u a c u l t u r e 2 9 : 1-32. H i g g s , D.A., F a g e r l u n d , U.H.M., M c B r i d e , J.R., P l o t n i k o f f , M.D., D o s a n j h , B.S., M a r k e r t , J.R. a n d D a v i d s o n , J . ( 1 9 8 3 ) . P r o t e i n q u a l i t y o f a l t e x c a n o l a meal f o r j u v e n i l e chinook salmon (Oncorhynchus tshawytscha) c o n s i d e r i n g d i e t a r y p r o t e i n and 3 , 5 , 3 ' - t r i i o d o - l - t h y r o n i n e c o n t e n t . A q u a c u l t u r e , 34: 214-238. H i g g s , D.A., M c B r i d e , J.R., D o s a n j h , B.S. a n d F a g e r l u n d , U.H.M. (1990). P o t e n t i a l f o r u s i n g c a n o l a meal and o i l i n f i s h d i e t s . In: F i s h P h y s i o l o g y , F i s h T o x i c o l o g y , and F i s h e r i e s Management, R.C. R y a n s ( e d ) , P r o c e e d i n g s o f a n I n t e r n a t i o n a l Symposium G u a n g z h o u , PRC, S e p t e m b e r 14-16, 1988. p p 88-107. H i l t o n , J.W. a n d A t k i n s o n , J . L . ( 1 9 8 2 ) . R e s p o n s e s o f r a i n b o w t r o u t (Salmo gairdneri) t o increased levels of available c a r b o h y d r a t e i n p r a c t i c a l t r o u t d i e t s . B r . J . N u t r . , 47: 596607. H i l t o n , J.W., A t k i n s o n , J . L . a n d S l i n g e r , S . J . ( 1 9 8 2 ) . Maximum t o l e r a n c e l e v e l , d i g e s t i o n and metabolism o f D-glucose ( c e r e l o s e ) i n r a i n b o w t r o u t (Salmo gairdneri) r e a r e d on a p r a c t i c a l d i e t . C a n . J . F i s h . A q u a t . S c i . , 3 9 : 1229-1234.  165 H i l t o n , J.W. and S l i n g e r , S . J . (1981). N u t r i t i o n and F e e d i n g o f rainbow t r o u t . Canadian S p e c i a l P u b l . o f F i s h e r i e s and A q u a t i c S c i e n c e s . , Dept. o f F i s h e r i e s and Oceans, Ottawa, 55pp. H i l t o n , J.W. and S l i n g e r , S . J . (1983) . E f f e c t o f wheat bran replacement o f wheat m i d d l i n g s i n e x t r u s i o n p r o c e s s e d ( f l o a t i n g ) d i e t s on t h e growth o f j u v e n i l e rainbow t r o u t {Salmo gairdneri). A q u a c u l t u r e , 35: 201-210. H i l t o n , J.W. and S l i n g e r , S . J . (1986). D i g e s t i b i l i t y and u t i l i z a t i o n o f c a n o l a meal i n p r a c t i c a l - t y p e d i e t s f o r rainbow t r o u t (Salmo gairdneri) . Can. J . F i s h . Aquat. S c i . 43: 11491155. f  H o f e r , R. (1982). P r o t e i n d i g e s t i o n and p r o t e o l y t i c a c t i v i t y i n t h e d i g e s t i v e t r a c t o f an omnivorous c y p r i n i d . Comp. Biochem. P h y s i o l . [ A ] 7 2 ( 1 ) : 55-63. H o f e r , R. and Sturmbauer, C. (1985). I n h i b i t i o n o f t r o u t and c a r p amylase by wheat. A q u a c u l t u r e , 48: 277-283. Hudon, B. and De l a Noue, J . (1985). I n f l u e n c e o f meal frequency on apparent n u t r i e n t d i g e s t i b i l i t y i n rainbow t r o u t , Salmo gairdneri. B u l l . F r . P i s c i c , 293-4: 49-51. Inaba, D., Ogino, C , Takamatsu, C , Sugano, S. and Hata, H. (1962) . D i g e s t i b i l i t y o f d i e t a r y components i n f i s h e s . I . D i g e s t i b i l i t y o f d i e t a r y p r o t e i n i n rainbow t r o u t . B u l l . J a p . Soc. S c i . F i s h . , 29: 242-244. Inaba, D., Ogino, C , Takamatsu, C , Ueda, T. and Kurokawa, K. (1963) . D i g e s t i b i l i t y o f d i e t a r y components i n f i s h e s . 2. D i g e s t i b i l i t y o f d i e t a r y p r o t e i n and s t a r c h i n rainbow t r o u t . B u l l . J p n . Soc. S c i . F i s h . , 29: 242-244. J o b l i n g , M., (1983). A s h o r t r e v i e w and c r i t i q u e o f methodology used i n f i s h growth and n u t r i t i o n s t u d i e s . J . F i s h . B i o l . , 23: 685-703 Jones, J.D. (1979). Rapeseed p r o t e i n c o n c e n t r a t e p r e p a r a t i o n e v a l u a t i o n . J . Am. O i l Chem. Soc. 56: 716-721.  and  Kane, E.A., Jacobson, W.C. and Moore, L.A. (1950). A comparison of t e c h n i q u e s used i n d i g e s t i b i l i t y s t u d i e s w i t h d a i r y c a t t l e . J . N u t r . , 41: 583- 589. Kapoor, B.G., Smit, H. and V e r i g h i n a , I.A. (1975). The a l i m e n t a r y c a n a l and d i g e s t i o n i n t e l e o s t s . Adv. Mar. B i o l . , 13: 109-239. K a u s h i k , S . J . and Luquet, P. (1976). Estude de l a d i g e s t i b i l i t e des a c i d e s amines de regimes a base de z e i n e chez l a t r u i t a r c - e n - c i e l . [Study o f amino a c i d d i g e s t i b i l i t y o f z e i n d i e t s i n rainbow t r o u t ] . Annd. H y d r o b i o l . , 7: 11-19.  166 K a u s h i k , S . J , a n d O l i v a - T e l e s , A. (1985). E f f e c t s o f d i g e s t i b l e e n e r g y on n i t r o g e n a n d e n e r g y b a l a n c e i n r a i n b o w t r o u t . A q u a c u l t u r e , 50: 89-101. K a u s h i k , S . J . , M e d a l e , F., F a u c o n n e a u , B. a n d B l a n c , D. (1989). E f f e c t o f d i g e s t i b l e c a r b o h y d r a t e s on p r o t e i n / e n e r g y u t i l i z a t i o n a n d on g l u c o s e m e t a b o l i s m i n r a i n b o w t r o u t (Salmo gairdneri R . ) . A q u a c u l t u r e , 79: 63-74. K e t o l a , H.G. ( 1 9 8 0 ) . I n f l u e n c e o f v a r i o u s d i e t a r y f i s h m e a l s on t h e g r o w t h o f f r y o f A t l a n t i c s a l m o n . F i s h H e a l t h News 9 ( 1 ) : 1-2. K e t o l a , H.G. ( 1 9 8 2 ) . Amino a c i d n u t r i t i o n o f f i s h e s : r e q u i r e m e n t s and s u p p l e m e n t a t i o n o f d i e t s . Comp. B i o c h e m . P h y s i o l . , 73B: 17-24. K e u l s , M. ( 1 9 5 2 ) . The u s e o f t h e s t u d e n t i z e d r a n g e i n c o n n e c t i o n w i t h an a n a l y s i s o f v a r i a n c e . E u p h y t i c a 1: 112-122. K i r c h g e s s n e r , M., K u e r z i n g e r , H. a n d S c h w a r t z , F . J . ( 1 9 8 6 ) . D i g e s t i b i l i t y o f crude n u t r i e n t s i n d i f f e r e n t f e e d s and e s t i m a t i o n o f t h e i r e n e r g y c o n t e n t f o r c a r p (Cyprinus carpio L . ) . A q u a c u l t u r e , 58: 185-194. K i t a m i k a d o , M. a n d T a c h i n o , S. ( 1 9 6 0 a ) . S t u d i e s on t h e d i g e s t i v e enzymes o f r a i n b o w t r o u t . I . C a r b o h y d r a s e s . B u l l . J a p . S o c . S c i . F i s h . , 26: 679-684. T r a n s l . S e r . , F i s h . R e s . Bd Can. (2193) . K i t a m i k a d o , M. a n d T a c h i n o , S. (1960b). S t u d i e s on t h e d i g e s t i v e enzymes o f r a i n b o w t r o u t . I I I . E s t e r a s e s . B u l l . J a p . S o c . S c i . F i s h . , 26: 691-694. T r a n s l . S e r . , F i s h . R e s . Bd Can. ( 2 1 9 4 ) . K i t c h i n , S.E. a n d M o r r i s , D. ( 1 9 7 1 ) . The e f f e c t o f - a c c l i m a t i o n t e m p e r a t u r e on amino a c i d t r a n s p o r t i n t h e g o l d f i s h i n t e s t i n e . Comp. B i o c h e m . P h y s i o l . , 40A: 431-443. Knapka, J . J . , B a r t , K.M., Brown, D.G. a n d C r a g l e , R.G. ( 1 9 6 7 ) . E v a l u a t i o n o f p o l y e t h y l e n e , c h r o m i c o x i d e , a n d c e r i u m - 1 4 4 as d i g e s t i b i l i t y i n d i c a t o r s i n b u r r o s . J . N u t r i t i o n , 92: 79-85. L a l l , S.P., Adams, N . J . a n d H i n e s , J.A. ( 1 9 8 4 ) . D i g e s t i b i l i t y measurement i n f e e d s t u f f s f o r A t l a n t i c salmon i n f r e s h w a t e r and s e a w a t e r . P r o c . Symposium o f F i s h N u t r i t i o n , Aberdeen. L a l l , S P. a n d B i s h o p , F . J . ( 1 9 7 7 ) . S t u d i e s on m i n e r a l a n d p r o t e i n u t i l i z a t i o n by A t l a n t i c salmon (Salmo salar) grown i n s e a w a t e r . F i s h . Mar. S e r v . R e s . Dev. T e c h . Rep. 688: 16p.  167 L a l l , S.P. a n d B i s h o p , F . J . ( 1 9 7 9 ) . S t u d i e s on t h e n u t r i e n t r e q u i r e m e n t s o f r a i n b o w t r o u t , Salmo gairdneri, grown i n s e a w a t e r a n d f r e s h w a t e r . I n : A d v a n c e s i n A q u a c u l t u r e , T.V.R. P i l l a y a n d W.A. D i l l . ( E d s . ) , p p . 580-584. F a r n h a m , E n g l a n d : F i s h i n g News B o o k s . L e e , D . J . a n d Putnam, G.B. ( 1 9 7 3 ) . The r e s p o n s e o f r a i n b o w t r o u t to v a r y i n g protein/energy r a t i o s i n a t e s t d i e t . J . Nutr., 103: 916-922. L e e , J . , F i s h e r , M.T. a n d M a r e , B. ( 1 9 8 6 ) . C o m p a r i s o n o f t e c h n i q u e s f o r chromium s e s q u i o x i d e a n a l y s i s i n marker s t u d i e s . J . S c i . F o o d A g r i c , 37: 3 6 6 - 3 7 2 . L e e , P.G. a n d L a w r e n c e , A . L . ( 1 9 8 5 ) . E f f e c t s o f d i e t a n d s i z e on g r o w t h , f e e d d i g e s t i b i l i t y a n d d i g e s t i v e enzyme a c t i v i t i e s o f t h e m a r i n e s h r i m p , Penaeus s e t i f e r u s L i n n a e u s . J . W o r l d M a r i c u l . S o c . 16: 2 7 5 - 2 8 7 . L e g e r , C , B e r g o t , P., L u q u e t , P., F r a n z y , J . a n d M e u r o t , J . (1977). S p e c i f i c d i s t r i b u t i o n o f f a t t y a c i d s i n t h e t r i g l y c e r i d e s o f rainbow t r o u t adipose t i s s u e : I n f l u e n c e o f t e m p e r a t u r e . L i p i d s 12: 538-543. L e h n i n g e r , A . L . ( 1 9 8 2 ) . P r i n c i p l e s o f b i o c h e m i s t r y . W o r t h , New York. L i e d , E., J u l s h a m n , K. a n d B r a e k k a n , O.R. ( 1 9 8 2 ) . D e t e r m i n a t i o n o f p r o t e i n d i g e s t i b i l i t y i n A t l a n t i c c o d (Gadus morhua) w i t h i n t e r n a l a n d e x t e r n a l i n d i c a t o r s . C a n . J . F i s h . A q u a t . S c i . 39: 854-861. L i n d s a y , G.J.H. ( 1 9 8 4 ) . D i s t r i b u t i o n a n d f u n c t i o n o f d i g e s t i v e t r a c t c h i t i n o l y t i c enzymes i n f i s h . J . F i s h . B i o l . , 2 4 : 5 2 9 536. L o v e l l , R.T. ( 1 9 7 7 ) . F e e d i n g p r a c t i c e s . I n : N u t r i t i o n a n d f e e d i n g o f c h a n n e l c a t f i s h , S o u t h e r n C o o p e r a t i v e S e r i e s 2 1 8 , pp 5 0 - 5 5 . L u q u e t , P. a n d B e r g o t , F. ( 1 9 7 6 ) . E v a l u a t i o n de d i v e r s t r a i t m e n t s t e c h n o l o g i q u e s d e s c e r e a l e s . V I I . U t i l i s a t i o n de m a i s p r e s s e , f l o c o n n e , e x p a n s e e t e x t r u d e d a n s 1 ' a l i m e n t a t i o n de l a t r u i t e a r c ^ - e n - c i e l . A n n . Z o o t e c h . , 2 5 : 63-69. Lyman, 0. ( 1 9 8 4 ) . An i n t r o d u c t i o n t o s t a t i s t i c a l m e t h o d s a n d d a t a a n a l y s i s . 2 n d e d i t i o n . PWS P u b l i s h e r s . B o s t o n M a s s a c h u s e t t s . M a r t i n e z - P a l a c i o s , C A . (1988) . D i g e s t i b i l i t y s t u d i e s i n j u v e n i l e s o f t h e M e x i c a n c i c h l i d , Cichlasoma urophthalmus ( G u n t h e r ) . A q u a c u l t u r e a n d F i s h e r i e s management, 19; 55-62 M a c L e o d , M.G. ( 1 9 7 7 ) . E f f e c t s o f s a l i n i t y on f o o d a b s o r p t i o n and c o n v e r s i o n i n t h e rainbow t r o u t g a i r d n e r i i . M a r . B i o l . 4 3 : 93-102.  intake, Salmo  168 Maynard, L.A. and L o o s l i , J.K. (1969). A n i m a l N u t r i t i o n . 6th e d i t i o n . M c G r a w - H i l l , New York, S t . L o u i s , San F r a n c i s c o , London, Sydney, Toronto, Mexico, Panama, 613 pp. Mazeaud, M.M. and Mazeaud, F. (1981). A d r e n e r g i c responses t o s t r e s s i n f i s h . I n : A.D. P i c k e r i n g (Ed.), S t r e s s and F i s h . Academic P r e s s , London, pp. 4 9-75. McCallum, I.M. and H i g g s , D.A. (1989). An assessment o f p r o c e s s i n g e f f e c t s on t h e n u t r i t i v e v a l u e o f marine p r o t e i n s o u r c e s f o r j u v e n i l e chinook salmon (Oncorhynchus tshawytscha) . A q u a c u l t u r e , 77: 181-200. M i l l e r , E.L. and De Boer, F. (1988). F e e d s t u f f s 6. B y - p r o d u c t s o f a n i m a l o r i g i n . L i v e s t . P r o d . S c i . , 19: 159-195. M i t a r u , B.N., B l a i r , R., R e i c h e r t , R.D. and Roe, W.E. (1984). Dark and y e l l o w rapeseed h u l l s , soybean h u l l s and a p u r i f i e d f i b e r s o u r c e : t h e i r e f f e c t on d r y m a t t e r , energy, p r o t e i n and amino a c i d d i g e s t i b i l i t i e s i n c a n n u l a t e d p i g s . J . Anim. S c i . , 59: 1510-1518. M o r r i s o n , F.B. I t h a c a , New  (1949). Feeds and f e e d i n g . M o r r i s o n p u b l i s h i n g York, 1207 pp.  Co.  Mowlah, A.H., Sakata, T. and Kakimoto, D. (1979). M i c r o f l o r a i n the a l i m e n t a r y t r a c t o f gray m u l l e t V. S t u d i e s on t h e c h i t i n o l y t i c enzymes o f E n t e r o j b a c t e r and Vibrio. B u l l . Jpn. Soc. S c i . F i s h 45: 1313-1317. M u r a i , T., Ogata, H., V i l l a n e d a , A. and Watanabe, T. (1989). U t i l i z a t i o n o f soy f l o u r by f i n g e r l i n g rainbow t r o u t h a v i n g d i f f e r e n t body s i z e . Nippon S u i s a n G a k k a i s h i 55 ( 6 ) : 10671073. N a t i o n a l Research C o u n c i l . (1978). N u t r i e n t r e q u i r e m e n t s o f l a b o r a t o r y a n i m a l s , 3 r d ed. N a t i o n a l Acadamy o f S c i e n c e s , Washington, D.C. N a t i o n a l Research C o u n c i l . (1981). Subcommitee on c o l d water n u t r i t i o n . N u t r i e n t r e q u i r e m e n t s o f c o l d water f i s h e s . N a t i o n a l Academy P r e s s , Washington, D.C.  fish  N a t i o n a l Research C o u n c i l . (1983). N u t r i e n t r e q u i r e m e n t s o f warmwater f i s h e s and s h e l l f i s h e s . N a t i o n a l Academy p r e s s , Washington, D.C. Naczk, M., S h a h i d i , F., Diosady, L.L. and Rubin, L . J . (1986). Removal o f g l u c o s i n o l a t e s from midas rapeseed and mustard seed • by methanol-ammonia. Canadian I n s t i t u t e o f Food S c i e n c e Technology. 75-77.  169 Noaillac-Depeyre, J . and Gas, N. (1976). E l e c t r o n m i c r o s c o p i c study on gut e p i t h e l i u m o f t h e t e n c h (Tinea tinea L.) w i t h r e s p e c t t o i t s a b s o r p t i v e f u n c t i o n s . T i s s u e C e l l . 8: 511-530. Nose, T. ( i 9 6 0 ) . On t h e d i g e s t i o n o f f o o d p r o t e i n by g o l d f i s h (Carassius auratus L.) and rainbow t r o u t (Salmo irideus G.). B u l l . Freshwat. F i s h . Res. Lab., Tokyo 10: 11-22. Nose, T. (1967). On t h e m e t a b o l i c f e c a l n i t r o g e n i n young rainbow t r o u t . B u l l . F r e s h w a t e r F i s h . Res. Lab. Tokyo 17: 97-105. Nose, T. and Mamiya, H. (1963). P r o t e i n d i g e s t i b i l i t y o f f l a t f i s h meal i n rainbow t r o u t . B u l l . F r e s h w a t e r F i s h . Res. Lab. 12 (2): 1-4. Oestemer, G.A., Hanson, L.E. and Meade, R . J . (1973). L e u c i n e i s o l e u c i n e i n t e r r e l a t i o n s h i p i n t h e young p i g . J . Anim. S c i . , 36: 674-678. Ogata, H., A r a i , S. and Nose, T. (1983). Growth response o f c h e r r y salmon Oncorhynchus masou and mago salmon 0. rhodurus f r y f e d p u r i f i e d c a s e i n d i e t s supplemented w i t h amino a c i d s . B u l l . J a p . Soc. S c i . F i s h . , 49: 1381- 1385. Ogino, C. and Chen, M.S. (1973). P r o t e i n n u t r i t i o n i n f i s h - ^ I I I . Apparent and t r u e d i g e s t i b i l i t y o f d i e t a r y p r o t e i n i n c a r p . B u l l . J p n . Soc. S c i . F i s h . , 39: 649-651. Ogino, C , C h i o u , J.Y. and T a k e u c h i , T. (1976). P r o t e i n n u t r i t i o n i n f i s h . V I . E f f e c t s o f d i e t a r y energy s o u r c e s on t h e • u t i l i z a t i o n o f p r o t e i n by rainbow t r o u t and c a r p . B u l l . J a p . Soc. S c i . F i s h . 42: 213-218. Ogino, C , K a k i n o , J . and Chen, M.S. (1973). P r o t e i n n u t r i t i o n i n f i s h . I I . D e t e r m i n a t i o n o f m e t a b o l i c f e c a l n i t r o g e n and endogenous n i t r o g e n e x c r e t i o n s o f c a r p . B u l l . J a p . Soc. S c i . F i s h . , 39: 519-523. Ohnesorge, F.K. and Rauch, R. (1968). P h a r m a c o l o g i c a l i n v e s t i g a t i o n s on t h e p e r i s t a l t i c movements o f t h e gut o f t h e t r e n c h (Tinea vulgaris). Z. V e r g l . P h y s i o l . , 58: 153-171. Opstvedt, J . , M i l l e r , R., Hardy, R.W. and S p i n e l l i , J . (1984). H e a t - i n d u c e d changes i n s u l f h y d r y l groups and d i s u l f i d e bands i n f i s h p r o t e i n and t h e i r e f f e c t on p r o t e i n and amino a c i d d i g e s t i b i l i t y i n rainbow t r o u t . J . A g r i c . Food. Chem., 32: 929-935. Page, A.W. and Andrews, J.W. (1973). I n t e r a c t i o n s o f d i e t a r y l e v e l s o f p r o t e i n and energy on c h a n n e l c a t f i s h , Ictalurus punctatus. J . N u t r . 103: 1339-1346.  170 P a l m e r , T.N. and Ryman, B.E. ( 1 9 7 2 ) . S t u d i e s on o r a l i n t o l e r a n c e i n f i s h . J . F i s h . B i o l . , 4: 311-319.  glucose  Pandey, H.S. and S i n g h , R.P. ( 1 9 8 0 ) . P r o t e i n d i g e s t i b i l i t y by K h o s t i f i s h (Colisa fasciatus) under the i n f l u e n c e o f c e r t a i n f a c t o r s . A c t a H y d r o c h e i m . H y d r o b i o l . 8: 583-589. Pfeffer, fish.  E . ( 1 9 8 2 ) . U t i l i z a t i o n o f d i e t a r y p r o t e i n by Comp. B i o c h e m . P h y s i o l . , 73B: 51-57.  salmonid  P h i l l i p s , A.M. ( 1 9 6 9 ) . N u t r i t i o n , d i g e s t i o n , and e n e r g y u t i l i z a t i o n . I n . Hoar, W.S. and R a n d a l l , D.J. ( e d s . ) , F i s h P h y s i o l o g y , I . E x c r e t i o n , i o n i c r e g u l a t i o n , and m e t a b o l i s m , pp391-432. New Y o r k : A c a d e m i c P r e s s . P h i l l i p s , A.M., T u n i s o n , A.V. and Brockway, D.R. (1948). U t i l i z a t i o n o f c a r b o h y d r a t e s by t r o u t . I n : F i s h . Res. B u l l . No.11. S t a t e o f New Y o r k C o n s e r v a t i o n D e p a r t m e n t , A l b a n y . P h i l l i p s o n , J . (1964). A m i n i a t u r e bomb c a l o r i m e t e r b i o l o g i c a l s a m p l e s . O i k o s , 15: 130-139.  for small  P i c k e r i n g , A.D., P o t t i n g e r , T.G. and C h r i s t i e , P. ( 1 9 8 2 ) . R e c o v e r y o f brown t r o u t , Salmo trutta L . , from a c u t e h a n d l i n g s t r e s s : a t i m e - c o u r s e s t u d y . J . F i s h B i o l . , 20: 229-234. P l a n t i k o w , A. and P l a n t i k o w , H. ( 1 9 8 5 ) . A l a n i n e a m i n o p e p t i d a s e (AAP) a c t i v i t y i n t h e m i d g u t o f r a i n b o w t r o u t (Salmo gairdneri R . ) : The i n f l u e n c e o f f e e d q u a n t i t y and q u a l i t y , t e m p e r a t u r e and o s m o l a r i t y . A q u a c u l t u r e , 48: 261-76. P o s t , G., Shanks, W.E. and S m i t h , R.R. ( 1 9 6 5 ) . A method f o r c o l l e c t i n g m e t a b o l i c e x c r e t i o n s from f i s h . P r o g . F i s h C u l t . , 27: 132-134. P o t t e r , N.N. ( 1 9 8 4 ) . F o o d S c i e n c e . ( 3 r d e d i t i o n ) . The A v i P u b l i s h i n g Company, I n c . W e s t p o r t C o n n e c t i c u t . 7 80pp. R a n d a l l , D.J. and W r i g h t , P.A. ( 1 9 8 7 ) . Ammonia d i s t r i b u t i o n and e x c r e t i o n i n f i s h . F i s h P h y s i o l , and Biochem., 3: 107-120. R i c h a r d s o n , N. L., H i g g s , D.A., Beames, R.M. and M c B r i d e , J.R. ( 1 9 8 5 ) . I n f l u e n c e o f d i e t a r y c a l c i u m , p h o s p h o r u s , z i n c and sodium p h y t a t e l e v e l on c a t a r a c t i n c i d e n c e , g r o w t h and h i s t o p a t h o l o g y i n j u v e n i l e c h i n o o k salmon (Oncorhynchus tshawytscha). J . N u t r . 115: 553-567. R i n g r o s e , R.C. (Salvelinus 1117.  (1971). C a l o r i e - t o - p r o t e i n fontinalis). J . F i s h . Res.  r a t i o f o r brook Bd. Can. 2 8 ( 8 ) :  trout 1113-  Rumsey, G.L. (1981). S i g n i f i c a n c e o f n i t r o g e n m e t a b o l i s m : Why does t h e s a l m o n i d r e q u i r e a h i g h p r o t e i n d i e t ? S a l m o n i d , 5: 20-24.  171 Rumsey, G.L. a n d K e t o l a , H.G. ( 1 9 7 5 ) . Amino a c i d s u p p l e m e n t a t i o n o f c a s e i n i n d i e t s o f A t l a n t i c s a l m o n (Salmo salar) f r y and o f s o y b e a n m e a l f o r r a i n b o w t r o u t (Salmo gairdneri) fingerlings. J . F i s h . R e s . B d C a n . , 3 2 : 422-426. R y c h l y , J . a n d S p a n n h o f , L. ( 1 9 7 9 ) . N i t r o g e n b a l a n c e i n t r o u t I . Digestibility of diets containing varying levels of protein and c a r b o h y d r a t e . A q u a c u l t u r e , 16: 39-46. SAS U s e r s G u i d e : S t a t i s t i c s v e r s i o n 5. ( 1 9 8 5 ) . SAS I n s t i t u t e I n c . , C a r e y , N. C. S a t i a , B.P. ( 1 9 7 4 ) . Q u a n t i t a t i v e p r o t e i n r e q u i r e m e n t s t r o u t . P r o g . F i s h . C u l t . 3 6 : 80-85  o f rainbow  S c h m i t z , 0., G r e u e l , E. a n d P f e f f e r , E. ( 1 9 8 3 ) . A m e t h o d o f determining d i g e s t i b i l i t y of nutrients i n eels. Aquaculture, 32: 71-78. Schneeman, B.O., R i c h t e r , D.B. a n d J a c o b s , L.R. ( 1 9 8 2 ) . R e s p o n s e t o d i e t a r y wheat b r a n i n t h e e x o c r i n e p a n c r e a s a n d i n t e s t i n e o f r a t s . J . N u t r . , 1 1 2 : 283-286. S c h n e i d e r , B.H. a n d F l a t t , W.P. ( 1 9 7 5 ) . The e v a l u a t i o n o f f e e d s through d i g e s t i b i l i t y experiments. U n i v e r s i t y o f Georgia P r e s s , A t h e n s , GA. 1 6 8 p . S e i d m a n , E.R. a n d L a w r e n c e , A . L . ( 1 9 8 5 ) . G r o w t h , f e e d d i g e s t i b i l i t y and p r o x i m a t e body c o m p o s i t i o n o f - j u v e n i l e Penaeus vannamei a n d Panaeus monodon grown a t d i f f e r e n t d i s s o l v e d o x y g e n l e v e l s . J . W o r l d M a r i c u l . S o c . 16: 333-346. S i l k , D.B.A., H e g a r t y . J . E . , F a i r c l o u g h , P.D. a n d C l a r k , M.L. (1982). C h a r a c t e r i z a t i o n and n u t r i t i o n a l s i g n i f i c a n c e o f p e p t i d e t r a n s p o r t i n man. A n n . N u t r . M e t a b . 2 6 : 3 3 7 - 3 5 2 . S i n g h , R.P. a n d N o s e , T. ( 1 9 6 7 ) . D i g e s t i b i l i t y o f c a r b o h y d r a t e s i n y o u n g r a i n b o w t r o u t . B u l l . F r e s h w a t e r F i s h . R e s . L a b . 17: 21-25. S i r e , M.F., L u t t o n , C. a n d V e r n i e r , J.M. ( 1 9 8 1 ) . New v i e w s on i n t e s t i n a l a b s o r p t i o n o f l i p i d s i n t e l e o s t e a n f i s h e s : an u l t r a s t r u c t u r a l and b i o c h e m i c a l study i n t h e rainbow t r o u t . J . L i p i d R e s . 2 2 : 81-94.. S k r e d e , A., K r o g d a h l , A. a n d A u s t r e n g , E. ( 1 9 8 0 ) . D i g e s t i b i l i t y o f amino a c i d s i n r a w f i s h a n d meat a n d bone m e a l f o r t h e c h i c k e n , f o x , mink a n d r a i n b o w t r o u t . Z. T i e r p h y s i c a l . T i e r e r n a h r . F u t t e r m i t t e l k . , 43: 92-101. S m i t h , B.W. a n d L o v e l l , R.T. ( 1 9 7 1 ) . D i g e s t i b i l i t y o f n u t r i e n t s i n s e m i - p u r i f i e d r a t i o n s by channel c a t f i s h i n s t a i n l e s s s t e e l t r o u g h s . P r o c . A n n . C o n f . S o u t h e a s t . A s s o c . Game F i s h Commrs, 25: 4 5 2 - 4 5 9 .  172 Smith, B.W. and L o v e l l , R.T. (1973). D e t e r m i n a t i o n o f apparent p r o t e i n d i g e s t i b i l i t y i n feeds f o r channel c a t f i s h . Trans. Am. F i s h . S o c , 102: 831-835. Smith, L.S. (1989). D i g e s t i v e F u n c t i o n s i n t e l e o s t f i s h . I n : J.E. H a l v e r (ed.) F i s h N u t r i t i o n . Second e d i t i o n ; Academic P r e s s . London. Smith, R.L. (1969). I n t e s t i n a l a m i n o - a c i d t r a n s p o r t i n t h e t e l e o s t Haemulon plumieri. Comp. Biochem. P h y s i o l . , 30: 23.  marine 1115-  Smith, R.R. (1971). A method o f measuring d i g e s t i b i l i t y and m e t a b o l i z a b l e energy o f f i s h f e e d s . P r o g . F i s h C u l t . 33: 132134. Smith, R.R. (1976). M e t a b o l i z a b l e energy o f f e e d s t u f f s f o r t r o u t . F e e d s t u f f s 48 (23): 16-17. Smith, R.R., P e t e r s o n , M.C. and A l l r e d A.C. (1980). E f f e c t o f l e a c h i n g on apparent d i g e s t i o n c o e f f i c i e n t s o f f e e d s t u f f s f o r s a l m o n i d s . P r o g . F i s h C u l t . 42: 195-199. Smith, R.R., Rumsey, G.L. and S c o t t , M.L. (1978a). Net energy, maintenance r e q u i r e m e n t s o f salmonids as measured by d i r e c t c a l o r i m e t r y . E f f e c t o f body s i z e and e n v i r o n m e n t a l t e m p e r a t u r e . J . N u t r . , .108: 1017-1024. Smith, R.R., Rumsey, G.L. and S c o t t , M.L. (1978b). Heat increment a s s o c i a t e d w i t h d i e t a r y p r o t e i n , f a t and c a r b o h y d r a t e and complete d i e t s i n s a l m o n i d s : comparative e n e r g e t i c e f f i c i e n c y . J . N u t r . , 108: 1025-1032. Spannhof, L. and Kiihne, H. (1977) . Untersuchungen z u r Verwertung v e r s c h i e d e n e r F u t t e r m i s c h u n g e n durch e u r o p a i s c h e A a l e (Anguilla anguilla). A r c h . T i e r e r n a h r . , 27: 517-531. Spannhof, L. and P l a n t i k o w , H. (1983). S t u d i e s on c a r b o h y d r a t e d i g e s t i o n i n rainbow t r o u t . A q u a c u l t u r e , 30: 95-108. S p i n e l l i , J . , Mahnken, C. and S t e i n b e r g , M. (1979). A l t e r n a t i v e s o u r c e s o f p r o t e i n f o r f i s h meal i n s a l m o n i d d i e t s . I n : F i n f i s h n u t r i t i o n and f i s h f e e d t e c h n o l o g y , J.E. H a l v e r and K. Tiews (Eds), V o l . I I . Heenemann V e r l a g s g e s e l l s c h a f t MbH. B e r l i n , pp 131-142. S p y r i d a k i s , P., M e t a i l l e r , R., Gabaudan, J . and R i a z a , A. (1989). S t u d i e s on n u t r i e n t d i g e s t i b i l i t y i n european sea bass (Dicentrarchus labrax) 1. M e t h o d o l o g i c a l a s p e c t s c o n c e r n i n g f a e c e s c o l l e c t i o n . A q u a c u l t u r e , 77: 61-70. S t e e l , R.G.D. and T o r r i e , J.H. (1960). P r i n c i p l e s and of s t a t i s t i c s . McGraw H i l l , New York, NY..  procedures  173 Stevenson, A.E. and De Langen, H. (1960). Measurement o f f e e d i n t a k e by g r a z i n g c a t t l e and sheep. N. Z. J . A g r i c . Res. 5: 314-319. S t i c k n e y , R.R. and Andrews, J.W. (1972). E f f e c t s o f d i e t a r y l i p i d s on growth, food c o n v e r s i o n , l i p i d and f a t t y a c i d c o m p o s i t i o n o f channel c a t f i s h . J . N u t r . , 102: 249-258. Storebakken, T. (1985). B i n d e r s i n f i s h f e e d s . I . E f f e c t o f a l g i n a t e and Guar gum on growth, d i g e s t i b i l i t y , f e e d i n t a k e and passage t h r o u g h t h e g a s t r o i n t e s t i n a l t r a c t o f rainbow t r o u t . A q u a c u l t u r e , 47: 11-2 6. Storebakken, T. and A u s t r e n g , E. (1987). R a t i o n l e v e l s f o r s a l m o n i d s . I I . Growth, f e e d i n t a k e , p r o t e i n d i g e s t i b i l i t y , body c o m p o s i t i o n , and f e e d c o n v e r s i o n i n rainbow t r o u t w e i g h i n g 0.5-1.0 kg. A q u a c u l t u r e , 60: 207-221. Swarup, C. and G o e l , K.A. (1975). H i s t o c h e m i c a l s t u d y o f t h e a c t i v i t y o f l i p a s e i n t h e d i g e s t i v e system o f some t e l e o s t f i s h e s . A c t a Histochem. 54: 10-15. Tacon, A.G.J, and Cowey, C.B. (1985). P r o t e i n and amino a c i d r e q u i r e m e n t s . I n : T y l e r , P. and Calow, P. ( e d s . ) , F i s h E n e r g e t i c s - New P e r s p e c t i v e s . John Hopkins U n i v . P r e s s . B a l t i m o r e , MD. Tacon, A.G.J., H a a s t e r , J.V., F e a t h e r s t o n e , P.H., K e r r . K. and J a c k s o n , A . J . (1984). S t u d i e s on t h e u t i l i z a t i o n o f f u l l - f a t soybean and s o l v e n t e x t r a c t e d soybean meal i n a complete d i e t f o r rainbow t r o u t . B u l l . Jpn. Soc. S c i . F i s h . , 49: 1437-1443. Tacon, A.G.J, and J a c k s o n , A . J . (1985). U t i l i z a t i o n o f c o n v e n t i o n a l and u n c o n v e n t i o n a l p r o t e i n s o u r c e s i n p r a c t i c a l f i s h f e e d s . I n : Cowey, C.B., Mackie, A.M. and B e l l , J.G.. ( e d s , ) , N u t r i t i o n and F e e d i n g o f F i s h . Academic P r e s s , London. Tacon, A.G.J, and R o d r i g u e s , A.M.P. (1984). Comparison o f chromic o x i d e , crude f i b r e , p o l y e t h y l e n e and a c i d - i n s o l u b l e ash as d i e t a r y markers f o r t h e e s t i m a t i o n o f 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 i n rainbow t r o u t . A q u a c u l t u r e , 43: 391-399. Takeda, M., Shimeno, S., Hosokawa, H., Kajiyama, H. and K a i s y o , T. (1975). The e f f e c t o f d i e t a r y c a l o r i e - t o - p r o t e i n r a t i o on t h e growth, f e e d c o n v e r s i o n and body c o m p o s i t i o n o f young y e l l o w t a i l . B u l l . Jap. Soc. S c i . F i s h . , 41: 443-447. T a k e u c h i , T., Watanabe, T. and Ogino, C. (1978). Optimum r a t i o o f p r o t e i n t o l i p i d i n t h e d i e t o f rainbow t r o u t . B u l l . Jap. Soc. S c i . F i s h . , 44: 633-638. T a k e u c h i , T., Watanabe, T. and Ogino, C. (1979). Optimum r a t i o o f d i e t a r y energy t o p r o t e i n f o r c a r p . B u l l . Jap. Soc. S c i . F i s h . , 45: 983-987.  174 T a l b o t , C. (1985). L a b o r a t o r y methods i n f i s h f e e d i n g and n u t r i t i o n a l s t u d i e s . I n : F i s h E n e r g e t i c s , New P e r s p e c t i v e s , P . T y t l e r and P.Calow ( e d s . ) , Croom Helm, London. T a r r , H.L.A. and B i e l y , J . (1973). E f f e c t o f p r o c e s s i n g on t h e n u t r i t i o n a l v a l u e o f f i s h meal and r e l a t e d p r o d u c t s . I n : E f f e c t o f p r o c e s s i n g on t h e n u t r i t i o n a l v a l u e o f f e e d s . N a t i o n a l Academy o f S c i e n c e s , W a s h i n g t o n , D.C, pp. 252-281. T a y l o r , S . J . , C o l e , D.J.A. and L e w i s , D. (1977). An i n t e r a c t i o n o f l e u c i n e , i s o l e u c i n e and v a l i n e i n t h e d i e t o f t h e g r o w i n g p i g . P r o c . N u t r . Soc. 36,36A. T i e w s , K., optimal 1-29.  Gropp, J . and Koops, H. (1976). On t h e d e v e l o p m e n t o f r a i n b o w t r o u t p e l l e t f e e d s . A r c h . F i s h W i s s . , 27 ( 1 ) :  T r u s t , T . J . and Sparrow, R.A.H. (1974). The b a c t e r i a l f l o r a t h e a l i m e n t a r y t r a c t o f f r e s h w a t e r s a l m o n i d f i s h e s . Can. M i c r o b i o l . , 20: 1219-1228.  in J.  Tookey, H.L., V a n E t t e n , C H . and D a x e n b i c h l e r , M.E. (1980). G l u c o s i n o l a t e s . In: L i e n e r , I.E. (ed.), T o x i c C o n s t i t u e n t s o f P l a n t F o o d s t u f f s . A c a d e m i c P r e s s , London and New Y o r k , pp 103142. T u n i s o n , A.V., Brockway, D., M a x w e l l , J . , D o r r , A. and McCay, C. (1942). P r o t e i n u t i l i z a t i o n by b r o o k t r o u t . I n : The n u t r i t i o n o f t r o u t . F i s h Res. B u l l . No. 4. U.S. F i s h and W i l d l . S v c . C o r t l a n d H a t c h . Rep. No. 11. U g a j i n , M. (1979). S t u d i e s on t h e taxonomy o f m a j o r m i c r o f l o r a t h e i n t e s t i n a l c o n t e n t s o f s a l m o n i d s . B u l l . J p n . Soc. S c i . F i s h . 45 ( 6 ) : 721-731. U s h e r , M.L., Talbot, C t r a n s f e r t o seawater s a l m o n s m o l t s (Salmo V e g a s - V e l e z , M. d i g e s t i f des  on  and Eddy, F.B., (1990). E f f e c t s of on d i g e s t i o n and g u t f u n c t i o n i n A t l a n t i c salar L . ) . A q u a c u l t u r e , 90: 85-96.  (1972). L a s t r u c t u r e h i s t o l o g i q u e due t u b e p o i s s o n s t e l e o s t e e n s . T e t h y s . 4: 163-174.  V e n s - C a p p e l l , B. (1985). M e t h o d i c a l s t u d i e s on d i g e s t i o n i n t r o u t . 1. 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 methods f o r f a e c e s c o l l e c t i o n . A q u a c u l t u r a l Eng. 4: 33-4 9. W a l t o n , M.J. and Cowey, C B . (1982) . A s p e c t s of' i n t e r m e d i a t e m e t a b o l i s m i n s a l m o n i d f i s h . Comp. B i o c h e m . P h y s i o l . , 73B: 79. Watanabe, T. (1977). S p a r i n g a c t i o n o f l i p i d s on d i e t a r y i n f i s h -Low p r o t e i n d i e t w i t h h i g h c a l o r i c c o n t e n t . T e c h n o c r a t 1 0 ( 8 ) : 34-39.  to  59-  protein  175 Watanabe, T. Physiol.,  ( 1 9 8 2 ) . L i p i d n u t r i t i o n i n f i s h . Comp. B i o c h e m . 73B, 3-15.  Watanabe, T., N a r i , H., S a t o h , S., T a k e u c h i , M. and N o s e , T. ( 1 9 8 3 ) . N u t r i t i o n a l e v a l u a t i o n o f b r o w n m e a l s as a p r o t e i n s o u r c e i n d i e t s f o r r a i n b o w t r o u t . B u l l . J a p . Soc. S c i . F i s h . , 49 ( 7 ) : 1 0 8 3 - 1 0 8 7 . Watanabe, T., O g i n o , C , K o s h i i s h i , Y. and M a t s u n a g a , T. ( 1 9 7 4 ) . Requirement o f rainbow t r o u t f o r e s s e n t i a l f a t t y a c i d s . B u l l . J p n . Soc. S c i . F i s h . 4 0 ( 5 ) : 493-499. Watanabe, T., T a k e u c h i , T. and O g i n o , C. (1979) •. S t u d i e s on t h e s p a r i n g e f f e c t o f l i p i d on d i e t a r y p r o t e i n i n r a i n b o w t r o u t (Salmo g a i r d n e r i ) . I n : F i n f i s h n u t r i t i o n a n d f i s h f e e d t e c h n o l o g y , J . E . H a l v e r a n d K. T i e w s ( E d s ) , V o l . I . Heenemann V e r l a g s g e s e l l s c h a f t MbH. B e r l i n , pp. 1 1 3 - 1 2 5 . W a t t s , R.L. a n d W a t t s , D.C. (1974). N i t r o g e n metabolism f i s h e s . Chem. Z o o l . , 8: 369-446.  in  Wedemeyer, G.A. ( 1 9 7 6 ) . P h y s i o l o g i c a l r e s p o n s e s o f j u v e n i l e coho s a l m o n (Oncorhynchus kisutch) and r a i n b o w t r o u t (Salmo gairdneri) t o h a n d l i n g and c r o w d i n g s t r e s s i n i n t e n s i v e f i s h c u l t u r e . J . F i s h . R e s . Bd. Can., 33: 2699-2710. W i l s o n , R.P. ( 1 9 8 5 ) . Amino a c i d and 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 . I n : Cowey, C.B., M a c k i e , A.M. a n d B e l l , J.G. ( e d s . ) , N u t r i t i o n and F e e d i n g o f F i s h . A c a d e m i c P r e s s , L o n d o n . W i l s o n , R.P. and Cowey, C.B. ( 1 9 8 5 ) . Amino a c i d c o m p o s i t i o n o f whole body t i s s u e o f r a i n b o w t r o u t and A t l a n t i c salmon. A q u a c u l t u r e , 48: 373-376. W i l s o n , R.P. and Poe, W.E. (1985). Apparent d i g e s t i b l e p r o t e i n and e n e r g y c o e f f i c i e n t s o f common f e e d i n g r e d i e n t s f o r c h a n n e l c a t f i s h . P r o g . F i s h C u l t . , 47: 154-158. W i n b e r g , G.G. ( 1 9 6 1 ) . New i n f o r m a t i o n on m e t a b o l i c r a t e i n f i s h e s . V o p r . I k h t i o l . , 1 ( 1 ) , 157-65. T r a n s l . S e r . , F i s h . Bd. Can., ( 3 6 2 ) , 1961.  Res.  W i n d e l l , J.T., F o l t z , J.W. and S a r o k o n , J.A. ( 1 9 7 8 a ) . M e t h o d s o f f e c a l c o l l e c t i o n and n u t r i e n t l e a c h i n g i n d i g e s t i b i l i t y s t u d i e s . P r o g . F i s h C u l t . , 40: 5 1 - 5 5 . W i n d e l l , J.T., F o l t z , J.W. and f i s h s i z e , t e m p e r a t u r e , and d i g e s t i b i l i t y of a p e l l e t e d gairdneri. T r a n s . Am. F i s h .  S a r o k o n , J.A. ( 1 9 7 8 b ) . E f f e c t amount o f f e e d on n u t r i e n t d i e t by r a i n b o w t r o u t , Salmo S o c , 107: 613-616.  Wood, J.D. ( 1 9 5 8 ) . N i t r o g e n e x c r e t i o n i n some m a r i n e Can. J . B i o c h e m . P h y s i o l . , 36: 1 2 3 7 - 1 2 4 2 .  of  teleosts.  176 Yu,  T.C. and S i n n h u b e r , R.O. (1972). E f f e c t of d i e t a r y l i n o l e n i c a c i d and d o c o s a h e x a e n o i c a c i d on g r o w t h and f a t t y a c i d c o m p o s i t i o n o f r a i n b o w t r o u t (Salmo g a i r d n e r i ) . L i p i d s 7 ( 7 ) : 450-454.  Y u r k o w s k i , M., B a i l e y , J.K., E v a n s , R.E., Tabackek, J.L. and A y l e s . G.B. (1978). A c c e p t a b i l i t y of rapeseed p r o t e i n s i n d i e t s f o r r a i n b o w t r o u t (Salmo g a i r d n e r i ) . J . F i s h . Res. B o a r d Can. 35: 951-962. Z e i t o u n , I.H.,- H a l v e r , J . E . , U l l r e y . D.E. and T a c k . P . I . (1973). I n f l u e n c e o f s a l i n i t y on p r o t e i n r e q u i r e m e n t s o f r a i n b o w t r o u t (Salmo gairdneri) f i n g e r l i n g s . J . F i s h . Res. Bd. Can. 30: 1867-1873. Z e i t o u n , I.H., U l l r e y . D.E., H a l v e r , J . E . , T a c k . P . I . and Magee, W.T. ( 1 9 7 4 ) . I n f l u e n c e o f s a l i n i t y on p r o t e i n r e q u i r e m e n t s o f c o h o s a l m o n (Oncorhynchus kisutch) s m o l t s . J . F i s h . Res. Bd. Can. 31: 1 1 4 5 - 1 1 4 8 .  177 6.0 APPENDICES APPENDIX 1. Procedures f o l l o w e d f o r t h e q u a n t i t a t i v e o r t o t a l c o l l e c t i o n o f f i s h f e c e s w i t h t h e "Guelph system" i n experiment I .  A) O r g a n i z a t i o n o f equipment p r i o r t o c o l l e c t i o n  procedure.  1.  Turn on c e n t r i f u g e r e f r i g e r a t i o n u n i t and s e t temperature t o 5°C.  2.  L a b e l c l e a n 250 ml c e n t r i f u g e b o t t l e s w i t h tank number, date and time o f c o l l e c t i o n u s i n g permanent marker.  B) F e c a l c o l l e c t i o n procedures t o be performed two t a n k s a t a time a t 0830 hours d a i l y f o r t h r e e weeks. 1.  U s i n g a f i n e s p a t u l a , c a r e f u l l y scrape any f e c e s which may have been c a r r i e d over t h e tank o v e r f l o w onto t h e f u n n e l s c r e e n mesh and p l a c e i t i n t o t h e a p p r o p r i a t e c e n t r i f u g e bottle.  2.  I n c r e a s e t h e water f l o w i n t o t h e two t a n k s by f u l l y t h e f l o w v a l v e s (10-12 l i t e r s / m i n u t e ) .  3.  As t h e water f l o w i n g i n t o t h e t a n k s i n c r e a s e s , f e c e s r e s t i n g on t h e h o r i z o n t a l p i p e c o n n e c t i n g t h e tank w i t h t h e c o l l e c t i o n column w i l l be d i s l o d g e d and w i l l e n t e r t h e v e r t i c a l s t a n d p i p e (see tank F i g u r e 1 ) . C l o s e l y m o n i t o r t h e movement o f t h e f e c a l m a t t e r as i t i s c a r r i e d by t h e u p w e l l i n g water.  4.  U s i n g a 3.81 cm rubber s t o p p e r , p l u g t h e o v e r f l o w d r a i n b e f o r e any f e c a l p a r t i c l e s a r e c a r r i e d over t h e s i d e .  5.  A l l o w t h e water l e v e l i n t h e t a n k s t o r i s e an a d d i t i o n a l 3 t o 5 cm and t h e shut o f f t h e water c o m p l e t e l y . Interruption of the u p w e l l i n g water movement i n t h e s t a n d p i p e w i l l a l l o w suspended f e c a l matter t o s e t t l e i n t o t h e 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 t o s e t t l e i n t o t h e c o l l e c t i o n column, s t e p s 1 t o 4 a r e r e p e a t e d on t h e next p a i r o f t a n k s u n t i l a l l t a n k s have been f l u s h e d c l e a r o f f e c e s from t h e c o n n e c t i n g p i p e . A t t h i s s t a g e , a l l tank d r a i n s s h o u l d be plugged and water f l o w i n t o t h e t a n k s i n t e r r u p t e d .  7.  Remove t h e d r a i n p l u g from t h e f i r s t p a i r o f t a n k s . The h i g h e r "head" i n t h e tank w i l l cause a surge o f water f l o w t o c a r r y any r e m a i n i n g f e c a l p a r t i c l e s r e s t i n g on t h e c o n n e c t i n g p i p e t o t h e v e r t i c a l s t a n d p i p e area (see F i g u r e 1 ) .  opening  178 APPENDIX 1.  (cont'd)  8.  Once a g a i n , p l u g the o v e r f l o w d r a i n b e f o r e any p a r t i c l e s r e a c h t h i s l e v e l and r e p e a t the procedure on the r e m a i n i n g tanks.  9.  Once the water l e v e l i n the t a n k s has reached t h e l e v e l o f the o v e r f l o w d r a i n , p l u g the d r a i n as b e f o r e and open t h e water v a l v e i n the f i r s t tank u n t i l a f l o w r a t e o f a p p r o x i m a t e l y 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 t o 5 cm tank head has been a c h i e v e d , 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 s m a l l clamp which i s connected t o t h e incoming water line. T h i s w i l l c r e a t e a h i g h v e l o c i t y j e t o f water t o come out o f t h e f a r end o f t h e 0.64 cm hose. 11. At the same t i m e , remove the p l u g from the tank o v e r f l o w 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 c o n n e c t i n g p i p e which connects t h e 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 i n c r e a s e i n water f l o w caused by the drop i n water l e v e l from t h e t a n k , c o u p l e d w i t h the h i g h v e l o c i t y water j e t out o f t h e 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 d e p o s i t s are d i s l o d g e d from the c o n n e c t i n g p i p e and are c a r r i e d t o t h e 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 f e c e s has s e t t l e d i n t h e c o l l e c t i o n column and none remains i n t h e c o n n e c t i n g p i p e , the 0.64 cm s e m i - r i g i d hose i s p u l l e d out o f the tank and the water i s once a g a i n t u r n e d o f f . 13. P r i o r t o c o l l e c t i n g t h e f e c e s 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 l o n g aluminum r o d (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 s t a n d p i p e and t h e t i p i s used t o d i s l o d g e f e c a l p a r t i c l e s which may have become s t u c k i n the p i p e 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 p l u g w i t h a spout i n the m i d d l e . 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 o f 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 t h e p l u g spout. 15. G e n t l y squeeze the f l e x i b l e t u b i n g above the clamp a few t i m e s t o a l l o w f e c e s i n t h e column t o s l i d e i n t o the t u b i n g . 16. While f i r m l y s q u e e z i n g the f l e x i b l e t u b i n g , remove t h e clamp and a l l o w f e c e s t o 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 o f 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 d u r i n g t h i s procedure as t h i s w i l l cause s e r i o u s leaching losses.  179 APPENDIX 1.  (cont'd)  17. Once a l l f e c a l m a t t e r has been c o l l e c t e d i n t o t h e c e n t r i f u g e b o t t l e , r e p l a c e t h e clamp and squeeze t h e t u b i n g t i g h t l y t o p r e v e n t water from l e a k i n g o u t . 18. Open t h e water v a l v e and a d j u s t t h e water f l o w i n t o t h e t a n k . 19. Repeat s t e p s 9 t o 18 f o r a l l o t h e r t a n k s . 20. C a r e f u l l y t a k e c e n t r i f u g e b o t t l e s t o 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 a t 10,000 x g. 21. The same procedure weeks.  i s r e p e a t e d a t 1430 hours d a i l y f o r t h r e e  180  APPENDIX 2. Procedures f o l l o w e d f o r t h e q u a n t i t a t i v e feed consumption d e t e r m i n a t i o n by chinook salmon i n Experiment I . A) P r e - f e e d i n g p r e p a r a t i o n s t o be c a r r i e d out i n the l a b o r a t o r y . 1.  Sieve feed p e l l e t s t o remove p e l l e t s length.  s m a l l e r than 3 mm i n  2.  Weigh empty p r e - l a b e l l e d feed v i a l s (Wl).  3.  Add feed r a t i o n t o each corresponding weight (W2). Thus, d a i l y feed r a t i o n W2 - Wl.  feed v i a l and r e c o r d ( i f f u l l y consumed) i s  B) Q u a n t i t a t i v e f e e d i n g procedure t o be f o l l o w e d after fecal collections.  immediately  1.  Increase the water flow i n t o each tank t o approximately 10 l i t e r s per minute t o ensure t h a t r e f u s e d feed p e l l e t s are q u i c k l y f l u s h e d out o f the tank.  2.  I n s e r t s p e c i a l l y made p e r f o r a t e d cup i n t o t h e c o l l e c t i o n column v i a the v e r t i c a l stand p i p e . The cup should r e s t below t h e l e v e l o f t h e pipe connecting t h e tank bottom d r a i n with t h e c o l l e c t i o n column t o ensure t h a t r e f u s e d feed p e l l e t s drop i n t o the cup a f t e r b e i n g f l u s h e d out o f the tank.  3.  Using a spoon, take some p e l l e t s out o f t h e f i r s t feed v i a l and feed t h i s t o the a p p r o p r i a t e f i s h , one p e l l e t at a time. When f i s h l o s e i n t e r e s t i n the feed, move t o t h e next tank and feed those f i s h with t h e i r feed r a t i o n and r e t u r n t o t h e f i r s t tank once these f i s h l o s e t h e i r f e e d i n g enthusiasm. Feed only two tanks at a time.  4.  Attempt t o feed at l e a s t one h a l f the d a i l y r a t i o n t o each tank i n t h e morning and save t h e remainder f o r t h e afternoon feeding.  5.  Once f e e d i n g t h e f i r s t two tanks has been completed, i n c r e a s e the water flow i n t o these two tanks t o maximum flow (approx. 12 l i t e r s / m i n ) .  6.  I n s e r t t h i n 0.64 cm s e m i - r i g i d tube i n t o t h e tank bottom s l o t and open the clamp t o allow water under pressure t o flow through the tube.  181 APPENDIX 2. (cont'd)  7.  S l i d e t h e 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 t h e p i p e c o n n e c t i n g t h e tank bottom s l o t and t h e c o l l e c t i o n column. The water j e t a t t h e end o f t h e s e m i - r i g i d t u b e , c o u p l e d w i t h t h e i n c r e a s e d f l o w r a t e i n t o t h e tank w i l l ensure t h a t a l l f e e d p e l l e t s a r e f l u s h e d out o f t h e t a n k and drop i n t o t h e cup s i t t i n g a t t h e bottom o f t h e c o l l e c t i o n column.  8.  C l o s e r i g i d tube hose clamp and p u l l out t h e tube from t h e tank.  9.  A d j u s t water f l o w i n t o t h e t a n k and t h e n b r i n g up t h e cup from t h e c o l l e c t i o n column.  10. Q u i c k l y count and r e c o r d t h e number o f f e e d p e l l e t s i n t h e cup b e f o r e t h e y d i s i n t e g r a t e .  collected  11. The same p r o c e d u r e i s r e p e a t e d f o r e v e r y t a n k . 12. Feed t h e remainder o f p e l l e t s i n t h e a f t e r n o o n as d e s c r i b e d above and i f p e l l e t s remain i n t h e f e e d v i a l s , reweigh each v i a l a g a i n (W3). 13. C a l c u l a t e t h e amount o f d r y f e e d consumed f o r t h e day as follows: % d r y m a t t e r x [ (W2 - WI) - (W3 - WI) - (W4)] where W4 = number o f f e e d p e l l e t s r e c o v e r e d from cup x mean f e e d p e l l e t weight  (0.0465  grams).  182 APPENDIX 3 . I n f o r m a t i o n on t h e 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 a s d e s c r i b e d b y t h e 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 t h e code g i v e n t o t h e f e e d s t u f f i n this thesis).  1.  British Gilley,  C o l u m b i a h e r r i n g meal (HM) s u p p l i e d b y Mr. B r i a n D. B r i t i s h Columbia Packers L t d .  a) The meal was p r e p a r e d u s i n g whole male a n d f e m a l e (minus t h e roe) h e r r i n g (Clupea harengus pallasi) c a r c a s s e s from t h e 1988 h e r r i n g r o e s e a s o n . b) Once c a u g h t , t h e 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 b e i n g deep f r o z e n (-20°C) f o u r d a y s l a t e r . c) The f i s h were thawed a t 4 t o 7°C p r i o r t o r o e e x t r a c t i o n . d) C a r c a s s e s were p r o c e s s e d w i t h i n two t o t h r e e d a y s o f r o e extraction. e) The m i n c e d h e r r i n g was c o o k e d b y i n d i r e c t steam a t 95°C, and was t h e n p a s s e d t h r o u g h a s i n g l e s c r e w a u g e r t o remove s t i c k water. f) The t e m p e r a t u r e i n t h e s t e a m - j a c k e t r o t a r y d r y e r was s e t a t 77°C. g) The f i n a l h e r r i n g meal was g r o u n d i n a hammer m i l l b e f o r e being s t a b i l i z e d with antioxidant.  A n c h o v y meal (AM) from C h i l e s u p p l i e d b y Mr. J a c k M a r k e r t , M o o r e - C l a r k Co. (Canada) I n c . No f u r t h e r p r o c e s s i n g  3.  information  available.  Menhaden meal (MM) o f a s p e c i a l s e l e c t g r a d e from L o u i s i a n a s u p p l i e s b y Mr. K e l s e y S h o r t , Z a p a t a H a y n i e C o r p . a) M e a l p r e p a r e d u s i n g whole, f r e s h menhaden w i t h a TVN o f l e s s t h a n 0.05%. b) T e m p e r a t u r e o f meal a t t i m e o f e x i t f r o m t h e d r y e r was l e s s t h a n 54°C.  4.  Low t e m p e r a t u r e N o r w e g i a n h e r r i n g meal J a s o n Mann, EWOS Canada L t d .  (LT) s u p p l i e d b y Mr.  a) U s e d whole f r e s h h e r r i n g a n d c a p e l i n f r o m s o u t h e r n Norway w i t h a TVN o f l e s s t h a n 0.05%. b) P r o c e s s i n g t e m p e r a t u r e s n e v e r r e a c h 100°C. c) 200-400 ppm e t h o x y q u i n 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 b y - p r o d u c t meal from Kansas (PMK) manufactured by N a t i o n a l B y - p r o d u c t s I n c . (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 C e n t e r , 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 t h e d r y meal was t h e n passed t h r o u g h 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 o f bone.. No f u r t h e r p r o c e s s i n g 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 b y - p r o d u c t meal from B r i t i s h Columbia (PMB) manufactured by West Coast R e d u c t i o n L t d . , Vancouver, Columbia.  British  a) P o u l t r y b y - p r o d u c t s a r e p r o c e s s e d c o n t i n u o u s l y by means o f a 2 stage vacuum e v a p o r a t i o n system. b) Maximum t e m p e r a t u r e a t t a i n e d i s 135°C f o r 20 m i n u t e s .  7.  H y d r o l y z e d f e a t h e r meal (FM) manufactured by West Coast R e d u c t i o n L t d . , Vancouver, B r i t i s h Columbia. a) Raw f e a t h e r s a r e b a t c h h y d r o l y z e d at 40 p s i and 142°C f o r 4 0 minutes. b) The f e a t h e r s are t h e n d r i e d i n a steam tube d r y e r . c) The maximum t e m p e r a t u r e i n t h e d r y e r i s a p p r o x i m a t e l y 100°C w i t h t h e c o n t a c t t e m p e r a t u r e b e i n g 170°C.  8.  B l o o d meal (BM) manufactured by West Coast R e d u c t i o n L t d . , Vancouver, B r i t i s h Columbia. a) Raw b l o o d p r o c e s s i n g c o n s i s t s o f c o n t i n u o u s c o a g u l a t i o n a t a p p r o x i m a t e l y 98°C by means o f l i v e steam. b) S e p a r a t i o n i s a c c o m p l i s h e d by means o f a c e n t r i f u g e , and d r y i n g t o t h e f i n a l m o i s t u r e c o n t e n t i s performed i n a continuous disk dryer. c) The maximum p r o d u c t t e m p e r a t u r e i n t h e d r y e r i s a p p r o x i m a t e l y 100°C w i t h t h e c o n t a c t t e m p e r a t u r e b e i n g 143°C.  184 APPENDIX 3. (cont'd)  9.  D r i e d whey powder (DW) was o b t a i n e d from Ms. Nancy R i c h a r d s o n , White C r e s t M i l l s . a) L i q u i d whey, s e p a r a t e d from cheese c u r d s , i s f i r s t e v a p o r a t e d t o b r i n g s o l i d s down t o 32%. b) T h i s l i q u i d i s t h e n pumped under p r e s s u r e t h r o u g h spray nozzles into a dryer unit. c) When t h e m i s t h i t s t h e h o t a i r i n t h e d r y e r , t h e s o l i d s drop t o t h e bottom o f t h e d r y e r and a r e augered o u t .  10. Commercial c a n o l a meal (CM) o b t a i n e d t h r o u g h R i t c h i e - S m i t h Feeds, I n c . , A b b o t s f o r d B.C. and p r o c e s s e d by Canbra Foods Ltd., Lethbridge, A l b e r t a . a) T h i s c a n o l a meal i s a b l e n d o f b o t h t y p e s o f seed ( P o l i s h and A r g e n t i n i a n ) t o a r r i v e a t a p r o t e i n l e v e l o f 36%. b) The c l e a n seed i s f l a k e d i n r o l l e r m i l l s as t h e f i r s t s t e p i n b r e a k i n g t h e seed s t r u c t u r e t o a l l o w s e p a r a t i o n o f o i l and meal. c) Many o i l b e a r i n g c e l l s a r e made permeable t o o i l by c o o k i n g which a l s o c o a l e s c e s t h e o i l i n t o l a r g e r d r o p l e t s . d) Cooking i s done (temperature n o t given) f o r 20 t o 30 minutes i n s t a c k cookers heated i n d i r e c t l y by steam. e) The method o f o i l e x t r a c t i o n i s performed t h r o u g h an e x p e l l e r / s o l v e n t or mechanical/chemical operation using hexane as t h e s o l v e n t . f) The s o l v e n t i s s t r i p p e d from t h e meal u s i n g l i v e steam. g) The meal i s d r i e d and i s t h e n t o a s t e d f o r complete s o l v e n t e x t r a c t i o n (temperatures n o t g i v e n ) .  11. G l u c o s i n o l a t e f r e e c a n o l a meal (GCMa) o b t a i n e d t h r o u g h Dr. J.M. B e l l , Dept. o f A n i m a l S c i e n c e , U n i v e r s i t y o f Saskatchewan, Saskatoon. a) T h i s 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. D a v i d Hutcheson and i s a s e l e c t i o n d e r i v e d from t h e 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 c a n o l a i s r e f e r r e d t o as BC86-18 and i s n o t a l i c e n s e d v a r i e t y as y e t . b) The seed was grown on v e r y c l e a n l a n d and was f r e e o f w i l d mustard and o t h e r weed seed c o n t a m i n a n t s . c) The seed was c r u s h e d i n t h e POS P i l o t P l a n t Corp. f a c i l i t i e s u s i n g a p r e - p r e s s s o l v e n t (hexane) e x t r a c t i o n method d e s i g n e d t o s i m u l a t e t y p i c a l commercial p r o c e d u r e s i n use i n Canada, t h a t i s c l e a n i n g , t e m p e r i n g , f l a k i n g ,  185 APPENDIX 3.  d) e)  (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 p r 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 . The maximum t e m p e r a t u r e i n t h e c o o k i n g / c o n d i t i o n i n g p h a s e a r e 90 t o 1 0 0 ° C . Steam i s u s e d i n t h e 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 m e a l (GCMb) p r o v i d e d b y D r . R u b i n D i o s a d y a n d sample was e x t r a c t e d w i t h a c o m b i n a t i o n o f methanol/ammonia t o r e d u c e t h e g l u c o s i n o l a t e c o n t e n t o f t h e 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 n o t made a v a i l a b l e since t h i s i s a patented process.  13.  R a p e s e e d p r o t e i n c o n c e n t r a t e (RP) s u p p l i e d by D r . J o h n D. J o n e s , F o o d R e s e a r c h C e n t r e , A g r i c u l t u r e Canada, O t t a w a . a)  Rapeseed p r o t e i n c o n c e n t r a t e s h i p p e d t o t h e Department o f F i s h e r i e s a n d Oceans i n West V a n c o u v e r i n A u g u s t 1987, a n d s t o r e d a t -20°C a t t h i s f a c i l i t y . b) L o t number o f p r o d u c t t e s t e d was 343 w h i c h i d e n t i f i e s b o t h t h e s e e d a n d t h e method o f p r e p a r a t i o n [BRONOWSKI F R I - 7 3 5(343)]. c) P r e p a r e d u s i n g d e h u l l e d , s o l v e n t a n d w a t e r e x t r a c t e d rapeseed f l o u r . d) D e h u l l e d s e e d i s t r e a t e d t w i c e by e x t r a c t i n g w i t h b o i l i n g water f o r 2 minutes. 14.  Soybean m e a l (SM) o b t a i n e d t h r o u g h A b b o t s f o r d B.C.  Ritchie  Smith Feeds, I n 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) o b t a i n e d R o g e r s L t d . A b b o t s f o r d B.C.  f r o m Van W a t e r s a n d  -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.  E x t r u d e wheat (EW) o b t a i n e d t h r o u g h White C r e s t M i l l s , Campbell R i v e r .  Ms. Nancy  Richardson,  a) U s e d h a r d wheat o f p r a i r i e o r i g i n . b) M o i s t u r e i s added i n t h e form o f steam t o h a r d wheat. c) The wheat i s c o o k e d i n an e x t r u d e r a t 14 9 t o 193°C a n d i s then f o r c e d through a dye. 17.  Wheat m i d d l i n g s (WM) o b t a i n e d from V a n W a t e r s a n d R o g e r s , 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 o r g a n i c m a t t e r d i g e s t i b i l i t y based on t h e d i r e c t o r i n d i r e c t method. (Experiment I ) . Source WEEK METHOD MANAGEMENT WEEK*METHOD WEEK*MANAGEMENT METHOD*MANAGEMENT WEEK*METHOD*MANAGEMENT Error Corrected Total  DF  Mean Square  2 8.1305833 1 15.8802083 2 218.2812500 2 2.3355833 3 10.6902315 2 2.5112500 3 2.2446759 32 6.2233333 47  F Value  P r >F  1.31 2.55 35.07 0.38 1.72 0.40 0.36  0.2848 0.1141 0.0001 0.6901 0.1830 0.6713 0.7818  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 t h ed i r e c t o r i n d i r e c t method. (Experiment I ) . Source DF Mean Square F Value Pr > F WEEK METHOD MANAGEMENT WEEK*METHOD WEEK*MANAGEMENT METHOD ^MANAGEMENT WEEK*METHOD*MANAGEMENT Error Corrected Total  2 10.6989167 1 16.6352083 2 173.3805833 2 0.6935833 3 4.3375926 2 1.1072500 3 0.5070370 32 5.2829167 47  2.03 3.14 32. 65 0.13 0.82 0.21 0.10  0.1485 0.0828 0.0001 0.8774 0.4919 0.8120 0.9617  ANOVA f o r g r o s s energy d i g e s t i b i l i t y based on t h e d i r e c t o r i n d i r e c t method. (Experiment I ) . Source DF Mean Square F Value Pr > WEEK METHOD MANAGEMENT WEEK*METHOD WEEK*MANAGEMENT METHOD *MANAGEMENT WEEK*METHOD*MANAGEMENT Error Corrected Total  2 8.7622500 1 11.8133333 2 197.7112500 2 4.7605833 3 4.7706019 2 7.5179167 3 4.0868981 32 6.5475000 47  1.34 1.80 30.19 0.73 0.73 1.15 0.62  0.2766 0.1966 0.0001 0.4911 0.5425 0.3299 0.6046  187  APPENDIX 4. C o n t i n u e d . ANOVA f o r o r g a n i c m a t t e r d i g e s t i b i l i t y based on t h e morning and afternoon f e c a l c o l l e c t i o n s . (Experiment I ) . Source  DF  WEEK METHOD MANAGEMENT WEEK*METHOD WEEK*MANAGEMENT METHOD*MANAGEMENT WEEK*METHOD*MANAGEMENT Error Corrected Total  2 1 2 2 3 2 3 32 47  Mean Square 5.815139 769.600833 228.405069 11.451389 8.258750 35.970625 4.006157 4.43896  F Value 1.30 173.37 51.45 2.58 1.86 8.10 0.90  Pr > 0 .2857 0 .0001 0 . 0001 0 .0915 0 .1562 0 .0014 0 .4507  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 t h e 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  WEEK METHOD MANAGEMENT WEEK*METHOD WEEK*MANAGEMENT METHOD ^MANAGEMENT WEEK*METHOD*MANAGEMENT Error Corrected Total  2 13. .29503 1..64 1 1406.,23521 173. ,80 2 318. ,11951 . 39. ,32 2 23. .96837 2..96 14. ,58912 3 1.,80 2 120, .98951 14, .95 2,.34 3 18, . 90245 32 8,.09125 47  Pr > F 0,.2113 0..0001 0..0001 0,.0796 0,.1665 0,.0001 0,.0923  ANOVA f o r g r o s s energy d i g e s t i b i l i t y based on t h e 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  WEEK METHOD MANAGEMENT WEEK*METHOD WEEK * MANAGEMENT METHOD *MANAGEMENT WEEK*METHOD*MANAGEMENT Error Corrected Total  2 1 2 2 3 2 3 32 47  19. ,123090 642. ,210208 248. . 685625 16. , 607535 8,. 172454 58, .967292 5,.586157 6,.71937  F Value 2,.85 95, .56 37, .01 2,.47 1,.22 8,.78 0,.83  Pr > F 0..0867 0,.0001 0..0001 0,.1033 0,.3197 0,.0009 0,.4865  188 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 i n t a k e d a t a from experiment I . Source  DF  MANAGEMENT Error Total  2 6 8  Mean Sq. ( 1 0 ) - 5  49.398 4.998  F Value 9.88  Pr > F 0.012 6  A n a l y s i s o f v a r i a n c e o f s p e c i f i c growth r a t e o f chinook salmon i n experiment I . Source  DF  Model Error Total  2 6 8  Mean Sq. (10~ ) 5  72.218 1.504  F Value 48.01  Pr > F 0.0002  A n a l y s i s of v a r i a n c e 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. Source  DF  Feedstuff Error Total  19 42 61  Mean Square 869.1843 32.0596  F Value 27.11  Pr > F 0.0001  A n a l y s i s o f v a r i a n c e f o r crude p r o t e i n d i g e s t i b i l i t y o f t h e 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. Source  DF  Feedstuff Error Total  19 42 61  Mean Square 595.0453 9.8698  F Value 60.29  Pr > F 0.0001  A n a l y s i s o f v a r i a n c e f o r g r o s s energy d i g e s t i b i l i t y o f t h e 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. Source  DF  Feedstuff Error Total  19 42 61  Mean Square 690.1523 50.4111  F Value 13.69  Pr > F 0.0001  189 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 f o r d i g e s t i b l e energy o f t h e f e e d s t u f f s t e s t e d i n e x p e r i m e n t s I I , T i l and IV. Source  DF  Feedstuff Error Total  19 42 61  Analysis of variance Source  DF  DIET Error Total  5 12 17  Analysis of variance II. Source DIET Error Total  DF 5 12 17  Analysis of variance Experiment I I . Source  DF  DIET Error Total  5 12 17  Mean Square  F Value  0.00822056 0.00041478  19.82  o f weight g a i n Mean Square 4.4512667 0.1340056  Pr > F 0.0001  ( g / f i s h ) f o r Experiment I I .  F Value 33.22  Pr > F 0.0001  o f s p e c i f i c growth r a t e Mean Square 0.35170343 0.01111144  F Value  Pr > F  31.65  0.0001  o f d a i l y d r y feed i n t a k e Mean Square 0.00053960 0.00034183  (SGR) f o r Experiment  F Value 1.58  (DFI) f o r  Pr > F 0.2392  A n a l y s i s o f v a r i a n c e o f d a i l y d r y feed i n t a k e as a p e r c e n t o f B.W. (FI) f o r Experiment I I . Source  DF,  DIET Error Total  5 12 17  Mean Square 0.01094222 0.01535556  F Value 0.71  Pr > F 0.6257  190 APPENDIX 4. C o n t i n u e d 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 II. Source  DF  DIET Error Total  5 12 17  Mean Square 0.15511222 0.00631111  (FE) f o r E x p e r i m e n t  F Value  Pr > F  24.58  0.0001  Analysis of variance of protein efficiency r a t i o Experiment I I . Source  DF  DIET Error Total  5 12 17  Mean Square 0.36289889 0.01802222  Analysis of variance of digestible Experiment I I . Source  DF  DIET Error Total  5 12 17  Mean Square 0.00083289 0.00016578  Analysis of variance of digestible Experiment I I . Source  DF  DIET Error Total  5 12 17  Mean Square 0.00068413 0.00007661  Analysis of variance of digestible II. Source  DF  DIET Error Total  5 12 17  Mean Square 0.68997423 0.10778794  (PER) f o r  F Value  Pr > F  20.14  0.0001  organic matter per f i s h f o r  F Value 5.02  Pr > F 0.0100  protein per f i s h f o r  F Value 8.93  Pr > F 0.0010  energy per f i s h  F Value 6.40  f o r Experiment  Pr > F 0.0040  191 APPENDIX 4. C o n t i n u e d Analysis of variance Source  DF  DIET Error Total  8 18 26  Analysis of variance III. Source  DF  DIET Error Total  8 18 26  Analysis of variance Experiment I I I . Source  DF  DIET Error Total  8 18 26  o f f i s h weight g a i n f o r Experiment I I I . Mean Square 3.0449204 0.1374444  F Value 22.15  Pr > F 0.0001  o f s p e c i f i c growth r a t e Mean Square 0.13321233 0.00684156  (SGR) f o r Experiment  F Value 19.47  Pr > F 0.0001  of d a i l y dry feed intake Mean Square 0.01656420 0.00083444  (DFI) p e r f i s h f o r  F Value 19.85  Pr > F 0.0001  A n a l y s i s o f v a r i a n c e o f d a i l y d r y feed i n t a k e o f body w e i g h t f o r Experiment I I I . Source  DF  DIET Error Total  8 18 26  Analysis of variance III. Source  DF  DIET Error Total  8 18 26  Mean Square 0.22575648 0.01072593  F Value  Pr > F  21.05  0.0001  o f feed e f f i c i e n c y r a t i o Mean Square 0.07175833 0.00182222  (FI) as a p e r c e n t  F Value 39.38  (FE) f o r Experiment  Pr > F 0.0001  192 APPENDIX  4.  Continued  Analysis of variance Experiment I I I . Source  DF  DIET Error Total  8 18 26  Analysis of variance Experiment I I I . Source  DF  DIET Error Total  8 18 26  of protein Mean Square  efficiency ratio  F Value  0.2109.8333 0.00488889  43.16  DF  DIET Error Total  8 18 26  Analysis III.  DF  DIET Error Total  8 18 26  Analysis  Pr > F 0.0001  Mean Square  F Value  0.00897970 0.00037411  24.00  Mean Square  F Value  0.00449975 0.00019526  23.05  Pr > F 0.0001  per f i s h f o r  Pr > F 0.0001  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  Source  of variance  Source  DF  DIET Error Total  7 18 25  for  of d i g e s t i b l e organic matter per f i s h f o r  Analysis of variance of digestible protein Experiment I I I . Source  (PER)  Mean Square 6.5478872 0.2699349  o f weight Mean Square 6.0356907 0.3614556  F Value ,24.26  gain  f o r Experiment  Pr > F 0.0001  f o r Experiment IV.  F Value  Pr > F  16.70  0.0001  193  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 s p e c i f i c growth r a t e (SGR) f o r Experiment IV. Source DIET  Error Total  DF 7  18 25  Mean Square 0.02285464  0.00276025  F Value 8.28  Pr > F 0.0001  A n a l y s i s o f v a r i a n c e f o r d a i l y d r y f e e d i n t a k e (DFI) f o r Experiment I V . Source DIET  Error Total  DF 7  18 25  Mean Square 0.01459376  0.00221345  F Value 6.59  Pr > F 0.0006  A n a l y s i s o f v a r i a n c e o f d a i l y d r y f e e d i n t a l e as a p e r c e n t o f body weight (FI) f o r experiment IV. Source DIET  Error Total  DF 7  18 25  Mean Square 0.09586190  0.01453426  F,Value 6.60  Pr > F 0.0006  T - t e s t between t h e o r g a n i c m a t t e 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 d e t e r m i n e d from f e c e s c o l l e c t e d w i t h t h e "Guelph system" and from f e c e s 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 3  Mean 7.3066667  Std Error 1.1134980  T 6.5619038  Prob>|T 0.0224  T - t e s t between t h e o r g a n i c m a t t e 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 d e t e r m i n e d from f e c e s c o l l e c t e d w i t h t h e "Guelph system" and from f e c e s 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. C o n t i n u e d REF-a DIET N Obs 3  Mean 4.1166667  Std Error 0.9249617  T  Prob>|T|  4.4332249  0.0376  T - t e s t between t h e o r g a n i c m a t t e 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 d e t e r m i n e d from f e c e s c o l l e c t e d w i t h t h e "Guelph system" and from f e c e s 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 3  Mean 4.2933333  Std Error 1.1743130  T 3.6560382  Prob>|T| 0.0673  T - t e s t between t h e o r g a n i c m a t t e 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 o f t h e t e s t i n g r e d i e n t s i n e x p e r i m e n t s I I , I I I and IV, d e t e r m i n e d from f e c e s c o l l e c t e d w i t h t h e "Guelph system" and from f e c e s c o l l e c t e d d i r e c t l y from t h e f i s h . INGR=RP N Obs 3  Mean 3.0600000  Std Error 2.2670024  T 1.3498001  Prob>|T| 0.3096  INGR=CMN Obs 3  N Obs 3  Mean 1 .5833333  Mean 2 .3800000  Std E r r o r 4. 0896835  T 0.3871530  Std E r r o r 2. 2908805  0.7360  T 1.0389019  Prob>|T  Prob>|T| 0.4080  195 APPENDIX  N Obs  4.  Continued INGR=HM Mean Std Error  3. 9866667  3  N Obs 3  Mean 4. 9633333  N Obs 3  N Obs 3  N Obs 3  N Obs 3  N Obs 3  N Obs 3  Mean 8.9400000  0.1971745  Mean 5. 5633333  Mean 4 .6433333  Mean 4 .2066667  Mean 6.2033333  20 .2189784  Std Error 3.3772589  4.7207203  1 .4696336  1.2581777  1.8937788  0.7056990  6 .1702996  0.7822262  Std  7. 8834364  1.3520396  Std  5 .9360496  1.3131937  3 .1113487  Prob>|T| 0.0896  T 4.7238524  Prob>IT| 0.0272  T  Error  Prob>|T| 0.0157  T  Error  Prob>IT| 0.0253  T  Std Error  Prob>|T| 0.1988  T  Std Error  Prob>|T| 0.2794  T  Error  Prob>|T| 0.0024  T  Std Error  INGR=PMB Mean Std 7 .7633333  T  Prob>|T| 0.0316  196 APPENDIX 4. C o n t i n u e d .  N Obs 6  Mean 10 .0166667  Std  2.7072676 TVI/'-T->  N Obs 3  8 .4400000  3  N Obs 3  N Obs 3  Mean -0 .7266667  Mean 1 .3233333  Mean 0.6966667  3 .6999175  Hi  »V  T  Error -0 .5615816  0.6309  0 .3377241  Error  2.5172097  Prob>IT|  . T Prob>|T|  Error  3.9183854  Std  Prob>IT| 0.3282  1 .2827439  1.2939646  Std  0.0140  T  Error  JUT)  Std  Prob>|T|  TPT.T  6.5796454 -L IN  N Obs  ICQ.  Std  Mean  T  Error  0.7677  T 0.2767615  Prob>|T| 0.8079  

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0098489/manifest

Comment

Related Items