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Cataracts, growth and histopathology in juvenile chinook salmon (Oncoryhnchus tshawytscha) as influenced… Richardson, Nancy L. 1986

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CATARACTS, GROWTH AND HISTOPATHOLOGY  IN JUVENILE  CHINOOK SALMON (Oncorhynchus t s h a w y t s c h a ) AS INFLUENCED BY DIETARY CALCIUM, PHOSPHORUS, ZINC AND SODIUM PHYTATE  By NANCY L. RICHARDSON B.Sc.  ( A g r . ) , 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 , 1982 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES Department of Animal S c i e n c e We accept t h i s t h e s i s as conforming to the r e q u i r e d standard  THE UNIVERSITY OF BRITISH COLUMBIA January 1986 ©  Nancy L. R i c h a r d s o n , 1986  In p r e s e n t i n g requirements  this thesis f o r an  of  British  it  freely available  agree that for  Library  s h a l l make  for reference  and  study.  I  for extensive copying of  h i s or  be  her  copying or  f i n a n c i a l gain  g r a n t e d by  publication  s h a l l not  The U n i v e r s i t y o f B r i t i s h 1956 Main Mall V a n c o u v e r , Canada V6T 1Y3  DE-6  (3/81)  the  be  of  further this  this  my  It is thesis  a l l o w e d w i t h o u t my  Columbia  thesis  head o f  representatives.  permission.  Date  University  the  s c h o l a r l y p u r p o s e s may  understood that  the  the  I agree that  permission  by  f u l f i l m e n t of  advanced degree at  Columbia,  department or  for  in partial  written  -  ii  -  ABSTRACT In  1981  stocks  at  a severe  several  hypothesized levels  of  dietary  consisting in  that  of  juvenile  British the  chinook  salmon.  study)  dietary  of  on  efficiency, conducted cataract diets  to  all  acid  at  studies  (g/kg  Diets  close  when  to In  unity  growth,  times  I  ranged  and  food  III,  and  cataracts  dietary  levels  c a l c i u m and  phytate  and  zinc  however,  when  cataract  formation  dietary  cataractogenic fish  which  on  were  was  diet. fed  (zinc  For  phytic  conversion,  example,  cataract  more  III in  sodium protein II to  history. of  The  calcium,  and  phosphorus  ratio  increased  mortality  and  g/kg).  Moreover,  the  Cataracts  effects  were  dependent  upon  the  opacities  did  42  and  0.06-25.8,  g/kg  days  test  zinc  (21.1-25.8  not  was  of  contribution.  diet.  between  and  (as  high  possibility  susceptible  life  g/kg  diet  this  was  thesis,  Experiment  Levels  exacerbated  MD.27  to  content  0.05-0.100  were  due  conversion,  a calcium to  acid  It  variations  acid  0.034-0.400  incidence.  be  wide  early  salmon  This  study)  salmon.  based.  have  phosphorus  to  zinc.  phytate-phosphorus  at  levels  noted  the  high  cataract  zinc  to  to  food  were  their  4.4-53,  the  protein  bilateral  low  from  formulated  induced  of  salmon in  deficiency  phytic  chinook  casein-gelatin  disregarding  experiments  depressed  different  of  coho  hatcheries.  (preliminary  and  and  investigate  appetite,  in  chinook  were  I  to  influence  incidence,  State  relation  zinc  whether  diet)  respectively.  the  phosphorus,  were  in  chinook  a zinc  designed  histopathology  determine  from  Experiments  cataract and  formation  for  phytic  health  was  in  Washington  phosphorus  investigated  calcium,  growth,  and  occurred  cataracts resulted  experiments,  (comprehensive  phytate)  outbreak  Columbia  c a l c i u m and  three  levels  cataract  In  time  and  high of  high  prevented experiment of  appear  diet)  exposure  until  84.  day  This  II, to 126  a in  knowledge  of  an  when  apparent  delay  investigating Diets  anomalies  salmon.  the  containing in  containing  pyloric  Plasma,  related  to  dietary  It  is  concluded  _>11.6 caeca! 25  for  and  phytic that  eye  study,  cataracts could  ratios  of  (zinc)-binding  (or  agent  structure. g Ca/kg and  and, acid  level.  high  dietary of  be  also  and  to  29.1  zinc  levels  zinc  present.  hatchery  g calcium/kg  levels plasma  of  the  managers  chinook unless  and  salmon  a strong  are that  related  high  mineral  to  inversely  detrimental zinc  conditions  fed  diets  chinook  blood,  and  the  in  of  directly  acid  salmon  under  induced  ingestion  were  phytic  chinook  Further, in  to  nephrocalcinosis  case of  juvenile  induced  useful  problem.  produced  the  be  Furthermore,  kidney  in  phosphorus) was  acid  development.  not  may  a cataract  g phytic  performance  normal  calcium  of  liver  concentration  health  essential  cause  blood,  zinc  the  cataract formation  a minimum of  dietary  to  in  is  of  dietary  this  iv  TABLE  OF  -  CONTENTS  Section  Page  ABSTRACT L I S T OF T A B L E S  ii ix  L I S T OF F I G U R E S ACKNOWLEDGEMENTS  xi xi i CHAPTER  1.0  INTRODUCTION  .... CHAPTER  2.0  LITERATURE  2.1  The  Fish  2.1.3 2.1.4  Growth Diseases The  2  Eye  Structure Mineral Composition  2.2.1 2.2.2 2.2.3 2.2.4 2.2.5 2.2.5.1 2.2.5.2 2.2.5.3 2.2.6  1  REVIEW  2.1.1 2.1.2  2.2  1  3 3 3 '  Lens  4 5 5  Lens Lens Lens Lens Lens  Capsule Epithelium Substance Function Composition P r o t e i n and w a t e r Major inorganic constituents Trace minerals Lens M e t a b o l i s m  6 6 7 7 8 8 9 9 10  2.3. 2.3.1 2.3.2  C l a s s i f i c a t i o n of Cataracts Developmental C a t a r a c t s Acquired Cataracts  10 10 11  2.4  Causes  12  2.5 2.5.1 2.5.2 2.5.3 2.5.4 2.5.5  Cataract Formation Gross Changes Areas Affected Membrane P e r m e a b i l i t y . . Lens P r o t e i n s Lens M i n e r a l s  2.6 2.6.1 2.6.2 2.6.3  Hatchery Diets W h i t e f i s h Meal H e r r i n g Meal Alternative Protein  2.7 2.7.1 2.7.2  C a l c i u m and P h o s p h o r u s R e q u i r e m e n t s and A v a i l a b i l i t y Properties  of  Cataracts  Sources..  13 14 14 14 15 15  V  TABLE  OF  CONTENTS  cont  Page  2.7.3  Interaction  19  2.7.4 2.7.4.1  Mineral Interactions Magnesium  19 19  2.7.4.2 2.8 2.8.1  Z i n c . . . Zinc Requirements  2.8.2 2.9  20  and  Availability  Properties Phytic  2.9.1 2.9.2 2.9.3  22  Acid...'  23  Structure Occurrence Properties  23 24 24  2.10 2.10.1 2.10.2 2.10.3  Phytates Structure Properties Interactions  2.11  Fibre  2.12 ' 2.12.1 2.12.2 2.12.3  Calcium-Zinc-Phytates Swine Studies Rat S t u d i e s Poultry Studies  2.13  The  2.14  Phytic  Acid  2.15  Growth  in  25 25 26 27  Diets-Effects  Effect  of  Gut  on  Mineral  pH o n  Studies  with  Availability  ,  Phytate  GENERAL  Activity  Salmonids  MATERIALS  27 29 30 30 31  Salmonids  31 32 33  CHAPTER 3.0  21 21  3  AND METHODS  3.1  Experimental  3.2 3.3 3.4 3.5 3.5.1 3.5.2 3.5.3 3.5.4 3.5.5 3.5.6 3.5.7 3.5.8  Culture Conditions Diet Formulations 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 S a m p l i n g Diet allocation Feeding Fish weight Cataract assessment Proximate analysis F i s h h e a l t h and h i s t o l o g i c a l e x a m i n a t i o n P l a s m a and b l o o d a n a l y s i s Tissue analysis  Fish  36 36 36 36 37 45 45 45 45 45 46 46 46 47  -  TABLE  OF C O N T E N T S  vi  -  cont  Page CHAPTER  4.0  4  EXPERIMENT I. P r e l i m i n a r y study of the e f f e c t s of dietary c a l c i u m , p h o s p h o r u s , z i n c and p h y t i c a c i d 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 chinook salmon.  4.1  Introduction  48  4.2 4.2.1  M a t e r i a l s and Methods Experimental Fish  49 49  4.2.2 4.2.3 4.2.4 4.2.4.1  Experimental Diets Feeding Sampling Procedures F i s h w e i g h t and  cataract  49 49 49 49  incidence  4.2.4.2  Proximate  analysis  49  4.2.4.3  Histology  samples  49  4.2.4.4 4.2.4.5  Plasma samples Statistical analysis  4.3 4.3.1 4.3.2  Results Influence Influence  4.3.2.1 4.3.2.2  of of  diet diet  treatment treatment  50 50  on- c a t a r a c t i n c i d e n c e on c h i n o o k p e r f o r m a n c e  51 51 51  4.3.3 4.3.4 4.3.5  F i s h growth Food i n t a k e , f o o d c o n v e r s i o n and p r o t e i n e f f i c i e n c y r a t i o (PER) I n f l u e n c e o f d i e t t r e a t m e n t on p r o x i m a t e c o m p o s i t i o n I n f l u e n c e o f d i e t t r e a t m e n t on g e n e r a l h e a l t h I n f l u e n c e o f d i e t t r e a t m e n t on h i s t o p a t h o l o g y . . . . .  52 52 53 61  4.3.5.1 4.3.5.2 4.3.5.3 4.3.6  Kidney P y l o r i c c a e c a and stomach Thyroid I n f l u e n c e o f d i e t t r e a t m e n t on  61 62 64 64  4.4 4.4.1 4.4.2 4.4.2.1 4.4.2.2 4.4.3 4.4.4 4.4.5 4.4.5.1 4.4.5.2 4.4.5.3 4.4.6 4.5  ,  Discussion Cataract Incidence Chinook Performance F i s h growth Food i n t a k e , f o o d Proximate Composition Fish Health Histopathology.... Kidney P y l o r i c c a e c a and Thyroid Plasma Minerals Conclusion  conversion  -. plasma mineral  and  levels  PER  stomach  CHAPTER  51  66 66 67 67 67 69 6.9 69 69 70 71 71 72  5  5.0  EXPERIMENT I I . The s u s c e p t i b i l i t y of j u v e n i l e c h i n o o k salmon t o c a t a r a c t formation i n r e l a t i o n to d i e t a r y changes i n e a r l y life.  5.1  Introduction  74  -  TABLE  OF  CONTENTS  5.2  vii  -  cont  Materials  Page  and  Methods  74  5.2.1  Experimental  Fish  74  5.2.2 5.2.3  Experimental Feeding  Diets  74 75  5.2.4 5.2.5  Sampling Procedures Statistical Analysis  5.3 5.3.1 5.3.2 5.4 5.4.1 5.4.2 5.5  Results Influence Influence .  of of  diet diet  77 77  treatment treatment  on on  77 77 77  cataract incidence f i s h growth..!  Discussion  78  Cataract Incidence F i s h Growth  78 81  Conclusion  81 CHAPTER  6  6.0  EXPERIMENT I I I . Comprehensive study of the e f f e c t s of dietary c a l c i u m , p h o s p h o r u s , z i n c and p h y t i c a c i d on c a t a r a c t s , 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 s a l m o n .  6.1 6.2 6.2.1 6.2.2 6.2.3 6.2.4 6.2.4.1 6.2.4.2  Introduction M a t e r i a l s and Methods Experimental Fish Experimental Diets Feeding Sampling Procedures F i s h w e i g h t and c a t a r a c t Proximate analysis  6.2.4.3 6.2.4.4 6.2.4.5 6.2.5 6.3 6.3.1 6.3.2  82 82 82 82 82 83 83 83  incidence  H i s t o l o g y samples Blood samples T i s s u e samples Statistical Analysis Results Influence Influence  of of  diet diet  treatment treatment  83 83 83 84  on on  cataract incidence chinook performance  84 84 84  6.3.2.1 6.3.2.2 6.3.3 6.3.4  F i s h growth F o o d i n t a k e , f o o d c o n v e r s i o n a n d PER I n f l u e n c e o f d i e t t r e a t m e n t on p r o x i m a t e c o m p o s i t i o n I n f l u e n c e o f d i e t t r e a t m e n t on g e n e r a l h e a l t h  84 85 91 91  6.3.5  Influence  93  6.3.5.1 6.3.5.2 6.3.6 6.3.6.1 6.3.6.2 6.3.6.3  of  diet  treatment  on  histopathology...  Kidney P y l o r i c caeca Influence of d i e t treatment levels Whole blood Liver Kidney  93 93 on  blood  and  tissue  mineral 93 93 96 98  -  TABLE  OF  6.4  CONTENTS  Page  Discussion  98  Cataract  6.4.2 6.4.2.1  Chinook Performance F i s h growth  6.4.2.2 6.4.3 6.4.4 6.4.5 6.4.5.1 6.4.5.2 6.4.6 6.4.6.1 6.4.6.2 6.4.6.3  Food i n t a k e , f o o d Proximate Composition Fish Health Histopathology Kidney P y l o r i c caeca Mineral Analysis Whole b l o o d Liver Kidney  Incidence  98 100 100 conversion  Conclusion  SUMMARY AND  and  PER  101 102 102 103 103 103 104 104 105 107 108  CHAPTER 7.0  -  cont  6.4.1  6.5  vi i i  CONCLUSIONS  7 110  BIBLIOGRAPHY  113  APPENDIX  124  -  LIST  ix  OF  -  TABLES  Table  Page  1.  Nutritional  and  2.  Formulation experiments  of  3.  3a.  4.  4a.  5.  6.  7.  8.  9.  10.  11.  12.  13.  non-nutritional basal  fed  to  juvenile  cataracts chinook  in  Composition of the nine mineral-phytate j u v e i l e chinook salmon i n Experiment 1  salmon  supplements  in  fed  12  all  Proximate  composition,  mineral  nine  diets  juvenile  fed  to  and  phytic  chinook  to 39  Composition of the eighteen m i n e r a l - p h y t a t e supplements 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 s II and I I I  test  animals  38  acid  content  salmon  in  fed  to 40-41  of  the  Experiment  I....  P r o x i m a t e c o m p o s i t i o n , m i n e r a l and p h y t i c a c i d c o n t e n t o f t h e e i g h t e e n t e s t d i e t s fed to j u v e n i l e chinook salmon i n Experiments II and I I I • Cataract incidence in j u v e n i l e chinook l e v e l s of phytate in Experiment 1  salmon  fed  diets  with  F i n a l whole body proximate c o m p o s i t i o n of j u v e n i l e s a l m o n f e d t e s t d i e t s f o r 105 d a y s i n E x p e r i m e n t 1  j u v e n i l e chinook salmon f e d f o r a p e r i o d o f 126 d a y s i n  Mean w e t juvenile III Mean  chinook salmon Experiment III  56  test 58  chinook 60  F i n a l mean p l a s m a c o n c e n t r a t i o n s o f v a r i o u s m i n e r a l s chinook salmon f e d the t e s t d i e t s i n Experiment 1  Cataract incidence in juvenile 2 1 , 4 2 , 6 3 , 8 4 , 105 and 119 i n  43-44  54  Mean d a i l y f o o d c o n s u m p t i o n , f o o d c o n v e r s i o n and p r o t e i n e f f i c i e n c y r a t i o f o r groups of j u v e n i l e chinook salmon f e d d i e t s b e t w e e n d a y s 22 and 105 i n E x p e r i m e n t 1  Mean w e t w e i g h t s o f at 42-day i n t e r v a l s  42  high  S p e c i f i c g r o w t h r a t e s and o b s e r v e d m o r t a l i t i e s f o r juvenile c h i n o o k s a l m o n f e d t e s t d i e t s f o r 105 d a y s i n E x p e r i m e n t 1  in  juvenile 65  the test diets E x p e r i m e n t II  examined  on  days  80 0, 86  w e i g h t s , s p e c i f i c g r o w t h r a t e s and m o r t a l i t i e s f o r c h i n o o k salmon f e d t e s t d i e t s f o r 84 days i n E x p e r i m e n t  daily  87 food  consumption,  efficiency ratio for d i e t s between days 0 14.  diet  causes of  food  groups of and 84 i n  conversion  and  j u v e n i l e chinook Experiment III  protein salmon  F i n a l whole body proximate c o m p o s i t i o n of j u v e n i l e salmon f e d t e s t d i e t s f o r 84 days i n E x p e r i m e n t I I I  fed  test 90  chinook  92  X  LIST 15.  16.  17.  18.  OF  TABLES c o n t  Page  The % i n c i d e n c e o f n e p h r o c a l c i n o s i s and v a c u o l i z a t i o n o f t h e p y l o r i c c a e c a i n j u v e n i l e c h i n o o k s a l m o n a t d a y s 50 and 8 4 i n Experiment III  94  F i n a l whole blood concentrations of various minerals chinook salmon f e d the t e s t d i e t s in Experiment III  95  in  juvenile  F i n a l l i v e r concentrations of various minerals in juvenile chinook salmon fed the t e s t d i e t s i n Experiment III  97  Final kidney concentrations chinook salmon f e d the t e s t  99  of various minerals diets in Experiment  in III  juvenile  - xi  -  LIST OF FIGURES Figure  Page  1.  S t r u c t u r e of a f i s h eye  4  2.  C r o s s - s e c t i o n of a l e n s showing f i b r e f o r m a t i o n  7  3.  The s t r u c t u r e of p h y t i c a c i d  24  4.  The s t r u c t u r e of a p h y t a t e complex  26  5.  Growth c u r v e of f i s h under c o n s t a n t e n v i r o n m e n t a l c o n d i t i o n s  35  6.  J u v e n i l e chinook salmon f e d a h i g h p h y t a t e d i e t  55  7.  Geometric mean w e i g h t s of j u v e n i l e chinook salmon f e d t e s t f o r 105 d a y s - i n Experiment 1  8.  diets 57  Food i n t a k e , f o o d c o n v e r s i o n and p r o t e i n e f f i c i e n c y r a t i o f o r r e p r e s e n t a t i v e groups of j u v e n i l e chinook salmon f e d d i e t s c o n t a i n i n g low and h i g h p h y t i c a c i d at f o u r 21-day i n t e r v a l s i n Experiment 1  59  9.  Kidney s e c t i o n of a chinook salmon f e d a d i e t w i t h a h i g h l e v e l c a l c i u m and phosphorus i n Experiment 1  10a.  C r o s s - s e c t i o n of p y l o r i c c a e c a from a h i g h p h y t a t e - f e d chinook salmon i n Experiment 1  62  C r o s s - s e c t i o n of p y l o r i c caeca from a low p h y t a t e - f e d chinook salmon i n Experiment 1  63  C r o s s - s e c t i o n of t h e c a r d i a c stomach and p y l o r i c c a e c a of a chinook salmon i n Experiment I  63  T h y r o i d f o l l i c l e s of a chinook salmon f e d a h i g h p h y t a t e d i e t and a low p h y t a t e d i e t i n Experiment 1  64  12.  Schematic r e p r e s e n t a t i o n of d i e t a r y t r e a t m e n t s g i v e n t o chinook salmon from swim-up t o day 126 i n Experiment II  76  13.  Geometric mean w e i g h t s of chinook salmon f e d t e s t d i e t s f o r 84 days i n Experiment I I I  88  R e p r e s e n t a t i v e p a i r s of f i s h f e d d i e t s 1, 5 and 7 (15a) and d i e t s 1 1 , 15 and 12 (15b) f o r 84 days i n Experiment I I I  89  10b. 10c. 11.  14.  of  61  - xii  -  ACKNOWLEDGEMENTS Many p e o p l e were i n v o l v e d i n t h i s t h e s i s .  To b e g i n w i t h I would l i k e t o  thank my a d v i s o r , D r . Beames, f o r h i s h e l p f u l comments and e s p e c i a l l y f o r a l l o w i n g me t o s w i t c h t h e s u b j e c t of my t h e s i s from dogs t o f i s h .  I would  a l s o l i k e t o thank my committee members: D r . H i g g s , P r o f . March, D r . T a i t and D r . P e t e r s o n f o r t h e i r h e l p i n making t h i s a b e t t e r t h e s i s .  I would  e s p e c i a l l y l i k e t o thank Dr. Higgs f o r h i s c o n s t a n t e n t h u s i a s m and encouragement d u r i n g my s o j o u r n at t h e West Vancouver L a b o r a t o r y .  There are  s e v e r a l o t h e r s t a f f members of t h e West Van L a b , both p a s t and p r e s e n t , whom I owe t h a n k s . Helen Dye.  Most n o t a b l y , Dianne P l o t n i k o f f ,  Jack M a r k e r t , Andy Lamb and  T h e i r h e l p i n t h e t e c h n i c a l and maintenance a s p e c t s of  e x p e r i m e n t s was g r e a t l y a p p r e c i a t e d . w o r k i n g w i t h them.  the  More i m p o r t a n t l y , i t was a p l e a s u r e  Thanks a r e a l s o owed t o Jack M c B r i d e f o r t h e h i s t o l o g i c a l  p h o t o g r a p h s ; t o Morva Booth f o r t e a c h i n g me some of t h e m y s t e r i e s of  the  Micom and t o D r . P i e t Dejong (UBC F a c u l t y of Commerce) f o r t h e s t a t i s t i c a l a n a l y s e s i n E x p e r i m e n t s I and I I . S p e c i a l t h a n k s t o N e i l Holman (EPS) f o r h i s f r i e n d s h i p and e n d l e s s p a t i e n c e i n p h o t o g r a p h i n g f i s h f o r t h i s t h e s i s and i n r e l a t e d p r o j e c t s .  And  t h a n k s t o a l l t h e p e o p l e at t h e Lab who h e l p e d t o make my t i m e t h e r e so enjoyable. N e e d l e s s t o s a y , I w i s h t o thank my p a r e n t s f o r t h e i r l o v e and support and f o r a l l t h e Sunday d i n n e r s and f r e e l u n c h e s .  A n d , o f c o u r s e , my  acknowledgements would not be complete w i t h o u t e x p r e s s i n g s i n c e r e thanks t o A l l e n Mayes.  H i s l o v e , "humour" and s u p p o r t have c a r r i e d me a long way from  t h e f r o z e n wastes of W i n n i p e g .  - 1 -  CHAPTER 1  1.0  INTRODUCTION Exophthalmos (pop-eye) and c a t a r a c t s a r e , r e s p e c t i v e l y , t h e f i r s t  second most f r e q u e n t eye d i s o r d e r s i n c u l t u r e d f i s h (Dukes 1 9 7 5 ) .  and  Reports  from h a t c h e r i e s worldwide i n d i c a t e t h a t c a t a r a c t s of d i e t a r y o r i g i n  are  widespread and c o s t l y i n terms of l o s t f i s h p r o d u c t i o n (Poston e t a l . 1 9 7 8 ) . Japan had i t s f i r s t major c a t a r a c t outbreak i n rainbow t r o u t  (Salmo  g a i r d n e r i ) s t o c k s i n 1971 (Kubota 1976), and subsequent l o s s e s of f i s h due t o c a t a r a c t s have i n c r e a s e d a n n u a l l y i n Japan as has t h e number of salmon and trout species affected.  In t h e Zomba P l a t e a u of M a l a w i , C e n t r a l A f r i c a , an  average of 10% of t h e rainbow t r o u t s t o c k s are l o s t a n n u a l l y due t o from c a t a r a c t s (Lee et a l . 1976). i n Great B r i t a i n ,  Salmon and t r o u t have a l s o been  I c e l a n d and t h e U n i t e d S t a t e s ( K e t o l a 1 9 7 9 ) .  blindness affected  A local  example of f i s h c a t a r a c t s o c c u r r e d i n 1981 when s e v e r a l B r i t i s h Columbia and Washington S t a t e h a t c h e r i e s e x p e r i e n c e d severe o u t b r e a k s i n t h e i r (Oncorhynchus t s h a w y t s c h a ) and coho ( 0 . k i s u t c h ) salmon s t o c k s .  chinook Despite  t h i s , t h e f i s h were r e l e a s e d and i t has been e s t i m a t e d t h a t f u t u r e l o s s e s t o t h e B.C. f i s h e r y of chinook salmon from t h i s r e l e a s e w i l l  be at l e a s t 30,000  f i s h , which r e p r e s e n t s a 13% r e d u c t i o n i n t h e t o t a l a d u l t r e t u r n alone 1981).  At t h e t i m e of t h e outbreak i n B.C. and Washington, a l l  (Perry  affected  h a t c h e r i e s were u s i n g t h e same commerical d i e t , namely, Oregon M o i s t  Pellets  (OMP), which c o n t a i n e d a h i g h p r o p o r t i o n of a North American E a s t Coast h e r r i n g meal.  T h i s meal had a high ash c o n t e n t due t o f i l l e t removal from  the f i s h before p r o c e s s i n g .  C o n s e q u e n t l y , t h e l e v e l s of c a l c i u m (Ca) and  phosphorus (P) i n t h i s d i e t were h i g h .  On t h e b a s i s of work conducted on  -  rainbow  trout  it  was  an  induced  to  Zn.  fry  postulated zinc  Also,  are  played such  an  as  that  thesis  was  incidence  interrelationships  the  role  from  of  the  high  dietary  acid,  the  cereal  products  of  commercial its  strong  Barash  et  cataract  phytic  into  because  cause  by  that  of  incorporated  important  corroborated  arising  a constituent  commonly  and  probable  hypothesized  designed  understanding  cataract  food  most  deficiency  was  and  the  (1979)  -  the  (1982),  outbreak  ratios  of  salmonid  oilseed  foods,  to  was  Ca  hexaphosphate and  ability  al.  and  P  of  meals  may  chelate  have  minerals  Zn.  This clearer  Ketola  (Zn)  it  myo-inositol, which  by  2  in  of  between  protein  To  of  effects  species,  or  best  phytic  the  above  efficiency,  of  the  acid  major  of  chinook  my k n o w l e d g e ,  of  two  effects  juvenile  conversion, the  the  with  per  dietary salmon,  dietary and  there  s e on  goals  in  Ca, and  mind  Zn 2)  factors  and to  on  histopathology are  no  reports  cataract formation  histopathological  effects  literature  is  of  high  1)  to  obtain  phytic  acid  determine growth, in  in  food  have any  dietary  intake,  salmon. investigated  animal  levels  of  acid. The knowledge  following on  nutritional  the  lens,  the  s i g n i f i c a n c e of  review  causes Ca,  Zn  and and  designed  effects phytic  of  to  provide  cataracts,  acid.  background and  on  the  chinook  that  a  on  the  phytic  -  3  -  CHAPTER  2.0  LITERATURE  2.1  THE  FISH  Little  REVIEW  EYE  research  has  been  but  much o f  compared  to  mammals,  research  is  applicable to  2.1.1  structural  other  with  structural  the  the  the  ophthalmic  information  pathology  derived  from  of  fishes  as  mammalian  fishes.  lens,  The  important  one  respect change  to  shape  lens  lens  much  lens  further  is  being  like  living  eye  and  as  the  (e.g.  human  and  between  (van  Duijn  accommodate  of  a fixed  and  into  the  arm o f globe  and  In  the  human  which  are  near  choroid  a camera.  of  the  eye  which  or  the  or  far,  moves  but  to  retina, 1).  is  with  can  in  the  there  displace  appendix  it  same  lens  Instead,  acts  lens  the  eye  the  of  is  (Fig.  human  eye  eyes  choroid,  humours  changed.  The  has  sclera,  eye  the  thesis  which  vitreous  be  among  this  trout)  nerve,  and  cannot  the  than  fish  1973).  attached to  focusing  the  objects  shape  and  optic  aqueous  eyes  However,  salmon  eye:  the  to  is  a  the  withdraws  the  forward.  Composition  in  is  all  the  significant  iris.  this  eye  difference  contains  matter  unknown,  1975).  pupil,  the  back  present  choroid  (Dukes  appendix  Mineral The  combined  teleost  iris,  sickle-shaped  2.1.2  among f i s h  accomodation  its  the  exists  components  cornea,  fish.  on  variation  vertebrates  concerned  fish  done  Structure More  all  2  The in  the  the  amounts  highest choroid  concentration  Although  essential  is  minerals  in  the  higher  concentrations  of  both  Zn  and  c e r t a i n t i s s u e s , most  concentration of  with  eye  of  Zn known  (Underwood  in  freshwater  Zn  and  Cu  in  notably  exist  1971),  fish the  to  and  than  eye  copper  are  in  (Cu)  the  normally for  reasons  saltwater  very  in  high,  - 4 -  their function has not been determined.  There have been, however, some  suggestions to explain the high Zn concentration.  For example, a study by  Bowness et a l . (1952) with coloured and albino rabbits showed that the high Zn concentration in the i r i s and choroid was due to the presence of the pigment melanin.  Also, Weitzel et a l . (1953) reported that carnivores have  higher Zn concentrations than herbivores.  This was attributed to the  presence of a tapetum lucidum which reflects stray light back through the retina (Munz 1971).  Both melanin and a tapetum lucidum are present in some  fishes, but no correlation has yet been made between this and the high Zn concentration in the eye.  CHOROID  VITREOUS  Fig. 2.1.3  1.  Structure of a fish eye (van Duijn, J r . 1973).  Growth Unlike mammals, fish continue to grow throughout their lives, and as the  body grows the eye grows as well.  Thus factors which determine rate of body  - 5 -  growth w i l l  a l s o determine eye g r o w t h .  The most common f a c t o r s i n c l u d e f o o d  q u a l i t y and a v a i l a b i l i t y , water t e m p e r a t u r e , f i s h d e n s i t y and f i s h F i s h eyes g r e a t l y i n c r e a s e i n s i z e d u r i n g j u v e n i l e l i f e D u r i n g t h e f i r s t y e a r of l i f e ,  age.  (Johns 1981).  l e n s e s i n t e l e o s t f i s h may i n c r e a s e i n volume  by a t h o u s a n d f o l d ( F e r n a l d and Wright 1983).  Under h a t c h e r y c o n d i t i o n s  great  c a r e must be t a k e n t o ensure t h a t t h e aforementioned f a c t o r s are c a r e f u l l y c o n s i d e r e d i n o r d e r t o ensure proper l e n s g r o w t h . 2.1.4  Diseases The two most common eye d i s e a s e s i n f i s h are exophthalmos (pop-eye)  cataracts.  These can be due t o many f a c t o r s , both n u t r i t i o n a l  non-nutritional. confined f i s h ,  and  and  A l t h o u g h exophthalmos i s t h e most f r e q u e n t eye d i s e a s e  i t i s not t h e most s e r i o u s .  When an outbreak of  in  cataracts  o c c u r s i t i n v a r i a b l y l e a d s t o b l i n d n e s s , whereas exophthalmos does n o t . B l i n d f i s h cannot s u c c e s s f u l l y f i n d f o o d o r evade p r e d a t o r s . they have l i t t l e chance of s u r v i v a l .  Consequently,  T h i s may have d r a s t i c e f f e c t s on a d u l t  r e t u r n s t o commercial and r e c r e a t i o n a l f i s h e r i e s and t o h a t c h e r i e s . A c a t a r a c t , a l s o known as w h i t e eye o r opaque e y e , i s d e f i n e d as a l e n s opacity.  The f i r s t E n g l i s h usage of " c a t a r a c t " was t o d e s c r i b e f l o o d g a t e s ,  and i n t o d a y ' s usage t h e " g a t e " r e f e r s t o t h e o p a c i t y which descends on t h e eye (Lerman 1966).  I t s h o u l d be noted t h a t t h e r e are a l s o o p a c i t i e s of  the  c o r n e a but t h e s e are not d e f i n e d as c a t a r a c t s , t h e r e f o r e c a r e must be t a k e n when d i a g n o s i n g an o p a c i t y as being e i t h e r l e n t i c u l a r o r c o r n e a l . As c a t a r a c t s are a d i s e a s e of t h e l e n s , any f u r t h e r d i s c u s s i o n of them must.be preceded by a d i s c u s s i o n of t h e l e n s 2.2  itself.  THE LENS The v e r t e b r a t e l e n s , a n o n - v a s c u l a r t i s s u e d e r i v e d from t h e  surface  -  ectoderm  of  the  the  is  spherical,  lens  tends  to  contain lens  eye,  protrude three  a relatively occupies  through  basic  the  regions:  a  simple  large  pupil the  -  part  (Dukes  lens  epithelial of  the  1975).  capsule,  structure.  inside All  the  of  In  the  globe  vertebrate  lens  fish and  lenses  epithelium  and  the  substance.  2.2.1  Lens The  capsule  the  epithelial 1966).  Capsule  lens  surrounds  cells  The  and  surface  portion  nutrient  exchanges  aqueous  2.2.2  of  Lens  the  epithelial become  of  elastic  the  lens.  a double-1ayered  contains  acid  slightly  thicker  occur  between  covering It  is  the  than  the  anterior  lens  capsule  is  anterior  portion  aids  derived  structure  polysaccharides  in  in and  the  capsule  completely from  lens  acid.  portion.  and  permeable  achieving  modified  adult  sialic  posterior  selectively it  which  the  (Lerman The  Almost  all  adjacent  and  the  increased  this.  Epithelium to  lens cells  crowded  elongation  into  cells  the  than  a partially  surface  is  The  the  Posterior to  is  humour.  thickness  is  exterior  anterior  down  is  6  the  lens  equator which  together lens adult  capsule  is  the  lens  continues at  the  fibres. lens  and  to  extending  epithelium grow  periphery The  (Manery  young 1961).  from (Fig.  throughout of lens  the  the  anterior  2),  a single  life.  equator  contains  The  of  layer  cells  undergo  more  portion  a  these  of  which  gradual nucleated  - 7-  LENS  EPITHELIUM  NUCLEUS  F i g . 2.  ANTERIOR  CAPSULE  C r o s s - s e c t i o n o f a lens showing f i b r e f o r m a t i o n ( P i r i e and van Heyningen 1956).  2.2.3  Lens Substance I n c l u d e d i n t h e lens substance a r e t h e f i b r e c e l l s and t h e i r  p r o t e i n s , and t h e i n t e r s t i t i a l m a t e r i a l which can be d i v i d e d i n t o and n u c l e a r r e g i o n s .  constituent cortical  Fibre formation i s a continuous process r e s u l t i n g i n  t h e e a r l i e r (primary) f i b r e s being b u r i e d .  The most d e e p l y b u r i e d f i b r e s  c o n s t i t u t e t h e nucleus of the lens and they a r e t h i n n e r and l a r g e r than t h e peripheral  (secondary) f i b r e s .  The young, p e r i p h e r a l f i b r e s o f t h e l e n s  c o r t e x r e t a i n t h e i r n u c l e i and are t h e most a c t i v e m e t a b o l i c a l l y (Jubb and Kennedy 1 9 7 0 ) .  The mature, c e n t r a l f i b r e s l o s e t h e i r n u c l e i but s t i l l  active. 2.2.4  Lens F u n c t i o n The primary f u n c t i o n o f t h e lens i s t o a c h i e v e and m a i n t a i n  transparency.  For t h i s reason t h e l e n s i s a v a s c u l a r and t r a n s p a r e n t .  remain  -  Transparency protein. lens.  is  Such  achieved  cataracts function  is  the  from  is  the  1.33  composition  within  upon  to  the  1.53  lens  is  swelling  the and  lens,  accomodation  the  difference  in  refractive  In  and  vitreous  make  of  which  the is  Protein  humours.  lens,  and  index  deformation  does  not  fish of  birds  lenses water of  lens  differences softer  the  any  in  index  What  namely,  the  lens  percentage  in  the  majority  in  the  lens  index  Thus the  the  lens  requires  the  will  lens  a  linear  index  (Pumphrey  internal  from the  puncture of  an  of  increases  refractive  centre  at  the  1961).  chemical  aqueous  upset  humour.  this  (Grant  the  light  is  concentration  their  Due  difference  1974).  fish  lenses  between  determines  the  amount  is  the  lens  this  of  primarily and  the  difference  water  and  dependent surrounding  is  highly  the  gross  refractile  water  the  result,  throughout  phenomenon  and  present.  s p e c i e s where  refractive  index  different  opaqueness  of  2.2.5.1  refractive  increased  at  index  of  cells  Protein  refractive which  fibre  in  nucleus.  to  the  result  refractive  efficiency  protein  index  of  Composition  composition  are  The  The  aqueous  to  a constant  important  increases  the  spacing  concentration.  cortex  proper  a rapid Lens  most  -  regular  refractive  1973).  non-vascularity  causing 2.2.5  the  and  the  the  the  protein  Maintaining  to  in  (Philipson  gradually outside  which  of  the  spacing maintains  Alterations  scattering  by  8  have  prey  (e.g.  and more  occur,  of  is  the  the  any whose  accomodated  by  mammals),  there  deformable. lens  highest  species lenses  must  In  be  as  percentage  studied. contain  At about  deformation will  the  be  fish,  as w e l l  more w a t e r where  refractile  as  of  and  the  protein  opposite  80% w a t e r  as present  lens  possible. the  end  of  compared  As  a  lowest the to  scale 50%  in  - 9 -  t r o u t ( K l e t h i and Mandel 1965).  T h i s high water c o n c e n t r a t i o n makes l e n s  t r a n s p a r e n c y maintenance an e a s i e r t a s k and may be a major reason why b i r d s are not as prone t o c a t a r a c t s as o t h e r s p e c i e s (Kuck, J r . 2.2.5.2  Major i n o r g a n i c  1975).  constituents  The l e n s c o n t a i n s a s u b s t a n t i a l amount of potassium (K) which i s common i n a t i s s u e c o n t a i n i n g l a r g e amounts of p r o t e i n and i n t r a c e l l u l a r s p a c e . i  Sodium (Na) o c c u r s i n t h e e x t r a c e l l u l a r space and ranges i n  concentration  f r o m .10 t o .50 of t h a t of K depending on t h e age and v i a b i l i t y of t h e l e n s . That i s , Na i n c r e a s e s w i t h age and l e n s d e g e n e r a t i o n .  Calcium i s present  low l e v e l s i n t h e young l e n s but i t i s thought t o be h i g h l y s i g n i f i c a n t  in  in  m a i n t a i n i n g normal l e n s membrane p e r m e a b i l i t y ( T h o f t and K i n o s h i t a 1965). C a l c i u m c o n t e n t i n c r e a s e s w i t h age and i s h i g h e r i n t h e n u c l e u s than i n t h e c o r t e x ( B e l l o w s 1944).  Other major c o n s t i t u e n t s i n c l u d e : magnesium (Mg),  phosphate, s u l p h a t e , c h l o r i d e and b i c a r b o n a t e .  Bicarbonate i s very important  i n m a i n t a i n i n g l e n s pH at 7.4; a t a s k i t shares w i t h l a c t i c a c i d which c o n t i n u a l l y d i f f u s e s out from the l e n s i n t o t h e aqueous humour. 2.2.5.3  Trace m i n e r a l s  Lenses of a l l v e r t e b r a t e s c o n t a i n numerous m i n e r a l s which are p r e s e n t v a r y i n g amounts depending upon t h e s p e c i e s i n v o l v e d and t h e c o n d i t i o n of lens.  the  Iron ( F e ) , Cu and Zn are c l o s e l y a s s o c i a t e d w i t h r e s p i r a t i o n , amino  a c i d m e t a b o l i s m , pH r e g u l a t i o n , energy metabolism and c e l l membrane p e r m e a b i l i t y (Murata and Taura 1975). which t h e y are p r e s e n t w i l l  Consequently, the c o n c e n t r a t i o n s  be i m p o r t a n t t o t h e i n t e g r i t y of t h e l e n s .  in As  p r e v i o u s l y mentioned, t h e r o l e of Zn i n t h e l e n s i s unknown, but i t has t h e p o t e n t i a l t o be i m p o r t a n t i n many f u n c t i o n s , e s p e c i a l l y l e n s growth and t h e maintenance of t h e o p t i m a l c o n f i g u r a t i o n of s o l u b l e l e n s p r o t e i n s  in  (Baldwin  - 10 -  and B e n t l e y 1980). 2.2.6  Lens Metabolism The l e n s d e r i v e s i t s nourishment e n t i r e l y from t h e aqueous humour v i a  t h e s e m i - p e r m e a b i l i t y o f t h e l e n s c a p s u l e and l e n s e p i t h e l i u m .  This  dependence on t h e aqueous humour makes the lens v e r y s u s c e p t i b l e t o a l t e r a t i o n s i n t h e aqueous c o m p o s i t i o n .  Any d i s r u p t i o n of t h e c a p s u l a r  membrane r e s u l t s i n an i n f l u x of f l u i d and t h e l e n s t u r n s opaque  (Miller  1978). The energy needs of t h e l e n s are met c h i e f l y by c a r b o h y d r a t e metabolism which d i f f e r s 2.3  l i t t l e from t h e c a r b o h y d r a t e metabolism of o t h e r t i s s u e s .  CLASSIFICATION OF CATARACTS C l i n i c a l c l a s s i f i c a t i o n of c a t a r a c t s i s based upon t h e appearance of  o p a c i t y ( i e s ) as seen through a s l i t - l a m p b i o m i c r o s c o p e . l o c a t i o n s i n t h e l e n s i n which c a t a r a c t s may o r i g i n a t e : cortical,  l a m e l l a r and n u c l e a r (Jubb and Kennedy 1970).  the  There are f o u r major subcapsular, The s i z e , shape and  l o c a t i o n of t h e o p a c i t y i s determined and an attempt i s then made t o  classify  t h e i n f o r m a t i o n i n t o a broad c a t e g o r y , e i t h e r developmental c a t a r a c t s o r acquired c a t a r a c t s . 2.3.1  Developmental C a t a r a c t s T h i s t y p e of c a t a r a c t i s v e r y common i n a l l animal s p e c i e s and may t a k e  on many f o r m s .  Under t h e s l i t - l a m p beam, t h e l e n s e s of most people w i l l  show  v e r y s m a l l o p a c i t i e s of t h i s t y p e , but t h e y are u s u a l l y too s m a l l t o be significant  ( M i l l e r 1978).  As p r e v i o u s l y mentioned, t h e l e n s i s formed i n l a y e r s w i t h t h e e a r l i e s t f i b r e s f o r m i n g t h e nucleus and t h e l a t e r f i b r e s c o n s t i t u t i n g t h e c o r t e x . With d e v e l o p m e n t a l c a t a r a c t s , u s u a l l y o n l y t h a t p a r t i c u l a r zone which  is  -  being  formed  Therefore, and in  clear. the  2.3.2  lens  at  the If and  Acquired These  the  time  fibres the  when  formed  opacity  have  no  the  -  opacity  before  occurs  1 1  and  early  detrimental  is  caused w i l l  after in  the  life  effects  on  it  be  affected will  affected. site  are  normal  quickly  become  buried  of  fibres  which  vision.  Cataracts  c a t a r a c t s are  are  already  formed.  may  involve  the  the  Unlike  entire  lens.  result  of  the  developmental  degeneration cataracts they  lens  are  progressive  and  - 12 -  2.4  CAUSES OF CATARACTS C a t a r a c t s may be caused by a v a r i e t y of f a c t o r s  classifications  are n u t r i t i o n a l  (Table 1 ) .  Two major  and n o n - n u t r i t i o n a l .  TABLE 1 Nutritional  and n o n - n u t r i t i o n a l  Nutritional Deficiency  causes of c a t a r a c t s  in  animals.1  Non-nutritional Excess  Radi a t i o n e.g. U l t r a v i o l e t  Vitamin D  Xylose  Vitamin E  Galactose  X-rays  Vitamin C  Fructose  Gamma r a y s  Riboflavin  Lactose  Bacterial  Calcium  Calcium  Parasitic  Zinc  Phosphorus  Cold s t r e s s  Selenium  Phytic  Physical  Magnesi um  Tyrosine  acid  light  infection  infection  trauma  Naphthalene  Manganese  Thioacetamide  Iodine  Electricity  Tryptophan  Steroids  Phenylalanine  Hereditary  Hi s t i d i n e  e.g. Alport's  disease syndrome  Methionine  Homocystinuri a  Protein  Fabry's  disease  1 The f o l l o w i n g r e f e r e n c e s were used: M i t c h e l l and Dodge 1935; Darby 1939; H a l l et a l . 1948; A l l i s o n 1962; Lerman 1966; Von Sallman et a l . 1 9 6 6 ; Grant 1974; Dukes 1975; Hockwin and Koch 1975; McLaren and H a l a s a 1975; Whanger and Weswig 1975; Bunce and Hess 1976; Lee et aT. 1976; M i l l e r 1978; K e t o l a 1979; Poston et a l . 1977; Satoh et a l . 1983a,b; R i c h a r d s o n e t a l . 1985.  - 13 -  Of t h e causes l i s t e d i n T a b l e 1, t h e f o l l o w i n g have been r e p o r t e d  in  fish: 1.  poor q u a l i t y p r o t e i n  2.  riboflavin  3.  tryptophan d e f i c i e n c y  4.  methionine  5.  zinc  6.  magnesium d e f i c i e n c y  7.  manganese d e f i c i e n c y  8.  iodine  9.  excess c a l c i u m and phosphorus  deficiency  deficiency  deficiency  deficiency  10.  excess p h y t i c  11.  thioacetamide  12.  p h y s i c a l trauma  13.  parasitic  14.  ultraviolet  acid  infection light  A l t h o u g h T a b l e 1 i n d i c a t e s a number of f a c t o r s which may cause c a t a r a c t s , a b s o l u t e statements cannot be made.  T h i s i s because, f o r t h e most p a r t ,  it  i s not known whether t h e s e f a c t o r s are major i n d u c e r s of c a t a r a c t f o r m a t i o n , o r whether t h e y j u s t seem t o be the most l i k e l y cause based on t h e experimental design.  The most e x t e n s i v e l y s t u d i e d c a t a r a c t s are t h e "sugar  c a t a r a c t s " caused by e x c e s s e s of g a l a c t o s e , x y l o s e and l a c t o s e .  Cataracts  r e s u l t i n g from g a l a c t o s a e m i a may be t h e o n l y ones i n which t h e b i o c h e m i c a l and m e t a b o l i c a l t e r a t i o n s t h a t occur have been e l u c i d a t e d . 2.5  CATARACT FORMATION The purpose of t h i s s e c t i o n i s t o d e s c r i b e t h e v a r i o u s changes which  - 14 -  o c c u r i n the l e n s d u r i n g c a t a r a c t f o r m a t i o n .  As mentioned p r e v i o u s l y , i t  is  p o s s i b l e t o s t a t e what changes o c c u r , but w i t h t h e e x i s t i n g s t a t e of knowledge i t i s not p o s s i b l e t o s t a t e w i t h much c o n v i c t i o n how such changes occur. 2.5.1  Gross Changes  Macroscopically,  a c a t a r a c t o u s l e n s appears w h i t i s h and c l o u d y .  Lenses  w i t h p a r t i a l c a t a r a c t s are g e n e r a l l y c l o u d y i n t h e c e n t r e whereas severe c a t a r a c t s are c h a r a c t e r i z e d by c l o u d i n e s s a l l over t h e l e n s .  Upon c l o s e r  e x a m i n a t i o n , d i s t i n c t c h a r a c t e r i s t i c s can be seen such as t h e ground g l a s s appearance of sugar c a t a r a c t s . 2.5.2  Areas A f f e c t e d There i s c o n s i d e r a b l e v a r i a t i o n i n t h e changes which o c c u r i n t h e l e n s  during cataract formation.  D e f i c i e n c i e s of m e t h i o n i n e , r i b o f l a v i n and Zn i n  s a l m o n i d d i e t s have i n i t i a t e d c a t a r a c t s w i t h d i s t i n c t p a t t e r n s of (Poston and K e t o l a 1981).  development  In m e t h i o n i n e - d e f i c i e n t salmon and t r o u t ,  the  f i r s t n o t i c e a b l e l e n s changes o c c u r r e d i n t h e s u b c a p s u l a r c o r t e x of t h e anterior region.  The o p a c i t i e s p r o g r e s s e d through t h e p e r i n u c l e a r r e g i o n t o  the nucleus i t s e l f .  Changes i n r i b o f l a v i n - d e f i c i e n t rainbow t r o u t began i n  t h e p o s t e r i o r s u b c a p s u l a r c o r t e x and p r o g r e s s e d t o t h e p e r i n u c l e u s and nucleus.  In Z n - d e f i c i e n t t r o u t , the e a r l i e s t changes appeared i n the  perinucleus. 2.5.3  Membrane P e r m e a b i l i t y An i n c r e a s e i n membrane p e r m e a b i l i t y r e s u l t s i n c e l l u l a r s w e l l i n g and i t  has been suggested t h a t a p o s s i b l e u n i v e r s a l cause of c a t a r a c t s i s p e r m e a b i l i t y t o Na (Duncan and Croghan 1 9 6 9 ) .  increased  The l e n s must r e g u l a t e  i n t e r n a l e l e c t r o l y t e balance a g a i n s t s i g n i f i c a n t i o n c o n c e n t r a t i o n  its  - 15 -  gradients.  T h i s i s accomplished by a c a t i o n pump (Na-K-ATPase) which  l o c a t e d p r i m a r i l y i n t h e l e n s e p i t h e l i u m , j u s t beneath t h e a n t e r i o r capsule.  is  lens  Once t h e i n t e g r i t y of t h e l e n s membrane i s d i s t u r b e d , t h e pump  breaks down and Na begins t o accumulate i n t h e l e n s . 2.5.4  Lens P r o t e i n s In t h e ageing l e n s the water c o n t e n t d e c r e a s e s , and t h e most p r o b a b l e  cause of t h i s decrease i s b e l i e v e d t o be r e l a t e d t o t h e changes i n t h e c o n c e n t r a t i o n s of s o l u b l e and i n s o l u b l e l e n s p r o t e i n s  (Lerman 1966).  Most of  t h e d r y weight of the l e n s i s composed of t h e s t r u c t u r a l p r o t e i n s : a l p h a - , b e t a - , and gamma-crystal 1 i n s and a l b u m i n o i d .  S t u d i e s on ageing r a t and  d o g f i s h l e n s e s have shown a sharp decrease i n t h e s y n t h e s i s of s o l u b l e  lens  p r o t e i n s and a steady i n c r e a s e i n a l b u m i n o i d p r o t e i n ( M a l i k e t a l . 1 9 6 9 ) . T h i s i s comparable t o a c a t a r a c t l e n s .  In Z n - d e f i c i e n t l a k e t r o u t and  rainbow t r o u t , both t h e a l p h a - and b e t a - c r y s t a l l i n p r o t e i n s were reduced i n t h e c a t a r a c t l e n s (Barash et a l . 1 9 8 2 ) .  The d e c l i n e i n s t r u c t u r a l  proteins  i s b e l i e v e d t o be a r e s u l t of t h e i r t r a n s f o r m a t i o n i n t o i n s o l u b l e p r o t e i n s . A l t h o u g h l i t t l e i s known about t h i s p r o c e s s , once t h e s t r u c t u r a l  proteins  have been a l t e r e d the r e s u l t i n g c a t a r a c t s are i r r e v e r s i b l e (N. B u s s a n i c h p e r s . comm.). 2.5.5  Lens M i n e r a l s C a l c i u m i s of s p e c i a l i n t e r e s t i n l e n s r e s e a r c h because of  p o s s i b i l i t y of Ca-phosphate p r e c i p i t a t e s c o n t r i b u t i n g t o l e n s (Manery 1961).  the  opacities  S t u d i e s w i t h human l e n s e s have shown t h e c o n c e n t r a t i o n s of Ca  and Zn t o be much h i g h e r i n t h e c a t a r a c t o u s l e n s than i n t h e normal (Murata and Taura 1975).  lens  The c o n c e n t r a t i o n s of Ca and Zn were a l s o h i g h e r  complete c a t a r a c t s v e r s u s p a r t i a l c a t a r a c t s .  L e v e l s of Mg, Fe and Cu were  in  - 16 -  a l s o r e p o r t e d t o be h i g h e r , but 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  noted  between t h e l e v e l s p r e s e n t i n p a r t i a l c a t a r a c t s and t h o s e i n complete cataracts.  The v a l u e s r e p o r t e d by Murata and Taura (1975) were on a wet  weight b a s i s which may e x p l a i n the l a c k of s i g n i f i c a n t i n c r e a s e s i n t h e c o n c e n t r a t i o n s of Mg, Fe and Cu i n t h e c o m p l e t e l y c a t a r a c t o u s l e n s .  Other  s t u d i e s have shown normal human l e n s e s t o c o n t a i n c o n s i d e r a b l e Zn and ageing c a t a r a c t o u s l e n s e s t o c o n t a i n i n c r e a s e d l e v e l s of Fe and Cu but l e s s Zn (Kuck, J r . 1975; Baldwin and B e n t l e y 1980).  significantly  An e i g h t f o l d i n c r e a s e  Ca (on a d r y weight b a s i s ) has been observed i n c a t a r a c t o u s l e n s e s of salmon ( R i c h a r d s o n unpub.) 2.6  in  chinook  S m a l l e r i n c r e a s e s i n Cu, Fe, Zn and Mg were s e e n .  HATCHERY DIETS The o p e r a t i o n and maintenance of a h a t c h e r y i s an e x p e n s i v e  undertaking.  As w i t h o t h e r forms of animal husbandry f e e d c o s t s are a major  i t e m i n t h e h a t c h e r y budget.  F i s h d i e t s have t o be h i g h i n p r o t e i n (40-55%)  and t h e most s u c c e s s f u l commercial d i e t s have been t h o s e based on f i s h m e a l as t h e major p r o t e i n s o u r c e .  There are two major types of f i s h m e a l used i n  d i e t s f o r salmon h a t c h e r i e s .  The f i r s t u t i l i z e s f i l l e t i n g wastes from the  human f o o d f i s h e r y and t h e second i s made from whole f i s h .  In North America  t h e s e two t y p e s are commonly c a l l e d w h i t e f i s h meal (WFM) and h e r r i n g meal (HM), r e s p e c t i v e l y . 2.6.1  W h i t e f i s h Meal W h i t e f i s h meal i s c h a r a c t e r i z e d by a h i g h ash ( 22%) and low o i l  c o n t e n t on a dry weight b a s i s . and hake.  Common t y p e s of w h i t e f i s h are c o d ,  ( 2%)  halibut  The h i g h ash i n the r e s u l t i n g f i s h meal i s not a c h a r a c t e r i s t i c of  the f i s h i t s e l f ,  but of t h e way i t i s p r o c e s s e d .  Because w h i t e f i s h  is  d e s i r e d f o r human consumption, t h e f i l l e t s are removed and what i s l e f t  goes  -17  i n t o t h e manufacture of f i s h m e a l .  -  C o n s e q u e n t l y , the f i s h m e a l  contains  r e l a t i v e l y l a r g e amounts of bone and c a r t i l a g e and t h e r e f o r e , a h i g h ash content.  D i e t s based on WFM have been i m p l i c a t e d i n c a t a r a c t s i n h a t c h e r y  f i s h due t o t h e i r high ash c o n t e n t s 2.6.2  (Ogino and Yang 1978; K e t o l a 1979).  H e r r i n g Meal H e r r i n g meal i s the most common p r o t e i n source used i n commerical  salmonid d i e t s .  Two types of HM are c u r r e n t l y being used i n North A m e r i c a .  These are a West Coast HM and an East Coast HM.  The l e s s abundant West Coast  h e r r i n g i s c o m p l e t e l y rendered down t o f i s h f o o d ( a f t e r t h e removal o f  roe)  and t h e meal c o n t a i n s a p p r o x i m a t e l y 12% ash and 7% l i p i d ( d r y w e i g h t ) .  By  c o n t r a s t , E a s t Coast h e r r i n g may be f i l l e t e d b e f o r e meal p r o d u c t i o n . C o n s e q u e n t l y , t h i s HM may have s i m i l a r ash c o n t e n t t o WFM. 2.6.3  A l t e r n a t i v e P r o t e i n Sources Due t o t h e high c o s t of animal p r o t e i n and t h e f l u c t u a t i n g  of such p r o t e i n , a g r e a t deal of r e s e a r c h i s being done t o f i n d p l a n t p r o t e i n sources ( e . g . soybean and c a n o l a ) f o r f i s h d i e t s .  availability suitable T e s t s by  Poston e t a l . (1977) w i t h i s o l a t e d soy p r o t e i n r e s u l t e d i n an 80-90% i n c i d e n c e of b i l a t e r a l lens c a t a r a c t s i n t r o u t and salmon.  However, t h e same  soy p r o t e i n d i e t supplemented w i t h m e t h i o n i n e s u c c e s s f u l l y prevented c a t a r a c t formation.  Soybean products are u s u a l l y d e f i c i e n t  i n m e t h i o n i n e and such a  d e f i c i e n c y has been shown t o cause c a t a r a c t s . 2.7  CALCIUM AND PHOSPHORUS The r e q u i r e m e n t s f o r Ca and P by s a l m o n i d s are g e n e r a l l y c o n s i d e r e d  t o g e t h e r because t h e i r metabolism i s i n t i m a t e l y c o n n e c t e d . 2.7.1  Requirements and A v a i l a b i l i t y Q u a n t i t a t i v e d i e t a r y r e q u i r e m e n t s f o r Ca and P i n chinook salmon have  not been e s t a b l i s h e d however, t h e Ca t o P r a t i o f o r f i s h i s g e n e r a l l y than one ( B . E . March p e r s . comm.).  less  T h i s r a t i o may d i f f e r depending on t h e  s p e c i e s , the water c h e m i s t r y and t h e a v a i l a b l e P l e v e l i n t h e d i e t . F i s h can absorb Ca v i a t h e g i l l s and f i n s and through t h e d r i n k i n g of water ( L a l l 1979).  Thus t h e d i e t a r y r e q u i r e m e n t s are q u i t e low.  For  example, Rumsey (1977) e s t a b l i s h e d a Ca requirement f o r rainbow t r o u t of  only  0.2% of t h e d i e t i n both s o f t w a t e r (3 mg/L Ca) and hard water (45 mg/L C a ) . In v e r t e b r a t e s , c h o l e c a l c i f e r o l  ( V i t a m i n D 3 ) r e g u l a t e s Ca i n t h e blood and  bone m i n e r a l i z a t i o n by a i d i n g a b s o r p t i o n from t h e gut and by m o b i l i z i n g Ca i o n s from bone ( v i a p a r a t h y r o i d hormone).  In salmonids however, V i t . D3 does  not appear t o be n e c e s s a r y f o r Ca a b s o r p t i o n ( B a r n e t t e t a l . 1 9 7 9 ) . The a v a i l a b l e P r e q u i r e m e n t f o r rainbow t r o u t i s 0.7-0.8% of t h e d i e t (NRC 1981).  Phosphate i s low i n both f r e s h w a t e r and s e a w a t e r , t h e r e f o r e f o o d  c o n s t i t u t e s the main s o u r c e of P.  Phosphorus i s r e a d i l y a v a i l a b l e from  animal sources but i f p l a n t s o u r c e s are used supplements of i n o r g a n i c P are required.  T h i s i s due t o p l a n t - P being t i e d up i n p h y t i c a c i d complexes  which cannot be broken down i n m o n o g a s t r i c s because of the absence, o r i n s u f f i c i e n t q u a n t i t y , of t h e enzyme phytase i n t h e g a s t r o - i n t e s t i n a l  tract.  For example, K e t o l a (1975) f e d A t l a n t i c salmon 0.7% p l a n t - P and determined t h a t a minimum of 0.6% i n o r g a n i c P was a l s o r e q u i r e d t o a c h i e v e adequate f i s h growth. 2.7.2  Properties C a l c i u m and P are e s s e n t i a l t o bone f o r m a t i o n w i t h Ca-phosphate and  C a - c a r b o n a t e f o r m i n g t h e major m i n e r a l c o n s t i t u e n t s of bone.  In mammals t h e  r a t i o of Ca3(P04)2 t o CaC03 i s about 7:1 and i n f i s h i t i s 11:1 ( P r o s s e r and Brown 1950).  T h i s suggests a g r e a t e r demand f o r P by f i s h than by mammaTs.  - 19 -  C a l c i u m i s a l s o r e q u i r e d f o r c a r t i l a g e f o r m a t i o n , o s m o r e g u l a t i o n , muscle c o n t r a c t i o n , b l o o d c l o t t i n g and as a c o f a c t o r i n enzymatic r e a c t i o n s . C a l c i u m d e f i c i e n c y has not been d e f i n e d i n s a l m o n i d s . Phosphorus i s important i n c a r b o h y d r a t e m e t a b o l i s m , f a t m e t a b o l i s m , and i s a c o n s t i t u e n t of p h o s p h o l i p i d s .  Phosphorus d e f i c i e n c y i n salmonids  is  c h a r a c t e r i z e d by reduced growth, poor f o o d c o n v e r s i o n and decreased bone m i n e r a l i z a t i o n (NRC 1981). 2.7.3  Interaction Numerous s t u d i e s have i n v e s t i g a t e d t h e Ca-P r e l a t i o n s h i p i n  animals.  land  R e s u l t s have shown t h a t each m i n e r a l d i r e c t l y i n f l u e n c e s  the  g a s t r o i n t e s t i n a l a b s o r p t i o n , s k e l e t a l u t i l i z a t i o n and e x c r e t i o n of t h e o t h e r ( N i c o l a y s o n e t a l . 1953).  For example, excess Ca i n t h e duodenum combines  w i t h P t o form i n s o l u b l e t r i c a l c i u m phosphate which i n h i b i t s P a b s o r p t i o n (Yano e t a l . 1 9 7 9 ) . always e x c e p t i o n s .  Some g e n e r a l i z a t i o n s can be made a l t h o u g h t h e r e are B r i e f l y , high Ca i n t a k e r e s u l t s i n reduced P a b s o r p t i o n ,  whereas low d i e t a r y Ca s t i m u l a t e s P a b s o r p t i o n (Cohen 1 9 8 0 ) .  Similarly,  P i n t a k e i n c r e a s e s Ca a b s o r p t i o n from t h e small i n t e s t i n e and i s  low  associated  w i t h h y p e r c a l c a e m i a (Hughes e t a l . 1975; Fox and Care 1978; A b d e l - H a f e z 1982). 2.7.4  Mineral  Interactions  C a l c i u m and P are i n v o l v e d i n a number of i n t e r a c t i o n s w i t h o t h e r minerals.  In f i s h t h e most i m p o r t a n t of t h e s e i n t e r a c t i o n s are those  involving  Mg and Z n .  2.7.4.1  Magnesium  S t u d i e s w i t h t e r r e s t r i a l animals have demonstrated t h a t a h i g h Ca:P intake w i l l  aggravate Mg d e f i c i e n c y symptoms ( O ' D e l l 1960; L i k u s k i and Forbes  - 20 -  1965; Woodard and Jee 1984).  M a g n e s i u m - d e f i c i e n t d i e t s c o n t a i n i n g 0.004% Mg  and 2.6% Ca w i t h a Ca:P r a t i o of 1:1, r e s u l t e d i n r e n a l c a l c i n o s i s when f e d t o rainbow t r o u t (Cowey 1 9 7 6 ) .  T h i s d i d not occur i n t r o u t f e d a d i e t  c o n t a i n i n g 1.4% Ca, o r when Mg was i n c r e a s e d t o 0.1% (Cowey e t a l . 1977). 2.7.4.2  Zinc  The i n t e r a c t i o n between Ca and Zn has r e c e i v e d a g r e a t deal attention.  of  Tucker and Salmon (1955) observed a Zn d e f i c i e n c y syndrome  ( p a r a k e r a t o s i s ) i n swine f e d a h i g h Ca d i e t .  T h i s syndrome suggested t h a t  t h e h i g h Ca l e v e l i n t e r f e r e d w i t h Zn a b s o r p t i o n and t h i s r e s u l t e d e x t e n s i v e r e s e a r c h i n t o t h e Ca-Zn r e l a t i o n s h i p .  in  Subsequent experiments  performed by v a r i o u s r e s e a r c h e r s on d i f f e r e n t animals have y i e l d e d s i m i l a r r e s u l t s ( e . g . Leucke e t a l . 1957; Hoefer et a l . 1 9 6 0 ) .  Contrary to t h i s  are  experiments which have shown p u r i f i e d c a s e i n o r egg p r o t e i n d i e t s , w i t h h i g h Ca c o n c e n t r a t i o n s , not t o induce Zn d e f i c i e n c i e s u n l e s s t h e Zn c o n c e n t r a t i o n was i n i t i a l l y l e s s than 10 mg/kg ( D a v i s 1966).  A l s o , some swine s t u d i e s  i n d i c a t e Zn a b s o r p t i o n t o be normal i n s p i t e of p a r a k e r a t o s i s ( W h i t i n g and Bezeau 1958; Forbes  1960).  P o u l t r y s t u d i e s have shown t h a t an i n c r e a s e i n t h e d i e t a r y Ca c o n t e n t causes a decrease i n t h e Zn c o n t e n t of bone, l i v e r and f e a t h e r s a l . 1958).  (Pensack et  Z i n c s u p p l e m e n t a t i o n of t h e b i r d s ' d i e t s i n c r e a s e d t i s s u e Zn  l e v e l s and a s i m i l a r e f f e c t was a c h i e v e d w i t h the combined a d d i t i o n o f Ca and Zn. K e t o l a (1979) demonstrated t h a t WFM-based d i e t s c o n t a i n i n g high Ca and P, r e l a t i v e t o Zn, caused b i l a t e r a l trout f r y .  l e n s c a t a r a c t s and poor growth i n rainbow  But a Zn c o n c e n t r a t i o n of 150 mg/kg i n t h e f i s h m e a l  p r e v e n t e d c a t a r a c t f o r m a t i o n and improved growth.  diets  - 21 -  2.8  ZINC Zinc occurs i n a l l  turnover.  l i v i n g c e l l s and undergoes c o n t i n u a l d e p o s i t i o n and  Bone, by v i r t u e of sheer b u l k , c o n t a i n s a major p o r t i o n of body Zn  (Bergman 1970).  In t h e b l o o d , 75% of the Zn i s found i n t h e e r y t h r o c y t e s , 3%  i n t h e l e u c o c y t e s and 22% i n t h e plasma o r serum.  Zinc i s present i n red  b l o o d c e l l s m a i n l y as c a r b o n i c . a n h y d r a s e and i n w h i t e b l o o d c e l l s as a i  p r o t e i n complex.  In plasma most of t h e Zn i s bound t o a l p h a - g l o b u l i n s .  As  mentioned p r e v i o u s l y , t h e h i g h e s t Zn c o n c e n t r a t i o n i n a normal t i s s u e i s  in  t h e c h o r o i d w i t h c o n c e n t r a t i o n s r a n g i n g from 139 ( c a t t l e ) t o 69,000 mg/kg t i s s u e ( f o x ) on a dry weight b a s i s (Underwood 1971). G e n e r a l l y , Zn l e v e l s i n t h e body are h a l f t h a t of t h e Fe c o n t e n t and 10-15 t i m e s t h a t of t h e Cu c o n t e n t 2.8.1  (Orten 1966).  Requirements and A v a i l a b i l i t y As w i t h most m i n e r a l s , Zn r e q u i r e m e n t s are d i f f i c u l t t o determine  because Zn a v a i l a b i l i t y i s i n f l u e n c e d by o t h e r d i e t a r y i n g r e d i e n t s , i n t e s t i n a l f u n c t i o n and f a c t o r s which i n f l u e n c e m i n e r a l and t i s s u e metabolism.  The d i e t a r y Zn requirement of rainbow t r o u t i s suggested as  b e i n g between 15-30 mg/kg (Ogino and Yang 1978).  However, rainbow t r o u t  fed  WFM d i e t s c o n t a i n i n g 60 mg Zn/kg developed c a t a r a c t s ( K e t o l a 1979) due t o t h e h i g h c o n c e n t r a t i o n of o t h e r m i n e r a l s such as Ca and P.  In a d d i t i o n t o  its  a s s o c i a t i o n w i t h c a t a r a c t s , Zn d e f i c i e n c y i n salmonids has been c h a r a c t e r i z e d by poor growth, h i g h m o r t a l i t y , decreased a p p e t i t e and f o o d c o n v e r s i o n , and e r o s i o n of t h e f i n s and s k i n (Ogino and Yang 1978; K e t o l a 1979; Watanabe e t a l . 1980; Satoh et a l . 1983b).  Because of t h i s danger, i t i s recommended  t h a t commercial f i s h d i e t s f o r salmonids c o n t a i n 150 mg Zn/kg ( K e t o l a 1982). T h i s i s c o n s i d e r a b l y below l e v e l s which might be c o n s i d e r e d t o be harmful as  - 22 -  rainbow t r o u t have been f e d d i e t s c o n t a i n i n g up t o 1700 mg Zn/kg w i t h no effects  (Wekell e t a l . 1 9 8 3 ) .  duodenum.  ill  Z i n c i s absorbed t o a l i m i t e d e x t e n t from the  Less than 10% of t h e d i e t a r y Zn i n t a k e i s absorbed ( D a v i s 1966)  which i s p r o b a b l y why Zn t o x i c i t y i n any animal i s r a r e . Z i n c a b s o r p t i o n may be enhanced by t h e a d d i t i o n of c e r t a i n c h e l a t o r s . For example, s u p p l e m e n t a t i o n of d i e t s w i t h e t h y l e n e d i a m i n e t r a a c e t i c  acid  (EDTA) r e s u l t e d i n i n c r e a s e d Zn a v a i l a b i l i t y i n p o u l t r y ( K r a t z e r e t a l . 1959).  I t was suggested t h a t EDTA combined w i t h , o r i s o l a t e d , t h e Zn and  e i t h e r through a b s o r p t i o n of t h e complex, o r through a change i n  intestinal  pH, a l l o w e d t h e Zn t o be a b s o r b e d . The a b i l i t y o f bone Zn t o be u t i l i z e d d u r i n g Zn d e f i c i e n c y i s a controversial issue.  H u r l e y and Swenerton (1971) argue t h a t bone Zn i s  f i r m l y bound, and t h e r e f o r e u n a v a i l a b l e .  C o n v e r s e l y , o t h e r s r e p o r t bone Zn  t o be a v a i l a b l e , at l e a s t i n growing animals (Harland e t a l . 1975; Brown et a l . 1978).  Murray and Messer (1981) i n v e s t i g a t e d bone Zn t u r n o v e r i n growing  r a t s and they too concluded t h a t bone does not s e r v e as a r e s e r v o i r of a v a i l a b l e Zn.  However, t h e r e was i n c r e a s e d bone Zn d e p o s i t i o n when t h e  a n i m a l s were C a - d e f i c i e n t , s u g g e s t i n g a p a r t i a l s u b s t i t u t i o n of Zn f o r Ca i n bone m i n e r a l . 2.8.2  Properties Z i n c i s a component of a number of metalloenzymes w i t h i n t h e body.  Because of t h i s i t has been suggested t h a t any ion competing w i t h Zn f o r b i n d i n g s i t e s w i t h i n t h e p r o t e i n of t h e enzymes would i n f l u e n c e t h e a v a i l a b i l i t y of t h e Zn t o t h e animal  (Davis 1 9 6 6 ) .  t h e f o l l o w i n g metalloenzymes: 1.  c a r b o n i c anhydrase (0.3% Zn)  Zinc i s associated with  - 23 -  2.  pancreatic carboxypeptidase  3.  l i v e r dehydrogenase  4.  a l k a l i n e phosphatase  5.  t r y p t o p h a n desmolase  6.  m a l i c dehydrogenase  7.  g l u t a m i c dehydrogenase  8.  lactic  9.  retinol  dehydrogenase dehydrogenase  Of t h e above enzymes, c a r b o n i c anhydrase c o n t a i n s t h e h i g h e s t c o n c e n t r a t i o n of Z n .  However, t h e l e n s c o n t a i n s much more c a r b o n i c  than, t h e c h o r o i d , but f a r l e s s Zn ( G a l i n e t a l . 1 9 6 2 ) .  anhydrase  T h i s suggests t h a t  t h e s o u r c e of Zn i n t h e c h o r o i d may be something o t h e r than c a r b o n i c anhydrase. 2.9  PHYTIC ACID An important c o n s i d e r a t i o n i n m i n e r a l b i o a v a i l a b i l i t y s t u d i e s i s t h e  presence of d i e t a r y f a c t o r s which may i n h i b i t a b s o r p t i o n of minerals.  certain  Such a f a c t o r i s p h y t i c a c i d , a w a t e r - s o l u b l e o r g a n i c a c i d of  myo-inositol.  P h y t i c a c i d i s a s t r o n g c h e l a t i n g agent which may b i n d w i t h  p r o t e i n , P and both mono- and d i v a l e n t c a t i o n s ( e s p e c i a l l y Ca and Zn) t o f o r m , at i n t e s t i n a l pH, i n s o l u b l e s a l t complexes c a l l e d p h y t a t e s . 2.9.1  Structure The chemical s t r u c t u r e of p h y t i c a c i d i s s t i l l under d e b a t e , but t h e  most w i d e l y accepted i s t h e Anderson s t r u c t u r e c o n t a i n i n g 12 t i t r a t a b l e hydrogen i o n s as opposed t o 18 i n t h e second most p l a u s i b l e f o r m , t h e Neuberg s t r u c t u r e (Evans et a l . 1 9 8 2 ) .  P r e s e n t l y , t h e proper chemical  designation  f o r p h y t i c a c i d i s m y o - i n o s i t o l 1 , 2 , 3 , 4 , 5 , 6 - h e x a k i s dihydrogen phosphate  (Siy  - 24 -  and T a l b o t  1982).  The s t r u c t u r a l c o n f i g u r a t i o n i s presented i n F i g u r e 3.  F i g . 3. 2.9.2  The s t r u c t u r e of p h y t i c a c i d (Erdman, J r .  1979).  Occurrence P h y t i c a c i d o c c u r s i n a l l foods of p l a n t o r i g i n ( L l o y d e t a l . 1 9 7 8 ) ,  p r i m a r i l y seeds and whole g r a i n s , and i s e s p e c i a l l y h i g h i n o i l s e e d s such as soybean and c o t t o n s e e d , which are commonly i n c o r p o r a t e d i n t o salmonid f o o d s . On a d r y b a s i s , whole o i l s e e a s c o n t a i n a p p r o x i m a t e l y 1.5% p h y t i c a c i d , o v e r 7% has been found i n some p r o t e i n c o n c e n t r a t e s  but  (Erdman, J r . 1979).  l o c a t i o n of p h y t i c a c i d v a r i e s w i t h t h e type of seed or g r a i n .  The  For example,  p h y t i c a c i d i n peanuts, s u n f l o w e r seeds and c o t t o n s e e d i s c o n c e n t r a t e d w i t h i n t h e p r o t e i n body membrane.  In c o n t r a s t , soybean p h y t i c a c i d has no s p e c i f i c  s i t e of accumulation although i t  i s s t i l l concentrated within protein  bodies  (Erdman, J r . 1979). 2.9.3  Properties P h y t i c a c i d i s the main s t o r a g e form of phosphate and i n o s i t o l i n mature  - 25 -  seeds.  In mature c e r e a l g r a i n s , 60-80% of t h e t o t a l P i s c o n t a i n e d w i t h i n  t h e p h y t i c a c i d component, but v a l u e s up t o 99% have been r e p o r t e d f o r whole wheat f l o u r (Nahapetian and Young 1980) and v a l u e s as low as 30.4% f o r n i g e r s e e d (Eklund 1975).  I t i s g e n e r a l l y accepted t h a t p l a n t s u t i l i z e p h y t i c  a c i d as a source of P d u r i n g g e r m i n a t i o n (Erdman, J r . 1979).  This  is  accomplished by p l a n t phytases which h y d r o l y s e p h y t a t e t o p h o s p h o r i c a c i d and inositol.  Chen and Pan (1977) r e p o r t e d a t w o f o l d i n c r e a s e i n p h y t a s e  a c t i v i t y f i v e days a f t e r g e r m i n a t i o n of soybeans and a t h i r t y - s e v e n f o l d i n c r e a s e i n a c t i v i t y f i v e days a f t e r g e r m i n a t i o n of a pea v a r i e t y . P h y t i c a c i d i s a l s o a source of h i g h - e n e r g y phosphoryl g r o u p s , a c e l l w a l l p r e c u r s o r , and i s b e l i e v e d t o a c t as an a n t i o x i d a n t d u r i n g seed s t o r a g e (Graf 1983). 2.10  PHYTATES As p h y t i c a c i d accumulates i n t h e v a r i o u s p l a n t s t o r a g e s i t e s , m i n e r a l s  c h e l a t e t o i t t o form s a l t complexes commonly c a l l e d p h y t a t e s .  Phytate  u s u a l l y o c c u r s as a mixed Ca-Mg-K s a l t c a l l e d p h y t i n (Graf 1 9 8 3 ) . 2.10.1  Structure  A h y p o t h e t i c a l p h y t a t e ' c o n t a i n i n g Ca, Mg and Zn i o n s i s p r e s e n t e d i n F i g u r e 4.  T h i s f i g u r e i l l u s t r a t e s t h a t c a t i o n s may form s t r o n g bonds between  two phosphate groups o r weak bonds w i t h i n a phosphate group.  - 26 -  F i g . 4. 2.10.2  The s t r u c t u r e of a p h y t a t e complex (Erdman, J r .  1979).  Properties  The s t r o n g c h e l a t i n g a b i l i t y of p h y t i c a c i d has i m p l i c a t e d i t numerous m i n e r a l d e f i c i e n c i e s .  in  S t u d i e s have shown s e v e r a l m i n e r a l - p h y t a t e  complexes to be i n s o l u b l e at i n t e s t i n a l pH (5-7) t h e r e b y r e n d e r i n g t h e c h e l a t e d m i n e r a l s u n a v a i l a b l e f o r a b s o r p t i o n u n l e s s phytase i s p r e s e n t  in  s u f f i c i e n t c o n c e n t r a t i o n t o break the p h y t a t e complex.  little  Ruminants have  d i f f i c u l t y i n h y d r o l y z i n g p h y t a t e due to the l a r g e m i c r o b i a l p o p u l a t i o n t h e rumen ( J e n k i n s 1965; Nelson et a l . 1976). l i t t l e or no p h y t a s e .  M o n o g a s t r i c s however, have  Phytase has not been i d e n t i f i e d i n f i s h but i t has  been d e t e c t e d i n t h e i n t e s t i n a l mucosa of dogs ( J e n k i n s and P h i l i p s rodents  in  1960),  (Roberts and Yudkin 1961), p o u l t r y (Nelson et a l . 1971) and i n humans  ( B i t a r and R e i n h o l d 1 9 7 2 ) . The high s t a b i l i t y of m i n e r a l - p h y t a t e complexes at pH 6.5 ( i n d e s c e n d i n g  - 27 -  o r d e r ) has been r e p o r t e d t o be Z n , Cu, n i c k e l al.  ( N i ) , Mg and Ca (Maddaiah e t  1964).  2.10.3  Interactions  At i n t e s t i n a l pH Zn forms t h e most s t a b l e ( i n s o l u b l e ) p h y t a t e complex. C o n s e q u e n t l y , i t has been t h e f o c u s of many p h y t i c a c i d s t u d i e s .  For  example, Oberleas e t a l . (1966) observed poor Zn u t i l i z a t i o n and 40% growth r e d u c t i o n i n r a t s f e d 1% p h y t i c a c i d (as % of t h e d i e t ) and suggested t h a t t h i s was due t o t h e f o r m a t i o n of an i n s o l u b l e Ca-Zn p h y t a t e i n t h e i n t e s t i n a l lumen.  However, poor m i n e r a l u t i l i z a t i o n may not be a r e s u l t o f  phytates  alone.  F a c t o r s such as f i b r e c o n t e n t , p r o t e i n s o u r c e o r a v a i l a b i l i t y ,  i n t e s t i n a l pH, t h e presence of o t h e r m i n e r a l s and method of p r o c e s s i n g may a l s o i m p a i r m i n e r a l a v a i l a b i l i t y ( I s m a i 1 - B e i g i e t a l . 1977; Oberleas and H a r l a n d 1977; Hardie-Muncy and Rasmussen 1979; Nahapetian and Young 1980; Smith and Rotruck 1981). 2.11  FIBRE DIETS - EFFECTS ON MINERAL  AVAILABILITY  The r e s u l t s from numerous s t u d i e s c o n d u c t e d , both i n v i t r o and i n v i v o , t o a s s e s s m i n e r a l a v a i l a b i l i t y from f i b r e d i e t s have been v a r i e d .  Reinhold  e t a l . (1975) performed i n v i t r o s t u d i e s w i t h wheat breads and c o n c l u d e d t h a t p h y t a t e s had l i t t l e e f f e c t upon t h e u t i l i z a t i o n of Ca o r Z n .  In f a c t ,  p h y t a t e removal from bran ( v i a phytase o r a c i d e x t r a c t i o n ) i n c r e a s e d the amount of bound Zn r e p o r t e d l y due t o an i n c r e a s e i n ' f i b r e  concentration.  N e v e r t h e l e s s , they d i d f i n d p h y t a t e t o b i n d Ca and Zn but t h e y p r e d i c t e d t h a t t h i s compound would e v e n t u a l l y be d i g e s t e d , whereas t h e Ca and Zn bound i n t h e f i b r e f r a c t i o n would n o t .  Hence m i n e r a l a b s o r p t i o n would be i m p a i r e d .  S i m i l a r r e s u l t s were o b t a i n e d by I s m a i l - B e i g i e t a l . (1977) who i n v e s t i g a t e d t h e b i n d i n g c a p a b i l i t i e s of Tanok, an unleavened wholemeal wheat  - 28 -  bread c o n t a i n i n g 0.7% p h y t i c a c i d .  Tanok i s a s t a p l e f o o d i n M i d d l e  Eastern  v i l l a g e s whose i n h a b i t a n t s s u f f e r a h i g h i n c i d e n c e of Zn d e f i c i e n c y c h a r a c t e r i z e d by r e t a r d e d growth and hypogonadal dwarfism ( L l o y d et a l . 1978).  M i n e r a l b i n d i n g i s a pH dependent a c t i o n (Thompson and Weber 1979)  and I s m a i 1 - B e i g i ' s group found t h a t at pH 6 . 5 , 54% of t h e Zn was bound i n Tanok v e r s u s 88% which was bound i n a p h y t a t e - f r e e Tanok.  The g r e a t e r  a f f i n i t y f o r Zn by t h e l a t t e r bread was a t t r i b u t e d t o i t s i n c r e a s e d f i b r e c o n t e n t as a r e s u l t o f p h y t a t e r e m o v a l . The e x t e n t t o which d i e t a r y f i b r e i n h i b i t s m i n e r a l i s g e n e r a l l y a f u n c t i o n of t h e chemical s t r u c t u r e of t h e  utilization indigestible  p o l y s a c c h a r i d e , p a r t i c u l a r l y i t s ion-exchange p r o p e r t i e s and t h e degree of m i c r o b i a l d e g r a d a t i o n o c c u r r i n g i n t h e gut (Harmouth-Hoene and S c h e l e n z 1980). In c o n t r a s t t o t h e above e x p e r i m e n t s , t h e r e s u l t s of Davies e t a l . (1977) who performed i n v i v o s t u d i e s w i t h r a t s , i n d i c a t e d p h y t a t e , r a t h e r than f i b r e , t o be t h e major cause of reduced Zn a v a i l a b i l i t y .  Franz e t a l .  (1980) f e d r a t s s e m i - p u r i f i e d d i e t s c o n t a i n i n g e i t h e r c o r n , r i c e , wheat, beans o r w h i t e beans.  lima  With t h e e x c e p t i o n of the legume d i e t s , t h e p h y t i c  a c i d c o n c e n t r a t i o n appeared t o be i n v e r s e l y r e l a t e d t o Zn u t i l i z a t i o n w i t h t h e lowest a v a i l a b i l i t i e s o c c u r r i n g i n t h e whole c o r n and brown r i c e  diets.  They p o s t u l a t e d t h a t Zn i n legumes may e x i s t i n a d i f f e r e n t complex than Zn i n c e r e a l s , t h e r e f o r e i t would not be a f f e c t e d by p h y t i c a c i d i n t h e same way. C h i c k s f e d soybean d i e t s w i t h an a d d i t i o n a l 6% d i e t a r y f i b r e (as wheat b r a n , corn b r a n , soy b r a n , oat h u l l s o r c e l l u l o s e ) e x h i b i t e d no s i g n s o f reduced growth o r m i n e r a l d e f i c i e n c i e s  (Thompson and Weber 1 9 8 1 ) .  However,  - 29 -  c h i c k s f e d d i e t s c o n t a i n i n g 6% r i c e bran had s i g n i f i c a n t l y lower growth r a t e s , f e e d i n t a k e s , t i b i a w e i g h t s , and t i b i a Z n , Fe and Mn c o n c e n t r a t i o n s . A n a l y s i s showed t h a t the r i c e bran d i e t c o n t a i n e d 1.3% p h y t i c a c i d whereas t h e next h i g h e s t c o n c e n t r a t i o n was found i n t h e wheat bran d i e t c o n t a i n e d o n l y 0.42% p h y t i c a c i d .  which  I t was concluded t h a t t h e p h y t i c a c i d had  i n t e r f e r e d with mineral metabolism. i  The f i n d i n g s of van der Aar et a l . (1983) i n d i c a t e d t h a t t h e m i n e r a l s t a t u s i n c h i c k s f e d d i e t a r y f i b r e was dependent upon t h e t y p e , amount and p a r t i c l e s i z e of f i b r e .  The i n c l u s i o n of c o r n , oat o r wheat bran at 4% and  8% of t h e d i e t reduced Zn a b s o r p t i o n but i t c o u l d not be d e t e r m i n e d i f was l a r g e l y due t o p h y t a t e s .  this  In any c a s e , t h e r e appeared t o be some  c o r r e l a t i o n between p h y t i c a c i d and Zn a v a i l a b i l i t y because, o f t h e t h r e e f i b r e s o u r c e s , Zn a b s o r p t i o n was l e a s t a f f e c t e d by corn which c o n t a i n e d 0.24% phytic acid.  By c o n t r a s t , Zn a b s o r p t i o n was most a f f e c t e d by t h e wheat bran  which c o n t a i n e d 4.6% p h y t i c  acid.  A more r e c e n t study (Navert et a l . 1985) i n v o l v e d t h e f e e d i n g of leavened wheat bran t o human v o l u n t e e r s .  P h y t i c a c i d c o n t e n t was reduced by  v a r y i n g t h e l e a v e n i n g t i m e from 0-120 h o u r s .  The r e s u l t s showed t h a t Zn  absorption increased with fermentation time i . e .  reduced p h y t i c a c i d c o n t e n t  was a s s o c i a t e d w i t h enhanced Zn a b s o r p t i o n . 2.12  CALCIUM-ZINC-PHYTATES R e c e n t l y , t h e r e has been emphasis p l a c e d on employing p h y t a t e t o Zn  molar r a t i o s as d e t e r m i n a n t s o f Zn s t a t u s (Franz e t a l . 1980; Lo et a l . 1981; House e t a l . 1982; Forbes e t a l . 1983; Meyer e t a l . 1983).  However, t h e  l i t e r a t u r e suggests t h a t a more u s e f u l determinant, may a l s o i n c l u d e t h e l e v e l o f d i e t a r y Ca.  - 30 -  2.12.1  Swine S t u d i e s  P a r a k e r a t o s i s , o r Zn d e f i c i e n c y syndrome, has been induced i n swine (Oberleas e t a l . 1962) by t h e a d d i t i o n of 0.7% p h y t i c a c i d t o a c a s e i n d i e t . Z i n c d e f i c i e n c y has a l s o been observed i n swine f e d a soybean d i e t 0.5% p h y t i c a c i d . deficiency.  containing  I n c r e a s i n g t h e Ca c o n t e n t from 0.8 to 1.5% aggravated t h e  The s k i n l e s i o n s c h a r a c t e r i s t i c of Zn d e f i c i e n c y were r e v e r s e d  when t h e d i e t s were supplemented w i t h Z n . 2.12.2  Rat S t u d i e s  Forbes (1964) observed reduced growth and femur Zn c o n c e n t r a t i o n i n r a t s f e d a soy p r o t e i n d i e t and t h e s e e f f e c t s became more marked as t h e d i e t a r y Ca c o n c e n t r a t i o n was i n c r e a s e d .  P h y t i c a c i d c o n c e n t r a t i o n was not determined  but Forbes suggested t h a t t h e e f f e c t of Ca on Zn u t i l i z a t i o n may have been mediated by t h e presence of p h y t i c a c i d .  L i k u s k i and Forbes (1965) used a  f a c t o r i a l experiment i n which r a t s were f e d c a s e i n d i e t s c o n t a i n i n g (on a dry b a s i s ) 0 . 4 , 0.8 and 1.2% Ca; 12 and 65 mg Z n / k g ; and 0 . 0 , 0.4 and 2% p h y t i c acid.  The a d d i t i o n of 0.4% p h y t i c a c i d d i d not a f f e c t weight g a i n , but the  a d d i t i o n of 2% p h y t i c a c i d s i g n i f i c a n t l y reduced d a i l y weight g a i n by 45% when t h e Zn l e v e l was 12 mg/kg and Ca was 0.4%.  Weight g a i n was f u r t h e r  reduced by 76% and 79% when d i e t a r y Ca was i n c r e a s e d t o 0.8% and 1.2%, respectively.  O b e r l e a s et a l . (1966) concluded t h a t N a - p h y t a t e , added t o  d i e t s t o o b t a i n 1% p h y t i c a c i d , i n h i b i t e d Zn a b s o r p t i o n i n r a t s and reduced growth by 40% compared t o r a t s f e d n o n - p h y t a t e d i e t s .  T h i s growth  was f u r t h e r aggravated by i n c r e a s i n g d i e t a r y Ca from 0.8% t o 1.6%.  inhibition These  e f f e c t s were e l i m i n a t e d when t h e d i e t a r y Zn l e v e l was i n c r e a s e d t o 55 mg from 6-8 mg/kg.  S i m i l a r r e s u l t s were o b t a i n e d by Davies and N i g h t i n g a l e  (1975).  For example, r a t s f e d p u r i f i e d d i e t s supplemented w i t h 15 mg Z n / k g , 1% p h y t i c  a c i d and 1.3% C a , had s i g n i f i c a n t l y lower weight g a i n s (84% r e d u c t i o n ) the non-phytate fed r a t s .  than  F u r t h e r m o r e , t h e r e were s i g n i f i c a n t decreases  c a r c a s s r e t e n t i o n of Fe, Cu, Zn and Mn.  in  Moreover, when t h e Zn supplement was  reduced t o 0.5 mg/kg, t h e s e v e r i t y of t h e r e s u l t s i n c r e a s e d .  In a d d i t i o n t o  n e g a t i v e e f f e c t s on growth, i t was concluded t h a t t h e p h y t i c a c i d i m p a i r e d both t h e a b s o r p t i o n of d i e t a r y Zn and t h e r e a b s o r p t i o n of endogenously-secreted Zn. 2.12.3  Poultry Studies  Reduced Zn b i o a v a i l a b i l i t y has been demonstrated i n c h i c k s  fed  corn-soybean d i e t s and t h i s has been a t t r i b u t e d t o t h e p h y t i c a c i d c o n t e n t of t h e d i e t s ( O ' D e l l and Savage 1960).  Chicks fed c a s e i n - g e l a t i n d i e t s with  0.5% p h y t a t e and 1.2% Ca e x h i b i t e d s i g n i f i c a n t growth r e d u c t i o n s when t h e d i e t s were m a r g i n a l i n Zn ( O ' D e l l e t a l . 1964).  I n c r e a s i n g Ca t o 2.4% o f  the  d i e t had no e f f e c t on growth o r Zn a b s o r p t i o n when p h y t i c a c i d was a b s e n t . Bafundo e t a l . (1984) f e d c h i c k s an adequate corn-soybean meal d i e t Ca supplements of 0.91% and 1.82%.  with  C h i c k s f e d d i e t s w i t h 1.82% Ca showed  reduced growth and food c o n v e r s i o n , and depressed l e v e l s of t i s s u e and plasma Zn.  The a d d i t i o n of 1.2% Na-phytate y i e l d e d s i m i l a r r e s u l t s , but o n l y i n the  presence of excess Ca and t h e absence of supplemental Z n .  The f i n d i n g s  of  O ' D e l l e t a l . (1964) showed t h a t Zn a v a i l a b i l i t y t o c h i c k s was reduced o n l y i n t h e presence of p h y t i c a c i d and t h a t d i e t a r y Ca e x a c e r b a t e d t h e 2.13  effect.  THE EFFECT OF GUT pH ON PHYTATE ACTIVITY Most s o l u b i l i t y s t u d i e s have been conducted i n v i t r o and t h e r e s u l t s  g e n e r a l l y i n d i c a t e t h a t p h y t a t e s are most s o l u b l e at g a s t r i c pH (3-5) and l e a s t s o l u b l e at small i n t e s t i n a l pH ( 5 - 7 ) .  The n u t r i t i o n a l a c t i v i t y of  p h y t i c a c i d i s a l s o dependent upon t h e presence of c a t i o n s .  Oberleas et a l .  - 32 -  (1966) found Z n - p h y t a t e s t o be h i g h l y i n s o l u b l e at small i n t e s t i n a l pH and t h i s e f f e c t was aggravated by t h e a d d i t i o n of Ca.  Using a 1:1:1  C a : Z n : p h y t a t e complex, 77% of t h e Zn was p r e c i p i t a t e d .  Increasing the r a t i o  t o 2:1:1 r e s u l t e d i n p r e c i p i t a t i o n of 97% of t h e Zn and 84% of t h e C a .  The  absence o r low c o n c e n t r a t i o n of Ca reduced t h e a f f i n i t y of p h y t i c a c i d f o r Z n , c o n s e q u e n t l y r e n d e r i n g Zn more a v a i l a b l e f o r a b s o r p t i o n .  Therefore,  it  appears t h a t t h e maximum b i n d i n g of Zn t o p h y t i c a c i d r e q u i r e s Ca and an optimum pH (Oberleas and Harland 1977). 2.14  PHYTIC ACID STUDIES WITH SALMONIDS L i t t l e r e s e a r c h has been done t o determine t h e e f f e c t s of  p h y t i c a c i d i n f i s h as compared t o o t h e r a n i m a l s .  dietary  S p i n e l 1i e t a l . (1979) f e d  rainbow t r o u t OMP t h a t had t h e p h y t a t e s removed from t h e soybean p o r t i o n of t h e d i e t and t h e y found t h a t growth performance was s i g n i f i c a n t l y i m p r o v e d . F u r t h e r experiments by S p i n e l l i e t a l . (1983) were conducted t o more a c c u r a t e l y assess t h e e f f e c t s of p h y t a t e s on growth and f o o d c o n v e r s i o n rainbow t r o u t .  The f i s h were f e d a c a s e i n - g e l a t i n d i e t c o n t a i n i n g 0.5% of  e i t h e r Na- or C a - p h y t a t e and i n c r e a s i n g l e v e l s of Ca (0.92 t o 1.2%). l e v e l i n the d i e t was kept c o n s t a n t at 54 mg/kg. p h y t a t e reduced weight g a i n by 10%. the response.  in  The Zn  The a d d i t i o n of 0.5%  I n c r e a s e d l e v e l s of Ca d i d not aggravate  The added p h y t a t e s d i d not s i g n i f i c a n t l y a f f e c t Fe and Zn  l e v e l s i n t h e b l o o d , l i v e r or k i d n e y .  However, b l o o d Cu i n c r e a s e d w i t h t h e .  a d d i t i o n of p h y t i c a c i d but then decreased when t h e d i e t s were supplemented w i t h Ca or Mg.  In v i t r o t e s t s showed t h a t t h e c a s e i n - p h y t a t e complex was  p o o r l y h y d r o l y z e d by p e p s i n .  Subsequent i n v i v o t e s t s , u s i n g a  c a s e i n - p h y t a t e complex ( v s . c a s e i n a l o n e ) , r e v e a l e d t h a t d i e t was reduced by 6.6%.  digestibility  T h e r e f o r e , t h e y concluded t h a t t h e reduced growth was  - 33 -  due t o decreased p r o t e i n a v a i l a b i l i t y r a t h e r than t o decreased m i n e r a l availability.  However, i t i s c o n c e i v a b l e t h a t the low l e v e l o f p h y t i c  employed by S p i n e l l i  acid  ( r e l a t i v e t o t h e c o n c e n t r a t i o n s used i n s t u d i e s on o t h e r  a n i m a l s ) was i n s u f f i c i e n t t o a f f e c t growth and m i n e r a l u t i l i z a t i o n  especially  when t h e d i e t a r y Zn l e v e l was two t o t h r e e t i m e s h i g h e r than t h e known requirement of salmonids. 2.15  -  GROWTH IN SALMONIDS The l i f e c y c l e of the chinook salmon may be d i v i d e d i n t o s i x g e n e r a l  s t a g e s : eggs, a l e v i n s , f r y , s m o l t s , m a t u r i n g and mature a d u l t s .  Given a  water t e m p e r a t u r e of 10-11°C, chinook eggs hatch 48-54 days a f t e r fertilization.  The newly hatched f i s h are r e f e r r e d t o as a l e v i n s and t h e y  p o s s e s s y o l k sacs from which t h e y d e r i v e nourishment f o r another 42-46 d a y s . A f t e r t h e y o l k sacs have been absorbed t h e f i s h begin t o a c t i v e l y seek f o o d . At t h i s s t a g e t h e y are commonly c a l l e d swim-up f r y .  A p p r o x i m a t e l y 90 days  a f t e r swim-up, t h e f r y e n t e r t h e " s i l v e r i n g " o r s m o l t i n g stage and are now ready t o m i g r a t e t o t h e sea as s m o l t s . are n o r m a l l y r e l e a s e d .  At t h i s s t a g e , h a t c h e r y - r e a r e d salmon  Some chinook salmon remain i n f r e s h w a t e r f o r  1-2  y e a r s b e f o r e heading downstream, but most m i g r a t e a few months a f t e r swim-up.  Chinook salmon may spend up t o 8 y e a r s m a t u r i n g at sea but t h e  m a j o r i t y r e t u r n t o spawn a f t e r 4-5 y e a r s (Hart 1973). Growth of h a t c h e r y f i s h i s dependent upon s e v e r a l f a c t o r s .  The most  i m p o r t a n t are f o o d q u a l i t y , p a r t i c l e s i z e and a v a i l a b i l i t y , f i s h s i z e and water t e m p e r a t u r e ( S t a u f f e r 1973).  F i g u r e 5 d e p i c t s t h e growth c u r v e f o r a  f i s h under c o n s t a n t environmental c o n d i t i o n s from 0-500 days post swim-up. T h i s t h e s i s i s concerned w i t h t h e p e r i o d r a n g i n g from 0-150 d a y s .  As  t h i s r e p r e s e n t s a v e r y small p o r t i o n i n t h e growth c u r v e , the d a t a have been  - 34 -  transformed to a log s c a l e .  The s o l i d l i n e s show e x p e r i m e n t a l l y observed  growth stages and t h e broken l i n e s show what the growth phase would look if  a g i v e n growth r a t e p e r s i s t e d .  like  The most r a p i d stage of growth o c c u r s at  t h e y o u n g e s t , s m a l l e s t stage ( B r e t t 1979) w i t h t h e r a t e d e c l i n i n g s h a r p l y at t h e onset of m a t u r i t y .  - 35 -  FISH G R O W T H 1000  -  100  2 0 0 DAYS  Fig. 5.  CURVE  POST  300  400  500  SWIM-UP  Growth curve of fish under constant environmental conditions.  - 36 -  CHAPTER 3  3.0  GENERAL MATERIALS AND METHODS The f o l l o w i n g s e c t i o n s d e s c r i b e m a t e r i a l s and methods which were common  t o t h e t h r e e experiments conducted i n t h i s 3.1  Experimental  study.  Fish  Chinook salmon f r y were o b t a i n e d from B i g Qualicum h a t c h e r y (Vancouver I s l a n d , B.C.) broodstock i n February 1982 and February 1983.  The f i s h were  s e l e c t e d f o r u n i f o r m s i z e and were d i s t r i b u t e d randomly i n t o 29-L  (experiment  I I ) and 150-L t a n k s (experiments I and I I I ) . 3.2  Culture Conditions The f i s h were m a i n t a i n e d i n an i n d o o r f a c i l i t y c o n t a i n i n g two rows of  tanks.  A c o n t i n u o u s s u p p l y o f a e r a t e d w e l l water (4-6 L/min) m a i n t a i n e d t h e  water t e m p e r a t u r e at 10-11°C. mg/L were as f o l l o w s :  Some c h e m i c a l parameters of t h e w e l l water  CaC03 (hardness) 3 6 . 8 - 3 8 . 1 ; Ca 1 1 . 9 - 1 2 . 3 ; Cu <0.005;  Mg 1.7; Na 1 0 . 1 - 1 1 . 6 ; l e a d <0.002; Zn <0.002. 6.6-6.7.  The pH of t h e water was  Throughout the s t u d y , overhead d a y l i g h t f l u o r e s c e n t  ( V i t a l i t e , D u r o t e s t 40W) p r o v i d e d a n a t u r a l 3.3  in  lights  photoperiod.  Diet Formulations The d i e t s were c a s e i n - g e l a t i n based w i t h v a r y i n g l e v e l s of C a , Zn and  p h y t i c a c i d , as sodium p h y t a t e .  A l t h o u g h t h e Na-phytate c o n t a i n e d 22% Na,  d i e t a r y l e v e l s of Na d i d not exceed t h o s e found n o r m a l l y i n OMP, a p r i n c i p a l commerical h a t c h e r y d i e t (Higgs et a l . 1982a).  Thus, t h e d i f f e r e n c e s  d i e t s i n Na c o n t e n t was not c o n s i d e r e d t o be a confounding f a c t o r .  between  Dietary  l e v e l s o f C a , Zn and p h y t i c a c i d were s e l e c t e d t o b r a c k e t t h e range t h a t c o u l d a c t u a l l y o r p o t e n t i a l l y be p r e s e n t i n p r a c t i c a l salmonid d i e t s  - 37 -  depending on t h e e x t e n t o f i n c o r p o r a t i o n o f v a r i o u s types o f animal and p l a n t p r o t e i n s and t h e c o m p o s i t i o n o f t h e m i n e r a l supplement.  D i e t s were  f o r m u l a t e d t o have a Ca t o P r a t i o o f c l o s e t o u n i t y , independent o f t h e phytate-P c o n t r i b u t i o n . 3a.  D i e t a r y f o r m u l a t i o n s a r e l i s t e d i n Tables 2, 3 and  P r o x i m a t e c o m p o s i t i o n and d i e t a r y m i n e r a l l e v e l s a r e l i s t e d i n T a b l e s 4  and 4 a . 3.4  Diet  Preparation  D i e t s were prepared i n May 1982 (experiment I ) and February 1983 (experiments I I and I I I ) . hours i n a b a l l m i l l Ohio).  ( N o r t o n , Chemical P r o c e s s P r o d u c t s D i v i s i o n , A k r o n ,  The m i n e r a l - p h y t a t e mixes were homogenized by v i g o r o u s s h a k i n g i n  p l a s t i c bags. bags.  The b a s a l m i n e r a l supplement was mixed f o r 24  They were then added t o t h e b a s a l m i n e r a l mix and reshaken i n  V i t a m i n s were combined f o r 60 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 C o . , D i v i s i o n o f Harsco C o r p . , P e n n s y l v a n i a ) .  Ingredients  f o r t h e b a s a l d i e t s were mixed t o g e t h e r ( 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 mixer (Hobart M a n u f a c t u r i n g C o . , T r o y ,  Ohio)  f o r experiment I and f o r 30 minutes i n a Marion mixer (Rapids Machinery C o . , Iowa) f o r experiments I I and I I I .  V i t a m i n and m i n e r a l p r e - m i x e s were added  t o t h e basal d i e t s and combined i n a Hobart mixer f o r 20 m i n u t e s . was then c o l d - p e l l e t e d i n a CL-type 2 l a b o r a t o r y p e l l e t m i l l  Each d i e t  (California  P e l l e t M i l l C o . , 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 1.59 mm d i e .  After  c o o l i n g , t h e p e l l e t s were crumbled and hand-screened t o a p p r o p r i a t e s i z e d crumbles.  The remainder o f t h e h e r r i n g o i l was sprayed on t o t h e d i e t s  a .hand-held s y r i n g e (needle s i z e 18) and mixed i n w i t h a spoon. were s t o r e d at -40°C u n t i l r e q u i r e d .  Stock d i e t s  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 r e f r i g e r a t e d n i g h t l y at 3-4°C.  using  - 38 -  TABLE 2 Formulation of basal d i e t fed to j u v e n i l e chinook salmon in a l l experiments. Concentration Ingredient*  (g/kg dry d i e t )  Casein (92.43% crude protein)  476.23  G e l a t i n (99.65% crude-protein)  50.00  Dextrin  20.31  Herring o i l - s t a b i l i z e d  2  120.00  Vitamin supplement^  20.00  Mineral supplement^  20.00  Choline chloride (60%)  8.33  Ascorbic acid  1.00  Amino acid mixS Mineral-phytate + a - c e l l u l o s e mix^ 1 D i e t a r y ingredients were commercially o b t a i n e d .  29.35 264.29 The vitamin-free c a s e i n ,  g e l a t i n , d e x t r i n and a - c e l l u l o s e were from IC.N N u t r i t i o n a l Biochemicals, C l e v e l a n d , Ohio.  Sodium phytate was obtained from Sigma Chemical C o . , S t . L o u i s ,  M i s s o u r i , U.S.A. 2  S t a b i l i z e d with 0.33% BHA-BHT (1:1).  3 The vitamin supplement provided the f o l l o w i n g amounts per kg of dry d i e t : DL-ct -tocopherol 300 IU; thiamin mononitrate 50 mg; r i b o f l a v i n 200 mg; n i a c i n 500 mg; b i o t i n 5 mg; 0-calcium pantothenate 300 mg; pyridoxine-HCl 50 mg; f o l i c a c i d 15 mg; menadione (as hetrazeen) 39.6 mg; vitamin B-12 0.2 mg; i n o s i t o l 2000 mg; p-aminobenzoic acid 400 mg; r e t i n o l acetate 5000 I U ; c h o l e c a l c i f e r o l 2400 IU (1500 i n expt. I ) ; BHT 22 mg. 4 The mineral supplement provided the f o l l o w i n g (mg/kg of dry d i e t ) : c i t r i c  acid  9 0 . 3 ; copper (as CU2C5H4O7.2 1/2 H2O) 6.5; i r o n (as FeCsH507.5H20) 50; manganese (as MnS04.H20) 74.8; iodine (as KI) 4 . 6 ; potassium (as K2SO4) 2675; sodium c h l o r i d e 1594; cobalt (as CoCl2.6H20) 3.6; aluminum (as A1C13.6H20) 0.67; sodium f l u o r i d e 0.08; magnesium (as MgS04.7H20) 603. 5 Amino a c i d mix provided (g/kg dry d i e t ) : L-arginine-HCl 7.0; DL-methionine 2.85; carboxymethylcellulose (CMC) 14; a - c e l l u l o s e 5 (10 in expt. I - t o t a l amino a c i d mix 33.85). 6  Total concentration was 250.29 g/kg i n e x p t . I .  mineral-phytate mixes are presented i n Table 3.  The composition of the  TABLE 3 C o m p o s i t i o n o f t h e n i n e m i n e r a l - p h y t a t e supplements f e d t o j u v e n i l e chinook salmon i n Experiment I .  D I E T Ingredient  1  2  3  4  5  6  7  8  9  9.52  9.52  9.52  9.52  67.30  19.24  111.76  31.94  7.84  7.84  g/kg d r y d i e t Calcium carbonate (CaC0 )  9.52  3  9.52  9.52  9.52  9.52  C a l c i u m phosphate monobasic (CaH4(P04) .H 0) 2  2  .  .  -  •  67.30  -  67.30  -  67.30  111.76  -  111.76  -  C a l c i u m phosphate dibasic (CaHP0 .2H20)  111.76  4  -  '•  Sodium phosphate monobasic (NaH2P04.H 0) 2  Phytic  7.84  7.84  7.84  7.84  2.5  2.5  2.5  2.5  0.088  0.088  1.41  1.41  229.02  49.96  7.844  7.84  7.84  acid  sodium s a l t (64.6% p h y t i c Zinc  acid)  40.0  40.0  40.0  40.0  10.0  sulphate  (ZnS0 .7H 0) 4  a-cellulose  2  230.34  51.28  0,088 192.84  0.088 13.78  •1.41  1.41  191.52  12.46  0.352 171.40  TABLE 3a C o m p o s i t i o n of t h e e i g h t e e n m i n e r a l - p h y t a t e (A, B and C) and I I I  supplements f e d t o j u v e n i l e chinook  salmon i n Experiments  II  (1-15).  D I E T Ingredient  A  B  C  1  2 g/kg d r y  Calcium  3  4  5  6  diet  carbonate  (CaC0 ) 3  Calcium  12.09  12.09  12.09  12.09  12.09  12.09  12.09  12.09  12.09  -  73.27  73.27  6.48  66.79  6.48  66.79  6.48  66.79  -  121.72  121.72  10,77  110.95  10.77  110.95  .10.77  110.95  6.86  6.86  6.86  6.86  6.86  6.86  6.86  6.86  3.27  3.27  3.27  3.27  32.73  32.73  0.106  0.106  1.073  1.073  phosphate  monobasic (CaH4(P0 ) .H 0) 4  Calcium  2  2  phosphate  dibasic (CaHP04.2H20) Sodium  phosphate  monobasic 15.77  (NaH P0 .H 0) 2  Phytic  4  2  acid  sodium  salt  (64.6% p h y t i c Zinc  acid)  2.5  2.5  0.106  0.106  40.0  sulphate  (ZnS0 .7H 0) 4  a-cellulose  2  223.82  37.74  0.106 0.244  214.71  54:22  213.75  53.26  0.106 185.25  0.106 24.76  TABLE 3a c o n t ' d . . . . .  -•'  • 7  Ingredient  D I  g/kg d r y Calcium  carbonate  (CaC0 ) 3  Calcium  12  11  10  8  ET 13  14  15  diet  ..,  12.09  12.09  12.09  12.09  12.09  12.09  12.09  12.09  12.09  6.48  66.79  -  73.27  36.64  36.64  36.64  36.64  36.64  10.77  110.95  -  121.72  60.86  60.86  60.86  60.86  60.86  6.86  6.86  6.86  6.86  6.86  6.86  phosphate  monobasic (CaH4(P04) .H 0) 2  Calcium  2  phosphate  dibasic (CaHP04.2H 0) 2  Sodium  phosphate  monobasic (NaH P0 .H 0) 2  Phytic  4  :  2  6.86  15.77  32.73  32.73  18.0  acid  sodium s a l t  ,  (64.6% p h y t i c Zinc  .6.86  acid)  18.0  18.0  18.0  0.10.  35.9  18.0  sulphate  (ZnS0 .7H 0) 4  a-cellulose  2  1.073 184.29  1.073 23.80  0.589 207.84  1.178  0.589 21.76  119.84  118.66  0.589 137.15  0.589 101.35  0.589 119.25  V  TABLE 4 Proximate composition, mineral and phytic acid content of the nine test diets fed to juvenile chinook salmon In Experiment  I. DIET 1  2  3  4  Protein (N x 6.25)  539  520  536  532  Crude lipid (Bllgh-Dyer)  128  131  131  130  36  194  35  104  96  103  6  7  8  9  542  531  526  537  521  111  128  109  108  107  193  62  225  62  226  92  102  97  100  96  94  52.6  17.7  57.5  19.3  Proximate composition  Ash Moisture (as fed)  5 g/kg dry diet  v  86  Mineral content* Calcium (Ca)  4.37  49.1  4.52  47.8  Phosphorus (P)  5.92  46.2  6.01  45.3  Magnesium (Mg)  0.65  0.68  0.65  0.71  0.76  0.77  0.69  0.77  0.65  Copper (Cu)  0.010  0.008  0.009  0.008  0.020  0.013  0.010  0.010  0.009 0.091  5.14 14.7  51.3 55.9  5.29 14.6  Iron (Fe)  0.113  0.107  0.098  0.102  0.107  0.096  0.118  0.088  Manganese (Mn)  0.072  0.067  0.077  0.064  0.085  0.076  0.070  0.081  0.074  Zinc (Zn)  0.054  0.050  0.354  0.403  0.055  0.051  0.396  0.391  0.152  Cobalt (Co)  0.003  0.003  0.004  0.004  0.004  0.002  0.004  0.003  0.003  Sodium (Na)  3.94  3.90  4.14  3.90  Ca:P2  0.74  1.06  0.75  1.06  Ca:Mg  6.7  P:Mg Ca:Zn Phytic add  72.2  7.0  15.1 0.35  67.3  6.8  13.9 0.92  15.2 0.36  66.6  7.7  14.8 0.91 68.3  5.66 0.92 27.4  9.1  67.9  9.2  63.8  19.3  72.6  21.2  74.7  29.8  80.9  982.0  12.8  118.6 :  93.4  1005.8  13.4  134.5  116.4  25.8  25.8  25.8  25.8  1.62  1.62  1.62  1.62  :  6.46  1 Determined by plasma spectroscopy (Hlggs et a l . 1982). 2  Diets were formulated to have a calcium to phosphorus ratio of close to unity when considering phosphorus  sources other than sodium phytate.  v  ,  TABLE 4a Proximate composition, mineral and phytic acid content of the eighteen test diets fed to Juvenile chinook salmon in Experiments II  (A, B and C) and III  (1-15). D I E T  A  B  C  1  Protein (N x 6.25)  512  520  512  518  Crude lipid (Bllgh-Dyer)  110  116  102  121  47  222  260  3  4  5  6  514  506  509  509  514  116  114  118  117  116  58  178  60  177  83  110  105  110  . 226  102  99  101  100  106  47.1  10.2  50.0  44.4  16.2  50.7  g/kg dry diet  Proximate composition  Ash Moisture (as fed)  2  105;  Mineral content^ Calcium (Ca)  5.48  73.1  66.5  9.63  46.0  Phosphorus (P)  7.34  51.1  64.9  9.84  44.4  9.94 10.6  Magnesium (Mg)  1.11  1.06  1.02  0.84  0.90  0.78  0.96  0.74  1.08  Copper (Cu)  0.009  0.008  0.009  0.008  0.008  0.008  0.008  0.008  0.009 0.130  Iron (Fe)  0.085  0.085  0.081  0.090  0.105  0.090  0.109  0.094  Manganese (Mn)  0.099  0.096  0.094  0.076  0.093  0.085  0.086  0.071  0.067  Zinc (Zn)  0.055  0.058  0.061  0.056  0.081  0.271  0.224  0.052  0.056  Cobalt (Co)  0.004  0.004  0.003  0.005  0.004  0.004  0.004  Sodium (Na)  5.34  4.10  3.83  4.60  4.46  3.99  Ca:P2  0.75  Ca:Mg  4.94  P:Mg Ca:Zn Phytic acid  6.61 99.6 1.62  1.43 69.0  14.3 1.02 65.2  0.98 11.5  1.04  0.94  51.1  12.7  1.06 49.1  0.005 12.6 0.63 13.8  0.005 12.5 0.99 46.3  48.2  63.6  11.1  49.3  13.6  46.2  21.9  46.9  1260.0  1090.0  171.9  567.9  36.7  210.3  196.1  892.9  21.1  21.1  1.62  25.8  2.10  2.10  2.10  2.10  1 Determined by plasma spectroscopy (Higgs et a l . 1982). 2  Oiets were formulated to have a calcium to phosphorus ratio of close to unity when considering phosphorus  sources other than sodium phytate.  TABLE 4a cont'd.  DIET 7  8  9  10  11  12  13  14  15  509  g/kg dry diet  Proximate composition Protein (N x 6.25)  498  509  506  503  500  495  508  503  Crude lipid (Bligh-Dyer)  117  103  104  112  99  98  102  106  115  Ash  80  224  61  259  144  128  108  198  136  Moisture (as fed)  98  107  97  104  100  105  98  96  100  9.80  48.6  5.60  52.1  25.2  29.1  28.3  29.1  29.1  52.6  31.1  30.1  26.4  34.8  30.3  Mineral content* Calcium (Ca) Phosphorus (P)  15.8  51.0  11.0  Magnesium (Mg)  0.73  0.89  0.78  0.97  0.71  0.76  0.77  0.84  0.85  Copper (Cu)  0.008  0.008  0.008  0.008  0.008  0.008  0.009  0.008  0.009  Iron (Fe)  0.081  0.108  0.077  0.119  0.094  0.091  0.110  0.091  .096  Manganese (Mn)  0.094  0.074  0.078  0.076  0.091  0.076  0.092  0.076  0.077  Zinc (Zn)  0.269  0.316  0.149  0.157  0.034  0.279  0.150  0.100  0.169  Cobalt (Co)  0.004  0.004  0.004  0.004  0.004  0.005  0.005  0.004  0.004  9.64  7.96  8.15  3.32  Sodium (Na) Ca:P2  12.4 0.62  11.7 0.95  0.99  0.81  0.97  1.07  13.8 0.85  8.25 0.96  53.7  35.5  38.3  36.7  35.1  34.2  14.1  34.2  43.8  39.6  39.3  41.4  35.6  153.8  37.6  331.8  • 741.2  104.3  188.7  173.5  172.2  21.1  11.6  11.6  11.6  11.6  23.2  11.6  Ca:Mg  13.4  54.6  P:Mg  21.6  57.3  Ca:Zn  36.4 21.1  Phytic acid  0.51  13.2  7.20  0.06  * Determined by plasma spectroscopy (Higgs et a l . 1982). 2 Diets were formulated to have a calcium to phosphorus ratio of close to unity when considering phosphorus sources other than sodium phytate.  - 45 -  3.5 3.5.1  E x p e r i m e n t a l Procedures and Sampling Diet  allocation  D i e t s were a s s i g n e d randomly t o each row of t a n k s i n a complete randomized b l o c k d e s i g n w i t h b l o c k s as r e p l i c a t e s .  T h e r e f o r e , b l o c k s were  not a n a l y z e d as such but were i n c l u d e d i n t h e e r r o r t e r m . 3.5.2  Feeding  . • r.-  F i s h were f e d by hand t o s a t i a t i o n t h r e e times d a i l y , seven days a week, e x c e p t on w e i g h i n g d a y s . were f e d s i x t i m e s d a i l y .  For t h e f i r s t 28 days of experiment I I t h e f i s h Food p a r t i c l e s i z e was a d j u s t e d i n r e l a t i o n  f i s h s i z e (Fowler and Burrows 1971).  to  D a i l y food consumption was r e c o r d e d f o r  experiments I and I I I . 3.5.3  F i s h weight On day 0 of t h e experiments and e v e r y 21 days t h e r e a f t e r ( e v e r y 42 days  i n e x p t . I I ) , a l l f i s h were removed from t h e tanks and random samples of 60 f i s h / t a n k were a n a 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 ) ,  placed  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 0.01 g . day.  F i s h were s t a r v e d f o r 24 hours p r i o r t o each w e i g h i n g  Removal of a l l f i s h from each tank a l l o w e d f o r tank c l e a n i n g and an  a c c u r a t e check of f i s h numbers.  The . l a t t e r c o u l d then be checked a g a i n s t  d a i l y m o r t a l i t y r e c o r d s ( e x p t s . I and I I I ) .  Throughout t h e e x p e r i m e n t s ,  numbers were p e r i o d i c a l l y t h i n n e d out t o reduce f i s h 3.5.4  fish  density.  C a t a r a c t assessment On each w e i g h i n g day, t h e eyes of 60 f i s h / t a n k were checked  m i c r o s c o p i c a l l y ( i . e . unaided eye) f o r any s i g n s of c a t a r a c t s .  If  cataracts  were observed a l l f i s h i n t h e r e s p e c t i v e t a n k ( s ) were then examined f o r cataracts.  - 46  3.5.5  -  Proximate a n a l y s i s F i s h were sampled randomly at t h e b e g i n n i n g and end of e x p e r i m e n t s I and  I I I f o r proximate a n a l y s i s .  The f i s h were k i l l e d w i t h 2 - p h e n o x y e t h a n o l ,  b l o t t e d d r y on absorbent c l o t h , p l a c e d i n h e a t - s e a l e d p l a s t i c bags and s t o r e d at -40°C f o r subsequent a n a l y s i s .  P r i o r t o a n a l y s i s , f i s h samples were  p a r t i a l l y thawed, p l a c e d i n a b l e n d e r and blended t o a f i n e homogenate. Samples were a n a l y z e d f o r m o i s t u r e , ash (AOAC 1975), l i p i d ( B l i g h and Dyer 1959) and t o t a l n i t r o g e n (Technicon I n s t r u m e n t a l C o . , L t d . , methods 369-75A/A and 334-74 W/B). crude p r o t e i n . 3.5.6  industrial  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  Test d i e t s were s i m i l a r l y a n a l y z e d .  F i s h h e a l t h and h i s t o l o g i c a l  examination  At t h e end of experiments I and I I I , 10-20 f i s h / d i e t t r e a t m e n t were assessed f o r general h e a l t h by personnel from t h e P a c i f i c B i o l o g i c a l D i a g n o s t i c S e r v i c e s B r a n c h , Nanaimo, B.C.  Station,  The f i s h were examined f o r  b a c t e r i a l d i s e a s e and e x t e r n a l and i n t e r n a l a b n o r m a l i t i e s .  Haematocrits,  haemoglobin c o n t e n t , r e d b l o o d c e l l count and shape and s i z e of b l o o d c e l l s were measured. the g i l l ,  A l s o f o r experiments I and I I I , h i s t o l o g i c a l e x a m i n a t i o n s o f  l i v e r , k i d n e y , p y l o r i c caeca and stomach were conducted by t h e  methods of McBride and van Overbeeke (1971) and van Overbeeke and M c B r i d e (1971). 3.5.7  Plasma and blood a n a l y s i s Plasma samples were taken at the end of experiment I and whole blood  samples at t h e end of experiment I I I f o r m i n e r a l a n a l y s i s .  The samples were  prepared and analyzed on an I n d u c t i v e l y Coupled Argon Plasma-Atomic E m i s s i o n Spectrometer (ICAP-AES, J a r r e l l - A s h Model 975 Plasma Atomcomp; F i s h e r S c i e n t i f i c C o . , Vancouver, B . C . ) .  7  - 47 -  3.5.8  Tissue  analysis  At t h e end of experiment I I I , samples of f i s h were k i l l e d and s t o r e d at -40°C f o r l a t e r m i n e r a l a n a l y s i s of l i v e r s and k i d n e y s .  - 48 -  CHAPTER 4  4.0  EXPERIMENT I - PRELIMINARY STUDY OF THE EFFECTS OF DIETARY CALCIUM, PHOSPHORUS, ZINC AND PHYTIC ACID LEVEL ON CATARACT INCIDENCE, GROWTH AND HISTOPATHOLOGY IN JUVENILE CHINOOK SALMON  4.1  INTRODUCTION Because of t h e l a c k of i n f o r m a t i o n on t h e n u t r i t i o n a l b a s i s f o r  cataract  f o r m a t i o n i n P a c i f i c salmon, t h i s s t u d y was conducted t o determine t h e e f f e c t s of wide v a r i a t i o n s i n d i e t a r y l e v e l s of Ca, P, Zn and p h y t i c a c i d on c a t a r a c t s as w e l l as on growth, f o o d i n t a k e , f o o d c o n v e r s i o n and p r o t e i n e f f i c i e n c y r a t i o i n chinook salmon.  In a d d i t i o n , t h e h i s t o p a t h o l o g i c a l  e f f e c t s of d i e t a r y p h y t i c a c i d were a s s e s s e d .  P h y t i c a c i d , as sodium  p h y t a t e , was i n c l u d e d as a d i e t a r y treatment t o assess 1) whether c a t a r a c t f o r m a t i o n c o u l d o r i g i n a t e from i n g e s t i o n of a d i e t c o n t a i n i n g a h i g h r a t i o  of  Ca t o Zn and a low l e v e l of a m i n e r a l - c h e l a t i n g agent, o r 2) whether t h e s i m u l t a n e o u s presence of high d i e t a r y r a t i o s of c a l c i u m t o z i n c and of a s t r o n g m i n e r a l - c h e l a t i n g agent were r e q u i r e d .  P h y t i c a c i d i s known t o  s t r o n g l y c h e l a t e d i v a l e n t m i n e r a l s , such as Z n , t o form i n s o l u b l e p h y t a t e s  in  t h e i n t e s t i n a l lumen, thus l o w e r i n g Zn b i o a v a i l a b i l i t y (Oberleas and H a r l a n d 1977; Erdman, J r . 1 9 7 9 ) .  In a d d i t i o n , the presence of high d i e t a r y Ca i s  thought t o enhance t h e a f f i n i t y between p h y t i c a c i d and Zn i n t h e i n t e s t i n e , thus a g g r a v a t i n g t h e d e p r e s s i o n of growth and Zn u t i l i z a t i o n .  Conversely,  d i e t a r y Zn s u p p l e m e n t a t i o n has been shown t o c o u n t e r a c t t h e e f f e c t s  of  p h y t a t e s , and t h i s p o s s i b i l i t y was a l s o i n v e s t i g a t e d i n chinook salmon.  - 49 -  4.2  MATERIALS AND METHODS  4.2.1  Experimental  Fish  Chinook salmon f r y were s e l e c t e d f o r u n i f o r m weight and d i s t r i b u t e d randomly i n t o 18 150-L F i b e r g l a s t a n k s r e s u l t i n g i n 113 f i s h / t a n k .  Initial  mean weight of t h e f i s h was 1.09 ± 0.03 g (SEM). 4.2.2  Experimental Diets  '  Nine d r y d i e t s (Tables 2-4) c o n t a i n i n g v a r y i n g l e v e l s of C a , Zn and p h y t i c a c i d were prepared at t h e West Vancouver L a b o r a t o r y as p r e v i o u s l y described. 4.2.3  Feeding Each d i e t was f e d t o two t a n k s o f f i s h f o r 105 d a y s .  F i s h were f e d t o  s a t i a t i o n t h r e e t i m e s d a i l y w i t h t h e p o i n t of s a t i a t i o n b e i n g reached when a c t i v e f e e d i n g ceased ( a p p r o x i m a t e l y one h o u r ) . 4.2.4  Sampling Procedures  4.2.4.1  F i s h weight and c a t a r a c t  incidence  Random samples of 60 f i s h / t a n k were weighed i n d i v i d u a l l y on days 0, 2 1 , 42, 63, 84 and 105.  Eyes were examined f o r c a t a r a c t s on each w e i g h i n g day as  described in section 3.5.4. 4.2.4.2  Proximate a n a l y s i s  P r i o r t o t h e s t a r t of t h e e x p e r i m e n t , 113 f i s h were s e l e c t e d randomly f o r proximate a n a l y s i s .  At t e r m i n a t i o n of t h e s t u d y , 6-20 f i s h per d i e t  t r e a t m e n t were sampled and pooled i n t o two groups per t r e a t m e n t f o r subsequent a n a l y s i s . 4.2.4.3  H i s t o l o g y samples  S i x f i s h from each d i e t a r y t r e a t m e n t were s e l e c t e d randomly f o r h i s t o l o g i c a l e x a m i n a t i o n of t h e g i l l ,  l i v e r , k i d n e y , t h y r o i d , stomach and  - 50 -  p y l o r i c caeca. 4.2.4.4  Plasma samples  Blood was withdrawn from t h e caudal v e s s e l o f at l e a s t 25 f i s h / d i e t treatment. centrifuged.  Blood was c o l l e c t e d i n h e p a r i n i z e d c a p i l l a r y tubes and A f t e r c e n t r i f u g a t i o n , t h e plasma from each tube was t r a n s f e r r e d  i n t o one of f o u r p o o l s per d i e t t r e a t m e n t f o r a n a l y s i s . 4.2.5  S t a t i s t i c a l Analysis On t h e b a s i s of t h e growth p a t t e r n o b s e r v e d , t h e experiment was d i v i d e d  i n t o two stages of e x p o n e n t i a l growth f o r d a t a a n a l y s i s .  Stages 1 and 2  corresponded t o days 0-63 and days 64-105, r e s p e c t i v e l y . Geometric mean wet w e i g h t s were a n a l y z e d by a one-way a n a l y s i s of v a r i a n c e (ANOVA).  S p e c i f i c growth r a t e s ( B r e t t 1979) were determined and  a n a l y z e d by a one-way a n a l y s i s of c o v a r i a n c e of t h e n a t u r a l l o g o f t h e wet w e i g h t s w i t h day as t h e c o v a r i a t e . c o v a r i a t e s l o p e (e ^°P s  body weight per day.  e  Growth r a t e s were d e r i v e d f r o m t h e  - 1) x 100 and e x p r e s s e d as a p e r c e n t i n c r e a s e  in  Food i n t a k e was e x p r e s s e d as a percentage of body  weight and was c a l c u l a t e d f o r a g i v e n p e r i o d of t i m e ( t , t + l ) a c c o r d i n g t o t h e following formula:  DFI x 100 * (logn^+l  d a i l y d r y f o o d i n t a k e per f i s h  +  logn^ * 2 ) P where DFI i s mean e x  ( t , t + l ) and l o g n t , lognt+1 are t h e averaged  n a t u r a l l o g wet w e i g h t s at t h e s t a r t ( t ) and t h e end (t+1) of t h e p e r i o d . The d a t a were a n a l y z e d by a one- o r two-way ANOVA.  Food c o n v e r s i o n and  p r o t e i n e f f i c i e n c y r a t i o s were c a l c u l a t e d ( i n grams) as wet weight g a i n * dry f o o d i n t a k e and wet weight g a i n *• p r o t e i n i n t a k e , r e s p e c t i v e l y , and were a n a l y z e d by a one- ( d i e t ) and two-way ( d i e t , p e r i o d ) ANOVA. m i n e r a l l e v e l s were a n a l y z e d by a one-way ANOVA. performed by u s i n g t h e program UBC GENLIN.  Data f o r plasma  S t a t i s t i c a l a n a l y s e s were  See Appendix A f o r ANOVA t a b l e s .  - 51 -  When a p p r o p r i a t e , Newman-Keuls'  ( K e u l s 1952) t e s t and S c h e f f e ' s  1959) t e s t (growth r a t e s o n l y ) w i t h P=0.05 were used t o d e t e c t d i f f e r e n c e s between t r e a t m e n t means.  (Scheffe*  significant  The s t a n d a r d e r r o r of t h e mean (SEM)  was d e r i v e d by u s i n g t h e e r r o r mean square from t h e ANOVA as t h e e s t i m a t e of sample v a r i a n c e . 4.3  RESULTS  4.3.1  . •  I n f l u e n c e of D i e t Treatment on C a t a r a c t T a b l e 5 shows t h a t b i l a t e r a l  f e d d i e t s 5 and 6.  Incidence  l e n s c a t a r a c t s were d e t e c t e d o n l y i n groups  W i t h i n t h e s e groups t h e c a t a r a c t s were o f v a r y i n g degrees  of s e v e r i t y r a n g i n g from a w h i t e p i n p o i n t dot c e n t r a l l y l o c a t e d i n t h e l e n s of each eye i n t h e l e a s t a f f e c t e d f i s h , t o complete opaqueness o f each l e n s i n extreme cases ( F i g . 6 ) . then i n f i s h f e d d i e t 5. f e d d i e t 6.  C a t a r a c t s were d e t e c t e d i n f i s h f e d d i e t 6 and C a t a r a c t s were not observed u n t i l day 63 i n f i s h  •  Of t h e 195 f i s h examined from t h i s t r e a t m e n t , 26% e x h i b i t e d some  degree of l e n s o p a c i t y .  Due t o t h e h i g h m o r t a l i t y t h a t o c c u r r e d i n  fish  r e c e i v i n g d i e t 6, t h e eyes were checked f o r t h e l a s t t i m e on day 84 r a t h e r than on day 105.  On day 84, c a t a r a c t s were noted i n 34% of t h e r e m a i n i n g 140  f i s h f e d d i e t 6 and i n 25% of the 183 f i s h f e d d i e t 4.3.2  I n f l u e n c e of D i e t Treatment on Chinook  4.3.2.1  5.  Performance  F i s h growth  T a b l e 6 shows t h a t a l l groups had more r a p i d growth i n s t a g e 1 than i n stage 2.  The p o o r e s t s p e c i f i c growth r a t e i n both stages o c c u r r e d i n f i s h  i n g e s t i n g d i e t 6, which c o n t a i n e d 51 g C a , 25.8 g p h y t i c a c i d and 0.05 g Zn per k i l o g r a m .  By day 105, t h e mean w e i g h t s of f i s h f e d d i e t s 5-8 w i t h 25.8 g  of p h y t i c a c i d / k g were 54-73% l e s s than t h o s e of c o r r e s p o n d i n g groups f e d d i e t s 1-4 w i t h 1.62 g of p h y t i c a c i d / k g ( F i g . 7; P<0.001).  Further,  the  - 52 -  f i n a l w e i g h t s of low p h y t a t e groups g i v e n 48-49 g Ca/kg ( d i e t s 2 and 4) were  30.-38% l e s s than t h o s e g i v e n 4 . 4 - 4 . 5 g Ca/kg ( d i e t s 1 and 3; P<0.001).  High  d i e t a r y Ca a l s o decreased growth i n h i g h p h y t a t e - f e d f i s h when d i e t a r y Zn was 0.05 g/kg d i e t . 4.3.2.2  Food i n t a k e , f o o d c o n v e r s i o n and p r o t e i n e f f i c i e n c y r a t i o  (PER)  Table 7 shows t h a t t h e o v e r a l l f o o d i n t a k e s of groups f e d h i g h Ca d i e t s ( 2 , 4 and 8) were reduced s i g n i f i c a n t l y (P<0.001) compared t o t h o s e of f e d d i e t s low i n Ca ( 1 , 3 and 7 ) .  groups  The one e x c e p t i o n t o t h e f o r e g o i n g  g e n e r a l i z a t i o n was noted i n f i s h f e d d i e t 6 which d i d not have a reduced i n t a k e compared t o f i s h f e d d i e t 5.  D i e t a r y l e v e l s of Ca and Zn d i d not  appear t o i n f l u e n c e f o o d c o n v e r s i o n and PER i n t h e low p h y t a t e - f e d  fish.  However, f i s h f e d t h e h i g h p h y t a t e d i e t s had s i g n i f i c a n t l y lower f o o d c o n v e r s i o n and PER (Zn at 0.05 g/kg) when Ca was a l s o h i g h ( d i e t 6 v s . 5 ) . F u r t h e r m o r e , h i g h d i e t a r y Zn s i g n i f i c a n t l y i n c r e a s e d f o o d c o n v e r s i o n and PER i n s p i t e of t h e presence of excess Ca ( d i e t 8 v s . 7 ) .  I n c r e a s e d Zn c o n t e n t  a l s o improved PER i n h i g h p h y t a t e - f e d f i s h when Ca was low ( d i e t 7 v s . 5 ) . O v e r a l l , high l e v e l s o f p h y t i c a c i d d r a m a t i c a l l y reduced f o o d c o n v e r s i o n and PER i n chinook salmon. F i g u r e 8 i l l u s t r a t e s t h e changes which o c c u r r e d i n f o o d i n t a k e , f o o d c o n v e r s i o n and PER of r e p r e s e n t a t i v e groups of low and h i g h p h y t a t e - f e d  fish  over f o u r 21-day p e r i o d s . 4.3.3  I n f l u e n c e of D i e t Treatment on P r o x i m a t e Composition D i e t a r y t r e a t m e n t m a r k e d l y i n f l u e n c e d t e r m i n a l l e v e l s of  proximate c o n s t i t u e n t s i n the f i s h  (Table 8 ) .  whole-body  For example, h i g h p h y t a t e  groups c o n s i s t e n t l y had h i g h e r p e r c e n t a g e s of m o i s t u r e , p r o t e i n and ash and lower percentages of l i p i d than noted i n t h e o t h e r g r o u p s .  Moreover,  fish  f e d d i e t s w i t h h i g h Ca and P (ash) c o n t e n t g e n e r a l l y had g r e a t e r l e v e l s of p r o t e i n and ash and a lower l i p i d c o n t e n t than observed i n r e s p e c t i v e c o u n t e r p a r t s f e d d i e t s w i t h low l e v e l s of Ca and P.  The l e v e l s o f  proximate  c o n s t i t u e n t s i n f i s h f e d d i e t 9 g e n e r a l l y f e l l w i t h i n t h e range found f o r  low  phytate groups. 4.3.4  I n f l u e n c e of D i e t Treatment on General  Health  A p a t h o l o g i c a l e x a m i n a t i o n of 20 f i s h from each o f t h e n i n e t r e a t m e n t groups at t e r m i n a t i o n of t h e s t u d y r e v e a l e d no s i g n i f i c a n t d i f f e r e n c e s a b n o r m a l i t i e s i n h a e m a t o c r i t , haemoglobin, blood c e l l count o r b l o o d shape and s i z e .  or  cell  F u r t h e r , no e v i d e n c e of b a c t e r i a l o r v i r a l pathogens or of  p r o t o z o a n p a r a s i t e s was f o u n d .  However, oedema, o r c o l l e c t i o n o f f l u i d  in  t h e stomach, was v i s u a l l y d e t e c t e d i n s e v e r a l of t h e f i s h f e d h i g h p h y t a t e diets. M o r t a l i t y was n e g l i g i b l e i n a l l groups except t h o s e r e c e i v i n g d i e t s c o n t a i n i n g high p h y t i c a c i d .  H e r e , m o r t a l i t y ranged from 9.3% f o r f i s h  fed  d i e t 5, which c o n t a i n e d low Ca and Z n , t o 44.7% f o r f i s h f e d d i e t 6 where Zn was low but Ca h i g h (Table 6 ) .  TABLE 5 C a t a r a c t i n c i d e n c e i n j u v e n i l e chinook salmon f e d d i e t s w i t h h i g h l e v e l s of p h y t a t e and examined on days 0, 2 1 , 42, 63 and 84 i n Experiment Phytic Ca Diet  5 6  7 8  Zn  acid  I.  1  % lens o p a c i t y  Examination Fish  day c a t a r a c t s < 25  < 50  > 75  75  22  1  2  26  74  15  7  4  34  66  13  3  18  (g/kg d r y  diet)  examined  detected  % cataracts  5.1  0.05  25.8  183  84  25  0.05  25.8  195  63  140  84  51  5.3 53  2  0.40  25.8  120  0  0.39  25.8  120  0  1 C a t a r a c t s were not d e t e c t e d i n groups f e d d i e t s c o n t a i n i n g low o r i n t e r m e d i a t e l e v e l s o f p h y t a t e 1-4,  9).  2 % l e n s o p a c i t y r e f e r s t o t h e percentage of the l e n s s u r f a c e e s t i m a t e d t o be opaque by v i s u a l only.  (diets  observation  - 55 -  F i g . 6a.  F i g . 6b.  Fig.  6.  J u v e n i l e chinook salmon f e d a high p h y t a t e d i e t , c a t a r a c t i n 6a and p a r t i a l c a t a r a c t i n 6b.  Note advanced  - 56 -  TABLE 6 S p e c i f i c growth rates and m o r t a l i t i e s f o r j u v e n i l e chinook salmon fed t e s t d i e t s f o r 105 days in Experiment I. Phytic Ca Diet  1 2 3 4 5 6 7  4.4 49 4.5 48 5.1 51 5.3  acid  Zn  (g/kg dry  Growth rate (% wet-wt/day)*  diet)  . Mortality Day 0-63  0.05  1.62  2.40  0.05  1.62  1.84  0.35  1.62  2.32  0.40  1.62  1.88&C  d  b  0.05  25.8  1.19  0.05  25.8  0.86  0.40  25.8  1.19  25.8  1.08  8  53  0.39  9  17.7  0.15 2 SEM  6.46  2  c d  a  a  a  a  . bcd 1 9  0.11  Day 64-105  Day 0-105  1.64C  2.14  1.50  1.72  c  1.80  b  b  (%)  3.9 4.4  b  2.4  1.37 c  1.71b  4.4  0.52  0.91  9.3  2.15  c  b  a  0.023 0.64 0.68  a  ab  a b  0.52 1.00 0.97  a  a  a  a  1.41 c  1.94  0.25  0.071  b  1 A one-factor a n a l y s i s of covariance of l o g  e  b  44.7 15.0 17.5 2.4  wet weights with d i e t as the  f a c t o r and time as the l i n e a r c o v a r i a t e , i n t e r a c t i o n between the f a c t o r and c o v a r i a t e , and two sources of e r r o r , r e p l i c a t i o n s (nested w i t h i n d i e t s and i n t e r a c t e d with time) and measurement e r r o r ( r e s i d u a l ) , i n d i c a t e d a s i g n i f i c a n t d i e t e f f e c t with P<0.001 f o r day 0-63, day 64-105 and o v e r a l l . Within a column, d i e t groups with the same s u p e r s c r i p t l e t t e r form a homogeneous subset ( S c h e f f e ' s m u l t i p l e range t e s t with P=0.05).  - 57 -  F i g . 7.  Geometric mean weights of j u v e n i l e chinook salmon f e d t e s t d i e t s 105 days i n Experiment I .  for  - 58 -  TABLE 7 Mean daily food consumption, food conversion and protein efficiency ratio for groups of juvenile chinook salmon fed test diets between days 22 and 105 in Experiment Ca  1 2 3 4 5 6 7  Zn  Phytic acid  (g/kg dry diet)  Diet  4.4 49 4.5 48 5.1 51 5.3  I.*  Protein efficiency Food intake  Food conversion  ratio  0.05  1.62  2.10Cd  1.33  d  2.47e  0.05  1.62  1.64*  1.37  d  2.63e  0.35  1.62  2.12Cd  1.31  d  2.44e  0.40  1.62  1.68  1.28  d  2.40  e  b  a  0.05  25.8  2.07C  0.42°  0.78  0.05  25.8  2.30**  0.19  0.36*  0.40  25.8  2.18Cd  0.51°  0.96C ; , .-•  25.8  1.83  0.65C  1.20  1.98C  1.30*  2.50«  0.094  0.14  0.27  8  53  0.39  9  17.7  0.15  6.46  2 SEM  b  a  d  1 A two factor analysis of variance (diet, period) with interaction and two sources of error, replication (nested within diet) and measurement error (residual) indicated, indicated P<0.001 for diet.  This analysis was based on 9x4x2=72 observations,  comprising all diet, period and replication combinations.  Significant differences were  noted in food intake P<0.001), food conversion (P<0.05) and PER (P<0.05) between the 21-day periods and a significant interaction between diet and period (P<0.01) was observed for food intake.  Within a column, diet groups with the same superscript  were not significantly different (Newman-Keuls test with P=0.05).  letter  - 59 -  B  FOOD  3«r  22-42  r~i Our  64-34  day  83-103  INTAKE  J>31>:| c : y t::3 -*J  r.-.i  &j $ tej  1  :  FOOD CONVERSION  be  b  T  m  "•> rXs  WOTE1N  EFFICIENCY  -  RATIO  aufa  l i i i i  3  *A  *9  Z n .03  £3  2  3  J J 31 .03  .03  F b y t i c a c i d 1.42 1 4 2 2 3 3 Z3JB  Fig.  8.  Food i n t a k e , food c o n v e r s i o n and p r o t e i n e f f i c i e n c y r a t i o f o r r e p r e s e n t a t i v e groups o f j u v e n i l e chinook salmon f e d d i e t s c o n t a i n i n g low and h i g h p h y t i c a c i d a t f o u r 21-day i n t e r v a l s i n Experiment I .  A one-way ANOVA w i t h d i e t as t h e f a c t o r i n d i c a t e d a  s i g n i f i c a n t d i e t e f f e c t f o r f o o d i n t a k e : P<0.05 (day 2 2 - 4 2 ) , P<0.01 (day 4 3 - 6 3 , 64-84) and P<0.001 (day 8 5 - 1 0 5 ) .  In r e g a r d t o food  c o n v e r s i o n and PER, ANOVA i n d i c a t e d P<0.01 (day 64-84) and P<0..001 d u r i n g a l l o t h e r 21-day i n t e r v a l s .  - 60 -  TABLE 8 Final whole body proximate composition of juvenile chinook salmon fed test diets for 105 days in Experiment 1.1  • $ of dry matter  Ca Diet  49  2  4.5  3  4  48  5.1  5  6  51  7  8  9  Phytic acid  (g/kg dry diet)  4.4  1  Zn  5.3  53  17.7  0.05  0.05  0.35  0.40  0.05  0.05  0.40  0.39  0.15  2  1.52  1.62  1.62  1.62  25.8  25.8  25.8  25.8  6.46  Moisture(S)  Protein  Lipid  Ash  73.0  61.6  27.7  7.7  74.4  62.5  28.9  8.3  75.2  65.3  21.7  10.4  75.9  65.8  23.3  12.9  73.8  61.1  29.7  7.6  75.1  65.6  28.1  7.1  74.9  64.4  25.4  11.1  74.2 .  63.7  24.1  10.4  80.9  73.3  11.3  11.1  80.0  66.2  14.5  14.5  82.0  73.5  10.3  19.4  80.4  70.0  11.4  16.7  80.9  78.2  13.2  11.2  81.2  71.3  12.5  13.2  79.2  76.2  16.6  15.6  80.3  72.8  17.4  15.2  74.7  66.0  26.6  9.8  74.9  67.1  24.8  10.0  1 Twenty gram samples of fish were removed from each replicate group per diet treatment (3 to 10 fish/group) and carcasses were stored at -40*C Defore analysis. 2  Mean percentages ± 2SEM for body moisture, protein, lipid and ash of chinook at the  beginning of the study (n=3) were, respectively, 80.5 ± 0 . 3 0 , 75.2 ± 1.69, 14.2 ± 0.39 and 11.4 > 1.71.  - 61 -  4.3.5  I n f l u e n c e of D i e t Treatment on H i s t o p a t h o l o g y G i l l and l i v e r s t r u c t u r e i n r e p r e s e n t a t i v e f i s h from a l l  groups were judged t o be n o r m a l .  treatment  A b n o r m a l i t i e s were d e t e c t e d i n samples of  k i d n e y of high Ca-fed f i s h ( d i e t s 2,4,6 and 8 ) , and i n t h e p y l o r i c c a e c a , stomach and t h y r o i d of h i g h p h y t a t e - f e d f i s h ( d i e t s 5 - 8 ) .  These are  d i s c u s s e d below. 4.3.5.1  Kidney  N e p h r o c a l c i n o s i s was observed i n a l l groups f e d h i g h d i e t a r y l e v e l s of Ca ( F i g . 9 ) .  F i g . 9.  Kidney s e c t i o n of a chinook salmon f e d a h i g h l e v e l of Ca and P (diet 2).  Note n e p h r o c a l c i n o s i s c h a r a c t e r i z e d by c a s t s , p r e s u m p t i v e  calcium d e p o s i t s , w i t h i n degenerating tubules or ducts ( a r r o w s ) . Hematoxylin and e o s i n , x50.  - 62 -  4.3.5.2  P y l o r i c caeca and stomach  The p y l o r i c c a e c a l e p i t h e l i a l c e l l s of f i s h f e d h i g h p h y t a t e d i e t s were c h a r a c t e r i z e d by h y p e r t r o p h y and marked v a c u o l i z a t i o n of t h e c y t o p l a s m ( F i g . 10a).  By c o n t r a s t , t h o s e of f i s h f e d low p h y t a t e d i e t s were s l e n d e r ,  columnar-shaped c e l l s w i t h a f i n e g r a n u l a r c y t o p l a s m ( F i g . 1 0 b ) .  The  a b e r r a n t p y l o r i c c a e c a l s t r u c t u r e and hence presumed abnormal f u n c t i o n was most e v i d e n t i n chinook salmon f e d d i e t 6 w i t h 50-70% of t h e p y l o r i c caeca affected.  I t was a l s o noted t h a t t h e mucous l i n i n g i n t h e p y l o r i c c a e c a was  markedly t h i n n e r than t h a t i n t h e stomach ( F i g . 1 0 c ) . epithelial  Moreover,  the  l a y e r of t h e stomach, i n c o n t r a s t t o t h a t of t h e p y l o r i c c a e c a ,  was a f f e c t e d o n l y m a r g i n a l l y i n f i s h f e d high l e v e l s of p h y t a t e .  The stomach  d i d e x h i b i t a modest a t r o p h y i n v o l v i n g the m u s c u l a t u r e as w e l l as g l a n d u l a r and e p i t h e l i a l components, but t h e r e was no e v i d e n c e of v a c u o l i z a t i o n of  the  epithelial  cells.  F i g . 10a.  C r o s s - s e c t i o n of p y l o r i c caeca from a h i g h p h y t a t e - f e d ( d i e t 5) chinook salmon.  Note h y p e r t r o p h y of t h e e p i t h e l i a l c e l l s  and marked v a c u o l i z a t i o n of the c y t o p l a s m .  (arrow)  Masson T r i c h o m e , x40.  - 63 -  F i g . 10b.  C r o s s - s e c t i o n o f p y l o r i c c a e c a from a low p h y t a t e - f e d ( d i e t 1) chinook salmon.  Note s l e n d e r , columnar-shaped e p i t h e l i a l  cells  w i t h a f i n e g r a n u l a r c y t o p l a s m (arrow) t y p i c a l of normal j u v e n i l e chinook salmon.  F i g . 10c.  Masson Trichome, x40.  C r o s s - s e c t i o n o f a p o r t i o n of c a r d i a c stomach ( l e f t ) and p y l o r i c caeca ( r i g h t ) of a chinook salmon ( f e d d i e t 6 ) .  Note t h e  sharp c o n t r a s t between t h e r e l a t i v e l y t h i c k l a y e r of mucous (arrows) on t h e s u r f a c e of t h e e p i t h e l i a l c e l l s i n t h e stomach and t h e t h i n l a y e r of mucous c o a t i n g t h e s e c e l l s i n t h e p y l o r i c caeca.  Periodic acid Schiff  (PAS)-hematoxylin, x40.  - 64 -  4.3.5.3  Thyroid  H i s t o l o g i c a l e x a m i n a t i o n r e v e a l e d a b n o r m a l i t i e s of t h y r o i d t i s s u e i n f i s h fed the high phytate d i e t s .  A p p r o x i m a t e l y 30-40% of the t h y r o i d f o l l i c l e s  t h e h i g h p h y t a t e groups were a t r o p h i e d ( F i g . 1 1 a ) . squamous i n shape and 2-3 um i n h e i g h t .  in  The e p i t h e l i a l c e l l s were  By c o n t r a s t , t h y r o i d  follicle  e p i t h e l i a l c e l l s i n t h e r e m a i n i n g groups were c u b o i d a l i n shape and 4-6 um i n height ( F i g . l i b ) .  F i g . 11.  T h y r o i d f o l l i c l e s of chinook salmon f e d a high p h y t a t e d i e t 11a and a low p h y t a t e d i e t  (1) i n l i b .  e p i t h e l i a l c e l l h e i g h t of f o l l i c l e s  (5)  Note marked r e d u c t i o n  in  in  i n t h e high p h y t a t e - f e d chinook  salmon compared t o t h e low p h y t a t e - f e d chinook salmon ( a r r o w s ) . PAS-hematoxylin, 4.3.6  xl60.  I n f l u e n c e of D i e t Treatment on Plasma M i n e r a l  Levels  D i e t a r y t r e a t m e n t s i g n i f i c a n t l y a f f e c t e d plasma l e v e l s of C a , P, Mg, Zn (P<0.001) and Mn (P<0.05), but not l e v e l s o f Fe o r Cu (Table 9 ) .  Plasma  - 65 -  TABLE 9 F i n a l mean plasma concentrations of various minerals i n j u v e n i l e chinook salmon f e d the t e s t d i e t s i n Experiment I . Phytic Ca Diet  Zn  acid  Mn  (g/kg dry d i e t )  P  Mg  (mg/kg dry weight)  1  .4.4  0.05  1.62  0.06a  107.5Cd  28.8C  368.3  2  49  0.05  1.62  0.073b  112.0  29.4C  355.5C  12.0  0.35  1.62  0.08  108.5Cd  33.2  380.OC  19.3C  1.62  108.8Cd  35.3  353.5C  10.3  103.3 c  18.5  93.0  14.5a  199.3  b  28.6C  254.3  103.3 c  b  27.4C  258.8  111.3  29. OC  391.0  1.86  18.6  3  4.5  •  4  48  0.40  5  ' 5.1  0.05  25.8  0.08  6  51  0.05  25.8  0.08  0.40  25.8  0.06  25.8  0.10  7  5.3  8  53  0.39  9  17.7  0.15  6.46  2 SEM  1  Zn  Ca  0.09  a b  a b  a b  a  b  0.08 0.02  a b  a b  d  b  99.0  a  d  3.6  d  d  b  248.5  C  b  a  b  b  C  18.3C a  a  15.3b 17.0 10.3 14.5 11.5  b c  a  b  a  1.52  Blood was withdrawn from the caudal vessels of at l e a s t 25 f i s h per d i e t  treatment.  Following c e n t r i f u g a t i o n , the plasma from each f i s h was t r a n s f e r r e d  i n t o one of f o u r pools per d i e t treatment f o r a n a l y s i s . 2 A one f a c t o r a n a l y s i s of variance with d i e t as the f a c t o r i n d i c a t e d a s i g n i f i c a n t d i e t e f f e c t with P<0.001 f o r Ca, Mg, P and Zn, and P<0.05 f o r Mn.  Within a column,  values with a common s u p e r s c r i p t l e t t e r were not. s i g n i f i c a n t l y d i f f e r e n t (Newman-Keuls t e s t with P=0.05).  - 66 -  l e v e l s of Zn and P were i n f l u e n c e d the most.  The observed plasma Zn l e v e l s  were h i g h e s t i n t h e low p h y t a t e groups g i v e n 0 . 3 5 - 0 . 4 0 g/kg of Zn and lowest i n f i s h f e d d i e t 6.  supplemental  Plasma P l e v e l s were h i g h e r i n low p h y t a t e  groups and i n f i s h f e d d i e t 9, which c o n t a i n e d i n t e r m e d i a t e l e v e l s of C a , P, Zn and p h y t i c a c i d , than i n h i g h p h y t a t e g r o u p s , p a r t i c u l a r l y i n f i s h diet  fed  6. Plasma Mg l e v e l s were s i g n i f i c a n t l y depressed i n t h e low p h y t a t e - f e d  f i s h when d i e t a r y l e v e l s of Ca and P were h i g h ( d i e t s 2 and 4 ) . 4.4 4.4.1  DISCUSSION Cataract  Incidence  The r e s u l t s i n d i c a t e t h a t h i g h d i e t a r y r a t i o s of Ca and P t o Zn a l o n e (~1000:1) were not s u f f i c i e n t t o i n i t i a t e c a t a r a c t f o r m a t i o n i n j u v e n i l e chinook salmon ( T a b l e 5 ) .  However, c a t a r a c t f o r m a t i o n d i d o c c u r when d i e t s  c o n t a i n e d a low l e v e l of Zn (0.05 g/kg) c o u p l e d w i t h a h i g h c o n c e n t r a t i o n (25.8 g/kg) of p h y t i c a c i d which i s a s t r o n g m i n e r a l c h e l a t i n g a g e n t .  This  e f f e c t was e x a c e r b a t e d by a h i g h d i e t a r y c o n c e n t r a t i o n of Ca. Supplementation of t h e h i g h p h y t a t e d i e t s w i t h Zn (0.40 v s . 0.05 p r e v e n t e d c a t a r a c t f o r m a t i o n at both Ca c o n c e n t r a t i o n s .  g/kg)  These r e s u l t s are  c o n s i s t e n t w i t h t h e v i e w p o i n t t h a t p h y t i c a c i d decreases Zn b i o a v a i l a b i l i t y i n t h e i n t e s t i n a l lumen, p a r t i c u l a r l y when excess Ca i s p r e s e n t . Even though t h e low l e v e l of Zn ( 0 . 0 5 g/kg) employed i n t h i s  study  exceeded t h e suggested r e q u i r e m e n t of Zn by s a l m o n i d s (Ogino and Yang 1978), t h i s l e v e l proved t o be inadequate when t h e d i e t a r y l e v e l of p h y t i c a c i d was high.  Hence, g r e a t c a r e s h o u l d be t a k e n i n t h e f o r m u l a t i o n of  practical  d i e t s f o r salmonids based on c o m b i n a t i o n s of animal and p l a n t p r o t e i n s ensure an adequate a v a i l a b l e Zh c o n t e n t .  to  - 67 -  4.4.2  Chinook  4.4.2.1  Performance  F i s h growth  The more r a p i d growth of f i s h which was observed i n s t a g e 1 compared t o stage 2 (Table 6) was p r o b a b l y a consequence of a p r o g r e s s i v e d e c r e a s e i n growth r a t e i n stage 2 due t o i n c r e a s e d s i z e ( B r e t t 1979) f o r t h o s e f i s h consuming d i e t s w i t h low ( d i e t s 1 and 3) o r i n t e r m e d i a t e ( d i e t 9) C a , P and phytic acid content.  However, i n t h e r e m a i n i n g groups high d i e t a r y l e v e l s of  p h y t i c a c i d and, t o a l e s s e r degree, o f Ca and P a l s o p l a y e d a r o l e .  Indeed,  throughout t h e study t h e r a t e of f i s h growth was d r a m a t i c a l l y depressed by a high d i e t a r y phytic acid c o n c e n t r a t i o n .  Moreover, h i g h d i e t a r y l e v e l s of Ca  and P r e s u l t e d i n a c o n s t a n t d e c l i n e i n t h e growth r a t e of low p h y t a t e groups which was s i g n i f i c a n t f o r f i s h i n g e s t i n g d i e t 2 i n stage 1.  The  significant  r e d u c t i o n i n t h e mean weight of f i s h f e d 25.8 g of p h y t i c a c i d w i t h 51 g Ca/kg ( d i e t 6) and i n f i s h f e d 1.62 g of p h y t i c a c i d c o u p l e d w i t h 48-49 g/kg Ca ( d i e t s 2 and 4) a l s o demonstrates t h e g r o w t h - d e p r e s s i n g e f f e c t of d i e t a r y l e v e l s o f Ca and/or P ( F i g . 7 ) .  high  T h i s both c o n f i r m s and extends t h e  f i n d i n g s of S p i n e l l i et a l . (1983) f o r rainbow t r o u t where a growth r e d u c t i o n of 5% o c c u r r e d i n f i s h f e d d i e t s c o n t a i n i n g more than 1% C a . In t h i s study d i e t a r y Zn c o n c e n t r a t i o n per se d i d not  significantly  i n f l u e n c e t h e growth r a t e of chinook salmon. 4.4.2.2  Food i n t a k e , f o o d c o n v e r s i o n and PER  High d i e t a r y l e v e l s o f Ca and P depressed a p p e t i t e i n f i s h f e d t h e low p h y t a t e d i e t s 2 and 4 ( T a b l e 7 ) .  However, t h e f o o d consumed by t h e s e f i s h  was u t i l i z e d e f f i c i e n t l y based on t h e f o o d c o n v e r s i o n and PER r e s u l t s . t h e i r energy demands were l i k e l y met.  The decreased a p p e t i t e of f i s h  Thus fed  d i e t s 2 and 4 compared t o d i e t s 1 and 3 may be a r e f l e c t i o n of t h e lower  - 68 -  p a l a t a b i l i t y of t h e former d i e t s as a r e s u l t of t h e i r high ash c o n t e n t . A l s o , t h e d i m i n i s h e d a p p e t i t e may have been due t o i n s u f f i c i e n t Mg r e l a t i v e t o t h e h i g h d i e t a r y c o n c e n t r a t i o n s of Ca and P even though t h e known Mg r e q u i r e m e n t of f i s h was met (Ogino e t a l . 1978; Knox e t a l . 1981)".  S i g n s of  e x c e s s i v e d i e t a r y l e v e l s of Ca and P i n r e l a t i o n t o Mg o r Mg d e f i c i e n c y  in  t r o u t i n c l u d e poor g r o w t h , l o s s of a p p e t i t e and c a l c i n o s i s of t h e k i d n e y and muscle (Cowey et a l . 1977; Knox et a l . 1 9 8 3 ) .  Although the f i s h fed d i e t s 2  and 4 d i d not s u f f e r poor growth t h e y d i d s u f f e r  nephrocalcinosis.  The d a t a i n d i c a t e t h a t growth r e s t r i c t i o n i n t h e high p h y t a t e groups was due t o a p r o g r e s s i v e r e d u c t i o n i n t h e i r c a p a c i t y t o c o n v e r t f o o d i n t o f l e s h ( F i g . 8; T a b l e 7 ) .  (protein)  Some of t h e lowered f o o d and p r o t e i n  c o n v e r s i o n i n t h e h i g h p h y t a t e - f e d f i s h p r o b a b l y stemmed from d i m i n i s h e d Zn bioavailability.  For example, i n c r e a s i n g Zn i n t h e h i g h p h y t a t e d i e t s  did  p a r t i a l l y o f f s e t t h e d i m i n i s h e d f o o d and p r o t e i n c o n v e r s i o n e s p e c i a l l y when Ca c o n t e n t was a l s o h i g h .  However, because p h y t i c a c i d can b i n d w i t h s e v e r a l  m i n e r a l s o t h e r than Z n , such as Mg, P, C u , Fe and Mn, and cause imbalances amongst them (Erdman, J r . 1979; Hartman, J r . 1979), i t i s c o n c e i v a b l e t h a t t h e lowered f o o d and p r o t e i n c o n v e r s i o n i n t h e h i g h p h y t a t e groups may a l s o have been p a r t l y caused by reduced b i o a v a i l a b i l i t y of m i n e r a l s such as P ( K e t o l a 1975) and Mg (Ogino et a l . 1 9 7 8 ) .  Other p o s s i b l e f a c t o r s i n c l u d e 1)  impairment of d i e t d i g e s t i b i l i t y owing t o t h e f o r m a t i o n of  phytic  a c i d / p r o t e i n complexes which i n h i b i t e n z y m a t i c d i g e s t i o n of t h e p r o t e i n (Hartman, J r . 1979; Graf 1983; S p i n e l l i et a l . 1983) and 2) depressed a b s o r p t i o n o f n u t r i e n t s i n t h e p y l o r i c c a e c a l r e g i o n of t h e i n t e s t i n e ( F i g . 10a).  T h i s l a t t e r . c a u s e w i l l be d i s c u s s e d i n s e c t i o n 4 . 4 . 5 . 2 .  In r e g a r d t o  f a c t o r one, Ogino and Yang (1978) r e p o r t e d reduced p r o t e i n d i g e s t i b i l i t y  in  - 69 -  rainbow t r o u t f e d Z n - d e f i c i e n t d i e t s .  They h y p o t h e s i z e d t h a t t h e low Zn  c o n t e n t may have reduced p r o t e a s e a c t i v i t y such as c a r b o x y p e p t i d a s e which c o n t a i n s Zn as an important p a r t of i t s s t r u c t u r e .  Similarly,  Spinelli  (1983) r e p o r t e d c a s e i n - p h y t i c a c i d complexes t o be p o o r l y h y d r o l y z e d by p e p s i n and suggested t h i s t o be a cause f o r poor p r o t e i n e f f i c i e n c y  in  rainbow t r o u t f e d p h y t a t e d i e t s . 4.4.3  P r o x i m a t e Composition A l t h o u g h s t a t i s t i c a l a n a l y s e s were not performed on p r o x i m a t e  composition data, the r e s u l t s i n d i c a t e that f i s h fed the high phytate d i e t s (5-8) had h i g h e r l e v e l s of m o i s t u r e , p r o t e i n and ash and much lower l e v e l s of l i p i d compared t o t h e o t h e r groups (Table 8 ) .  The low l i p i d c o n t e n t was  p r o b a b l y due t o t h e poor f o o d c o n v e r s i o n such t h a t t h e r e was not enough s u r p l u s energy f o r f a t d e p o s i t i o n .  The h i g h ash c o n t e n t i n f i s h f e d d i e t s  2,4,6 and 8 may be a s c r i b a b l e t o t h e h i g h ash c o n t e n t of t h e s e d i e t s 4).  (Table  However, t h e ash c o n t e n t s of f i s h f e d t h e low ash d i e t s 5 and 7 were  s i m i l a r t o t h o s e of f i s h f e d the h i g h ash d i e t s 2 and 4.  T h i s may have been  due t o t h e presence of high p h y t i c a c i d i n d i e t s 5 and 7. 4.4.4  F i s h Health The o n l y gross a b n o r m a l i t y found i n t h e h e a l t h e x a m i n a t i o n  which was seen i n s e v e r a l of t h e h i g h p h y t a t e - f e d f i s h .  was oedema  The cause of t h i s  is  not c l e a r , but i t may have r e s u l t e d from p a r t i a l a t r o p h y of t h e stomach (Dorland 1965). 4.4.5  Histopathology  4.4.5.1  Kidney  F i s h which i n g e s t e d t h e high Ca (P) d i e t s e x h i b i t e d n e p h r o c a l c i n o s i s , phenomenon seen i n rainbow t r o u t which have consumed h i g h d i e t a r y l e v e l s of  a  - 70 -  Ca i n r e l a t i o n t o Mg.  The h i g h Ca d i e t s used i n t h e p r e s e n t s t u d y c o n t a i n e d  Ca t o Mg r a t i o s r a n g i n g from 67:1 t o 72:1 (Table 4 ) .  A l t h o u g h t h e known Mg  requirement of f i s h was met, i t may be t h a t t h e chinook salmon i n t h i s  study  s u f f e r e d c a l c i n o s i s of t h e k i d n e y due t o i n s u f f i c i e n t Mg r e l a t i v e t o t h e d i e t a r y Ca and P l e v e l .  Knox et a l . (1981) r e p o r t e d t h a t t h e Mg r e q u i r e m e n t  of t r o u t does not i n c r e a s e as t h e d i e t a r y l e v e l s o f Ca and P are r a i s e d , but chinook salmon may respond d i f f e r e n t l y and a d d i t i o n a l work i s w a r r a n t e d i n t h i s area. in t h i s 4.4.5.2  D i e t a r y p h y t i c a c i d d i d not a g g r a v a t e t h e i n c i d e n c e of  calcinosis  study. P y l o r i c caeca and stomach  The f u n c t i o n of t h e p y l o r i c c a e c a i s t o f a c i l i t a t e n u t r i e n t a b s o r p t i o n (Fange and Grove 1979).  (e.g.  lipid)  However, t h e s t r u c t u r e of t h e p y l o r i c  caeca i n f i s h f e d d i e t s c o n t a i n i n g h i g h l e v e l s of p h y t i c a c i d was not t y p i c a l of normal j u v e n i l e salmon.  The h y p e r t r o p h y and marked v a c u o l i z a t i o n of t h e  p y l o r i c e p i t h e l i a l c e l l s i n t h e h i g h p h y t a t e - f e d f i s h may have r e s u l t e d from a t o x i c e f f e c t o f p h y t i c a c i d on the e p i t h e l i a l  l a y e r , i m p a i r e d Mg  b i o a v a i l a b i l i t y (Ogino e t a l . 1978) due t o c o m p l e x a t i o n of Mg w i t h p h y t i c a c i d (Erdman, J r . 1 9 7 9 ) , o r b o t h . With r e g a r d t o t h e p o s s i b i l i t y of p h y t i c a c i d t o x i c i t y , i t was r e p o r t e d i n s e c t i o n 4 . 3 . 5 . 2 t h a t t h e mucous l i n i n g i n t h e p y l o r i c caeca was much t h i n n e r than t h a t i n t h e stomach.  T h i s i s normal i n salmonids and t h i s  d i f f e r e n c e i n t h i c k n e s s may account f o r t h e d i s s i m i l a r response t o p h y t i c acid concentration.  P o s s i b l y t h e i n c r e a s e d mucous l a y e r i n t h e stomach  p r o t e c t e d a g a i n s t t h e t o x i c i t y of p h y t i c a c i d .  A l t e r n a t i v e l y , the strong  a c i d i c environment of t h e stomach (pH 3 . 5 ) , i n c o n t r a s t t o t h e n e u t r a l  to  a l k a l i n e c o n d i t i o n i n t h e i n t e s t i n e ( 6 - 8 ) , may have p r o t e c t e d t h e stomach  - 71 -  from p h y t i c a c i d t o x i c i t y . 4.4.5.3  Thyroid  S t r u c t u r a l a b e r r a t i o n s o f t h e t h y r o i d i n t h e h i g h p h y t a t e groups  (Fig.  11a) may have been caused by p r o t e i n (amino a c i d ) and/or m i n e r a l deprivation.  These c o n d i t i o n s are known t o suppress t h y r o i d a l f u n c t i o n  in  s a l m o n i d s (Higgs et a l . 1982b) and p h y t i c a c i d i s c a p a b l e of f o r m i n g p r o t e i n - p h y t a t e and m i n e r a l - p h y t a t e complexes. 4.4.6  Plasma M i n e r a l s T a b l e 9 i l l u s t r a t e s t h a t plasma Zn l e v e l s i n j u v e n i l e chinook salmon  were d i r e c t l y r e l a t e d t o d i e t a r y Zn c o n c e n t r a t i o n and i n v e r s e l y r e l a t e d t o dietary phytic acid l e v e l .  D i e t a r y Ca c o n t e n t had no i n f l u e n c e on plasma Zn  c o n c e n t r a t i o n except when t h e d i e t s i m u l t a n e o u s l y c o n t a i n e d a h i g h l e v e l p h y t i c a c i d and a low l e v e l o f Z n .  of  These r e s u l t s h e l p t o c o n f i r m t h a t 25.8 g  o f p h y t i c a c i d / k g d i e t d i d i m p a i r Zn a b s o r p t i o n i n chinook salmon and t h a t t h i s e f f e c t was aggravated by 51 g Ca/kg d i e t .  I t i s not known whether t h e  h i g h d i e t a r y l e v e l of p h y t i c a c i d i n f l u e n c e d t h e a b s o r p t i o n o f both exogenous and endogenous Zn i n chinook salmon.  However, Davies and N i g h t i n g a l e  (1975)  performed c a r a c a s s a n a l y s e s i n r a t s and c o n c l u d e d t h a t t h e a d d i t i o n of 10 g of p h y t i c a c i d per k i l o g r a m d i e t i m p a i r e d not o n l y t h e a b s o r p t i o n of  dietary  Z n , but a l s o t h e r e s o r p t i o n of endogenously s e c r e t e d Z n . I t i s noteworthy t h a t t h e h i g h p h y t a t e - f e d f i s h which developed c a t a r a c t s ( d i e t s 5 and 6) a l s o e x h i b i t e d s i g n i f i c a n t l y reduced plasma Zn levels.  T h i s s t r e n g t h e n s t h e h y p o t h e s i s t h a t low Zn a v a i l a b i l i t y r e s u l t s  cataract formation. Plasma P was i n v e r s e l y r e l a t e d t o d i e t a r y p h y t i c a c i d c o n c e n t r a t i o n . F i s h f e d 25.8 g p h y t i c a c i d / k g d i e t had s i g n i f i c a n t l y reduced plasma P  in  - 72 -  levels.  As w i t h Z n , p h y t i c a c i d b i n d s w i t h P t o form s t r o n g p h y t a t e  complexes which render P u n a v a i l a b l e f o r a b s o r p t i o n at t h e i n t e s t i n a l pH of f i s h e s (NRC 1981). The s i g n i f i c a n t r e d u c t i o n of plasma Mg i n f i s h f e d low l e v e l s of p h y t i c a c i d c o u p l e d w i t h h i g h l e v e l s of d i e t a r y Ca and P s u p p o r t s t h e c o n t e n t i o n t h a t t h e reduced a p p e t i t e and n e p h r o c a l c i n o s i s i n c i d e n c e i n f e d d i e t s 2 and 4 may have stemmmed from reduced Mg a b s o r p t i o n .  fish  However,  plasma Mg l e v e l s i n t h e h i g h p h y t a t e groups d i d not bear any c o n s i s t e n t r e l a t i o n s h i p t o d i e t a r y Ca and P c o n c e n t r a t i o n which makes i t d i f f i c u l t  to  account f o r n e p h r o c a l c i n o s i s i n f i s h f e d d i e t s 6 and 8 s o l e l y on t h e b a s i s of Mg d e f i c i e n c y .  The measurement of C a , P and Mg l e v e l s i n t h e k i d n e y would  have f a c i l i t a t e d d a t a i n t e r p r e t a t i o n i n t h i s c a s e . S p i n e l l i e t a l . (1983) demonstrated t h a t b l o o d Cu l e v e l s i n rainbow t r o u t f e d p u r i f i e d d i e t s i n c r e a s e d when t h e d i e t a r y p h y t a t e l e v e l was i n c r e a s e d t o 5.0 g from 0.0 g / k g .  F u r t h e r m o r e , b l o o d Cu decreased i n t h e  p h y t a t e groups when d i e t a r y Ca and Mg were r a i s e d t o 13 g and 0.85 g / k g , respectively.  The marked v a r i a t i o n s of d i e t a r y p h y t a t e and Ca i n t h e p r e s e n t  e x p e r i m e n t , however, d i d not a f f e c t plasma Cu l e v e l s i n chinook salmon. Whether t h i s i s a s p e c i e s - s p e c i f i c response cannot be deduced from t h e s e two e x p e r i m e n t s , but i t does bear c o n s i d e r a t i o n . 4.5  CONCLUSION A l t h o u g h t h i s s t u d y d i d not show t h a t the 1981 c a t a r a c t problem i n  Canadian and American s t o c k s of chinook and coho salmon o r i g i n a t e d from an induced Zn d e f i c i e n c y ,  i t does show t h a t Zn i s i m p o r t a n t f o r normal  i n t e g r i t y i n chinook salmon.  lens  The f i n d i n g s suggest t h a t , when p r a c t i c a l  l e v e l s of Zn are employed and when d i e t a r y c o n c e n t r a t i o n s o f Ca and P are a t ,  - 73 -  o r near t h e maximum l e v e l s found i n p r a c t i c a l salmonid f o o d s , a Zn d e f i c i e n c y w i l l be induced o n l y when a m i n e r a l - b i n d i n g agent such as p h y t i c a c i d i s present in the d i e t in s u f f i c i e n t c o n c e n t r a t i o n .  also  Hence, h i g h d i e t a r y r a t i o s  o f Ca t o Zn per se w i l l not induce c a t a r a c t f o r m a t i o n i n chinook salmon under c o n d i t i o n s s i m i l a r t o t h o s e used i n t h e p r e s e n t s t u d y .  - 74 -  CHAPTER 5  5.0  EXPERIMENT I I - THE SUSCEPTIBILITY OF JUVENILE CHINOOK SALMON TO CATARACT FORMATION IN RELATION TO DIETARY CHANGES IN EARLY LIFE  5.1  INTRODUCTION Due t o t h e d e s i g n of t h e p r e v i o u s experiment i t was not p o s s i b l e t o  measure t h e degree of s u s c e p t i b i l i t y t o c a t a r a c t s at d i f f e r e n t t i m e s i n t h e e a r l y l i f e of chinook salmon. obtain t h i s information.  The main o b j e c t i v e of t h e p r e s e n t s t u d y was t o  The r a t i o n a l e f o r t h i s goal i s d e r i v e d from  mammalian s t u d i e s which have shown t h a t c a t a r a c t s can be induced more r e a d i l y i n v e r y young animals (Grant 1 9 7 4 ) . confirmed in f i s h . juvenile l i f e  To my knowledge t h i s has not been  However, f i s h eyes i n c r e a s e i n s i z e g r e a t l y d u r i n g  (Johns 1981) and thus t h e y may be more prone t o l e n s damage  d u r i n g such t i m e .  Three d i e t s , based on experiment I , were f e d i n e i g h t  d i f f e r e n t sequences t o determine whether t h e f i s h were most s u s c e p t i b l e t o c a t a r a c t development d u r i n g t h e f i r s t , second o r t h i r d 42-day p e r i o d  after  swim-up. 5.2 5.2.1  MATERIALS AND METHODS Experimental  Fish  Swim-up chinook f r y were s e l e c t e d v i s u a l l y f o r u n i f o r m s i z e and were d i s t r i b u t e d randomly i n t o 16 29-L F i b e r g l a s t a n k s r e s u l t i n g i n 250 f i s h / t a n k . A f t e r 30 days a l l groups of f i s h were t r a n s f e r r e d i n t o 150-L t a n k s t o reduce f i s h density. 5.2.2  The i n i t i a l mean weight of t h e f i s h was 0.49 ± 0.01 g (SEM).  Experimental D i e t s The t h r e e d i e t s (A,B and C; Tables 2,3a and 4 a ) , were prepared as  - 75 -  d e s c r i b e d i n Chapter 3 ( s e c t i o n 3 . 4 ) . IU (NRC 1981).  C h o l e c a l c i f e r o l was i n c r e a s e d t o 2400  The d i e t s were a d m i n i s t e r e d t o two groups of f i s h  accordance w i t h t h e s c h e d u l e shown i n F i g u r e 12.  in  D i e t s A and B both  c o n t a i n e d low l e v e l s of sodium p h y t a t e and low and high l e v e l s , r e s p e c t i v e l y , of Ca and P.  D i e t C c o n t a i n e d high l e v e l s of sodium p h y t a t e , Ca and P.  All  d i e t s c o n t a i n e d 0.06 g Zn/kg which i s above t h e recommended r e q u i r e m e n t f o r salmonids (Ogino and Yang 1978).  T h i s l e v e l was s e l e c t e d because t h e  c a t a r a c t outbreak i n some B.C. h a t c h e r y s t o c k s of coho and chinook salmon i n 1981 was e x p e r i e n c e d w i t h a commerical d i e t c o n t a i n i n g a s i m i l a r l e v e l Zn.  Therefore,  of  i t was hoped t h a t t h e f i n d i n g s from t h i s study would be of  p r a c t i c a l s i g n i f i c a n c e t o h a t c h e r y managers. 5.2.3  Feeding The f i s h were f e d s i x t i m e s d a i l y t o s a t i a t i o n f o r t h e f i r s t 28 days and  t h r e e t i m e s d a i l y f o r t h e r e m a i n i n g 98 days of the 126-day s t u d y .  Periods  of  42 days were chosen t o s i m u l a t e h a t c h e r y c o n d i t i o n s i n which a p a r t i c u l a r b a t c h of f e e d i s l i k e l y t o be r e p l a c e d e v e r y 28-42 d a y s .  It also  allowed  t i m e f o r t h e chinook salmon t o a d j u s t t o changes i n d i e t c o m p o s i t i o n and t o resume a c t i v e f e e d i n g .  E i g h t t r e a t m e n t s , out of a p o s s i b l e t w e n t y - s e v e n ,  were s e l e c t e d t o s i m u l a t e s i t u a t i o n s which may occur i n a h a t c h e r y environment, namely,  (1) s h o r t - t e r m exposure t o a c a t a r a c t o g e n i c d i e t  A-C-A and A - A - C ) , (2) c o n t i n u o u s exposure t o a n u t r i t i o n a l l y w e l l diet  (C-A-A,  balanced  ( A ) , (3) s h o r t - t e r m exposure t o a low p h y t a t e d i e t c o n t a i n i n g h i g h  l e v e l s of Ca and P i n r e l a t i o n t o Zn (B-A-A, A-B-A and A - A - B ) , and l a s t l y , (4) c o n t i n u o u s exposure t o d i e t B.  With r e g a r d t o t h i s l a s t t r e a t m e n t ,  it  was p o s t u l a t e d , based on t h e f i n d i n g s of K e t o l a (1979) f o r rainbow t r o u t , t h a t t h e exposure t i m e of chinook salmon t o a d i e t c o n t a i n i n g a h i g h Ca and P  - 76 -  TREATMENT 1  DIETS  I  -  I  A —  1  »  A  4  |  A  5  r — - S  6  H  3  A  J  -  1  C  j  A  ,  A  i  C  j  A  j_  A  j  A  j  A  .  B  .  A  i  1  1  7  ,  A  ,  A  j  B  8  I  §  i  4  \  S  0  42 DAYS  F i g . 12.  :  .  84 POST  126  SWIM-UP  Schematic r e p r e s e n t a t i o n of d i e t a r y treatments given t o salmon from swim-up t o day 126 i n Experiment  II.  chinook  - 77 -  t o Zn r a t i o i n experiment I may have been too s h o r t t o i n d u c e c a t a r a c t s . T h e r e f o r e , d i e t B was f e d f o r 126 days i n t h i s study which was t h e same d u r a t i o n as t h a t employed by K e t o l a . 5.2.4  Sampling Procedures Random samples of 60 f i s h / t a n k were weighed i n d i v i d u a l l y t o t h e n e a r e s t  0.01 gram on days 0, 42, 84 and 126.  F i s h eyes were checked m a c r o s c o p i c a l l y  as d e s c r i b e d p r e v i o u s l y ( s e c t i o n 3 . 2 . 5 . 4 ) . 5.2.5  Statistical Analysis Wet weights were a n a l y z e d u s i n g a one-way ANOVA (UBC GENLIN) and  d i f f e r e n c e s between means were t e s t e d u s i n g Duncan's New M u l t i p l e Range Test (Duncan 1955) w i t h P=0.05. 5.3 5.3.1  See Appendix B f o r ANOVA t a b l e .  RESULTS I n f l u e n c e of D i e t Treatment on C a t a r a c t  Incidence  B i l a t e r a l l e n s c a t a r a c t s were observed on day 126 i n 44 out of 360 f i s h (12%) which r e c e i v e d t r e a t m e n t 2.  These f i s h were exposed t o t h e  c a t a r a c t o g e n i c d i e t (C) between days 42 and 84 ( F i g . 1 2 ) .  N e i t h e r of  the  groups f e d d i e t C d u r i n g t h e f i r s t 42 days ( t r e a t m e n t 1) o r t h e l a s t 42 days (treatment 3) e x h i b i t e d c a t a r a c t s .  C a t a r a c t s were not d e t e c t e d at any t i m e  i n f i s h f e d d i e t B. 5.3.2  I n f l u e n c e of D i e t Treatment on F i s h Growth Growth was i n f l u e n c e d s i g n i f i c a n t l y by d i e t c o m p o s i t i o n and by t h e  p e r i o d when t h e d i e t s were f e d .  T a b l e 10 shows t h a t f i s h f e d d i e t C f o r  the  f i r s t 42 days post swim-up ( t r e a t m e n t 1) had s i g n i f i c a n t l y lower mean w e i g h t s at day 42 than those on a l l o t h e r t r e a t m e n t s .  With one e x c e p t i o n  (treatment  6 ) , t h e mean w e i g h t s of f i s h f e d d i e t A ( t r e a t m e n t s 2,3,4 and 7) were h i g h e r than those f e d d i e t B ( t r e a t m e n t s 5 and 8 ) .  On day 84 however, f i s h on  - 78 -  t r e a t m e n t 6, which were changed from d i e t A t o d i e t B on day 42, were s i g n i f i c a n t l y l i g h t e r i n w e i g h t than t h o s e on t r e a t m e n t s 3,4 and 7 which remained on d i e t A.  Moreover, c o n t i n u o u s a d m i n i s t r a t i o n of d i e t B ( t r e a t m e n t  8) d r a m a t i c a l l y reduced growth t o day 84 as compared w i t h the growth of  fish  which had r e c e i v e d d i e t A from day 0 t o day 84 ( t r e a t m e n t s 3,4 and 7 ) . The g r e a t e s t f i n a l mean w e i g h t s were observed f o r f i s h f e d d i e t A throughout t h e 126 d a y - s t u d y ( t r e a t m e n t 4) and i n f i s h f e d d i e t A i n c o m b i n a t i o n w i t h d i e t B ( t r e a t m e n t s 5,6 and 7 ) .  F i s h f e d d i e t B f o r 126  days, o r v a r i o u s c o m b i n a t i o n s of d i e t A w i t h C, had d r a m a t i c a l l y reduced f i n a l mean w e i g h t s , w i t h t h e p o o r e s t growth b e i n g observed f o r f i s h on t r e a t m e n t 1. 5.4 5.4.1  DISCUSSION Cataract  Incidence  The o n l y treatment which induced c a t a r a c t s i n j u v e n i l e chinook salmon was t r e a t m e n t 2 i n which t h e h i g h p h y t a t e d i e t was f e d between days 42 and 84.  The i n s p e c t i o n system used i n t h i s study i n d i c a t e d t h a t t h e r e was a l a g  p e r i o d between exposure t o t h e c a t a r a c t o g e n i c d i e t and t h e m a n i f e s t a t i o n of t h e c a t a r a c t s , s i n c e o p a c i t i e s were not r e a d i l y d i s c e r n i b l e u n t i l day 126. F a i l u r e of d i e t C t o i n i t i a t e c a t a r a c t s d u r i n g t h e f i r s t and t h i r d 42-day p e r i o d may have been r e l a t e d t o t h e s t a t u s of Zn r e s e r v e s i n t h e f i s h those t i m e s .  For example, i t may be p o s t u l a t e d t h a t t h e swim-up f r y  during acquired  s u f f i c i e n t m i n e r a l (Zn) r e s e r v e s from t h e absorbed y o l k sacs t o c o u n t e r a c t decreased Zn b i o a v a i l a b i l i t y from t h e d i e t d u r i n g t h e f i r s t 42 days a f t e r swim-up.  S i m i l a r l y , f i s h f e d d i e t A f o r t h e f i r s t 84 days may have a c q u i r e d  adequate Zn r e s e r v e s such t h a t a subsequent 42 days on d i e t C was not s u f f i c i e n t t o v i s i b l y harm l e n s t i s s u e .  However, t h e f a i l u r e t o d e t e c t  -.79 -  c a t a r a c t s i n f i s h r e c e i v i n g d i e t C between days 84 and 126 cannot be viewed as c o n c l u s i v e e v i d e n c e t h a t t h e p r o v i s i o n of the h i g h p h y t a t e d i e t at t h a t t i m e had no e f f e c t on c a t a r a c t i n c i d e n c e .  I f t h e d e l a y i n onset was of a  s i m i l a r d u r a t i o n t o t h a t found when d i e t C was f e d between days 42 and 84, t h e t e r m i n a t i o n of t h e experiment at day 126 would not have a l l o w e d f o r cataract detection.  I t i s a l s o c o n c e i v a b l e t h a t , w i t h t h e c o n c e n t r a t i o n of  Zn i n t h e eye changing i n r e l a t i o n t o body s i z e (Shearer 1984), t h e d i e t a r y c o n c e n t r a t i o n of Zn needed t o m a i n t a i n normal l e n s growth between days 84 and 126 may have been l e s s than t h a t which was r e q u i r e d between days 42 and 8 4 . The r e s u l t s of t h i s study c o n f i r m and extend t h o s e of experiment  I.  They suggest t h a t h i g h d i e t a r y l e v e l s of Ca and P t o Zn per se do not i n i t i a t e c a t a r a c t o g e n e s i s i n j u v e n i l e chinook salmon under t h e c o n d i t i o n s t h i s study even a f t e r 126 days of e x p o s u r e .  This i s in contrast to the  f i n d i n g s of K e t o l a (1979) w i t h rainbow t r o u t and suggests t h a t chinook may respond d i f f e r e n t l y from rainbow t r o u t .  of  salmon  A l t h o u g h K e t o l a d i d not a n a l y z e  h i s d i e t s f o r p h y t i c a c i d c o n t e n t , o r suggest a m i n e r a l - p h y t a t e i n t e r a c t i o n , i t i s p o s s i b l e t h a t t h e r e was s u f f i c i e n t p h y t i c a c i d p r e s e n t i n t h e soybean and wheat m i d d l i n g s p o r t i o n s of h i s b a s a l d i e t t o induce c a t a r a c t s . A c c o r d i n g t o t h e r e s u l t s of experiment I and t h o s e of t h e p r e s e n t s t u d y , c a t a r a c t s can be a n t i c i p a t e d i f the d i e t s i m u l t a n e o u s l y c o n t a i n s h i g h of Ca and P i n r e l a t i o n t o Zn and t h e presence of a s t r o n g m i n e r a l agent.  binding  T h e r e f o r e , p h y t i c a c i d may have been an i m p o r t a n t c o n t r i b u t o r  Ketola's results.  levels  to  - 80 -  TABLE 10 Mean wet weights (g * 2 SEM) o f j u v e n i l e Chinook salmon fed the t e s t d i e t s a t 42-day i n t e r v a l s f o r a period of 126 days i n Experiment I I . 1  Treatment  Day 0  Day 42  Day 84  Day 126  0.75a  1.62*  5.2ia  1.49Cd  2.13  - CAA  0.48  ! - ACA  0.48  1 - AAC  0.49  1.58Cd  3.96d  t - AAA  0.50  1.55Cd  3.90  i - BAA  0.49  1.17b  3.07C  8.56b  i - ABA  0.50  1.36°c  2.87C  8.39b  ' - AAB  0.51  1.58<i  4.09  ! - BBB  0.49  1.26"  2.37b  5.56  2 SEM2  0.01  0.06  0.16  0.42  •  d  d  d  6.11  a  5.38  a  9.45b  8.82  b  a  1 A one-way ANOVA indicated a s i g n i f i c a n t treatment efect P<0.001 f o r days 42 and 84 and P<0.01 f o r day 126. Within a column, values with the same s u p e r s c r i p t l e t t e r were not s i g n i f i c a n t l y d i f f e r e n t (Duncan's m u l t i p l e range t e s t with 2  P=0.05).  Due t o missing values, the 2 SEM f o r treatments 3 and 5 f o r days 42, 84 and 126  were 0.08, 0.22 and 0.60, r e s p e c t i v e l y .  - 81 -  5.4.2  F i s h Growth The changes observed i n t h e growth p a t t e r n of j u v e n i l e chinook salmon i n  r e l a t i o n t o d i e t a r y t r e a t m e n t were a n t i c i p a t e d based on t h e r e s u l t s of experiment I .  The p r e s e n t r e s u l t s r e i n f o r c e t h e concept t h a t h i g h d i e t a r y  c o n c e n t r a t i o n s of Ca and P depress t h e growth of chinook salmon.  Therefore,  when f o r m u l a t i n g d i e t s f o r j u v e n i l e chinook salmon, c a r e s h o u l d be t a k e n i n t h e 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 ( e . g . f i s h meals) t o ensure low ash content.  Otherwise t h e r e d u c t i o n i n smolt s i z e at t h e t i m e of ocean r e l e a s e  c o u l d compromise marine s u r v i v a l ( B i l t o n 1 9 8 4 ) . 5.5  CONCLUSION The f i n d i n g s suggest t h a t t h e r e may be a " c r i t i c a l window" i n t h e e a r l y  development of chinook salmon d u r i n g which i t t h i s species to a cataractogenic d i e t .  i s i m p e r a t i v e not t o expose  However, a d d i t i o n a l work i s  required  t o c o n f i r m whether chinook salmon are most prone t o c a t a r a c t development between 42 and 84 days a f t e r swim-up and t o v e r i f y whether a f u l l  42 days  exposure i s n e c e s s a r y . From a p r a c t i c a l , s t a n d p o i n t , a knowledge of t h e apparent l a g e f f e c t  in  exposure t o a c a t a r a c t o g e n i c d i e t and u l t i m a t e m a n i f e s t a t i o n of t h e c a t a r a c t s may be u s e f u l t o h a t c h e r y managers who f i n d themselves c o n f r o n t e d w i t h a c a t a r a c t problem.  I f t h e a n a l y s e s of t h e h a t c h e r y d i e t being used at the  t i m e of c a t a r a c t appearance y i e l d r e s u l t s which i n d i c a t e no a b n o r m a l i t i e s  in  n u t r i e n t l e v e l s or no e x c e s s e s of a n t i - n u t r i t i o n a l f a c t o r s ( e . g . p e s t i c i d e s ) , i t may be n e c e s s a r y t o a n a l y z e samples of f o o d which had been p r o v i d e d s e v e r a l weeks b e f o r e t h e problem was d e t e c t e d .  CHAPTER 6  6.0  EXPERIMENT I I I - A COMPREHENSIVE STUDY OF THE EFFECTS OF DIETARY CALCIUM, PHOSPHORUS, ZINC AND PHYTIC ACID ON CATARACTS, GROWTH AND HISTOPATHOLOGY IN JUVENILE CHINOOK SALMON.  6.1  INTRODUCTION Experiment I e s t a b l i s h e d t h a t i n t e r a c t i o n s between C a , P, Zn and p h y t i c  a c i d d i d a f f e c t c a t a r a c t f o r m a t i o n , growth 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 chinook salmon.  However, t h e d e s i g n d i d not p e r m i t t h e t e s t i n g of  i n t e r m e d i a t e l e v e l s of t h e above d i e t a r y f a c t o r s .  T h e r e f o r e , t h i s s t u d y was  conducted as a t h r e e f a c t o r composite d e s i g n t o more t h o r o u g h l y  investigate  t h e e f f e c t s of t h e s e d i e t a r y f a c t o r s on c a t a r a c t f o r m a t i o n and performance chinook salmon.  I t i s noteworthy t h a t a t h r e e f a c t o r composite d e s i g n  in  (with  15 t r e a t m e n t c o m b i n a t i o n s ) has t h e same s t a t i s t i c a l power as a 3 x 3 x 3 factorial 6.2 6.2.1  (Davies 1954).  MATERIALS AND METHODS Experimental  Fish  Chinook salmon f r y , s e l e c t e d f o r u n i f o r m w e i g h t , were d i s t r i b u t e d at random i n t o 30 150-L tanks u n t i l each c o n t a i n e d 140 f i s h .  I n i t i a l mean  weight was 1.28 ± 0.03 g (SEM). 6.2.2  Experimental  Diets  The 15 dry d i e t s used i n t h e s t u d y are d e s c r i b e d i n T a b l e s 2, 3a and 4 a . 6.2.3  Feeding Each d i e t was f e d t o two groups of f i s h t h r e e t i m e s d a i l y f o r 84 d a y s .  - 83 -  6.2.4  Sampling Procedures  6.2.4.1  F i s h weight and c a t a r a c t  incidence  On days 0, 2 1 , 42, 63 and 84, random samples of 60 f i s h / t a n k were 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 0.01 g . as d e s c r i b e d i n s e c t i o n 3 . 5 . 4 .  Eyes were examined f o r  cataracts  A l t h o u g h t h e growth study p o r t i o n of  this  experiment was t e r m i n a t e d at day 84, due t o h i g h m o r t a l i t y i n groups f e d d i e t 11, t h e r e m a i n i n g f i s h from a l l groups were m a i n t a i n e d on t h e i r  respective  d i e t s f o r an a d d i t i o n a l 35 days f o r t h e purpose of o b s e r v i n g c a t a r a c t incidence. 6.2.4.2  Proximate a n a l y s i s  At t h e b e g i n n i n g of t h e e x p e r i m e n t , 125 f i s h were s e l e c t e d at random and p o o l e d i n t o f o u r groups f o r p r o x i m a t e a n a l y s i s .  At the end of t h e s t u d y a  minimum of 20 f i s h / t r e a t m e n t were s e l e c t e d randomly and pooled i n t o f o u r groups per d i e t a r y t r e a t m e n t f o r p r o x i m a t e a n a l y s i s . 6.2.4.3  H i s t o l o g y samples  On day 50, s i x f i s h per t r e a t m e n t were k i l l e d f o r  histological  e x a m i n a t i o n , and at t e r m i n a t i o n of t h e study 6-10 f i s h per t r e a t m e n t were killed.  S t r u c t u r e s examined i n c l u d e d t h e g i l l ,  kidney, l i v e r ,  pancreas,  stomach and p y l o r i c c a e c a . 6.2.4.4  Blood samples  At t h e end of t h e e x p e r i m e n t , b l o o d was withdrawn from t h e caudal v e s s e l s of at l e a s t 20 f i s h / d i e t t r e a t m e n t .  The samples were pooled  into  t h r e e g r o u p s / t r e a t m e n t and were r e f r i g e r a t e d f o r subsequent m i n e r a l a n a l y s i s . 6.2.4.5  T i s s u e samples  A minimum of 20 f i s h / t r e a t m e n t were k i l l e d at t h e end of t h e s t u d y and s t o r e d at -40°C f o r f u t u r e t i s s u e e x t r a c t i o n .  A f t e r p a r t i a l thawing, the  - 84 -  l i v e r s and k i d n e y s were removed f o r m i n e r a l a n a l y s i s and were p o o l e d , respectively, 6.2.5  i n t o f o u r and t h r e e groups per d i e t t r e a t m e n t .  S t a t i s t i c a l Analysis One-way ANOVA's were used t o a n a l y z e growth r a t e , geometric mean  w e i g h t s , f o o d i n t a k e , f o o d c o n v e r s i o n , PER, 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 a n a l y s e s (Appendix C ) .  The f o l l o w i n g f o r m u l a was used t o c a l c u l a t e  specific  growth r a t e : ( l o g n t + i - logn^ * time) x 100 where logn^ and logn^+i are t h e averaged n a t u r a l l o g wet w e i g h t s at t h e s t a r t ( t ) and t h e end (t+1) of period.  Time was measured i n d a y s .  Duncan's m u l t i p l e range t e s t and  S c h e f f e ' s t e s t (growth r a t e s ) w i t h P=0.05 were used t o t e s t f o r between means.  the  differences  The a n a l y s e s were performed u s i n g t h e s t a t i s t i c a l package SAS  (SAS 1 9 8 2 ) . 6.3  RESULTS  6.3.1  I n f l u e n c e of D i e t Treatment on C a t a r a c t  Incidence  C a t a r a c t s were observed f i r s t on day 63 i n f i s h which r e c e i v e d d i e t s t h a t c o n t a i n e d h i g h Ca and p h y t a t e ( 6 ) , low d i e t a r y Zn and i n t e r m e d i a t e p h y t a t e (11) and medium d i e t a r y Ca and h i g h p h y t a t e ( 1 4 ) , and on day 84 i n f i s h f e d d i e t 5 which c o n t a i n e d low Ca and h i g h p h y t a t e .  T a b l e 11 shows t h e  i n c i d e n c e of c a t a r a c t s on day 63 and on subsequent e x a m i n a t i o n days up t o day 119.  C a t a r a c t s were not observed i n f i s h f e d low p h y t a t e d i e t s  (1-4 and 13)  or i n f i s h f e d medium and h i g h p h y t a t e d i e t s when t h e d i e t a r y Zn l e v e l was more than 0.140 g/kg ( d i e t s 7-10, 12 and 1 5 ) . 6.3.2  I n f l u e n c e of D i e t Treatment on Chinook  6.3.2.1  Performance  F i s h growth  D i e t a r y c o m p o s i t i o n s i g n i f i c a n t l y a f f e c t e d t h e s p e c i f i c growth r a t e s and mean w e i g h t s of j u v e n i l e chinook salmon (P<0.001). " T a b l e 12 and F i g u r e 13 show t h a t growth r a t e s were reduced s i g n i f i c a n t l y i n f i s h f e d d i e t s 5-8,11  85 -  and 14.  These d i e t s , w i t h t h e e x c e p t i o n of d i e t 11, c o n t a i n e d h i g h l e v e l s of  phytic acid.  D i e t 11 c o n t a i n e d an i n t e r m e d i a t e l e v e l of p h y t i c a c i d and t h e  lowest l e v e l of d i e t a r y Z n .  W i t h i n t h e h i g h p h y t a t e groups, growth improved  when t h e d i e t a r y Zn l e v e l was i n c r e a s e d from 0.05 and 0.06 g t o 0.27 and 0.32 g/kg d i e t ( F i g s . 13 and 1 4 a ) .  Mean wet w e i g h t s of i n t e r m e d i a t e p h y t a t e - f e d  f i s h i n c r e a s e d as d i e t a r y Zn was i n c r e a s e d (Table 12; F i g s . 13 and 1 4 b ) . Moreover, h i g h d i e t a r y l e v e l s of Ga g e n e r a l l y depressed mean weight  in  chinook salmon f e d h i g h and low p h y t a t e d i e t s ( e . g . d i e t 1 v s . 2; d i e t 5 v s . 6). 6.3.2.2  Food i n t a k e , food c o n v e r s i o n and PER  D i e t a r y treatment s i g n i f i c a n t l y a f f e c t e d f i s h a p p e t i t e (P<0.05).  Fish  f e d d i e t 11 had a s i g n i f i c a n t l y h i g h e r f o o d i n t a k e than a l l o t h e r groups except f o r f i s h f e d d i e t s 1,3 and 10 ( T a b l e 1 3 ) .  O t h e r w i s e , f o o d i n t a k e was  s i m i l a r between t r e a t m e n t g r o u p s . D i e t a r y t r e a t m e n t a l s o a f f e c t e d f o o d c o n v e r s i o n and PER (Table 13; P<0.001).  F i s h f e d the high p h y t a t e d i e t s  (5-8 and 14) and t h e low Zn d i e t  (11) had lower v a l u e s f o r both v a r i a b l e s compared t o t h e o t h e r g r o u p s . D i e t a r y l e v e l s o f C a , P and Zn i n f l u e n c e d f o o d c o n v e r s i o n and PER when t h e d i e t s a l s o c o n t a i n e d i n t e r m e d i a t e or h i g h l e v e l s of p h y t i c a c i d .  For  example, i n f i s h f e d t h e low Zn, h i g h p h y t a t e d i e t s (5 and 6 ) , f o o d c o n v e r s i o n and PER were reduced when t h e d i e t s a l s o c o n t a i n e d h i g h d i e t a r y Ca (diet 5 vs. 6 ) .  F u r t h e r m o r e , when d i e t a r y Zn l e v e l s were i n c r e a s e d , f o o d  c o n v e r s i o n and PER improved at both low and high Ca l e v e l s ( d i e t 5 v s . 7; d i e t 6 v s . 8)  TABLE 11 Cataract Incidence In j u v e n i l e chinook salmon examined on days 0, 21, 42, 63, 84, 105 and 119 of Experiment 111.' Examination  Phytic Ca Diet  5  6  11  Zn  (g/kg dry  10.2  50  0.05  0.06  % lens o p a c i t y  2  acid  Fish  diet)  examined  detected  % cataracts  0  £ 25  < 50  > 75  21.1  247  84  5  95  5  -  -  129  105  16  84  10  6  -  123  119  23  77  11  9  3  230  63  2  98  2  -  197  84  6  94  6  54  105  22  78  14  8  2  54  119  26  74  12  12  2  21.1  day cataracts  -  25.2  0.034 11.6  186  63  2  98  2  -  -  29.1  0.10  189  63  6  94  6  -  -  176  84  6  94  5  1  -  59  105  14  86  8  6  -  59  119  H  86  8  6  -  23.2  1 Cataracts were not detected i n groups fed low phytate diets. (1-4 and 13), or medium t o high z i n c d i e t s (7-10, 12 and 15). 2  % lens opacity r e f e r s to the percentage of the lens surface estimated t o be opaque by v i s u a l  only.  observation  TABLE 12 F i n a l geometric mean wet weights, s p e c i f i c growth rates and m o r t a l i t i e s f o r j u v e n i l e chinook salmon fed t e s t d i e t s f o r 84 days 1n Experiment III.1,2  Ca Diet  1 2  Zn  Phytic  acid  (g/kg dry d i e t )  9.6 46  Weight  Growth rate  Mortality  (9) .  (* wet wt/day)  (*)  0.06  2.1  7.363  2.123°  0.08  2.1  6.03b  1.89  ab  3  9.9  0.27  2.1  7.58a  2.14a  4  47.1  0.22  2.1  5.17Cd  1.71  5  10.2  0.05  21.1  2.22  6  50  0.06  21.1  1.649  0.35  f  0.72  ab  d  0.76 1.1 2.3 1.1 3.0  de  17.8  7  9.8  0.27  21.1  2.92e  1.04C  5.3  8  48.6  0.32  21.1  2.70e  1.02C  7.2  9  5.6  0.15  11.6  5.86b  1.85  ab  10  52.1  0.16  11.6  4.73  1.71  ab  11  25.2  0.034  11.6  1.419  12  29.1  0.28  11.6  5.22C  1.73  13  28.3  0.15  5.29C  1.76ab  2.3  14  29.1  0.10  23.2  2.54ef  0.89C  7.9  15  29.1  0.17  11.6  4.72"  1.66b  2.3  0.30  0.01  2 SF.M  0.06  cd  0.14e ab  3.8 37.3 1.5  1 A one-way ANOVA with d i e t as the f a c t o r indicated a s i g n i f i c a n t d i e t e f f e c t with P<0.001 f o r mean weight and growth r a t e . superscript  Within a column, d i e t groups with the same  l e t t e r form a homogeneous subset.  (Duncan's m u l t i p l e range t e s t with  P=0.05; Scheffe's test with P=0.05 f o r growth r a t e s ) . 2  I n i t i a l mean weight was 1.28 t 0.03 g.  WEIGHT OF CHINOOK  1  2  3  4  5  6  7  8  9  SALMON  10  11  12  13  14  IS  DIET  Fig. 13. Wet weight* of chinook aabmon at day» and 84 H . Group* with a common euperecrip letter were not eignifiecuitly different Note that fieh fed diet 11 (*) lott weight between day* 42 an  14a. F i g . 14a.  14b.  R e p r e s e n t a t i v e p a i r s of f i s h f e d d i e t s 1,5 and 7 (top t o bottom) f o r 84 days i n Experiment I I I .  Note marked r e d u c t i o n i n s i z e of f i s h f e d h i g h l e v e l s of p h y t i c  r e l a t i v e t o f i s h f e d d i e t 1 (low p h y t i c a c i d ) .  A l s o note t h e a m e l i o r a t i n g e f f e c t  acid of  h i g h d i e t a r y z i n c w i t h i n t h e h i g h p h y t a t e f i s h (7 v s . 5 ) . F i g . 14b.  R e p r e s e n t a t i v e p a i r s o f f i s h f e d i n t e r m e d i a t e p h y t a t e d i e t s 11,15 and 12 (top t o bottom) f o r 84 days i n Experiment I I I .  Note i n c r e a s e d f i s h s i z e as d i e t a r y z i n c (g/kg d i e t ) was  i n c r e a s e d from 0.034 t o 0.17 t o 0 . 2 8 .  TABLE 13 Mean d a i l y food consumption, food conversion and p r o t e i n e f f i c i e n c y r a t i o f o r groups of j u v e n i l e chinook salmon fed t e s t d i e t s between days 0 and 84 i n Experiment I I I . l  Ca Diet  1 2  Zn  Phytic acid  (g/kg dry d i e t )  9.6 46  Protein efficiency Food intake  Food conversion  ratio  0.06  2.10  1.66ab  1.173b  2.363b  0.08  2.1  1.58bc  1.133b  2.193bc  3  9.9  0.27  2.1  1.66ab  1.243  2.443  4  47.1  0.22  2.1  l,47bcd  1.073b  2.093bc  5  10.2  0.05  21.1  1.4lbcd  0.43d  0.85  6  50  0.06  21.1  1.52bcd  0.20e  0.38f  e  7  9.8  0.27  21.1  1.3icd  0.69  8  48.6  0.32  21.1  1.26d  0.71C  1.39d  9  5.6  0.15  11.6  1.38bcd  1.243  2.453  c  1.38d  10  52.1  0.16  11.6  1.67ab  0.96  11  25.2  0.034  11.6  1.903  0.06e  0.12f  12  29.1  0.28  11.6  l,44bcd  1.113b  2.24 bc  13  28.3  0.15  1.45bcd  1.123b  2.203bc  14  29.1  0.10  23.2  1.45bcd  0.53Cd  1.05de  15  29.1  0.17  11.6  l^gbcd  1.01b  l.ggbc  0.18  0.12  0.24  2 SEM  0.06  b  1.90C  a  1 A one-way ANOVA with d i e t as the f a c t o r indicated a s i g n i f i c a n t d i e t e f f e c t with P<0.05 f o r food Intake and P<0.001 f o r food conversion and p r o t e i n e f f i c i e n c y r a t i o . Within a column, d i e t groups with the same s u p e r s c r i p t l e t t e r were not s i g n i f i c a n t l y d i f f e r e n t (Duncan's m u l t i p l e range t e s t with P=0.05).  - 91 -  6.3.3  I n f l u e n c e of D i e t Treatment on P r o x i m a t e Composition The proximate c o m p o s i t i o n of chinook salmon was i n f l u e n c e d by d i e t a r y  t r e a t m e n t (P<0.001).  T a b l e 14 shows t h a t f i s h f e d d i e t s c o n t a i n i n g h i g h  p h y t i c a c i d ( d i e t s 5-8 and 1 4 ) , o r i n t e r m e d i a t e p h y t i c a c i d c o u p l e d w i t h  low  d i e t a r y z i n c ( d i e t 1 1 ) , had s i g n i f i c a n t l y h i g h e r l e v e l s (%) of m o i s t u r e and p r o t e i n and lower l i p i d l e v e l s i n t h e body than noted i n t h e r e m a i n i n g groups.  The two e x c e p t i o n s were f o r f i s h f e d d i e t s 6 and 8 i n which p r o t e i n  was not h i g h e r than t h a t i n t h e o t h e r g r o u p s . i n f i s h f e d d i e t s 6 and 1 1 .  Body ash c o n t e n t was h i g h e s t  F i s h f e d d i e t s c o n t a i n i n g h i g h l e v e l s of Ca and  P w i t h 21.1 g p h y t i c a c i d / k g ( d i e t s 6 and 8 ) , o r medium Ca w i t h 23.2 g p h y t i c a c i d / k g ( d i e t 14) had h i g h e r ash l e v e l s than t h e i r r e s p e c t i v e c o u n t e r p a r t s . In t h e low p h y t a t e groups (1-4 and 1 3 ) , ash l e v e l s were h i g h e s t i n f i s h  fed  high d i e t a r y l e v e l s of Ca ( d i e t s 2 and 4 ) . 6.3.4  I n f l u e n c e of D i e t Treatment on General  Health  A p a t h o l o g i c a l e x a m i n a t i o n of 10 f i s h from each of t h e 15 t r e a t m e n t groups r e v e a l e d no a b n o r m a l i t i e s i n h a e m a t o c r i t , haemoglobin, b l o o d c e l l count or s i z e and shape of b l o o d c e l l s .  Gross e x a m i n a t i o n showed no i n t e r n a l  or external a b n o r m a l i t i e s . M o r t a l i t y was h i g h e s t i n t h e h i g h p h y t a t e - f e d groups ( d i e t s 6-8 and 14) and i n f i s h f e d d i e t 1 1 , w i t h m o r t a l i t y r a n g i n g from 5.3% i n f i s h f e d d i e t 7 t o 37.3% i n f i s h f e d d i e t 11 (Table 1 2 ) . r e m a i n i n g groups.  M o r t a l i t y was n e g l i g i b l e i n t h e  - 92 -  TAELE 14 F i n a l whole body proximate composition of j u v e n i l e chinook salmon f e d t e s t d i e t s f o r 84 days i n Experiment I I I . l % of dry matter2 Zn  Ca Diet  1 2  3 4  Phytic acid  (g/kg d r y d i e t )  9.6 46 9.9 47.1  Moisture(Z)  Protein  Lipid  Ash  0.06  2.1  76.09  66.9^  21.9"  0.08  2.1  76.7efg  68.0°cd  20.8^  11.3  0.27  2.1  75.99  66.6 *  d  2i.8«  lO.ie  2.1  77.4de  65. l  e  19.2=  a  72.9  a  11.49  11.9*.  b  10.89"  17.4a  0.22  5  10.2  0.05  21.1  81.8  6  50  0.06  21.1  80.7b  68.8  7  9.8  0.27  21.1  79.7C  72.1*  8  48.6  0.32  21.1  79.7C  68.6 =  9  5.6  0.15  11.6  78.2  d  15. ie  b  b  68.1 <b  0.16  11.6  76.8 9  67.6 «l  11  25.2  0.034  11.6  81.63  72.8a  12  29.1  0.28  11.6  77.3 ef  13  28.3  0.15  9.8"  14.6  b  11.4  d  e  20.1°c  f  65.8  e  20.1  71.5  a  11.49  b  18.9  29.1  0.10  23.2  79.6  15  29.1  0.17  11.6  77.6 e.  69.0  0.62  1.1  C  d  13.2C  12.0«»  d  65.2  14  2 SEM3  12.2  d  76.4 9  11.8*  - lO.ie  d  52.1  0.06  d  d  13.7'  10  ef  9.8*  bc  12. I d 13.2=  cd  0.90  10.9*6 0.78  1 Twenty gram samples of f i s h were removed from each r e p l i c a t e group per d i e t treatment (10 t o 20 f i s h / g r o u p ) and carcasses were stored a t -40*C before a n a l y s i s . 2 A one f a c t o r ANOVA with d i e t as the f a c t o r i n d i c a t e d a s i g n i f i c a n t d i e t e f f e c t w i t h P<0.001 f o r m o i s t u r e , p r o t e i n l i p i d and ash. W i t h i n a column values w i t h t h e same superscript  l e t t e r were not s i g n i f i c a n t l y d i f f e r e n t (Duncan's m u l t i p l e range t e s t w i t h  P-0.05). 3 Mean percentages ± 2SEM f o r body moisture, p r o t e i n , l i p i d and ash of Chinook a t t h e beginning of t h e study (n=4) were, r e s p e c t i v e l y , 80.8 ± 0.59, 76.5 i 3.4. 12.1 ± 1.5 and 9.7 * 0.76.  - 93 -  6.3.5  I n f l u e n c e of D i e t Treatment on H i s t o p a t h o l o g y No p a t h o l o g i c a b n o r m a l i t i e s were noted i n t h e s t r u c t u r e of t h e l i v e r ,  g i l l or pancreas.  S t r u c t u r a l anomalies were seen i n t h e k i d n e y and p y l o r i c  caeca. 6.3.5.1  Kidney  N e p h r o c a l c i n o s i s was observed on days 50 and 84 i n f i s h f e d more than 25.0 g Ca/kg d i e t  (Table 1 5 ) .  Except f o r f i s h f e d d i e t 15, t h e i n c i d e n c e of  c a l c i n o s i s i n c r e a s e d between days 50 and 8 4 . 6.3.5.2  Pyloric  caeca  The p y l o r i c c a e c a l e p i t h e l i a l c e l l s of f i s h f e d d i e t s c o n t a i n i n g >^ 11.6 g p h y t i c a c i d t o g e t h e r w i t h >_ 9.8 g Ca/kg e x h i b i t e d h y p e r t r o p h y and v a c u o l i z a t i o n of t h e c y t o p l a s m (Table 1 5 ) . 84 i n f i s h f e d d i e t s 5 - 8 , 1 0 , 1 1 , 1 4 and 15.  T h i s was observed on days 50 and F i s h f e d d i e t 12 showed e f f e c t s on  day 84 o n l y . 6.3.6  I n f l u e n c e of D i e t Treatment on Blood and T i s s u e M i n e r a l  6.3.6.1  Levels  Whole blood  D i e t a r y t r e a t m e n t s i g n i f i c a n t l y a f f e c t e d whole b l o o d l e v e l s of P, Mg, Zn and Fe (P<0.001); Mn (P<0.01) and Ca (P<0.05) w i t h t h e most marked d i f f e r e n c e s being observed i n l e v e l s of b l o o d Zn and P (Table 1 6 ) .  TABLE 15 The % i n c i d e n c e of n e p h r o c a l c i n o s i s and v a c u o l i z a t i o n of t h e p y l o r i c c a e c a i n j u v e n i l e chinook salmon at days 50 and 84 i n Experiment  Ca Diet 1 2  Zn  Phytic  Fish  acid  examined  (g/kg d r y d i e t ) 9.6 46  III. V a c u o l i z a t i o n of Nephrocalcinosis*  d 50  d 84  d 50  d 84  pyloric caeca d 50  d 84  0.06  2.1  4  6  -  -  -  0.08  2.1  4  6  100  100  -  -  -  -  •-  100  -  -  -  50  50  100  33  60  3  9.9  0.27  2.1  4  6  -  4  47.1  0.22  2.1  6  6  80  5  10.2  0.05  21.1  6  10  -  6  50  0.06  21.1  6  10  66  7  9.8  0.27  21.1  6  10  -  -  33  40  8  48.6  0.32  21.1  6  10  100  100  66  70  9  5.6  0.15  11.6  5  10  -  -  -  -  10  52.1  0.16  11.6  5  10  100  100  60  30  11  25.2  0.034 11.6  6  10  25  50  16  40  12  29.1  0.28  4  10  50  70  -  20  13  28.3  0.15  4  6  75  80  -  -  14  29.1  0.10  23.2  6  10  33  70  66  80  15  29.1  0.17  11.6  4  6  100  66  25  50  1  11.6 0.06  2  N e p h r o c a l c i n o s i s was c h a r a c t e r i z e d by c a l c i u m d e p o s i t s w i t h i n d e g e n e r a t i n g t u b u l e s o r d u c t s .  2 A minimum of 10% of the p y l o r i c caeca was a f f e c t e d w i t h 10-60% of t h e i r c e l l s showing v a c u o l i z a t i o n .  - 95 -  TABLE 16 F i n a l whole blood c o n c e n t r a t i o n s o f various minerals i n j u v e n i l e chinook salmon fed the t e s t d i e t s i n Experiment I I I . I ,  2  Phytic Ca Oiet  1 2  Zn  acid.  Mn  Ca  Zn  (g/kg d r y d i e t )  9.6 46  P  Fe  (mg/kg c r y weight)  0.06  2.1  0.24Cde  74.7 c  0.03  2.1  0.28  81.3 :  a o C ( l e  b  1287*  205.3*>  67.0° 59.0C  b(  16.1 e  1357^  205.0*  b  236.0  fl  3  9.9  0.27  2.1  0.35a  76.3 c  24.1*  14.33a  4  47.1  0.22  2.1  0.22 e  85.0°c  23.5"  1230  5  10.2  0.05  21.1  0.22  69.7=  9.99  6  50  0.06  21.1  0.24Cde  84.0°C  9.8  0.27  21.1  0.22°>  74.7 c  48.6  0.32  9  5.6  10  7  d  d e  a D C d e  187.7 c  10.29  1080  d  195.7 c  b  22.343  1070  d  b  18.3"  1130  80.3 c  0.15  ii.s  0.3oahcd  72.0*>c  52.1  0.16  11.6  0.32  a b c  85.0 c  11  25.2  0.034 11.6  0.23  d e  12  29.1  0.28  0.35  13  28.3  0.15  14  29.1  0.10  23.2  0-26  15  29.1  0.17  11.6  0.33  2 SEM  0.06  0.21  a  b c d e  a b  0.05  b  52.7 e  d  d  54.7Cde c d  159.0*  I300  b  204.7  1273  b  179.3C  49.7*  b  69.3ab  a  U.9 3  1045  d  193.7 c  58.0C  b  24.5*  1300  b  190.7*>c  59.3«i  25.2i  1287"  b  14.isf  1113C  b  20.5==  131oab  83.7 c 81.0 c 10.8  58.3C  20.2°=  81.7°c  e  22.3«  b  57.3Cd  b  b  108.3 88.7  c d  177.3  d  a  74.3  1020  0,29  11.6  b c  a  d  21.1  8  Mg  f  2.0  d  b  b  d  82.S  d  56.0<= e  198.3 =  58.0  190.7*>c  60.0=  b  d  c d  195.3 c  57.0«»  13.7  4.3  1 Blood was withdrawn from t h e caudal vessels o f at l e a s t 20 f i s h p e r d i e t treatment.  The whole blood from each f i s h was t r a n s f e r r e d  i n t o one o f t h r e e p o o l s  per d i e t treatment f o r a n a l y s i s . 2 A one f a c t o r a n a l y s i s o f v a r i a n c e with d i e t as the f a c t o r i n d i c a t e d a s i g n i f i c a n t d i e t e f f e c t w i t h P<0.001 f o r Zn, P. Fe and Mg; P<C31 f o r Kn and P<0.05 f o r Ca. W i t h i n a column, values w i t h a common s u p e r s c r i p t  l e t t e r were 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 (Duncan's new m u l t i p l e range t e s t w i t h ?=0.05).  -96  -  The lowest blood Zn l e v e l s were noted i n f i s h which were f e d d i e t s c o n t a i n i n g 0 . 0 5 - 0 . 0 6 g of Zn c o u p l e d w i t h 21.1 g of p h y t i c a c i d / k g ( d i e t s 5 and 6) and i n f i s h f e d d i e t 11 which c o n t a i n e d 0.034 g Zn and 11.6 g p h y t i c acid/kg.  The h i g h e s t b l o o d Zn c o n c e n t r a t i o n s were observed i n f i s h which  were f e d d i e t s c o n t a i n i n g at l e a s t 0.15 g Zn c o n c u r r e n t l y w i t h 2 . 1 - 1 1 . 6 g of p h y t i c a c i d / k g ( d i e t s 3 , 4 , 7 , 9 , 1 2 and 1 3 ) .  The two e x c e p t i o n s were i n f i s h  f e d d i e t s 10 and 15 where b l o o d Zn l e v e l s were s i g n i f i c a n t l y lower than Zn l e v e l s found i n f i s h f e d d i e t s 3,12 and 1 3 . F i s h f e d d i e t 11 or t h e h i g h p h y t a t e d i e t s (5-8 and 14) had s i g n i f i c a n t l y reduced P c o n c e n t r a t i o n i n t h e b l o o d . B l o o d Ca was not s i g n i f i c a n t l y a f f e c t e d except i n f i s h which d i e t 11.  ingested  These f i s h had a much h i g h e r c o n c e n t r a t i o n of Ca i n the blood  compared t o a l l o t h e r g r o u p s . Lowest b l o o d Mg l e v e l s were observed i n f i s h f e d t h e h i g h p h y t a t e d i e t s 6,7 and 8 and i n f i s h f e d d i e t 10.  The l a t t e r d i e t c o n t a i n e d a h i g h Ca and  an i n t e r m e d i a t e p h y t i c a c i d c o n t e n t .  F i s h f e d d i e t s c o n t a i n i n g low and  i n t e r m e d i a t e l e v e l s of p h y t i c a c i d t o g e t h e r w i t h low Ca ( d i e t s 1,3 and 9) had h i g h e r blood Mg l e v e l s than t h e i r c o u n t e r p a r t s where the d i e t a r y Ca l e v e l was high ( d i e t s 2,4 and 10, r e s p e c t i v e l y ) . 6.3.6.2  Liver  T a b l e 17 i l l u s t r a t e s t h a t d i e t a r y t r e a t m e n t s i g n i f i c a n t l y a f f e c t e d l e v e l s of P, Mg, Z n , Fe, Cu (P<0.001); Ca (P<0.01) and Mn (P<0.05).  liver  The most  marked d i f f e r e n c e s were observed i n c o n c e n t r a t i o n s of Z n , Fe and Cu. F i s h f e d d i e t s 3,4 and 12 had h i g h e r l i v e r Zn l e v e l s than a l l groups except those f e d d i e t s 7,13 and 14. f i s h f e d d i e t s 2,5,6 and 1 1 .  other  Lowest Zn l e v e l s were observed i n  - 97 -  TABLE 17 F i n a l l i v e r c o n c e n t r a t i o n s o f v a r i o u s m i n e r a l s i n j u v e n i l e chinook salmon f e d t h e t e s t d i e t s i n Experiment I I I . I ,  2  Phytic  (g/kg d r y d i e t )  MH V  9.6 0.06  2.1  5.2 c  872.5"c  0.08  2.1  4.5*>c  1873.3 bc  3  9.9 0.27  2.1  4.6"c  4  47.1 0.22  2.1  5  10.2 0.05  6  50  Ca Diet  1  46  2  Zn  acid  Ca  Zn  P  Cu  Fe  Mg  (mg/kg d r y weight)  b  a  88.6Cd  HQSQCde  12.8bc  I39.5ef 712.50C  8.7C  1 3 6 . 2 ^ 734.23b  H525bcd  I3.4&C  176.2<* 722.5bc  81.0Cde 12025OC  807.5bc  126.5  5.6°c  1642.50C  127.5*  134503  13.lbc  188.7de 7 9 7 . 5 3  21.1  6.6°c  1395.0°c  66.9e  10230«  34.5»  301.7C  715.Obc  0.06  21.1  5.6°c  2360.0^  79.a1e  10495de  30.0*  395.5°  607.5°"  7  9.8 0.27  21.1  8.0  1190.0bc  113.5 b  125003b  17.7b  245.5<1  717.5bc  8  48.6 0.32  21.1  6.1"=  1950. abc  96.0  12150bc  18.00  207.2<le 685.0°c  9  5.6 0.15  ills  6.1°c  665.0=  101.Sbc  •io  52.1 0.16  11.5  4.gbc  1152.50C  ab  0  a  a  87.7cd  11  25.2 0.034 11.6  3.9  12  29.1 0.28  4.4°c  1215.0°  13  28.3 0.15  4.8°c  2075.0 b= 1 1 6 . 2  14  29.1 0.10  23.2  15  29.1 0.17  11.6  11.6 0.06  10. l  a  4.5°c  83.7Cde  a  3303.3  C  c  125.7  a  3372.5  a  1847.5abc 937.5  o c d  a  aD  a  112.7 ° 90. I d  158.2e^ 705.0bc  1195QOC  110.2  I0.3  b c  f  732.  29.7*  607.7*  10307<*e  12.5bc  147.2** 480.O  12275bc  II.7DC  ISl.oef 702.5bc  121C0bc  I7.5  391Q  f  b  HZSObcde lo.abc 777  4.7  282.7C  465.7e e  730.03b  146.2ef 647.5cd 42.9  2 SEM  2.3  1 L i v e r s were e x t r a c t e d  from a minimum o f 20 f i s h per d i e t t r e a t m e n t .  from each f i s h was t r a n s f e r r e d  12.9  114C0bct!e 13.8bc  46.4  The l i v e r  i n t o one o f f o u r pools p e r d i e t treatment f o r  analysis. 2 A one f a c t o r a n a l y s i s o f v a r i a n c e w i t h d i e t as the f a c t o r i n d i c a t e d a s i g n i f i c a n t d i e t e f f e c t w i t h P<0.001 f o r Zn, P, Cu, Fe and Mg; P<0.01 f o r Ca and P<0.05 f o r Hn.  W i t h i n a column, values w i t h a common s u p e r s c r i p t  significantly  l e t t e r were n o t  d i f f e r e n t (Duncan's new m u l t i p l e ranee t e s t w i t h P=0.05).  - 98 -  L i v e r Fe l e v e l s were h i g h e s t i n f i s h f e d d i e t 11 which c o n t a i n e d t h e l o w e s t Zn content f o l l o w e d by f i s h which i n g e s t e d t h e h i g h p h y t a t e d i e t s  5,6  and 14. L i v e r Cu was h i g h e s t i n f i s h f e d d i e t s 5,6 and 1 1 . Magnesium l e v e l s were s i g n i f i c a n t l y reduced i n f i s h f e d d i e t s 11 and 12.  F i s h f e d d i e t 4 had h i g h e r l i v e r Mg c o n t e n t than a l l groups e x c e p t those  f e d d i e t s 2,10 and 14. 6.3.6.3  Kidney  D i e t a r y treatment s i g n i f i c a n t l y a f f e c t e d k i d n e y l e v e l s of C a , P, Z n , Mn (P<0.001), Fe (P<0.01) and Mg (P<0.05; T a b l e 1 8 ) . The h i g h e s t l e v e l s of k i d n e y Ca and P were seen i n f i s h f e d d i e t s c o n t a i n i n g 4 8 . 6 - 5 2 . 1 g Ca w i t h 1 1 . 6 - 2 1 . 1 g p h y t i c a c i d / k g ( d i e t s 6,8 and 10) and i n f i s h f e d d i e t 14 which c o n t a i n e d 29.1 g Ca and 23.2 g p h y t i c a c i d / k g . Kidney Zn l e v e l s were h i g h e s t i n f i s h f e d d i e t s 4,8 and 12, and lowest i n f i s h f e d d i e t s 1 and 5. L e v e l s of k i d n e y Mg and Fe were s i g n i f i c a n t l y h i g h e r i n f i s h f e d d i e t 11 compared t o a l l o t h e r g r o u p s . 6.4 6.4.1  DISCUSSION Cataract  Incidence  C a t a r a c t s appeared i n f i s h which were f e d 21-23 g p h y t i c a c i d w i t h 0.05-0.100 g Zn/kg d i e t  (Table 1 1 ) .  C a t a r a c t s were a l s o d e t e c t e d i n  f e d d i e t 11 which c o n t a i n e d 11.6 g p h y t i c a c i d and 0.034 g Z n / k g .  fish  The  i n c i d e n c e of c a t a r a c t s as a r e s u l t of d i e t 11 was o n l y 2% at day 63 w i t h no f u r t h e r c a t a r a c t s a p p e a r i n g at subsequent w e i g h i n g s .  However, between days  63 and 84 t h e r e was high m o r t a l i t y i n f i s h f e d d i e t 11 (Table 12) and 21 of  - 99 -  TABLE 18 F i n a l kidney concentrations  o f various minerals i n j u v e n i l e chinook salmon f e d t h e  t e s t d i e t s i n Experiment I I I . I ,  2  Phytic Ca  2  3  2.1  3.29  0.08  2.1  7.0  9.9 0.27  2.1  2.79  46  47.1  0.22  2.1  5  10.2  0.05  21.1  6  50  0.06  21.1  8 9  9.8 0.27 21.1  Zn  6.9  15606  def  def  99.76  5.16*9  18836  11.6  9.9=  29.1  0.28 11.6  13  28.3  0.15  14  29.1  0.10  29.1  2 SEH  0.06 23.2  0.17 11.6  21.0  de  3.8*9 d  9.7= 7  . def 9  2.5  b  d  d  1037= e  176.0 °  37800*  238.0= e  I453  b  142.7°cde  14133"  338.3°  1000  d  150.7°cd  28833°=  222.7 e  d  f  def  2100Q d  162.7°=  27733°  144.3  12773=  a  b c d  180.3 ° 26.6  ab  33433  b  d  b  d  9 468.0  1417  a  b  I800  295.7bcde  183006f9h 297.7 = e b  d  a  b  d  1240  b c d  1273 =  307.7bcd  1457°  21833 6r' 247.7= 6  1203  30667  b  d  3540  d  d  21600 6f  3  3783e  6379  b  1293 =  259.7bcde  208.3  d  215.7e  1153°=°  16700 9"  g.7bcce a  15333=  de  b c d e  d  1423  d  9.0=  298.0  14033"  b  1307 =  320.7°=  de  13300= U8.0= e  a  228.7^6  d  b  1380 =  1163 c  13  272670  d  Z4133=  de  267.6 = 6  34000 ° 122.3=  1037e  12  Ng  257.7°=  14900"  158339"  a  12.8°=  3.39  0.034 11.6  Fe  229QQ  ab  2000®  f  5.0e 9  11.6  25.2  155.0°= 184.0  5.6 0.15  11  c d e  121.3  9 14833=  41100*  0.16  d  11236  14.8°  52.1  P  109.5 e  9 15767=  0.32 21.1  48.6  10  15  Ca  (mg/kg dry weight)  0.06  9.6  4  7  Mn  acid  (g/kg d r y d i e t )  Diet  1  Zn  d  50.8  1 Kidneys were e x t r a c t e d from a minimum o f 20 f i s h per d i e t t r e a t m e n t .  b c d  211.5  The kidney  from each f i s h was t r a n s f e r r e d i n t o one o f three pools p e r d i e t t r e a t m e n t f o r analysis. 2 A one f a c t o r a n a l y s i s o f v a r i a n c e with d i e t as the f a c t o r i n d i c a t e d a s i g n i f i c a n t d i e t e f f e c t w i t h P<0.001 f o r Mn, Ca, Zn and P; P<0.01 f o r Fe and P<0.05 f o r Mg. W i t h i n a column, values with a common s u p e r s c r i p t l e t t e r were not s i g n i f i c a n t l y d i f f e r e n t (Duncan's new m u l t i p l e range t e s t w i t h P=0.C5).  - 100 -  '  t h e f i s h which d i e d d u r i n g t h i s t i m e ( r e p r e s e n t i n g 40% of t h e m o r t a l i t y ) had bilateral cataracts.  C a t a r a c t s c o u l d not be induced i n f i s h f e d h i g h r a t i o s  of Ca t o Zn a l o n e , but i t would appear t h a t an i n t e r m e d i a t e l e v e l of  phytic  a c i d (11.6 g/kg) can induce c a t a r a c t f o r m a t i o n i n j u v e n i l e chinook salmon  if  t h e d i e t s i m u l t a n e o u s l y c o n t a i n s a m a r g i n a l l e v e l of Z n . C a t a r a c t s d i d not appear i n t h e h i g h p h y t a t e groups which were f e d d i e t s c o n t a i n i n g 0.27-0.32 g Zn/kg ( d i e t s 7 and 8 ) .  T h i s c o n f i r m s t h e r e s u l t s of  experiment I and f u r t h e r demonstrates t h e importance of Zn i n m a i n t a i n i n g lens transparency. In both t h e p r e s e n t experiment and i n experiment I , c a t a r a c t s were seen f i r s t on day 63 i n f i s h f e d d i e t s c o n t a i n i n g h i g h Ca and h i g h p h y t i c a c i d and then on day 84 i n f i s h f e d low Ca and h i g h p h y t i c a c i d .  In t h e p r e s e n t  s t u d y , f i s h f e d a medium Ca d i e t w i t h h i g h p h y t i c a c i d ( d i e t 14) a l s o e x h i b i t e d c a t a r a c t s on day 6 3 .  These r e s u l t s support t h e c o n t e n t i o n t h a t  i n c r e a s e d l e v e l s of d i e t a r y Ca and P, i n t h e presence of h i g h p h y t i c will  acid,  aggravate Zn d e f i c i e n c y symptoms i n j u v e n i l e chinook salmon and t h u s  promote c a t a r a c t o g e n e s i s . 6.4.2  Chinook Performance  6.4.2.1  F i s h growth  Growth was reduced s i g n i f i c a n t l y i n chinook salmon which were f e d d i e t s c o n t a i n i n g at l e a s t 21.1 g p h y t i c a c i d / k g , o r 0.034 g Zn c o n c u r r e n t l y w i t h 11.6 g p h y t i c a c i d / k g .  Moreover, f i s h f e d >_ 28.3 g Ca/kg ( d i e t s  2,4,6,10-13  and 15) had s i g n i f i c a n t l y lower mean w e i g h t s than d i d t h e i r low Ca counterparts.  The one e x c e p t i o n was i n f i s h f e d d i e t 8.  In t h i s case  appeared t h a t the h i g h Zn c o n t e n t i n d i e t 8 (0.32 g/kg) compensated f o r e x c e s s Ca such t h a t growth was not f u r t h e r reduced r e l a t i v e t o t h a t of  it the fish  - 101 -  f e d d i e t 7.  D i e t a r y Zn l e v e l s ^> 0.17 g/kg appeared t o enhance growth  in  chinook salmon which were f e d d i e t s c o n t a i n i n g >^ 11.6 g p h y t i c a c i d / k g .  This  e f f e c t was most d r a m a t i c i n t h e i n t e r m e d i a t e p h y t a t e groups (11,12 and 1 5 ) . F u r t h e r , t h e r e s u l t s suggest t h a t i t i s p o s s i b l e (under t h e c o n d i t i o n s  of  t h i s s t u d y ) t o p a r t i a l l y compensate f o r t h e presence of c e r t a i n l e v e l s of p h y t i c a c i d by i n c r e a s i n g t h e Tevel of Zn s u p p l e m e n t a t i o n . 6.4.2.2  Food i n t a k e , food c o n v e r s i o n and PER  Chinook salmon which were f e d d i e t 11 had t h e h i g h e s t f o o d i n t a k e but t h e lowest f o o d c o n v e r s i o n and PER.  T h i s can l i k e l y be a t t r i b u t e d t o poor  Zn and p r o t e i n a v a i l a b i l i t y due t o t h e f o r m a t i o n of i n s o l u b l e Zn- and p r o t e i n - p h y t a t e complexes i n t h e i n t e s t i n a l  lumen.  For example,  increasing  Zn c o n c e n t r a t i o n from 0.034 g t o 0.170 g/kg ( d i e t 15) d r a m a t i c a l l y improved f o o d and p r o t e i n c o n v e r s i o n . high phytate groups.  Poor c o n v e r s i o n r a t e s were a l s o observed i n t h e  T h i s e f f e c t was a l l e v i a t e d somewhat by i n c r e a s i n g t h e  l e v e l of d i e t a r y Zn from 0.05 and 0.06 g t o 0.27 and 0.32 g/kg, r e s p e c t i v e l y ( d i e t 5 v s . 7; d i e t 6 v s . 8 ) .  It i s i n t e r e s t i n g that the poorest  conversion  r a t i o s were observed f o r f i s h which a l s o c o n t a i n e d t h e lowest l e v e l s of Zn and P.  blood  P h y t i c a c i d i s known t o b i n d w i t h both of t h e s e m i n e r a l s and  reduce t h e i r a v a i l a b i l i t y f o r a b s o r p t i o n .  F u r t h e r m o r e , Zn and P d e f i c i e n c i e s  i n rainbow t r o u t are c h a r a c t e r i z e d by poor growth and reduced f o o d c o n v e r s i o n (Watanabe e t a l . 1980; NRC 1981).  As mentioned i n experiment I  (section  4 . 4 . 2 . 2 ) , t h e lowered food and p r o t e i n c o n v e r s i o n i n t h e high p h y t a t e - f e d f i s h may a l s o have been p a r t l y due t o t h e f o r m a t i o n of  phytate-protein  complexes and/or poor n u t r i e n t a b s o r p t i o n i n t h e p y l o r i c c a e c a l  region.  - 102 -  6.4.3  Proximate Composition As i n experiment I , f i s h f e d t h e h i g h p h y t a t e d i e t s e x h i b i t e d the  h i g h e s t l e v e l s of body m o i s t u r e and p r o t e i n and t h e lowest l i p i d  levels.  P r e v i o u s s t u d i e s (Groves 1970; R e i n t z and H i t z e l 1980) have demonstrated t h a t body f a t  increased with increased f i s h s i z e .  Thus t h e marked d i f f e r e n c e s  in  f i s h s i z e and f o o d c o n v e r s i o n i n t h e h i g h p h y t a t e g r o u p s , compared t o t h e o t h e r g r o u p s , can l i k e l y account f o r t h i s d i s s i m i l a r response i n p r o x i m a t e composition. Ash l e v e l s were g e n e r a l l y r e l a t e d t o d i e t a r y ash c o n t e n t .  Fish fed  d i e t s which were h i g h i n Ca and P c o n t a i n e d h i g h e r ash c o n t e n t s than d i d t h e i r c o u n t e r p a r t s which were f e d d i e t s low i n Ca and P.  In most c a s e s , t h e  presence of h i g h p h y t i c a c i d s i g n i f i c a n t l y i n c r e a s e d body ash c o n c e n t r a t i o n compared t o t h e c o r r e s p o n d i n g low p h y t a t e d i e t s ( 1 , 2 and 4 v e r s u s 5,6 and 8, respectively). 6.4.4  Fish Health The g r o s s e x a m i n a t i o n of f i s h at t h e end of t h e t r i a l y i e l d e d no  abnormalities.  The i n c i d e n c e of oedema i n t h e high p h y t a t e - f e d f i s h  experiement I ( s e c t i o n 4 . 3 . 4 ) was not e v i d e n t i n t h i s s t u d y .  in  T h i s may have  been due t o t h e s l i g h t l y lower p h y t i c a c i d l e v e l s i n t h i s study ( 2 1 . 1 and 23.2 g/kg d i e t ) v e r s u s the 25.8 g/kg used i n experiment  I.  The amount of d i e t a r y Zn and p h y t i c a c i d appeared t o i n f l u e n c e m o r t a l i t y w i t h t h e h i g h e s t number of deaths o c c u r r i n g i n f i s h f e d t h e lowest l e v e l Zn (0.034 g/kg; T a b l e 1 0 ) .  of  In t h e h i g h p h y t a t e - f e d f i s h , m o r t a l i t y was 17.8%  when Ca was 50 g and Zn was 0.06 g/kg ( d i e t 6 ) , but when Zn was i n c r e a s e d t o 0.32 g/kg ( d i e t 8) t h e m o r t a l i t y f e l l  t o 7.2%.  T h i s was a l s o observed i n  experiment I where m o r t a l i t y dropped from 44.7% t o 17.5% when d i e t a r y Zn was  - 103 -  i n c r e a s e d from 0.05 t o 0.39 g / k g .  Thus chinook s u r v i v a l appeared t o be  dependent upon adequate Zn a b s o r p t i o n .  S i m i l a r r e s u l t s were o b t a i n e d by  Ogino and Yang (1978) f o r rainbow t r o u t i n which high m o r t a l i t y r a t e s were associated with Zn-deficient 6.4.5  diets.  Histopathology  6.4.5.1  Kidney  -  A f t e r 50 days n e p h r o c a l c i n o s i s was observed i n f i s h f e d 10 of t h e 15 diets.  Any d i e t which c o n t a i n e d more than 25 g Ca/kg produced k i d n e y  calcinosis.  F u r t h e r m o r e , t h e s e d i e t s a l s o c o n t a i n e d h i g h Ca t o Mg r a t i o s  which ranged from 34:1 t o 5 5 : 1 .  The most s e v e r e l y a f f e c t e d f i s h were t h o s e  f e d d i e t s 2 , 4 , 6 , 8 and 10 which c o n t a i n e d Ca t o Mg r a t i o s i n t h e range of 46:1 to 54:1.  As mentioned p r e v i o u s l y ( 4 . 4 . 5 . 1 ) ,  shown t o cause c a l c i n o s i s i n rainbow t r o u t .  i n s u f f i c e n t d i e t a r y Mg has been The f i n d i n g s of both experiments  I and I I I suggest t h a t h i g h d i e t a r y Ca and P i n r e l a t i o n t o Mg w i l l  cause  c a l c i n o s i s i n j u v e n i l e chinook salmon. D i e t a r y p h y t i c a c i d d i d not appear t o i n f l u e n c e t h e i n c i d e n c e of calcinosis.  I t i s i n t e r e s t i n g t h a t some r e s e a r c h has shown t h a t p h y t i c  acid  can reduce t h e i n c i d e n c e of r e n a l c a l c u l i by b i n d i n g t o and then d i s s o l v i n g any e x i s t i n g Ca d e p o s i t s (Graf 1983).  M o d l i n (1980) f e d p h y t a t e d i e t s  mice and r e p o r t e d an i n v e r s e r e l a t i o n s h i p between t h e amount o f p h y t i c consumed and t h e i n c i d e n c e of n e h p r o c a l c i n o s i s .  6.4.5.2  acid  No such c o r r e l a t i o n has been  r e p o r t e d f o r s a l m o n i d s , nor was one e v i d e n t i n t h i s experiment o r experiment  to  in  I.  Pyloric  caeca  In experiment I , t h e s t r u c t u r e of t h e p y l o r i c caeca was abnormal i n f e d 25.8 g p h y t i c acid/kg ( s e c t i o n 4 . 3 . 5 . 2 ) .  In t h e p r e s e n t s t u d y ,  fish  - 104 -  h y p e r t r o p h y of t h e e p i t h e l i a l c e l l s and v a c u o l i z a t i o n of t h e c y t o p l a s m was observed as e a r l y as day 50 i n f i s h f e d more than 21 g p h y t i c a c i d / k g 15).  The p y l o r i c c a e c a of f i s h f e d d i e t s c o n t a i n i n g low p h y t i c  acid,  r e g a r d l e s s of t h e Ca l e v e l , was normal f o r j u v e n i l e chinook salmon.  However,  t h e amount of d i e t a r y Ca may have e x e r t e d an i n f l u e n c e on t h e p y l o r i c when p h y t i c a c i d was g r e a t e r than 2.1 g/kg d i e t .  For i n s t a n c e ,  caeca  abnormality  of t h e p y l o r i c c a e c a was e v i d e n t i n f i s h f e d d i e t s w i t h 11.6 g p h y t i c ( d i e t s 10-12 and 15) w i t h t h e e x c e p t i o n of f i s h f e d d i e t 9.  (Table  acid/kg  The Ca c o n t e n t  i n t h e l a t t e r d i e t was 5.6 g compared t o t h e more than 25 g Ca/kg which was p r e s e n t i n the f o r m e r d i e t s .  F u r t h e r m o r e , i n t h e h i g h p h y t a t e groups  (diets  5-8) t h e r e was a g r e a t e r i n c i d e n c e of v a c u o l i z a t i o n of t h e c y t o p l a s m i n f e d d i e t s 6 and 8 which a l s o c o n t a i n e d high l e v e l s of d i e t a r y C a .  fish  Possible  e x p l a n a t i o n s f o r t h e e f f e c t s of p h y t i c a c i d on t h e p y l o r i c caeca have been outlined previously (section 4.4.5.2). 6.4.6  Mineral A n a l y s i s  6.4.6.1  Whole blood  T a b l e 16 i l l u s t r a t e s t h a t whole blood Zn was d i r e c t l y r e l a t e d t o d i e t a r y Zn c o n c e n t r a t i o n and i n v e r s e l y r e l a t e d t o d i e t a r y p h y t i c a c i d c o n c e n t r a t i o n . S i m i l a r f i n d i n g s were r e p o r t e d i n experiment I f o r plasma Zn ( s e c t i o n 4.3.6).  Blood Zn was a l s o i n f l u e n c e d by d i e t a r y Ca c o n t e n t i n t h e low and  high p h y t a t e groups when d i e t a r y Zn was low and h i g h , r e s p e c t i v e l y .  For  example, f i s h f e d d i e t 1 which c o n t a i n e d 9.63 g Ca/kg had s i g n i f i c a n t l y h i g h e r blood Zn than d i d f i s h which were f e d d i e t 2 where Ca was i n c r e a s e d t o 46 g / k g .  S i m i l a r l y , i n t h e h i g h p h y t a t e groups ( 5 - 8 ) , f i s h f e d d i e t 7 ( 9 . 8 g  Ca/kg) had s i g n i f i c a n t l y h i g h e r b l o o d Zn than d i d f i s h f e d d i e t 8 which c o n t a i n e d 48.6 g C a / k g .  These r e s u l t s support t h e t h e o r y t h a t p h y t i c  acid  - 105 -  i n h i b i t s Zn b i o a v a i l a b i l i t y and t h a t Zn a b s o r p t i o n i s f u r t h e r reduced when t h e d i e t a r y Ca c o n t e n t i s h i g h . As. w i t h t h e plasma P r e s u l t s r e p o r t e d i n s e c t i o n 4 . 4 . 6 , d i e t a r y P c o n c e n t r a t i o n per se d i d not i n f l u e n c e blood P l e v e l s .  Phytic  acid  c o n c e n t r a t i o n however, d i d s i g n i f i c a n t l y i n f l u e n c e b l o o d P l e v e l s w i t h h i g h d i e t a r y phytate hindering P absorption.  The i n t e r m e d i a t e l e v e l of  phytic  a c i d (11.6 g/kg d i e t ) s i g n i f i c a n t l y reduced b l o o d P when d i e t a r y Zn was at 0.034 g/kg d i e t  (diet 11).  However, t h i s appears t o be a d i r e c t r e s u l t of  t h e d i e t a r y Zn l e v e l because the blood P l e v e l was s i g n i f i c a n t l y  increased  when d i e t a r y Zn was g r e a t e r than 0.100 g/kg ( d i e t s 9,10,12 and 1 5 ) . The high c o n c e n t r a t i o n of blood Ca found i n f i s h f e d d i e t 11 a l s o seems t o be a r e f l e c t i o n of t h e low d i e t a r y Zn i n t a k e .  I n c r e a s i n g t h e d i e t a r y Zn  l e v e l from 0.034 g t o 0.050 g/kg and above s i g n i f i c a n t l y reduced t h e amount of Ca i n t h e b l o o d . Blood Mg l e v e l s were i n v e r s e l y r e l a t e d t o d i e t a r y c o n c e n t r a t i o n s of Ca and p h y t i c a c i d .  For example, i n t h e low p h y t a t e g r o u p s , b l o o d Mg was  reduced s i g n i f i c a n t l y i n f i s h f e d 46-47 g Ca/kg ( d i e t s 2 and 4) compared t o f i s h f e d 9 g Ca/kg ( d i e t s 1 and 3 ) . high p h y t a t e g r o u p s . absorption.  The r e s u l t s were not as c l e a r i n t h e  Here d i e t a r y Ca d i d not s i g n i f i c a n t l y depress Mg  T h i s i s c o n s i s t e n t w i t h t h e plasma Mg r e s u l t s r e p o r t e d  s e c t i o n 4 . 4 . 6 from experiment I .  However, at t h e i n t e r m e d i a t e l e v e l  in of  p h y t i c a c i d (11.6 g / k g ) , blood Mg was s i g n i f i c a n t l y h i g h e r i n f i s h f e d d i e t 9, where t h e d i e t a r y Ca c o n t e n t was 5.6 g, than i n f i s h f e d d i e t s w i t h more than 29 g C a / k g . 6.4.6.2  Liver  In g e n e r a l , l i v e r Zn c o n c e n t r a t i o n was a l s o d i r e c t l y r e l a t e d t o d i e t a r y  - 106 -  Zn i n t a k e (Table 1 7 ) .  C a l c i u m i n t a k e d i d not a f f e c t Zn d e p o s i t i o n but p h y t i c  a c i d d i d have some i n f l u e n c e .  For i n s t a n c e , f i s h f e d d i e t 1, which c o n t a i n e d  low C a , Zn and p h y t i c a c i d , had h i g h e r l i v e r Zn l e v e l s than d i d f i s h f e d 5 which a l s o c o n t a i n e d low Ca and Zn but h i g h p h y t i c a c i d .  Similar  diet  results  were o b t a i n e d i n f i s h f e d d i e t s 4 and 8 which c o n t a i n e d high Ca and Zn but d i f f e r e d in phytic acid content.  The h i g h p h y t a t e - f e d f i s h ( d i e t 8) had  s i g n i f i c a n t l y lower l i v e r Zn than t h e low p h y t a t e - f e d f i s h  (diet 4).  These  r e s u l t s suggest t h a t Zn was c h e l a t e d t o p h y t i c a c i d and rendered u n a v a i l a b l e for  absorption. L i v e r Fe was s i g n i f i c a n t l y i n f l u e n c e d by p h y t i c a c i d c o n c e n t r a t i o n w i t h  high Fe d e p o s i t i o n o c c u r r i n g i n f i s h f e d t h e high p h y t a t e d i e t s 5,6 and 14. I t would appear t h a t p h y t i c a c i d enhanced Fe a b s o r p t i o n i n t h i s c a s e .  The  b e n e f i c i a l e f f e c t of p h y t i c a c i d on Fe a b s o r p t i o n has been documented i n o t h e r s t u d i e s (Welch and van Campen 1975; Liebman and D r i s k e l l 1979). example, M o r r i s and E l l i s if  For  (1976) showed t h a t Fe was r e a d i l y absorbed by r a t s  i t was p r e s e n t as m o n o f e r r i c p h y t a t e due t o t h e s o l u b i l i t y of t h i s complex  at pH 7 and above. The e x t r e m e l y high l i v e r Fe c o n c e n t r a t i o n i n f i s h f e d d i e t 11 i s p r o b a b l y due t o t h e low Zn c o n c e n t r a t i o n of t h e d i e t . f o r t h e same b i n d i n g s i t e s . of t h e o t h e r .  Thus an excess of one w i l l  Both Fe and Zn compete reduce t h e a b s o r p t i o n  For example, r a t s t u d i e s have shown t h a t i n c r e a s e d l e v e l s of  d i e t a r y Zn h i n d e r e d Fe a b s o r p t i o n . r e l e a s e of Fe from f e r r i t i n  T h i s was l a r g e l y due t o Zn i n h i b i t i n g the  (a major s t o r a g e form of F e ) , and/or by  i n t e r f e r i n g w i t h t h e i n c o r p o r a t i o n of Fe i n t o f e r r i t i n  (Underwood 1971).  A  s i m i l a r s i t u a t i o n o c c u r r e d i n t h e p r e s e n t s t u d y i n f i s h f e d t h e high p h y t a t e d i e t s 7 and 8.  In t h i s c a s e , t h e l a c k of i n c r e a s e d Fe d e p o s i t i o n i n t h e s e  - 107 -  f i s h may be a r e f l e c t i o n of t h e high d i e t a r y Zn c o n t e n t . The amount of Cu i n the l i v e r was i n f l u e n c e d by d i e t a r y l e v e l s o f Zn and phytic acid.  For example, the i n g e s t i o n of d i e t s c o n t a i n i n g low Zn c o u p l e d  w i t h h i g h p h y t i c a c i d ( d i e t s 5 and 6 ) , or medium p h y t i c a c i d ( d i e t 1 1 ) , r e s u l t e d i n s i g n i f i c a n t l y high Cu d e p o s i t i o n .  P h y t i c a c i d has been shown t o  enhance Cu a b s o r p t i o n by i n h i b i t i n g Zn a b s o r p t i o n ( K l e v a y 1977) and t h i s may be what o c c u r r e d h e r e .  I n c r e a s i n g Zn i n d i e t s 7 and 8 t o 0.270 and 0.320  g/kg d i e t , r e s p e c t i v e l y , s i g n i f i c a n t l y reduced l i v e r Cu compared t o t h a t of f i s h f e d d i e t s 5 and 6 which c o n t a i n e d 0.05 and 0.06 g Z n / k g , r e s p e c t i v e l y . Knox e t a l . (1984) r e p o r t e d t h a t h e p a t i c Cu l e v e l s i n rainbow t r o u t were reduced s i g n i f i c a n t l y when t h e d i e t a r y Zn l e v e l was i n c r e a s e d t o 0.500 g from 0.034 g / k g .  A d i e t a r y Zn i n t a k e of 0.120 g/kg d i e t has been shown t o  m e t a l l o t h i o n e i n s y n t h e s i s i n r a t s ( F i s c h e r et a l . 1 9 8 3 ) . were o b t a i n e d i n w i n t e r f l o u n d e r  (Shears and F l e t c h e r  Similar  induce  results  1984).  M e t a l l o t h i o n e i n , which i s a l s o p r e s e n t i n salmonids (Ley, I I I 1984; M c C a r t e r and Roch 1983), i s a m e t a l - b i n d i n g p r o t e i n t h a t p r e f e r e n t i a l l y b i n d s w i t h Cu and r e n d e r s i t u n a v a i l a b l e f o r a b s o r p t i o n (Bremner 1980).  Therefore, i t  is  q u i t e p o s s i b l e t h a t i t may have been r e s p o n s i b l e f o r r e d u c i n g l i v e r Cu i n chinook salmon. 6.4.6.3  Kidney  K i d n e y Ca l e v e l s were d i r e c t l y r e l a t e d t o d i e t a r y l e v e l s of Ca (Table 18).  F i s h f e d any d i e t high i n Ca, r e g a r d l e s s of t h e p h y t i c a c i d  e x h i b i t e d e l e v a t e d c o n c e n t r a t i o n s of k i d n e y C a .  Thus i t i s not  t h a t t h e s e f i s h were a f f l i c t e d w i t h k i d n e y c a l c i n o s i s .  level,  surprising  The h i g h e s t l e v e l  k i d n e y Ca was observed i n f i s h which were f e d d i e t s t h a t c o n t a i n e d h i g h l e v e l s of Ca and p h y t i c a c i d .  T h i s suggests t h a t p h y t i c a c i d e x a c e r b a t e s  of  - 108 -  r e n a l c a l c i n o s i s i n chinook salmon.  F i s h f e d d i e t s low i n Ca ( 1 , 3 , 5 , 7 and 9)  had s i g n i f i c a n t l y reduced k i d n e y Ca. Kidney P l e v e l s were d i r e c t l y r e l a t e d t o d i e t a r y c o n c e n t r a t i o n s of C a , P and p h y t i c a c i d . concentration.  High Ca, P and p h y t i c a c i d i n t a k e r e s u l t e d i n h i g h k i d n e y P S i m i l a r l y , f i s h f e d low Ca and P d i e t s ( 1 , 3 , 5 , 7 and 9) had  s i g n i f i c a n t l y lower k i d n e y P l e v e l s . As w i t h t h e l e v e l s of b l o o d and plasma Z n , k i d n e y Zn l e v e l s i n chinook salmon were d i r e c t l y r e l a t e d t o d i e t a r y Zn i n t a k e .  F i s h f e d d i e t s 3 and 7  however, d i d not have s i g n i f i c a n t l y h i g h e r k i d n e y Zn even though t h e d i e t a r y Zn i n t a k e was h i g h (0.27 g / k g ) .  The d i e t a r y Zn c o n c e n t r a t i o n i n d i e t 8 was  0.50 g h i g h e r than t h a t i n d i e t 7 which may be why k i d n e y Zn was s i g n i f i c a n t l y h i g h e r i n t h i s group compared t o t h a t of f i s h f e d d i e t  7.  However, t h e r e i s no apparent e x p l a n a t i o n f o r t h e low Zn l e v e l s i n f i s h diet  fed  3. The high Mg l e v e l i n t h e k i d n e y s of f i s h f e d d i e t 11 appears t o be  r e l a t e d t o the low d i e t a r y Zn i n t a k e .  Kidney Mg was reduced s i g n i f i c a n t l y  in  c o m p a r a t i v e groups when t h e d i e t a r y Zn c o n t e n t was i n c r e a s e d from 0.034 g t o 0.170 and 0.279 g/kg ( d i e t s 15 and 12, r e s p e c t i v e l y ) . Kidney Fe was a l s o g r e a t l y e l e v a t e d i n f i s h f e d d i e t 11.  As w i t h  liver  Fe t h i s may be a r e s u l t of t h e low d i e t a r y Zn i n t a k e which reduces the c o m p e t i t i o n between Zn and Fe f o r b i n d i n g s i t e s . 6.5  CONCLUSION D i e t a r y p h y t i c a c i d at c o n c e n t r a t i o n s of 1 1 . 6 - 2 3 . 2 g/kg d i e t  c a t a r a c t f o r m a t i o n i n j u v e n i l e chinook salmon when the d i e t c o n t a i n e d 0.034-0.100 g Z n / k g .  induced  concurrently  Furthermore, cataractous f i s h contained the  l o w e s t l e v e l s of Zn i n t h e b l o o d and l i v e r .  This strengthens the contention  - 109 -  t h a t Zn p l a y s a major r o l e i n l e n s n u t r i t i o n .  In a d d i t i o n t o c a t a r a c t s ,  i n c r e a s e d m o r t a l i t y , decreased growth, f o o d c o n v e r s i o n and PER, and p y l o r i c caecal  i r r e g u l a r i t i e s were e v i d e n t i n f i s h f e d t h e high p h y t a t e d i e t s .  N e p h r o c a l c i n o s i s was observed i n chinook salmon f e d d i e t s c o n t a i n i n g at 25.2 g C a / k g .  least  - 110 -  CHAPTER 7 7.0  SUMMARY AND CONCLUSION Bilateral  l e n s c a t a r a c t s were induced i n j u v e n i l e chinook salmon which  were f e d d i e t s c o n t a i n i n g a minimum of 11.6 g p h y t i c a c i d / k g (18.0 g sodium p h y t a t e ) and a maximum of 0.100 g Z n / k g .  Moreover, t h e s e v e r i t y of  and t h e number of f i s h a f f e c t e d i n c r e a s e d w i t h t i m e .  opacities  High d i e t a r y l e v e l s of  Ca and P (50 g/kg) aggravated t h e e f f e c t s of p h y t i c a c i d on c a t a r a c t o g e n e s i s whereas a d i e t a r y Zn i n t a k e of at l e a s t 0.150 g/kg p r e v e n t e d c a t a r a c t formation. C a t a r a c t f o r m a t i o n a l s o appeared t o be dependent upon t h e t i m e and d u r a t i o n of exposure t o a c a t a r a c t o g e n i c d i e t .  For example, f i s h  fed  c a t a r a c t o g e n i c d i e t s f o r 12-15 weeks developed o p a c i t i e s by the n i n t h week. However, when t h e d i e t was f e d at d i f f e r e n t  i n t e r v a l s (experiment I I ) ,  was a d e l a y between exposure t o the d i e t and t h e appearance of cataracts.  there  the  Such a phenomenon may have o c c u r r e d i n t h e 1981 c a t a r a c t outbreak  i n B.C. and Washington S t a t e chinook salmon s t o c k s .  C a t a r a c t s were d e t e c t e d  i n t h e s e f i s h s h o r t l y b e f o r e t h e y were r e l e a s e d as s m o l t s .  Furthermore, the  s i z e of the a f f e c t e d f i s h was s i m i l a r t o t h e n o n - a f f e c t e d f i s h .  In c o n t r a s t ,  t h e c a t a r a c t o u s f i s h i n experiments I and I I I were d r a s t i c a l l y reduced s i z e compared t o t h e n o n - c a t a r a c t o u s f i s h .  The c a t a r a c t o u s f i s h  in  in  experiment I I however, were much l a r g e r than t h o s e of t h e o t h e r s t u d i e s . T h i s experiment showed t h a t growth r e c o v e r y was p o s s i b l e a f t e r exposure t o a poor q u a l i t y d i e t and t h e r e s u l t s suggest t h a t a s i m i l a r s i t u a t i o n may have o c c u r r e d i n 1981.  The 1981 f i s h a l s o m a i n t a i n e d f a i r l y good growth i n  of c a t a r a c t f o r m a t i o n .  spite  Thus i t i s p o s s i b l e t h a t t h e y may have been exposed  t o a poor q u a l i t y d i e t s e v e r a l weeks p r i o r t o t h e appearance of c a t a r a c t s .  - Ill -  F i s h growth, f o o d c o n v e r s i o n , PER and m o r t a l i t y were a l s o i n f l u e n c e d by p h y t i c a c i d c o n c e n t r a t i o n .  directly  F i s h which were f e d d i e t s  c o n t a i n i n g >_ 21.1 g p h y t i c a c i d / k g e x h i b i t e d reduced g r o w t h , f o o d and p r o t e i n c o n v e r s i o n and h i g h e r m o r t a l i t y .  The lowest v a l u e s o v e r a l l were observed i n  f i s h f e d 0.034 g Zn c o u p l e d w i t h 11.6 g p h y t i c a c i d / k g d i e t .  Thus c a r e f u l  c o n s i d e r a t i o n of t h e d i e t a r y Zn l e v e l i s e s p e c i a l l y i m p o r t a n t when m i n e r a l b i n d i n g agents ( e . g . p h y t i c a c i d and f i b r e ) are a l s o p r e s e n t i n t h e d i e t . The amount of Ca and P p r e s e n t i n a d i e t i s a l s o of c r i t i c a l  importance.  Based on mean weight d a t a , a Ca l e v e l of 28.3 g/kg i n a low p h y t a t e d i e t  will  s i g n i f i c a n t l y reduce growth under the c o n d i t i o n s of t h i s s t u d y .  Moreover,  e l e v a t e d l e v e l s of Ca and/or p h y t a t e w i l l compound t h i s e f f e c t .  However,  i n c r e a s i n g the d i e t a r y Zn c o n c e n t r a t i o n w i l l  a l l e v i a t e some of t h e growth  r e d u c t i o n i n f i s h f e d d i e t s c o n t a i n i n g a minimum of 11.6 g p h y t i c a c i d / k g . S t r u c t u r a l damage t o t h e p y l o r i c caeca may o c c u r i n chinook salmon which are f e d d i e t s c o n t a i n i n g >_ 21.1 g p h y t i c a c i d / k g .  Moreover, 11.6 g p h y t i c  a c i d i n t h e presence o f >^ 25 g Ca/kg may cause h y p e r t r o p h y of t h e p y l o r i c caeca.  S i m i l a r l y , n e p h r o c a l c i n o s i s may o c c u r i n chinook salmon when t h e y are  f e d d i e t s c o n t a i n i n g >1 25 g Ca/kg r e g a r d l e s s of t h e p h y t i c a c i d c o n t e n t . M i n e r a l a n a l y s e s of t h e b l o o d , plasma and l i v e r r e v e a l e d Zn t o be t h e o n l y t r a c e m i n e r a l which was s i g n i f i c a n t l y reduced i n c o n c e n t r a t i o n i n h i g h phytate-fed f i s h .  F u r t h e r m o r e , t h e lowest p l a s m a , b l o o d and l i v e r  l e v e l s occurred i n f i s h fed the c a t a r a c t o g e n i c  zinc  diets.  The f i n d i n g s suggest an e s s e n t i a l r o l e f o r Zn i n l e n s m e t a b o l i s m of j u v e n i l e chinook salmon, but more work i s r e q u i r e d t o e l u c i d a t e t h e n a t u r e of this role.  For example, s l i t - l a m p b i o m i c r o s c o p y of l e n s t i s s u e p r i o r  d u r i n g and a f t e r exposure t o a c a t a r a c t o g e n i c d i e t may r e v e a l  to,  information  -  112  -  which c o u l d p r o v i d e e a r l y warning s i g n s of c a t a r a c t f o r m a t i o n .  Further  s t u d i e s may a l s o be conducted t o more a c c u r a t e l y a s s e s s the c r i t i c a l t i m e of exposure t o a c a t a r a c t o g e n i c d i e t by chinook salmon and t o q u a n t i f y t h e maximum l e v e l s of d i e t a r y Ca, P and p h y t i c a c i d (and p o s s i b l y o t h e r m i n e r a l - b i n d i n g agents such as f i b r e ) which may be used i n chinook  salmon  d i e t s and s t i l l m a i n t a i n good growth, f o o d u t i l i z a t i o n and h e a l t h .  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A one-way ANOVA t a b l e of n a t u r a l l o g weight at day Source Diet Nrep(Diet) Residual Total  2.  DF  analyses 105.  P 0.00000 0.00883  8 9 1048 1065  A two-way ANOVA of mean wet w e i g h t s f o r days 0 t o 105 showing main e f f e c t s and i n t e r a c t i o n s . Source Ca Zn Phytic Ca*Zn Ca*Phytic Zn*Phytic Ca*Zn*Phytic Nrep(Ca*Zn*Phytic) Time Time*Ca Time*Zn Time*Phytic Time*Ca*Zn Time*Ca*Phytic Time*Zn*Phytic Time*Ca*Zn*Phytic Residual Total  DF 1 1 1 1 1 1 1 9 1 1 1 1 1 1 1 1 81 107  P .  0.00005 0.21846 0.00000 0.70450 0.00072 0.30216 0.15041 0.64290 0.00000 0.00000 0.21578 0.00000 0.78912 0.00003 0.24688 0.23852  all  - 125 -  3.  A one-way a n a l y s i s of c o v a r i a n c e t a b l e f o r t h e n a t u r a l l o g of wet w e i g h t s f o r days 0-105. Source  DF  Diet Nrep(Diet) Time Time*Diet Time*Nrep(Diet) Residual Total 4.  5.  8 9 1 8 9 6430 6465  P 0.00000 0.00028 0.00000 0.00000 0.00009  A one-way ANOVA f o r f o o d c o n v e r s i o n ( F C ) , p r o t e i n e f f i c i e n c y (PER) and f o o d i n t a k e ( F I ) f o r days 0 - 2 1 . Source  DF  P (FC)  P (PER)  P (FI)  Diet Nrep(Diet) Total  8 9 17  0.01475 1.00000  0.01591 1.00000  0.01426 1.00000  A two-way ANOVA f o r FC, PER and FI f o r days 22-105. Source  DF  P (FC)  P (PER)  P (FI)  Diet Nrep(Diet) Period Period*Diet Residual Total  8 9 3 24 27 71  0.00000 0.99770 0.01133 0.56179  0.00000 0.99821 0.01278 0.58285  0.00003 0.52809 0.00000 0.00152  ratio  - 126 -  A two-way ANOVA t a b l e f o r FC, PER and FI f o r days 22-105 showing t h e main e f f e c t s and i n t e r a c t i o n s . Source  DF  P (FC)  Ca Zn Phytic Ca*Zn Ca*Phytic Zn*Phytic Ca*Zn*Phytic Nrep(Ca*Zn*Phytic) Time Time*Ca Time*Zn Time*Phytic Time*Ca*Zn Time*Ca*Phytic Time*Zn*Phytic Time*Ca*Zn*Phytic Time*Nrep(Ca*Zn*Phytic) Residual Total  1 1 1 1 1 1 1 9 1 1 1 1 1 1 1 1 9 36 71  0.26421 0.00032 0.00000 0.00335 0.22736 0.00001 0.00024 0.99893 0.00002 0.91231 0.84803 0.00011 0.17260 0.42076 0.03622 0.10244 0.99863  P  (PER)  0.62848 0.00039 0.00000 0.01068 0.06155 0.00001 0.00066 0.99917 0.00002 0.91566 0.88971 0.00010 0.18452 0.40664 0.03526 0.08739 0.99889  A one-way ANOVA f o r plasma z i n c l e v e l s of c h i n o o k s a l m o n . Source  DF  Diet Nrep(Diet) Total  8 27 35  P 0.00000 1.00000  P  (FI)  0.00002 0.04682 0.00010 0.00189 0.00017 0.01122 0.00101 0.89534 0.00000 0.02221 0.55261 0.01167 0.08653 0.81290 0.16154 0.30325 0.68631  - 127 -  8.  B.  A one-way ANOVA of plasma z i n c l e v e l s showing main e f f e c t s interactions. Source  DF  P  Ca Zn Phytic Ca*Zn Ca*Phytic Zn*Phytic Ca*Zn*Phytic Nrep(Ca*Zn*Phytic) Residual Total  1 1 1 1 1 1 1 27 -0 35  0.37947 0.00000 0.00000 0.10971 0.00596 0.00005 0.61192 1.00000  The f o l l o w i n g t a b l e i s an example of t h e s t a t i s t i c s used i n Experiment II. 9.  A one-way ANOVA t a b l e of wet w e i g h t s at day 0 i n Experiment Source Treat Nrep(Treat) Residual Total  C.  and  DF  P  7 8 944 959  0.32230 0.09191  II.  The f o l l o w i n g ANOVA t a b l e s are examples of t h e a n a l y s e s performed i n Experiment I I I . 10.  A one-way ANOVA t a b l e of t h e n a t u r a l l o g wet w e i g h t s at day 84. Source Diet Residual Total  DF 14 1781 1795  P 0.2274  - 128 -  11.  12.  13.  14.  15.  A one-way ANOVA of s p e c i f i c growth r a t e (SGR), FC, PER and FI f o r days 0-84. Source  DF  P (SGR)  P (FC)  P (PER)  Diet Residual Total  14 15 29  0.0001  0.0001  0.0001  P  (FI)  0.0145  A one-way ANOVA f o r p r o t e i n c o n t e n t of chinook salmon at day 8 4 .  Source  DF  Diet Residual Total  14 45 59  P 0.0001  A one-way ANOVA o f t h e b l o o d z i n c l e v e l s i n chinook salmon a f t e r 84 days. Source  DF  Diet Residual Total  14 30 44  P 0.0001  A one-way ANOVA o f k i d n e y z i n c c o n t e n t o f chinook salmon a f t e r 84 days. Source  DF  Diet Residual Total  14 28 42  P 0.0001  A one-way ANOVA o f l i v e r z i n c l e v e l s i n chinook salmon a f t e r 84 d a y s . Source  DF  Diet Residual Total  14 44 58  P 0.0001  

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