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UBC Theses and Dissertations

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 tshawytscha) 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 Columbia, 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 Sc ience We accept t h i s t h e s i s as conforming to the requ i r ed standard THE UNIVERSITY OF BRITISH COLUMBIA January 1986 © Nancy L. R i chardson , 1986 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree at the U n i v e r s i t y o f B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and study. I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e copying o f t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the head o f my department or by h i s or her r e p r e s e n t a t i v e s . I t i s understood t h a t copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be allowed without my w r i t t e n p e r m i s s i o n . The U n i v e r s i t y of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date DE-6 (3/81) - i i -A B S T R A C T I n 1 9 8 1 a s e v e r e c a t a r a c t o u t b r e a k o c c u r r e d i n c h i n o o k a n d c o h o s a l m o n s t o c k s a t s e v e r a l B r i t i s h C o l u m b i a a n d W a s h i n g t o n S t a t e h a t c h e r i e s . I t w a s h y p o t h e s i z e d t h a t t h e c a t a r a c t s r e s u l t e d f r o m a z i n c d e f i c i e n c y d u e t o h i g h l e v e l s o f d i e t a r y c a l c i u m a n d p h o s p h o r u s i n r e l a t i o n t o z i n c . T h i s t h e s i s , c o n s i s t i n g o f t h r e e e x p e r i m e n t s , w a s d e s i g n e d t o i n v e s t i g a t e t h i s p o s s i b i l i t y i n j u v e n i l e c h i n o o k s a l m o n . E x p e r i m e n t s I ( p r e l i m i n a r y s t u d y ) a n d I I I ( c o m p r e h e n s i v e s t u d y ) i n v e s t i g a t e d t h e i n f l u e n c e o f w i d e 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 o f c a l c i u m , p h o s p h o r u s , z i n c a n d p h y t i c a c i d ( a s s o d i u m p h y t a t e ) o n g r o w t h , c a t a r a c t i n c i d e n c e , a p p e t i t e , f o o d c o n v e r s i o n , p r o t e i n e f f i c i e n c y , h e a l t h a n d h i s t o p a t h o l o g y i n c h i n o o k s a l m o n . E x p e r i m e n t I I w a s c o n d u c t e d t o d e t e r m i n e w h e t h e r c h i n o o k s a l m o n w e r e m o r e s u s c e p t i b l e t o c a t a r a c t f o r m a t i o n a t d i f f e r e n t t i m e s i n t h e i r e a r l y l i f e h i s t o r y . T h e t e s t d i e t s f o r a l l s t u d i e s w e r e c a s e i n - g e l a t i n b a s e d . L e v e l s o f c a l c i u m , z i n c a n d p h y t i c a c i d ( g / k g d i e t ) r a n g e d f r o m 4 . 4 - 5 3 , 0 . 0 3 4 - 0 . 4 0 0 a n d 0 . 0 6 - 2 5 . 8 , r e s p e c t i v e l y . D i e t s w e r e f o r m u l a t e d t o h a v e a c a l c i u m t o p h o s p h o r u s r a t i o o f c l o s e t o u n i t y w h e n d i s r e g a r d i n g t h e p h y t a t e - p h o s p h o r u s c o n t r i b u t i o n . I n e x p e r i m e n t s I a n d I I I , h i g h p h y t i c a c i d c o n t e n t ( 2 1 . 1 - 2 5 . 8 g / k g d i e t ) d e p r e s s e d g r o w t h , f o o d a n d p r o t e i n c o n v e r s i o n , i n c r e a s e d m o r t a l i t y a n d i n d u c e d b i l a t e r a l c a t a r a c t s ( z i n c a t 0 . 0 5 - 0 . 1 0 0 g / k g ) . M o r e o v e r , h i g h d i e t a r y l e v e l s o f c a l c i u m a n d p h o s p h o r u s e x a c e r b a t e d t h e e f f e c t s o f h i g h p h y t a t e a n d l o w z i n c o n c a t a r a c t i n c i d e n c e . C a t a r a c t s w e r e p r e v e n t e d h o w e v e r , w h e n d i e t a r y z i n c l e v e l s w e r e M D . 2 7 g / k g d i e t . I n e x p e r i m e n t I I , c a t a r a c t f o r m a t i o n w a s n o t e d t o b e d e p e n d e n t u p o n t h e t i m e o f e x p o s u r e t o a c a t a r a c t o g e n i c d i e t . F o r e x a m p l e , o p a c i t i e s d i d n o t a p p e a r u n t i l d a y 126 i n f i s h w h i c h w e r e f e d t h e c a t a r a c t d i e t b e t w e e n d a y s 4 2 a n d 8 4 . T h i s k n o w l e d g e o f a n a p p a r e n t d e l a y i n c a t a r a c t f o r m a t i o n may b e u s e f u l t o h a t c h e r y m a n a g e r s w h e n i n v e s t i g a t i n g t h e c a u s e o f a c a t a r a c t p r o b l e m . 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 a n d 2 9 . 1 g c a l c i u m / k g i n d u c e d a n o m a l i e s i n p y l o r i c c a e c a ! s t r u c t u r e . F u r t h e r m o r e , t h e i n g e s t i o n o f d i e t s c o n t a i n i n g a m i n i m u m o f 2 5 g C a / k g p r o d u c e d n e p h r o c a l c i n o s i s i n c h i n o o k s a l m o n . P l a s m a , b l o o d , l i v e r a n d k i d n e y z i n c l e v e l s w e r e d i r e c t l y r e l a t e d t o d i e t a r y z i n c c o n c e n t r a t i o n a n d , i n t h e c a s e o f p l a s m a a n d b l o o d , 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 l e v e l . I t i s c o n c l u d e d t h a t h i g h d i e t a r y l e v e l s o f p h y t i c a c i d a r e d e t r i m e n t a l t o t h e h e a l t h a n d p e r f o r m a n c e o f j u v e n i l e c h i n o o k s a l m o n a n d t h a t z i n c i s e s s e n t i a l f o r n o r m a l e y e d e v e l o p m e n t . F u r t h e r , u n d e r t h e c o n d i t i o n s o f t h i s s t u d y , c a t a r a c t s c o u l d n o t b e i n d u c e d i n c h i n o o k s a l m o n f e d h i g h d i e t a r y r a t i o s o f c a l c i u m ( o r p h o s p h o r u s ) t o z i n c u n l e s s a s t r o n g m i n e r a l ( z i n c ) - b i n d i n g a g e n t w a s a l s o p r e s e n t . i v -T A B L E OF CONTENTS S e c t i o n P a g e A B S T R A C T i i L I S T OF T A B L E S i x L I S T OF F I G U R E S x i ACKNOWLEDGEMENTS x i i CHAPTER 1 1 . 0 INTRODUCTION . . . . 1 CHAPTER 2 2 . 0 L I T E R A T U R E R E V I E W 2 . 1 T h e F i s h E y e 3 2 . 1 . 1 S t r u c t u r e 3 2 . 1 . 2 M i n e r a l C o m p o s i t i o n 3 2 . 1 . 3 G r o w t h 4 2 . 1 . 4 D i s e a s e s ' 5 2 . 2 T h e L e n s 5 2 . 2 . 1 L e n s C a p s u l e 6 2 . 2 . 2 L e n s E p i t h e l i u m 6 2 . 2 . 3 L e n s S u b s t a n c e 7 2 . 2 . 4 L e n s F u n c t i o n 7 2 . 2 . 5 L e n s C o m p o s i t i o n 8 2 . 2 . 5 . 1 P r o t e i n a n d w a t e r 8 2 . 2 . 5 . 2 M a j o r i n o r g a n i c c o n s t i t u e n t s 9 2 . 2 . 5 . 3 T r a c e m i n e r a l s 9 2 . 2 . 6 L e n s M e t a b o l i s m 10 2 . 3 . C l a s s i f i c a t i o n o f C a t a r a c t s 10 2 . 3 . 1 D e v e l o p m e n t a l C a t a r a c t s 10 2 . 3 . 2 A c q u i r e d C a t a r a c t s 11 2 . 4 C a u s e s o f C a t a r a c t s 12 2 . 5 C a t a r a c t F o r m a t i o n 13 2 . 5 . 1 G r o s s C h a n g e s 14 2 . 5 . 2 A r e a s A f f e c t e d 14 2 . 5 . 3 M e m b r a n e P e r m e a b i l i t y . . 14 2 . 5 . 4 L e n s P r o t e i n s 15 2 . 5 . 5 L e n s M i n e r a l s 1 5 2 . 6 H a t c h e r y D i e t s 2 . 6 . 1 W h i t e f i s h M e a l 2 . 6 . 2 H e r r i n g M e a l 2 . 6 . 3 A l t e r n a t i v e P r o t e i n S o u r c e s . . 2 . 7 C a l c i u m a n d P h o s p h o r u s 2 . 7 . 1 R e q u i r e m e n t s a n d A v a i l a b i l i t y 2 . 7 . 2 P r o p e r t i e s V T A B L E OF CONTENTS c o n t P a g e 2 . 7 . 3 I n t e r a c t i o n 19 2 . 7 . 4 M i n e r a l I n t e r a c t i o n s 19 2 . 7 . 4 . 1 M a g n e s i u m 19 2 . 7 . 4 . 2 Z i n c . . . 2 0 2 . 8 Z i n c 2 1 2 . 8 . 1 R e q u i r e m e n t s a n d A v a i l a b i l i t y 21 2 . 8 . 2 P r o p e r t i e s 22 2 . 9 P h y t i c A c i d . . . ' 2 3 2 . 9 . 1 S t r u c t u r e 23 2 . 9 . 2 O c c u r r e n c e 2 4 2 . 9 . 3 P r o p e r t i e s 24 2 . 1 0 P h y t a t e s 25 2 . 1 0 . 1 S t r u c t u r e 2 5 2 . 1 0 . 2 P r o p e r t i e s 26 2 . 1 0 . 3 I n t e r a c t i o n s 27 2 . 1 1 F i b r e D i e t s - E f f e c t s o n M i n e r a l A v a i l a b i l i t y 27 2 . 1 2 ' C a l c i u m - Z i n c - P h y t a t e s , 2 9 2 . 1 2 . 1 S w i n e S t u d i e s 3 0 2 . 1 2 . 2 R a t S t u d i e s 3 0 2 . 1 2 . 3 P o u l t r y S t u d i e s 31 2 . 1 3 T h e E f f e c t o f G u t pH on P h y t a t e A c t i v i t y 31 2 . 1 4 P h y t i c A c i d S t u d i e s w i t h S a l m o n i d s 32 2 . 1 5 G r o w t h i n S a l m o n i d s 33 CHAPTER 3 3 . 0 G E N E R A L M A T E R I A L S AND METHODS 36 3 . 1 E x p e r i m e n t a l F i s h 36 3 . 2 C u l t u r e C o n d i t i o n s 3 6 3 . 3 D i e t F o r m u l a t i o n s 36 3 . 4 D i e t P r e p a r a t i o n 37 3 . 5 E x p e r i m e n t a l P r o c e d u r e s a n d S a m p l i n g 4 5 3 . 5 . 1 D i e t a l l o c a t i o n 4 5 3 . 5 . 2 F e e d i n g 4 5 3 . 5 . 3 F i s h w e i g h t 4 5 3 . 5 . 4 C a t a r a c t a s s e s s m e n t 4 5 3 . 5 . 5 P r o x i m a t e a n a l y s i s 4 6 3 . 5 . 6 F i s h h e a l t h a n d h i s t o l o g i c a l e x a m i n a t i o n 4 6 3 . 5 . 7 P l a s m a a n d b l o o d a n a l y s i s 4 6 3 . 5 . 8 T i s s u e a n a l y s i s 47 - v i -T A B L E OF CONTENTS c o n t P a g e CHAPTER 4 4 . 0 E X P E R I M E N T I . P r e l i m i n a r y s t u d y o f t h e e f f e c t s o f d i e t a r y c a l c i u m , p h o s p h o r u s , z i n c a n d p h y t i c a c i d l e v e l o n c a t a r a c t i n c i d e n c e , g r o w t h a n d 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 . 4 . 1 I n t r o d u c t i o n 4 8 4 . 2 M a t e r i a l s a n d M e t h o d s 49 4 . 2 . 1 E x p e r i m e n t a l F i s h 4 9 4 . 2 . 2 E x p e r i m e n t a l D i e t s 4 9 4 . 2 . 3 F e e d i n g 4 9 4 . 2 . 4 S a m p l i n g P r o c e d u r e s 4 9 4 . 2 . 4 . 1 F i s h w e i g h t a n d c a t a r a c t i n c i d e n c e 4 9 4 . 2 . 4 . 2 P r o x i m a t e a n a l y s i s 49 4 . 2 . 4 . 3 H i s t o l o g y s a m p l e s 4 9 4 . 2 . 4 . 4 P l a s m a s a m p l e s 50 4 . 2 . 4 . 5 S t a t i s t i c a l a n a l y s i s 5 0 4 . 3 R e s u l t s 5 1 4 . 3 . 1 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- c a t a r a c t i n c i d e n c e 51 4 . 3 . 2 I n f l u e n c e o f d i e t t r e a t m e n t o n c h i n o o k p e r f o r m a n c e 5 1 4 . 3 . 2 . 1 F i s h g r o w t h 51 4 . 3 . 2 . 2 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 p r o t e i n e f f i c i e n c y r a t i o ( P E R ) 52 4 . 3 . 3 I n f l u e n c e o f d i e t t r e a t m e n t o n p r o x i m a t e c o m p o s i t i o n 5 2 4 . 3 . 4 I n f l u e n c e o f d i e t t r e a t m e n t o n g e n e r a l h e a l t h 5 3 4 . 3 . 5 I n f l u e n c e o f d i e t t r e a t m e n t o n h i s t o p a t h o l o g y . . . . . 6 1 4 . 3 . 5 . 1 K i d n e y 6 1 4 . 3 . 5 . 2 P y l o r i c c a e c a a n d s t o m a c h -. 6 2 4 . 3 . 5 . 3 T h y r o i d 64 4 . 3 . 6 I n f l u e n c e o f d i e t t r e a t m e n t o n p l a s m a m i n e r a l l e v e l s 6 4 4 . 4 D i s c u s s i o n 66 4 . 4 . 1 C a t a r a c t I n c i d e n c e 66 4 . 4 . 2 C h i n o o k P e r f o r m a n c e 6 7 4 . 4 . 2 . 1 , F i s h g r o w t h 67 4 . 4 . 2 . 2 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 67 4 . 4 . 3 P r o x i m a t e C o m p o s i t i o n 69 4 . 4 . 4 F i s h H e a l t h 6.9 4 . 4 . 5 H i s t o p a t h o l o g y . . . . 69 4 . 4 . 5 . 1 K i d n e y 6 9 4 . 4 . 5 . 2 P y l o r i c c a e c a a n d s t o m a c h 70 4 . 4 . 5 . 3 T h y r o i d 7 1 4 . 4 . 6 P l a s m a M i n e r a l s 71 4 . 5 C o n c l u s i o n 7 2 CHAPTER 5 5 . 0 E X P E R I M E N T I I . T h e s u s c e p t i b i l i t y o f j u v e n i l e c h i n o o k s a l m o n t o c a t a r a c t f o r m a t i o n i n r e l a t i o n t o d i e t a r y c h a n g e s i n e a r l y l i f e . 5 . 1 I n t r o d u c t i o n 74 - v i i -T A B L E OF CONTENTS c o n t P a g e 5 . 2 M a t e r i a l s a n d M e t h o d s 74 5 . 2 . 1 E x p e r i m e n t a l F i s h 74 5 . 2 . 2 E x p e r i m e n t a l D i e t s 74 5 . 2 . 3 F e e d i n g 7 5 5 . 2 . 4 S a m p l i n g P r o c e d u r e s 77 5 . 2 . 5 S t a t i s t i c a l A n a l y s i s 77 5 . 3 R e s u l t s 77 5 . 3 . 1 I n f l u e n c e o f d i e t t r e a t m e n t o n c a t a r a c t i n c i d e n c e 77 5 . 3 . 2 I n f l u e n c e o f d i e t t r e a t m e n t o n f i s h g r o w t h . . ! 77 5 . 4 . D i s c u s s i o n 7 8 5 . 4 . 1 C a t a r a c t I n c i d e n c e 78 5 . 4 . 2 F i s h G r o w t h 8 1 5 . 5 C o n c l u s i o n 81 CHAPTER 6 6 . 0 E X P E R I M E N T I I I . C o m p r e h e n s i v e s t u d y o f t h e e f f e c t s o f d i e t a r y c a l c i u m , p h o s p h o r u s , z i n c a n d p h y t i c a c i d o n c a t a r a c t s , g r o w t h a n d 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 I n t r o d u c t i o n 8 2 6 . 2 M a t e r i a l s a n d M e t h o d s 8 2 6 . 2 . 1 E x p e r i m e n t a l F i s h 8 2 6 . 2 . 2 E x p e r i m e n t a l D i e t s 8 2 6 . 2 . 3 F e e d i n g 8 2 6 . 2 . 4 S a m p l i n g P r o c e d u r e s 8 3 6 . 2 . 4 . 1 F i s h w e i g h t a n d c a t a r a c t i n c i d e n c e 8 3 6 . 2 . 4 . 2 P r o x i m a t e a n a l y s i s 8 3 6 . 2 . 4 . 3 H i s t o l o g y s a m p l e s 8 3 6 . 2 . 4 . 4 B l o o d s a m p l e s 8 3 6 . 2 . 4 . 5 T i s s u e s a m p l e s 8 3 6 . 2 . 5 S t a t i s t i c a l A n a l y s i s 8 4 6 . 3 R e s u l t s 8 4 6 . 3 . 1 I n f l u e n c e o f d i e t t r e a t m e n t o n c a t a r a c t i n c i d e n c e 8 4 6 . 3 . 2 I n f l u e n c e o f d i e t t r e a t m e n t o n c h i n o o k p e r f o r m a n c e 8 4 6 . 3 . 2 . 1 F i s h g r o w t h 8 4 6 . 3 . 2 . 2 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 8 5 6 . 3 . 3 I n f l u e n c e o f d i e t t r e a t m e n t o n p r o x i m a t e c o m p o s i t i o n 91 6 . 3 . 4 I n f l u e n c e o f d i e t t r e a t m e n t o n g e n e r a l h e a l t h 9 1 6 . 3 . 5 I n f l u e n c e o f d i e t t r e a t m e n t o n h i s t o p a t h o l o g y . . . 9 3 6 . 3 . 5 . 1 K i d n e y 9 3 6 . 3 . 5 . 2 P y l o r i c c a e c a 9 3 6 . 3 . 6 I n f l u e n c e o f d i e t t r e a t m e n t o n b l o o d a n d t i s s u e m i n e r a l l e v e l s 9 3 6 . 3 . 6 . 1 W h o l e b l o o d 9 3 6 . 3 . 6 . 2 L i v e r 9 6 6 . 3 . 6 . 3 K i d n e y 98 - v i i i -T A B L E OF CONTENTS c o n t P a g e 6 . 4 D i s c u s s i o n 9 8 6 . 4 . 1 C a t a r a c t I n c i d e n c e 9 8 6 . 4 . 2 C h i n o o k P e r f o r m a n c e 1 0 0 6 . 4 . 2 . 1 F i s h g r o w t h 1 0 0 6 . 4 . 2 . 2 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 101 6 . 4 . 3 P r o x i m a t e C o m p o s i t i o n 1 0 2 6 . 4 . 4 F i s h H e a l t h 102 6 . 4 . 5 H i s t o p a t h o l o g y 1 0 3 6 . 4 . 5 . 1 K i d n e y 1 0 3 6 . 4 . 5 . 2 P y l o r i c c a e c a 1 0 3 6 . 4 . 6 M i n e r a l A n a l y s i s 104 6 . 4 . 6 . 1 W h o l e b l o o d 1 0 4 6 . 4 . 6 . 2 L i v e r 105 6 . 4 . 6 . 3 K i d n e y 1 0 7 6 . 5 C o n c l u s i o n 108 CHAPTER 7 7 . 0 SUMMARY AND CONCLUSIONS 1 1 0 B I B L I O G R A P H Y 113 A P P E N D I X 1 2 4 - i x -L I S T OF T A B L E S T a b l e P a g e 1 . N u t r i t i o n a l a n d n o n - n u t r i t i o n a l c a u s e s o f c a t a r a c t s i n a n i m a l s 12 2 . F o r m u l a t i o n o f b a s a l d i e t f e d t o j u v e n i l e c h i n o o k s a l m o n i n a l l e x p e r i m e n t s 3 8 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 s u p p l e m e n t s f e d t o j u v e 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 1 39 3 a . C o m p o s i t i o n o f 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 s u p p l e m e n t s f e d t o j u v e n i l e c h i n o o k s a l m o n i n E x p e r i m e n t s I I a n d I I I 4 0 - 4 1 4 . 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 a n d p h y t i c a c i d c o n t e n t o f t h e n i n e t e s t d i e t s f e d t o j u v e n i l e c h i n o o k s a l m o n i n E x p e r i m e n t I . . . . 4 2 4 a . 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 a n d 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 f e d t o j u v e n i l e c h i n o o k s a l m o n i n E x p e r i m e n t s I I a n d I I I • 4 3 - 4 4 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 c h i n o o k s a l m o n f e d d i e t s w i t h h i g h l e v e l s o f p h y t a t e i n E x p e r i m e n t 1 5 4 6 . S p e c i f i c g r o w t h r a t e s a n d o b s e r v e d m o r t a l i t i e s f o r j u v e n i l e c h i n o o k s a l m o n f e d t e s t d i e t s f o r 1 0 5 d a y s i n E x p e r i m e n t 1 56 7 . M e a n 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 a n d 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 g r o u p s o f j u v e n i l e 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 b e t w e e n d a y s 22 a n d 105 i n E x p e r i m e n t 1 58 8 . F i n a l w h o l e b o d y p r o x i m a t e c o m p o s i t i o n o f j u v e n i l e 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 1 0 5 d a y s i n E x p e r i m e n t 1 6 0 9 . F i n a l m e a n 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 i n j u v e n i l e c h i n o o k s a l m o n f e d t h e t e s t d i e t s i n E x p e r i m e n t 1 65 1 0 . M e a n w e t w e i g h t s o f j u v e n i l e c h i n o o k s a l m o n f e d t h e t e s t d i e t s a t 4 2 - d a y i n t e r v a l s f o r a p e r i o d o f 126 d a y s i n E x p e r i m e n t I I 8 0 1 1 . 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 c h i n o o k s a l m o n e x a m i n e d o n d a y s 0 , 2 1 , 4 2 , 6 3 , 8 4 , 1 0 5 a n d 119 i n E x p e r i m e n t I I I 86 1 2 . M e a n w e t 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 a n d m o r t a l i t i e s f o r j u v e n i l e c h i n o o k s a l m o n f e d t e s t d i e t s f o r 8 4 d a y s i n E x p e r i m e n t I I I 87 1 3 . M e a n 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 a n d 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 g r o u p s o f j u v e n i l e 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 b e t w e e n d a y s 0 a n d 8 4 i n E x p e r i m e n t I I I 9 0 1 4 . F i n a l w h o l e b o d y p r o x i m a t e c o m p o s i t i o n o f j u v e n i l e 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 8 4 d a y s i n E x p e r i m e n t I I I 9 2 X L I S T OF T A B L E S c o n t P a g e 1 5 . T h e % 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 a n d 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 a n d 8 4 i n E x p e r i m e n t I I I 9 4 1 6 . F i n a l w h o l e b l o o d 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 c h i n o o k s a l m o n f e d t h e t e s t d i e t s i n E x p e r i m e n t I I I 9 5 1 7 . 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 c h i n o o k s a l m o n f e d t h e t e s t d i e t s i n E x p e r i m e n t I I I 9 7 1 8 . F i n a l k i d n e y 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 c h i n o o k s a l m o n f e d t h e t e s t d i e t s i n E x p e r i m e n t I I I 9 9 - x i -LIST OF FIGURES F igu re Page 1. S t ruc tu re of a f i s h eye 4 2. C ros s - sec t i on of a lens showing f i b r e format ion 7 3. The s t r u c t u r e of phy t i c ac id 24 4. The s t r u c t u r e of a phytate complex 26 5. Growth curve of f i s h under constant environmental c ond i t i on s 35 6. J uven i l e chinook salmon fed a high phytate d i e t 55 7. Geometric mean weights of 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 1 57 8. Food i n t a k e , food convers ion 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 ep r e sen t a t i v e groups of j u v e n i l e chinook salmon fed d i e t s con ta in i ng low and high phy t i c a c i d at f ou r 21-day i n t e r v a l s i n Experiment 1 59 9. Kidney se c t i on of a chinook salmon fed a d i e t w i th a high l e v e l of ca l c ium and phosphorus i n Experiment 1 61 10a. C ro s s - se c t i on of p y l o r i c caeca from a high phyta te- fed chinook salmon i n Experiment 1 62 10b. C ros s - sec t i on of p y l o r i c caeca from a low phyta te- fed chinook salmon i n Experiment 1 63 10c. C ros s - sec t i on of the ca rd i a c stomach and p y l o r i c caeca of a chinook salmon i n Experiment I 63 11. Thyro id f o l l i c l e s of a chinook salmon f ed a h igh phytate d i e t and a low phytate d i e t i n Experiment 1 64 12. Schematic r ep re sen ta t i on of d i e t a r y treatments g iven to chinook salmon from swim-up to day 126 i n Experiment II 76 13. Geometric mean weights of chinook salmon fed t e s t d i e t s f o r 84 days i n Experiment I I I 88 14. Representa t ive p a i r s of f i s h fed d i e t s 1, 5 and 7 (15a) and d i e t s 11, 15 and 12 (15b) f o r 84 days i n Experiment I I I 89 - x i i -ACKNOWLEDGEMENTS Many people were i nvo l ved i n t h i s t h e s i s . To begin w i th I would l i k e to thank my a d v i s o r , Dr. 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 ow ing me t o sw i t ch the sub jec t of my t h e s i s from dogs to f i s h . I would a l s o l i k e t o thank my committee members: Dr . H iggs, P r o f . March, Dr. T a i t and Dr. Peterson f o r t h e i r he lp 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 constant enthusiasm and encouragement du r ing my sojourn at the West Vancouver Labora to ry . There are seve ra l o ther s t a f f members of the West Van Lab, both past and present , whom I owe thanks . Most no tab l y , Dianne P l o t n i k o f f , Jack Marker t , Andy Lamb and Helen Dye. The i r he lp i n the t e c h n i c a l and maintenance aspects of the exper iments was g r e a t l y app re c i a t ed . More impo r t an t l y , i t was a p leasure working w i th them. Thanks are a l s o owed to Jack McBride f o r the h i s t o l o g i c a l photographs; to Morva Booth f o r t each ing me some of the mys te r i e s of the Micom and to Dr . P i e t Dejong (UBC Fa cu l t y of Commerce) f o r the s t a t i s t i c a l ana lyses i n Exper iments I and I I . Spec i a l thanks t o Ne i l Holman (EPS) f o r h i s f r i e n d s h i p and endless pa t i ence i n photographing 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 thanks t o a l l the people at the Lab who helped to make my t ime there so en joyab l e . Needless t o say , I wish to thank my parents f o r t h e i r l ove and support and f o r a l l t he Sunday d inners and f r e e lunches . And, of cou r se , my acknowledgements would not be complete w i thout exp ress ing s i n c e r e thanks to A l l e n Mayes. H is l o v e , "humour" and support have c a r r i e d me a long way from the f r o z en wastes of Winnipeg. - 1 -CHAPTER 1 1.0 INTRODUCTION Exophthalmos (pop-eye) and ca ta ra c t s a re , r e s p e c t i v e l y , the f i r s t and second most f requent eye d i s o rde r s in c u l t u r ed f i s h (Dukes 1975). Reports from ha t che r i e s worldwide i n d i c a t e tha 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 product ion (Poston et a l . 1978). 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 tocks i n 1971 (Kubota 1976), and subsequent l o s ses of f i s h due to c a t a r a c t s have increased annua l l y in Japan as has the number of salmon and t r o u t spec ies a f f e c t e d . In the Zomba P lateau of Malawi , Cen t ra l A f r i c a , an average of 10% of the rainbow t r ou t stocks are l o s t annua l l y due to b l i ndness from ca t a r a c t s (Lee et a l . 1976). Salmon and t r ou t have a l so been a f f e c t ed i n Great B r i t a i n , Ice land and the United S ta tes (Ke to la 1979). A l o c a l example of f i s h c a t a r a c t s occurred in 1981 when severa l B r i t i s h Columbia and Washington S ta te ha t che r i e s exper ienced severe outbreaks i n t h e i r chinook (Oncorhynchus tshawytscha) and coho (0. k i s u t ch ) salmon s t o c k s . Desp i te t h i s , the f i s h were re l eased and i t has been est imated tha t f u t u r e l osses to the B.C. f i s h e r y of chinook salmon from t h i s r e l ease w i l l be at l e a s t 30,000 f i s h , which represents a 13% reduc t i on in the t o t a l adu l t r e t u rn alone (Perry 1981). At the t ime of the outbreak in B.C. and Washington, a l l a f f e c t ed ha t che r i e s were us ing the same commerical d i e t , namely, Oregon Moist P e l l e t s (OMP), which conta ined a h igh p ropor t i on of a North American East Coast he r r i ng meal . Th is meal had a high ash content due to f i l l e t removal from the f i s h before p ro ces s i ng . Consequent ly, the l e v e l s of ca l c ium (Ca) and phosphorus (P) i n t h i s d i e t were h i gh . On the bas i s of work conducted on - 2 -r a i n b o w t r o u t f r y b y K e t o l a ( 1 9 7 9 ) a n d c o r r o b o r a t e d b y B a r a s h e t a l . ( 1 9 8 2 ) , i t w a s p o s t u l a t e d t h a t t h e m o s t p r o b a b l e c a u s e o f t h e c a t a r a c t o u t b r e a k w a s an i n d u c e d z i n c ( Z n ) d e f i c i e n c y a r i s i n g f r o m h i g h d i e t a r y r a t i o s o f C a a n d P t o Z n . A l s o , i t w a s h y p o t h e s i z e d t h a t p h y t i c a c i d , t h e h e x a p h o s p h a t e o f m y o - i n o s i t o l , a n d a c o n s t i t u e n t o f t h e c e r e a l p r o d u c t s a n d o i l s e e d m e a l s w h i c h a r e c o m m o n l y i n c o r p o r a t e d i n t o c o m m e r c i a l s a l m o n i d f o o d s , may h a v e p l a y e d a n i m p o r t a n t r o l e b e c a u s e o f i t s s t r o n g a b i l i t y t o c h e l a t e m i n e r a l s s u c h a s Z n . T h i s t h e s i s w a s d e s i g n e d w i t h t w o m a j o r g o a l s i n m i n d 1 ) t o o b t a i n a c l e a r e r u n d e r s t a n d i n g o f t h e e f f e c t s o f d i e t a r y C a , Z n a n d p h y t i c a c i d o n 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 c h i n o o k s a l m o n , a n d 2 ) t o d e t e r m i n e t h e i n t e r r e l a t i o n s h i p s b e t w e e n t h e a b o v e d i e t a r y f a c t o r s o n g r o w t h , f o o d i n t a k e , f o o d c o n v e r s i o n , p r o t e i n e f f i c i e n c y , a n d h i s t o p a t h o l o g y i n c h i n o o k s a l m o n . T o t h e b e s t o f my k n o w l e d g e , t h e r e a r e no r e p o r t s t h a t h a v e i n v e s t i g a t e d t h e e f f e c t s o f p h y t i c a c i d p e r s e o n c a t a r a c t f o r m a t i o n i n a n y a n i m a l s p e c i e s , o r o f 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 o f h i g h d i e t a r y l e v e l s o f p h y t i c a c i d . T h e f o l l o w i n g l i t e r a t u r e r e v i e w i s d e s i g n e d t o p r o v i d e b a c k g r o u n d k n o w l e d g e o n t h e l e n s , t h e c a u s e s a n d e f f e c t s o f c a t a r a c t s , a n d o n t h e n u t r i t i o n a l s i g n i f i c a n c e o f C a , Z n a n d p h y t i c a c i d . - 3 -CHAPTER 2 2 . 0 L I T E R A T U R E REVIEW 2 . 1 THE F I S H EYE L i t t l e r e s e a r c h h a s b e e n d o n e o n t h e o p h t h a l m i c p a t h o l o g y o f f i s h e s a s c o m p a r e d t o m a m m a l s , b u t m u c h o f t h e i n f o r m a t i o n d e r i v e d f r o m m a m m a l i a n r e s e a r c h i s a p p l i c a b l e t o f i s h e s . 2 . 1 . 1 S t r u c t u r e M o r e s t r u c t u r a l v a r i a t i o n e x i s t s among f i s h e y e s t h a n among t h e e y e s o f a l l o t h e r v e r t e b r a t e s c o m b i n e d ( D u k e s 1 9 7 5 ) . H o w e v e r , t h i s t h e s i s i s c o n c e r n e d w i t h t h e t e l e o s t e y e ( e . g . s a l m o n a n d t r o u t ) w h i c h h a s t h e s a m e s t r u c t u r a l c o m p o n e n t s a s t h e human e y e : o p t i c n e r v e , s c l e r a , c h o r o i d , r e t i n a , c o r n e a , l e n s , i r i s , p u p i l , a n d t h e a q u e o u s a n d v i t r e o u s h u m o u r s ( F i g . 1 ) . T h e o n e i m p o r t a n t d i f f e r e n c e b e t w e e n t h e f i s h e y e a n d t h e human e y e i s w i t h r e s p e c t t o a c c o m o d a t i o n ( v a n D u i j n 1 9 7 3 ) . I n t h e human e y e t h e l e n s c a n c h a n g e i t s s h a p e t o a c c o m m o d a t e o b j e c t s w h i c h a r e n e a r o r f a r , b u t i n t h e f i s h t h e l e n s i s o f a f i x e d s h a p e a n d c a n n o t b e c h a n g e d . I n s t e a d , t h e r e i s a s i c k l e - s h a p e d a p p e n d i x a t t a c h e d t o t h e c h o r o i d w h i c h a c t s t o d i s p l a c e t h e l e n s much l i k e t h e f o c u s i n g a r m o f a c a m e r a . T h e l e n s a p p e n d i x w i t h d r a w s t h e l e n s f u r t h e r b a c k i n t o t h e g l o b e o f t h e e y e o r m o v e s i t f o r w a r d . 2 . 1 . 2 M i n e r a l C o m p o s i t i o n T h e e y e c o n t a i n s a l l t h e e s s e n t i a l m i n e r a l s w i t h b o t h Z n a n d c o p p e r ( C u ) b e i n g p r e s e n t i n s i g n i f i c a n t a m o u n t s i n c e r t a i n t i s s u e s , m o s t n o t a b l y t h e c h o r o i d a n d i r i s . T h e h i g h e s t c o n c e n t r a t i o n o f Z n k n o w n t o e x i s t n o r m a l l y i n l i v i n g m a t t e r i s i n t h e c h o r o i d o f t h e e y e ( U n d e r w o o d 1 9 7 1 ) , a n d f o r r e a s o n s u n k n o w n , t h i s c o n c e n t r a t i o n i s h i g h e r i n f r e s h w a t e r f i s h t h a n i n s a l t w a t e r f i s h . A l t h o u g h t h e c o n c e n t r a t i o n s o f Z n a n d Cu i n t h e e y e a r e v e r y h i g h , - 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 ir is 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. Fig. 1. Structure of a fish eye (van Duijn, Jr . 1973). 2.1.3 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 CHOROID VITREOUS - 5 -growth w i l l a l so determine eye growth. The most common f a c t o r s i n c l ude food q u a l i t y and a v a i l a b i l i t y , water temperature, f i s h dens i t y and f i s h age. F i sh eyes g r e a t l y inc rease i n s i z e dur ing j u v e n i l e l i f e (Johns 1981). During the f i r s t year of l i f e , lenses i n t e l e o s t f i s h may inc rease in volume by a thousandfo ld (Ferna ld and Wright 1983). Under hatchery c ond i t i on s great care must be taken to ensure tha t the aforementioned f a c t o r s are c a r e f u l l y cons idered in order to ensure proper lens growth. 2.1.4 Diseases The two most common eye d i seases i n f i s h are exophthalmos (pop-eye) and c a t a r a c t s . These can be due to many f a c t o r s , both n u t r i t i o n a l and n o n - n u t r i t i o n a l . Although exophthalmos i s the most f requent eye d i sease i n con f ined f i s h , i t i s not the most s e r i o u s . When an outbreak of c a t a r a c t s occurs i t i n v a r i a b l y leads to b l i ndnes s , whereas exophthalmos does no 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 food or evade p reda to r s . Consequent ly, they have l i t t l e chance of s u r v i v a l . Th i s may have d r a s t i c e f f e c t s on adu l t r e tu rns to commercial and r e c r e a t i o na l f i s h e r i e s and to ha t c he r i e s . A c a t a r a c t , a l so known as whi te eye or opaque eye, i s de f ined as a lens o p a c i t y . The f i r s t Eng l i sh usage of " c a t a r a c t " was to desc r i be f l o odga t e s , and i n today ' s usage the "gate" r e f e r s to the opac i t y which descends on the eye (Lerman 1966). I t should be noted tha t there are a l so o p a c i t i e s of the cornea but these are not de f ined as c a t a r a c t s , t he re f o re care must be taken when d iagnos ing an opac i t y as being e i t h e r l e n t i c u l a r or c o r n e a l . As c a t a r a c t s are a d i sease of the l ens , any f u r t h e r d i s cu s s i on of them must.be preceded by a d i s cu s s i on of the lens i t s e l f . 2.2 THE LENS The ve r teb ra te l en s , a non-vascu lar t i s s u e der ived from the sur face - 6 -e c t o d e r m o f t h e e y e , i s a r e l a t i v e l y s i m p l e e p i t h e l i a l s t r u c t u r e . I n f i s h t h e l e n s i s s p h e r i c a l , o c c u p i e s a l a r g e p a r t o f t h e i n s i d e o f t h e g l o b e a n d t e n d s t o p r o t r u d e t h r o u g h t h e p u p i l ( D u k e s 1 9 7 5 ) . A l l v e r t e b r a t e l e n s e s c o n t a i n t h r e e b a s i c r e g i o n s : t h e l e n s c a p s u l e , t h e l e n s e p i t h e l i u m a n d t h e l e n s s u b s t a n c e . 2 . 2 . 1 L e n s C a p s u l e T h e l e n s c a p s u l e i s a p a r t i a l l y e l a s t i c c o v e r i n g w h i c h c o m p l e t e l y s u r r o u n d s t h e e x t e r i o r s u r f a c e o f t h e l e n s . I t i s d e r i v e d f r o m m o d i f i e d e p i t h e l i a l c e l l s a n d i s a d o u b l e - 1 a y e r e d s t r u c t u r e i n t h e a d u l t l e n s ( L e r m a n 1 9 6 6 ) . T h e s u r f a c e c o n t a i n s a c i d p o l y s a c c h a r i d e s a n d s i a l i c a c i d . T h e a n t e r i o r p o r t i o n i s s l i g h t l y t h i c k e r t h a n t h e p o s t e r i o r p o r t i o n . A l m o s t a l l n u t r i e n t e x c h a n g e s o c c u r b e t w e e n t h e a n t e r i o r c a p s u l e a n d t h e a d j a c e n t a q u e o u s h u m o u r . T h e l e n s c a p s u l e i s s e l e c t i v e l y p e r m e a b l e a n d t h e i n c r e a s e d t h i c k n e s s o f t h e a n t e r i o r p o r t i o n a i d s i t i n a c h i e v i n g t h i s . 2 . 2 . 2 L e n s E p i t h e l i u m P o s t e r i o r t o t h e l e n s c a p s u l e a n d e x t e n d i n g f r o m t h e a n t e r i o r p o r t i o n down t o t h e l e n s e q u a t o r i s t h e l e n s e p i t h e l i u m ( F i g . 2 ) , a s i n g l e l a y e r o f e p i t h e l i a l c e l l s w h i c h c o n t i n u e s t o g r o w t h r o u g h o u t l i f e . T h e c e l l s w h i c h b e c o m e c r o w d e d t o g e t h e r a t t h e p e r i p h e r y o f t h e e q u a t o r u n d e r g o a g r a d u a l e l o n g a t i o n i n t o l e n s f i b r e s . T h e y o u n g l e n s c o n t a i n s m o r e o f t h e s e n u c l e a t e d c e l l s t h a n t h e a d u l t l e n s ( M a n e r y 1 9 6 1 ) . - 7 -LENS EPITHELIUM ANTERIOR NUCLEUS CAPSULE F i g . 2. Cross-sec t ion of a lens showing f i b r e format ion ( P i r i e and van Heyningen 1956). 2 .2 .3 Lens Substance Inc luded in the lens substance are the f i b r e c e l l s and t h e i r c o n s t i t u e n t p r o t e i n s , and the i n t e r s t i t i a l mate r i a l which can be d i v i d ed i n t o c o r t i c a l and nuc lear r eg i ons . F i b re format ion i s a cont inuous process r e s u l t i n g i n the e a r l i e r (pr imary) f i b r e s being bu r i e d . The most deeply bur i ed f i b r e s c o n s t i t u t e the nucleus of the lens and they are t h i nne r and l a r ge r than the pe r i phe r a l (secondary) f i b r e s . The young, pe r i phe ra l f i b r e s of the lens co r tex r e t a i n t h e i r nuc le i and are the most a c t i v e m e t a b o l i c a l l y (Jubb and Kennedy 1970). The mature, c en t r a l f i b r e s l o se t h e i r nuc l e i but s t i l l remain a c t i v e . 2 .2 .4 Lens Funct ion The pr imary func t i on of the lens i s to achieve and mainta in t ransparency . For t h i s reason the lens i s avascu la r and t r anspa ren t . - 8 -T r a n s p a r e n c y i s a c h i e v e d b y t h e r e g u l a r s p a c i n g o f t h e f i b r e c e l l s a n d t h e i r p r o t e i n . S u c h s p a c i n g m a i n t a i n s a c o n s t a n t r e f r a c t i v e i n d e x t h r o u g h o u t t h e l e n s . A l t e r a t i o n s i n t h e r e f r a c t i v e i n d e x r e s u l t i n i n c r e a s e d l i g h t s c a t t e r i n g w h i c h i s t h e m o s t i m p o r t a n t p h e n o m e n o n i n t h e m a j o r i t y o f c a t a r a c t s ( P h i l i p s o n 1 9 7 3 ) . T h e r e f r a c t i v e i n d e x i n t h e l e n s i s a l i n e a r f u n c t i o n o f t h e p r o t e i n c o n c e n t r a t i o n . P r o t e i n c o n c e n t r a t i o n i n c r e a s e s g r a d u a l l y f r o m t h e c o r t e x t o t h e n u c l e u s . T h u s t h e r e f r a c t i v e i n d e x a t t h e o u t s i d e i s 1 . 3 3 a n d i n c r e a s e s t o 1 . 5 3 a t t h e l e n s c e n t r e ( P u m p h r e y 1 9 6 1 ) . M a i n t a i n i n g t h e p r o p e r r e f r a c t i v e i n d e x r e q u i r e s an i n t e r n a l c h e m i c a l c o m p o s i t i o n w i t h i n t h e l e n s w h i c h i s d i f f e r e n t f r o m t h e a q u e o u s h u m o u r . Due t o t h e n o n - v a s c u l a r i t y o f t h e l e n s , a n y p u n c t u r e w i l l u p s e t t h i s d i f f e r e n c e c a u s i n g a r a p i d s w e l l i n g a n d o p a q u e n e s s o f t h e l e n s ( G r a n t 1 9 7 4 ) . 2 . 2 . 5 L e n s C o m p o s i t i o n T h e e f f i c i e n c y o f a c c o m o d a t i o n i n f i s h l e n s e s i s p r i m a r i l y d e p e n d e n t u p o n t h e d i f f e r e n c e i n r e f r a c t i v e i n d e x b e t w e e n t h e l e n s a n d t h e s u r r o u n d i n g a q u e o u s a n d v i t r e o u s h u m o u r s . What d e t e r m i n e s t h i s d i f f e r e n c e i s t h e g r o s s c o m p o s i t i o n o f t h e l e n s , n a m e l y , t h e a m o u n t o f w a t e r a n d h i g h l y r e f r a c t i l e p r o t e i n w h i c h i s p r e s e n t . 2 . 2 . 5 . 1 P r o t e i n a n d w a t e r I n s p e c i e s w h e r e t h e l e n s i s a c c o m o d a t e d b y d e f o r m a t i o n a s w e l l a s r e f r a c t i v e i n d e x d i f f e r e n c e s ( e . g . m a m m a l s ) , t h e r e w i l l b e m o r e w a t e r p r e s e n t t o m a k e t h e l e n s s o f t e r a n d m o r e d e f o r m a b l e . I n t h e f i s h , w h e r e l e n s d e f o r m a t i o n d o e s n o t o c c u r , t h e l e n s m u s t b e a s r e f r a c t i l e a s p o s s i b l e . A s a r e s u l t , f i s h l e n s e s h a v e t h e h i g h e s t p e r c e n t a g e o f p r o t e i n a n d t h e l o w e s t p e r c e n t a g e o f w a t e r o f a n y s p e c i e s s t u d i e d . A t t h e o p p o s i t e e n d o f t h e s c a l e a r e t h e b i r d s o f p r e y w h o s e l e n s e s c o n t a i n a b o u t 80% w a t e r c o m p a r e d t o 50% i n - 9 -t r ou t ( K l e t h i and Mandel 1965). Th is high water concent ra t i on makes lens t ransparency maintenance an eas i e r task and may be a major reason why b i r d s are not as prone to ca t a ra c t s as other spec ies (Kuck, J r . 1975). 2 .2 .5 .2 Major inorgan i c cons t i t uen t s The lens conta ins a subs t an t i a l amount of potassium (K) which i s common i n a t i s s u e con ta in i ng l a rge amounts of p r o t e i n and i n t r a c e l l u l a r space. i Sodium (Na) occurs i n the e x t r a c e l l u l a r space and ranges in concen t ra t i on from .10 to .50 of that of K depending on the age and v i a b i l i t y of the l en s . That i s , Na inc reases wi th age and lens degenerat ion . Calc ium i s present i n low l e v e l s i n the young lens but i t i s thought to be h i g h l y s i g n i f i c a n t i n ma in ta in ing normal lens membrane pe rmeab i l i t y (Thoft and K i n o s h i t a 1965). Calc ium content inc reases wi th age and i s h igher i n the nuc leus than i n the co r tex (Bel lows 1944). Other major c ons t i t uen t s i n c l ude : magnesium (Mg), phosphate, su lpha te , c h l o r i d e and b i ca rbonate . B icarbonate i s very important i n ma in ta in ing lens pH at 7.4; a task i t shares w i th 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 lens i n t o the aqueous humour. 2 .2 .5 .3 Trace minera ls Lenses of a l l ve r tebra tes conta in numerous minera l s which are present i n va ry ing amounts depending upon the spec ies invo lved and the c ond i t i o n of the l e n s . Iron (Fe ) , Cu and Zn are c l o s e l y assoc ia ted wi th r e s p i r a t i o n , amino ac id metabol ism, pH r e g u l a t i o n , energy metabol ism and c e l l membrane pe rmeab i l i t y (Murata and Taura 1975). Consequent ly, the concen t ra t i ons in which they are present w i l l be important to the i n t e g r i t y of the l e n s . As p r e v i o u s l y mentioned, the r o l e of Zn i n the lens i s unknown, but i t has the p o t e n t i a l to be important i n many f u n c t i o n s , e s p e c i a l l y lens growth and the maintenance of the opt imal c on f i gu r a t i on of so l ub l e lens p r o t e i n s (Baldwin - 10 -and Bent ley 1980). 2.2.6 Lens Metabol ism The lens de r i ves i t s nourishment e n t i r e l y from the aqueous humour v i a the semi -pe rmeab i l i t y o f the lens capsu le and lens ep i t h e l i um . This dependence on the aqueous humour makes the lens very s u s cep t i b l e to a l t e r a t i o n s i n the aqueous compos i t i on . Any d i s r up t i o n of the capsu lar membrane r e s u l t s i n an i n f l u x of f l u i d and the lens turns opaque ( M i l l e r 1978). The energy needs of the lens are met c h i e f l y by carbohydrate metabol ism which d i f f e r s l i t t l e from the carbohydrate metabolism of o ther t i s s u e s . 2.3 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 the appearance of the o p a c i t y ( i e s ) as seen through a s l i t - l a m p b iomicroscope. There are f ou r major l o c a t i o n s i n the lens i n which c a t a r a c t s may o r i g i n a t e : subcapsu lar , c o r t i c a l , l ame l l a r and nuc lear (Jubb and Kennedy 1970). The s i z e , shape and l o c a t i o n of the opac i t y i s determined and an attempt i s then made to c l a s s i f y the in fo rmat ion i n t o a broad ca tegory , e i t h e r developmental c a t a r a c t s or acqu i red c a t a r a c t s . 2.3.1 Developmental Ca ta rac t s Th is type of ca ta rac t i s very common in a l l animal spec ies and may take on many forms. Under the s l i t - l a m p beam, the lenses of most people w i l l show very smal l o p a c i t i e s of t h i s t ype , but they are u sua l l y too smal l to be s i g n i f i c a n t ( M i l l e r 1978). As p r ev i ou s l y mentioned, the lens i s formed i n l aye r s w i th the e a r l i e s t f i b r e s forming the nucleus and the l a t e r f i b r e s c o n s t i t u t i n g the c o r t e x . With developmental c a t a r a c t s , u s ua l l y on ly that p a r t i c u l a r zone which i s - 1 1 -b e i n g f o r m e d a t t h e t i m e w h e n t h e o p a c i t y i s c a u s e d w i l l b e a f f e c t e d . T h e r e f o r e , t h e f i b r e s f o r m e d b e f o r e a n d a f t e r t h e a f f e c t e d s i t e a r e n o r m a l a n d c l e a r . I f t h e o p a c i t y o c c u r s e a r l y i n l i f e i t w i l l q u i c k l y b e c o m e b u r i e d i n t h e l e n s a n d h a v e no d e t r i m e n t a l e f f e c t s o n v i s i o n . 2 . 3 . 2 A c q u i r e d C a t a r a c t s T h e s e c a t a r a c t s a r e t h e r e s u l t o f t h e d e g e n e r a t i o n o f l e n s f i b r e s w h i c h a r e a l r e a d y f o r m e d . U n l i k e d e v e l o p m e n t a l c a t a r a c t s t h e y a r e p r o g r e s s i v e a n d m a y i n v o l v e t h e e n t i r e l e n s . - 12 -2.4 CAUSES OF CATARACTS Catarac ts may be caused by a v a r i e t y of f a c t o r s (Table 1 ) . Two major c l a s s i f i c a t i o n s are n u t r i t i o n a l and n o n - n u t r i t i o n a l . TABLE 1 N u t r i t i o n a l and non -nu t r i t i o na l causes of c a t a ra c t s in an imals .1 N u t r i t i o n a l Non -nu t r i t i o na l De f i c i ency Excess Radi a t i on V i tamin D Xylose e .g . U l t r a v i o l e t l i g h t V i tamin E Ga lactose X-rays V i tamin C Fructose Gamma rays R i b o f l a v i n Lactose B a c t e r i a l i n f e c t i o n Calc ium Calc ium P a r a s i t i c i n f e c t i o n Z inc Phosphorus Cold s t r e s s Selenium Phy t i c a c i d Phys i ca l trauma Magnesi um Tyros ine Naphthalene Manganese Thioacetamide Iodine E l e c t r i c i t y Tryptophan S te ro i d s Pheny la lan ine Hered i ta ry d isease Hi s t i d i n e e .g . A l p o r t ' s syndrome Methionine Homocystinuri a P ro t e i n Fabry ' s d i sease 1 The f o l l ow i n g re ferences were used: M i t c h e l l and Dodge 1935; Darby 1939; Ha l l et a l . 1948; A l l i s o n 1962; Lerman 1966; Von Sallman et a l . 1966; Grant 1974; Dukes 1975; Hockwin and Koch 1975; McLaren and Halasa 1975; Whanger and Weswig 1975; Bunce and Hess 1976; Lee et aT. 1976; M i l l e r 1978; Ke to l a 1979; Poston et a l . 1977; Satoh et a l . 1983a,b; Richardson et a l . 1985. - 13 -Of the causes l i s t e d i n Table 1, the f o l l o w i n g have been repor ted in f i s h : 1. poor q u a l i t y p r o t e i n 2. r i b o f l a v i n d e f i c i e n c y 3. t ryptophan d e f i c i e n c y 4. methionine d e f i c i e n c y 5. z i n c d e f i c i e n c y 6. magnesium de f i c i e n c y 7. manganese d e f i c i e n c y 8. i od i ne d e f i c i e n c y 9. excess ca lc ium and phosphorus 10. excess phy t i c a c i d 11. th ioacetamide 12. phys i c a l trauma 13. p a r a s i t i c i n f e c t i o n 14. u l t r a v i o l e t l i g h t Although Table 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 , abso lu te statements cannot be made. Th is i s because, f o r the most pa r t , i t i s not known whether these f a c t o r s are major inducers of c a t a r a c t f o rma t i on , or whether they j u s t seem to be the most l i k e l y cause based on the exper imental des i gn . The most e x t en s i v e l y s tud ied c a t a r a c t s are the "sugar c a t a r a c t s " caused by excesses of ga l a c to se , xy l o se and l a c t o s e . Ca ta rac t s r e s u l t i n g from ga lac tosaemia may be the on ly ones in which the b iochemica l and metabo l i c a l t e r a t i o n s tha 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 on i s to desc r i be the var ious changes which - 14 -occur i n the lens dur ing ca t a ra c t f o rmat i on . As mentioned p r e v i o u s l y , i t i s p o s s i b l e to s t a t e what changes occur , but w i th the e x i s t i n g s t a t e of knowledge i t i s not po s s i b l e to s t a t e w i th much c onv i c t i o n how such changes occur . 2.5.1 Gross Changes Mac r o s cop i c a l l y , a ca tarac tous lens appears wh i t i s h and c loudy . Lenses wi th p a r t i a l c a t a r a c t s are gene ra l l y c loudy in the cent re whereas severe c a t a r a c t s are cha ra c t e r i z ed by c loud iness a l l over the l en s . Upon c l o s e r examinat ion, 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 the ground g l a ss appearance of sugar c a t a r a c t s . 2.5.2 Areas A f f e c t ed There i s cons ide rab le v a r i a t i o n i n the changes which occur i n the lens dur ing ca ta rac t f o rma t i on . D e f i c i e n c i e s of meth ion ine , r i b o f l a v i n and Zn i n salmonid d i e t s have i n i t i a t e d ca t a r a c t s w i th d i s t i n c t pa t te rns of development (Poston and Ke to l a 1981). In me th i on i ne -de f i c i en t salmon and t r o u t , the f i r s t no t i c eab l e lens changes occurred i n the subcapsular co r tex of the an t e r i o r r e g i on . The o p a c i t i e s progressed through the pe r i nu c l e a r reg ion to 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 in the p o s t e r i o r subcapsular cor tex and progressed to the pe r inuc l eus and nuc l eus . In Zn -de 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 pe r i nu c l eu s . 2.5.3 Membrane Pe rmeab i l i t y An inc rease in membrane pe rmeab i l i t y r e s u l t s i n c e l l u l a r swe l l i n g and i t has been suggested tha t a po s s i b l e un i v e r s a l cause of c a t a r a c t s i s inc reased pe rmeab i l i t y to Na (Duncan and Croghan 1969). The lens must r egu l a te i t s i n t e r n a l e l e c t r o l y t e balance aga inst s i g n i f i c a n t ion concen t ra t i on - 15 -g r ad i en t s . Th is i s accomplished by a c a t i on pump (Na-K-ATPase) which i s l o ca ted p r i m a r i l y in the lens ep i t h e l i um , j u s t beneath the a n t e r i o r lens capsu l e . Once the i n t e g r i t y of the lens membrane i s d i s t u r bed , the pump breaks down and Na begins to accumulate i n the l e n s . 2.5.4 Lens P ro te ins In the ageing lens the water content decreases, and the most probable cause of t h i s decrease i s be l i eved to be r e l a t e d to the changes in the concen t ra t i ons of so lub l e and i n s o l u b l e lens p ro t e i n s (Lerman 1966). Most of the dry weight of the lens i s composed of the s t r u c t u r a l p r o t e i n s : a l pha - , b e t a - , and gamma-crystal 1 ins and a lbumino id . Stud ies on ageing r a t and dog f i sh lenses have shown a sharp decrease i n the syn thes i s of s o l ub l e lens p ro t e i n s and a steady inc rease i n a lbuminoid p ro t e i n (Mal ik et a l . 1969). Th is i s comparable to a ca ta rac t l e n s . In Zn - de f i c i e n t lake t r ou t and rainbow t r o u t , both the a lpha- and b e t a - c r y s t a l l i n p ro t e i n s were reduced in the ca t a ra c t lens (Barash et a l . 1982). The de c l i n e i n s t r u c t u r a l p r o t e i n s i s be l i eved to be a r e s u l t of t h e i r t r ans fo rmat i on i n t o i n s o l u b l e p r o t e i n s . Although l i t t l e i s known about t h i s process , once the s t r u c t u r a l p r o t e i n s have been a l t e r ed 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. Bussanich pe r s . comm.). 2.5.5 Lens M inera ls Calc ium i s of spec i a l i n t e r e s t i n lens research because of the 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 to lens o p a c i t i e s (Manery 1961). S tud ies w i th human lenses have shown the concen t ra t i ons of Ca and Zn to be much h igher i n the ca ta rac tous lens than i n the normal lens (Murata and Taura 1975). The concen t ra t i ons of Ca and Zn were a l so h igher i n complete c a t a r a c t s versus p a r t i a l c a t a r a c t s . Leve ls of Mg, Fe and Cu were - 16 -a l so repor ted to be h igher , but there were no s i g n i f i c a n t d i f f e r en c e s noted between the l e v e l s present i n p a r t i a l c a t a r a c t s and those i n complete c a t a r a c t s . The va lues reported by Murata and Taura (1975) were on a wet weight bas i s which may exp l a i n the lack of s i g n i f i c a n t inc reases in the concent ra t i ons of Mg, Fe and Cu i n the complete ly ca ta rac tous l e n s . Other s t ud i e s have shown normal human lenses to con ta in cons ide rab l e Zn and ageing ca ta rac tous lenses to conta in increased l e v e l s of Fe and Cu but s i g n i f i c a n t l y l e s s Zn (Kuck, J r . 1975; Baldwin and Bent ley 1980). An e i g h t f o l d inc rease in Ca (on a dry weight bas i s ) has been observed i n ca ta rac tous lenses of chinook salmon (Richardson unpub.) Smal ler i nc reases i n Cu, Fe, Zn and Mg were seen. 2.6 HATCHERY DIETS The opera t ion and maintenance of a hatchery i s an expens ive under tak ing . As w i th other forms of animal husbandry feed cos t s are a major i tem i n the hatchery budget. F i sh d i e t s have to be h igh i n p r o t e i n (40-55%) and the most success fu l commercial d i e t s have been those based on f i shmea l as the major p r o t e i n source. There are two major types of f i shmea l used i n d i e t s f o r salmon ha t che 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 food f i s h e r y and the second i s made from whole f i s h . In North America these two types are commonly c a l l e d w h i t e f i s h meal (WFM) and he r r i ng meal (HM), r e s p e c t i v e l y . 2.6.1 Wh i t e f i sh Meal Wh i t e f i sh meal i s cha ra c t e r i z ed by a high ash ( 22%) and low o i l ( 2%) content on a dry weight b a s i s . Common types of w h i t e f i s h are cod, h a l i b u t and hake. The high 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 the way i t i s processed. Because w h i t e f i s h i s de s i r ed f o r human consumption, the f i l l e t s are removed and what i s l e f t goes - 1 7 -i n t o the manufacture of f i s hmea l . Consequent ly, the f i shmea l conta ins r e l a t i v e l y l a rge amounts of bone and c a r t i l a g e and t he r e f o r e , a high ash conten t . D i e t s based on WFM have been imp l i c a t ed in c a t a r a c t s i n hatchery f i s h due to t h e i r high ash contents (Ogino and Yang 1978; Ke to l a 1979). 2.6.2 Her r ing Meal Her r ing 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 Amer ica . These are a West Coast HM and an East Coast HM. The l e s s abundant West Coast he r r i ng i s complete ly rendered down to f i s h food ( a f t e r the removal of roe) and the meal conta ins approximately 12% ash and 7% l i p i d (dry we i gh t ) . By c on t r a s t , East Coast he r r i ng may be f i l l e t e d before meal p r oduc t i on . Consequent ly, t h i s HM may have s i m i l a r ash content to WFM. 2.6.3 A l t e r n a t i v e P ro te in Sources Due to the high cost of animal p r o t e i n and the f l u c t u a t i n g a v a i l a b i l i t y of such p r o t e i n , a great deal of research i s being done to f i n d s u i t a b l e p l an t p r o t e i n sources ( e .g . soybean and cano la) f o r f i s h d i e t s . Tests by Poston et a l . (1977) with i s o l a t e d soy p r o t e i n r e su l t ed i n an 80-90% inc idence 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, the same soy p r o t e i n d i e t supplemented wi th methionine s u c c e s s f u l l y prevented c a t a r a c t f o rma t i on . Soybean products are u s u a l l y d e f i c i e n t i n methionine and such a d e f i c i e n c y has been shown to cause c a t a r a c t s . 2.7 CALCIUM AND PHOSPHORUS The requirements f o r Ca and P by salmonids are gene ra l l y cons idered together because t h e i r metabolism i s i n t i m a t e l y connected. 2.7.1 Requirements and A v a i l a b i l i t y Quan t i t a t i v e d i e t a r y requirements f o r Ca and P i n chinook salmon have not been e s t ab l i s hed however, the Ca to P r a t i o f o r f i s h i s g ene r a l l y l e s s than one (B.E. March pe r s . comm.). Th is r a t i o may d i f f e r depending on the spec i e s , the water chemist ry and the a v a i l a b l e P l eve l i n the d i e t . F i sh can absorb Ca v i a the g i l l s and f i n s and through the d r i n k i n g of water ( L a l l 1979). Thus the d i e t a r y requirements are qu i t e low. For example, Rumsey (1977) e s t ab l i s h ed a Ca requirement f o r rainbow t r ou t of on ly 0.2% of the d i e t i n both so f twater (3 mg/L Ca) and hard water (45 mg/L Ca ) . In ve r t eb ra t e s , c h o l e c a l c i f e r o l (V i tamin D 3 ) r egu la tes Ca i n the blood and bone m i n e r a l i z a t i o n by a i d i ng absorpt ion from the gut and by m o b i l i z i n g Ca ions from bone ( v i a pa ra thy ro i d hormone). In salmonids however, V i t . D3 does not appear to be necessary f o r Ca absorpt ion (Barnett et a l . 1979). The a v a i l a b l e P requirement f o r rainbow t r ou t i s 0.7-0.8% of the d i e t (NRC 1981). Phosphate i s low in both f reshwater and seawater, t h e r e f o r e food c o n s t i t u t e s the main source 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 an t sources are used supplements of i no rgan i c P are r e q u i r e d . Th is i s due to p lan t -P being t i e d up i n phy t i c a c i d complexes which cannot be broken down i n monogastr ics because of the absence, or i n s u f f i c i e n t quan t i t y , of the enzyme phytase in the g a s t r o - i n t e s t i n a l t r a c t . For example, Ke to l a (1975) fed A t l a n t i c salmon 0.7% p lant-P and determined tha t a minimum of 0.6% ino rgan i c P was a l so requ i red t o ach ieve adequate f i s h growth. 2.7.2 P rope r t i e s Calc ium and P are e s s e n t i a l to bone format ion w i th Ca-phosphate and Ca-carbonate forming the major minera l c ons t i t uen t s of bone. In mammals the r a t i o of Ca3(P04)2 to CaC03 i s about 7:1 and i n f i s h i t i s 11:1 (Prosser and Brown 1950). Th is suggests a g rea te r demand f o r P by f i s h than by mammaTs. - 19 -Calc ium i s a l so requ i red f o r c a r t i l a g e f o rma t i on , osmoregu la t ion, muscle c o n t r a c t i o n , b lood 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 . Calc ium d e f i c i e n c y has not been def ined i n sa lmonids. Phosphorus i s important i n carbohydrate metabol ism, f a t metabol ism, and i s a c ons t i t uen t of phospho l i p i ds . Phosphorus d e f i c i e n c y in salmonids i s c ha r a c t e r i z ed by reduced growth, poor food convers ion and decreased bone m i n e r a l i z a t i o n (NRC 1981). 2.7.3 I n t e r a c t i o n Numerous s tud i e s have i n ve s t i ga t ed the Ca-P r e l a t i o n s h i p i n land an ima ls . Resu l t s have shown that each minera l d i r e c t l y i n f l uences the g a s t r o i n t e s t i n a l abso rp t i on , s k e l e t a l u t i l i z a t i o n and ex c r e t i on of the other (N ico layson et a l . 1953). For example, excess Ca i n the duodenum combines w i th P t o form i n s o l ub l e t r i c a l c i u m phosphate which i n h i b i t s P absorpt ion (Yano et a l . 1979). Some gene r a l i z a t i o n s can be made although there are always excep t i ons . B r i e f l y , high Ca i n take r e s u l t s i n reduced P abso rp t i on , whereas low d i e t a r y Ca s t imu la tes P absorpt ion (Cohen 1980). S i m i l a r l y , low P i n take inc reases Ca absorpt ion from the smal l i n t e s t i n e and i s assoc ia ted w i th hyperca lcaemia (Hughes et a l . 1975; Fox and Care 1978; Abdel-Hafez 1982). 2.7.4 M inera l I n t e ra c t i ons Calc ium and P are invo lved in a number of i n t e r a c t i o n s w i th other m i ne r a l s . In f i s h the most important of these i n t e r a c t i o n s are those i n v o l v i n g Mg and Zn. 2 .7 .4 .1 Magnesium Stud ies w i th t e r r e s t r i a l animals have demonstrated tha t a h igh Ca:P i n t ake w i l l aggravate Mg de f i c i e n c y symptoms (O 'De l l 1960; L i k u s k i and Forbes - 20 -1965; Woodard and Jee 1984). Magnes ium-def ic ient d i e t s con ta i n i ng 0.004% Mg and 2.6% Ca w i th a Ca:P r a t i o of 1:1, r e su l t e d in rena l c a l c i n o s i s when fed to rainbow t r ou t (Cowey 1976). Th is d i d not occur i n t r o u t f ed a d i e t con ta i n i ng 1.4% Ca, or when Mg was inc reased to 0.1% (Cowey et a l . 1977). 2 .7 .4 .2 Z inc The i n t e r a c t i o n between Ca and Zn has rece ived a great deal of a t t e n t i o n . Tucker and Salmon (1955) observed a Zn d e f i c i e n c y syndrome (pa rake ra t o s i s ) i n swine fed a high Ca d i e t . This syndrome suggested tha t the h igh Ca l e ve l i n t e r f e r e d w i th Zn absorpt ion and t h i s r e s u l t e d i n ex tens i ve research i n t o the Ca-Zn r e l a t i o n s h i p . Subsequent experiments performed by va r i ous researchers 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 et a l . 1957; Hoefer et a l . 1960). Cont rary to t h i s are experiments which have shown p u r i f i e d case in or egg p r o t e i n d i e t s , w i th high Ca concen t r a t i ons , not to induce Zn d e f i c i e n c i e s un less the Zn concen t ra t i on was i n i t i a l l y l e s s than 10 mg/kg (Davis 1966). A l s o , some swine s tud ies i n d i c a t e Zn absorpt ion to be normal i n s p i t e of pa rake ra tos i s (Whit ing and Bezeau 1958; Forbes 1960). P ou l t r y s tud ies have shown that an increase i n the d i e t a r y Ca content causes a decrease in the Zn content of bone, l i v e r and f ea the r s (Pensack et a l . 1958). Z inc supplementat ion of the b i r d s ' d i e t s increased 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 achieved w i th the combined add i t i on of Ca and Zn. Ke t o l a (1979) demonstrated tha t WFM-based d i e t s con ta i n i ng high Ca and P, r e l a t i v e to Zn, caused b i l a t e r a l lens ca ta rac t s and poor growth i n rainbow t r ou t f r y . But a Zn concen t ra t i on of 150 mg/kg i n the f i shmea l d i e t s prevented ca t a ra c t fo rmat ion and improved growth. - 21 -2.8 ZINC Z inc occurs i n a l l l i v i n g c e l l s and undergoes con t i nua l depos i t i on and tu rnove r . Bone, by v i r t u e of sheer bu lk , conta ins a major p o r t i o n of body Zn (Bergman 1970). In the b lood, 75% of the Zn i s found i n the e r y t h r o c y t e s , 3% in the leucocytes and 22% in the plasma or serum. Z inc i s present i n red b lood c e l l s main ly as carbon ic .anhydrase and i n whi te b lood c e l l s as a i p ro t e i n complex. In plasma most of the Zn i s bound to a l p h a - g l o b u l i n s . As mentioned p r e v i ou s l y , the h ighest Zn concent ra t i on i n a normal t i s s u e i s i n the choro id w i th concent ra t i ons ranging from 139 ( c a t t l e ) t o 69,000 mg/kg t i s s u e ( fox) on a dry weight bas i s (Underwood 1971). Gene r a l l y , Zn l e v e l s i n the body are h a l f tha t of the Fe content and 10-15 t imes tha t of the Cu content (Orten 1966). 2.8.1 Requirements and A v a i l a b i l i t y As w i th most m ine ra l s , Zn requirements are d i f f i c u l t to determine because Zn a v a i l a b i l i t y i s i n f l uenced by other 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 un c t i on and f a c t o r s which i n f l uence minera l and t i s s u e metabol ism. The d i e t a r y Zn requirement of rainbow t r ou t i s suggested as being between 15-30 mg/kg (Ogino and Yang 1978). However, rainbow t r ou t fed WFM d i e t s con ta in ing 60 mg Zn/kg developed ca t a r a c t s (Ke to l a 1979) due to the h igh concen t ra t i on of other minera l s such as Ca and P. In add i t i o n to i t s a s so c i a t i on w i th 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 ha r a c t e r i z ed by poor growth, high m o r t a l i t y , decreased appe t i t e and food conve r s i on , and e ros i on of the f i n s and sk i n (Ogino and Yang 1978; Ke t o l a 1979; Watanabe et a l . 1980; Satoh et a l . 1983b). Because of t h i s danger, i t i s recommended tha t commercial f i s h d i e t s f o r salmonids con ta in 150 mg Zn/kg (Ke to l a 1982). Th is i s cons ide rab ly below l e v e l s which might be cons idered to be harmful as - 22 -rainbow t r ou t have been fed d i e t s con ta in i ng up to 1700 mg Zn/kg w i th no i l l e f f e c t s (Wekell et a l . 1983). Z inc i s absorbed to a l i m i t e d extent from the duodenum. Less than 10% of the d i e t a r y Zn in take i s absorbed (Davis 1966) which i s probably why Zn t o x i c i t y i n any animal i s r a r e . Z inc absorpt ion may be enhanced by the add i t i on of c e r t a i n c h e l a t o r s . For example, supplementat ion of d i e t s w i th e thy l ened i am ine t raace t i c a c i d (EDTA) r e su l t ed i n increased Zn a v a i l a b i l i t y i n pou l t r y (K ra t ze r et a l . 1959). I t was suggested tha t EDTA combined w i t h , or i s o l a t e d , the Zn and e i t h e r through absorpt ion of the complex, or through a change i n i n t e s t i n a l pH, a l lowed the Zn to be absorbed. The a b i l i t y of bone Zn to be u t i l i z e d dur ing Zn d e f i c i e n c y i s a c o n t r o v e r s i a l i s s u e . Hur ley and Swenerton (1971) argue tha t bone Zn i s f i r m l y bound, and t he r e f o r e unava i l a b l e . Converse ly , o thers repo r t bone Zn to be a v a i l a b l e , at l e a s t i n growing animals (Harland et a l . 1975; Brown et a l . 1978). Murray and Messer (1981) i n ve s t i g a t ed bone Zn turnover i n growing r a t s and they too concluded tha t bone does not serve as a r e s e r v o i r of a v a i l a b l e Zn. However, the re was increased bone Zn depos i t i on when the animals were C a - d e f i c i e n t , suggest ing 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 in bone m i ne r a l . 2.8.2 P rope r t i e s Z inc i s a component of a number of metalloenzymes w i t h i n the body. Because of t h i s i t has been suggested tha t any ion competing w i th Zn f o r b ind ing s i t e s w i t h i n the p r o t e i n of the enzymes would i n f l u ence the a v a i l a b i l i t y of the Zn to the animal (Davis 1966). Z inc i s assoc ia ted wi th the f o l l o w i n g metal loenzymes: 1. ca rbon i c anhydrase (0.3% Zn) - 23 -2. panc rea t i c carboxypept idase 3. l i v e r dehydrogenase 4. a l k a l i n e phosphatase 5. t ryptophan desmolase 6. ma l i c dehydrogenase 7. g lutamic dehydrogenase 8. l a c t i c dehydrogenase 9. r e t i n o l dehydrogenase Of the above enzymes, ca rbon i c anhydrase conta ins the h ighes t concen t ra t i on of Zn. However, the lens conta ins much more carbon ic anhydrase than, the cho ro i d , but f a r l e s s Zn (Ga l i n et a l . 1962). Th is suggests t ha t the source of Zn i n the choro id may be something other than carbon ic anhydrase. 2.9 PHYTIC ACID An important c ons i de ra t i on i n minera l b i o a v a i l a b i l i t y s tud ies i s the 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 absorpt ion of c e r t a i n m ine r a l s . Such a f a c t o r i s phy t i c a c i d , a wa te r - so lub le organ ic a c i d of m y o - i n o s i t o l . Phy t i c ac id i s a s t rong che l a t i ng agent which may b ind w i th p r o t e i n , P and both mono- and d i v a l en t ca t i ons ( e s p e c i a l l y Ca and Zn) to form, 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 phy ta tes . 2.9.1 S t ruc tu re The chemical s t r u c t u r e of phy t i c a c i d i s s t i l l under debate, but the most w ide ly accepted i s the Anderson s t r u c t u r e con ta in ing 12 t i t r a t a b l e hydrogen ions as opposed to 18 i n the second most p l a u s i b l e form, the Neuberg s t r u c t u r e (Evans et a l . 1982). P r e s en t l y , the proper chemical des igna t i on f o r phy t i c ac id i s myo- i nos i t o l 1 ,2 ,3 ,4 ,5 ,6-hexak i s dihydrogen phosphate (S iy - 24 -and Ta lbo t 1982). The s t r u c t u r a l con f i gu ra t i on i s presented in F igure 3. F i g . 3. The s t r u c t u r e of phy t i c a c i d (Erdman, J r . 1979). 2 .9 .2 Occurrence Phy t i c ac id occurs in a l l foods of p l an t o r i g i n (L loyd et a l . 1978), 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 igh i n o i l s eeds such as soybean and cottonseed, which are commonly incorpora ted i n to salmonid f oods . On a dry b a s i s , whole o i l s eeas conta in approximate ly 1.5% phy t i c a c i d , but over 7% has been found in some p ro te in concentrates (Erdman, J r . 1979). The l o c a t i o n of phyt i c ac id va r i e s w i th the type of seed or g r a i n . For example, p h y t i c a c i d i n peanuts, sunf lower seeds and cottonseed i s concentrated w i t h i n the p r o t e i n body membrane. In c on t r a s t , soybean phy t i c ac id 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 w i t h i n p ro te i n bodies (Erdman, J r . 1979). 2.9.3 P roper t i es P hy t i c ac id i s the main storage form of phosphate and i n o s i t o l i n mature - 25 -seeds. In mature ce rea l g r a i n s , 60-80% of the t o t a l P i s conta ined w i t h i n the phy t i c ac id component, but va lues up to 99% have been repor ted f o r whole wheat f l o u r (Nahapetian and Young 1980) and va lues as low as 30.4% f o r n igerseed (Eklund 1975). I t i s g ene r a l l y accepted tha t p l an t s u t i l i z e phy t i c ac id as a source of P dur ing germinat ion (Erdman, J r . 1979). Th is i s accompl ished by p lan t phytases which hydro lyse phytate to phosphor ic a c i d and i n o s i t o l . Chen and Pan (1977) repor ted a two fo ld inc rease i n phytase a c t i v i t y f i v e days a f t e r germinat ion of soybeans and a t h i r t y - s e v e n f o l d inc rease in a c t i v i t y f i v e days a f t e r germinat ion of a pea v a r i e t y . Phy t i c ac id i s a l so a source of h igh-energy phosphoryl groups, a c e l l wa l l p recu rso r , and i s be l i eved to act as an an t i ox i dan t dur ing seed storage (Graf 1983). 2.10 PHYTATES As phy t i c ac id accumulates i n the va r i ous p l an t s torage s i t e s , m ine ra l s che l a t e to i t to form s a l t complexes commonly c a l l e d phy ta tes . Phytate u s u a l l y occurs as a mixed Ca-Mg-K s a l t c a l l e d phyt in (Graf 1983). 2.10.1 S t ruc tu re A hypo the t i ca l phy t a t e ' c on t a i n i ng Ca, Mg and Zn ions i s presented in F igure 4. Th is f i g u r e i l l u s t r a t e s t ha t c a t i on s may form s t rong bonds between two phosphate groups or weak bonds w i t h i n a phosphate group. - 26 -F i g . 4. The s t r u c t u r e of a phytate complex (Erdman, J r . 1979) . 2.10.2 P rope r t i e s The s t rong c h e l a t i n g a b i l i t y of phy t i c a c i d has imp l i c a ted i t i n numerous minera l d e f i c i e n c i e s . Stud ies have shown severa l m ine ra l -phy ta te 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) thereby render ing the che la ted minera l s unava i l ab l e f o r absorpt ion un less phytase i s present i n s u f f i c i e n t concent ra t i on to break the phytate complex. Ruminants have l i t t l e d i f f i c u l t y i n hyd ro l y z i ng phytate due to the l a rge m i c rob i a l popu la t i on i n the rumen (Jenk ins 1965; Nelson et a l . 1976). Monogastr ics however, have l i t t l e or no phytase. Phytase has not been i d e n t i f i e d in f i s h but i t has been detected i n the i n t e s t i n a l mucosa of dogs (Jenkins and P h i l i p s 1960), rodents (Roberts and Yudkin 1961), pou l t r y (Nelson et a l . 1971) and i n humans ( B i t a r and Reinhold 1972). The high s t a b i l i t y of m inera l -phyta te complexes at pH 6.5 ( i n descending - 27 -order) has been repor ted to be Zn, Cu, n i c k e l ( N i ) , Mg and Ca (Maddaiah et a l . 1964). 2.10.3 I n t e r a c t i on s At i n t e s t i n a l pH Zn forms the most s t ab l e ( i n s o l u b l e ) phytate complex. Consequent ly, i t has been the focus of many phy t i c a c i d s t u d i e s . For example, Oberleas et a l . (1966) observed poor Zn u t i l i z a t i o n and 40% growth reduc t i on i n r a t s fed 1% phy t i c a c i d (as % of the d i e t ) and suggested tha t t h i s was due to the format ion of an i n s o l u b l e Ca-Zn phytate i n the i n t e s t i n a l lumen. However, poor minera 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 a lone . Fac tors such as f i b r e content , p r o t e i n source or a v a i l a b i l i t y , i n t e s t i n a l pH, the presence of o ther minera l s and method of p rocess ing may a l so impa i r minera l a v a i l a b i l i t y ( Isma i1-Be ig i et a l . 1977; Oberleas and Har land 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 tud ies conducted, both in v i t r o and in v i v o , to assess minera 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 . Re inho ld et a l . (1975) performed in v i t r o s tud ies w i th wheat breads and concluded tha t phytates had l i t t l e e f f e c t upon the u t i l i z a t i o n of Ca or Zn. In f a c t , phytate removal from bran ( v i a phytase or a c i d e x t r a c t i o n ) increased the amount of bound Zn r epo r t ed l y due to an inc rease i n ' f i b r e c on cen t r a t i on . Never the less , they d id f i n d phytate to b ind Ca and Zn but they p red i c t ed tha t t h i s compound would even tua l l y be d i ges t ed , whereas the Ca and Zn bound i n the f i b r e f r a c t i o n would not . Hence minera l absorpt ion would be impa i red . S i m i l a r r e s u l t s were obta ined by I sma i l - Be i g i et a l . (1977) who i n v e s t i g a t ed the b ind ing c a p a b i l i t i e s of Tanok, an unleavened wholemeal wheat - 28 -bread con ta in i ng 0.7% phy t i c a c i d . Tanok i s a s t ap l e food i n Midd le Eastern v i l l a g e s whose i nhab i t an t s s u f f e r a high inc idence of Zn d e f i c i e n c y cha r a c t e r i z ed by re tarded growth and hypogonadal dwarf ism (L loyd et a l . 1978). M inera l b ind ing i s a pH dependent a c t i on (Thompson and Weber 1979) and I sma i1 -Be i g i ' s group found tha t at pH 6 .5 , 54% of the Zn was bound i n Tanok versus 88% which was bound i n a phy ta te - f r ee Tanok. The g rea te r a f f i n i t y f o r Zn by the l a t t e r bread was a t t r i b u t e d to i t s inc reased f i b r e content as a r e s u l t o f phytate removal . The extent to which d i e t a r y f i b r e i n h i b i t s minera l u t i l i z a t i o n i s g ene r a l l y a f un c t i o n of the chemical s t r u c t u r e of the i n d i g e s t i b l e po l y sac cha r i de , p a r t i c u l a r l y i t s ion-exchange p rope r t i e s and the degree of m i c r ob i a l degradat ion o c cu r r i ng i n the gut (Harmouth-Hoene and Schelenz 1980). In con t ras t to the above exper iments, the r e s u l t s of Davies et a l . (1977) who performed i n v i vo s tud ies w i th r a t s , i n d i c a t ed phy ta te , r a the r than f i b r e , to be the major cause of reduced Zn a v a i l a b i l i t y . Franz et a l . (1980) fed r a t s s em i - pu r i f i e d d i e t s con ta i n i ng e i t h e r co rn , r i c e , wheat, l ima beans or whi te beans. With the except ion of the legume d i e t s , the phy t i c ac id concen t ra t i on appeared to be i n v e r s e l y r e l a t e d to Zn u t i l i z a t i o n w i th the lowest a v a i l a b i l i t i e s o c cu r r i ng i n the whole corn and brown r i c e d i e t s . They pos tu l a ted tha t Zn in 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 he r e f o r e i t would not be a f f e c t ed by phy t i c a c i d i n the same way. Ch icks fed soybean d i e t s w i th an add i t i o na l 6% d i e t a r y f i b r e (as wheat bran, corn bran, soy bran, oat h u l l s or c e l l u l o s e ) e xh i b i t e d no s igns of reduced growth or minera l d e f i c i e n c i e s (Thompson and Weber 1981). However, - 29 -ch i cks fed d i e t s con ta in i ng 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 , feed i n t akes , t i b i a we ights , and t i b i a Zn, Fe and Mn concen t ra t i ons . Ana l y s i s showed tha t the r i c e bran d i e t conta ined 1.3% phy t i c a c i d whereas the next h ighest concen t ra t i on was found i n the wheat bran d i e t which conta ined on ly 0.42% phy t i c a c i d . I t was concluded tha t the phy t i c ac id had i n t e r f e r e d w i th minera l metabol ism. 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 ed tha t the minera l s t a tus i n ch i cks fed d i e t a r y f i b r e was dependent upon the t ype , 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 co rn , oat or wheat bran at 4% and 8% of the d i e t reduced Zn absorpt ion but i t cou ld not be determined i f t h i s was l a r g e l y due to phy ta tes . In any case , there appeared t o be some c o r r e l a t i o n between phy t i c a c i d and Zn a v a i l a b i l i t y because, of the three f i b r e sources , Zn absorpt ion was l e a s t a f f e c t ed by corn which conta ined 0.24% phy t i c a c i d . By c on t r a s t , Zn absorpt ion was most a f f e c t ed by the wheat bran which conta ined 4.6% phy t i c a c i d . A more recent study (Navert et a l . 1985) i nvo lved the feed ing of leavened wheat bran to human vo l un tee r s . Phy t i c ac id content was reduced by va ry i ng the leaven ing t ime from 0-120 hours. The r e s u l t s showed tha t Zn absorpt ion increased w i th fe rmentat ion t ime i . e . reduced phy t i c ac id content was assoc ia ted wi th enhanced Zn abso rp t i on . 2.12 CALCIUM-ZINC-PHYTATES Recent l y , there has been emphasis p laced on employing phytate to Zn molar r a t i o s as determinants of Zn s ta tus (Franz et a l . 1980; Lo et a l . 1981; House et a l . 1982; Forbes et a l . 1983; Meyer et a l . 1983). However, the l i t e r a t u r e suggests tha t a more use fu l determinant, may a l so i n c lude the l e v e l o f d i e t a r y Ca. - 30 -2.12.1 Swine S tud ies P a r ake r a t o s i s , or Zn d e f i c i e n c y syndrome, has been induced i n swine (Oberleas et a l . 1962) by the add i t i o n of 0.7% phyt i c a c i d to a case in d i e t . Z inc d e f i c i e n c y has a l so been observed i n swine fed a soybean d i e t con ta i n i ng 0.5% phy t i c a c i d . Inc reas ing the Ca content from 0.8 to 1.5% aggravated the d e f i c i e n c y . The sk i n l e s i on 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 reversed when the d i e t s were supplemented w i th Zn. 2.12.2 Rat S tud ies Forbes (1964) observed reduced growth and femur Zn concen t ra t i on i n r a t s f ed a soy p ro t e i n d i e t and these e f f e c t s became more marked as the d i e t a r y Ca concent ra t i on was i n c reased . Phy t i c a c i d concent ra t i on was not determined but Forbes suggested tha t the 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 the presence of phy 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 fed case in d i e t s c on t a i n i ng (on a dry bas i s ) 0.4, 0.8 and 1.2% Ca; 12 and 65 mg Zn/kg; and 0.0, 0.4 and 2% phy t i c a c i d . The add i t i on of 0.4% phy t i c a c i d d i d not a f f e c t weight g a i n , but the add i t i on of 2% phy t i c ac id s i g n i f i c a n t l y reduced d a i l y weight ga in by 45% when the Zn l e v e l was 12 mg/kg and Ca was 0.4%. Weight ga in was f u r t h e r reduced by 76% and 79% when d i e t a r y Ca was increased to 0.8% and 1.2%, r e s p e c t i v e l y . Oberleas et a l . (1966) concluded tha t Na-phytate, added to d i e t s to ob ta in 1% phy t i c a c i d , i n h i b i t e d Zn absorpt ion i n r a t s and reduced growth by 40% compared to r a t s fed non-phytate d i e t s . Th is growth i n h i b i t i o n was f u r t h e r aggravated by i n c r ea s i ng d i e t a r y Ca from 0.8% to 1.6%. These e f f e c t s were e l im ina ted when the d i e t a r y Zn l e v e l was inc reased to 55 mg from 6-8 mg/kg. S i m i l a r r e s u l t s were obta ined by Davies and N i gh t i nga l e (1975). For example, r a t s f ed p u r i f i e d d i e t s supplemented wi th 15 mg Zn/kg, 1% phy t i c ac id and 1.3% Ca, had s i g n i f i c a n t l y lower weight gains (84% reduc t i on) than the non-phytate fed r a t s . Furthermore, there were s i g n i f i c a n t decreases in carcass r e t en t i on of Fe, Cu, Zn and Mn. Moreover, when the Zn supplement was reduced to 0.5 mg/kg, the s e v e r i t y of the r e s u l t s i n c reased . In add i t i o n to negat ive e f f e c t s on growth, i t was concluded tha t the phy t i c a c i d impaired both the absorpt ion of d i e t a r y Zn and the reabsorp t ion of endogenously-secreted Zn. 2.12.3 Pou l t r y Stud ies 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 f ed corn-soybean d i e t s and t h i s has been a t t r i b u t e d to the phy t i c a c i d content of the d i e t s (O 'De l l and Savage 1960). Ch icks fed c a s e i n - g e l a t i n d i e t s w i th 0.5% phytate and 1.2% Ca e xh i b i t e d s i g n i f i c a n t growth reduc t i ons when the d i e t s were marginal i n Zn (O 'De l l et a l . 1964). Inc reas ing Ca to 2.4% of the d i e t had no e f f e c t on growth or Zn absorpt ion when phy t i c a c i d was absent. Bafundo et a l . (1984) fed ch i c k s an adequate corn-soybean meal d i e t w i th Ca supplements of 0.91% and 1.82%. Ch icks fed d i e t s wi th 1.82% Ca showed reduced growth and food conve r s i on , and depressed l e v e l s of t i s s u e and plasma Zn. The add i t i on 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 on ly i n the presence of excess Ca and the absence of supplemental Zn. The f i n d i n g s of O 'De l l et a l . (1964) showed tha t Zn a v a i l a b i l i t y to ch i cks was reduced on ly in the presence of phy t i c ac id and tha t d i e t a r y Ca exacerbated the e f f e c t . 2.13 THE EFFECT OF GUT pH ON PHYTATE ACTIVITY Most s o l u b i l i t y s tud ies have been conducted i n v i t r o and the r e s u l t s g ene r a l l y i n d i c a t e tha t phytates are most so l ub l e at g a s t r i c pH (3-5) and l e a s t so l ub 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 phy t i c ac id i s a l so dependent upon the presence of c a t i o n s . Oberleas et a l . - 32 -(1966) found Zn-phytates to be h i g h l y i n s o l u b l e at smal l 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 the add i t i on of Ca. Using a 1:1:1 Ca:Zn:phytate complex, 77% of the Zn was p r e c i p i t a t e d . Inc reas ing the r a t i o to 2:1:1 r e su l t ed i n p r e c i p i t a t i o n of 97% of the Zn and 84% of the Ca. The absence or low concent ra t i on of Ca reduced the a f f i n i t y of phy t i c ac id f o r Zn, consequent ly render ing Zn more a v a i l a b l e f o r abso rp t i on . There fo re , i t appears tha t the maximum b ind ing of Zn to phy t i c ac id r equ 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 research has been done to determine the e f f e c t s of d i e t a r y phy t i c ac id i n f i s h as compared to other an ima ls . Sp ine l 1i et a l . (1979) fed rainbow t r ou t OMP that had the phytates removed from the soybean po r t i on of the d i e t and they found tha t growth performance was s i g n i f i c a n t l y improved. Fur ther experiments by S p i n e l l i et a l . (1983) were conducted to more a c cu ra te l y assess the e f f e c t s of phytates on growth and food convers ion i n rainbow t r o u t . The f i s h were f ed a c a s e i n - g e l a t i n d i e t c on ta i n i ng 0.5% of e i t h e r Na- or Ca-phytate and i n c r ea s i ng l e v e l s of Ca (0.92 to 1.2%). The Zn l e v e l i n the d i e t was kept constant at 54 mg/kg. The add i t i o n of 0.5% phytate reduced weight gain by 10%. Increased l e v e l s of Ca d id not aggravate the response. The added phytates 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 the b lood, l i v e r or k idney . However, b lood Cu increased w i th the . add i t i o n of phy t i c ac id but then decreased when the d i e t s were supplemented wi th Ca or Mg. In v i t r o t e s t s showed tha t the case in -phy ta te complex was poo r l y hydro lyzed by peps in . Subsequent in v i vo t e s t s , us ing a case in-phyta te complex ( v s . case in a l one ) , revea led tha t d i e t d i g e s t i b i l i t y was reduced by 6.6%. There fore , they concluded tha t the reduced growth was - 33 -due to decreased p r o t e i n a v a i l a b i l i t y r a the r than to decreased minera l a v a i l a b i l i t y . However, i t i s conce ivab le t ha t the low l e ve l o f phy t i c ac id employed by S p i n e l l i ( r e l a t i v e to the concen t ra t i ons used i n s tud i e s on other animals) was i n s u f f i c i e n t to a f f e c t growth and minera l u t i l i z a t i o n e s p e c i a l l y when the d i e t a r y Zn l e v e l was two to th ree t imes h igher than the known requ i rement of sa lmonids. -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 ded i n t o s i x general s tages: eggs, a l e v i n s , f r y , smol ts , matur ing and mature a du l t s . Given a water temperature of 10-11°C, chinook eggs hatch 48-54 days a f t e r f e r t i l i z a t i o n . The newly hatched f i s h are r e f e r r e d to as a l e v i n s and they possess yo l k sacs from which they de r i ve nourishment f o r another 42-46 days. A f t e r the yo l k sacs have been absorbed the f i s h begin to a c t i v e l y seek f ood . At t h i s stage they are commonly c a l l e d swim-up f r y . Approx imate ly 90 days a f t e r swim-up, the f r y enter the " s i l v e r i n g " or smo l t ing stage and are now ready t o migrate t o the sea as smo l t s . At t h i s s tage, ha tchery- reared salmon are normal ly r e l e a sed . Some chinook salmon remain i n f reshwater f o r 1-2 years before heading downstream, but most migra te a few months a f t e r swim-up. Chinook salmon may spend up to 8 years matur ing at sea but the m a j o r i t y r e tu rn to spawn a f t e r 4-5 years (Hart 1973). Growth of hatchery f i s h i s dependent upon severa l f a c t o r s . The most important are food 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 temperature (S t au f f e r 1973). F i gu re 5 dep i c t s the growth curve f o r a f i s h under constant environmental c ond i t i on s from 0-500 days post swim-up. Th i s t h e s i s i s concerned wi th the pe r i od rang ing from 0-150 days. As t h i s represents a very smal l po r t i on i n the growth curve , the data have been - 34 -transformed to a log s c a l e . The s o l i d l i n e s show expe r imen ta l l y observed growth stages and the broken l i n e s show what the growth phase would look l i k e i f a g iven growth r a t e p e r s i s t e d . The most r ap i d stage of growth occurs at the youngest, sma l l e s t stage (B re t t 1979) w i th the r a t e d e c l i n i n g sharp ly at the onset of ma tu r i t y . - 35 -F I S H G R O W T H C U R V E 1 0 0 0 -1 0 0 2 0 0 3 0 0 D A Y S P O S T S W I M - U P 4 0 0 5 0 0 Fig. 5. 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 sec t i ons desc r ibe ma t e r i a l s and methods which were common to the th ree experiments conducted i n t h i s s tudy. 3.1 Exper imenta l F i sh Chinook salmon f r y were obta ined from B ig Qualicum hatchery (Vancouver I s l a n d , B.C.) broodstock in February 1982 and February 1983. The f i s h were s e l e c t ed f o r un i form 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 tanks (experiments I and I I I ) . 3.2 Cu l t u r e Cond i t i ons The f i s h were mainta ined i n an indoor f a c i l i t y con ta i n i ng two rows of t anks . A cont inuous supply of aerated we l l water (4-6 L/min) mainta ined the water temperature at 10-11°C. Some chemical parameters of the we l l water i n mg/L were as f o l l o w s : CaC03 (hardness) 36 .8 -38 .1 ; Ca 11 .9-12.3; Cu <0.005; Mg 1.7; Na 10.1-11.6; lead <0.002; Zn <0.002. The pH of the water was 6 .6 -6 .7 . Throughout the study, overhead day l i g h t f l u o r e s cen t l i g h t s ( V i t a l i t e , Durotest 40W) prov ided a na tu ra l photoper iod . 3.3 D ie t Formulat ions The d i e t s were c a s e i n - ge l a t i n based w i th va ry ing l e v e l s of Ca, Zn and phy t i c a c i d , as sodium phyta te . Al though the Na-phytate conta ined 22% Na, d i e t a r y l e v e l s of Na d id not exceed those found normal ly i n OMP, a p r i n c i p a l commerical hatchery d i e t (Higgs et a l . 1982a). Thus, the d i f f e r en c e s between d i e t s i n Na content was not cons idered to be a confounding f a c t o r . D i e t a r y l e v e l s of Ca, Zn and phy t i c ac id were s e l e c t ed to bracket the range tha t cou ld a c t u a l l y o r p o t e n t i a l l y be present i n p r a c t i c a l salmonid d i e t s - 37 -depending on the extent of i n co rpo ra t i on of var ious types of animal and p lan t p r o t e i n s and the composit ion of the minera l supplement. D ie t s were formulated to have a Ca to P r a t i o of c l o se to un i t y , independent of the phytate-P c o n t r i b u t i o n . D i e ta ry f o rmu la t i ons are l i s t e d i n Tables 2, 3 and 3a . Proximate composit ion and d i e t a r y minera l l e v e l s are l i s t e d i n Tables 4 and 4a . 3.4 D ie t P repara t i on D i e t s were prepared i n May 1982 (experiment I ) and February 1983 (experiments II and I I I ) . The basal minera l supplement was mixed f o r 24 hours i n a b a l l m i l l (Norton, Chemical Process Products D i v i s i o n , Akron, Oh io ) . The minera l -phyta te mixes were homogenized by v igorous shaking i n p l a s t i c bags. They were then added to the basal minera l mix and reshaken i n bags. V i tamins were combined f o r 60 minutes i n a Twin She l l Dry Blender ( Pa t t e r son -Ke l l e y Co. , D i v i s i o n of Harsco Corp . , Pennsy l van ia ) . Ingred ients f o r the basal d i e t s were mixed together (wi th a po r t i on of the o i l ) f o r 20 minutes i n a Hobart commercial mixer (Hobart Manufactur ing Co . , Troy, Ohio) f o r experiment I and f o r 30 minutes i n a Marion mixer (Rapids Machinery Co . , Iowa) f o r experiments I I and I I I . V i tamin and minera l pre-mixes were added to the basal d i e t s and combined i n a Hobart mixer f o r 20 minutes. Each d i e t was then c o l d - p e l l e t e d in a CL-type 2 l abo ra to ry p e l l e t m i l l ( C a l i f o r n i a P e l l e t M i l l Co . , San F ranc i s co , C a l i f o r n i a ) with a 1.59 mm d i e . A f t e r c o o l i n g , the p e l l e t s were crumbled and hand-screened to appropr i a te s i zed crumbles. The remainder of the he r r i ng o i l was sprayed on to the d i e t s us ing a .hand-held sy r inge (needle s i z e 18) and mixed i n w i th a spoon. Stock d i e t s were s to red at -40°C u n t i l r e qu i r e d . D i e t s i n use were kept i n a i r t i g h t con ta ine 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. - 38 -TABLE 2 Formulation of basal d iet fed to juven i l e chinook salmon in a l l experiments. Ingredient* Concentration (g/kg dry diet) Casein (92.43% crude protein) 476.23 Ge lat in (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 chlor ide (60%) 8.33 Ascorbic acid 1.00 Amino acid mixS 29.35 Mineral-phytate + a-ce l lu lose mix^ 264.29 1 D ietary ingredients were commercially obtained. The vitamin-free case in , g e l a t i n , dextr in and a-cel lu lose were from IC.N Nut r i t i ona l Biochemicals, C leveland, Ohio. Sodium phytate was obtained from Sigma Chemical Co., S t . Lou is , M i s sour i , U.S.A. 2 S t ab i l i z ed with 0.33% BHA-BHT (1:1). 3 The vitamin supplement provided the fo l lowing amounts per kg of dry d i e t : DL-ct -tocopherol 300 IU; thiamin mononitrate 50 mg; r i bo f l av i n 200 mg; n iac in 500 mg; b i o t i n 5 mg; 0-calcium pantothenate 300 mg; pyridoxine-HCl 50 mg; f o l i c acid 15 mg; menadione (as hetrazeen) 39.6 mg; vitamin B-12 0.2 mg; inos i to l 2000 mg; p-aminobenzoic acid 400 mg; ret ino l acetate 5000 I U ; cho leca lc i fe ro l 2400 IU (1500 in expt. I ) ; BHT 22 mg. 4 The mineral supplement provided the fo l low ing (mg/kg of dry d i e t ) : c i t r i c ac id 90.3; copper (as CU2C5H4O7.2 1/2 H2O) 6.5; i ron (as FeCsH507.5H20) 50; manganese (as MnS04.H20) 74.8; iodine (as KI) 4.6; potassium (as K2SO4) 2675; sodium ch lo r ide 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 acid mix provided (g/kg dry d i e t ) : L-arginine-HCl 7.0; DL-methionine 2.85; carboxymethylcel lulose (CMC) 14; a-ce l lu lose 5 (10 in expt. I - tota l amino ac id mix 33.85). 6 Total concentration was 250.29 g/kg in expt. I . The composition of the mineral-phytate mixes are presented in Table 3. TABLE 3 Composi t ion of the nine m ine ra l -phy ta te supplements fed to j u v e n i l e chinook salmon i n Experiment I . D I E T Ingred ient 1 2 3 4 5 6 7 8 9 g/kg dry d i e t Ca lc ium carbonate (CaC0 3 ) 9.52 9.52 9.52 9.52 9.52 9.52 9.52 9.52 9.52 Calc ium phosphate monobasic (CaH4(P04) 2 .H 2 0) . . - • 67.30 - 67.30 - 67.30 - 67.30 19.24 Ca lc ium phosphate d i b a s i c (CaHP0 4.2H20) 111.76 111.76 - 111.76 - '• 111.76 31.94 Sodium phosphate monobasic (NaH2P04.H 20) 7.84 7.84 7.84 7.84 7.844 7.84 7.84 7.84 7.84 Phy t i c ac id sodium s a l t (64.6% phy t i c ac id) 2.5 2.5 2.5 2.5 40.0 40.0 40.0 40.0 10.0 Z inc su lphate (ZnS0 4 .7H 2 0) 0.088 0.088 1.41 1.41 0,088 0.088 •1.41 1.41 0.352 a - c e l l u l o s e 230.34 51.28 229.02 49.96 192.84 13.78 191.52 12.46 171.40 TABLE 3a Composit ion of the e ighteen m ine ra l -phy ta te supplements fed to j u v e n i l e chinook salmon i n Experiments II (A, B and C) and I I I (1-15) . D I E T Ingred ien t A B C 1 2 3 4 5 6 g/kg d ry d i e t Calc ium carbonate (CaC0 3) 12.09 12.09 12.09 12.09 12.09 12.09 12.09 12.09 12.09 Calc ium phosphate monobasic (CaH4(P0 4 ) 2 .H 2 0 ) - 73.27 73.27 6.48 66.79 6.48 66.79 6.48 66.79 Calc ium phosphate d i b a s i c (CaHP04.2H20) - 121.72 121.72 10,77 110.95 10.77 110.95 .10.77 110.95 Sodium phosphate monobasic (NaH 2 P0 4 .H 2 0) 15.77 6.86 6.86 6.86 6.86 6.86 6.86 6.86 6.86 Phy t i c ac id sodium s a l t (64.6% phy t i c ac id) 2.5 2.5 40.0 3.27 3.27 3.27 3.27 32.73 32.73 Z inc su lphate (ZnS0 4 .7H 2 0) 0.106 0.106 0.106 0.106 0.106 1.073 1.073 0.106 0.106 a - c e l l u l o s e 223.82 37.74 0.244 214.71 54:22 213.75 53.26 185.25 24.76 TABLE 3a c o n t ' d . . . . . -•' • D I E T Ingred ient 7 8 10 11 12 13 14 15 g/kg dry d i e t Calc ium carbonate .., (CaC0 3 ) 12.09 12.09 12.09 12.09 12.09 12.09 12.09 12.09 12.09 Calc ium phosphate monobasic (CaH4(P04) 2 .H 2 0) 6.48 66.79 - 73.27 36.64 36.64 36.64 36.64 36.64 Calc ium phosphate d i b a s i c (CaHP04.2H 20) 10.77 110.95 - 121.72 60.86 60.86 60.86 60.86 60.86 Sodium phosphate monobasic (NaH 2 P0 4 .H 2 0) : .6.86 6.86 15.77 6.86 6.86 6.86 6.86 6.86 6.86 Phy t i c ac id sodium s a l t , (64.6% phy t i c ac id ) 32.73 32.73 18.0 18.0 18.0 18.0 0.10. 35.9 18.0 Z inc su lphate (ZnS0 4 .7H 2 0) 1.073 1.073 0.589 0.589 1.178 0.589 0.589 0.589 a - c e l l u l o s e 184.29 23.80 207.84 21.76 119.84 118.66 137.15 101.35 119.25 V TABLE 4 Proximate composition, mineral and phytic acid content of the nine test diets fed to juvenile chinook salmon In Experiment I. D I E T 1 2 3 4 5 6 7 8 9 Proximate composition g/kg dry diet Protein (N x 6.25) 539 520 536 532 542 531 526 537 521 Crude lipid (Bllgh-Dyer) 128 131 131 130 111 128 109 108 107 Ash 36 194 35 193 62 225 62 226 v 86 Moisture (as fed) 104 96 103 92 102 97 100 96 94 Mineral content* Calcium (Ca) 4.37 49.1 4.52 47.8 5.14 51.3 5.29 52.6 17.7 Phosphorus (P) 5.92 46.2 6.01 45.3 14.7 55.9 14.6 57.5 19.3 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 Iron (Fe) 0.113 0.107 0.098 0.102 0.107 0.096 0.118 0.088 0.091 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 15.1 13.9 15.2 14.8 5.66 Ca:P2 0.74 1.06 0.75 1.06 0.35 0.92 0.36 0.91 0.92 Ca:Mg 6.7 72.2 7.0 67.3 6.8 66.6 7.7 68.3 27.4 P:Mg 9.1 67.9 9.2 63.8 19.3 72.6 21.2 74.7 29.8 Ca:Zn 80.9 982.0 12.8 118.6 : 93.4 1005.8 13.4 134.5 116.4 Phytic add 1.62 1.62 1.62 1.62 : 25.8 25.8 25.8 25.8 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 2 3 4 5 6 Proximate composition g/kg dry diet Protein (N x 6.25) 512 520 512 518 514 506 509 509 514 Crude lipid (Bllgh-Dyer) 110 116 102 121 116 114 118 117 116 Ash 47 222 260 58 178 60 177 83 . 226 Moisture (as fed) 110 105 110 102 99 101 100 106 105; Mineral content^ Calcium (Ca) 5.48 73.1 66.5 9.63 46.0 9.94 47.1 10.2 50.0 Phosphorus (P) 7.34 51.1 64.9 9.84 44.4 10.6 44.4 16.2 50.7 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 Iron (Fe) 0.085 0.085 0.081 0.090 0.105 0.090 0.109 0.094 0.130 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 0.005 0.005 Sodium (Na) 5.34 4.10 14.3 3.83 4.60 4.46 3.99 12.6 12.5 Ca:P2 0.75 1.43 1.02 0.98 1.04 0.94 1.06 0.63 0.99 Ca:Mg 4.94 69.0 65.2 11.5 51.1 12.7 49.1 13.8 46.3 P:Mg 6.61 48.2 63.6 11.1 49.3 13.6 46.2 21.9 46.9 Ca:Zn 99.6 1260.0 1090.0 171.9 567.9 36.7 210.3 196.1 892.9 Phytic acid 1.62 1.62 25.8 2.10 2.10 2.10 2.10 21.1 21.1 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. D I E T 7 8 9 10 11 12 13 14 15 Proximate composition g/kg dry diet Protein (N x 6.25) 498 509 506 503 500 495 508 503 509 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 Mineral content* Calcium (Ca) 9.80 48.6 5.60 52.1 25.2 29.1 28.3 29.1 29.1 Phosphorus (P) 15.8 51.0 11.0 52.6 31.1 30.1 26.4 34.8 30.3 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 Sodium (Na) 12.4 11.7 9.64 7.96 13.2 8.15 3.32 13.8 8.25 Ca:P2 0.62 0.95 0.51 0.99 0.81 0.97 1.07 0.85 0.96 Ca:Mg 13.4 54.6 7.20 53.7 35.5 38.3 36.7 35.1 34.2 P:Mg 21.6 57.3 14.1 34.2 43.8 39.6 39.3 41.4 35.6 Ca:Zn 36.4 153.8 37.6 331.8 • 741.2 104.3 188.7 173.5 172.2 Phytic acid 21.1 21.1 11.6 11.6 11.6 11.6 0.06 23.2 11.6 * Determined by plasma spectroscopy (Higgs et al . 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 Exper imental Procedures and Sampling 3.5.1 D ie t a l l o c a t i o n D ie t s were ass igned randomly to each row of tanks i n a complete randomized b lock des ign w i th b locks as r e p l i c a t e s . There fore , b l ocks were not analyzed as such but were inc luded i n the e r r o r term. 3.5.2 Feeding . • r.-F i sh were fed by hand to s a t i a t i o n three t imes d a i l y , seven days a week, except on weighing days. For the f i r s t 28 days of experiment I I the f i s h were fed s i x t imes d a i l y . Food p a r t i c l e s i z e was adjusted i n r e l a t i o n to f i s h s i z e (Fowler and Burrows 1971). D a i l y food consumption was recorded f o r experiments I and I I I . 3.5.3 F i s h weight On day 0 of the experiments and every 21 days t h e r e a f t e r (every 42 days i n exp t . I I ) , a l l f i s h were removed from the tanks and random samples of 60 f i s h / t a n k were anaesthet i zed w i th 2-phenoxyethanol (0.5 ml/L wa t e r ) , p laced on an absorbent c l o t h to remove excess mo i s tu re , and i n d i v i d u a l l y weighed to the nearest 0.01 g. F i s h were s ta rved f o r 24 hours p r i o r to each weighing day. Removal of a l l f i s h from each tank al lowed f o r tank c l ean i ng and an accurate check of f i s h numbers. The . l a t t e r cou ld then be checked aga ins t d a i l y m o r t a l i t y records (exp t s . I and I I I ) . Throughout the exper iments, f i s h numbers were p e r i o d i c a l l y th inned out to reduce f i s h d en s i t y . 3.5.4 Cata rac t assessment On each weighing day, the eyes of 60 f i s h / t ank 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 igns of c a t a r a c t s . I f c a t a r a c t s were observed a l l f i s h i n the r e spe c t i v e tank(s ) were then examined f o r c a t a r a c t s . - 46 -3.5.5 Proximate ana l y s i s F i sh were sampled randomly at the beginning and end of experiments 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 th 2-phenoxyethanol, b l o t t e d dry on absorbent c l o t h , p laced i n heat -sea led p l a s t i c bags and s tored at -40°C f o r subsequent a n a l y s i s . P r i o r to 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 laced i n a b lender and blended to a f i n e homogenate. Samples were analyzed f o r mo i s tu re , 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 rogen (Technicon Instrumental Co . , L t d . , i n d u s t r i a l methods 369-75A/A and 334-74 W/B). N i t rogen was m u l t i p l i e d by 6.25 to ob ta i n crude p r o t e i n . Test d i e t s were s i m i l a r l y ana lyzed. 3.5.6 F i s h hea l th and h i s t o l o g i c a l examinat ion At the end of experiments I and I I I , 10-20 f i s h / d i e t t reatment were assessed f o r general hea l th by personnel from the P a c i f i c B i o l o g i c a l S t a t i o n , D iagnos t i c Se rv i ces Branch, Nanaimo, B.C. The f i s h were examined f o r b a c t e r i a l d i sease and ex te rna l and i n t e r n a l abno rma l i t i e s . Haematocr i t s , haemoglobin content , red blood c e l l count and shape and s i z e of b lood c e l l s were measured. A l so f o r experiments I and I I I , h i s t o l o g i c a l examinat ions of the g i l l , l i v e r , k idney, p y l o r i c caeca and stomach were conducted by the methods of McBride and van Overbeeke (1971) and van Overbeeke and McBride (1971). 3.5.7 Plasma and blood ana l y s i s Plasma samples were taken at the end of experiment I and whole blood samples at the end of experiment I I I f o r minera l a n a l y s i s . The samples were prepared and analyzed on an I ndu c t i v e l y Coupled Argon Plasma-Atomic Emission Spectrometer (ICAP-AES, J a r r e l l - A s h Model 975 Plasma Atomcomp; F i s he r S c i e n t i f i c Co . , Vancouver, B .C . ) . 7 - 47 -3.5.8 T i s sue ana l y s i s At the end of experiment I I I , samples of f i s h were k i l l e d and s tored at -40°C f o r l a t e r minera l a na l y s i s of l i v e r s and k i dneys . - 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 the lack of i n fo rmat ion on the n u t r i t i o n a l bas i s f o r c a t a r a c t fo rmat ion i n P a c i f i c salmon, t h i s study was conducted to determine the 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 phy t i c a c i d on c a t a r a c t s as we l l as on growth, food i n t ake , food convers ion 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 , the h i s t o pa 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 phy t i c ac id were assessed. Phy t i c a c i d , as sodium phy ta te , was inc luded as a d i e t a r y treatment to assess 1) whether c a t a r a c t fo rmat ion cou ld o r i g i n a t e from i nges t i on of a d i e t con ta i n i ng a h igh r a t i o of Ca t o Zn and a low l e v e l of a m i ne r a l - c he l a t i n g agent, or 2) whether the s imultaneous presence of high d i e t a r y r a t i o s of ca lc ium to z i n c and of a s t rong m ine r a l - che l a t i ng agent were r equ i r ed . Phy t i c ac id i s known to s t r ong l y che la te d i v a l e n t m ine ra l s , such as Zn, to form i n s o l u b l e phytates in the i n t e s t i n a l lumen, thus lower ing Zn b i o a v a i l a b i l i t y (Oberleas and Harland 1977; Erdman, J r . 1979). In a d d i t i o n , the presence of high d i e t a r y Ca i s thought to enhance the a f f i n i t y between phy t i c ac id and Zn i n the i n t e s t i n e , thus aggravat ing the depress ion of growth and Zn u t i l i z a t i o n . Converse ly , d i e t a r y Zn supplementat ion has been shown to counteract the e f f e c t s of phy ta tes , and t h i s p o s s i b i l i t y was a l so i n ve s t i ga t ed i n chinook salmon. - 49 -4.2 MATERIALS AND METHODS 4 .2 .1 Exper imental F i sh Chinook salmon f r y were s e l e c t ed f o r un i form 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 tanks r e s u l t i n g i n 113 f i s h / t a n k . I n i t i a l mean weight of the f i s h was 1.09 ± 0.03 g (SEM). 4 .2 .2 Exper imental D ie t s ' Nine dry d i e t s (Tables 2-4) c on t a i n i ng va ry i ng l e v e l s of Ca, Zn and phy t i c a c i d were prepared at the West Vancouver Laboratory as p r e v i ou s l y de s c r i bed . 4 .2 .3 Feeding Each d i e t was fed to two tanks of f i s h f o r 105 days. F i s h were fed to s a t i a t i o n three t imes d a i l y w i th the po in t of s a t i a t i o n being reached when a c t i v e feed ing ceased (approx imate ly one hou r ) . 4 .2 .4 Sampling Procedures 4 .2 .4 .1 F i s h weight and ca t a ra c t inc idence 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, 21, 42, 63, 84 and 105. Eyes were examined f o r c a t a r a c t s on each weighing day as desc r i bed i n s e c t i on 3 .5 .4 . 4 .2 .4 .2 Proximate ana l y s i s P r i o r to the s t a r t of the exper iment, 113 f i s h were se l e c t ed randomly f o r proximate a n a l y s i s . At t e rm ina t i on of the s tudy, 6-20 f i s h per d i e t t reatment were sampled and pooled i n t o two groups per treatment f o r subsequent a n a l y s i s . 4 . 2 . 4 . 3 H i s to l ogy samples S i x f i s h from each d i e t a r y treatment were se l e c ted randomly f o r h i s t o l o g i c a l examinat ion of the g i l l , l i v e r , k idney, 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 the caudal vesse l o f at l e a s t 25 f i s h / d i e t t rea tment . Blood was c o l l e c t e d i n hepa r i n i zed c a p i l l a r y tubes and c en t r i f u ged . A f t e r c e n t r i f u g a t i o n , the plasma from each tube was t r a n s f e r r e d i n t o one of f ou r pools per d i e t treatment 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 Ana l y s i s On the bas i s of the growth pa t te rn observed, the experiment was d i v i ded i n to two stages of exponent ia l growth f o r data a n a l y s i s . Stages 1 and 2 corresponded to days 0-63 and days 64-105, r e s p e c t i v e l y . Geometric mean wet weights were analyzed by a one-way ana l y s i s of va r i ance (ANOVA). S p e c i f i c growth r a te s (B re t t 1979) were determined and analyzed by a one-way ana l y s i s of covar iance of the na tu ra l log of the wet weights w i th day as the c o v a r i a t e . Growth r a t e s were de r i ved from the c o va r i a t e s lope (e s^°P e - 1) x 100 and expressed as a percent inc rease i n body weight per day. Food i n take was expressed as a percentage of body weight and was c a l c u l a t e d f o r a g iven per iod of t ime ( t , t + l ) accord ing to the f o l l o w i n g fo rmu la: DFI x 100 * (logn^+l + logn^ * 2 ) e x P where DFI i s mean d a i l y dry food in take per f i s h ( t , t + l ) and l ogn t , lognt+1 are the averaged na tu ra l log wet weights at the s t a r t ( t ) and the end (t+1) of the pe r i o d . The data were analyzed by a one- or two-way ANOVA. Food convers ion 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 gain * dry food i n take and wet weight ga in *• p ro t e i n i n t a ke , r e s p e c t i v e l y , and were analyzed by a one- ( d i e t ) and two-way ( d i e t , per iod) ANOVA. Data f o r plasma minera l l e v e l s were analyzed by a one-way ANOVA. S t a t i s t i c a l ana lyses were performed by us ing the program UBC GENLIN. See Appendix A f o r ANOVA t a b l e s . - 51 -When app rop r i a t e , Newman-Keuls' (Keuls 1952) t e s t and S che f f e ' s (Scheffe* 1959) t e s t (growth ra tes on ly) w i th P=0.05 were used to detec t s i g n i f i c a n t d i f f e r en c e s between treatment means. The standard e r r o r of the mean (SEM) was de r i ved by us ing the e r r o r mean square from the ANOVA as t h e e s t i m a t e of sample va r i an ce . 4.3 RESULTS . • 4 .3 .1 In f luence of D ie t Treatment on Ca ta rac t Inc idence Table 5 shows tha t b i l a t e r a l lens c a t a r a c t s were detected on l y i n groups fed d i e t s 5 and 6. Wi th in these groups the c a t a r a c t s were of va ry i ng degrees of s e v e r i t y rang ing from a wh i te p i npo in t dot c e n t r a l l y l o ca ted i n the lens of each eye i n the l e a s t a f f e c t ed f i s h , t o complete opaqueness of each lens i n extreme cases ( F i g . 6 ) . Ca ta rac t s were detected i n f i s h f ed d i e t 6 and then i n f i s h fed d i e t 5. Ca ta rac t s were not observed u n t i l day 63 i n f i s h • f ed d i e t 6. Of the 195 f i s h examined from t h i s t reatment , 26% exh i b i t e d some degree of lens o pa c i t y . Due to the h igh m o r t a l i t y tha t occurred i n f i s h r e c e i v i n g d i e t 6, the eyes were checked f o r the l a s t t ime on day 84 r a the r than on day 105. On day 84, c a t a r a c t s were noted i n 34% of the remaining 140 f i s h f ed d i e t 6 and in 25% of the 183 f i s h fed d i e t 5. 4 .3 .2 In f luence of D ie t Treatment on Chinook Performance 4 .3 .2 .1 F i s h growth Table 6 shows tha t a l l groups had more r ap i d growth in stage 1 than i n stage 2. The poorest s p e c i f i c growth r a t e i n both stages occurred i n f i s h i nges t i ng d i e t 6, which conta ined 51 g Ca, 25.8 g phy t i c a c i d and 0.05 g Zn per k i l og ram. By day 105, the mean weights of f i s h fed d i e t s 5-8 wi th 25.8 g of phy t i c ac id/kg were 54-73% l e s s than those of corresponding groups f ed d i e t s 1-4 wi th 1.62 g of phy t i c ac id /kg ( F i g . 7; P<0.001). Fu r t he r , the - 52 -f i n a l weights of low phytate groups g iven 48-49 g Ca/kg ( d i e t s 2 and 4) were 30.-38% l e s s than those g iven 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 so decreased growth in h igh phyta te- fed 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 ake , food convers ion and p ro te i n e f f i c i e n c y r a t i o (PER) Table 7 shows tha t the o v e r a l l food in takes of groups f ed h igh 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 to those of groups f ed d i e t s low i n Ca (1,3 and 7 ) . The one except ion to the f o rego ing g ene r a l i z a t i o n was noted i n f i s h fed d i e t 6 which d i d not have a reduced i n take compared to f i s h f ed d i e t 5. D i e t a r y l e v e l s of Ca and Zn d id not appear to i n f l uence food convers ion and PER in the low phyta te- fed f i s h . However, f i s h fed the high phytate d i e t s had s i g n i f i c a n t l y lower food convers ion and PER (Zn at 0.05 g/kg) when Ca was a l so high ( d i e t 6 v s . 5 ) . Furthermore, high d i e t a r y Zn s i g n i f i c a n t l y inc reased food convers ion and PER in s p i t e of the presence of excess Ca ( d i e t 8 v s . 7 ) . Increased Zn content a l so improved PER i n high phyta te- fed 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 of phy t i c ac id d r ama t i c a l l y reduced food convers ion and PER i n chinook salmon. F igure 8 i l l u s t r a t e s the changes which occurred i n food i n t a k e , food convers ion and PER of r ep r e sen t a t i v e groups of low and high phyta te- fed f i s h over f ou r 21-day pe r i od s . 4 .3 .3 In f luence of D ie t Treatment on Proximate Composit ion D i e ta ry treatment markedly i n f l uenced te rmina l l e v e l s of whole-body proximate c on s t i t u en t s i n the f i s h (Table 8 ) . For example, h igh phytate groups c o n s i s t e n t l y had h igher percentages of mo i s tu re , p r o t e i n and ash and lower percentages of l i p i d than noted i n the other groups. Moreover, f i s h f ed d i e t s w i th h igh Ca and P (ash) content gene ra l l y had g rea te 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 content than observed i n r e s pe c t i v e counterpar ts fed d i e t s w i th low l e v e l s of Ca and P. The l e v e l s of proximate c on s t i t u en t s i n f i s h fed d i e t 9 gene ra l l y f e l l w i t h i n the range found f o r low phytate groups. 4 .3 .4 In f luence of D ie t Treatment on General Health A pa tho l og i c a l examinat ion of 20 f i s h from each of the n ine treatment groups at t e rm ina t i on of the study revea led no s i g n i f i c a n t d i f f e r e n c e s or abnorma l i t i e s i n haematocr i t , haemoglobin, blood c e l l count or b lood c e l l shape and s i z e . Fu r t he r , no evidence of b a c t e r i a l or v i r a l pathogens or of protozoan pa r a s i t e s was found. However, oedema, or c o l l e c t i o n o f f l u i d i n the stomach, was v i s u a l l y detected i n severa l of the f i s h fed h igh phytate d i e t s . 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 those r e c e i v i n g d i e t s con ta i n i ng high phy t i c a c i d . Here, m o r t a l i t y ranged from 9.3% f o r f i s h f ed d i e t 5, which conta ined low Ca and Zn, to 44.7% f o r f i s h fed d i e t 6 where Zn was low but Ca h igh (Table 6 ) . TABLE 5 Cata rac t inc idence i n j u v e n i l e chinook salmon fed d i e t s w i th high l e v e l s of phytate and examined on days 0, 21, 42, 63 and 84 in Experiment I . 1 Phy t i c Examination Ca Zn ac id F i sh day ca ta ra c t s D ie t (g/kg dry d i e t ) examined detected % ca ta rac t s % lens o p a c i t y 2 < 25 < 50 > 75 5 5.1 0.05 25.8 6 51 0.05 25.8 7 5.3 0.40 25.8 8 53 0.39 25.8 183 195 140 120 120 84 63 84 25 26 34 0 0 75 22 74 15 66 13 1 7 3 2 4 18 1 Ca ta rac t s were not detected i n groups fed d i e t s con ta in ing low or i n te rmed ia te l e v e l s of phytate ( d i e t s 1-4, 9 ) . 2 % lens opac i t y r e f e r s to the percentage of the lens sur face est imated t o be opaque by v i s u a l obse rva t i on on l y . - 55 -F i g . 6a . F i g . 6b. F i g . 6. J uven i l e chinook salmon fed a high phytate 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 Spec i f i c growth rates and mor ta l i t i e s fo r juven i le chinook salmon fed tes t d iets fo r 105 days in Experiment I. Phyt ic Growth rate (% wet-wt/day)* Ca Zn acid . Mor ta l i t y Diet (g/kg dry d ie t ) Day 0-63 Day 64-105 Day 0-105 (%) 1 4.4 0.05 1.62 2.40 d 1.64C 2.14 b 3.9 2 49 0.05 1.62 1.84 b 1.50 c 1.72 b 4.4 3 4.5 0.35 1.62 2 . 32 c d 1.80 c 2.15 b 2.4 4 48 0.40 1.62 1.88&C 1.37 bc 1.71b 4.4 5 5.1 0.05 25.8 1.19 a 0.52 a 0 .91 a 9.3 6 51 0.05 25.8 0.86 a 0.023 a 0.52 a 44.7 7 5.3 0.40 25.8 1.19 a 0 .64 a b 1.00 a 15.0 8 53 0.39 25.8 1.08 a 0 .68 a b 0.97 a 17.5 9 17.7 0.15 6.46 2 . 1 9 b c d 1.41 bc 1.94 b 2.4 2 SEM 0.11 0.25 0.071 1 A one-factor analysis of covariance of l o g e wet weights with d ie t as the fac to r and time as the l i near covar iate, in teract ion between the fac to r and covar iate, and two sources of e r ro r , rep l i ca t i ons (nested wi th in d ie ts and interacted with time) and measurement error ( res idua l ) , indicated a s i gn i f i c an t d iet e f fec t with P<0.001 for day 0-63, day 64-105 and o v e r a l l . Within a column, d iet groups with the same superscr ipt l e t t e r form a homogeneous subset (Scheffe 's mul t ip le range tes t with P=0.05). - 57 -F i g . 7. Geometric mean weights of 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 i n Experiment I . - 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 I.* Ca Zn Phytic acid Protein efficiency Diet (g/kg dry diet) Food intake Food conversion ratio 1 4.4 0.05 1.62 2.10Cd 1.33d 2.47e 2 49 0.05 1.62 1.64* 1.37d 2.63e 3 4.5 0.35 1.62 2.12Cd 1.31 d 2.44e 4 48 0.40 1.62 1.68 a 1.28d 2.40 e 5 5.1 0.05 25.8 2.07C 0.42° 0.78 b 6 51 0.05 25.8 2.30** 0.19 a 0.36* 7 5.3 0.40 25.8 2.18Cd 0.51° 0.96C ; , .-• 8 53 0.39 25.8 1.83b 0.65C 1.20d 9 17.7 0.15 6.46 1.98C 1.30* 2.50« 2 SEM 0.094 0.14 0.27 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 letter were not significantly different (Newman-Keuls test with P=0.05). - 59 -F O O D I N T A K E B 3 « r 2 2 - 4 2 r~i Our 6 4 - 3 4 d a y 8 3 - 1 0 3 -*J r.-.i J>31>:| c :y t:::3 & j $ tej FOOD CONVERSION 1 be b T m "•> rXs -W O T E 1 N EFFICIENCY R A T I O l i i i aufa 2 3 i *A *9 J J 31 3 Z n .03 £ 3 . 0 3 . 0 3 F b y t i c a c i d 1.42 1 4 2 2 3 3 Z3JB F i g . 8. Food i n t ake , food convers ion 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 rep resen ta t i ve groups o f j u v e n i l e chinook salmon fed d i e t s conta in ing low and h igh phy t i c a c i d at fou r 21-day i n t e r v a l s i n Experiment I . A one-way ANOVA w i th d i e t as the f a c t o r i n d i c a t ed 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 food i n t ake : P<0.05 (day 22-42) , P<0.01 (day 43-63, 64-84) and P<0.001 (day 85-105). In regard to food convers ion and PER, ANOVA i nd i c a t ed P<0.01 (day 64-84) and P<0..001 dur ing a l l o ther 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 • Diet Ca Zn Phytic acid (g/kg dry diet) Moisture(S) Protein $ of dry matter2 Lipid Ash 1 4.4 0.05 1.52 73.0 61.6 27.7 7.7 74.4 62.5 28.9 8.3 2 49 0.05 1.62 75.2 65.3 21.7 10.4 75.9 65.8 23.3 12.9 3 4.5 0.35 1.62 73.8 61.1 29.7 7.6 75.1 65.6 28.1 7.1 4 48 0.40 1.62 74.9 64.4 25.4 11.1 74.2 . 63.7 24.1 10.4 5 5.1 0.05 25.8 80.9 73.3 11.3 11.1 80.0 66.2 14.5 14.5 6 51 0.05 25.8 82.0 73.5 10.3 19.4 80.4 70.0 11.4 16.7 7 5.3 0.40 25.8 80.9 78.2 13.2 11.2 81.2 71.3 12.5 13.2 8 53 0.39 25.8 79.2 76.2 16.6 15.6 80.3 72.8 17.4 15.2 9 17.7 0.15 6.46 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, l ipid 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 Inf luence of D ie t Treatment on H is topatho logy G i l l and l i v e r s t r u c t u r e in r ep resen ta t i ve f i s h from a l l t reatment groups were judged to be normal. Abnorma l i t i e s were detected i n samples of k idney of high Ca-fed f i s h (d i e t s 2,4,6 and 8 ) , and i n the p y l o r i c caeca, stomach and t hy r o i d of h igh phyta te- fed f i s h ( d i e t s 5-8) . These are d i scussed below. 4 .3 .5 .1 Kidney Nephroca l c inos i s was observed i n a l l groups f ed high d i e t a r y l e v e l s of Ca ( F i g . 9 ) . F i g . 9. Kidney sec t i on of a chinook salmon fed a high l e v e l of Ca and P (d i e t 2 ) . Note nephroca l c i nos i s c ha r a c t e r i z ed by c a s t s , presumptive ca lc ium depos i t s , w i t h i n degenerat ing tubu les or ducts (a r rows) . 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 caeca l e p i t h e l i a l c e l l s of f i s h fed high phytate d i e t s were cha r a c t e r i z ed by hypertrophy and marked v a c u o l i z a t i o n of the cytoplasm ( F i g . 10a) . By c on t r a s t , those of f i s h fed low phytate d i e t s were s l ende r , columnar-shaped c e l l s w i th a f i n e g ranu la r cytoplasm ( F i g . 10b) . The aberrant p y l o r i c caeca l s t r u c t u r e and hence presumed abnormal f un c t i o n was most ev ident i n chinook salmon fed d i e t 6 w i th 50-70% of the p y l o r i c caeca a f f e c t e d . I t was a l so noted tha t the mucous l i n i n g i n the p y l o r i c caeca was markedly t h i nne r than tha t i n the stomach ( F i g . 10c ) . Moreover, the e p i t h e l i a l l aye r of the stomach, i n con t ras t t o tha t of the p y l o r i c caeca, was a f f e c t ed on ly ma rg i na l l y i n f i s h fed high l e v e l s of phy ta te . The stomach d id e x h i b i t a modest atrophy i n v o l v i n g the muscu lature as we l l as g l andu la r and e p i t h e l i a l components, but there was no evidence of v a c u o l i z a t i o n of the e p i t h e l i a l c e l l s . F i g . 10a. C r o s s - s e c t i on of p y l o r i c caeca from a high phy ta te - fed ( d i e t 5) chinook salmon. Note hypertrophy of the e p i t h e l i a l c e l l s (arrow) and marked v a cuo l i z a t i o n of the cy top lasm. Masson Tr ichome, x40. - 63 -F i g . 10b. C ross - sec t i on of p y l o r i c caeca from a low phyta te- fed (d i e t 1) chinook salmon. Note s l ende r , columnar-shaped e p i t h e l i a l c e l l s w i th a f i n e granu la r cytoplasm (arrow) t y p i c a l of normal j u v e n i l e chinook salmon. Masson Trichome, x40. F i g . 10c. C ross - sec t i on of a po r t i on of ca rd i ac stomach ( l e f t ) and p y l o r i c caeca ( r i g h t ) of a chinook salmon (fed d i e t 6 ) . Note the sharp con t ras t between the r e l a t i v e l y t h i c k l aye r of mucous (arrows) on the sur face of the e p i t h e l i a l c e l l s i n the stomach and the t h i n l aye r of mucous coa t ing these c e l l s i n the p y l o r i c caeca. P e r i o d i c ac id S c h i f f (PAS)-hematoxy l in , x40. - 64 -4 .3 .5 .3 Thyro id H i s t o l o g i c a l examinat ion revea led abnorma 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 . Approx imate ly 30-40% of the t h y r o i d f o l l i c l e s i n the h igh phytate groups were a t roph ied ( F i g . 11a) . The e p i t h e l i a l c e l l s were squamous i n shape and 2-3 um i n he i gh t . By c on t r a s t , t h y r o i d f o l l i c l e e p i t h e l i a l c e l l s i n the remaining groups were cubo ida l i n shape and 4-6 um i n he ight ( F i g . l i b ) . F i g . 11. Thyro id f o l l i c l e s of chinook salmon f ed a high phytate d i e t (5) i n 11a and a low phytate d i e t (1) i n l i b . Note marked reduc t i on i n e p i t h e l i a l c e l l he ight of f o l l i c l e s i n the high phy ta te - fed chinook salmon compared to the low phyta te- fed chinook salmon (a r rows) . PAS-hematoxyl in, x l 6 0 . 4 .3 .6 In f luence of D ie t Treatment on Plasma Minera l Leve ls 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 ed plasma l e v e l s of Ca, P, Mg, Zn (P<0.001) and Mn (P<0.05), but not l e v e l s of Fe or Cu (Table 9 ) . Plasma - 65 -TABLE 9 F ina l mean plasma concentrations of various minerals in juven i l e chinook salmon fed the tes t d ie ts in Experiment I . Phytic Ca Zn acid Mn Ca Zn P Mg Diet (g/kg dry d ie t ) (mg/kg dry weight) 1 .4.4 0.05 1.62 0.06a 107.5Cd 28.8C 368.3 C 18.3C 2 49 0.05 1.62 0.073b 112.0 d 29.4C 355.5C 12 .0 a 3 4.5 0.35 1.62 • 0 . 08 a b 108.5Cd 33 .2 d 380.OC 19 .3C 4 48 0.40 1.62 0 . 09 a b 108.8Cd 35 .3 d 353.5C 10 .3 a 5 ' 5.1 0.05 25.8 0 . 08 a b 103.3 bc 18.5 b 248.5 b 15.3b 6 51 0.05 25.8 0 . 08 a b 93 .0 a 14.5a 199.3 a 1 7 . 0 b c 7 5.3 0.40 25.8 0 .06 a 99 .0 b 28.6C 254.3 b 10 .3 a 8 53 0.39 25.8 0.10 b 103.3 bc 27 .4C 258.8 b 14.5 b 9 17.7 0.15 6.46 0 . 08 a b 111.3 d 29. OC 391.0C 11 .5 a 2 SEM 0.02 3.6 1.86 18.6 1.52 1 Blood was withdrawn from the caudal vessels of at least 25 f i s h per d ie t treatment. Fol lowing cent r i fugat ion , the plasma from each f i s h was transferred into one of four pools per d ie t treatment fo r ana lys i s . 2 A one fac tor analys is of variance with d iet as the fac to r indicated a s i gn i f i c an t d iet e f fec t with P<0.001 f o r Ca, Mg, P and Zn, and P<0.05 fo r Mn. Within a column, values with a common superscr ipt 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 ren t (Newman-Keuls tes t with P=0.05). - 66 -l e v e l s of Zn and P were i n f l uenced the most. The observed plasma Zn l e v e l s were h ighest i n the low phytate groups g iven 0.35-0.40 g/kg of supplemental Zn and lowest i n f i s h fed d i e t 6. Plasma P l e v e l s were h igher i n low phytate groups and i n f i s h f ed d i e t 9, which conta ined in te rmed ia te l e v e l s of Ca, P, Zn and phy t i c a c i d , than i n high phytate groups, p a r t i c u l a r l y i n f i s h fed d i e t 6. Plasma Mg l e v e l s were s i g n i f i c a n t l y depressed in the low phy ta te - fed f i s h when d i e t a r y l e v e l s of Ca and P were high ( d i e t s 2 and 4 ) . 4.4 DISCUSSION 4.4 .1 Catarac t Inc idence The r e s u l t s i n d i c a t e tha t high d i e t a r y r a t i o s of Ca and P t o Zn alone (~1000:1) were not s u f f i c i e n t to i n i t i a t e c a t a r a c t fo rmat ion i n j u v e n i l e chinook salmon (Table 5 ) . However, c a t a r a c t fo rmat ion d id occur when d i e t s conta ined a low l e v e l of Zn (0.05 g/kg) coupled w i th a high concen t ra t i on (25.8 g/kg) of phy t i c a c i d which i s a s t rong minera l c he l a t i ng agent. Th i s e f f e c t was exacerbated by a high d i e t a r y concen t ra t i on of Ca. Supplementat ion of the high phytate d i e t s w i th Zn (0.40 v s . 0.05 g/kg) prevented c a t a r a c t fo rmat ion at both Ca concen t r a t i on s . These r e s u l t s are cons i s t en t w i th the v iewpo int tha t phy 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 the 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 resent . Even though the low l e v e l of Zn (0.05 g/kg) employed in t h i s study exceeded the suggested requirement of Zn by salmonids (Ogino and Yang 1978), t h i s l e v e l proved to be inadequate when the d i e t a r y l e ve l of phy t i c a c i d was h i g h . Hence, great care should be taken i n the f o rmu la t i on of p r a c t i c a l d i e t s f o r salmonids based on combinat ions of animal and p lant p r o t e i n s to ensure an adequate a v a i l a b l e Zh con ten t . - 67 -4.4.2 Chinook Performance 4 .4 .2 .1 F i sh growth The more r ap i d growth of f i s h which was observed i n stage 1 compared to stage 2 (Table 6) was probably a consequence of a p rog ress i ve decrease i n growth r a t e i n stage 2 due to increased s i z e (B re t t 1979) f o r those f i s h consuming d i e t s w i th low (d i e t s 1 and 3) o r in termed ia te ( d i e t 9) Ca, P and phy t i c a c i d content . However, i n the remaining groups high d i e t a r y l e v e l s of phy t i c ac id and, to a l e s s e r degree, of Ca and P a l so p layed a r o l e . Indeed, throughout the study the r a t e of f i s h growth was d r ama t i c a l l y depressed by a h igh d i e t a r y phy t i c ac id c on cen t r a t i on . Moreover, h igh d i e t a r y l e v e l s of Ca and P r e su l t e d i n a constant de c l i ne i n the growth r a t e of low phytate groups which was s i g n i f i c a n t f o r f i s h i nges t i ng d i e t 2 in stage 1. The s i g n i f i c a n t reduc t i on i n the mean weight of f i s h fed 25.8 g of phy t i c a c i d w i th 51 g Ca/kg ( d i e t 6) and i n f i s h fed 1.62 g of phy t i c ac id coupled wi th 48-49 g/kg Ca ( d i e t s 2 and 4) a l so demonstrates the growth-depress ing e f f e c t of high d i e t a r y l e v e l s of Ca and/or P ( F i g . 7 ) . Th is both conf i rms and extends the 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 ou t where a growth reduc t i on of 5% occurred i n f i s h fed d i e t s c on ta i n i ng more than 1% Ca. In t h i s study d i e t a r y Zn concen t ra t i on per se d i d not s i g n i f i c a n t l y i n f l u en ce the growth r a t e of chinook salmon. 4 .4 .2 .2 Food i n t ake , food convers ion and PER High d i e t a r y l e ve l s o f Ca and P depressed appe t i t e i n f i s h fed the low phytate d i e t s 2 and 4 (Table 7 ) . However, the food consumed by these 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 the food convers ion and PER r e s u l t s . Thus t h e i r energy demands were l i k e l y met. The decreased appe t i t e of f i s h fed d i e t s 2 and 4 compared to d i e t s 1 and 3 may be a r e f l e c t i o n of the lower - 68 -p a l a t a b i l i t y of the former d i e t s as a r e s u l t of t h e i r high ash con ten t . A l s o , the d imin i shed appe t i t e may have been due to i n s u f f i c i e n t Mg r e l a t i v e to the high d i e t a r y concen t ra t i ons of Ca and P even though the known Mg requirement of f i s h was met (Ogino et a l . 1978; Knox et a l . 1981)". S igns of excess ive 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 to Mg or Mg d e f i c i e n c y i n t r o u t i n c l ude poor growth, l o s s of appe t i t e and c a l c i n o s i s of the k idney and muscle (Cowey et a l . 1977; Knox et a l . 1983). 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 they d i d s u f f e r n eph r o ca l c i n o s i s . The data i n d i c a t e tha t growth r e s t r i c t i o n i n the high phytate groups was due to a p rog ress i ve reduc t i on i n t h e i r c apa c i t y to convert food (p ro t e i n ) i n t o f l e s h ( F i g . 8; Table 7 ) . Some of the lowered food and p r o t e i n convers ion i n the high phyta te- fed f i s h probab ly stemmed from d imin i shed Zn b i o a v a i l a b i l i t y . For example, i n c r ea s i ng Zn i n the high phytate d i e t s d i d p a r t i a l l y o f f s e t the d imin i shed food and p r o t e i n convers ion e s p e c i a l l y when Ca content was a l so h i g h . However, because phy t i c ac id can b ind w i th seve ra l m inera l s o ther than Zn, such as Mg, P, Cu, Fe and Mn, and cause imbalances amongst them (Erdman, J r . 1979; Hartman, J r . 1979), i t i s conce ivab le tha t the lowered food and p r o t e i n convers ion i n the high phytate groups may a l so 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 inera l s such as P (Ke to la 1975) and Mg (Ogino et a l . 1978). Other p o s s i b l e f a c t o r s i n c lude 1) impairment of d i e t d i g e s t i b i l i t y owing to the format ion of phy t i c a c i d / p r o t e i n complexes which i n h i b i t enzymatic d i g e s t i on of the p ro 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 absorpt ion of n u t r i e n t s i n the p y l o r i c caeca l reg ion of the i n t e s t i n e ( F i g . 10a) . Th i s l a t t e r . c a u s e w i l l be d i scussed i n s e c t i on 4 . 4 . 5 . 2 . In regard to f a c t o r one, Ogino and Yang (1978) repor ted reduced p ro t e i n d i g e s t i b i l i t y i n - 69 -rainbow t r o u t fed Zn -de f i c i e n t d i e t s . They hypothes ized tha t the low Zn content may have reduced protease a c t i v i t y such as carboxypept idase which conta ins Zn as an important par t of i t s s t r u c t u r e . S i m i l a r l y , S p i n e l l i (1983) repor ted c a se i n -phy t i c a c i d complexes to be poo r l y hydro lyzed by pepsin and suggested t h i s to be a cause f o r poor p ro t e i n e f f i c i e n c y i n rainbow t r o u t fed phytate d i e t s . 4 .4 .3 Proximate Composit ion Although s t a t i s t i c a l analyses were not performed on proximate composi t ion da ta , the r e s u l t s i n d i c a t e tha t f i s h fed the high phytate d i e t s (5-8) had h igher l e v e l s of mo is tu re , 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 to the other groups (Table 8 ) . The low l i p i d content was probab ly due to the poor food convers ion such tha t the re was not enough surp lus energy f o r f a t d epo s i t i o n . The high ash content i n f i s h fed d i e t s 2,4,6 and 8 may be a s c r i bab l e to the h igh ash content of these d i e t s (Table 4 ) . However, the ash contents of f i s h f ed the low ash d i e t s 5 and 7 were s i m i l a r t o those of f i s h fed the high ash d i e t s 2 and 4. Th is may have been due to the presence of high phy t i c ac id in d i e t s 5 and 7. 4 .4 .4 F i s h Health The on l y gross abnormal i ty found i n the hea l th examinat ion was oedema which was seen i n severa l of the high phyta te- fed f i s h . The cause of t h i s i s not c l e a r , but i t may have r e su l t ed from p a r t i a l atrophy of the stomach (Dorland 1965). 4 .4 .5 H is topatho logy 4 .4 .5 .1 Kidney F i s h which ingested the high Ca (P) d i e t s e xh i b i t e d neph r o ca l c i n o s i s , a phenomenon seen i n rainbow t r ou t which have consumed high d i e t a r y l e v e l s of - 70 -Ca i n r e l a t i o n to Mg. The h igh Ca d i e t s used i n the present study conta ined Ca to Mg r a t i o s rang ing from 67:1 to 72:1 (Table 4 ) . Although the known Mg requirement of f i s h was met, i t may be t ha t the chinook salmon i n t h i s study su f f e red c a l c i n o s i s of the k idney due to i n s u f f i c i e n t Mg r e l a t i v e t o the d i e t a r y Ca and P l e v e l . Knox et a l . (1981) repor ted tha t the Mg requirement of t r o u t does not inc rease as the d i e t a r y l e v e l s of 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 dd i t i o n a l work i s warranted i n t h i s a rea . D i e ta ry phy t i c a c i d d i d not aggravate the inc idence of c a l c i n o s i s i n t h i s s tudy . 4 .4 .5 .2 P y l o r i c caeca and stomach The f un c t i o n of the p y l o r i c caeca i s to f a c i l i t a t e nu t r i e n t ( e . g . l i p i d ) absorpt ion (Fange and Grove 1979). However, the s t r u c t u r e of the p y l o r i c caeca i n f i s h f ed d i e t s con ta i n i ng h igh l e v e l s of phy 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 hypertrophy and marked v a c u o l i z a t i o n of the p y l o r i c e p i t h e l i a l c e l l s i n the high phyta te- fed 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 of phy t i c a c i d on the e p i t h e l i a l l a y e r , impaired Mg b i o a v a i l a b i l i t y (Ogino et a l . 1978) due to complexat ion of Mg w i th phy t i c a c i d (Erdman, J r . 1979), or both . With regard to the p o s s i b i l i t y of phy t i c a c i d t o x i c i t y , i t was repor ted i n s e c t i on 4 .3 .5 .2 tha t the mucous l i n i n g i n the p y l o r i c caeca was much t h i nne r than tha t i n the stomach. Th is 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 kness may account f o r the d i s s i m i l a r response t o phy t i c ac id c on cen t r a t i o n . P o s s i b l y the inc reased mucous l aye r i n the stomach p ro tec ted aga ins t the t o x i c i t y of phy t i c a c i d . A l t e r n a t i v e l y , the s t rong a c i d i c environment of the stomach (pH 3 . 5 ) , i n c on t r a s t to the neu t ra l to a l k a l i n e c ond i t i o n i n the i n t e s t i n e ( 6 - 8 ) , may have pro tec ted the stomach - 71 -from phy t i c a c i d t o x i c i t y . 4 . 4 . 5 . 3 Thyro id S t r u c t u r a l abe r ra t i ons of the t h y r o i d i n the high phytate groups ( F i g . 11a) may have been caused by p r o t e i n (amino ac i d ) and/or minera l d e p r i v a t i o n . These cond i t i on s are known to suppress t h y r o i d a l f u n c t i o n i n salmonids (Higgs et a l . 1982b) and phy t i c ac id i s capable of forming p ro te i n -phy ta te and m ine ra l -phy ta te complexes. 4 .4 .6 Plasma M inera l s Table 9 i l l u s t r a t e s t ha 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 to d i e t a r y Zn concen t ra t i on and i n v e r s e l y r e l a t e d to d i e t a r y phy t i c ac id l e v e l . D i e t a r y Ca content had no i n f l u en ce on plasma Zn concen t ra t i on except when the d i e t s imu l taneous ly conta ined a high l e v e l of phy t i c ac id and a low l e v e l o f Zn . These r e s u l t s help to con f i rm tha t 25.8 g of p h y t i c ac id/kg d i e t d i d impa i r Zn absorpt ion i n chinook salmon and tha 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 the high d i e t a r y l e v e l of phy t i c ac id i n f l uenced the absorpt ion of both exogenous and endogenous Zn i n chinook salmon. However, Davies and N i gh t i nga l e (1975) performed caracass analyses i n r a t s and concluded tha t the add i t i o n of 10 g of phy t i c ac id per k i l ogram d i e t impaired not on l y the absorpt ion of d i e t a r y Zn, but a l so the r e so rp t i on of endogenously sec re ted Zn. I t i s noteworthy tha t the high phyta te- fed 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 xh i b i t e d s i g n i f i c a n t l y reduced plasma Zn l e v e l s . Th is strengthens the hypothes is tha t low Zn a v a i l a b i l i t y r e s u l t s i n c a t a r a c t f o rmat i on . 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 phy t i c a c i d c on cen t r a t i o n . F i s h fed 25.8 g phy t i c a c i d /kg d i e t had s i g n i f i c a n t l y reduced plasma P - 72 -l e v e l s . As w i th Zn, phy t i c ac id binds w i th P to form s t rong phytate complexes which render P unava i l ab l e f o r absorp t ion at the 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 reduc t i on of plasma Mg i n f i s h fed low l e v e l s of phy t i c a c i d coupled w i th high l e v e l s of d i e t a r y Ca and P supports the conten t ion tha t the reduced appe t i t e and neph roca l c i nos i s i n c i dence i n f i s h fed d i e t s 2 and 4 may have stemmmed from reduced Mg abso rp t i on . However, plasma Mg l e v e l s i n the h igh phytate groups d i d not bear any c on s i s t en t r e l a t i o n s h i p to d i e t a r y Ca and P concen t ra t i on which makes i t d i f f i c u l t to account f o r neph roca l c i nos i s i n f i s h fed d i e t s 6 and 8 s o l e l y on the bas i s of Mg d e f i c i e n c y . The measurement of Ca, P and Mg l e v e l s i n the k idney would have f a c i l i t a t e d data i n t e r p r e t a t i o n i n t h i s case . S p i n e l l i et a l . (1983) demonstrated tha t blood Cu l e v e l s i n rainbow t r o u t fed p u r i f i e d d i e t s increased when the d i e t a r y phytate l e v e l was increased to 5.0 g from 0.0 g/kg. Furthermore, blood Cu decreased i n the phytate groups when d i e t a r y Ca and Mg were r a i s e d to 13 g and 0.85 g/kg, r e s p e c t i v e l y . The marked v a r i a t i o n s of d i e t a r y phytate and Ca i n the present exper iment, 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 these two exper iments, but i t does bear c on s i d e r a t i o n . 4.5 CONCLUSION Although t h i s study d id not show tha t the 1981 ca t a r a c t problem in Canadian and American s tocks 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 tha t Zn i s important f o r normal lens i n t e g r i t y i n chinook salmon. 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 concen t ra t i ons of Ca and P are a t , - 73 -or near the maximum l e v e l s found i n p r a c t i c a l salmonid foods , a Zn d e f i c i e n c y w i l l be induced on l y when a m ine ra l -b i nd i ng agent such as phy t i c a c i d i s a l so present i n the d i e t i n s u f f i c i e n t c on cen t r a t i on . Hence, high d i e t a r y r a t i o s of Ca to Zn per se w i l l not induce c a t a r a c t fo rmat ion i n chinook salmon under cond i t i ons s i m i l a r to those used in the present s tudy. - 74 -CHAPTER 5 5.0 EXPERIMENT II - THE SUSCEPTIBILITY OF JUVENILE CHINOOK SALMON TO CATARACT FORMATION IN RELATION TO DIETARY CHANGES IN EARLY LIFE 5.1 INTRODUCTION Due to the des ign of the prev ious experiment i t was not p o s s i b l e to measure the 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 imes i n the e a r l y l i f e of chinook salmon. The main o b j e c t i v e of the present study was to ob ta i n t h i s i n f o rma t i on . The r a t i o n a l e f o r t h i s goal i s de r i ved from mammalian s tud ies which have shown tha t c a t a r a c t s can be induced more r e a d i l y i n very young animals (Grant 1974). To my knowledge t h i s has not been conf i rmed in f i s h . However, f i s h eyes i nc rease i n s i z e g r e a t l y dur ing j u v e n i l e l i f e (Johns 1981) and thus they may be more prone to lens damage dur ing such t ime . Three d i e t s , based on experiment I , were fed in e i gh t d i f f e r e n t sequences t o determine whether the f i s h were most s u s c ep t i b l e to c a t a r a c t development dur ing the f i r s t , second or t h i r d 42-day per iod a f t e r swim-up. 5.2 MATERIALS AND METHODS 5.2.1 Experimental F i sh Swim-up chinook f r y were se l e c t ed v i s u a l l y f o r un i form 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 tanks r e s u l t i n g in 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 tanks to reduce f i s h d en s i t y . The i n i t i a l mean weight of the f i s h was 0.49 ± 0.01 g (SEM). 5.2.2 Experimental D ie t s The three d i e t s (A,B and C; Tables 2,3a and 4a ) , were prepared as - 75 -desc r ibed i n Chapter 3 ( s e c t i on 3 . 4 ) . C h o l e c a l c i f e r o l was inc reased to 2400 IU (NRC 1981). The d i e t s were admin is tered to two groups of f i s h in accordance wi th the schedule shown in F igure 12. D ie t s A and B both conta ined low l e v e l s of sodium phytate 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 ie t C conta ined high l e v e l s of sodium phyta te , Ca and P. A l l d i e t s conta ined 0.06 g Zn/kg which i s above the recommended requirement f o r salmonids (Ogino and Yang 1978). Th is l e ve l was se l e c ted because the c a t a r a c t outbreak i n some B.C. hatchery s tocks of coho and chinook salmon in 1981 was exper ienced w i th a commerical d i e t con ta i n i ng a s i m i l a r l e v e l of Zn. There fore , i t was hoped tha t the 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 to hatchery managers. 5.2.3 Feeding The f i s h were fed s i x t imes d a i l y to s a t i a t i o n f o r the f i r s t 28 days and th ree t imes d a i l y f o r the remain ing 98 days of the 126-day s tudy. Per iods of 42 days were chosen to s imu la te hatchery cond i t i on s in which a p a r t i c u l a r batch of feed i s l i k e l y to be rep laced every 28-42 days. I t a l so a l lowed t ime f o r the chinook salmon to ad jus t to changes in d i e t composi t ion and to resume a c t i v e f eed i ng . E ight t rea tments , out of a p o s s i b l e twenty-seven, were se l e c t ed to s imu la te s i t u a t i o n s which may occur i n a hatchery environment, namely, (1) shor t - te rm exposure to a ca ta rac togen i c d i e t (C-A-A, A-C-A and A-A-C) , (2) cont inuous exposure to a n u t r i t i o n a l l y we l l balanced d i e t (A) , (3) shor t - te rm exposure to a low phytate d i e t con ta i n i ng high l e v e l s of Ca and P i n r e l a t i o n to Zn (B-A-A, A-B-A and A-A-B) , and l a s t l y , (4) cont inuous exposure to d i e t B. With regard to t h i s l a s t t reatment , i t was po s tu l a t ed , based on the 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 ha t the exposure t ime of chinook salmon to a d i e t con ta i n i ng a high Ca and P - 76 -T R E A T M E N T D IETS 1 I - J - 1 I A — C j A 3 1 A , A i C » i : 4 | A j A j _ A 5 r — - S j A j A 6 H A . B . A 1 1 7 , A , A j B 8 I § i 4 \ S . 0 4 2 8 4 1 2 6 D A Y S P O S T S W I M - U P F i g . 12. Schematic r ep r e sen t a t i on of d i e t a r y treatments g iven to ch inook salmon from swim-up to day 126 i n Experiment I I . - 77 -to Zn r a t i o i n experiment I may have been too shor t to induce c a t a r a c t s . There fore , d i e t B was fed f o r 126 days i n t h i s study which was the same dura t i on as tha t employed by K e t o l a . 5.2.4 Sampling Procedures Random samples of 60 f i s h / t a nk were weighed i n d i v i d u a l l y to the nearest 0.01 gram on days 0, 42, 84 and 126. F i sh eyes were checked mac roscop i ca l l y as descr ibed p r ev i ou s l y ( se c t i on 3 . 2 . 5 . 4 ) . 5.2.5 S t a t i s t i c a l A na l y s i s Wet weights were analyzed us ing a one-way ANOVA (UBC GENLIN) and d i f f e r en ce s between means were t e s ted using Duncan's New M u l t i p l e Range Test (Duncan 1955) wi th P=0.05. See Appendix B f o r ANOVA t a b l e . 5.3 RESULTS 5.3.1 In f luence of D ie t Treatment on Ca ta rac t Inc idence B i l a t e r a l lens c a t a r a c t s were observed on day 126 in 44 out of 360 f i s h (12%) which rece ived treatment 2. These f i s h were exposed to the ca ta rac togen i c d i e t (C) between days 42 and 84 ( F i g . 12) . Ne i ther of the groups fed d i e t C dur ing the f i r s t 42 days (treatment 1) or the l a s t 42 days (treatment 3) e xh i b i t e d c a t a r a c t s . Ca ta rac t s were not detected at any t ime in f i s h fed d i e t B. 5.3.2 Inf luence of D ie t Treatment on F i sh Growth Growth was i n f l uenced s i g n i f i c a n t l y by d i e t composi t ion and by the per iod when the d i e t s were f e d . Table 10 shows tha t f i s h fed d i e t C f o r the f i r s t 42 days post swim-up (treatment 1) had s i g n i f i c a n t l y lower mean weights at day 42 than those on a l l o ther t rea tments . With one except ion (treatment 6 ) , the mean weights of f i s h fed d i e t A (treatments 2,3,4 and 7) were h igher than those fed d i e t B (treatments 5 and 8 ) . On day 84 however, f i s h on - 78 -treatment 6, which were changed from d i e t A to 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 weight than those on treatments 3,4 and 7 which remained on d i e t A. Moreover, cont inuous adm in i s t r a t i on of d i e t B (treatment 8) d r ama t i c a l l y reduced growth to day 84 as compared w i th the growth of f i s h which had rece ived d i e t A from day 0 to day 84 (treatments 3,4 and 7 ) . The greates t f i n a l mean weights were observed f o r f i s h fed d i e t A throughout the 126 day-study (treatment 4) and in f i s h fed d i e t A i n combinat ion wi th d i e t B ( treatments 5,6 and 7 ) . F i sh fed d i e t B f o r 126 days, or va r ious combinat ions of d i e t A w i th C, had d r ama t i c a l l y reduced f i n a l mean we ights , w i th the poorest growth being observed f o r f i s h on treatment 1. 5.4 DISCUSSION 5.4.1 Cata rac t Inc idence The on ly treatment which induced ca t a r a c t s i n j u v e n i l e chinook salmon was treatment 2 in which the high phytate d i e t was fed between days 42 and 84. The i n spec t i on system used in t h i s study i nd i c a t ed tha t there was a lag per iod between exposure t o the ca ta rac togen i c d i e t and the man i f e s t a t i on of the c a t a r a c t s , s i nce 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 to i n i t i a t e c a t a r a c t s dur ing the f i r s t and t h i r d 42-day per iod may have been r e l a t e d to the s ta tus of Zn reserves in the f i s h dur ing those t imes . For example, i t may be pos tu la ted tha t the swim-up f r y acqu i red s u f f i c i e n t minera l (Zn) reserves from the absorbed yo lk sacs to counterac t decreased Zn b i o a v a i l a b i l i t y from the d i e t dur ing the 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 ed d i e t A f o r the f i r s t 84 days may have acqu i red adequate Zn reserves such tha 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 lens t i s s u e . However, the f a i l u r e to detect -.79 -ca 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 conc l u s i ve ev idence tha t the p r o v i s i o n of the high phytate d i e t at tha t t ime had no e f f e c t on ca ta rac t i n c i dence . I f the de lay i n onset was of a s i m i l a r du ra t i on to tha t found when d i e t C was fed between days 42 and 84, the t e rm ina t i on of the experiment at day 126 would not have a l lowed f o r c a t a r a c t d e t e c t i o n . I t i s a l so conce ivab le t h a t , w i th the concen t ra t i on of Zn i n the eye changing in r e l a t i o n to body s i z e (Shearer 1984), the d i e t a r y concen t ra t i on of Zn needed to ma inta in normal lens growth between days 84 and 126 may have been l e s s than tha t which was requ i r ed between days 42 and 84. The r e s u l t s of t h i s study con f i rm and extend those of experiment I . They suggest tha t high d i e t a r y l e v e l s of Ca and P to Zn per se do not i n i t i a t e ca ta rac togenes i s i n j u v e n i l e chinook salmon under the c ond i t i o n s of t h i s study even a f t e r 126 days of exposure. Th is i s i n c on t r a s t t o the f i n d i n g s of Ke to l a (1979) w i th rainbow t r o u t and suggests t ha t chinook salmon may respond d i f f e r e n t l y from rainbow t r o u t . Although Ke to l a d i d not ana lyze h i s d i e t s f o r phy t i c a c i d content , or suggest a m ine ra l -phy ta te i n t e r a c t i o n , i t i s po s s i b l e t ha t there was s u f f i c i e n t phy t i c ac id present i n the soybean and wheat m idd l ings po r t i ons of h i s basal d i e t to induce c a t a r a c t s . Accord ing to the r e s u l t s of experiment I and those of the present s tudy, 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 imu l taneous ly con ta ins h igh l e v e l s of Ca and P in r e l a t i o n to Zn and the presence of a s t rong minera l b ind ing agent. There fore , phy t i c a c i d may have been an important c o n t r i b u t o r to K e t o l a ' s r e s u l t s . - 80 -TABLE 10 Mean wet weights (g * 2 SEM) of juvenile Chinook salmon fed the test diets at 42-day intervals for a period of 126 days i n Experiment II.1 Treatment Day 0 Day 42 Day 84 Day 126 - CAA 0.48 0.75a 1.62* 5.2ia ! - ACA 0.48 • 1.49Cd 2.13d 6.11a 1 - AAC 0.49 1.58Cd 3.96d 5.38a t - AAA 0.50 1.55Cd 3.90d 9.45b 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 4.09d 8.82b ! - BBB 0.49 1.26" 2.37b 5.56a 2 SEM2 0.01 0.06 0.16 0.42 1 A one-way ANOVA indicated a s i g n i f i c a n t treatment efect P<0.001 for days 42 and 84 and P<0.01 for day 126. Within a column, values with the 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 multiple range test with P=0.05). 2 Due to missing values, the 2 SEM for treatments 3 and 5 for days 42, 84 and 126 were 0.08, 0.22 and 0.60, respectively. - 81 -5.4.2 F i sh Growth The changes observed in the growth pa t t e rn of j u v e n i l e chinook salmon in r e l a t i o n t o d i e t a r y treatment were an t i c i p a t e d based on the r e s u l t s of experiment I . The present r e s u l t s r e i n f o r c e the concept tha t high d i e t a r y concen t ra t i ons of Ca and P depress the growth of chinook salmon. There fo re , when f o rmu la t i ng d i e t s f o r j u v e n i l e chinook salmon, care should be taken i n the s e l e c t i o n of d i e t a r y i ng red i en t s ( e . g . f i s h meals) to ensure low ash con ten t . Otherwise the reduc t i on i n smolt s i z e at the t ime of ocean r e l ease cou ld compromise marine s u r v i v a l ( B i l t o n 1984). 5.5 CONCLUSION The f i n d i n g s suggest tha t there may be a " c r i t i c a l window" in the e a r l y development of chinook salmon dur ing which i t i s imperat ive not to expose t h i s spec ies to a ca ta rac togen i c d i e t . However, a dd i t i o na l work i s r equ i r ed t o con f i rm whether chinook salmon are most prone to c a t a ra c t development between 42 and 84 days a f t e r swim-up and to v e r i f y whether a f u l l 42 days exposure i s necessary. From a p r a c t i c a l , s tandpo in t , a knowledge of the apparent lag e f f e c t i n exposure to a ca ta rac togen i c d i e t and u l t ima t e man i f e s ta t i on of the c a t a r a c t s may be use fu l to hatchery managers who f i n d themselves conf ronted w i th a c a t a r a c t problem. I f the analyses of the hatchery d i e t being used at the t ime 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 abno rma l i t i e s i n n u t r i e n t l e v e l s or no excesses 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 necessary to analyze samples of food which had been prov ided seve ra l weeks before the problem was de tec ted . 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 ab l i s hed tha t i n t e r a c t i o n s between Ca, P, Zn and phy t i c ac id d i d a f f e c t c a t a r a c t f o rmat i on , growth and h i s topa tho logy in j u v e n i l e chinook salmon. However, the design d id not permit the t e s t i n g of in termed ia te l e v e l s of the above d i e t a r y f a c t o r s . There fore , t h i s study was conducted as a th ree f a c t o r composite des ign to more thorough ly i n v e s t i g a t e the e f f e c t s of these d i e t a r y f a c t o r s on c a t a r a c t fo rmat ion and performance in chinook salmon. I t i s noteworthy tha t a th ree f a c t o r composite design (with 15 treatment combinat ions) has the same s t a t i s t i c a l power as a 3 x 3 x 3 f a c t o r i a l (Davies 1954). 6.2 MATERIALS AND METHODS 6.2.1 Exper imental F i sh Chinook salmon f r y , se l e c ted f o r un i form weight, were d i s t r i b u t e d at random in to 30 150-L tanks u n t i l each conta ined 140 f i s h . I n i t i a l mean weight was 1.28 ± 0.03 g (SEM). 6.2.2 Exper imental D ie t s The 15 dry d i e t s used in the study are descr ibed i n Tables 2, 3a and 4a. 6.2.3 Feeding Each d i e t was fed to two groups of f i s h three t imes d a i l y f o r 84 days. - 83 -6.2.4 Sampling Procedures 6 .2 .4 .1 F i s h weight and ca ta rac t inc idence On days 0, 21, 42, 63 and 84, random samples of 60 f i s h / t a nk were i n d i v i d u a l l y weighed to the nearest 0.01 g. Eyes were examined f o r c a t a r a c t s as descr ibed in s e c t i on 3 .5 .4 . Although the growth study po r t i on of t h i s experiment was terminated at day 84, due to high m o r t a l i t y i n groups fed d i e t 11, the remaining f i s h from a l l groups were mainta ined on t h e i r r e spe c t i v e d i e t s f o r an add i t i o na l 35 days f o r the purpose of observ ing c a t a r a c t i n c i dence . 6 .2 .4 .2 Proximate ana l y s i s At the beg inn ing of the exper iment, 125 f i s h were s e l e c t ed at random and pooled i n t o f ou r groups f o r proximate a n a l y s i s . At the end of the study a minimum of 20 f i s h / t r ea tmen t were se l e c ted randomly and pooled i n t o f ou r groups per d i e t a r y treatment f o r proximate a n a l y s i s . 6 .2 .4 .3 H i s t o l ogy samples On day 50, s i x f i s h per treatment were k i l l e d f o r h i s t o l o g i c a l examinat ion, and at t e rm ina t i on of the study 6-10 f i s h per treatment were k i l l e d . S t ruc tu res examined inc luded the g i l l , k idney, l i v e r , pancreas, stomach and p y l o r i c caeca. 6 .2 .4 .4 Blood samples At the end of the exper iment, b lood was withdrawn from the caudal ve s se l s of at l e a s t 20 f i s h / d i e t t reatment . The samples were pooled i n t o three groups/treatment and were r e f r i g e r a t e d f o r subsequent minera l a n a l y s i s . 6 .2 .4 .5 T i ssue samples A minimum of 20 f i s h / t r ea tmen t were k i l l e d at the end of the study and s to red 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 idneys were removed f o r minera l a na l y s i s and were poo led, r e s p e c t i v e l y , i n to f ou r and three groups per d i e t t reatment . 6.2.5 S t a t i s t i c a l Ana l y s i s One-way ANOVA's were used to analyze growth r a t e , geometr ic mean we ights , food i n t a k e , food conve r s i on , PER, proximate compos i t ion and minera l analyses (Appendix C ) . The f o l l o w i n g fo rmu la was used to c a l c u l a t e s p e c i f i c growth r a t e : ( lognt+i - logn^ * t ime) x 100 where logn^ and logn^+i are the averaged na tura l log wet weights at the s t a r t ( t ) and the end (t+1) of the p e r i o d . Time was measured i n days. Duncan's m u l t i p l e range t e s t and S che f f e ' s t e s t (growth r a t e s ) w i th P=0.05 were used to t e s t f o r d i f f e r en ce s between means. The analyses were performed us ing the s t a t i s t i c a l package SAS (SAS 1982). 6.3 RESULTS 6.3.1 In f luence of D ie t Treatment on Catarac t Inc idence Ca ta rac t s were observed f i r s t on day 63 i n f i s h which r e ce i ved d i e t s tha t conta ined high Ca and phytate (6 ) , low d i e t a r y Zn and in te rmed ia te phytate (11) and medium d i e t a r y Ca and high phytate (14) , and on day 84 in f i s h fed d i e t 5 which conta ined low Ca and high phy ta te . Table 11 shows the inc idence of c a t a r a c t s on day 63 and on subsequent examinat ion days up to day 119. Ca ta rac ts were not observed in f i s h fed low phytate d i e t s (1-4 and 13) or i n f i s h fed medium and high phytate d i e t s when the d i e t a r y Zn l e ve l was more than 0.140 g/kg ( d i e t s 7-10, 12 and 15) . 6.3.2 In f luence of D ie t Treatment on Chinook Performance 6 .3 .2 .1 F i sh growth D i e ta ry composi t ion s i g n i f i c a n t l y a f f e c t ed the s p e c i f i c growth r a te s and mean weights of j u v e n i l e chinook salmon (P<0.001). "Tab le 12 and F igure 13 show tha t growth ra te s were reduced s i g n i f i c a n t l y i n f i s h fed d i e t s 5-8,11 85 -and 14. These d i e t s , w i th the except ion of d i e t 11, conta ined high l e v e l s of phy t i c a c i d . Diet 11 conta ined an in te rmed ia te l e v e l of phy t i c ac id and the lowest l e ve l of d i e t a r y Zn. Wi th in the high phytate groups, growth improved when the d i e t a r y Zn l e v e l was inc reased from 0.05 and 0.06 g to 0.27 and 0.32 g/kg d i e t ( F i g s . 13 and 14a). Mean wet weights of i n te rmed ia te phy ta te - fed f i s h increased as d i e t a r y Zn was inc reased (Table 12; F i g s . 13 and 14b) . Moreover, h igh 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 i n chinook salmon fed h igh and low phytate d i e t s ( e . g . d i e t 1 v s . 2; d i e t 5 vs . 6 ) . 6 .3 .2 .2 Food i n t ake , food convers ion and PER D i e ta ry treatment s i g n i f i c a n t l y a f f e c t ed f i s h appe t i t e (P<0.05). F i s h f ed d i e t 11 had a s i g n i f i c a n t l y h igher food i n take than a l l o ther groups except f o r f i s h fed d i e t s 1,3 and 10 (Table 13) . Otherwise, food i n t ake was s i m i l a r between treatment groups. D i e ta ry treatment a l so a f f e c t ed food convers ion and PER (Table 13; P<0.001). F i sh fed the high phytate d i e t s (5-8 and 14) and the low Zn d i e t (11) had lower va lues f o r both v a r i a b l e s compared to the other groups. D i e ta ry l e v e l s of Ca, P and Zn i n f l uenced food convers ion and PER when the d i e t s a l so conta ined in te rmed ia te or high l e v e l s of phy t i c a c i d . For example, i n f i s h fed the low Zn, h igh phytate d i e t s (5 and 6 ) , food convers ion and PER were reduced when the d i e t s a l so conta ined high d i e t a r y Ca ( d i e t 5 vs . 6 ) . Furthermore, when d i e t a r y Zn l e v e l s were i n c reased , food convers ion 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 juvenile chinook salmon examined on days 0, 21, 42, 63, 84, 105 and 119 of Experiment 111.' Diet Ca Zn (g/kg dry Phytic acid diet) Fish examined Examination day cataracts detected % cataracts 0 % lens o p a c i t y 2 £ 25 < 50 > 75 5 10.2 0.05 21.1 247 84 5 95 5 - -129 105 16 84 10 6 -123 119 23 77 11 9 3 6 50 0.06 21.1 230 63 2 98 2 -197 84 6 94 6 -54 105 22 78 14 8 2 54 119 26 74 12 12 2 11 25.2 0.034 11.6 186 63 2 98 2 - -29.1 0.10 23.2 189 63 6 94 6 - -176 84 6 94 5 1 -59 105 14 86 8 6 -59 119 H 86 8 6 -1 Cataracts were not detected in groups fed low phytate diets. (1-4 and 13), or medium to high zinc diets (7-10, 12 and 15). 2 % lens opacity refers to the percentage of the lens surface estimated to be opaque by visual observation only. TABLE 12 Final 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 juvenile chinook salmon fed test diets for 84 days 1n Experiment III.1,2 Ca Zn Phytic acid Weight Growth rate M o r t a l i t y Diet (g/kg dry diet) (9) . (* wet wt/day) (*) 1 9.6 0.06 2.1 7.363 2.123° 0.76 2 46 0.08 2.1 6.03b 1.89 a b 1.1 3 9.9 0.27 2.1 7.58a 2.14a 2.3 4 47.1 0.22 2.1 5.17Cd 1.71 a b 1.1 5 10.2 0.05 21.1 2.22f 0.72d 3.0 6 50 0.06 21.1 1.649 0.35 d e 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 a b -10 52.1 0.16 11.6 4.73 c d 1.71 a b 3.8 11 25.2 0.034 11.6 1.419 0.14e 37.3 12 29.1 0.28 11.6 5.22C 1.73 a b 1.5 13 28.3 0.15 0.06 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 2 SF.M 0.30 0.01 1 A one-way ANOVA with diet as the factor indicated a s i g n i f i c a n t diet effect with P<0.001 for mean weight and growth rate. Within a column, diet groups with the same superscript l e t t e r form a homogeneous subset. (Duncan's multiple range test with P=0.05; Scheffe's test with P=0.05 for growth rates). 2 I n i t i a l mean weight was 1.28 t 0.03 g. WEIGHT OF CHINOOK SALMON 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 1 4 I S D I E T Fig. 13. Wet weight* of chinook aabmon at day» and 84 H . Group* with a common euperecript letter were not eignifiecuitly different Note that fieh fed diet 11 (*) lott weight between day* 42 and 84. 14a. 14b. F i g . 14a. Representa t i ve p a i r s of f i s h fed d i e t s 1,5 and 7 (top t o bottom) f o r 84 days in Experiment I I I . Note marked r educ t i on in s i z e of f i s h fed high l e v e l s of phy t i c a c i d r e l a t i v e to f i s h f ed d i e t 1 (low phy t i c a c i d ) . A l so note the ame l i o r a t i ng e f f e c t of h igh d i e t a r y z i n c w i t h i n the h igh phytate f i s h (7 v s . 5 ) . F i g . 14b. Representa t i ve p a i r s of f i s h fed in te rmed ia te phytate d i e t s 11,15 and 12 (top to bottom) f o r 84 days in Experiment I I I . Note increased 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 inc reased from 0.034 to 0.17 to 0 .28. TABLE 13 Mean da i l y food consumption, food conversion and protein e f f i c i e n c y r a t i o f o r groups of juvenile chinook salmon fed test diets between days 0 and 84 in Experiment I I I . l Ca Zn Phytic acid Protein e f f i c i e n c y Diet (g/kg dry diet) Food intake Food conversion r a t i o 1 9.6 0.06 2.10 1.66ab 1.173b 2.363b 2 46 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 0.85e 6 50 0.06 21.1 1.52bcd 0.20e 0.38f 7 9.8 0.27 21.1 1.3icd 0.69c 1.38d 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 10 52.1 0.16 11.6 1.67ab 0.96b 1.90C 11 25.2 0.034 11.6 1.903 0.06e 0.12f 12 29.1 0.28 11.6 l,44bcd 1.113b 2.24abc 13 28.3 0.15 0.06 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^gb c d 1.01b l.ggbc 2 SEM 0.18 0.12 0.24 1 A one-way ANOVA with diet as the factor indicated a s i g n i f i c a n t diet effect with P<0.05 for food Intake and P<0.001 for food conversion and protein e f f i c i e n c y r a t i o . Within a column, diet groups with the same superscript l e t t e r were not s i g n i f i c a n t l y different (Duncan's multiple range test with P=0.05). - 91 -6.3.3 Inf luence of D ie t Treatment on Proximate Composit ion The proximate composi t ion of chinook salmon was i n f l uenced by d i e t a r y treatment (P<0.001). Table 14 shows tha t f i s h fed d i e t s con ta i n i ng high phy t i c ac id ( d i e t s 5-8 and 14) , or i n te rmed ia te phy t i c a c i d coupled w i th low d i e t a r y z i n c ( d i e t 11) , had s i g n i f i c a n t l y h igher l e v e l s (%) of mo is ture and p r o t e i n and lower l i p i d l e v e l s i n the body than noted i n the remaining groups. The two except ions were f o r f i s h fed d i e t s 6 and 8 in which p r o t e i n was not h igher than tha t i n the other groups. Body ash content was h ighest in f i s h fed d i e t s 6 and 11. F i s h fed d i e t s con ta i n i ng high l e v e l s of Ca and P wi th 21.1 g phy t i c ac id/kg ( d i e t s 6 and 8 ) , or medium Ca w i th 23.2 g phy t i c ac id/kg (d i e t 14) had h igher ash l e v e l s than t h e i r r e spe c t i v e coun te rpa r t s . In the low phytate groups (1-4 and 13) , ash l e v e l s were h ighest 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 In f luence of D ie t Treatment on General Health A pa tho l og i ca l examinat ion of 10 f i s h from each of the 15 treatment groups revea led no abno rma l i t i e s i n haematoc r i t , haemoglobin, b lood c e l l count or s i z e and shape of b lood c e l l s . Gross examinat ion showed no i n t e r n a l or ex te rna l abno rma l i t i e s . M o r t a l i t y was h ighest i n the high phy ta te - fed groups ( d i e t s 6-8 and 14) and in f i s h fed d i e t 11, w i th m o r t a l i t y rang ing from 5.3% i n f i s h fed d i e t 7 to 37.3% in f i s h fed d i e t 11 (Table 12) . M o r t a l i t y was n e g l i g i b l e i n the remaining groups. - 92 -TAELE 14 Fi n a l whole body proximate composition of j u v e n i l e chinook salmon fed test d i e t s f o r 84 days i n Experiment I I I . l Ca Zn Phytic acid (g/kg dry d i e t ) % of dry matter2 Diet Moisture(Z) Protein L i p i d Ash 1 9.6 0.06 2.1 76.09 6 6 . 9 ^ 21.9" 9.8* 2 46 0.08 2.1 76.7efg 68.0°cd 20.8^ 11.3 d 3 9.9 0.27 2.1 75.99 66.6 d* 2i.8« lO.ie 4 47.1 0.22 2.1 77.4de 65. l e 19.2=d 11.8* 5 10.2 0.05 21.1 81.8 a 72.9 a 11.49 11.9*. 6 50 0.06 21.1 80.7b 68.8 b 10.89" 17.4a 7 9.8 0.27 21.1 79.7C 72.1* 13.7' 8 48.6 0.32 21.1 79.7C 68.6b= 15. ie 13.2C 9 5.6 0.15 11.6 78.2 d 68.1b<-d - lO.ie 10 52.1 0.16 11.6 76.8 e f9 67.6b«l 12.2 d 12.0«» 11 25.2 0.034 11.6 81.63 72.8a 9.8" 14.6 b 12 29.1 0.28 11.6 77.3 def 65.2 e 20.1°c 11.4 d 13 28.3 0.15 0.06 76.4 f9 65.8 e 20.1 b c 12. I d 14 29.1 0.10 23.2 79.6 C 71.5 a 11.49 13.2= 15 29.1 0.17 11.6 77.6de. 69.0 b 18.9 c d 10.9*6 2 SEM3 0.62 1.1 0.90 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 to 20 fish/group) and carcasses were stored at -40*C before analysis. 2 A one factor ANOVA with d i e t as the factor indicated 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 moisture, protein l i p i d and ash. Within a column values with the 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 multiple range t e s t with 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 at the beginning of the study (n=4) were, re 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 In f luence of D ie t Treatment on H is topatho logy No pa tho log i c abnorma l i t i e s were noted i n the s t r u c t u r e of the 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 the k idney and p y l o r i c caeca. 6 .3 .5 .1 Kidney Nephroca l c inos i s was observed on days 50 and 84 i n f i s h fed more than 25.0 g Ca/kg d i e t (Table 15) . Except f o r f i s h fed d i e t 15, the inc idence of c a l c i n o s i s increased between days 50 and 84. 6 .3 .5 .2 P y l o r i c caeca The p y l o r i c caeca l e p i t h e l i a l c e l l s of f i s h fed d i e t s c on t a i n i ng >^  11.6 g phy t i c a c i d together wi th >_ 9.8 g Ca/kg e xh i b i t e d hypertrophy and v a c u o l i z a t i o n of the cytoplasm (Table 15) . Th is was observed on days 50 and 84 i n f i s h fed d i e t s 5-8,10,11,14 and 15. F i s h fed d i e t 12 showed e f f e c t s on day 84 on l y . 6.3.6 In f luence of D ie t Treatment on Blood and T i ssue Minera l Leve l s 6 .3 .6 .1 Whole blood 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 ed whole blood 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 th the most marked d i f f e r en c e s being observed in l e v e l s of b lood Zn and P (Table 16) . TABLE 15 The % inc idence of nephroca l c i nos i s and v a cuo l i z a t i o n of the p y l o r i c caeca i n j u v e n i l e chinook salmon at days 50 and 84 in Experiment I I I . Phy t i c F i sh Va cuo l i z a t i o n of Ca Zn ac id examined Nephroca l c inos i s* p y l o r i c c a e ca 2 Die t (g/kg dry d i e t ) d 50 d 84 d 50 d 84 d 50 d 84 1 9.6 0.06 2.1 4 6 - - -2 46 0.08 2.1 4 6 100 100 - -3 9.9 0.27 2.1 4 6 - - - • -4 47.1 0.22 2.1 6 6 80 100 - -5 10.2 0.05 21.1 6 10 - - 50 50 6 50 0.06 21.1 6 10 66 100 33 60 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 11.6 4 10 50 70 - 20 13 28.3 0.15 0.06 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 Nephroca l c inos i s was cha rac te r i z ed by ca lc ium depos i t s w i t h i n degenerat ing tubu les or duc t s . 2 A minimum of 10% of the p y l o r i c caeca was a f f e c ted wi th 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 concentrations of various minerals in j u v e n i l e chinook salmon fed the test d i e t s i n Experiment I I I . I , 2 Phytic Ca Zn a c i d . Mn Ca Zn P Fe Mg Oiet (g/kg dry d i e t ) (mg/kg cry weight) 1 9.6 0.06 2.1 0.24Cde 74.7 bc 1287* 205.3*> 67.0° 2 46 0.03 2.1 0 . 2 8 a o C ( l e 81.3 b (: 16.1fle 1357^ 205.0* 59.0C d 3 9.9 0.27 2.1 0.35a 76.3 bc 24.1* 14.33a 236.0 a 74.3 a 4 47.1 0.22 2.1 0.22 de 85.0°c 23.5" 1230 b c 177.3 c d 57.3Cd 5 10.2 0.05 21.1 0.22 d e 69.7= 9.99 1020 d 187.7 bc 58.3C d 6 50 0.06 21.1 0.24Cde 84.0°C 10.29 1080 d 195.7 bc 52.7 de 7 9.8 0.27 21.1 0.22°> 74.7 bc 22.343 1070 d 54.7Cde 8 48.6 0.32 21.1 0 , 2 9 a D C d e 80.3 bc 18.3" 1130 c d 159.0* 49.7* 9 5.6 0.15 i i . s 0.3oahcd 72.0*>c 22.3«b I300 b 204.7 b 69.3ab 10 52.1 0.16 11.6 0 . 3 2 a b c 85.0 bc 20.2°= 1273 b 179.3C d 56.0<=de 11 25.2 0.034 11.6 0 . 2 3 d e 108.3 a U.9 f3 1045 d 193.7 bc 58.0C d 12 29.1 0.28 11.6 0.35 a 88.7 b 24.5* 1300 b 190.7*>c 59.3«i 13 28.3 0.15 0.06 0.21 e 81.7°c 25.2i 1287" 198.3 b= 58.0 c d 14 29.1 0.10 23.2 0 - 2 6 b c d e 83.7 bc 14.isf 1113C d 190.7*>c 60.0= 15 29.1 0.17 11.6 0.33 a b 81.0 bc 20.5== 131oab 195.3 bc 57.0«» 2 SEM 0.05 10.8 2.0 82.S 13.7 4.3 1 Blood was withdrawn from the caudal vessels of at least 20 f i s h per d i e t treatment. The whole blood from each f i s h was transferred i n t o one o f three pools per d i e t treatment f o r an a l y s i s . 2 A one fa c t o r analysis of variance with d i e t as the fa 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 Zn, P. Fe and Mg; P<C31 f o r Kn and P<0.05 f o r Ca. Within a column, values with a common 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 new multiple range test with ?=0.05). - 9 6 -The lowest blood Zn l e v e l s were noted in f i s h which were f ed d i e t s c on ta i n i ng 0.05-0.06 g of Zn coupled w i th 21.1 g of phy t i c ac id /kg ( d i e t s 5 and 6) and in f i s h fed d i e t 11 which conta ined 0.034 g Zn and 11.6 g phy t i c a c i d / k g . The h ighest b lood Zn concen t ra t i ons were observed in f i s h which were fed d i e t s con ta in i ng at l e a s t 0.15 g Zn concu r ren t l y w i th 2.1-11.6 g of phy t i c ac id/kg (d i e t s 3 ,4 ,7 ,9 ,12 and 13 ) . The two except ions were i n f i s h fed d i e t s 10 and 15 where b lood 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 fed d i e t s 3,12 and 13. F i s h fed d i e t 11 or the h igh phytate 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 concen t ra t i on i n the b lood . Blood Ca was not s i g n i f i c a n t l y a f f e c t ed except i n f i s h which ingested d i e t 11. These f i s h had a much h igher concent ra t i on of Ca i n the blood compared to a l l o ther groups. Lowest blood Mg l e v e l s were observed i n f i s h fed the h igh phytate d i e t s 6,7 and 8 and i n f i s h fed d i e t 10. The l a t t e r d i e t conta ined a high Ca and an in te rmed ia te phy t i c ac id con ten t . F i s h fed d i e t s con ta i n i ng low and in te rmed ia te l e v e l s of phy t i c a c i d together w i th low Ca ( d i e t s 1,3 and 9) had h igher blood Mg l e v e l s than t h e i r counterpar ts 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 L i v e r Table 17 i l l u s t r a t e s tha t 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 ed l i v e r l e v e l s of P, Mg, Zn, Fe, Cu (P<0.001); Ca (P<0.01) and Mn (P<0.05). The most marked d i f f e r ences were observed i n concen t ra t i ons of Zn, Fe and Cu. F i sh fed d i e t s 3,4 and 12 had h igher l i v e r Zn l e v e l s than a l l other groups except those fed d i e t s 7,13 and 14. Lowest Zn l e v e l s were observed i n f i s h fed d i e t s 2,5,6 and 11. - 97 -TABLE 17 Fi n a l l i v e r concentrations of various minerals in j u v e n i l e chinook salmon fed the test diets i n Experiment I I I . I , 2 Phytic Ca Zn acid MH Ca Zn P Cu Fe Mg Diet (g/kg dry die t ) V (mg/kg dry weight) 1 9.6 0.06 2.1 5.2 bc 872.5"c 88.6Cd HQSQCde 12.8bc I39.5ef 712 .50C 2 46 0.08 2.1 4.5*>c 1873.3 abc 81.0Cde 12025OC 8.7C 136. 2 ^ 734.23b 3 9.9 0.27 2.1 4.6"c 807.5bc 126.5 a H525bcd I3.4&C 176.2<* 722.5bc 4 47.1 0.22 2.1 5.6°c 1642 .50C 127.5* 134503 13.lbc 188.7de 797 .53 5 10.2 0.05 21.1 6.6°c 1395.0°c 66.9e 10230« 34.5» 301.7C 715.Obc 6 50 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 a b 1190.0bc 113.5 ab 125003b 17.7b 245.5<1 717.5bc 8 48.6 0.32 21.1 6.1"= 1950. 0abc 9 6 . 0 o c d 12150bc 18.00 207.2<le 685.0°c 9 5.6 0.15 i l l s 6.1°c 665.0= 101.Sbc 114C0bct!e 13.8bc 158.2e^ 705.0bc •io 52.1 0.16 11.5 4.gbc 1152.50C 87.7cd 1195QOC I 0 . 3 b c 110.2 f 732. 11 25.2 0.034 11.6 3.9C 3303.3 a 83.7Cde 391Q f 29.7* 607 .7* 465.7e 12 29.1 0.28 11.6 4.4°c 1215.0°c 125.7 a 10307<*e 12.5bc 147.2** 480.O e 13 28.3 0.15 0.06 4.8°c 2075.0ab= 116.2 a D 12275bc II.7DC ISl.oef 702.5bc 14 29.1 0.10 23.2 10. l a 3372.5 a 112.7a° 121C0bc I 7 . 5 b 282.7C 730.03b 15 29.1 0.17 11.6 4.5°c 1847.5abc 90. I d HZSObcde lo.abc 146.2ef 647.5cd 2 SEM 2.3 937.5 12.9 777 4.7 42.9 46.4 1 Livers were extracted from a minimum of 20 f i s h per d i e t treatment. The l i v e r from each f i s h was transferred i n t o one of four pools per d i e t treatment f o r an a l y s i s . 2 A one f a c t o r analysis 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 Zn, P, Cu, Fe and Mg; P<0.01 f o r Ca and P<0.05 f o r Hn. Within a column, values with a common 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 new multiple ranee t e s t with P=0.05). - 98 -L i v e r Fe l e v e l s were h ighes t i n f i s h fed d i e t 11 which conta ined the lowest Zn content f o l l owed by f i s h which ingested the h igh phytate d i e t s 5,6 and 14. L i v e r Cu was h ighest i n f i s h fed d i e t s 5,6 and 11. 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 fed d i e t s 11 and 12. F i s h fed d i e t 4 had h igher l i v e r Mg content than a l l groups except those fed d i e t s 2,10 and 14. 6 .3 .6 .3 Kidney D i e ta ry treatment s i g n i f i c a n t l y a f f e c t ed k idney l e v e l s of Ca, P, Zn, Mn (P<0.001), Fe (P<0.01) and Mg (P<0.05; Table 18) . The h ighest l e v e l s of k idney Ca and P were seen i n f i s h fed d i e t s con ta in i ng 48.6-52.1 g Ca wi th 11.6-21.1 g phy t i c ac id /kg ( d i e t s 6,8 and 10) and i n f i s h fed d i e t 14 which conta ined 29.1 g Ca and 23.2 g phy t i c a c i d / k g . Kidney Zn l e v e l s were h ighest i n f i s h fed d i e t s 4,8 and 12, and lowest i n f i s h fed d i e t s 1 and 5. Leve ls of k idney Mg and Fe were s i g n i f i c a n t l y h igher i n f i s h f ed d i e t 11 compared to a l l o ther groups. 6.4 DISCUSSION 6.4.1 Cataract Inc idence Catarac ts appeared i n f i s h which were fed 21-23 g phy t i c a c i d w i th 0.05-0.100 g Zn/kg d i e t (Table 11) . Ca ta rac t s were a l so detected i n f i s h fed d i e t 11 which conta ined 11.6 g phy t i c ac id and 0.034 g Zn/kg. The inc idence of c a t a ra c t s as a r e s u l t of d i e t 11 was on l y 2% at day 63 wi th no f u r t h e r ca ta rac t s appearing at subsequent we igh ings. However, between days 63 and 84 there was high m o r t a l i t y i n f i s h fed d i e t 11 (Table 12) and 21 of - 99 -TABLE 18 Fi n a l kidney concentrations of 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 Diet Phytic Ca Zn acid (g/kg dry d i e t ) Mn Ca Zn P (mg/kg dry weight) Fe Ng 1 9.6 0.06 2.1 3.29 15606 109.5 de 14900" 257.7°= d e 1380b= 2 46 0.08 2.1 7.0 d e f9 15767= 1 2 1 . 3 c d e Z4133= d 228.7^6 1307 b= d 3 9.9 0.27 2.1 2.79 11236 155.0°= 14033" 2 9 8 . 0 b c d e 1153°=° 4 47.1 0.22 2.1 6.9 d e f9 14833= 184.0 a b 229QQ d e 215.7e 1293 b= d 5 10.2 0.05 21.1 5.0e f9 2000® 99.76 158339" 267.6 b= d6 1163 bc d 6 50 0.06 21.1 12.8°= 34000a° 122.3= d e 33433 a b 320.7°= 1423 b 7 9.8 0.27 21.1 5.16*9 18836 1 3g.7bcce 16700 f9" 259.7bcde 1037=de 8 48.6 0.32 21.1 14.8° 41100* 176.0a° 37800* 238.0= de I453 b 9 5.6 0.15 11.6 3.39 1037e 142.7°cde 14133" 338.3° 1000 d 10 52.1 0.16 11.6 9.9=d 272670 150.7°cd 28833°= 222.7 de 1417 b 11 25.2 0.034 11.6 21.0 a 13300=d U8.0= de 2100Q d e f9 468.0 a I800 a 12 29.1 0.28 11.6 9.0=de 15333= 208.3 3 21600 d6f 295.7bcde 1273 b= d 13 28.3 0.15 0.06 3.8*9 3783e 162.7°= 183006f9h 297.7 b= de 1 2 4 0 b c d 14 29.1 0.10 23.2 9.7=d 27733° 144.3 b c d 30667 b 307.7bcd 1457° 15 29.1 0.17 11.6 7 . 9 d e f 12773=d 180.3a° 21833d6r' 247.7=d6 1 2 0 3 b c d 2 SEH 2.5 6379 26.6 3540 50.8 211.5 1 Kidneys were extracted from a minimum of 20 f i s h per d i e t treatment. The kidney from each f i s h was transferred i n t o one of three 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 analysis 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 Mn, Ca, Zn and P; P<0.01 f o r Fe and P<0.05 f o r Mg. Within a column, values with a common 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 new mul t i p l e range t e s t with P=0.C5). - 100 - ' the f i s h which d ied dur ing t h i s t ime ( rep resen t ing 40% of the m o r t a l i t y ) had b i l a t e r a l c a t a r a c t s . Ca ta rac t s cou ld not be induced i n f i s h fed high r a t i o s of Ca to Zn a lone, but i t would appear t ha t an in te rmed ia te l e v e l of phy t i c a c i d (11.6 g/kg) can induce c a t a r a c t fo rmat ion i n j u v e n i l e chinook salmon i f the d i e t s imu l taneous ly conta ins a marginal l e v e l of Zn. Ca ta rac ts d i d not appear i n the h igh phytate groups which were fed d i e t s con ta i n i ng 0.27-0.32 g Zn/kg ( d i e t s 7 and 8 ) . Th is conf i rms the r e s u l t s of experiment I and f u r t h e r demonstrates the importance of Zn in ma in ta in i ng lens t ransparency . In both the present experiment and in experiment I , c a t a r a c t s were seen f i r s t on day 63 in f i s h fed d i e t s con ta i n i ng high Ca and high phy t i c a c i d and then on day 84 i n f i s h fed low Ca and high phy t i c a c i d . In the present s tudy, f i s h fed a medium Ca d i e t w i th high phy t i c a c i d ( d i e t 14) a l so e x h i b i t e d ca ta rac t s on day 63. These r e s u l t s support the conten t ion tha t inc reased l e v e l s of d i e t a r y Ca and P, i n the presence of h igh phy t i c a c i d , w i l l 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 thus promote ca ta rac togenes i s . 6 .4.2 Chinook Performance 6 .4 .2 .1 F i sh growth Growth was reduced s i g n i f i c a n t l y i n chinook salmon which were fed d i e t s con ta i n i ng at l e a s t 21.1 g phy t i c a c i d / kg , or 0.034 g Zn concu r r en t l y w i th 11.6 g phy t i c a c i d / kg . Moreover, f i s h fed >_ 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 weights than d i d t h e i r low Ca coun te rpa r t s . The one except ion was i n f i s h fed d i e t 8. In t h i s case i t appeared tha t the high Zn content i n d i e t 8 (0.32 g/kg) compensated f o r the excess Ca such tha t growth was not f u r t h e r reduced r e l a t i v e to t ha t of f i s h - 101 -f ed d i e t 7. D i e t a r y Zn l e v e l s >^ 0.17 g/kg appeared to enhance growth in chinook salmon which were fed d i e t s c on t a i n i ng >^  11.6 g phy t i c a c i d / kg . Th is e f f e c t was most dramatic i n the in te rmed ia te phytate groups (11,12 and 15) . Fu r t he r , the r e s u l t s suggest tha t i t i s p o s s i b l e (under the cond i t i on s of t h i s study) t o p a r t i a l l y compensate f o r the presence of c e r t a i n l e v e l s of phy t i c a c i d by i n c reas i ng the Tevel of Zn supplementat ion. 6 .4 .2 .2 Food i n t ake , food convers ion and PER Chinook salmon which were f ed d i e t 11 had the h ighest food i n take but the lowest food convers ion and PER. Th 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 to the fo rmat ion of i n s o l u b l e Zn- and p ro te i n -phy ta te complexes i n the i n t e s t i n a l lumen. For example, i n c r ea s i ng Zn concen t ra t i on from 0.034 g to 0.170 g/kg (d i e t 15) d r ama t i c a l l y improved food and p r o t e i n convers i on . Poor convers ion r a t e s were a l so observed i n the h igh phytate groups. Th is e f f e c t was a l l e v i a t e d somewhat by i n c r ea s i ng the l e ve 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 vs . 8 ) . I t i s i n t e r e s t i n g tha t the poorest convers ion r a t i o s were observed f o r f i s h which a l so conta ined the lowest l e v e l s of b lood Zn and P. Phy t i c ac id i s known to b ind w i th both of these minera l s and reduce t h e i r a v a i l a b i l i t y f o r ab so rp t i on . Furthermore, Zn and P d e f i c i e n c i e s i n rainbow t r ou t are cha ra c t e r i z ed by poor growth and reduced food convers ion (Watanabe et a l . 1980; NRC 1981). As mentioned i n experiment I ( sec t i on 4 . 4 . 2 . 2 ) , the lowered food and p ro t e i n convers ion in the high phyta te- fed f i s h may a l so have been p a r t l y due to the fo rmat ion of phy ta te -p ro te i n complexes and/or poor nu t r i e n t absorp t ion i n the p y l o r i c caeca l r eg i on . - 102 -6.4.3 Proximate Composit ion As i n experiment I , f i s h f ed the h igh phytate d i e t s e xh i b i t e d the h ighes t l e v e l s of body mois ture and p r o t e i n and the lowest l i p i d l e v e l s . P rev ious s tud ies (Groves 1970; Re in tz and H i t z e l 1980) have demonstrated tha t body f a t increased w i th increased f i s h s i z e . Thus the marked d i f f e r en ce s i n f i s h s i z e and food convers ion i n the h igh phytate groups, compared to the other groups, 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 in proximate compos i t i on . Ash l e v e l s were gene r a l l y r e l a t e d to d i e t a r y ash con ten t . F i s h fed d i e t s which were h igh i n Ca and P conta ined h igher ash contents than d id t h e i r counterpar ts which were fed d i e t s low i n Ca and P. In most cases , the presence of h igh phy t i c ac id s i g n i f i c a n t l y inc reased body ash concen t ra t i on compared to the corresponding low phytate d i e t s (1,2 and 4 versus 5,6 and 8, r e s p e c t i v e l y ) . 6.4.4 F i sh Health The gross examinat ion of f i s h at the end of the t r i a l y i e l d e d no abno rma l i t i e s . The inc idence of oedema in the high phyta te- fed f i s h in experiement I ( s e c t i on 4.3.4) was not ev ident i n t h i s s tudy. Th is may have been due to the s l i g h t l y lower phy t i c a c i d l e v e l s i n t h i s study (21.1 and 23.2 g/kg d i e t ) versus the 25.8 g/kg used in experiment I . The amount of d i e t a r y Zn and phy t i c a c i d appeared to i n f l u ence m o r t a l i t y w i th the h ighest number of deaths o c cu r r i ng i n f i s h fed the lowest l e v e l of Zn (0.034 g/kg; Tab le 10) . In the high phy ta te - fed 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 inc reased to 0.32 g/kg ( d i e t 8) the m o r t a l i t y f e l l t o 7.2%. Th is was a l so observed i n experiment I where m o r t a l i t y dropped from 44.7% to 17.5% when d i e t a r y Zn was - 103 -inc reased from 0.05 to 0.39 g/kg. Thus chinook s u r v i v a l appeared to be dependent upon adequate Zn abso rp t i on . S i m i l a r r e s u l t s were obta ined 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 assoc i a ted w i th Zn - de f i c i e n t d i e t s . 6.4.5 H is topatho logy 6 .4 .5 .1 Kidney -A f t e r 50 days neph roca l c i nos i s was observed in f i s h fed 10 of the 15 d i e t s . Any d i e t which conta ined more than 25 g Ca/kg produced k idney c a l c i n o s i s . Furthermore, these d i e t s a l so conta ined high Ca to Mg r a t i o s which ranged from 34:1 to 55:1 . The most seve re l y a f f e c t ed f i s h were those fed d i e t s 2 ,4 ,6 ,8 and 10 which conta ined Ca to Mg r a t i o s i n the range of 46:1 to 54:1 . As mentioned p r ev i ou s l y ( 4 . 4 . 5 . 1 ) , i n s u f f i c e n t d i e t a r y Mg has been shown to cause c a l c i n o s i s i n rainbow t r o u t . The f i n d i n g s of both experiments I and I I I suggest tha t high d i e t a r y Ca and P i n r e l a t i o n to 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 ie ta ry phy t i c ac id d i d not appear to i n f l u ence the inc idence of c a l c i n o s i s . I t i s i n t e r e s t i n g tha t some research has shown that phy t i c a c i d can reduce the inc idence of rena l c a l c u l i by b ind ing t o and then d i s s o l v i n g any e x i s t i n g Ca depos i t s (Graf 1983). Modl in (1980) fed phytate d i e t s to mice and repor ted an inverse r e l a t i o n s h i p between the amount of phy t i c ac id consumed and the inc idence of n ehp r o c a l c i n o s i s . No such c o r r e l a t i o n has been repor ted f o r sa lmonids, nor was one ev ident i n t h i s experiment or i n experiment I . 6 .4 .5 .2 P y l o r i c caeca In experiment I , the s t r u c t u r e of the p y l o r i c caeca was abnormal i n f i s h f e d 25.8 g phy t i c ac id/kg ( se c t i on 4 . 3 . 5 . 2 ) . In the present s tudy, - 104 -hypertrophy of the 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 the cytoplasm was observed as e a r l y as day 50 i n f i s h fed more than 21 g phy t i c ac id /kg (Table 15) . The p y l o r i c caeca of f i s h fed d i e t s con ta in i ng low phy t i c a c i d , r ega rd l e s s of the Ca l e v e l , was normal f o r j u v e n i l e chinook salmon. However, the amount of d i e t a r y Ca may have exerted an i n f l uence on the p y l o r i c caeca when phy t i c ac id was g rea te r than 2.1 g/kg d i e t . For i n s t ance , abnormal i ty of the p y l o r i c caeca was ev ident i n f i s h fed d i e t s w i th 11.6 g phy t i c ac id /kg ( d i e t s 10-12 and 15) w i th the except ion of f i s h fed d i e t 9. The Ca content i n the l a t t e r d i e t was 5.6 g compared to the more than 25 g Ca/kg which was present i n the former d i e t s . Furthermore, i n the high phytate groups ( d i e t s 5-8) there was a g rea te r inc idence of v a cuo l i z a t i o n of the cytoplasm i n f i s h fed d i e t s 6 and 8 which a l so conta ined high l e v e l s of d i e t a r y Ca. P o s s i b l e exp lanat ions f o r the e f f e c t s of phy t i c ac id on the p y l o r i c caeca have been o u t l i n e d p r ev i ou s l y ( s e c t i on 4 . 4 . 5 . 2 ) . 6.4.6 Minera l A n a l y s i s 6 .4 .6 .1 Whole blood Table 16 i l l u s t r a t e s tha t whole blood Zn was d i r e c t l y r e l a t e d to d i e t a r y Zn concent ra t i on and i n v e r s e l y r e l a t ed to d i e t a r y phy t i c ac id c on cen t r a t i o n . S i m i l a r f i n d i n g s were repor ted i n experiment I f o r plasma Zn ( se c t i on 4 . 3 . 6 ) . Blood Zn was a l so i n f l uenced by d i e t a r y Ca content i n the low and high phytate groups when d i e t a r y Zn was low and h i gh , r e s p e c t i v e l y . For example, f i s h fed d i e t 1 which conta ined 9.63 g Ca/kg had s i g n i f i c a n t l y h igher blood Zn than d id f i s h which were fed d i e t 2 where Ca was increased to 46 g/kg. S i m i l a r l y , i n the h igh phytate groups (5 -8 ) , f i s h fed 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 igher blood Zn than d id f i s h fed d i e t 8 which conta ined 48.6 g Ca/kg. These r e s u l t s support the theory tha t phy t i c ac id - 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 tha t Zn absorpt ion i s f u r t h e r reduced when the d i e t a r y Ca content i s h i gh . As. w i th the plasma P r e s u l t s repor ted in s e c t i on 4 .4 .6 , d i e t a r y P concen t ra t i on per se d i d not i n f l uence blood P l e v e l s . Phy t i c ac id concen t ra t i on however, d i d s i g n i f i c a n t l y i n f l u en ce blood P l e v e l s w i th high d i e t a r y phytate h inde r ing P abso rp t i on . The in te rmed ia te l e v e l of phy t i c ac id (11.6 g/kg d i e t ) s i g n i f i c a n t l y reduced b lood P when d i e t a r y Zn was at 0.034 g/kg d i e t ( d i e t 11) . However, t h i s appears to be a d i r e c t r e s u l t of the d i e t a r y Zn l e ve l because the blood P l e ve 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 rea te r than 0.100 g/kg ( d i e t s 9,10,12 and 15) . The high concent ra t i on of blood Ca found i n f i s h fed d i e t 11 a l so seems to be a r e f l e c t i o n of the low d i e t a r y Zn i n t a k e . Inc reas ing the d i e t a r y Zn l e ve l from 0.034 g to 0.050 g/kg and above s i g n i f i c a n t l y reduced the amount of Ca i n the b lood . 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 to d i e t a r y concen t ra t i ons of Ca and phy t i c a c i d . For example, i n the low phytate groups, blood Mg was reduced s i g n i f i c a n t l y i n f i s h fed 46-47 g Ca/kg ( d i e t s 2 and 4) compared to f i s h fed 9 g Ca/kg (d i e t s 1 and 3 ) . The r e s u l t s were not as c l e a r i n the high phytate groups. Here d i e t a r y Ca d id not s i g n i f i c a n t l y depress Mg abso rp t i on . Th is i s cons i s t en t w i th the plasma Mg r e s u l t s repor ted in s e c t i on 4.4.6 from experiment I . However, at the in te rmed ia te l e ve l of phy t i c ac id (11.6 g/kg) , blood Mg was s i g n i f i c a n t l y h igher in f i s h fed d i e t 9, where the d i e t a r y Ca content was 5.6 g, than i n f i s h fed d i e t s w i th more than 29 g Ca/kg. 6 .4 .6 .2 L i v e r In gene ra l , l i v e r Zn concent ra t i on was a l so d i r e c t l y r e l a t e d to d i e t a r y - 106 -Zn i n take (Table 17 ) . Ca lc ium i n t ake d id not a f f e c t Zn depos i t i on but phy t i c ac id d i d have some i n f l u en c e . For i n s t ance , f i s h fed d i e t 1, which conta ined low Ca, Zn and phy t i c a c i d , had h igher l i v e r Zn l e v e l s than d id f i s h fed d i e t 5 which a l so conta ined low Ca and Zn but h igh phy t i c a c i d . S i m i l a r r e s u l t s were obta ined in f i s h fed d i e t s 4 and 8 which conta ined high Ca and Zn but d i f f e r e d i n phy t i c ac id con ten t . The high phyta te- fed 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 the low phyta te- fed f i s h ( d i e t 4 ) . These r e s u l t s suggest tha t Zn was che la ted to phy t i c ac id and rendered unava i l ab l e f o r abso rp t i on . L i v e r Fe was s i g n i f i c a n t l y i n f l uenced by phy t i c ac id concen t ra t i on w i th high Fe depos i t i on o c cu r r i ng i n f i s h fed the high phytate d i e t s 5,6 and 14. I t would appear tha t phy t i c a c i d enhanced Fe absorpt ion in t h i s case . The b e n e f i c i a l e f f e c t of phy t i c ac id on Fe absorpt ion has been documented in o ther s tud ies (Welch and van Campen 1975; Liebman and D r i s k e l l 1979). For example, Mor r i s and E l l i s (1976) showed tha t Fe was r e a d i l y absorbed by r a t s i f i t was present as monofer r i c phytate due to the s o l u b i l i t y of t h i s complex at pH 7 and above. The extremely high l i v e r Fe concen t ra t i on in f i s h fed d i e t 11 i s probably due to the low Zn concen t ra t i on of the d i e t . Both Fe and Zn compete f o r the same b ind ing s i t e s . Thus an excess of one w i l l reduce the absorpt ion of the o the r . For example, r a t s tud i e s have shown tha t inc reased l e v e l s of d i e t a r y Zn hindered Fe abso rp t i on . Th is was l a r g e l y due to Zn i n h i b i t i n g the r e l ease of Fe from f e r r i t i n (a major storage form of Fe ) , and/or by i n t e r f e r i n g wi th the i n co rpo r a t i on 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 occurred i n the present study in f i s h fed the high phytate d i e t s 7 and 8. In t h i s case , the lack of increased Fe depos i t i on i n these - 107 -f i s h may be a r e f l e c t i o n of the high d i e t a r y Zn conten t . The amount of Cu in the l i v e r was i n f l uenced by d i e t a r y l e v e l s of Zn and phy t i c a c i d . For example, the i nges t i on of d i e t s con ta i n i ng low Zn coupled w i th h igh phy t i c ac id (d i e t s 5 and 6 ) , or medium phy t i c a c i d (d i e t 11) , r e su l t e d in 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 . Phy t i c ac id has been shown to enhance Cu absorpt ion by i n h i b i t i n g Zn absorpt ion (Klevay 1977) and t h i s may be what occurred here . Increas ing Zn i n d i e t s 7 and 8 to 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 to tha t of f i s h fed d i e t s 5 and 6 which conta ined 0.05 and 0.06 g Zn/kg, r e s p e c t i v e l y . Knox et a l . (1984) repor ted tha t hepat i c Cu l e v e l s i n rainbow t r ou t were reduced s i g n i f i c a n t l y when the d i e t a r y Zn l e v e l was increased to 0.500 g from 0.034 g/kg. A d i e t a r y Zn in take of 0.120 g/kg d i e t has been shown to induce me ta l l o t h i one i n syn thes i s i n r a t s ( F i s che r et a l . 1983). S i m i l a r r e s u l t s were obta ined i n w in te r f l ounder (Shears and F l e t che r 1984). M e t a l l o t h i o n e i n , which i s a lso present i n salmonids (Ley, I I I 1984; McCarter and Roch 1983), i s a meta l -b ind ing p r o t e i n tha t p r e f e r e n t i a l l y b inds w i th Cu and renders i t unava i l ab l e f o r absorpt ion (Bremner 1980). There fo re , i t i s q u i t e p o s s i b l e tha t i t may have been r e spons i b l e f o r reduc ing l i v e r Cu in chinook salmon. 6 .4 .6 .3 Kidney Kidney Ca l e v e l s were d i r e c t l y r e l a t e d to d i e t a r y l e v e l s of Ca (Table 18) . F i s h fed any d i e t high i n Ca, r ega rd l e s s of the phy t i c a c i d l e v e l , e xh i b i t e d e levated concent ra t ions of k idney Ca. Thus i t i s not s u r p r i s i n g tha t these f i s h were a f f l i c t e d w i th k idney c a l c i n o s i s . The h ighes t l e ve l of k idney Ca was observed in f i s h which were f ed d i e t s tha t conta ined high l e v e l s of Ca and phy t i c a c i d . Th is suggests tha t phy t i c ac id exacerbates - 108 -rena l c a l c i n o s i s i n chinook salmon. F i s h fed d i e t s low in Ca (1 ,3 ,5 ,7 and 9) had s i g n i f i c a n t l y reduced k idney 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 to d i e t a r y concent ra t i ons of Ca, P and phy t i c a c i d . High Ca, P and phy t i c ac id in take r e su l t e d i n high k idney P concen t r a t i on . S i m i l a r l y , f i s h fed 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 idney P l e v e l s . As wi th the l e v e l s of b lood and plasma Zn, k idney 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 to d i e t a r y Zn i n t a k e . F i sh fed 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 igher k idney Zn even though the d i e t a r y Zn i n take was high (0.27 g/kg) . The d i e t a r y Zn concent ra t i on i n d i e t 8 was 0.50 g h igher than tha t i n d i e t 7 which may be why k idney Zn was s i g n i f i c a n t l y h igher i n t h i s group compared to tha t of f i s h fed d i e t 7. However, there i s no apparent exp lana t i on f o r the low Zn l e v e l s i n f i s h fed d i e t 3. The high Mg l e ve l i n the k idneys of f i s h fed d i e t 11 appears t o be r e l a t e d to 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 comparat ive groups when the d i e t a r y Zn content was increased from 0.034 g to 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 so g r e a t l y e leva ted in f i s h fed d i e t 11. As w i th l i v e r Fe t h i s may be a r e s u l t of the low d i e t a r y Zn in take which reduces the compet i t ion between Zn and Fe f o r b ind ing s i t e s . 6.5 CONCLUSION D ie ta ry phy t i c a c i d at concen t ra t i ons of 11.6-23.2 g/kg d i e t induced c a t a r a c t fo rmat ion in j u v e n i l e chinook salmon when the d i e t c oncu r r en t l y conta ined 0.034-0.100 g Zn/kg. Furthermore, ca ta rac tous f i s h conta ined the lowest l e v e l s of Zn i n the blood and l i v e r . Th is strengthens the content ion - 109 -tha t Zn p lays a major r o l e i n lens n u t r i t i o n . In add i t i on t o c a t a r a c t s , i nc reased m o r t a l i t y , decreased growth, food convers ion and PER, and p y l o r i c caeca l i r r e g u l a r i t i e s were ev ident i n f i s h fed the high phytate d i e t s . Neph roca l c i nos i s was observed i n chinook salmon fed d i e t s con ta i n i ng at l e a s t 25.2 g Ca/kg. - 110 -CHAPTER 7 7.0 SUMMARY AND CONCLUSION B i l a t e r a l lens 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 fed d i e t s con ta in i ng a minimum of 11.6 g phy t i c ac id/kg (18.0 g sodium phytate) and a maximum of 0.100 g Zn/kg. Moreover, the s e v e r i t y of o p a c i t i e s and the number of f i s h a f f e c t ed increased w i th t ime . High d i e t a r y l e v e l s of Ca and P (50 g/kg) aggravated the e f f e c t s of phy t i c ac id on ca ta rac togenes i s whereas a d i e t a r y Zn in take of at l eas t 0.150 g/kg prevented c a t a r a c t f o rma t i on . Catarac t fo rmat ion a l so appeared to be dependent upon the t ime and dura t i on of exposure to a ca ta rac togen i c d i e t . For example, f i s h fed ca ta rac togen 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 in th week. However, when the d i e t was fed at d i f f e r e n t i n t e r v a l s (experiment I I ) , t he re was a de lay between exposure to the d i e t and the appearance of the c a t a r a c t s . Such a phenomenon may have occurred i n the 1981 ca ta rac t outbreak i n B.C. and Washington S ta te chinook salmon s t o c k s . Ca ta rac t s were detected in these f i s h s h o r t l y before they were re l eased as smo l t s . Furthermore, the s i z e of the a f f e c ted f i s h was s i m i l a r to the non-a f fec ted f i s h . In c on t r a s t , the ca tarac tous f i s h in experiments I and I I I were d r a s t i c a l l y reduced i n s i z e compared to the non-cataractous f i s h . The ca ta rac tous f i s h i n experiment II however, were much l a r ge r than those of the other s t u d i e s . Th i s experiment showed tha t growth recovery was p o s s i b l e a f t e r exposure to a poor q u a l i t y d i e t and the r e s u l t s suggest tha t a s i m i l a r s i t u a t i o n may have occur red in 1981. The 1981 f i s h a l so mainta ined f a i r l y good growth i n s p i t e of c a t a r a c t f o rmat i on . Thus i t i s p o s s i b l e tha t they may have been exposed to a poor q u a l i t y d i e t severa l weeks p r i o r to the appearance of c a t a r a c t s . - I l l -F i sh growth, food conve r s i on , PER and m o r t a l i t y were a l so d i r e c t l y i n f l uenced by phy t i c a c i d c oncen t r a t i on . F i s h which were fed d i e t s con ta in i ng >_ 21.1 g phy t i c ac id/kg exh i b i t e d reduced growth, food and p r o t e i n convers ion and h igher m o r t a l i t y . The lowest va lues o v e r a l l were observed i n f i s h fed 0.034 g Zn coupled wi th 11.6 g phy t i c ac id/kg d i e t . Thus c a r e f u l c ons i de ra t i on of the d i e t a r y Zn l e ve l i s e s p e c i a l l y important when minera l b ind ing agents ( e . g . phy t i c a c i d and f i b r e ) are a l so present i n the d i e t . The amount of Ca and P present i n a d i e t i s a l so of c r i t i c a l importance. Based on mean weight da ta , a Ca l e v e l of 28.3 g/kg i n a low phytate d i e t w i l l s i g n i f i c a n t l y reduce growth under the cond i t i on s of t h i s s tudy. Moreover, e levated l e v e l s of Ca and/or phytate w i l l compound t h i s e f f e c t . However, i n c r ea s i ng the d i e t a r y Zn concent ra t i on w i l l a l l e v i a t e some of the growth reduc t i on in f i s h fed d i e t s con ta in i ng 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 to the p y l o r i c caeca may occur i n chinook salmon which are f ed d i e t s con ta in i ng >_ 21.1 g phy t i c a c i d / kg . Moreover, 11.6 g phy t i c ac id i n the presence of >^  25 g Ca/kg may cause hypertrophy of the p y l o r i c caeca. S i m i l a r l y , neph roca l c i nos i s may occur i n chinook salmon when they are fed d i e t s con ta in i ng >1 25 g Ca/kg rega rd less of the phy t i c a c i d con ten t . M inera l analyses of the b lood, plasma and l i v e r revea led Zn to be the on ly t r a ce minera l which was s i g n i f i c a n t l y reduced i n concen t ra t i on in high phyta te- fed f i s h . Furthermore, the lowest plasma, b lood and l i v e r z i n c l e v e l s occurred i n f i s h fed the ca ta rac togen i c d i e t s . 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 lens metabol ism of j u v e n i l e chinook salmon, but more work i s r equ i r ed to e l u c i d a t e the nature of t h i s r o l e . For example, s l i t - l a m p biomicroscopy of lens t i s s u e p r i o r t o , dur ing and a f t e r exposure to a ca ta rac togen i c d i e t may revea l i n fo rmat i on - 112 -which cou ld prov ide e a r l y warning s igns of c a t a r a c t f o rma t i on . Fur ther s t ud i e s may a l so be conducted to more a c cu ra t e l y assess the c r i t i c a l t ime of exposure to a ca ta rac togen i c d i e t by chinook salmon and to quan t i f y the maximum l e v e l s of d i e t a r y Ca, P and phy t i c ac id (and p o s s i b l y other m ine ra l -b i nd ing 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 ma inta in good growth, food u t i l i z a t i o n and h e a l t h . The r e s u l t s of such research would a id f i s h c u l t u r i s t s i n p revent ing c a t a r a c t outbreaks and would help to ensure adequate f i s h s i z e at the t ime of r e l ea se i n t o the ocean and thus maximize s u r v i v a l . A l s o , i n s i g h t may be gained i n t o u t i l i z i n g p lan t p r o t e i n in salmonid d i e t s as a means f o r reduc ing d i e t c o s t s . - 113 -BIBLIOGRAPHY Abdel-Hafez, H.M., M. Manas-Almendros, R. Ross and A.D. Care . 1982. E f f e c t s of d i e t a r y phosphorus and ca lc ium on the i n t e s t i n a l absorp t ion of ca lc ium i n sheep. B r i t . J . Nutr: 47:69-77. A l l i s o n , L.N. 1962. Cata rac t among hatchery-reared lake t r o u t . (Abs t rac t ) The Prog. F i s h - C u l t . 24:155. AOAC. 1975. O f f i c i a l methods of a n a l y s i s . A s s o c i a t i o n of O f f i c i a l A n a l y t i c a l Chemists (12th e d i t i o n ) , Washington, D.C. 1094 pp. Bafundo, K.W., D.H. Baker and P.R. F i t z g e r a l d . 1984. Z inc u t i l i z a t i o n i n the ch ick as i n f l uenced by d i e t a r y concent ra t i ons of ca l c i um and phytate and by E imer i a a ce r vu l i n a i n f e c t i o n . P ou l t r y S c i . 63:2430-2437. Ba ldwin , G. and P . J . Ben t l ey . 1980. The z i n c metabol ism of the amphibian l ens . Exp. Eye Res. 30(4):333-343. Barash, H., H.A. Poston and G.L. Rumsey. 1982. D i f f e r e n t i a t i o n of s o l ub l e p ro te ins in c a t a r a c t s caused by d e f i c i e n c i e s of meth ion ine , r i b o f l a v i n or z i n c i n d i e t s fed to A t l a n t i c salmon, Salmo s a l a r , rainbow t r o u t , Salmo g a i r d n e r i , and lake t r o u t , S a l v e l i n u s namaycush. Co rne l l Vet . 72:361-371. Ba rne t t , B . J . , C.Y. Cho and S . J . S l i n g e r . 1979. The e s s e n t i a l i t y of c h o l e c a l c i f e r o l i n the d i e t s of rainbow t r ou t (Salmo g a i r d n e r i ) . Comp. Biocehm. P h y s i o l . 63A:291-297. Be l l ows , J .G . 1944. Cata rac t and Anomalies of the Lens. S t . L ou i s , Mosby, M i s s o u r i , p. 17-31. Bergman, B. 1970. The z i n c concent ra t ion in hard and s o f t t i s s u e s of the r a t . A c t a . Odont. Scand. 28:425-440. B i l t o n , H.T. 1984. Returns of chinook salmon i n r e l a t i o n to j u v e n i l e s i z e at r e l e a s e . Can. Tech. Rep. F i s h . Aquat. S c i . 1245, 33p. B i t a r , K. and J .G . Re i nho l d . 1972. Phytase and a l k a l i n e phosphatase a c t i v i t i e s i n i n t e s t i n a l mucosae of r a t , c h i c ken , c a l f and man. Biochem. B iophys . A c t a . 268:442-452. B l i g h , E.G. and W.J. Dyer. 1959. A rap id method of t o t a l l i p i d e x t r a c t i o n and p u r i f i c a t i o n . Can. J . Biochem. P h y s i o l . 37:911-917. Bowness, J .M . , R.A. Morton, M.H. Shak i r and A . L . Stubbs. 1952. D i s t r i b u t i o n of copper and z i n c in mammalian eyes. Biochem. J . 51:521-530. Bremner, V.M. 1980. Abso rp t i on , t r anspor t and d i s t r i b u t i o n of copper. In: B i o l o g i c a l Roles of Copper. Excerpta Med ica , Amsterdam, p. 23-48. B r e t t , J .R. 1979. Environmental f a c t o r s and growth. In: F i sh Phys io logy Vo l . V I I I . W.S. Hoar, D.J . Randal l and J .R. B r e t t ( e d s . ) . Academic P ress , Inc . , New York, N.Y. p. 599-675. - 114 -Brown, E.D., W. Chan and J .C . Smith. 1978. Bone m i n e r a l i z a t i o n dur ing a developing z i n c d e f i c i e n c y . Proc . Soc. Exp. B i o l . Med. 157:211-214. Bunce, G.E. and J . L . Hess. 1976. L en t i c u l a r o p a c i t i e s i n young r a t s as a consequence of maternal d i e t s low in t ryptophan and/or v i t am in E. J . Nutr . 106:222-229. Bussan i ch , N. 1981. Ve te r i na ry ophtha lmolog is t (pe r s . comm.). Chen, L.H. and S.H. Pan. 1977. Decrease of phytates dur ing germinat ion of pea seeds (P is ium s a t i v a ) . Nutr . Rep. I n t . 16:125-131. Cohen, R.D.H. 1980. Phosphorus in rangeland ruminant n u t r i t i o n a rev iew. L i v e s t . Prod. S c i . 7:25-27. Cowey, C B . 1976. Use of s yn the t i c d i e t s and b iochemica l c r i t e r i a i n the assessment of n u t r i e n t requirements of f i s h . J . F i s h . Res. Bd. Can. 33:1040-1045. Cowey, C .B . , D. Knox, J.W. Adron, S. George and B. P i r i e . 1977. The product ion of rena l c a l c i n o s i s by magnesium d e f i c i e n c y i n rainbow t r ou t (Salmo g a i r d n e r i ) . B r i t . J . Nut r . 38:127-135. Darby, W.J. and P.L. Day. 1939. Xylose as a ca ta rac togen i c agent. P roc . Soc. Exp. B i o l . Med. 41:507-508. Dav ies , O.L. 1954. The determinat ion of optimum c o n d i t i o n s . In: The Design and Ana l y s i s of I n du s t r i a l Exper iments. O l i v e r and Boyd (Pub.) f o r Imper ia l Chemical Indus t r i e s L t d . p. 495-551. Dav ies , N.T. and R. N i gh t i n ga l e . 1975. The e f f e c t s of phytate on i n t e s t i n a l absorpt ion and se c re t i on of z i n c , and whole-body r e t e n t i o n of z i n c , copper, i r on and manganese in r a t s . B r i t . J . Nu t r . 34:243-258. Dav ies , N.T., V. H r i s t i c and A. F l e t t . 1977. Phytate r a t he r than f i b r e in bran as a major detr iment of z i n c a v a i l a b i l i t y to r a t s . Nu t r . Rep. In t . 15:207-214. Dav i s , G.K. 1966. Fac to rs i n f l u enc i ng z i n c a v a i l a b i l i t y i n an ima ls . In: Z inc Metabols im. A .S . Prasad ( e d . ) . Char les C. Thomas Pub. I l l i n o i s , USA. p. 215-223. Dor land ' s I l l u s t r a t e d Medical D i c t i ona ry ( 2 4 t h E d . ) . 1965. W.B. Saunders Co. Dukes, T.W. 1975. Ophthalmic pathology of f i s h e s . In: The Patho logy of F i s he s . W.E. R i b e l i n and G. Migaki (eds.) U n i v e r s i t y of Wiscons in Press , p. 383-398. Duncan, D.B. 1955. M u l t i p l e range and m u l t i p l e F - t e s t s . B i omet r i c s 11:1-42. Duncan, G. and P.C. Croghan. 1969. Mechanisms f o r the r e g u l a t i o n of c e l l volume w i th p a r t i c u l a r re ference to the l e n s . Exp. Eye Res. 8:421-428. - 115 -Ek lund, A. 1975. The contents of phy t i c ac id in p r o t e i n concent ra tes prepared from n igerseed, sunf lower seed, rapeseed and poppy seed. Upsala J . Med. S c i . 80:5-6. Erdman, J r . , J.W. 1979. O i l seed p h y t a t e s : n u t r i t i o n a l i m p l i c a t i o n s . J . Am. O i l Chem. Soc. 56:736-741. Evans, W.J . , E . J . McCourtney and R . I . Shrager. 1982. T i t r a t i o n s tud i e s of phy t i c a c i d . J . Am. O i l Chem. Soc. 59:189. Fange, R. and D. Grove. 1979. D i g e s t i o n . In: F i sh Phys io logy V o l . V I I I . W.S. Hoar, D.J . Randal l and J . R . B r e t t ( e d s . ) . Academic Press I n c . , New York, N.Y. p.162-260. Fe rna l d , R.D. and S.E. Wr ight . 1983. Maintenance of o p t i c a l q u a l i t y dur ing c r y s t a l l i n e lens growth. Nature 301:618-620. F i s c h e r , P.W.F., A. Giroux and M.R. L'Abbe. 1983. E f f e c t s of z i n c on mucosal copper b ind ing and on the k i n e t i c s of copper ab so rp t i on . J . Nut r . 113:462-469. Forbes, R.M. 1960. N u t r i t i o n a l i n t e r a c t i o n s of z i n c and c a l c i um . Fed. Proc .:643-647. Forbes, R.M. 1964. M inera l u t i l i z a t i o n in the r a t I I I . E f f e c t s of c a l c i um, phosphorus, l a c t o se and source of p r o t e i n i n z i n c - d e f i c i e n t and in z inc-adequate d i e t s . J . Nut r . 83:225-233. Forbes, R.M., J.W. Erdman, J r . , H.M. Pa rker , H. Kondo and S.M. Ke t e l s en . 1983. B i o a v a i l a b i l i t y of z i n c in coagulated soy p r o t e i n (Tofu) to r a t s and e f f e c t of d i e t a r y ca l c ium at a constant phy t a t e : z i n c r a t i o . J . Nutr . 113:205-210. Fowler , L.G. and R.E. Burrows. 1971. The Abernathy salmon d i e t . Prog. F i s h - C u l t . 33:67-75. Fox, J . and A.D. Care . 1978. E f f e c t of low ca l c ium and low phosphorus d i e t s on the i n t e s t i n a l absorpt ion of phosphate i n i n t a c t and parathyro idectomized p i g s . J . Endocr. 77:225-231. F ranz , K .B. , B.M. Kennedy and D.A. F e l l e r s . 1980. R e l a t i v e b i o a v a i l a b i l i t y of z i n c from se l ec ted ce rea l s and legumes us ing r a t growth. J . Nut r . 110:2272-2283. G a l i n , M.A., H.D. Nano and T. H a l l . 1962. Ocular z i n c c on c en t r a t i o n . Inves t . Ophth. 1:142-148. Gra f , E. 1983. App l i c a t i o n s of phy t i c a c i d . J . Am. O i l . Chem. Soc. 60:1861-1867. Grant , W.M. 1974. Types of t o x i c e f f e c t s i n v o l v i n g the eyes . In: Tox i co logy of the Eye, 2 n d Ed . , Char les C. Thomas Pub., S p r i n g f i e l d , I l l i n o i s , p. 24-29. - 116 -Groves, T.D.D. 1970. Body compos i t ion changes du r ing growth i n young sockeye salmon (Oncorhynchus nerka) i n f r e s h wate r . J . F i s h . Res. Board Can. 27:929-94?: H a l l , W.K., L .L . Bowles, V .P . Sydens t r i c ke r and H.L. Schmidt , J r . 1948. Ca ta rac t s due to d e f i c i e n c i e s of pheny l a l an ine and of h i s t i d i n e i n the r a t . A comparison w i th o ther types of c a t a r a c t s . J . Nu t r . 36:277-295. Hardie-Muncy, D.A. and A . I . Rasmussen. 1979. I n t e r r e l a t i o n s h i p s between z i n c and p r o t e i n l e v e l and source i n weanl ing r a t s . J . N u t r . 109:321-329. Ha r l and , B .F . , M.R. Sp ivey Fox and B.E. F r y . 1975. P r o t e c t i o n aga ins t z i n c d e f i c i e n c y by p r i o r excess d i e t a r y z i n c i n young Japanese q u a i l . J . Nu t r . 105:1509-1518. Harmuth-Hoene, A. and R. Sche lenz . 1980. E f f e c t of d i e t a r y f i b e r on minera l abso rp t i on i n growing r a t s . J . Nu t r . 110:1774-1784. Ha r t , J . L . 1973. P a c i f i c F i shes of Canada. B u l l e t i n 180, F i s h e r i e s Research Board of Canada, p. 125. Hartman, J r . , G.H. 1979. Removal of phyta te from soy p r o t e i n . J . Am. O i l . Chem. Soc . 56:731-735. H iggs, D.A., J .R . McBr ide , J .R . Marke r t , B.S. Dosanjh, M.D. P l o t n i k o f f and W.C. C l a r k e . 1982a. Eva l ua t i on of Tower and Candle Rapeseed (Canola) Meal and Bronowski Rapeseed p r o t e i n concen t ra te as p r o t e i n supplements i n p r a c t i c a l d ry d i e t s f o r j u v e n i l e chinook salmon (Oncorhynchus  t shawytscha) . Aquacu l tu re 29:1-31. H iggs , D.A., U.H.M. Fager lund , J . G . Ea les and J .R . McB r i de . 1982b. A p p l i c a t i o n of t h y r o i d and s t e r o i d hormones as a nabo l i c agents i n f i s h c u l t u r e . Comp. Biochem. P h y s i o l . 738:143-176. Hockwin, 0 . and H. Koch. 1975. Combined nox ious i n f l u e n c e s . I n : Ca t a r a c t s and Abno rma l i t i e s of the Lens. J .G . Be l low ( ed . ) Grune and S t r a t t o n I n c . , New York . p. 243-254. Hoefer , J . A . , E.R. M i l l e r , D.E. U l l r e y , H.D. R i t c h i e and R.w". Leucke. 1960. I n t e r r e l a t i o n s h i p s between c a l c i um , z i n c , i r o n and copper i n swine f e e d i n g . J . Anim. S c i . 19:249-259. House, W.A., R.M. Welch and D.R. Van Campen. 1982. E f f e c t of p h y t i c a c i d on the a b s o r p t i o n , d i s t r i b u t i o n and endogenous e x c r e t i o n o f z i n c i n r a t s . J . Nu t r . 112:941-953. Hughes, M.R., P.F. Brumbaugh, M.R. Hauss l e r , J . E . Wergedal and D.J . B a y l i n k . 1975. Regu l a t i on of serum l - a l pha , 25 -d i h yd r oxy v i t a m i n D3 by ca l c i um and phosphate i n the r a t . Sc ience 190:578-580. Hur l ey , L .S . and H. Swenerton. 1971. Lack of m o b i l i z a t i o n of bone and l i v e r z i n c under t e r a t o gen i c c ond i t i o n s of z i n c d e f i c i e n c y i n r a t s . J . Nu t r . 101:597-604. - 117 -I sma i1 -Be i g i , F., B. F a r a j i and J .G . Re inho ld . 1977. B ind ing of z i n c and i r on to wheat bread, wheat bran and t h e i r components. Am J . C l i n . Nut r . 30:1721-1725. J enk in s , K . J . and P.H. P h i l l i p s . 1960. The minera l requirements of the dog. 1. Phosphorus requirement and a v a i l a b i l i t y . J . Nut r . 70:235-240. J enk ins , N.K. 1965. Phytate metabolism i n an ima ls . Nature 205:89. Johns, P.R. 1981. Growth of f i s h r e t i n a s . Amer. Z o o l . 21:447-458. Jubb, K.V.F. and P.C. Kennedy. 1970. Pathology of Domestic An ima ls , V o l . 2, 2nd Ed. Academic Press , p. 495-514. K e t o l a , H.G. 1975. Requirements of A t l a n t i c salmon f o r d i e t a r y phosphorus. Trans. Am. F i s h . Soc. 3:548-551. K e t o l a , H.G. 1979. In f luence of d i e t a r y z i n c on c a t a r a c t s i n rainbow t r ou t (Salmo g a i r d n e r i ) . J . Nut r . 109:965-969. K e t o l a , H.G. 1982. Z inc supplements prevent c a t a r a c t s i n salmon and t r o u t f ed d i e t s con ta i n i ng high-ash f i s h meal . Salmonid 5 (5 ) :15 . Keu l s , M. 1952. The use of the s tudent i zed range i n connect ion w i th an ana l y s i s of v a r i an ce . Euphyt i ca 1:112-122. K l e t h i , J . and P. Mandel. 1965. Eye lens nuc l eo t i des of d i f f e r e n t spec ies of v e r t eb r a t e s . Nature 205:1114. K levay , L.M. 1977. Hypocho lestero lemia due to sodium phy ta te . Nu t r . Rep. I n t . 15:587-595. Knox, D., C B . Cowey and J.W. Adron. 1981. S tud ies on the n u t r i t i o n of salmonid f i s h . The magnesium requirement of rainbow t r o u t (Salmo  g a i r d n e r i ) . B r i t . J . Nutr: 45:137-148. Knox, D., C B . Cowey and J.W. Adron. 1983. S tud ies on the n u t r i t i o n of rainbow t r ou t (Salmo g a i r d n e r i ) . Magnesium d e f i c i e n c y : the e f f e c t of feed ing wi th a magnesium-supplemented d i e t . B r i t . J . Nu t r . 50:121-127. Knox, D., C B . Cowey and J.W. Adron. 1984. E f f e c t s of d i e t a r y z i n c i n take upon copper metabol ism i n rainbow t r ou t (Salmo g a i r d n e r i ) . Aquacu l ture 40:199-207. K r a t z e r , F .H. , J . B . A l l r e d , P.N. Dav i s , B . J . Marsha l l and P. Vohra. 1959. E f f e c t of au toc l av ing soybean p r o t e i n and the add i t i o n of e thy l ened iamine t raace t i c ac id on the b i o l o g i c a l a v a i l a b i l i t y of d i e t a r y z i n c f o r tu rkey p o u l t s . J . Nut r . 68:313-322. Kubota, S .S . 1976. Catarac t i n f i s h e s : Pa tho l og i c a l changes i n the l e n s . F i sh Pathology 10:191-197. - 118 -Kuck, J r . , J . F .R . 1975. Composit ion of the l en s . In :Ca ta rac t s and Abnorma l i t i es of the Lens. J .G . Bel lows ( e d . ) . Grune and S t r a t t e n , I n c . , New York. p. 69-98. L a l l , S.P. 1979. M inera l s i n f i n f i s h n u t r i t i o n . In: Proceedings of the World Symposium on F i n f i s h N u t r i t i o n and Feed Technology, Hamburg, Germany, June 20-23. p. 85-97. Lee, W.R., R.J . Roberts and C . J . Shepherd. 1976. Ocu lar patho logy in rainbow t r ou t i n Malawi (Zomba D i sease ) . J . Comp. Path . 86:221-233. Lerman, S. 1966. The l en s . In: Bas ic Ophthalmology, McGraw-Hi l l Book Co. p. 195-259. Leucke, R.W., J .A . Hoefer, W.S. Brammell and D.A. Schmidt. 1957. Calc ium and z i n c i n pa rake ra tos i s of swine. J . Anim. S c i . 16:3-11. Ley, I I I , H.L., M.L. Fai11a and D.S. Cher ry . 1983. I s o l a t i o n and c h a r a c t e r i z a t i o n of hepa t i c me t a l l o t h i one i n from rainbow t r o u t (Salmo  g a i r d n e r i ) . Comp. Biochem. P h y s i o l . 74B:507-513. Liebman, M. and J . D r i s k e l l . 1979. D i e ta ry phytate and l i v e r i r on r e p l e t i o n i n i r on-dep le ted r a t s . Nut r . Rep. I n t . 19:281-287. L i k u s k i , H.J.A. and R.M. Forbes. 1965. M inera l u t i l i z a t i o n i n the r a t : E f f e c t s of ca lc ium and phy t i c ac id on the u t i l i z a t i o n of d i e t a r y z i n c . J . Nut r . 85:230-234. L i p s c h i t z , D.A., K.M. Simpson, J .D . Cook and E.R. M o r r i s . 1978. Absorp t ion of monoferr i c phytate by dogs. J . Nut r . 109:1154-1160. L l o yd , L .E . , B.E. McDonald and E.W. Crampton. 1978. E s s e n t i a l t r a ce e lements. In: Fundamentals of N u t r i t i o n , 2nd Ed. W.H. Freeman and Co. p. 248-270. Lo, G . , S .L . S e t t l e , F.H. S te inke and D.T. Hopkins. 1981. E f f e c t of phy ta te : z i n c molar r a t i o and i s o l a t e d soybean p r o t e i n on z i n c b i o a v a i l a b i l i t y . J . Nutr . 111:2223-2235. Maddaiah, V.T. , A.A. Kurnick and B.L. Re i d . 1964. Phy t i c a c i d s t u d i e s . P roc . Soc. Exp. B i o l . Med. 115:391-393. Ma l i k , S.R.K. and A.K. Gupta, S. C h a t t e r j i and P.S. Agarwa l . 1969. So lub l e p ro t e i n s i n normal and ca ta rac tous human l enses . Exp. Eye Res. 8:93-98. Manery, J . F . 1961. M ine ra l s i n nonosseous connec t ive t i s s u e s ( i n c l u d i n g the b lood , lens and co rnea) . In: M inera l Metabol ism an Advanced T r e a t i s e . V o l . I P r i n c i p l e s , Processes and Systems Par t B. C.L . Comar and F. Bronner ( e d s . ) . Academic P ress , I n c . , New York. p. 551-608. March, B.E. 1982. P ro f e s so r , Dept. of P ou l t r y Sc ience , U n i v e r s i t y of B r i t i s h Columbia, (pers.comm.). - 119 -McBr ide, J .R . and A .P . van Overbeeke. 1971. E f f e c t s of androgens, estrogens and Cortisol on the s k i n , stomach, l i v e r , pancreas and k idney i n gonadectomized adu l t sockeye salmon (Oncorhynchus ne r ka ) . J . F i s h . Res. Board Can. 28:485-490. McCarter , J .A . and M. Roch. 1983. Hepatic me t a l l o t h i one i n and r e s i s t a n c e to copper i n j u v e n i l e coho salmon. Comp. Biochem. P h y s i o l . 74C:133-137. McLaren, D.S. and A. Ha lasa . 1975. N u t r i t i o n a l and metabo l i c c a t a r a c t . In : Catarac ts and Abnorma l i t i e s of the Lens. J .G . Bel lows ( e d . ) , Grune and S t r a t t o n , Inc . New York. p. 255-263. Meyer, N.R., M.A. S t ua r t and C M . Weaver. 1983. B i o a v a i l a b i l i t y of z i n c from de fa t ted soy f l o u r , soy h u l l s and whole eggs as determined by i n t r i n s i c and e x t r i n s i c l a b e l l i n g techn iques . J . Nu t r . 113:1255-1264. M i l l e r , S . J . 1978. The l e n s . In: Parson 's Diseases of the Eye ( 1 6 t h E d . ) . Chu r ch i l l L i v i n g s t one , p. 276-295. M i t c h e l l , H.S. and W.M. Dodge. 1935. Catarac t i n r a t s fed on h igh l ac tose r a t i o n s . J . Nu t r . 9:37-49. Mod l i n , M. 1980. U r i na r y phosphorylated i n o s i t o l s and rena l s tone . Lancet 8204:1113-1114. M o r r i s , E.R. and R. E l l i s . 1976. I s o l a t i o n of monofer r i c phytate from wheat bran and i t s b i o l o g i c a l va lue as an i r on source to the r a t . J . Nu t r . 106:753-760. Munz, F.W. 1971. V i s i o n : v i s u a l pigments. In: F i s h Phys io logy V o l . V. W.S. Hoar and D.J. Randal l ( ed s . ) . Academic P ress , I n c . , New York, N.Y. p. 1-32. Murata, T. and Y. Taura. 1975. Study of t r a ce m e t a l l i c elements i n the l en s . Ophth. Res. 7:8-14. Murray, E . J . and H.H. Messer. 1981. Turnover of bone z i n c dur ing normal and acce le ra ted bone l o s s i n r a t s . J . Nut r . 111:1641-1647. Nahapet ian, A. and V.R. Young. 1980. Metabol ism of 14 c - phy t a t e in r a t s : E f f e c t of low and high d i e t a r y ca l c ium i n t a k e s . J . Nu t r . 110:1458-1472. NRC 1981. Nu t r i en t requirements of co ldwater f i s h e s . No. 16. Nat iona l Academy P r e s s , Washington, D .C 63 pp. Naver t , B., B. Sandstrom and A. Cederb lad. 1985. Reduct ion of the phytate content of bran by leaven ing bread and i t s e f f e c t on z i n c absorp t ion in man. B r i t . J . Nu t r . 53:47-53. Ne lson, T . S . , T.R. Sh i eh , R . J . Wodzinski and J . H . Ware. 1971. E f f e c t of supplemental phytase on the u t i l i z a t i o n of phytate P by c h i c k s . J . Nutr . 101:1289-1294. - 120 -Ne lson, T . S . , L.B. Dan i e l s , J .R . Ha l l and L.G. S h i e l d s . 1976. Hyd ro l y s i s of na tu ra l phytate-phosphorus i n the d i g e s t i v e t r a c t of c a l v e s . J . Anim. S c i . 42:1509-1512. N i co l ayson , R., N. Eeg-Larsen and O.J . Malm. 1953. Phys i o l ogy of ca l c ium metabol ism. P h y s i o l . Rev. 33:424-444. Ober leas , D., M.E. Muhrer and B.L. O ' D e l l . 1962. E f f e c t s of p h y t i c a c i d on z i n c a v a i l a b i l i t y and pa rake ra tos i s i n swine. J . Anim. S c i . 21:57-61. Ober leas , D., M.E. Muhrer, E. Mer le and B.L. O ' D e l l . 1966. D i e t a r y meta l-complex ing agents and z i n c a v a i l a b i l i t y i n the r a t . J . Nut r . 90:56-61. Ober leas , D. and B.F. Har land . 1977. N u t r i t i o n a l agents which a f f e c t metabo l i c z i n c s t a t u s . In: Z inc Metabol ism: Current Aspects i n Heal th and D isease . G . J . Brewer and A .S . Prasad ( e d s . ) . A lan R. L i s s , I n c . , New York. p. 11-24. O ' D e l l , B.L. 1960. Magnesium requirement and i t s r e l a t i o n t o o ther d i e t a r y c o n s t i t u e n t s . Fed. P roc . 19:654-668. O ' D e l l , B.L. and J . E . Savage. 1960. E f f e c t of phy t i c a c i d on z i n c a v a i l a b i l i t y . P roc . Soc. Exp. B i o l , and Med. 103:304-306. O ' D e l l , B.L . , J .M. Yohe and J . E . Savage. 1964. Z in c a v a i l a b i l i t y i n the ch i ck as a f f e c t ed by phyta te , ca l c ium and e t hy l ened i am ine - t e t r a -ace ta te . P ou l t r y S c i . 43:415-419. Ogino, C , F. Takashime amd J .Y . Ch iou. 1978. Requirement of rainbow t r ou t f o r d i e t a r y magnesium. B u l l . Jpn . Soc. S c i . F i s h . 44:1105-1108. Ogino, C. and G. Yang. 1978. Requirement of rainbow t r o u t f o r d i e t a r y z i n c . B u l l . Jpn. Soc. S c i . F i s h . 44:1015-1018. Orten, J .M. 1966. B iochemica l aspects of z i n c metabo l i sm. In: Z inc Metabol ism. A . S . Prasad ( e d . ) . Char les C. Thomas (Pub.) , p. 38-45. Pensack, J .M . , J .M. Henson and P.D. Bogdonoff. 1958. The e f f e c t s of ca l c ium and phosphorus on the z i n c requirements of growing ch i c kens . (Abs t rac t ) Pou l t r y S c i . 37:1232-1233. Pe r r y , T. 1982. Ca ta rac t s i n B.C. chinook and coho: cos t s and cu re s . Proceedings of the 3 2 n d annual Northwest F i s h Cu l t u r e Conference, Dec. 1-3, 1981, Tumwater, Washington, USA. p. 77-82. P h i l i p s o n , B. 1973. Changes i n the lens r e l a t e d to the r educ t i on of t ransparency . Exp. Eye Res. 16:29-39. P i r i e , A. and R. van Heyningen. 1956. Ocu lar e f f e c t s of n u t r i t i o n a l d i s ease . In: B iochemis t ry of the Eye. B l a ckwe l l S c i e n t i f i c P u b l i c a t i o n s , Ox fo rd . - 121 -Poston, H.A., R.C. R i i s , G.L. Rumsey and H.G. K e t o l a . 1977. The e f f e c t of supplemental d i e t a r y amino a c i d s , m inera l s and v i tamins on salmonids fed ca ta rac togen i c d i e t s . Co rne l l Vet . 67:472-509. Poston, H.A., R.C. R i i s , G.L. Rumsey and H.G. K e t o l a . 1978. N u t r i t i o n a l l y induced ca t a r a c t s i n salmonids fed p u r i f i e d and p r a c t i c a l d i e t s . Mar. F i s h . Rev. 1348:45-46. Poston, H.A. and H.G. K e t o l a . 1981. In f luence of n u t r i t i o n on development of c a t a ra c t s i n salmon and t rou t (Abs t rac t #172). XII I n t e r na t i ona l Congress of N u t r i t i o n , Aug. 16-21, San Diego, C a l i f . , USA. P ro s se r , C.L . and F.A. Brown, J r . 1950. Inorgan ic i o n s . In: Comparative Animal Phys io logy ( 2 n d E d . ) . W.B. Saunders Co. p. 57-80. Pumphrey, R . J . 1961. Concerning v i s i o n . In: The c e l l and the organism. Essays presented to S i r James Gray. J .A . Ramsay and V.B. Wigglesworth ( e d s . ) . Cambridge Un i v e r s i t y P ress , London, p. 193-208. Re inho ld , J . G . , F. I sma i l -Be i g i and B. F a r a d j i . 1975. F i b r e versus phytate as determinant of the a v a i l a b i l i t y of ca l c i um, z i n c and i r on of b r e ad s t u f f s . Nut r . Rep. In t . 12:75-85. R e i n t z , G. and F. H i t z e l . 1980. Formulat ion of p r a c t i c a l d i e t s f o r rainbow t r ou t based on des i red performance and body compos i t i on . Aquacu l ture 19:243-252. R i chardson, N.L. 1982. N u t r i t i o n a l c a t a r a c t s i n ha tchery- reared sa lmonids. B.Sc. t h e s i s , U n i v e r s i t y of B r i t i s h Columbia. 82 p. R i chardson, N.L., D.A. Higgs, R.M. Beames and J .R . McBr ide. 1985. In f luence of d i e t a r y ca l c i um, phosphorus, z i n c and sodium phytate l e v e l on ca ta rac t i n c i dence , growth and h i s topa tho logy i n j u v e n i l e chinook salmon (Oncorhynchus tshawytscha). J . Nut r . 115:553-567. Rober ts , A .H. and J . Yudk in. 1961. E f f e c t of phytate and o ther d i e t a r y f a c t o r s on i n t e s t i n a l phytase and bone c a l c i f i c a t i o n i n the r a t . B r i t . J . Nut r . 15:457-471. Rumsey, G.L. 1977. F i sh n u t r i t i o n recent advances. Proceedings of the I n t e rna t i ona l Symposium on Disease of Cu l tu red Sa lmonids. A p r i l 4-7, S e a t t l e , Washington, p. 16-40. SAS. 1982. SAS User ' s Guide: S t a t i s t i c s . SAS I n s t i t u t e I n c . , Cary, North C a r o l i n a , USA. 584 pp. Satoh, S . , H. Yamamoto, T. Takeuchi and T. Watanabe. 1983a. E f f e c t s on growth and minera l composi t ion of rainbow t r ou t of d e l e t i o n of t r a c e elements or magnesium from f i s h meal d i e t . B u l l . Jpn . Soc. S c i . F i s h . 49:425-429. Satoh, S . , T. Takeuch i , Y. Narabe and T. Watanabe. 1983b. E f f e c t s of d e l e t i o n of severa l t r a ce elements from f i s h meal d i e t s on growth and minera l composi t ion of rainbow t r ou t f i n g e r l i n g s . B u l l . Jpn . Soc. S c i . F i s h . 49:1909-1916. - 122 -Sche f f e , H. 1959. The ana l y s i s of va r i ance . John Wi ley and Sons, I n c . , New York. 477 pp. Shearer , K.D. 1984. Changes in elemental compos i t ion of ha tchery- reared rainbow t r o u t , Salmo g a i r d n e r i , assoc ia ted w i th growth and rep roduc t i on . Can. J l F i s h . Aquat. S c i . 41:1592-1600. Shears, M.A. and G.L. F l e t c h e r . 1984. The r e l a t i o n s h i p between me ta l l o t h i one i n and i n t e s t i n a l z i n c absorpt ion i n the w in te r f l o unde r . Can. J . Z oo l . 62:2211-2220. S i y , R.D. and F.D.F. T a l b o t . 1982. P repara t i on of low-phytate rapeseed p ro t e i n by u l t r a f i l t r a t i o n : 1. The aqueous e x t r a c t i o n of phytate from deo i l ed rapeseed meals. J . Am. O i l Chem. Soc. 59:191-194. Smith, K.T. and J .T . Rot ruck . 1981. Chemical and b i o l o g i c a l s tud i e s of phytate-minera l i n t e r a c t i o n s . J . Am. O i l Chem. Soc. 573A (Abs t rac t 35 ) . S p i n e l l i , J . , C. Mahnken and M. S t e i nbe rg . 1979. A l t e r n a t e sources of p ro te i n s f o r f i s h meal i n salmonid d i e t s . Proceedings of World Symposium on F i n f i s h N u t r i t i o n and F i sh feed Technology, V o l . I I . J . Halver and K. Tiews (eds.) June 20-23, Hamburg, B e r l i n , p. 131-142. S p i n e l l i , J . , C.R. Houle and J .C . Weke l l . 1983. The e f f e c t of phytates on the growth of rainbow t r ou t (Salmo g a i r d n e r i ) f ed p u r i f i e d d i e t s con ta in i ng va ry ing q u a n t i t i e s of ca l c ium and magnesium. Aquacu l ture 30:71-83. S t a u f f e r , G.D. 1973. A growth model f o r salmonids reared in hatchery environments. Ph.D. t h e s i s , U n i v e r s i t y of Washington, S e a t t l e . Tho f t , R.A. and J .H . K i n o s h i t a . 1965. The e f f e c t of ca l c ium on r a t lens p e rmeab i l i t y . I nves t . Ophthalmol. 4:122. Thompson, S.A. and C.W. Weber. 1979. In f luence of pH on the b ind ing of copper, z i n c and i r o n in s i x f i b r e sources . J . Food S c i . 44:752-754. Thompson, S.A. and C.W. Weber. 1981. E f f e c t of d i e t a r y f i b r e sources on t i s s u e minera l l e v e l s i n c h i c k s . P ou l t r y S c i . 60:840-845. Tucker, H.F. and W.D. Salmon. 1955. Pa rake ra to s i s or z i n c d e f i c i e n c y d i sease i n the p i g . P roc . Soc. Exp. B i o . Med. 88:613-636. Underwood, E . J . 1971. Trace Elements i n Human and Animal N u t r i t i o n ( 3 r d Ed.) Academic P ress , New York. van der Aar , P . J . , G.C. Fahey, J r . , S.C. R i c k e , S .E . A l l e n and L.L. Berger . 1983. E f f e c t s of d i e t a r y f i b e r s on minera l s t a tus of c h i c k s . J . Nut r . 113:653-661. - 123 -van Du i j n , J r . , C. 1973. Diseases of F i shes ( 3 r d E d . ) . Char les C. Thomas, Pub., I l l i n o i s , USA. 372 pp. van Overbeeke, A .P . and J .R. McBr ide. 1971. H i s t o l o g i c a l e f f e c t s of 11-ke to tes tos te rone , 17a-methy l tes tos terone, e s t r a d i o l cyp ionate and C o r t i s o l on the i n t e r r e n a l t i s s u e , t h y r o i d gland and p i t u i t a r y g land of gonadectomized sockeye salmon (Oncorhynchus ne rka ) . J . F i s h . Res. Board Can. 28:477-484. von Sal lmann, L., J . E . Ha l ve r , E. C o l l i n s and P. Gr imes. 1966. Thioacetamide- induced ca t a ra c t w i th i n va s i v e p r o l i f e r a t i o n of the lens ep i the l i um i n rainbow t r o u t . Cancer Res. 26:1819-1825. Watanabe, T. T. Takeuchi and C. Ogino. 1980. E f f e c t on rainbow t r o u t and chum salmon of d e l e t i o n of t r a ce elements from f i s h meal d i e t . B u l l . Jpn. Soc. S c i . F i s h . 46:1521-1521. W e i t z e l , G . , A . -M. F r e t z d o r f f and A. Eberhagen. 1953. Zink i n den Augen von Sauget ie ren . Z. P h y s i o l . Chem. Hoppe-Sey ler 's 292:221. Weke l l , J . C . , K.D. Shearer and C.R. Houle. 1983. High z i n c supplementat ion of rainbow t r ou t d i e t s . Prog. F i s h - C u l t . 45:144-147. Welch, R.M. and D.R. van Campen. 1975. Iron a v a i l a b i l i t y to r a t s from soybeans. J . Nut r . 105:253-256. Whanger, P.O. and P.H. Weswig. 1975. E f f e c t s of Se, Cr and an t i o x i d an t s on growth, eye c a t a r a c t s , plasma cho l e s t e r o l and blood g lucose i n Se d e f i c i e n t , V i tamin E supplemented r a t s . Nut r . Rep. I n t . 12:345-358. Wh i t i ng , F. and L.M. Bezeau. 1958. Ca lc ium, phosphorus and z i n c ba lance in p igs as i n f l uenced by the weight of p i g and the l e v e l of c a l c i um , z i n c and v i tamin D i n the r a t i o n . Can. J . Anim. Sc. 38:109. Woodard, J .C . and S.S.W. Jee. 1984. E f f e c t s of d i e t a r y c a l c i um, phosphorus and magnesium on i n t r a -neph ron i c c a l c i n o s i s i n r a t s . J . Nu t r . 114:2331-2338. Yano, H., H. Matsui and R. Kawashima. 1979. E f f e c t of d i e t a r y ca l c ium l e v e l s on concent ra t i on and s o l u b i l i t y of macro m ine ra l s i n the d i g e s t i v e t r a c t of sheep. J . Anim. S c i . 48:954-960. - 124 -APPENDIX A. The f o l l o w i n g t a b l e s con ta in examples of the s t a t i s t i c a l ana lyses performed i n Experiment I . 1. A one-way ANOVA t a b l e of natura l log weight at day 105. Source DF P D ie t 8 0.00000 Nrep(Diet) 9 0.00883 Res idua l 1048 Tota l 1065 2. A two-way ANOVA of mean wet weights f o r days 0 to 105 showing a l l main e f f e c t s and i n t e r a c t i o n s . Source DF P Ca 1 . 0.00005 Zn 1 0.21846 Phy t i c 1 0.00000 Ca*Zn 1 0.70450 Ca*Phyt ic 1 0.00072 Zn*Phyt ic 1 0.30216 Ca*Zn*Phytic 1 0.15041 Nrep(Ca*Zn*Phytic) 9 0.64290 Time 1 0.00000 Time*Ca 1 0.00000 Time*Zn 1 0.21578 Time*Phyt ic 1 0.00000 Time*Ca*Zn 1 0.78912 Time*Ca*Phytic 1 0.00003 Time*Zn*Phytic 1 0.24688 Time*Ca*Zn*Phytic 1 0.23852 Res idua l 81 Tota l 107 - 125 -3. A one-way ana l y s i s of covar iance t a b l e f o r the na tu ra l log of wet weights f o r days 0-105. Source DF P D iet 8 0.00000 Nrep(Diet) 9 0.00028 Time 1 0.00000 Time*Diet 8 0.00000 Time*Nrep(Diet) 9 0.00009 Res idua l 6430 Tota l 6465 4. A one-way ANOVA f o r food convers ion (FC) , p r o t e i n e f f i c i e n c y r a t i o (PER) and food in take (FI) f o r days 0-21. Source DF P (FC) P (PER) P (FI) D ie t 8 0.01475 0.01591 0.01426 Nrep(Diet) 9 1.00000 1.00000 1.00000 Tota l 17 5. 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) D iet 8 0.00000 0.00000 0.00003 Nrep(Diet) 9 0.99770 0.99821 0.52809 Per iod 3 0.01133 0.01278 0.00000 Per iod*Diet 24 0.56179 0.58285 0.00152 Res idua l 27 Tota l 71 - 126 -A two-way ANOVA t a b l e f o r FC, PER and FI f o r days 22-105 showing the 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) P (PER) P (FI) Ca 1 0.26421 0.62848 0.00002 Zn 1 0.00032 0.00039 0.04682 Phy t i c 1 0.00000 0.00000 0.00010 Ca*Zn 1 0.00335 0.01068 0.00189 Ca*Phyt ic 1 0.22736 0.06155 0.00017 Zn*Phyt ic 1 0.00001 0.00001 0.01122 Ca*Zn*Phytic 1 0.00024 0.00066 0.00101 Nrep(Ca*Zn*Phyt ic) 9 0.99893 0.99917 0.89534 Time 1 0.00002 0.00002 0.00000 Time*Ca 1 0.91231 0.91566 0.02221 Time*Zn 1 0.84803 0.88971 0.55261 T ime*Phyt ic 1 0.00011 0.00010 0.01167 Time*Ca*Zn 1 0.17260 0.18452 0.08653 Time*Ca*Phytic 1 0.42076 0.40664 0.81290 Time*Zn*Phytic 1 0.03622 0.03526 0.16154 Time*Ca*Zn*Phytic 1 0.10244 0.08739 0.30325 Time*Nrep(Ca*Zn*Phytic) 9 0.99863 0.99889 0.68631 Res idua l 36 Tota l 71 A one-way ANOVA f o r plasma z i n c l e v e l s of chinook salmon. Source DF P D iet 8 0.00000 Nrep(Diet) 27 1.00000 Tota l 35 - 127 -8. 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 and i n t e r a c t i o n s . Source DF P Ca 1 0.37947 Zn 1 0.00000 Phyt i c 1 0.00000 Ca*Zn 1 0.10971 Ca*Phyt ic 1 0.00596 Zn*Phyt ic 1 0.00005 Ca*Zn*Phytic 1 0.61192 Nrep(Ca*Zn*Phytic) 27 1.00000 Res idual -0 Tota l 35 B. The f o l l o w i n g t ab l e i s an example of the s t a t i s t i c s used i n Experiment I I . 9. A one-way ANOVA t a b l e of wet weights at day 0 in Experiment I I . Source DF P Treat 7 0.32230 Nrep(Treat) 8 0.09191 Res idual 944 Tota l 959 C. The f o l l o w i n g ANOVA t ab l e s are examples of the analyses performed in Experiment I I I . 10. A one-way ANOVA t a b l e of the na tura l log wet weights at day 84. Source DF P D ie t 14 0.2274 Res idua l 1781 Tota l 1795 - 128 -11 . 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) P (FI) D ie t 14 0.0001 0.0001 0.0001 0.0145 Res idual 15 Tota l 29 12. A one-way ANOVA f o r p ro te i n content of chinook salmon at day 84. Source DF P Diet 14 0.0001 Residual 45 Tota l 59 13. A one-way ANOVA of the blood z i n c l e v e l s i n chinook salmon a f t e r 84 days. Source DF P Diet 14 0.0001 Residual 30 Tota l 44 14. A one-way ANOVA of k idney z i n c content of chinook salmon a f t e r 84 days. Source DF P Diet 14 0.0001 Residual 28 Tota l 42 15. A one-way ANOVA of l i v e r z i n c l e v e l s in chinook salmon a f t e r 84 days. Source DF P Diet Res idual Tota l 14 0.0001 44 58 

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