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UBC Theses and Dissertations

Textural and chemical changes in the muscle of chum salmon (oncorhynchus keta) during spawning migration Reid, Rebecca Anne 1991

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TEXTURAL AND CHEMICAL CHANGES THE MUSCLE OF CHUM SALMON (ONCORHYNCHUS KETA) DURING SPAWNING MIGRATION By REBECCA ANNE REID B.S.Ag., The U n i v e r s i t y o f S a s k a t c h e w a n THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES ( D e p a r t m e n t o f Fo o d S c i e n c e ) We a c c e p t t h i s t h e s i s a s c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA May 1991 (c) R e b e c c a Anne R e i d , 1991 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of ^hrrcyi S'CAS^ r<?^ The University of British Columbia Vancouver, Canada DE-6 (2788) ABSTRACT The s pawning m i g r a t i o n o f chum salmon r e s u l t s i n a d e t e r i o r a t i o n of the e a t i n g q u a l i t y of the f l e s h . There i s a l o s s i n t h e c o l o u r o f t h e m u s c l e f r o m r e d - p i n k t o w h i t e - g r e y , a d i s t i n c t o f f - f l a v o u r d e v e l o p s and t h e f i r m t e x t u r e becomes s o f t and watery. The reduced appeal of the canned and f r e s h product r e s u l t s i n an economic l o s s of t h i s important B.C. commodity. Chum salmon were sampled a t f o u r s t a g e s d u r i n g t h e i r s p a w n i n g m i g r a t i o n i n o r d e r t o d e s c r i b e t h e p h y s i c a l , chemical and s t r u c t u r a l changes which o c c u r r e d as the f i s h matured. The f i s h were c a t e g o r i z e d i n t o f o u r grades based on t h e i r e x t e r n a l c h a r a c t e r i s t i c s . G r a d e o n e , s i l v e r - b r i g h t s , were t h e l e a s t mature f i s h , g r a d e two, i n t e r m e d i a t e s , and grade t h r e e , d a r k s , were p r o g r e s s i v e l y more mature. Grade f o u r , spawning, were the most mature. Proximate a n a l y s i s of the raw muscle r e v e a l e d a s i g n i f i c a n t decrease i n p r o t e i n and l i p i d and i n c r e a s e i n moisture. The l e v e l of a s t a x a n t h i n and t h e Hunter v a l u e s a/L, a and a/b decreased s i g n i f i c a n t l y w i t h i n c r e a s i n g grade. T h e t e x t u r e o f t h e c a n n e d f i s h was m e a s u r e d i n s t r u m e n t a l l y w i t h an I n s t r o n U n i v e r s a l T e s t i n g Machine u s i n g the t e x t u r e p r o f i l e a n a l y s i s (TPA) method. A panel of s e m i - t r a i n e d judges a l s o e v a l u a t e d the canned f l e s h based on i t s f i r m n e s s , f i b r o u s n e s s , dryness, and chewiness. The - i i s e n s o r y p a n e l d e t e r m i n e d t h a t t h e s i l v e r - b r i g h t f i s h were s i g n i f i c a n t l y more f i r m , f i b r o u s , d r y , a n d chewy t h a n t h e s p a w n i n g f i s h . Of t h e t e n i n s t r u m e n t a l TPA p a r a m e t e r s , c o h e s i v e n e s s a n d s l o p e 2 were a b l e t o d i s t i n g u i s h b e t w e e n g r a d e o n e a n d f o u r . P o o r c o r r e l a t i o n b e t w e e n t h e i n s t r u m e n t a l and s e n s o r y measure o f t e x t u r e s u g g e s t e d t h a t t h e s o f t e n i n g c h a r a c t e r i z e d b y t h e s e n s o r y p a n e l was n o t b e i n g m e a s u r e d by t h e I n s t r o n . T h i n a n d u l t r a t h i n s e c t i o n s o f t h e m u s c l e w e r e p h o t o g r a p h e d a t t h e l i g h t a n d e l e c t r o n m i c r o s c o p i c l e v e l . The l i g h t m i c r o g r a p h s c l e a r l y showed an a p p a r e n t l o s s i n t h e m y o f i b r i l l a r d i a m e t e r o r number w i t h i n m u s c l e f i b r e s a s t h e f i s h became more m a t u r e . F o c a l d e g e n e r a t i o n o f m y o f i b r i l s was e v i d e n t i n t h e e l e c t r o n m i c r o g r a p h s . C h a n g e s w e r e d e s c r i b e d b y f o u r c r i t e r i a , s m e a r i n g , t h e l o s s o f a d i s t i n c t a p p e a r a n c e o f t h e a c t i n and m y o s i n , s p l i t t i n g , t h e t e a r i n g o f m y o f i b r i l b u n d l e s , t h i n n i n g , a l o s s i n m y o s i n o r a c t i n f i l a m e n t s , and an i n c r e a s e i n i n t e r m y o f i b r i l s p a c e . P r o t e i n a s e a c t i v i t y o f t h e d o r s a l m u s c l e was a s s a y e d a t pH 3.5, 6.2 a n d 7.0. T h e r e was a s i g n i f i c a n t i n c r e a s e i n a c t i v i t y a t pH 3.5 b u t no c h a n g e a t pH 6.2 o r 7.0. A c i d p h o s p h a t a s e , a l y s o s o m a l m a r k e r , a l s o i n c r e a s e d s i g n i f i c a n t l y w i t h g r a d e . T h e s e f i n d i n g s s u g g e s t e d t h a t l y s o s o m a l enzymes l a r g e l y c o n t r i b u t e d t o t h e d e g r a d a t i o n i n t h e m u s c l e . i i i TABLE OF CONTENTS ABSTRACT . i i L I S T OF TABLES v i i L I S T OF FIGURES i x ACKNOWLEDGMENTS , x i i LITERATURE REVIEW 1 1.1. I n t r o d u c t i o n 1 1.2. B i o c h e m i c a l c h a n g e s i n t h e m u s c l e o f chum s a l m o n 2 1.2.1. C o l o u r 4 1.3. T e x t u r e 8 1.3.1. I n s t r u m e n t a l a n a l y s i s 8 1.3.2. S e n s o r y a n a l y s i s 18 1.4. U l t r a s t r u c t u r e o f t h e m u s c l e o f chum s a l m o n .. 23 1.4.1. S t r u c t u r e o f m u s c l e 23 1.4.2. M u s c l e d e g e n e r a t i o n * 29, 1.4.3. S t a r v a t i o n 33 1.5. P r o t e i n a s e a c t i v i t y 36 1.5.1. L y s o s o m a l s y s t e m 36 1.5.2. P r o t e i n t u r n o v e r , 37 1.5.3. S t a r v a t i o n 39 CHEMICAL ANALYSIS 42 2.1. I n t r o d u c t i o n 42 - i v -2.2. M a t e r i a l s and Methods 43 2.2.1. Sampling 43 2.2.2. Grading 43 2.2.3. Proximate a n a l y s i s 44 2.2.3.1. T o t a l l i p i d a n a l y s i s ........ 44 2.2.3.2. Mo i s t u r e a n a l y s i s 46 2.2.3.3. P r o t e i n d e t e r m i n a t i o n 46 2.2.4. Colour 47 2.2.4.1. A s t a x a n t h i n measurement 47 2.2.4.2. Hunter Lab measurements 47 2.3. R e s u l t s and D i s c u s s i o n 48 2.3.1. A s t a x a n t h i n content 48 2.3.2. Hunter Lab v a l u e s 50 2.3.3. Proximate a n a l y s i s 54 3. TEXTURE ANALYSIS 57 3.1. I n t r o d u c t i o n 57 3.2. M a t e r i a l s and Methods 60 3.2.1. Instrumental a n a l y s i s o f t e x t u r e .... 61 3.2.2. Sensory a n a l y s i s o f t e x t u r e 62 3.3. R e s u l t s and D i s c u s s i o n 65 3.3.1. Instrumental 65 3.3.2. Sensory a n a l y s i s o f t e x t u r e 73 -v-3.3.3. S e n s o r y and i n s t r u m e n t a l c o r r e l a t i o n 78 4. PROTEINASE A C T I V I T Y 84 4.1. I n t r o d u c t i o n 84 4.2. M a t e r i a l s and Methods 87 4.2.1. Measurement o f p r o t e i n a s e a c t i v i t y .. 87 4.2.2. Measurement o f a c i d p h o s p h a t a s e a c t i v i t y 89 4.3. R e s u l t s and D i s c u s s i o n 90 4.3.1. P r o t e i n a s e a c t i v i t y 90 4.3.2. A c i d p h o s p h a t a s e a c t i v i t y 93 5. STRUCTURAL CHANGES IN MUSCLE 99 5.1. I n t r o d u c t i o n 99 5.2. M a t e r i a l s a n d Methods 100 5.2.1. L i g h t m i c r o s c o p y 100 5.2.2. E l e c t r o n m i c r o s c o p y 100 5.3. R e s u l t s and D i s c u s s i o n 101 5.3.1. L i g h t m i c r o s c o p y 101 5.3.2. E l e c t r o n m i c r o s c o p y , 104 6. CONCLUSIONS 120 7 . REFERENCES . 125 8. APPENDIXES 134 v i * L I S T OF TABLES T a b l e l . l . C o n t e n t a n d p e r c e n t a g e c o m p o s i t i o n o f .... 6 t h e m a j o r c a r o t e n o i d s i n t h e c h u m s a l m o n m u s c l e d u r i n g s p a w n i n g m i g r a t i o n (Ando a n d H a t a n o , 1 9 8 7 ) . T a b l e 2.1 Mean v a l u e s f o r a s t a x a n t h i n c o n t e n t i n .... 48 t h e d o r s a l m u s c l e o f chum s a l m o n . T a b l e 2.2 H u n t e r L a b m e a n v a l u e s f o r t h e .... 51 d o r s a l m u s c l e o f chum s a l m o n . T a b l e 2.3 R e s u l t s f r o m t h e c o r r e l a t i o n a n a l y s i s .... 53 between g r a d e , H u n t e r L a b v a l u e s and a s t a x a n t h i n c o n t e n t . T a b l e 2.4 Mean v a l u e s f o r p e r c e n t a g e p r o t e i n , . — 55 m o i s t u r e a n d l i p i d c o n t e n t o f d o r s a l m u s c l e o f chum s a l m o n on a wet b a s i s . T a b l e 3.1 Mean i n s t r u m e n t a l T P A p a r a m e t e r s o f .... 67 t h e f o u r g r a d e s o f chum s a l m o n . T a b l e 3.2 P r o b a b i l i t y v a l u e s p r o d u c e d f r o m .... 69 o n e - w a y A N O V A s o n t h e s g u a r e r o o t t r a n s f o r m e d I n s t r o n TPA d a t a . T a b l e 3.3 Mean u n t r a n s f o r m e d r e s u l t s f r o m t h e .... 74 s e n s o r y p a n e l ' s e v a l u a t i o n o f t h e t e x t u r a l d i f f e r e n c e s b e t w e e n t h e f o u r g r a d e s o f chum s a l m o n . T a b l e 3.4 R e s u l t s f r o m t h e a n a l y s i s o f v a r i a n c e .... 77 o f t h e s e n s o r y a n a l y s i s t o d e t e r m i n e d i f f e r e n c e s b e t w e e n g r a d e s a n d s e q u e n t i a l r e j e c t i v e B o n f e r r o n i p r o c e d u r e f o r t h e m u l t i p l e c o m p a r i s o n o f t h e d a t a . T a b l e 3.5 C o r r e l a t i o n v a l u e s ( r ) e x t r a c t e d f r o m a P e a r s o r i c o r r e l a t i o n m a t r i x o f t h e c o m p a r i s o n b e t w e e n t h e i n s t r u m e n t a l p a r a m e t e r s a n d t h e s e n s o r y a t t r i b u t e s . 79 T a b l e 4.1 P r o t e i n a s e a c t i v i t y a t pH 3.5, 6.2 and 7.0 d u r i n g t h e d i f f e r e n t s t a g e s o f s p a w n i n g m i g r a t i o n . 91 - v i i -Table 4.2 Tabl e 5.1 Table 8.1. Tabl e 8.2. Tabl e 8.3. Table 8.4. A c i d p h o s p h a t a s e a c t i v i t y (mM .... 95 phosp h a t e ) i n d o r s a l m u s c l e o f chum salmon d u r i n g spawning m i g r a t i o n . P e r c e n t a g e o f s i l v e r - b r i g h t a n d .... 119 spawning f i s h a s s i g n e d t o each s c o r e u s e d t o j u d g e m i c r o g r a p h s o f chum d o r s a l muscle. Absorbance a t U.V. 280 o f s o l u b l e N .... 134 v e r s u s g r a d e a s a m e a s u r e o f t h e p r o t e i n a s e a c t i v i t y o f chum d o r s a l muscle a t pH 3.5. Absorbance a t U.V. 280 o f s o l u b l e N .... 135 v e r s u s g r a d e a s a m e a s u r e o f t h e p r o t e i n a s e a c t i v i t y o f chum d o r s a l muscle a t pH 6.2. A b s o r b a n c e a t U.V. 280 o f s o l u b l e N .... 136 v e r s u s g r a d e a s a m e a s u r e o f t h e p r o t e i n a s e a c t i v i t y o f chum d o r s a l muscle a t pH 7.0. C o n c e n t r a t i o n o f phosphate (mM) v e r s u s .... 137 g r a d e a s a m e a s u r e o f t h e a c i d p h o s p h a t a s e a c t i v i t y o f chum m u s c l e d u r i n g spawning m i g r a t i o n . - v i i i -LIST OF FIGURES Figure 1.1 Figure 1.2 Figure 1.3 Figure 1.4 Figure 2.1 Figure 3.1 Figure 3.2 Figure 3.3 Figure 4.1 Figure 4.2 Figure 5.1 Generalized texture p r o f i l e a n a l y s i s .... 15 (TPA) curve (Bourne, 1982). Texture p r o f i l e score sheet (Bourne, .... 22 1982) A p p e a r a n c e o f m u s c u l a t u r e o f a .... 25 t y p i c a l f i s h . White spaces represent the p a r a l l e l muscle c e l l s , " d i v i d i n g " l i n e s represent the myocommata which separate the myomeres (Love, 1980). Major s u b c e l l u l a r components o f .... 27 myotomal muscle (Peter, 1973). I l l u s t r a t i o n of the f o u r grades of .... 45 chum salmon. Example o f t y p i c a l t e x t u r e s c o r e .... 64 sheet used to analyze texture of the f o u r g r a d e s o f t h e c a n n e d chum samples. Example of a t y p i c a l Instron texture .... 66 p r o f i l e a n a l y s i s curve f o r a chum sample. Standardized sensory scores f o r the .... 76 four grades of chum salmon f o r the a t t r i b u t e s f i r m n e s s , c h e w i n e s s , dryness arid fibrousness. Flow diagram of the method used to .... 88 measure the p r o t e i n a s e a c t i v i t y i n the dorsal muscle of four grades of chum muscle. Standard curve of the absorbance at .... 94 4p5 nm versus the c o n c e n t r a t i o n of E-nitrophenol; L i g h t micrograph of a cross s e c t i o n .... 102 of s i l v e r - b r i g h t chum salmon s t a i n e d w i t h p h o s p h o t u n g s t i c a c i d haematoxylin. (Magnification 1500). - i x -F i g u r e 5.2 F i g u r e 5.3 F i g u r e 5.4 F i g u r e 5.5 F i g u r e 5.6 F i g u r e 5.7 F i g u r e 5.8 F i g u r e 5.9 F i g u r e 5.10 L i g h t m i c r o g r a p h o f a c r o s s s e c t i o n .... 103 of s i l v e r - b r i g h t chum salmon s t a i n e d w i t h p h o s p h o t u n g s t i c a c i d haematoxylin. ( M a g n i f i c a t i o n 600). L i g h t m i c r o g r a p h o f a l o n g i t u d i n a l .... 105 s e c t i o n o f a s i l v e r - b r i g h t chum salmon s t a i n e d w i t h p h o s p h o t u n g s t i c a c i d h a e m a t o x y l i n . ( M a g n i f i c a t i o n 1500). L i g h t m i c r o g r a p h o f a c r o s s s e c t i o n .... 106 o f a s p a w n i n g chum s a l m o n s t a i n e d w i t h p h o s p h o t u n g s t i c a c i d haematoxylin. ( M a g n i f i c a t i o n 1500). L i g h t m i c r o g r a p h o f a c r o s s s e c t i o n .... 107 o f a s p a w n i n g chum s a l m o n s t a i n e d w i t h p h o s p h o t u n g s t i c a c i d haematoxylin. ( M a g n i f i c a t i o n 600). L i g h t m i c r o g r a p h o f a l o n g i t u d i n a l .... 108 s e c t i o n o f a s p a w n i n g chum s a l m o n s t a i n e d w i t h p h o s p h o t u n g s t i c a c i d haematoxylin. ( M a g n i f i c a t i o n 1500). E l e c t r o n m i c r o g r a p h o f t h e d o r s a l .... 109 m u s c l e o f a s i l v e r - b r i g h t chum salmon. ( M a g n i f i c a t i o n 27000). E l e c t r o n m i c r o g r a p h o f t h e d o r s a l .... 110 m u s c l e o f a s i l v e r - b r i g h t chum salmon. ( M a g n i f i c a t i o n 27000). E l e c t r o n m i c r o g r a p h o f t h e d o r s a l .... I l l m u s c l e o f a s i l v e r - b r i g h t chum salmon. ( M a g n i f i c a t i o n 27000). E l e c t r o n m i c r o g r a p h o f t h e d o r s a l .... 112 m u s c l e o f a s i l v e r - b r i g h t chum salmon. ( M a g n i f i c a t i o n 27000). -x-F i g u r e 5.11 E l e c t r o n m i c r o g r a p h o f t h e d o r s a l m u s c l e o f s p a w n i n g chum s a l m o n . Arrow i n d i c a t e s a r e a o f " s m e a r i n g " . ( M a g n i f i c a t i o n 26000). F i g u r e 5.12 E l e c t r o n m i c r o g r a p h o f t h e d o r s a l m u s c l e o f s p a w n i n g chum s a l m o n . Arrows i n d i c a t e a r e a s o f " s p l i t t i n g " o f t h e m y o f i b r i l s . ( M a g n i f i c a t i o n 26000). F i g u r e 5.13 E l e c t r o n m i c r o g r a p h o f t h e d o r s a l m u s c l e o f a s p a w n i n g chum s a l m o n . N o t i c e a r e a s w i t h " i n t e r m y o f i b r i l l a r " spaces. ( M a g n i f i c a t i o n 26000). F i g u r e 5.14 E l e c t r o n m i c r o g r a p h o f t h e d o r s a l m u s c l e o f s p a w n i n g chum s a l m o n . A r r o w s p o i n t t o a r e a s w h i c h h a v e m y o f i b r i l " t h i n n i n g " . ( M a g n i f i c a t i o n 26000). - x i -ACKNOWLEDGMENTS No one c o u l d ever c l a i m t h a t a t h e s i s was e n t i r e l y an i n d i v i d u a l e f f o r t . Of the many people who's encouragement and a d v i c e helped me complete t h i s p r o j e c t , I would l i k e t o extend my most s i n c e r e thanks towards my s u p e r v i s o r , Dr. T. D u r a n c e , f o r h i s p a t i e n c e , k n o w l e d g e a n d c o n s t a n t encouragement. I would a l s o l i k e t o thank Dr. D. Walker f o r s h a r i n g h i s i n v a l u a b l e knowledge o f e l e c t r o n microscopy and f o r the Saturdays he spent h e l p i n g me wit h my work. I thank Dr. D. K i t t s and Dr. W. P o w r i e f o r f i t t i n g c o m m i t t e e meetings i n t o t h e i r busy s c h e d u l e s and f o r t h e i r h e l p f u l i d e a s and comments. I would a l s o l i k e t o thank my f a t h e r f o r h i s h e l p i n the l a b and f o r h i s c o n f i d e n c e i n my work. I t i s f i t t i n g t o end t h i s s e c t i o n w i t h a quote from the a u t h o r D o u g l a s Adams, "So Long, and Thanks f o r a l l t h e F i s h " . - x i i -1. LITERATURE REVIEW 1.1. I n t r o d u c t i o n Chum s a l m o n f O n c o r h y n c h u s k e t a ) i s an i m p o r t a n t commer-c i a l c o m m o d i t y i n B.C., w h e r e i t r a n k s a s t h e #3 s a l m o n f i s h e r y . I n 1987, 11,000 t o n n e s were c a u g h t f o r a t o t a l l a n d e d v a l u e o f 27 m i l l i o n d o l l a r s . Chum a r e h a t c h e d i n t h e g r a v e l b e d s o f f a s t r u n n i n g s t r e a m s a f t e r i n c u b a t i n g o v e r t h e w i n t e r . W i t h i n a few d a y s o f e m e r g i n g f r o m t h e s p a w n i n g b e d s , t h e f r y d r i f t downstream t o t h e s e a , where t h e y may s p e n d t h r e e t o f i v e y e a r s f e e d i n g on p l a n k t o n and s m a l l c r u s t a c e a n . Spawning m i g r a t i o n b e g i n s f o r 90 t o 99% o f t h e s t o c k i n t h e i r t h i r d o r f o u r t h y e a r and may b e g i n a s e a r l y a s J u l y , o r e x t e n d a s l a t e a s J a n u a r y , d e p e n d i n g on t h e s t o c k (Beacham and S t a r r , 1 9 8 2 ) . Chum a r e o f t e n i n an a d v a n c e d s t a g e o f s e x u a l m a t u r i t y a s t h e y e n t e r t h e i r n a t a l s t r e a m s , a n d o f t e n spawn n o t f a r f r o m t h e s e a (Neave, 1 9 6 6 ) . E v e n b e f o r e t h e s a l m o n e n t e r f r e s h w a t e r , t h e y s t o p f e e d i n g , and must s u r v i v e on s t o r e d l i p i d and p r o -t e i n i n t h e i r w h i t e m u s c l e u n t i l t h e y have spawned. A r e s u l t o f t h e s e p h y s i o l o g i c a l c h a n g e s i n t h e s a l m o n i s a l o s s i n t h e e a t i n g q u a l i t y o f t h e f i s h . The m u s c l e l o s e s i t s r e d - p i n k c o l o u r , a n d u l t i m a t e l y b e c o m e s a w h i t e - g r e y c o l o u r . The t e x t u r e c h a n g e s f r o m f i r m and d e s i r -a b l e t o s o f t a n d w a t e r y . T h e s e c h a n g e s w e r e n o t e d b y B i l i n s k i e t a l . ( 1 9 8 4 ) who s t u d i e d t h e e f f e c t o f s e x u a l - 1 -m a t u r a t i o n o n t h e q u a l i t y o f t h e f l e s h o f c o h o s a l m o n . T h e s e w o r k e r s n o t i c e d c h a n g e s i n t h e c a n n e d p r o d u c t s u c h a s a n i n c r e a s e i n f r e e l i q u i d , a d e c r e a s e i n f r e e o i l , d i s c o l o u r a t i o n and s o f t e n i n g o f t h e f l e s h a n d a d e t e r i o r a -t i o n o f t h e f l a v o u r and o d o u r . The q u a l i t y c h a n g e s w h i c h o c c u r i n t h e chum d u r i n g mat-u r a t i o n c o u l d be a v o i d e d b y h a r v e s t i n g o n l y t h e o c e a n - r u n f i s h . However, b e c a u s e f i s h o f d i f f e r e n t s t o c k s c a n n o t be a d e q u a t e l y d i s t i n g u i s h e d u n t i l t h e y b e g i n t h e i r s p a w n i n g r u n , m i x e d s t o c k f i s h e r i e s may r e s u l t i n t h e o v e r h a r v e s t i n g o f weaker s t o c k s . T e r m i n a l f i s h e r i e s management t e c h n i q u e s r e d u c e t h i s p r o b l e m a n d a l l o w t h e f i s h i n g f l e e t t o t a r g e t s p e c i f i c s t o c k s (Holmes, 1 9 8 2 ) . 1.2. B i o c h e m i c a l c h a n g e s i n t h e m u s c l e o f chum s a l m o n Ando and H a t a n o (1986a) m e a s u r e d c h a n g e s i n t h e c o l o u r , p r o t e i n , f a t and m o i s t u r e c o n t e n t o f t h e m u s c l e o f chum i n o r d e r t o a s s e s s t h e p h y s i o l o g i c a l s t a t e o f t h e s a l m o n t h r o u g h o u t t h e s p a w n i n g p e r i o d . A t t h e f i r s t s t a g e t e s t e d ( f e e d i n g m i g r a t i o n ) , t h e m u s c l e was h i g h i n t o t a l a n d s a r c o p l a s m i c p r o t e i n , a n d l o w i n n i n h y d r i n p o s i t i v e , n o n - p r o t e i n , n i t r o g e n o u s compounds. As t h e s p a w n i n g p r o -g r e s s e d , t h i s r e l a t i o n s h i p r e v e r s e d s o t h a t t h e t o t a l a n d s a r c o p l a s m i c p r o t e i n d e c r e a s e d , and t h e n i n h y d r i n p o s i t i v e s u b s t a n c e s i n c r e a s e d . The l i p i d c o n t e n t was m a r k e d l y r e -d u c e d d u r i n g t h e e a r l y s t a g e s o f m i g r a t i o n , i n d i c a t i n g i t was u s e d as a p r i m a r y e n e r g y s o u r c e f o r g o n a d d e v e l o p m e n t . -2-I d l e r and B i t n e r s ( I 960) m e a s u r e d a 200% i n c r e a s e i n t h e f a t c o n t e n t o f t h e o v a r i e s , an 11% i n c r e a s e i n o v a r y m o i s t u r e , and no c hange i n o v a r y p r o t e i n , by t h e end o f t h e m i g r a t o r y r u n . A n a l y s i s o f t h e m i l t r e v e a l e d a 33% i n c r e a s e i n p r o -t e i n , a s l o w , s t e a d y i n c r e a s e i n f a t , and l i t t l e c hange i n m o i s t u r e c o n t e n t . C h a n g a n d I d l e r ( 1 9 6 0 ) m o n i t o r e d t h e l e v e l o f l i v e r g l y c o g e n as an i n d i c a t o r o f t h e p h y s i o l o g i c a l c o n d i t i o n and n u t r i t i o n a l s t a t e o f t h e s p a w n i n g s o c k e y e s a l m o n . G l y c o g e n l e v e l s i n t h e f e m a l e were s i g n i f i c a n t l y l o w e r t h a n i n t h e m ale, however f o r b o t h s e x e s t h e l e v e l d e c r e a s e d d u r i n g t h e e a r l y m i g r a t o r y p h a s e , a n d i n c r e a s e d i n t h e l a t e r s t a g e . Mommsen e t a l . (1980) f o u n d t h a t t h e c a r b o h y d r a t e s t o r e s i n t h e m u s c l e were m a i n t a i n e d t h r o u g h o u t m i g r a t i o n , a n d f u n c -t i o n e d a s a s o u r c e o f s h o r t t e r m e m e r g e n c y e n e r g y i n t h e m u s c l e . Duncan and T a r r (1958) s t u d i e d t h r e e p r o t e i n f r a c t i o n s , a c t o m y o s i n , m y o a l b u m i n a n d m y o g e n s , a n d c o l l a g e n , a n d n o n - p r o t e i n n i t r o g e n o u s m a t e r i a l i n s o c k e y e m u s c l e . A d e -c r e a s e i n t h e t o t a l n i t r o g e n c o n t e n t was a t t r i b u t e d p r i m a -r i l y t o t h e d e c r e a s e i n a c t o m y o s i n and s t r o m a f r a c t i o n . A n d o a n d H a t a n o ( 1 9 8 6 b ) f o l l o w e d t h e d e g r e e o f m y o f i b r i l l a r p r o t e i n d e g r a d a t i o n d u r i n g s p a w n i n g m i g r a t i o n by m e a s u r i n g N - m e t h y l h i s t i d i n e by i o n e x c h a n g e c h r o m a t o g r a -phy. T h i s amino a c i d i s u s e f u l a s an i n d e x o f m y o f i b r i l l a r p r o t e i n d e g r a d a t i o n b e c a u s e i t i s c o n t a i n e d o n l y i n a c t i n - 3 -and m y o s i n , i s n o t r e - u t i l i z e d f o r p r o t e i n s y n t h e s i s , and i s e x c r e t e d q u a n t i t a t i v e l y i n t o u r i n e . I n t h i s manner, t h e s e w o r k e r s were a b l e t o c l e a r l y show t h e d e g r e e o f m y o f i b r i l l a r p r o t e i n d e g r a d a t i o n d u r i n g s p a w n i n g m i g r a t i o n o f chum s a l m o n . 1.2.1. C o l o u r C a r o t e n o i d s a r e a g r o u p o f p i g m e n t s w h i c h , i n a n i m a l s s u c h a s P a c i f i c s a l m o n , c o n t r i b u t e t o t h e y e l l o w , o r a n g e , an d r e d c o l o u r s f o u n d i n t h e i n t e g u m e n t , m u s c l e , and o v a -r i e s . A s t a x a n t h i n i s an i m p o r t a n t c a r o t e n o i d , a n d i s t h e m ain p i g m e n t f o u n d i n chum s a l m o n (Simpson, 1 9 8 2 ) . B e c a u s e s a l m o n a r e u n a b l e t o s y n t h e s i z e t h i s c a r o t e n o i d de n o v o , a s t a x a n t h i n a n d o t h e r r e l a t e d c a r o t e n o i d s must be o b t a i n e d f r o m t h e d i e t . T h u s t h e d e g r e e o f p i g m e n t a t i o n i n t h e a n i m a l i s a r e f l e c t i o n o f t h e i n g e s t i o n a n d m e t a b o l i s m o f s p e c i f i c d i e t a r y p i g m e n t s o u r c e s (Ando e t a l . , 1 9 8 9 ) . The d i f f e r e n c e s i n t h e m u s c l e and s k i n c o l o u r o f chum s a l m o n a r e c l o s e l y a s s o c i a t e d w i t h t h e p h y s i o l o g i c a l s t a t e o f t h e f i s h (Ando e t a l . 1 9 8 5 ) . Ando e t a l . (1989) u s e d HPLC t e c h n i q u e s t o f o l l o w t h e c h a n g e i n c o l o u r o f chum s a l m o n t h r o u g h o u t t h e s p a w n i n g m i g r a t i o n . A s t a x a n t h i n was t h e p r i m a r y c a r o t e n o i d f o u n d i n i m m a t u r e chum s a l m o n . D u r i n g m i g r a t i o n t h e r e was an i n c r e a s e i n serum c a r o t e n o i d l e v e l s , and an i n c r e a s e i n t h e c a r o t e n o i d l e v e l s i n t h e o v a -r i e s . W i t h s e x u a l m a t u r i t y , a c o n s i d e r a b l e p r o p o r t i o n o f 4 - k e t o - z e a x a n t h i n and z e a x a n t h i n were d e t e c t e d i n t h e m u s c l e -4-o f t h e s p e n t c h u m . T h e m e c h a n i s m p r o p o s e d f o r t h i s c h a n g e was v i a a r e d u c t i v e m e t a b o l i c p a t h w a y f r o m a s t a x a n t h i n t o 4 - k e t o - z e a x a n t h i n t o z e a x a n t h i n . T a b l e 1 . 1 d e p i c t s t h i s l o s s i n a s t a x a n t h i n , a n d t h e r e l a t i v e i n c r e a s e i n z e a x a n t h i n a n d 4 - k e t o - z e a x a n t h i n i n t h e chum d u r i n g f o u r s t a g e s o f i t s m i g r a t i o n ( A n d o a n d H a t a n o , 1 9 8 7 ) . T h e c o l o u r o f t h e d i f f e r e n t c a r o t e n o i d s i s a r e s u l t o f t h e p r e s e n c e o f a s y s t e m o f c o n j u g a t e d d o u b l e b o n d s . A s t h e n u m b e r o f c o n j u g a t e d d o u b l e b o n d s i n c r e a s e , t h e max imum a b s o r b a n c e o f t h e compound o c c u r s a t t h e l o n g e r w a v e l e n g t h s , a n d t h e h u e o f t h e c a r o t e n o i d becomes r e d d e r . I n g e n e r a l , t h e c a r o t e n o i d s i n f o o d a r e f o u n d i n t h e t r a n s c o n f i g u r a -t i o n . T h e s h i f t t o a c i s i s o m e r a s a r e s u l t o f h e a t , l i g h t , o r a c i d i t y , c a u s e s a g r a d u a l l i g h t e n i n g o f c o l o u r . B e c a u s e a s t a x a n t h i n i s a n u n s t a b l e p i g m e n t , i t i s i m p o r t a n t d u r i n g a n y q u a n t i f i c a t i o n t o p r o t e c t i t f r o m e x c e s s h e a t , a c i d , e x -p o s u r e t o l i g h t , o x y g e n , o r l i p o x y g e n a s e s . T h e r e i s a n e e d f o r i n s t r u m e n t a l t e c h n i q u e s t o p r o v i d e c o n s i s t e n t q u a l i t a t i v e a n d q u a n t i t a t i v e i n f o r m a t i o n o n c o l o u r ( S k r e d e a n d S t o r e b a k k e n , 1 9 8 6 ) . T h e c o l o u r p e r c e i v e d when t h e e y e v i e w s a n i l l u m i n a t e d o b j e c t i s r e l a t e d t o t h r e e f a c t o r s , t h e s p e c t r a l c o m p o s i t i o n o f t h e l i g h t s o u r c e , t h e c h e m i c a l a n d p h y s i c a l c h a r a c t e r i s t i c s o f t h e o b j e c t , a n d t h e s p e c t r a l s e n s i t i v i t y p r o p e r t i e s o f t h e e y e . A s y s t e m d e v e l -o p e d t o q u a n t i t a t e t h e c o l o u r r e s p o n s e m e c h a n i s m o f t h e human e y e was p r o p o s e d b y t h e C o m m i s s i o n I n t e r n a t i o n a l d e -5-T a b l e 1.1. C o n t e n t and p e r c e n t a g e c o m p o s i t i o n o f t h e m a j o r c a r o t e n o i d s i n t h e chum s a l m o n m u s c l e d u r i n g s p a w n i n g m i g r a t i o n (Ando and H a t a n o , 1 9 8 7 ) . G r a d e Sex A s t a x a n t h i n Z e a x a n t h i n 4 - k e t o -z e a x a n t h i n 1 M 2 14.5 ( 8 9 ) 1 0.19 (1.2) 0.34 (2.1) F 11.4 (91) 0.10 (0.8) 0.20 (1.2) 2 M 10.7 (86) 0.32 (2.6) 0.45 (3.7) F 9.4 (84) 0.31 (2.8) 0.48 (4.3) 3 M 0.4 (73) 0.07 (11.8) 0.03 (5.0) F 0.4 (78) 0.04 (8.4) 0.02 (4.3) 4 M 0.7 (64) 0.26 (23.7) 0.05 (4.9) F 1.0 (70) 0.29 (20.0) 0.05 (3.4) B r a c k e t s i n d i c a t e p e r c e n t a g e o f t o t a l c a r o t e n o i d . V a l u e s a r e m e a s u r e d i n mg/100 g wh o l e m u s c l e . - 6 -l ' E c l a i r a g e ( C I E ) (de Man, 1 9 8 0 ) . However b e c a u s e t h e C I E s c a l e s were n o t a b l e t o p r o v i d e a r e a s o n a b l y u n i f o r m e s t i -mate o f c o l o u r a s p e r c e i v e d by t h e human e y e , o t h e r c o l o u r s c a l e s were d e v e l o p e d b a s e d on t h e o p p o n e n t c o l o u r t h e o r y o f c o l o u r v i s i o n . The o p p o n e n t c o l o u r t h e o r y o f c o l o u r v i s i o n was p r o -p o s e d by E w a l d H e r i n g i n 1879, and was l a t e r r e f i n e d by G.E. M u e l l e r i n 1930 (de Man, 1 9 8 0 ) . The t h e o r y s t a t e d t h a t i n t h e human e y e t h e r e was a n i n t e r m e d i a t e s i g n a l - s w i t c h i n g s t a g e b e t w e e n t h e l i g h t r e c e p t o r s i n t h e r e t i n a a n d t h e o p t i c n e r v e , w h i c h t o o k c o l o u r s i g n a l s t o t h e b r a i n . I n t h e s w i t c h i n g s t a g e , t h e r e d c o l o u r r e s p o n s e s were compared w i t h g r e e n t o g e n e r a t e a r e d - g r e e n c o l o u r d i m e n s i o n , d e n o t e d a s " a " . T h i s r e d - g r e e n r e s p o n s e was t h e n compared w i t h b l u e t o g e n e r a t e a y e l l o w t o b l u e c o l o u r d i m e n s i o n , d e n o t e d a s "b". A t h i r d d i m e n s i o n c o n s i d e r e d w a s l i g h t n e s s , ( L ) , a n o n - l i n e a r f u n c t i o n w h i c h r e l a t e d t o t h e p e r c e n t r e f l e c t a n c e o f an o b j e c t . S e v e r a l s y s t e m s o f c o l o u r c l a s s i f i c a t i o n have b e e n d e v e l o p e d t o p r o v i d e q u a n t i t a t i v e i n f o r m a t i o n . The H u n t e r L a b s y s t e m has been u s e d s u c c e s s f u l l y t o d e s c r i b e a n d measure c o l o u r . The modern model o f t h i s ma-c h i n e makes u s e o f a r e f l e c t a n c e s p e c t r o p h o t o m e t e r w h i c h i s i n t e r f a c e d w i t h an IBM p e r s o n a l c o m p u t e r . V i s i b l e l i g h t r e -f l e c t e d f r o m t h e sample i s a n a l y z e d and t h e r e s u l t a n t s p e c -t r u m i s u s e d t o c a l c u l a t e t h e s p e c t r a l v a l u e s L, a, and b. -7 -0 1.3. Texture 1.3.1. Instrumental analysis The texture of a food product i s commonly considered the most d i f f i c u l t organoleptic property to describe accu-r a t e l y . Szczesniak, (1963) suggested t h i s problem was caused f i r s t by the lack of an adequate bridge between theo-r e t i c a l rheology and p r a c t i c a l applications, and second be-cause most of the reported research had only considered spe-c i f i c t e xtural c h a r a c t e r i s t i c s of i n d i v i d u a l foods. A need was thus i d e n t i f i e d to develop a r a t i o n a l system, and nomen-cl a t u r e to describe and translate textural properties into p r e c i s e l y defined, measurable properties (Friedman et a l . , 1963). Texture and consistency were defined by two elements, f i r s t the physical structure of the material ( i . e . i t s geom-etry) , and second the way the material f e l t i n the mouth ( i . e . i t s mechanical and surface properties). Szczesniak, (1963) grouped texture into three main c l a s s e s , mechanical c h a r a c t e r i s t i c s - those manifested by the reaction of food to s t r e s s , geometric c h a r a c t e r i s t i c s - the arrangement of the constituents of food, and other c h a r a c t e r i s t i c s - the mouth f e e l f a c t o r s . This analysis of texture lead to the acceptance of f i v e primary independent mechanical parame-te r s , hardness, cohesiveness, adhesiveness, v i s c o s i t y , and e l a s t i c i t y , and three secondary dependent parameters, b r i t -tleness, chewiness, and gumminess. Hardness was defined as - 8 -t h e f o r c e n e c e s s a r y t o a t t a i n a g i v e n d e f o r m a t i o n , c o h e s i v e -n e s s was t h e s t r e n g t h o f i n t e r n a l b o n d s w h i c h made up t h e body o f t h e p r o d u c t , v i s c o s i t y was t h e r a t e o f f l o w p e r u n i t f o r c e , e l a s t i c i t y ( l a t e r c h a n g e d t o s p r i n g i n e s s ) was t h e r a t e a t w h i c h d e f o r m e d m a t e r i a l r e t u r n e d t o an u n d e f o r m e d c o n d i t i o n a f t e r t h e d e f o r m i n g f o r c e was r e m o v e d , an d a d h e -s i v e n e s s was t h e work n e c e s s a r y t o overcome t h e a t t r a c t i v e f o r c e s between t h e s u r f a c e o f f o o d and t h e s u r f a c e o f o t h e r m a t e r i a l s w i t h w h i c h t h e f o o d came i n c o n t a c t . B r i t t l e n e s s , o r f r a c t u r a b i l i t y , was t h e f o r c e w i t h w h i c h t h e m a t e r i a l f r a c t u r e d , a n d was r e l a t e d t o t h e p r i m a r y p a r a m e t e r s o f h a r d n e s s and c o h e s i v e n e s s . C h e w i n e s s was t h e e n e r g y r e -q u i r e d t o m a s t i c a t e a s o l i d f o o d t o a s t a t e r e a d y f o r s w a l -l o w i n g , and was r e l a t e d t o h a r d n e s s , c o h e s i v e n e s s , and e l a s -t i c i t y . Gumminess was t h e e n e r g y r e q u i r e d t o d i s i n t e g r a t e a s e m i s o l i d f o o d p r o d u c t t o a s t a t e r e a d y f o r s w a l l o w i n g . T h e g e o m e t r i c c h a r a c t e r i s t i c s d e s c r i b e d t h e r e l a t i v e s i z e and s h a p e o f t h e p a r t i c l e s , and t h e i r r e l a t i v e o r i e n t a -t i o n . O t h e r c h a r a c t e r i s t i c s d e s c r i b e d mouth f e e l q u a l i t y and were r e l a t e d t o t h e p e r c e p t i o n o f m o i s t u r e a n d f a t i n f o o d . A p p l i c a t i o n o f t h e s e p a r a m e t e r s r e q u i r e d t h e d e s i g n o f a n i n s t r u m e n t a l u n i t c a p a b l e o f t r a n s l a t i n g t h e s e d e f i n i -t i o n s i n t o a n u n b i a s e d , r e c o r d e d p h y s i c a l m e a s u r e m e n t . F r i e d m a n e t a l . , ( 1 9 6 3 ) r e c o g n i z e d t h a t i n o r d e r t o - 9 -c h a r a c t e r i z e t e x t u r e , a c o m p r e h e n s i v e o b j e c t i v e t e s t was n e e d e d . The o b j e c t i v e m e a s u r e m e n t o f t e x t u r e was d i v i d e d i n t o t h r e e g e n e r a l c a t e g o r i e s , f u n d a m e n t a l t e s t s , e m p i r i c a l t e s t s , and i m i t a t i v e t e s t s . The f i r s t c a t e g o r y m e a s u r e d t h e f u n d a m e n t a l r h e o l o g i c a l p r o p e r t i e s o f f o r c e , d e f o r m a t i o n , and t i m e f r o m w h i c h i t was p o s s i b l e t o c a l c u l a t e p a r a m e t e r s s u c h as u l t i m a t e s t r e n g t h , P o i s s o n ' s r a t i o , Young's modulus and s h e a r / b u l k modulus ( B o u r n e , 1 9 7 8 ) . T e x t u r e m e a s u r i n g i n s t r u m e n t s c a u s e t h e m e c h a n i c a l and s t r u c t u r a l f a i l u r e o f f o o d s by i m p o s i n g one o r more f o r c e s . T h e s e h a v e i n c l u d e d t e n s i o n , t h e a p p l i c a t i o n o f u n i a x i a l p a r a l l e l f o r c e s t o c a u s e e x t e n s i o n , c o m p r e s s i o n , t h e u n i a x i a l p a r a l l e l f o r c e w h i c h c a u s e s f l a t t e n i n g , and s h e a r , a u n i a x i a l t a n g e n t i a l f o r c e w h i c h c a u s e s s e p a r a t i o n o r c u t -t i n g . C o m p r e s s i o n , an i m p o r t a n t measure o f t e x t u r e , c a n be t e s t e d i n two ways. The f i r s t t e s t i s a b u l k c o m p r e s s i o n , i n w h i c h t h e s a m p l e i s c o m p r e s s e d i n t h r e e d i m e n s i o n s by means o f h y d r a u l i c p r e s s u r e . The s e c o n d t e s t i s u n i a x i a l c o m p r e s s i o n w h e r e b y t h e sample i s c o m p r e s s e d i n one d i r e c -t i o n , a n d u n r e s t r a i n e d i n t h e o t h e r t w o d i m e n s i o n s . The method o f t e x t u r e p r o f i l e a n a l y s i s u t i l i z e s t h e p r i n c i -p l e o f u n i a x i a l c o m p r e s s i o n t o c r u s h f o o d . The t h e o r e t i c a l a c t i o n o f u n i a x i a l c o m p r e s s i o n was a p p l i e d t o Hookean s o l i d s o f u n i f o r m c r o s s - s e c t i o n a l a r e a a t a s m a l l s t r a i n ( i . e . 1%) b e f o r e r u p t u r e , i s d e f i n e d by Young's modulus o f e l a s t i c i t y (E) ( B o u r n e , 1 9 8 2 ) . -10-E = C o m p r e s s i v e s t r e s s = F/A  C o m p r e s s i v e s t r a i n d e l t a L / L Where: S t r e s s = f o r c e p e r u n i t a r e a S t r a i n = d e f o r m a t i o n p e r u n i t d i m e n s i o n F = a p p l i e d f o r c e A = c r o s s s e c t i o n a l a r e a L = u n s t r e s s e d h e i g h t d e l t a L = change i n h e i g h t due t o F • -Thus " E" i s t h e s l o p e o f t h e s t r e s s / s t r a i n c u r v e , and a m e a s u r e o f s t i f f n e s s ( B o u r n e , 1 9 8 2 ) . F o r most f o o d s how-e v e r , t h e p u r e d e f i n i t i o n o f " E " r a r e l y a p p l i e s . T h i s i s b e c a u s e f i r s t , t h e s a m p l e i s u s u a l l y s u b j e c t e d t o a l a r g e a m o u n t o f s t r a i n , a n d s e c o n d , b e c a u s e m o s t f o o d s a r e v i s c o e l a s t i c , n o t e l a s t i c . V i s c o e l a s t i c i t y o c c u r s when a w e i g h t p l a c e d on a f o o d c a u s e s i m m e d i a t e c o m p r e s s i o n ( " i n -s t a n t a n e o u s e l a s t i c d e f o r m a t i o n " ) , f o l l o w e d by a p r o l o n g e d , c o n t i n u o u s d e c e l e r a t i n g r a t e o f d e f o r m a t i o n c a l l e d " r e t a r d e d d e f o r m a t i o n " . When t h e w e i g h t i s removed, t h e i n s t a n t a n e o u s p a r t i a l e l a s t i c r e c o v e r y i s f o l l o w e d by a f u t u r e r e c o v e r y w i t h r e s p e c t t o t i m e , c a l l e d " r e t a r d e d r e c o v e r y " . Thus i n a permanent d e f o r m a t i o n , t h e sample n e v e r r e t u r n s t o i t s o r i g -i n a l h e i g h t . I n o r d e r t o m a i n t a i n t h e " p u r i t y " o f t h e d e f i -n i t i o n o f Young's M o d u l u s , most f o o d s a r e d e f i n e d i n s t e a d by a "modulus o f d e f o r m a b i l i t y " ( J o h n s o n e t a l . , 1 9 8 0 ) . The modulus o f d e f o r m a b i l i t y i s r e p r e s e n t a t i v e o f a m a t e r i a l ' s o v e r a l l r e s i s t a n c e t o d e f o r m a t i o n . A c t u a l m e a s u r e d v a l u e s a r e d e p e n d e n t on t e s t c o n d i t i o n s , e s p e c i a l l y s p e c i f i c d i m e n -s i o n s o f t h e m a t e r i a l , and d e f o r m a t i o n r a t e s . I t s h o u l d be -11-n o t e d however t h a t i f a h i g h d e g r e e o f u n i a x i a l c o m p r e s s i o n c a u s e d a p r o d u c t t o r u p t u r e , s p r e a d , f r a c t u r e , o r b r e a k , t h e n t h e modulus o f d e f o r m a b i l i t y a l s o w o u l d no l o n g e r a p p l y ( B o u r n e , 1 9 8 2 ) . F u n d a m e n t a l t e s t s s u c h a s Young's modulus have t e n d e d t o c o r r e l a t e p o o r l y w i t h s e n s o r y e v a l u a t i o n , a n d a r e o f t e n n o t m e a n i n g f u l i n t h e m s e l v e s b e c a u s e f o o d d o e s n o t h a v e s i m p l e r h e o l o g i c a l p r o p e r t i e s t h a t a r e i n d e p e n d e n t o f s t r e s s and s t r a i n c o n d i t i o n s . Thus t h e p r i m a r y f u n c t i o n o f f u n d a -m e n t a l t e s t s h a s o f t e n b e e n t o p r o v i d e a b a s i s f r o m w h i c h e m p i r i c a l t e s t s may be d e v e l o p e d ( B r i n t o n and B o u r n e , 1 9 7 2 ) . E m p i r i c a l t e s t s measure p a r a m e t e r s t h a t h a v e been f o u n d t o be r e l a t e d t o t e x t u r a l q u a l i t y , b u t t h e y do n o t i n c l u d e a l l t h e p a r a m e t e r s n e c e s s a r y f o r a c o m p l e t e d e s c r i p t i o n o f t e x t u r e . I n s t r u m e n t s u s e d f o r t h e s e t e s t s i n c l u d e p e n e t r o m -e t e r s , c o m p r e s s o r s , c o n s i s t o m e t e r s , t e n d e r o m e t e r s , a n d s h e a r i n g d e v i c e s . B e c a u s e t h e s e i n s t r u m e n t s have t e n d e d t o c o n c e n t r a t e o n l y on a s m a l l p h a s e o f t h e m e c h a n i c a l p r o p e r -t i e s i n v o l v e d i n c h e w i n g , t h e y have n o t p r o v i d e d a c o m p l e t e p i c t u r e o f how t h e m e c h a n i c a l p a r a m e t e r s o f t e x t u r e w e r e s e n s e d i n t h e mouth ( F r i e d m a n e t a l . , 1 9 6 3 ) . I m i t a t i v e t e s t s were d e v e l o p e d t o i m i t a t e t h e a c t u a l c o n d i t i o n s t o w h i c h f o o d was s u b j e c t e d i n t h e mouth. I n -s t r u m e n t s t h a t f a l l i n t o t h i s c a t e g o r y i n c l u d e t h e B r a b e n d e r F a r i n o g r a p h , A m y l o g r a p h , a n d A l v e o g r a p h , a n d t h e I n s t r o n U n i v e r s a l T e s t i n g M a c h i n e ( S z c z e s n i a k , 1 9 6 3 ) . One o f t h e -12-f i r s t i n s t r u m e n t s d e s i g n e d t o s i m u l a t e m a s t i c a t i o n was t h e d e n t u r e t e n d e r o m e t e r d e v e l o p e d by t h e F o o d T e c h n o l o g y L a b o -r a t o r y o f t h e M a s s a c h u s e t t s I n s t i t u t e o f T e c h n o l o g y ( F r i e d m a n e t a l . , 1 9 6 3 ) . T h i s i n s t r u m e n t was l a t e r u s e d a s a b a s i c p r o t o t y p e by F r i e d m a n e t a l . , (1963) f o r t h e c o n -s t r u c t i o n o f t h e i r " t e x t u r o m e t e r " . T h e i r t e x t u r o m e t e r c o n -s i s t e d o f f o u r b a s i c u n i t s , an a r t i c u l a t o r w i t h a m o t o r , v a r i a b l e - p u l l e y d r i v e , a n d s t r a i n - g a u g e s e n s i n g p l a t e , a f a s t r e s p o n s e pen r e c o r d e r , a v a r i a b l e - v o l t a g e power s u p p l y , a n d a m e t h o d t o m e a s u r e t h e s t r a i n - g a u g e c i r c u i t . T h e s y s t e m p r o d u c e d a f o r c e - d i s t a n c e c u r v e w h i c h r e p r e s e n t e d an " i n t e g r a l p i c t u r e o f t h e t e x t u r a l c h a r a c t e r i s t i c s o f t h e p r o d u c t s a m p l e u n d e r t e s t " , and was t h u s c a l l e d a " t e x t u r e p r o f i l e " ( F r i e d m a n e t a l . , 1 9 6 3 ) . Bourne f i r s t u s e d t h e I n s t r o n U n i v e r s a l T e s t i n g M a c h i n e t o measure t h e d e f o r m a t i o n o f f o o d s w i t h a h i g h d e g r e e o f p r e c i s i o n ( B r i n t o n and B o u r n e , 1 9 7 2 ) . T h i s m a c h i n e i s d i -v i d e d i n t o two b a s i c p a r t s , a mechanism w h i c h d r i v e s a h o r i -z o n t a l c r o s s - h e a d i n a v e r t i c a l d i r e c t i o n a t s e l e c t e d s p e e d s w i t h i n t h e r a n g e o f 0.2 t o 50 cm p e r m i n u t e , an d a f o r c e s e n s i n g and r e c o r d i n g s y s t e m w h i c h c o n s i s t s o f a l o a d c e l l a nd a s t r i p c h a r t r e c o r d e r . By u s e o f a s e n s i t i v i t y s e l e c -t o r s w i t c h and s e v e r a l d i f f e r e n t l o a d c e l l s , i t i s p o s s i b l e t o o b t a i n a f u l l s c a l e d e f l e c t i o n o f t h e p e n o v e r a f o r c e r a n g e o f 0.2 t o 500 k g f o r c e (2N t o 5kN) , and t o p r o d u c e a r e c o r d o f a s a m p l e ' s f o r c e - d i s t a n c e r e l a t i o n s h i p . S i n c e i t s -13-i n t r o d u c t i o n , t h e I n s t r o n has g a i n e d w i d e s p r e a d u s e i n t h e e v a l u a t i o n o f t e x t u r a l p r o p e r t i e s o f s o l i d f o o d . T e x t u r e p r o f i l e a n a l y s i s i s a u s e f u l t e c h n i q u e f o r t h e e v a l u a t i o n o f t e x t u r a l p a r a m e t e r s o f a w i d e r a n g e o f f o o d s . B o u r n e (1982) u s e d t h i s t e c h n i q u e t o e v a l u a t e t h e t e x t u r e p r o f i l e p a r a m e t e r s d e r i v e d f r o m f o r c e - d e f o r m a t i o n c u r v e s ob-t a i n e d f r o m t h e I n s t r o n . F l a t p l a t e s were a t t a c h e d t o t h e I n s t r o n and u s e d t o com p r e s s b i t e - s i z e d p i e c e s o f a s a m p l e t w i c e i n a r e c i p r o c a t i n g m o t i o n t o i m i t a t e t h e a c t i o n o f t h e jaw. The c r o s s - h e a d , d r i v e n a t a c o n s t a n t s p e e d , a p p r o a c h e d t h e e n d o f t h e c o m p r e s s i o n s t r o k e , t h e n a b r u p t l y r e v e r s e d , and c o n t i n u e d w i t h an upward s t r o k e a t t h e same s p e e d . T h i s a c t i o n r e s u l t e d i n t h e p r o d u c t i o n o f a f o r c e - t i m e c u r v e f r o m w h i c h a number o f t e x t u r a l p a r a m e t e r s c o u l d be m e a s u r e d w h i c h c o r r e l a t e d w e l l w i t h s e n s o r y e v a l u a t i o n s ( B o u r n e , 1 9 8 2 ) . T h i s t e c h n i q u e was a r e f l e c t i o n o f t h e n e e d f o r m u l -t i d i m e n s i o n a l a n a l y s i s o f t e x t u r e . F i g u r e 1.1 d e p i c t s a g e n e r a l i z e d t e x t u r e p r o f i l e c u r v e . B o u r n e e t a l . (1978) d e s c r i b e d F i g u r e 1.1 as f o l l o w s . A sample was p l a c e d between two f l a t p l a t e s on t h e I n s t r o n . The c r o s s - h e a d d e s c e n d e d a t c o n s t a n t s p e e d a n d c o m p r e s s e d t h e s a m p l e . The f o r c e a t t h e f i r s t s i g n i f i c a n t p e a k was known a s f r a c t u r a b i l i t y . As t h e c o m p r e s s i o n c o n t i n u e d , a s e c o n d peak, h a r d n e s s 1, was f o r m e d a t t h e p o i n t o f maximum c o m p r e s s i o n . The I n s t r o n c r o s s - h e a d t h e n r e v e r s e d -14-FIRST BITE SECOND BITE ADHESIVE FORCE 1 TIME-* F i g u r e 1.1. G e n e r a l i z e d T e x t u r e P r o f i l e A n a l y s i s ( T P A ) c u r v e ( B o u r n e , 1 9 8 2 ) . -15-d i r e c t i o n , and t h e f o r c e r e t u r n e d t o z e r o . A r e a 1 was t h e a r e a u n d e r t h e c u r v e up t o t h e maximum p o i n t o f c o m p r e s s i o n , a n d r e p r e s e n t e d t h e work done t o c o m p r e s s a n d c r u s h f o o d d u r i n g t h e f i r s t b i t e . The p o s i t i v e a r e a t o t h e r i g h t o f t h e d o t t e d l i n e ( i n A r e a 1) was t h e o r e t i c a l l y t h e work r e -t u r n e d by t h e f o o d t o t h e m a c h i n e d u r i n g c o m p r e s s i o n . How-e v e r , a s i g n i f i c a n t p a r t o f t h e s m a l l a r e a was p o s s i b l y c a u s e d by pen r e s p o n s e and was a s s o c i a t e d w i t h s u b s t a n t i a l e r r o r . T h e n e g a t i v e p o r t i o n o f t h e c u r v e u n d e r t h e z e r o f o r c e l i n e r e p r e s e n t e d t h e a d h e s i o n o f t h e f o o d t o t h e com-p r e s s i o n p l a t e a s i t moved upward d u r i n g t h e d e c o m p r e s s i o n p h a s e o f t h e f i r s t b i t e . A r e a 3 was d e f i n e d a s t h e wor k done t o p u l l t h e f o o d a p a r t , and was t h e a r e a u n d e r t h e z e r o f o r c e l i n e . The a d h e s i v e f o r c e was d e f i n e d a s t h e maximum n e g a t i v e f o r c e . H a r d n e s s 2 was t h e f o r c e a t t h e p o i n t o f t h e maximum s e c o n d c o m p r e s s i o n . A g a i n , t h e p o s i t i v e a r e a t o t h e r i g h t o f t h e d o t t e d l i n e was t h e work r e t u r n e d t o t h e machine d u r i n g d e c o m p r e s s i o n . A r e a 2 was t h e work done on t h e f o o d by t h e machin e d u r i n g t h e s e c o n d c o m p r e s s i o n . The s p r i n g i n e s s was d e f i n e d a s " t h e d i s t a n c e t h e s a m p l e r e c o v -e r e d i n h e i g h t b e t w e e n t h e e n d o f t h e f i r s t r i s e i n f o r c e g r e a t e r t h a n z e r o i n t h e s e c o n d b i t e , and i t s p o i n t o f m a x i -mum c o m p r e s s i o n " . B ourne e t a l . (1978) a l s o d e f i n e d s e v e r a l p a r a m e t e r s t h a t c o u l d n o t be d i r e c t l y m e a s u r e d f r o m t h e t e x -t u r e p r o f i l e . C o h e s i v e n e s s was d e r i v e d by d i v i d i n g a r e a 2 b y a r e a 1, g u m m i n e s s was t h e p r o d u c t o f h a r d n e s s a n d -16-c o h e s i v e n e s s , and c h e w i n e s s was t h e p r o d u c t o f gumminess and s p r i n g i n e s s . To o b t a i n i n f o r m a t i o n f r o m f o r c e - t i m e g r a p h s m a n u a l l y was t i m e c o n s u m i n g , d i f f i c u l t a n d o f l i m i t e d a c c u r a c y ( A b b o t t e t a l . , 1 9 8 2 ) . As a s o l u t i o n t o t h i s p r o b l e m , com-p u t e r a t t a c h m e n t s w i t h a u t o m a t i c r e a d o u t s o f f o r c e - d e f o r m a t i o n p a r a m e t e r s have been d e v e l o p e d . By i n t e r -f a c i n g t h e I n s t r o n w i t h a m i c r o c o m p u t e r , a d u a l s y s t e m was f o r m e d , c a p a b l e o f m e a s u r i n g and r e c o r d i n g l o a d c e l l o u t p u t s i g n a l s and f o r c e - d e f o r m a t i o n c u r v e s . Use o f t h e c o m p u t e r a l l o w e d t h e a n a l y s i s o f t h e d a t a f r o m a l a r g e number o f sam-p l e s i n a r e l a t i v e l y s h o r t t i m e , and t h e a n a l y s i s o f a l a r g e number o f v a r i a b l e s . However, A b b o t t e t a l . , (1982) an d B o u r n e e t a l . , (1978) c a u t i o n e d t h a t t h e o r i g i n a l a u t o m a t i c t y p e s y s t e m s were n o t a p p r o p r i a t e f o r t e x t u r e p r o f i l e c u r v e s b e c a u s e t h e v a r i a t i o n common i n t h e s e c u r v e s r e s u l t e d i n s e -r i o u s e r r o r s i n t h e i r i n t e r p r e t a t i o n . A b b o t t e t a l . , (1982) p r o v i d e d a p r o g r a m w h i c h t h e y c l a i m e d was a b l e t o r e c o r d t h e l o a d c e l l o u t p u t s i g n a l , c o n t r o l t h e I n s t r o n c r o s s - h e a d s p e e d a n d d i r e c t i o n , a n d a u t o m a t i c a l l y e x t r a c t d a t a f r o m t e x t u r e p r o f i l e t y p e c o m p r e s s i o n t e s t s . A s o l u t i o n t o t h e p r o b l e m s w i t h c o m p u t e r a n a l y s i s o f t h e v a r i a b l e t e x t u r e p r o -f i l e c u r v e s was t o a l l o w t h e o p e r a t o r t o s e l e c t t h e p e a k s , a r e a s , and d i s t a n c e s w h i c h t h e c omputer t h e n c a l c u l a t e d . D e s p i t e t h e a p p a r e n t s i m p l i c i t y o f t h e I n s t r o n , Shama a n d Sherman (1973) c a u t i o n e d t h a t i n s t r u m e n t a l e v a l u a t i o n -17-o f t e x t u r a l p r o p e r t i e s a s a method t o p r e d i c t s e n s o r y r e -s p o n s e s r e q u i r e d t h a t t h e i n s t r u m e n t a l t e s t c o n d i t i o n s be s e l e c t e d c a r e f u l l y . The m a g n i t u d e and r a t e o f t h e a p p l i e d l o a d i n f l u e n c e d t h e i n s t r u m e n t a l e v a l u a t i o n o f t e x t u r a l p r o p e r t i e s d i f f e r e n t l y f o r e a c h f o o d d e p e n d i n g on how i t was n o r m a l l y m a s t i c a t e d . I n t h e I n s t r o n , a s t h e r a t e o f l o a d i n g ( t h e c r o s s - h e a d s p e e d ) i n c r e a s e d , t h e f o r c e t o a c h i e v e com-p r e s s i o n a l s o i n c r e a s e d a n d t h e r e f o r e i n f l u e n c e d t h e f o r c e - d e f o r m a t i o n d a t a . Thus i t was s u g g e s t e d t h a t when a c o m p a r i s o n was made b e t w e e n f o o d s w i t h d i f f e r e n t t e x t u r a l p r o p e r t i e s , d i f f e r e n t i n s t r u m e n t a l t e s t c o n d i t i o n s may be n e c e s s a r y i n o r d e r t o s i m u l a t e r e l e v a n t m a s t i c a t o r y p a t -t e r n s . 1.3.2. S e n s o r y a n a l y s i s O r g a n o l e p t i c t e s t s h a v e f r e q u e n t l y b e e n u s e d t o s t u d y t h e i m p a c t o f a p r o c e s s o p e r a t i o n on q u a l i t y a t t r i b u t e s o f a f o o d . A g u i l e r a and S t a n l e y (1990) d e s c r i b e d t h r e e t y p e s o f s e n s o r y p a n e l s ; consumer p a n e l s , w h i c h m e a s u r e d t h e r e s p o n s e o f a " t y p i c a l " c o n s u m e r t o t h e p r o d u c t ; a c c e p t a n c e p a n e l s w h i c h a s k e d h e d o n i c q u e s t i o n s , i . e . how much t h e sample was l i k e d / d i s l i k e d and why; and a n a l y t i c a l p a n e l s w h i c h m e a s u r e d i n t e n s i t y d i f f e r e n c e s i n t h e t e x t u r a l a t t r i b u t e s o f t h e sam-p l e s . The o b j e c t o f t h e a n a l y t i c a l p a n e l s was t o o b t a i n n u-m e r i c a l d a t a w h i c h c o u l d be a n a l y z e d s t a t i s t i c a l l y . T h e p r o b l e m w i t h t h e f i r s t two t e c h n i q u e s was t h a t commonly " q u a l i t y was i n t h e m i n d o f t h e o b s e r v e r " , a n d a m e a s u r e d -18-r e s p o n s e was a r e s u l t o f t h e j u d g e ' s p r e v i o u s c o n d i t i o n i n g , and was s u b j e c t t o many v a r i a b l e s o u t s i d e t h e c o n t r o l o f a f o o d p r o c e s s o r ( L u n d , 1 9 8 2 ) . The d e v e l o p m e n t o f a c o m p r e h e n s i v e and a n a l y t i c a l s e n -s o r y method t o e v a l u a t e f o o d t e x t u r e was t h u s n e e d e d t o p r o -d u c e a r a t i o n a l , w e l l - d e f i n e d s y s t e m f o r c l a s s i f y i n g t e x -t u r e . S z c z e s n i a k e t a l . , (1963) n o t e d t h e common p r o b l e m o f p o o r c o r r e l a t i o n b e t w e e n i n s t r u m e n t a l a n d s e n s o r y e v a l u a -t i o n s a n d s u g g e s t e d t h a t t h e m a c h i n e s were n o t e v a l u a t i n g t h e same p r o p e r t y a s t h e s e n s o r y t e s t s . S z c z e s n i a k e t a l . , (1963) d e v e l o p e d a t e c h n i q u e w h i c h was d e m o n s t r a t e d t o p r o -d u c e v e r y g o o d c o r r e l a t i o n s w i t h i n s t r u m e n t a l e v a l u a t i o n s . W i t h t h i s method, t e x t u r e o f a f o o d p r o d u c t was o b j e c t i v e l y a n a l y z e d by t r a i n i n g an e x p e r t t a s t e p a n e l t o c r e a t e a " s e n -s o r y t e x t u r e p r o f i l e " o f any f o o d p r o d u c t . The t e r m " t e x t u r e p r o f i l i n g " was t h u s u s e d t o d e s c r i b e t h e o r g a n o l e p t i c m u l t i d i m e n s i o n a l a n a l y s i s o f a s a m p l e i n t e r m s o f t h e d e g r e e and o r d e r o f a p p e a r a n c e o f m e c h a n i c a l , g e o m e t r i c , f a t and m o i s t u r e c h a r a c t e r i s t i c s , f r o m t h e f i r s t b i t e t h r o u g h c o m p l e t e m a s t i c a t i o n . R e q u i r e m e n t s f o r t h i s c o m p r e h e n s i v e s e n s o r y m e t h o d i n c l u d e d a p a n e l o f t r a i n e d j u d g e s , a s t a n d a r d r a t i n g s c a l e , and p r o p e r p a n e l p r o c e d u r e s ( B r a n t e t a l . , 1 9 6 3 ) . D i f f e r e n t t e x t u r a l c h a r a c t e r i s t i c s were c a t e g o r i z e d by s t a n d a r d r a t i n g s c a l e s composed o f f o o d s w i t h d i f f e r e n t t e x -t u r a l i n t e n s i t i e s . T h e s e f o o d s p r o v i d e d t h e b a s i s f o r t h e -19-e v a l u a t i o n o f t h e m e c h a n i c a l p a r a m e t e r s o f t e x t u r e . Spe-c i f i c f o o d s f o r e a c h p o i n t on t h e s c a l e s were s e l e c t e d s o t h a t p r o p e r r e f e r e n c e s t a n d a r d s w e r e a l w a y s a v a i l a b l e . R a t i n g s c a l e s were d e v e l o p e d a n d i n c l u d e d : h a r d n e s s - t h e f o r c e r e q u i r e d t o p e n e t r a t e a s u b s t a n c e w i t h m o l a r t e e t h ; b r i t t l e n e s s - a s e c o n d a r y p a r a m e t e r w h i c h encompassed h a r d -n e s s a n d c o h e s i v e n e s s , d e f i n e d a s t h e e a s e o r f o r c e w i t h w h i c h a sample c r u m b l e d , c r a c k e d , o r s h a t t e r e d ; c h e w i n e s s -t h e l e n g t h o f t i m e i n s e c o n d s r e q u i r e d t o m a s t i c a t e a sample a t t h e r a t e o f one chew p e r s e c o n d i n o r d e r t o r e d u c e i t t o a s t a t e s a t i s f a c t o r y f o r s w a l l o w i n g ; gumminess - a s e c o n d a r y p a r a m e t e r d e f i n e d as d e n s i t y t h a t p e r s i s t e d t h r o u g h o u t mas-t i c a t i o n , a d h e s i v e n e s s - t h e f o r c e r e q u i r e d t o remove t h e m a t e r i a l t h a t a d h e r e d t o t h e mouth d u r i n g n o r m a l e a t i n g ; and v i s c o s i t y - t h e f o r c e r e q u i r e d t o draw a l i q u i d f r o m a s p o o n o v e r t h e t o n g u e . Use o f t h e s e r a t i n g s c a l e s e n a b l e d t h e e v a l u a t i o n o f a g i v e n t e x t u r a l p a r a m e t e r i n an "unknown" p r o d u c t i n t e r m s o f known p r o d u c t s . A c o m p r e h e n s i v e s e n s o r y m ethod was t h e n d e v e l o p e d t o e v a l u a t e t h e t e x t u r e o f a g i v e n f o o d p r o d u c t . B r a n d t e t a l . , (1963) u s e d t h e t h r e e main c l a s s e s o f t e x t u r e ; m e c h a n i -c a l , g e o m e t r i c a n d t h o s e p r o p e r t i e s r e l a t i n g t o f a t a n d m o i s t u r e c o n t e n t as a s y s t e m a t i c means o f r e c o r d i n g t h e t e x -t u r a l c h a r a c t e r i s t i c s o f a g i v e n f o o d by o r d e r o f a p p e a r -a n c e . T e x t u r e was c o n s i d e r e d t o have a d e f i n i t e p a t t e r n r e -g a r d i n g t h e o r d e r i n w h i c h c h a r a c t e r i s t i c s were p e r c e i v e d . -20-F i r s t b i t e , m a s t i c a t o r y , and r e s i d u a l p h a s e s were d e s c r i b e d . The f i r s t b i t e , o r i n i t i a l p h a s e , i n c l u d e d t h e m e c h a n i c a l c h a r a c t e r i s t i c s o f h a r d n e s s , b r i t t l e n e s s , v i s c o s i t y , a n d o t h e r g e o m e t r i c c h a r a c t e r i s t i c s i n i t i a l l y o b s e r v e d . T h e s e c o n d b i t e , o r m a s t i c a t o r y p h a s e , i n c l u d e d t h e m e c h a n i c a l c h a r a c t e r i s t i c s o f gumminess, c h e w i n e s s , a d h e s i v e n e s s , a n d o t h e r g e o m e t r i c c h a r a c t e r i s t i c s o b s e r v e d d u r i n g c h e w i n g . The t h i r d b i t e , t h e r e s i d u a l p h a s e , encompassed c h a n g e s i n -d u c e d i n t h e m e c h a n i c a l a n d g e o m e t r i c c h a r a c t e r i s t i c s t h r o u g h o u t m a s t i c a t i o n . P a n e l s e l e c t i o n was b a s e d on i n t e r e s t a n d a v a i l a b i l i t y o f p e r s o n n e l . The s e n s o r y p a n e l was t r a i n e d t o f a m i l i a r i z e t h e j u d g e s w i t h t h e s t a n d a r d r a t i n g s c a l e s , u n t i l unknown f o o d s a m p l e s c o u l d be a c c u r a t e l y r a t e d i n d i v i d u a l l y a n d t h r o u g h g r o u p d i s c u s s i o n . The c l a s s i f i c a t i o n o f t h e t e x -t u r a l c h a r a c t e r i s t i c s were s t u d i e d , and t h e g e o m e t r i c c h a r -a c t e r i s t i c s o f f o o d s were e v a l u a t e d . The p a n e l t h e n d e v e l -o p e d a c o m p l e t e t e x t u r e p r o f i l e f o r a sample u s i n g t h e p r e -d e t e r m i n e d s t a n d a r d r a t i n g s c a l e . A t y p i c a l t e x t u r e p r o f i l e s c o r e s h e e t i s shown i n F i g u r e 1.2. P r o p e r l y t r a i n e d p a n e l i s t s c o m p l i e d w i t h t h e two c r i t e -r i a t h a t d e f i n e d an o b j e c t i v e m e t hod; f i r s t t h e d a t a o b -t a i n e d was i n d e p e n d e n t o f i n d i v i d u a l o b s e r v a t i o n , a n d - 2 1 -T e x t u r e P r o f i l e S c o r e S h e e t P r o d u c t : D a t e : Time: 1. I n i t i a l ( p e r c e i v e d on f i r s t b i t e ) a) M e c h a n i c a l - H a r d n e s s - F r a c t u r a b i l i t y - V i s c o s i t y b) G e o m e t r i c c ) O t h e r 2. M a s t i c a t o r y ( p e r c e i v e d d u r i n g c h e w i n g ) a) M e c h a n i c a l - Gumminess - C h e w i n e s s - A d h e s i v e n e s s b) G e o m e t r i c c ) O t h e r c h a r a c t e r i s t i c s 3) R e s i d u a l - R a t e o f breakdown - Type o f breakdown - M o i s t u r e a b s o r p t i o n - Mouth c o a t i n g F i g u r e 1.2. T e x t u r e p r o f i l e s c o r e s h e e t ( B o u r n e , 1 9 8 2 ) . -22-s e c o n d , t h e r e s u l t s were r e p e a t a b l e , and v e r i f i e d by o t h e r p a n e l s h e l d a t d i f f e r e n t t i m e s and p l a c e s . T h i s m e thod o f s e n s o r y t e x t u r e p r o f i l e a n a l y s i s p r o -v i d e d a means o f h e l p i n g f o o d r e s e a r c h e r s o b t a i n d e s c r i p t i v e a n d q u a n t i t a t i v e s e n s o r y d a t a on t e x t u r a l c h a r a c t e r i s t i c s o f f o o d p r o d u c t s . I t h a d t h e a d v a n t a g e o f f l e x i b i l i t y , w i t h a p p l i c a t i o n t o any f o o d p r o d u c t o r t e x t u r a l c h a r a c t e r i s t i c , and o b j e c t i v i t y t h r o u g h r i g i d l y d e f i n e d p o i n t s o f r e f e r e n c e a n d n o m e n c l a t u r e . One l i m i t a t i o n o f t h e method was r e l a t e d t o t h e v a r i a b l e d e g r e e o f t h e p a n e l i s t s s k i l l when a p p l y i n g t h e method. 1.4. U l t r a s t r u c t u r e o f t h e m u s c l e o f chum s a l m o n 1.4.1. S t r u c t u r e o f m u s c l e A l t h o u g h t h e r e a r e d i f f e r e n c e s i n t h e g r o s s o r g a n i z a -t i o n b e t w e e n f i s h m u s c l e a n d t e r r e s t r i a l a n i m a l s , B r e m n e r and H a l l e t t , (1985) f o u n d t h a t t h e r e were e n o u g h s i m i l a r i -t i e s a t t h e u l t r a s t r u c t u r a l l e v e l t h a t mammalian m u s c l e c o u l d be u s e d as a model t o d e s c r i b e t h e m u s c l e o f many f i s h s p e c i e s . S h i n d o e t a l . , (1986) compared t h e u l t r a s t r u c t u r e o f s k e l e t a l m u s c l e o f e i g h t s p e c i e s o f f r e s h and s a l t w a t e r f i s h , a n d f o u n d them s i m i l a r t o e a c h o t h e r and s i m i l a r t o t h a t d e s c r i b e d f o r mammalian s k e l e t a l m u s c l e . D i f f e r e n c e s w h i c h e x i s t b e t w e e n f i s h a n d t e r r e s t r i a l a n i m a l s i n c l u d e d t h e a r r a n g e m e n t o f m u s c l e f i b r i l s , t h e amount o f s a r c o p l a s m , t h e r e l a t i o n s h i p o f n u c l e i and m i t o c h o n d r i a t o o t h e r m u s c l e -23-c o m p o n e n t s , a n d t h e a r r a n g e m e n t o f t h e s a r c o p l a s m i c r e t i c u l u m ( B i s h o p and Odense, 1 9 6 7 ) . The g r o s s s t r u c t u r e o f f i s h was d e s c r i b e d b y H a l l e t t a nd Bremner, (1988) a s c o n s i s t i n g o f l o n g s h e e t s o f m u s c l e w h i c h e x t e n d e d f r o m h e a d t o t a i l on b o t h s i d e s o f t h e body. A d j a c e n t m u s c l e b l o c k s c a l l e d myotomes, were s e p a r a t e d f r o m e a c h o t h e r b y t h i n t r a n s v e r s e c o l l a g e n o u s s h e e t s c a l l e d myocommata ( H a l l e t t and Bremner, 1 9 8 8 ) . F i g u r e 1.3 d e p i c t s t h e s t r u c t u r e o f t h i s c o n n e c t i v e t i s s u e . The myocommata i n -t e r c o n n e c t e d w i t h t h e myotomes, and were r e s p o n s i b l e f o r t h e t r a n s m i s s i o n o f t h e m u s c l e ' s c o n t r a c t i l e f o r c e s . T h e myotomes on b o t h s i d e s o f t h e a x i a l s k e l e t o n t a k e t h e s h a p e o f d o u b l e c o n e s w h i c h f i t t o g e t h e r a l o n g t h e l o n g a x i s o f t h e f i s h body, and g i v e t h e a p p e a r a n c e a t t h e s u r f a c e o f a s e r i e s o f "W- s h a p e d " s e c t i o n s r e s t i n g o n t h e i r s i d e s . W i t h i n e a c h myotome, t h e m u s c l e f i b r e s (myomeres) r u n a p -p r o x i m a t e l y p a r a l l e l t o e a c h o t h e r , b u t a t v a r y i n g a n g l e s t o t h e myocommatal s h e e t s . E a c h myomere i s a s i n g l e c e l l , and i s c omposed o f h u n d r e d s o f m y o f i b r e s w h i c h o c c u p y most o f t h e i n t e r i o r o f m u s c l e f i b r e s . M y o f i b r e s a r e e n v e l o p e d and s e p a r a t e d f r o m t h e e x t r a c e l l u l a r e n v i r o n m e n t b y t h e s a r c o l e m m a , a d o u b l e m e m b r a n e c o m p o s e d o f a n i n n e r s e m i - p e r m e a b l e membrane (plasmalemma) and an o u t e r d i f f u s e b a sement l a y e r (Howgate, 1 9 7 9 ) . M y o f i b r i l s a r e t h e b a s i c c o n t r a c t i l e u n i t s o f t h e c e l l s , a n d a r e c l o s e p a c k e d , n o n - b r a n c h i n g , e l o n g a t e d , c y l i n d r i c a l s t r u c t u r e s o f a b o u t - 2 4 -F i g u r e 1.3. A p p e a r a n c e o f m u s c u l a t u r e o f a t y p i c a l f i s h . W h i t e s p a c e s r e p r e s e n t t h e p a r a l l e l m u s c l e c e l l s , " d i v i d i n g " l i n e s r e p r e s e n t t h e myocommata w h i c h s e p a r a t e t h e myomeres. ( L o v e , 1 9 8 0 ) . - 2 5 -o n e m i c r o n i n d i a m e t e r , w h i c h e x t e n d t h e l e n g t h o f t h e m u s c l e f i b r e . S u r r o u n d i n g t h e m y o f i b r i l s i s a l a c e - l i k e n e t w o r k o f t u b u l e s , t h e s a r c o p l a s m i c r e t i c u l u m , a n d t h e T - s y s t e m . P e t e r (1973) d e s c r i b e d t h e T - s y s t e m a s an i n v a g i n a t i o n o f t h e c e l l ' s p l a s m a membrane w h i c h s u r r o u n d e d t h e m y o f i b r i l s midway b e t w e e n t h e Z - l i n e s and t h e c e n t r e s o f t h e A b a n d s . I t s f u n c t i o n was t o c o n d u c t e l e c t r i c a l i m p u l s e s f r o m c e l l membranes i n t o t h e s a r c o p l a s m i c r e t i c u l u m o f m u s c l e d u r i n g c o n t r a c t i o n and r e l a x a t i o n . The s a r c o p l a s m i c r e t i c u l u m l i e s o n e i t h e r s i d e o f t h e T - t u b u l e s . The s a r c o p l a s m was d e -s c r i b e d by D u b o w i t z , (1985) a s t h e c y t o p l a s m o f t h e f i b r e , a n d c o n t a i n s m i t o c h o n d r i a , t h e t u b u l e membrane s y s t e m , m i c r o t u b u l e s , i n t e r m e d i a t e f i l a m e n t s , G o l g i a p p a r a t u s , g l y -c o g e n , a n d f r e e l i p i d d r o p l e t s . F i g u r e 1.4 d e s c r i b e s t h e m a j o r s u b c e l l u l a r components o f a m u s c l e c e l l . O b s e r v a t i o n o f a l o n g i t u d i n a l s e c t i o n o f a m u s c l e f i b r e r e v e a l e d a r e p e t i t i o u s p a t t e r n o f l i g h t a n d d a r k b a n d s , w h i c h was t h e r e s u l t o f a r e g u l a r o v e r l a p p i n g a r r a n g e m e n t o f t h i c k and t h i n f i l a m e n t s w h i c h made up t h e m y o f i b r i l . T h e s e r e p e t i t i o u s s t r u c t u r e s , s a r c o m e r e s , were a r e s u l t o f t h e r e g u l a r a l t e r n a t i o n o f d i f f e r e n t p r o t e i n s , and were c h a r a c -t e r i s t i c o f t h e s t r i a t e d p a t t e r n o f s k e l e t a l m u s c l e ( D u b o w i t z , 1 9 8 5 ) . S h i n d o , e t a l . (1986) f o u n d a marked d i f -f e r e n c e i n t h e r e l a t i v e w i d t h o f I , a n d A b a n d s o f f i s h m u s c l e a s c o m p a r e d t o m a m m a l i a n m u s c l e . I n b o t h w h i t e -26-Sarcolemma N u c l e i , r i b o s o m e s M y o f i b r i l s I n t e r m y o f i b r i l l a r y - t u b u l a r complex T - t r a n s v e r s e l — s y s t e m S a r c o p l a s m i c 1 — r e t i c u l u m C o n t r a c t i l e p r o t e i n s o f m y o f i l a m e n t s i . e . m y o s i n , - a c t i n , t r o p o m y o s i n , t r o p o n i n , a c t i n i n , M - p r o t e i n s M i t o c h o n d r i a L ysosomes G o l g i a p p a r a t u s G l y c o g e n p a r t i c l e s , l i p i d b o d i e s S o l u b l e p r o t e i n s and enzymes o f c a r b o h y d r a t e m e t a b o l i s m F i g u r e 1.4. M a j o r s u b c e l l u l a r components o f myotomal m u s c l e . ( P e t e r , 1973) -27-and d a r k m u s c l e , t h e r e l a t i v e w i d t h o f t h e I bands was l e s s t h a n t h a t u s u a l l y i l l u s t r a t e d f o r m a m m a l i a n s t r i a t e d m u s c l e s . The w o r k e r s were n o t c o n v i n c e d however, t h a t t h i s f i n d i n g was a c h a r a c t e r i s t i c f e a t u r e o f a l l f i s h m u s c l e , and s u g g e s t e d t h a t i t m i g h t be due t o s h r i n k a g e w h i c h o c c u r r e d d u r i n g p r e p a r a t i o n o f t h e s p e c i m e n s . B i s h o p a n d O d e n s e (1967) d e s c r i b e d two h i s t o l o g i c a l l y d i s t i n c t t y p e s o f s t r i a t e d m u s c l e i n a m a r i n e t e l e o s t . The b u l k o f t h i s t i s s u e was a l i g h t brown, o r p i n k c o l o u r e d mass c a l l e d w h i t e m u s c l e . T h i s t i s s u e was t h o u g h t t o be s i m i l a r t o t y p e I I f i b r e s i n humans, and was c h a r a c t e r i z e d by l a r g e f i b r e s i z e , l e s s s a r c o p l a s m , l e s s g l y c o g e n , a n d f e w e r m i t o c h o n d r i a t h a n t h e s e c o n d t y p e o f s t r i a t e d m u s c l e . W i t h few b l o o d c a p i l l a r i e s , i t was t h o u g h t t o be c o n s t r u c t e d t o f a c i l i t a t e t h e l a r g e s t p o s s i b l e number o f c o n t r a c t i l e u n i t s , and g e a r e d f o r a n a e r o b i c p r o d u c t i o n o f e n e r g y d u r i n g swim-ming a t h i g h s p e e d s (Dunn e t a l . , 1 9 8 1 ) . A t t h e s u r f a c e o f t h e w h i t e m u s c l e , l y i n g i n a b a n d a l o n g t h e f i s h ' s m i d - l i n e , was a s t r i p o f brown c o l o u r e d t i s s u e c a l l e d d a r k , o r r e d m u s c l e . T h e s e m u s c l e f i b r e s were s i m i l a r t o t y p e I f i b r e s i n h u m ans, a n d w e r e s m a l l e r i n d i a m e t e r , w i t h a l o n g e r s a r c o m e r e t h a n t h e w h i t e f i b r e s . T h e y w e r e r i c h l y v a s c u l a r i z e d , h a d a l a r g e number o f m i t o c h o n d r i a w i t h w e l l d e v e l o p e d i n n e r c r i s t a e , and t e n d e d t o f a t i g u e and c o n t r a c t m o r e s l o w l y t h a n w h i t e m u s c l e ( F r a n z i n i - A r m s t r o n g a n d P o r t e r , 1964; P e t e r , 1 9 7 3 ) . A c r o s s s e c t i o n o f t h e w h i t e -28-m u s c l e f i b r e s r e v e a l e d t h a t t h e m y o f i b r i l s were e l l i p t i c a l , o r n e a r l y c i r c u l a r i n t h e c e n t r e o f m u s c l e f i b r e s , b u t r e c t -a n g u l a r , o r m o re a s y m m e t r i c a t t h e p e r i p h e r y o f m u s c l e f i b r e s . D ark m u s c l e f i b r e s were p o l y g o n a l , a n d l e s s asym-m e t r i c a t any p o r t i o n o f i t s c r o s s s e c t i o n ( S h i n d o e t a l . , 1 9 8 6 ) . P r o c t o r e t a l . , (1980) n o t e d t h e p r e s e n c e o f a t h i r d f i b r e t y p e c a l l e d p i n k f i b r e s i n t h e m u s c u l a t u r e o f Salmo t r u t t a , brown t r o u t . The p i n k f i b r e s were s i t u a t e d between t h e r e d a n d w h i t e f i b r e s , w e r e i n t e r m e d i a t e i n s i z e a n d c h a r a c t e r between t h e l a r g e w h i t e , and s m a l l r e d f i b r e d i a m -e t e r , and were t h o u g h t t o h a v e a r o l e d u r i n g i n t e r m e d i a t e swimming s p e e d s . 1.4.2. M u s c l e d e g e n e r a t i o n Kakwas a n d Adams, (1985) u s e d a number o f c r i t e r i a t o d i s t i n g u i s h m u s c l e d i s e a s e s . F a c t o r s i n c l u d e d t h e c h a r a c t e r and d i s t r i b u t i o n o f t h e c h a n g e s o f t h e m u s c l e f i b r e and s u p -p o r t i n g t i s s u e , t h e f u n c t i o n a l e f f e c t o f t h e l e s i o n s , a n d t h e p r e s e n c e o r a b s e n c e o f g e n e t i c , o r o t h e r f a c t o r s . P a t h o l o g i c a l c h a n g e s f o u n d i n m u s c l e b i o p s i e s may a f f e c t t h e m y o f i b r i l s , o r t h e i r s u p p o r t i n g membranes ( D u b o w i t z , 1 9 8 5 ) . F o r e x a m p l e , i n t e r r u p t i o n s o r b r e a k s i n t h e c o n t i n u i t y o f t h e sarcolemma o f t e n o c c u r r e d w i t h n e c r o t i z i n g i n j u r i e s , b u t were n o t n e c e s s a r i l y l e t h a l t o t h e c e l l . The s u r f a c e o f t h e m u s c l e c e l l s may a p p e a r i r r e g u l a r , p o s s i b l y a r e s u l t o f a d e c r e a s e i n f i b r e v o l u m e ( P e t e r , 1 9 7 3 ) . F o l d i n g o f t h e -29-s a r c o l e m m a i s a l s o common, a n d t h e b a s e m e n t membrane may s p l i t away f r o m t h e p l a s m a membrane, c r e a t i n g e x t e n s i v e f o l d s i n t h e e x t r a c e l l u l a r s p a c e . L o s s , o r a l t e r a t i o n o f m y o f i l a m e n t s was c o n s i d e r e d t h e m o s t common a b n o r m a l i t y i n d i s e a s e d m u s c l e ( D u b o w i t z , 1 9 8 5 ) . I t was a l s o one o f t h e most d i f f i c u l t c h a n g e s t o a s s e s s , b e c a u s e e v e n n o r m a l m u s c l e o f t e n d e p a r t s f r o m t h e " c l a s s i c a l a p p e a r a n c e " . P a t h o l o g i c a l c h a n g e s o b s e r v e d i n m u s c l e b i o p s i e s i n c l u d e d a l t e r a t i o n s o f t h e f i b r e s i z e a n d d i s t r i b u t i o n , s p l i t t i n g , b r a n c h i n g , and l o s s o f m y o f i b r i l s . I n s e v e r e n e c r o s i s , c h a r a c t e r i s t i c m y o f i l a m e n t s t r u c t u r e may be l o s t and r e p l a c e d by an amor-phous g r a n u l a r m a t e r i a l . S e l e c t i v e l o s s , o r a b n o r m a l i t i e s o f a p a r t i c u l a r r e g i o n may i n c l u d e I o r A b a n d l o s s a n d Z - l i n e a l t e r a t i o n s s u c h a s s t r e a m i n g , b r o a d e n i n g ( t h i c k e n -i n g ) , i r r e g u l a r i t i e s ( z i g z a g a p p e a r a n c e ) , o r c o m p l e t e d i s i n -t e g r a t i o n . Kakwas and Adams, (1985) c a t e g o r i z e d c h a n g e s i n m u s c l e f i b r e s i n t o t h r e e g r o u p s . The f i r s t g r o u p was c a l l e d s e g -m e n t a l n e c r o s i s i n w h i c h t h e r e was a c l u s t e r i n g o f a few o r many n e c r o t i c s a r c o m e r e s . The s e c o n d g r o u p , m y o f i b r i l l a r d i s f i g u r e m e n t , was u s e d t o c l a s s i f y a s e r i e s o f s u b c e l l u l a r l e s i o n s w h i c h were o f t e n d i f f i c u l t t o d i s t i n g u i s h f r o m e l e c -t r o n m i c r o g r a p h a r t i f a c t s . The t h i r d g r o u p , m o r p h o l o g i c a l c h a n g e s , i n c l u d e d c h a n g e s i n f i b r e t y p e s and a l t e r a t i o n s o f t h e o r g a n e l l e s . -30-B a s e d on mammalian s t u d i e s , i t i s r e a s o n a b l e t o b e l i e v e t h a t t h e p h y s i c a l s t r e s s t h a t o c c u r s d u r i n g s p a w n i n g m i g r a -t i o n c o n t r i b u t e s t o m u s c l e d e t e r i o r a t i o n o f chum. F a c t o r s w h i c h a r e l i k e l y r e s p o n s i b l e f o r c h a n g e s i n t h e u l t r a s t r u c t u r e o f s p a w n i n g s a l m o n a r e r e l a t e d t o f i r s t , t h e f a t i g u e a s s o c i a t e d w i t h b o t h r a p i d b u r s t s o f e n e r g y and p r o -l o n g e d , r e p e t i t i o u s , l o w f o r c e g a t h e r i n g t y p e s o f c o n t r a c -t i o n s ( B e l c a s t r o e t a l . , 1 9 8 5 ) , s e c o n d t h e e f f e c t s o f s t a r -v a t i o n d u r i n g m i g r a t i o n , and t h i r d , t h e h o r m o n a l c h a n g e s o c -c u r r i n g d u r i n g d e v e l o p m e n t o f t h e r e p r o d u c t i v e s y s t e m . B e l c a s t r o e t a l . , (1985) o b s e r v e d c h a n g e s i n r a t m u s c l e w h i c h h a d u n d e r g o n e p r o l o n g e d , r e p e t i t i o u s c o n t r a c t i o n s . The s a r c o m e r e a p p e a r e d wavy and d i s o r i e n t e d . M y o f i l a m e n t d i s s o l u t i o n was o b s e r v e d a t t h e l a s t s t a g e o f t h e f a t i g u e p r o c e s s , and was o n l y a s s o c i a t e d w i t h e x t r e m e c o n d i t i o n s o f m u s c l e u s e . F r i d e n e t a l . , ( 1 9 8 3 ) o b s e r v e d m y o f i b r i l l a r damage f o l l o w i n g i n t e n s e e c c e n t r i c e x e r c i s e i n man. L i g h t m i c r o g r a p h s o f s e m i - t h i n s e c t i o n s r e v e a l e d f o c a l d i s t u r -b a n c e s o f t h e c r o s s - s t r i a t e d b a n d p a t t e r n o f t h e m y o f i l a m e n t . A t t h e e l e c t r o n m i c r o s c o p i c l e v e l , t h e s e f o c a l d i s t u r b a n c e s were f o u n d t o be a r e s u l t o f marked s t r e a m i n g , b r o a d e n i n g , and t o t a l d i s r u p t i o n o f m y o f i b r i l Z-bands. Gaps i n t h e r e g u l a r l a t t i c e p a t t e r n o f t h e Z-bands, and an a p p a r -e n t l o s s o f t h e t h i c k m y o f i l a m e n t s were a l s o o b s e r v e d . I t was f o u n d t h a t e x e r c i s e t o t h e p o i n t o f e x h a u s t i o n r e s u l t e d i n f i b r e n e c r o s i s a n d an i n c r e a s e i n t h e a c i d h y d r o l y t i c - 3 1 -a c t i v i t y o f m u s c l e enzymes. No mechanism f o r t h e s e c h a n g e s was known, however i t was s u g g e s t e d t h a t l y s o s o m a l enzymes were l i b e r a t e d i n t h e damaged f i b r e s , and may have been r e -s p o n s i b l e f o r t h e i n i t i a t i o n o f t h e d e g r a d a t i o n o f m y o f i b r i l l a r m a t e r i a l . G e o r g e e t a l . , ( 1 9 8 7 ) s t u d i e d u l t r a s t r u c t u r a l c h a n g e s i n t h e b r e a s t m u s c l e o f t h e C a n a d a g o o s e a f t e r m i g r a t i o n , a t i m e o f e x c e s s m u s c l e u s e and f a -t i g u e . E x c e s s Z - l i n e s t r e a m i n g , a n d a n i n c r e a s e i n t h e number o f l y s o s o m e - l i k e b o d i e s were a p p a r e n t i n t h e m u s c l e . The h i s t o p a t h o l o g i c a l c h a n g e s i n t h e m u s c l e were c o r r e l a t e d t o l y s o s o m a l e n z y m e a c t i v i t y , a n d i t was n o t e d t h a t f l i g h t - i n d u c e d n e c r o t i c c h a n g e s i n t h e m u s c l e m i m i c k e d t h e symptoms o f human and a n i m a l m y o p a t h i e s . P a l e s o f t e x u d a t i v e (PSE) p o r k i s a w e l l known phenome-non whereby s t r e s s e d a n i m a l s e x h i b i t an a c c e l e r a t e d r a t e o f p o s t mortem g l y c o l y i s , w h i c h c a u s e s r i g o r m o r t i s t o o c c u r a t a l o w pH w h i l e t h e c a r c a s s t e m p e r a t u r e i s s t i l l h i g h . The u l t r a s t r u c t u r a l d i f f e r e n c e s b e t w e e n n o r m a l a n d PSE m u s c l e were i n v e s t i g a t e d by C a s s e n s e t a l . ( 1 9 6 3 ) . W i t h i n 40 m i n -u t e s p o s t m o r t e m , s a r c o p l a s m i c m a t e r i a l was d i s r u p t e d , I - b a nds were "clumped" t o g e t h e r , and f i b r e b u n d l e s a p p e a r e d d i s c o n n e c t e d . No marked a l t e r a t i o n i n m y o f i b r i l s were o b -s e r v e d i n t h e f i r s t 24 h o u r s o f n o r m a l p o r k m u s c l e . C l o k e e t a l . (1981) a l s o o b s e r v e d an u n u s u a l d e g r e e o f d i s r u p t i o n i n t h e PSE m u s c l e a f t e r d e a t h , i n c l u d i n g m y o f i b r i l l a r b r e a k -down w i t h l o s s o f m a t e r i a l a t t h e Z - l i n e s . -32-1.4.3. S t a r v a t i o n The e n e r g y t h a t n o n - f e e d i n g , m a t u r i n g s a l m o n r e q u i r e f o r s u r v i v a l , m i g r a t i o n , a n d g onad d e v e l o p m e n t i s o b t a i n e d f i r s t by u t i l i z i n g a v a i l a b l e l i p i d s t o r e s i n t h e l i v e r a n d m u s c l e s . When t h i s s u p p l y becomes d e p l e t e d , t h e r e i s e x t e n -s i v e u s e o f t h e p r o t e i n s f r o m t h e w h i t e m u s c l e f o r e n e r g y ( L o v e , 1 9 8 0 ) . B e a r d a l l and J o h n s t o n (1985) s t u d i e d t h e u l t r a s t r u c t u r e o f myotomal m u s c l e o f t h e low f a t f i s h , P o l l a c h i u s v i r e n s L. ( s a i t h e ) d u r i n g a p e r i o d o f s t a r v a t i o n . A f i f t y p e r c e n t l o s s i n body w e i g h t , an 80% l o s s i n t i s s u e g l y c o g e n s t o r e s , a n d a t r o p h y o f t h e f a s t ( w h i t e ) a n d s l o w ( r e d ) t w i t c h myotomal m u s c l e f i b r e s was o b s e r v e d . A t r o p h y o f t h e m u s c l e r e s u l t e d i n a d e c r e a s e i n t h e m y o f i b r i l c r o s s - s e c t i o n a l a r e a . A f t e r 74 d a y s o f s t a r v a t i o n , t h e v o l u m e o f t h e m y o f i b r i l r e d u c e d f r o m 84 t o 62% o f t h e m u s c l e c e l l . A f t e r 3 m o n t h s , t h e mean c r o s s - s e c t i o n a l a r e a o f t h e m y o t o m a l m u s c l e ha d d e c r e a s e d by 50%, and t h e volume d e n s i t y o f t h e m y o f i b r i l h a d d e c r e a s e d by 25%. I n a d d i t i o n , a p r e f e r e n t i a l d e g e n e r a t i o n o f M - l i n e p r o t e i n s , and o f t h i c k f i l a m e n t s a t t h e p e r i p h e r y o f t h e m y o f i b r i l was o b s e r v e d . I n s e v e r e l y a t r o p h i e d m u s c l e f i b r e s , t h e m y o s i n i n m y o f i b r i l s and w h o l e f i b r e s w e r e d e g r a d e d , h o w e v e r t h e s a r c o m e r e s s t i l l m a i n -t a i n e d t h e i r a l i g n m e n t . I n 1981, J o h n s t o n s t u d i e d t h e e f -f e c t s o f s t a r v a t i o n on t h e u l t r a s t r u c t u r e o f s k e l e t a l m u s c l e f r o m a m a r i n e f l a t f i s h . A t r o p h y o f w h i t e m u s c l e f i b r e s was -33-a s s o c i a t e d w i t h a d e c r e a s e i n t h e d i a m e t e r a n d f r a c t i o n a l v olume o c c u p i e d by m y o f i b r i l s . A l s o common was p e r i p h e r a l d e g e n e r a t i o n o f Z - d i s k s , w h i c h c a u s e d t h e u n r a v e l i n g o f t h e t h i n f i l a m e n t l a t t i c e . J o h n s t o n s u g g e s t e d t h a t t h i s e f f e c t c a u s e d a p a r t i a l d e c r e a s e i n m y o f i b r i l l a r d i a m e t e r , and t h e m a i n t e n a n c e o f c o n t r a c t i l e f u n c t i o n i n s t a r v e d f i s h . I n s e -v e r e l y d e g e n e r a t i n g w h i t e f i b r e s , d i s o r g a n i z e d t h i c k a n d t h i n f i l a m e n t s , a n d numerous m u l t i - m e m b r a n e l y s o s o m e - l i k e v e s i c l e s were o b s e r v e d . I t was s u g g e s t e d t h a t t h i s g r a d u a l r e d u c t i o n i n m y o f i b r i l l a r d i a m e t e r p r o v i d e d a m e c h a n i s m w h e r e b y m y o f i b r i l s c o u l d be u s e d a s an e n e r g y s o u r c e w h i l e s t i l l m a i n t a i n i n g some c o n t r a c t i l e f u n c t i o n s . T h i s h y p o t h e -s i s was s u p p o r t e d b y L o v e (1980) who s t u d i e d h i s t o l o g i c a l c h a n g e s i n t h e m u s c l e c e l l s o f t h e f i s h C y p r i n u s c a r p i o . E v e n a f t e r 14 months o f s t a r v a t i o n , t h e p a r a l l e l o r i e n t a t i o n o f m y o f i l a m e n t s i n t h e s e f i s h , a l t h o u g h l a r g e l y d e s t r o y e d , s t i l l h a d some r e c o g n i z a b l e m y o f i b r i l s . B e a r d a l l a n d J o h n s t o n ( 1 9 8 5 ) l i s t e d f o u r s t a g e s o f s t a r v a t i o n - i n d u c e d d e g e n e r a t i o n t h a t c o u l d be f o u n d i n t h e w h i t e m u s c l e o f m a r i n e f l a t f i s h . A t s t a g e o n e , t h e m y o f i b r i l s h a d a n o r m a l a p p e a r a n c e . I n t h e s e c o n d s t a g e , t h e d i a m e t e r o f t h e m y o f i b r i l s were r e d u c e d , and t h e p e r i p h -e r a l t h i c k f i l a m e n t s ( m y o s i n ) were a b s e n t . I n t h e t h i r d s t a g e , t h i c k m y o f i l a m e n t s were a b s e n t , l e a v i n g t h e t h i n f i l -a ments and Z - d i s c i n t a c t . I n t h e f o u r t h s t a g e o f d e g e n e r a -t i o n , t h e m y o f i b r i l h a d b e e n c o m p l e t e l y , o r l a r g e l y -34-d i s r u p t e d , and p h a g o c y t o s e d . H a a r d (1987) o b s e r v e d t h a t b e t w e e n 20 t o 40% o f t h e c a t c h o f t h e A m e r i c a n p l a i c e ( H i p p o g l o s s o i d e s p l a t e s s o i d e s ) was u n s u i t a b l e f o r m a r k e t b e c a u s e o f a j e l l i e d c o n d i t i o n w h i c h was u n a c c e p t a b l e t o c o n s u m e r s . I n c o n t r a s t t o t h e n o r m a l g r e y i s h c o l o u r a n d f i r m t e x t u r e o f t h e m u s c l e , t h e f i l l e t s o f t h e s e f i s h were j e l l y - l i k e , g l o s s y , and o p a l e s -c e n t i n a p p e a r a n c e . The c o n n e c t i v e t i s s u e s e p t a was w i d e and t r a n s l u c e n t , and t h e c o n n e c t i v e t i s s u e i t s e l f was l o o s e , and i n d e f i n i t e i n n a t u r e . The c u t f a c e o f t h e f i l l e t s was s m o o t h , a n d t h e myotomes were p u l l e d away f r o m e a c h o t h e r (Templeman and A n d r e w s , 1 9 5 6 ) . P r o x i m a t e a n a l y s i s o f t h e n o r m a l a n d j e l l i e d f i l l e t s r e v e a l e d t h a t t h e w a t e r c o n t e n t i n t h e j e l l i e d f i l l e t s i n c r e a s e d f r o m t h e n o r m a l 78%, t o 89%, and t h e c r u d e p r o t e i n was r e d u c e d f r o m 18 % t o 9%. G e l e l e c t r o p h o r e s i s o f t h e m y o f i b r i l l a r p r o t e i n showed r e d u c e d l e v e l s o f a c t i n and m y o s i n ( H a a r d , 1 9 8 7 ) . H i s t o l o g i c a l e x -a m i n a t i o n r e v e a l e d t h a t t h e r e w e r e l a r g e s p a c e s b e t w e e n m u s c l e f i b r e s , and l e s s m u s c l e m a t e r i a l p e r u n i t a r e a . L o n g s e c t i o n o f t h e f i b r e s e x h i b i t e d w a v i n e s s , p o s s i b l y a r e s u l t o f f i x a t i o n on t h e l o o s e r s t r u c t u r e o f t h e j e l l i e d t i s s u e (Templeman and Andrews, 1 9 5 6 ) . T h i s j e l l i e d c o n d i t i o n was t h o u g h t t o be due p a r t l y t o p r o t e i n e m a c i a t i o n d u r i n g s e x u a l m a t u r a t i o n , and a l s o t o s t a r v a t i o n o f t h e f i s h . -35-1.5. P r o t e i n a s e a c t i v i t y 1.5.1. L y s o s o m a l s y s t e m Dean and B a r r e t t , (1976) d e s c r i b e d t h e l y s o s o m a l s y s t e m a s s e d i m e n t a b l e i n t r a c e l l u l a r m e m b r a n e - l i m i t e d v e s i c l e s o f v a r y i n g s i z e and s h a p e . S u r r o u n d e d by a s i n g l e t h i c k mem-b r a n e , t h e y c o n t a i n e d a d i v e r s e a r r a y o f enzymes w h i c h g e n -e r a l l y h a d a pH optimum i n t h e a c i d r a n g e , a l t h o u g h some w e r e a c t i v e a t n e u t r a l p H . T h e s e e n z y m e s i n c l u d e d p r o t e i n a s e s , g l y c o s i d a s e s , n u c l e a s e s , p h o s p h o l i p a s e s , p h o s p h a t a s e s , a n d s u l p h a t a s e s . T h e f u n c t i o n o f t h e s e o r g a n e l l e s was t h e d i g e s t i o n o f complex m a c r o m o l e c u l e s . F o r e x a m p l e , c e l l c o m p o n e n t p r o t e i n s w e r e d e g r a d e d t o s m a l l p e p t i d e s a n d a m i n o a c i d s , c o m p l e x l i p i d s t o f r e e f a t t y a c i d s , c a r b o h y d r a t e s t o m o n o s a c c h a r i d e s , a n d n u c l e i c a c i d s t o n u c l e o s i d e s and p h o s p h a t e s . A l t h o u g h a n o v e r a l l i n c r e a s e i n t h e n u m b e r o f l y s o s o m e s , and t h e amount o f c a t h e p t i c a c t i v i t y h a s b e e n r e -p o r t e d d u r i n g p e r i o d s o f r a p i d p r o t e i n d e g r a d a t i o n , t h e d i r e c t i n v o l v e m e n t o f l y s o s o m a l enzymes i n t h e i n i t i a l d e g -r a d a t i o n o f m y o f i b r i l l a r p r o t e i n s i s u n p r o v e n . T h e r e a r e many o t h e r p r o t e o l y t i c enzymes i m p l i c a t e d i n m y o f i b r i l l a r b r e a k d o w n . B e s i d e s t h e enzymes i n t h e l y s o s o m a l s y s t e m , t h e r e a r e a l s o a l k a l i n e p r o t e i n a s e s , s e r i n e p r o t e i n a s e s , and c a l c i u m a c t i v a t e d n e u t r a l p r o t e i n a s e s (CANP) ( S e g a l a n d D o y l e , 1 9 7 8 ) . However, c i r c u m s t a n t i a l e v i d e n c e w h i c h i m p l i -c a t e d l y s o s o m e s a s an i n t r a c e l l u l a r s i t e f o r p r o t e i n b r e a k --36-down i n c l u d e d a number o f p h y s i o l o g i c a l a n d p a t h o l o g i c a l s t u d i e s i n w h i c h t h e o v e r a l l r a t e s o f p r o t e o l y s i s c o r r e l a t e d w i t h t h e a c t i v i t y o f l y s o s o m a l p r o t e i n a s e s , o r w i t h t h e s i z e and number o f l y s o s o m e s . 1.5.2. P r o t e i n t u r n o v e r S e g a l e t a l . (1978) d e s c r i b e d t h e p r o c e s s o f p r o t e i n t u r n o v e r as t h e t r a n s f o r m a t i o n o f a p r o t e i n t o a s u s c e p t i b l e f o r m , i t s u p t a k e t o a l y s o s o m a l d i g e s t i v e s y s t e m , a n d i t s b r e a k d o w n b y p r o t e o l y s i s . I n a n o r m a l e u k a r y o t i c c e l l , i t i s a c c e p t e d t h a t t h e r e a r e two d i s t i n c t f e a t u r e s o f t h e d e g r a d a t i v e p r o c e s s o f p r o t e i n t u r n o v e r . F i r s t , t h e r e i s a r e l a t i o n s h i p between t h e m o l e c u l a r w e i g h t o f t h e p o l y p e p t i d e a n d i t s h a l f - l i f e , f o r example l a r g e m o l e c u l a r w e i g h t p r o -t e i n s b r e a k d o w n more r a p i d l y t h a n s m a l l m o l e c u l a r w e i g h t p r o t e i n s . S e c o n d , t h e p r o t e i n ' s i s o e l e c t r i c p o i n t a n d h a l f - l i f e a r e r e l a t e d , f o r example a c i d i c p r o t e i n s a r e d e -g r a d e d m o r e r a p i d l y t h a n t h e n e u t r a l o r b a s i c o n e s . P r o t e o l y s i s w i t h i n t h e l y s o s o m a l s y s t e m i s i n i t i a t e d b y e n d o p e p t i d a s e s w h i c h c l e a v e bonds w i t h i n a p e p t i d e , and i s c o m p l e t e d by e x o p e p t i d a s e s w h i c h r e l e a s e n e w l y e x p o s e d t e r -m i n a l r e s i d u e s (Dean and B a r r e t t , 1 9 7 6 ) . C o n t r o l o f p r o t e i n t u r n o v e r , a n d t h e d e g r a d a t i o n o f b o t h i n t r a a n d e x t r a c e l l u l a r p r o t e i n was b r o u g h t a b o u t t h r o u g h c h a n g e s i n t h e s p e c i f i c e n z y m e b i n d i n g o f t h e l y s o s o m a l membrane a n d o t h e r s o l i d s u p p o r t s (Dean, 1 9 7 8 ) . The p r e c i s e i n t e r a c t i o n between t h e membrane and t h e enzyme -37-was t h e r e f o r e i m p o r t a n t t o i t s a c t i v i t y . F o r example, t h e c o n v e r s i o n o f f r u c t o s e 1,6 d i p h o s p h a t a s e f r o m a f o r m w i t h a n e u t r a l pH optimum t o a f o r m w i t h an a l k a l i n e pH optimum o c -c u r r e d most r a p i d l y a t pH 6.5 when bound t o a membrane, b u t a t pH 4.5 when s o l u b i l i z e d . T o u n d e r s t a n d t h e r o l e a n d m e c h a n i s m o f l y s o s o m a l enzyme a c t i o n i n c a t a b o l i c p r o c e s s e s , i t i s n e c e s s a r y t o i n -v e s t i g a t e t h e i r c h a r a c t e r i s t i c s . A m a j o r e n d o p e p t i d a s e f o u n d o n l y i n t h e l y s o s o m e i s c a t h e p s i n D. I t s p o i n t s o f c l e a v a g e were f o u n d t o be s t r o n g l y c l u s t e r e d i n more h y d r o -p h o b i c p a r t s o f t h e p o l y p e p t i d e c h a i n s , p r e f e r a b l y a d j a c e n t t o a r o m a t i c s i d e c h a i n s . T h i s enzyme was u n a b l e t o c l e a v e l o w m o l e c u l a r w e i g h t s u b s t r a t e s , a n d r e q u i r e d p e p t i d e s o f f i v e o r more r e s i d u e s . Maximum a c t i v i t y a g a i n s t t h e s u b -s t r a t e h e m o g l o b i n was between t h e pH 2.8-4.0, a l t h o u g h p r e -c i s e optimum pH d e p e n d e d on t h e s o u r c e o f t h e enzyme a n d s u b s t r a t e ( B a r r e t t , 1 9 7 8 ) . T h i s r e q u i r e m e n t f o r a c i d i c c o n -d i t i o n s s u g g e s t e d t h a t i n v i v o f u n c t i o n c o u l d o n l y o c c u r w i t h i n c e l l s , o r i n v e r y r e s t r i c t e d e x t r a c e l l u l a r l o c a t i o n s . I n c u b a t i o n o f c a t h e p s i n D w i t h m y o f i b r i l l a r p r o t e i n s c a u s e d e x t e n s i v e d e g r a d a t i o n o f h i g h m o l e c u l a r w e i g h t (> 200K) p r o -t e i n s s u c h a s c o n n e c t i n , n e b u l i n , f i l a m i n , and m y o s i n h e a v y c h a i n . C a l c i u m a c t i v a t e d n e u t r a l p r o t e i n a s e s (CANP) c o u l d n o t d e g r a d e t h e h i g h m o l e c u l a r w e i g h t p r o t e i n s o r t h e m y o s i n h e a v y c h a i n t o t h e same e x t e n t , h o w e v e r d e s m i n , a p r o t e i n w h i c h s u r r o u n d s t h e m y o f i b r i l a t t h e Z - l i n e was r e m o v e d . -38-The i n c u b a t i o n w i t h CANP f o l l o w e d by c a t h e p s i n D p r o d u c e d an e l e c t r o p h o r e t i c p a t t e r n s i m i l a r t o t h a t o f c a t h e p s i n D. B i r d e t a l . , (1978) s u g g e s t e d t h a t c a t h e p s i n D c a u s e d s u b -s t a n t i a l c o n f o r m a t i o n a l c h a n g e s i n t h e m o l e c u l e , w h i c h e n -a b l e d CANP t o t h e n f u r t h e r d e g r a d e t h e m o l e c u l e . However, i t was n o t e s t a b l i s h e d c o n c l u s i v e l y t h a t t h e two e n z y m e s a c t e d i n a c o o p e r a t i v e f a s h i o n t o d e g r a d e m y o f i b r i l l a r p r o -t e i n s . A n o t h e r e n d o p e p t i d a s e o f i m p o r t a n c e t o t h e f o r m a t i o n o f m y o f i b r i l l a r b r e a k d o w n p r o d u c t s was t h e t h i o l p r o t e i n a s e c a t h e p s i n B. Maximum a c t i v i t y w i t h most s u b s t r a t e s was a t pH 6.0, a l t h o u g h l o w e r pH optimums were f o u n d w i t h some s u b -s t r a t e s b e c a u s e o f t h e d e n a t u r i n g e f f e c t o f t h e a c i d i t y ( B a r r e t t , 1 9 7 8 ) . The i n c u b a t i o n o f c a t h e p s i n B a t pH 5.2 w i t h p u r i f i e d a c t i n and m y o s i n c a u s e d t h e d e g r a d a t i o n o f d e -n a t u r e d m y o s i n t o p r o g r e s s i v e l y s m a l l e r p o l y p e p t i d e f r a g -m ents t o an u l t i m a t e m o l e c u l a r s i z e o f l e s s t h a n 30,000. The d e g r a d a t i o n o f n a t i v e m y o s i n was l e s s e x t e n s i v e , and r e -l e a s e d a h e t e r o g e n e o u s g r o u p o f p o l y p e p t i d e s w i t h m o l e c u l a r w e i g h t s f r o m 10,000-50,000. I n a d d i t i o n , a compound o f mo-l e c u l a r w e i g h t 150,000 was a l s o p r o d u c e d w h i c h was r e s i s -t a n t t o f u r t h e r p r o t e o l y s i s . 1.5.3. S t a r v a t i o n M i l l w a r d e t a l . , (1978) f o u n d t h a t t h e m u s c l e l y s o s o m a l s y s t e m s d u r i n g s t a r v a t i o n were i m p l i c a t e d i n t h e c a t a b o l i c i n c r e a s e s i n p r o t e i n b r e a k d o w n . F o r e x a m p l e , d u r i n g -39-n u t r i e n t d e p r i v a t i o n , p r o t e i n c a t a b o l i s m was e n h a n c e d 30-150% i n s k e l e t a l m u s c l e , and m o r p h o l o g i c a l s t u d i e s showed an i n c r e a s e i n s i z e a n d f r a g i l i t y o f t h e l y s o s o m e s . T h e c h a r a c t e r i s t i c s o f i n t r a c e l l u l a r p r o t e i n d e g r a d a t i o n d u r i n g s t a r v a t i o n w e r e d e s c r i b e d b y D i c e a n d W a l k e r , (1978) who s u g g e s t e d t h a t t h e i n c r e a s e d p r o t e i n c a t a b o l i s m was f u n d a -m e n t a l l y d i f f e r e n t f r o m n o r m a l p r o t e i n d e g r a d a t i o n , and t h a t t h e m e c h a n i s m was n o t s i m p l y a n a c c e l e r a t i o n o f n o r m a l p r o t e o l y s i s . S e v e r a l b i o c h e m i c a l e x p l a n a t i o n s have been p u t f o r w a r d f o r t h i s e n h a n c e d c a t a b o l i s m . The f i r s t s u g g e s t i o n was t h a t t h e c e l l p r o t e i n s b e c a m e more s u s c e p t i b l e t o p r o t e o l y t i c a t t a c k . The n o r m a l l y s t a b l e p r o t e i n s became p r e f e r e n t i a l l y p r o t e i n a s e - s e n s i t i v e , and t h u s t h e a c c e p t e d c o r r e l a t i o n b e t w e e n m o l e c u l a r s i z e a n d h a l f - l i f e , a n d n e t c h a r g e and h a l f - l i f e was r e d u c e d . The s e c o n d s u g g e s t i o n was t h a t t h e s u s c e p t i b i l i t y o f c e l l p r o t e i n s r e m a i n e d t h e same, b u t t h e a c t i v i t y o f t h e d e g r a d a t i v e s y s t e m was e n h a n c e d . Thus t h e s y s t e m i n v o l v e d t h e n o r m a l p r o t e i n d e g r a d a t i v e ma-c h i n e r y , a n d t h e n o r m a l l y s o s o m a l u p t a k e o f c e l l u l a r c o n -s t i t u e n t s . A l l t y p e s o f p r o t e i n s w o u l d be d e g r a d e d a t e n -h a n c e d b u t p r o p o r t i o n a l r a t e s . O t h e r e x p l a n a t i o n s f o r t h e e n h a n c e d d e g r a d a t i o n w e r e h y p o t h e s i z e d t o i n c l u d e t h e i n v o l v e m e n t o f n o n - l y s o s o m a l p r o t e i n a s e s . D i c e and W a l k e r , 1978 o b s e r v e d t h e p r e s e n c e o f n o n - l y s o s o m a l p r o t e i n a s e s i n m u s c l e , a n d t h e i n c r e a s e i n t h e i r a c t i v i t y d u r i n g s t a r v a t i o n . I f t h e s e enzymes were t o -40-u s e s m a l l a n d b a s i c p r o t e i n s a s t h e i r s u b s t r a t e , t h e n t h e n o r m a l c o r r e l a t i o n o f p r o t e i n b r e a k d o w n w i t h i s o e l e c t r i c p o i n t and m o l e c u l a r w e i g h t w o u l d be r e d u c e d . D i c e a n d W a l k e r ( 1 9 7 8 ) commented t h a t w h a t e v e r t h e m e c h a n i s m o c c u r r i n g , d u r i n g s t a r v a t i o n c o n d i t i o n s o r g a n i s m s b e n e f i t f r o m t h e i n c r e a s e d p r o d u c t i o n o f amino a c i d s f o r u s e a s a s o u r c e o f e n e r g y . B e c a u s e t h e c r u c i a l r e g u l a t o r y e n -zymes t e n d n o t t o be t h e s t a b l e p r o t e i n s , i t w o u l d be a d v a n -t a g e o u s t o d e g r a d e t h e s e s t a b l e p r o t e i n s , b e c a u s e t h e y c o u l d be s a c r i f i c e d w i t h t h e l e a s t d i s r u p t i o n o f t h e c e l l ' s a b i l -i t y t o a d a p t m e t a b o l i c a l l y . O t h e r w o r k e r s h a v e a l s o s u g g e s t e d t h e p o s s i b i l i t y o f n o n - l y s o s o m a 1 p r o t e i n a s e s i n t h e i n i t i a l s t a g e s o f m y o f i b r i l l a r d e g r a d a t i o n ( A s g h a r and B h a t t i , 1 9 8 7 ) . F o r e x -a m p l e , CANP h a v e b e e n shown t o d e g r a d e i n t a c t m y o f i b r i l s , remove Z-bands, and d e g r a d e t r o p o n i n c o m p l e x , t r o p o m y o s i n , and C - p r o t e i n . T h i s enzyme was u n a b l e t o d e g r a d e a c t i n and m y o s i n . However, i t was t h o u g h t t o p l a y an i m p o r t a n t r o l e i n t h e d i s a s s e m b l y o f t h i c k and t h i n f i l a m e n t s p r i o r t o t h e s p e c i f i c c l e a v a g e o f m y o s i n l i g h t a n d h e a v y c h a i n s b y m y o f i b r i l l a r p r o t e i n a s e s . F i n a l d e g r a d a t i o n was a c c o m -p l i s h e d v i a t h e l y s o s o m a l pathway. -41-2. CHEMICAL ANALYSIS 2.1. I n t r o d u c t i o n The s p a w n i n g m i g r a t i o n o f chum s a l m o n i s a p e r i o d i n t h e i r l i f e c y c l e i n w h i c h t h e f i s h must e n d u r e s e v e r e e n v i -r o n m e n t a l a n d p h y s i o l o g i c a l c h a n g e s , a n d u n a c c u s t o m e d s t r e s s e s . T h e s e i n c l u d e t h e movement f r o m s a l t t o f r e s h w a t e r , an o f t e n l e n g t h y f r e s h w a t e r m i g r a t i o n , b a c t e r i a l and f u n g a l d i s e a s e , e l e v a t e d w a t e r t e m p e r a t u r e s , s t a r v a t i o n , and s e x u a l m a t u r a t i o n ( M c B r i d e e t a l . , 1 9 6 5 ) . The b i o c h e m i c a l c h a n g e s w h i c h o c c u r i n t h e f i s h i m p a c t upon t h e i r c o m m e r c i a l q u a l i t y . By q u a n t i t a t i n g t h e s e c h a n g e s a t d i f f e r e n t p o i n t s o f t h e m i g r a t i o n , t h e s t a t e o f t h e f i s h may be a c c u r a t e l y d e s c r i b e d , and i t s e c o n o m i c v a l u e a s s e s s e d . The e x t e r n a l a p p e a r a n c e o f t h e f i s h a p p r o x i m a t e l y f o l -l o w s t h e p h y s i o l o g i c a l s t a t e o f t h e a n i m a l , and i s commonly u s e d t o i n d i c a t e t h e s t a g e o f m a t u r i t y (Ando a n d H a t a n o , 1 9 8 6 a ) . I d l e r and T s u y a k i (1958) m e a s u r e d p h y s i c a l c h a n g e s s u c h a s s n o u t l e n g t h , s k i n t h i c k n e s s , a n d w e i g h t , a n d r e -l a t e d t h e s e t o b i o c h e m i c a l i n d i c e s s u c h as c h a n g e s i n f a t , p r o t e i n , a n d m o i s t u r e , and v i s c e r a l w e i g h t . O t h e r c h a n g e s i n c l u d e d a n e a r c o m p l e t e w a s t i n g o f t h e a l i m e n t a r y t r a c t , a n d o t h e r s i g n i f i c a n t a l t e r a t i o n s i n i n t e r n a l o r g a n s ( I d l e r and B i t n e r s , 1 9 6 0 ) . B o t h t h e male and f e m a l e f i s h e x p e r i -e n c e d l o s s i n p r o t e i n and l i p i d and an i n c r e a s e i n m o i s t u r e however, I d l e r and B i t n e r s (1960) r e p o r t e d t h a t t h e f e m a l e s -42-h a d 22% m o r e f a t l o s s a n d 25% more p r o t e i n l o s s i n t h e m u s c l e . H a t a n o e t a l . (1983) compared t h e c h e m i c a l c o m p o s i t i o n o f f a l l ( l a t e ) a n d summer ( e a r l y ) s p a w n i n g chum s a l m o n i n J a p a n . The p r o t e i n c o n t e n t d e c r e a s e d f r o m 20 t o 16%, t h e l i p i d d r o p p e d f r o m 8.5 t o 1.6%, and t h e m o i s t u r e c o n t e n t i n -c r e a s e d f r o m 72 t o 80%. The o b j e c t o f t h i s s t u d y was t o f i r s t a s s e s s t h e e f f i -c i e n c y o f a g r a d i n g s y s t e m w h i c h d i v i d e d t h e f i s h i n t o f o u r g r a d e s b a s e d o n t h e i r e x t e r n a l a p p e a r a n c e . T h e s e c o n d o b j e c t was t o measure t h e c h a n g e s i n c r u d e p r o t e i n , f a t , and m o i s t u r e i n t h e m u s c l e o f t h e m i g r a t i n g chum s a l m o n . 2.2. M a t e r i a l s and Methods 2.2.1. S a m p l i n g F i s h were s a m p l e d o v e r a 2 y e a r p e r i o d . I n t h e f i r s t y e a r , a l l f i s h w e r e o b t a i n e d f r o m a l o c a l p a c k i n g p l a n t e x c e p t f o r t h e s p a w n i n g f i s h w h i c h were c o l l e c t e d f r o m t h e C h i l l i w a c k R i v e r H a t c h e r y ( C h i l l i w a c k , B . C . ) . A l l t h e f i s h w ere g r a d e d , s a m p l e d , washed, d r e s s e d , p a c k a g e d i n d o u b l e h e a v y gauge p o l y e t h y l e n e b a g s , and s t o r e d a t - 3 0 ° C . 2.2.2. G r a d i n g The f i s h were g r a d e d b y c o m p a r i n g t h e i r e x t e r n a l a p -p e a r a n c e t o a s e t o f 9 p i c t u r e s o f chum composed by Anon. ( 1 9 8 6 ) . A t e n t h g r a d e was a d d e d t o i n c l u d e s p a w n i n g f i s h . The 10 g r a d e s were t h e n r e d u c e d t o f o u r g r a d e s by a s s i g n i n g g r a d e s A t o D as g r a d e 1, g r a d e s E t o G as g r a d e 2, g r a d e s H t o I a s g r a d e 3, and " g r a d e J " , any f i s h more ma t u r e t h a n I , a s g r a d e 4. A b r i e f d e s c r i p t i o n o f t h e c r i t e r i a u s e d t o g r a d e t h e f i s h were a s f o l l o w s . G r a d e 1, s i l v e r - b r i g h t s , were b l u e - b l a c k i n c o l o u r w i t h a s i l v e r y s h e e n , a n d no n o -t i c e a b l e c o l o u r c h a n g e s o r w a t e r marks on t h e f i s h . G r a d e 2, i n t e r m e d i a t e s , showed l o s s o f t h e u n i f o r m s i l v e r s k i n c o l o u r , a n d d i s t i n c t c o l o u r c h a n g e s i n t h e f o r m o f p e r p e n -d i c u l a r c o l o u r b a r s w h i c h were l i g h t r e d t o d a r k g r e e n o r b l a c k i n c o l o u r , f r o m t h e v e n t r a l t o d o r s a l s u r f a c e . G r a d e 3, d a r k f i s h , h a d i n t e n s i v e , d i s t i n c t , p e r p e n d i c u l a r c o l o u r b a r s o f d a r k g r e e n o r b l a c k , and t h e s k i n was t h i c k and c o v -e r e d w i t h a l a y e r o f s l i m e . G r a d e 4, s p a w n i n g f i s h , h a d v e r y d a r k g r e e n , b l a c k o r y e l l o w s k i n c o l o u r , v e r y t h i c k s k i n and a h e a v y s l i m e c o v e r . F i g u r e 2.1 i l l u s t r a t e s t h e a p p e a r a n c e o f t h e f o u r g r a d e s o f s a l m o n u s e d i n t h i s s t u d y . 2.2.3. P r o x i m a t e a n a l y s i s 2.2.3.1. T o t a l l i p i d a n a l y s i s T h e t o t a l l i p i d was d e t e r m i n e d b y s l i g h t l y m o d i f y i n g t h e m e t h o d o f B l i g h a n d D y e r ( 1 9 5 9 ) . T h e l i p i d was e x -t r a c t e d f r o m t h e w h i t e m u s c l e b y h o m o g e n i z i n g 25 g o f t h e sample w i t h 25 mL c h l o r o f o r m and 50 mL m e t h a n o l f o r two m i n -u t e s i n a s p a r k f r e e b l e n d e r . 25 mL o f c h l o r o f o r m was t h e n a d d e d t w i c e t o t h e m i x t u r e and h o m o g e n i z e d e a c h t i m e f o r 30 s e c o n d s . The homogenate was f i l t e r e d t h r o u g h Whatman No. 1 f i l t e r p a p e r w i t h a B u c h n e r f u n n e l . A d d i t i o n a l l i p i d was -44-Grade 1 - Silver bright Grade 3 - Dark Grade 4 - Spawning F igure 2 .1. I l l u s t r a t i o n of the appearance of the four grades of chum salmon used in the study. - 4 5 -r e c o v e r e d b y r e - h o r a o g e n i z i n g and r e - f i l t e r i n g t h e r e s i d u a l t i s s u e . The f i l t r a t e was t h e n a l l o w e d t o s e t t l e i n a g r a d u -a t e d c y l i n d e r , t h e volume o f t h e c h l o r o f o r m l a y e r was mea-s u r e d , a n d t h e m e t h a n o l l a y e r was removed. The l i p i d c o n -t e n t was f o u n d by e v a p o r a t i n g t o d r y n e s s 5 mL o f t h e c h l o r o -f o r m m i x t u r e i n a t a r e d r o u n d b o t t o m f l a s k . The c h l o r o f o r m was removed u s i n g a r o t o e v a p o r a t o r i n a w a t e r b a t h a t 4 5°C, a n d d r y i n g t h e r e s i d u e w i t h a s t r e a m o f n i t r o g e n . T h e f l a s k s were s t o r e d i n t h e d a r k i n a vacuum d e s i c c a t o r w i t h g r a n u l a r s i l i c a g e l u n t i l t h e y were w e i g h e d . T o t a l l i p i d was f o u n d u s i n g t h e f o l l o w i n g e q u a t i o n : [ T o t a l l i p i d = W * V1/V2] Where: W = w e i g h t o f l i p i d i n a l i q u o t V I = volume o f c h l o r o f o r m l a y e r V2 = volume o f a l i q u o t 2.2.3.2. M o i s t u r e a n a l y s i s The m o i s t u r e c o n t e n t o f t h e f i s h was f o u n d b y d r y i n g t r i p l i c a t e 5 g s a m p l e s o f t h e chum m u s c l e i n a vacuum o v e n a t 70°C f o r 6 h o u r s . The s a m p l e s were w e i g h e d and r e - d r i e d f o r 1 h o u r t o e n s u r e c o n s t a n t w e i g h t . 2.2.3.3. P r o t e i n d e t e r m i n a t i o n The c r u d e p r o t e i n c o n t e n t o f t h e chum m u s c l e was f o u n d u s i n g a method f o r m i c r o - K j e l d a h l d i g e s t i o n s (Anon., 1 9 7 0 ) . A p p r o x i m a t e l y 9 g o f d r i e d chum m u s c l e ( 0 . 5 - 2 . 5 mg N) was d i g e s t e d i n 30 mL K j e l d a h l d i g e s t i o n f l a s k s w i t h c o n c e n -t r a t e d s u l f u r i c a c i d a n d HgO c a t a l y s t . T h e m i x t u r e was -46-h e a t e d f o r 15 t o 30 s e c o n d s and 1 mL o f 30% h y d r o g e n p e r o x -i d e was added a s an o x i d a n t t o s p e e d up t h e d e c o m p o s i t i o n o f t h e o r g a n i c m a t e r i a l . Once t h e d i g e s t i o n was c o m p l e t e d , t h e s o l u t i o n was d i l u t e d i n 25 mL v o l u m e t r i c f l a s k s , and t h e am-m o n i a c o n t e n t o f t h e s a m p l e s w a s d e t e r m i n e d c o l o u r i m e t r i c a l l y w i t h a T e c h i c o n A u t o a n a l y z e r I I . The p e r -c e n t p r o t e i n was d e t e r m i n e d by m u l t i p l y i n g t h e ppm N by t h e c o n s t a n t 6.25. 2.2.4. C o l o u r 2.2.4.1. A s t a x a n t h i n measurement The a s t a x a n t h i n c o n t e n t was m e a s u r e d by r e s o l u b i l i z i n g t h e l i p i d e x t r a c t e d by t h e B l i g h and D y e r method (1959) i n r e a g e n t g r a d e a c e t o n e , and f i l t e r i n g i t t h r o u g h Whatman No. 2 f i l t e r p a p e r . A b s o r b a n c e r e a d i n g s were m e a s u r e d i n 1 cm p a t h l e n g t h q u a r t z c u v e t t e s u s i n g a S h i m a d z u UV 160 U . V . - V i s i b l e R e c o r d i n g S p e c t r o p h o t o m e t e r ( S h i m a d z u C o r p . , K y o t o , J a p a n ) . The c o n c e n t r a t i o n o f t h e c a r o t e n o i d was c a l -c u l a t e d a s s u m i n g an e x t i n c t i o n c o e f f i c i e n t ( 1 % ) o f 1900 a t 475 nm ( S k r e d e a n d S t o r e b a k k e n , 1 9 8 6 ) . 2.2.4.2. H u n t e r L a b measurements C o l o u r p a r a m e t e r s L , a , a n d b i n t h e raw chum f l e s h were c a l c u l a t e d f r o m t h e r e f l e c t a n c e s p e c t r a r e c o r d e d by a H u n t e r L a b L a b s c a n 6000 S p e c t r o c o l o u r i m e t e r ( H u n t e r A s s o c i -a t e s L a b . I n c . , V i r g i n i a ) i n t h e r e g i o n o f 400-700 nm. A 1/4 i n c h a p e r t u r e was u s e d . Raw m u s c l e segments o f t h e a p -p r o x i m a t e d i m e n s i o n s 2 cm X 1 cm X 0.5 cm were p r e s e n t e d t o -47-t h e i n s t r u m e n t on g l a s s c o v e r s l i p s . T r i p l i c a t e s a m p l e s o f t i s s u e f r o m e a c h f i s h were m e a s u r e d t h r e e t i m e s e a c h a t d i f -f e r e n t l o c a t i o n s , and t h e v a l u e s were a v e r a g e d t o o b t a i n L, a, a n d b r e a d i n g s f o r e a c h s a m p l e . 2.3. R e s u l t s and D i s c u s s i o n 2.3.1. Astaxanthin content The g r a d i n g o f t h e f i s h was v e r y i m p o r t a n t b e c a u s e i t f o r m e d t h e b a s i s o f c o m p a r i s o n f r o m w h i c h a l l c o n c l u s i o n s were made a b o u t d i f f e r e n c e s between g r a d e s . F o r t h i s r e a s o n i t was i m p o r t a n t t o c o n f i r m t h a t t h e e x t e r i o r a p p e a r a n c e o f t h e f i s h c o r r e s p o n d e d t o o t h e r c h a n g e s t h a t o c c u r r e d a s t h e f i s h m a t u r e d . I n s a l m o n i d s , c a r o t e n o i d s a r e o f d i e t a r y o r i g i n a nd b e c a u s e t h e y a r e m a i n l y d e p o s i t e d i n t h e m u s c l e , t h e y a r e r e s p o n s i b l e f o r i t s t y p i c a l r e d - p i n k c o l o u r . W i t h s e x u a l m a t u r a t i o n h o w e v e r , t h e c a r o t e n o i d s a r e m o b i l i z e d f r o m t h e m u s c l e and t r a n s p o r t e d t o t h e i n t e g u m e n t a n d o v a -r i e s v i a t h e s e r u m . A s t a x a n t h i n i s t h e m a i n c a r o t e n o i d f o u n d i n chum, a n d i s t h e one o f t e n m e a s u r e d when f o l l o w i n g t h e l o s s i n c o l o u r o f t h e f i s h (Ando e t a l . , 1 9 8 5 ) . T h e mean v a l u e s o b t a i n e d f r o m t h e a n a l y s i s o f t h e a s t a x a n t h i n c o n t e n t o f t h e f i s h a r e g i v e n i n T a b l e 2.1. A n a l y s i s o f v a r i a n c e o f t h e c o m p l e t e l y r a n d o m i z e d d e s i g n d e -t e r m i n e d t h a t t h e r e was a s i g n i f i c a n t d i f f e r e n c e ( a < 0.05) b e t w e e n a t l e a s t o ne o f t h e g r a d e s . M u l t i p l e c o m p a r i s o n s between t h e g r a d e s were made w i t h s i n g l e d e g r e e o f f r e e d o m -48-T a b l e 2.1. M e a n v a l u e s f o r a s t a x a n t h i n c o n t e n t i n t h e d o r s a l m u s c l e o f chum s a l m o n (mg/kg ) . G r a d e n A s t a x a n t h i n (mg/kg m u s c l e ) 1 5 6.53 (2.6) 3 a 2 6 6.07 (3.3) ab 3 10 2.78 (1.8) be 4 6 1.08 (0.7) c Mean ( s t a n d a r d d e v i a t i o n ) , "n" d e n o t e s sample s i z e . D i f f e r e n t l e t t e r s s i g n i f y a s i g n i f i c a n t d i f f e r e n c e . 1 2 3 -49-c o n t r a s t s ( W i l k i n s o n , 1988) and c o r r e c t e d w i t h t h e i m p r o v e d s e q u e n t i a l r e j e c t i v e B o n f e r r o n i p r o c e d u r e ( H o l l a n d a n d D i P o n z i o C o p e n h a v e r , 1 9 8 7 ) . G r a d e 1 was d i f f e r e n t f r o m g r a d e s 3 and 4. G r a d e 2 was s i g n i f i c a n t l y d i f f e r e n t ( a < 0.05) f r o m g r a d e 4. G r a d e s 3 was s i g n i f i c a n t l y d i f e r e n t rom g r a d s 1 and 4. 2.3.2. H u n t e r L a b v a l u e s B e c a u s e t h e a s t a x a n t h i n was e x t r a c t e d w i t h o u t r i g o r o u s p r o c e d u r e s t o p r e v e n t o x i d a t i o n , t h e H u n t e r c o l o u r i m e t e r was a l s o u s e d t o m e a s u r e c o l o u r c h a n g e s i n t h e m u s c l e i t s e l f . L, a and b v a l u e s were measured, and a r e shown i n T a b l e 2.2. A n a l y s i s o f v a r i a n c e o f g r a d e s i n d i c a t e d t h a t d i f f e r e n c e s e x i s t e d i n t h e p a r a m e t e r s L, a, and b. M u l t i p l e c o m p a r i s o n b y s i n g l e d e g r e e o f f r e e d o m c o n t r a s t s a n d t h e r e j e c t i v e B o n f e r r o n i p r o c e d u r e d e t e c t e d t h e f o l l o w i n g d i f f e r e n c e s b e -tween t h e g r a d e s . ' L ' v a l u e s were f o u n d t o i n c r e a s e w i t h i n c r e a s i n g g r a d e , and g r a d e 1, 2, and 3 were d i f f e r e n t f r o m g r a d e 4. The ' a ' v a l u e s d e c r e a s e d w i t h i n c r e a s i n g g r a d e , g r a d e s 1 and 2 were d i f f e r e n t f r o m 3 and 4, and g r a d e 3 was d i f f e r e n t f r o m g r a d e 4. The 'b' v a l u e s d e c r e a s e d w i t h i n -c r e a s i n g g r a d e , a n d g r a d e s 1 a n d 2 w e r e d i f f e r e n t f r o m g r a d e 3 and 4. The r a t i o s a / L , and a/b p r o v e d t o be s i g -n i f i c a n t l y d i f f e r e n t between e a c h g r a d e e x c e p t between 1 and 2. E x a m i n a t i o n o f T a b l e s 2.1 a n d 2.2 r e v e a l e d t h a t mean a s t a x a n t h i n c o n t e n t was r o u g h l y p r o p o r t i o n a l t o ' a ' v a l u e s . -50-T a b l e 2.2. H u n t e r L a b mean v a l u e s f o r t h e d o r s a l m u s c l e o f chum s a l m o n . G r a d e n L v a l u e 1 6 40.02 (0.51) a 3 2 7 35.82 (0.18) a 3 11 41.59 (0.41) a 4 11 47.08 (0.48) b a v a l u e b v a l u e 10.05 (0.07) a 11.45 (0.16) a 7.80 (0.13) a 10.52 (1.46) a 4.40 (0.09) b 9.40 (0.08) b -1.30 (0.10) c 7.67 (0.13) b "n" d e n o t e s sample s i z e . Mean ( s t a n d a r d e r r o r o f mean). D i f f e r e n t l e t t e r s s i g n i f y a s i g n i f i c a n t (P < 0.05) d i f f e r e n c e . - 51-A more d e t a i l e d l o o k a t t h e r e l a t i o n s h i p was p e r f o r m e d by c o r r e l a t i o n a n a l y s i s w h i c h was c a r r i e d o u t b e t w e e n t h e f o u r g r a d e s o f s a l m o n , t h e c o n c e n t r a t i o n o f a s t a x a n t h i n , t h e H u n t e r L, a and b c o l o u r v a l u e s and t h e r a t i o s a/L and a/b f o r i n d i v i d u a l f i s h . The c o r r e l a t i o n s were t e s t e d f o r t h e i r s i g n i f i c a n c e , a n d t h e r e s u l t s o f t h i s t e s t a r e shown i n T a b l e 2.3. The c o l o u r v a l u e s a/L, a s t a x a n t h i n , a, and a/b were s i g n i f i c a n t l y n e g a t i v e l y c o r r e l a t e d t o g r a d e a t t h e 1% l e v e l , s o t h a t a s t h e g r a d e i n c r e a s e d , t h e i r v a l u e s d e -c r e a s e d . The p a r a m e t e r s b and L were n o t s i g n i f i c a n t . The r a t i o a/L had t h e l a r g e s t r v a l u e . A l t h o u g h t h i s r a t i o i s n o t commonly u s e d i n t h e l i t e r a t u r e a s a measure o f c o l o u r , i t was a l o g i c a l c h o i c e i n t h i s c a s e . The d e c r e a s e i n ' a ' r e f l e c t e d t h e l o s s i n c a r o t e n o i d , s p e c i f i c a l l y a s t a x a n t h i n , a n d t h e i n c r e a s e i n ' L ' v a l u e may h a v e r e -f l e c t e d t h e r e l a t i v e i n c r e a s e i n c o n n e c t i v e t i s s u e a s t h e c o n c e n t r a t i o n o f c o n t r a c t i l e p r o t e i n i n t h e m u s c l e was d i -m i n i s h e d . The c o l o u r o f t h e m u s c l e o f s a l m o n i s an i n d i c a t o r com-monly u s e d i n i n d u s t r y t o a s s e s s i t s q u a l i t y a n d m a t u r i t y . I n t h i s s t u d y i t was f o u n d t h a t t h e l o s s i n a s t a x a n t h i n i n t h e d o r s a l m u s c l e d i d n o t c l e a r l y d i f f e r e n t i a t e between e a c h g r a d e o f chum. The ' a ' H u n t e r v a l u e however, was a b l e t o d i s t i n g u i s h b e t w e e n a l l o f t h e g r a d e s e x c e p t one a n d two. A k s n e s e t a l . (1986) v i s u a l l y a s s e s s e d t h e l o s s o f c o l o u r i n m a t u r i n g A t l a n t i c s a l m o n , Salmo s a l a r . C h a n g e s i n c o l o u r -52-T a b l e 2.3. R e s u l t s f r o m t h e c o r r e l a t i o n a n a l y s i s b e t w e e n g r a d e , H u n t e r L a b v a l u e s , a n d a s t a x a n t h i n c o n t e n t . I n d e p e n d e n t v a l u e 1 r v a l u e a/L -0.796 * * 2 a s t a x a n t h i n -0.746 ** a -0.736 ** a/b -0.750 ** b -0.468 NS L 0.566 NS _ 2 'n', t h e number o f s a m p l e s , was 24. S i g n i f i c a n t a t t h e 1% l e v e l , NS = n o t s i g n i f i c a n t a t t h e 5% l e v e l . -53-were f o u n d t o o c c u r l a t e i n t h e m a t u r a t i o n p r o c e s s , t h u s t h e c o l o u r o f t h e m a t u r i n g f i s h was s i g n i f i c a n t l y l e s s v i v i d t h a n t h e immature f i s h . I t i s i n t e r e s t i n g t o o b s e r v e t h e l a c k o f d i s c r i m i n a t i o n o f t h e a s t a x a n t h i n i n d i s t i n g u i s h i n g between t h e g r a d e s o f t h e s a l m o n , e s p e c i a l l y b e c a u s e t h e ' a ' H u n t e r v a l u e was a m e a s u r e o f t h e r e d n e s s o f t h e f i s h , w h i c h i s p r i m a r i l y due t o a s t a x a n t h i n c o n t e n t . An e x p l a n a t i o n o f t h i s i n c o n s i s -t e n c y i s t h a t t h e i s o l a t i o n t e c h n i q u e f o r a s t a x a n t h i n r e -s u l t e d i n l o s s o f t h e c a r o t e n o i d . T h i s c o u l d h a v e o c c u r r e d f r o m p h y s i c a l l o s s e s d u r i n g t h e f a t e x t r a c t i o n p r o c e s s , o r f r o m c h e m i c a l o x i d a t i o n o f t h e c a r o t e n o i d a t any t i m e d u r i n g t h e e x t r a c t i o n , d r y i n g , o r r e s o l u b i l i z i n g p r o c e s s . B e c a u s e H u n t e r v a l u e s a r e s o e a s i l y o b t a i n e d , and do n o t c a u s e o x i -d a t i o n o f t h e c a r o t e n o i d , t h e y were f o u n d t o be a b e t t e r method o f d e t e r m i n i n g t h e g r a d e o f t h e f i s h by i t s c o l o u r . 2.3.3. P r o x i m a t e a n a l y s i s The r e s u l t s o f t h e a n a l y s i s o f p r o t e i n , m o i s t u r e , a n d l i p i d a r e g i v e n i n T a b l e 2.4. A one-way a n a l y s i s o f v a r i -a n c e was c o m p l e t e d t o d e t e r m i n e w h e t h e r t h e r e was a d i f f e r -e n c e between t h e f i s h g r a d e s f o r t h e t h r e e p a r a m e t e r s . The r e s u l t s f o r c r u d e p r o t e i n r e v e a l e d s i g n i f i c a n t (P < 0.05) d i f f e r e n c e s b e t w e e n t h e g r a d e s . G r a d e s 1, 2, a n d 3 were s i g n i f i c a n t l y d i f f e r e n t (P < 0.05) f r o m g r a d e 4. M o i s t u r e was a l s o s i g n i f i c a n t l y d i f f e r e n t (P < 0.05) between g r a d e s . G r a d e s 1, 2, and 3 were d i f f e r e n t (P < 0.05) f r o m g r a d e 4. -54-T a b l e 2.4. M e a n v a l u e s (% o n a w e t b a s i s ) o f p r o t e i n , m o i s t u r e and l i p i d c o n t e n t o f d o r s a l m u s c l e o f chum s a l m o n . G r a d e P r o t e i n n M o i s t u r e n L i p i d n 1 17 .9 a ( 0 .96) 6 74. 17 a ( 0 . 29) 9 4 .9 a ( 2 . 0 ) 6 2 19 .0 a ( 0 .04) 6 75. 02 a ( 0 . 09) 7 3 .8 a b ( 2 . 1 ) 9 3 18 .3 a ( 0 .13) 11 75. 58 a ( 0 . 27) 11 4 .3 a b ( 2 . 3 ) 6 4 15 .8 b ( 0 .45) 10 79. 47 b ( 0 . 32) 11 1 .0 b ( 0 . 9 2 ) 4 Mean v a l u e s ( s t a n d a r d . e r r o r o f mean), "n" d e n o t e s sample s i z e o f mean. D i f f e r e n t l e t t e r s s i g n i f y s i g n i f i c a n t d i f f e r e n c e . -55-The a n a l y s i s o f l i p i d c o n t e n t r e v e a l e d a s i g n i f i c a n t d i f f e r -e n c e ( P < 0.05) b e t w e e n mean s c o r e s f o r t o t a l l i p i d s , however o n l y g r a d e s 1 and 4 d i f f e r e d s i g n i f i c a n t l y f r o m e a c h o t h e r . T h e s e r e s u l t s s u g g e s t t h a t t h e f i s h e x a m i n e d u n d e r -went a g r a d u a l l o s s o f p r o t e i n and l i p i d , however i t was n o t u n t i l t h e f i s h was a t an a d v a n c e d s t a g e o f s e x u a l m a t u r i t y t h a t t h e c h a n g e s became d r a m a t i c . A k s n e s e t a l . ( 1 9 8 6 ) f o u n d t h a t d e s p i t e d i f f e r e n t e n e r g y n e e d s , c h e m i c a l c h a n g e s i n t h e p r o x i m a t e a n a l y s i s o f male and f e m a l e f a r m e d A t l a n t i c s a l m o n s a m p l e s were t h e same. B i l i n s k i e t a l . (1984) s t u d -i e d t h e e f f e c t o f m a t u r a t i o n on c o h o s a l m o n and f o u n d a s i g -n i f i c a n t c o r r e l a t i o n between t h e m o i s t u r e c o n t e n t o f t h e raw f l e s h and t h e f i n a l q u a l i t y o f t h e c a n n e d p r o d u c t . I t was c o n c l u d e d t h a t m o i s t u r e c o n t e n t o f raw f l e s h was o f p o t e n -t i a l v a l u e a s a q u a l i t y i n d i c a t o r t o d e t e r m i n e t h e a c c e p t -a b i l i t y o f s a l m o n f o r c a n n i n g d u r i n g p r o g r e s s i v e i n c r e a s e i n s e x u a l m a t u r i t y . Samples f o r t h e p r o x i m a t e a n a l y s i s o n l y f r o m t h e d o r s a l m u s c l e o f t h e f i s h . T h i s was done i n o r d e r t o r e d u c e e x p e r -i m e n t a l e r r o r s by a n a l y z i n g m u s c l e f r o m d i f f e r e n t p a r t s o f t h e f i s h . K a r r i c k and T h u r s t o n (1964) t o o k s a m p l e s f r o m t h e nape, c e n t r e , and t a i l p o r t i o n s o f s i l v e r s a l m o n t o compare t h e v a l u e s f r o m t h e p r o x i m a t e a n a l y s i s . The nape ha d t h e h i g h e s t l i p i d and t h e l o w e s t p r o t e i n a n d m o i s t u r e c o n t e n t . The t a i l c o n t a i n e d t h e l o w e s t l i p i d v a l u e s , b u t t h e amount o f p r o t e i n was s i m i l a r t o t h e c e n t r e s e c t i o n . -56-3. TEXTURE ANALYSIS 3.1. I n t r o d u c t i o n The t e x t u r a l c h a r a c t e r i s t i c s o f f i s h m u s c l e a r e a r e -f l e c t i o n o f t h e p r o p e r t i e s and c o n c e n t r a t i o n s o f t h e s t r u c -t u r a l e l e m e n t s o f t h e t i s s u e and o f t h e i r c o m p l e x a r r a n g e -ment i n t h e m u s c l e ( D u n a j s k i , 1 9 7 9 ) . The f i b r o u s p r o t e i n s c o n s t i t u t e up t o 70 t o 80% o f t h e t o t a l p r o t e i n c o n t e n t i n t h e m u s c l e , a n d a r e r e s p o n s i b l e f o r t h e s t r u c t u r e a n d r h e o l o g i c a l p r o p e r t i e s o f t h e f i s h meat. U n l i k e mammalian t i s s u e , c o l l a g e n o u s t i s s u e s a r e p r e s e n t i n r e l a t i v e l y low c o n c e n t r a t i o n s i n f i s h , a r e e a s i l y g e l a t i n i z e d by c o o k i n g , a n d do n o t c o n t r i b u t e t o t h e t e x t u r e o f t h e c o o k e d p r o d u c t ( L o v e e t a l . , 1 9 7 4 ) . H a t a e e t a l . (1990) f o u n d raw f i s h w i t h a f i r m t e x t u r e h a d a s i g n i f i c a n t l y h i g h e r c o l l a g e n c o n -t e n t t h a n s o f t t e x t u r e d f i s h . Once t h e f i s h was c o o k e d how-e v e r , no r e l a t i o n s h i p was f o u n d between c o l l a g e n c o n t e n t and t e n d e r n e s s . I s o e t a l . , (1984) s t a t e d t h a t any c h a n g e s i n t h e t e x t u r e o f f i s h f l e s h d u r i n g h e a t p r o c e s s i n g were p r i m a -r i l y due t o t h e h e a t d e n a t u r a t i o n o f f i b r o u s p r o t e i n i n t h e t i s s u e . L o v e e t a l . (1974) i n v e s t i g a t e d d i f f e r e n c e s i n t e x t u r e o f s t e a m - c o o k e d c o d m u s c l e i n r e l a t i o n t o a number o f param-e t e r s s u c h a s pH, body l e n g t h , and p h y s i o l o g i c a l c o n d i t i o n o f t h e f i s h . A n e g a t i v e c o r r e l a t i o n was d e m o n s t r a t e d b e -tween pH and t e x t u r e . As t h e pH o f t h e m u s c l e i n c r e a s e d , -57-t h e f l e s h became more t e n d e r . Body l e n g t h was a l s o f o u n d t o c o r r e l a t e n e g a t i v e l y w i t h pH, however t h i s r e l a t i o n s h i p was n o t t r u e f o r f i s h d u r i n g t h e i r s p a w n i n g c y c l e . The s t r e s s o f s p a w n i n g c a u s e d t h e d e p l e t i o n o f p r o t e i n , f a t , a n d m o s t i m p o r t a n t , g l y c o g e n i n t h e c o d m u s c l e . Lower p o s t - m o r t e m g l y c o g e n l e v e l s r e s u l t e d i n l e s s l a c t i c a c i d b e i n g f o r m e d , t h u s t h e pH o f t h e m u s c l e d i d n o t d r o p a s much as w o u l d be e x p e c t e d . Huynh (1989) m e a s u r e d t h e c hange i n pH i n spawn-i n g chum s a l m o n and n o t e d an i n c r e a s e i n p o s t mortem pH f r o m 6.2 i n t h e s i l v e r - b r i g h t s t o pH 6.5 f o r t h e d a r k f i s h and pH 7.0 f o r t h e spawned f i s h . H a t a e e t a l . (1990) u s e d l i g h t m i c r o s c o p y and s c a n n i n g e l e c t r o n m i c r o s c o p y t o o b s e r v e t h e r o l e o f m u s c l e f i b r e s i n t h e t e x t u r e o f c o o k e d f i s h . C r o s s s e c t i o n s o f c o o k e d m u s c l e f i b r e s w e r e c o m p a r e d w i t h f i r m n e s s a s m e a s u r e d b y a T e x t u r o m e t e r . A f i r m , d r y t e x t u r e was c o r r e l a t e d w i t h t h i n -n e r , s h o r t e r f i b r e s , and a s o f t , f r i a b l e t e x t u r e was c o r r e -l a t e d w i t h t h i c k e r , l o n g e r m u s c l e f i b r e s . O b s e r v a t i o n o f t h e m u s c l e s a m p l e s a f t e r b e i n g t e s t e d on a T e x t u r o m e t e r r e v e a l e d t h a t t h e f i b r e s were n o t b r o k e n o r d e f o r m e d . I n s t e a d , t h e b u n d l e s a p p e a r e d t o h a v e s l i d , o r s h i f t e d o v e r e a c h o t h e r a n d s e p a r a t e d i n t o i n d i v i d u a l f i b r e s . I n f i r m t e x t u r e d f i s h , t h e r e was c o n s i d e r a b l y l e s s s h i f t i n g o f t h e b u n d l e s t h a n i n t h e s o f t t e x t u r e d f i s h . D u r i n g m a s t i c a t i o n , d i s c r i m i n a t i o n o f t e x t u r e was t h o u g h t t o -58-be a c h i e v e d b y d e t e c t i n g d i f f e r e n c e s i n t h e s l i d i n g movement o f m u s c l e f i b r e s . H a t a e e t a l . (1990) s u g g e s t e d t h a t d u r i n g t h e c o o k i n g p r o c e s s , s a r c o p l a s m i c p r o t e i n s were s q u e e z e d i n t o i n t e r s t i -t i a l s p a c e s , and o n c e c o a g u l a t e d , a c t e d t o impede t h e s l i d -i n g o f m u s c l e f i b r e s o v e r e a c h o t h e r . Thus t h e p r e s e n c e o r a b s e n c e o f h e a t c o a g u l a t e d p r o t e i n i n t h e i n t e r - a n d i n t r a - c e l l u l a r s p a c e s o f t h e m u s c l e f i b r e s d e t e r m i n e d t h e f i r m n e s s and f r i a b i l i t y o f c o o k e d m u s c l e t i s s u e . The I n s t r o n U n i v e r s a l T e s t i n g M a c h i n e i s an i m i t a t i v e t y p e i n s t r u m e n t t h a t i s a b l e t o m e a s u r e t h e d e f o r m a t i o n c h a r a c t e r i s t i c s o f f o o d w i t h a h i g h d e g r e e o f p r e c i s i o n . T h i s m a c h i n e i s a b l e t o m e a s u r e t h e m e c h a n i c a l f a i l u r e o f f o o d s i n many d i f f e r e n t ways d e p e n d i n g o n t h e t e s t c e l l u s e d . T e x t u r e p r o f i l e a n a l y s i s (TPA) i s a t e c h n i q u e t h a t h a s b e e n u s e d s u c c e s s f u l l y t o measure a w i d e v a r i e t y o f t e x -t u r a l a t t r i b u t e s . T h i s t e s t i n g method s i m u l a t e s t h e mouth's c h e w i n g m o t i o n by c o m p r e s s i n g b i t e s i z e s a m p l e s w i t h two f l a t p l a t e s , t w i c e i n a r e c i p r o c a t i n g m o t i o n . B a s e d on t h e f o r c e - t i m e c u r v e o b t a i n e d , a number o f t e x t u r a l p a r a m e t e r s a r e d e r i v e d , o r c a l c u l a t e d t o d e s c r i b e t h e t e x t u r e o f t h e f o o d . A k s n e s e t a l . (1986) s t u d i e d t h e c h a n g e s i n t h e s e n s o r y p r o p e r t i e s o f m a t u r i n g f a r m e d A t l a n t i c s a l m o n . S i g n i f i c a n t c h a n g e s i n o d o u r , f l a v o u r , and t e x t u r e were o b s e r v e d between t h e immature, and m a t u r e f i s h . The t e x t u r e o f t h e immature -59-f i s h was d e s c r i b e d as f i l a m e n t o u s , w i t h a f i r m c o n s i s t e n c y , w h i l e t h e m a t u r e f i s h became w a t e r y , t o u g h a n d " d i s t i n c t l y s o f t " . The b i o c h e m i c a l c h a n g e s , and p h y s i c a l s t r e s s o f s t a r v a -t i o n d u r i n g t h e s p a w n i n g m i g r a t i o n o f chum s a l m o n a d v e r s e l y a f f e c t s t h e t e x t u r e o f t h e raw a n d c o o k e d f l e s h . As t h e f i s h becomes more m a t u r e , t h e m u s c l e l o s e s i t s f i r m , d e s i r -a b l e p r o p e r t i e s , and becomes s o f t and w a t e r y . T h i s d e t e r i o -r a t i o n i n t e x t u r e h a s a s i g n i f i c a n t e f f e c t on consumer a c -c e p t a b i l i t y o f t h i s i m p o r t a n t B r i t i s h C o l u m b i a n commodity. The o b j e c t o f t h i s work was t o d e v e l o p an i n s t r u m e n t a l and s e n s o r y method t o m e a s u r e t h e t e x t u r a l c h a n g e s i n t h e c a n n e d f i s h . M easurements were made w i t h t h e I n s t r o n U n i -v e r s a l T e s t i n g M a c h i n e , and w i t h a s e m i - t r a i n e d t a s t e p a n e l . 3.2. M a t e r i a l s and Methods The f i s h u s e d f o r t h i s work were o b t a i n e d d u r i n g t h e summer a n d f a l l o f 1989 f r o m B.C. P a c k e r s L t d . (Richmond, B . C . ) , and f r o m t h e C h i l l i w a c k h a t c h e r y ( C h i l l i w a c k , B . C . ) . The f i s h w ere g r a d e d , washed, d r e s s e d , and f r o z e n a t - 3 0 ° C f o r s e v e r a l m o n t h s . A f t e r s t o r a g e , t h e f i s h w e r e t h a w e d o v e r n i g h t a t 4 ° C , washed i n c o l d w a t e r , t h e s k i n and m a j o r b o n e s were removed, and t h e f i l l e t s were b u t c h e r e d i n t o 1.5 i n c h t h i c k s t e a k s . T h r e e p i e c e t i n p l a t e c a n s o f t h e d i m e n -s i o n s 307 X 200 ( W e l l s Can Company L i m i t e d , B u r n a b y , B.C.) were p a c k e d w i t h 213 g o f t h e s a l m o n , and 1% u n - i o d i z e d s a l t -60-was a d d e d . The c a n s were r e t o r t e d i n p u r e s t e a m a t 248°F f o r an o p e r a t o r ' s p r o c e s s t i m e o f 65 m i n u t e s i n an FMC 500 W L a b o r a t o r y r e t o r t (FMC C o r p o r a t i o n , S a n t a C l a r a , C A ) , w i t h w a t e r c o o l i n g . T h i s p r o c e s s was b a s e d on known c o m m e r c i a l p r a c t i c e s ( C o l l i n s , 1 9 8 9 ) . The c a n s were h e l d f o r one month a t room t e m p e r a t u r e b e f o r e b e i n g t e s t e d f u r t h e r . Cans f r o m e a c h o f t h e f o u r g r a d e s o f s a l m o n were exam-i n e d b y a s e n s o r y p a n e l a n d a l s o w e r e t e s t e d o b j e c t i v e l y u s i n g a n I n s t r o n U n i v e r s a l T e s t i n g M a c h i n e ( M o d e l 1122, I n s t r o n C o r p . , C a n t o n , MA.). T h r e e c a n s f r o m e a c h f i s h were o p e n e d , d r a i n e d o f f r e e l i q u i d , a n d c o m b i n e d t o r e p r e s e n t one e x p e r i m e n t a l u n i t . The c a n n e d s a l m o n was t h e n f l a k e d u s i n g two f o r k s , i n t o p i e c e s o f a b o u t 3-5 mm. H a l f o f e a c h sample was u s e d f o r s e n s o r y e v a l u a t i o n and h a l f f o r i n s t r u -m e n t a l e v a l u a t i o n o f t e x t u r e . The numbers o f e x p e r i m e n t a l u n i t s and f i s h were a s f o l l o w s : g r a d e 1 c o n s i s t e d o f 11 e x -p e r i m e n t a l u n i t s , 33 c a n s , and 8 f i s h ; g r a d e 2, 4 e x p e r i m e n -t a l u n i t s , 12 c a n s , a n d 5 f i s h ; g r a d e 3, 16 e x p e r i m e n t a l u n i t s , 48 c a n s , and 7 f i s h ; and g r a d e 4 was r e p r e s e n t e d by 7 e x p e r i m e n t a l u n i t s , 21 c a n s , and 8 f i s h . 3.2.1. I n s t r u m e n t a l a n a l y s i s o f t e x t u r e I n s t r u m e n t a l a n a l y s i s was p e r f o r m e d u s i n g t h e I n s t r o n U n i v e r s a l T e s t i n g M a c h i n e b y t h e T e x t u r e P r o f i l e A n a l y s i s (TPA) m ethod o f B o u r n e ( 1 9 7 8 ) . S a m p l e s w e r e p r e p a r e d b y s e v e r i n g t h e end o f a 50 c c s y r i n g e a t i t s z e r o l i n e , p l a c -i n g a n o t c h e d n y l o n d i s k i n t h e b o t t o m o f t h e c y l i n d e r , - 6 1 -a d d i n g 10 g o f t h e f l a k e d f i s h a n d l i g h t l y c o m p r e s s i n g i t w i t h a n o t h e r n y l o n d i s k t o a h e i g h t o f 2 cm and a d i a m e t e r o f 2.5 cm. T h i s s y s t e m e n s u r e d a s t r i c t , c y l i n d r i c a l s a m p le g e o m e t r y a n d a l l o w e d f o r c o r r e c t u n i a x i a l c o m p r e s s i o n . A 5.5 cm f l a t - f a c e d , t e f l o n c o a t e d p l a t e was u s e d t o c o m p r e s s t h e b i t e - s i z e c y l i n d e r s o f f i s h t o 25% o f t h e i r o r i g i n a l h e i g h t , t w i c e , i n a r e c i p r o c a t i n g m o t i o n a t a c r o s s - h e a d s p e e d o f 100 mm/min. F i v e r e p l i c a t e m e a s u r e s o f e a c h sample were c o l l e c t e d . The I n s t r o n was i n t e r f a c e d t o a c o m p u t e r t o a l l o w r e c o r d i n g o f f o r c e / d e f o r m a t i o n c u r v e s i n d i g i t i z e d f o r m a t i n t e r v a l s o f 0.02 s e c o n d s . H a r d n e s s , c o h e s i v e n e s s , c h e w i n e s s , a n d gumminess w e r e c a l c u l a t e d a s d e s c r i b e d b y B o u r n e ( 1 9 7 8 ) . I n c r e m e n t a l v a l u e s o f t h e s l o p e s o f t h e f o r c e / d e f o r m a t i o n c u r v e s were c a l c u l a t e d f r o m t h e d i g i t i z e d d a t a o v e r i n t e r v a l s o f 0.06 s e c o n d s . The maximum s l o p e a s -s o c i a t e d w i t h e a c h c o m p r e s s i o n was e q u a t e d t o i n s t r u m e n t a l f i r m n e s s . 3.2.2. S e n s o r y a n a l y s i s o f t e x t u r e T e x t u r e a n a l y s i s o f t h e s a l m o n was m o d e l e d a f t e r t h e G e n e r a l F o o d s T e x t u r e P r o f i l e method ( B r a n d t e t a l . , 1963; S z c z e s n i a k e t a l . 1 9 6 3 ) . N i n e p a n e l i s t s were s e l e c t e d b a s e d o n p r i o r p a n e l e x p e r i e n c e a n d i n t e r e s t i n t h e p r o j e c t . T h r e e p r e l i m i n a r y t r a i n i n g s e s s i o n s were c o n d u c t e d i n o r d e r t o f a m i l i a r i z e t h e p a n e l i s t s w i t h t h e r a n g e o f t e x t u r e s t o be e x p e c t e d , and t o d e v e l o p by c o n s e n s u s a l i s t o f t e x t u r e a t t r i b u t e s and d e f i n i t i o n s t o d e s c r i b e t h e c a n n e d f i s h . The -62-d e f i n i t i o n s c h o s e n i n c l u d e d f i r m n e s s , t h e f o r c e r e q u i r e d t o co m p r e s s t h e m a t e r i a l between t h e m o l a r s ; d r y n e s s , t h e s e n -s a t i o n o f a p r o g r e s s i v e i n c r e a s e i n f r e e f l u i d s i n t h e o r a l c a v i t y d u r i n g m a s t i c a t i o n ( i . e . t h e o v e r a l l w e t n e s s o f t h e s a m p l e ) ; f i b r o u s n e s s , t h e p e r c e i v e d number o f f i b r e s e v i d e n t d u r i n g m a s t i c a t i o n ; and c h e w i n e s s , t h e t o t a l e f f o r t r e q u i r e d t o p r e p a r e t h e sample t o a s t a t e r e a d y f o r s w a l l o w i n g . The p a n e l i s t s a s s e s s e d t h e f o u r t e x t u r a l a t t r i b u t e s by m a r k i n g a v e r t i c a l l i n e f o r e a c h sample on a 6 i n c h l i n e a r s c a l e w h i c h was a n c h o r e d on b o t h e n d s w i t h r e f e r e n c e s a m p l e s a n d d e -s c r i p t i o n s . T h i s mark r e p r e s e n t e d t h e p a n e l i s t ' s judgement o f t h e i n d i v i d u a l a t t r i b u t e f o r a s a m p l e , r e l a t i v e t o o t h e r s a m p l e s . F i g u r e 3.1 i s an e x a m p l e o f t h e r a t i n g s h e e t s u s e d . Ten h a l f h o u r s e s s i o n s were h e l d t o a s s e s s t h e t e x t u r e o f t h e f i s h a nd i n e a c h s e s s i o n e a c h j u d g e was g i v e n f o u r unknown f i s h s a m p l e s t o a n a l y z e . P a n e l i s t s were a s k e d t o compare t h e t e x t u r e o f t h e s a l m o n s a m p l e s t o two r e f e r e n c e s a m p l e s , f l a k e d ham and c a n n e d t u n a . The f l a k e d ham r e p r e -s e n t e d a p r o d u c t w i t h a s o f t , j u i c y f l e s h , v e r y few f i b r e s p r e s e n t , and l i t t l e e f f o r t n e e d e d t o chew. The c a n n e d t u n a r e p r e s e n t e d a f i s h p r o d u c t w h i c h was f i r m , d r y , f i b r o u s and r e q u i r e d c o n s i d e r a b l e c h e w i n g e f f o r t . B e c a u s e many c a n s o f e a c h r e f e r e n c e p r o d u c t were n e e d e d o v e r t h e t e n s e s s i o n s , an e f f o r t was made t o p u r c h a s e t h e same l o t numbers f o r t h e t u n a a n d ham. T h i s h e l p e d r e d u c e v a r i a t i o n w i t h i n t h e -63-Name: D a t e : EVALUATION OF TEXTURE ATTRIBUTES P l e a s e , e v a l u a t e t h e f i r m n e s s , j u i c i n e s s , c h e w i n e s s and f i b r o u s n e s s o f t h e s e s a m p l e s by m a r k i n g a v e r t i c a l l i n e f o r e a c h s a m p l e o n t h e h o r i z o n t a l l i n e s t o i n d i c a t e y o u r r a t i n g s . L a b e l e a c h v e r t i c a l l i n e w i t h t h e code number o f t h e s ample i t r e p r e s e n t s . FIRMNESS s o f t f i r m DRYNESS wet d r y FIBROUSNESS few many CHEWINESS l i t t l e e f f o r t much e f f o r t COMMENTS: F i g u r e 3.1. Example o f t y p i c a l t e x t u r e s c o r e s h e e t u s e d t o a n a l y z e t e x t u r e o f t h e 4 g r a d e s o f t h e c a n n e d chum s a m p l e s . -64-r e f e r e n c e p o i n t s which would a f f e c t the p a n e l i s t s judgement of the t e s t samples. The judges were asked not t o smoke or d r i n k f o r one hour p r i o r t o the p a n e l . Sensory a n a l y s i s was performed i n i n d i v i d u a l booths under red l i g h t s t o mask any v i s u a l n o n - u n i f o r m i t y between the samples. Water, u n s a l t e d soda water, and u n s a l t e d c r a c k e r s were a v a i l a b l e f o r c l e a n s -i n g the mouth a f t e r t a s t i n g each sample. F o u r a t t r i b u t e s o f t e x t u r e were c o n s i d e r e d by t h e judges and t h e r e was concern t h a t b i a s would occur from the "halo e f f e c t " (Larmond, 1977) i n which a g e n e r a l impression would be formed about the t e x t u r e of the samples. To e l i m i -nate t h i s e f f e c t , the judges were presented w i t h d i f f e r e n t l y coded samples f o r each of the f o u r a t t r i b u t e s and were not t o l d when the samples were a c t u a l l y the same f i s h . The sam-p l e s o f f i s h were p r e s e n t e d t o the p a n e l i s t s i n a random orde r . 3.3. R e s u l t s and D i s c u s s i o n 3.3.1. Instrumental F i g u r e 3.2 shows a t y p i c a l t e x t u r e p r o f i l e c u r v e ob-t a i n e d from the I n s t r o n f o r the canned chum sample. Analy-s i s of the c u r v e s produced by t h e d a t a c o l l e c t i o n program ar e g i v e n i n T a b l e 3.1. T h i s t a b l e r e p r e s e n t s t h e mean v a l u e s o f the parameters f o r each o f the f o u r grades, and t h e i r s t a n d a r d e r r o r s . S t a t i s t i c a l a n a l y s i s by a one-way a n a l y s i s o f v a r i a n c e (ANOVA) was performed t o t e s t the hy-p o t h e s i s t h a t t h e r e was no d i f f e r e n c e between grades f o r the - 6 5 -' I I I ! • M i l l ' M i l l M ; i | i M i ; : * : ' i i i • ,: i M I • • I i I i * Hardness 1 Direction of chart F i g u r e 3.2. Example o f a t y p i c a l I n s t r o n t e x t u r e p r o f i l e a n a l y s i s curve f o r a chum sample. -66-T a b l e 3.1. Mean i n s t r u m e n t a l TPA p a r a m e t e r s o f t h e f o u r g r a d e s o f chum s a l m o n . P a r a m e t e r G r a d e 1 G r a d e 2 Gra d e 3 Gra d e 4 A r e a 1 202 (25 • 0) 152 (32. 2) 170 (20 .0) 139 (29. 3) A r e a 2 78. 5 (9 .58) 56. 6 ( 1 1 . 5) 70 .9(15 .7) 48 .7(11. 0) H a r d 1 8. 33(1 .02) 5. 2 3 ( 1 . 20) 6 .74(0 .85) 5 .0 9 ( 1 . 13) H a r d 2 5. 55(0 .72) 3. 4 8 ( 0 . 85) 4 .54(0 .59) 3 .49(0. 57) C o h e s . 1. 06(0 .04) 1. 0 8 ( 0 . 03) 1 .04(0 .02) 1 .16(0. 0 3 ) 2 S p r i n g . 1. 83(0 .13) 1. 9 9 ( 0 . 07) 2 .00(0 .11) 1 .64(0. 18) S l o p e 1 36. 3 (5 .89) 19. 2 ( 4 . 82) 28 .8 (4 .41) 20 .4 ( 4 . 74) S l o p e 2 38. 2 (6 .03) 20. 6 ( 5 . 79) 30 .2 (4 .92) 20 .5 ( 6 . 13) Gummi 21. 5 (2 .74) 14. 1 ( 3 . 39) 17 .9 (2 .38) 14 .6 ( 3 . 13) Chewi 96. 4(14 .1) 70. 9 ( 1 7 . 6) 84 .1(12 .3) 60 .6(16. 0) 3 n 12 06 14 05 S t a n d a r d e r r o r s a r e p r e s e n t e d i n b r a c k e t s . Two o u t l i e r s were removed f r o m t h e d a t a s e t . "n" = number o f e x p e r i m e n t a l u n i t s . -67-t e x t u r e p a r a m e t e r s . Upon t e s t i n g a s s u m p t i o n s o f ANOVA, a s y s t e m a t i c i n c r e a s e i n s t a n d a r d e r r o r s w i t h i n c r e a s i n g mean was d i s c o v e r e d . The l a c k o f h o m o g e n e i t y o f v a r i a n c e s was a r e f l e c t i o n o f t h e l a r g e r b i o l o g i c a l v a r i a t i o n i n h e r e n t i n t h e t e x t u r e o f l e s s m a t u r e f i s h , t h a n i n s p a w n i n g f i s h . A s q u a r e r o o t t r a n s f o r m a t i o n was p e r f o r m e d o n t h e d a t a i n o r d e r t o i m p r o v e t h e h o m o g e n e i t y o f t h e v a r i a n c e s . Two f i s h w e r e d e l e t e d f r o m t h e d a t a s e t a s o u t l i e r s b e c a u s e t h e i r s t a n d a r d d e v i a t i o n s b e t w e e n r e p l i c a t e m e a s u r e s w e r e much h i g h e r t h a n t h e o t h e r f i s h . The r e s u l t s o f t h e a n a l y s i s o f t h e t r a n s f o r m e d d a t a a r e shown i n T a b l e 3.2. S l o p e 2 and c o h e s i v e n e s s were t h e o n l y p a r a m e t e r s w h i c h were s i g n i f i -c a n t l y d i f f e r e n t b e t w e e n a t l e a s t one o f t h e g r a d e s ( a < 0 . 0 5 ) , b u t h a r d n e s s 1 a n d 2, a n d s l o p e 1 were s i g n i f i c a n t a t a < 0 . 1 0 . A r e a 1 a n d 2, s p r i n g i n e s s , g u m m i n e s s , a n d c h e w i n e s s were n o t d i f f e r e n t between t h e f o u r chum g r a d e s . M u l t i p l e c o m p a r i s o n s b e t w e e n g r a d e s w e r e p e r f o r m e d w i t h s i n g l e d e g r e e o f f r e e d o m c o n t r a s t s , a n d c o r r e c t e d w i t h t h e i m p r o v e d s e q u e n t i a l r e j e c t i v e B o n f e r r o n i p r o c e d u r e . F o r t h e p a r a m e t e r c o h e s i v e n e s s , g r a d e 4 was s i g n i f i c a n t l y d i f f e r e n t b e tween g r a d e s 1, 2, and 3 ( a < 0 . 0 5 ) . G r a d e s 1 and 4 were s i g n i f i c a n t l y d i f f e r e n t f o r s l o p e 2 ( a < 0 . 0 5 ) , a n d f o r h a r d n e s s 1 a n d 2, and s l o p e 1 ( a < 0 . 1 0 ) . The h i g h d e g r e e o f b i o l o g i c a l s c a t t e r i n t h e i n s t r u m e n t a l measurements w h i c h l e a d t o h i g h s t a n d a r d d e v i a t i o n s o f t h e mean v a l u e s c o n t r i b u t e d t o t h e l a c k o f s i g n i f i c a n c e f o r TPA v a r i a b l e s . -68-T a b l e 3.2 P r o b a b i l i t y v a l u e s p r o d u c e d f r o m one-way ANOVAs on t h e s q u a r e r o o t t r a n s f o r m e d I n s t r o n TPA d a t a . TPA P a r a m e t e r P r o b a b i l i t y V a l u e A r e a 1 0.28 (NS) A r e a 2 0.10 (NS) H a r d n e s s 1 0.06 2 H a r d n e s s 2 0.08 2 C o h e s i v e n e s s •''O.OOl 3 S p r i n g i n e s s 0.26 (NS) S l o p e 1 0.06 2 S l o p e 2 0.04 3 Gumminess 0.15 (NS) C h e w i n e s s 0.24 (NS) Two o u t l i e r s removed f r o m t h e d a t a s e t S i g n i f i c a n t a t t h e 10% l e v e l S i g n i f i c a n t a t t h e 5% l e v e l -69-O b s e r v a t i o n o f g e n e r a l t r e n d s showed t h a t g r a d e 1 r e -q u i r e d t h e most f o r c e , o r e n e r g y t o c o m p r e s s t h e s a m p l e , b o t h on t h e f i r s t , and s e c o n d c o m p r e s s i o n , w h i l e g r a d e 4 r e -q u i r e d t h e l e a s t f o r c e . An e x c e p t i o n t o t h i s o b s e r v a t i o n was t h e TPA p a r a m e t e r c o h e s i v e n e s s , w h i c h was f o u n d t o i n -c r e a s e s i g n i f i c a n t l y f r o m g r a d e one t o g r a d e f o u r . T h i s f i n d i n g c a n be e x p l a i n e d b y c o n s i d e r i n g t h e p h y s i o l o g i c a l c h a n g e s i n t h e s a l m o n d u r i n g s p a w n i n g , a n d t h e e f f e c t s o f h e a t p r o c e s s i n g . As t h e f i s h became more m a t u r e , t h e r e was a l o s s i n m y o f i b r i l l a r p r o t e i n s , a r e l a t i v e i n c r e a s e i n c o n -n e c t i v e t i s s u e p r o t e i n s , a n d an i n c r e a s e i n t h e m o i s t u r e c o n -t e n t o f t h e m u s c l e . A k s n e s e t a l . (1986) f o u n d an i n c r e a s e i n t h e h y d r o x y p r o l i n e c o n t e n t o f m a t u r i n g f a r m e d A t l a n t i c s a l m o n . I t was c o n c l u d e d t h a t t h e m e t a b o l i z i n g o f p r o t e i n f r o m t h e m u s c l e t o o k p l a c e a t t h e e x p e n s e o f n o n - c o l l a g e n o u s , and c e l l u l a r p r o t e i n s . Upon r e t o r t i n g t h e f i s h , t h e c o n n e c t i v e t i s s u e was g e l a t i n i z e d , a n d w i t h t h e i n c r e a s e d m o i s t u r e c o n t e n t , i t c o n t r i b u t e d t o f o r m a t i o n o f a s t i c k y , more c o h e s i v e f i n a l p r o d u c t . R e p r o d u c i b i l i t y o f o b j e c t i v e t e x t u r e m e a s u r e m e n t s was a f f e c t e d b y t h e p r e p a r a t i o n o f e x p e r i m e n t a l u n i t s . J o h n s t o n , (1981) f o u n d t h a t t h e o b j e c t i v e e v a l u a t i o n o f f i s h t e x t u r e was d i f f i c u l t b e c a u s e o f v a r i a t i o n s w h i c h e x i s t e d i n t h e f l e s h c o m p o s i t i o n and s t r u c t u r e a l o n g t h e l e n g t h o f t h e f i s h . A k s n e s e t a l . (1986) r e d u c e d v a r i a t i o n by u s i n g o n l y t h e m i d d l e o n e t h i r d o f t h e f i l l e t s f o r o r g a n o l e p t i c - 7 0 -a n a l y s i s . I n t h e p r e s e n t s t u d y , t h r e e c a n s were s a m p l e d r a n d o m l y f r o m d i f f e r e n t l o c a t i o n s a l o n g a f i s h , a n d w e r e c o m b i n e d t o o b t a i n one e x p e r i m e n t a l u n i t . T h i s t e c h n i q u e g r e a t l y r e d u c e d t h e p o s s i b i l i t y o f i n t r o d u c i n g e r r o r b a s e d on d i f f e r e n c e s i n t e x t u r e between t h e v a r i o u s l o c a t i o n s o f t h e f i s h t h a t were c a n n e d . I n o r d e r t o r e d u c e e r r o r due t o v a r i a t i o n i n m u s c l e t e x t u r e w i t h i n t h e c a n s due t o p a c k i n g o f t h e c a n , o r l o c a -t i o n o f c o n n e c t i v e t i s s u e i n t h e m u s c l e , a f l a k e d p r o d u c t was u s e d f o r t h e i n s t r u m e n t a l and s e n s o r y e v a l u a t i o n s . The d e c i s i o n t o u s e f l a k e d s a m p l e s was b a s e d on two o b s e r v a -t i o n s . F i r s t , c a n n e d s a l m o n i s n o t commonly s e r v e d a s a w h o l e c h u n k , t h e r e f o r e f l a k i n g w o u l d more c l o s e l y s i m u l a t e t h e s t a t e i n w h i c h c a n n e d s a l m o n i s c o m m o n l y c o n s u m e d . S e c o n d , i n a s t u d y by B o r d e r i a s e t a l . ( 1 9 8 3 ) , i n s t r u m e n t a l a n d s e n s o r y c o m p a r i s o n s o f c o o k e d f i l l e t s a n d c o o k e d m i n c e o f 5 f i s h s p e c i e s w e r e o b s e r v e d . No c o r r e l a t i o n b e t w e e n s e n s o r y and i n s t r u m e n t a l d a t a was f o u n d f o r t h e c o o k e d f i l -l e t s , b u t s i g n i f i c a n t c o r r e l a t i o n was f o u n d f o r t h e m i n c e d p r o d u c t s . I n a d d i t i o n , t h e c o e f f i c i e n t o f v a r i a t i o n i n t h e raw a n d c o o k e d f i l l e t s was h i g h e r t h a n i n t h e m i n c e d f i s h . B o r d e r i a s e t a l . (1983) f o u n d t h a t t h e a p p l i c a t i o n o f a com-p r e s s i o n t e s t o n t o t h e f i l l e t s c a u s e d t h e myomere l a y e r s t o s l i d e away f r o m t h e f o r c e , t h u s m a k i n g i t i m p o s s i b l e t o r e -p r o d u c e t h e a n a l y s i s . S z c z e s n i a k (1987) s t a t e d t h a t m i n c i n g t h e f i l l e t s e f f e c t i v e l y e l i m i n a t e d t h e h e t e r o g e n e i t y o f t h e - 7 1 -t e s t m a t e r i a l w h i c h was t h e c a u s e o f t h e h i g h d e g r e e o f s c a t t e r i n t h e i n s t r u m e n t a l t e s t . A d i s a d v a n t a g e o f t h e m i n c i n g however was t h a t some t e x t u r a l p a r a m e t e r s c o u l d t h e n n o t be d e t e c t e d . D u n a j s k i (1979) s t a t e d t h a t a d i f f i c u l t y e n c o u n t e r e d i n m e a s u r i n g t h e r h e o l o g i c a l p r o p e r t i e s o f c o o k e d f i s h , was t h a t common i n s t r u m e n t a l m e t h o d s w e r e o f t e n i n e f f e c t i v e . B e c a u s e c o o k i n g o f most f i s h r e s u l t e d i n t o t a l g e l a t i n i z a -t i o n o f t h e c o l l a g e n , t h e s t r u c t u r a l i n t e g r i t y o f t h e myocommata was d i s r u p t e d , and t h e m u s c l e f i b r e s became t h e o n l y e l e m e n t s o f r e s i s t a n c e . I t was recommended t h a t i n -s t r u m e n t a l t e s t s s h o u l d be d e s i g n e d t o m e a s u r e t h e r e s i s -t a n c e o f t h e f i b r e s t o m e c h a n i c a l s t r e s s . However b e c a u s e o f t h e o r g a n i z a t i o n o f t h e f i s h ' s myomeres, p o s i t i o n i n g t h e s a m p l e t o o r i e n t t h e f i b r e s t h e same d i r e c t i o n e a c h t i m e became a n e a r l y i m p o s s i b l e t a s k . T h e t e c h n i q u e o f f o r m i n g t h e s a m p l e s i n a 50 c c s y -r i n g e , was f o u n d t o be v e r y e f f e c t i v e i n p r e p a r i n g i d e n t i c a l c y l i n d e r s o f f i s h . However d e s p i t e h a v i n g 5 r e p l i c a t e sam-p l e s f o r e a c h e x p e r i m e n t a l u n i t , t h e s t a n d a r d d e v i a t i o n s w i t h i n e x p e r i m e n t a l u n i t s were s t i l l q u i t e h i g h . I n a d d i -t i o n t o i n h e r e n t d i f f e r e n c e s w i t h i n and between f i s h o f t h e same g r a d e , i n c o m p l e t e d r a i n i n g o f t h e c a n c o n t e n t s , o r d r y i n g o f t h e f i s h c o u l d a l s o h a v e c a u s e d v a r i a t i o n s i n t h e m e a s u r e m e n t o f t e x t u r e . B e c a u s e s a m p l e s f o r b o t h t h e i n -s t r u m e n t a l a n d s e n s o r y a n a l y s i s came f r o m t h e same t h r e e -72-c a n s , i n s t r u m e n t a l measurements o f t e x t u r e were o n l y p o s s i -b l e a f t e r s e n s o r y a n a l y s i s was c o m p l e t e d . T h i s d e l a y r e -s u l t e d i n some d r y i n g o f t h e f i s h m u s c l e , and may have c o n -t r i b u t e d t o t h e h i g h s t a n d a r d d e v i a t i o n s . 3.3.2. S e n s o r y a n a l y s i s o f t e x t u r e T a b l e 3.3 d i s p l a y s t h e mean r e s u l t s and s t a n d a r d d e v i a -t i o n s f r o m t h e s e n s o r y p a n e l ' s e v a l u a t i o n o f t h e t e x t u r a l d i f f e r e n c e s b e t w e e n t h e f o u r g r a d e s o f chum s a l m o n . An a n a l y s i s o f v a r i a n c e was p e r f o r m e d t o t e s t t h e h y p o t h e s i s t h a t t h e r e was no d i f f e r e n c e b e t w e e n t h e f o u r g r a d e s f o r e a c h a t t r i b u t e . T h i s h y p o t h e s i s was r e j e c t e d however, b e -c a u s e e a c h a t t r i b u t e h a d a t l e a s t one g r a d e d i f f e r e n t f r o m t h e o t h e r t h r e e . O b s e r v a t i o n o f t h e s c o r i n g s y s t e m u s e d by t h e i n d i v i d -u a l j u d g e s l e a d t o t h e c o n c l u s i o n t h a t d e s p i t e t h e i r t r a i n -i n g , d i f f e r e n t j u d g e s t e n d e d t o u s e d i f f e r e n t p a r t s o f t h e l i n e a r s c a l e t o mark t h e s a m p l e s . F o r e xample, j u d g e "X" t e n d e d t o u s e o n l y t h e f i r s t h a l f o f t h e s c a l e , j u d g e "Y" g r o u p e d a l l t h e e v a l u a t i o n s i n t h e m i d d l e o f t h e s c a l e , and j u d g e " Z " m a r k e d t h e r e s u l t s f u r t h e r t o t h e e n d o f t h e s c a l e . The e f f e c t o f t h i s n o n - u n i f o r m i t y was t h a t when c a l -c u l a t i n g an a v e r a g e f o r e a c h t e x t u r a l a t t r i b u t e , t h e e x t r e m e v a l u e s c a n c e l l e d e a c h o t h e r o u t , a n d t h e r e a p p e a r e d t o be l e s s d i f f e r e n c e b e t w e e n t h e g r a d e s t h a n was e x p e c t e d . To e l i m i n a t e t h e n o n - u n i f o r m u s e o f t h e s c a l e w h i c h r e s u l t e d i n non-homogeneous v a r i a n c e between j u d g e s , a Z - t r a n s f o r m a t i o n T a b l e 3.3. Mean, u n t r a n s f o r m e d r e s u l t s f r o m t h e s e n s o r y p a n e l ' s e v a l u a t i o n o f t h e t e x t u r a l d i f f e r e n c e s between t h e f o u r g r a d e s o f chum s a l m o n . G r a d e S e n s o r y A t t r i b u t e s F i r m n e s s D r y n e s s F i b r o u s n e s s C h e w i n e s s n 1 7 . 6 9 ( 2 . 6 ) 1 8.34 (2.3) 8.60 (2.3) 7.41 (2.8) 97 2 6.81(3.5) 7.16 (3.2) 7.87 (3.0) 7.09 (3.0) 43 3 6.54(2.8) 5.22 (2.7) 7.00 (2.8) 7.42 (2.7) 150 4 3.15(2.3) 3.76 (2.5) 2.82 (1.6) 3.15 (2.2) 62 S t a n d a r d d e v i a t i o n a r e shown i n b r a c k e t s . n= t o t a l number o f o b s e r v a t i o n s f o r a p a r t i c u l a r g r a d e . -74-was p e r f o r m e d on e a c h o f t h e t e x t u r a l a t t r i b u t e s f o r e a c h j u d g e i n d i v i d u a l l y s o t h a t t h e mean o f e a c h o f t h e i r s c o r e s was e q u a l t o z e r o and t h e v a r i a n c e was e q u a l t o one. T h i s was j u s t i f i e d b e c a u s e e a c h j u d g e t e s t e d t h e same s a m p l e s . F i g u r e 3.3 i s a g r a p h i c a l r e p r e s e n t a t i o n o f t h e s t a n d a r d i z e d v a l u e s f o r e a c h g r a d e o f t h e 4 a t t r i b u t e s . The b a r g r a p h showed a c l e a r t r e n d t o w a r d s a d e c r e a s e i n f i r m n e s s , d r y -n e s s , c h e w i n e s s , and f i b r o u s n e s s . A n a l y s i s o f v a r i a n c e r e -v e a l e d a s i g n i f i c a n t d i f f e r e n c e between a t l e a s t one o f t h e 4 g r a d e s . The s t a t i s t i c a l p r o g r a m S y s t a t ( W i l k i n s o n , 1988) was u s e d t o p e r f o r m s i n g l e d e g r e e o f f r e e d o m c o n t r a s t s w i t h t h e d a t a . M u l t i p l e c o m p a r i s o n s o f t h e d a t a were t e s t e d w i t h t h e s e q u e n t i a l l y r e j e c t i v e B o n f e r r o n i p r o c e d u r e t o c o n t r o l t h e o v e r a l l c o m p a r i s o n e r r o r r a t e f o r m u l t i p l e h y p o t h e s e s ( H o l l a n d a n d D i P o n z i o C o p e n h a v e r , 1 9 8 7 ) . The r e s u l t s o f t h i s t e s t a r e g i v e n i n T a b l e 3.4. The j u d g e s were a b l e t o d i s t i n g u i s h b e t w e e n e a c h o f t h e 4 g r a d e s f o r t h e a t t r i b u t e d r y n e s s . G r a d e 1 was s i g n i f i c a n t l y d r y e r t h a n g r a d e 2, g r a d e 2 was d r y e r t h a n g r a d e 3, and g r a d e 4 was t h e l e a s t d r y . F o r t h e a t t r i b u t e f i b r o u s n e s s , g r a d e s 1 a n d 2 c o u l d n o t be d i s t i n g u i s h e d , and g r a d e 3 and 4 were s i g n i f i c a n t l y l e s s f i b r o u s t h a n any o t h e r g r a d e . F o r t h e a t t r i b u t e f i r m -n e s s , no d i f f e r e n c e was f o u n d b e t w e e n g r a d e s 1 v e r s u s 2 w h i c h were t h e most f i r m . G r a d e s 3 a n d 4 c o u l d a l s o n o t be d i s t i n g u i s h e d , and were l e s s f i r m t h a n g r a d e s 1 and 2. F o r t h e a t t r i b u t e c h e w i n e s s , g r a d e s 1, 2, a n d 3 w e r e n o t -75-- 2 1 1 G R A D E 1 G R A D E 2 G R A D E 3 G R A D E 4 S E X U A L M A T U R I T Y F i g u r e 3 . 3 . S t a n d a r d i z e d s e n s o r y s c o r e s f o r t h e f o u r g r a d e s o f chum s a l m o n f o r t h e a t t r i b u t e s f i r m n e s s , c h e w i n e s s , d r y n e s s and f i b r o u s n e s s . - 7 6 -T a b l e 3.4. R e s u l t s f r o m a n a l y s i s o f v a r i a n c e o f t h e s e n s o r y s c o r e s t o d e t e r m i n e d i f f e r e n c e s between g r a d e s , a n d s e q u e n t i a l r e j e c t i v e B o n f e r r o n i p r o c e d u r e f o r t h e m u l t i p l e c o m p a r i s o n o f t h e d a t a . E f f e c t 1 v s 2 D i f f e r e n c e w i t h i n t h e 1 v s 3 1 v s 4 2 v s 3 g r a d e s 2 v s 4 3 v s 4 F i r m n e s s NS * * NS * D r y n e s s * * * * * * F i b r o u s n e s s NS * * * * * C h e w i n e s s NS NS * NS * * = s i g n i f i c a n t l y d i f f e r e n t a t a = 0.05. NS = n o t s i g n i f i c a n t l y d i f f e r e n t a t a = 0.05. -77-s i g n i f i c a n t l y d i f f e r e n t , b u t t h e y were c h e w i e r t h a n g r a d e 4. 3.3.3. S e n s o r y and i n s t r u m e n t a l c o r r e l a t i o n C o r r e l a t i o n a n a l y s i s was p e r f o r m e d between t h e i n s t r u -m e n t a l a n d s e n s o r y v a l u e s u s i n g a P e a r s o n c o r r e l a t i o n b y S y s t a t ( W i l k i n s o n , 1 9 8 8 ) . T h e r e was a s i g n i f i c a n t c o r r e l a -t i o n between t h e p a r a m e t e r c o h e s i v e n e s s a n d t h e a t t r i b u t e s f i r m n e s s , f i b r o u s n e s s , c h e w i n e s s , and j u i c i n e s s ( a = 0 . 0 5 ) . T a b l e 3.5 i s p a r t o f t h e m a t r i x c r e a t e d f r o m c o m p a r i n g t h e i n s t r u m e n t a l and s e n s o r y a t t r i b u t e s w i t h a P e a r s o n c o r r e l a -t i o n m a t r i x . The u s e o f s e n s o r y p a n e l s t o e v a l u a t e t h e a c c e p t a b i l i t y o r q u a l i t y o f a p r o d u c t h a s t h e d i s a d v a n t a g e t h a t t h e r e may be a l o s s o f a c c u r a c y due t o f a t i g u e i f a l a r g e number o f s a m p l e s a r e e x a m i n e d i n a s h o r t t i m e , and u n a v o i d a b l e p a n e l c h a n g e s w i l l mean t h e l o s s o f members o f t h e team. L u n d ( 1 9 8 2 ) c o m m e n t e d t h a t o f t e n s e n s o r y r e s p o n s e s w e r e n o n - l i n e a r l y r e l a t e d t o t h e p h y s i c a l a n d c h e m i c a l c h a n g e s t h a t t h e y r e f l e c t . A l t h o u g h many s t u d i e s h a v e u s e d s e n s o r y a n a l y s i s , few h a v e g e n e r a t e d d a t a w h i c h c o u l d be q u a n t i f i e d i n t o a model u s e f u l t o p r e d i c t t h e e f f e c t s o f p r o c e s s i n g . The p r o b l e m s a s s o c i a t e d w i t h s e n s o r y p a n e l s h a v e p r o m p t e d much work on d e v e l o p i n g e f f e c t i v e i n s t r u m e n t a l t e c h n i q u e s . L e v i t t , (1974) d e s c r i b e d t h r e e s t e p s u s e d t o d e t e r m i n e w h e t h e r an i n s t r u m e n t a l t e s t c o u l d be u s e d i n p l a c e o f s e n -s o r y e v a l u a t i o n s . F i r s t t h e s e n s o r y v a r i a b l e s were s e l e c t e d t o d e s c r i b e t h e o r g a n o l e p t i c c h a r a c t e r i s t i c . I n s t r u m e n t a l -78-T a b l e 3.5. C o r r e l a t i o n v a l u e s ( r ) e x t r a c t e d f r o m a P e a r s o n c o r r e l a t i o n m a t r i x o f t h e c o m p a r i s o n between t h e i n s t r u m e n t a l p a r a m e t e r s , a n d t h e s e n s o r y a t t r i b u t e s . I n s t r u m e n t a l F i r m n e s s S e n s o r y A t t r i b u t e s 1 C h e w i n e s s P a r a m e t e r s D r y n e s s F i b r o u s . A r e a 1 0.347 0.239 0.324 0.366 A r e a 2 0.432 0.235 0.404 0.451 H a r d 1 0. 387 0.197 0.323 0.375 H a r d 2 0.376 0.182 0.316 0.378 C o h e s i v e n e s s 0.788* 0.521* 0.827* 0.815* S p r i n g i n e s s 0.411 0.325 0.538 0.489 S l o p e 1 0.388 0.176 0.292 0.379 S l o p e 2 0.410 0.182 0.328 0.413 Gumminess 0.314 0.143 0.237 0.324 C h e w i n e s s 0.344 0.200 0.319 0.385 Sample s i z e 'n' = 19. * S i g n i f i c a n t a t t h e 5% l e v e l . -79-v a r i a b l e s were t h e n s e l e c t e d s o t h a t t h e y c o u l d d e s c r i b e t h e i n f o r m a t i o n c o n t a i n e d i n t h e s e n s o r y v a r i a b l e s . The t h i r d s t e p was t o j u d g e w h e t h e r i n s t r u m e n t a l v a r i a b l e s a d e q u a t e l y d e s c r i b e d t h e u n d e r l y i n g d i m e n s i o n s o f t h e o r g a n o l e p t i c c h a r a c t e r i s t i c . I f i t c o u l d be shown t h a t v a r i a b l e s mea-s u r e d by i n s t r u m e n t a l methods f u l f i l l e d t h e same f u n c t i o n a s t h e s e n s o r y p a n e l s , i t w o u l d be p o s s i b l e t o o b t a i n a d e -s c r i p t i o n o f o r g a n o l e p t i c c h a r a c t e r i s t i c o f t h e p r o d u c t more q u i c k l y , a c c u r a t e l y and e c o n o m i c a l l y . A g u i l e r a a n d S t a n l e y , ( 1990) c o n s i d e r e d m u l t i v a r i a t e a n a l y s i s t o be t h e b e s t a p p r o a c h f o r a n a l y s i s o f s e n s o r y and i n s t r u m e n t a l m e a s u r e m e n t s o f t e x t u r e . T h e t e c h n i q u e s o f c l u s t e r a n a l y s i s , p r i n c i p l e component a n a l y s i s , and s t e p w i s e d i s c r i m i n a n t a n a l y s i s a l l o w e d f o r e f f i c i e n t s i m p l i f i c a t i o n a n d i n t e r p r e t a t i o n o f a l a r g e number o f v a r i a b l e s s i m u l t a -n e o u s l y . O t h e r m e t h o d s s u c h a s s i m p l e l i n e a r r e g r e s s i o n were l i m i t e d b e c a u s e t h e y o n l y i n d i c a t e d how one s e t o f num-b e r s v a r i e d w i t h a n o t h e r , and assumed p a r a m e t r i c d a t a i . e . n o r m a l d i s t r i b u t i o n . M u l t i p l e r e g r e s s i o n a n d c o r r e l a t i o n was l i m i t e d b e c a u s e r e s u l t s were a f f e c t e d b y c o r r e l a t i o n o f v a r i a b l e s w i t h i n e a c h s e t ( L u n d , 1 9 8 2 ) . The m a g n i t u d e o f an a p p l i e d l o a d and t h e r a t e t h a t t h e l o a d was a p p l i e d i n f l u e n c e d i n s t r u m e n t a l e v a l u a t i o n o f t e x -t u r a l p r o p e r t i e s . Shama and Sherman (1973) f o u n d t h a t w h i l e c h e w i n g o f t e n o c c u r r e d i n a s i n u o s o i d a l p a t t e r n , t h e I n s t r o n ' s f o r c e c o m p r e s s i o n t e s t was a l i n e a r a p p l i c a t i o n o f -80-s t r e s s t o t h e s a m p l e . T h u s , i t was s p e c u l a t e d t h a t c o r r e l a -t i o n b e t w e e n i n s t r u m e n t a l a n d s e n s o r y d a t a was o f t e n m i s -l e a d i n g . However, by e v a l u a t i n g t e x t u r a l p r o p e r t i e s d u r i n g t h e down s t r o k e o f t h e c h e w i n g c y c l e ( a n a p p r o x i m a t e l y l i n e a r m o t i o n ) , t h e I n s t r o n was a b l e t o m e a s u r e h a r d n e s s , b r i t t l e n e s s , and c r i s p n e s s . H a t a e e t a l . (1984) s t u d i e d t h e t e x t u r e o f 5 s p e c i e s o f f i s h by d e t e r m i n i n g d r i p , r e s i d u a l w e i g h t , f i r m n e s s , c o h e -s i v e n e s s , p e n e t r a t i o n , f i b r e v o l u m e , f i b r e d i a m e t e r , a n d f i b r e l e n g t h . S t a t i s t i c a l a n a l y s i s was p e r f o r m e d u s i n g a n a l y s i s o f v a r i a n c e and d i s c r i m i n a n t a n a l y s i s . I t was c o n -c l u d e d t h a t t e x t u r a l d i f f e r e n c e s o f v a r i o u s f i s h c o u l d be d e t e c t e d by d i s c r i m i n a n t a n a l y s i s b u t n o t by e v a l u a t i o n o f i n d i v i d u a l i t e m s o f measurement. The c o r r e l a t i o n s b e t w e e n s e n s o r y a n d i n s t r u m e n t a l t e x t u r e measurements were u s u a l l y o f an a s s o c i a t i v e , i n d i r e c t n a t u r e , and r e f l e c t e d u n d e r l y i n g e f f e c t s w h i c h m i g h t h ave o p e r a t e d i n t h e same o r d i f f e r e n t d i r e c t i o n s o f b o t h s i d e s o f t h e c o r r e l a t i o n . T h e r e f o r e c o r -r e l a t i o n a n d p r e d i c t i o n c o u l d n o t i m p l y e q u a l i t y o r an u n -d e r s t a n d i n g o f t h e mechanism b e h i n d t h e a s s o c i a t i o n . B o r d e r i a s e t a l . , (1983) s t u d i e d d i f f e r e n t i n s t r u m e n t a l methods t o f i n d w h i c h c o r r e l a t e d b e s t w i t h s e n s o r y a n a l y s i s . Raw a n d c o o k e d s k i n n e d f i l l e t s and m i n c e d f i s h were t e s t e d by s e n s o r y TPA methods. T h e s e r e s u l t s were t h e n compared t o t h e d a t a g e n e r a t e d b y s h e a r , c o m p r e s s i o n , a n d j u i c i n e s s t e s t s p e r f o r m e d u s i n g a n I n s t r o n T e x t u r o m e t e r . T h e f i s h - 8 1 -m i n c e s c o r r e l a t e d b e t t e r w i t h t h e s e n s o r y and i n s t r u m e n t a l t e c h n i q u e s t h a n d i d t h e f i s h f i l l e t s . E f f e c t i v e i n s t r u m e n -t a l t e s t i n g m e t h o d s i n c l u d e d t h e K r a m e r s h e a r c e l l , t h e p u n c t u r e t e s t , and e v a l u a t i o n o f j u i c i n e s s . K a r l and S c h r e i b e r (1985) u s e d c a n n e d b l u e w h i t i n g and m a c k e r e l t o d e t e r m i n e w h e t h e r s e n s o r y e v a l u a t i o n o f t h e t e x -t u r e c o u l d be c o r r e l a t e d w i t h t h e d a t a o b t a i n e d f r o m a n I n s t r o n w i t h a K r a m e r s h e a r c e l l , a n d a B r a b e n d e r F a r i n o g r a p h . The v a l u e a t t h e b e g i n n i n g o f t h e B r a b e n d e r c u r v e c o r r e l a t e d w i t h t h e peak h e i g h t o f t h e I n s t r o n c u r v e ( r = 0 . 8 3 ) . T h e I n s t r o n was m o r e s u c c e s s f u l t h a n t h e B r a b e n d e r i n p r e d i c t i n g t h e s e n s o r y p a n e l ' s " r e s p o n s e t o f i r s t b i t e " , and " s t r u c t u r e a f t e r p r o l o n g e d c h e w i n g " . D u l c e e t a l . (1987) compared t h e t e x t u r e o f d e - b o n e d , f l a k e d , c a n n e d f i s h e v a l u a t e d w i t h a s e m i - t r a i n e d s e n s o r y p a n e l a n d w i t h an O t t a w a t e x t u r o m e t e r e x t r u s i o n c e l l a t -t a c h e d t o an I n s t r o n U n i v e r s a l t e s t i n g m a c h i n e . F o r c e mea-s u r e m e n t s were h i g h l y c o r r e l a t e d w i t h b o t h t e n d e r n e s s ( r = 0.91) a n d j u i c i n e s s ( r = 0 . 8 7 ) . I t was c o n c l u d e d t h a t t h e O t t awa t e x t u r o m e t e r was a g o od p r e d i c t o r o f t e x t u r e a s p e r -c e i v e d by a t a s t e p a n e l . I n t h i s s t u d y , s e n s o r y a n a l y s i s was c l e a r l y s u p e r i o r t o i n s t r u m e n t a l TPA i n d i s c r i m i n a t i n g power. None o f t h e 10 TPA p a r a m e t e r s were c o n s i s t e n t l y d i f f e r e n t b e t w e e n g r a d e s , w h i l e s e n s o r y d r y n e s s was s i g n i f i c a n t l y d i f f e r e n t b e t w e e n a l l f o u r g r a d e s . C o r r e l a t i o n o f t h e i n s t r u m e n t a l p a r a m e t e r s -82-a n d s e n s o r y a t t r i b u t e s showed t h a t c o h e s i v e n e s s c o r r e l a t e d w i t h a l l f o u r s e n s o r y p a r a m e t e r s . H o w e v e r t h e r e w e r e no o t h e r s i g n i f i c a n t c o r r e l a t i o n s . -83-4. PROTEINASE A C T I V I T Y 4.1. I n t r o d u c t i o n The d e c r e a s e i n e a t i n g q u a l i t y o f chum s a l m o n d u r i n g t h e s p a w n i n g m i g r a t i o n i s now w e l l e s t a b l i s h e d . T h e r e i s however, c o n s i d e r a b l e i n t e r e s t i n u n d e r s t a n d i n g t h e r e a s o n s f o r t h i s d e t e r i o r a t i o n . Konagaya (1983) f o u n d no e v i d e n c e o f t h e p r e s e n c e o f s p o r o z o a n o r o t h e r p a r a s i t e s i n t h e f i s h , a nd c o n c l u d e d t h a t b i o c h e m i c a l c h a n g e s were r e s p o n s i b l e f o r i t s p o o r t e x t u r e . H a t a n o e t a l . ( 1 9 8 3 ) , and Konagaya (1983) u s e d p r o x i m a t e a n a l y s i s t o c h a r a c t e r i z e chum i n J a p a n e s e w a t e r s a t d i f f e r e n t m a t u r i t y s t a g e s and showed a d e c r e a s e i n t h e p r o t e i n c o n t e n t o f t h e m u s c l e f r o m 20% t o 16%, a d e -c r e a s e i n l i p i d f r o m 8.5% t o 1.6%, and an i n c r e a s e i n m o i s -t u r e f r o m 72% t o 80% f o r e a r l y a n d l a t e - r u n chum r e s p e c -t i v e l y . Mommsen e t a l . (1980) m e a s u r e d t h e a c t i v i t y o f 13 meta-b o l i c enzymes, and t h e c o n c e n t r a t i o n o f s o l u b l e and i n s o l u -b l e p r o t e i n o f s o c k e y e d u r i n g s p a w n i n g m i g r a t i o n . By t h e e n d o f s p a w n i n g m i g r a t i o n , t h e a c t i v i t y o f e l e v e n o f t h e t h i r t e e n enzymes was r e d u c e d t o 30% o f t h e i r o r i g i n a l l e v e l . The s o l u b l e a n d i n s o l u b l e p r o t e i n was d e c r e a s e d by 70%, i n -d i c a t i n g i t s u s e i n d e v e l o p m e n t o f t h e g o n a d s , a n d a s an e n e r g y s o u r c e . Two m e t a b o l i c enzymes showed no c h a n g e i n l e v e l o r a c t i v i t y , a l a n i n e a m i n o t r a n s f e r a s e ( G T P ) , and m a l i c enzyme. I t was t h o u g h t t h a t t h e s e enzymes were s e l e c t i v e l y -84-m a i n t a i n e d i n o r d e r t o s u s t a i n e s s e n t i a l b i o c h e m i c a l o r p h y s i o l o g i c a l f u n c t i o n t h r o u g h o u t m i g r a t i o n . P r o t e i n a s e a c -t i v i t y i n m u s c l e b u f f e r e d t o pH 4.05 i n c r e a s e d l i n e a r l y w i t h t h e d i s t a n c e t h e s a l m o n m i g r a t e d s o t h a t e n d m i g r a t i o n l e v e l s w e r e s e v e n t i m e s t h e p r e - m i g r a t o r y l e v e l s . No c h a n g e , o r s l i g h t d e c r e a s e s i n a c t i v i t y were d e t e c t e d a t pH 6.8 and 8.5. B a s e d o n i n h i b i t i o n s t u d i e s w i t h p e p s t a t i n , t h e s e w o r k e r s c o n c l u d e d t h a t t h e p r o t e i n a s e a c -t i v i t y a t pH 4.0 was due t o t h e p r e s e n c e o f c a t h e p s i n D. The a c t i v i t y a t t h e l o w pH i n d i c a t e d t h e i n v o l v e m e n t o f m u s c l e l y s o s o m e s . Two l y s o s o m a l e n z y m e s , a c i d i c c a r b o x y p e p t i d a s e A, and B - N - a c e t y l g l u c o s a m i n i d a s e were a l s o a s s a y e d . No c h a n g e i n a c t i v i t y d u r i n g m i g r a t i o n was d e -t e c t e d f o r t h e s e two enzymes, t h u s i t was c o n c l u d e d t h a t t h e t o t a l number o f l y s o s o m e s d i d n o t i n c r e a s e . P a r t i a l p u r i f i -c a t i o n o f c a t h e p s i n D l e a d t o t h e c o n c l u s i o n t h a t t h e o b -s e r v e d i n c r e a s e i n a c t i v i t y was due t o an i n c r e a s e i n t h e number o f enzyme m o l e c u l e s , a n d n o t a r e s u l t o f enzyme a c t i -v a t i o n . A n d o a n d H a t a n o ( 1 9 8 6 b ) u s e d t h e a m i n o a c i d N - m e t h y l h i s t i d i n e ( M e H i s ) a s a n i n d e x o f m a t u r i t y o f m y o f i b r i l l a r d e g r a d a t i o n i n v i v o . T h i s amino a c i d i s c o n -t a i n e d o n l y i n a c t i n a n d m y o s i n , i s n o t u t i l i z e d f o r e i t h e r p r o t e i n s y n t h e s i s o r e n e r g y , and i s e x c r e t e d q u a n t i t a t i v e l y i n t o t h e u r i n e . I t was s u g g e s t e d t h a t t h e l e v e l o f t h i s amino a c i d i n t h e m u s c l e e x t r a c t s o f m a t u r i n g chum s a l m o n -85-r e f l e c t e d t h e d e g r e e o f m y o f i b r i l l a r p r o t e i n d e g r a d a t i o n . T h i s a p p r o a c h c l e a r l y showed t h a t t h e d e g r e e o f m y o f i b r i l l a r d e g r a d a t i o n i n c r e a s e d d u r i n g s p a w n i n g m i g r a t i o n . I t was a l s o f o u n d t h a t t h e l e v e l o f M e H i s was h i g h e r f o r f e m a l e s t h a n f o r m a l e s . Ando e t a l . (1986) f o u n d t h a t serum s e x s t e r o i d s s u c h a s a n d r o g e n s a n d e s t r o g e n s were c l o s e l y r e l a t e d t o p r o t e i n d e g r a d a t i o n a n d t o h i g h l e v e l s o f p r o t e i n a s e a c t i v i t y o f chum s a l m o n m u s c l e d u r i n g s p a w n i n g m i g r a t i o n . E n h a n c e d p r o -t e i n c a t a b o l i s m was i n i t i a t e d b y h o r m o n e l e v e l s w h i c h c h a n g e d i n r e s p o n s e t o n u t r i e n t d e p l e t i o n and i n c r e a s e d a c -t i v i t y l e v e l s , i n o r d e r t o p r o v i d e t h e e n e r g y n e c e s s a r y f o r m i g r a t i o n . A l l t h e a v a i l a b l e e v i d e n c e , s u c h a s t h e d e c r e a s e i n t o t a l p r o t e i n , t h e i n c r e a s e i n M eHis, and t h e a p p a r e n t l o s s o f m y o f i l a m e n t s , s u g g e s t e d t h a t m y o f i b r i l l a r p r o t e i n s , i n -c l u d i n g a c t i n a n d m y o s i n , may b e d e g r a d e d b y m u s c l e p r o t e i n a s e s a c t i v a t e d d u r i n g s p a w n i n g m i g r a t i o n . I n o r d e r t o u n d e r s t a n d t h e c h a n g e s i n t h e chum, i t was n e c e s s a r y t o c o n s i d e r p o s s i b l e mechanisms o f t h e p h y s i c a l d e g r a d a t i o n o f t h e f i s h ' s body. The o b j e c t o f t h i s s t u d y was t o d e t e r m i n e t h e c h a n g e s i n t h e a c t i v i t y l e v e l o f m u s c l e p r o t e i n a s e s b u f f e r e d t o pH 3.5, 6.2 a n d 7.0 d u r i n g s p a w n i n g m i g r a t i o n . B e c a u s e l y s o s o m a l enzymes have an i m p o r t a n t r o l e i n t h e d e g r a d a t i o n o f t h e m y o f i b r i l l a r component o f t h e t o t a l b o d y ' s p r o t e i n , -86-t h e a c t i v i t y o f a c i d p h o s p h a t a s e , a l y s o s o m a l m a r k e r , was a l s o a s s a y e d . 4.2. M a t e r i a l s and Methods The d e c i s i o n t o c o l l e c t chum s a m p l e s i n t h e y e a r 1990 was made when i t was d i s c o v e r e d t h a t some o f t h e w o r k p l a n n e d was n o t p o s s i b l e on t h e s a m p l e s c o l l e c t e d i n 1989 w h i c h were p o s t - r i g o r f i s h w h i c h h a d u n d e r g o n e 3-5 d a y s o f p o s t - m o r t e m c h a n g e s . I n t h e s e c o n d y e a r , e m p h a s i s was p l a c e d on o b t a i n i n g t h e f i s h i n a p r e - r i g o r s t a t e . S i l v e r - b r i g h t f i s h were c o l l e c t e d by t r a v e l l i n g t o t h e N.E. s i d e o f V a n c o u v e r I s l a n d ( P o r t M c N e i l ) , and t a k i n g t h e f i s h s a m p l e s i n J o h n s t o n e S t r a i t a b o a r d a D e p a r t m e n t o f F i s h e r i e s and Oceans (DFO) t e s t f i s h e r y v e s s e l . I n t e r m e d i -a t e and d a r k f i s h were o b t a i n e d on a n o t h e r DFO t e s t f i s h e r y v e s s e l , t h i s t i m e on t h e F r a s e r R i v e r . Spawning f i s h were o b t a i n e d f r o m t h e C h i l l i w a c k R i v e r H a t c h e r y . F i s h d o r s a l m u s c l e was c o l l e c t e d , s e a l e d i n z i p l o c k f r e e z e r b a g s a n d s t o r e d a t - 3 0 ° C . 4.2.1. Measurement o f p r o t e i n a s e a c t i v i t y The p r o t e i n a s e a c t i v i t y o f t h e m u s c l e was m e a s u r e d b y t h e m e t h o d o f K o n a g a y a ( 1 9 8 3 ) , a n d i s d e s c r i b e d i n F i g u r e 4.1. The d o r s a l m u s c l e o f t h e chum was h o m o g e n i z e d i n t h r e e p a r t s d i s t i l l e d w a t e r / i c e m i x t u r e a t 0 ° C f o r 30 s e c o n d s i n an U l t r a - T u r r a x h o m o g e n i z e r . Two grams o f t h e h o m o g e n a t e was a d d e d t o e a c h o f 2 s e t s o f 3 t r i p l i c a t e t e s t t u b e s . The t u b e s were t h e n d i l u t e d w i t h 2 mL o f 0.2 M c i t r a t e - p h o s p h a t e -87-Muscle + ice, 1:3 w/w Test homogenize at 0°C, 30s 2 g homogenate 2 mL citrate-phoshate buffer, pH 3.5, 6.2, or 7.0. (6 aliquaots / pH) Blank incubate 1 h at 37° C 1 Add cold TCA solution to 15% i Hold at 4°C, 1 h. Clarify by filtration I Read absorbance at 280 nm Add cold TCA stock to 15% Hold at 4°C. 1 h. I Clarify by filtration I Read absorbance at 280 nm F i g u r e 4.1. F l o w d i a g r a m o f t h e method u s e d t o measure t h e p r o t e i n a s e a c t i v i t y i n t h e d o r s a l m u s c l e o f f o u r g r a d e s o f chum m u s c l e . - 8 8 -b u f f e r a d j u s t e d t o e i t h e r pH 3.5, 6.2, o r 7.0. Two s e t s o f t h r e e t u b e s were d i l u t e d w i t h pH 3.5 b u f f e r , 2 s e t s o f t h r e e t u b e s w i t h pH 6.2 b u f f e r , and 2 s e t s o f 3 t u b e s w i t h pH 7.0. One s e t o f t h e t u b e s a t e a c h pH were i n c u b a t e d a t 37 °C i n a w a t e r - b a t h s h a k e r a t a p p r o x i m a t e l y 100 RPM. A f t e r 1 h o u r , t h e i n c u b a t e d ( t e s t ) s a m p l e s w e r e r e m o v e d f r o m t h e w a t e r b a t h a n d 8 mL o f c o l d 15% t r i c h l o r o a c e t i c a c i d (TCA) was ad d e d t o t h e i n c u b a t e d homogenate. The m i x t u r e was h e l d f o r 1 h o u r i n t h e c o l d b e f o r e b e i n g f i l t e r e d t h r o u g h Whatman #2 f i l t e r p a p e r . The a b s o r b a n c e o f t h e f i l t r a t e was m e a s u r e d a t 280 nm on a S h i m a d z u U.V. 160 U.V - V i s i b l e R e c o r d i n g S p e c t r o p h o t o m e t e r ( S h i m a d z u C o r p . K y o t o , J a p a n ) . The s e c o n d s e t o f t u b e s ( b l a n k s ) a t t h e t h r e e pH v a l u e s were n o t i n c u -b a t e d . I n s t e a d , 8 mL o f c o l d 15% TCA was adde d i m m e d i a t e l y . T h e t u b e s were v o r t e x m i x e d , h e l d f o r one h o u r i n an i c e b a t h , a n d t h e n f i l t e r e d t h r o u g h Whatman #2 f i l t e r p a p e r . The a b s o r b a n c e o f t h e f i l t r a t e was me a s u r e d a t 280 nm. The mean v a l u e s f r o m t h e t r i p l i c a t e s a m p l e s o f t h e ' t e s t ' were s u b t r a c t e d f r o m t h e mean ' b l a n k ' v a l u e s t o o b t a i n a d e l t a a b s o r b a n c e v a l u e f o r e a c h o f t h e f o u r g r a d e s a t t h e t h r e e pH v a l u e s . 4.2.2. Measurement o f a c i d p h o s p h a t a s e a c t i v i t y T h e a c i d p h o s p h a t a s e a c t i v i t y o f t h e m u s c l e was mea-s u r e d u s i n g t h e method o f Beeken and R o e s s n e r ( 1 9 7 2 ) . The d o r s a l m u s c l e o f t h e chum was h o m o g e n i z e d (0.5 g t i s s u e i n 18 g d i s t i l l e d w a t e r / i c e ) f o r 30 s e c o n d s i n an U l t r a - T u r r a x -89-h o m o g e n i z e r , a n d t h e n f i l t e r e d t h r o u g h Whatman #2 f i l t e r p a p e r . T h i s enzyme s o l u t i o n (0.2 mL) was a d d e d t o 1 mL o f t h e s u b s t r a t e s o l u t i o n , p - n i t r o p h e n y l p h o s p h a t e (PNPP). The s u b s t r a t e s o l u t i o n was p r e p a r e d by m i x i n g e q u a l v o l u m e s o f r e a g e n t 1, 0.4% d i s o d i u m p - n i t r o p h e n y l p h o s p h a t e i n 0.001N HC1, a n d r e a g e n t 2, 0.1M Na c i t r a t e b u f f e r pH 4.8. T h e s u b s t r a t e - e n z y m e m i x t u r e was i n c u b a t e d a t 37°C f o r 30 m i n -u t e s i n a w a t e r - b a t h s h a k e r a t 100 RPM. A s s a y s were p e r -f o r m e d i n t r i p l i c a t e . T he s a m p l e s were t h e n removed f r o m t h e b a t h , 3 mL o f 0.1 N NaOH was added, and t h e t u b e s were r e a d a t 405 nm. S t a n d a r d s o l u t i o n s w e r e p r e p a r e d w i t h p - n i t r o p h e n o l (PNP) o f c o n c e n t r a t i o n s 0.2, 0.4, 0.6, 0.8, and 1.0 mM. A s t a n d a r d c u r v e was p r e p a r e d by t r e a t i n g 0.15 mL o f e a c h s o l u t i o n w i t h 3 mL o f NaOH, a n d r e a d i n g a b s o r b a n c e a t 405 nm. 4.3. R e s u l t s and D i s c u s s i o n 4.3.1. P r o t e i n a s e a c t i v i t y T h e a c t i v i t y o f m u s c l e p r o t e i n a s e s was m e a s u r e d a t t h r e e pH v a l u e s . A pH o f 3.5 r e p r e s e n t e d t h e a c i d i c c o n d i -t i o n s p r e s e n t i n t h e l y s o s o m a l s y s t e m o f m u s c l e , pH 6.2 was t h e t y p i c a l m u s c l e pH m e a s u r e d a f t e r r e s o l u t i o n o f r i g o r , a n d pH 7.0 was u s e d t o d e t e c t t h e a c t i v i t y o f n e u t r a l , o r s l i g h t l y a l k a l i n e p r o t e i n a s e s . The r e s u l t s o f t h e e x p e r i -ment a r e g i v e n i n T a b l e 4.1. The b a r g r a p h o f a c t i v i t y a t pH 3.5 v e r s u s i n d e x o f m a t u r i t y i s shown i n t h e A p p e n d i x i n T a b l e 8.1. T h e r e was a s i g n i f i c a n t c o r r e l a t i o n ( a < 0.05) -90-T a b l e 4.1. P r o t e i n a s e a c t i v i t y a t pH 3 . 5 , 6.2 a n d 7.0 d u r i n g t h e d i f f e r e n t s t a g e s o f s p a w n i n g m i g r a t i o n . G r a d e P r o t e i n a s e A c t i v i t y pH 3.5 pH 6.2 pH 7.0 1 0. 0226 ( 0 . 018) -0 .0163 (0 .017) 0 .0219 (0 .014) 1 0. 0369 ( 0 . 024) -0 .0041 (0 .006) 0 .0344 (0 .006) 1 - 0 . 0129 (0. 010) -0 .0310 (0 .034) 0 .0256 (0 .005) 2 0. 1453 (0. 025) 0 .0799 (0 .005) 0 .0468 (0 .080) 3 0. 0792 (0. 049) 0 .0347 (0 .049) 0 .0180 (0 .011) 3 0. 2365 ( 0 . 028) -0 .0856 (0 .002) 0 .0976 (0 .001) 4 0. 1295 (0. 013) -0 .0015 (0 .012) 0 .0719 (0 .013) 4 0. 1060 (o. 008) 0 .0144 (0 .024) 0 .0719 (0 .013) 4 0. 2811 (0. 008) 0 .0714 (0 .014) 0 .0976 (0 .001) M e a n s o f t r i p l i c a t e d e t e r m i n a t i o n s , s t a n d a r d d e v i a t i o n s a r e p r e s e n t e d i n b r a c k e t s . -91-between i n c r e a s e d a c t i v i t y and i n c r e a s e d m a t u r i t y , w i t h an r v a l u e o f 0.701, and a sample s i z e o f 9. The b a r g r a p h o f t h e p r o t e i n a s e a c t i v i t y a t pH 6.2 v e r s u s i n d e x o f m a t u r i t y i s shown i n t h e A p p e n d i x , T a b l e 8.2, however no s i g n i f i c a n t c o r r e l a t i o n was f o u n d b e t w e e n t h e s e two f a c t o r s . T h e p r o t e i n a s e a c t i v i t y a t pH 7.0 i s shown i n t h e A p p e n d i x , T a b l e 8.3, however no c o r r e l a t i o n between a c t i v i t y and matu-r i t y was f o u n d a t t h i s pH. The f i n d i n g s o f h i g h p r o t e i n a s e a c t i v i t y i n t h e a c i d r a n g e a r e c o n s i s t e n t w i t h t h e s t u d y b y K o n a g a y a ( 1 9 8 2 , 1 9 8 3 ) . T h i s w o r k e r compared t h e optimum pH a c t i v i t y o f chum d o r s a l m u s c l e a t t h r e e m i g r a t o r y s t a g e s . The a u t o l y t i c a c -t i v i t y was h i g h e s t a t pH 3.5 a t e a c h o f t h e 3 s t a g e s . T h e r e was a f o u r f o l d i n c r e a s e i n a c t i v i t y f r o m s t a g e 1 t o s t a g e 2, and a s i x f o l d i n c r e a s e f r o m s t a g e 1 t o s t a g e 3. B e c a u s e l i t t l e p r o t e i n a s e a c t i v i t y was d e t e c t e d i n t h e s l i g h t l y a l -k a l i n e r e g i o n , e v e n i n t h e p r e s e n c e o f 1- mM c a l c i u m , and 2 mM c y s t e i n e , c a l c i u m a c t i v a t e d n e u t r a l p r o t e i n a s e s (CANP) w e r e n o t c o n s i d e r e d t o c o n t r i b u t e t o t h e c h a n g e s i n t h e m u s c l e . I n mammalian s y s t e m s , s k e l e t a l m u s c l e i s v e r y l o w i n l y s o s o m a l c o n t e n t . However, R e d d i e t a l . ( 1 9 7 2 ) r e p o r t e d t h a t t h e c a t h e p t i c a c t i v i t y o f f i s h m u s c l e was t e n t i m e s g r e a t e r t h a n t h a t o f m a m m a l i a n t i s s u e . T o s t u d y t h e i n t r a c e l l u l a r l o c a l i z a t i o n o f c a t h e p t i c a c t i v i t y i n t h e s k e l e t a l m u s c l e o f f l o u n d e r , R e d d i e t a l . ( 1 9 7 2 ) u s e d -92-c e n t r i f u g a t i o n t o s e p a r a t e t h e m u s c l e p r o t e i n i n t o f i v e f r a c t i o n s . T h e s e f r a c t i o n s i n c l u d e d ( P I ) , u n b r o k e n c e l l s a n d n u c l e i , ( P 2 ) , m i t o c h o n d r i a , ( P 3 ) , l y s o s o m e s , ( P 4 ) , m i c r o s o m e s , a n d ( S 4 ) , f i n a l s u p e r n a t a n t . T h e h i g h e s t c a t h e p t i c a c t i v i t y was f o u n d i n t h e P3, l y s o s o m a l f r a c t i o n , a l t h o u g h t h e P2, a n d P4 f r a c t i o n s a l s o h a d some a c t i v i t y . A t pH 4.0 t h e P3 a c t i v i t y was optimum, a t pH 6.5 t h e r e was l e s s a c t i v i t y , and a t pH 7.5, no a c t i v i t y c o u l d be d e t e c t e d . 4.3.2. A c i d p h o s p h a t a s e a c t i v i t y The e n h a n c e d a c t i v i t y o f p r o t e i n a s e s a t pH 3.5 m i g h t be a t t r i b u t e d t o l y s o s o m a l c a t h e p s i n s f o u n d i n and a r o u n d t h e m u s c l e c e l l s . A c i d p h o s p h a t a s e has been commonly r e c o g n i z e d a s a l y s o s o m a l m a r k e r s u c h t h a t i n c r e a s i n g l e v e l s o f t h e enzyme i n d i c a t e d i n c r e a s i n g c o n c e n t r a t i o n o f l y s o s o m e s (Chayen e t a l . 1 9 7 3 ) . T h e r e f o r e , i t s a c t i v i t y was m o n i t o r e d a t t h e d i f f e r e n t m a t u r i t y l e v e l s . I n t h i s a s s a y , i n c u b a t i o n o f t h e enzyme w i t h PNPP r e -s u l t e d i n f o r m a t i o n o f t h e breakdown p r o d u c t s PNP and i n o r -g a n i c p h o s p h a t e . A d d i t i o n o f NaOH c o n v e r t e d t h e PNP i n t o t h e y e l l o w p h e n o x i d e i o n , w h i c h w a s m e a s u r e d b y c o l o u r i m e t r i c a n a l y s i s . A s t a n d a r d c u r v e o f PNP was c o n -s t r u c t e d t o c o n v e r t a b s o r b a n c e a t 405 nm t o p h o s p h a t e c o n -c e n t r a t i o n , a n d i s shown i n F i g u r e 4.2. The a c t i v i t y o f a c i d p h o s p h a t a s e was r e p o r t e d i n t h e A p p e n d i x , T a b l e 4.2 and a s a g r a p h i n A p p e n d i x , T a b l e 8.4. -93-1.00 0.80 > 0.60 D 0.40 0.20 0.00 0.0 0.2 0.4 0.6 0.8 1.0 Concentration of PNP Figure 4.2 St a n d a r d curve of the absorbance at 405 nm versus the concentration of £-nitrophenol. -94-T a b l e 4.2. A c i d p h o s p h a t a s e a c t i v i t y (mM p h o s p h a t e ) i n d o r s a l m u s c l e o f chum s a l m o n d u r i n g s p a w n i n g m i g r a t i o n . G r a d e P h o s p h a t e R e l e a s e (mM) 1 0.2541 ( 0 . 0 0 0 ) 2 1 0.2770 (0.011) 1 0.2826 (0.005) 2 0.2903 (0.003) 2 0.2943 (0.007) 3 0.3116 (0.010) 3 0.3071 (0.016) 4 0.3401 (0.022) 4 0.4060 (0.001) 4 0.4611 (0.003) Means o f t r i p l i c a t e d e t e r m i n a t i o n s . S t a n d a r d d e v i a t i o n s a r e shown i n b r a c k e t s . -95-C o r r e l a t i o n a n a l y s i s s h o w e d t h e r e was a s i g n i f i c a n t c o r r e l a t i o n ( a < 0.05) between t h e l e v e l o f m a t u r i t y and t h e i n c r e a s e d a c t i v i t y o f a c i d p h o s p h a t a s e , w i t h an r v a l u e o f 0.829. The a c t i v i t y o f t h e enzyme i n c r e a s e d s i g n i f i c a n t l y b etween g r a d e s 1 and 4. The i n c r e a s e d l e v e l s o f p r o t e i n a s e a c t i v i t y a t pH 3.5, t o g e t h e r w i t h t h e o b s e r v e d i n c r e a s e i n a c i d p h o s p h a t a s e a c t i v i t y w i t h i n c r e a s i n g m a t u r i t y , s u g -g e s t e d t h a t l y s o s o m a l c a t h e p s i n s may be r e s p o n s i b l e f o r t h e s o f t e n i n g o f t h e chum s a l m o n m u s c l e t i s s u e d u r i n g s p a w n i n g m i g r a t i o n . The e f f e c t o f c o l d s t o r a g e on t h e p r o t e i n a s e a c t i v i t y i n t h e m u s c l e i s an i m p o r t a n t c o n s i d e r a t i o n . W a r r i e r e t a l . (1988) s t u d i e d t h e c h a n g e i n a c t i v i t y o f a m i n o p e p t i d a s e and c a t h e p s i n B i n t h e m u s c l e e x t r a c t o f 7 t r o p i c a l f i s h s p e c i e s d u r i n g s t o r a g e a t 0°C and f r o z e n s t o r a g e . S i g n i f i c a n t d i f -f e r e n c e s between t h e s p e c i f i c a c t i v i t i e s o f t h e enzymes were f o u n d i n t h e d i f f e r e n t s p e c i e s o f f i s h . T h e r e was a g e n e r a l d e c r e a s e i n t h e a c t i v i t y o f t h e a m i n o p e p t i d a s e and c a t h e p s i n B d u r i n g 4 months o f c o l d s t o r a g e , however t h e r a t e o f d e -c r e a s e w i d e l y v a r i e d w i t h e a c h f i s h s p e c i e s . I t was e x p e c t e d t h a t t h e a c t i v i t y o f chum's p r o t e o l y t i c enzymes w o u l d a l s o d e c r e a s e w i t h t i m e . However b e c a u s e o f t h e r e l a t i v e l y s h o r t t i m e i n f r o z e n s t o r a g e , l e s s t h a n 2 months, and t h e low s t o r a g e t e m p e r a t u r e o f - 3 0 ° C , t h e l o s s i n a c t i v i t y was n o t e x p e c t e d t o i n v a l i d a t e t h e r e s u l t s o f t h e e x p e r i m e n t . -96-T h e r e h a s been much d i s a g r e e m e n t among w o r k e r s r e g a r d -i n g t h e n a t u r a l o c c u r r e n c e o f l y s o s o m e s i n m u s c l e c e l l s . S t e i n e r e t a l . , (1984) s u g g e s t e d t h a t l y s o s o m a l enzymes i n m u s c l e t i s s u e m i g h t be l o c a t e d e i t h e r i n macrophages o f c o n -n e c t i v e t i s s u e s , o r w i t h i n m u s c l e c e l l s . I n o r d e r t o o b t a i n i n f o r m a t i o n a b o u t t h e o c c u r r e n c e a n d l o c a t i o n o f l y s o s o m e s i n t h e c e l l s o f n o r m a l s t r i a t e d m u s c l e , S t e i n e r e t a l . (1984) u s e d h i s t o c h e m i c a l t e c h n i q u e s f o r l i g h t a nd e l e c t r o n m i c r o s c o p y t o d e t e c t a c i d p h o s p h a t a s e i n t h e m u s c l e o f c o h o s a l m o n . L i g h t m i c r o g r a p h s o f 10 m i c r o n c r y o s t a t s e c t i o n s r e v e a l e d a n o n - u n i f o r m d i s t r i b u t i o n o f l y s o s o m e s i n t h e m u s c l e . A l l o f t h e a c i d p h o s p h a t a s e a c t i v i t y a p p e a r e d t o be l i m i t e d t o t h e c o n n e c t i v e t i s s u e a r e a o f t h e endomysium and p e r i m y s i u m . No a c t i v i t y was f o u n d c e n t r a l l y l o c a t e d among t h e m y o f i b r i l s . I n o r d e r t o d e t e r m i n e w h e t h e r t h e l y s o s o m e s were w i t h i n t h e m u s c l e c e l l s a t t h e p e r i p h e r a l c e l l membrane o r i n t h e c o n n e c t i v e t i s s u e c o m partments, Epon embedded t i s -s u e s were e x a m i n e d by e l e c t r o n m i c r o s c o p y . T h r e e p o p u l a -t i o n s o f l y s o s o m e s c o u l d be d i s t i n g u i s h e d on t h e b a s i s o f t h e i r l o c a t i o n w i t h i n t h e t i s s u e . The l y s o s o m e s were f o u n d i n t h e c o n n e c t i v e t i s s u e c e l l s ( i d e n t i f i e d a s m a c r o p h a g e s ) , a t t h e p e r i p h e r y o f , b u t w i t h i n , m u s c l e c e l l s , a n d w i t h i n t h e m u s c l e c e l l s d i s t r i b u t e d among t h e m y o f i b r i l s . I t was c o n c l u d e d t h a t l y s o s o m e s o c c u r r e d i n s a l m o n m u s c l e t i s s u e , b u t w e r e n o t u n i f o r m l y d i s t r i b u t e d . T he c o n c l u s i o n t h a t t h e r e was d i s c r e t e l o c a l i z a t i o n o f l y s o s o m e s i n c o h o m u s c l e -97-p r o v i d e d f u r t h e r e v i d e n c e t h a t l y s o s o m a l enzymes may be i m -p o r t a n t i n t h e d e t e r i o r a t i o n o f chum m u s c l e d u r i n g s p a w n i n g . I t w o u l d be i n t e r e s t i n g t o d e t e r m i n e t h e numbers and s i z e o f l y s o s o m e s i n i n t a c t chum m u s c l e d u r i n g d i f f e r e n t s t a g e s o f s p a w n i n g m i g r a t i o n . T h i s w o u l d p r o v i d e more e v i d e n c e o f t h e i n v o l v e m e n t o f l y s o s o m a l p r o t e i n a s e s i n m u s c l e d e g r a d a t i o n d u r i n g s p a w n i n g m i g r a t i o n . - 9 8 -5. STRUCTURAL CHANGES IN MUSCLE 5.1. I n t r o d u c t i o n E l e c t r o n m i c r o s c o p y i s a t e c h n o l o g y by w h i c h t h e s t r u c -t u r e o f b o t h n o r m a l and p a t h o l o g i c a l s t r i a t e d m u s c l e d i s o r -d e r s c a n be s t u d i e d . As a r e s u l t , t h e r e i s a w e l l e s t a b -l i s h e d body o f knowledge a v a i l a b l e r e g a r d i n g t h e b i o l o g i c a l s t r u c t u r e o f m u s c l e t i s s u e and a w i d e r a n g e o f u s e f u l t e c h -n i q u e s w h i c h h a v e b e e n e s t a b l i s h e d t o s t u d y c h a n g e s w h i c h o c c u r i n m u s c l e a t t h e u l t r a s t r u c t u r a l l e v e l . I n 1976, S t a n l e y and S w a t l a n d commented t h a t t h e r e was a c e r t a i n r e -l u c t a n c e on t h e p a r t o f f o o d s c i e n t i s t s i n v e s t i g a t i n g meat t e x t u r e t o b o r r o w t e c h n i q u e s f r o m s o u r c e s s u c h a s h i s t o c h e m i s t r y and e l e c t r o n m i c r o s c o p y . I n s t e a d , much r e -s e a r c h t i m e was d i r e c t e d t o t h e complex s t u d y o f t h e mechan-i c a l , o r r h e o l o g i c a l p r o p e r t i e s o f m u s c l e . The d e s i r e t o i m p r o v e t h e t e x t u r e o f t h e f l e s h o f chum s a l m o n h a s b e e n a d d r e s s e d by e m p i r i c a l m e t h o d s . D i f f e r e n t p r o c e s s i n g t e c h n i q u e s s u c h a s r e m o v i n g s k i n a n d bone, b r i n e t r e a t m e n t s , and h e a t p r o c e s s i n g i n t h i n p r o f i l e r e t o r t a b l e p o u c h e s have b e e n t e s t e d ( C o l l i n s , 1 9 8 9 ) . L i g h t , and e l e c -t r o n m i c r o s c o p y may be e m p l o y e d t o o b s e r v e c h a n g e s i n m u s c l e d u r i n g s p a w n i n g m i g r a t i o n a t t h e u l t r a s t r u c t u r a l l e v e l . T h e r e a r e many f a c t o r s w h i c h c o m b i n e t o a f f e c t t h e u l t r a s t r u c t u r a l a p p e a r a n c e o f t h e f i s h ' s s k e l e t a l m u s c l e . T h e p h y s i c a l s t r e s s e s o f m i g r a t i o n , t o g e t h e r w i t h t h e -99-p r o c e s s o f s e x u a l m a t u r a t i o n a n d s t a r v a t i o n c o n t r i b u t e t o m y o f i b r i l l a r d e g r a d a t i o n . The o b j e c t o f t h i s e x p e r i m e n t was t o o b s e r v e t h e p h y s i -c a l a p p e a r a n c e o f m u s c l e f r o m t h e s p a w n i n g chum s a l m o n a t t h e u l t r a s t r u c t u r a l l e v e l and compare t h i s p a r a m e t e r t o t h a t o f an o c e a n - r u n f i s h . 5.2. M a t e r i a l s and Methods 5.2.1. L i g h t m i c r o s c o p y M u s c l e f r o m t h e f o u r g r a d e s o f chum s a l m o n were s a m p l e d i n a p r e - r i g o r s t a t e . T i s s u e was e x c i s e d f r o m t h e d o r s a l m u s c l e , t r i m m e d i n t o 5 mm b l o c k s a n d a l l o w e d t o r e s t f o r s e v e r a l m i n u t e s i n a d r y p e t r i p l a t e . The m u s c l e was t h e n f i x e d f o r one week i n a 10% f o r m a l i n s o l u t i o n . A f t e r f i x a -t i o n , t h e t i s s u e was d e h y d r a t e d w i t h an a s c e n d i n g s e r i a l d i -l u t i o n o f e t h a n o l and x y l e n e , and embedded i n p a r a f f i n wax. S i x m i c r o n s e c t i o n s were c u t , t h e t i s s u e was dewaxed w i t h x y l e n e , and r e h y d r a t e d w i t h a d e s c e n d i n g s e r i a l d i l u t i o n o f e t h a n o l and w a t e r ( K i e r n a n , 1 9 8 1 ) . The s e c t i o n s were t h e n - s t a i n e d u s i n g p h o s p h o t u n g s t i c a c i d h a e m a t o x y l i n ( L i l l i e , 1 9 7 7 ) . P h o t o g r a p h s were t a k e n u s i n g a Z e i s s MC 100 l i g h t m i c r o s c o p e . 5.2.2. E l e c t r o n m i c r o s c o p y G r a d e s one and f o u r o f t h e chum s a l m o n were s a m p l e d i n a p r e - r i g o r s t a t e . A 3 mm d i a m e t e r c o r e o f t i s s u e was t a k e n f r o m t h e d o r s a l m u s c l e o f t h e f i s h u s i n g a p u n c h b i o p s y i n -s t r u m e n t . T h e t i s s u e was a l l o w e d t o r e s t f o r s e v e r a l -100-m i n u t e s , and r i n s e d w i t h 0.1 M s o d i u m c a c o d y l a t e b u f f e r (pH 7 . 3 ) . The t i s s u e was f i x e d w i t h 2.5% g l u t a r a l d e h y d e i n 0.1M s o d i u m c a c o d y l a t e b u f f e r , pH 7.3, a t 4°C o v e r n i g h t . The m u s c l e was r i n s e d i n b u f f e r and p o s t f i x e d i n a 1% OsO 4/0.1 M s o d i u m c a c o d y l a t e b u f f e r m i x t u r e f o r 1 h o u r , r i n s e d i n d i s t i l l e d w a t e r t h r e e t i m e s , a n d s e r i a l l y d e h y d r a t e d i n e t h a n o l and p r o p y l e n e o x i d e . The t i s s u e was i n f i l t r a t e d and embedded w i t h S p u r r ' s m e d i a . R i b b o n s o f u l t r a t h i n " s i l v e r " s e c t i o n s (500-600 A n g s t r o m s ) were c u t f r o m t h e b l o c k s w i t h an Om U3 u l t r a m i c r o t o m e ( R e i c h e r t , A u s t r i a ) , a n d p l a c e d on c o p p e r g r i d s . The g r i d s were s t a i n e d f o r 20 m i n u t e s w i t h 2% aqueous u r a n y l a c e t a t e and f o r 10 m i n u t e s w i t h S a t o ' s l e a d c i t r a t e ( K i e r n a n , 1 9 8 1 ) . The g r i d s were v i e w e d and p h o t o -g r a p h e d w i t h a t r a n s m i s s i o n e l e c t r o n m i c r o s c o p e . 5.3. R e s u l t s and D i s c u s s i o n 5.3.1. L i g h t m i c r o s c o p y T h e p h o s p h o t u n g s t i c a c i d h a e m a t o x y l i n h i s t o c h e m i c a l s t a i n was u s e d t o i n v e s t i g a t e t h e c h a n g e s i n t h e m o r p h o l o g y o f t h e m u s c l e d u r i n g s p a w n i n g m i g r a t i o n . M i c r o g r a p h s o f s e c t i o n s f r o m t h e d o r s a l m u s c l e c l e a r l y showed a c hange be -t w e e n t h e s i l v e r - b r i g h t a n d s p a w n i n g f i s h . W i t h i n t h e m u s c l e c e l l s o f t h e g r a d e 1 f i s h , t h e m y o f i b r i l s were q u i t e c l o s e l y p a c k e d , a s i l l u s t r a t e d i n t h e c r o s s s e c t i o n , F i g u r e 5.1, F i g u r e 5.2 a n d t h e l o n g i t u d i n a l s e c t i o n , F i g u r e 5.3. -101-Figure 5.1. Light micrograph of a s i l ve r -b r i gh t chum salmon stained with phosphotungstic acid haematoxylin. Bar represents 10 microns. (Magnification 1500). -102-Figure 5.2. Light micrograph of a cross section of a silver-bright chum salmon stained with phosphotungstic acid haematox-y l i n . Bar represents 20 microns. (Magnification 600). -103-The g r a d e 2 a n d 3 f i s h w ere s i m i l a r , b u t a p p e a r e d t o h a ve p r o g r e s s i v e l y more s p a c e between t h e m y o f i b r i l b u n d l e s . I n t h e s p a w n i n g f i s h , d r a m a t i c c h a n g e s were s e e n i n t h e m u s c l e c e l l s . F i g u r e 5.4 and F i g u r e 5.5 show a c r o s s s e c t i o n o f m u s c l e c e l l s i n w h i c h t h e l a r g e s p a c e s w i t h i n t h e c e l l s i n -d i c a t e a l o s s o f m y o f i b r i l s . F i g u r e 5.6 was a l o n g i t u d i n a l s e c t i o n o f t h e s p a w n i n g m u s c l e and d e m o n s t r a t e d m y o f i b r i l l a r t e a r i n g , and l o s s i n i n t e g r i t y o f t h e m u s c l e s t r u c t u r e . The c h a n g e s o b s e r v e d by t h e l i g h t m i c r o g r a p h s were a l s o f o u n d b y G r e e n e ( 1 9 2 6 ) who d e s c r i b e d t h e s t a t e o f m y o f i b r i l l a r c h e e k m u s c l e i n t h e s p a w n i n g c h i n o o k s a l m o n . M u s c l e f i b r e s were e x t r e m e l y d i s o r g a n i z e d , and h a d l o s t a l l s t r u c t u r a l c h a r a c t e r i s t i c s . S t r i a t i o n s had d i s a p p e a r e d f r o m t h e f i b r e s , a n d t h e c e l l s a p p e a r e d u n s t r u c t u r e d and s w o l l e n . S i m i l a r c h a n g e s were f o u n d i n t h e w h i t e m u s c l e , however t h e y were l e s s d r a m a t i c . 5.3.2. E l e c t r o n m i c r o s c o p y T h i r t y - s i x e l e c t r o n m i c r o g r a p h i c f i e l d s o f d i f f e r e n t m u s c l e c e l l s i n two g r a d e 1 and two g r a d e 4 chum f i s h were o b s e r v e d . A l t h o u g h i n many m i c r o g r a p h s t h e r e were d i s t i n c t d i f f e r e n c e s between t h e s i l v e r - b r i g h t and s p a w n i n g chum, i n o t h e r m i c r o g r a p h s i t was d i f f i c u l t t o d i f f e r e n t i a t e between t h e two g r a d e s . F i g u r e s 5.7 t o 5.10 a r e e l e c t r o n m i c r o -g r a p h s o f t h e m u s c l e o f s i l v e r - b r i g h t chum s a l m o n . I t was i n t e r e s t i n g t o n o t e t h a t t h e m i c r o g r a p h s o f t h e s i l v e r -b r i g h t f i s h e x h i b i t e d many o f t h e same f o r m s o f d e g e n e r a t i o n -104-Figure 5.3. Light micrograph of a longitudinal section of a silver-bright chum salmon stained with phosphotungstic acid haematoxylin. Bar represents 10 microns. (Magnification 1500). -105-Figure 5.4. Light micrograph of a cross section of a spawning chum salmon stained with phosphotungstic acid haematoxylin. Bar represents 10 microns. (Magnification 1500). -106-Figure 5.5. Light micrograph of a cross section of a spawning chum salmon stained with phosphotungstic acid haematoxylin. Bar represents 20 microns. (Magnification 600). -107-Figure 5.6. Light micrograph of a longitudinal section of a spawning chum salmon stained with phosphotungstic acid haematox-y l i n . Bar represents 10 microns. (Magnification 1500). -108-F i g u r e 5.7. E l e c t r o n m i c r o g r a p h o f t h e d o r s a l m u s c l e o f a s i l v e r - b r i g h t s a l m o n . B a r o n u p p e r r i g h t c o r n e r o f p h o t o g r a p h r e p r e s e n t s 1 m i c r o n . ( M a g n i f i c a t i o n = 2 7 , 0 0 0 ) . -109-F i g u r e 5.8. E l e c t r o n m i c r o g r a p h o f t h e d o r s a l m u s c l e o f a s i 1 v e r - b r i g h t s a l m o n . B a r o n u p p e r r i g h t c o r n e r o f p h o t o g r a p h r e p r e s e n t s 1 m i c r o n . ( M a g n i f i c a t i o n = 2 7 , 0 0 0 ) . -110-F i g u r e 5.9. E l e c t r o n m i c r o g r a p h o f t h e d o r s a l m u s c l e o f a s i l v e r - b r i g h t s a l m o n . B a r o n u p p e r r i g h t c o r n e r o f p h o t o g r a p h r e p r e s e n t s 1 m i c r o n . ( M a g n i f i c a t i o n = 2 7 , 0 0 0 ) . - I l l -F i g u r e 5.10. E l e c t r o n m i c r o g r a p h o f t h e d o r s a l m u s c l e o f a s i 1 v e r - b r i g h t s a l m o n . B a r o n u p p e r r i g h t c o r n e r o f p h o t o g r a p h r e p r e s e n t s 1 m i c r o n . ( M a g n i f i c a t i o n = 2 7 , 0 0 0 ) . -112-a s t h e s p a w n i n g f i s h , b u t were p r e s e n t much l e s s f r e q u e n t l y . As a r e s u l t , a l i s t o f c r i t e r i a t o d i s t i n g u i s h b e t w e e n t h e m i c r o g r a p h s o f s i l v e r - b r i g h t a n d s p a w n i n g f i s h was d e v e l o p e d . F o r e a c h m i c r o g r a p h , f o u r c r i t e r i a w ere s c o r e d b y a s s i g n i n g a v a l u e o f 0 (none o b s e r v e d ) , o r 1 ( o b s e r v e d somewhere on t h e p h o t o g r a p h ) . The f i n a l s c o r e f o r e a c h p h o t o g r a p h was c a l c u l a t e d as t h e sum of, t h e 4 c r i -t e r i a a n d t h u s t h e s c o r e s r a n g e d between 0 t o 4. The c r i t e -r i a u s e d were d e s c r i b e d a s f o l l o w s . S m e a r i n g , F i g u r e 5.11, t h e p a r t i a l o r c o m p l e t e l o s s o f a d i s t i n c t p a t t e r n o f a c t i n and m y o s i n f i l a m e n t s . I f a c t i n and m y o s i n c o u l d be d i s t i n -g u i s h e d , a s c o r e o f 0 was a s s i g n e d . S p l i t t i n g , F i g u r e 5.12, w ere b r e a k s o r t e a r s w i t h i n t h e m y o f i b r i l b u n d l e s . I f any b r e a k s o r t e a r s were o b s e r v e d , a s c o r e o f 1 was a s s i g n e d . I n t e r - m y o f i b r i l s p a c e s , F i g u r e 5.13, was an i n c r e a s e i n t h e s p a c e n o r m a l l y f o u n d b e t w e e n m u s c l e f i b r i l s . S w o l l e n T - t u b u l e s w e r e n o t s u f f i c i e n t f o r a s c o r e o f 1. I f t h e s p a c e s between b u n d l e s were s m a l l and r e g u l a r , a s c o r e o f 0 was a s s i g n e d e v e n i f t h e t u b u l e s a p p e a r e d s w o l l e n . The e n t i r e p h o t o g r a p h was c o n s i d e r e d t o d e t e r m i n e w h e t h e r t h e s p a c e s w e r e u n u s u a l . T h i n n i n g , F i g u r e 5.14, was t h e d e -c r e a s e d d e n s i t y o f t h e m y o f i l a m e n t s w i t h i n a f i b r i l . S m a l l s p a c e s b e t w e e n t h e a c t i n and m y o s i n i n t h e m y o f i b r i l s were a s s i g n e d a s c o r e o f 1. F i v e j u d g e s were a s k e d t o c a t e g o r i z e t h e m i c r o g r a p h s u s i n g t h e s c o r i n g s y s t e m , and t h e i r r e s u l t s a r e g i v e n i n -113-F i g u r e 5.11. E l e c t r o n m i c r o g r a p h o f t h e d o r s a l m u s c l e o f s p a w n i n g chum s a l m o n . " S m e a r i n g " i s p r e s e n t on t h e u p p e r p o r t i o n o f t h e p h o t o g r a p h . B a r on u p p e r r i g h t c o r n e r o f p h o t o g r a p h r e p r e s e n t s 1 m i c r o n . ( M a g n i f i c a t i o n = 2 6 , 0 0 0 ) . -114-F i g u r e 5.12. E l e c t r o n m i c r o g r a p h o f t h e d o r s a l m u s c l e o f s p a w n i n g chum s a l m o n . " S p l i t t i n g " i s p r e s e n t t o t h e r i g h t o f t h e p h o t o g r a p h . B a r on u p p e r r i g h t c o r n e r o f p h o t o g r a p h r e p r e s e n t s l m i c r o n . ( M a g n i f i c a t i o n = 2 6 , 0 0 0 ) . -115-F i g u r e 5.13. E l e c t r o n m i c r o g r a p h o f t h e d o r s a l m u s c l e o f s p a w n i n g c h u m s a l m o n . N o t i c e a r e a s w i t h " i n t e r - m y o f i b r i l s p a c e s " B a r on u p p e r r i g h t c o r n e r o f p h o t o g r a p h r e p r e s e n t s 1 m i c r o n . ( M a g n i f i c a t i o n = 2 6 , 0 0 0 ) . - 1 1 6 -F i g u r e 5.14. E l e c t r o n m i c r o g r a p h o f t h e d o r s a l m u s c l e o f s p a w n i n g chum s a l m o n . " T h i n n i n g " w i t h i n t h e m y o f i b r i l b u n d l e s i s a p p a r e n t . B a r on u p p e r r i g h t c o r n e r o f p h o t o g r a p h r e p r e s e n t s l m i c r o n . ( M a g n i f i c a t i o n = 2 6 , 0 0 0 ) . -117 T a b l e 5.1. The j u d g e s h a d a 72 t o 87% s u c c e s s r a t e i n a s -s i g n i n g t h e s i l v e r - b r i g h t s a m p l e s a c u m u l a t i v e s c o r e o f z e r o , o r one. I t was c o n c l u d e d t h a t i t was n o t p o s s i b l e t o c o n s i s t e n t l y d i s t i n g u i s h b e t w e e n t h e s i l v e r - b r i g h t a n d s p a w n i n g s a m p l e s b a s e d on a s i n g l e m i c r o s c o p i c f i e l d . How-e v e r , t h i s i n a b i l i t y o f t h e g r a d i n g s y s t e m s h o u l d n o t be c o n s i d e r e d a s a f a i l u r e o f t h e method. I n s t e a d , i t was a r e f l e c t i o n o f t h e f o c a l n a t u r e o f m u s c l e d e g r a d a t i o n w h i c h o c c u r e d d u r i n g s e x u a l m a t u r a t i o n . A l t h o u g h some a r e a s i n t h e m u s c l e o f s p a w n i n g chum a p p e a r e d s e v e r e l y d e g r a d e d , o t h e r a r e a s r e t a i n e d t h e s t r u c t u r e and i n t e g r i t y o f a s i l v e r b r i g h t s a m p l e . C o n v e r s e l y , i t was n o t c o r r e c t t o a ssume t h a t t h e s i l v e r - b r i g h t f i s h w o u l d n o t h a v e a n y a r e a s o f m u s c l e d e g r a d a t i o n . I t was n e c e s s a r y t o c o n s i d e r a number o f m i c r o g r a p h s i n o r d e r t o c o r r e c t l y a s s e s s t h e d e g r e e o f m a t u r i t y o f t h e f i s h . I t i s e x p e c t e d t h e r e f o r e , t h a t g i v e n a number o f m i c r o g r a p h s f r o m t h e same f i s h , t h i s s c o r i n g s y s t e m c o u l d s u c c e s s f u l l y d i s t i n g u i s h between m a t u r e and im-m a t u r e f i s h . -118-T a b l e 5.1. P e r c e n t a g e o f s i l v e r - b r i g h t a n d s p a w n i n g f i s h a s s i g n e d t o e a c h s c o r e u s e d t o j u d g e m i c r o g r a p h s o f chum d o r s a l m u s c l e . J u d g e G r a d e 0 1 S c o r e 2 3 4 T o t a l 1 1 S i l v e r 28 13 6 3 0 87% Spawn 1 3 13 19 12 2 S i l v e r 20 20 3 9 0 77% Spawn 6 9 10 11 11 3 S i l v e r 10 19 19 7 0 72% Spawn 0 3 13 19 8 4 S i l v e r 22 22 12 0 0 73% Spawn 5 10 10 17 2 5 S i l v e r 7 44 5 0 0 75% Spawn 3 17 12 12 0 T o t a l c o r r e c t c l a s s i f i c a t i o n . S i l v e r - b r i g h t f i s h were c l a s s i f i e d a s h a v i n g s c o r e s l e s s t h a n 2. -119-6. CONCLUSIONS T h e s p a w n i n g m i g r a t i o n o f chum s a l m o n ( O n c o r h y n c h u s k e t a ) a d v e r s e l y a f f e c t e d t h e a p p e a r a n c e a n d e a t i n g q u a l i t y o f t h e c a n n e d p r o d u c t . A l t h o u g h h a r v e s t i n g t h e chum o n l y as s i l v e r - b r i g h t s i n t h e o c e a n w o u l d i m p r o v e t h e q u a l i t y o f t h e f i n a l p r o d u c t , o t h e r p r o b l e m s p r e v e n t t h i s p r a c t i c e . I n o r d e r t o p r o t e c t weaker s t o c k s o f f i s h , a l a r g e p o r t i o n o f t h e chum a r e h a r v e s t e d a s t h e y a p p r o a c h t h e mouth o f t h e r i v e r . T h e o b j e c t o f t h i s w o r k was t o a s s e s s t h e p h y s i c a l , c h e m i c a l a n d t e x t u r a l c h a n g e s w h i c h o c c u r r e d i n t h e chum t h r o u g h o u t t h e i r s p a w n i n g m i g r a t i o n . T h i s was a c c o m p l i s h e d u s i n g methods s u c h a s p r o x i m a t e a n a l y s i s , c o l o u r a n a l y s i s , p r o t e i n a s e a s s a y s , l i g h t a n d t r a n s m i s s i o n e l e c t r o n m i c r o s -c o p y . I n a d d i t i o n , s e n s o r y p a n e l s a n d a n i n s t r u m e n t a l method were u s e d t o measure t e x t u r e . T h e f i s h w e r e g r a d e d i n t o f o u r c a t e g o r i e s b a s e d o n t h e i r e x t e r n a l a p p e a r a n c e . The s k i n c o l o u r , t h i c k n e s s and s l i m e l a y e r were i m p o r t a n t c o n s i d e r a t i o n s i n t h e d e t e r m i n a -t i o n o f g r a d e . C o l o u r o f t h e raw f l e s h was m e a s u r e d w i t h a H u n t e r L a b c o l o u r i m e t e r , and by c a l c u l a t i n g t h e c o n c e n t r a -t i o n o f a s t a x a n t h i n i n t h e f a t - s o l u b l e p o r t i o n o f t h e m u s c l e b a s e d on i t s a b s o r b a n c e a t 475 nm. The v i s u a l change i n t h e m u s c l e f r o m a r e d - p i n k c o l o u r t o a w h i t e - g r e y c o l o u r was a r e f l e c t i o n o f t h e l o s s o f - 1 2 0 -a s t a x a n t h i n i n t h e m u s c l e and t h e c h a n g e s i n t h e H u n t e r L a b v a l u e s . The c o n t e n t o f a s t a x a n t h i n d e c r e a s e d s i g n i f i c a n t l y w i t h i n c r e a s i n g g r a d e . The H u n t e r a and b v a l u e s a l s o d e -c r e a s e d s i g n i f i c a n t l y , b u t t h e L v a l u e i n c r e a s e d . C o r r e l a -t i o n a n a l y s i s between g r a d e and t h e c o l o u r v a l u e s i n d i c a t e d t h a t a/L, a s t a x a n t h i n , ' a ' , and a/b were s i g n i f i c a n t l y n e g a -t i v e l y c o r r e l a t e d w i t h g r a d e . The b e s t i n d i c a t o r o f g r a d e was t h e r a t i o a/L. P r o x i m a t e a n a l y s i s c o n f i r m e d l i t e r a t u r e f i n d i n g s t h a t t h e r e were s i g n i f i c a n t d e c r e a s e s i n p r o t e i n , f r o m 17.9 % t o 15.8% and l i p i d f r o m 4.9% t o 1.0% and i n c r e a s e s i n m o i s t u r e f r o m 74% t o 79% a s t h e f i s h m a t u r e d . T h e s e r e s u l t s demon-s t r a t e d t h e s e v e r e d e p l e t i o n o f t h e e n e r g y s o u r c e s i n t h e m u s c l e a s t h e f i s h p r o g r e s s e s i n i t s m i g r a t i o n . I n s t r u m e n t a l t e x t u r e p r o f i l e a n a l y s i s (TPA) was n o t e f -f e c t i v e i n m e a s u r i n g t h e p r o g r e s s i v e s o f t e n i n g o f t h e s a l m o n m u s c l e . The o n l y two p a r a m e t e r s t o d e t e c t a d i f f e r e n c e i n g r a d e ( a = 0.05) were c o h e s i v e n e s s , t h e r a t i o o f t h e work done d u r i n g t h e f i r s t b i t e ( A r e a 1) and t h e work done on t h e s e c o n d b i t e ( A r e a 2 ) , and s l o p e 2, t h e maximum s l o p e o f t h e f i r s t c o m p r e s s i o n . C o h e s i v e n e s s i n c r e a s e d s i g n i f i c a n t l y w i t h g r a d e and s l o p e 2 d e c r e a s e d w i t h g r a d e . T h e s e n s o r y p a n e l i s t s w e r e more s u c c e s s f u l i n t h e i r a b i l i t y t o d i s t i n g u i s h t h e f o u r g r a d e s o f s a l m o n . A l l o f t h e a t t r i b u t e s d e c r e a s e d s i g n i f i c a n t l y w i t h g r a d e , however d i s c r i m i n a t i o n between e a c h g r a d e was most s u c c e s s f u l w i t h -121-t h e s e n s o r y a t t r i b u t e d r y n e s s . B y p e r f o r m i n g Z - t r a n s f o r m a t i o n s on t h e d a t a f r o m e a c h j u d g e , i t was p o s s i -b l e t o e l i m i n a t e t h e n o n - h o m o g e n e i t y o f v a r i a n c e s b e t w e e n j u d g e s t h u s t h e a n a l y s i s o f v a r i a n c e was more e f f e c t i v e i n d i s t i n g u i s h i n g between t h e g r a d e s f o r e a c h a t t r i b u t e . C o r -r e l a t i o n a n a l y s i s between s e n s o r y and i n s t r u m e n t a l m e a s u r e s o f t e x t u r e i n d i c a t e d t h a t c o h e s i v e n e s s was s i g n i f i c a n t l y n e g a t i v e l y c o r r e l a t e d w i t h t h e f o u r s e n s o r y a t t r i b u t e s , and t h a t f i b r o u s n e s s b e s t c o r r e l a t e d w i t h c o h e s i v e n e s s . T he n e g a t i v e c o r r e l a t i o n between s e n s o r y a n d i n s t r u m e n t a l mea-s u r e s i n d i c a t e d t h a t a l t h o u g h t h e r e was a d e f i n i t e s o f t e n i n g o f t h e t e x t u r e o f t h e c a n n e d p r o d u c t c a u s e d by t h e breakdown o f m y o f i b r i l l a r p r o t e i n s , t h e I n s t r o n was n o t m e a s u r i n g t h i s c h a n g e . B e c a u s e t h e m u s c l e f i b r e s were n o t o r i e n t e d i n any p a r t i c u l a r d i r e c t i o n i n t h e sample p r e s e n t e d t o t h e I n s t r o n , i t was n o t p o s s i b l e t o m e a s u r e t h e l o s s o f m y o f i b r i l l a r s t r e n g t h . I t i s p o s s i b l e however t h a t t h e I n s t r o n was mea-s u r i n g t h e f o r c e n e c e s s a r y f o r t h e m u s c l e f i b r e s t o s l i d e a c r o s s e a c h o t h e r . S t a r v a t i o n i n t h e m i g r a t i n g s a l m o n w o u l d r e s u l t i n a l o s s t h e m y o f i b r i l l a r p r o t e i n s a n d an i n c r e a s e i n t h e m o i s t u r e c o n t e n t and t h e r e l a t i v e amount o f c o n n e c -t i v e t i s s u e . As t h e c o n t e n t o f g e l a t i n i z e d c o l l a g e n , m o i s -t u r e and s a r c o p l a s m i c p r o t e i n s i n c r e a s e d w i t h i n c r e a s i n g ma-t u r i t y o f t h e f i s h , t h e f o r c e n e c e s s a r y t o s l i d e t h e m u s c l e f i b r e s a c r o s s e a c h o t h e r w o u l d a l s o i n c r e a s e . -122-The p r o t e i n a s e a c t i v i t y o f t h e m u s c l e was m e a s u r e d a t pH 3.5, 6.2 and 7.0. T h e r e was a s i g n i f i c a n t i n c r e a s e i n a c t i v i t y w i t h g r a d e a t pH 3.5. T h e a c t i v i t y o f a c i d p h o s p h a t a s e , a l y s o s o m a l m a r k e r , a l s o i n c r e a s e d s i g n i f i -c a n t l y w i t h g r a d e . T h e s e r e s u l t s i n d i c a t e d t h e p o s s i b l e c o n t r i b u t i o n o f l y s o s o m a l e n z y m e s t o t h e d e g r a d a t i o n o f m u s c l e d u r i n g m i g r a t i o n . F u r t h e r work w o u l d be u s e f u l t o i s o l a t e and i d e n t i f y t h e s e enzymes. L i g h t a n d e l e c t r o n m i c r o g r a p h s w e r e u s e d t o o b s e r v e v i s i b l e c h a n g e s i n t h e s t r u c t u r e o f t h e m u s c l e . L i g h t m i -c r o g r a p h s r e v e a l e d a n a p p a r e n t l o s s i n t h e d i a m e t e r , o r number o f m y o f i b r i l s i n t h e m u s c l e c e l l s . L o n g i t u d i n a l s e c -t i o n s showed an i n c r e a s e d o c c u r r e n c e o f m y o f i b r i l l a r t e a r -i n g , and l o s s o f i n t e g r i t y o f t h e m u s c l e . E l e c t r o n m i c r o -g r a p h s o f t h e m u s c l e r e v e a l e d a r e a s o f f o c a l d e g e n e r a t i o n . T h i s d e g e n e r a t i o n was d e s c r i b e d b y f o u r c r i t e r i a i n c l u d i n g s m e a r i n g , a p a r t i a l o r c o m p l e t e l o s s t h e o r g a n i z a t i o n o f t h e m y o f i b r i l s , s p l i t t i n g , t e a r s o r b r e a k s i n m y o f i b r i l b u n d l e s , i n t e r m y o f i b r i l s p a c e s , e x c e s s c y t o p l a s m i c f l u i d i n t h e c e l l , a nd t h i n n i n g , a d e c e a s e i n t h e d e n s i t y o f t h e m y o f i l a m e n t s w i t h i n a f i b r i l . Use o f t h e s e f o u r c r i t e r i a h e l p e d d e t e r -m i n e w h e t h e r t h e m i c r o g r a p h was f r o m a s p a w n i n g o r s i l v e r - b r i g h t f i s h . However, i t was n o t p o s s i b l e t o p r e d i c t t h e m a t u r i t y o f a f i s h b a s e d on one p h o t o g r a p h b e c a u s e o f t h e f o c a l n a t u r e o f t h e d e g e n e r a t i o n . I n a d d i t i o n , m i c r o -g r a p h s o f s i l v e r - b r i g h t f i s h a l s o d e m o n s t r a t e d some o f t h e -123-same c h a n g e s , b u t t o a l e s s e r d e g r e e . I t w o u l d be n e c e s s a r y t o l o o k a t a number o f p h o t o g r a p h s t o a c c u r a t e l y d i s t i n g u i s h t h e f i s h . -124-7. REFERENCES A b b o t t , J . A., M a s s i e , D. R. , a n d Watada, A. E. 1982. The u s e o f a co m p u t e r w i t h an I n s t r o n f o r t e x t u r a l mea-s u r e m e n t s . J o u r n a l o f T e x t u r e S t u d i e s . 13:413. A g u i l e r a , J.M. a n d S t a n l e y , D.W. 1990. " M i c r o s t r u c t u r a l P r i n c i p l e s o f F o o d P r o c e s s i n g a n d E n g i n e e r i n g " . E l s e l v i e r A p p l i e d S c i e n c e . New Y o r k . p. 161-192. A k s n e s , A., G j e r d e , B. , and O l a R o a l d , S. 1986. B i o l o g i c a l , c h e m i c a l , and o r g a n o l e p t i c c h a n g e s d u r i n g m a t u r a t i o n o f f a r m e d A t l a n t i c s a l m o n , Salmo s a l a r . A q u a c u l t u r e . 53:7. Ando, S., and H a t a n o , M. 1986a. B i o c h e m i c a l c h a r a c t e r i s t i c s o f chum s a l m o n m u s c l e d u r i n g s p a w n i n g m i g r a t i o n . B u l -l e t i n o f t h e J a p a n e s e S o c i e t y o f S c i e n t i f i c F i s h e r -i e s . 5 2 ( 7 ) : 1 2 2 9 . A n d o , S., a n d H a t a n o , M. 1 9 8 6 b . M y o f i b r i l l a r p r o t e i n d e g r a d a t i o n i n s p a w n i n g m i g r a t i n g chum s a l m o n , a s e v a l u a t e d by e x t r a c t i v e N - m e t h y l h i s t i d i n e l e v e l . B u l -l e t i n o f t h e J a p a n e s e S o c i e t y o f S c i e n t i f i c F i s h e r -i e s . 5 2 ( 7 ) : 1 2 3 7 . A n d o a n d H a t a n o M. 1 9 8 7 . M e t a b o l i c p a t h w a y s o f c a r o t e n o i d s i n chum s a l m o n O n c o r h y n c h u s k e t a d u r i n g s p a w n i n g m i g r a t i o n . C o m p a r a t i v e B i o c h e m i s t r y a n d P h y s i o l o g y . 8 7 B ( 2 ) : 4 1 1 . Ando, S., H a t a n o , M. , a n d Zama, K. 1985. A c o n s u m p t i o n o f m u s c l e l i p i d d u r i n g s p a w n i n g m i g r a t i o n o f chum s a l m o n O n c o r h y n c h u s k e t a . B u l l e t i n o f t h e J a p a n e s e S o c i e t y o f S c i e n t i f i c F i s h e r i e s . 5 1 ( 1 1 ) : 1 8 1 7 . A n d o , S., O s a d a , K. , H a t a n o , M., a n d S a n e y o s h i , M. 1989. C o m p a r i s o n o f c a r o t e n o i d s i n m u s c l e a n d o v a r y f r o m f o u r g e n e r a o f s a l m o n i d f i s h e s . C o m p a r a t i v e B i o c h e m -i s t r y and P h y s i o l o g y . 9 3 B ( 3 ) : 5 0 3 . A n d o , S., Y a m a z a k i , F. , H a t a n o , M. , a n d Zama, K. 1 9 8 6 . D e t e r i o r a t i o n o f chum s a l m o n f O n c o r h y n c h u s k e t a ) m u s c l e d u r i n g s p a w n i n g m i g r a t i o n - I I I . C h a n g e s i n p r o t e i n c o m p o s i t i o n and p r o t e a s e a c t i v i t y o f j u v e n i l e chum s a l m o n m u s c l e upon t r e a t m e n t w i t h s e x s t e r o i d s . C o m p a r a t i v e B i o c h e m i s t r y and P h y s i o l o g y . 8 3 B ( 2 ) : 3 2 5 . A n o n y m o u s . 1 9 8 6 . " C o l o u r E v a l u a t i o n G u i d e f o r P a c i f i c S a l m o n . A l a s k a S e a f o o d M a r k e t i n g I n s t i t u t e . P . O. Box DX, J u n e a u , A l a s k a . -125-A n o n y m o u s . 1 9 7 0 . " O f f i c i a l M e t h o d s o f A n a l y s i s o f t h e A s s o c i a t i o n o f O f f i c i a l A n a l y t i c a l C h e m i s t s " . 1 1 t h e d i t i o n . M e t h o d No. 2051. A s s o c i a t i o n o f O f f i c i a l A n a l y t i c a l C h e m i s t s . W a s h i n g t o n , D.C. A s g h a r , A., a n d B h a t t i , A.R. 1987. E n d o g e n o u s p r o t e o l y t i c e n z y m e s i n s k e l e t a l m u s c l e : t h e i r s i g n i f i c a n c e i n m u s c l e p h y s i o l o g y and d u r i n g p o s t m o r t e m a g i n g e v e n t s i n c a r c a s s e s . A d v a n c e s i n F o o d R e s e a r c h . 31:343. B a r r e t t , A. J . 1 9 7 8 . L y s o s o m a l p r o t e i n a s e s a n d t h e i r s p e c i f i c i t y . I n : " P r o t e i n T u r n o v e r and Lysosome F u n c -t i o n . " S e g a l , H. L . , a n d D o y l e D. J . ( E d . ) . 295 p. A c a d e m i c P r e s s , New Y o r k . Beacham, T.D., a n d S t a r r , P. 1982. P o p u l a t i o n b i o l o g y o f c h um s a l m o n , O n c o r h y n c h u s k e t a , f r o m t h e F r a s e r R i v e r , B r i t i s h C o l u m b i a . U.S. N a t i o n a l M a r i n e F i s h e r -i e s S e r v i c e . F i s h e r i e s B u l l e t i n . 8 0 ( 4 ) : 8 1 3 . B e a r d a l l , C H . , and J o h n s t o n , I.A. 1985. The u l t r a s t r u c t u r e o f m y o t o mal m u s c l e s o f s a i t h e P o l l a c h i u s v i r e n s L . f o l l o w i n g s t a r v a t i o n and r e f e e d i n g . E u r o p e a n J o u r n a l o f C e l l B i o l o g y . 3 9 ( 1 ) : 1 0 5 . Beeken, W.L. a n d R o e s s n e r , K.D. 1972. I n v i v o l a b e l i n g o f h e p a t i c l y s o s o m e s b y i n t r a g a s t r i c a d m i n i s t r a t i o n o f a c r i d i n e o r a n g e . L a b o r a t o r y I n v e s t i g a t i o n s . 2 6 ( 2 ) : 1 7 3 . B e l c a s t r o , A.N., M a c l e a n , I . , a n d G i l c h r i s t , J . 1 9 8 5 . B i o c h e m i c a l b a s i s o f m u s c u l a r f a t i g u e a s s o c i a t e d w i t h r e p e t i t i o u s c o n t r a c t i o n s o f s k e l e t a l m u s c l e . I n t e r n a -t i o n a l J o u r n a l o f B i o c h e m i s t r y . 1 7 ( 4 ) : 4 4 7 . B i l i n s k i , E . , J o n a s , R. E . E . , P e t e r s , M. D., a n d C h o r o m a n s k i , E. M. 1984. E f f e c t s o f s e x u a l m a t u r a t i o n on t h e q u a l i t y o f c o h o s a l m o n ( O n c o r h y n c h u s k i s u t c h ) f l e s h . C a n a d i a n I n s t i t u t e o f F o o d S c i e n c e and T e c h -n o l o g y J o u r n a l . 1 7 ( 4 ) : 2 7 1 . B i r d , J . W. C. , S p a n i e r , A. M. , a n d S c h w a r t z , W. N. 1978. C a t h e p s i n s B a n d D: p r o t e o l y t i c a c t i v i t y a n d u l t r a s t r u c t u r a l l o c a l i z a t i o n i n s k e l e t a l m u s c l e . I n : " P r o t e i n T u r n o v e r a n d L y s o s o m e F u n c t i o n . " S e g a l , H. L. , and D o y l e , D. J . ( E d s . ) . 589 p. A c a d e m i c P r e s s , New Y o r k . B i s h o p , C M . a n d O d e n s e , P.H. 1967. The u l t r a s t r u c t u r e o f t h e w h i t e s t r i a t e d myotomal m u s c l e o f t h e c o d , Gadus morhua. J o u r n a l o f F i s h e r i e s R e s e a r c h B o a r d , Canada. 2 4 ( 1 2 ) : 2 5 4 9 . -126-B l i g h , E.G. a n d D y e r , W.J. 1959. A r a p i d m e thod o f t o t a l l i p i d e x t r a c t i o n a n d p u r i f i c a t i o n . C a n a d i a n J o u r n a l o f B i o c h e m i s t r y and P h y s i o l o g y . 3 7 ( 8 ) : 9 1 1 . B o r d e r i a s , A . J . , Lamua, M. , a n d T e j a d a , M. 1983. T e x t u r e a n a l y s i s o f f i s h f i l l e t s and m i n c e d f i s h by b o t h s e n -s o r y and i n s t r u m e n t a l methods. J o u r n a l o f F o o d T e c h -n o l o g y . 18:85. B o u r n e , M.C. 1 9 7 8 . T e x t u r e p r o f i l e a n a l y s i s . F o o d T e c h n o l o g y . J u l y : 6 2 . B o u r n e , M.C. 1982. "Food T e x t u r e and V i s c o s i t y : C o n c e p t and Measurement. A c a d e m i c P r e s s , New Y o r k . B o u r n e , M. C.; K e n n y , J . F . ; a n d B a r n a r d , J . 1 9 7 8 . C o m p u t e r - a s s i s t e d r e a d o u t o f d a t a f r o m t e x t u r e p r o -f i l e a n a l y s i s c u r v e s . J o u r n a l o f T e x t u r e S t u d i e s . 9:481. B r a n d t , M. A.; S k i n n e r , E . Z., a n d C o l e m a n , J . A. 1963. T e x t u r e p r o f i l e m e t h o d . J o u r n a l o f F o o d S c i e n c e . 28:404. B r e m n e r , H.A., a n d H a l l e t t , I . C . 1 9 8 5 . M u s c l e f i b e r -c o n n e c t i v e t i s s u e j u n c t i o n s i n t h e f i s h b l u e g r e n a -d i e r ( M a c r u r o n u s n o v a e z e l a n d i a e ) . A s c a n n i n g e l e c t r o n m i c r o s c o p e s t u d y . J o u r n a l o f F o o d S c i e n c e . 50:975. B r i n t o n , R. H. J r . a n d B o u r n e , M. C. 1 9 7 2 . D e f o r m a t i o n t e s t i n g o f f o o d s . J o u r n a l o f T e x t u r e S t u d i e s . 3:284. C a s s e n s , R.G., B r i s k e y , E . J ; , a n d H o e k s t r a , W.G. 1 9 6 3 . E l e c t r o n m i c r o s c o p y o f p o s t - m o r t e m c h a n g e s i n p o r c i n e m u s c l e . J o u r n a l o f F o o d S c i e n c e . 28:680. Chang, V.M., a n d I d l e r , D.R. 1960. B i o c h e m i c a l s t u d i e s on s o c k e y e s a l m o n d u r i n g s p a w n i n g m i g r a t i o n . X I I . L i v e r g l y c o g e n . C a n a d i a n J o u r n a l o f B i o c h e m i s t r y and P h y s i -o l o g y . 38:553. Chayen, J . , B i t e n s k y , L. , and B u t c h e r , R. G. ( E d s . ) . 1973. " P r a c t i c a l H i s t o c h e m i s t r y . " J o h n W i l e y a n d S o n s , London. C l o k e , J.D., D a v i d , E.A., G o r d o n , J . , H s i e h , S . I . , G r i d e r , J . , A d d i s , P.B., and M c G r a t h , G.J. 1981. S c a n n i n g and t r a n s m i s s i o n e l e c t r o n m i c r o s c o p y o f n o r m a l a n d PSE p o r c i n e m u s c l e . S c a n n i n g E l e c t r o n M i c r o s c o p y . 3:435. -127-C o l l i n s , L . S . 1989. Q u a l i t y Enhancement o f Canned L a t e - R u n Chum ( O n c o r h y n c h u s k e t a ) . MSc. U n i v e r s i t y o f B r i t i s h C o l u m b i a . D e a n , R. T . 1 9 7 8 . L y s o s o m a l m e c h a n i s m s o f p r o t e i n d e g r a d a t i o n . I n : " P r o t e i n T u r n o v e r and Lysosome F u n c -t i o n . " S e g a l , H. L. , and D o y l e , D. J . ( E d s . ) . 29 p. A c a d e m i c P r e s s , New Y o r k . Dean, R. T., and B a r r e t t , A. J . 1976. L y s o s o m e s . I n : " E s s a y s i n B i o c h e m i s t r y . " V o l . 1 2 . C a m p b e l l , P.N., a n d A l d r i d g e W. N. ( E d s . ) 1 p. A c a d e m i c P r e s s New Y o r k . d e M a n , J.M. 1 9 8 0 . " P r i n c i p l e s o f F o o d C h e m i s t r y " . A V I P u b l i s h i n g Co. W e s t p o r t . D i c e , J . F . , a n d W a l k e r , C. D. 1 9 7 8 . T h e g e n e r a l c h a r a c t e r i s t i c s o f i n t r a c e l l u l a r p r o t e i n d e g r a d a t i o n i n d i a b e t e s and s t a r v a t i o n . I n : " P r o t e i n T u r n o v e r and L y s o s o m e F u n c t i o n . " S e g a l , H. L . , a n d D o y l e , D. J . ( E d s . ) . 105 p. A c a d e m i c P r e s s , New Y o r k . D u b o w i t z , V. 1985. " M u s c l e b i o p s y : a p r a c t i c a l a p p r o a c h " . 2nd e d s . B a i l l i e r e T i n d a l l , S u s s e x . D u l c e , M., P a r a d e s , C., a n d B a k e r , R.C. 1 9 8 7 . A r e s e a r c h n o t e : p h y s i c a l , c h e m i c a l a n d s e n s o r y c h a n g e s d u r i n g t h e r m a l p r o c e s s i n g o f t h r e e s p e c i e s o f c a n n e d f i s h . J o u r n a l o f F o o d P r o c e s s i n g and P r e s e r v a t i o n . 12:71. D u n a j s k i , E . 1 9 7 9 . T e x t u r e o f f i s h m u s c l e . J o u r n a l o f T e x t u r e S t u d i e s . 10:301. Duncan, D.W., and T a r r , H.L.A. 1958. B i o c h e m i c a l s t u d i e s on s o c k e y e s a l m o n d u r i n g s p a w n i n g m i g r a t i o n . I I I . C h a n g e s i n t h e p r o t e i n a n d n o n - p r o t e i n n i t r o g e n o u s f r a c t i o n s i n m u s c l e s o f m i g r a t i n g s o c k e y e s a l m o n . C a -n a d i a n J o u r n a l o f B i o c h e m i s t r y a n d P h y s i o l o g y . 36:799. Dunn, J . F . , D a v i s o n , W. , M a l o i y , G.M., H o c h a c h k a , P.W., a n d G u p p y , M. 1 9 8 1 . A n u l t r a s t r u c t u r a l a n d h i s t o c h e m i c a l s t u d y o f t h e a x i a l m u s c u l a t u r e i n t h e A f r i c a n l u n g f i s h . F r a n z i n i - A r m s t r o n g , C , and P o r t e r , D.R. 1964. S a r c o l e m m a l i n v a g i n a t i o n s c o n s t i t u t i n g t h e T - s y s t e m i n f i s h m u s c l e f i b r e s . J o u r n a l o f C e l l B i o l o g y . 22:675. -128-F r i e d m a n , H.H., W h i t n e y , J . E . , and S z c z e s n i a k , A. S. 1963. The t e x t u r o m e t e r - a new i n s t r u m e n t f o r o b j e c t i v e t e x t u r e m e a s u r e m e n t . J o u r n a l o f F o o d S c i e n c e . 28:390-396. F r i d e n , J . , S j o s t r o m , M., and Ekblom, B. 1983. M y o f i b r i l l a r damage f o l l o w i n g i n t e n s e e c c e n t r i c e x e r c i s e i n man. I n t e r n a t i o n a l J o u r n a l o f S p o r t s M e d i c i n e . 4 ( 3 ) : 1 7 0 . G e o r g e , J . C , J o h n , T.M., a n d M i n h a s , K . J . 1987. S e a s o n a l d e g r a d a t i v e , r e p a r a t i v e a n d r e g e n e r a t i v e u l t r a s t r u c t u r a l c h a n g e s i n t h e b r e a s t m u s c l e o f t h e m i g r a t o r y Canada g o o s e . C y t o b i o s . 52:109. G r e e n e , C. W. 1926. The p h y s i o l o g y o f s p a w n i n g m i g r a t i o n . P h y s i o l o g i c a l R e v i e w s . V o l . V I ( 2 ) : 2 0 1 . H a a r d , N . F . 1 9 8 7 . P r o t e i n a n d n o n - p r o t e i n n i t r o g e n c o n s t i t u e n t s i n j e l l i e d A m e r i c a n p l a i c e H i p p o g l o s s o i d e s p l a t e s s o i d e s . C a n a d i a n I n s t i t u t e o f F o o d S c i e n c e and T e c h n o l o g y J o u r n a l 2 0 ( 2 ) : 9 8 . H a l l e t t , I.C., and Bremner, H.A. 1988. F i n e s t r u c t u r e o f t h e m y o c o m m a t a - m u s c l e f i b r e j u n c t i o n i n h o k i ( M a c r u r o n u s n o v a e z e l a n d i a e ) . J o u r n a l o f t h e S c i e n c e o f F o o d and A g r i c u l t u r e . 44:245. H a t a e , K., Y o s h i m a t s u , F . , a n d M a t s u m o t o , J . J . 1984 . D i s c r i m i n a t i v e c h a r a c t e r i z a t i o n o f d i f f e r e n t t e x t u r e p r o f i l e s o f v a r i o u s c o o k e d f i s h m u s c l e . J o u r n a l o f F o o d S c i e n c e . 49:721. H a t a e , K., Y o s h i m a t s u , F., and Matsumoto, J . J . 1990. R o l e o f m u s c l e f i b r e s i n c o n t r i b u t i n g f i r m n e s s o f c o o k e d f i s h . J o u r n a l o f F o o d S c i e n c e . 5 5 ( 3 ) : 6 9 3 . H a t a n o , M. , T a k a m a , K. , K o j i m a H., a n d Zama K. 1983 . P r o x i m a t e c o m p o s i t i o n o f f a l l chum s a l m o n . B u l l e t i n o f t h e J a p a n e s e S o c i e t y o f S c i e n t i f i c F i s h e r i e s . 4 9 ( 2 ) : 2 1 3 . H o l l a n d , B.S., and D i P o n z i o C o p e n h a v e r , M. 1987. An i m p r o v e d s e q u e n t i a l l y r e j e c t i v e B o n f e r r o n i t e s t p r o c e d u r e . B i o m e t r i c s . 43:417-423. H o l m e s , A.W. 1 9 8 2 . S a l m o n Q u a l i t y C o n s i d e r a t i o n s f o r F i s h e r i e s M anagement. D e p a r t m e n t o f F i s h e r i e s a n d O c e a n s , F i e l d S e r v i c e s B r a n c h . V i c t o r i a , B.C. Howgate, P. 1979. F i s h . I n : "Food M i c r o s c o p y " . Vaughan, J.G. ( E d s . ) A c a d e m i c P r e s s . London, p. 353. -129-Huynh, M.D. 1989. U t i l i z a t i o n o f l a t e - r u n chum s a l m o n - a q u a l i t y s t u d y . T e c h n i c a l r e p o r t No. 2 1 . A p p l i e d B i o l o g y D i v i s i o n , B r i t i s h C o l u m b i a R e s e a r c h C o r p o r a t i o n . V a n c o u v e r , B.C. I d l e r , D.R., a n d B i t n e r s , I . 1960. B i o c h e m i c a l s t u d i e s on s o c k e y e s a l m o n d u r i n g s p a w n i n g m i g r a t i o n . I X . F a t , p r o t e i n a n d w a t e r i n t h e m a j o r i n t e r n a l o r g a n s a n d c h o l e s t e r o l i n t h e l i v e r and gonads o f t h e s t a n d a r d f i s h . J o u r n a l F i s h e r i e s R e s e a r c h B o a r d o f C a n a d a . 1 7 ( 1 ) : 1 1 3 . I d l e r , D.R., a n d T s u y a k i , H. 1958. B i o c h e m i c a l s t u d i e s on s o c k e y e s a l m o n d u r i n g s p a w n i n g m i g r a t i o n . I . P h y s i c a l m e a s u r e m e n t s , p l a s m a c h o l e s t e r o l , a n d e l e c t r o l y t e l e v e l s . C a n a d i a n J o u r n a l o f B i o c h e m i s t r y and P h y s i o l -ogy. 3 6 ( 8 ) : 7 8 3 . I s o , N., M i z u n o , H., S a i t o , T., O h z e k i , F., a n d Wang, Z. 1984. S t u d i e s on t h e r h e o l o g i c a l p r o p e r t i e s o f t h e h e a t e d y e l l o w t a i l meat. B u l l e t i n o f t h e J a p a n e s e So-c i e t y o f S c i e n t i f i c F i s h e r i e s . 5 0 ( 1 2 ) : 2 0 6 1 . J o h n s o n , E. A.; S e g a r s , R.A.; K a p s a l i s , J . G.; Normand, M. D.; a n d P e l e g , M. 1980. E v a l u a t i o n o f t h e compres-s i v e d e f o r m a b i l i t y modulus o f f r e s h a n d c o o k e d f i s h f l e s h . J o u r n a l o f F o o d S c i e n c e . 45:1318. J o h n s t o n , I . A . 1 9 8 1 . Q u a n t i t a t i v e a n a l y s i s o f m u s c l e b r e a k d o w n d u r i n g s t a r v a t i o n i n t h e m a r i n e f l a t f i s h P l e u r o n e c t e s p l a t e s s a . C e l l a n d T i s s u e R e s e a r c h . 214:369. Kakwas, B.A. and Adams, D.A. 1985. I n : " D i s e a s e s o f M u s c l e s " 4 t h e d . 256 p . H a r p e r and Row P u b l . I n c . P h i l a d e l p h i a . K a r l , H., and S c h r e i b e r , W. 1985. T e x t u r e a n a l y s i s o f c a n n e d f i s h . J o u r n a l o f T e x t u r e S t u d i e s . 16:271. K a r r i c k , N . L . , a n d T h u r s t o n , C . E . 1 9 6 4 . P r o x i m a t e c o m p o s i t i o n o f s i l v e r s a l m o n . J o u r n a l o f A g r i c u l t u r a l a nd F o o d C h e m i s t r y . 1 2 ( 3 ) : 2 8 2 . K i e r n a n , J.A. 1981. " H i s t o l o g i c a l and H i s t o c h e m i c a l M e thods: T h e o r y and P r a c t i c e " . Pergamon P r e s s , O x f o r d . K o n a g a y a , S. 1982. E n h a n c e d p r o t e a s e a c t i v i t y i n m u s c l e o f chum s a l m o n O n c o r h y n c h u s k e t a d u r i n g s p a w n i n g m i g r a -t i o n . B u l l e t i n o f t h e J a p a n e s e S o c i e t y o f S c i e n t i f i c F i s h e r i e s . 4 8 ( 1 0 ) : 1 5 0 3 . -130-Konagaya, S. 1983. E n h a n c e d p r o t e a s e a c t i v i t y i n t h e m u s c l e o f chum s a l m o n d u r i n g s p a w n i n g m i g r a t i o n w i t h r e f e r -e n c e t o s o f t e n i n g o f l y s i n g phenomenon o f t h e meat. B u l l . T o k a i Reg. F i s h . R e s . L a b . 109:41. Larmond, E. 1977. L a b o r a t o r y Methods f o r S e n s o r y E v a l u a t i o n o f F o o d . R e s e a r c h B r a n c h Canada D e p a r t m e n t o f A g r i -c u l t u r e , P u b l i c a t i o n 1637. O t t a w a . L e v i t t , D . . J . 1974. T h e u s e o f s e n s o r y a n d i n s t r u m e n t a l a s s e s s m e n t o f o r g a n o l e p t i c c h a r a c t e r i s t i c s v i a m u l t i v a r i a t e s t a t i s t i c a l methods. J o u r n a l o f T e x t u r e S t u d i e s . 5:183. L i l l i e , R.D. 1977. "H.J. Conn's B i o l o g i c a l S t a i n s " . 473 p. W i l l i a m s and W i l k i n s Co. B a l t i m o r e , p. 473. L o v e , M. 1 9 8 0 . "The C h e m i c a l B i o l o g y o f F i s h " . A c a d e m i c P r e s s . New Y o r k . L o v e , R.M., R o b e r t s o n , I . , S m i t h , G.L., a n d W h i t t l e , K . J . 1974. The t e x t u r e o f c o d m u s c l e . J o u r n a l o f T e x t u r e S t u d i e s . 5:201. Lund, D. 1982. Q u a n t i f y i n g r e a c t i o n s i n f l u e n c i n g q u a l i t y o f f o o d s : t e x t u r e , f l a v o u r , a n d a p p e a r a n c e . J o u r n a l o f F o o d P r o c e s s i n g and P r e s e r v a t i o n . 6:133. M i l l w a r d , D. J . , B a t e s P. C. , L a u r e n t , G. J . , and L o , C. C. 1978. F a c t o r s a f f e c t i n g p r o t e i n breakdown i n s k e l e t a l m u s c l e . I n : " P r o t e i n T u r n o v e r and Lysosome F u n c t i o n . " S e g a l , H. L . , a n d D o y l e , D. J . ( E d s . ) . 619 p . A c a -d e m i c P r e s s , New Y o r k . M c B r i d e , J.R., F a g e r l u n d , U.H.M., S m i t h , M. , and T o m l i n s o n , N. 1 9 6 5 . P o s t - s p a w n i n g d e a t h o f P a c i f i c s a l m o n : s o c k e y e s a l m o n ( O n c o r h y n c h u s n e r k a ) m a t u r i n g a n d s p a w n i n g i n c a p t i v i t y . J o u r n a l F i s h e r i e s R e s e a r c h B o a r d o f Canada. 2 2 ( 3 ) : 7 7 5 . Mommsen, T . P . , F r e n c h , C . J . , a n d H o c h a c h k a , P.W. 1980. S i t e s and p a t t e r n s o f p r o t e i n and amino a c i d u t i l i z a -t i o n d u r i n g t h e s p a w n i n g m i g r a t i o n o f s a l m o n . C a n a -d i a n J o u r n a l o f Z o o l o g y . 5 8 ( 1 0 ) : 1 7 8 5 . Neave, F. 1966. Salmon o f t h e n o r t h P a c i f i c o c e a n . P a r t I I I . A r e v i e w o f t h e l i f e h i s t o r y o f n o r t h P a c i f i c s a l m o n . 6. Chum s a l m o n i n B r i t i s h C o l u m b i a . I n t e r n a t i o n a l N o r t h P a c i f i c F i s h e r i e s C o m m i s s i o n B u l l e t i n 18:81. -131-P e t e r , J . B . 1973. S k e l e t a l m u s c l e : d i v e r s i t y and m u t a b i l i t y o f i t s h i s t o c h e m i c a l , e l e c t r o n m i c r o s c o p i c , b i o c h e m i -c a l , a n d p h y s i o l o g i c p r o p e r t i e s . I n : "The S t r i a t e d M u s c l e " , l p . C M . P e a r s o n a n d F.K M o s t o f i ( E d s . ) . The W i l l i a m s and w i l k i n s Co. B a l t i m o r e . P r o c t o r , C , M o s s e , P.R.L., a n d H u d s o n , R.C.L. 1980. A h i s t o c h e m i c a l and u l t r a s t r u c t u r a l s t u d y o f t h e d e v e l -o p m e n t o f t h e p r o p u l s i v e m u s c u l a t u r e o f t h e brown t r o u t , Salmo t r u t t a L ., i n r e l a t i o n t o i t s swimming b e h a v i o u r . J o u r n a l o f F i s h B i o l o g y . 16:309. R e d d i , P. K. r C o n s t a n t i n i d e s , S.M., and Dymsza, H. A. 1972. C a t h e p t i c a c t i v i t y o f f i s h m u s c l e . J o u r n a l o f F o o d S c i e n c e . 37:643. S e g a l , H. L. , Brown, J . A . , Dunaway G. A., W i n k l e r , J . R., M a d n i c k , H. M., a n d R o t h s t e i n , D. M. 1978. F a c t o r s i n v o l v e d i n t h e r e g u l a t i o n o f p r o t e i n t u r n o v e r . I n : " P r o t e i n T u r n o v e r a n d L y s o s o m e F u n c t i o n . " S e g a l , H. L., a n d D o y l e , D. J . ( E d s . ) . 9 p . A c a d e m i c P r e s s , New Y o r k . S e g a l , H. L . , and D o y l e D* 1978. I n t r o d u c t i o n . I n : " P r o t e i n T u r n o v e r a n d L y s o s o m e F u n c t i o n . " S e g a l , H. L . , a n d D o y l e , D. J . ( E d s . ) . 1 p. A c a d e m i c P r e s s , New Y o r k . Shama, F., a n d Sherman, P. 1973. E v a l u a t i o n o f some t e x t u r a l p r o p e r t i e s o f f o o d s w i t h t h e I n s t r o n u n i v e r s a l t e s t -i n g m a c h i n e . J o u r n a l o f T e x t u r e S t u d i e s , 4:344. S h i n d o , K., T s u c h i y a , T . , a n d M a t s u m o t o , J . J . 1 9 8 6 . H i s t o l o g i c a l s t u d y on w h i t e and d a r k m u s c l e s o f v a r i -o us f i s h e s . B u l l e t i n o f t h e J a p a n e s e S o c i e t y o f S c i -e n t i f i c F i s h e r i e s . 5 2 ( 8 ) : 1 3 7 7 . S i m p s o n , K . L . 1982. C a r o t e n o i d p i g m e n t s i n s e a f o o d . " I n : C h e m i s t r y a n d B i o c h e m i s t r y o f M a r i n e F o o d P r o d u c t s " . M a r t i n , R.E., F l i c k , G . J ; , H e b a r d , C.E., a n d Ward, D.R. ( E d s . ) . 115 p. AVI P u b l i s h i n g Co. W e s t p o r t . S k r e d e , G., a n d S t o r e b a k k e n , T. 1986. C h a r a c t e r i s t i c s o f c o l o r i n raw, b a k e d a n d smoked w i l d a n d p e n - r e a r e d A t l a n t i c s a l m o n . J o u r n a l o f F o o d S c i e n c e . 5 1 ( 3 ) : 8 0 4 . S t a n l e y , D.W,, a n d S w a t l a n d , H.J. 1976. The m i c r o s t r u c t u r e o f m u s c l e t i s s u e - a b a s i s f o r meat t e x t u r e m e a s u r e -ment. J o u r n a l o f T e x t u r e S t u d i e s . 7:65. -132-S t e i n e r , P. H. , B r o d e r s o n , S. H., a n d L i s t o n J . 1 9 8 4 . U l t r a s t r u c t u r a l l o c a l i z a t i o n o f l y s o s o m e s i n c o h o s a l m o n a n d s t e e l h e a d t r o u t m u s c l e . J o u r n a l o f F o o d S c i e n c e . 49:975. S z c z e s n i a k , A. S. 1 9 6 3 . C l a s s i f i c a t i o n o f t e x t u r a l c h a r a c t e r i s t i c s . J o u r n a l o f F o o d S c i e n c e . 28:385-389. S z c z e s n i a k , A.S. 1987. C o r r e l a t i n g s e n s o r y w i t h i n s t r u m e n t a l t e x t u r e m e a s u rements- an o v e r v i e w o f r e c e n t d e v e l o p -ments. J o u r n a l o f T e x t u r e S t u d i e s . 18:1-15. S z c z e s n i a k , A. S.; B r a n d t , M. A., and F r i e d m a n , H. H. 1963. D e v e l o p m e n t o f s t a n d a r d r a t i n g s c a l e s f o r m e c h a n i c a l p a r a m e t e r s o f t e x t u r e and c o r r e l a t i o n between t h e o b -j e c t i v e a n d s e n s o r y m e t h o d s o f t e x t u r e e v a l u a t i o n . J o u r n a l o f F o o d S c i e n c e . 28:397. Templeman, W., and Andrews, G.L. 1956. J e l l i e d c o n d i t i o n i n t h e A m e r i c a n p l a i c e H i p p o g l o s s o i d e s p l a t e s s o i d e s ( F a b r i c u s ) . J o u r n a l o f t h e F i s h e r i e s R e s e a r c h B o a r d o f C a n ada. 1 3 ( 2 ) : 1 4 7 . W a r r i e r , S.B., G h a d i , S.V., and N i n j o o r , V. 1988. E f f e c t o f c o l d s t o r a g e o n t h e p r o t e o l y t i c e n z y m e s o f f i s h m u s c l e . J o u r n a l o f F o o d S c i e n c e a n d T e c h n o l o g y , I n d i a . 2 5 ( 5 ) : 3 1 3 . W i l k i n s o n , L . 1 9 8 8 . " S y s t a t : t h e s y s t e m f o r s t a t i s t i c s " . S y s t a t I n c . E v a n s t o n , I L . 8. APPENDIXES T a b l e 8 . 1 . A b s o r b a n c e a t U . V . 2 8 0 o f s o l u b l e N v e r s u s g r a d e a s a m e a s u r e o f t h e p r o t e i n a s e a c t i v i t y o f chum d o r s a l m u s c l e a t pH 3 . 5 . 0.30 O oo CM > ZD <D O c CO _Q i_ O U) j Q < 0.20 0.10 0.00 - 0 . 1 0 ' — ; 1 1 1 2 3 3 4 4 4 - Index of Maturity - 1 3 4 -T a b l e 8.2. A b s o r b a n c e a t U.V. 280 o f s o l u b l e N v e r s u s g r a d e a s a measure o f t h e p r o t e i n a s e a c t i v i t y o f chum d o r s a l m u s c l e a t pH 6.2. O 03 OJ > D 0) u c (rj -Q i_ O CO n < 0.30 0.22 0.14 0.06 -0.02 - 0 . 1 0 ' ' 1 1 1 2 3 3 4 4 4 Index of Maturity - 1 3 5 -T a b l e 8.3. A b s o r b a n c e a t U.V. 280 o f s o l u b l e N v e r s u s g r a d e a s a measure o f t h e p r o t e i n a s e a c t i v i t y o f chum d o r s a l m u s c l e a t pH 7.0. 0 . 3 0 O 00 CM > 0) o c iTj n o (f) < 0 . 2 2 h 0 . 1 4 0 . 0 6 - 0 . 0 2 h - 0 . 1 0 1 1 1 2 3 3 4 4 4 Index of Maturity . -136-Table 8.4. C o n c e n t r a t i o n of phosphate (mM) v e r s u s grade as a measure of the a c i d phosphatase a c t i v i t y of chum muscle during spawning migration. 0 . 4 6 OJ 1 1 1 2 2 3 3 4 4 4 Index o f M a t u r i t y - 1 3 7 -

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