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UBC Theses and Dissertations

Frozen storage and cryoprotection of eggs from chum salmon (Oncorhynchus keta) Craig, Cheryl Lynne 1984

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F R O Z E N S T O R A G E A N D C R Y O P R O T E C T I O N O F E G G S F R O M C H U M S A L M O N [ONCORHYNCHUS KETA) b y C H E R Y L L Y N N E C R A I G B . S c . H o n o u r s , U n i v e r s i t y o f S a s k a t c h e w a n A T H E S I S S U B M I T T E D IN P A R T I A L F U L F I L L M E N T O F T H E R E Q U I R E M E N T S F O R T H E D E G R E E O F M A S T E R O F S C I E N C E in T H E F A C U L T Y O F G R A D U A T E S T U D I E S D e p a r t m e n t o f F o o d S c i e n c e We a c c e p t th i s t h e s i s as c o n f o r m i n g to t h e r e q u i r e d stajxd-artt T H E 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 A p r i l 1 984 © C h e r y l L y n n e C r a i g , 1984 I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r an advanced degree a t t h e 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 , I a g r e e t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by t h e head o f my department o r by h i s o r h e r r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department o f FboD SaflVjCE The U n i v e r s i t y o f B r i t i s h C olumbia 1956 Main Mall V ancouver, Canada V6T 1Y3 DE-6 (3/81) A B S T R A C T T h i s s t u d y was i n i t i a t e d to a s s e s s t h e i n f l u e n c e o f f r o z e n s t o r a g e o f e g g s f rom s e x u a l l y m a t u r e female c h u m s a l m o n (Oncorhynchus keta) o n t h e p h y s i c a l p r o p e r t i e s o f t h e m e m b r a n e a n d t h e y o l k . T h e e f f e c t i v e n e s s o f s e v e r a l s a l t s , s u c r o s e , g l y c e r o l a n d e t h y l e n e g l y c o l as c r y o p r o t e c t i v e a g e n t s f o r p r e v e n t i n g a n y o b s e r v e d c r y o d a m a g e to t h e e g g s was d e t e r m i n e d . E l e c t r o n m i c r o s c o p y was e m p l o y e d to g a i n an i n s i g h t in to t h e m i c r o -s t r u c t u r e o f t h e c h u m e g g e n v e l o p e . In a d d i t i o n , who le e g g s w e r e s u b j e c t e d to f lat p l a t e c o m p r e s s i v e l o a d i n g in o r d e r to d e t e r m i n e t h e s t r e n g t h o f t h e m e m b r a n e . T h e m e m b r a n e a p p e a r e d to be a c o m p l e x s t r u c t u r e w h i c h e n a b l e d i n t a c t e g g s to w i t h s t a n d c o m p r e s s i o n f o r c e s o f 0 .47 to 1.22 N e w t o n s . T h e m e m b r a n e r u p t u r e e n e r g y r a n g e d f rom 2 .36 to 7 .99 (x 10 "*) J o u l e s , a n d was i n d e p e n d e n t o f e g g s i z e . U p o n f r e e z i n g f r e s h e g g s at -10°C f o r 7 d a y s , t h e n t h a w i n g in a i r at 25°C, a d r o p o f 46% in m e m b r a n e r u p t u r e e n e r g y was e x p e r i e n c e d . T h e f low b e h a v i o u r o f y o l k e x p r e s s e d f rom f r e s h c h u m e g g s , as e x a m i n e d w i t h t h e a i d o f a B r a b e n d e r r o t a t i o n a l v i s c o m e t e r , d i s p l a y e d a n o n - N e w t o n i a n p s e u d o p l a s t i c n a t u r e . A n a l y s e s b y d i f f e r e n t i a l s c a n n i n g c a l o r i m e t r y a n d p o l y a c r y l a m i d e ge l e l e c t r o p h o r e s i s i n d i c a t e d t h e p r e s e n c e o f s e v e r a l major p r o t e i n s . When t h e y o l k m a t e r i a l h a d b e e n f r o z e n at -10°C f o r 7 d a y s , t h e n t h a w e d in a i r at 25°C, l i t t le v a r i a t i o n was o b s e r v e d to o c c u r b y a n y o f t h e t e c h n i q u e s u s e d . I m m e r s i o n in a 2 .5 M s o l u t i o n o f s o d i u m c h l o r i d e f o r 30 min at 25°C was t h e most e f f e c t i v e a n d c o n s i s t e n t c r y o p r o t e c t i v e a g e n t o u t o f all o f t h e p o t e n t i a l a g e n t s t e s t e d . C a v i a r p r o d u c e d f rom p r e v i o u s l y f r o z e n , t h e n t h a w e d e g g s t h a t h a d b e e n so t r e a t e d b e f o r e f r e e z i n g d i f f e r e d f rom t h a t p r o d u c e d f r o m _ b o t h ii f r e s h a n d u n t r e a t e d f r o z e n a n d t h a w e d e g g s in t h e r a t e o f s o d i u m c h l o r i d e u p t a k e a n d y o l k a p p a r e n t v i s c o s i t y f o r t h e same c h l o r i d e c o n t e n t . M e m b r a n e s t r e n g t h o f s u c h c a v i a r was s i m i l a r to t h a t p r o d u c e d f rom f r e s h e g g s , b e i n g g r e a t e r t h a n t h a t p r o d u c e d f rom u n t r e a t e d p r e v i o u s l y f r o z e n a n d t h a w e d e g g s . S e n s o r y e v a l u a t i o n s t u d i e s i n d i c a t e d t h a t c a v i a r f rom e g g s t h a t h a d b e e n c r y o p r o t e c t e d w e r e r a n k e d s i m i l a r to c a v i a r f rom u n t r e a t e d f r o z e n a n d t h a w e d e g g s i n t h e p a r a m e t e r s o f y o l k v i s c o s i t y a n d m e m b r a n e s t r e n g t h . B o t h w e r e s i g n i f i c a n t l y d i f f e r e n t f rom h i g h q u a l i t y c a v i a r p r o d u c e d f rom f r e s h e g g s . H o w e v e r , t h e h e d o n i c r a t i n g s i n d i c a t e d a p r e f e r e n c e f o r c a v i a r f rom c r y o -p r o t e c t e d e g g s o v e r t h a t p r o c e s s e d f rom u n t r e a t e d f r o z e n a n d t h a w e d e g g s . R e s u l t s o f t h i s s t u d y i n d i c a t e t h a t c r y o p r o t e c t i o n o f c h u m e g g s to r e d u c e t h e d e f e c t s c a u s e d b y f r o z e n s t o r a g e is f e a s i b l e . Wi th f u r t h e r r e s e a r c h , t h i s t e c h n i q u e h o l d s p o t e n t i a l a d v a n t a g e s f o r t h e c a v i a r i n d u s t r y . i i i-TABLE OF CONTENTS Page ABSTRACT ii LIST OF TABLES . . . v i i LIST OF FIGURES ix ACKNOWLEDGEMENTS x i i INTRODUCTION 1 LITERATURE REVIEW 3 A. Yolk Composition 3 1. Lipovitellin 4 2. Phosvitin . 4 3. Livetins 6 4. 3' component 6 B. Membrane Structure 8 C. Mechanism of Ice Formation 10 D. Mechanism of Ice Damage 13 E. Cryoprotectants 14 F. Processing 17 MATERIALS AND METHODS 20 A. Sample Preparation 20 1. Whole egg blend • • 20 2. Egg magma 20 3. Egg yolk 20 4. Membrane 22 B. Proximate Analysis 22 1. Determination of moisture 22 i v Page 2. Determination of protein 22 3. Determination of crude lipid 2 3 4. Determination of chloride content 2 3 5. Determination of water activity 23 C. Scanning Electron Microscopy 2 3 D. Poly aery lamide Gel Electrophoresis 24 E. Differential Scanning Calorimetry 27 F. Physical Properties 27 1. Flow behaviour studies 28 2. Membrane strength studies 29 G. Experimental Procedures 30 1. Effect of egg size on membrane strength 30 2. Frozen storage effects 31 3. Cryoprotectant agent effects ; 33 4. Effectiveness of sodium chloride as a cryoprotectant. . . . "32-a) Objective analysis - sample preparation 34 b) Subjective analysis - sample preparation 37 c) Subjective analysis - sensory evaluation 37 * H. Statistical Analysis 38 1. Analysis of variance 38 a) Effect of cryoprotectants 38 b) Effect of pre-freezing treatment with sodium chloride . 39 i . Membrane strength data 39 ii. Sensory evaluation 40 2. Analysis of regression lines 40 v Page RESULTS AND DISCUSSION 42 A. Proximate Analysis 42 B. Differential Scanning Calorimetry 44 C. Poly acrylamide Gel Electrophoresis 46 D. Scanning Electron Microscopy 50 E. Flow Behaviour Studies 56 F. Force Deformation Analysis 61 G. Effect of Egg Size on Membrane Rupture Energy 67 H. Effects of Frozen Storage 67 1. Effects on yolk 69 a) Polyacrylamide gel electrophoresis 69 b) Differential scanning calorimetry 71 c) Viscometric analysis 73 2. Effect on membrane rupture energy 7 7 I. Effects of Cryoprotectants , . 7 7 J. Effectiveness of Cryoprotection by Sodium Chloride 89-1. Effect of cryoprotection on rate of sodium chloride uptake . . . 89 2. Effect of cryoprotection on development of apparent viscosity . 9 3 3. Effect of cryoprotection on membrane rupture energy 1 1 0 K. Sensory Evaluation 113 -CONCLUSIONS 1 2 2 LITERATURE CITED 125' APPENDIX 1 3 5 v i LIST OF TABLES Table Page 1 Coding system for egg sample identification 21 2 Identity and quantity of yolk sample applied to electrophoresis gels 26 3 Proximate analysis of chum salmon eggs (n-3) 43 4 Enthalpy of denaturation and denaturation temperature of proteins of fresh chum egg yolk of two codes . . . 47 5 Mean values of Power-law flow parameters from^ rheogrdms of liquid yolk prepared from fresh chum eggs (n=3) 58 6 Mean membrane rupture energy and rupture force for fresh chum eggs of several codes (n=25) 63 7 Means of parameters calculated from values of rupture force and energy for membranes of fresh eggs of three codes (n-25) 65 8 Enthalpy of denaturation for individual proteins of fresh and frozen /thawed chum egg yolk 7 4 9 Denaturation temperatures of individual proteins of fresh and frozen/thawed chum egg yolk of two codes 75 10 Effect of frozen storage on membrane rupture energy 78 11 Effect of cryoprotective treatment on membrane rupture energy of eggs before and after freeze/thawing 85 12 Parameters obtained from linear regression of salting curves for chum eggs 92 13 Mean values of Power-law flow parameters of liquid yolk prepared from fresh, frozen /thawed and cryoprotected eggs of various chloride contents from three codes (n=3) 103 v i i Table Page -4 14 Mean membrane rupture energies (J x 10 ) for salted fresh, frozen/thawed and cryoprotected eggs (n=25) 111 15 Analysis of variance of membrane rupture energy for eggs of various chloride contents 112 16 Mean ratings (nine point scale) for yolk viscosity and membrane strength judged by sensory panel for each caviar sample presented (n=8) 115 17 Analysis of variance of ratings from sensory analysis of membrane strength 116 18 Analysis of variance of ratings from sensory analysis of yolk viscosity 117 19 Mean ratings from nine point hedonic scale for each caviar sample presented 119 20 Analysis of variance of ratings from nine point hedonic scale 120 v i i i LIST OF FIGURES Figure Page 1 Flow diagram of procedure for investigation into frozen storage effects 32 2 Flow diagram of procedure for investigation into effective-ness of sodium chloride as a cry op rote ct ant 35 3 Typical DSC thermogram of fresh yolk from eggs of Code VI 45 4 Densitometer tracing of poly acrylamide gel from electrophoresis of yolk from fresh chum eggs 48 5-9 Scanning electron micrographs of a chum egg 52 5 Cross-section of fresh egg membrane as revealed by a rupture in the egg surface (pw=50 \i.m) 53 6 Cross-section of frozen /thawed membrane as revealed by a rupture in the egg surface (pw=490 ym) 53 7 Similar view to Figure 5 (pw=140 pm) 54 8 Similar view to Figure 5 (pw=620 ym) 54 9 Similar view to Figure 5 (pw=1350 ym) 55 10 Rheograms for yolk of fresh chum eggs from four codes /57 11 Typical force deformation curve resulting from compression of a single chum egg 62 12 Scatter diagram of membrane rupture energy as a function of egg surface area 68 13 Electrophoretic patterns of fresh and frozen/thawed native and salted chum egg yolk of two codes 70 14 Typical DSC thermograms of fresh and frozen /thawed yolk from eggs of Code VI 72 ix Figure Page 15 Rheograms of yolk from fresh and frozen/thawed eggs of two codes 76 . 16 Effect of treatment with various potential cryoprotective agents on membrane rupture energy of eggs from Code III • • • • 80 17 Effect of treatment with various potential cryoprotective agents on membrane rupture energy of eggs from Code IV . . . . 81 18 Effect of treatment with various potential cryoprotective agents on membrane rupture energy of eggs from Code VII- . . . 82 19 Effect of treatment with various potential cryoprotective agents on membrane rupture energy of eggs from Code VIII . . . 83 20 Effect of immersion in a saturated sodium chloride solution for various periods on the chloride content of fresh eggs, eggs previously frozen/thawed, and cryoprotected eggs of three codes 90 21 Rheograms of yolk from fresh eggs of Code IX immersed for various periods in a saturated sodium chloride solution 94 22 Rheograms of yolk from fresh eggs of Code X immersed for various periods in a saturated sodium chloride solution 95 23 Rheograms of yolk from fresh eggs of Code XI immersed for various periods in a saturated sodium chloride solution 96 24 Rheograms of yolk from frozen/thawed eggs of Code IX for immersed for various periods in a saturated sodium chloride solution 9 7 25 Rheograms of yolk from frozen/thawed eggs of Code X immersed for various periods in a saturated sodium chloride solution 98 Figure Page 26 Rheograms of yolk from frozen/thawed eggs of Code XI immersed for various periods in a saturated sodium chloride solution 99 27 Rheograms of yolk from cryoprotected eggs of Code IX immersed for various periods in a saturated sodium chloride solution 100 28:': Rheograms of yolk from cryoprotected eggs of Code X immersed for various periods in a saturated sodium chloride solution 101 29 Rheograms of yolk from cryoprotected eggs of Code XI immersed for various periods in a saturated sodium chloride solution 102 30 Effect of chloride content of eggs on apparent viscosity of yolk from fresh, frozen/thawed and cryoprotected eggs of Code IX ~ 106. 31 Effect of chloride content of eggs on apparent viscosity of yolk from fresh, frozen /thawed and cryoprotected eggs of Code X 107 32 Effect of chloride content of eggs on apparent viscosity of yolk from fresh, frozen/thawed and cryoprotected * eggs of Code XI 108 x i ACKNOWLEDGEMENTS The author wishes to express her sincere appreciation to Dr. W. D. Powrie for his invaluable support of this study and his review of the manuscript. Special thanks to Dr. M. A. Tung for his many helpful suggestions and also to Dr. S. Nakai and Dr. T. Howard for all their interest in, patience and review of this thesis. The author is also very grateful for the support and aid extended by the Fisheries Technology Division, B. C. Research. The invaluable technical advice from S. Yee, R. Yada and L. Robinson is also gratefully acknowledged. Appreciation is extended as well for the financial support of the Science Council of British Columbia. This thesis is dedicated to my mother and father, without whom I would never have been able to begin, and to Mr. Tom Tautorus, without whom I would never have been able to finish. x i i 1. I N T R O D U C T I O N T h e t e r m " c a v i a r " is u s e d to d e s c r i b e s e p a r a t e d a n d s a l t e d f i s h e g g s . T h e r o e o f m a n y t e l e o s t s p e c i e s a r e e m p l o y e d f o r t h i s p u r p o s e ; most p r i z e d a r e t h o s e o f s t u r g e o n a n d s a l m o n , b u t c a r p , w h i t e f i s h , p i k e - p e r c h , p i k e , g r e y m u l l e t , c o d a n d h e r r i n g a r e a l so u t i l i z e d ( Z a i t s e v et a l . , 1 9 6 9 ) . T h e U S S R p r e s e n t l y l e a d s in w o r l d s a l m o n c a v i a r p r o d u c t i o n . S t e r n i n a n d H o r i (1 982) r e p o r t t h a t S o v i e t p r o d u c t i o n o f c a v i a r r e c e n t l y r e a c h e d 600 t o n n e s p e r y e a r , w h i l e C a n a d a p r o d u c e s o n l y a smal l f r a c t i o n o f t h i s a m o u n t . Most o f t h e roe r e s u l t i n g f rom t h e s a l m o n h a r v e s t in C a n a d a is c o n s i d e r e d as o f f a l , h o w e v e r , t h e a m o u n t t h a t was u s e d fo r h u m a n c o n s u m p t i o n in r e c e n t y e a r s c o m m a n d e d p r i c e s t h a t r a n g e d f rom s e v e n to e i g h t d o l l a r s p e r k i l o g r a m ( F i s h e r i e s P r o d u c t i o n S t a t i s t i c s o f B r i t i s h C o l u m b i a , 1 9 8 1 ) , s e c o n d in v a l u e o n l y to m i l d , c u r e d a n d s m o k e d s a l m o n . A l t h o u g h p r e s e n t l y t h e m a j o r i t y o f r o e i n t e n d e d f o r p r o c e s s i n g is u s e d f o r " s u j i k o " , a J a p a n e s e d e l i c a c y i n v o l v i n g h i g h l y s a l t e d i n t a c t o v a r i e s , t h e r e is i n c r e a s i n g i n t e r e s t in s a l t i n g t h e s e p a r a t e e g g s to p r o d u c e a E u r o p e a n s t y l e c a v i a r . T h e r e d d i s h o r a n g e roe o f t h e s p e c i e s Oncorhynchus keta is most c o m m o n l y u t i l i z e d f o r s a l m o n c a v i a r p r o d u c t i o n ( S t e r n i n a n d H o r i , 1 9 8 2 ) . C o m m o n l y c a l l e d " c h u m " , t h i s sa lmon p o s s e s s e s t h e l a r g e s t e g g s o f a n y o f t h e o t h e r f i s h s p e c i e s u s e d , a v e r a g i n g 200 mg in w e i g h t p e r e g g ( S m i r n o v et a l . , 1 968) , a n d o c c u p y i n g 10-11% o f t h e tota l b o d y w e i g h t o f t h e f i s h ( Z a i t s e v et a l . , 1 9 6 9 ) . C h u m s a l m o n a r e a m o n g t h e last o f t h e P a c i f i c s a l m o n to e n t e r t h e f r e s h w a t e r t r i b u t a r i e s to s p a w n a n d r o e o f m a t u r i t y s u i t a b l e f o r c a v i a r p r o d u c t i o n is f o u n d o n l y in t h e m o n t h s o f A u g u s t to N o v e m b e r ( S t e r n i n a n d H o r i , 1 9 8 2 ) . F i s h p r o c e s s o r s w o r k i n g w i t h r o e a r e f a c e d w i t h t h e s e a s o n a l p r o b l e m o f l a r g e i n f l u x e s o f f r e s h , h i g h l y p e r i s h a b l e f i s h r o e . A l t h o u g h f r o z e n s t o r a g e f o r l a t e r p r o c e s s i n g is o n e s o l u t i o n to e v e n o u t t h e f l u c t u a t i o n s in 2. raw p r o d u c t s u p p l y , it has b e e n f o u n d t h a t s u c h h a n d l i n g r e s u l t s in t h e a l t e r a t i o n o f s e v e r a l a s p e c t s o f e n d p r o d u c t q u a l i t y . O n e o f t h e most i m p o r -t a n t o f t h e s e is t h e t e x t u r e o f t h e s e p a r a t e c a v i a r g r a i n s ( S t e r n i n a n d H o r i , 1 982; Z a i t s e v et a l . , 1 969) . T h e p u r p o s e o f t h i s t h e s i s was t w o f o l d : to d e t e r m i n e t h e l o c a t i o n a n d e x t e n t o f d a m a g e r e s u l t i n g f rom a f r e e z e / t h a w c y c l e in i n d i v i d u a l e g g s a n d to i n v e s t i g a t e t h e p o t e n t i a l u s e o f c r y o p r o t e c t a n t s in a l l e v i a t i n g a n y s u c h d e f e c t s . T h e u l t imate goa l was to p r o d u c e a c r y o p r o t e c t e d e g g t h a t c o u l d y i e l d a c a v i a r m o r e n e a r l y r e s e m b l i n g c a v i a r p r e p a r e d w i t h f r e s h e g g s t h a n t h a t p r o d u c e d f rom f r o z e n e g g s . In t h e c o u r s e o f t h e i n v e s t i g a t i o n , t h e c o m p o s i t i o n a n d p h y s i c a l p r o p e r t i e s o f t h e c h u m e g g w e r e d e t e r m i n e d t h r o u g h p r o x i m a t e a n a l y s i s , 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 , e l e c t r o p h o r e s i s , d i f f e r e n t i a l s c a n n i n g c a l o r i m e t r y , v i s c o m e t r y a n d f o r c e d e f o r m a t i o n a n a l y s i s . In a d d i t i o n , s i n c e t h e s u c c e s s o f a n y m o d i f i e d p r o c e s s i n g t e c h n i q u e d e p e n d s u p o n a c c e p t a n c e o f t h e f i na l p r o d u c t , s e n s o r y e v a l u a t i o n was e m p l o y e d to d e t e r m i n e t h e q u a l i t y o f t h e c a v i a r u l t i m a t e l y p r o d u c e d f rom c r y o p r o t e c t e d e g g s . 3. L I T E R A T U R E R E V I E W A . Y o l k C o m p o s i t i o n C e n t r i f u g a t i o n o f t h e i n t e r i o r f l u i d o f c o h o s a l m o n (O. kisutch) e g g s b y M a r k e r t a n d V a n s t o n e (1968) r e s u l t e d in t h e f o r m a t i o n o f t h r e e d i s t i n c t l a y e r s : a c l e a r h i g h d e n s i t y p i g m e n t e d s o l u t i o n (98%), a w a x y m a t e r i a l o f l ower d e n s i t y (1.2%) a n d a f l o a t i n g p i g m e n t e d oi l (0 .8%) . C o m p o s i t i o n o f t h e l a t t e r two l a y e r s has not b e e n t h e s u b j e c t o f m a n y s t u d i e s ; h o w e v e r , a c o n s i d e r a b l e a m o u n t o f a t t e n t i o n h a s b e e n f o c u s s e d o n t h e p r o t e i n s t h a t f o rm t h e p r i n c i p a l l a y e r a n d a r e t h e p r e d o m i n a n t c o n s t i t u e n t s o f a l l e g g y o l k ( N a k a g a w a a n d T s u c h i y a , 1972) . P r e v i o u s r e s e a r c h has r e v e a l e d t h a t h i g h d e n s i t y p r o t e i n s l o c a t e d in t h e e g g y o l k o f m a n y v e r t e b r a t e o v i p a r o u s s p e c i e s a r e c o m p a r a b l e ( C o o k a n d M a r t i n , 1 9 6 9 ) . I n t e r s p e c i e s c h a r a c t e r i s t i c s o f t h e s e p r o t e i n s a r e s i m i l a r , to t h e p o i n t t h a t Wal lace et a l . (1 967) s u g g e s t e d t h e same names be u s e d , as l o n g as t h e s p e c i e s is i d e n t i f i e d , in o r d e r to p r e v e n t u n n e c e s s a r y c o m p l i c a t i o n s in n o m e n c l a t u r e . In t e l e o s t s , as in o t h e r e g g l a y i n g v e r t e b r a t e s , l i p o p r o t e i n s a r e c o n s i d e r e d to be t h e p r i m a r y e n e r g y s o u r c e f o r d e v e l o p i n g e m b r y o s ( N a k a g a w a a n d T s u c h i y a , 1969) . S e v e r a l o t h e r p r o t e i n s , o n e o f w h i c h is a p h o s p h o p r o t e i n , h a v e a l so b e e n i d e n t i f i e d in u n f e r t i l i z e d t e l e o s t e g g y o l k . O n e o f t h e s i m p l e p r o t e i n s , c o n t a i n i n g no l i p i d o r p h o s p h o r u s c o m p o n e n t s has b e e n l a b e l l e d B" c o m p o n e n t b y M a r k e r t a n d V a n s t o n e (1 968) , w h i l e t h e l i p o p r o t e i n , p h o s p h o -p r o t e i n a n d r e m a i n i n g s i m p l e p r o t e i n s h a v e b e e n i d e n t i f i e d o n t h e b a s i s o f e l e c t r o p h o r e t i c m o b i l i t y , s t a i n i n g c h a r a c t e r i s t i c s a n d p r e c i p i t a t i o n w i t h ammonium s u l f a t e as a n a l o g o u s to t h e l i p o v i t e l l i n , p h o s v i t i n a n d l i v e t i n s , r e s p e c t i v e l y , f o u n d in a v i a n e g g y o l k ( M a r k e r t a n d V a n s t o n e , 1 9 6 8 ) . 4. 1. L i p o v i t e l l i n S t u d i e s b y M a r k e r t a n d V a n s t o n e ( 1 9 7 1 ) , r e v e a l e d t h e l i p o v i t e l l i n in c o h o (O. kisutch) as p o s s e s s i n g 12.5% n i t r o g e n , 24.3% l i p i d a n d 0.56% p h o s p h o r u s (al l p r e s e n t in t h e l i p i d p o r t i o n ) , o n a d r y w e i g h t b a s i s . T h e y s u g g e s t e d a m o l e c u l a r w e i g h t o f 2 5 0 , 0 0 0 , w h i c h a g r e e s wel l w i t h t h e v a l u e o b t a i n e d b y A n d o (1 965) , o f 240 ,000 to 260 ,000 f o r t h e l i p o p r o t e i n f o u n d in r a i n b o w t r o u t (Salmo irideus). N a k a g a w a a n d T s u c h i y a (1 969) i s o l a t e d a " c o m p o n e n t I" , t h a t was t h e ma in f r a c t i o n in t r o u t (S. gairdnerii irideus) e g g y o l k , a n d was s i m i l a r to t h e l i p o v i t e l l i n M a r k e r t a n d V a n s t o n e (1971) f o u n d in c o h o e g g y o l k , in c o n t a i n i n g 11.9% n i t r o g e n , 0.5% p h o s p h o r u s a n d 22.8% l i p i d , o f w h i c h some 50% was p h o s p h o l i p i d . T h e l i p o v i t e l l i n f o u n d in t h e s e t e l e o s t e g g y o l k s is most p r o b a b l y p r e s e n t in t h e fo rm o f a m o l e c u l e a n d not t h a t o f a m i c e l l e , a c c o r d i n g to t h e t h e o r y e x p r e s s e d b y C o o k a n d M a r t i n (1 962) . T h i s t h e o r y p o s t u l a t e s t h a t a l i p o p r o t e i n w i t h a l i p i d c o n t e n t o f l ess t h a t 40% a n d a n e u t r a l l i p i d r p h o s p h o l i p i d r a t i o o f a b o u t 1:1 o r l ess w o u l d p o s s e s s a s t r u c t u r e c o n -s i s t i n g o f a p r o t e i n m a t r i x w h o s e m o l e c u l a r i n t e g r i t y is not d i s t u r b e d b y t h e r e l a t i v e l y smal l a m o u n t o f l i p i d p r e s e n t . S u c h a n o r g a n i z a t i o n w o u l d t h e n b e e x p e c t e d to r e p o n d i n a d i f f e r e n t m a n n e r t h a n a mice l le to v a r i a t i o n s in e n v i r o n -menta l c o n d i t i o n s . 2. P h o s v i t i n T h e c o m p o s i t i o n o f t e l e o s t p h o s v i t i n has b e e n s t u d i e d b y s e v e r a l g r o u p s o f r e s e a r c h e r s . A p h o s p h o r u s c o n t e n t o f 8.5% was o b t a i n e d fo r c o h o sa lmon p h o s v i t i n b y M a r k e r t a n d V a n s t o n e ( 1 9 7 1 ) , a n d a v a l u e o f 8.33% was o b t a i n e d f o r b r o w n t r o u t p h o s v i t i n (S. trutta) b y S c h m i d t et a l . , ( 1 9 6 5 ) . M a r k e r t a n d V a n s t o n e (1971) a l so r e p o r t e d p h o s v i t i n n i t r o g e n c o n t e n t to be 12.5% f o r c o h o p h o s v i t i n a n d n o t e d t h e a b s e n c e o f a n y l i p i d c o m p o n e n t . A s u g g e s t e d v a l u e o f 27 ,000 f o r t h e m o l e c u l a r w e i g h t was somewhat h i g h e r t h a n o t h e r v a l u e s o f 5. 2 4 , 0 0 0 a n d 19,350 o b t a i n e d f o r r a i n b o w t r o u t p h o s v i t i n b y Wal lace et a l . , (1966) a n d M a n o a n d Y o s h i d a ( 1 9 6 9 ) , r e s p e c t i v e l y , as wel l as 19 ,000 f o r c h u m s a l m o n a n d 18 ,500 f o r b r o w n t r o u t p h o s v i t i n o b t a i n e d b y S c h m i d t et a l . , (1 965) . A n N/P r a t i o o f 3.31 was o b t a i n e d b y M a r k e r t a n d V a n s t o n e (1971) f o r c o h o p h o s v i t i n , w h i c h a g r e e s wel l w i t h t h a t o f 2 . 7 9 - 3 . 4 4 o b t a i n e d f o r r a i n b o w t r o u t b y Wal lace et a l . , (1 966) . A s t u d y p e r f o r m e d b y Mano a n d L i p m a n n (1 966) , w h e r e i n p h o s v i t i n o b -t a i n e d f rom t h e e g g s o f s e v e r a l t e l e o s t s p e c i e s was e l u t e d f rom a D E A E c e l l u l o s e c o l u m n b y a s e r i e s o f sa l t g r a d i e n t s o l u t i o n s , r e v e a l e d t h e p h o s v i t i n f o u n d in s a l m o n ( s p e c i e s u n i d e n t i f i e d ) y o l k to v a r y somewhat in o r g a n i z a t i o n f rom t h a t f o u n d i n o t h e r t e l e o s t s . A l l t e l eos t p h o s v i t i n s , w i t h t h e e x c e p t i o n o f t h a t o b t a i n e d f rom s a l m o n , y i e l d e d a s e r i e s o f d i s t i n c t p h o s p h o p r o t e i n s v a r y i n g in t h e a m o u n t o f p h o s p h o r u s p r e s e n t . T h e s e w e r e p o s t u l a t e d to h a v e b e e n f o r m e d b y a s t e p w i s e , d i s c o n t i n u o u s p h o s p h o r y l a t i o n to d i s t i n c t l e v e l s , ( b u t t h e p o s s i b i l i t y o f a s i m i l a r s t e p w i s e d e g r a d a t i o n d u e to s a m p l e p r e p a r a -t i o n t e c h n i q u e was not r u l e d o u t . ) H o w e v e r , D E A E c h r o m a t o g r a p h y o f s a l m o n p h o s v i t i n p r e p a r a t i o n y i e l d e d o n l y a s i n g l e p h o s p h o p r o t e i n o f 9.9% p h o s p h o r u s . P h o s v i t i n a n d l i p o v i t e l l i n i n v i r t u a l l y al l v e r t e b r a t e e g g s o c c u r in t h e fo rm o f c o m p l e x e s (Wal lace et a l . , 1966) . It has b e e n s u g g e s t e d t h a t t h e s e two p r o t e i n s a r e la id d o w n in t h e te leos t e g g b y t h e same s e r u m p r o t e i n c o m p l e x , " v i t e l l o g e n i n " , t h a t has b e e n o b s e r v e d i n o t h e r e g g l a y i n g v e r t e b r a t e s ( H a r a et a l . , 1980) . H a r a a n d H i r a i ( 1 9 7 8 ) , w o r k i n g w i t h r a i n b o w t r o u t e g g s , p e r f o r m e d an i m m u n o l o g i c a l p r e c i p i t a t i o n s t u d y t h a t s u g g e s t e d t h a t t h i s s e r u m p r o t e i n was a c t u a l l y a c o m p l e x o f t h e two e g g y o l k p r o t e i n s , l i p o v i t e l l i n a n d p h o s v i t i n . P h o s v i t i n , two t y p e s o f l i p o v i t e l l i n a n d a smal l a m o u n t o f low d e n s i t y l i p o p r o t e i n a r e c o n d e n s e d as i n s o l u b l e " g r a n u l e s " in a v i a n y o l k , a n d in f r o g e g g y o l k , p h o s v i t i n a n d a s i n g l e t y p e o f l i p o v i t e l l i n a r e c o n d e n s e d in to " p l a t e l e t s " (Wal lace , 1 965) . In ; t e l e o s t s , h o w e v e r , t h i s is not t h e c a s e (Wal lace 6. et a l . , 1966) , w h e r e a n o n - g r a n u l a r f l u i d y o l k is t h e r u l e , w i t h p h o s v i t i n a n d l i p o v i t e l l i n p r e s e n t in s o l u b l e f o r m s . T h e y o l k p r o t e i n s o f t e l e o s t s a p p e a r to b e l e s s p h o s p h o r y l a t e d t h a n t h e c o r r e s p o n d i n g p r o t e i n s o f o t h e r v e r t e d b r a t e s , a n d t h i s d i f f e r e n c e may b e a c a u s e o f t h e h i g h e r d e g r e e o f w a t e r s o l u b i l i t y ( J a r e d a n d Wa l l ace , 1 9 6 8 ) . L e s s p r o t e i n k i n a s e a c t i v i t y has b e e n f o u n d in a c e t o n e p o w d e r s o f t r o u t a n d k i l l i f i s h o v a r i e s t h a n t h o s e o f f r o g , s u g g e s t i n g a l ess a c t i v e p h o s p h o r y l a t i n g s y s t e m e x i s t i n g in t e l e o s t s , w h i c h w o u l d r e s u l t in i n c o m p l e t e l y p h o s p h o r y l a t e d y o l k p r o t e i n s ( J a r e d a n d Wa l lace , 1 968) . 3. L i v e t i n s A n i m m u n o d i f f u s i o n s t u d y b y M a r k e r t a n d V a n s t o n e (1971) r e v e a l e d t h a t o n l y t r a c e a m o u n t s o f l i v e t i n s a r e p r e s e n t in c o h o e g g y o l k . T h i s h a d b e e n p r e v i o u s l y s u g g e s t e d b y t h e a u t h o r s ' , e l e c t r o p h o r e t i c w o r k (1968) o n t h e h i g h d e n s i t y l a y e r o f f i v e s p e c i e s o f P a c i f i c s a l m o n w h i c h f a i l e d to y i e l d v i s i b l e p r o o f o f l i v e t i n s o n t h e r e s u l t a n t e l e c t r o p h e r o g r a m s . E l e c t r o p h o r e s i s o f a c o n c e n t r a t e d w a t e r s o l u b l e e x t r a c t o f l i v e t i n s f rom e g g y o l k h a d p r o d u c e d f i v e to e i g h t z o n e s , d e p e n d i n g u p o n t h e s p e c i e s . C h u m s a l m o n p o s s e s s e d o n e v e r y major a n d s e v e r a l m i n o r z o n e s , a n d t h e mobr l i t y o f t h e major l i v e t i n z o n e c o r r e s p o n d e d to t h a t o f t h e 3' c o m p o n e n t . 4. 3' c o m p o n e n t T h e r e is some d o u b t as to t h e n a t u r e o f t h e t h i r d major p r o t e i n o f t e l e o s t e g g y o l k , v a r i o u s l y l a b e l l e d 3' c o m p o n e n t ( J a r e d a n d W a l l a c e , 1968; M a r k e r t a n d V a n s t o n e , 1968; 1971) o r "x p r o t e i n " ( N a k a g a w a a n d T s u c h i y a , 1 9 6 9 ) . A m o l e c u l a r w e i g h t f o r t h i s s i m p l e p r o t e i n o f 3 0 , 0 0 0 , w i t h 14.7% n i t r o g e n was r e p o r t e d b y M a r k e r t a n d V a n s t o n e ( 1 9 7 1 ) . J a r e d a n d Wal lace (1968) f o u n d t h a t e x t e n s i v e d i l u t i o n o f a s a l i n e e x t r a c t o f r a i n b o w t r o u t e g g s r e m o v e d p h o s v i t i n , b u t f a i l e d to p r e c i p i t a t e v t h i s c o m p o n e n t , a n d so s u g g e s t e d i ts 7. p o s s i b l e i d e n t i t y as t h a t o f a s e r u m p r o t e i n o r l i v e t i n . C o n v e r s e l y , M a r k e r t a n d V a n s t o n e ( 1 9 7 1 ) , w o r k i n g w i t h c o h o s a l m o n , w e r e u n a b l e to r e p e a t s u c h a s e p a r a t i o n b y d i l u t i o n . T h e y p e r f o r m e d an i m m u n o d i f f u s i o n s t u d y in w h i c h no p r e c i p i t a t i o n l i n e s o c c u r r e d b e t w e e n p l a s m a o f male c o h o s a l m o n a n d t h o s e o f a n t i - g " c o m p o n e n t s e r u m , i n d i c a t i n g t h a t £' c o m p o n e n t is not a l i v e t i n , b u t an u n i q u e e g g p r o t e i n . F e w e r s t u d i e s h a v e b e e n c e n t e r e d o n t h e r e m a i n i n g c o m p o n e n t s o f t e l e o s t e g g f l u i d . N a k a g a w a a n d T s u c h i y a (1971) i n v e s t i g a t e d t h e c o m p o s i t i o n o f t h e o i l g l o b u l e o f r a i n b o w t r o u t a n d f o u n d it to be v e r y r i c h in c a r o t e n o i d s , l a c k i n g in p h o s p h o r u s a n d c o m p r i s e d m o s t l y o f t r i g l y c e r i d e s , w i t h a smal l a m o u n t o f c h l o e s t e r o l , c h o l e s t e r o l e s t e r s a n d h i g h e r f a t t y a c i d s . It is t h o u g h t t h a t t h e s e l i p i d s d o p r o v i d e c o n s i d e r a b l e e n e r g y f o r t h e d e v e l o p i n g o r g a n i s m ( N a k a g a w a a n d T s u c h i y a , 1969) , b u t t h e o i l g l o b u l e is m a i n l y u t i l i z e d o n l y a f t e r h a t c h i n g , i n a l a t e r p h a s e o f t h e y o l k s a c p e r i o d ( N a k a g a w a a n d T s u c h i y a , 1 972; A n d o , 1 965) . C z e c z u g a (1979) i n v e s t i g a t e d t h e s o u r c e o f t h e i n t e n s e r e d c o l o r o f c h u m e g g s a n d f o u n d t h e to ta l c a r o t e n o i d c o n t e n t to r a n g e f rom 2. 505 to 2 .798 U g / g f r e s h w e i g h t . O f t h i s , a s t a x a n t h i n e s t e r is t h e major c o n s t i t u e n t (67 .8%). A p p a r e n t l y , r o e p o s s e s s i n g h i g h e r c a r o t e n o i d c o n t e n t s a r e m o r e r e s i s t a n t to e n v i r o n m e n t a l f a c t o r s , p a r t i c u l a r l y low o x y g e n c o n t e n t s , a n d t e n d to h a t c h f r y t h a t s h o w a l ower m o r t a l i t y r a t e . R e s i s t a n c e to b a c t e r i a may b e c o n f e r r e d b y an i n c r e a s e in m e m b r a n e s t r e n g t h c a u s e d b y c e r t a i n c a r o t e n o i d s a n d low e n v i r o n m e n t a l o x y g e n may p o s s i b l y be c o m b a t t e d b y t h e p o t e n t i a l o f c a r o t e n o i d d o u b l e b o n d s to be r e v e r s i b l y s a t u r a t e d w i t h o x y g e n . 8. B.' M e m b r a n e S t r u c t u r e A l l o v i p a r o u s s p e c i e s m u s t d e v e l o p s t r u c t u r e s to p r o t e c t a n d m a i n t a i n f o r m i n g e m b r y o s a n d t h e t e l e o s t s a r e no e x c e p t i o n . D u r i n g t h e c o u r s e o f d e v e l o p m e n t , e m b r y o s o f t h e b o n y f i s h e s a r e s u r r o u n d e d w i t h p r o t e c t i v e m e m b r a n e s a n d c u s h i o n e d b y t h e f l u i d - f i l l e d p e r i v i t e l l i n e s p a c e t h a t f o r m s a f t e r f e r t i l i z a t i o n ( L a a l e , 1 9 8 0 ) . O b s e r v a t i o n o f t h e e g g s o f m a n y s p e c i e s o f f i s h h a v e r e v e a l e d s p e c i e s s p e c i f i c d i f f e r e n c e s in t h e e g g m e m b r a n e , b o t h in t h e i n t e r n a l s t r u c t u r e o f t h e m e m b r a n e , a n d in t h e e x t e r n a l s u r f a c e f e a t u r e s ( R i e h l et a l . , 1 9 8 0 ) . L o n n i n g (1972) h a s s u g g e s t e d t h a t t h e s t r u c ^ t u r e o f t h e e g g m e m b r a n e may be d e t e r m i n e d m o r e b y e x t e r n a l e n v i r o n m e n t a l i n f l u e n c e s t h a n b y t a x o n o m i c a f f i n i t i e s . S a l m o n s p e c i e s p o s s e s s d e m e r s a l e g g s t h a t a r e h e a v y a n d t e n d to s i n k , w i t h a s p e c i f i c g r a v i t y g r e a t e r t h a n t h a t o f w a t e r ( L a g l e r et a l . , 1 9 6 2 ) . T h i s is a d i s t i n c t a d v a n t a g e to s p e c i e s who lay t h e i r e g g s in s t r e a m s o f fas t r u n n i n g w a t e r . O n t h e o t h e r h a n d , e g g s w h i c h a r e d e p o s i t e d in m a s s e s in a m a r i n e e n v i r o n m e n t a r e p e l a g i c , t e n d i n g to f loat ( S t e h r a n d H a w k e s , 1 979) . D i f f e r e n c e s in t ime r e q u i r e d f o r d e v e l o p m e n t p r i o r to h a t c h i n g may a l so be r e f l e c t e d in s u c h c h a r a c t e r i s t i c s as s i z e o f e g g a n d t h i c k n e s s o f m e m b r a n e . P i n k s a l m o n ( Oncorhynchus gorbuscha ) e g g s t h a t r e q u i r e f i v e to s i x m o n t h s to d e v e l o p a r e 7 .0 mm in a v e r a g e d i a m e t e r , w h i l e e g g o f t h e s t a r r y f l o u n d e r (Platichthys stellatus (Pa l las )) a r e o n l y 0 . 9 mm in d i a m e t e r a n d r e q u i r e o n l y 14 d a y s to d e v e l o p ( S t e h r a n d H a w k e s , 1 9 7 9 ) . P e l a g i c e g g s h a v e a l so b e e n f o u n d to p o s s e s s m e m b r a n e s t h a t a r e s i m p l e r a n d t h i n n e r in s t r u c t u r e t h a n t h e d e m e r s a l e g g s o f s p e c i e s s u c h as t h e s a l m o n , w h o s e e g g m e m b r a n e must p r o v i d e p r o t e c t i o n f rom m e c h a n i c a l s t r e s s e s d u r i n g a l o n g d e v e l o p m e n t w i t h i n a g r a v e l n e s t ( S t e h r a n d H a w k e s , 1979; L o n n i n g , 1 9 7 2 ) . H a m o r a n d C a r s i d e (1977) p u b l i s h e d t h e f i r s t d e t a i l e d c o m p o s i t i o n o f 9. t h e e g g m e m b r a n e o f a t e l e o s t , u s i n g t h e s p e c i e s Salmo salar L . M a d e u p o f 92% w a t e r , t h e m a j o r i t y o f t h e r e m a i n d e r was d i v i d e d b e t w e e n a s h , c a r b o -h y d r a t e a n d p r o t e i n in a 1:3:1 r a t i o , w i t h l i p i d p r e s e n t i n a f r a c t i o n a l a m o u n t . P r e v i o u s l y , t h e m e m b r a n e h a d b e e n t h o u g h t to be c o m p o s e d e s s e n t i a l l y o f a p r o t e i n c a l l e d " i c t h u l o k e r a t i n " ( B e l l et a l . , 1969) . T h i s p r o t e i n was s h o w n to b e r e s i s t a n t to t h e a c t i o n o f most p r o t e o l y t i c e n z y m e s , b u t r e a d i l y d i g e s t e d b y t h e h a t c h i n g e n z y m e r e l e a s e d f rom t h e m a t u r e e m b r y o , l a b e l l e d " c h o r i o n a s e " b y l u c h i a n d Y a m a g a m i ( 1 9 7 6 ) . A more c o m p l e x m e m b r a n e c o m p o s i t i o n t h a n p r e v i o u s l y t h o u g h t was s u g g e s t e d b y H a m o r a n d G a r s i d e ( 1 9 7 3 ) , w h o d e m o n -s t r a t e d t h e p r e s e n c e o f p h o s p h o l i p i d s , c h o l e s t e r o l , D N A a n d R N A , as wel l as e n z y m e b e a r i n g m i c r o b o d i e s , in a d d i t i o n to t h e s t r u c t u r a l p r o t e i n s . Be l l et a l . (1 969) o b s e r v e d t h a t b o t h t h e h a t c h i n g e n z y m e a n d t h e p r o -t e o l y t i c e n z y m e " p r o n a s e " a c t e d o n l y o n t h e i n n e r s u r f a c e o f t h e m e m b r a n e o f O . keta e g g s ; t h o s e b a t h e d e x t e r n a l l y w e r e u n a f f e c t e d . A v e r y d e l i c a t e f i lm s h a p e d l i k e t h e o r i g i n a l who le s t r u c t u r e a l w a y s r e m a i n e d a f t e r d i g e s t i o n o f m e m b r a n e s i m m e r s e d in e n z y m e s o l u t i o n a n d c o u l d not be f u r t h e r d i g e s t e d w i t h t h e a d d i t i o n o f f r e s h e n z y m e . T h e y s u g g e s t e d a p o s s i b l e f u n c t i o n o f a p r o t e c t i v e c o a t i n g a g a i n s t t h e a c t i o n o f p r o t e o l y t i c b a c t e r i a a n d p r e m a t u r e e m e r g e n c e b y t h e h a t c h i n g e n z y m e o f s u r r o u n d i n g e g g s . M i c r o s t r u c t u r a l s t u d i e s p e r f o r m e d b y n u m e r o u s r e s e a r c h e r s h a v e r-:r. e s t a b l i s h e d t h a t s a l m o n e g g m e m b r a n e s c o n s i s t o f t h r e e ma in l a y e r s : T h e o u t e r m o s t l a y e r is a m u c o u s c o a t , p o l y s a c c h a r i d e in n a t u r e ( K o b a y a s h i , 1 982) . Be low t h i s l a y e r l ies t h e i n t e r m e d i a t e l a y e r d i v e r s e l y t e r m e d c o r t e x r a d i a t u s e x t e r n u s ( R i e h l et a l . , 1980) , a l so k n o w n as t h e p r i m a r y o o c y t e m e m b r a n e , p r i m a r y c h o r i o n ( G r i e r s o n a n d N e v i l l e , 1982) o r z o n a p e l l u c i d a e x t e r n u s ( K o b a y a s h i , 1 9 8 2 ) . T h i s l a y e r has a l so b e e n s h o w n to be m o d e r a t e l y p o l y s a c -c h a r i d e in n a t u r e ( K o b a y a s h i , 1 982) , a n d n e i t h e r t h e m u c o u s coat n o r t h e c o r t e x r a d i a t u s e x t e r n u s ( C R E ) a r e d i s s o l v a b l e in a c i d ( K o b a y a s h i , 1 982) . 10. In c o n t r a s t , t h e i n n e r m o s t l a y e r , n u m e r o u s l y l a b e l l e d t h e c o r t e x r a d i a t u s i n t e r n u s ( C R I ) ( R i e h l et a l . , 1 9 8 0 ) , c o r t i c a l m e m b r a n e , s e c o n d a r y c h o r i o n ( G r i e r s o n a n d N e v i l l e , 1981) o r z o n a p e l l u c i d a i n t e r n u s has b e e n s h o w n to p a r t i a l l y s w e l l a n d d i s s o l v e in 5 x 10" 3 N h y d r o c h l o r i c a c i d ( K o b a y a s h i , 1 982) . T h e C R E a n d C R I h a v e b e e n r e p o r t e d b y s e v e r a l r e s e a r c h e r s to be r a d i a l l y p e r f o r a t e d w i t h a s e r i e s o f c a n a l s ( L a a l e , 1 9 8 0 ) . T h e s e c a n a l s p o s s e s s m i c r o v i l l i t h a t a r e a c t u a l l y e x t e n s i o n s o f b o t h t h e o o c y t e m e m b r a n e a n d t h e f o l l i c u l a r c e l l s , w h i c h s h r i n k as t h e e g g m a t u r e s ( R i e h l et a l . , 1 9 8 0 ) . G e n e r a l l y , t h e C R E a p p e a r s h o m o g e n e o u s a n d e l e c t r o n d e n s e in 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 ( G r i e r s o n a n d N e v i l l e , 1981; K o b a y a s h i , 1982; H u r l e y a n d F i s h e r , 1 9 6 6 ) , w h i l e t h e C R I r e v e a l s a h i g h l y c o m p l e x s t r u c t u r e , a h e r r i n g b o n e p a t t e r n b e i n g most p r e v a l e n t a m o n g t h e t e l e o s t s p e c i e s s t u d i e d . G r i e r s o n a n d N e v i l l e (1981) h a v e i n t e r p r e t e d t h i s p a t t e r n as r e p r e s e n t i n g s e c t i o n s o f a l aminate c o m p o s e d o f s u c c e s s i v e l a y e r s o f p r o t e i n m i c r o f i b r i l s w h i c h c h a n g e in d i r e c t i o n t h r o u g h a smal l a n g l e in s u c c e s s i v e l a y e r s , r e s s u i t i n g in a h e l i c o i d a l a r r a n g e m e n t . T h e n u m b e r o f l a y e r s o r lamel lae in t h i s a r r a n g e m e n t v a r i e s b e t w e e n s p e c i e s , f rom 17-1 8 o b s e r v e d in Centrolabrus exoletus to 5 in Gadus morrhua ( L o n n i n g , 1 972) . It has b e e n o b s e r v e d t h a t t h e e g g s o f o v o v i v i p a r o u s f i s h h a v e f e w e r lamel lae t h a n o v i p a r o u s s p e c i e s ( G r i e r s o n a n d N e v i l l e , 1981) , w h i c h a g a i n may r e f l e c t d i f f e r e n t r e g u i r e m e n t s f o r s t r u c t u r a l d u r a b i l i t y . A c c o r d i n g l y , s t u d i e s o n t h e e g g m e m b r a n e s o f P a c i f i c s a l m o n h a v e r e v e a l e d a c o m p l e x l a m i n a r s t r u c t u r e ( S t e h r a n d H a w k e s , 1979; K o b a y a s h i , 1 982) t h a t is in k e e p i n g w i t h t h e r e l a t i v e l y h a r s h c o n d i t i o n s u n d e r w h i c h e m b r y o d e v e l o p m e n t m u s t t a k e p l a c e . C . M e c h a n i s m o f Ice F o r m a t i o n T h e d e c e p t i v e l y s i m p l e p h a s e t r a n s i t i o n o f l i q u i d w a t e r to s o l i d ice 11. lies well within the realm of the most important biochemical processes. Its effect on the viability and/or physical characteristics of biological tissues has been well documented in the literature, however, the causes of altera-tions brought about by freezing still remain incompletely understood. The mechanism of freezing has been the object of much study and is presently thought to occur in two discrete stages: nucleation and crystal growth. Nucleation is essential for any further continuation of the process and describes the condensation of a group of water molecules into a con-figuration enabling successive water molecules to attach themselves with a resulting reduction in their chemical potential (Franks, 1981). In order for this growth to occur, these "ice embryos" must be of a certain critical size, defined as the point at which there is an equal probability of the structure growing or vanishing (Franks, 1981; Meryman, 1 960), and of a sufficient lifetime to allow crystal growth. At the melting point, water and ice are in equilibrium (Fennema, 1973; Franks, 1981) and any reduction in temperature results in further formation of ice. If there is no ice already present in the system, crystals will not begin to form until the temperature drops to several degrees below 0°C (Fennema, 1973). This phenomenon, known as "supercooling", is thought to be due to the presence of an energy barrier based on the changes in free energy involved in the aggregation of sufficient water molecules to form nuclei of critical size. The fact that tissue systems may be cooled to tempera-tures well below 0°C and then rewarmed without any alteration in their structure indicates that any damage caused by the freezing process is not due solely to lowered temperatures (Fennema, 1 973), The temperature of the system has a direct effect on both the critical size and lifetime of nuclei and it follows then that the degree of supercooling 12. in a n y s y s t e m is d e p e n d e n t u p o n t h e t e m p e r a t u r e o f t h a t s y s t e m . L o w e r t e m p e r a t u r e s r e s u l t in a d e c r e a s e in c r i t i c a l s i z e a n d a l e n g t h e n i n g in t h e l i f e t ime o f w a t e r m o l e c u l e c l u s t e r s ( F r a n k s , 1981) . T h e r a t e o f n u c l e a t i o n i n c r e a s e s b y m a n y o r d e r s o f m a g n i t u d e o v e r a n a r r o w r a n g e o f t e m p e r a t u r e s . A t - 3 9 ° C , t h e c r i t i c a l s i z e becomes c o m p a r a b l e to t h e s i z e o f r a n d o m a g g r e -g a t i o n s o f w a t e r m o l e c u l e s a n d t h i s t e m p e r a t u r e is t h o u g h t to be t h e l imit f o r s u p e r c o o l i n g ( M e r y m a n , 1 960) . T w o t y p e s o f n u c l e a t i o n h a v e b e e n d e s c r i b e d ( F e n n e m a , 1 9 7 3 ) . H o m o -g e n e o u s , t h r e e d i m e n s i o n a l n u c l e a t i o n i n v o l v e s t h e c h a n c e f o r m a t i o n o f w a t e r m o l e c u l e s in to a c r i t i c a l l y s i z e d n u c l e u s t h r o u g h r a n d o m d e n s i t y a n d e n e r g y f l u c t u a t i o n s . D u r i n g h e t e r o g e n e o u s , two d i m e n s i o n a l n u c l e a t i o n , t h e a g g r e -g a t i o n o f w a t e r m o l e c u l e s o c c u r s o n t h e s u r f a c e o f n o n - a q u e o u s s u b s t a n c e s , s u c h as s u s p e n d e d f o r e i g n b o d i e s , s u r f a c e f i lms o r e v e n t h e wal ls o f c o n -t a i n e r s ( F e n n e m a , 1 973; F r a n k s , 1981) . T h e f o r m e r p r o c e s s r e q u i r e s v e r y low t e m p e r a t u r e s a n d o c c u r s i n o n l y p u r e w a t e r ( F e n n e m a , 1973) . O n t h e o t h e r h a n d , h e t e r o g e n e o u s n u c l e a t i o n , t h o u g h u n l i k e l y at 0°C , is m u c h m o r e p r o b a b l e at h i g h e r f r e e z i n g t e m p e r a t u r e s , a n d is t h o u g h t to lower t h e a c t i v a t i o n e n e r g y o f n u c l e a t i o n b y e i t h e r d e c r e a s i n g t h e n u m b e r o f w a t e r m o l e c u l e s r e q u i r e d f o r a c r i t i c a l l y s i z e d n u c l e u s , o r b y s t a b i l i z i n g o r d e r e d c l u s t e r s o f w a t e r m o l e c u l e s in t h e immedia te v i c i n i t y ( F e n n e m a , 1 9 7 3 ) . D u e to t h e c o n d i t i o n s a n d h i g h e r t e m p e r a t u r e s at w h i c h t h i s p r o c e s s o c c u r s , it is t h o u g h t to fo rm t h e p r e d o m i n a n t mode o f n u c l e a t i o n in food s y s t e m s ( F e n n e m a , 1 9 7 3 ) . C r y s t a l l i z a t i o n is t h e s e c o n d s t a g e o f t h e p h a s e t r a n s i t i o n o f w a t e r in to i ce a n d i n v o l v e s t h e s u b s e q u e n t b u i l d - u p o f s u c c e s s i v e w a t e r m o l e c u l e s o n t o t h e i ce n u c l e u s . T h i s p r o c e s s , d e p e n d e n t o n t h e r a t e o f d i f f u s i o n o f w a t e r m o l e c u l e s to t h e a c t i v e n u c l e u s , is t e m p e r a t u r e d e p e n d e n t as well., w i t h t e m -p e r a t u r e s c l o s e to t h e m e l t i n g p o i n t f a v o u r i n g c r y s t a l l i z a t i o n ( F r a n k s , 1 9 8 1 ) . 13. D u e to t h e c o n t r a s t i n g d e p e n d e n c e o f c r y s t a l l i z a t i o n a n d n u c l e a t i o n o n t e m -p e r a t u r e , low f r e e z i n g t e m p e r a t u r e s r e s u l t in m a n y smal l i ce c r y s t a l s , w h i l e t e m p e r a t u r e s c l o s e r to t h e m e l t i n g p o i n t c r e a t e f e w e r , l a r g e r c r y s t a l s . D . M e c h a n i s m s o f Ice D a m a g e A l t e r a t i o n s i n f r o z e n m a t e r i a l a r e a r e s u l t o f o n e o r m o r e p r o c e s s e s i n v o l v e d in t h e t r a n s i t i o n o f l i q u i d w a t e r to s o l i d i ce c r y s t a l s . T h e f i r s t o f t h e s e l ies in t h e p h y s i c a l f o rm o f i ce c r y s t a l s a c t i n g t h r o u g h m e c h a n i c a l d i s r u p t i o n . D e p e n d e n t u p o n t h e s t r u c t u r e o f i n d i v i d u a l w a t e r m o l e c u l e s , i ce c r y s t a l s m u s t fo rm in t h e s h a p e o f a h e x a g o n a l p r i s m ( L u y e t , 1960) . In idea l c o n d i t i o n s , t h e s i x c o r n e r s o f t h e f o r m i n g c r y s t a l g r o w f a s t e r t h a n t h e f a c e s , p r o d u c i n g s t a r s a n d h e x a g o n a l r o s e t t e s t h a t , w i t h c o n t i n u e d g r o w t h a n d a d d e d b r a n c h i n g y i e l d s a " s k e l e t o n " - a m a n y b r a n c h e d f o r m , s t i l l r e t a i n i n g t h e b a s i c h e x a g o n a l s h a p e ( L u y e t , 1 9 6 0 ) . T h e t r a n s i t i o n o f w a t e r to i ce at 0°C r e s u l t s i n a v o l u m e e x p a n s i o n o f a p p r o x i m a t e l y 9% ( F e n n e m a , 1 966) . In a n a q u e o u s s y s t e m , d e p e n d i n g o n c o m p o s i t i o n , t h e a m o u n t o f u n f r e e z a b l e w a t e r a n d t h e t e m p e r a t u r e r a n g e e m p l o y e d , t h e r e wi l l be r e s t r i c t e d a r e a s o f e x p a n s i o n a n d c o n t r a c t i o n l e a d i n g to local s t r e s s e s . T h e p h y s i c a l n a t u r e o f t h e t r a n s f o r m a t i o n o f w a t e r to ice t h u s may lead to m e c h a n i c a l d a m a g e . D i r e c t l y r e l a t e d to t h i s m e c h a n i c a l d a m a g e , is t h e p r o c e s s t h a t o c c u r s d u r i n g f r o z e n s t o r a g e . U n l e s s f r o z e n v e r y s l o w l y , i ce c r y s t a l s a r e m e t a -s t a b l e ( F r a n k s , 1 9 8 1 ) , r e s u l t i n g f r o m a s u r f a c e : v o l u m e r a t i o too l a r g e f o r t h e s m a l l e r c r y s t a l s f o r m e d ( L u y e t , 1969) , a n d so r e c r y s t a l l i z a t i o n may o c c u r . T h i s p r o c e s s may t a k e p l a c e in o n e o r m o r e o f s e v e r a l d i f f e r e n t w a y s , b u t t h e d r i v i n g f o r c e is t o w a r d s f e w e r , l a r g e r c r y s t a l s w i t h an a c c o m p a n y i n g m i n i -m i z a t i o n o f f r e e e n e r g y ( F e n n e m a , 1 973) . 14. T h e s e c o n d c a u s e o f d a m a g e to a s y s t e m r e s u l t s f rom t h e e f f e c t o f i ce c r y s t a l g r o w t h o n t h e s u r r o u n d i n g a q u e o u s s y s t e m a n d is t h e d i r e c t r e s u l t o f c o n c e n t r a t i o n c h a n g e s . B e c a u s e t h i s p r o c e s s r e m o v e s m a i n l y w a t e r f r o m t h e s y s t e m , t h e n o n - a q u e o u s s o l u t e s b e c o m e more c o n c e n t r a t e d in t h e r e -m a i n i n g u n f r o z e n w a t e r ( F r a n k s , 1981) , a n d t h i s c a n h a v e a n i m p o r t a n t e f f e c t i f c o m p o u n d s s e n s i t i v e to h i g h l e v e l s o f e l e c t r o l y t e s a r e p r e s e n t . T h e e x t e n t o f t h i s c o n c e n t r a t i o n d e p e n d s u p o n t h e f r e e z i n g r a t e , t h e u l t imate t e m p e r a t u r e a c h i e v e d a n d t h e n a t u r e o f t h e s y s t e m ( F e n n e m a , 1966) . T h e r a t e o f f r e e z i n g has b e e n s h o w n to h a v e a d i r e c t e f f e c t o n t h e n u m b e r , s i z e a n d l o c a t i o n o f i ce c r y s t a l s w i t h s low f r e e z i n g r e s u l t i n g i n f e w e r , l a r g e r c r y s t a l s in e x t r a c e l l u l a r s p a c e s , w h i l e v e r y r a p i d f r e e z i n g c r e a t e s n u m e r o u s smal l i n t r a c e l l u l a r c r y s t a l s ( L u y e t , 1 969; F e n n e m a , 1966) . S low f r e e z i n g t h e n , b y c o n c e n t r a t i n g e x t r a c e l l u l a r f l u i d w i t h t h e f o r m a t i o n o f l a r g e i ce c r y s t a l s l eads to t h e s e c o n d t y p e o f c o n c e n t r a t i o n d a m a g e , r e s u l t i n g i n t h e c r e a t i o n o f a n o s m o t i c g r a d i e n t a c r o s s t h e ce l l m e m b r a n e ( M e r y m a n et a l . , 1 9 7 7 ) . S u b s e q u e n t m o v e m e n t o f w a t e r o u t o f t h e ce l l in a n a t tempt to a c h i e v e o s m o t i c e q u i l i b r i u m c a n r e s u l t i n d e h y d r a t i o n o f t h e c e l l , l e a v i n g it s h r u n k e n a n d d i s t o r t e d . B o t h o f t h e s e e f f e c t s a r e a r e s u l t o f r e m o v a l o f w a t e r f rom t h e s y s t e m a n d a p a r a l l e l has b e e n d r a w n b e t w e e n t h e s y m p t o m s o f f r e e z e d a m a g e a n d t h o s e c a u s e d b y d r o u g h t a n d h i g h sa l t c o n c e n t r a t i o n s ( F r a n k s , 1 9 8 1 ) . E . C r y o p r o t e c t a n t s It is c l e a r t h a t p r e v e n t i o n o f d a m a g e d u e to f r e e z i n g must d e c r e a s e e i t h e r t h e d e h y d r a t i o n o r t h e e f f e c t s o f d e h y d r a t i o n t h a t o c c u r w i t h t h e r e m o v a l o f w a t e r c a u s e d b y i c e c r y s t a l g r o w t h ( D o e b b l e r , 1966; M e r y m a n , 1 9 6 0 ) , a n d to a g r e a t e x t e n t as p o s s i b l e , p r e v e n t t h e f o r m a t i o n o f l a r g e d i s r u p t i v e i ce c r y s t a l s . E v e r s i n c e t h e b r e a k t h r o u g h w o r k o f P o l g e et a l . , ( 1 9 4 9 ) , p r e -15. v e n t i o n o f t h e s e e f f e c t s h a s b e e n a t t e m p t e d w i t h t h e u s e o f v a r i o u s c o m p o u n d s , c o l l e c t i v e l y k n o w n as c r y o p r o t e c t a n t s , t h a t a p p e a r to w o r k b y a l t e r i n g t h e p h y s i c a l c o n d i t i o n o f t h e ice a n d s o l u t i o n s in t h e s y s t e m ( F a r r a n t , 1 9 8 0 ) . In a s t u d y b y L u s e n a ( 1 9 5 5 ) , e f f e c t i v e s o l u t e s w e r e o b s e r v e d to a l t e r o n l y 2 s l i g h t l y t h e a b i l i t y o f w a t e r to n u c l e a t e h e t e r o g e n e o u s l y , b u t w e r e a b l e to m a r k e d l y r e t a r d t h e r a t e o f i ce c r y s t a l g r o w t h . G e n e r a l c h a r a c t e r i s t i c s o f al l c r y o p r o t e c t a n t s i n c l u d e a h i g h s o l u b i l i t y i n w a t e r t h a t e n a b l e s t h e c o m p o u n d to r e m a i n in s o l u t i o n e v e n a f t e r t h e f i r s t i ce is f o r m e d , a n d a low t o x i c i t y to t h e s y s t e m b e i n g p r o t e c t e d ( F a r r a n t , 1980) . T h e e f f e c t i v e n e s s o f a n y p a r t i c u l a r c r y o p r o t e c t a n t has b e e n l i n k e d to i ts c a p a c i t y to f o rm h y d r o g e n b o n d s a n d t h u s b i n d w a t e r m o l e c u l e s , i n t e r f e r i n g w i t h t h e i r i n c o r p o r a t i o n in to i ce c r y s t a l s ( P a r k e r , 1972; F a r r a n t , 1 9 8 0 ) . T h i s is o f p a r t i c u l a r i m p o r t a n c e f o r s y s t e m s in w h i c h w a t e r e x i s t s i n n a r r o w c a p i l l a r i e s o r p o r e s ( F r a n k s , 1981) . T h e c o r r e l a t i o n o f c r y o p r o t e c t a n t b e h a v i o u r w i t h m o l e c u l a r s t r u c t u r e has b e e n p r o p o s e d ( D o e b b l e r , 1966; L i n g , 1967), b a s e d o n t h e e f f e c t o n w a t e r s t r u c t u r e o f a p a r t i c u l a r s o l u t e ' s m o l e c u l e s . T h e p r e s e n c e in a mo lecu le o f i o n i c g r o u p s , w h i c h act as w a t e r s t r u c t u r e b r e a k e r s ( L i n g , 1967) , o r o f n o n - p o l a r g r o u p s t h a t o r i e n t w a t e r m o l e c u l e s a n d i n t e r f e r e w i t h a d j a c e n t h y d r o g e n b o n d i n g ( D o e b b l e r , 1 9 6 6 ) , d e c r e a s e a n y c o m p o u n d ' s a b i l i t y to act as a c r y o p r o t e c t a n t . H o w e v e r , t h e m o r e n u m e r o u s t h e h y d r o g e n b o n d i n g s i t e s o n a c o m p o u n d , t h e more m a g n i f i e d t h e p r o t e c t i v e c a p a c i t y ( D o e b b l e r , 1 9 6 6 ) . In t h e same w a y , it has b e e n n o t e d t h a t e x c e s s i v e h y d r o g e n b o n d i n g s i t e s o n a n u c l e a t i n g agent| .s s u r f a c e r e d u c e c r y s t a l f o r m a t i o n . A g e n t s t h a t p o s s e s s more t h a n o n e t y p e o f f u n c t i o n a l g r o u p w o u l d a p p a r e n t l y t h e n b e h a v e a c c o r d i n g to t h e o v e r a l l a d d i t i v e e f f e c t ( L i n g , 1967) . T h e r e a r e two b a s i c c a t e g o r i e s o f c r y o p r o t e c t a n t s , b a s e d o n t h e i r c a p a c i t y to p e n e t r a t e in to t h e i n t e r i o r o f a ce l l a n d t h e t y p e o f f r e e z i n g f o r w h i c h 16. p r o t e c t i o n is o f f e r e d ( M c G a n n , 1978) . T h e fact t h a t a g e n t s e f f e c t i v e in s low f r e e z i n g c o n d i t i o n s a r e not n e c e s s a r i l y e f f e c t i v e d u r i n g r a p i d f r e e z i n g , w h e n a d d e d to t h e v a r i a t i o n in p e n e t r a b i l i t y e x p r e s s e d b y s e v e r a l s u c c e s s f u l c r y o p r o t e c t a n t s , s u g g e s t s two d i f f e r e n t modes o f a c t i o n ( M e r y m a n , 1 9 6 0 ) . P e n e t r a t i n g a g e n t s s u c h as g l y c e r o l , e t h y l e n e g l y c o l , a n d d i m e t h y l s u l f o x i d e act in m u l t i m o l a r c o n c e n t r a t i o n s (3 to 4 m o l a r ) ( M e r y m a n , 1971) t h a t u s u a l l y r e s u l t in l a r g e e x t r a c e l l u l a r c r y s t a l s a n d ce l l d e h y d r a t i o n . P e n e t r a t i o n o f t h e s e a g e n t s in to t h e ce l l is o f u tmost i m p o r t a n c e o r f r e e z i n g i n j u r i e s w o u l d be s i m u l a t e d b y h i g h e x t r a c e l l u l a r c o n c e n t r a t i o n s , r e s u l t i n g in d e h y d r a t i o n o f t h e c e l l . T h e r a t e o f p e n e t r a t i o n o f d i f f e r e n t c r y o p r o t e c t a n t s v a r i e s , w i t h d i m e t h y l -s u l f o x i d e b e i n g p a r t i c u l a r l y s u c c e s s f u l . S u c h e f f e c t i v e n e s s becomes o f i n -c r e a s i n g i m p o r t a n c e w h e n c r y o p r o t e c t i o n o f w h o l e t i s s u e s a n d o r g a n s a r e c o n s i d e r e d in w h i c h a c r y o p r o t e c t i v e a g e n t m u s t p e n e t r a t e in to t h e i n t e r i o r o f t h e s y s t e m . N o n - t o x i c i t y is a l so v e r y i m p o r t a n t , c o n s i d e r i n g t h e l a r g e mo lar c o n c e n -t r a t i o n s r e q u i r e d f o r e f f e c t i v e n e s s . T h e m e c h a n i s m o f a c t i o n t h a t has b e e n p r o p o s e d f o r p e n e t r a t i n g c r y o -p r o t e c t a n t s i n v o l v e s a r e d u c t i o n in t h e a m o u n t o f e x t r a c e l l u l a r i ce f o r m e d . S u c h an e f f e c t c a n b e b r o u g h t a b o u t b y a c o l l i g a t i v e b i n d i n g o f w a t e r m o l e c u l e s ( M e r y m a n , 1971) , o r t h r o u g h an i n c r e a s e in t h e v i s c o s i t y o f t h e s y s t e m , r e s u l t i n g in a d e c r e a s e d d i f f u s i o n r a t e o f w a t e r m o l e c u l e s to t h e ice c r y s t a l s u r f a c e s ( M e r y m a n et a l . , 1977) . B o t h m e c h a n i s m s s e r v e to i n t e r f e r e w i t h t h e f o r m a t i o n a n d g r o w t h o f i ce c r y s t a l s . T h e o t h e r c a t e g o r y o f c r y o p r o t e c t a n t s i n c l u d e s h i g h m o l e c u l a r w e i g h t n o n -p e n e t r a t i n g c o m p o u n d s s u c h as p o l y v i n y l p y r i d o n e , s u g a r s a n d s u g a r a l c o h o l s t h a t , u n l i k e p e n e t r a t i n g a g e n t s , a p p e a r to b e a b l e to p r o t e c t a g a i n s t f r e e z i n g d a m a g e in q u i t e low molar c o n c e n t r a t i o n s u n d e r more r a p i d f r e e z i n g c o n d i t i o n s 17. (Meryman, 1971). The exact source of cryoprotection for these compounds is still uncertain, however, a theory has been proposed involving a mechanism acting through the temporary alteration of the cell membrane permeability to solutes. This prevents excessive cellular dehydration beyond a maximum tolerated volume (Meryman, 1971), and rapid cooling rates are then necessary to prevent overly excessive leakage of cell contents before freezing occurs. F. Processing The processing of roe for caviar involves several steps that have been described in detail by Zaitsev et al. (1969). The eggs are contained in two ovaries formed by two flattened sacs extending along either side of the spinal column in the abdominal cavity of the female fi s h . Initially, the ovaries are removed from the fish d uring dressing of the latter, then washed in water at a temperature under 5°C to remove blood and other extraneous matter. During this washing step, grading takes place, with firm and elastic ovaries of a light orange to orange colour receiving top grade status (Sternin and Hori, 1982). "Screening" follows grading of the roe, wherein ovaries are manually split open lengthwise, then rotated gently by hand on a stainless steel or cord screen with 1 cm openings. The ovaries are covered by a thin trans-parent film to which immature eggs adhere firmly. However, as maturity increases, the eggs are less securely attached and separate easily. Eggs fall through the first screen onto a second (also with 1 cm mesh openings), then finally onto a slanting screen of 4 mm mesh, labelled the "strainer". This final screen strains out any water and retains fragments of membranes and burst grains, allowing intact eggs to roll down its surface. Roe that are mature and of grade 1 quality separate very easily, with little breakage 18. o f t h e g r a i n s , a n d t h e s c r e e n i n g l o s s e s a r e smal l ( S t e r n i n a n d H o r i , 1982) . S a l t i n g o f t h e loose e g g s t a k e s p l a c e t h r o u g h i m m e r s i o n in a p r e h e a t e d s a t u r a t e d sa l t ( s o d i u m c h l o r i d e ) s o l u t i o n ( 1 . 2 g / c m 3 ) at a t e m p e r a t u r e l e s s t h a n 15°C, a n d a r o e : b r i n e ra t io o f a p p r o x i m a t e l y 1 :3 . S a l t i n g is p e r f o r m e d i n l a r g e v a t s , w i t h c o n t i n u a l g e n t l e a g i t a t i o n o v e r a n 8-18 m i n u t e p e r i o d , d e p e n d i n g u p o n t h e q u a l i t y o f t h e r o e a n d t h e t e m -p e r a t u r e o f t h e b r i n e . S c u m , b u r s t m e m b r a n e s a n d b l o o d c l o t s f loat o n t h e s u r f a c e o f t h e b r i n e a n d c a n be s k i m m e d o f f w i t h a m e s h n e t . W h e n t h e e g g s h a v e a c h i e v e d t h e d e s i r e d sa l t c o n t e n t ( m e a s u r e d e i t h e r o r g a n o l e p t i c a l l y o r w i t h t h e u s e o f a c h l o r i d e ion e l e c t r o d e ( S t e r n i n a n d H o r i , 1 9 8 2 ) ) , t h e y a r e r e m o v e d a n d p l a c e d in b a s k e t s o r s t r a i n e r s to al low t h e b r i n e to d r a i n o f f . T h i s p r o c e d u r e may t a k e f rom 2-12 h o u r s , a g a i n d e p e n d i n g u p o n t h e q u a l i t y o f t h e o r i g i n a l raw p r o d u c t . T i m e is an i m p o r t a n t f a c t o r , as e g g s left too l o n g b e c o m e s t i c k y , w i t h o u t t h e p r o p e r loose c o n s i s t e n c y . P r e s e r v a t i v e s o f 0.1% h e x a m e t h y l e n e t e t r a m i n e ( Z a i t s e v et a l . , 1969) o r 0.1% b e n z o i c a c i d ( S t e r n i n a n d H o r i , 1982) may t h e n b e a d d e d to e n s u r e l o n g e r s h e l f - l i f e . V e g e t a b l e oi l (0.6%) a n d / o r g l y c e r i n e (0.015%) a r e a d d e d to p r e v e n t s t i c k i n e s s o f t h e g r a i n s a n d to l e n d a l u s t e r to t h e f i na l p r o d u c t . A c c e p t a b l e p r o c e s s e d c a v i a r is d i v i d e d in to two g r a d e s , w i t h g r a d e 1 c a v i a r p o s s e s s i n g a s a l i n i t y o f b e t w e e n 4-6%, w h i l e g r a d e 2 c a n be as h i g h as 8% ( Z a i t s e v et a l . , 1969) . T h e h i g h e s t q u a l i t y , w h e n j u d g e d o r g a n o l e p -t i c a l l y , c o n s i s t s o f f i r m , loose g r a i n s t h a t do not s t i c k t o g e t h e r w h e n p r e s s e d in t h e pa lm o f t h e h a n d . If s q u e e z e d b e t w e e n f i n g e r a n d t h u m b , a s i n g l e g r a i n wi l l n o t r b u r s t e a s i l y a n d t h e l i q u i d e x p r e s s e d wil l r e t a i n t h e s h a p e o f a d r o p l e t . W h e n s q u e e z e d , p o o r e r q u a l i t y e g g s wil l e i t h e r b u r s t r e a d i l y , r e v e a l i n g a t h i n i n n e r f l u i d , o r wi l l h a v e a w a x y a p p e a r a n c e a n d fo rm a s o l i d m a s s . O t h e r q u a l i t y f a c t o r s o f t h e f i na l p r o d u c t i n c l u d e c o l o u r , s h a p e a n d s i z e o f t h e loose g r a i n s ( S t e r n i n a n d H o r i , 1982) . T a s t e is a lso o f p r i m e 19. i m p o r t a n c e . T o p q u a l i t y c a v i a r l a c k s a n y s o u r s m e l l , may p o s s e s s a s l i g h t b i t t e r a n d / o r s h a r p , b u t no " s k i n " a f t e r t a s t e . T h e l a t t e r s e n s a t i o n r e s u l t s f r o m c a v i a r p r o c e s s e d f rom e g g s o f a m o r e m a t u r e s t a g e t h a n d e s i r a b l e , w i t h a v e r y wel l d e v e l o p e d m e m b r a n e . T h e f i na l y i e l d o f c a v i a r f r o m h i g h q u a l i t y o v a r i e s is 60-65% (w/w) o r 80-85% (w/w) o f t h e s c r e e n e d roe ( S t e r n i n a n d H o r i , 1982) . 20. M A T E R I A L S A N D M E T H O D S A . S a m p l e P r e p a r a t i o n O v a r i e s f r o m s e x u a l l y m a t u r e female c h u m s a l m o n (Oncorhynchus keta) w e r e o b t a i n e d f rom loca l f i s h p r o c e s s o r s i m m e d i a t e l y a f t e r e v i s c e r a t i o n . S a m p l e i d e n t i f i c a t i o n was f a c i l i t a t e d b y a s s i g n i n g a c o d e n u m b e r to e a c h d a t e o f c o l l e c t i o n as s h o w n in T a b l e 1. L o o s e e g g s w e r e o b t a i n e d b y " s c r e e n i n g " , a p r o c e d u r e c o m m o n l y u s e d in c a v i a r p r o d u c t i o n ( Z a i t s e v et a l . , 1969) . In t h i s m e t h o d , w h o l e o v a r i e s w e r e m a n u a l l y s p l i t o p e n l e n g t h w i s e , t h e n p l a c e d o n a s t a i n l e s s s t e e l w i r e d s c r e e n w i t h 1 cm s q u a r e o p e n i n g s a n d g e n t l y r o t a t e d w i t h l i g h t p r e s s u r e b y h a n d . I n i d i v i d u a l e g g s w e r e s e p a r a t e d f rom t h e o v a r y i n t h i s m a n n e r a n d w e r e c o l l e c t e d in a t r a y be low t h e s c r e e n . T h e s e p a r a t e e g g s w e r e t h e n p r e p a r e d in o n e o f t h e f o l l o w i n g m a n n e r s , d e p e n d i n g u p o n t h e a n a l y s i s i n t e n d e d : 1. Whole e g g b l e n d S a m p l e s w e r e m a c e r a t e d b y b l e n d i n g at least 20 e g g s w i t h a P o l y t r o n h o m o g e n i z e r (Mode l N o . P T 1 0 - 3 5 , B r i n k m a n I n s t r u m e n t s I n c . , W e s t b u r y , N Y ) at 3 ,000 r p m f o r 2 min at w h i c h t ime t h e l i q u i d a p p e a r e d h o m o g e n e o u s , w i t h no w h o l e e g g s o r v i s i b l e p i e c e s o f m e m b r a n e p r e s e n t . 2. E g g magma P r e p a r a t i o n i n v o l v e d p l a c i n g at least 20 e g g s i n a p l a s t i c s t r a i n e r (1 mm s q u a r e o p e n i n g s ) a n d c r u s h i n g them w i t h a r u b b e r s p a t u l a . T h e f r e e y o l k was t h e n a l l owed to d r a i n t h r o u g h t h e s t r a i n e r in to a c o n t a i n e r b e l o w . 3- E g g y o l k T h e i m m i s c i b l e l i p i d p o r t i o n o f t h e magma was r e m o v e d b y c e n t r i f u g i n g T a b l e 1. C o d i n g s y s t e m f o r e g g s a m p l e i d e n t i f i c a t i o n . D a t e c o l l e c t e d C o d e 1981 O c t o b e r 15 I 1982 J u l y 14 II A u g u s t 11 III A u g u s t 21 IV S e p t e m b e r 3 V S e p t e m b e r 9 VI S e p t e m b e r 16 VII S e p t e m b e r 27 VIII O c t o b e r 8 IX O c t o b e r 12 X O c t o b e r 14 XI O c t o b e r 21 XII 22. t h e l a t t e r f o r 90 min at 4°C at 23 ,500 x g in a S o r v a l l R C 2 - B r o t o r . T h e h i g h e r d e n s i t y l a y e r , l a b e l l e d t h e " y o l k " , was t h e n r e m o v e d f rom t h e bot tom o f t h e t u b e , u s i n g a 10 ml_ d i s p o s a b l e t u b e r c u l i n s y r i n g e . 4. M e m b r a n e T h e r e s i d u e r e m a i n i n g in t h e p l a s t i c s t r a i n e r a f t e r p r e p a r a t i o n o f e g g magma was w a s h e d w i t h 2% ( w / v ) s o d i u m c h l o r i d e , t h e n b l e n d e d in 2% ( w / v ) s o d i u m c h l o r i d e w i t h t h e P o l y t r o n at 3,000 r p m u n t i l no i n t a c t m e m b r a n e f r a g m e n t s r e m a i n e d . T h e s u s p e n s i o n was c e n t r i f u g e d ( 2 3 , 5 0 0 x g f o r 15 min o n a S o r v a l l R C 2 - B r o t o r ) a n d w a s h e d 3 t imes w i t h an e q u a l v o l u m e o f 2% ( w / v ) s o d i u m c h l o r i d e , a n d t h e f i n a l r e s i d u e was d r i e d o v e r n i g h t i n a v a c u u m o v e n at 80°C. B . P r o x i m a t e A n a l y s i s 1. D e t e r m i n a t i o n o f m o i s t u r e S a m p l e s o f w h o l e e g g b l e n d o r e g g magma (5 g ) w e r e m i x e d w i t h 7 g o f w a s h e d s a n d i n a n a l u m i n u m s a m p l e p a n . T h e s e w e r e p r e - d r i e d o n low h e a t , s t i r r i n g w i t h a g l a s s r o d to p r e v e n t s u r f a c e c a s e h a r d e n i n g . F i n a l d r y i n g was c a r r i e d o u t in a v a c u u m o v e n at 105 k P a v a c u u m a n d 80°C f o r 18 h . T h e m o i s t u r e c o n t e n t s w e r e c a l c u l a t e d as a p e r c e n t a g e (wet b a s i s ) a n d r e s u l t s p r e s e n t e d as t h e a v e r a g e o f a t r i p l i c a t e a n a l y s e s . 2. D e t e r m i n a t i o n o f p r o t e i n " S a m p l e s o f f r e e z e - d r i e d who le e g g b l e n d , e g g magma o r m e m b r a n e (12 mg) w e r e p l a c e d in a 30 m L m i c r o - K j e l d a h l f l a s k a n d d i g e s t e d u s i n g t h e m e t h o d o f C o n c o n a n d S o l t e s s (1 9 7 3 ) . D i g e s t e d s a m p l e s w e r e a n a l y z e d f o r n i t r o g e n c o n t e n t b y means o f a T e c h n i c o n A u t o a n a l y z e r ( T e c h n i c o n I n s t r u m e n t s C o r p . , T a r r y t o w n , N Y ) . R e s u l t s w e r e c o n v e r t e d in to c r u d e p r o t e i n c o n t e n t b y m u l t i p l y i n g % N b y 6.25 ( P o m e r a n z a n d M e l o a n , 1 9 7 1 ) . T r i p l i c a t e s a m p l e s w e r e d i g e s t e d a n d t h e a v e r a g e e x p r e s s e d as c r u d e p r o t e i n (% wet b a s i s ) . 3. D e t e r m i n a t i o n o f c r u d e l i p i d F r e e z e - d r i e d s a m p l e s o f w h o l e e g g b l e n d o r e g g magma (10 g ) w e r e p l a c e d in to 500 m L b e a k e r s a n d c a r r i e d t h r o u g h a m o d i f i e d B l i g h - D y e r e x t r a c t i o n ( L e e et a l . , 1 9 6 6 ) . A l i q u o t s (25 m L ) o f t h e c h l o r o f o r m l a y e r w e r e d r i e d u n d e r n i t r o g e n in a 50°C w a t e r b a t h , t h e n s t o r e d o v e r n i g h t in a d e s i c c a t o r o v e r p h o s p h o r u s p e n t o x i d e b e f o r e w e i g h i n g . E x t r a c t i o n s w e r e p e r f o r m e d in t r i p l i c a t e a n d t h e a v e r a g e c r u d e l i p i d c o n t e n t e x p r e s s e d as a p e r c e n t a g e (wet b a s i s ) . 4. D e t e r m i n a t i o n o f c h l o r i d e c o n t e n t O n e g r a m s a m p l e s o f who le e g g b l e n d w e r e m i x e d w i t h 30 m L o f 0.1 N s i l v e r n i t r a t e ( E n g e l h a r d I n d . , A u r o r a , O N ) f o r t h e t i t r a t i o n m e t h o d 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 ( A O A C ) # 1 8 . 0 3 4 - 1 8 . 0 3 5 . T h e c h l o r i d e c o n t e n t was c a l c u l a t e d as a p e r c e n t a g e (wet b a s i s ) , a n d r e s u l t s w e r e r e p o r t e d as an a v e r a g e o f d u p l i c a t e a n a l y s e s . 5. D e t e r m i n a t i o n o f w a t e r a c t i v i t y S a m p l e s o f e g g y o l k (10 m L ) w e r e p l a c e d o n a R o t r o n i c H y g r o s k o p D T D M S 1 0 0 w a t e r a c t i v i t y m e a s u r i n g s t a t i o n ( R o t r o n i c A g , Z u r i c h , S w i t z e r l a n d ) , a n d a l l owed to r e m a i n at room t e m p e r a t u r e f o r 45 m i n , at w h i c h t ime w a t e r a c t i v i t y r e a d i n g s r e m a i n e d e s s e n t i a l l y c o n s t a n t . R e a d i n g s w e r e o b t a i n e d o n 3-10 m L p o r t i o n s o f y o l k a n d t h e a v e r a g e v a l u e was c a l c u l a t e d . T h e r e f e r e n c e s a m p l e was a s a t u r a t e d b a r i u m c h l o r i d e s o l u t i o n . C . 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 F r e s h e g g s a n d s e v e r a l t h a t h a d b e e n s t o r e d at -10°C f o r 7 d , t h e n t h a w e d at room t e m p e r a t u r e w e r e f i x e d w i t h 4.0% ( v / v ) g l u t a r a l d e h y d e ( J . E . EM S e r v i c e s I n c . , P o i n t C l a i r e , D o r v a l l , P Q ) in 0 .066 M p h o s p h a t e b u f f e r p H 7.0 (75 ml_ 0 .066 M s o d i u m p h o s p h a t e + 4 8 . 5 m L 0 .066 M p o t a s s i u m p h o s p h a t e ) " o v e r n i g h t . T h e s a m p l e s w e r e r i n s e d 3 t imes (5 min p e r c h a n g e ) in 0 .066 M p h o s p h a t e b u f f e r ( p H 7 .0) f o r 1 h . T h e e g g s w e r e t h e n a g a i n r i n s e d 3 t imes w i t h a 0 .066 M p h o s p h a t e b u f f e r ( p H 7 . 0 ) , a n d d e h y d r a t e d t h r o u g h a n a s c e n d i n g e t h a n o l s e r i e s : 5 min e a c h o f 50, 70 a n d 80% ( v / v ) e t h a n o l , f o l l o w e d b y 2 c h a n g e s o f 10 min e a c h , w i t h 100% e t h a n o l . S u b s e q u e n t i n f i l t r a t i o n w i t h amyl a c e t a t e ( F i s h e r C h e m i c a l s L t d . , p u r i f i e d g r a d e ) was a c h i e v e d w i t h 10 min c h a n g e s o f 25 , 50 a n d 75% ( v / v ) amy l a c e t a t e d i l u t e d w i t h a b s o l u t e e t h a n o l , f o l l owed b y a 1 h s o a k in 100% amy l a c e t a t e . S a m p l e s w e r e t h e n c r i t i c a l l y p o i n t d r i e d in a P a r r 5770 C r i t i c a l Po in t D r y i n g B o m b ( P a r r I n s t r u m e n t C o . , M o l i n e , IL) u s i n g c a r b o n d i o x i d e as t h e t r a n s i t i o n a l f l u i d ( c r i t i c a l t e m p e r a t u r e a n d p r e s s u r e : 30°C, 7468 k P a ) . A f t e r m o u n t i n g o n a l u m i n u m s t u b s w i t h s i l v e r p a s t e a n d g o l d c o a t i n g b y v a c u u m e v a p o r a t i o n , t h e s a m p l e s w e r e o b s e r v e d w i t h a C a m b r i d g e S t e r e o s c a n 250 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 . Images w e r e r e c o r d e d w i t h a P o l a r o i d 545 l a n d f i lm c a m e r a o n p o s i t i v e / n e g a t i v e f i lm ( T y p e 5 5 ) . A n o p e r a t i n g v o l t a g e o f 20 k v was e m p l o y e d . D . E l e c t r o p h o r e s i s P o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s was p e r f o r m e d o n s a l m o n e g g y o l k in an u p r i g h t s l a b g e l e l e c t r o p h o r e s i s s y s t e m ( # S - 1 0 6 0 S C , A T T O C o r p o r a t i o n , T o k y o ) . U s i n g t h e s t o c k s o l u t i o n s f o r t h e z o n e e l e c t r o p h o r e s i s t e c h n i q u e as d e s c r i b e d b y D a v i s ( 1 9 6 4 ) , a 7.5% ( w / v ) a c r y l a m i d e ( S i g m a C h e m i c a l C o r p . , S t . L o u i s , MO) s e p a r a t i o n g e l was p o l y m e r i z e d as a 10 cm x 13 .5 cm x 0.2 cm s l a b , l e a v i n g a s p a c e o f 3 cm at t h e t o p o f t h e ge l m o l d . A c o n c e n t r a t i o n g e l o f 3.125% a c r y l a m i d e was c a r e f u l l y p i p e t t e d a r o u n d t h e c o m b . F o l l o w i n g p o l y -m e r i z a t i o n , t h e s p a c e r c o m b was c a r e f u l l y r e m o v e d a n d a n y u n p o l y m e r i z e d a c r y l a m i d e was r e m o v e d w i t h a s y r i n g e . M a r k e r d y e was p r e p a r e d b y d i s -p e r s i n g 10 mg b r o m o p h e n o l b l u e ( M a t h e s o n , C o l e m a n a n d B e l l , N o r w e e d , O H ) in a s o l u t i o n o f 0.2 m L 0.5 M T r i s ( F i s h e r c e r t i f i e d A C S A k a l i m e t r i c S t a n d a r d ) in h y d r o c h l o r i c a c i d b u f f e r ( p H 6.8) a n d 2 m L g l y c e r o l w i t h w a t e r to 10 m L . T h i s was a p p l i e d to e a c h s lo t w i t h a s y r i n g e to a d e p t h o f 1 mm, fo l l owed b y t h e s a m p l e s o l u t i o n s in t h e o r d e r a n d a m o u n t s i n d i c a t e d in T a b l e 2. A l l e l e c t r o p h o r e s i s e x p e r i m e n t s w e r e p e r f o r m e d o n t h e e g g y o l k f r a c t i o n . F o r e a c h s a m p l e 0.1 m L was m i x e d w i t h 1.25 mL g l y c e r o l a n d 5 mL o f t h e T r i s ( 6 . 0 m L ) : g l y c i n e ( 2 8 . 8 g ) : w a t e r (to 100 m L ) r u n n i n g b u f f e r f r o m D a v i s ( 1 9 6 4 ) . S i n c e some y o l k p r o t e i n s w e r e i n s o l u b l e in t h e T r i s b u f f e r , t h e d e t e r g e n t s s o d i u m d o d e c y l s u l f a t e ( S D S ) a n d d e o x y c h o l a t e ( D O C ) ( B D H C h e m i c a l s , L t d . , P o o l e , E n g l a n d ) as wel l as s o d i u m c h l o r i d e w e r e e v a l u a t e d f o r t h e i r e f f e c t i v e n e s s to c o m p l e t e l y s o l u b i l i z e t h e s a m p l e . S u c c e s s i v e r u n s w e r e p e r f o r m e d w i t h t h e r u n n i n g b u f f e r c o n t a i n i n g e i t h e r 0.5% S D S , 6% D O C o r 1.0% s o d i u m c h l o r i d e . T h e s e c o n c e n t r a t i o n s w e r e p r e v i o u s l y f o u n d to be t h e lowest c o n c e n t r a t i o n f o r c o m p l e t e s o l u b i l i z a t i o n o f t h e y o l k . D O C was f o u n d to p r o d u c e t h e bes t w e l l -d e f i n e d s e p a r a t i o n ; t h u s al l s u b s e q u e n t e l e c t r o p h o r e t i c r u n s w e r e p e r f o r m e d u t i l i z i n g t h i s d e t e r g e n t in t h e s a m p l e a n d r u n n i n g b u f f e r s . E a c h s lo t c o n t a i n i n g m a r k e r d y e a n d s a m p l e s o l u t i o n was o v e r l a y e r e d w i t h r u n n i n g b u f f e r a n d t h e e n t i r e s l a b c a r e f u l l y p l a c e d in to t h e lower r e s e r v o i r t a n k o f t h e e l e c t r o p h o r e t i c a p p a r a t u s . B o t h u p p e r a n d l ower r e s e r v o i r t a n k s h a d p r e v i o u s l y b e e n f i l l e d w i t h t h e r u n n i n g b u f f e r . E l e c t r o p h o r e s i s was p e r -f o r m e d at 20-25°C w i t h a n in i t i a l a p p l i c a t i o n o f 120 mV u n t i l t h e m a r k e r d y e m i g r a t e d to t h e f r o n t o f t h e s e p a r a t i o n g e l . T h e r e a f t e r , t h e ge l was s u b j e c t e d to a 100 mV p o t e n t i a l u n t i l t h e m a r k e r d y e was a p p r o x i m a t e l y 0 .5 cm f rom t h e bo t tom o f t h e g e l . F i x i n g a n d s t a i n i n g w e r e a c c o m p l i s h e d a f t e r a 1 h i m m e r s i o n o f t h e ge l in T a b l e 2. I d e n t i t y a n d q u a n t i t y o f y o l k sampIe~;solutions a p p l i e d to e l e c t r o p h o r e s i s g e l s . C o d e C o n d i t i o n n o . o f y o l k Q u a n t i t y o f s a m p l e s o l u t i o n a p p l i e d ( l iL) G e l S lo t n o . n o . II f r o z e n / t h a w e d 5 1 1 10 2 5 3 10 4 f r e s h ( S ) a 5 5 10 6 f r e s h 5 7 10 8 f r o z e n / t h a w e d ( S ) 5 9 10 10 f r o z e n / thawed 5 11 10 12 VI f r o z e n / t h a w e d 10 2 1 f r o z e n / thawed ( S ) 10 2 f r e s h 10 3 f r e s h ( S ) 10 4 f r o z e n / t h a w e d 10 5 5 6 f r o z e n / thawed (S) 10 7 5 8 f r e s h 10 9 5 10 f r e s h (S) 10 11 5 12 a Y o l k f rom c a v i a r , i e . e g g s p r e v i o u s l y s u b j e c t e d to a s a t u r a t e d s o d i u m c h l o r i d e c o l u t i o n u n t i l c h l o r i d e c o n t e n t r e a c h e d 4-5% ( m e a s u r e d b y c h l o r i d e ion e l e c t r o d e ) . a s t a i n i n g s o l u t i o n o f 0.025% ( w / v ) C o o m a s s i e B r i l l i a n t B l u e R250 ( C o l a b L a b o r a t o r i e s I n c . , C h i c a g o H e i g h t , IL) in 50% ( v / v ) : 10% ( v / v ) g l a c i a l a c e t i c a c i d . D e s t a i n i n g was a c c o m p l i s h e d b y a n o v e r n i g h t i m m e r s i o n in 25% ( v / v ) m e t h a n o l : 7% ( v / v ) g l a c i a l a c e t i c a c i d , a n d t h e g e l s w e r e s t o r e d in f r e s h d e s t a i n i n g s o l u t i o n . G e l s w e r e s c a n n e d in a d e n s i t o m e t e r ( K o n t e s F i b e r O p t i c S c a n n e r Mode l 800, V i n e l a n d , N J ) a n d d a t a r e c o r d e d b y a Mode l "42B V a r i c o r d v a r i a b l e r e s p o n s e r e c o r d e r ( P h o t o v o l t C o r p . , New Y o r k C i t y , N Y ) . P h o t o g r a p h s o f t h e g e l s w e r e r e c o r d e d o n K o d a c h r o m e A S A - 4 0 0 , u s i n g a n A s a h i - P e n t a x S p o t m a t i c F c a m e r a . . E . D i f f e r e n t i a l S c a n n i n g C a l o r i m e t r y ( D S C ) D i f f e r e n t i a l s c a n n i n g c a l o r i m e t r y was p e r f o r m e d w i t h a P e r k i n E l m e r D S C -2C ( N o r w a l k , C T ) . I n d i u m s a m p l e s w e r e u s e d to c a l i b r a t e f o r t e m p e r a t u r e a n d to o b t a i n t h e m a c h i n e c o n s t a n t u s e d in c a l c u l a t i o n o f heat o f e n t h a l p y . T h e in i t i a l t e m p e r a t u r e was a d j u s t e d to 340°K, fo l l owed b y a p r o g r a m m e d s c a n (at 5 d e g r e e s / m i n ) u p to 420°K. T h e r m a l s c a n s w e r e c o n t r o l l e d , r e c o r d e d a n d a n a l y z e d b y a P e r k i n E l m e r T h e r m a l A n a l y s i s D a t a S t a t i o n a n d p r i n t o u t o f r e s u l t s was o b t a i n e d o n a n i n t e r f a c e d P e r k i n E l m e r A S P l o t t e r . 2 S a m p l e s o f 70-80 mg o f e g g y o l k w e r e p r e s s u r e s e a l e d in s t a i n l e s s s t e e l p a n s . A s a m p l e o f l ead o f s u f f i c i e n t w e i g h t f o r t h e a p p r o p r i a t e heat c a p a c i t y was s e a l e d in a s t a i n l e s s s tee l p a n a n d u s e d f o r a r e f e r e n c e . A l l a n a l y s e s w e r e p e r f o r m e d in t r i p l i c a t e a n d v a l u e s e x p r e s s e d as an a v e r a g e . F . P h y s i c a l P r o p e r t i e s S i n c e t h e c h u m s a l m o n e g g s w e r e d i v i d e d in to two d i s c r e t e c o m p o n e n t s , t h e m e m b r a n e a n d t h e m a g m a , s e p a r a t e i n v e s t i g a t i o n s w e r e c a r r i e d o u t o n t h e s e 28. f r a c t i o n s . A v i s c o m e t r i c s t u d y o f t h e l i q u i d y o l k ( i e . magma w i t h t h e i m m i s c i b l e l i p i d p o r t i o n r e m o v e d ) was u s e d to c h a r a c t e r i z e i ts s t e a d y s h e a r f low b e h a v i o u r a n d a c o m p r e s s i o n t e s t o f t h e i n t a c t e g g b e t w e e n p a r a l l e l f lat p l a t e s was e m -p l o y e d to f i n d t h e c h a r a c t e r i s t i c s o f t h e e g g m e m b r a n e u p o n a p p l i c a t i o n o f f o r c e . 1. F low b e h a v i o u r s t u d i e s A B r a b e n d e r R h e o t r o n ( S e r i a l N o . 802102, C . W. B r a b e n d e r I n s t r u m e n t s I n c . , S o u t h H a c k e n s a c k , N J ) r o t a t i o n a l v i s c o m e t e r was e m p l o y e d i n t h i s s t u d y o f t h e f low b e h a v i o u r o f t h e e g g y o l k s a m p l e s . S p r i n g A was u s e d w i t h a c o u e t t e a t t a c h m e n t ( C - 5 ) at a c o n t r o l l e d t e m p e r a t u r e o f 25°C. T h e f i x t u r e was l o a d e d w i t h 10 m L o f t h e s a m p l e a n d p l a c e d in to t h e t h e r m o s t a t e d j a c k e t f o r t e m p e r a t u r e e q u i l i b r a t i o n . A t t h a t t ime ( a p p r o x i m a t e l y 5 m i n ) , a s h e a r r a t e o f 5 r p m ( 4 . 5 s 1 ) was a p p l i e d , f o l l o w e d b y 10 s i n t e r v a l s at s u c c e s s i v e l y h i g h e r s h e a r r a t e s , u p to 904 r p m (814 s 1 ) . T h i s r a t e was m a i n t a i n e d f o r 30 s , t h e n d e c r e a s e d s t e p w i s e b a c k to 5 r p m , a g a i n at 10 s i n t e r v a l s . A f t e r a n o t h e r 10 s at t h e lowest r o t a t i o n a l s p e e d , t h e same i n c r e a s e / d e c r e a s e c y c l e was c o n -d u c t e d . T h e t o r s i o n s i g n a l was r e c o r d e d f rom t h e m e t e r f o r e a c h g e a r s e t t i n g . T h r e e r e p l i c a t e s w e r e u s e d f o r e a c h s a m p l e t e s t e d a n d t h e c o r r e s p o n d i n g s c a l e r e a d i n g s f o r t h e a p p r o p r i a t e r o t a t i o n a l s p e e d w e r e a v e r a g e d f o r t h e u p c u r v e s a n d d o w n c u r v e s s e p a r a t e l y . F o r e a c h r o t a t i o n a l s p e e d , t h e s h e a r r a t e (y, s 1 ) . was c a l c u l a t e d b y t h e e q u a t i o n : Y = N x B (1 ) w h e r e N is t h e r o t a t i o n a l s p e e d in r p m a n d B is t h e s h e a r r a t e c o n s t a n t f o r t h e C - 5 f i x t u r e . ( T h e K r e i g e r - M a r o n c o r r e c t i o n f o r d e v i a t i o n f rom N e w t o n i a n f low was not a p p l i e d as t h e r a t i o o f t h e r a d i u s o f t h e i n n e r c y l i n d e r o v e r t h a t o f t h e o u t e r c y l i n d e r is v e r y c l o s e to 1 f o r t h e C - 5 c o u e t t e a t t a c h m e n t o f t h e v i s c o m e t e r ) . 29. S h e a r s t r e s s (a, mPa) was c a l c u l a t e d b y t h e e q u a t i o n : a = S R x A (2) w h e r e S R is t h e a v e r a g e s c a l e r e a d i n g a n d A is c a l c u l a t e d f rom t h e f o l l o w i n g e q u a t i o n : A = 596.1 - 1 . 943SR + 0 . 0 0 4 1 0 8 S R 2 - 0. 00000325SR 3 (3) T h e e q u a t i o n f o r t h e A f a c t o r was o b t a i n e d t h r o u g h m u l t i p l e r e g r e s s i o n a n a l y s e s o f c o r r e s p o n d i n g v a l u e s o f t h e s c a l e r e a d i n g s at v a r i o u s s h e a r r a t e s , u s i n g s t a n d a r d o i l s o f k n o w v i s c o s i t y ( N o . S - 6 0 , S - 6 0 0 a n d S - 2 0 0 0 , C a n n o n I n s t r u -ments C o . , S t a t e C o l l e g e , P A ) . A p p a r e n t v i s c o s i t y ( n , m P a - s ) was t h e n o b t a i n e d f r o m t h e e q u a t i o n : n = CT /1 (4) A M o n r o e p r o g r a m m a b l e c a l c u l a t o r (Mode l 1880) was u s e d w i t h l i n e a r r e g r e s s i o n to d e t e r m i n e t h e f it o f t h e P o w e r Law mode l to t h e d a t a , u s i n g t h e f o r m : logri = log m + ( n - 1 ) l o g y (5) w h e r e m is t h e c o n s i s t e n c y c o e f f i c i e n t ( m P a - s n ) a n d n is t h e f low b e h a v i o u r i n d e x . 2. M e m b r a n e - s t r e n g t h s t u d i e s O n e i n t a c t e g g was p l a c e d b e t w e e n p a r a l l e l f lat s m o o t h s u r f a c e f i x t u r e s m o u n t e d o n an I n s t r o n Mode l 1122 U n i v e r s a l T e s t i n g M a c h i n e ( I n s t r o n I n c . , C a n t o n , M A ) , e g u i p p e d w i t h a 50 k g load c e l l . M o v e m e n t o f t h e c r o s s h e a d was c o n t r o l l e d s u c h t h a t t h e e g g was c o m p r e s s e d at 10 mm/min u p to r u p t u r e u s i n g t h e 0 .20 k g f u l l s c a l e s e n s i t i v i t y f o r t h e load c e l l . T h e f o r c e d e f o r m a t i o n c u r v e d e s c r i b i n g t h e c o m p r e s s i o n b e h a v i o u r o f t h e e g g was o b t a i n e d o n t h e I n s t r o n s t r i p c h a r t r e c o r d e r ( c h a r t s p e e d set at 50:1 p r o p o r t i o n a l s p e e d w i t h r e s p e c t to t h e c r o s s h e a d s p e e d ) . T w e n t y - f i v e i n d i v i d u a l e g g s w e r e u s e d f o r e a c h t r e a t m e n t t e s t e d . 30. R u p t u r e e n e r g y was o b t a i n e d f rom a r e a s u n d e r 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 f rom t h e p o i n t o f f i r s t r e s i s t a n c e to m e m b r a n e d e f o r m a t i o n , as d e t e r m i n e d w i t h a p l a n i m e t e r ( N u m o n i c s C o r p . , N o r t h W a l e s , P A ) a n d c o n v e r t e d i n t o u n i t s o f e n e r g y ( j o u l e s ) . P e a k h e i g h t was m e a s u r e d a n d c o n v e r t e d in to u n i t s o f f o r c e ( N e w t o n s ) to o b t a i n ' t h e r u p t u r e f o r c e . T o t a l d e f o r m a t i o n ( c m ) , f i r m n e s s (N/m) a n d c o m p l i a n c e (m/Ni) w e r e c a l c u l a t e d f rom t h e a r e a a n d p e a k h e i g h t as p r e v i o u s l y o b t a i n e d . T o t a l d e f o r m a t i o n r e f l e c t s t h e c r o s s h e a d m o v e m e n t f rom p o i n t o f f i r s t c o n t a c t to r u p t u r e o f t h e e g g m e m b r a n e a n d was e s t i m a t e d b y t h e f o r m u l a f o r t r i a n g u l a r a r e a , i e . , b = 2 ( a r e a ) 7 h e i g h t . T h e v a l u e f o r b was e x p r e s s e d as d e f o r m a t i o n o f t h e e g g u p to t h e p o i n t o f r u p t u r e . F i r m n e s s is d e f i n e d as t h e r e s i s t a n c e to a p p l i e d f o r c e as r e f l e c t e d b y t h e s l o p e o f t h e c u r v e , a n d was e s t i m a t e d b y t h e v a l u e o f p e a k h e i g h t / d e f o r m a t i o n , e x p r e s s e d in u n i t s o f N/m. F i n a l l y , t h e r e c i p r o c a l o f f i r m n e s s was d e f i n e d as c o m p l i a n c e , a m e a s u r e o f y i e l d i n g to a p p l i e d f o r c e . G . E x p e r i m e n t a l P r o c e d u r e s 1. E f f e c t o f e g g s i z e o n m e m b r a n e s t r e n g t h A n i n v e s t i g a t i o n was c o n d u c t e d i n t o t h e r e l a t i o n s h i p b e t w e e n t h e s i z e o f e g g a n d m e m b r a n e s t r e n g t h to d e t e r m i n e w h e t h e r t h e v a r i a t i o n in m e m b r a n e s t r e n g t h m e a s u r e m e n t s c o u l d be r e l a t e d to t h e s i z e o f t h e e g g . If s o , t h i s c o u l d be a f a c t o r to c o n s i d e r in s u b s e q u e n t e x p e r i m e n t s . F i f t y f r e s h e g g s ( C o d e V I ) w e r e t e s t e d f o r m e m b r a n e s t r e n g t h , u t i l i z i n g t h e I n s t r o n m a c h i n e , a c c o r d i n g to t h e p r o c e d u r e p r e v i o u s l y o u t l i n e d . J u s t p r i o r to t h e t e s t , m e a s u r e m e n t s o f e g g h e i g h t a n d w i d t h (+ 0.1 mm) w e r e t a k e n w i t h t h e a id o f d i v i d e r s a n d a s c a l e w h i l e t h e e g g was r e s t i n g o n a f lat s u r -f a c e . T h e e g g s u r f a c e a r e a was t h e n c a l c u l a t e d w i t h t h e f o r m u l a f o r s u r f a c e a r e a ( S A ) o f an o b l a t e s p h e r o i d ( B e y e r , 1978) : S A = 2 i r a 2 + i r b 2 / e l o g e ( 1 + e / 1 - E ) (6) 31. w h e r e a is t h e major a x i s o f t h e s p h e r o i d , b is t h e m i n o r a x i s a n d e is e q u a l to / a 2 - b 2 / a . 2. F r o z e n s t o r a g e e f f e c t s . A s p r e v i o u s l y s t a t e d , t h e p h y s i c a l s t r u c t u r e o f c h u m sa lmon e g g s w e r e c o n s i d e r e d as two d i s c r e t e s y s t e m s : t h e i n n e r f l u i d magma a n d t h e s u r r o u n d i n g e l a s t i c m e m b r a n e . E v a l u a t i o n o f t h e e f f e c t s o f f r o z e n s t o r a g e o n p h y s i c a l p r o p e r t i e s w e r e e v a l u a t e d in t e r m s o f y o l k f low b e h a v i o u r a n d m e m b r a n e r u p -t u r e e n e r g y b y s e p a r a t e a n a l y s e s o n f r e s h e g g s a n d t h o s e t h a w e d a f t e r s t o r a g e at -10°C f o r 7 d . T h e p r o c e d u r e fo l l owed is o u t l i n e d in F i g u r e 1, in w h i c h a s a m p l e o f f o u r o v a r i e s w e r e s c r e e n e d w i t h i n 2 h o f e v i s c e r a t i o n to p r o d u c e s e p a r a t e e g g s , h a l f o f w h i c h w e r e c r u s h e d to o b t a i n e g g m a g m a , w h i c h was t h e n c e n t r i f u g e d to i so la te t h e e g g y o l k . T h i s was c o l l e c t e d f r o m t h e c e n t r i -f u g e t u b e s , p o o l e d a n d a g a i n d i v i d e d . In e a c h o f s i x 50 m L p o l y p r o p y l e n e s c r e w - t o p d i s p o s a b l e c e n t r i f u g e t u b e s , 15 m L a m o u n t s w e r e p l a c e d , a n d t h r e e o f t h e s e w e r e s t o r e d at -10°C f o r 7 d . T h e o t h e r t h r e e t u b e s w e r e k e p t u n f r o z e n a n d u s e d d i r e c t l y f o r v i s c o m e t r y . T w o lots o f a p p r o x i m a t e l y 50 e g g s w e r e p i c k e d f rom t h e r e m a i n i n g who le e g g s a n d p l a c e d in s c r e w - t o p p o l y p r o p y l e n e c e n t r i f u g e t u b e s . O n e o f t h e t u b e s was s t o r e d f o r 7 d at - 1 0 ° C , w h i l e 25 e g g s f rom t h e o t h e r t u b e w e r e i m m e d i a t e l y a n a l y z e d b y t h e I n s t r o n c o m p r e s s i o n t e s t . A f t e r 7 d , t h e f r o z e n s a m p l e s w e r e p l a c e d at room t e m p e r a t u r e to t h a w , t h e n t r e a t e d in t h e same m a n n e r as t h e f r e s h s a m p l e s . T h i s e x p e r i m e n t was p e r f o r m e d w i t h C o d e II s a m p l e s , a n d l a t e r w i t h C o d e VI in o r d e r to t e s t w h e t h e r v a r i a t i o n may be o a t t r i b u t e d to s a m p l e s f rom d i f f e r e n t s e a s o n s o f t h e y e a r . T h e r m a l a n a l y s i s was c o n d u c t e d o n y o l k f rom C o d e s II a n d VI in t h e m a n n e r p r e v i o u s l y d e s c r i b e d , h o w e v e r , t h e P e r k i n - E l m e r D a t a S t a t i o n was not a v a i l a b l e f o r a n a l y s i s o f C o d e II y o l k s a m p l e s . ' . I n s t e a d , . d a t a f r o m t h e c a l o r i m e t e r - w e r e r e c o r d e d o n a P e r k i n - E l m e r S i n g l e C h a n n e l , M u l t i r a n g e T h e r m a l A n a l y s i s i g u r e 1. F low d i a g r a m o f p r o c e d u r e f o r i n v e s t i g a t i o n in to f r o z e n s t o r a g e e f f e c t s . o v a r i e s " s c r e e n e d " loose e g g s d i v i d e d in to 2 lots _L " s i e v e d " magma c e n t r i f u g e d 90 m i n / 2 3 , 500 x g y o l k d i v i d e d i n t o 2 lots d i v i d e d in to 2 lots s t o r e d -10°C/7 d a y s t h a w e d 25°C F R O Z E N / T H A W E D F R E S H F R O Z E N / T H A W E D F R E S H Y O L K Y O L K W H O L E E G G S W H O L E E G G S s t o r e d -10°C/7 d a y s t h a w e d 25°C R h e o l o g i c a l a n a l y s i s F o r c e / d e f o r m a t i o n a n a l y s i s 33. r e c o r d e r w i t h t e m p e r a t u r e m a r k e r (#0056-1003) . T h e a r e a s u n d e r c u r v e s w e r e o b t a i n e d w i t h an e l e c t r o n i c p l a n i m e t e r a n d t h e f o l l o w i n g e q u a t i o n u s e d f o r c a l c u l a t i o n o f t r a n s i t i o n e n e r g y : A Heat = (K x R x A ) / ( W x S) (7) w h e r e AHeat is t h e t r a n s i t i o n e n e r g y ( c a l / g ) , K is t h e c a l i b r a t e d i n s t r u m e n t c o n s t a n t ( 1 . 0 ) , R is t h e r a n g e s e n s i t i v i t y ( m c a l - i n / s ) , A is t h e p e a k a r e a ( i n 2 ) , W is t h e s a m p l e w e i g h t (mg) a n d S is t h e r e c o r d e r c h a r t s p e e d ( i n / s ) . E l e c t r o p h o r e t i c a n a l y s e s w e r e c a r r i e d o u t o n y o l k f rom C o d e s II a n d V I , f o l l o w i n g t h e p r o c e d u r e f o r m e r l y o u t l i n e d . In a d d i t i o n , to d e t e r m i n e w h e t h e r s a l t i n g o f t h e e g g s (an i n t e g r a l p a r t o f c a v i a r p r o d u c t i o n ; Z a i t s e v et a l . , 1969) p r o d u c e d a n e f f e c t o n y o l k p r o t e i n s , c a v i a r was p r o c e s s e d f rom f r e s h e g g s ( C o d e s II a n d V I ) b y i m m e r s i o n in a s a t u r a t e d sa l t s o l u t i o n u n t i l t h e c h l o r i d e c o n t e n t r e a c h e d 4-5%, as d e t e r m i n e d b y a c h l o r i d e ion e l e c t r o d e (Mode l 96 -17-00, O r i o n R e s e a r c h , I n c . , C a m b r i d g e , M A ) . Y o l k was t h e n o b t a i n e d f rom t h e s e s a l t e d e g g s a n d s u b j e c t e d to e l e c t r o p h o r e s i s , b o t h as f r e s h m a t e r i a l a n d t h a t t h a w e d a f t e r 7 d s t o r a g e at -10°C/ 3. C r y o p r o t e c t a n t a g e n t e f f e c t s In o r d e r to e v a l u a t e t h e e f f e c t i v e n e s s o f v a r i o u s c r y o p r o t e c t a n t s as a means o f m a i n t a i n i n g t h e m e m b r a n e p r o p e r t i e s o f f r e s h e g g s t h r o u g h f r o z e n s t o r a g e , s e l e c t e d c r y o p r o t e c t a n t s w e r e a p p l i e d at d i f f e r e n t l e v e l s to f r e s h e g g s a n d t h e m e m b r a n e e n e r g y d e t e r m i n e d b e f o r e a n d a f t e r f r o z e n s t o r a g e . T h e p r o c e d u r e i n v o l v e d o b t a i n i n g 4 o v a r i e s f r o m 4 female c h u m s a l m o n a n d s c r e e n i n g t h e s e w i t h i n 2 h o f e v i s c e r a t i o n . T h i r t y m L o f t h e r e s u l t a n t s e p a r a t e e g g s w e r e i m m e r s e d in 50 m L o f t h e c r y o p r o t e c t a n t s o l u t i o n , f o r a p e r i o d o f 30 m i n , w i t h g e n t l e a g i t a t i o n a f t e r 15 m i n . T h e s a m p l e s w e r e d r a i n e d f o r 1 m i n , t h e n d i v i d e d in to two p o r t i o n s . O n e h a l f was p l a c e d in a 50 m L d i s p o s a b l e s c r e w - t o p c e n t -r i f u g e t u b e , p l a c e d at -10°C a n d s t o r e d f o r 7 d . F r o m t h e r e m a i n i n g h a l f , 25 34: e g g s w e r e i m m e d i a t e l y a n a l y z e d w i t h t h e I n s t r o n c o m p r e s s i o n t e s t as p r e v i o u s l y d e s c r i b e d . A f t e r 7 d , t h e f r o z e n s a m p l e s w e r e t h a w e d in a i r at room t e m p e r a -t u r e , t h e n a n a l y z e d as f o r t h e f r e s h . T h e c r y o p r o t e c t a n t s o l u t i o n s u s e d w e r e : 0 . 2 , 1 .0 , 2 .0 a n d 3.0 M c o n c e n t r a t i o n s o f s o d i u m c h l o r i d e , 1.0 a n d 3.0 M c o n c e n t r a t i o n s o f s o d i u m p h o s p h a t e , g l y c e r o l , e t h y l e n e g l y c o l a n d c a l c i u m c h l o r i d e , a n d 0 .5 a n d 1.0 M c o n c e n t r a t i o n s o f s u c r o s e . In a d d i t i o n , s o d i u m p h o s p h a t e a n d s o d i u m c h l o r i d e w e r e c o m b i n e d in c o n c e n t r a t i o n s o f 0 . 3 M : 0.2 M, r e s p e c t i v e l y , a n d 0. 9 M : 0 .5 M, r e s p e c t i v e l y . 4. E f f e c t i v e n e s s o f s o d i u m c h l o r i d e as a c r y o p r o t e c t a n t T h e e f f i c a c y o f a c r y o p r o t e c t a n t f o u n d to be e f f e c t i v e in t h e p r e v i o u s s e c t i o n is u l t i m a t e l y d e t e r m i n e d b y t h e q u a l i t y o f t h e c a v i a r p r o d u c e d f rom e g g s r e s u l t i n g f rom s u c h t r e a t m e n t . In o r d e r f o r t h e c r y o p r o t e c t i v e t r e a t m e n t to be j u d g e d s a t i s f a c t o r y , t h e r e s u l t i n g c a v i a r m u s t be s i m i l a r in c h a r a c t e r i s t i c s s u c h as t a s t e , a p p e a r a n c e a n d t e x t u r e to t h a t p r o d u c e d f rom f r e s h e g g s . M o r e o v e r , t h e p r o d u c t m u s t b e s u p e r i o r to c a v i a r p r o d u c e d f r o m f r o z e n e g g s . 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 w e r e a g a i n c a r r i e d ou t in two p a r t s : t h e a n a l y s i s o f y o l k f low b e h a v i o u r a n d t h a t o f m e m b r a n e s t r e n g t h . E v a l u a t i o n was p e r f o r m e d b o t h o b j e c t i v e l y b y I n s t r o n a n a l y s e s a n d t h e R h e o t r o n , a n d s u b j e c t i v e l y , u t i l i z i n g s e n s o r y p a n e l a n a l y s i s . C a v i a r f rom f r e s h , f r o z e n / t h a w e d a n d c r y o p r o t e c t e d e g g s was a n a l y z e d a n d t h e r e s u l t s c o m p a r e d . a) O b j e c t i v e a n a l y s i s - s a m p l e p r e p a r a t i o n T h e p r o c e d u r e fo l l owed is o u t l i n e d in F i g u r e 2 a n d i n v o l v e d s c r e e n i n g 12 o v a r i e s w i t h i n 2 h o f e v i s c e r a t i o n , a n d d i v i d i n g t h e e g g s in to t h r e e p o r t i o n s . O n e p o r t i o n was l a b e l l e d " F r o z e n / t h a w e d " , p l a c e d i n t o a p o l y p r o p y l e n e f r e e z e r b a g , a n d s e a l e d so t h a t t h e mass r e t a i n e d a f lat s h a p e o f w i d t h a p p r o x i m a t e l y 2 c m . A n o t h e r p o r t i o n w h i c h was l a b e l l e d " C r y o p r o t e c t e d " , was i m m e r s e d in a 10°C s o l u t i o n o f 15% ( w / v ) s o d i u m c h l o r i d e ( r a t i o o f e g g s to sa l t s o l u t i o n , 3 to 5 ) F i g u r e 2. F low d i a g r a m o f p r o c e d u r e f o r i n v e s t i g a t i o n in to e f f e c t i v e n e s s o f s o d i u m c h l o r i d e as a c r y o p r o t e c t a n t . o v a r i e s " s c r e e n e d " loose e g g s d i v i d e d in to 3 lots i m m e r s i o n in to 15% NaCl/10°C f o r 30 min -20°C/10 d a y s F R E S H C R Y O P R O T E C T E D F R O Z E N I m m e r s i o n i n t o s a t u r a t e d NaCl/10°C f o r 0, 3, 4, 5, 6, 10, 15 min d r a i n e d overnight/10°C s t o r e d at 4°C in g l a s s j a r s 36. f o r 30 m i n , a l l o w e d to d r a i n in a m e s h b a s k e t f o r 4 min i n a l a y e r 2 e g g s d e e p , t h e n p a c k e d in a p o l y p r o p y l e n e f r e e z e r b a g a n d s e a l e d in t h e same m a n n e r as t h e " F r o z e n / t h a w e d " s a m p l e . It is i m p o r t a n t h e r e to s t r e s s t h a t t h e t e r m " C r y o p r o t e c t e d " r e f e r s to e g g s t r e a t e d w i t h a p o t e n t i a l c r y o p r o t e c t a n t , t h e n f r o z e n a n d t h a w e d in t h e same m a n n e r as t h e " F r o z e n / t h a w e d " e g g s . B o t h o f t h e s e w e r e t h e n p l a c e d i n a s t i l l a i r f r e e z e r at - 2 0 ° C . T h e f i na l p o r t i o n was l a b e l l e d " F r e s h " a n d p r o c e s s e d i m m e d i a t e l y in to c a v i a r . S i n c e t h e i m m e r s i o n t ime in s a t u r a t e d s o d i u m c h l o r i d e n e e d e d to p r o d u c e e g g s w i t h t h e a p p r o p r i a t e c h l o r i d e c o n t e n t f o r c a v i a r ( i e . , 4-5%, Z a i t s e v et a l . , 1969) , v a r i e s s u b s t a n -t i a l l y f rom s a m p l e to s a m p l e ( H o r i , 1982) , a r a n g e o f s a l t i n g t imes w e r e e m p l o y e d : 0 min ( u n s a l t e d e g g s ) , 3, 4, 5, 6, 10 a n d 15 m i n . A p p r o x i m a t e l y o n e s e v e n t h o f t h e f r e s h e g g s was r e s e r v e d a n d t h e r e m a i n d e r was i m m e r s e d in a s a t u r a t e d sa l t s o l u t i o n at 10°C. A t i n t e r v a l s o f 3, 4, 5, 6, 10 a n d 15 m i n , 250 m L o f e g g s w e r e r e m o v e d a n d set to d r a i n in a l a y e r 2 cm d e e p in a n e t - l i n e d b a s k e t . T h e u n s a l t e d p o r t i o n was s t o r e d in a g l a s s j a r at 5°C d u r i n g t h e d r a i n i n g p e r i o d (18 h ) . T h e r e a f t e r , t h e s a l t e d e g g s w e r e a lso p l a c e d in g l a s s j a r s a n d s t o r e d at 5°C u n t i l a n a l y s i s . A n a l y s i s o f t h e s a m p l e s so o b t a i n e d i n c l u d e d c h l o r i d e c o n t e n t , f low b e -h a v i o u r a n d m e m b r a n e s t r e n g t h , as d e s c r i b e d ( w i t h t h e e x c e p t i o n t h a t f o r v i s c o s i t y a n a l y s i s , o n l y e v e r y s e c o n d s h e a r r a t e a v a i l a b l e o n t h e B r a b e n d e r R h e o t r o n was u s e d a n d t h e s e w e r e a p p l i e d o n l y o n c e , in i n c r e a s i n g p r o g r e s s i o n ) . B o t h t h e v i s c o s i t y a n a l y s i s a n d m e m b r a n e s t r e n g t h a n a l y s i s w e r e c o m p l e t e d w i t h i n 48 h p o s t - s a l t i n g . A f t e r a s t o r a g e p e r i o d o f 10 d , t h e f r o z e n / t h a w e d a n d c r y o p r o t e c t e d p o r -t i o n s w e r e t h a w e d o v e r n i g h t at 5°C , t h e n t r e a t e d a n d a n a l y z e d in t h e same m a n n e r as t h e f r e s h p o r t i o n . T h e p r o c e d u r e was r e p e a t e d 3 t imes u s i n g s a m p l e C o d e s I X , X a n d X I , w i t h t h e e x c e p t i o n t h a t a s a l t i n g t ime o f 15 min was not i n c l u d e d f o r t h e f i r s t 37. r e p l i c a t e ( i e . C o d e I X ) , d u e to u n c e r t a i n t y o f t h e e x t e n t o f s a l t i n g t ime n e e d e d to p r o d u c e d e t e c t a b l e d i f f e r e n c e s in e g g c h a r a c t e r i s t i c s . b ) S u b j e c t i v e a n a l y s i s - s a m p l e p r e p a r a t i o n A s a m p l e o f 4 o v a r i e s w e r e s c r e e n e d w i t h i n 2 h e v i s c e r a t i o n a n d t h e e g g s d i v i d e d in to 3 p o r t i o n s . F r o z e n / t h a w e d a n d c r y o p r o t e c t e d p o r t i o n s w e r e p r o d u c e d i n t h e s a m e m a n n e r as o u t l i n e d i n t h e p r e v i o u s s e c t i o n o n s a m p l e p r e p a r a t i o n f o r o b j e c t i v e a n a l y s e s , i n c l u d i n g f r o z e n s t o r a g e at -20°C in a s t i l l a i r f r e e z e r . T h e f i n a l t h i r d , l a b e l l e d " F r e s h " , was i m m e r s e d in a s a t u r a t e d s o d i u m c h l o r i d e s o l u t i o n at 10°C f o r 8 m i n , w h i c h p r o d u c e d e g g s p o s s e s s i n g a c h l o r i d e c o n t e n t o f a b o u t 4%, as i n d i c a t e d b y t h e c h l o r i d e c o n t a c t e l e c t r o d e . T h i s p o r t i o n was t h e n p a c k e d i n a g l a s s ja r a n d h e l d at 5°C >. A t t h e e n d o f 7 w e e k s , t h e f r o z e n / t h a w e d a n d c r y o p r o t e c t e d p o r t i o n s w e r e e a c h t h a w e d o v e r n i g h t at 5°C , t h e n d i v i d e d in to t h r e e . T h e 3 p o r t i o n s o f t h e f r o z e n / t h a w e d s a m p l e : w e r e s a l t e d at 10°C f o r 4 . 0 , 4 .5 a n d 5.0 m i n , a n d t h e 3 por t ions 1 , o f t h e c r y o p r o t e c t e d s a m p l e w e r e s a l t e d f o r 0 . 0 , 1 .0 , a n d 2 .0 m i n . S a l t i n g t imes w e r e d e t e r m i n e d b y s u b j e c t i v e d e t e r m i n a t i o n o f s a l i n i t y t h r o u g h s a m p l e t a s t i n g . A l l b r i n e d e g g s w e r e a l l owed to d r a i n f o r 18 h in a l a y e r 2 e g g s d e e p in a n e t - l i n e d b a s k e t . O n t h e f o l l o w i n g d a y , t h e y w e r e p a c k e d in to g l a s s j a r s a n d h e l d at 5°C . A f t e r a s t o r a g e p e r i o d o f 6 w e e k s , mold was d i s c o v e r e d in t h e j a r h o l d i n g t h e f r e s h s a m p l e a n d it was d i s c a r d e d f o r s e n s o r y p a n e l a n a l y s i s . R e p l a c e m e n t o f t h i s r e f e r e n c e was f u r n i s h e d b y c a v i a r o b t a i n e d f rom t h e F i s h e r i e s T e c h n o -l o g y D i v i s i o n at B . C . R e s e a r c h t h a t h a d b e e n d e t e r m i n e d as b e i n g o f h i g h q u a l i t y ( H o r i , 1 9 8 2 ) . c ) S u b j e c t i v e a n a l y s i s - s e n s o r y e v a l u a t i o n A p a i r e d c o m p a r i s o n tes t ( L a r m o n d , 1977) was c h o s e n f o r s e n s o r y e v a l u a t i o n o f m e m b r a n e s t r e n g t h a n d v i s c o s i t y . A n i n e p o i n t s c a l e was u s e d r a n g i n g f rom 38. " e x t r e m e l y t h i c k e r (o r t o u g h e r ) " to " e x t r e m e l y t h i n n e r (or w e a k e r ) " , w i t h a s c o r i n g o f 5 i n d i c a t i n g a s a m p l e no d i f f e r e n t t h a n t h e r e f e r e n c e . T h e 4 .5 min s a l t i n g t ime o f t h e f r o z e n / t h a w e d s a m p l e s a n d t h e 1.0 min s a l t i n g t ime o f t h e c r y o p r o t e c t e d s a m p l e s w e r e c h o s e n f o r p r e s e n t a t i o n to t h e p a n e l o n t h e b a s i s o f p o s s e s s i n g o p t i m u m t a s t e in t e r m s o f s a l i n i t y a n d low r a n c i d i t y . T h r e e s a m p l e s e a c h o f t h e 4 .5 min s a l t i n g t ime o f f r o z e n / t h a w e d e g g s a n d o f t h e 1.0 min s a l t i n g t ime o f t h e c r y o p r o t e c t e d e g g s , a n d o n e s a m p l e o f t h e h i g h q u a l i t y f r e s h c a v i a r made u p t h e s e v e n r a n d o m l y p r e s e n t e d s a m p l e s ( l a b e l l e d w i t h 3 d i g i t n u m b e r s ) t h a t w e r e p r e s e n t e d to e a c h o f t h e p a n e l m e m b e r s f o r c o m p a r i -s o n w i t h t h e r e f e r e n c e ( f r e s h h i g h q u a l i t y c a v i a r ) . E i g h t p a n e l m e m b e r s w e r e c h o s e n , who h a d b e e n p r e s e n t at c a v i a r t a s t e p a n e l s at least o n c e b e f o r e a n d so w e r e f a m i l i a r w i t h t h e p r o d u c t . In a d d i t i o n , a n i n e p o i n t h e d o n i c s c a l e , r a n g i n g f rom " l i k e e x t r e m e l y " to " d i s l i k e e x t r e m e l y " (L iarmond, 1977) was i n c l u d e d as a m e a s u r e o f t h e o v e r a l l a c c e p t a b i l i t y o f t h e s a m p l e s . H . S t a t i s t i c a l A n a l y s e s I . A n a l y s i s o f v a r i a n c e U s i n g t h e a n a l y s i s o f v a r i a n c e ( A N O V A ) p r o g r a m p a c k a g e ( * M F A V ) ( L e , 1980) , a v a i l a b l e t h r o u g h t h e U B C c o m p u t e r ( A m d a h l 470 V/8) s y s t e m , d a t a f r o m s e v e r a l e x p e r i m e n t s w e r e a n a l y z e d . A l eve l o f p < 0 . 0 5 was e m p l o y e d in al l a n a l y s e s to d e t e r m i n e s i g n i f i c a n c e , a n d i f s i g n i f i c a n c e was f o u n d , a N e w m a n -K e u l 1 s m u l t i p l e r a n g e t e s t , a v a i l a b l e w i t h t h e * M F A V p r o g r a m was a p p l i e d . a) E f f e c t o f c r y o p r o t e c t a n t T h e p u r p o s e o f t h e a n a l y s i s was to d e t e r m i n e w h i c h , i f a n y , c r y o p r o t e c t i v e t r e a t m e n t s r e s u l t e d in t h a w e d e g g s w i t h m e m b r a n e s t r e n g t h not s i g n i f i c a n t l y d i f f e r e n t f r o m t h a t o f t h e f r e s h . A o n e - w a y a n a l y s i s o f v a r i a n c e w i t h T r e a t m e n t 39. as t h e s i n g l e f a c t o r was p e r f o r m e d s e p a r a t e l y f o r e a c h c o d e d l o t . P r e a n d p o s t f r e e z i n g r e s u l t s o f a n y c r y o p r o t e c t a n t t r e a t m e n t w e r e c o n s i d e r e d as s e p a r a t e t r e a t m e n t s ( r e s u l t i n g in a n u m b e r o f l e v e l s in t h e a n a l y s i s t w i c e t h e n u m b e r o f a c t u a l c r y o p r o t e c t i v e t r e a t m e n t s ) . N e w m a n - K e u l 1 s m u l t i p l e r a n g e t e s t t h e n e n a b l e d g r o u p i n g o f means not s i g n i f i c a n t l y d i f f e r e n t f rom e a c h o t h e r in c o d e s w h e r e i n t h e f a c t o r o f T r e a t m e n t h a d b e e n e v a l u a t e d as a s i g n i f i c a n t s o u r c e o f v a r i a t i o n . b) E f f e c t o f p r e - f r e e z i n g t r e a t m e n t w i t h s o d i u m c h l o r i d e : i . M e m b r a n e s t r e n g t h d a t a A M o d e l III m u l t i f a c t o r i a l A N O V A was a p p l i e d to m e m b r a n e r u p t u r e e n e r g y d a t a w i t h C o d e (3 l e v e l s ) as a r a n d o m f a c t o r , a n d C o n d i t i o n o f e g g b e f o r e s a l t i n g (3 l e v e l s ) a n d M i n u t e s in s a t u r a t e d s o d i u m c h l o r i d e s o l u t i o n (5 l e v e l s ) as f i x e d f a c t o r s . ( O n l y 5 l e v e l s o f t h e M i n u t e s f a c t o r , r e p r e s e n t i n g t h e s a l t i n g t imes o f 3-10 m i n , w e r e u s e d f o r s t a t i s t i c a l a n a l y s i s , d u e to m i s s i n g v a l u e s f o r t h e 0 m i n s a l t i n g t ime o f C o d e XI f r o z e n / t h a w e d a n d c r y o p r o t e c t e d e g g s , a n d t h e 15 min s a l t i n g t ime o f C o d e IX e g g s . ) D u e to t h e r a n d o m n a t u r e o f o n e o r m o r e f a c t o r s i n h e r e n t in a Mode l III A N O V A , t h e m e t h o d o f c a l c u l a t i o n d i f f e r s f rom a n A N O V A w h e r e i n al l f a c t o r s a r e f i x e d ( i e . . Mode l I A N O V A ) ( Z a r , 1 9 7 4 ) . In t h e p r e s e n t s t u d y . C o d e r e p r e s e n t s a r a n d o m f a c t o r , as t h e t h r e e d a t e s o f s a m p l i n g w e r e a r b i t r a r i l y c h o s e n . A n u l l h y p o t h e s i s (H Q=y ^ =y 2=y 3 ) as a p p l i e d to t h i s f a c t o r w o u l d be m o r e a p t l y e x p r e s s e d as " H Q = t h e r e is no d i f f e r e n c e in m e m b r a n e s t r e n g t h b e t w e e n d a y s " . B e c a u s e o f t h i s d i f f e r e n c e , t h e r e s i d u a l e r r o r mean s q u a r e is not u s e d as t h e d e n o m i n a t o r o f t h e F s t a t i s t i c f o r t e s t i n g e v e r y s o u r c e o f v a r i a t i o n , r a t h e r , t e s t i n g t h e main e f f e c t s o f C o n d i t i o n a n d M i n u t e s is m o r e a p p r o p r i a t e l y d o n e b y u s i n g t h e r e s p e c t i v e i n t e r a c t i o n mean s g u a r e s o f C o n d i t i o n x C o d e a n d M i n u t e s x C o d e as d e n o m i n a t o r s o f F - v a l u e s . L i k e w i s e , 40. t h e i n t e r a c t i o n e f f e c t o f C o n d i t i o n x M i n u t e s is most a p t l y a s s e s s e d b y t e s t i n g t h e c o r r e s p o n d i n g mean s g u a r e a g a i n s t t h e C o n d i t i o n x M i n u t e s x C o d e mean s q u a r e ( T a b l e C 2 ; p 3 4 2 ; Z a r , 1 9 7 4 ) . i i . S e n s o r y e v a l u a t i o n T w o - w a y a n a l y s e s o f v a r i a n c e , w i t h S a m p l e s (7 l e v e l s r e p r e s e n t i n g t h e 7 s a m p l e s p r e s e n t e d in t h e s e n s o r y p a n e l ) a n d J u d g e s (8 l e v e l s r e p r e s e n t i n g t h e 8 j u d g e s ) as s o u r c e s o f v a r i a t i o n , w e r e p e r f o r m e d f o r d a t a f r o m t h e v i s c o s i t y a n d t h e m e m b r a n e s t r e n g t h a s s e s s m e n t s . T h e S a m p l e f a c t o r was f u r t h e r d i v i d e d i n t o t h r e e s o u r c e s o f v a r i a t i o n e x p r e s s e d as c o m p a r i s o n s b e t w e e n : a) t h e c o n t r o l s a m p l e a n d t h e r e m a i n d e r o f t h e s a m p l e s p r e s e n t e d , b) c a v i a r f rom t h e c r y o p r o t e c t e d a n d t h e f r o z e n / t h a w e d s a m p l e s , a n d c ) r e p l i c a t e s w i t h i n e a c h t r e a t m e n t ( i e . t r i p l i c a t e s e a c h o f c a v i a r f rom c r y o -p r o t e c t e d a n d f r o z e n / t h a w e d e g g s ) . A t w o - w a y A N O V A was u s e d to a n a l y z e d a t a f rom t h e a s s e s s m e n t o f p r e -f e r e n c e y i e l d e d b y t h e h e d o n i c s c a l e , w i t h S a m p l e s a n d J u d g e s as t h e ma in e f f e c t s . B e c a u s e t h e i n t e r e s t in t h i s a n a l y s i s lay o n l y w i t h t h e c o m p a r a t i v e p r e f e r e n c e s o f t h e j u d g e s f o r e a c h s e p a r a t e s a m p l e , no f u r t h e r d i v i s i o n o f t h e S a m p l e t e r m was c a r r i e d o u t . 2. A n a l y s i s o f r e g r e s s i o n l i n e s C o m p a r i s o n s b e t w e e n r h e o g r a m s w e r e a c c o m p l i s h e d b y u t i l i z i n g t h e p r e -v i o u s l y d e s c r i b e d P o w e r law f low model as a r e g r e s s i o n e q u a t i o n f o r d a t a p o i n t s i n c l u d e d w i t h i n t h e r a n g e 36 to 300 s 1 . A p r e - w r i t t e n p r o g r a m , t i t l e d " N L I N " , a v a i l a b l e o n t h e U B C F o o d S c i e n c e D e p a r t m e n t c o m p u t i n g ID ( T u n g , 1983) , was t h e n u s e d to d e t e r m i n e t h e p r e s e n c e o f a s i g n i f i c a n t d i f f e r e n c e b e t w e e n two r e g r e s s i o n l i n e s . T h i s c o v a r i a n c e p r o g r a m c o m p a r e s two s e t s o f d a t a c o m p r i s i n g t h e two r e g r e s s i o n l i n e s o n t h e b a s e s o f h o m o g e n e i t y o f r e s i d u a l v a r i a n c e s , s l o p e s a n d l e v e l s . T h e s i g n i f i c a n c e o f t h e s e c o m p a r i s o n s is b a s e d o n an F - v a l u e g e n e r a t e d in e a c h c a s e . In t h i s c o m p a r i s o n o f P o w e r law -41. r h e o g r a m s , a d i f f e r e n c e in s l o p e s w o u l d i n d i c a t e t h a t n v a l u e s d i f f e r a n d a d i f f e r e n c e in l e v e l s w o u l d i n d i c a t e t h a t m v a l u e s d i f f e r . R E S U L T S A N D D I S C U S S I O N A . P r o x i m a t e A n a l y s i s R e s u l t s o f a p r o x i m a t e a n a l y s i s o f C o d e s V , IX a n d XII a r e p r e s e n t e d in T a b l e 3. V a l u e s f o r p r o t e i n a n d m o i s t u r e a g r e e wel l w i t h f i g u r e s r e p o r t e d in t h e l i t e r a t u r e f o r s a l m o n s p e c i e s ( H a m o r a n d G a r s i d e , 1977; S m i r n o v et a l . , 1968) , as wel l as o t h e r s p e c i e s o f f i s h ( L i n k o et a l . , 1 980; K a i t a r a n t a , 1 9 8 1 ) . T h e l i p i d c o n t e n t , h o w e v e r , as y i e l d e d b y t h e m e t h o d e m p l o y e d in t h i s s t u d y , e x c e e d s t h a t p r e v i o u s l y r e p o r t e d b y S m i r n o v et a l . , (1968) o f 4.54% (wet b a s i s ) f o r c h u m s a l m o n , i n s t e a d l y i n g c l o s e r to t h o s e v a l u e s o b t a i n e d b y Ham o r a n d G a r s i d e (1 977) o f 13.00% ( w h o l e e g g , wet b a s i s ) a n d 16.00% ( y o l k , wet b a s i s ) f o r A t l a n t i c sa lmon (Salmo salar). V a r i a t i o n f rom S m i r n o v ' s v a l u e s may b e d u e to m e t h o d s u s e d to a n a l y z e l i p i d c o n t e n t . T h e a b i l i t y o f t h e S o x h l e t e x t r a c t i o n m e t h o d , as u s e d b y S m i r n o v et a l . , to e x t r a c t l i p o p r o t e i n s is k n o w n to be i n e f f i c i e n t ( L e e et a l . , 1966) . T h e m e t h o d u s e d b y Ham o r a n d G a r s i d e i n v o l v e d l i p i d e x t r a c t i o n a c c o r d i n g to t h e t e c h n i q u e o f B l i g h a n d D y e r (1 959) , a n d t h i s p r o c e d u r e may t h e n h a v e p r o d u c e d h i g h e r y i e l d s o f l i p i d m a t e r i a l . T h e m e t h o d u s e d in t h i s s t u d y was a m o d i f i c a t i o n o f t h e B l i g h - D y e r p r o c e d u r e ( L e e et a l . , 1966) a n d c o u l d t h e n be e x p e c t e d to g i v e s i m i l a r h i g h y i e l d s . In a d d i t i o n , it has b e e n s h o w n t h a t t h e l i p i d c o n t e n t o f t e l e o s t e g g s m a y i n c r e a s e w i t h a d v a n c i n g m a t u r i t y ( V u o r e l a et a l . , 1 9 7 9 ) , so t h a t e g g s o f C o d e s V , IX a n d XII c o l l e c t e d late in t h e s e a s o n , m a y a c t u a l l y h a v e p o s s e s s e d a h i g h e r l i p i d c o n t e n t . T h e l a r g e a m o u n t s o f p r o t e i n a n d l i p i d f o u n d in t h e sa lmon e g g r e f l e c t t h e n e e d o f a g r o w i n g e m b r y o f o r an a d e g u a t e s u p p l y o f e n e r g y to e n a b l e d e v e l o p m e n t ( H o l l e t t a n d H a y e s , 1946) . A v a l u e o f 0 .97 was d e t e r m i n e d f o r t h e w a t e r a c t i v i t y o f c h u m e g g y o l k o f C o d e I, a v a l u e w h i c h i n d i c a t e s a s u s c e p t i b i l i t y to b a c t e r i a , y e a s t s a n d T a b l e 3. P r o x i m a t e a n a l y s i s o f c h u m sa lmon e g g s ( n = 3 ) . F r a c t i o n C o d e h o . M o i s t u r e (%wb) C r u d e l i p i d (%wb) C r u d e o r o t e i n (%wb) Whole e g g V 56.9 14.3 27 . 8 ( 0 . 2 ) a ( 0 . 3 ) ( 0 . 6 ) IX 56. 4 12. 9 28 .2 ( 0 . 1 ) ( 1 . 8 ) ( 0 . 3 ) XII 57. 3 15 .0 26. 8 ( 0 . 2 ) (!3.8) ( 1 . 3 ) Magma V 5 6 . 6 14.1 2 4 . 4 ( 1 . 0 ) ( 0 . 2 ) ( 2 . 3 ) IX 56 .0 15. 9 26. 5 ( 0 . 4 ) ( 1 . 8 ) ( 2 . 9 ) XII 56 .4 15 .3 2 5 . 8 ( 1 . 0 ) ( 1 . 0 ) ( 3 . 3 ) S t a n d a r d d e v i a t i o n 44. m o l d s ( K a r e l , 1 975) , a n d n e c e s s i t a t e s t h e u s e o f low t e m p e r a t u r e , s t e r i l e c o n -d i t i o n s a n d t h e a d d i t i o n o f p r e s e r v a t i v e s in i n d u s t r i a l a p p l i c a t i o n to e n s u r e a d e q u a t e p r e s e r v a t i o n . B . D i f f e r e n t i a l S c a n n i n g C a l o r i m e t r y F i g u r e 3 r e p r e s e n t s a t y p i c a l d i f f e r e n t i a l s c a n n i n g c a l o r i m e t r y ( D S C ) t h e r m o g r a m o f c h u m e g g y o l k , r e v e a l i n g t h r e e o b v i o u s p e a k s . P r e v i o u s re^ i s e a r c h e r s s t u d y i n g t h e r m a l a n a l y s i s o f p r o t e i n s ( D o n o v a n et a l . , 1975; W r i g h t et a l . , 1977) h a v e p o s t u l a t e d t h a t e a c h major p e a k in a D S C t h e r m o g r a m r e -p r e s e n t s o n e t h e r m a l p r o c e s s ( i e . t h e d e n a t u r a t i o n o f o n e p r o t e i n ) . S u p p o r t f o r t h i s t h e o r y has come f rom c o m p a r i s o n s o f t h e r m o g r a m s o f t h e e n t i r e p r o t e i n s y s t e m w i t h t h o s e o f i n d i v i d u a l p u r i f i e d p r o t e i n s ( e g . t h e t h e r m o g r a m o f w h o l e a v i a n e g g w h i t e was c o m p a r e d w i t h t h e r m o g r a m s o f s e p a r a t e p r o t e i n s i n c l u d i n g o v a l b u m i n , c o n a l b u m e n a n d l y s o z y m e in t h e s t u d y b y D o n o v a n et a l . , 1 975) . It w o u l d a p p e a r t h e n , t h a t t h e r e a r e at least t h r e e t h e r m a l p r o c e s s e s t a k i n g p l a c e i n t h e t h e r m o g r a m s o b t a i n e d in t h i s s t u d y , r e p r e s e n t i n g t h e d e n a t u r a t i o n o f t h r e e p r o t e i n s . T h i s a g r e e s wel l w i t h t h e p r e s e n c e o f t h r e e major p r o t e i n s ( l i p o v i t e l l i n , p h o s v i t i n a n d g' c o m p o n e n t ) in c h u m e g g y o l k . P o s i t i v e i d e n t i f i c a t i o n o f e a c h p e a k in t e r m s o f t h e p r o t e i n it r e p r e s e n t s is d u b i o u s w i t h o u t c o m p a r i s o n s w i t h t h e r m o g r a m s o f t h e c o r r e s p o n d i n g i s o l a t e d p r o t e i n . H o w e v e r , t h e s i z e o f t h e p e a k o b t a i n e d is p r o p o r t i o n a l to t h e a m o u n t o f p r o t e i n p r e s e n t ( D o n o v a n et a l . , 1975) so t h a t it c o u l d at least be s u g g e s t e d t h a t t h e l a r g e s t p e a k is r e p r e s e n t a t i v e o f l i p o v i t e l l i n , w h i c h has b e e n s h o w n to be t h e d o m i n a n t p r o t e i n in t e l e o s t e g g y o l k ( M a r k e r t a n d V a n s t o n e , 1968; N a k a g a w a a n d T s u c h i y a , 1969) . T h e o t h e r two p e a k s c o u l d t h e n r e p r e s e n t t h e 8 ' c o m p o n e n t a n d p h o s v i t i n . T h e t e m p e r a t u r e s o f d e n a t u r a t i o n , as d e f i n e d b y t h e p e a k m a x i m a , a r e Figure 3. Typical DSC thermogram of fresh yolk from eggs of Code VI. 46. r e p r e s e n t e d in T a b l e 4. ( T h e m a x i m u m f o r P e a k 3 o f C o d e II was e s t i m a t e d f rom t h e c u r v e s s h o w n b y t h e P e r k i n E l m e r T h e r m a l A n a l y s i s r e c o r d e r . ) T h e s e v a l u e s r e v e a l h i g h r e s i s t a n c e s to h e a t . P r e v i o u s r e s e a r c h y i e l d e d d e n a -t u r a t i o n t e m p e r a t u r e s o f 84.0°C f o r o v a l b u m i n , 69. 5°C f o r c o n a l b u m i n ( D o n o v a n et a l . , 1975) a n d 60-80°C as a r a n g e o f d e n a t u r a t i o n t e m p e r a t u r e s f o r r a b b i t m u s c l e p r o t e i n s ( W r i g h t et a l . , 1 9 7 7 ) . In c o m p a r i s o n , c u r r e n t v a l u e s o f a p p r o x i m a t e l y 87°C a n d 107°C f o r c h u m y o l k p r o t e i n s a p p e a r to be s i g n i -f i c a n t l y h i g h e r . L i k e w i s e , t h e v a l u e s f o r t h e e n t h a l p i e s o f t h e v a r i o u s p e a k s ( T a b l e 4) a p p e a r d i m i n i s h e d in c o m p a r i s o n w i t h v a l u e s o b t a i n e d f o r e g g w h i t e p r o t e i n s ( 3 . 6 4 c a l / g f o r o v a l b u m i n ; 3 .63 c a l / g f o r c o n a l b u m i n ( D o n o v a n et a l . , 1975)) a n d r a b b i t m u s c l e p r o t e i n s ( 3 . 4 6 c a l / g f o r a c t i n ; 3 .33 c a l / g f o r m y o s i n ( W r i g h t et a l . , 1 9 7 7 ) ) . H o w e v e r , t h e l a t t e r e f f e c t may well be d u e to v a l u e s f o r e n t h a l p y b e i n g g i v e n as c a l o r i e s p e r g r a m o f y o l k d r y m a t t e r in t h e p r e s e n t s t u d y , a n d not g r a m o f p r o t e i n s , s i n c e t h e e x a c t p r o p o r t i o n o f e a c h p r o t e i n p r e s e n t is u n k n o w n . A n y rea l d i f f e r e n c e s t h a t e x i s t r e f l e c t v a r i a t i o n s i n t h e s t r u c t u r e o f t h e p r o t e i n s . A n e x a m p l e o f t h e i n f l u e n c e o f p r o t e i n s t r u c t u r e o n t h e r m a l a n a l y s i s r e s u l t s is f o u n d in t h e c a s e o f o v o m u c i n , a m i x t u r e o f e g g p r o t e i n s h a v i n g s i m i l a r p r o p e r t i e s , a n d p o s s e s s i n g a v e r y s i m p l e r a n d o m co i l c o n f o r m a t i o n w h i c h y i e l d s no p e a k s in a D S C t h e r m o g r a m ( D o n o v a n et a l . , 1 9 7 5 ) . C . P o l y a c r y l a m i d e G e l E l e c t r o p h o r e s i s In t h e p r e s e n t s t u d y , an a n i o n i c d e t e r g e n t , d e o x y c h o l a t e ( D O C ) was a d d e d to t h e s a m p l e a n d r u n n i n g b u f f e r to e n h a n c e s o l u b i l i z a t i o n o f t h e y o l k p r o t e i n s . T h e d e n s i t o m e t e r t r a c e r e s u l t i n g f rom p o l y a c r y l a m i d e ge l e l e c t r o p h o r e s i s ( P A G E ) o f c h u m e g g y o l k is d e p i c t e d in F i g u r e 4 a n d r e v e a l s t h e s e p a r a t i o n o f f o u r major d i s c r e t e b a n d s , l a b e l l e d I, II , III a n d I V . P r e v i o u s e l e c t r o p h o r e t i c s t u d i e s o f t e l e o s t e g g y o l k b y o t h e r r e s e a r c h e r s h a v e a l so i n d i c a t e d t h e p r e -T a b l e 4. E n t h a l p y o f d e n a t u r a t i o n a n d d e n a t u r a t i o n t e m p e r a t u r e o f p r o t e i n s o f f r e s h c h u m e g g y o l k o f two c o d e s . D e n a t u r a t i o n C o d e n o . P e a k n o . A Heat ( c a l / g y o l k , d b ) P e a k n o . t e m p e r a t u r e ( ° c ) II 1 0 . 2 9 ( 0 . 0 1 ) a 1 86.0 f( 1 TO) VI 1 0 .27 ( 0 . 0 2 ) 1 86.4 ( 0 . 2 ) II 3 2 .60 ( 0 . 0 3 ) 2 106 .7 ( 1 . 1 ) VI 2 & 3 2. 95 (0 .04) 2 107 .5 ( 0 . 4 ) II VI 3 3 110. 8 ( 1 . 1 ) b a S t a n d a r d d e v i a t i o n ( n = 3 ) . v a l u e u n a v a i l a b l e f rom P e r k i n E l m e r T h e r m a l A n a l y s i s S t a t i o n . 4 9 r s e n c e o f s e v e r a l p r o t e i n s . T i s e l i u s a n d s t a r c h ge l e l e c t r o p h o r e t i c p a t t e r n s o f r a i n b o w t r o u t e g g y o l k b y N a k a g a w a a n d T s u c h i y a ( 1 9 6 9 ) r e v e a l e d t h r e e p r o -t e i n s : a l i p o p r o t e i n , a p h o s p h o p r o t e i n a n d a s i m p l e p r o t e i n w h i c h w e r e p r e -s u m a b l y a n a l o g o u s to t h e a v i a n e g g y o l k l i p o v i t e l l i n , p h o s v i t i n a n d l i v e t i n , r e s p e c t i v e l y . W o r k i n g w i t h f i v e s p e c i e s o f P a c i f i c s a l m o n , M a r k e r t a n d V a n s t o n e ( 1 9 6 8 ) s e p a r a t e d t h r e e y o l k p r o t e i n s o n c e l l u l o s e ace ta te a n d i d e n t i f i e d t h e m o n t h e b a s i s o f d y e - b i n d i n g c a p a c i t y , r e t e n t i o n o n h y d r o x y a p a t i t e a n d t h e d e -g r e e o f p r e c i p i t a t i o n w i t h ammonium s u l f a t e . In a d d i t i o n , t h e y w e r e a b l e to i so la te a f o u r t h s i m p l e p r o t e i n t h a t m i g r a t e d t h e same d i s t a n c e as l i v e t i n s b u t c o u l d be p r e c i p i t a t e d o u t o f y o l k s o l u t i o n b y d i l u t i o n w i t h w a t e r . T h i s c o m -p o n e n t was s u b s e q u e n t l y n a m e d "B 1 c o m p o n e n t " . Wi th b o t h s t a r c h g e l a n d c e l l u l o s e a c e t a t e e l e c t r o p h o r e s i s , t h e l i p o v i t e l l i n f r a c t i o n r e m a i n e d at t h e o r i g i n , f o l l owed b y o n e o r m o r e b a n d s o f l i v e t i n s a n d t h e B' c o m p o n e n t , a n d f i n a l l y t h e p h o s v i t i n . T h e same p a t t e r n o c c u r r e d in p o l y a c r y l a m i d e ge l e l e c t r o p h o r e s i s a p p l i e d to t h e s e p a r a t i o n o f a v i a n y o l k h i g h d e n s i t y p r o t e i n s ( C h a n g et a l . , 1 9 7 0 ; M c B e e a n d C o t t e r i l l , 1 9 7 9 ; D i x o n a n d C o t t e r i l l , 1 9 8 1 ) , i e . l a r g e l i p o p r o t e i n m o l e c u l e s . l i p o v i t e l l i n ( a a n d g B ) r e m a i n e d c l o s e to t h e o r i g i n , w i t h t h e l i v e t i n s n e x t p r e s e n t in a s e r i e s o f b a n d s , a n d f i n a l l y , t h e p h o s v i t i n as a s i n g l e b a n d m i g r a t i n g t h e f u r t h e s t . P o l y a c r y l a m i d e a n d s t a r c h ge l e l e c t r o p h o r e s i s o f p r o t e i n s b r i n g a b o u t t h e f o r m a t i o n o f s e p a r a t e z o n e s o n t h e b a s i s o f e l e c t r i c c h a r g e a n d m o l e c u l a r s i z e o f t h e p r o t e i n s ( D e y l et a l . , 1 9 7 9 ) . T h e a d d i t i o n o f D O C in t h e p r e s e n t s t u d y c a u s e s t h e e f f e c t o f v a r y i n g m o l e c u l a r c h a r g e s o n p r o t e i n m o v e m e n t to be c a n c e l l e d o u t , m a k i n g m o l e c u l a r s i z e t h e major f a c t o r in s e p a r a t i o n s '.' ( H j e l m e l a n d a n d C h r a m b a c h , 1 9 8 1 ) . No s u c h d e t e r g e n t s w e r e e m p l o y e d in t h e p r e v i o u s l y r e f e r r e d to s t u d i e s o n a v i a n y o l k , u s i n g p o l y a c r y l a m i d e g e l , a n d so c h a r g e w o u l d be e x p e c t e d to p l a y a r o l e in t h e s e p a r a t i o n o b t a i n e d . H o w e v e r , t h e r e s u l t s r e m a i n what may be e x p e c t e d i f o n l y t h e m o l e c u l a r s i z e , b a s e d o n 50. m o l e c u l a r w e i g h t is c o n s i d e r e d . A v i a n l i p o v i t e l l i n s (a a n d 0) h a v e b e e n e s t i m a t e d as b e i n g a p p r o x i m a t e l y 400,000 d a l t o n s ( d i m e r f o r m s ) ( B e r n a r d i a n d C o o k , 1 960) , l i v e t i n s ( < 8 a n d 7) as r a n g i n g b e t w e e n w e i g h t s o f 45 ,000 to 150,000 ( M a r t i n et a l . , 1 9 5 7 ) , a n d p h o s v i t i n as 40 ,000 d a l t o n s ( W i n d h o l z et a l . , 1 9 7 6 ) . If it is a s s u m e d t h a t m o l e c u l a r s i z e fo l lows m o l e c u l a r w e i g h t , t h e n t h e o r d e r o f t h e p r o t e i n b a n d s a c h i e v e d w i t h a v i a n y o l k h i g h d e n s i t y p r o t e i n fo l lows t h a t w h i c h c o u l d be e x p e c t e d . L i k e w i s e , t h e m o l e c u l a r w e i g h t o f c o h o l i p o v i t e l l i n , p h o s v i t i n a n d 6' c o m p o n e n t h a v e b e e n e s t i m a t e d as 390 ,000 , 27 ,000 a n d 3 0 , 0 0 0 , r e s p e c t i v e l y ( M a r k e r t a n d V a n s t o n e , 1 9 6 8 ) , a n d t h e c o m p a r a t i v e m a g n i t u d e s t h u s d i s p l a y e d a r e r e f l e c t e d in t h e m o v e m e n t o f c h u m y o l k p r o t e i n s o n c e l l u -lose a c e t a t e , as o b s e r v e d b y M a r k e r t a n d V a n s t o n e ( 1 9 6 8 ) . T h e r e f o r e , b a s e d o n t h e p o s t i o n o f t h e b a n d s , t e n t a t i v e i d e n t i f i c a t i o n o f -the' p r o t e i n s in t h e p r e s e n t s t u d y may be m a d e . T h e l a r g e p e a k n e a r t h e o r i g i n ( P e a k I) is most p r o b a b l y l i p o v i t e l l i n , t h e 8' c o m p o n e n t a n d l i v e t i n m o l e c u l e s a r e w i t h i n P e a k s II a n d III a n d f i n a l l y , t h e s m a l l e s t , f u r t h e s t m i g r a t i n g p e a k ( P e a k IV) p r o b a b l y r e p r e s e n t s p h o s v i t i n . D . 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 A s c a n n i n g e l e c t r o n m i c r o g r a p h o f t h e c r o s s s e c t i o n o f a c h u m s a l m o n e g g m e m b r a n e is d e p i c t e d i n F i g u r e 5. T h i s a n d all s u b s e q u e n t v i e w s r e p r e s e n t i n g t h e m e m b r a n e c r o s s s e c t i o n , a r e i n fac t a c t u a l s p l i t s in t h e e g g m e m b r a n e , most l i k e l y t h e r e s u l t o f s t r e s s a p p l i e d to t h e e g g d u r i n g r e m o v a l f r o m t h e o v a r y b y t h e t e c h n i q u e o f s c r e e n i n g . O b s e r v a t i o n o f F i g u r e 5 c l e a r l y r e v e a l s two l a y e r s , t h e i n n e r o f w h i c h a p p e a r s to b e f i b r o u s in n a t u r e . S u c h a s t r u c t u r e a lso a p p e a r s in t h e m i c r o -g r a p h s o f K o b a y a s h i (1982) who d e s c r i b e d t h i s i n n e r p o r t i o n o f t h e c h u m m e m -b r a n e as a d i s t i n c t d i s c r e t e z o n e o f i n t e r t w i s t i n g f i b e r s , g r a d u a l l y g i v i n g 51. w a y to a m o r e c o n t i n u o u s h o m o g e n e o u s r e g i o n in t h e o u t e r p o r t i o n . S t e h r a n d H a w k e s (1 979) p r o d u c e d s c a n n i n g m i c r o g r a p h s o f t h e e g g s o f p i n k s a l m o n O. gorbuscha (Wa lbaum) a n d i n t e r p r e t e d t h e e g g m e m b r a n e to b e c o m p o s e d o f s h o r t , d i s c o n t i n u o u s lame l lae , w i t h p o r e c a n a l s p e n e t r a t i n g t h e z o n a r a d i a t a , p r o d u c i n g a c o l u m n a r l i k e a p p e a r a n c e . S u c h an a p p e a r a n c e is i n d i c a t e d in t h e c r o s s s e c t i o n o f t h e f r o z e n / t h a w e d e g g m e m b r a n e ( F i g u r e 6) b u t is not as a p p a r e n t in t h e f r e s h e g g m e m b r a n e ( F i g u r e 7 ) . It is not k n o w n w h e t h e r t h i s d i f f e r e n c e is d u e to t h e f r o z e n s t o r a g e o f t h e e g g , o r m e r e l y a n a r t i f a c t o f p r e p a r a t i v e t e c h n i q u e . T h e d i s c r e t e n e s s o f t h e f i b e r s o b s e r v e d b y b o t h K o b a y a s h i (1 982) a n d S t e h r a n d H a w k e s (1979) is m i s s i n g in t h e m i c r o g r a p h s o f t h e p r e s e n t s t u d y , h o w e v e r , t h i s may b e e x p l a i n e d b y t h e fac t t h a t i f t h e c r o s s s e c t i o n a l c r a c k s a r e i n d e e d p r o d u c e d in t h e e g g m e m b r a n e b y t h e s c r e e n i n g p r o c e s s , it is g u i t e l i k e l y t h a t i n n e r y o l k may h a v e l e a k e d o u t o n t o t h e m e m b r a n e c r o s s s e c t i o n b e f o r e f i x a t i o n o c c u r r e d , t h u s o b s c u r i n g f i n e d e t a i l . D i s c r e t e f i b e r s c o v e r e d b y a coat o f y o l k m a t e r i a l , a r e t h e n r e p r e s e n t e d b y i n t e r - t w i s t i n g r i d g e s . T h e w i d t h o f t h i s p a r t o f t h e m e m b r a n e a p p e a r s to be f a i r l y u n i f o r m ( F i g u r e 8 ) , a n d m e a s u r e s a b o u t 40 y m , in c l o s e a g r e e m e n t w i t h v a l u e s p r e v i o u s l y o b t a i n e d f o r c h u m s a l m o n o f 50 y m ( K o b a y a s h i , 1 982) , 55 to 70 y m f o r p i n k s a l m o n ( S t e h r a n d H a w k e s , 1 979) a n d 1 8 to 55 y m fo r o t h e r t e l e o s t s p e c i e s ( C r i e r s o n a n d N e v i l l e , 1981; H u r l e y a n d F i s h e r , 1 9 6 6 ) . T h e s u r f a c e o f t h e c h u m e g g , as r e v e a l e d in F i g u r e 9, a p p e a r s as a f i b -r i l l a r l a y e r s i m i l a r to t h a t r e p o r t e d b y K o b a y a s h i (1 982) . T h i s l a y e r was f o u n d to be p o l y s a c c a r i d e in n a t u r e b y K o b a y a s h i (1 982) a n d may act b o t h to a b s o r b i m p a c t s a n d p r o t e c t t h e e g g , as wel l as a p o s s i b l e a d h e s i v e a p p a r a t u s , a n a d a p t a t i o n f a v o u r a b l e to t h e d e m e r s a l e g g s c h a r a c t e r i s t i c o f t h o s e s p e c i e s tha t depos i t ' t h e i r e g g s i n f a s t r u n n i n g s t r e a m s ( L a g l e r et a l . , 1 962; C r i e r s o n a n d N e v i l l e , 1 9 8 1 ) . u r e s 5 to 9. S c a n n i n g e l e c t r o n m i c r o g r a p h s o f s a l m o n c h u m e g g s . u r e 5. C r o s s - s e c t i o n o f f r e s h e g g m e m b r a n e as r e - F i g u r e 6. C r o s s - s e c t i o n o f f r o z e n / t h a w e d e g g m e m -v e a l e d by a r u p t u r e in t h e e g g s u r f a c e b r a n e as r e v e a l e d by a r u p t u r e in t h e e g g (pw = 50 y m ) . s u r f a c e (pw = 490 y m ) . F i g u r e 9. S i m i l a r v i e w to F i g u r e 5 (pw = 1 350 u m ) . 56. A t h i r d l a y e r has b e e n r e p o r t e d to e x i s t in t h e m e m b r a n e o f t h e e g g s o f t e l e o s t s p e c i e s a n d l a b e l l e d t h e c o r t e x r a d i a t u s e x t e r n u s b y R i e h l et a l . . ( 1 9 8 0 ) . T h e s e r e s e a r c h e r s f o u n d t h i s l a y e r to be not r e a d i l y a p p a r e n t t h r o u g h 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 , b u t r e q u i r e d t r a n s m i s s i o n EM to al low d i f f e r e n t i a t i o n f r o m t h e c o r t e x r a d i a t u s i n t e r n u s . T h e s c a n n i n g e l e c t r o n m i c r o g r a p h s o b t a i n e d in t h e p r e s e n t s t u d y l i k e w i s e d i d not r e v e a l t h e p r e s e n c e o f t h i s l a y e r . E . F low B e h a v i o u r S t u d i e s T h e f low b e h a v i o u r o f c h u m y o l k f r o m e g g s o f f o u r c o d e d s a m p l e s is d e m o n -s t r a t e d b y t h e r h e o g r a m s d e p i c t e d in F i g u r e 10. T h e c h a n g e in a p p a r e n t v i s c o s i t y w i t h s h e a r r a t e is t y p i c a l o f a n o n - N e w t o n i a n f l u i d , a n d it a p p e a r s t h a t b o t h s h e a r t h i n n i n g at l ower r a t e s o f s h e a r a n d , in y o l k s o f g r e a t e r a p p a r e n t v i s c o s i t i e s (VI a n d I I I ) , s h e a r t h i c k e n i n g at h i g h e r r a t e s o f s h e a r a r e o c c u r r i n g . A c c o r d i n g l y , d i s c u s s i o n o f t h e r h e o g r a m s wi l l d e a l w i t h t h e s e two p h e n o m e n a s e p a r a t e l y : f i r s t w i t h t h e s h e a r t h i n n i n g p o r t i o n f r o m 36.0 to 300 s 1 . A d e c r e a s e in s h e a r r a t e a f t e r a c h i e v i n g a max imum d i d not r e s u l t in t h e h y s t e r e s i s loop i n d i c a t i v e o f t ime d e p e n d e n t b e h a v i o u r ; t h e r e f o r e , t h e f low b e h a v i o u r o f c h u m y o l k in t h e s h e a r t h i n n i n g r e g i o n was s e e n to be p s e u d o ^ p l a s t i c in n a t u r e . R e s u l t s o f a p p l i c a t i o n o f t h e P o w e r - l a w f low mode l to t h e b e h a v i o u r o f y o l k s f rom t h e s e f o u r d i f f e r e n t c o d e d s a m p l e s c a n b e s e e n f rom T a b l e 5, w h i c h l i s t s t h e c o n s i s t e n c y c o e f f i c i e n t s ( m ) , f low b e h a v i o u r i n d i c e s ( n ) , a n d c o e f f i c i e n t s o f d e t e r m i n a t i o n ( r 2 ) . V a l u e s f o r r 2 i n d i c a t e t h e P o w e r -law mode l to be a g o o d d e s c r i p t i o n o f t h e f low c h a r a c t e r i s t i c s o f c h u m e g g y o l k . P s e u d o p l a s t i c b e h a v i o u r is c o n f i r m e d b y v a l u e s o f l e s s t h a n 1.0 f o r f low b e -h a v i o u r i n d i c e s , a n d it c a n be s e e n t h a t b o t h t h e d e g r e e o f p s e u d o p l a s t i c i t y (as i n d i c a t e d b y t h e m a g n i t u d e s o f h) a n d t h e " t h i c k n e s s " (as r e p r e s e n t e d b y t h e c o n s i s t e n c y c o e f f i c i e n t s ) v a r y b e t w e e n y o l k s a m p l e s , w i t h t h e t h i n n e s t y o l k e x h i b i t i n g b e h a v i o u r c l o s e s t to N e w t o n i a n . 57. F igure 1 0 . Rheograms of yolk from f resh chum eggs from four codes . T a b l e 5. M e a n v a l u e s o f P o w e r H a w f low p a r a m e t e r s f rom r h e o g r a m s o f l i q u i d y o l k p r e p a r e d f rom f r e s h c h u m e g g s ( n = 3 ) . C o n s i s t e n c y F low b e h a v i o u r c o e f f i c i e n t i n d e x 2 C o d e n o . m, (mPa s ) n r II 330 0 .88 0. 95 III 525 0 .84 0 .96 VI 1000 0.81 0 .96 VIII 363 0.87 0. 92 59. S u c h t ime i n d e p e n d e n t s h e a r t h i n n i n g is t y p i c a l o f p r o t e i n s o l u t i o n s a n d c a n b e p o s t u l a t e d as r e s u l t i n g f rom i n c r e a s i n g a l i g n m e n t o f t h e l a r g e p r o t e i n m o l e c u l e s in to lamel lae w i t h i n c r e a s i n g s h e a r r a t e s . F o l l o w i n g t h e t e n d e n c y to m a x i m i z e e n t r o p y , at low r a t e s o f s h e a r , t h e m o l e c u l e s a r e o r i e n t e d at r a n d o m , c a u s i n g a g r e a t e r r e s i s t a n c e to f l o w . H o w e v e r , w i t h h i g h e r r a t e s o f s h e a r , t h e m o l e c u l e s b e c o m e i n c r e a s i n g l y more a l i g n e d a l o n g t h e p l a n e s o f s h e a r a n d t h i s p r o g r e s s i v e l y m o r e o r d e r e d a r r a n g e m e n t r e s u l t s in less r e s i s t a n c e to f l o w , b y d e f i n i t i o n a d e c r e a s e in v i s c o s i t y ( R e i n e r a n d B l a i r , 1 967; T u n g , 1978; P r a d i p a s e n a a n d R h a , 1 9 7 7 ) . T h e s e c o n d p o r t i o n o f t h e r h e o g r a m in two o f t h e f o u r s a m p l e s ( C o d e s III a n d V I ) e x h i b i t s a n a p p a r e n t i n c r e a s e in v i s c o s i t y w i t h h i g h e r r a t e s o f s h e a r . T i m e d e p e n d e n c y is a g a i n l a c k i n g , as i n d i c a t e d b y t h e a b s e n c e o f a h y s t e r e s i s l o o p . S u c h b e h a v i o u r has b e e n k n o w n to r e s u l t f r o m t u r b u l e n t f l o w , a c o n d i -t i o n t h a t o c c u r s at h i g h r a t e s o f s h e a r w h e n t h e o r d e r l y m o v e m e n t o f a d j o i n i n g lamel la a r e d i s r u p t e d , w h o r l s a n d v o r t i c e s a r e i n t r o d u c e d in to t h e f l o w , a n d r e s i s t a n c e to s h e a r i n c r e a s e s ( v a n W a z e r et a l . , 1963) . A v a l u e c a n be c a l c u -l a ted u s i n g an e q u a t i o n b a s e d o n f l u i d d e n s i t y , v i s c o s i t y , r a d i u s o f m e a s u r i n g b o d y a n d mean v e l o c i t y w h i c h , w h e n e x c e e d i n g a c r i t i c a l v a l u e o f 2100, i n d i c a t e s t h e p r e s e n c e o f t u r b u l e n c e i n a N e w t o n i a n f l u i d . T h i s v a l u e , k n o w n as t h e R e y n o l d ' s N u m b e r h a s b e e n m o d i f i e d f o r u s e w i t h n o n - N e w t o n i a n f l u i d s ( W h o r l o w , 1980) a n d r e - l a b e l l e d t h e G e n e r a l i z e d R e y n o l d ' s N u m b e r . T h e e q u a t i o n w h e n a p p l i e d to t h e c o a x i a l c y l i n d e r s y s t e m as u s e d in t h i s s t u d y , g a v e a r o u g h e s t i m a t i o n , s u f f i c i e n t f o r a s s e s s i n g t h e p r e s e n c e o r a b s e n c e o f t u r b u l e n t f low at h i g h s h e a r r a t e s . G e n e r a l i z e d R e y n o l d ' s N u m b e r s f o r y o l k s o f C o d e III a n d VI w e r e c a l c u l a t e 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 : C D = ( 8 D n v 2 _ n p ) / ( 1 / n + 3 ) n 2 n m ( 8 ) K6 w h e r e D is t h e w i d t h o f t h e g a p ( c m ) , V is t h e m a s s a v e r a g e v e l o c i t y o f f l u i d 60. ( c m / s ) a n d p is t h e d e n s i t y o f t h e f l u i d ( 1 . 6 g / m L , o b t a i n e d f r o m t h e a v e r a g e o f w e i g h t / v o l u m e m e a s u r e m e n t s f o r 4 s a m p l e s o f y o l k ( C o d e V I I I ) ) . V a l u e s c a l c u l a t e d w e r e 11 .8 a n d 9.1 f o r C o d e III a n d V I , r e s p e c t i v e l y , so t h a t it c a n b e s t a t e d t h a t a n i n c r e a s e in v i s c o s i t y n o t e d at h i g h e r s h e a r r a t e s f o r t h e s e c o d e s is p r o b a b l y not d u e to t u r b u l e n t f l o w . A p o s s i b l e e x p l a n a t i o n f o r s u c h b e h a v i o u r may i n s t e a d l ie in t h e momentum t r a n s f e r t h e o r y f o r d i l a t a n t f l u i d s d i s c u s s e d b y B a u e r a n d C o l l i n s (1 967) . T h i s c o n c e p t i n v o l v e s t h e s i d e w a y s m o v e m e n t o f p a r t i c l e s f rom o n e a d j a c e n t lamel la in to t h e n e x t , w i t h al l p a r t i c l e s i n t h e d i s r u p t e d l a y e r e x p e r i e n c i n g a c h a n g e i n v e l o c i t y . S i n c e a s h e a r r a t e i n c r e a s e r e s u l t s in m o r e p a r t i c l e s b e -c o m i n g a l i g n e d in to l ame l l ae , p r o g r e s s i v e l y m o r e p a r t i c l e s a r e a f f e c t e d w i t h t h e m o v e m e n t o f a p a r t i c l e f rom o n e lamel la in to t h e n e x t , a n d i n c r e a s e d s h e a r r a t e s w o u l d t h e n r e s u l t in a c o r r e s p o n d i n g v i s c o s i t y i n c r e a s e . S u c h a t h e o r y w o u l d s u g g e s t t h a t s h e a r t h i c k e n i n g s h o u l d o c c u r at l ower s h e a r r a t e s in f l u i d s o f o v e r a l l h i g h e r a p p a r e n t v i s c o s i t y . T h i s b e h a v i o u r is n o t e d in y o l k s f rom C o d e s III a n d VI w i t h t h e in i t i a l o c c u r r e n c e o f s h e a r t h i c k e n i n g at a s h e a r r a t e o f 400 s 1 f o r y o l k f rom C o d e VI w h o s e o v e r a l l a p p a r e n t v i s c o s i t y is h i g h e r t h a n y o l k f r o m C o d e III , in w h i c h s h e a r t h i c k e n i n g d o e s not b e g i n u n t i l 804 s 1 . It is p o s s i b l e t h a t y o l k s f rom C o d e s II a n d V I I I , e a c h o f l ower o v e r a l l a p p a r e n t v i s c o s i t y t h a n C o d e III , may y e t show s h e a r t h i c k e n i n g i f h i g h e r r a t e s o f s h e a r w e r e e m p l o y e d . B e c a u s e t h i s s h e a r t h i c k e n i n g p h e n o m e n o n d o e s not a p p e a r in al l s a m p l e s , b u t o n l y i n t h o s e a b o v e a c e r t a i n v a l u e o f a p p a r e n t v i s c o s i t y , a n d in o r d e r to s i m p l i f y f u r t h e r a n a l y s e s , al l s u b s e q u e n t d i s c u s s i o n o f f low b e h a v i o u r d e a l s w i t h t h e s h e a r r a t e i n t e r v a l o f 36 to 300 s 1 , r e p r e s e n t i n g t h e P o w e r - l a w p s e u d o p l a s t i c p o r t i o n o f t h e r h e o g r a m . T h i s r e g i o n is a l so m o r e c o m p a r a b l e to s h e a r r a t e s i n v o l v e d in s e n s o r y a n a l y s i s ( R a o , 1977) . 61. F . F o r c e D e f o r m a t i o n A n a l y s i s A t y p i c a l f o r c e d e f o r m a t i o n c u r v e r e s u l t i n g f rom c o m p r e s s i o n o f o n e e g g o n 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 is d e p i c t e d in F i g u r e 11. T h e c u r v e a p p e a r s to be o f a f o rm t y p i c a l l y o b t a i n e d f rom c o m p r e s s i o n o f f ood p r o d u c t s , r e f l e c t i n g an e v e r i n c r e a s i n g r e s i s t a n c e to f o r c e a p p l i e d , f o l l owed b y a s u d d e n d e c r e a s e at w h a t has b e e n r e f e r r e d to as t h e b i o y i e l d p o i n t ( M o h s e n i n a n d M i t t a l . , 1 977) . T h e p o r t i o n o f t h e c u r v e l y i n g b e t w e e n t h e p o i n t o f f i r s t c o n t a c t ( C ) a n d t h e p o i n t o f f i r s t r e s i s t a n c e ( R ) is a m e a s u r e o f s e a t i n g a c c o m m o d a t i o n , o r t h e e x t e n t to w h i c h t h e e g g c o u l d be d e f o r m e d p r i o r to a n y r e s i s t a n c e b y t h e m e m b r a n e , a n d t e n d e d to v a r y d e p e n d i n g u p o n t h e f l a c c i d i t y o f t h e e g g . S u c h b e h a v i o u r was a l so n o t e d b y P o m e r a n z (1974) in h is s t u d y o n p l a i c e e g g s w h e r e i n it was f o u n d t h a t an e g g c o u l d be d e f o r m e d 25% b e f o r e a n y a p p r e c i a b l e r e s i s t a n c e was r e c o r d e d . T h e s u b s e g u e n t g r a d u a l i n c r e a s e in r e s i s t a n c e r e s u l t e d f rom a s t r e t c h i n g o f t h e e g g m e m b r a n e a r o u n d t h e i n c o m p r e s s i b l e y o l k , to a p o i n t w h e r e t h e d e -f o r m a t i o n l imit was r e a c h e d a n d r u p t u r e o c c u r r e d . Immediate c e s s a t i o n in r e -s i s t a n c e f o l l o w e d ; ( a n y d e v i a t i o n f rom t h e i n s t a n t a n e o u s n a t u r e o f t h i s e v e n t , as d e p i c t e d b y t h e c u r v e in F i g u r e 11, r e s u l t e d f rom r e c o r d e r p e n r e s p o n s e t i m e . ) T h e two p a r a m e t e r s o f r u p t u r e e n e r g y ( j o u l e s ) a n d r u p t u r e f o r c e ( N e w t o n s ) w e r e o b t a i n e d d i r e c t l y f rom 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 a n d t h e s e a r e p r e s e n -t e d in T a b l e 6 f o r f r e s h e g g s o f s e v e r a l c o d e s . A l t h o u g h a t t e m p t s h a v e b e e n made in t h e p a s t to d e t e r m i n e t h e b u r s t i n g s t r e n g t h o f t e l e o s t e g g s , r e s u l t s r e p o r t e d in t h e l i t e r a t u r e a r e not in g o o d a g r e e m e n t . H a y e s a n d A r m s t r o n g (1942) u t i l i z e d a s y s t e m i n v o l v i n g an o r d i n a r y k i t c h e n s p r i n g b a l a n c e to o b t a i n v a l u e s f o r t h e f o r c e in k i l o g r a m s r e q u i r e d to r u p t u r e e g g s o f t h e s p e c i e s Salmo salar. T h e i r e x p e r i m e n t s y i e l d e d a n a v e r a g e v a l u e o f 0 .14 k g ( 1 . 3 7 N) w h i c h l ies c l o s e to h i g h e r v a l u e s w i t h i n t h e r a n g e 1.22 to 0 .47 N o b t a i n e d f o r e g g s i n 1.2 DEFORMATION (mm) F igure 1 1 . Typ ica l force deformation curve resul t ing from compression of a s ing le chum egg. T a b l e 6. M e a n m e m b r a n e r u p t u r e e n e r g y a n d r u p t u r e f o r c e f o r f r e s h c h u m e g g s o f s e v e r a l c o d e s ( n = 2 5 ) . C o d e n o . M e a n m e m b r a n e r u p t u r e e n e r c y (J x 10 k ) M e a n m e m b r a n e r u p t u r e f o r c e ( N ) II 3 . 1 4 0 ,56 ( 0 . 9 6 ) a ( 0 . 1 9 ) III 2 .36 0 .47 ( 0 . 9 4 ) ( 0 . 1 6 ) IV 3 .34 0 .64 ( 1 . 9 4 ) ( 0 . 1 9 ) IX 7. 99 1.22 ( 3 . 3 4 ) ( 0 . 3 8 ) X 5.82 0.92 ( 3 . 6 3 ) ( 0 . 4 3 ) XI 4 . 1 7 0 .74 ( 2 . 0 0 ) ( 0 . 3 0 ) S t a n d a r d d e v i a t i o n 64. t h e p r e s e n t s t u d y . H o w e v e r , e g g s o f Sdlmo gairdnerii w e r e r e p o r t e d b y G r i e r s o n a n d N e v i l l e (1981) to be c a p a b l e o f w i t h s t a n d i n g f o r c e s o f 1 k g ( 9 . 8 N) b e f o r e b u r s t i n g . T h i s v a l u e was o b t a i n e d b y s q u a s h i n g an e g g b e t w e e n two g l a s s s l i d e s p l a c e d u n d e r a n u m b e r o f b a l a n c e w e i g h t s . T h e a u t h o r s do s t a t e t h a t o t h e r f i s h s p e c i e s w e r e o b s e r v e d to be f a r l ess s t r o n g , so t h e a p p a r e n t d i f f e r e n c e b e t w e e n t h i s r e s u l t a n d t h o s e o b t a i n e d f o r c h u m s a l m o n in t h e p r e s e n t s t u d y may b e d u e to s p e c i e s v a r i a t i o n . T h e d i s p a r i t y b e t w e e n t h e r e s u l t s o f H a y e s a n d A r m s t r o n g a n d G r i e r s o n a n d N e v i l l e , w o r k i n g w i t h t h e same s p e c i e s , c o u l d wel l be a r e s u l t o f t h e d i v e r s e m e t h o d s e m p l o y e d . A c c o r d i n g to G r i e r s o n a n d N e v i l l e ( 1 9 8 1 ) , t h e a b i l i t y to r e s i s t l a r g e f o r c e s o f c o m p r e s s i o n , as e x h i b i t e d b y t h e c h u m e g g s s t u d i e d in t h i s i n v e s t i g a t i o n , is d u e to t h e h e l i c o i d a r r a n g e m e n t o f t h e f i b r o u s laminate o f t h e f i s h e g g m e m -b r a n e . S u c h s t r e n g t h w o u l d b e o f o b v i o u s a d v a n t a g e in t h e p r o t e c t i o n o f t h e d e v e l o p i n g o r g a n i s m ; e g g s o f t h e P a c i f i c sa lmon m u s t w i t h s t a n d b o t h t h e p r e s s u r e o f t h e s u r r o u n d i n g e g g m a s s , as wel l as t h e g r a v e l t h a t is p i l e d o v e r t o p o f al l i n t h e fo rm o f a n e s t . T h r e e o t h e r p a r a m e t e r s w e r e o b t a i n e d f rom f o r c e d e f o r m a t i o n a n a l y s i s o f t h e e g g s a n d t h e s e a r e p r e s e n t e d in T a b l e 7. T h e s e p a r a m e t e r s w e r e al l c a l -c u l a t e d f rom v a l u e s f o r max imum f o r c e a n d t h e a r e a u n d e r t h e f o r c e d e f o r m a t i o n c u r v e , a n d r e p r e s e n t f r e s h e g g s f rom c o d e s I X , X a n d X I . A s c a n be s e e n , a d e f o r m a t i o n o f a p p r o x i m a t e l y 20% r e s u l t e d in e g g r u p t u r e ( u s i n g a n a v e r a g e e g g h e i g h t o f 5.7 m m , as m e a s u r e d in t h e s t u d y o n t h e e f f e c t o f e g g s i z e o n m e m b r a n e r u p t u r e e n e r g y ) , a l t h o u g h t h i s v a l u e was u n d e r e s t i m a t e d somewhat b y t h e m e t h o d o f c a l c u l a t i o n e m p l o y e d to a c h i e v e i t . T h e a b i l i t y to w i t h s t a n d s u b s t a n t i a l d e f o r m a t i o n o f s t r u c t u r e b e f o r e r u p t u r e is a l so r e f l e c t e d in v a l u e s f o r e g g f i r m n e s s r a n g i n g f r o m 670 to 960 N/m, a n d c o m p l i a n c e f rom 0.(3011 to 0 .0018 m A N . T h e s e v a l u e s , l i k e t h a t o f t h e r u p t u r e f o r c e , a r e i n d i c a t i v e o f t h e e s s e n t i a l r e q u i r e m e n t s f o r a s t r u c t u r e w h o s e p u r p o s e is to p r o t e c t i n t e r i o r 65. T a b l e 7. M e a n s o f p a r a m e t e r s c a l c u l a t e d f rom v a l u e s o f r u p t u r e f o r c e a n d e n e r g y f o r m e m b r a n e s o f f r e s h e g g s o f t h r e e c o d e s ( n = 2 5 ) . M o d i f i e d m o d u l u s C o d e D e f o r m a t i o n F i r m n e s s o f c o m p l i a n c e (mm) (N/m) (m/N x 10 2 ) IX 1.3 960 0.11 ( 0 . 2 ) a (260) ( 0 . 0 3 ) X 1.2 770 0.15 ( 0 . 4 ) (320) ( 0 . 0 6 ) XI 1.1 670 0 .18 ( 0 . 2 ) (250) ( 0 . 0 9 ) S t a n d a r d d e v i a t i o n 66. c o n t e n t s a g a i n s t p h y s i c a l s t r e s s . T h e t e r m " f i r m n e s s " is u s e d in k e e p i n g w i t h M o h s e n i n a n d Mi t ta l (1 977) who d i f f e r e n t i a t e b e t w e e n t h e " m o d u l u s o f d e f o r m a b i l i t y " , a v a l u e c a l c u l a t e d as t h e s l o p e o f a f o r c e - d e f o r m a t i o n c u r v e in t h e a r e a o f i n i t i a l r e s i s t a n c e w h e n t h e m a t e r i a l is u n d e r smal l s t r a i n , a n d t h e " m o d u l u s o f e l a s t i c i t y " r e p r e s e n t i n g t h e r e g i o n c l o s e r to t h e b i o y i e l d p o i n t w h e r e a h i g h e r amount o f s t r a i n is o c c u r -r i n g . In t h i s i n v e s t i g a t i o n , a s s u m i n g t h e f o r c e d e f o r m a t i o n c u r v e to be a r i g h t a n g l e t r i a n g l e , a v a l u e l y i n g i n t e r m e d i a t e to t h e two modu l i as e x p r e s s e d b y M o h s e n i n a n d M i t ta l was o b t a i n e d f rom t h e s l o p e o f t h e h y p o t e n u s e , a n d t e r m e d t h e " f i r m n e s s " o f t h e e g g . C o r r e l a t i o n c o e f f i c i e n t s o f 0. 95, 0 .87 a n d 0 .96 w e r e c a l c u l a t e d f o r t h e p a r a m e t e r s o f m e m b r a n e r u p t u r e e n e r g y a n d f o r c e f o r C o d e s I X , X a n d X I , r e s p e c t i v e l y . A c c o r d i n g l y to M o h s e n i n a n d Mi t ta l (1 977) , m e c h a n i c a l p r o p e r t i e s r e f l e c t i n g y i e l d i n g a n d f r a c t u r e o f t h e m a t e r i a l s h o u l d c o r r e l a t e wel l w i t h e a c h o t h e r , b u t not n e c e s s a r i l y w i t h t h o s e i n d i c a t i n g t h e b e h a v i o u r o f a m a t e r i a l u n d e r a smal l s t r a i n . T h e s i g n i f i c a n t c o r r e l a t i o n f o u n d b e t w e e n r u p t u r e f o r c e a n d r u p t u r e e n e r g y is t h e n not u n e x p e c t e d as b o t h a r e i n t i m a t e l y i n v o l v e d w i t h t h e m a t e r i a l u n d e r h i g h s t r e s s , a l t h o u g h t h e r u p t u r e e n e r g y is m o r e an i n d i c a t i o n o f a h i s t o r y o f r e s i s t a n c e f o r c e s o v e r t h e e n t i r e p e r i o d o f d e f o r m a t i o n . C o r r e l a t i o n c o e f f i c i e n t s f o r t h e o t h e r p a r a m e t e r s w e r e not c o m p u t e d , as t h e s e v a l u e s w e r e a c t u a l l y c a l c u l a t e d f r o m m e a s u r e m e n t s u s e d to o b t a i n r u p t u r e f o r c e a n d e n e r g y . A s t h e y w e r e not o b t a i n e d d i r e c t l y f rom f o r c e d e f o r m a t i o n c u r v e s , t h e v a l i d i t y o f a n y c o n c l u s i o n s d r a w n c o n c e r n i n g i n t e r r e l a t i o n s h i p s is d u b i o u s . 67. C . E f f e c t o f E g g S i z e o f M e m b r a n e R u p t u r e E n e r g y O f a l l o f t h e p a r a m e t e r s o b t a i n e d f rom t h e f o r c e d e f o r m a t i o n c u r v e , t h a t o f m e m b r a n e r u p t u r e e n e r g y was c o n s i d e r e d to be most n e a r l y r e p r e s e n t a t i v e o f t h e o v e r a l l r e s p o n s e o f t h e e g g m e m b r a n e t h r o u g h o u t t h e e n t i r e p e r i o d o f c o m p r e s s i o n , a n d so was u s e d in all s u c c e s s i v e e x p e r i m e n t s to e v a l u a t e t r e a t -ment e f f e c t s . P r e l i m i n a r y t e s t i n g was n e c e s s a r y , h o w e v e r , to e v a l u a t e t h e r e l a t i o n s h i p b e t w e e n t h e r u p t u r e e n e r g y a n d e g g s i z e , as a c c o r d i n g to V o i s e y ( 1 9 7 1 ) , s a m p l e s a n a l y z e d in c o m p r e s s i o n s t u d i e s m e a s u r i n g o n l y f o r c e a n d d e f o r m a t i o n m u s t be o f i d e n t i c a l d i m e n s i o n s to m a k e m e a n i n g f u l c o m p a r i s o n s . If t h i s is not t h e c a s e , r e s u l t s m u s t be c a l c u l a t e d b a s e d o n d i m e n s i o n s o f t h e s a m p l e s a n a l y z e d . In t h e a n a l y s i s o f f i s h e g g s , b i o l o g i c a l v a r i a t i o n m a k e s t h e f i r s t c o n d i t i o n u n f e a s i b l e . M e a s u r e m e n t o f e v e r y e g g t e s t e d w o u l d p r o v i d e i n f o r m a t i o n o f s a m p l e d i m e n s i o n s , h o w e v e r , t h i s p r o c e d u r e is a l so v e r y t e d i o u s a n d t ime ". c o n s u m i n g w h e n t h e n u m b e r s o f s a m p l e s a n a l y z e d a r e c o n s i d e r e d . T h e r e f o r e , t h i s p r e s e n t i n v e s t i g a t i o n was u n d e r t a k e n in o r d e r to a s c e r t a i n w h e t h e r t h e e f f e c t o f e g g s i z e o n m e a s u r e m e n t o f r u p t u r e e n e r g y w o u l d b e o f s u f f i c i e n t m a g n i t u d e to m e r i t c o n s i d e r a t i o n i n f u r t h e r s t u d i e s . A s c a t t e r d i a g r a m o f t h e r e s u l t s o b t a i n e d is s h o w n i n F i g u r e 12 w h e r e i n r u p t u r e e n e r g y is p l o t t e d a g a i n s t e g g s u r f a c e a r e a . F r o m t h i s it c a n b e s e e n t h a t no a p p a r e n t c o r r e l a t i o n e x i s t e d b e t w e e n t h e s e two c h a r a c t e r i s t i c s . S u c h a f i n d i n g a g r e e s w i t h t h a t o f H a y e s a n d A r m s t r o n g (1942) who r e p o r t e d t h a t no c h a n g e i n t h e w e i g h t o f a n e g g f r o m t h e s p e c i e s Salmo salar L . c o u l d be a s s o c i a t e d w i t h a c h a n g e i n t h e f o r c e r e q u i r e d f o r m e m b r a n e r u p t u r e . H . E f f e c t s o f F r o z e n S t o r a g e S u b j e c t i v e a s s e s s m e n t o f c a v i a r m a d e f rom e g g s t h a t h a d b e e n p r e v i o u s l y 68. >• o ce ui z UJ UJ ec => t-0. 3 OC UJ z < IX CO 7 13.0 1 2 . 0 - 1 11.0 H 1 0 . 0 H 9.0-1 8.0-1 7.OH 6.0-1 5 . 0 H SURFACE AREA (mm2) F i g u r e 12. S c a t t e r d i a g r a m o f m e m b r a n e r u p t u r e e n e r g y as f u n c t i o n o f e o q s u r f a c e a r e a . 6-9. f r o z e n i n d i c a t e d t h a t s u c h c a v i a r was o f a d i f f e r e n t q u a l i t y t h a n t h a t made f r o m f r e s h e g g s ( S t e r n i n a n d H o r i , 1 982; Z a i t s e v , 1 969) . A c c o r d i n g l y , an i n v e s t i g a t i o n was c o n d u c t e d o n b o t h f r e s h a n d f r o z e n / t h a w e d u n s a l t e d e g g s to d i s c o v e r w h e r e t h e s o u r c e o f t h i s d i f f e r e n c e may l i e . S i n c e f i s h e g g s a r e r e a d i l y d i v i d e d in to t h e two p h y s i c a l s y s t e m s o f t h e i n t e r i o r f l u i d a n d t h e s u r r o u n d i n g m e m b r a n e , e v a l u a t i o n o f a n y e f f e c t s f r o z e n s t o r a g e may h a v e h a d was a p p l i e d to e a c h s y s t e m s e p a r a t e l y . 1. E f f e c t s o n y o l k It was c o n s i d e r e d u n l i k e l y t h a t t h e i m m i s c i b l e l i p i d d r o p l e t f o r m i n g a b o u t 1% ( M a r k e r t a n d V a n s t o n e , 1968) o f t h e i n t e r i o r f l u i d o f s a l m o n e g g s a n d c o n -s i s t i n g o f m a i n l y t r i g l y c e r i d e s ( N a k a g a w a a n d T s u c h i y a , 1971) w o u l d h a v e a l a r g e e f f e c t o n y o l k c o n s i s t e n c y . I n s t e a d , i f a n y e f f e c t w e r e to o c c u r as a r e s u l t o f s t o r a g e at low t e m p e r a t u r e s , it w o u l d most p r o b a b l y be f o u n d i n t h e p r o t e i n s o l u t i o n f o r m i n g a p p r o x i m a t e l y 98% ( v / v ) o f t h e e g g y o l k ( M a r k e r t a n d V a n s t o n e , 1 968) . S u c h an a l t e r a t i o n in p r o t e i n has b e e n n o t e d f o r a v i a n e g g y o l k u p o n s t o r a g e at t e m p e r a t u r e s below - 6 ° C (Kamat et a l . , 1976) . O n t h i s a s s u m p t i o n , a n a l y t i c a l m e t h o d s o f p o l y a c r y l a m i d e ge l e l e c t r o p h o r e s i s , d i f f e r e n -t ia l s c a n n i n g c a l o r i m e t r y a n d v i s c o m e t r i c a n a l y s i s w e r e a p p l i e d to c h u m y o l k in a n a t t e m p t to d i s c o v e r i f t h e p r o t e i n s o f t h e y o l k h a d u n d e r g o n e a n y m o d i -f i c a t i o n d u r i n g the , p e r i o d o f f r o z e n s t o r a g e . a) P o l y a c r y l a m i d e ge l e l e c t r o p h o r e s i s T h e r e s u l t s o f p o l y a c r y l a m i d e ge l e l e c t r o p h o r e s i s (PAGE) a r e r e p r e s e n t e d in g e l s d e p i c t e d in F i g u r e 13a a n d b . F r o m an e x a m i n a t i o n o f t h e s e g e l s , it c a n be s e e n t h a t no d i f f e r e n c e in t h e m o b i l i t y o f t h e f o u r p r o t e i n s o c c u r r e d a f t e r s e v e n d a y s s t o r a g e at - 1 0 ° C . T h e r e f o r e , no b r e a k d o w n o r a g g r e g a t i o n o f a n y p a r t i c u l a r p r o t e i n t o o k p l a c e , as was n o t e d fo r a v i a n e g g y o l k p r o t e i n s ( C h a n g et a l . , 1 9 7 7 ) . T h e l a t t e r r e s u l t was f o u n d to be d u e m a i n l y to t h e • # 1 t 1 a) 1 2 3 4 5 6 7 8 9 10 11 12 minium b ) 1 2 3 4 5 6 7 8 9 10 11 12 F i g u r e 13. E l e c t r o p h o r e t i c p a t t e r n s o f f r e s h a n d f r o z e n / t h a w e d n a t i v e a n d s a l t e d c h u m e g g y o l k o f two c o d e s . ( R e f e r to T a b l e 2 f o r m e t h o d o f o b t a i n i n g s a l t e d y o l k a n d a m o u n t s o f s a m p l e a p p l i e d to g e l . ) a) C o d e II - s lot 1, 2, 3, 4, 11, 12 : f r o z e n / t h a w e d y o l k ; s lot 5, 6 : f r e s h , s a l t e d y o l k ; s lo t 7, 8 : f r e s h y o l k ; s lot 9, 10 : f r o z e n / t h a w e d , s a l t e d y o l k . b ) C o d e VI - s lo t 1, 5, 6 : f r o z e n / t h a w e d y o l k ; s lo t 2, 7, 8 : f r o z e n / t h a w e d s a l t e d y o l k ; s lot 3, 9, 10 : f r e s h y o l k ; s lot 4, 11, 12 : f r e s h , s a l t e d y o l k . 71 . b r e a k i n g u p o f t h e m i c e l l a r s t r u c t u r e o f low d e n s i t y l i p o p r o t e i n s p r e s e n t in a v i a n y o l k as a r e s u l t o f a n 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 s a l t s c a u s e d b y t h e i n c o r p o r a t i o n o f w a t e r in to i ce c r y s t a l s t h r o u g h f r e e z i n g ( P o w r i e , 1 9 6 8 ) . T h e d i f f e r e n c e in b e h a v i o u r b e t w e e n t e l e o s t a n d a v i a n y o l k is most r e a d i l y e x p l a i n e d b y t h e a b s e n c e o f s u c h low d e n s i t y l i p o p r o t e i n s in t e l e o s t y o l k ( C o o k a n d M a r t i n , 1 9 6 9 ) . A s d i s c u s s e d p r e v i o u s l y , t h e a d d i t i o n o f d e o x y c h o l a t e to t h e r u n n i n g b u f f e r in t h e s y s t e m i n v o l v e d in t h i s s t u d y t e n d e d to o v e r c o m e s e p a r a t i o n o n t h e b a s i s o f c h a r g e a n d so s e p a r a t i o n w o u l d h a v e o c c u r r e d m a i n l y t h r o u g h d i f f e r e n c e s in m o l e c u l a r s i z e . It is a p p a r e n t t h e n f r o m r e s u l t s o f t h e e l e c t r o p h o r e s i s p e r -f o r m e d in t h i s s t u d y t h a t f r o z e n s t o r a g e at -10°C f o r s e v e n d a y s h a d no e f f e c t o n t h e m o l e c u l a r s i z e o f t h e p r o t e i n s , n o r d i d it i n d u c e a n y a g g r e g a t i o n o r b r e a k d o w n o f t h e m o l e c u l e s . A s m e n t i o n e d i n t h e M e t h o d s a n d M a t e r i a l s s e c t i o n , s a m p l e s o f y o l k f r o m s a l t e d e g g s w e r e a l so a n a l y z e d in t h e same m a n n e r as f r e s h a n d f r o z e n / t h a w e d y o l k a n d it a p p e a r s f r o m F i g u r e 1 3 t h a t t h e a d d i t i o n o f sa l t l i k e w i s e h a d no i n f l u e n c e o n t h e y o l k p r o t e i n s , as d e t e c t a b l e b y P A C E . b ) D i f f e r e n t i a l s c a n n i n g c a l o r i m e t r y T h e r e s u l t s o f a t y p i c a l t h e r m a l a n a l y s i s o f y o l k f rom f r e s h a n d f r o z e n / t h a w e d y o l k a r e p r e s e n t e d i n F i g u r e 14. A n a l y s i s b y d i f f e r e n t i a l s c a n n i n g c a l o r i m e t r y ( D S C ) p r o d u c e s a c u r v e i n d i c a t i n g t h e d i f f e r e n c e in t h e amount o f heat t h a t m u s t be s u p p l i e d to a s a m p l e i n o r d e r to m a i n t a i n i t s t e m p e r a t u r e e q u i v a l e n t w i t h t h a t o f a r e f e r e n c e ( D a n i e l s , 1 973) . V a r i a t i o n s in t h e amount o f heat t h a t is r e q u i r e d c a n be c a u s e d b y e n d o t h e r m i c r e a c t i o n s , s u c h as d e -n a t u r a t i o n . In t h e p r e s e n t s t u d y , t h e d e n a t u r a t i o n o f p r o t e i n s p r e s e n t in c h u m y o l k a r e b e i n g a n a l y z e d a n d it c a n b e s e e n f r o m F i g u r e 14 t h a t t h e c u r v e s o b t a i n e d f r o m f r e s h a n d f r o z e n / t h a w e d y o l k a r e v e r y s i m i l a r . A S t u d e n t ' s t 2.00 I A 1 1 1 1 1 1 340.00 350.00 360.00 370.00 380.00 390.00 400.00 T E M P E R A T U R E O O Figure 14. Typical DSC thermograms of fresh and frozen/thawed yolk from eggs of Code VI ({ ) = fresh yolk; ( ) = frozen/thawed yolk). 73. t es t p e r f o r m e d o n t h e d a t a r e p o r t e d in T a b l e s 8 a n d 9 i n d i c a t e d t h a t o n l y t h e e n t h a l p y o f d e n a t u r a t i o n ( T a b l e 9} s i g n i f i c a n t l y d i f f e r e d in o n l y o n e p e a k o f C o d e II a n d t h i s s i g n i f i c a n t d i f f e r e n c e was not r e p e a t e d in t h e s e c o n d c o d e a n a l y z e d . B a s e d o n t h e s e r e s u l t s , it a p p e a r s t h a t t h e c o n f o r m a t i o n o f t h e p r o t e i n s o f c h u m y o l k a r e a f f e c t e d v e r y l i t t l e , i f at a l l , b y f r o z e n s t o r a g e , s i n c e c o n f o r m a t i o n a l d i f f e r e n c e s s h o u l d a f f e c t t h e a m o u n t o f heat r e q u i r e d f o r d e n a t u r a t i o n to o c c u r . c ) V i s c o m e t r i c a n a l y s i s R h e o g r a m s o f f r e s h a n d f r o z e n / t h a w e d y o l k f o r two c o d i n g d a t e s a r e p r e -s e n t e d in F i g u r e 15. S t a t i s t i c a l a n a l y s i s o f t h e s l o p e s a n d l e v e l s o f t h e s e c u r v e s r e v e a l e d no s i g n i f i c a n t d i f f e r e n c e ( p < 0 . 0 5 ) , a n d so it a p p e a r s t h a t t h e r h e o l o -g i c a l b e h a v i o u r o f t h e y o l k was u n a f f e c t e d b y f r o z e n s t o r a g e . S i n c e t h e b e h a -v i o u r o f a s o l u t i o n is d e p e n d e n t u p o n i ts m o l e c u l a r s t r u c t u r e ( P r a d i p a s e n a a n d R h a , 1977; H e r m a n s s o n , 1975)', t h i s is a f u r t h e r i n d i c a t i o n t h a t no l a s t i n g c h a n g e has o c c u r r e d in t h e p r o t e i n s . A l l o f t h e a n a l y s e s p e r f o r m e d s u p p o r t t h e c o n c l u s i o n t h a t l i t t le o r no a l t e r a t i o n in p r o t e i n s as d e t e c t a b l e b y a n y o f t h e m e t h o d s e m p l o y e d , h a d o c c u r r e d as a r e s u l t o f s t o r a g e at -10°C f o r s e v e n d a y s . I n s t a b i l i t y in s t r u c t u r e o f t h e p r o t e i n s d u r i n g t h e p e r i o d o f f r o z e n s t o r a g e c o u l d be e x p e c t e d s i n c e t h e o v e r -al l b a l a n c e o f f o r c e s t h a t m a i n t a i n c o n f o r m a t i o n may h a v e b e e n d i s t u r b e d . L o w e r t e m p e r a t u r e s c o u l d h a v 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 s t r e n g t h o f h y d r o -p h o b i c i n t e r a c t i o n s , w h i l e i n c r e a s i n g t h e s t r e n g t h o f h y d r o g e n b o n d i n g ( T a b o r s k y 1970) . H o w e v e r , it a p p e a r s t h a t a n y d i s t o r t i o n in s t r u c t u r e t h a t m a y h a v e o c c u r r e d d u r i n g s t o r a g e at low t e m p e r a t u r e s was r e v e r s i b l e a n d t h e p r o t e i n s r e v e r t e d b a c k to n a t i v e fo rm w h e n h i g h e r t e m p e r a t u r e s w e r e r e s t o r e d . T a b l e 8. E n t h a l p y o f d e n a t u r a t i o n f o r i n d i v i d u a l p r o t e i n s o f f r e s h a n d f r o z e n / t h a w e d c h u m e g g y o l k . C o d e n o . C o n d i t i o n o f y o l k AHeat ( c a l / g y o l k , d b ) P e a k 1 Peak 2 & 3 II F r e s h 0 .29 2. 60 F r o z e n / t h a w e d 0.27 2 .41** VI F r e s h 0 .27 2. 95 F r o z e n / t h a w e d 0 .27 3.00 * * S i q n i f i c a n t l y d i f f e r e n t f rom v a l u e f o r P e a k 2 & 3 o f f r e s h y o l k f r o m t h e same c o d e ( p < 0 . 0 1 ) . 75. T a b l e 9. D e n a t u r a t i o n t e m p e r a t u r e s o f i n d i v i d u a l p r o t e i n s o f f r e s h a n d f r o z e n / t h a w e d c h u m e g g y o l k o f two c o d e s . D e n a t u r a t i o n t e m p e r a t u r e s (°C) C o d e n o . C o n d i t i o n o f y o l k P e a k 1 P e a k 2 P e a k 3 II F r e s h 86 .0 106. 7 110. 8 F r o z e n / t h a w e d 8 5 . 5 106. 6 110.7 VI F r e s h 86.4 107. 5 a F r o z e n / t h a w e d 8 1 . 3 104. 5 v a l u e s u n a v a i l a b l e f rom P e r k i n E l m e r T h e r m a l A n a l y s i s S t a t i o n . 220 0 100 200 300 «00 500 600 SHEAR RATE ($"') Figure 15. Rheograms of yolk from f resh and f rozen/ thawed eggs of two codes ( • = Code II; A -Code VI; ( ) = f resh e g g s ; ( ) = f rozen / thawed e g g s ) . 77. 2. E f f e c t ori m e m b r a n e r u p t u r e e n e r g y T h e r e s u l t s o f f o r c e d e f o r m a t i o n a n a l y s i s o f f r e s h a n d f r o z e n / t h a w e d i n t a c t e g g s a r e p r e s e n t e d in T a b l e 10. A S t u d e n t ' s t t e s t i n d i c a t e d a h i g h l y s i g n i f i c a n t d i f f e r e n c e in t h e r u p t u r e e n e r g y o f e g g m e m b r a n e s b e f o r e a n d a f t e r f r e e z e / t h a w i n g w i t h a n a v e r a g e 46% d e c r e a s e in p o s t f r o z e n / t h a w e d e g g m e m -b r a n e r u p t u r e e n e r g y . It a p p e a r s t h e n t h a t t h e a l t e r a t i o n t h a t has b e e n o b s e r v e d to o c c u r i n c h u m e g g s as a r e s u l t o f f r o z e n s t o r a g e r e s i d e s m a i n l y in t h e m e m b r a n e . T h i s c o u l d be e x p e c t e d , w h e n t h e c o m p o s i t i o n o f t h e m e m b r a n e is c o n s i d e r e d . C h u m s a l m o n e g g m e m b r a n e s , l i k e t h o s e o f a l l t e l e o s t s s t u d i e d , a r e c o m p l e x , at least p a r t i a l l y l aminate s t r u c t u r e s ( S t e h r a n d H a w k e s , 0 1 979; L a a l e , 1 9 8 0 ) . Hamor a n d C a r s i d e (1977) f o u n d t h e m e m b r a n e o f A t l a n t i c s a l m o n Salmo salar e g g s to b e made u p o f 92% w a t e r , a n d s i n c e t h e m e m b r a n e o f t h e r e l a t e d P a c i f i c s a l m o n is in all p r o b a b i l i t y o f s i m i l a r h i g h w a t e r c o n t e n t , be low f r e e z i n g p o i n t t e m p e r a t u r e s c o u l d be e x p e c t e d to p r o d u c e i ce c r y s t a l s t h a t w o u l d -r e a d i l y d i s r u p t m e m b r a n e s t r u c t u r e a n d t h u s w e a k e n i t . I. E f f e c t s o f c r y o p r o t e c t a n t s A n i m p o r t a n t a s p e c t t h a t m u s t be b o r n in m i n d w h e n c o n s i d e r i n g t h e e f f e c t o f c r y o p r o t e c t a n t s o n c h u m e g g s is t h e d i f f e r e n c e in s t r u c t u r e i n v o l v e d b e t w e e n t h e c e l l u l a r s u s p e n s i o n s , p r o t e i n s o l u t i o n s a n d t i s s u e s y s t e m s p r e -v i o u s l y r e p o r t e d in t h e l i t e r a t u r e d e a l i n g w i t h c r y o p r o t e c t a n t s , a n d t h e c h u m e g g m e m b r a n e . I n d i v i d u a l an ima l c e l l s a r e s u r r o u n d e d b y a n u l t r a t h i n p l a s m a m e m b r a n e a n d d u e to t h e i r s i z e a n d m e m b r a n e p e r m e a b i l i t y , a r e h i g h l y s u b j e c t to o s m o t i c s h o c k . V e g e t a b l e c e l l s a r e e q u a l l y f r a g i l e , b u t d o p o s s e s s t h e a d d e d a d v a n t a g e o f a r i g i d ce l l wal l w h i c h o f f e r s some p r o t e c t i o n a g a i n s t h y p o o s m o s i s . S u c h T a b l e 10. E f f e c t o f f r o z e n s t o r a g e o n m e m b r a n e r u p t u r e e n e r g y . M e a n m e m b r a n e r u p t u r e e n e r q y (J x 10^") C o d e n o . F r e s h Pos t f r e e z e / t h a w i n g II 3 .14 1.84** VI 5.87 3 .15** * * s i g n i f i c a n t at p<0.01 f r a g i l i t y is not r e f l e c t e d to a l a r g e d e g r e e in t h e c h u m e g g , w h i c h p o s s e s s e s a m e a s u r a b l y t h i c k e r m e m b r a n e o f g r e a t e r s t r u c t u r a l c o m p l e x i t y , m o r e l i k e l y c a p a b l e o f w i t h s t a n d i n g g r e a t e r i n t e r i o r s t r e s s t h a n t h e c e l l u l a r p l a s m a m e m -b r a n e . T h e r e f o r e , t h e p r o t e c t i o n a g a i n s t o s m o t i c s h o c k o f f e r e d b y t h e p e n e t r a t i n g c l a s s o f c r y o p r o t e c t a n t s is p r o b a b l y not a s i g n i f i c a n t a id in t h e f r e e z i n g o f c h u m e g g s . M o r e i m p o r t a n t is t h e i n h i b i t i o n o f i ce c r y s t a l f o r m a t i o n a f f o r d e d b y t h e w a t e r b i n d i n g c a p a c i t y o f t h e s e c r y o p r o t e c t i v e c o m p o u n d s u s e d . T h e r e s u l t s o f c r y o p r o t e c t i v e t r e a t m e n t s o n t h e r u p t u r e e n e r g y o f c h u m e g g s f rom f o u r d i f f e r e n t c o d e s a r e d e p i c t e d g r a p h i c a l l y in F i g u r e s 16, 17, 18 a n d 19. T h e i n d i v i d u a l v a r i a b i l i t y o f t h e e g g s c a n be s e e n f r o m t h e l a r g e s t a n d a r d d e v i a t i o n s . S u c h v a r i a b i l i t y was a lso e n c o u n t e r e d b y H a y e s (1942) f o r e g g s o f Salmo salar. W h e n t e n r e c e n t l y f e r t i l i z e d e g g s w e r e s u b j e c t e d to c o m p r e s s i o n , t h e s a m p l e mean was f o u n d to p o s s e s s a c o e f f i c i e n t o f v a r i a t i o n o f 20%. In a d d i t i o n , v a r i a t i o n i n m e m b r a n e r u p t u r e e n e r g y in r e s p o n s e to f r o z e n s t o r a g e b e t w e e n t h e d i f f e r e n t c o d e s c a n be s e e n f rom a c o m p a r i s o n o f v a l u e s f o r t h e c o n t r o l e g g s o f e a c h l o t . T h e p e r c e n t d e c r e a s e f rom f r e s h to frozen'£thawed c o n t r o l r a n g e d f rom 9 to 56% in t h e e g g m e m b r a n e s t r e n g t h o f C o d e s IV a n d V I I , r e s p e c t i v e l y . A s i m i l a r v a r i a t i o n in r e a c t i o n o f t h e e g g m e m b r a n e to o n e p a r t i c u l a r c r y o p r o t e c t a n t c a n a l so be s e e n f rom a c o m p a r i s o n o f e g g s f rom d i f f e r e n t c o d e s t r e a t e d w i t h s o d i u m c h l o r i d e . S u c h v a r i a t i o n r e v e a l s t h e i n f l u e n c e o f f a c t o r s o n r e s u l t s o b t a i n e d o t h e r t h a n t h e c r y o p r o t e c t i v e t r e a t m e n t i t s e l f , s u c h as t h e c o n d i t i o n a n d m a t u r i t y o f t h e e g g . A n y c o n c l u s i o n s d r a w n r e g a r d i n g t h e e f f e c t i v e n e s s o f a c r y o p r o -t e c t i v e t r e a t m e n t m u s t t h e n b e c o n s i d e r e d as s u b j e c t to a c e r t a i n a m o u n t o f v a r i a b i l i t y . P r e a n d p o s t f r e e z e / t h a w i n g r e s u l t s o f a n y c r y o p r o t e c t a n t w e r e c o n s i d e r e d as s e p a r a t e t r e a t m e n t s a n d w i t h i n e a c h l o t , t h e f a c t o r o f T r e a t m e n t was f o u n d 7.0 * 6.0 -: s.o >-O ac ui z U l U l oc 3 3 AC ui z < a: m z «.o -3.0 • 2.0 -1.0 -0.0 1.0M ETHYLENE CLYCOL 3.0M ETHYLENE CLYCOL 1.0M SODIUM PHOSPHATE 3.0M SODIUM PHOSPHATE TREATMENT Figure 16. Effect of treatment with var ious potential c ryopro tec t ive agents on membrane r u p t u r e energy of eggs from Code III. ( •= before f reez ing ; 0 = post f reeze / thawing ) . CO o > o IX u i u i IX 3 0. 3 IX UI z < IX CD s u i s 9.0 8.0 7.0 -6.0 5.0 -*.0 3.0 2.0 -1.0 ' 0.0 CONTROL 0.2M SODIUM CHLORIDE I.OM SODIUM CHLORIDE 2.0M SODIUM CHLORIDE 3.0M SODIUM CHLORIDE 0.3M SODIUM PHOSPHATE 0. 3M:0.2M SODIUM PHOSPHATE: SODIUM CHLORIDE 0.9M:O.SM SODIUM PHOSPHATE: SODIUM CHLORIDE TREATMENT F i g u r e 17 . E f f e c t o f t r e a t m e n t w i t h v a r i o u s p o t e n t i a l c r y o p r o t e c t i v e a g e n t s o n m e m b r a n e r u p t u r e e n e r g y o f e g g s f r o m C o d e I V . ( • = b e f o r e f r e e z i n g ; 0 = p o s t f reeze / t h a w i n g ) . CO 3 1 . 0 • T I ((±18.0) 3 0 . 0 T 7 . 0 " x 6 . 0 ->-O g 5 - 0 Z UI UI AC 3 H o. 3 et «.o z < 3 . 0 et CD s ui 3 2 . 0 • 1 .0 0 . 0 C O N T R O L 1.0M S O D I U M C H L O R I D E 2 . 0 M S O D I U M C H L O R I D E 3 .0M S O D I U M C H L O R I D E 0 .SM S U C R O S E 1 .0M S U C R O S E 1 .0M C A L C I U M C H L O R I D E 3 . 0 M C A L C I U M C H L O R I D E T R E A T M E N T Figure 18. Effect of treatment with various potential cryoprotective agents on membrane runture enemy of egas from Code VII (•= before freezing; 0= post freeze/ thawing). CO NJ 2.0M (5°C) 3.0M (5°C) TREATMENT Figure 19. Effect of treatment with various potential cryoprotective agents on membrane rupture energy of eggs from Code VIII (•= before freezing; O = post freeze/ thawing). CO OJ 84. was f o u n d to be a s i g n i f i c a n t s o u r c e o f v a r i a t i o n . A N e w m a n - K e u l ' s m u l t i p l e r a n g e t e s t t h e n e n a b l e d t h e g r o u p i n g o f means not s i g n i f i c a n t l y d i f f e r e n t f rom e a c h o t h e r . T o i n d i c a t e t h e i n i t i a l e f f e c t o f t h e c r y o p r o t e c t i v e t r e a t m e n t o n t h e m e m b r a n e r u p t u r e e n e r g y , as wel l as t h e d e g r e e to w h i c h m e m b r a n e r u p t u r e e n e r g y was m a i n t a i n e d t h r o u g h f r e e z e / t h a w i n g . T a b l e 11 r e p o r t s t h e p e r c e n t d i f f e r e n c e s in m e m b r a n e r u p t u r e e n e r g y b e t w e e n t h e f r e s h a n d p r e - f r e e z e / t h a w t r e a t e d e g g s , as wel l as t h e p e r c e n t d i f f e r e n c e b e t w e e n t h e p r e a n d p o s t f r e e z e / t h a w e d m e m -b r a n e r u p t u r e e n e r g i e s . N o n s i g n i f i c a n t d i f f e r e n c e s a r e i n d i c a t e d b y t h e l e t t e r s " N . S . " . O f p a r t i c u l a r i m p o r t a n c e a r e t h e t r e a t m e n t s w h i c h m a i n t a i n e d a m e m b r a n e r u p t u r e e n e r g y b o t h b e f o r e a n d a f t e r f r e e z e / t h a w i n g t h a t was not s i g n i f i c a n t l y d i f f e r e n t f rom t h a t o f t h e f r e s h c o n t r o l . T h e s e c o m p o u n d s w e r e e f f e c t i v e c r y o p r o t e c t a n t s in t h i s c o n t e x t . B o t h e t h y l e n e g l y c o l a n d g l y c e r o l s i g n i f i c a n t l y d e c r e a s e d t h e r u p t u r e e n e r g y o f e g g s b e f o r e f r o z e n s t o r a g e ( T a b l e 1 1 ) , most p r o b a b l y t h e r e s u l t o f a r e a c t i o n b e t w e e n t h e s e c o m p o u n d s a n d t h e p r o t e i n s p r e s e n t in t h e m e m b r a n e . Wi th t h e e x c e p t i o n o f e g g s t r e a t e d w i t h 1.0 M g l y c e r o l , no f u r t h e r d a m a g e r e s u l t e d f r o m f r o z e n s t o r a g e o f e g g s , w h i c h may i n d i c a t e some d e g r e e o f c r y o p r o t e c t i o n , h o w e v e r , t h e in i t i a l loss o f m e m b r a n e s t r e n g t h r e n d e r s t h e s e c o m p o u n d s u n s u i t a b l e as c r y o p r o t e c t a n t s . T h e f a i l u r e o f t h e 1.0 M g l y c e r o l t r e a t m e n t to p r e s e r v e a n y d e g r e e o f m e m b r a n e r u p t u r e e n e r g y may b e a r e f l e c t i o n o f t h e m u l t i m o l a r i t y r e q u i r e d f o r e f f e c t i v e n e s s o f some a g e n t s ( M e r y m a n , 1971) . M e m b r a n e r u p t u r e e n e r g i e s a f t e r t r e a t m e n t w i t h s o d i u m p h o s p h a t e v a r i e d s o m e w h a t . In e g g s o f C o d e III , no in i t i a l m e m b r a n e w e a k e n i n g o c c u r r e d w h e n t r e a t m e n t was w i t h s o d i u m p h o s p h a t e a l o n e . H o w e v e r , n e i t h e r was c r y o p r o t e c t i o n a p p a r e n t a n d t h i s l a c k may be a r e s u l t o f d a m a g e to m e m b r a n e c o m p o n e n t s c a u s e d b y t h e g r e a t e r c o n c e n t r a t i o n s o f p h o s p h a t e o c c u r r i n g i n t h e l i g u i d p h a s e as a r e s u l t o f t h e f r e e z i n g p r o c e s s . C o d e IV e g g s , t r e a t e d w i t h a l ower in i t i a l c o n -T a b l e 1 1 . . E f f e c t o f c r y o p r o t e c t i v e t r e a t m e n t s o n m e m b r a n e r u p t u r e e n e r g y o f e g g s b e f o r e a n d a f t e r f r e e z e / t h a w i n g . P e r c e n t d i f f e r e n c e in mean m e m b r a n e r u p t u r e e n e r g y C o d e T r e a t m e n t L e v e l b F r e s h e g g s a f t e r c r y o p r o t e c t i v e t r e a t m e n t C r y o p r o t e c t e d e g g s a f t e r f r o z e n s t o r a g e III N o n e - 4 6 . 2 S o d i u m 1.0M - 3 7 . 0 +11.8 N.S 3 - , c h l o r i d e 3 .0M - 2 .6 N. 'S . +17. 8 N . S . - • G l y c e r o l 1 .OM - 3 8 . 2 - 4 5 . 1 3 .0M - 4 3 . 6 - 4.1 N . S . S o d i u m 1 .OM + 8 .4 N . S . - 6 5 . 4 p h o s p h a t e 3.0M - 1 0 . 4 N . S . - 3 4 . 5 E t h y l e n e 1.0M - 3 2 . 3 - 1 5 . 8 N . S . g l y c o l 3.0M - 3 8 . 4 - 1.2 N . S . IV N o n e - 9. 3 N . S . S o d i u m 0. 2M - 2 7 . 2 N . S . + 5.4 N . S . c h l o r i d e 1 .OM - 8. 9 N . S . - 8. 3 N . S . 2 .0M +19.4 N . S . - 3. 6 N . S . 3.0M +24. 5 N . S . +53.0 S o d i u m 0. 3M +50.2 - 1 6 . 7 N . S . p h o s p h a t e S o d i u m p h o s - 0 . 3 M : 0 . 2M +50. 3 - 3 1 . 8 N . S . p h a t e ^sodium c h l o r i d e 0. 9 M : 0 . 5M +18.1 N. 'S . - 4 3 . 5 N . S . C o n t i n u e d . . . 86. T a b l e 11. C o n t i n u e d . P e r c e n t d i f f e r e n c e in mean m e m b r a n e r u p t u r e e n e r g y i F r e s h e g g s a f t e r C r y o p r o t e c t e d e g g s c r y o p r o t e c t i v e a f t e r f r o z e n C o d e T r e a t m e n t L e v e l t r e a t m e n t s t o r a g e VII N o n e - 5 6 . 7 N . S . S o d i u m 1.0M - 8 .7 N . S . - 1 5 . 8 N . S . c h l o r i d e 2.0M - 1 5 . 4 N . S . + 8. 9 N . S . 3.0M + 7.9 N . S . +12.0 N . S . S u c r o s e 0. 5M - 3 2 . 0 N . S . - 3.0 N . S . 1.0M - 1 8 . 8 N . S . - 2 .3 N . S . C a l c i u m 1.0M +788.0 - 7 6 . 5 c h l o r i d e 3.0M +15.2 N . S . - 1 0 . 1 N . S . VIII N o n e - 4 6 . 3 S o d i u m 1. 0M +4. 9 N . S . - 3 4 . 0 c h l o r i d e (25°C) 2. 0M - 4 . 5 N . S . - 8 .0 N . S . 3 .0M - 3. 9 N . S . - 3.5 N . S . S o d i u m 1.0M - 1 1 . 4 N . S . - 4 . 3 c h l o r i d e (5°C) 2 .0M - 8. 9 N . S . - 2 1 . 4 N . S . 3. 0M + 1.8 N . S . - 6.4 N . S . 9 N . S . i n d i c a t e s p e r c e n t d i f f e r e n c e s t h a t a r e not s i g n i f i c a n t ( p ^ O . 0 5 ) . 1 0 0 ( f r e s h u n t r e a t e d merrbrane r u p t u r e e n e r g y - f r e s h c r y o p r o t e c t e d m e m b r a n e r u p t u r e e n e r g y ) / f r e s h u n t r e a t e d m e m b r a n e r u p t u r e e n e r g y c 1 0 0 ( f r e s h c r y o p r o t e c t e d m e m b r a n e r u p t u r e e n e r g y - f r o z e n / t h a w e d c r y o p r o t e c t e d m e m b r a n e r u p t u r e e n e r g y ) / f r e s h c r y o p r o t e c t e d m e m b r a n e r u p t u r e e n e r g y . 87. c e n t r a t i o n ( i e . 0 . 3 M as c o m p a r e d to 1.0 M a n d 3.0 M) h a d a s i g n i f i c a n t s t r e n g t h e n i n g e f f e c t o n t h e m e m b r a n e , t h e c a u s e o f w h i c h is u n k n o w n , b u t p r o -b a b l y d u e to a n i n t e r a c t i o n w i t h t h e m e m b r a n e p r o t e i n s t h a t p r o v e s f a v o u r a b l e to m e m b r a n e s t r e n g t h o n l y at low c o n c e n t r a t i o n s o f s o d i u m p h o s p h a t e . F r o m T a b l e 11, it c a n be s e e n t h a t no s i g n i f i c a n t d e c r e a s e in t h i s e n h a n c e d m e m b r a n e r u p t u r e e n e r g y o c c u r r e d as a r e s u l t o f f r o z e n s t o r a g e . T h e u s e o f s u c r o s e as a c r y o p r o t e c t a n t d i d a p p e a r to h a v e f a v o u r a b l e r e s u l t s ( C o d e V I I I ) , w i t h m e m b r a n e r u p t u r e e n e r g y p r e s e r v e d b o t h b e f o r e a n d a f t e r f r o z e n s t o r a g e at t h e 1.0 M l e v e l . B a s e d o n i ts p o l y h y d r o x y n a t u r e , s u c r o s e c o u l d be e x p e c t e d to e x h i b i t c r y o p r o t e c t i v e p r o p e r t i e s a n d has b e e n i d e n t i f i e d as d o i n g so i n o t h e r s y s t e m s ( S o l i m a n a n d V a n d e n B e r g , 1971; M e r y m a n , 1 9 7 1 ) . T h e 1.0 M c o n c e n t r a t i o n o f c a l c i u m c h l o r i d e a d d e d h a d a s u b s t a n t i a l e f f e c t o n t h e m e m b r a n e r u p t u r e e n e r g y o f e g g s o f C o d e V I I , c a u s i n g a d r a s t i c i n -c r e a s e in m e m b r a n e t o u g h n e s s b e f o r e f r e e z e / t h a w i n g . T h e d i v a l e n t c a t i o n o f c a l c i u m has b e e n s h o w n to i n t e r a c t w i t h b o t h p r o t e i n a n d p o l y s a c c h a r i d e s , two o f t h e ma in c o m p o n e n t s o f c h u m e g g m e m b r a n e ( H a m o r a n d C a r s i d e , 1 977; K o b a y a s h i , 1 9 8 2 ) . I n t e r a c t i o n w i t h a p r o t e i n has b e e n e s t a b l i s h e d in t h e c a s e o f t h e mi lk p r o t e i n c a s e i n ( B r u n n e r , 1976) ar id is i n t e g r a l to mice l le s t r u c t u r e . A g e l o r p r e c i p i t a t e may fo rm o n t h e a d d i t i o n o f c a l c i u m to some p o l y s a c c h a r i d e s a n d is a r e s u l t o f a s s o c i a t i o n o f t h e d i v a l e n t ion w i t h a c i d i c g r o u p s o f t h e p o l y -s a c c h a r i d e ( H o d g e a n d O s m a n , 1 9 7 6 ) . S u c h i n t e r a c t i o n s may h a v e o c c u r r e d b e t w e e n t h e c a l c i u m a d d e d a n d t h e p r o t e i n a n d / o r p o l y s a c c h a r i d e c o m p o n e n t s o f t h e m e m b r a n e to p r o d u c e t h e i n i t i a l i n c r e a s e in s t r e n g t h n o t e d . H o w e v e r , no c r y o p r o t e c t i o n was s u b s e g u e n t l y i n d i c a t e d , w i t h a 76% d e c r e a s e in m e m b r a n e r u p t u r e e n e r g y a f t e r f r e e z e / t h a w i n g , i n d i c a t i n g t h a t s u c h i n t e r -a c t i o n s m u s t h a v e b e e n d i s r u p t e d . T h e h i g h e r c o n c e n t r a t i o n o f c a l c i u m c h l o r i d e ( 3 . 0 M) u t i l i z e d a p p e a r e d to 88. be o f v a l u e , h a v i n g b o t h a s m a l l e r i n i t i a l e f f e c t o n , a n d p r e s e r v i n g m o r e o f , t h e m e m b r a n e r u p t u r e e n e r g y d u r i n g f r e e z e / t h a w i n g . H o w e v e r , e x a m i n a t i o n o f t h e e g g s p r o d u c e d b y t h i s t r e a t m e n t r e v e a l e d t h a t t h e i n n e r f l u i d y o l k h a d i n c r e a s e d in c o n s i s t e n c y to an u n a c c e p t a b l e e x t e n t . A n y a p p a r e n t c r y o p r o -t e c t i v e e f f e c t was p r o b a b l y c a u s e d not b y a p r e s e r v a t i o n o f m e m b r a n e r u p t u r e e n e r g y b u t b y t h e r e s i s t a n c e to f o r c e o f f e r e d b y a s e m i - s o l i d y o l k . G e n e r a l l y , t h e a p p l i c a t i o n o f s o d i u m c h l o r i d e not o n l y a p p e a r e d c a p a b l e o f i n i t i a l l y m a i n t a i n i n g m e m b r a n e r u p t u r e e n e r g y , b u t a c t u a l l y a f f o r d e d some d e g r e e o f c r y o p r o t e c t i o n ( C o d e s III , I V , V a n d V I ) . T h i s r e s u l t is somewhat u n e x p e c t e d , s i n c e t h i s c o m p o u n d is not g e n e r a l l y c o n s i d e r e d to p o s s e s s u s e f u l c r y o p r o -t e c t i v e q u a l i t i e s , r a t h e r it is t h o u g h t to be a m o n g t h e i n j u r i o u s c o m p o u n d s p r e s e n t d u r i n g t h e p r o c e s s o f f r e e z i n g ( S o l i m a n a n d V a n d e n B e r g , 1971) . H o w e v e r , s o d i u m c h l o r i d e has b e e n u s e d as a c r y o p r o t e c t a n t in t h e f o o d i n d u s t r y in t h e p r e s e r v a t i o n o f s e v e r a l f o o d s , i n c l u d i n g e g g y o l k a n d f r u i t t i s s u e . In t h e f o r m e r c a s e , it was f o u n d to act d u r i n g f r e e z i n g b y d e c r e a s i n g t h e c o n c e n t r a t i o n o f p h o s p h a t e in t h e u n f r o z e n w a t e r p h a s e ( S o l i m a n a n d V a n d e n B e r g , 1971) , h o w e v e r t h e s y s t e m d e s c r i b e d in t h a t i n v e s t i g a t i o n was a p r o t e i n s o l u t i o n a n d so t h e same mode o f a c t i o n may not a p p l y to p r e s e r v a t i o n o f m e m b r a n e s t r u c t u r e . In t h e p r e s e n t s t u d y , r e s u l t s f r o m t r e a t m e n t s i n v o l v i n g c o m b i n a t i o n s o f s o d i u m p h o s p h a t e a n d s o d i u m c h l o r i d e ( C o d e V I ) a r e i n d e c i s i v e , d u e to t h e v a r i a b i l i t y in r e a c t i o n o f t h e c h u m e g g m e m b r a n e to s o d i u m p h o s p h a t e , h o w e v e r , it a p p e a r s t h a t t h e a d d i t i o n o f s o d i u m c h l o r i d e h a d l i t t l e s i g n i f i c a n t i n f l u e n c e . O n e o f t h e m e c h a n i s m s t h o u g h t to be i n v o l v e d in p r e s e r v a t i o n o f f r u i t t i s s u e f i r m n e s s was d e h y d r a t i o n o f t h e ce l l wal l ( S t e r l i n g , 1968) a n d t h i s is a p l a u s i b l e e x p l a n a t i o n f o r t h e a c t i o n o f s o d i u m c h l o r i d e i n t h i s s t u d y . B y r e m o v i n g w a t e r m o l e c u l e s f rom t h e l a m i n a r s y s t e m o f t h e m e m b r a n e a n d i n t e r f e r i n g w i t h t h e 89. p r o d u c t i o n o f l a r g e ice c r y s t a l s d u r i n g t h e p r o c e s s o f f r e e z i n g , m e m b r a n e s t r u c t u r e c o u l d be e x p e c t e d to r e m a i n i n t a c t to a g r e a t e r e x t e n t . O f al l o f t h e c r y o p r o t e c t i v e t r e a t m e n t s t e s t e d , s u c r o s e a n d s o d i u m c h l o r i d e w e r e t h e bes t c h o i c e s in t e r m s o f r e l a t i v e i n e f f e c t i v e n e s s o n m e m b r a n e s t r e n g t h b e f o r e f r e e z i n g a n d m a i n t a i n e n c e o f r u p t u r e e n e r g y d u r i n g f r e e z i n g . T h e u s e o f a s u c r o s e s o l u t i o n w o u l d n e c e s s i t a t e a p o s t - f r e e z i n g t r e a t m e n t in o r d e r to r i d t h e e g g s o f t h e u n d e s i r a b l e s w e e t - t a s t e . S o d i u m c h l o r i d e , o n t h e o t h e r h a n d , is a c t u a l l y u s e d in t h e p r o c e s s i n g o f t h e e g g s in to c a v i a r , a n d so l i t t le o r no p o s t - f r e e z i n g t r e a t m e n t s h o u l d be r e q u i r e d . O f al l l e v e l s t e s t e d , ~ a 2 .5 M s o l u t i o n o f t h i s c o m p o u n d g a v e t h e most u n i f o r m r e s u l t s , a n d c o n s e q u e n t l y , t h i s c o n c e n t r a t i o n o f s o d i u m c h l o r i d e was c h o s e n as t h e most c o n s i s t e n t a n d c o n -v e n i e n t c r y o p r o t e c t a n t to be u s e d in s u c c e s s i v e e x p e r i m e n t s . R e s u l t s r e p o r t e d in T a b l e 11 f o r C o d e VIII r e v e a l t h a t no s i g n i f i c a n t d i f -f e r e n c e in m a i n t e n a n c e o f m e m b r a n e r u p t u r e e n e r g y o c c u r r e d at t r e a t m e n t l e v e l s o f 2 .0 M a n d 3.0 M s o d i u m c h l o r i d e , w h e t h e r t h e e g g s w e r e t r e a t e d at 25°C, as in al l p r e v i o u s t e s t s , o r at 5°C . T h i s i n d i c a t e d t h e n t h a t t h e 2.5 M s o d i u m c h l o r i d e c r y o p r o t e c t i v e t r e a t m e n t w o u l d be c a p a b l e o f b e i n g u s e d in i n d u s t r y w i t h no a l t e r a t i o n in t h e u s u a l p a c k i n g p l a n t t e m p e r a t u r e o f 10°C. J . E f f e c t i v e n e s s o f C r y o p r o t e c t i o n b y S o d i u m C h l o r i d e 1. E f f e c t o f c r y o p r o t e c t i o n o n r a t e o f s o d i u m c h l o r i d e u p t a k e T h e p e r c e n t c h l o r i d e c o n t e n t s as a f u n c t i o n o f s a l t i n g t ime f o r t h e f r e s h , f r o z e n a n d c r y o p r o t e c t e d t r e a t m e n t s o f t h e C o d e s I X , X a n d XI a r e d e p i c t e d g r a p h i c a l l y in F i g u r e 20 . F r o m t h i s , it c a n b e s e e n t h a t in al l t h r e e c o d e s t h e r e was a d i f f e r e n c e in sa l t u p t a k e b y t h e c r y o p r o t e c t e d e g g s , c o m p a r e d to t h e f r e s h a n d f r o z e n / t h a w e d e g g s , w i t h c r y o p r o t e c t e d e g g s h a v i n g a h i g h e r c h l o r i d e c o n t e n t f o r t h e same p e r i o d o f i m m e r s i o n in t h e s a t u r a t e d s o d i u m c h l o r i d e 90. 12.0 T - 1 1 1 1 1 1 1 2 » 6 . 8 10 12 IS IS MINUTES IN SATURATED SODIUM CHLORIDE SOLUTION F i g u r e 2 0 . E f f e c t o f i m m e r s i o n in a s a t u r a t e d s o d i u m c h l o r i d e s o l u t i o n f o r v a r i o u s p e r i o d s o n t h e c h l o r i d e c o n t e n t o f f r e s h e g g s , e g a s p r e v i o u s l y f r o z e n / t h a w e d , a n d c r y o p r o t e c t e d e g g s f rom t h r e e c o d e s ( • = C o d e I X ; A = C o d e X ; • = C o d e X I ; ( ) = f r e s h e g g s ; ( ) = f r o z e n / t h a w e d e g g s ; ( ) = c r y o p r o t e c t e d e g g s ) . 91. s o l u t i o n . T h e f r e s h a n d f r o z e n / t h a w e d t r e a t m e n t s a p p e a r e d s i m i l a r in t h e i r r a t e o f sa l t u p t a k e , a l t h o u g h e g g s p r e v i o u s l y f r o z e n c o n s i s t e n t l y p o s s e s s e d a h i g h e r c h l o r i d e c o n t e n t f o r t h e same a m o u n t o f s a l t i n g t i m e , e s p e c i a l l y f o r p e r i o d s l o n g e r t h a n s i x m i n u t e s . T h e c u r v e s d e s c r i b e d a l o g a r i t h m i c r e l a t i o n s h i p a n d T a b l e 12 p r e s e n t s t h e r e s u l t s o f l i n e a r r e g r e s s i o n 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 : %C1 = m ( l o g t ime) + b (9) T h e c o e f f i c i e n t s o f d e t e r m i n a t i o n o b t a i n e d i n d i c a t e t h a t t h i s e q u a t i o n is a g o o d d e s c r i p t i o n o f t h e r e l a t i o n s h i p b e t w e e n s a l t i n g t ime a n d c h l o r i d e c o n t e n t f o r al l o f t h e t r e a t m e n t s . C o m p a r i s o n o f s l o p e a n d i n t e r c e p t s f o r f r e s h , f r o z e n / t h a w e d a n d c r y o p r o t e c t e d e g g s s u p p o r t e a r l i e r o b s e r v a t i o n s o n t h e s i m i l a r i t y o f t h e b e h a v i o u r ( i e . sa l t c o n t e n t a n d r a t e o f sa l t u p t a k e ) o f f r e s h a n d f r o z e n / t h a w e d e g g s , as c o m p a r e d to c r y o p r o t e c t e d . E x p l a n a t i o n f o r t h e s l i g h t l y l a r g e r v a l u e f o r s l o p e ( i n d i c a t i n g a m o r e r a p i d u p t a k e o f s a l t ) r e p o r t e d f o r t h e p r e v i o u s l y f r o z e n e g g s c a n b e f o u n d in t h e p r o b a b l e d a m a g e to t h e m e m b r a n e r e s u l t i n g f r o m f r o z e n s t o r a g e . S u c h i n j u r y c o u l d t h e n a l t e r t h e p e r m e a b i l i t y o f t h e m e m b r a n e a n d al low a m o r e r a p i d p e n e t r a t i o n o f s a l t . T h e l a r g e d i f f e r e n c e in b e h a v i o u r o f t h e c r y o p r o t e c t e d e g g s o b s e r v e d in t h i s s t u d y c a n b e a t t r i b u t e d to t h e h i g h e r sa l t c o n t e n t p o s s e s s e d at t h e i n i t i a t i o n o f s a l t i n g , d u e to t h e p r e - f r e e z i n g t r e a t m e n t w i t h 2.5 M s o d i u m c h l o r i d e f o r 30 m i n . In f a c t , t h e c h l o r i d e c o n t e n t p r e s e n t in t h e c r y o p r o t e c t e d e g g s at t h e s t a r t o f t h e s a l t i n g p e r i o d is in t h e n o r m a l r a n g e f o r t h e f i n a l p r o d u c t o f c a v i a r a n d so l i t t le o r no f u r t h e r s a l t i n g is a c t u a l l y n e c e s s a r y . Wi th r e s p e c t to t h e u s e o f t h i s c r y o p r o t e c t i v e t r e a t m e n t in i n d u s t r y , t h e s e r e s u l t s i n d i c a t e t h a t t h e r e is a d i f f e r e n c e in t h e b r i n i n g p r o c e s s r e q u i r e d f o r f r e s h e g g s a n d f o r t h o s e p r e v i o u s l y t r e a t e d w i t h t h e c r y o p r o t e c t a n t , in o r d e r to a c h i e v e e g g s o f t h e c o r r e c t sa l t c o n t e n t f o r c a v i a r . F u r t h e r e x p e r i m e n t a t i o n is n e e d e d to d e t e r m i n e more e x a c t l y t h e o p t i m u m t i m i n g o f t h e in i t i a l c r y o p r o -T a b l e 12. P a r a m e t e r s o b t a i n e d f rom l i n e a r r e g r e s s i o n o f s a l t i n g c u r v e s f o r c h u m e g g s . C o n d i t i o n o f C o d e n o . S l o p e I n t e r c e p t r u n s a l t e d e g g s (% c h l o r i d e ) F r e s h IX 0 .38 2.1 0 .96 X 0.46 1.9 1.00 F r o z e n / thawed XI 0. 37 2.2 0. 98 F r o z e n / t h a w e d IX 0 .47 1.9 0. 98 X 0. 50 2.0 1.00 XI 0 .53 1.7 1.00 C r y o p r o t e c t e d IX 0.21 5.5 0. 86 X 0.22 5. 9 0. 98 XI 0 .20 5. 6 0 .96 93. t e c t i v e t r e a t m e n t to e n s u r e e f f e c t i v e p r o t e c t i o n t h r o u g h f r o z e n s t o r a g e , y e t s t i l l p r o d u c e h i g h q u a l i t y c a v i a r o f t h e r e q u i r e d sa l t c o n t e n t . It w o u l d be d e s i r a b l e to h a v e a c r y o p r o t e c t e d e g g w i t h a p o s t f r e e z e / t h a w e d sa l t c o n t e n t l o w e r t h a n t h a t r e g u i r e d f o r c a v i a r , so t h a t a p o s t f r e e z e / t h a w s a l t i n g c a n be u s e d to a d j u s t t h e e g g s to t h e d e s i r e d c h l o r i d e c o n t e n t . F u r t h e r i n v e s t i g a t i o n in to t h e l o g a r i t h m i c r e l a t i o n s h i p b e t w e e n s a l t i n g t ime a n d c h l o r i d e c o n t e n t may r e v e a l t h i s to b e e q u a l l y a p p l i c a b l e to e g g s o f a n y '-*':/•• in i t i a l k n o w n c h l o r i d e c o n t e n t . If s o , t h e e q u a t i o n c o u l d b e u s e d to c a l c u l a t e t h e t ime p e r i o d r e q u i r e d to p r o d u c e a n y d e s i r e d f i n a l c h l o r i d e c o n t e n t , t h u s r e m o v i n g some o f t h e c o n j e c t u r e c o n c e r n i n g s a l t i n g t ime p r e s e n t l y i n h e r e n t in t h e p r o c e d u r e . 2. E f f e c t o f c r y o p r o t e c t i o n o n d e v e l o p m e n t o f a p p a r e n t v i s c o s i t y R e s u l t s o f v i s c o m e t r i c a n a l y s i s p e r f o r m e d o n y o l k f r o m f r e s h , f r o z e n / t h a w e d a n d c r y o p r o t e c t e d e g g s o f C o d e s I X , X a n d XI t h a t w e r e s u b j e c t e d to 3 to 15 m i n u t e s i m m e r s i o n in s a t u r a t e d s o d i u m c h l o r i d e , a r e p r e s e n t e d in t h e f o r m o f r h e o g r a m s in F i g u r e s 21 to 29 . T h e r e a p p e a r s to b e e n o u g h v a r i a t i o n in q u a n -t i t a t i v e r e s p o n s e to t r e a t m e n t s a n d s a l t i n g t imes b e t w e e n t h e t h r e e c o d e s t e s t e d to m a k e c o m b i n i n g t h e r e s u l t s u n d e s i r a b l e . H o w e v e r , t h e f low b e h a v i o u r o f all s a m p l e s a p p e a r s to b e s i m i l a r in t h a t , al l y o l k s e x h i b i t a p s e u d o p l a s t i c n a t u r e t h a t is c l o s e to N e w t o n i a n a n d t h e l e v e l o f o v e r a l l a p p a r e n t v i s c o s i t y w i t h i n e a c h t r e a t m e n t a n d c o d e is a f f e c t e d b y t h e c h l o r i d e c o n t e n t . R e g r e s s i o n a n a l y s i s was p e r f o r m e d o n t h e r h e o g r a m s d e p i c t e d in F i g u r e s 21 to 29, u s i n g t h e P o w e r law f low b e h a v i o u r m o d e l , a n d t h e p a r a m e t e r s o f f low b e h a v i o u r a n d c o n s i s t e n c y c o e f f i c i e n t a r e p r e s e n t e d in T a b l e 13. It is a p p a r e n t t h a t a l l f low b e h a v i o u r i n d i c e s ( n ) w e r e o n l y s l i g h t l y l ess t h a n 1 .00 , i n d i c a t i n g a p s e u d o p l a s t i c , n e a r N e w t o n i a n b e h a v i o u r t h a t was s u p p o r t e d b y t h e a c t u a l o b s e r v e d r h e o g r a m s . In al l c o d e s , b o t h t h e f low b e h a v i o u r i n d i c e s a n d t h e 700 600-Figure 21. Rheograms of yolk from fresh eggs of Code IX immersed for various Periods in a saturated sodium chloride solution (a = 0 min; b = 3 min; c = 4 mm; d = 5 mm; e - 6 mm. f - 10 mm) 0 . B > O u Z U l < a. a. < 700 600 500 . 400 -300 -200 " 100 -600 Fiqure 23. Rheoarams of volk from fresh eoqs of Code XI immersed for various periods in a saturated sodium chloride solution (a = o min; b = 3 min; c = 4 min; d = 5 mm; e = 6 mm; f - 10 mm; c - 15 min). 700 0. E i/l O u < a. a. < 600 -500 -400 -300 " 200 -100 — r ~ 100 — i — 200 300 SHEAR RATE (» l) — I — 400 — I — 500 600 Figure 24. Rheoqrams of yolk from frozen/thawed eags of Code IX immersed for various periods in a saturated sodium chloride solution (a = 0 min; b = 3 min; c = 4 mm; d - 5 mm; e = 6 mm; f = 10 min). _ , — , 1 — 1 1 ,00 200 300 MO SOO 600 SHEAR RATE U"1) Rheoqrams of yolk from frozen/thawed eqqs of Code X immersed for various periods in a saturated sodium chloride solution (a = 0 min; b = 3 min; c = U mm; d - 5 mm; e = 6 mm f = 10 min; q = 15 min). 700 100 " 0 I • 1 1 1 1 — 1 0 100 200 300 400 500 600 SHEAR RATE (s ') Figure 26. Rheoarams of yolk from frozen/thawed eggs of Code XI immersed for various periods in a saturated sodium chloride solution (b = 3 min; c = 4 min; d = 5 min; e = 6 min; f - 10 min g = 15 min). I— 100 — f — 200 —I 300 I— MO - 1 — 500 • 600 SHEAR RATE (•'> Fioure 27. Rheocrams of yolk from cryoprotected egos of Code IX immersed for various periods in a saturated sodium chloride solution (a = 0 min; b = 3 min; c = 4 mm; d = 5 mm; e - 6 mm f = 10 min). Figure 28. Rheograms of yolk from cryoprotected eggs of Code X immersed for various periods in a saturated sodium chloride solution (a = 6 min; b = 3 min; c = 4 min; d = 5 min; e = 6 min; f = 10 min; g = 15 min). 700 Figure 29. Rheograms of yolk from cryoprotected eggs of Code XI immersed for various periods in a saturated sodium chloride solution (b = 3 min; c = 4 min; d = 5 mm; e = 6 mm; f - 10 mm; g = 15 min). © T a b l e 13. M e a n v a l u e s o f P o w e r - l a w flow p a r a m e t e r s o f l i q u i d y o l k p r e p a r e d f rom f r e s h , f r o z e n / t h a w e d a n d c r y o p r o t e c t e d e g g s o f v a r i o u s c h l o r i d e c o n t e n t s f rom t h r e e c o d e s (n=3). C o d e IX C o d e X C o d e XI S a l t i n g C h l o r i d e m n C h l o r i d e m n C h l o r i d e m n C o n d i t i o n o f p e r i o d c o n t e n t c o n t e n t c o n t e n t u n s a l t e d e g g s (min) (%) (mPa s n ) (%) (mPa s ) -4%) (mPa s ) F r e s h 0 .0.48 5555 0.85 0.49 317 0. 87 0.44 — 3 3v06 160 0. 95 3.16 137 0. 93 3.50 288 0.87 4 3.65 299 0.86 3.57 163 0. 91 3.73 451 0.86 5 3.83 310 0.88 4.14 163 0.92 3.94 525 0.88 6 3. 91 281 0.87 4.30 175 0. 91 4.41 571 0.85 10 4. 90 583 0. 85 5.42 236 0. 88 5.41 666 0.85 15 — - — 6.63 377 0.85 6.17 684 0.83 F r o z e n / t h a w e d 0 0.43 498 0.84 0.41 2 94 0.88 — - — 3 3.18 417 0.86 3.56 183 0. 93 3.12 552 0. 82 4 3. 63 347 0.86 4.08 315 0.86 3.60 650 0.83 5 3.80 519 0.84 4. 37 275 0.88 4.33 497 0.84 6 4.47 515 0. 84 4. 93 299 0.88 4. 35 62 9 0.86 C o n t i n u e d . T a b l e 13. C o n t i n u e d . C o d e IX C o d e X C o d e XI S a l t i n g C h l o r i d e m n C h l o r i d e m n C h l o r i d e m n C o n d i t i o n o f u n s a l t e d e g g s p e r i o d (min) c o n t e n t (%) (mPa s " ) c o n t e n t (%) (mPa sn ) c o n t e n t (%) (mPa sn ) F r o z e n / t h a w e d 10 5.58 600 0. 86 7.15 349 0. 84 5. 83 683 0. 91 15 — - -- 7.13 592 0 .84 7 .43 711 0. 93 C r y o p r o t e c t e d 0 4.07 92 0. 98 4. 63 50 0. 96 — - — 3 7.12 165 0. 92 7. 62 40 1.04 7.32 180 0. 90 4 7 .49 124 0. 95 8.07 47 1.02 7.40 218 0 .89 5 7 .44 125 0. 95 8. 40 47 1.01 7.70 277 0 .86 6 7.66 167 0. 94 8. 52 52 1.03 7 .96 314 0. 89 10 9. 30 367 0. 86 9 .59 T)01 0. 96 9 .10 338 0 .86 15 — - -- 11.08 109 0. 95 9.86 665 0 . 8 4 o 105. c o n s i s t e n c y c o e f f i c i e n t s c a l c u l a t e d i n d i c a t e d t h e y o l k o f c r y o p r o t e c t e d e g g s to b e d i s s i m i l a r in c o m p a r i s o n w i t h t h o s e o f t h e f r e s h a n d f r o z e n / t h a w e d t r e a t -m e n t s . T h e h i g h e r v a l u e s f o r n o b t a i n e d f o r c r y o p r o t e c t e d e g g y o l k s i n d i c a t e d a b e h a v i o u r c l o s e r to N e w t o n i a n , a n d t h e l ower v a l u e s o f m r e v e a l e d t h i n n e r c o n s i s t e n c i e s t h a n t h e y o l k f r o m t h e o t h e r two t r e a t m e n t s , f o r t h e same s a l t i n g t imes a n d c h l o r i d e c o n t e n t s . B o t h c h l o r i d e c o n t e n t a n d v i s c o s i t y a r e i m p o r t a n t to t h e g u a l i t y a s s e s s m e n t o f c a v i a r , a n d h i g h q u a l i t y c a v i a r m u s t t h e n p o s s e s s o p t i m u m v a l u e s f o r t h e s e two p a r a m e t e r s at t h e same t i m e . E g g s t h a t r e q u i r e too h i g h o r too low a sa l t c o n t e n t to a c h i e v e t h e o p t i m u m v i s c o s i t y wi l l n e v e r p r o d u c e c a v i a r t h a t wil l b e j u d g e d o f h i g h q u a l i t y . T h e r e l a t i o n s h i p b e t w e e n c h l o r i d e c o n t e n t a n d a p p a r e n t v i s c o s i t y in t h e s a l t e d e g g s p r o d u c e d f r o m f r e s h , f r o z e n a n d c r y o p r o t e c t e d t r e a t m e n t s f o r t h e t h r e e c o d e s was i n v e s t i g a t e d a n d is d e p i c t e d g r a p h i c a l l y in F i g u r e s 30, 31 a n d 32. T w o c o n c l u s i o n s a r e a p p a r e n t f r o m t h e c u r v e s o b t a i n e d . In t h e f i r s t p l a c e , f o r e a c h c o d e , t h e a p p a r e n t v i s c o s i t i e s as a f u n c t i o n o f p e r c e n t c h l o r i d e a r e s i m i l a r f o r t h e f r e s h a n d f r o z e n / t h a w e d e g g s , w h i l e t h o s e o f t h e c r y o p r o t e c t e d e g g s a r e c o n s i d e r a b l y l e s s . S e c o n d l y , t h e r e is a s i g n i f i c a n t d r o p in a p p a r e n t v i s c o s i t y f o r a l l c u r v e s at v a l u e s o f 3 to 4% c h l o r i d e , f o l l owed b y a r i s e w i t h f u r t h e r i n c r e a s i n g c h l o r i d e c o n t e n t s . T h e c a u s e s o f t h e s e e f f e c t s c a n o n l y be s u r m i s e d o n t h e i n f o r m a t i o n a v a i l a b l e . It is k n o w n t h a t t h e c h u m e g g m e m b r a n e is p e r m e a b l e to b o t h w a t e r a n d i n o r g a n i c i o n s , b u t r e l a t i v e l y i m p e r m e a b l e to h i g h m o l e c u l a r w e i g h t s u b s t a n c e s ( E d d y , 1 9 7 4 ) . I m m e r s i o n o f c h u m e g g s in to a s a t u r a t e d s o d i u m c h l o r i d e s o l u t i o n a p p e a r e d to r e s u l t in not o n l y a n immedia te i n t a k e o f s a l t , b u t a l so o f w a t e r , as is r e v e a l e d b y t h e p u f f i n g u p o f t h e e g g s t h a t was o b s e r v e d , as t h e f l a c c i d i t y c o m m o n l y o b s e r v e d in u n f e r t i l i z e d f r e s h e g g s was l o s t , ( E d d y , 1 9 7 4 ) . S u c h a n i n t a k e o f w a t e r may a p p e a r to be an u n l i k e l y f low a g a i n s t t h e o s m o t i c p r e s s u r e 400 • a. E 300-< >• H in o u </) z U l oc < a. a. < 200-100-PER CENT CHLORIDE Figure 30. Effect of chlor ide content of eggs on apparent v iscos i ty of yo lk from f r e s h , f r o z e n / thawed and cryoprotected eggs of Code IX ( •= f resh e g g s ; • = f rozen/ thawed e g g s ; A = cryoprotected e g g s ) . o 300-, 8 10 ,2 PER CENT CHLORIDE Figure 31. Effect of chloride content of eggs on apparent viscosity of yolk from fresh, frozen/ thawed and cryoprotected eggs of Code X (•= fresh eggs; •= frozen/thawed eggs; • = cryoprotected eggs). o 500 ,00 I , , , , , 0 2 « 6 t 10 12 PER CENT CHLORIDE O CO Figure 32. Effect of chloride content of eggs on apparent viscosity of yolk from fresh, frozen/ thawed and cryoprotected eggs of Code XI (#= fresh eggs; • = frozen/thawed eggs! • = cryoprotected eggs). 109. c a u s e d b y t h e e x t e r n a l c o n c e n t r a t i o n o f s o d i u m c h l o r i d e , b u t s i m i l a r b e h a v i o u r a c r o s s a s e m i - p e r m e a b l e m e m b r a n e has p r e v i o u s l y b e e n r e c o r d e d in t h e l i t e r a t u r e . M e s c h i a a n d S e t n i k a r (1958) o b s e r v e d t h e f l o w i n g o f w a t e r a c r o s s a s e m i - <••:-p e r m e a b l e m e m b r a n e in to a d e x t r a n s o l u t i o n f r o m a u r e a s o l u t i o n w i t h 87 t imes t h e o s m o l a l i t y . H a r g e n s (1972) f o u n d s i m i l a r r e s u l t s w h e n a 0 .0015 M d e x t r a n s o l u t i o n d r e w a c r o s s a d i a l y z i n g m e m b r a n e a 0.2 M s o d i u m c h l o r i d e s o l u t i o n a p p r o x i m a t e l y 800 t imes s t r o n g e r . S u c h b e h a v i o u r may be e x p l a i n e d b y t h e t h e o r y o f S c h o l a n d e r ( S c h b l a n d e r , 1967; Hammel a n d S c h o l a n d e r , 1976) w h i c h t a k e s i n t o a c c o u n t h y d r o s t a t i c p r e s s u r e in a n e x p l a n a t i o n o f s o l u t e / s o l v e n t m o v e m e n t s a c r o s s s e m i - p e r m e a b l e m e m b r a n e s . T h e m e c h a n i s m p o s t u l a t e s t h a t w h e n e v e r s o l u t e m o l e c u l e s a r e c o n -f i n e d in a s o l v e n t , t h e y e x e r t p r e s s u r e o n t h e s o l v e n t s u r f a c e . T h i s s t r e s s is t r a n s m i t t e d h y d r a u l i c a l l y t h r o u g h o u t t h e s o l v e n t , g i v i n g r i s e to a " p a r t i a l h y d r o s t a t i c s o l v e n t p r e s s u r e " . If p l a c e d o n o n e s i d e o f a r i g i d m e m b r a n e i m p e r -meab le to t h e s o l u t e , t h i s p r e s s u r e h y d r a u l i c a l l y p u l l s t h r o u g h al l m o l e c u l e s f rom t h e o t h e r s i d e t h a t a r e a b l e to p e n e t r a t e t h e m e m b r a n e , u n t i l a n e g u i l i b r i u m is r e a c h e d . T h e r e f o r e , h i g h m o l e c u l a r w e i g h t s u b s t a n c e s s u c h as y o l k p r o t e i n s c o n f i n e d w i t h i n a n e x p a n s i b l e s e m i - p e r m e a b l e m e m b r a n e , as is t h e c a s e w i t h t h e c h u m e g g m e m b r a n e , c o u l d c r e a t e a p r e s s u r e t h a t w o u l d p u l l i n m o l e c u l e s c a p a b l e o f p e n e t r a t i n g t h e m e m b r a n e ( s u c h as s o d i u m c h l o r i d e ions a n d w a t e r m o l e c u l e s ) u n t i l t u r g o r p r e s s u r e e q u a l s t h e i n t e r i o r s o l u t e p r e s s u r e . A t t h i s p o i n t , f u r t h e r i n w a r d m o v e m e n t o f s o d i u m c h l o r i d e c o u l d t h e n s t i l l be e x p e c t e d to o c c u r d u e to d i f f u s i o n . In t h e p r e s e n t s t u d y t h e n , w h e n t h e e g g s w e r e f i r s t i m m e r s e d in t h e s a t u r a t e d s o d i u m c h l o r i d e s o l u t i o n , t h e r e was a n in i t i a l i n f l u x o f w a t e r as wel l as s a l t . T h i s immed ia te i n r u s h o f w a t e r s e r v e d to d e c r e a s e t h e a p p a r e n t v i s c o s i t y o f t h e y o l k o f e g g s t h a t w e r e r e m o v e d f rom t h e s o l u t i o n at t h i s p o i n t . F o r t h e e g g s i m m e r s e d f o r l o n g e r p e r i o d s , w h i l e a n i n c r e a s i n g t u r g o r p r e s s u r e s l o w e d a n d f i n a l l y h a l t e d 110. t h e i n f l u x o f w a t e r , t h e c o n c e n t r a t i o n o f sa l t c o n t i n u e d to i n c r e a s e b y d i f f u s i o n , p r o d u c i n g a c o r r e s p o n d i n g i n c r e a s e in y o l k v i s c o s i t y . T h e i n c r e a s e in a p p a r e n t v i s c o s i t y in a p r o t e i n s o l u t i o n w i t h 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 s o d i u m c h l o r i d e has b e e n n o t e d b e f o r e ( H e r m a n s s o n , 1975) . S u c h e v e n t s may e x p l a i n t h e r e l a t i o n s h i p r e v e a l e d b y F i g u r e s 30, 31 a n d 32. E x p l a n a t i o n f o r t h e d i s s m i m i l a r i t y o f t h e c r y o p r o t e c t e d e g g y o l k may a l so b e f o u n d w i t h i n S c h o l a n d e r ' s t h e o r y . P r i o r to f r e e z i n g , t h e c r y o p r o t e c t e d e g g s w e r e i m m e r s e d f o r 30 m i n u t e s in a 2 .5 M s o l u t i o n o f s o d i u m c h l o r i d e a n d c o u l d h a v e t h u s i m b i b e d m o r e w a t e r f o r a g i v e n i n f l u x o f sa l t t h a n w o u l d b e e x p e c t e d to o c c u r in a s a t u r a t e d sa l t s o l u t i o n . T h e r e s u l t o f t h i s w o u l d b e a y o l k w i t h a lower a p p a r e n t v i s c o s i t y f o r t h e same c h l o r i d e c o n t e n t d u e to h i g h e r w a t e r c o n t e n t . T h e a p p a r e n t v i s c o s i t y w o u l d t h e n b e e x p e c t e d to i n c r e a s e w i t h f u r t h e r e x p o s u r e to a s a t u r a t e d s o l u t i o n o f s o d i u m c h l o r i d e . . T h e s i g n i f i c a n c e t h a t t h e s e r e s u l t s h a v e o n t h e p o s s i b l e u s e o f c r y o p r o -t e c t i o n w i t h s o d i u m c h l o r i d e in t h e c a v i a r i n d u s t r y is s u b s t a n t i a l . C a v i a r p r o d u c e d in t h i s s t u d y f r o m c r y o p r o t e c t e d e g g s p o s s e s s e d a n a p p r e c i a b l y l ower v i s c o s i t y t h a n t h a t p r o d u c e d f r o m f r e s h e g g s f o r t h e r a n g e o f 4 to 5% c h l o r i d e c o n t e n t d e s i r e d in c a v i a r , a n d t h i s p o s e s a l a r g e p r o b l e m in a c h i e v i n g a c a v i a r p o s s e s s i n g at t h e same t ime o p t i m u m v a l u e s o f c o n s i s t e n c y a n d s a l i n i t y . 3. E f f e c t o f c r y o p r o t e c t i o n o n m e m b r a n e r u p t u r e e n e r g y V a l u e s f o r m e m b r a n e r u p t u r e e n e r g y o b t a i n e d t h r o u g h f o r c e d e f o r m a t i o n a n a l y s i s o f f r e s h , f r o z e n / t h a w e d a n d c r y o p r o t e c t e d e g g s i m m e r s e d in a s a t u r a t e d s o d i u m c h l o r i d e s o l u t i o n f o r p e r i o d s r a n g i n g f r o m 3 to 15 m i n u t e s f o r 3 c o d e s a r e p r e s e n t e d in T a b l e 14. R e s u l t s o f a t h r e e w a y a n a l y s i s o f v a r i a n c e a r e r e p o r t e d in T a b l e 15. T h e f a c t o r s o f C o d e a n d t h e C o n d i t i o n o f e g g b e f o r e s a l t i n g w e r e c o m p u t e d to b e h i g h l y s i g n i f i c a n t s o u r c e s o f v a r i a t i o n ( p < 0 . 0 1 ) . T h e r e s u l t s o f a N e w m a n - K e u l ' s m u l t i p l e r a n g e t e s t r e v e a l e d e a c h c o d e to be s i g n i f i c a n t l y d i f f e r e n t f rom t h e o t h e r . T h i s a g r e e s w i t h t h e t r e n d n o t e d in p r e v i o u s e x p e r i -T a b l e 14. M e a n m e m b r a n e r u p t u r e e n e r g i e s (J x 10 ) f o r s a l t e d f r e s h , f r o z e n / t h a w e d a n d c r y o p r o t e c t e d e g g s (n=25) . C o n d i t i o n o f e g g b e f o r e s a l t i n g C o d e 0 3 ' 4 5 . 6 10 15 F r e s h 1 X 7. 99 6.11 9 .03 7.12 6. 64 7. 76 ( 0 . 4 8 ) a ( 3 . 0 6 ) ( 3 . 6 5 ) ( 3 . 83) ( 3 . 91) ( 4 . 90) X 5.82 5.68 4. 95 6.47 5. 72 6. 12 6 .70 (0 .49) ( 3 . 1 6 ) (3 .57) ( 4 . 1 4 ) ( 4 . 30) ( 5 . 42) ( 6 . 6 3 ) XI 4.17 4. 31 3. 96 •4.24 ,4. 04 4. 10 4 .70 ( 0 . 4 4 ) (3 .50) (3 .73) ( 3 . 9 4 ) ( 4 . 41) ( 5 . 41) ( 6 . 1 7 ) F r o z e n 1 X 3.01 4 .04 4. 94 5.21 4. 91 5. 76 (o .43) ( 3 . 1 8 ) ( 3 . 6 3 ) ( 3 . 8 0 ) ( 4 . 47) ( 5 . 58) X 2.94 4.16 3.86 3. 90 4. 43 4. 05 4 .65 (0 .41) ( 3 . 5 6 ) ( 4 . 0 8 ) 4 .37) ( 4 . 93) ( 7 . 15) ( 7 . 1 3 ) XI 2.66 2.46 2.01 2. 39 2. 95 3.26 (3 .12) (3 .60) ( 4 . 3 3 ) ( 4 . 35) ( 5 . 83) ( 7 . 4 3 ) C r y o p r o t e c t e d IX 6.00 6. 80 7 .79 9.25 7. 74 7. 61 (4 .07) (7 .12) (7 .49) ( 7 . 4 4 ) ( 7 . 66) ( 9 . 30) X 4.62 6.32 6. 35 6.42 6. 58 7. 71 7 .84 (4 .63) (7 .62) ( 8 . 0 7 ) ( 8 . 4 0 ) ( 8 . 52) ( 9 . 59) ( 1 1 . 0 8 ) XI 3.85 4 .64 4.47 5. 17 4. 88 4 .83 ( 7 . 3 2 ) (7 .40) ( 7 . 7 0 ) ( 7 . 96) ( 9 . 10) ( 9 . 8 6 ) a V a l u e s e x p r e s s e d in b r a c k e t s r e p r e s e n t mean p e r c e n t c h l o r i d e c o n t e n t ( n =3). 112. T a b l e 15. A n a l y s i s o f v a r i a n c e o f m e m b r a n e r u p t u r e e n e r g y f o r e g g s o f v a r i o u s c h l o r i d e c o n t e n t s . S o u r c e o f v a r i a t i o n D . F . Mean s q u a r e F - r a t i o C o d e 2 80. .92 X 1 0 7 1 39. C o n d i t i o n o f e g g b e f o r e s a l t i n g 2 65. . 52 x 1 0 7 74. 76** M i n u t e s in s a t u r a t e d s o d i u m c h l o r i d e s o l u t i o n 4 1. 60 x 1 0 7 1. 26 C o d e x c o n d i t i o n 4 0. 88 x 1 0 7 1. 51 C o d e x m i n u t e s 8 1. 26 x 1 0 7 2. 18* C o n d i t i o n x m i n u t e s 8 0. 36 x 1 0 7 0. 46 C o d e x c o n d i t i o n x m i n u t e s 16 0. 78 X 1 0 7 1. 34 E r r o r 1080 0. 58 X 1 0 7 T o t a l 1124 * s i g n i f i c a n t at p<0.05 * s i g n i f i c a n t at p<0.01 113. mer i t s , i s . t h a t t h e r e may b e c o n s i d e r a b l e v a r i a t i o n in r e s p o n s e to t r e a t m e n t s f r o m o n e s h i p m e n t o f r o e to t h e n e x t . T h e m u l t i p l e r a n g e t e s t o f t h e f a c t o r o f C o n d i t i o n r e v e a l e d no s i g n i f i c a n t d i f f e r e n c e b e t w e e n r e s u l t s o b t a i n e d f rom f r e s h a n d c r y o p r o t e c t e d t r e a t m e n t s , b u t s u c h a d i f f e r e n c e d i d e x i s t b e t w e e n t h e s e a n d r e s u l t s o b t a i n e d f rom f r o z e n / t h a w e d e g g s . S u c h a n o u t c o m e r e f l e c t s t h e s u c c e s s f u l c r y o p r o t e c t i v e a b i l i t y o f s o d i u m c h l o r i d e in c h u m e g g m e m b r a n e in y i e l d i n g e g g s t h a t c o u l d be p r o c e s s e d in to c a v i a r o f c o m p a r a b l e m e m b r a n e s t r e n g t h to t h a t p r o d u c e d f r o m f r e s h . A last s i g n i f i c a n t s o u r c e o f v a r i a t i o n was r e v e a l e d in t h e i n t e r a c t i o n o f C o d e a n d M i n u t e s in s a t u r a t e d s o d i u m c h o r i d e s o l u t i o n . It is u n k n o w n w h y t h i s i n t e r a c t i o n w o u l d a p p e a r as a s i g n i f i c a n t f a c t o r , s i n c e t h e r e is no a p p a r e n t r e a s o n w h y an i n t e r a c t i o n b e t w e e n e g g s h i p m e n t a n d l e n g t h o f s a l t i n g t ime s h o u l d o c c u r , w h e n a s i g n i f i c a n t i n t e r a c t i o n b e t w e e n e g g s h i p m e n t a n d C o n d i t i o n o f e g g ( i e . f r e s h , f r o z e n o r c r y o p r o t e c t e d ) is m i s s i n g . K. S e n s o r y E v a l u a t i o n T h e two c h a r a c t e r i s t i c s o f y o l k v i s c o s i t y a n d m e m b r a n e r u p t u r e e n e r g y h a v e f o r m e d t h e b a s i s o f al l t h e o b j e c t i v e r h e o l o g i c a l s t u d i e s in t h i s i n v e s t i g a -t i o n . B o u r n e (1975) s t a t e s t h a t u l t i m a t e l y , it is t h e s u b j e c t i v e h u m a n r e s p o n s e to a p r o d u c t t h a t d e t e r m i n e s i ts s u c c e s s o r f a i l u r e . A c c o r d i n g l y , a s e n s o r y p a n e l a n a l y s i s was c o n d u c t e d in a n a t t e m p t to d e t e r m i n e i f j u d g e s c o u l d d i s c e r n a n y d i f f e r e n c e in y o l k v i s c o s i t y a n d m e m b r a n e s t r e n g t h b e t w e e n c a v i a r p r o d u c e d f rom f r o z e n e g g s a n d t h a t f rom c r y o p r o t e c t e d e g g s . A c o n t r o l s a m p l e o f h i g h g u a l i t y c a v i a r was i n c l u d e d in t h e s a m p l e s p r e s e n t e d as b o t h t h e r e f e r e n c e a n d as o n e o f t h e t r i a l s a m p l e s . T h i s was p e r f o r m e d to p r o v i d e a b a s i s f o r d e t e r - " m i n i n g t h e j u d g e s ' a b i l i t y to m a t c h t h e two c o n t r o l s a m p l e s p r e s e n t e d , as wel l as to s e e how t h e c a v i a r f rom f r o z e n a n d c r y o p r o t e c t e d e g g s w o u l d be a s s e s s e d i n r e l a t i o n to t h e h i g h q u a l i t y c o n t r o l . 114. R e s u l t s o f s e n s o r y e v a l u a t i o n o f t h e c h a r a c t e r i s t i c s o f m e m b r a n e s t r e n g t h a n d o f y o l k v i s c o s i t y a r e p r e s e n t e d in T a b l e 16. In t h e n i n e p o i n t s c a l e u s e d f o r t h e p a i r e d c o m p a r i s o n t e s t , a v a l u e o f 5 was u s e d as t h e c e n t e r p o i n t a n d a n y s a m p l e so r a n k e d w o u l d b e c o n s i d e r e d as e q u a l to t h e r e f e r e n c e . A v a l u e o f 1 was g i v e n to s a m p l e s r a n k e d " e x t r e m e l y t o u g h e r o r t h i c k e r " , w h i l e a v a l u e o f 9 was a s s i g n e d to s a m p l e s r a n k e d at t h e o t h e r e n d o f t h e s c a l e : " e x t r e m e l y w e a k e r o r t h i n n e r " . I n t e r m e d i a t e v a l u e s w e r e t h e n g i v e n to t h e d e s c r i p t i o n s l y i n g b e t w e e n t h e s e two e x t r e m e s ( see s a m p l e t e s t s in A p p e n d i x A ) . V a l u e s o b t a i n e d f o r e a c h t e s t w e r e s u b j e c t e d to a n a n a l y s i s o f v a r i a n c e w h i c h is p r e s e n t e d in T a b l e s T7 a n d 18. T h e p a r t i c u l a r a n a l y s i s e m p l o y e d p e r m i t s p a r t i t i o n i n g o f t h e S a m p l e s f a c t o r to al low e v a l u a t i o n o f a n y v a r i a t i o n i n t r o d u c e d b y t h e s i n g l e c o n t r o l c a v i a r s a m p l e in c o m p a r i s o n w i t h t h e r e s t o f t h e t r i a l s a m p l e s p r e s e n t e d ( C o n t r o l v s . c a v i a r f r o m c r y o p r o t e c t e d a n d f r o z e n / t h a w e d s a m p l e s ) , o r c a v i a r p r o d u c e d f r o m f r o z e n e g g s in c o m p a r i s o n w i t h tha t p r o d u c e d f rom c r y o p r o t e c t e d e g g s ( F r o z e n / t h a w e d v s . c r y o p r o t e c t e d s a m p l e s ) , as wel l as v a r i a t i o n i n t r o d u c e d w i t h i n t h e t h r e e r e p l i c a t e s o f e a c h o f t h e two e x p e r i -menta l t r e a t m e n t s ( R e p l i c a t e s w i t h i n c r y o p r o t e c t e d a n d f r o z e n / t h a w e d s a m p l e s ) . O f t h e s o u r c e s o f v a r i a n c e e v a l u a t e d , o n l y J u d g e s a n d C o n t r o l v s . c a v i a r f r o m c r y o p r o t e c t e d a n d f r o z e n / t h a w e d s a m p l e s w e r e s i g n i f i c a n t . T h e s i g n i f i -c a n c e o f t h e f a c t o r J u d g e s w o u l d i n d i c a t e t h a t i n d i v i d u a l v a r i a b i l i t y was h i g h in t h e s e n s o r y p a n e l . S u c h a r e s u l t i l l u s t r a t e s t h e p r o b l e m s in s p e c i f y i n g to a t a s t e p a n e l t h e p a r t i c u l a r c r i t e r i a b e i n g e v a l u a t e d . T h e s i g n i f i c a n c e o f t h e f a c t o r C o n t r o l v s . c a v i a r f r o m c r y o p r o t e c t e d a n d f r o z e n / t h a w e d s a m p l e s s h o w s t h a t t h e h i g h q u a l i t y c o n t r o l s a m p l e made f r o m f r e s h e g g s was r e a d i l y d i s c e r n e d f r o m t h e r e m a i n i n g s a m p l e s o f c a v i a r , b a s e d o n b o t h m e m b r a n e s t r e n g t h a n d y o l k v i s c o s i t y . Some e x p e r i m e n t a l e r r o r may h a v e b e e n i n t r o d u c e d at t h i s p o i n t d u e to t h e r a n c i d i t y o f t h e c a v i a r p r o d u c e d f rom f r o z e n / t h a w e d a n d c r y o p r o t e c t e d e g g s . T h i s f l a v o u r was c o m m e n t e d o n f a i r l y c o n s i s t e n t l y in t h e g u e s t i o n n a i r e 115. T a b l e 16. Mean r a t i n g s ( n i n e p o i n t s c a l e ) f o r y o l k v i s c o s i t y a n d m e m b r a n e s t r e n g t h j u d g e d b y s e n s o r y p a n e l f o r e a c h c a v i a r s a m p l e p r e s e n t e d ( n = 8 ) . C o n d i t i o n o f u n s a l t e d e g g s R e p l i c a t e V i s c o s i t y M e m b r a n e s t r e n g t h F r o z e n / t h a w e d 1 5 .4 d . 7 ) a 5.6 ( 0 . 7 ) 2 5.0 ( 1 . 1 ) 5. 3 ( 0 . 7 ) 3 5. 9 ( 1 . 0 ) 5.1 ( 0 . 6 ) C r y o p r o t e c t e d 1 6.1 ( 1 . 0 ) 5. 9 ( 1 . 3 ) -2 5. 5 ( 1 . 2 ) 5. 8 ( 1 . 3 ) 3 6.0 ( 1 . 3 ) 5 .5 ( 1 . 7 ) F r e s h ( c o n t r o l ) 4 .5 ( 0 . 9) 4 .6 ( 0 . 5 ) S t a n d a r d d e v i a t i o n 116. T a b l e 17. A n a l y s i s o f v a r i a n c e o f r a t i n g s f rom s e n s o r y a n a l y s i s o f m e m b r a n e s t r e n g t h . S o u r c e o f v a r i a t i o n S a m p l e s C o n t r o l v s . c r y o p r o t e c t e d a n d f r o z e n s a m p l e s F r o z e n v s . c r y o p r o t e c t e d s a m p l e s R e p l i c a t e s w i t h i n c r y o p r o t e c t e d a n d f r o z e n s a m p l e s J u d g e s E r r o r T o t a l D . F . M e a n s q u a r e F - r a t i o 6 1.48 1.35 1 5.50 5 .02* 1 1.69 4 .05 4 0.42 0 .38 7 3.21 2 .93** 42 1.10 55 * s i g n i f i c a n t at p<0.05 * * s i g n i f i c a n t at p<0.01 T a b l e 18. A n a l y s i s o f v a r i a n c e o f r a t i n g s f rom s e n s o r y a n a l y s i s o f y o l k v i s c o s i t y . S o u r c e o f v a r i a t i o n S a m p l e s C o n t r o l v s . c r y o p r o t e c t e d a n d f r o z e n s a m p l e s F r o z e n v s . c r y o p r o t e c t e d s a m p l e s R e p l i c a t e s w i t h i n c r y o p r o t e c t e d a n d f r o z e n s a m p l e s J u d g e s E r r o r T o t a l D . F . M e a n s q u a r e F - r a t i o 6 2.72 2.11 1 9.00 6 .97** 1 2.52 2 .09 4 1.21 0 .94 7 2 .77 2 . 1 5 * 42 1.29 55 S i g n i f i c a n t at p<0.05 * * s i g n i f i c a n t at p<0.01 118. d i s t r i b u t e d , a n d c o u l d h a v e b e e n c a u s e d b y i n a d e q u a t e f r o z e n s t o r a g e ( d u e to f r e e z e r b r e a k d o w n t h a t o c c u r r e d d u r i n g t h e s t o r a g e p e r i o d ) . A h e d o n i c s c a l e was i n c l u d e d in t h e s e n s o r y e v a l u a t i o n , to a s c e r t a i n w h e t h e r t h e r e was o v e r a l l p r e f e r e n c e f o r a n y p a r t i c u l a r c a v i a r s a m p l e , a n d r e s u l t s a r e r e p o r t e d in T a b l e 19. O n t h e n i n e p o i n t s c a l e u s e d , v a l u e s o f 1 a n d 9 r e p r e s e n t e d t h e j u d g e m e n t s o f " d i s l i k e e x t r e m e l y " a n d " l i k e e x t r e m e l y " , r e s -p e c t i v e l y . F o r s t a t i s t i c a l a n a l y s i s , a s i m p l e two w a y 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 o n t h e s e r e s u l t s to d e t e r m i n e i f t h e r e was a n y s i g n i f i c a n t d i f f e r e n c e in p r e f e r e n c e f o r a n y p a r t i c u l a r s a m p l e / s . C a l c u l a t i o n s as r e p o r t e d in T a b l e 20 s h o w e d t h a t b o t h S a m p l e s a n d J u d g e s w e r e s i g n i f i c a n t s o u r c e s o f v a r i a t i o n . T h e s i g n i f i c a n c e o f t h e J u d g e s t e r m a g a i n r e f l e c t s i n d i v i d u a l v a r i a b i l i t y in p r e -f e r e n c e o f t h e s a m p l e s . A N e w m a n - K e u l ' s m u l t i p l e r a n g e t e s t was p e r f o r m e d o n t h e s i g n i f i c a n t t e r m S a m p l e s a n d it was f o u n d t h a t mean p r e f e r e n c e s c o r e s f o r t h e t h r e e s a m p l e s o f c a v i a r p r o c e s s e d f rom c r y o p r o t e c t e d e g g s w e r e not s i g -n i f i c a n t l y d i f f e r e n t t h a n t h a t o f t h e h i g h g u a l i t y c o n t r o l , w h i l e t h e c a v i a r p r o -c e s s e d f rom t h e f r o z e n / t h a w e d e g g s r a n k e d s i g n i f i c a n t l y l ower in p r e f e r e n c e s c o r e s f o r two o u t o f t h e t h r e e r e p l i c a t e s . T h i s i n d i c a t e s t h a t a l t h o u g h no d i f f e r e n c e was d i s c e r n e d in t h e m e m b r a n e s t r e n g t h a n d y o l k v i s c o s i t i e s b e t w e e n c a v i a r f rom t h e f r o z e n / t h a w e d a n d c r y o p r o t e c t e d e g g s , g e n e r a l p r e f e r e n c e was e v i n c e d f o r t h a t p r o d u c e d f r o m c r y o p r o t e c t e d e g g s . T h e r e a r e two l im i ta t i ons i n h e r e n t in a s t u d y o f t h i s s o r t . F i r s t l y , as s t a t e d b y B o u r n e ( 1 9 7 5 ) , d u r i n g t h e m a s t i c a t i o n o f a f o o d in o r d e r to a s s e s s q u a l i t y , m a n y s e q u e n t i a l j u d g e m e n t s t a k e p l a c e , a n d t h e r e is a c o n t i n u o u s c h a n g e in p h y s i c a l p r o p e r t i e s w i t h r e s p e c t to d e s t r u c t i o n o f s t r u c t u r e , m i x i n g w i t h s a l i v a a n d t e m p e r a t u r e e f f e c t . C o n s i d e r i n g o n l y t h e "one e v e n t " p a r a m e t e r s o f m e m b r a n e r u p t u r e s t r e n g t h a n d y o l k v i s c o s i t y is l i m i t i n g in a n a s s e s s m e n t o f c a v i a r g u a l i t y a n d not r e p r e s e n t a t i v e o f t h e e n t i r e n a t u r e o f t h e f i na l p r o d u c t . H o w e v e r , as t h e s e two p a r a m e t e r s f o r m e d t h e b a s i s o f t h e d e v e l o p m e n t o f t h e 119. T a b l e 19. M e a n r a t i n g s f rom n i n e p o i n t h e d o n i c s c a l e f o r e a c h c a v i a r s a m p l e p r e s e n t e d . C o n d i t i o n o f u n s a l t e d e g g s R e p l i c a t e P r e f e r e n c e r a t i n g F r o z e n / t h a w e d 1 z 3 . 8 a ( 1 . 8 ) " 2 5 . l a b C ( 2 . 0 ) 3 4 . 9 a b ( 2 . 2 ) C r y o p r o t e c t e d 1 5 . 9 b C ( 1 . 6 ) 2 5 . 6 a b C ( 1 . 3 ) 3 5 . 4 a b C ( 1 . 3 ) F r e s h ( c o n t r o l ) 7 . 0 C ( 1 . 1 ) s t a n d a r d d e v i a t i o n z m e a n s s h a r i n g t h e same s u p e r s c r i p t a r e not s i g n i f i c a n t l y d i f f e r e n t ( p < 0 . 0 5 ) as d e t e r m i n e d b y N e w m a n - K e u l , s m u l t i p l e r a n g e t e s t . T a b l e 20. A n a l y s i s o f v a r i a n c e o f r a t i n g s f rom n i n e - p o i n t h e d o n i c s c a l e S o u r c e o f v a r i a t i o n D . F . ( M e a n s g u a r e F - r a t i o i S a m p l e s 6 7.88 4_ 4 4 * * J u d g e s 7 8 .49 4 . 7 8 * * E r r o r 42 1.77 T o t a l 55 * * s i g n i f i c a n t at p<0 .01 12il. c r y o p r o t e c t i v e t e c h n i q u e d e v i s e d , s e n s o r y e v a l u a t i o n o f t h e s e two c h a r a c t e r i s -t i c s c a n b e s a i d to f o r m t h e f i r s t p h a s e o f f u t u r e , m o r e i n t e n s e i n v e s t i g a t i o n s . In a d d i t i o n , t h e r a n c i d i t y o f t h e t r i a l s a m p l e s was p r o b a b l y a n i n f l u e n c e in j u d g e m e n t o f p a r a m e t e r s . F u r t h e r e v a l u a t i o n is t h e n n e c e s s a r y to more s u r e l y s t a t e t h e s u c c e s s o f t h e c r y o p r o t e c t i v e t e c h n i g u e in p r o d u c i n g a h i g h e r q u a l i t y c a v i a r t h a n t h a t p r o c e s s e d f rom f r o z e n / t h a w e d e g g s . 122. C O N C L U S I O N S F r o z e n s t o r a g e f o r l a t e r p r o c e s s i n g w o u l d be a v i a b l e s o l u t i o n to e v e n o u t t h e l a r g e f l u c t u a t i o n s in r o e s u p p l y t h a t p r o c e s s o r s d e a l i n g w i t h t h i s c o m m o d i t y a r e s e a s o n a l l y p r e s e n t e d w i t h . T h i s s t u d y was p e r f o r m e d to d e t e r m i n e t h e e x t e n t a n d l o c a t i o n o f d a m a g e in f r o z e n / t h a w e d e g g s a n d to i n v e s t i g a t e t h e p o s s i b i l i t y o f u t i l i z i n g a c r y o p r o t e c t a n t to a l l e v i a t e a n y s u c h a l t e r a t i o n s as w e r e f o u n d to o c c u r . T h e f i r s t p a r t o f t h e s t u d y i n v e s t i g a t e d t h e p h y s i c a l a s p e c t s o f t h e c h u m e g g . P r e v i o u s r e p o r t s o f t h e o c c u r e n c e o f at l eas t t h r e e major p r o t e i n s in s a l m o n e g g y o l k ( M a r k e r t a n d V a n s t o n e , 1968; 1971) w e r e c o n f i r m e d . D i f f e r e n -t ia l s c a n n i n g c a l o r i m e t r y r e v e a l e d t h r e e major p r o t e i n s ; e l e c t r o p h o r e s i s i n d i c a t e d t h e p r e s e n c e o f m o r e , h o w e v e r , t h i s d i f f e r e n c e m a y h a v e b e e n d u e to e i t h e r t h e d i f f e r e n c e s in s e n s i t i v i t y o f t h e two a n a l y s e s , o r to a m a s k i n g o f t h e heat d e n a t u r a t i o n p e a k s o f m i n o r p r o t e i n s b y t h e l a r g e p e a k s o b s e r v e d in t h e D S C t h e r m o g r a m . T h e f low b e h a v i o u r o f c h u m e g g y o l k was n o n - N e w t o n i a n , . p s e u d o p l a s t i c in n a t u r e . T h e d e g r e e o f d e v i a t i o n f rom a N e w t o n i a n f l u i d a n d t h e c o n s i s t e n c y v a r i e d f rom o n e c o d e d lot o f e g g s to a n o t h e r . T h e p r e v i o u s l y r e p o r t e d ( K o b a y a s h i , 1982; S t e h r a n d H a w k e s , 1 979) c o m p l e x n a t u r e o f t h e c h u m m e m b r a n e was a lso c o n f i r m e d , c o n s i s t i n g o f a n i n n e r l a y e r o f i n t e r t w i s t i n g f i b e r s a n d an o u t e r f i b r o u s l a y e r . A t h i r d i n t e r -m e d i a t e l a y e r p o s t u l a t e d to be p r e s e n t in t e l e o s t e g g m e m b r a n e s was not r e a d i l y a p p a r e n t in t h e p r e s e n t s t u d y . A s in p r e v i o u s i n v e s t i g a t i o n s w i t h o t h e r te leos t e g g s , t h e m e m b r a n e was c a p a b l e o f w i t h s t a n d i n g l a r g e f o r c e s o f c o m p r e s s i o n , a f e a t u r e a d v a n t a g e o u s to s p e c i e s w h o s e e g g s l ie in a mass o n t h e bot tom o f s t r e a m b e d s u n d e r a n e s t o f g r a v e l . T h e s e c o n d p a r t o f t h e s t u d y r e v e a l e d t h a t f r o z e n s t o r a g e f o r 7 d at -T0°C f o l l o w e d b y t h a w i n g in a i r at room t e m p e r a t u r e h a d l i t t le o r no e f f e c t o n t h e 123. e99 y ° , k p r o t e i n s as d e t e c t a b l e b y e i t h e r heat o f d e n a t u r a t i o n o r d e n a t u r a t i o n t e m p e r a t u r e , e l e c t r o p h o r e t i c p a t t e r n s , o r f low b e h a v i o u r . H o w e v e r , an a v e r a g e 46% d e c r e a s e in m e m b r a n e r u p t u r e e n e r g y was o b s e r v e d , i n d i c a t i n g t h a t t h e m e m b r a n e is t h e s o u r c e o f t h e d i f f e r e n c e d e t e c t e d b y s u b j e c t i v e o b s e r v a t i o n s b e t w e e n t h e f r e s h a n d t h e f r o z e n / t h a w e d e g g s . A s t u d y o f t h e p o t e n t i a l c r y o p r o t e c t a n t s t e s t e d in t h i s i n v e s t i g a t i o n r e -v e a l e d t h a t c r y o p r o t e c t i o n o f t h e c h u m e g g m e m b r a n e i n v o l v e d d i f f e r e n t = p r i n c i p l e s t h a n t h a t o p e r a t i n g in t h e u s u a l c e l l u l a r s u s p e n s i o n s r e p o r t e d in t h e l i t e r a t u r e . In t h i s s t u d y , h i g h l y e f f e c t i v e c e l l u l a r c r y o p r o t e c t a n t s s u c h as e t h y l e n e g l y c o l a n d g l y c e r o l h a d an a d v e r s e e f f e c t o n m e m b r a n e r u p t u r e e n e r g y , e v e n p r i o r to f r e e z i n g . T h e most e f f e c t i v e c r y o p r o t e c t a n t s t u d i e d , in t e r m s o f c o n s i s t e n t i n e f f e c t i v e n e s s o n m e m b r a n e r u p t u r e e n e r g y b e f o r e , a n d m a i n -t e n a n c e d u r i n g , f r e e z e / t h a w i n g was a 30 min i m m e r s i o n in a 2 .5 M s o l u t i o n o f s o d i u m c h l o r i d e . R e s u l t s o f a n a l y s i s o f c a v i a r p r o d u c e d f rom t h i s c r y o p r o t e c t a n t t r e a t m e n t i n d i c a t e d t h a t e g g s so p r o d u c e d d i d i n d e e d r e s u l t in c a v i a r w i t h a m e m b r a n e r u p t u r e e n e r g y c l o s e r to t h a t p r o d u c e d f rom f r e s h e g g s , t h a n t h a t p r o d u c e d f rom f r o z e n e g g s . H o w e v e r , a m o d i f i c a t i o n in t h e u s u a l b r i n i n g p r o c e d u r e is c a l l e d f o r to p r o d u c e c a v i a r o f t h e p r o p e r c h l o r i d e c o n t e n t , as m u c h s h o r t e r s a l t i n g t imes a p p e a r e d to be r e g u i r e d , t h a n w e r e n e c e s s a r y f o r e i t h e r f r e s h o r f r o z e n / t h a w e d e g g s . In a d d i t i o n , t h e c o n s i s t e n c y o f t h e r e s u l t i n g c a v i a r a p p e a r e d to be a d v e r s e l y a f f e c t e d , w i t h a lower a p p a r e n t v i s c o s i t y f o r a g i v e n c h l o r i d e c o n t e n t t h a t o c c u r s w i t h c a v i a r f rom e i t h e r f r e s h o r f r o z e n / t h a w e d e g g s . F u r t h e r e x p e r i m e n t a t i o n w i t h t h e c r y o p r o t e c t i v e t r e a t m e n t u s i n g a m o r e c o n c e n t r a t e d s o d i u m c h l o r i d e s o l u t i o n f o r a s h o r t e r p e r i o d o f t ime may not e v e n b e n e c e s s a r y , s i n c e t h e r a n g e o f a c c e p t a b l e a p p a r e n t v i s c o s i t i e s r e v e a l e d b y p r e v i o u s s e n s o r y a n a l y s e s is o n t h e o r d e r o f 400 to 1 500 m P a - s (10s x ) ( S t e r n i n a n d H o r i , 1 9 8 2 ) . 124. S e n s o r y p a n e l a n a l y s i s i n d i c a t e d t h a t t h e f r e s h h i g h q u a l i t y c o n t r o l c a v i a r was r e a d i l y d i f f e r e n t i a t e d o n t h e b a s i s o f b o t h m e m b r a n e s t r e n g t h a n d yc lJe v i s c o s i t y f rom t h e c a v i a r p r o d u c e d f rom b o t h t h e c r y o p r o t e c t e d a n d t h e f r o z e n / t h a w e d e g g s t h a t h a d b e e n s t o r e d f o r s i x w e e k s a t -20°C "in a s t i l l a i r f r e e z e r . H o w e v e r , t h e r a n c i d i t y o f t h e c a v i a r f r o m t h e p r e - f r o z e n e g g s may wel l h a v e b e e n a l a r g e s o u r c e o f e r r o r . M o r e e x p e r i m e n t a t i o n w i t h t h e e f f e c t o f l o n g s t o r a g e t imes in a m o r e c o n t r o l l e d f r o z e n s t o r a g e c o n d i t i o n o n t h e q u a l i t y o f c a v i a r p r o d u c e d f rom c r y o p r o t e c t e d e g g s is n e e d e d . E v e n w i t h t h e p r e s e n c e o f a r a n c i d t a s t e , h e d o n i c s t u d i e s w e r e p r o m i s i n g , r e v e a l i n g a p r e f e r e n c e f o r c a v i a r f rom c r y o p r o t e c t e d e g g s o v e r t h a t o f t h a t p r o d u c e d f rom f r o z e n / t h a w e d e g g s . R e s u l t s o f t h i s s t u d y i n d i c a t e t h a t it is p o s s i b l e to t r e a t c h u m e g g s p r i o r to f r e e z e / t h a w i n g s u c h t h a t t h e m e m b r a n e , t h e major s i t e o f f r e e z i n g d a m a g e , is p r o t e c t e d . What r e m a i n s is t h e n e e d f o r m o r e e x t e n s i v e s t u d i e s to c o -o r d i n a t e t h e o p t i m u m c o n c e n t r a t i o n a n d d u r a t i o n o f c r y o p r o t e c t i v e t r e a t m e n t w i t h t h a t o f a n y m o d i f i c a t i o n s o f p r e s e n t c a v i a r p r o c e s s i n g t e c h n i q u e to p r o d u c e a h i g h e r g u a l i t y c a v i a r t h a n t h a t p r e v i o u s l y a t t a i n a b l e w i t h t h e u s e o f u n p r o -t e c t e d e g g s . L I T E R A T U R E C I T E D A n d o , K. 1 965. U l t r a c e n t r i f u g a l a n a l y s i s o f y o l k p r o t e i n s in r a i n b o w t r o u t e g g a n d t h e i r c h a n g e s d u r i n g d e v e l o p m e n t . C a n . J . B i o c h e m . 43: 373-379. A O A C ( 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 ) . 1980. " 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 " , 12th e d i t i o n , e d . H o r o w i t z , W . , W a s h i n g t o n , D C . B a u e r , W. H. a n d C o l l i n s , E . A . 1967. T h i x o t r o p y a n d d i l a t a n c y . " in " R h e o l o g y . T h e o r y a n d A p p l i c a t i o n s " , V o l u m e 4, e d . , E i r i c h , F . R . , A c a d e m i c P r e s s , New Y o r k , C h a p t e r 8. B e l l , G . R . , H o s k i n s , C . E . a n d B a g s h a w , J . W. 1969. O n t h e s t r u c t u r e a n d e n z y m a t i c d e g r a d a t i o n o f t h e e x t e r n a l m e m b r a n e o f t h e s a l m o n e g g . C a n . J . Z o o l . 4 7 : 1 4 6 - 1 4 8 . B e r n a r d i , G . a n d C o o k , W. H . 1960. S e p a r a t i o n a n d c h a r a c t e r i z a t i o n o f t h e two h i g h d e n s i t y 3 l i p o p r o t e i n s o f e g g y o l k , a a n d ; 3 l i p o v i t e l l i n . B i o c h i m . B i o p h y s . A c t a 44:96 B e y e r , W. H . ( E d . ) 1 9 7 8 . " H a n d b o o k o f M a t h e m a t i c a l S c i e n c e s " ; 5th e d . T h e hemica l R u b b e r C o . , C l e v e l a n d , O H . 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 t h o d o f to ta 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 . J . B i o c h e m . P h y s i o l . 3 7 : 9 1 1 . B o u r n e , M . C . 1975. Is r h e o l o g y e n o u g h f o r f o o d t e s t u r e m e a s u r e m e n t ? J . T e x t . S t u d i e s 6 : 2 5 9 - 2 6 2 . B r u n n e r , J . R. 1 976. C h a r a c t e r i s t i c s o f e d i b l e f l u i d s o f an ima l o r i g i n : m i l k . in " P r i n c i p l e s o f F o o d S c i e n c e , P a r t I. F o o d C h e m i s t r y " , e d . F e n n e m a , O . , M a r c e l D e k k e r , I n c . New Y o r k , p p . 619-658 . C h a n g , P . , P o w r i e , W. D . a n d F e n n e m a , O . 1970. D i s c ge l e l e c t r o p h o r e s i s o f p r o t e i n s i n n a t i v e a n d heat t r e a t e d a l b u m i n , y o l k a n d c e n t r i f u g e d 2 w h o l e e g g . J . 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J . T e x t . S t u d i e s 9 : 3 - 3 1 . T u n g , M . A . 1 983. P e r s o n a l c o m m u n i c a t i o n . D e p a r t m e n t o f F o o d S c i e n c e , 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 , V a n c o u v e r , B . C . V a n W a z e r , J . R . , L y o n s , J . W . , K i m , K. Y . a n d C o l w e l l , R. E . 1963. " V i s c o s i t y a n d F low M e a s u r e m e n t " , I n t e r s c i e n c e P u b l i s h e r s , New Y o r k . V o i s e y , P, W. 1971. M o d e r n i z a t i o n o f t e x t u r e i n s t r u m e n t a t i o n . J . T e x t . S t u d i e s 2 : 1 2 9 - 1 9 5 . V u o r e l a , R . , K a i t a r a n t a , J . a n d L i n k o , R. 1979. P r o x i m a t e c o m p o s i t i o n o f f i s h roe in r e l a t i o n to m a t u r i t y . J . I n s t . C a n . S c i . T e c h n o l . A l i m e n t 1 2 ( 4 ) : 1 8 6 - 1 8 8 . W a l l a c e , R. A . 1965. R e s o l u t i o n a n d i s o l a t i o n o f a v i a n a n d a m p h i b i a n y o l k g r a n u l e p r o t e i n s u s i n g T E A E c e l l u l o s e . A n a l . B i o c h e m . 1 1 : 2 9 6 - 3 1 1 . W a l l a c e , R. A . , J a r e d , D . W. a n d E i s e n , A . Z . 1966. A g e n e r a l m e t h o d f o r t h e i s o l a t i o n a n d p u r i f i c a t i o n o f p h o s v i t i n f rom v e r t e b r a t e e g g s . C a n . J . B i o c h e m . 4 4 : 1 6 4 7 - 1 6 5 5 . W a l l a c e , R. A . , W a l k e r , S . L . a n d H a u s c h k a , P . V . 1967. C r u s t a c e a n l i p o -v i t e l l i n . I so la t ion a n d c h a r a c t e r i z a t i o n o f t h e major h i g h - d e n s i t y l i p o p r o t e i n f rom t h e e g g s o f d e c a p o d s . B i o c h e m i s t r y 6 :1582-1590 . W h o r l o w , R. W. 1980. T u b e v i s c o m e t e r s in " R h e o l o g i c a l T e c h n i q u e s " , E l l i s H o r w o o d , L t d . , C h i c h e s t e r , p p . 75 . W i n d h o l z , M . , B u d a v a r i , S . , S t r o u m t s o s , L . Y . a n d F e r t i g , M . N . 1976. " T h e M e r c k I n d e x " , 9th e d . , M e r c k a n d C o . , I n c . , R a h w a y , N J . W r i g h t , D . J . , L e a c h , I. B . a n d W i l d i n g , P . 1977. D i f f e r e n t i a l s c a n n i n g c a l o r i m e t r i c s t u d i e s o f m u s c l e a n d i ts c o n s t i t u e n t p r o t e i n s . J . S c i . F o o d A g . 2 8 : 5 5 7 - 5 6 4 . Z a i t s e v , V . , K i z e v e t t e r , I . , L a g u n o v , L . , M a r a n o v a , T . , M i n d e r , L . a n d P o d s e v a l o v , V . 1969. P r o c e s s i n g roe i n " F i s h C u r i n g a n d P r o c e s s i n g " M i r P u b l i s h e r s , M o s c o w , p p . 261-292. Z a r , J . H . 1974. " B i o s t a t i s t i c a l A n a l y s i s " , P r e n t i c e - H a l l , I n c . , E n g l e w o o d C l i f f s , N J . 135. APPENDIX 136. MULTIPLE COMPARISONS TEST -CAVIAR-Name . Date You are receiving samples of caviar to compare for viscosity and membrane strength. You have been given a reference sample, marked R, with which you are to compare each sample. Make judgements on more than one egg and retest as necessary to be sure. Test the samples i n the order i n which they have been presented ( l e f t to right). 1. Viscosity - Bite eggs with front teeth; make judgement on the inner l i q u i d of the eggs for viscosity. Determine whether the yolk i s thicker than, comparable to, or thinner than the reference. Then mark the amount of difference that exists. Sample number Thicker than R Equal to R Thinner than R Amount of Difference: None Slight Moderate Much . Extreme 2. Membrane Strength - Bite eggs with front teeth; make judgement on force required to rupture eggs. Determine whether the mem-brane i s tougher than, comparable to or weaker than the reference. Then mark the amount of difference that exists. Sample number Tougher than R Equal to R Weaker than R Amount of Difference: None Slight Moderate Much Extreme 137. Hedonic S c a l e S c o r i n g Date T a s t e r P r o d u c t T a s t e t h e s e samples and check how much you l i k e o r d i s l i k e each one. Use t h e a p p r o p r i a t e s c a l e t o show your a t t i t u d e by c h e c k i n g a t the p o i n t t h a t b e s t d e s c r i b e s y o u r f e e l i n g about t h e sample. P l e a s e g i v e a r e a s o n f o r t h i s a t t i t u d e . Remember you a r e t h e o n l y one who can t e l l what you l i k e . An honest e x p r e s s i o n o f your p e r s o n a l f e e l i n g w i l l h e l p us. Code Code Code'! Cod§ L i k e L i k e L i k e L i k e e x t r e m e l y e x t r e m e l y e x t r e m e l y e x t r e m e l y L i k e L i k e L i k e L i k e v e r y much v e r y much v e r y much v e r y much L i k e L i k e L i k e L i k e m o d e r a t e l y m o d e r a t e l y m o d e r a t e l y m o d e r a t e l y L i k e L i k e " L i k e L i k e s l i g h t l y s l i g h t l y s l i g h t l y s l i g h t l y N e i t h e r V.]::..•: N e i t h e r N e i t h e r N e i t h e r l i k e n o r l i k e n o r l i k e nor l i k e nor d i s l i k e d i s l i k e d i s l i k e d i s l i k e D i s l i k e D i s l i k e D i s l i k e D i s l i k e s l i g h t l y s l i g h t l y s l i g h t l y s l i g h t l y D i s l i k e D i s l i k e D i s l i k e D i s l i k e m o d e r a t e l y m o d e r a t e l y m o d e r a t e l y m o d e r a t e l y D i s l i k e D i s l i k e D i s l i k e D i s l i k e v e r y much v e r y much v e r y much v e r y much D i s l i k e ? ; ^  D i s l i k e D i s l i k e D i s l i k e e x t r e m e l y e x t r e m e l y e x t r e m e l y e x t r e m e l y Reason: Reason: Reason: Reason: 

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