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Measurement of plasma cortisol and histometry of the interrenal gland of juvenile pre-smolt coho salmon… Allan , Gerald D. 1971

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MEASUREMENT OF PLASMA CORTISOL AND HISTOMETRY OF THE INTERRSKAL GLAND OF JUVENILE. PRE-SMOLT COHO SALMON (ONCORHYNCHUS KISUTCH WALBAUM) DURING COLD TEMPERATURE ACCLIMATION • b y GERALD D. ALLAN B.Sc. (1964) U n i v e r s i t y o f B r i t i s h Columbia A T h e s i s Submitted i n P a r t i a l F u l f i l m e n t of the Requirements f o r the Degree of Master of Science In the Department of Zoology We accept t h i s t h e s i s as conforming t o the r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA August, 1371 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r an advanced degree at 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 a g r e e 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 the Head o f my Department o r by h i s 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 not 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 The U n i v e r s i t y o f B r i t i s h C o l umbia Vancouver 8, Canada • Date 2f>/ ABSTRACT Juvenile, pre-smolt coho salmon were subjected to a decrease in environmental temperature from 12°C (temperature of acclimation) to 2°C over a period of approximately 72 hours. During t h i s time, plasma C o r t i s o l values were estimated by the competitive protein binding (CPB) technique. In addition, an histometric analysis of the i n t e r r e n a l tissues of these fishes was performed as a measure of i n t e r r e n a l a c t i v i t y . ..Experimental r e s u l t s indicated that fluctuations in plasma C o r t i s o l concentrations occurred within 120 hours of the i n i t i a t i o n of temperature a l t e r a t i o n . Control leve l s for plasma C o r t i s o l were 2.9 +_ 0.75 ug c o r t i s o 1/100 ml plasma (mean jhS.D.). Maximum plasma C o r t i s o l concentration, observed at hour 84 a f t e r temperature a l t e r a t i o n , was 27.0 + 2.8 ug c o r t i s o l / 1 0 0 ml plasma (mean + S.E.). By hour 96 experimental C o r t i s o l values returned to a l e v e l j u s t s l i g h t l y above those of controls and did not change s i g n i f i c a n t l y a f t e r that time. Measurements of i n t e r r e n a l nuclear diameters showed a s i g n i f i c a n t increase in i n t e r r e n a l a c t i v i t y 14 days afte r i n i t i a t i o n of exposure to cold. This l e v e l of i n t e r r e n a l a c t i v i t y was maintained u n t i l the experiment was terminated (20 days exposure to cold). It i s concluded from this study t h a t during acclimation to cold temperature, plasma C o r t i s o l values of juvenile, p r e - s m o l t coho salmon d e m o n s t r a t e an e a r l y and r a p i d i n c r e a s e ( w i t h i n 120 hour s exposure t o c o l d e r t e m perature) f o l l o w e d by an e q u a l l y r a p i d d e c r e a s e t o a l e v e l j u s t s l i g h t l y above t h a t o f c o n t r o l s . F u r t h e r m o r e , i t i s c o n c l u d e d t h a t j u v e n i l e , p r e - s m o l t coho salmon t r e a t e d i n t h i s manner show no h i s t o l o g i c a l l y d e m o n s t r a b l e i n c r e a s e i n i n t e r r e n a l a c t i v i t y u n t i l w e l l a f t e r plasma C o r t i s o l v a l u e s have become s t a b i l i z e d a t a l e v e l s l i g h t l y above t h a t o f c o n t r o l s (14 days exposure t o c o l d ) . Abstract i L i s t of Figures v L i s t of Tables v i Acknowledgements v i i Introduction v i i i SECTION I - MATERIALS AND METHODS Experimental Animals 1 Description of Tanks 1 Holding Tank - Tank A 2 Control Tank - Tank B . . . 2 Experimental Tank - Tank C 2 Experimental Design Experiment One 5 Experiment Two 6 Autopsy Procedures H i s t o l o g i c a l Procedures H i s t o l o g i c a l technique 8 Karyometry . 8 S t a t i s t i c a l Procedures C o r t i s o l analysis 9 H i s t o l o g i c a l analysis 10 Quantification of Plasma C o r t i s o l by Competitive Protein Binding (CPB) Introduction 11 CPB - Fagerlund modification Materials 13 Method 14 Discussion 18 SECTION II - EXPERIMENTAL RESULTS Experiment One Experiment Two Karyometry SECTION III - DISCUSSION SECTION IV - CONCLUSIONS SECTION V - REFERENCES 8 Head kidney of Experiment One coho -Day 14 experimental f i s h Figure 1 Holding tank 4 2 Control tank 4 3 Experimental tank showing thermo- 4 regulating controls 4 Apparatus for d i s t r i b u t i o n of nitrogen gas 4 5 Head kidney of Experiment One coho - 23 Day 0 control f i s h 6. Head kidney of Experiment One coho - w 23 Day 0 experimental f i s h 7 Head kidney of Experiment One coho - 23 Day 14 control f i s h 23 9 Plasma C o r t i s o l concentration of juvenile coho salmon yearlings during cold temperature 26 acclimation - Experiment One 10 Plasma C o r t i s o l concentration of juvenile 30 coho salmon yearlings during cold temperature acclimation - Experiment Two 11 A c t i v i t y of the i n t e r r e n a l gland of juvenile coho salmon yearlings during cold temperature acclimation 34 vx Page TABLE I Values for the proportional standard i s deviation for duplicated non-chromato-graphed standard determinations. II Values for the proportional standard ]_g deviation for duplicate plasma sample determinations. I l l Plasma C o r t i s o l concentration of 24 juvenile coho salmon yearling during cold temperature acclimation -Experiment One. •* IV Plasma C o r t i s o l concentration of 28 juvenile coho salmon yearling during cold temperature acclimation -Experiment Two V A c t i v i t y of the i n t e r r e n a l gland of 32 juvenile coho salmon yearlings during cold temperature acclimation. VI Rates of temperature change for 43 Experiment One and Experiment Two. ACKNOWLEDGEMENTS Grateful thanks are due to my supervisor, Dr. Peter Ford, for his i n t e r e s t and f i n a n c i a l assistance during the inve s t i g a t i o n and throughout the preparation of t h i s manuscript. For h i s generous advice, assistance and encouragement throughout t h i s study, I extend a very s p e c i a l thanks to Dr. D. J . McLeay. I would l i k e to extend my thanks to Mr. U. H. M. Fagerlund for h i s advice and assistance. The author would also l i k e to express his thanks to Drs. C. V. Finnegan, H. C. Nordan, J. E. P h i l l i p s and W. S. Hoar for o f f e r i n g h e l p f u l suggestions and for reading the manuscript. My appreciation i s extended to Mr. D. Anderson, Mr. K. Hoogendyke, Mr. A. Koppel and Mr. E. McCulloch for t h e i r help with the day to day problems. I am also g r a t e f u l to Mr. S. Borden and hi s s t a f f f o r t h e i r assistance and guidance i n s t a t i s t i c a l matters F i n a l l y , I would l i k e to express my appreciation to my wife, I r i s , for her encouragement and to my children, Craig and Rachel, for t h e i r refreshing spontaneity. INTRODUCTION This introduction includes: a consideration of the supporting evidence for the homology between t e l e o s t i n t e r -renal tissue and the mammalian adrenal cortex, a discussion of the t e l e o s t p i t u i t a r y - i n t e r r e n a l axis, a review of the general role of t h i s axis i n response to diverse environmental conditions, and a consideration of some of the biochemical events associated with adrenocortical stimulation. In addition, i n terms of the response of plasma C o r t i s o l i n the juvenile coho salmon during cold temperature acclimation, there i s present a discussion of the events associated 5with thermal acclimation. HOMOLOGY The i n t e r r e n a l gland of t e l e o s t fishes i s , i n most species, a somewhat d i f f u s e tissue l y i n g along the posterior cardinal veins or t h e i r branches within the anterior kidney (Overbeeke, 1960; Nandi, 1962). On the basis of i t s embryology, histology and biochemistry, numerous studies have established that the interrenal gland i s homologous to the mammalian adrenal cortex (Pickford and Atz, 1957; Chester Jones and P h i l l i p s , 1960; Bern and Nandi, 1964). Valuable evidence favouring t h i s homology involves histochemical (Chavin and Kovacevic, 1961; Chavin, 1966) and f i n e - s t r u c t u r a l (Yamamoto and Onozato, 1965; Ogawa, 1967) studies of i n t e r -renal tissue and i_n v i t r o incubation studies with l a b e l l e d precursors of s t e r o i d synthesis ( P h i l l i p s and Mulrow, 1959; Nandi and Bern, 1960; Butler, 1965; A r a i et a l . , 1969). Incubation of teleostean head-kidneys, as well as d i r e c t extraction of t h i s kidney region (Fontaine and Leloup-Hatey, 1959), have demonstrated that the portion of the kidney containing i n t e r r e n a l c e l l s i s a source of c o r t i c o s t e r o i d s . Adrenocorticosteroids have been c l e a r l y demonstrated and i d e n t i f i e d i n the plasma of tel e o s t fishes (Bondy et al_. , 1957; Chester Jones and P h i l l i p s , 1960; Nandi and Bern, 1965; Chester Jones et a_l. , 1970) . C o r t i s o l i s shown to be a major c o r t i c o s t e r o i d i n the plasma of salmonids (Hane and Robertson, 1959; Schmidt and Idler, 1962; Sandor et a l . , 1966, 1967; Donaldson et a l . , 1968; Sandor, 1969) and plasma C o r t i s o l l e v e l s have been determined as an index of i n t e r r e n a l a c t i v i t y (Fagerlund, 1967; Donaldson and McBride, 1967; Fagerlund et a l . , 1968; Fagerlund and McBride, 1969). PITUITARY-INTERRENAL AXIS A number of recent investigations provide evidence for a p i t u i t a r y regulation of the i n t e r r e n a l tissue i n t e l e o s t s . Following hypophysectomy, atrophy of i n t e r r e n a l tissue occurs (Fontaine and Hatey, 195 3; Chavin and Kovacevic, 1961; Basu et a l . , 1965; Overbeeke and Ahsan, 1966; Donaldson and McBride, 1967). Hypertrophy of the i n t e r r e n a l tissue of teleosts subjected to ACTH treatment has been reported (Rasquin, 1951; Fontaine and Hatey, 1953; Chavin, 1956; Krauter, 1958; Basu et a l . , 1965; Fagerlund et. al_. , 1968). Evidence for the presence of a co r t i c o t r o p h i c hormone i n the t e l e o s t p i t u i t a r y has been provided by observations of i n t e r r e n a l hypertrophy following implantation of t e l e o s t p i t u i t a r i e s into i n t a c t or hypophysectomized Astyanax mexicanus (Rasquin, 1951) and Carassius auratus (Chavin, 1956) . S i m i l a r l y , Overbeeke and Ahsan (1966) demonstrated that i n j e c t i o n of p i t u i t a r y extracts of P a c i f i c salmon (Oncorhynchus kisutch and 0. tshawytscha) into hypophysectomized Couesius plumbeus reverses the atrophy of i n t e r r e n a l t i s s u e . Furthermore, a crude extract of p i t u i t a r y glands from the P a c i f i c salmon, Oncorhynchus keta has been shown to deplete rat adrenal ascorbic acid i n a manner s i m i l a r to that of mammalian ACTH (Rinfret and Hane, 1955). AXIS AND ENVIRONMENT It i s well established that the p i t u i t a r y - a d r e n o c o r t i c a l axis of mammals i s stimulated by numerous, diverse environ-mental conditions referred to as " s t r e s s f u l " (Gorbman and Bern, 1962). For example, many mammalian species show increased mean adrenal weight when subjected to increased population pressure (Gorbman and Bern, 1962; C h r i s t i a n and Davis, 1964). Other studies report a d i s t i n c t r e l a t i o n between p o s i t i o n i n a dominance hierarchy and adrenocortical a c t i v i t y (Louch and Higginbotham, 1967). In addition, i t has been observed that merely t r a n s f e r r i n g rats from one cage to another r e s u l t s i n a s i g n i f i c a n t r i s e i n plasma C o r t i s o l l e v e l s (Friedman and Ader, 1967). These reports, and others, indicate the se n s i t i v e response of the p i t u i t a r y - a d r e n o c o r t i c a l axis of mammals to environmental a l t e r a t i o n s . According to W. S. Hoar (1966): "„<, .although the l i t e r a t u r e i s s t i l l meagre, i t i s probable that the pi t u i t a r y - a d r e n a l system of the lower vertebrates i s activated by stress and that differences between the f i s h e s . . . and mammals are differences in degree rather than kind..." E f f e c t s which might be interpreted in terms of the stress concept have been noted among fishes under a variety of conditions. Marked hyperplasia of the in t e r r e n a l tissue in P a c i f i c salmon accompanying the spawning migration has been reported by Robertson and Wexler (1959). The adrenal tissue of the go l d f i s h i s markedly depleted of glucocorticoids by handling (Chavin and Kovacevic, 1961) . C i r c u l a t i n g glucocorticoids (Leloup-Hatey, 1958) and catecholamines (Nakano and Thomlinson, 1967) are increased a f t e r "stress". Both groups of adrenal hormones, e s p e c i a l l y catecholamines in very low doses, produce hyperglycemia (Falkmer, 1961; Robertson e_t a_l., 1963; Mazeaud, 1965; Young and Chavin, / mediated, in part, by the adrenal hormones^ i s present in tele o s t s . In addition, i t has been reported that holding adult rainbow trout in an aquarium with the water l e v e l lowered to 2 to 3 cm resulted in a s i g n i f i c a n t increase in plasma C o r t i s o l l e v e l s (Donaldson and McBride, 1967). Fagerlund (1967) , a f t e r studying the eff e c t s of handling adult sockeye salmon on plasma C o r t i s o l levels, concluded 1965). Thus, a pattern of "stress-induced" hyperglycemia that the pitu i t a r y - a d r e n a l system of adult salmon i s highly sen s i t i v e to various "stressors" and reacts with a manifold increase i n the concentration of C o r t i s o l i n the peripheral plasma. From the foregoing, several major points are evident. I t i s clear that the i n t e r r e n a l gland i s homologous to the mammalian adrenal cortex. Secondly, i t i s established that i n t e l e o s t fishes a p i t u i t a r y - i n t e r r e n a l axis e x i s t s that operates i n a manner s i m i l a r to the same axis i n mammals. F i n a l l y , i t i s suggested that the p i t u i t a r y - i n t e r r e n a l axis of t e l e o s t fishes responds i n a se n s i t i v e way to a v a r i e t y of "stressors". These relationships frame the t h e o r e t i c a l basis of t h i s t h e s i s . In the current investigation, the fundamental question being asked i s : how does the adrenocortical tissue of the juvenile coho salmon respond to the rigors of cold temperature acclimation? This question i s experimentally considered i n terms of the c o r r e l a t i o n between the concentration of a s p e c i f i c c i r c u l a t i n g g l u c o c o r t i c o i d , C o r t i s o l , and h i s t o l o g i c -a l l y demonstrable a l t e r a t i o n s i n the i n t e r r e n a l c e l l s of f i s h undergoing cold temperature acclimation. In answering t h i s question, an important q u a l i f i c a t i o n i s required, namely, a consideration of the conditions i n f e r r e d i n the terms adaptation, acclimation and "stress response". STRESS AND ACCLIMATION Fishes, with a few exceptions - tuna (Kishiouye, 1923) and lamnid sharks (Carey and Teal, 1969) - are poikilothermic. Although th e i r body temperature varies, these animals adjust their p h y s i o l o g i c a l state in order to compensate for such temperature changes. This process of acclimation involves predominantly behavioural and metabolic adjustments that require one to two weeks (Fry and Hochachka, 1970), depending upon the i n t e n s i t y of the required adjustment. In the present study, juvenile coho.salmon were subjected to a lowering of the environmental temperature o o 1 from 12 C (temperature of acclimation) to 2 C. This temperature range i s within the zone of thermal tolerance established by Brett (1952) and others. The adjustments of poikilotherms to a temperature change are multiple and exhibit d i f f e r e n t response times. The rate of temperature v a r i a t i o n in these experiments i s too rapid to allow for complete acclimation (approximately 1.7°C every 12 hours); however, the process of acclimation i s probably i n i t i a t e d during t h i s time period and may involve c i r c u l a t o r y and resp i r a t o r y adjustments, changing levels of c i r c u l a t i n g hormones, and others. These i n i t i a l changes may involve either p h y s i o l o g i c a l v a r i a t i o n s directed towards completion of the process of acclimation or adjustments to the p h y s i o l o g i c a l state r e s u l t i n g from a temperature s h i f t away from the i n i t i a l temperature of acclimation. Such a l t e r a t i o n s may be of the type c l a s s i f i e d i n the i n i t i a l alarm stage as defined by Selye (1950). These w i l l eventually disappear as acclimation to the new temperature i s achieved. Thus, i n the study reported here, the experimental conditions could evoke two categories of response; those directed towards acclimation and those concerned with maintaining a stable state i n an animal i n a non-acclimated condition. Selye (1950) employs the term "stress" to ref e r to the type of injurious environmental change which, i f sustained, w i l l lead to a somewhat standardized c o l l e c t i o n of responses referre d to as the general adaptation syndrome. This syndrome i s viewed as being t r i p h a s i c ; that i s , as occurring i n three definable stages. The f i r s t stage, which occurs immediately upon exposure to an injurious stimulus i s termed the alarm stage. This i s the response by the organism to a sudden exposure to a stimulus for which i t i s not adapted. I f the organism i s capable of withstanding t h i s i n i t i a l phase i t then enters the "stage of resistance" which may continue for a long period of time (weeks or months). When the sustained i n j u r i o u s agent f i n a l l y begins to have i t s f u l l e f f e c t upon the organism, there i s an apparent loss of adaptation, and the t h i r d stage, the "phase of exhaustion" occurs, which may r e s u l t i n death. Gorbman and Bern (1962) suggest that the "stress-concept" proposed by Selye (1950) has been somewhat maligned. In fact, terms such as stress, stressor, alarm reaction and others have achieved legitimacy they do not deserve. This has resulted i n a chronic misuse of the term "stress" and i t s associated synonyms. As a point of c l a r i f i c a t i o n , stress, as applied to t h i s investigation, i s to be considered in terms of the Selye general adaptation syndrome. Because the thermal treatment of experimental animals in t h i s study i s s t i l l within t h e i r c e n t r a l zone of thermal tolerance as defined by Fry (1947) and others, the presumption isjmade that the stage of exhaustion as outlined by Selye (1950) w i l l not be achieved. For t h i s reason, any response by these animals to the environmental parameter of temperature i s not, in the c l a s s i c a l sense, a stress response. If not a stress response then, what i s the basis for the change? From the point of view of the organism, any a l t e r a t i o n of an environmental factor which requires i t to expend metabolic energy in order to compensate, i s s t r e s s f u l . From the observer's point of view, such a response i s distinguishable in terms of the f i n a l outcome: i f the stimulus i s such that the animal achieves the stage of exhaustion (a state that usually r e s u l t s in.death) then stress i s the cause of death; i f the animal survives and i s able to compensate then the short-term e f f e c t i s that of adaptation and the long-term e f f e c t i s that of acclimation. BIOCHEMICAL VIEW OF ACCLIMATION TO COLD A great deal has been written concerning the p h y s i o l o g i c a l , biochemical and behavioural adjustments to thermal change by teleosts (for major reviews see Precht, 1968; Fry and Hochachka, 1970). Because of the complexity and d i v e r s i t y of the general l i t e r a t u r e with regard to the topic of temperature acclimation, t h i s introduction i s p r i m a r i l y concerned with a discussion of biochemical changes in animals undergoing acclimation to cold temperature. Adrenocortical hormones are known to influence mammalian intermediary metabolism, stimulating protein catabolism in extrahepatic tissues, p a r t i c u l a r l y s k e l e t a l muscle, and promoting gluconeogenesis with a concomitant increase in blood glucose and l i v e r glycogen levels (Long et. a_l., 1940; Cannon et_ a l . , 1956; Bellamy and Leonard, 1964). Evidence for the metabolic r o l e of c o r t i c o s t e r o i d s in fishes, although s t i l l scarce, i s gradually emerging. Except for minor modifications, te l e o s t fishes and mammals share a s i m i l a r c o r t i c o s t e r o i d action (Black et a l . , 1961; Storer, 1967; Chester Jones et a l . , 1969). In several species of teleosts, an increase in blood glucose or l i v e r glycogen levels follows the administration of ACTH or c o r t i c o s t e r o i d s (Nace, 1955; Falkmer, 1961; Robertson et a l . , 1963; Kumer et a_l. , 1966; Oguri and Nace, 1966; Butler, 1968). Butler (1968) reported that hypophysectomy or administration of a metabolic i n h i b i t o r of c o r t i c o s t e r o i d synthesis caused a s i g n i f i c a n t decrease in l i v e r glycogen lev e l s in Anquilla r o s t r a t a . Storer (1967) found that adminstration of C o r t i s o l to intact g o l d f i s h produced a decrease in body weight, an increase in ammonia secretion and an elevation of l i v e r glycogen phosphatase a c t i v i t y . These studies demonstrate that c o r t i s o l - t y p e steroids in teleosts, as in mammals, promote gluconeogenesis. Plasma c o r t i c o s t e r o i d l e v e l s of salmonids are markedly elevated during the spawning migration (Hane and Robertson, 1959; Robertson et a l . , 1961; Schmidt and Idler, 1962), and are accompanied by an obvious hyperplasia and hypertrophy of the i n t e r r e n a l tissue (Robertson and Wexler, 1959) . These changes are associated with a substantial catabolism of p a r i e t a l muscle protein (Idler and Clemens, 1959; Robertson et a l . , 1961), elevation of l i v e r glycogen (Chang and Idler, 1960) and hyperglycemia (Robertson e_t a_l., 1961) . Similar changes are observed when immature rainbow trout are fed C o r t i s o l acetate p e l l e t s . This evidence suggests that in some way the c i r c u l a t i n g glucocorticoids mediate the conversion of protein to carbohydrates, at least in f a s t i n g f i s h during t h e i r spawning migration. Furthermore, the inference i s clear that the carbohydrate so produced may allow the animal to maintain a more normal metabolism during t h i s highly s t r e s s f u l period. In general, tissues of cold acclimated f i s h , compared with the tissues of warm acclimated ones, may be described as follows: the rate of g l y c o l y s i s i s increased up to f i v e -f o l d (Hochachka and Hayes, 1962; Hochachka, 1967); the p a r t i c i p a t i o n of the pentose shunt may be increased from a n e g l i g i b l e contribution to as much as 10% of the t o t a l glucose metabolism,(Hochachka and Hayes, 1962; Hochachka, 1967); depending upon the tissue and.species, the Krebs cycle may be decreased, unchanged or possibly increased s l i g h t l y , whereas the electron transfer functions are c h a r a c t e r i s t i c a l l y increased (Hochachka and Hayes, 1962; Freed, 1969; Caldwell, 1969); lipogenesis i s activated, in some cases by only a small factor (Hochachka and Hayes, 1962; Dean, 1969), but in other cases the activations of synthesis of unique f a t t y acids may be increased by as much as'twelve-fold (Knipprath and Mead, 1968); glycogen synthesis rate is increased (Hochachka and Hayes, 1962); the rate of protein synthesis appears to be generally higher, at lea s t in certa i n species and tissues (Das and Prosser, 1967; Haschemeyer, 1969); an increase in the rate of synthesis and turnover of nu c l e i c acids (especially RNA) (Das, 1967); and, an a l t e r a t i o n of the ioni c microenvironment (Heiniche and Houston, 1965). It i s evident that in any given tissue not a l l of the above processes necessarily occur. Most of them probably take place in l i v e r , in which metabolic organization i s unusually complex. In tissue such as brain, g i l l and muscle, exergonic reactions are coupled to highly s p e c i a l i z e d work functions; hence metabolic organization may be abbreviated (Fry and Hochachka, 1970). A mechanistic model attempting to account for changes of the processes outlined above has been proposed (Hochachka, 1967). This model suggests that metabolic adjustments during cold temperature acclimation depends upon an i n i t i a l induction of a number of new isozymes which are s u f f i c i e n t l y s e n s i t i v e to control so as to be activated or i n h i b i t e d by preexisting modulators. Although t h i s model has been successful in s a t i s f y i n g a large amount of data, i t has been severely c r i t i c i z e d (Lardy e_t a_l. , 1965; Lardy, 1965) as i t does not account for two antagonistic events- gluconeogenesis and lipogenesis-occuring simultaneously. According to the model proposed by Lardy (1965), glucose metabolism i s regulated by g l u c o c o r t i c o i d s . These glucocorticoids i n i t i a t e two basic events. The f i r s t i s to release gluconeogenic precursors, such as free amino acids, from the peripheral tissues into the general c i r c u l a t i o n (an observation consistent with the data reported by Cannon e_t a_l., 1956; Bellamy and Leonard, 1964; Storer, 1967; and others). These gluconeogenic precursors then activate preexisting gluconeogenic enzymes in the l i v e r , thereby y i e l d i n g an increased product without measureable synthesis of enzymes. Secondly, glucocorticoids induce de novo synthesis of key gluconeogenic enzymes and thus further activate gluconeogenesis. In summary, i t i s evident that cold adapting tel e o s t fishes have imposed upon them a metabolic demand that has to be solved in order for them to survive. During the i n i t i a l stages of adaptation, the primary response systems function over the short-term to maintain a stable i n t e r n a l state. These responses may involve behavioural adjustments (such as seeking a more favourable environment) or p h y s i o l o g i c a l changes (such as a l t e r a t i o n s in r e s p i r a t i o n , c i r c u l a t i o n , hormonal l e v e l s and so on). During t h i s early period of adaptation the events as outlined by Lardy (1965) are i n i t i a t e d . These should produce an increase in gluconeogenic precursors, a r i s e in c i r c u l a t i n g g l u c o c o r t i c o i d concentrations, an increase in l i v e r glycogen and others. The net e f f e c t of these metabolic adjustments i s that over the long-term the organism becomes acclimated to the new conditions and i s capable of surviving. From the foregoing, i t i s apparent that C o r t i s o l and other glucocoticoids play an important r o l e in the regulation of glucose metabolism during cold temperature acclimation. Hence, t h e p u r p o s e s o f t h i s i n v e s t i g a t i o n a r e : (1) t o e s t a b l i s h i f j u v e n i l e coho salmon a c c l i m a t e d t o 12°C show a s i g n i f i c a n t i n c r e a s e i n plasma C o r t i s o l c o n c e n t r a t i o n w h i l e u n d e r g o i n g a c c l i m a t i o n t o 2°C. (2) t o d e t e r m i n e by h i s t o m e t r i c a n a l y s i s i f a r e l a t i o n s h i p e x i s t s between i n t e r r e n a l a c t i v i t y and plasma C o r t i s o l c o n c e n t r a t i o n under the c o n d i t i o n s o f a c c l i m a t i o n j u s t d e s c r i b e d . Material and Methods Experimental Animals The experimental animal selected for thi s investigation was the juvenile coho salmon yearling, Oncorhynchus kisutch. This salmonid, considered a pr i m i t i v e teleost, resides in l o c a l streams for a period of one year from the time of emergence from the gravel to the seaward migration. The juvenile form was chosen for i t s abundance in l o c a l streams, i t s sexual immaturity, i t s a b i l i t y to tolera t e a wide range of temperatures, and i t s reported hardy c h a r a c t e r i s t i c s when maintained under laboratory conditions. Five hundred coho salmon f r y were taken from Bertrand Creek (Langley, B.C.) on September 12, 1970 and transferred to the laboratory where they were placed in a holding tank (Tank A) . A l l f r y were fed frozen brine shrimp (Artemia, sp.) d a i l y at 0 900 hours. The water temperature of tank A was that of the inflow and varied from 12°C on September 12, 1970 to 7°C on January 11, 1971. Description of Tanks Holding tank: tank A The semi-circular f i b r e - g l a s s holding tank, 111 cm by 51 cm, had a water depth of 35 cm. This 200 l i t r e tank was illuminated by fluorescent lamps that provided an in t e n s i t y of 150 lux at the water surface. Light i n t e n s i t y measurements for a l l tanks were made with a model 200 Photovolt photometre. Tank A was covered with several sheets of transparent p l a s t i c . The water supply consisted of continuously aerated, dechlorinated water. The water temperature was that of the inflow. Control tank: tank B The rectangular, s t a i n l e s s s t e e l control tank, 61 by 182 cm, had a water depth of 38 cm and a t o t a l volume of 480 l i t r e s . The flow rate of the continuously aerated, dechlorinated water was approximately 1.5 l i t r e s per minute'. Tank B was covered by a retangular box whose dimensions matched the perimeter of the tank i t s e l f . This top was 18 cm deep and had enclosed within i t three equally spaced fluorescent lamps that provided a l i g h t i n t e n s i t y at the water surface of 400 lux. The control tank was heated by a 1000 watt, f l e x i b l e , nickle-chromium heating unit (Canlab H1960-1) connected in series with a stepdown transformer (Potter and BrumfieId-Model KA11BY) that regulated a mechanical relay (Potter and BrumfieId- Model KA9). This apparatus was capable of maintaining a constant temperature of 12 _+ 0.5°C. Experimental tank: tank C The dimensions of the experimental tank were similar to those of the control tank, tank B. An e f f o r t was made to ensure that a minimum of v a r i a t i o n existed between the experimental and control tanks. Such variables as water flow Figure 1: Holding tank - tank A Figure 2: Control tank, tank B, showing thermosensor and heating u n i t . Figure 3: Experimental, Tank C, tank showing e l e c t r o n i c thermoregulating controls and heating unit.. Figure 4: Apparatus for d i s t r i b u t i o n of nitrogen. rate, l i g h t i n t e n s i t y at the water surface, water depth and size of heating assembly were c a r e f u l l y controlled to make tanks B and C as s i m i l a r as possible. The temperature control device employed in tank C consisted of a r e f r i g e r a t o r component and a heating unit. The r e f r i g e r a t o r unit was set in series with a mechanical relay housed in a thermosensor l o g i c box (Versatherm-Model 2156) . This l o g i c box controlled a secondary mercury relay (Ebert E l e c t r i c a l Corp.) capable of accepting a surge amperage of 30 amperes. The response time of t h i s temperature control unit- that i s , the time between one unit shutting o f f and the other being activated was set at 30 seconds and provided an accuracy at 2°C of + 0.1°C. The heating unit was i d e n t i c a l to that in tank B. Experimental Design Experiment One At the beginning of Experiment One, 150 f r y were taken from the holding tank and 75 f i s h were each placed in tanks B and C. These f i s h were then held for three weeks at 12°C and a twelve hour photoperiod. After this period of acclimation, the f i s h in the experimental tank were subjected to a gradual"*" change in water temperature, u n t i l "*" The lowering of the temperature was spread over four days and was effected i n three stages: one, from 12°C to 7°C; two, from 7°C to 4°C and three, from 4°C to 2°C. b the present temperature of 2 C was achieved. Day 0 was defined as the time of i n i t i a l temperature a l t e r a t i o n . Experiment One was terminated on Day 19. From Day 0 to Day 19, f i s h were sampled i n l o t s of eight every four days. Control f i s h were sampled within 30 minutes of the sampling of the experimental animals. Sampling was performed at the same time d a i l y (between 1500 and 1600 hours) to reduce possible diurnal e f f e c t s . The photoperiod for both control and experimental f i s h was set at twelve hours. Experiment Two As for experiment One, Day 0 represented the time of gradual temperature a l t e r a t i o n . The rate of temperature decrease was the same as i n Experiment One. Experiment Two was terminated on Day 6. From Day 0 to Day 4, f i s h were sampled at twelve hour i n t e r v a l s . Sampling was performed at 0800 and 2000 hours respectively. From Day 4 to Day 6, f i s h were sampled every twenty-four hours i n i d e n t i c a l fashion. The photoperiod was set at twelve hours. No controls were used. Autopsy Procedures: Experiment One and Two: To obtain samples, several f i s h were l i g h t l y netted and ^ ( placed i n a large stacking dish f i l l e d with water. An i n d i v i d u a l f i s h was then removed and "damped-dried". Wet weight was then measured to the nearest tenth of a gram on a Mettler balance (Model P1200). The fork length was measured to the nearest millimetre. The caudal peduncle was wiped clean with 95% ethanol, dried and severed with a s c a l p e l . Blood was c o l l e c t e d from the caudal vessels into a heparinized .microhematocrit tube (Fisher S c i e n t i f i c ) and centrifuged at 12,000 RPM for 5 minutes in an Adams Microhematocrit Centrifuge (Clay-Adams Model CT 2900) and the hematocrit determined. The centrifuged blood samples were then placed on dry ice for rapid freezing and subsequently stored in a freezer. It has been shown by Fagerlund (1967) that simply passing a net over adult sockeye salmon produced a rapid and marked increase in plasma C o r t i s o l concentration. In a personal communication he suggested that the response time for an .elevated plasma C o r t i s o l l e v e l i n i t i a t e d by netting would be 10 to 15 minutes. For t h i s reason, s p e c i a l sampling procedures were developed to minimize t h i s e f f e c t . When sampling was begun, eight f i s h were removed from the tank and placed in a large stacking dish f i l l e d with water. This procedure reduced the ef f e c t s of netting to a minimum as i t required only a few seconds. In the subsequent handling of the i n d i v i d u a l animals, two people were involved. U t i l i z i n g t h i s system, the time for processing was shortened to a maximum of 15 minutes. Fish plasmas c o l l e c t e d in thi s manner showed no systematic v a r i a t i o n i n plasma C o r t i s o l concentration when correlated with the order of processing. Once the blood sample had been taken, the peritoneal c a v i t y was opened and the sex recorded. The head kidney containing the i n t e r r e n a l tissue was then removed with the surrounding muscle mass and fixed i n Bouin 1s f l u i d . The t o t a l handling time from the netting of the f i s h to f i x a t i o n of i n t e r r e n a l tissue was no more than 15 minutes. The in t e r r e n a l tissue was then taken to another laboratory where the histometric analysis was performed with the assistance of Dr. D. J . McLeay. H i s t o l o g i c a l Procedures H i s t o l o g i c a l technique: Following f i x a t i o n i n Bouin 1s f l u i d f or one week, the head kidneys were dissected from surrounding tissues, dehydrated i n ethanol, cleared i n benzene and embedded i n wax (Paraplast) i n the usual manner. S e r i a l transverse sections were cut at 5 microns. These preparations were then post-chromed i n 3% potassium dichromate overnight, followed by st a i n i n g with Mallory Heidenhain's Azan (Humason, 1962). Karyometry From each head kidney, f i v e sections were selected for measurement providing an anterior to poster i o r cross-section of the d i s t r i b u t i o n of i n t e r r e n a l c e l l s . Under low power magnification, a cluster of i n t e r r e n a l c e l l s was randomly chosen for measurement of 10 n u c l e i . Diameters of the i n t e r r e n a l c e l l n u c l e i were measured d i r e c t l y with an ocular micrometer at 1000X magnification ( o i l immersion). By changing the focus of the microscope, a series of cross-sections through the preparation i s seen. The area of the largest o p t i c a l cross-section of each c e l l nucleus was measured. Since coho. i n t e r r e n a l c e l l n u c l e i are e l i p t i c a l , mutually perpendicular diameters (longest and shortest) of each nucleus were measured. A computer program was written that computed the average of these two measurements and converted that average to microns. A t o t a l of 50 i n t e r r e n a l n u c l e i from the head kidney of each f i s h were measured. S t a t i s t i c a l Procedures C o r t i s o l Analysis Means and standard deviations were calculated for each set of C o r t i s o l values at a p a r t i c u l a r treatment time. A Student's " t " test was employed to determine i f a s i g n i f i c a n t difference existed between control and experimental values. Analysis of variance was made to e s t a b l i s h i f variances between means were s t a t i s t i c a l l y more s i g n i f i c a n t than variances within means. B a r t l e t t ' s test was applied to determine the p r o b a b i l i t y of variances being homogeneous. Scheffe's test for multiple comparisons with unequal sample sizes was used only when variances were homogeneous (Brownlee, 1965). In addition, regression analysis of Experiment One f i s h lengths, weights, sex and hematocrit against obtained C o r t i s o l values was performed. Means were considered s i g n i f i c a n t l y d i f f e r e n t i f p = 0.05 and very s i g n i f i c a n t l y d i f f e r e n t i f p = 0.01. H i s t o l o g i c a l analysis As described under methods (H i s t o l o g i c a l Karyometry, p. 8) each i n t e r r e n a l nuclear measurement required the estimate of two mutually perpendicular diameters. A computer program was written that: (1) averaged these two measurements and converted them to micron units; (2) calculated the mean and standard deviations for the 10 n u c l e i measured i n each i n t e r r e n a l clump; (3) calculated a grand mean and standard deviations for the 50 nuclei measured i n a l l the i n t e r r e n a l c e l l s counted i n an i n d i v i d u a l f i s h ; and (4) computed (by means of a Student's " t " t e s t ) , the s i g n i f i c a n c e of difference between nuclear diameters of experimental and control animals at the same treatment time. Graphical presentation of h i s t o l o g i c a l data u t i l i z e d grand mean and standard deviations. Means were considered to be s i g n i f i c a n t l y d i f f e r e n t i f p = 0.05, and very s i g n i f i c a n t l y d i f f e r e n t i f p = 0.01. In addition, analysis of variance was calculated for experimental and control treatment. B a r t l e t t ' s t e s t was applied and i f acceptable, a Scheffe's multiple comparison of unequal sample sizes was used. Quantification of Plasma C o r t i s o l by Competitive Protein Binding Introduction Many substances i n blood are bound to plasma proteins and i n some cases t h i s binding i s quite s p e c i f i c and shows a high degree of binding between the ligand and the protein (Berson and Yalow, 1957; Hunter and Greenwood, 1962; Murphy, 1964). Radioimmunoassay of these protein-bound ligands have been developed and include those for the determination of serum i n s u l i n (Berson and Yalow, 1957), serum glucagon (Barakat and Ekins, 1961) and C o r t i s o l and cortisone (Fagerlund, 1970). Most probably, s i m i l a r types of ligand-protein binding exists between many other substances and t h e i r c a r r i e r proteins. The radioimmunoassay procedures designed to determine the presence of these complexes i s currently ref e r r e d to as competitive protein binding (CPB). In order for t h i s method to be u t i l i z e d , two basic conditions must be met. The f i r s t is the a v a i l a b i l i t y of a l i g a n d - s p e c i f i c protein. The term " l i g a n d - s p e c i f i c " protein r e f e r s to a protein that binds only one ligand or a small group of chemically similar ligands. The second major consideration i s that a dynamic equilibrium exists between the ligand and i t s c a r r i e r protein. This requirement i n f e r s that the interactions of the ligand with i t s c a r r i e r protein follows stoichiometric laws. P r i n c i p l e of analysis by CPB If a r a d i o a c t i v e l y l a b e l l e d form of a substance, S*, i s added to a plasma containing an unlabelled, S, and limited amounts of i t s s p e c i f i c binding protein, P; and, i f a dynamic equilibrium e x i s t s between S and P, then S* w i l l d i s t r i b u t e i t s e l f evenly among the unlabelled S. If the binding a f f i n i t y between S and P i s very high, v i r t u a l l y a l l the S* added w i l l be bound u n t i l the P i s saturated. At equilibrium, SP + S*P w i l l equal S* P and SP . If further £ (S + S*) S* S S i s added i t w i l l also compete for the same binding s i t e s so that the S*P w i l l be reduced. From t h i s dynamic in t e r a c t i o n , the percentage bound S* can be plotted against the t o t a l S by simply increasing or decreasing the amount of S. Such a plot, for a given set of conditions, produces a standard curve. If, instead of pure, unlabelled S, a sample of plasma from which a l l the P has been removed and which contains an unknown amount of S i s added to the same system, i t may be quantified according to the f a l l in S*P i t causes. Competitive Protein Binding-Fagerlund Modification (Fagerlund, 1970) Mater i a l s Thin layer sheets were Eastman s i l i c a g e l Chromagrams 6060 which were used untreated. 3 Cortisol-1,2- H, s p e c i f i c a c t i v i t y 45.0 Ci/mM were supplied by New England Nuclear Corporation, Chicago I l l i n o i s , and we're used without p u r i f i c a t i o n . Purity was checked from time to time in the thin layer chromatography (TLC) system used in t h i s method. Standards of hydrocortisone (Cortisol) (BDH Laboratory Chemicals-0487320) were made up separately into aqueous solutions by d i l u t i o n of a stock standard containing 40 nanograms of steroid per m i l l i l i t r e . Chick serum was obtained from the Winley-Morris Company, Montreal, P. Q. The serum was mixed with enough t r i t i a t e d C o r t i s o l to give an a c t i v i t y of 125,000 to 250,000 counts per minute per m i l l i l i t r e and d i l u t e d with water to make a 4% stock solution that can be kept for several weeks i f r e f r i g e r a t e d . F l o r i s i l , 60-100 mesh (Fisher S c i e n t i f i c ) was screened by Endecott's test mesh (Fisher S c i e n t i f i c - 80 mesh) to obtain a p a r t i c l e range of 60-80 mesh, which gave a smaller deviation between duplicate determinations than the untreated material (Fagerlund, 1970) . The F l o r i s i l was dispensed by means of a s p e c i a l l y designed measurer made by d r i l l i n g a Teflon stopcock from a 250 ml separatory funnel and c a l i b r a t i n g the hole so d r i l l e d . Ten measurements with one such device gave the mean and standard deviation of 41.1 _+ 0.43 mg. A l l solvents were reagent grade and were d i s t i l l e d slowly through a d i s t i l l a t i o n r e f l u x apparatus. From each gallon d i s t i l l e d a 100 ml f o r e f r a c t i o n and endfraction was discarded. Method Plasma was obtained by thawing the heparinized tubes at room temperature and breaking the tubes just above the l e v e l of the compacted red blood cells.. The plasma was c o l l e c t e d by c a p i l l a r y action by means of a Lang-Levy pipette. The plasma was then placed in a 12 ml, glass-stoppered, c o n i c a l centrifuge tube (Fisher S c i e n t i f i c ) and d i l u t e d with 1 ml of d i s t i l l e d water dispensed from a 1 ml volumetric pipette (tolerance + 0 . 0 0 6 ml). To t h i s was added 8 ml of methylene chloride. Because of the v o l a t i l i t y of t h i s reagent, a s p e c i a l d e l i v e r y syringe was u t i l i z e d (Luer-Lock- 5 ml syringe; 5 ml metal p i p e t t i n g holder- Becton, Dickinson and Company, Rutherford, N.J.). The tubes were then manually shaken for 30 seconds and centrifuged at 2000 RPM in an International C l i n i c a l Centrifuge (Model CL) for 2 minutes. The aqueous phase was then removed by a s p i r a t i o n . Duplicate, 2 ml aliquots, were then removed by means of a s p e c i a l l y adapted volumetric pipette (Fisher S c i e n t i f i c -Pipette adaptor 13-682) and evaporated in centrifuge tubes. The evaporation step occured in a 45°C water bath under' nitrogen (Figure 4 ) . The evaporated extracts were then chrqmatographed in methylene c h l o r i d e - methanol- water (150: 9.0: 0.5) (Quesenberry e_t al_., 1965) on TLC sheets cut in h a l f and scored in channels 1 cm in width. The evaporated extracts were transferred to the chromatogram by r i n s i n g four times with two drops of a mixture of methanol and methylene chloride (10% methanol by volume) to points 1 cm apart along a baseline drawn across the longer dimension of the sheet. C o r t i s o l and cortisone markers were applied at the edges and the centre of the chromatogram. Development time i s 10 to 15 minutes and produces a solvent front approximately 8 cm from the baseline. The markers were located by u l t r a - v i o l e t l i g h t ( U l t r a - v i o l e t Products Inc., San Gabriel, C a l i f . ) . The corresponding regions containing the C o r t i s o l f r a c t i o n were then cut into squares, 1 cm by 1 cm, and dropped d i r e c t l y in centrifuge tubes containing 1 ml of d i s t i l l e d water. At t h i s stage, the samples can be stored overnight i f r e f r i g e r a t e d . One m i l l i l i t r e of the stock serum solution containing the C o r t i s o l binding globulin (CBG) and the t r i t i a t e d C o r t i s o l was added to each tube, giv i n g a f i n a l serum concentration of 2%. The tubes were covered with aluminum f o i l and warmed for 5 minutes in a 45°C water bath to remove any endogenous steroid from the CBG. The tubes were cooled for 2 minutes under a cold water tap and transferred to a cold room (4°c) and allowed three hours to e q u i l i b r a t e to that temperature. A l l remaining steps were carried out i n the 4°C cold room. -J A f t e r a minimum of two hours cooling, 80 mg of F l o r i s i l was added to each tube, which were then stoppered. The tube contents were mixed by simultaneously inverting a l l tubes. This was accomplished by means of a plywood box padded with foam rubber and pressed against the tube tops. The tube 2 contents were allowed 30 minutes to s e t t l e ; then 1 ml of the supernatant from each tube was added to 10 ml of Bray's f l u i d (Bray, 1960) and the r a d i o a c t i v i t y monitored on a Nuclear Chicago s c i n t i l l a t o r counter. For the purposes of determining mean recovery and concentration of unknowns, two kinds of standards are used: a non-chromatographed standard from which the standard curve i s made and a chromatographed standard from which mean recovery i s determined. For the former, usually three separate standards are s u f f i c i e n t to p l o t a standard curve. The standards selected are chosen so as to cover the anticipated range of plasma C o r t i s o l concentrations. Each of these standards i s determined in the following manner- a volume of standard equal to the volume of unknown used i s d i l u t e d to 1 ml with d i s t i l l e d water. To this i s added 1 ml of the CBG solution and the analysis continues from th i s point in the same way as for the plasma C o r t i s o l determinations. To determine recovery, a volume of standard, equal to.the product of the d i l u t i o n factor and the non-chromatographed standard in concentration, i s added to 1 ml of d i s t i l l e d water and subjected to the same procedures as are the unknowns.^ The amount of C o r t i s o l (ug/100 ml plasma) i s obtained by determining the counts per minute for the unknown plasma samples and p l o t t i n g t h i s value on the standard curve. The value i s then adjusted as indicated by the mean recovery of the chromatographed standard and m u l t i p l i e d by the d i l u t i o n factor (in t h i s procedure the d i l u t i o n factor i s 4) to give the number of micrograms per 100 ml of plasma. The concentration of the chromatographed standard used depends upon the d i l u t i o n factor for the plasma samples. For example, i f the d i l u t i o n factor i s 5, the concentration of the chromatographed standard would be 10 ug/100 ml for estimating the recovery of the 2 ug/100 ml non-chromatographed standard. Discussion of CPB Technique - Fagerlund Modification The r a t i o n a l e for the extraction and p u r i f i c a t i o n procedures are considered by Fagerlund (1970). As reported, duplicate samples are prepared for both non-chromatographed standards and chromatographed plasma samples. The values for the proportional standard deviation, S, (Bundy e_t a_l. , 1957) of duplicate determinations of standards and plasma samples are shown (Tables I and II resp e c t i v e l y . TABLE I VALUES FOR THE PROPORTIONAL STANDARD DEVIATION FOR DUPLICATED NON-CHROMATOGRAPHED STANDARD DETERMINATIONS Concentration of Standard Standard Deviation* Experiment I Experiment II 0 0.046 1 0.024 2 0.005 0.022 3 0.062 4 0.009 6 0. 175 0.015 8 0.012 0.010 * Values for the standard deviation, S, were obtained with the formula: S = J ?_51_2 and a = ( X l ~ * 2 ) where n 2 N X, + X 2 i s the number of duplicates and and X 2 are the r e s u l t s of duplicate determinations. VALUES FOR THE PROPORTIONAL STANDARD DEVIATION FOR DUPLICATE PLASMA SAMPLE DETERMINATIONS Range of Concentrations (ug/100 ml) 0.0-2.5 2.6-5.0 5.1-10.0 10.1-40.0 Standard Deviation 0.61 0.37 0.27 0.25 Number of Duplicates 17 11 15 11 Standard deviations calculations are based on a combination of Experiment I and Experiment II data. The proportional standard deviation values reported compare favourably with similar values published by Fagerlund (1970) with respect to non-chromatographed standard calculations (Table I). However, Table II values are considerably higher, e s p e c i a l l y in the lower concentration ranges. The observation that the variance among duplicate values decreases as the concentration of plasma C o r t i s o l increases i s supported by the fact that the percentage recovery of chromatographed standards improves as the concentration of that standard i s increased. When greater accuracy i s required in the low concentration range than i s obtained (SD 61% and 3 7%) i t i s of advantage to take a larger aliquot of methylene chloride extract for chromatography or to use a larger amount of plasma d i l u t e d with a proportionately smaller amount of d i s t i l l e d water. This was not done for two reasons: one, the anticipated order of response was within the range of greater accuracy (SD 27% and 25%); and two, the u t i l i z a t i o n of larger plasma volumes would require the pooling of a greater number of f i s h plasmas, thus reducing the s e n s i t i v i t y of the experimental design. The recovery of extracted and chromatographed standards when calculated from seven determinations of t r i p l i c a t e 2 ug/ 100 ml standards i s 85.2 _+ 10.5% and shows a mean and standard deviation of 1.89 _+ 0.64 ug/100 ml plasma. These values are close to the data reported by Fagerlund (1970), for the recovery of chromatographed standards, of 87.4 _+ 10.2% As mentioned, the solvent system used in t h i s technique was that developed by Quesenberry et_ al. , (1965). This system produced highly reproducible R f values for C o r t i s o l and cortisone. The R f c a l c u l a t i o n s showed a mean and standard deviation of 0.228 + 0.022 and 0.415 + 0.026 for C o r t i s o l and cortisone r e s p e c t i v e l y . In general, the technique of competitive protein binding, under the conditions of th i s procedure, i s q u a n t i t a t i v e l y suspect in the 0 to 5 ug range. However, I am confident that the system used has established an order of r e l a t i v e C o r t i s o l values. Because the order of response i s high (see Experimental Results, p.30), these r e l a t i v e values take on considerable experimental s i g n i f i c a n c e . Experimental Results Experiment One Experiment One was designed to determine i f a s i g n i f i c a n t change in plasma C o r t i s o l concentration occurs during cold temperature acclimation by juvenile coho salmon. Table III data indicates that such a response took place on Day 4 (p = 0.05). The mean and standard deviation for C o r t i s o l measurements determined from Day 4 plasmas were 1.8 _+ 1.00 and. 10.2 _+ 3.40 ug C o r t i s o l per 100 ml plasma for control and experimental animals respectively. As shown, (Table III) , th i s was the only measurement during the time course of Experiment One that a s i g n i f i c a n t difference between control and experimental C o r t i s o l values was obtained. Analysis of variances of C o r t i s o l values used to determine the mean C o r t i s o l value for each treatment time indicated a s i g n i f i c a n t difference (p ^0.05) between treatment times for both control and experimental groups. Variances within each treatment mean were shown to be non-homogeneous by B a r t l e t t ' s te s t . For t h i s reason, the Scheffe's multiple comparison test was not employed. P r o b a b i l i t y values for pj=0.05 were considered to be s i g n i f i c a n t and for p ^ 0.01 were considered to be very s i g n i f i c a n t . Figure 5: Head kidney of Day 0 control coho. Clusters of in t e r r e n a l c e l l s (IC) are d i s t r i b u t e d i r r e g u l a r l y throughout the hematopoietic tissue (HT). Azan. x 120 Figure 6: Head kidney of Day 0 experimental coho. Comparison with control f i s h (Figure 5) indicates approximately the same d i s t r i b u t i o n of i n t e r r e n a l c e l l c l usters (IC) . Azan. x 120 Figure 7: Head kidney of Day 14 control coho. D i s t r i b u t i o n of i n t e r r e n a l clumps i s s i m i l a r to that of Figure 5 and 6. Azan. x 120 Figure 8: Head kidney of Day 14 experimental coho. Note increase i n number of i n t e r r e n a l c e l l clumps (suggesting hyperplasia) r e l a t i v e to controls Azan x 120 TABLE III PLASMA CORTISOL CONCENTRATION OF JUVENILE COHO SALMON YEARLINGS DURING COLD TEMPERATURE ACCLIMATION (EXPERIMENT ONE) Number Plasma C o r t i s o l Concentration Water Temp. of C o r t i s o l Determinations* t Value t (ug/100 ml Plasma) Day Controls Exp . Controls Exp. Prob. Value Controls Mean + S.D. Experimental Mean + S.D. 0 12 12 3(7) 2(7) 2.33 0.10 2.9 + 0.75 1.05 + 1.06 4 12 2 4(9) 3(8) -4.78 0.005** 1.8 + 1.00 10.20 + 3.40 8 2 2(5) 0.40 + 0.00 14 12 2 2(3) 1(3) 0.99 0.50 2.1 + 0.14 2.00 + 0.00 20 12 2 4(8) 2(5) -1.42 0.20 0.1 + 0.25 1.25 + 1.76 : Numbers i n parenthesis represent the number of f i s h plasmas pooled for each set of determinations. :* S t a t i s t i c a l l y s i g n i f i c a n t at p = 0.01 l e v e l . Figure 9 Changes i n plasma C o r t i s o l concentrations of juvenile coho salmon yearlings during cold temperature acclimation (Experiment One). * Plasma C o r t i s o l values pl o t t e d are mean + standard deviation. ** Abscissa of plasma C o r t i s o l curve i s common to that of temperature p l o t . *** Day 4 represents time of s i g n i f i c a n t C o r t i s o l difference between controls and experimental animals o o OJ ZD •4—' CD 12 O Q_ ¥ 12 O if) o o rd £ 1 2 3 4 Control Experimental Control O (12°C) Exper. m ( 2°C) 20 BER OF DAYS Experiment Two Experiment Two was designed to investigate i n a more continuous manner the C o r t i s o l response shown by Experiment One. I t i s evident from the graphical presentation of Experiment Two data (Figure 10) that the fluctuations of plasma C o r t i s o l under the conditions of t h i s experiment can be broadly c l a s s i f i e d into three major orders of response: a minor increase, from hour 24 to hour 36; a major plasma C o r t i s o l elevation, from hour 72 to hour 84; and, r e l a t i v e l y constant pre-response and post-response values. * The pre-response and post-response l e v e l s for plasma C o r t i s o l have a mean and standard error of 5.8 + 1.4 ug C o r t i s o l per 100 ml plasma. The c a l c u l a t i o n of t h i s mean i s based upon values for plasma C o r t i s o l at hour 0, hour 12, hour 48, hour 96, hour 120 and hour 144. The mean calculated for pre-response and post-response C o r t i s o l values was higher than the plasma C o r t i s o l measurements from analysis of Experiment One controls (1.7 + 0.85 ug C o r t i s o l per 100 ml plasma). The region described as a minor response showed a mean and standard error of 11.6 + 2.2 ug C o r t i s o l and the region described as a major increase demonstrated a mean and standard error of 27.0 _+ 2.8 ug C o r t i s o l per 100 ml plasma. Figure 10 suggests that a r e l a t i o n s h i p exists between the timing of the increase i n plasma C o r t i s o l and the changes TABLE IV PLASMA CORTISOL CONCENTRATION OF JUVENILE COHO SALMON YEARLINGS DURING COLD TEMPERATURE ACCLIMATION (EXPERIMENT TWO) Plasma C o r t i s o l Concentration Number (ug/100 ml Plasma) of C o r t i s o l Determinations* Mean + S.E. 0 12 5 (8) 7.2 + 0. 9 12 7 3 (5) 2.0 + 0.9 24 7 2(4) 11.4 + 1. 6 36 7 4(6) 11.6 + 5.2 48 4 2(4) 5.5 + 1.3 60 2 3 (6) 8.2 + 0. 2 72 2 3 (7) 23.8 + 6.4 84 2 4(7) 27. 0 + 2.8 96 2 4(8) 6.9 + 3.1 120 2 2(5) 7.2 + 0.0 144 2 3 (7) 3.8 + 3.0 Water Temp. Hours ( C) * Numbers in parenthesis represent the number of f i s h plasmas pooled for each set of determinations. Figure 10 Changes i n plasma C o r t i s o l concentration of juvenile coho salmon yearlings during cold temperature acclimation (Experiment Two). * Values plotted for plasma C o r t i s o l are mean ± standard error. 3 ** Abscissa of plasma C o r t i s o l curve i s common to that of temperature p l o t . i n water temperature. The minor increase i n plasma C o r t i s o l , from hour 24 to hour 36, takes place immediately aft e r the environmental water temperature has s t a b i l i z e d at 7°C. S i m i l a r l y , the major increase i n plasma C o r t i s o l , from hour 72 to hour 84, occurs immediately aft e r the water temperature reaches 2°C. In addition, Figure 10 indicates that i n each instance a s i g n i f i c a n t increase i n plasma C o r t i s o l occurs, that concentration i s maintained for approximately 12 hours and then f a l l s quickly (within 12 hours) to the pre-response l e v e l . . g. Analysis of variances of C o r t i s o l values used to determine the mean C o r t i s o l measurement for each treatment time indicated a s i g n i f i c a n t difference (p Z. 0.01) between d i f f e r e n t treatment times. Variances within each treatment mean were shown to be non-homogeneous by B a r t l e t t ' s t e s t . Because no controls were kept the Student's t test could not be u t i l i z e d . Analysis of variances were considered to be s i g n i f i c a n t i f p 4: 0.05 and very s i g n i f i c a n t i f p^O.Ol. Karyometry Measurement of hypertrophy of i n t e r r e n a l nuclei was performed on Experiment One animals only. Table V indicates that control and experimental animals show s i g n i f i c a n t l y d i f f e r e n t values for mean in t e r r e n a l nuclear diameters only on Day 14 (p = 0.006). Levels of sig n i f i c a n c e were determined by a Student's t t e s t . P r o b a b i l i t i e s were considered s i g n i f i c a n t i f p ^ 0.05 and very s i g n i f i c a n t ACTIVITY OF THE INTERRENAL GLAND OF THE JUVENILE COHO SALMON (ONCORHYNCHUS KISUTCH) DURING COLD-TEMPERATURE ACCLIMATION Interrenal Nuclear Diameters (microns) Number of T T Controls Experimental Day Fish Va lue Prob. Mean + S.D.** Mean + S.D. 0 5 0. 78 0.484 6. 98 + 0.18 6.87 + 0.23 4 4 -0.62 0. 587 6. 70 + 0. 16 6.83 + 0.27 8 4 6. 78 + 0. 14 14 4 -5.75 0.006* 6.86 + 0. 16 7.51 + 0. 16 18 4 -1.16 0.315 6. 97 0.39 7. 26 + 0.31 20 4 -0.38 0. 717 7.07 + 0.29 7.44 + 0.22 * S t a t i s t i c a l l y s i g n i f i c a n t at p = 0.01 l e v e l ** Mean values for each f i s h are based on a measurement of ten n u c l e i from each of at least four i n t e r r e n a l clumps. The grand mean presented above represents the mean value for each group of f i v e f i s h . Figure 11 A c t i v i t y of i n t e r r e n a l gland of juvenile coho salmon yearlings during cold temperature acclimation. * Interrenal nuclear diameters are presented as mean + standard deviation. F I G U R E 1 1 £ 75 U J ACTIVITY OF INTERRENAL GLAND OF JUVENILE COHO SALMON YEARLINGS DURING COLD— TEMPERATURE ACCLIMATION Control • (12°C) Experiment. ESO ( 2°C) < | 70 LJJ I — S 6-5 T T T T J L 0 1 2 3 4 17 20 BER OF DAYS i f p = 0.01. Figure II c l e a r l y demonstrates the increase i n i n t e r r e n a l tissue of Day 14 experimental f i s h r e l a t i v e to Day 14 and Day 0 controls. Although the extent of hyperplasia was not quantified, examination of Figure 8 .suggests that considerable hyperplasia of in t e r r e n a l n u c l e i occurred i n Day 14 experimental f i s h . Analysis of variances of the i n d i v i d u a l measurements determining the mean for each treatment time indicated a s i g n i f i c a n t difference (pZ.0.01) between treatment times. Variances within each treatment time were shown to be homogeneous by B a r t l e t t ' s t e s t . Scheffe's test for multiple comparisons was u t i l i z e d for control and experimental data analysis. The results of Scheffe's tests indicated that the Day 14 treatment mean was s i g n i f i c a n t l y d i f f e r e n t (pZ.0.001) from a l l other experimental treatment means. DISCUSSION Straw and Fregly (1967) have reported that rats exposed to cold temperatures over a long period of time demonstrate an increase i n adrenocortical hormone concentrations that reached a maximum af t e r 7 days exposure to cold. I t i s also shown by these authors that adrenal weights, considered an index of adrenal a c t i v i t y , reached a maximum a f t e r 14 days i n the cold. This kind of response i s s i m i l a r to that obtained i n the karyometric analysis i n the present i n v e s t i g a t i o n , i n which a s i g n i f i c a n t increase between control and experimental i n t e r r e n a l nuclear diameters was noted on f i s h exposed to cold for 14 days, whereas maximum plasma C o r t i s o l values were obtained before 6 days of exposure to cold. Hypertrophy of i n t e r r e n a l n u c l e i i s generally considered an index of increased i n t e r r e n a l a c t i v i t y (McLeay, 1970) and others. Straw and Fregly (1967) suggest that i n rats such a r e s u l t i s not s u r p r i s i n g . The basis for t h i s p o s i t i o n i s that adrenal size i s most probably due to an average d a i l y secretion rate of ACTH over a long period of time rather than a series of sudden spikes i n ACTH release. As a r e s u l t , the lag i n the change of adrenal size r e l a t i v e to much e a r l i e r increases i n plasma adrenocortical hormone le v e l s i s understandable. Although no d i r e c t evidence, with respect to salmonids, is av a i l a b l e in support of th i s interpretation, the basic tenet is in agreement with the work of Dear and Guillemin (1960) who demonstrated that hypophysectomized r a t adrenals continued to decline in weight for as long as 28 days afte r hypophysectomy. The evidence is clear that the p i t u i t a r y - i n t e r r e n a l axis of salmonids functions by means of a negative feedback system (Donaldson and McBride, 1967) . Hypophysectomized adult trout demonstrate a rapid decrease in plasma C o r t i s o l within 24 hours of hypophysectomy (Donaldson and McBride, 1967) . This is thought to be due to a rapid turnover of endogenous ACTH. Figure 10 of the present investigation indicates a rapid increase and equally rapid decline in plasma C o r t i s o l concentration for each region of s i g n i f i c a n t response. These data may be interpretable in terms of the synthesis, release and apparently rapid turnover of endogenous ACTH. In addition, Fagerlund (1969) and others have shown that endogenous C o r t i s o l i s r a p i d l y converted to cortisone, although the reverse does not appear to be true. By combining these two pieces of information an interpretation of the r e s u l t s shown in Figure 10 as to the rate of change of plasma C o r t i s o l l e v e l s i s possible. I n i t i a l l y , as the animal responds to the metabolic demands of cold acclimation, the plasma C o r t i s o l concentration, mediated by adrenocortcotrophin, increase sharply. As the C o r t i s o l l e v e l r i s e s , the synthesis and release of ACTH i s inhibited, r e s u l t i n g in a decrease in endogenous adrenocorticotrophin and subsequently in a reduction in the i n t e r r e n a l production and release of g l u c o c o r t i c o i d s . In addition, the conversion of C o r t i s o l to cortisone and the normal clearance of these two hormones through kidney action causes a rapid decrease in the levels of these substances. Thus, the rates of fluctuations described by Figure 10 are possible 7'due to negative feedback, hormone interconversion and excretion. This interpretation, however, does not explain the i n t e n s i t y and timing of the changes in C o r t i s o l concentrations, but merely suggests why rapid elevations are followed by equally rapid declines. / The data presented in Figures 9 and 10 indicate^ that col acclimating juvenile coho salmon demonstrate a short-term increase in plasma C o r t i s o l concentration. The timing of this increase appears to be within 96 hours of the time of temperature a l t e r a t i o n . The r i s e in C o r t i s o l subsequent to temperature change i s suggested, by t h i s data, to be a r e l a t i v e l y rapid r i s e followed by an equally rapid decline to a l e v e l j u st s l i g h t l y above that of controls (Figures 9 and 10; Tables III and IV). Such a response by juvenile coho salmon i s complementary to the model proposed by Lardy (1965) and Lardy et. a_l. , (1965) According to their thesis, glucocorticoids i n i t i a t e two basic events during cold acclimation. The f i r s t of these i s t o r e l e a s e gluconeogenic p r e c u r s o r s t h a t a c t i v a t e p r e e x i s t i n g gluconeogenic enzymes i n the l i v e r , t h ereby y i e l d i n g an i n c r e a s e i n l i v e r g l y c o s e s y n t h e s i s . In a d d i t i o n , these same g l u c o c o r t i c o i d s induce de novo s y n t h e s i s of key gluconeogenic enzymes which a l s o r e s u l t s i n a net i n c r e a s e i n g l u c o s e s y n t h e s i s . The l e v e l s of plasma c o r t i c o s t e r o i d s and t h e i r concomitant e f f e c t s upon t e l e o s t f i s h e s i s documented, although somewhat s p a r s e l y . Responses.by t e l e o s t s t o i n c r e a s e i n plasma C o r t i s o l have been shown t o i n c l u d e obvious i n t e r r e n a l h y p e r p l a s i a (Robertson and Wexler, 1959) c a t a b o l i s m of p a r i e t a l muscle p r o t e i n (Robertson et al_., 1961), e l e v a t i o n of l i v e r glycogen (Chang and I d l e r , 1960) and hyperglycemia (Robertson et_ a l . , 1961) . These data are a l s o b r o a d l y s u p p o r t i v e o f the model pr e s e n t e d by Lardy (1965) of the p o s s i b l e r o l e of g l u c o c o r t i c o i d s i n the r e g u l a t i o n of g l u c o s e metabolism d u r i n g c o l d temperature a c c l i m a t i o n . In a d d i t i o n , a number of authors have pr e s e n t e d i n f o r m a t i o n t h a t suggests t h a t d u r i n g c o l d temperature a c c l i m a t i o n s e v e r a l of the i n t e r m e d i a r y m e t a b o l i c f u n c t i o n s undergo major r e o r g a n i z a t i o n (see " I n t r o d u c t i o n t o R e s u l t s " pp. XVIII t o XXI). These changes i n c l u d e an i n c r e a s e i n g l y c o l y s i s , an i n c r e a s e i n the events a s s o c i a t e d w i t h the o x i d a t i v e e l e c t r o n t r a n s f e r system and an i n c r e a s e i n glycogen s y n t h e s i s . A lthough the s i t u a t i o n i n fact i s far more complicated than that reported here, i t nonetheless appears that glucose oxidation increases during cold temperature acclimation with a subsequent r i s e i n the a v a i l a b i l i t y of high-energy phosphorylated compounds. If i t i s acceptable that during cold temperature acclimation, adjustments i n glucose metabolism as outlined above a c t u a l l y occur, and that these changes are mediated i n part by c i r c u l a t i n g glucocorticoids, then i t l o g i c a l l y follows to ask: of what benefit to the organism are these changes? As far as t h i s author i s aware, no precise answer to the question i s a v a i l a b l e . I t i s , however, reasonable to expect that an organism undergoing acclimation to a new environmental parameter would require some form of metabolic readjustment. Fundamentally, these changes might take the form of energy-requiring isozymic transformations (Hochachka, 1967; Fry and Hochachka, 1970) although supportive evidence i s somewhat confused i n t h i s regard. Su f f i c e i t to say that the experimental evidence c o l l e c t e d to date strongly suggests that these changes are energy requiring, although why t h i s i s so i s not at a l l c l e a r . An important q u a l i f i c a t i o n i n considering the e f f e c t s of glucocorticoids on the rearrangement of glucose metabolism of salmonids i s the fact that the bulk of evidence reported to date concerns adult or migrating adult f i s h . In the l a t t e r instance, conversion of body protein to more immediately u t i l i z a b l e energy forms has obvious short-term energy advantages. However, in the current investigation, the juvenile coho salmon used were in a p o s t - f i n g e r l i n g to an immediate pre-smolt stage-a stage where a major concern of the animal's metabolism is that of promoting growth. Fagerlund (1971) reported that juvenile coho salmon exposed to changes in ambient water temperatures-from 10°C in September, 1970 to 1°C in January, 1971-showed a r e l a t i v e l y high plasma C o r t i s o l concentration when compared with post-smolt juveniles. In addition, ., Fagerlund (1971) demonstrated that the values for plasma C o r t i s o l changed l i t t l e over the period of assay. The tentative conclusions, based on this evidence, i s that growth i s a more important factor in determining plasma C o r t i s o l levels in. pre-smolt juvenile coho salmon than i s acclimation to a lower water temperature. McLeay (1970) reported d i f f e r e n t findings. In his studies, i t was observed that juvenile pre-smolt coho salmon sampled in winter showed greater i n t e r r e n a l a c t i v i t y than did f i n g e r l i n g coho salmon, sampled from the same population, in the previous summer. From these data, McLeay (1970) suggests that i n t e r r e n a l a c t i v i t y varies inversely with the ambient water temperature although the conclusion that plasma C o r t i s o l levels follow the same patternwas only, inferred by that author. In the present investigation, i t i s noted (Table VI) that the rate of change of water temperature from the temperature of acclimation (12°C) to 2°C i s approximately 0.14 and 0.17 °C per hour for Experiments One and Two re s p e c t i v e l y . The differences in cooling rates were due p r i m a r i l y to variations in the temperature of the inflowing water. These rates of change are considerably more intense than that found in the natural environment. Because of t h i s , i t i s possible that the r e s u l t s of t h i s investigation more c l e a r l y r e f l e c t the response of pre-smolt juvenile coho salmon to a change in water temperature than would the designs as presented by McLeay (1970) and Fagerlund (1971), where factors such as predation, photoperiod, a v a i l a b i l i t y of food and others would come into e f f e c t . It is important to note, in interpreting the r e s u l t s of Experiments One and Two, that the variances within each treatment mean are of an order that makes i t s t a t i s t i c a l l y doubtful whether the reported i n t e n s i t i e s of responses are s i g n i f i c a n t . . It would appear that fluctuations in plasma C o r t i s o l , as reported for these experiments, should be thought of as an order of response rather than a p r e c i s e l y quantified l e v e l of plasma C o r t i s o l concentrations. Analysis of variances of treatment means for the karyometric data are of a l e v e l that allows for a more confident interpretation in t h i s respect. RATES OF TEMPERATURE CHANGE IN EXPERIMENT ONE AND EXPERIMENT TWO Temperature Interval ( C) Time Rate of Change * Experiment I Experiment II Interval (Hours) (°C/Hour)  Exp. I Exp. II Exp. I Exp. II 12.0-8 .0 12.0-7 .0 24 12 0.17 0.41 8.0-4 .0 7.0-4 .0 36 36 0.11 0.08 4.0-2 .0 4.0-2 .0 12 12 0.17 0.17 * The o v e r a l l rate of change (expressed as o C/Hours) i s 0.14 and 0.17 for Experiments I and II respe c t i v e l y . In the opinion, of t h i s investigator, the major reason for the nonhomogeneity of variances within treatment means for Experiment One and Experiment Two i s sample si z e . As outlined in "Material and Methods" (pp 7-8) the sample size used in these experiments was limited to a maximum of 8 animals. This r e s t r i c t i o n was intended to reduce the p o s s i b i l i t y of a handling-induced increase in plasma C o r t i s o l which was shown in a preliminary test to require 10 to 15 minutes before such a response became s i g n i f i c a n t . Another possible reason for variations .-> within treatment means may be the r e s u l t of the l i m i t a t i o n s imposed by the time required between taking sets of samples. Fagerlund (1967) demonstrated that increases in plasma C o r t i s o l , r e s u l t i n g from the movement of a net over the heads of adult salmon, require approximately 12 hours to return to pre-netting l e v e l s . Because of t h i s , i n d i v i d u a l variations of true plasma C o r t i s o l concentrations may be large for Experiment Two r e s u l t s ; that i s , differences in the rate of return of C o r t i s o l values to normal, among i n d i v i d u a l animals, may be great. In addition, a very p l a u s i b l e explanation for the divergency of measurements within each treatment mean may we l l be the technique of C o r t i s o l q u a n t i f i c a t i o n employed (see "Discussion of CPB"; ppl8-20). F i n a l l y , to test the p o s s i b i l i t y that variances within plasma C o r t i s o l treatment means were related to i n d i v i d u a l f i s h differences as weight, length, sex and hematocrit, a regression analysis of these parameters against plasma C o r t i s o l measurements was made. In each case, the slope of the curve so obtained was e s s e n t i a l l y zero, suggesting that i n d i v i d u a l variations among f i s h , with respect to the variables just mentioned, did not bias the reported C o r t i s o l values. Experiment Two d i f f e r e d from Experiment One in several aspects. No controls were used for two basic reasons: one, there was an i n s u f f i c i e n t number of f i s h a v a i l a b l e and two, the r e s u l t s of Experiment One suggested a consistent l e v e l of C o r t i s o l in control animals throughout the invest i g a t i o n . During the course of the second experiment, no f i s h weights, lengths and sex were recorded, although hematocrits were consistently taken as an index of f i s h v i t a l i t y . F i n a l l y , no h i s t o l o g i c a l treatment of Experiment Two interrenals was done. This decision was j u s t i f i e d by the r e s u l t s of the f i r s t experiment in which a clear karyometric picture, both s t a t i s t i c a l l y and with regard for similar published works, was obtained. The f a c t that Experiment One karyometric analysis indicated that hypertrophy of experimental i n t e r r e n a l n u c l e i r e l a t i v e to controls occurred a f t e r 14 days exposure to cold j u s t i f i e d not performing this analysis on Experiment Two animals, as the l a t t e r experiment was terminated aft e r only 6 days exposure to c o l d o The major sources of error in t h i s study were threefold. The f i r s t was the l i m i t a t i o n of the sample size to a maximum of 8 f i s h . The second, rel a t e d to the r e s t r i c t i o n s on sample size, was the tremendous i n d i v i d u a l variations in plasma constituents among d i f f e r e n t f i s h exposed to the same treatment. This observation has the support of several other investigators involved in similar research. The t h i r d major source of error was the- technique of C o r t i s o l q u a n t i f i c a t i o n where standard deviations between duplicate samples of the order of 20% were considered acceptable. Consistent to the basic question of t h i s thesis i s whether an acclimation response and not a stress response was investigated. The arguments in support of the pos i t i o n taken in t h i s study (see "Introduction to Results"; pp XIV-XVII) were predominantly academic and involved a measure of semantic i n t e r p r e t a t i o n . Although i t would be r e p e t i t i v e to restate the conditions suggesting that the changes investigated were, in fact, responses to acclimation, i t i s nonetheless admitted by th i s investigator that c r i t i c i s m of the view taken i s j u s t i f i e d . In retrospect, the study reported here has suggested many re l a t e d avenues for research into the process of acclimation to cold by juvenile coho salmon. Under si m i l a r experimental conditions i t would be useful to c o r r e l a t e changes in l i v e r glycogen, plasma glucose and plasma cortisone with that of plasma C o r t i s o l . In addition, a h i s t o l o g i c a l and histochemical study of the coho p i t u i t a r y , designed primarily to demonstrate ACTH a c t i v i t y , would also be of value, e s p e c i a l l y when compared with the biochemical investigations referred to above. Furthermore, a q u a n t i f i c a t i o n of i n t e r r e n a l hyperplasia, although time consuming and d i f f i c u l t , would c l a r i f y the picture. F i n a l l y , i t should be remembered that the technique of competitive protein binding i s r a p i d l y expanding and i s extremely usef u l in dealing with animals having the blood volume r e s t r i c t i o n s of juvenile salmonids. To t h i s end, investigation into the dynamics of C o r t i s o l , cortisone and other re l a t e d hormones i s possible. S i m i l a r l y , the a p p l i c a t i o n of t h i s technique to other important endocrinological factors such as ACTH appears to be a p o s s i b i l i t y in the very near future. These and other investigations would provide an important contribution in the c l a r i f i c a t i o n of the events involved in acclimation to cold temperature by juvenile coho salmon. CONCLUSIONS The following conclusions, based on the res u l t s t h i s i nvestigation, are made: (1) During acclimation to cold temperature, juvenile, pre-smolt coho salmon exhibit increases i n plasma C o r t i s o l concentration. (2) Under the experimental conditions imposed i n th i s study, plasma C o r t i s o l l e v e l s of cold-acclimating juvenile coho salmon achieve^ control values within 8 days of exposure to cold. (3) S i g n i f i c a n t hypertrophy of the i n t e r r e n a l n u c l e i of juvenile coho salmon occurs by Day 14 of exposure to cold temperature. 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