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Aspects of cortisol dynamics during the early ontogeny of three species of Pacific salmon (Oncorhynchus… Stratholt, Miles Linley 1995

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ASPECTS OF CORTISOL DYNAMICS DURING THE EARLY ONTOGENY OF THREE SPECIES OF PACIFIC SALMON {Oncorhynchus sp.) by Miles Linley Stratholt B.Sc. University of V i c t o r i a , 1990 A Thesis Submitted i n P a r t i a l Fulfilment of the Requirements for the Degree of Master of Science i n The Faculty of Graduate Studies Department of Zoology We accept t h i s thesis as conforming to the required standard The University of B r i t i s h Columbia August 1995 (c) Miles L. Stratholt, 1 9 9 5 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department The University of British Columbia Vancouver, Canada DE-6 (2/88) 11 Abstract Seven female coho salmon (Oncorhynchus kisutch), i n the f i n a l stages of oogenesis, were exposed to mechanical disturbance (chased for 60 seconds with hand net), twice d a i l y over a two week period. There was no s i g n i f i c a n t difference i n mean gonadosomatic index between disturbed (4.63 ± 0.19) and undisturbed (4.76 ± 0.34) females. Mean plasma C o r t i s o l l e v e l i n the disturbed females (227.52 ± 61.51 ng/ml) was higher, though not s i g n i f i c a n t l y , than that seen i n undisturbed females (140.99 ± 42.70 ng/ml). Mean oocyte C o r t i s o l content (22.13 ± 1.32 ng/gm) was s i g n i f i c a n t l y higher i n the disturbed, than i n the undisturbed females (9.90 ± 0.94 ng/gm). I t i s suggested that the C o r t i s o l content of fre s h l y ovulated oocytes i n salmonids i s r e f l e c t i v e of the l e v e l of c i r c u l a t i n g C o r t i s o l i n the adult female during l a t e oogenesis. Oocytes from 5 female coho salmon were s p l i t into paired groups and one half exposed to water containing exogenous C o r t i s o l . The C o r t i s o l immersed groups had a mean oocyte C o r t i s o l content (232.68 ± 13.93) that was s i g n i f i c a n t l y higher than that i n the paired untreated groups (37.03 ± 5.43). Mean oocyte C o r t i s o l content was monitored from 0 to 56 days post f e r t i l i z a t i o n (dpf). Oocyte C o r t i s o l content was seen to decline sharply a f t e r f e r t i l i z a t i o n , and there was no s i g n i f i c a n t difference between paired immersed and control groups by 16 dpf. The immersed groups demonstrated no s i g n i f i c a n t difference i n terms of mean time to hatch, mean synchrony of hatch, percent mortality, mean yolk sac to body weight r a t i o at hatch, or mean length and dry weight up to 56 I l l days post f e r t i l i z a t i o n , compared to the paired untreated groups. Steelhead (Oncorhynchus mykiss), embryos subjected to a 60 second emersion every f i f t e e n minutes f o r two hours, every f i v e days, from -4 to 32 days post hatch (dph) , demonstrated a measurable C o r t i s o l response beginning at 6 dph. The magnitude of t h i s response increased s i g n i f i c a n t l y at each sampling period thereafter with the exception of day 21 post hatch. Coho salmon embryos subjected to a sim i l a r treatment every seven days from -5 to 30 dph demonstrated a measurable C o r t i s o l response beginning 9 dph, and by 16 dph were demonstrating a C o r t i s o l response to emersion that, over an 8 hour period, was s i m i l a r to that reported for adult and juvenile salmonids to a va r i e t y of s t i m u l i . This onset of a measurable C o r t i s o l response to an environmental disturbance at 6 and 9 dph f o r steelhead and coho salmon respectively i s an ind i c a t i o n of the onset of a functional hypothalamic-pituitary-interrenal (HPI) axis. Embryonic chinook salmon (Oncorhynchus tshawytscha) exposed to a combination of a repeated emersion and exogenous C o r t i s o l treatment displayed reduced growth and s u r v i v a l compared to embryos receiving either treatment alone, or neither treatment. The combination of the two treatments was observed to r e s u l t i n a s i g n i f i c a n t increase i n whole body C o r t i s o l content i n chinook embryos beginning at 9 dph. This increase i n whole body C o r t i s o l content i s suggested as a factor i n the increased mortality. The reduction i n length and weight observed was att r i b u t e d to phys i o l o g i c a l responses to disturbance other than the C o r t i s o l i v response, as embryos treated with exogenous C o r t i s o l alone d i d not display reduced growth, while those receiving the emersion treatment alone did show some s i g n i f i c a n t reduction i n weight. V TABLE OF CONTENTS Page Abstract i i Table of Contents v L i s t of Tables v i L i s t of Figures v i i Acknowledgement ix Chapter 1 General Introduction 1 Chapter 2 Transfer of Maternal C o r t i s o l to Maturing Oocytes i n Female Coho Salmon (Oncorhynchus kisutch), and i t s E f f e c t s on Subsequent Embryos. 10 Introduction 11 Materials and Methods 14 Results 20 Discussion 32 Chapter 3 Ontogeny of the C o r t i s o l Response to an Environmental Disturbance i n Steelhead (Oncorhynchus mykiss), and Coho Salmon (O. kisutch), Embryos. 38 Introduction 39 Materials and Methods 43 Results 47 Discussion 58 Chapter 4 E f f e c t s of C o r t i s o l and/or an Environmental Disturbance on Growth and Survival of Chinook Salmon (Oncorhynchus tshawytscha) Embryos. 63 Introduction 64 Materials and Methods 66 Results 69 Discussion 75 Chapter 5 General Discussion 80 References 85 v i LIST OF TABLES Page Table 1. I n i t i a l coho salmon (O. kisutch) oocyte C o r t i s o l l e v e l s , percent m o r t a l i t i e s , time to 50% hatch, and time from 0 to 100% hatch f o r control and experimental groups. Experimental groups were immersed for two hours i n water containing 600 ug/L C o r t i s o l 23 v i i LIST OF FIGURES Page Fi g . 1. Competitive binding curves of C o r t i s o l standards and extracted oocyte tiss u e s e r i a l d i l u t i o n s 24 Fig . 2. Plasma C o r t i s o l content of disturbed and undisturbed adult female coho salmon, (O.kisutch) 25 Fig . 3. Gonadosomatic indices (GSI) of disturbed and undisturbed adult female coho salmon, (0. kisutch) 26 Fig . 4. Oocyte C o r t i s o l content from disturbed and undisturbed adult female coho salmon, (O. kisutch) 27 Fig . 5. Whole body C o r t i s o l content of C o r t i s o l immersed and control coho salmon, (O. kisutch) oocytes 28 Fig . 6. Body length of C o r t i s o l immersed and control coho salmon (O. kisutch), embryos 1, 9, and 17 days post hatch 29 Fig . 7. Body weight (dry) of C o r t i s o l immersed and control coho salmon (O. kisutch), embryos 1, 9, and 17 days post hatch 3 0 F i g . 8. Yolk sac to body weight r a t i o of C o r t i s o l immersed and control coho salmon (O. kisutch), embryos 1 day post hatch 31 Fig . 9. Whole body C o r t i s o l content of steelhead embryos (O. mykiss), exposed to emersion or undisturbed, from -4 to 3 days post hatch 51 Fig . 10. Whole body C o r t i s o l content of coho salmon (O. kisutch) embryos exposed to emersion or undisturbed, over 8 hours, at -5 days post hatch 52 Fig . 11. Whole body C o r t i s o l content of coho salmon (O. kisutch) embryos exposed to emersion or undisturbed, over 8 hours, at 2 days post hatch 53 Fig. 12. Whole body C o r t i s o l content of coho salmon (O. kisutch) embryos exposed to emersion or undisturbed, over 8 hours, at 9 days post hatch..... 54 Fig . 13. Whole body C o r t i s o l content of coho salmon (O. kisutch) embryos exposed to emersion or undisturbed, over 8 hours, at 16 days post hatch 55 v i i i F i g . 14. Whole body C o r t i s o l content of coho salmon (O. kisutch) embryos exposed to emersion or undisturbed, over 8 hours, at 23 days post hatch 56 Fig . 15. Whole body C o r t i s o l content of coho salmon (O. kisutch) embryos exposed to emersion or undisturbed, over 8 hours, at 30 days post hatch 57 Fig . 16. Whole body C o r t i s o l content of chinook salmon (O. tshawytscha), embryos either exposed to emersion, exogenous C o r t i s o l , given both treatments, or neither treatment (controls) 71 Fig . 17. Body length of chinook salmon (O. tshawytscha), embryos either exposed to emersion, exogenous C o r t i s o l , given both treatments, or neither treatment (controls) 72 Fig . 18. Body weight of chinook salmon (O. tshawytscha), embryos either exposed to emersion, exogenous C o r t i s o l , given both treatments, or neither treatment (controls) 73 Fig . 19. Percent Mortality of chinook salmon (O. tshawytscha), embryos either exposed to emersion, exogenous C o r t i s o l , given both treatments, or neither treatment (controls)... 74 i x A C K N O W L E D G E M E N T I would f i r s t l y l i k e to thank Dr. Edward Donaldson, my research supervisor, for giving me the opportunity to pursue graduate studies at the West Vancouver Laboratory, and for providing help, guidance, and encouragement over the past three years. I would also l i k e to express my gratitude to my committee members, Professors Robin L i l e y and Anthony Perks. I thank Dr. L i l e y for accepting me into h i s lab and for h i s advice, and I thank both professors for the valuable experience and information I obtained while attending t h e i r classes. At the West Vancouver Laboratory I wish to thank Ms. Helen Dye for her assistance i n the lab, and for her invaluable experience when negotiating the d i f f i c u l t waters of government paperwork. I would also l i k e to express my appreciation to the other students and s c i e n t i s t s that I had the good fortune to work with at West Van., including Andy Lamb, Luis Afonso, Amos Tandler, and Kyung Chung. This study was p a r t i a l l y funded by a DFO/NSERC grant to Dr. Robin L i l e y , a Rick Hansen Man i n Motion graduate fellowship, and by several Department of Zoology Teaching Assistantships. My f i n a l note of thanks I give to my fiancee, Andrea, who greeted me each evening with her b e a u t i f u l smile and good humour, and without whom these l a s t three years would have been unthinkable. CHAPTER 1 GENERAL I N T R O D U C T I O N 2 General Introduction Fish l i v i n g under natural or a r t i f i c i a l conditions w i l l , at almost a l l points i n t h e i r l i f e h istory, be exposed to changes and/or perturbations i n t h e i r surrounding environment. These can include general environmental s h i f t s i n temperature, pH, water q u a l i t y etc., or can come from d i r e c t i n t e r a c t i o n with predators, conspecifics, or i n the case of culture s i t u a t i o n s , with man. These s t i m u l i can be described as "stressors", and can act to bring about a combination of physiological and behavioural responses i n f i s h often described as "stress". Stress i s an ambiguous term, and one that lacks a cle a r d e f i n i t i o n , with i t s usage seeming to depend la r g e l y on the context i n which i t i s being applied. I t i s sometimes invoked to describe a p a r t i c u l a r stimulus or an adverse environmental condition, while at other times i t i s used to describe a response to the same. Selye (1976) described stress i n terms of the General Adaptation Syndrome (GAS). In i t he proposed three d i s t i n c t stages through which an animal progresses when exposed to stress. The f i r s t phase consists of an immediate phy s i o l o g i c a l response to the stress, and i s termed the alarm phase. This i s followed by a second, resistance phase, during which the animal attempts to escape or compensate for the adverse conditions which are invoking the stress response. The t h i r d and f i n a l phase occurs i f the animal i n unable to escape from or cope with the stress, and experiences exhaustion, and ultimately death. 3 A general underlying concept i n most d e f i n i t i o n s of stress i s proposed by Barton and Iwama (1991) , and that i s that stress represents a response reaction by f i s h to a stimulus, and t h i s response may somehow a l t e r the f i s h ' s homeostatic state. In t h i s thesis I w i l l r e f r a i n from using the term stress as much as possible when describing my own work, as t h i s study focused mainly on a single hormonal aspect of the so c a l l e d stress response. I w i l l instead simply describe the techniques I u t i l i z e d to t r y and invoke t h i s p a r t i c u l a r hormonal response. The terms stress, stress response, and stressor, w i l l s t i l l appear when r e f e r r i n g to the work of other investigators. Fish can react to environmental s t i m u l i i n a number of ways, both behaviourally and p h y s i o l o g i c a l l y , and the stress response can be separated into three d i s t i n c t phases using these c r i t e r i a . The ph y s i o l o g i c a l changes that take place i n f i s h that are exposed to p o t e n t i a l or applied stressors includes a neuroendocrine response which has been described as the primary e f f e c t , a secondary response, which i s described as the p h y s i o l o g i c a l consequences of the primary response, and a t e r t i a r y response, which encompasses such things as immune response, behaviour, growth and reproductive c a p a b i l i t i e s (Mazeaud et al., 1977). The primary stress response i s usually characterized by a rapid elevation of c i r c u l a t i n g l e v e l s of catecholamines and c o r t i c o s t e r o i d s . The major c o r t i c o s t e r o i d found i n the plasma of t e l e o s t s i s C o r t i s o l (Donaldson, 1981), and i t s synthesis and release from the in t e r r e n a l t i s s u e i s the culmination of a hormone 4 cascade that begins i n the brain. An applied or perceived stressor activates nerve impulses to the hypothalamus which i n turn releases c o r t i c o t r o p i n - r e l e a s i n g - f a c t o r (CRF). This hormone i s c a r r i e d by nerve f i b r e s to the anterior p i t u i t a r y gland where i t stimulates the corticotrophs of the pars d i s t a l i s to release the hormone adrenocorticotropin (ACTH) into c i r c u l a t i o n . I t i s ACTH which acts at the l e v e l of the i n t e r r e n a l to stimulate the synthesis and release of C o r t i s o l (Donaldson, 1981; Sumpter et al., 1986). The i n t e r r e n a l t i s s u e of t e l e o s t s i s located i n the pronephric, or head area of the kidney, i n close proximity to the posterior cardinal veins, and i s homologous to the adrenal gland found i n tetrapods. I t i s responsible f o r the synthesis and release of c o r t i c o s t e r o i d s into c i r c u l a t i o n . This region d i f f e r s from the tetrapod adrenal i n that i t does not possess d i s t i n c t medullary and c o r t i c a l tissues, and instead possesses two d i f f e r e n t c e l l types, the i n t e r r e n a l and chromaffin c e l l s , which are responsible f o r corticosteroidogenesis and catecholamine production respectively (Bonga, 1993). C o r t i s o l has been associated with many d i f f e r e n t e f f e c t s on the physiology of f i s h subjected to stressors, including a l t e r e d blood flow, heart rate, and the mobilization of glucose (hyperglycaemia). C o r t i s o l stimulates the mobilization of the animal's carbohydrate stores, and t h i s i n i t i a l reaction may be followed by the mobilization of proteins and l i p i d s as further sources of energy i n the form of glucose (Freeman and I d l e r , 1973; Leach and Taylor, 1980; Vijayan et al., 1994). I n i t i a l l y , the 5 increased c i r c u l a t i n g l e v e l s of C o r t i s o l are of a benefit to the f i s h i n that they f a c i l i t a t e i t s e f f o r t s to eithe r avoid, or accommodate, environmental disturbances. However, prolonged exposure to a adverse environmental conditions can cause chronic a c t i v a t i o n of the hypothalamic-pituitary-interrenal (HPI) axis and lead to extended elevation of c i r c u l a t i n g blood C o r t i s o l . This can cause long term mobilization, rather than storage, of glucose, and can ultimately lead to impaired growth and ti s s u e r e p a i r , and reduced immune and reproductive c a p a b i l i t i e s (Barton and Iwama, 1991; Donaldson, 1990). A decrease i n growth has been observed i n f i s h held under adverse environmental conditions, and t h i s reduction has been attr i b u t e d to the transfer of resources by the f i s h to catabolic p h y s i o l o g i c a l processes associated with the response to these conditions, and away from anabolic processes associated with growth. Brook trout (Salvelinus fontinalis) r a i s e d under conditions of low pH displayed reduced growth and elevated plasma C o r t i s o l l e v e l s (Tarn et al., 1988), while brook trout r a i s e d under high density also displayed reduced growth, but without elevated plasma C o r t i s o l l e v e l s (Vijayan et al. , 1990). A s i m i l a r decrease i n growth has been reported i n f i s h that were fed exogenous C o r t i s o l . One year old rainbow trout (Oncorhynchus mykiss) that were fed C o r t i s o l over a ten week period displayed reduced growth, condition factor, and l i v e r glycogen (Barton et al., 1987). Channel c a t f i s h (Ictalurus punctatus) fed food containing C o r t i s o l for the same period also displayed reduced growth and condition 6 factor, along with reduced hepatosomatic index (Davis et al., 1985) . Immunocompetence i n tel e o s t s has also been demonstrated to be adversely affected by environmental conditions, and has been associated with the accompanying r i s e i n blood C o r t i s o l l e v e l s (Barton and Iwama, 1991; Snieszko, 1974). The immune system of f i s h , and other vertebrates, consists of a number of non-specific components such as integument and b a c t e r i o l y t i c enzymes, as well as s p e c i f i c immune functions such as inflammation and antigen recognition and a c t i v a t i o n of immune eff e c t o r s (Campbell, 1992). Fish exposed to adverse environmental conditions have demonstrated increased s u s c e p t i b i l i t y to disease, increased mortality rates, as well as reduced amount of lysozyme i n various t i s s u e s , and reduced numbers of c i r c u l a t i n g lymphocytes and leucocytes (Barton et al., 1987; Maule et al., 1989; Maule and Schreck, 1990; Mock and Peters, 1990; Pottinger and Pickering, 1992). Similar indications of reduced immunocompetence have been evoked i n f i s h exposed to exogenous C o r t i s o l . Brown trout (Salmo trutta) treated with C o r t i s o l , either o r a l l y or by implantation, have demonstrated reduced numbers of c i r c u l a t i n g lymphocytes and increased m o r t a l i t i e s due to b a c t e r i a l and fungal i n f e c t i o n , while at the same time demonstrating s i g n i f i c a n t l y elevated l e v e l s of plasma C o r t i s o l (Pickering, 1984; Pickering and Pottinger, 1985; Pickering et al., 1989). Coho salmon (Oncorhyncus kisutch) exposed to emersion from water, or fed food containing C o r t i s o l , were seen to display s i g n i f i c a n t l y reduced numbers of c i r c u l a t i n g 7 lymphocytes, while at the same time having s i g n i f i c a n t l y higher lymphocyte numbers i n the thymus and anterior kidney, i n d i c a t i n g C o r t i s o l as a mediating agent i n lymphocyte t r a f f i c (Maule and Schreck, 1990). Reproductive a b i l i t y i s another process i n f i s h which has been shown to be adversely effected by exposure to adverse environmental conditions. The substantial physiological investment and rel i a n c e on c a r e f u l l y orchestrated endocrine events that characterizes reproductive processes make them prime candidates f o r disruption due to environmental perturbations. Elevated l e v e l s of plasma C o r t i s o l have been associated with suppressed plasma androgen l e v e l s i n mature male brown trout and t i l a p i a (Oreochromis mossambicus) (Foo and Lam, 1993a; Pickering et al., 1987), as well as with reduced 17B-estradiol and testosterone secretion i n cultured ovarian f o l l i c l e s of female brown trout (Carragher and Sumpter, 1990; Sumpter et al., 1987). They have also been linked with reduced hepatic estradiol-binding s i t e s i n female brown trout (Pottinger and Pickering, 1990) and reduced gonad s i z e , c i r c u l a t i n g sex s t e r o i d and v i t e l l o g e n i n l e v e l s , and p i t u i t a r y gonadotropin content i n brown and rainbow trout (Carragher et al. , 1989), suggesting that elevated plasma C o r t i s o l i n response to environmental disturbances can act at the l e v e l of the p i t u i t a r y , the l i v e r and the gonads, and may reduce reproductive a b i l i t y . C o r t i s o l , presumably of maternal o r i g i n , has been demonstrated to be present i n measurable l e v e l s i n the fr e s h l y ovulated oocytes of t e l e o s t s (de Jesus et al., 1991; de Jesus and Hirano, 1992; 8 Hwang et al., 1992; Yeoh et al., 1993), as have sex steroids (Feist et al., 1990; Rothbard et al., 1987) and thyroid hormones (Ayson and Lam, 1993; Leatherland et al., 1989a; Leatherland et al., 1989b). Despite the presence of C o r t i s o l i n t e l e o s t oocytes, i t s functional s i g n i f i c a n c e , i f any, has yet to be elucidated. Endogenous production of C o r t i s o l , along with the onset of a functional HPI axis i n tel e o s t s , i s another area of inv e s t i g a t i o n that has received l i t t l e attention. In mammals, and s p e c i f i c a l l y r a t s , the hypothalamic-pituitary-adrenal (HPA) axis has been demonstrated to be sensi t i v e to s t i m u l i in utero, and f e t a l corticosterone l e v e l s respond to hypoxia as early as day 18 of gestation (Ohkawa et al., 1991; Walker et al., 1991). Rats also demonstrate a period of non-responsiveness of the HPA axis that begins shortly a f t e r b i r t h ( G u i l l e t et al. , 1980; Haltmeyer et al., 1966; Sapolsky and Meaney, 1986), and i s suggested as being c r u c i a l for normal growth and d i f f e r e n t i a t i o n of tissues and organs, including the central nervous system (Moisan et al., 1992). Pottinger and Mosuwe (1994) found that rainbow trout embryos demonstrated increased whole body C o r t i s o l content i n response to mechanical disturbance and confinement beginning at 5 weeks post hatch, while Barry et al., (1995) observed a s i m i l a r increase i n rainbow trout i n response to emersion and low water temperature beginning at 2 weeks post hatch. No data i s availa b l e concerning what e f f e c t s t h i s early C o r t i s o l response to disturbance may have on growth or s u r v i v a l i n salmonids. 9 In summary i t can be concluded that exposure of te l e o s t s to adverse environmental conditions and/or disturbances, and the accompanying increase i n plasma C o r t i s o l that often accompanies t h i s exposure, can have deleterious e f f e c t s on growth, immunocompetence, and reproductive a b i l i t y . What i s less c l e a r i s the r o l e of C o r t i s o l i n the early ontogeny of t e l e o s t s . This in v e s t i g a t i o n was i n i t i a t e d to t r y and elucidate the re l a t i o n s h i p between disturbance induced C o r t i s o l production i n sexually mature female salmonids and the possible transfer of maternal C o r t i s o l to t h e i r developing oocytes, and what e f f e c t varying l e v e l s of oocyte C o r t i s o l may have on the v i a b i l i t y and ontogeny of t h e i r subsequent progeny. I also endeavoured to investigate the timing of the onset of endogenously produced C o r t i s o l i n response to environmental disturbance i n embryonic salmonids, as an indi c a t o r of the onset of the HPI axis, and what e f f e c t t h i s response may have on embryonic growth and s u r v i v a l . CHAPTER 2 Transfer of Maternal Cortisol to Maturing Oocytes in Female Coho Salmon corhynchus kisutch), and its Effects on Subsequent Embry< 11 Introduction: Physiological and behavioural responses to environmental disturbances, such as water borne pollutants and temperature extremes, s o c i a l interactions, or as a r e s u l t of aquaculture techniques (handling, sorting etc.), can have deleterious e f f e c t s on the reproductive c a p a b i l i t i e s of tel e o s t s (Barton and Iwama, 1991; B i l l a r d et al. , 1981; Donaldson and Schere, 1983; Donaldson, 1990; Greenberg and Wingfield, 1987). One of the aspects of f i s h physiology through which environmental perturbations may act to a f f e c t reproductive c a p a b i l i t i e s i s v i a the endocrine response to such environmental disturbances. A nearly universal reaction of tel e o s t s to environmental disturbances i s the response of the hypothalamic-pituitary-interrenal axis, a hormone cascade culminating i n the release, from the in t e r r e n a l c e l l s , of the c o r t i c o s t e r o i d C o r t i s o l into the bloodstream (Donaldson, 1981; Sumpter, 1993). The degree to which plasma C o r t i s o l l e v e l s are elevated i n response to an a change i n environmental homeostasis i s often used as a diagnostic t o o l to indicate a "stressed" condition among te l e o s t s (Barry et al., 1993; Barton et al., 1986; Davis and Parker, 1983; Davis and Parker, 1986; Flos et al., 1988; Foo and Lam, 1993; Mazur and Iwama, 1993; Pottinger and Pickering, 1992; Sumpter et al., 1986; Thorpe et al., 1987). Previous work has associated elevated plasma l e v e l s of C o r t i s o l i n salmonids, and other t e l e o s t s , with changes i n such reproductive parameters as c irculat ing levels of sex steroids, as well as with variable plasma gonadotropin and v i t e l l o g e n i n l e v e l s . 12 I t has also been linked with decline i n body s i z e , gonadosomatic index, oocyte s i z e , and p i t u i t a r y gonadotropin content (Carragher et al., 1989; Carragher and Sumpter, 1990; Foo and Lam, 1993; Pickering et al., 1987; Sumpter et al., 1987) Elevated C o r t i s o l l e v e l s have also been demonstrated to reduce hepatic estradiol-binding s i t e s i n salmonids (Pottinger and Pickering, 1990), and potentiate hepatic v i t e l l o g e n i n mRNA synthesis i n t i l a p i a (Ding et al., 1993), suggesting mechanisms by which C o r t i s o l l e v e l s may impact on oocyte q u a l i t y . Direct disturbance, i n the form of emersion from water, to maturing salmonids has also been demonstrated to negatively e f f e c t the q u a l i t y of gametes i n terms of subsequent v i a b i l i t y (Campbell et a l . , 1992). These r e s u l t s indicate a l i n k between the hypothalamic-pituitary-interrenal and hypothalamic-pituitary-gonadal axes, and suggest mechanisms by which external environmental disturbances may act to negatively impact on reproductive c a p a b i l i t i e s i n t e l e o s t s . Another less elucidated aspect of elevated plasma C o r t i s o l i n response to changes i n environmental homeostasis, one that could p o t e n t i a l l y have a bearing on reproductive success, i s the degree and the possible e f f e c t s of transfer of maternally derived C o r t i s o l into developing oocytes. C o r t i s o l has been found to be consistently present i n the fr e s h l y ovulated oocytes of t e l e o s t s (de Jesus et al., 1991; de Jesus and Hirano, 1992; Hwang et al., 1992; Yeoh et al., 1993), as have sex s t e r o i d hormones (de Jesus and Hirano, 1992; F e i s t and Schreck, 1993; F e i s t et al., 1990; 13 Rothbard et al., 1987; Yeoh et al., 1993) and thyroid hormones (T3 and T4) (Ayson and Lam, 1993; de Jesus et al., 1991; Leatherland et a l . , 1989a; Leatherland et al., 1989b). In anadromous salmonids elevated plasma C o r t i s o l l e v e l s have been associated with the migration into freshwater that accompanies sexual maturation and reproduction (Krieberg and Blackburn, 1994). Considering the highly variable nature of plasma C o r t i s o l l e v e l s i n response to the presence, or absence, of environmental disturbances; and assuming that oocyte C o r t i s o l i s of maternal o r i g i n , i t i s not unreasonable to speculate that elevated plasma C o r t i s o l i n an adult female during the period of oocyte growth and maturation may be r e f l e c t e d i n increased oocyte C o r t i s o l content. The purpose of t h i s investigation was to (1) , examine the r e l a t i o n s h i p between elevated maternal plasma C o r t i s o l l e v e l s and and subsequent C o r t i s o l content of oocytes, along with any e f f e c t s on ovary weight i n r e l a t i o n to body weight, and timing of ovulation (2), to chart the f l u x of C o r t i s o l during ontogeny i n oocytes containing natural and experimentally elevated l e v e l s of C o r t i s o l , and (3), to look for indications of possible e f f e c t s of elevated oocyte C o r t i s o l on a number of developmental parameters. 14 M a t e r i a l s a n d M e t h o d s : Applied Disturbance Experiment. Sixteen female coho salmon (Oncorhynchus kisutch), at the end of t h e i r spawning migration, were obtained from the Capilano River Hatchery (North Vancouver, B r i t i s h Columbia) and transported to the Dept. of Fishe r i e s and Oceans research f a c i l i t y at West Vancouver, B r i t i s h Columbia. Preovulatory females were selected by hatchery s t a f f using the degree of hardness of the abdomen as an i n d i c a t i o n of oocyte maturity, and those selected were deemed to be at least two weeks from ovulation. Fish were s p l i t into two equal groups and held outside i n adjacent, i d e n t i c a l 4,600 l i t r e c i r c u l a r f i b e r g l a s s tanks supplied with a combination of flowing Cypress Ck. and dechlorinated c i t y water at a temperature of 4.8 ± 0.6 °C. Fi s h were l e f t f o r 48 hours to acclimate to t h e i r surroundings before experimental manipulation was i n i t i a t e d . F i s h i n the experimental group were exposed to an environmental disturbance consisting of a mechanical a g i t a t i o n twice d a i l y over a period of two weeks. Every day, at randomly chosen times, f i s h were chased around the tank with a hand net for approximately 60 seconds. Fish i n the control group were l e f t undisturbed f o r the same two week period. A disturbance was not applied to the experimental group on the day of sampling. At the end of the two week period f i s h from both groups were netted and placed i n a l e t h a l dose of anaesthetic (200 mg/L MS222, buffered 1:1 with sodium bicarbonate). Blood was c o l l e c t e d from 15 each f i s h v i a the caudal vasculature and placed on i c e f o r < 30 min. p r i o r to being centrifuged f o r plasma separation. The ovaries were removed from each f i s h and ovulation state was determined. The f i s h were judged to have ovulated i f the majority of oocytes i n each ovary were detached and free i n the body cavity . Ovaries were then weighed and a sample of oocytes was taken from each. The carcass of each f i s h was also weighed i n order to determine gonadosomatic index (GSI). Plasma and oocyte samples were stored at -50° C for l a t e r C o r t i s o l determination. Immersion Experiment. Oocytes from f i v e female coho salmon (Oncorhynchus kisutch) were obtained from the Capilano River Hatchery (North Vancouver, B r i t i s h Columbia) a f t e r being selected as having ovulated by hatchery s t a f f . M i l t was also c o l l e c t e d by s t a f f from three male f i s h . Oocytes and m i l t were stored i n p l a s t i c bags f i l l e d with oxygen and placed on ice i n a cooler for transportation to the on-s i t e hatchery at the Dept. of Fishe r i e s and Oceans research f a c i l i t y at West Vancouver, B r i t i s h Columbia. Oocytes from each of the f i v e females were subsequently divided volumetericaly into equal control and experimental groups (n»300 to 700, depending on female). Oocytes were then placed i n seperate waxed paper containers before m i l t pooled from the three males was added. F e r t i l i z a t i o n was i n i t i a t e d by the addition of well water to each paired group simultaneously. This allowed for oocytes from each female to provide both experimental and control specimens. 16 Control and experimental groups were then placed i n two separate compartmentalized trays and placed i n 10 l i t r e s of well water at 11.7° C to which previously had been added 5 ml of 95% ethanol containing 6 mg of C o r t i s o l (Fisher). The concentration of ethanol was 0.05%, and the f i n a l C o r t i s o l concentration was 600 ug/L. The control group's water contained 5 ml 95% ethanol alone. Immersion of salmonid alevins i n sex steroids u t i l i z i n g ethanol as a solvent has been shown to be an e f f e c t i v e method of manipulating sex d i f f e r e n t i a t i o n i n salmonids ( P i f e r r e r and Donaldson, 1992). Trays were l e f t for 2 hours before being placed i n the hatchery stack and allowed to f l u s h with running well water (10 l i t e r min" 1) . The tray containing the immersed group was placed below the control group to avoid cross contamination with C o r t i s o l . Immediately a f t e r the 2 hour immersion time a sample of 10 oocytes was removed from each r e p l i c a t e , rinsed f o r f i v e minutes i n running well water to remove any exogenous C o r t i s o l , and stored at -50° C for l a t e r analysis of C o r t i s o l content. Sampling was repeated at 1, 3, and 8 days post f e r t i l i z a t i o n , and every eight days afterwards u n t i l 56 days post f e r t i l i z a t i o n . Three days a f t e r f e r t i l i z a t i o n , when there was believed to be no further threat of cross contamination, experimental and control groups were arranged so that a l l oocytes from each female were i n the same tray receiving water at the same temperature (11.2 ± 0.5° C) . This was done to standardize the conditions experienced by both control and experimental groups during development. 17 Developmental parameters such as timing and synchrony of hatch, percent mortality, yolk sac to body weight r a t i o at hatch, and subsequent dry weight and length of juveniles was monitored at in t e r v a l s during the extent of the experiment. Tissue Extraction. C o r t i s o l was extracted from t i s s u e u t i l i z i n g methods modified from F e i s t et al. , (1990). Frozen oocytes or juveniles were placed singly, or i n groups of f i v e , i n culture tubes and allowed to s i t for approx. 12 hours i n 0.1 N NaOH. Groups of 5 eggs were used when C o r t i s o l l e v e l s i n tissue f e l l below that detectable using a single oocyte. Tissue was then homogenized by hand i n a 1.5 ml glass t i s s u e homogenizer (Fisher). The homogenizer was rinsed with d i s t i l l e d water between each sample, and separate homogenizers were u t i l i z e d f or experimental and control groups. Homogenized ti s s u e was then vortexed f o r 30 seconds i n 10 or 15 ml of d i e t h y l ether (BDH), f o r single and pooled samples respectively, and the aqueous and organic layers were allowed to separate. The organic layer was decanted o f f a f t e r the aqueous layer was snap frozen by immersion i n acetone (BDH) cooled with dry ic e . This procedure was repeated for each sample and the organic layers were pooled i n a clean culture tube. The ether was then evaporated o f f by the application of a l i g h t stream of compressed a i r into each tube. After the tubes had dried, a further 1 ml of ether was used to wash down the sides of the tube and concentrate the extract i n the bottom. The ether was again evaporated o f f before the dried 18 extract was reconstituted i n 100 or 200 u l of phosphate buffered s a l i n e (pH 7.4) with 0.1 % g e l a t i n , again f o r singl e and pooled samples respectively, and stored at -50° C f o r l a t e r C o r t i s o l determination. C o r t i s o l Quantification. C o r t i s o l l e v e l s i n plasma and extracted t i s s u e samples was measured v i a a commercially available antibody coated tube radioimmunoassay k i t (INCstar). The protocol was modified by increasing the aliquot of standards and samples from 10 to 20 u l . This was done to increase the s e n s i t i v i t y of the assay (Sumpter, unpublished data). V a l i d a t i o n of the extraction procedure was done v i a a competitive binding curve u t i l i z i n g s e r i a l d i l u t i o n s of extracted samples that was seen to l i e p a r a l l e l to the standard curve (Fig. 1). Extraction e f f i c i e n c y was determined at each extraction i n t e r v a l as s p e c i f i e d by the k i t manufacturer (INCstar). E f f i c i e n c y was further validated by the use of [ H 3 ] C o r t i s o l . A l l values are adjusted for extraction e f f i c i e n c y . Extraction e f f i c i e n c y was determined to be 74.4 ± 1.3 % (n=20) f o r single samples, and 54.3 ± 1.9 % (n=20) for pooled samples (n=5). S t a t i s t i c a l Analysis. S t a t i s t i c a l tools u t i l i z e d were Student's t - t e s t , ANOVA, Mann-Whitney Rank Sum te s t , Fisher Exact t e s t and 2 X 2 Chi-square contingency table. The Levene Median and the Kolmogorov-Smirnov tes t s were used to t e s t f o r equal variance and normality 19 respectively. The Student-Newman-Keuls t e s t was u t i l i z e d to determine s i g n i f i c a n c e among means (SigmaStat™, Jandel S c i e n t i f i c Software) . Data were l o g 1 0 transformed to meet requirements of normality and equal variance where appropriate. 20 Results: Applied Disturbance Experiment. One of the undisturbed females demonstrated oocyte C o r t i s o l l e v e l s (46.84 ±7.37 ng/gm) that were higher than any other female, control or experimental. This female also demonstrated extremely high plasma C o r t i s o l l e v e l s (1032.3 ng/ml) when compared to the other females. This plasma C o r t i s o l l e v e l i s i n d i c a t i v e of a diseased or moribund f i s h (Barton and Iwama, 1991; Sumpter et al., 198 6), one that was most probably not exh i b i t i n g t y p i c a l C o r t i s o l dynamics, and as such was removed from the control group. One of the experimental females died within four hours of transport and was also removed. Mean plasma C o r t i s o l was seen to be higher, though not s i g n i f i c a n t l y , i n the disturbed group at the time of oocyte sampling compared to the undisturbed group ( t - t e s t , p > 0.05) (Fig. 2). Mean gonadosomatic index (GSI) of the disturbed and undisturbed groups was not found to be s i g n i f i c a n t l y d i f f e r e n t at the time of oocyte sampling ( t - t e s t , p > 0.05), (Fig. 3). Five of the seven females i n the disturbed group were seen to have ovulated, as compared to only one of the seven undisturbed females, though t h i s difference was not seen to be s i g n i f i c a n t l y d i f f e r e n t (Fisher Exact t e s t , p > 0.05). Mean oocyte C o r t i s o l l e v e l s i n the disturbed adult females was s i g n i f i c a n t l y higher than that observed i n the undisturbed ind i v i d u a l s (Fig. 4), (ANOVA, p < 0.05). Pairwise multiple comparison (Student-Newman-Keuls) revealed that a l l of the disturbed females had mean oocyte C o r t i s o l l e v e l s that were s i g n i f i c a n t l y higher than a l l of the undisturbed females, with the exception of the anomalous control f i s h . Immersion Experiment. The immersion protocol s i g n i f i c a n t l y elevated mean oocyte C o r t i s o l content i n each of the manipulated groups when compared to the untreated group from the same female ( t - t e s t , p < 0.0001), and a l l manipulated groups had mean oocyte C o r t i s o l l e v e l s that were s i g n i f i c a n t l y higher than those i n the untreated groups (Table 1) (ANOVA, p < 0.05). Mean oocyte C o r t i s o l content among each of the immersed groups declined over 24 hours (Fig. 5) , and were not s i g n i f i c a n t l y d i f f e r e n t from the i t s paired untreated group by 16 days post immersion ( t - t e s t , p > 0.05). Mortality rates did not d i f f e r s i g n i f i c a n t l y between paired immersed and untreated groups (Table 1) (Chi square contingency table, p > 0.05). Mean time to 50% hatch, and mean time from beginning to 100% hatch, also did not vary s i g n i f i c a n t l y between immersed and untreated groups (Table 1) (Mann-Whitney, p > 0.05). Body length and dry weight of each paired group was monitored for 24 days post hatch. Fish would have been followed further through development, but a power f a i l u r e at the f a c i l i t y resulted i n s i g n i f i c a n t m o r t a l i t i e s taking place among a l l groups. The experiment was terminated at t h i s point. There was no s i g n i f i c a n t difference seen i n mean body length (Fig. 6), or mean weight (Fig. 22 7) between paired immersed and untreated groups at any of the sampling times ( t - t e s t , p > 0.05). Mean yolk sac to body weight r a t i o was determined at the f i r s t sampling period a f t e r hatch for a l l groups, and no s i g n i f i c a n t difference was seen between paired immersed and untreated groups (Fig. 8) ( t - t e s t , p > 0.05). 23 Table 1. I n i t i a l oocyte C o r t i s o l l e v e l s , percent m o r t a l i t i e s , time to 50% hatch, and time from 0 to 100% hatch f o r control and experimental groups. Experimental groups were immersed f o r two hours i n water containing 600 ug/L C o r t i s o l . TREATMENT CONTROL EXPERIMENTAL I n i t i a l Oocyte C o r t i s o l Level (ng/g wet weight) (n=20) 37.03 ± 5.43 232.68 ± 13.93* Percent Mortality Female #1 #2 #3 #4 #5 41.88 45.96 46. 62 30.75 46.12 42.77 40.46 43 . 88 30.26 46.26 Time to 50% Hatch (hours) (n=5) 966.9 ± 1.07 962.1 ± 1.82 Time from 0 to 100% Hatch (hours) (n=5) 80.4 ± 1.82 80.4 ± 2.01 Values represent mean ± SE. * denotes s i g n i f i c a n t difference between control and immersed groups (p < 0.05). 24 1.00 0.90 0.80 0.70 0.60 0.50 0.40 0.30 0.20 0.10 0.00 10 - •+- S t a n d a r d s i i i i i i_ 100 C o r t i s o l L e v e l (ng/ml) — • — Di lu t ions 1000 Figure 1 . Competitive binding curves of C o r t i s o l standards and s e r i a l d i l u t i o n s of extracted coho salmon (Oncorhynchus kisutch), oocyte t i s s u e (n = 1 0 ) . 25 Figure 2. Plasma C o r t i s o l l e v e l s of adult female coho salmon (Oncorhynchus kisutch). Experimental f i s h were disturbed v i a mechanical a g i t a t i o n (chased with hand net f o r 60 sec.) twice d a i l y fo r two weeks. Control f i s h were l e f t undisturbed. No s i g n i f i c a n t difference was observed (p > 0.05). Bars represent mean ± SE (n=7) . 26 Figure 3. Gonadosomatic indices of adult female coho salmon (Oncorhynchus kisutch). Experimental f i s h were disturbed v i a mechanical a g i t a t i o n (chased with hand net for 60 sec.) twice d a i l y for two weeks. Control f i s h were l e f t undisturbed. No s i g n i f i c a n t difference was observed (p > 0.05). Bars represent mean ± SE (n=7). 27 Figure 4. Oocyte C o r t i s o l content of adult female coho salmon (Oncorhynchus kisutch). Experimental f i s h were disturbed v i a mechanical a g i t a t i o n (chased with hand net f o r 60 sec.) twice d a i l y fo r two weeks. Control f i s h were l e f t undisturbed. Bars represent mean ± SE (n=7). * indicates experimental groups which d i f f e r e d s i g n i f i c a n t l y from a l l control groups (p < 0.05). Figure 5. Whole body C o r t i s o l content of C o r t i s o l immersed and control coho salmon (Oncorhynchus kisutch), oocytes. Immersed oocytes were placed i n a solution of 600 ug/L C o r t i s o l f o r 2 hours immediately a f t e r f e r t i l i z a t i o n , during the water hardening process. Points indicate mean ± SE (n=20). * indicates immersed groups that d i f f e r e d s i g n i f i c a n t l y from controls (p < 0.05). 29 4 Figure 6. Body length of coho salmon (Oncorhynchus kisutch), embryos from the f i v e paired C o r t i s o l immersed and control groups of oocytes 40, 48, and 56 days post f e r t i l i z a t i o n (1, 9, and 17 days post hatch). No s i g n i f i c a n t difference was observed (p > 0.05). Bars represent mean ± SE (n=10). 30 Figure 7. Body weight (dry) of coho salmon (Oncorhynchus kisutch) , embryos from the f i v e paired C o r t i s o l immersed and control groups of oocytes 40, 48, and 56 days post f e r t i l i z a t i o n (1, 9, and 17 days post hatch). No s i g n i f i c a n t difference was observed (p > 0.05). Bars represent mean ± SE (n=10). 31 10 9 8 7 6 5 4 3 2 1 0 I I Cn1 Ex1 Cn2 Ex2 Cn3 Ex3 Cn4 Treatment Groups II Ex4 Cn5 Ex5 Figure 8. Ratio of yolk sac to body weight (dry) of coho salmon (Oncorhynchus kisutch), embryos from the f i v e paired C o r t i s o l immersed and control groups of oocytes 40 days post f e r t i l i z a t i o n (1 day post hatch). No s i g n i f i c a n t difference was observed (p > 0.05). Bars represent mean ± SE (n=10). 32 Discussion: Mean plasma C o r t i s o l l e v e l s among the disturbed females was higher than those observed i n the undisturbed group (Fig. 2) , though t h i s was not seen to be s t a t i s t i c a l l y s i g n i f i c a n t . My f a i l u r e to observe a s i g n i f i c a n t difference between the undisturbed and disturbed females i n terms of plasma C o r t i s o l may have been due to the combination of a number of factors. The experimental f i s h were not disturbed on the day of sampling and thus may have had r e l a t i v e l y lower plasma C o r t i s o l than would have been observed had they been disturbed, or the ag i t a t i o n experienced by the undisturbed f i s h during netting and app l i c a t i o n of anesthetic p r i o r to blood sampling may have elevated t h e i r plasma C o r t i s o l l e v e l s to those approaching that of the disturbed f i s h . I t may have also been the case that a f t e r 14 days of treatment, the disturbed f i s h were becoming acclimated to the mechanical a g i t a t i o n protocol and were not responding to the disturbance with as substantial a r i s e i n plasma C o r t i s o l . The proportion of females i n the disturbed group that had undergone ovulation over the two week treatment period (5 of 7) , appeared to be higher than that observed i n the undisturbed group (1 of 7), though t h i s difference was not seen to be s t a t i s t i c a l l y s i g n i f i c a n t . However, the p value observed (0.053), was very close to being considered s i g n i f i c a n t (0.05), suggesting that the disturbance protocol may have had some e f f e c t on the timing of ovulation. There i s some evidence suggesting a r o l e f o r C o r t i s o l i n ovulation. Bry (1985) reported that plasma C o r t i s o l l e v e l s were 33 low i n adult female rainbow trout p r i o r to ovulation, but showed a marked elevation during the immediate post-ovulatory period. I t has also been reported that plasma C o r t i s o l i n adult female rainbow trout attained maximal concentration on the day of ovulation (Bry, 1989) . I t may be that the female coho i n the disturbed group experienced elevated C o r t i s o l l e v e l s as a r e s u l t of the applied disturbance, and t h i s elevation i n c i r c u l a t i n g C o r t i s o l l e v e l s may have influenced the timing of ovulation. The applied disturbance protocol was seen to be e f f e c t i v e i n s i g n i f i c a n t l y elevating oocyte C o r t i s o l l e v e l s above those observed i n the undisturbed females (Fig. 4). The assumption was made that those females subjected to mechanical a g i t a t i o n would have experienced elevated l e v e l s of C o r t i s o l i n c i r c u l a t i o n . I t has been speculated that the hormones present i n the oocytes and early embryonic stages of tel e o s t s are of maternal o r i g i n (Greenblatt et al., 1989; Hwang et al., 1992). Injection of female r a b b i t f i s h (Siganus guttatus) with thyroxine (T4) has been demonstrated to increase T4 and triiodothyronine (T3) i n maternal c i r c u l a t i o n , and subsequently i n oocytes (Ayson and Lam, 1993). I n t r a a r t e r i a l i n j e c t i o n of labeled C o r t i s o l i n P a c i f i c salmon has been demonstrated to r e s u l t i n transfer of C o r t i s o l to almost a l l tissues very shortly a f t e r a p p l i c a t i o n (Donaldson and Fagerlund, 1972) . The increases i n C o r t i s o l content I observed i n the oocytes of those females which were subjected to mechanical a g i t a t i o n , as compared to those that were not, suggests that increased plasma 34 C o r t i s o l as a r e s u l t of an environmental disturbance may lead to increased deposition of C o r t i s o l i n the oocytes, even though I did not observe a s i g n i f i c a n t difference i n c i r c u l a t i n g C o r t i s o l l e v e l s . I t i s in t e r e s t i n g to note that the female that was excluded from the undisturbed group because of i t s extremely high plasma C o r t i s o l l e v e l , also demonstrated the highest oocyte C o r t i s o l content among a l l females. An applied disturbance during oogenesis i n female salmonids has been demonstrated to r e s u l t i n a decrease i n the q u a l i t y of the oocytes i n terms of s u r v i v a l to hatch (Campbell et al., 1992) . For t h i s reason I chose to a r t i f i c i a l l y elevate oocyte C o r t i s o l l e v e l s i n previously unmanipulated adult females i n an attempt to i s o l a t e the e f f e c t that C o r t i s o l may have on subsequent oocyte s u r v i v a l , as well as on the other measured developmental parameters. A l l groups of oocytes immersed i n C o r t i s o l exhibited s i g n i f i c a n t l y elevated C o r t i s o l l e v e l s when compared to controls immediately post immersion (Table 1) . Immersion treatment has been demonstrated to be e f f e c t i v e i n t r a n s f e r r i n g s t e r o i d hormones throughout coho salmon oocytes ( P i f e r r e r and Donaldson, 1994), and yolk and chorion were not seen to a f f e c t accumulation of radioactive steroids i n rainbow trout oocytes and embryos (Antila, 1984). In a l l groups, immersed and control, i n i t i a l oocyte C o r t i s o l l e v e l s measured immediately post immersion were higher than those l e v e l s observed 24 hours l a t e r (Fig. 5). This pattern of high oocyte hormone l e v e l s at f e r t i l i z a t i o n , followed by a rapid decline during early ontogeny has been observed 35 for C o r t i s o l i n chum salmon (Oncorhynchus keta) (de Jesus and Hirano, 1992), t i l a p i a (Oreocromis mossambicus) (Hwang et al., 1992) and Japanese flounder (Paralicthys olivaces) (de Jesus et a l . , 1991), as well as for other s t e r o i d hormones i n three species of t i l a p i a (O. niloticus, O. mossambicus, and Tilapia hornorum) (Rothbard et al., 1987), rainbow trout (Oncorhynchus mykiss) (Feist and Schreck, 1993; Yeoh et al., 1993), and coho salmon (Feist et al., 1990). This i s not the pattern reported for a l l hormones detected i n t e l e o s t oocytes. For example, the thyroid hormones, T4 and T3, have been observed to decrease gradually, or remain r e l a t i v e l y constant, i n the oocyte and subsequent larvae of r a b b i t f i s h (Ayson and Lam, 1993), t i l a p i a (O. mossambicus) (de Jesus et al. , 1991) and P a c i f i c salmon (Leatherland et al., 1989a; Leatherland et al., 1989b). My observation that there was no differences i n mortality rates between control and experimental groups suggests that, although an applied disturbance to adult female salmonids undergoing oogenesis has been demonstrated to decrease oocyte q u a l i t y (Campbell et al., 1992) and to increase oocyte C o r t i s o l content (this study), the decline i n q u a l i t y appears to be the r e s u l t of factors other than C o r t i s o l content alone. The f a i l u r e to observe any differences i n the measured parameters between treated and untreated groups (Table 1), coupled with the observed rapid decline i n oocyte C o r t i s o l a f t e r f e r t i l i z a t i o n (Fig. 5), seems to suggest that maternally derived 36 C o r t i s o l i s cleared from the oocytes early i n ontogeny, and does not have a s i g n i f i c a n t impact on l a t e r development. The mechanism underlying the clearance of C o r t i s o l from oocytes, whether by metabolic processes or simply by d i f f u s i o n , i s an area for further investigation. Yeoh (1993) has presented evidence supporting C o r t i s o l metabolism early i n salmonid ontogeny, reporting the presence of C o r t i s o l glucuronide i n steelhead embryos immediately post f e r t i l i z a t i o n . Yeoh (1993) also observed an increase i n the synchrony of hatch of salmonid oocytes immersed i n C o r t i s o l immediately post f e r t i l i z a t i o n . My experiment d i d not y i e l d a s i m i l a r r e s u l t , and t h i s could be due to a number of factors. Yeoh u t i l i z e d DMSO (di-methyl-sulfoxide) as a solvent for immersion treatments, and was thus able to r a i s e oocyte C o r t i s o l l e v e l s higher than I did u t i l i z i n g 95% ethanol. The increased oocyte C o r t i s o l l e v e l s may have effected synchrony of hatch, although Yeoh observed an increase i n hatch synchrony i n oocytes immersed i n any hormone ( C o r t i s o l , thyroxine, testosterone) dissolved i n DMSO, and i n oocytes immersed i n DMSO alone, suggesting that the solvent may have had some e f f e c t on the developing embyros. In conclusion my re s u l t s indicate that C o r t i s o l content i n fre s h l y ovulated oocytes of coho salmon, and probably a l l salmonids, i s r e f l e c t i v e of the c i r c u l a t i n g l e v e l s of C o r t i s o l i n the adult female during late oogenesis, and that an increase i n c i r u c l a t i n g C o r t i s o l l e v e l s as a r e s u l t of an environmental perturbation, i f sustained over a long enough period, can r e s u l t i n elevated oocyte C o r t i s o l content. My observation of rapid decline i n oocyte C o r t i s o l content during early ontogeny, coupled with my f a i l u r e to observe any differences i n the parameters monitored between oocytes with natural, and a r t i f i c i a l l y high, C o r t i s o l content suggests that C o r t i s o l i s not a major factor i n early salmonid development. CHAPTER 3 Ontogeny of the Cortisol Response to an Environmental Disturbance Steelhead {Oncorhynchus mykiss), and Coho Salmon {Oncorhynchus kisutch), Embryos. 39 Introduction: The a c t i v a t i o n of the hypothalamic-pituitary-interrenal (HPI) axis i s a nearly universal p h y s i o l o g i c a l response of t e l e o s t s to environmental disturbances or perturbations. A neuroendocrine response involving c o r t i c o t r o p i n releasing factor (CRF) that begins at the l e v e l of the hypothalamus and r e s u l t s i n the release of adrenocorticotropin (ACTH) from the p i t u i t a r y gland, which p r e c i p i t a t e s a subsequent release into c i r c u l a t i o n of the c o r t i c o s t e r o i d C o r t i s o l from the in t e r r e n a l t i s s u e (Donaldson, 1981; Mazeaud et al., 1977; Sumpter et al., 1986). This response has been commonly employed as a diagnostic t o o l to indicate a "stressed" condition i n tel e o s t s when c i r c u l a t i n g t i t e r s of C o r t i s o l are elevated above baseline l e v e l s (Barry et al., 1993; Barton et al., 1986; Davis and Parker, 1983; Davis and Parker, 1986; Flos et al. , 1988; Foo and Lam, 1993; Mazur and Iwama, 1993; Pottinger and Pickering, 1992; Sumpter et al., 1986; Thorpe et al., 1987), and has been speculated to have short term adaptive advantages, but may i n the long term prove deleterious (Barton and Iwama, 1991). Most investigations of t h i s response i n salmonids have dealt with f i s h that were either sexually mature, or i n the juvenile stage of development (Pickering et al., 1987; Carragher et al., 1989; Pickering et al., 1982; Pottinger and Pickering, 1990; Barton et al., 1980). There i s not a great deal of information a v a i l a b l e concerning the onset of response of the HPI axis i n reaction to 40 environmental perturbations during embryogenesis and early ontogeny. In birds there i s evidence that the pit u i t a r y - a d r e n a l axis i s established as early as day 14.5 of incubation (Woods et al. , 1971), and increased adrenal a c t i v i t y has been associated with hatching i n ducks (Tanabe et a l . , 1983). This has been speculated to be important i n adapting the developing animal to a new environment. Embryonic r a t adrenals secrete corticosterone i n response to a vari e t y of s t i m u l i beginning at the l a t e f e t a l period (Sapolsky and Meaney, 1986), and the r a t f e t a l hypothalamic-pituitary-adrenal (HPA) axis has been demonstrated to respond to f e t a l hypoxia as early as day 18 of gestation (Ohkawa et al., 1991). Neonatal rats exhibit basal and stimulated plasma corticosterone l e v e l s approaching those of adults at 1-2 days of age ( G u i l l e t et al., 1980), and the HPA axis has the functional capacity to respond to a vari e t y of s t i m u l i throughout neonatal l i f e (Walker et al., 1991) ACTH immunoreactive c e l l s have been observed to f i r s t appear i n the p i t u i t a r y of the rainbow trout (Oncorhynchus mykiss), approximately 10 days p r i o r to hatch (Saga, 1993) , and i n the cloudy dogfish (Scyliorhinus torazame) , and t i l a p i a (Oreochromis mossambicus) , at the time of, or just a f t e r , hatching (Chiba et al., 1992; Fu and Lock, 1990). A s i m i l a r condition has been reported i n the salamander (Hynobius nebulosus) (Oota and Saga, 1991). This suggests a possible r o l e of the HPI axis i n hatching, 41 although c o r t i c o s t e r o i d s did not modulate hatching i n the zebrafish (Brachydanio rerio) (Bogsnes and Walther, 1994). The conversion of steroids to metabolites has been observed i n oocytes and early embryos (cleavage to 20 pair-of-somites stage) of rainbow trout (Oncorhynchus mykiss) (Antila, 1984), and the conversion of l a b e l l e d progesterone to C o r t i s o l and cortisone has been demonstrated i n rainbow trout oocytes as early as 1 week post f e r t i l i z a t i o n ( P i l l a i et al., 1974). de Jesus and Hirano (1992) have observed changes i n whole body C o r t i s o l content of chum salmon (Oncorhynchus keta), approximately one week p r i o r to hatch, and si m i l a r changes i n whole body C o r t i s o l content have been reported for coho salmon (Oncorhynchus kisutch), (this t h e s i s , chpt 2). This suggests that embryonic salmonids possess the required enzymatic pathways needed to synthesize C o r t i s o l , and that the embryonic i n t e r r e n a l t i s s u e i s able to secrete C o r t i s o l . I t does not indicate at what point t h i s secretion i s e l i c i t e d i n response to environmental disturbance. Salmonids spend the early part of t h e i r l i f e h i s t o r y within the gravel redds of t h e i r natal streams. This period of time i s temperature dependent and includes incubation, hatch, and yolk sac absorption (Neitzel et al., 1985). During t h i s time embryonic salmonids can be exposed to po t e n t i a l environmental disturbances such as temperature extremes, fungal and protozoan i n f e c t i o n , v a r i a t i o n s i n water flow, dissolved oxygen concentration, pH, and deposition of sediments and organic matter (Fiss and Carline, 1993; Johnson, 1980; L i s l e and Lewis, 1992; Murray and Beacham, 1986; 42 Ne i t z e l and Becker, 1985; Reiser and White, 1988; Ringler and H a l l , 1988; Scrivener, 1988; Scrivener and Brownlee, 1989; Taylor and Bailey, 1979) . I t i s i n t e r e s t i n g to speculate whether an integrated HPI response, i n terms of C o r t i s o l synthesis, to these p o t e n t i a l environmental disturbances i s advantageous to embryonic salmonids. There i s some evidence for movement of salmonid larvae within the gravel redds i n response to drying and temperature s t i m u l i (de Leaniz et al., 1993; Mcdonald, Steve, perrs comm), and i t may be that the energy (glucose) mobilizing properties of C o r t i s o l may f a c i l i t a t e t h i s . The purpose of t h i s investigation was to examine whole body C o r t i s o l content i n embryonic steelhead and coho salmon f o r a period immediately p r i o r to, and approximately 30 days post hatch, and to u t i l i z e v a r i a t i o n i n C o r t i s o l content i n response to an applied disturbance as an in d i c a t i o n of a functional HPI axis. 43 M a t e r i a l s a n d M e t h o d s : Ontogeny of C o r t i s o l Response to Environmental Disturbance. Oocytes from a single female, and m i l t from three male, steelhead trout (Oncorhynchus mykiss) were obtained from the Chilliwack River Hatchery (Chilliwack, B r i t i s h Columbia). Gametes were stored on i c e i n p l a s t i c bags f i l l e d with oxygen and transported to the Department of Fishe r i e s and Oceans f a c i l i t y at West Vancouver, B r i t i s h Columbia. Oocytes were f e r t i l i z e d with m i l t pooled from the three males and were placed i n an incubation tray (Heath Techna. Corp.) i n the on s i t e hatchery and supplied with flowing well water (10 l i t r e s min - 1) at 13.1 ± 1.2° C. Oocytes were allowed to develop to a point approximately one week p r i o r to hatch, 20 dpf (days post f e r t i l i z a t i o n ) , as t h i s has been previously reported as the point where an increase i n oocyte C o r t i s o l has been observed i n salmonids (de Jesus and Hirano, 1992; Yeoh, 1993). A control and experimental set consisting of eight groups of f i v e oocytes each were removed and placed i n separate incubation trays. Oocytes were transferred a minimum of four days p r i o r to experimental manipulation, and the procedure was repeated every 5 days, at -4, 1, 6, 11, 16, 21, 26, an 32 days post hatch (dph). Embryos i n the experimental groups were subjected to a repeated environmental disturbance consisting of emersion out of water for 60 seconds, every f i f t e e n minutes over a period of 2 hours. The control group of oocytes or embryos was undisturbed. 44 Since C o r t i s o l q u a n t i f i c a t i o n required the measurement of whole body C o r t i s o l content, as opposed to plasma C o r t i s o l , i t was f e l t that repeated a p p l ication of an environmental disturbance over time was preferable to a single episode. This would allow f o r the de novo synthesis of C o r t i s o l from precursors, and for the accumulation of C o r t i s o l i n ti s s u e . At the end of the 2 hour period, a f t e r the eighth emersion, pre-hatch embryos i n both sets were k i l l e d by placement f o r 15 minutes i n a freezer at -50° C. Post-hatch embryos were k i l l e d by immersion i n an overdose of anaesthetic (200 mg/L MS-222 buffered 1:1 with sodium bicarbonate). Samples were frozen at -50° C for l a t e r assessment of whole body C o r t i s o l content. This protocol was repeated every f i v e days from the eyed egg stage, through hatching, up u n t i l yolk sac absorption. Time Course of C o r t i s o l Response to Environmental Disturbance During Ontogeny. Eggs from a single female coho salmon (Oncorhynchus kistuch) and m i l t from three males were obtained from the Capilano River Hatchery (North Vancouver, B r i t i s h Columbia). Gametes were stored on ice i n p l a s t i c bags f i l l e d with oxygen and transported to the Department of Fisheries and Oceans f a c i l i t y at West Vancouver. Oocytes were f e r t i l i z e d with m i l t pooled from the three males and placed i n an incubation tray (Heath Techna. Corp.) i n the on s i t e hatchery and supplied with flowing well water (10 l i t r e s min~ 1) at 10.7 ± 0.3° C. 45 After the oocytes were allowed to develop to a point approximately one week p r i o r to hatch, 38 dpf, a control and experimental set consisting of eight groups of f i v e embryos each were removed and placed i n separate incubation trays. Embryos were transferred a minimum of four days p r i o r to experimental manipulation, and the procedure was repeated every 7 days, instead of 5 as i n the previous experiment because increased routine a c t i v i t y by other workers at the f a c i l i t y necessitated that t r i a l s be c a r r i e d out on weekends to ensure minimal disturbance of the control groups. Oocytes or embryos i n the experimental groups were subjected to a repeated environmental disturbance as described above. The control group of oocytes or embryos was undisturbed. Both control and experimental groups were sampled at 1, 2, 4, and 8 hours a f t e r the commencement of the t r i a l , and embryos were k i l l e d i n the manner described above. Tissue Extraction and C o r t i s o l Quantification. Embryo tissue was extracted, and C o r t i s o l q u a n t i f i c a t i o n was determined, as described i n chapter 2. S t a t i s t i c a l Analysis. To determine whether there was a s i g n i f i c a n t difference i n whole body C o r t i s o l content between the experimental and control groups, a students t - t e s t was applied to data for each sampling period (Sigma Stat™, Jandel S c i e n t i f i c ) . 46 Whole body C o r t i s o l l e v e l s within control or experimental groups were analyzed by ANOVA, and pairwise comparisons were done u t i l i z i n g Student-Neuman-Keuls t e s t . The Levene Median and the Kolmogorov-Smirnov t e s t s were used to t e s t for equal variance and normality respectively. Data were l o g 1 0 transformed where assumptions of normality and homogeneity of variance were not met. 47 Results: Ontogeny of C o r t i s o l Response to Disturbance. No s i g n i f i c a n t difference i n whole body C o r t i s o l content was observed i n control and experimental steelhead embryos at -4 and 1 dph. At 6 dph there was a s i g n i f i c a n t increase observed i n the whole body C o r t i s o l content of those embryos that received the repeated disturbance ( t - t e s t , p<0.05) (Fig. 1). Over the next 5 sampling i n t e r v a l s (11, 16, 21, 2 6 and 3 2 dph), the whole body C o r t i s o l content of a l l steelhead embryo groups receiving the repeated disturbance were s i g n i f i c a n t l y higher than those observed i n the controls ( t - t e s t , p<0.05). The difference i n whole body C o r t i s o l content between the control and experimental groups progressed from a 1.5 f o l d increase at 6 dph, to a 4.2 f o l d increase at 32 dph. The whole body C o r t i s o l content i n the experimental groups increased as development progressed, from 5.88 ng/g at 6 dph, to 45.57 ng/g at 32 dph, and with the exception of the 21 dph sample, were s i g n i f i c a n t l y higher than those l e v e l s observed i n the previous sample i n t e r v a l (ANOVA, p<0.5). The whole body C o r t i s o l l e v e l s i n the control groups decreased s i g n i f i c a n t l y at 1 dph, and increased s i g n i f i c a n t l y at 11, 16, and 21 dph (ANOVA, p<0.05). After t h i s there was no s i g n i f i c a n t difference i n whole body C o r t i s o l content observed between control groups. 48 Time Course of C o r t i s o l Response to Disturbance During Ontogeny. There were no s i g n i f i c a n t differences i n whole body C o r t i s o l content observed between those coho embryos receiving the repeated disturbance, and those that were undisturbed, at -5 and 2 dph during the 8 hour duration of the t r i a l ( t - t e s t , p>0.05) (Fig. 2). There were no s i g n i f i c a n t differences between the Oh and any subsequent sampling time i n either the control or experimental groups (ANOVA, p>0.05). At 9 dph there was a s i g n i f i c a n t difference i n whole body C o r t i s o l content observed between those coho embryos rec e i v i n g the repeated disturbance and those that did not. There was no s i g n i f i c a n t difference observed at Oh ( t - t e s t , p>0.05), but a s i g n i f i c a n t difference was observed at the l h sampling i n t e r v a l , and at each subsequent sampling i n t e r v a l thereafter (2, 4, and 8h) (t - t e s t , p<0.05) (Fig. 3). There was no s i g n i f i c a n t difference i n whole body C o r t i s o l content observed between Oh and any other i n t e r v a l within the control group over the duration of the t r i a l (ANOVA, p>0.05). Whole body C o r t i s o l content i n the experimental embryos was s i g n i f i c a n t l y higher at l h than at Oh, and remained s i g n i f i c a n t l y elevated above the Oh l e v e l during the duration of the t r i a l (ANOVA, p<0.05). At 16 dph there was no s i g n i f i c a n t difference observed i n whole body C o r t i s o l content between experimental and control groups at 0 and 2h (t - t e s t , p>0.05). Whole body C o r t i s o l content was 49 s i g n i f i c a n t l y elevated i n the experimental groups at 1, 4, and 8h (t - t e s t , p<0.05) (Fig. 4). There was no s i g n i f i c a n t difference i n whole body C o r t i s o l content observed between the Oh and any other sampling i n t e r v a l within the control group over the duration of the t r i a l (ANOVA, p>0.05) . / Whole body C o r t i s o l content i n the experimental embryos was s i g n i f i c a n t l y higher at the 1, 2, and 4h sampling i n t e r v a l s , than at the Oh i n t e r v a l (ANOVA, p<0.05), before dropping to a l e v e l not s i g n i f i c a n t l y d i f f e r e n t at the 8h i n t e r v a l (ANOVA, p>0.05). At 23 dph there was no s i g n i f i c a n t difference observed i n whole body C o r t i s o l content between experimental and control groups at 0 and 8h ( t - t e s t , p>0.05). Whole body C o r t i s o l content was s i g n i f i c a n t l y elevated i n the experimental groups at 1, 2, and 4h, (t - t e s t , p<0.05) (Fig. 4). There was a s i g n i f i c a n t elevation i n whole body C o r t i s o l content observed between the Oh l e v e l and a l l subsequent l e v e l s i n both the control and experimental groups (ANOVA, p>0.05). At 3 0 dph i n i t i a l (Oh) whole body C o r t i s o l content was not s i g n i f i c a n t l y d i f f e r e n t between control and experimental groups ( t -t e s t , p>0.05). A s i g n i f i c a n t increase was observed i n the experimental groups at the 1, 2, 4, and 8h sampling i n t e r v a l ( t -te s t , p<0.05) (Fig. 5). There was a s i g n i f i c a n t increase over the Oh l e v e l i n whole body C o r t i s o l content observed i n the control embryos at 2h (ANOVA, p<0.05). There was no s i g n i f i c a n t difference observed between the 50 Oh whole body C o r t i s o l content and any of the other sampling i n t e r v a l s i n the control group (ANOVA, p>0.05). Among the experimental embryos there was a s i g n i f i c a n t difference observed i n whole body C o r t i s o l content between the Oh l e v e l and a l l of the subsequent sampling i n t e r v a l s (ANOVA, p<0.05). 51 Figure 9. Whole body C o r t i s o l content of steelhead salmon (Oncorhynchus mykiss), embryos receiving repeated emersion, and l e f t undisturbed, from -4 to 32 days post hatch (dph) . Bars represent mean ± SE (n=8). * denotes s i g n i f i c a n t difference between control and experimental groups (p < 0.05). + Denotes s i g n i f i c a n t differences within groups from previous value (p < 0.05). 52 10 — O — Contro l T ime (hours) — • — E m e r s e d Figure 10. Whole body C o r t i s o l content of coho salmon (Oncorhynchus kisutch), embryos over an 8 hour period receiving repeated emersion, and l e f t undisturbed, at -5 days post hatch (dph). No s i g n i f i c a n t difference was observed (p > 0.05). Points represent mean ± SE (n=8). 53 O — Control Time (hours) — • — Emersed Figure 11. Whole body C o r t i s o l content of coho salmon (Oncorhynchus kisutch), embryos, over an 8 hour period, receiving repeated emersion and l e f t undisturbed, at 2 days post hatch (dph). No s i g n i f i c a n t difference was observed (p > 0.05). Points represent mean ± SE (n=8). <D •*—' <D E cn c ~o to » o O 54 * Contro l T ime (hours) — • — E m e r s e d Figure 12. Whole body C o r t i s o l content of coho salmon (Oncorhynchus kisutch), embryos over an 8 hour period receiving repeated emersion, and l e f t undisturbed, at 9 days post hatch (dph). Points represent mean ± SE (n=8). * denotes s i g n i f i c a n t difference between control and experimental groups (p < 0.05). + denotes s i g n i f i c a n t differences from values at time 0 within groups (p < 0.05) . 55 CD CD E CD c "o w V_ o O — O— Control Time (hours) — Emersed Figure 13. Whole body C o r t i s o l content of coho salmon (Oncorhynchus kisutch), embryos over an 8 hour period receiving repeated emersion, and l e f t undisturbed, at 16 days post hatch (dph). Points represent mean ± SE (n=8). * denotes s i g n i f i c a n t difference between control and experimental groups (p < 0.05). + denotes s i g n i f i c a n t differences from values at time 0 within groups (p < 0.05). 56 cn a> CD E cn cn c " o w o O Contro l T ime (hours) — • — E m e r s e d Figure 14. Whole body C o r t i s o l content of coho salmon (Oncorhynchus kisutch), embryos over an 8 hour period receiving repeated emersion, and l e f t undisturbed, at 23 days post hatch (dph). Points represent mean ± SE (n=8). * denotes s i g n i f i c a n t difference between control and experimental groups (p < 0.05). + denotes s i g n i f i c a n t differences from values at time 0 within groups (p < 0.05) . 57 CD •*—< CD E CO c o CO o O Contro l T ime (hours) — • — E m e r s e d Figure 15. Whole body C o r t i s o l content of coho salmon (Oncorhynchus kisutch), embryos over an 8 hour period receiving repeated emersion, and l e f t undisturbed, at 3 0 days post hatch (dph). Points represent mean ± SE (n=8). * denotes s i g n i f i c a n t difference between control and experimental groups (p < 0.05). + denotes s i g n i f i c a n t differences from values at time 0 within groups (p < 0. 05) . 58 Discussion: C o r t i s o l l e v e l s have been observed to increase i n salmonid oocytes approximately 1 week p r i o r to hatch (de Jesus and Hirano, 1992; Yeoh, 1993). My r e s u l t s show a s i g n i f i c a n t increase i n whole body C o r t i s o l content of embryonic coho salmon between 5 days before, and 2 days af t e r , hatch i n both control and experimental groups (Figs. 10 and 11). This would indicate that the i n t e r r e n a l c e l l s i n the embryonic coho salmon are capable of synthesizing c o r t i c o s t e r o i d s at t h i s point i n development. The f a c t that I observed no s i g n i f i c a n t differences between undisturbed embryos and those receiving the repeated disturbance at both sampling periods indicates that some components of the HPI axis are immature, or inac t i v e , at t h i s point. In r a t s there i s a period of non-responsiveness of the HPA axis that begins shortly a f t e r b i r t h ( G u i l l e t et al., 1980; Haltmeyer et al., 1966; Sapolsky and Meaney, 1986; Walker et al., 1986) when application of a va r i e t y of s t i m u l i f a i l s to e l i c i t an increase i n c i r c u l a t i n g l e v e l s of c o r t i c o s t e r o i d s . I t has been suggested that t h i s "stress" non-responsive period (SNRP) i s due to enhanced brain and/or p i t u i t a r y s e n s i t i v i t y to g l u c o c o r t i c o i d feedback (Levin et al., 1988; Walker et al., 1986), and i s c r u c i a l f o r normal growth and d i f f e r e n t i a t i o n of a number of tissues and organs, including the central nervous system (Moisan et al., 1992). The HPA axis p r i o r to t h i s point can be activated by a va r i e t y of s t i m u l i . Fetal corticosterone l e v e l s are responsive to 59 environmental disturbance, or application of exogenous ACTH, i n mammals p r i o r to b i r t h (Ohkawa et al., 1991; Rose et al., 1982; Walker et al., 1986), and immunoreactive ACTH c e l l s appear i n the r a t and porcine p i t u i t a r y as early as days 17 and 40 of gestation respectively (Chatelain et al., 1980; Sasaki et al., 1992). I observed a s i g n i f i c a n t difference i n whole body C o r t i s o l content between those steelhead and coho salmon embryos that received a series of disturbances, compared to those that were undisturbed, beginning at 6 and 9 dph respectively (Figs. 9 and 12), but not at -5 and 2 dph for coho (Figs. 10 and 11), or -4 and 1 dph for steelhead (Fig. 9). This indicates the onset of de novo synthesis of C o r t i s o l i n response to environmental s t i m u l i , and i s evidence of a functional HPI axis. A d i s s e c t i b l e p i t u i t a r y gland mass i s f i r s t detectable i n rainbow trout approximately 15 days post f e r t i l i z a t i o n (Ballard, 1973) , but immunoreactive ACTH c e l l s do not appear u n t i l approximately 10 days p r i o r to hatch (Saga et al., 1993), which corresponds to the period of corticosteroidogenesis reported i n salmonid oocytes (de Jesus and Hirano, 1992; Yeoh, 1993). Pottinger and Mosuwe (1994), reported a measurable increase i n immunoreactive c o r t i c o s t e r o i d s i n rainbow trout, i n response to mechanical disturbance and confinement, beginning at 5 weeks post hatch. Barry et al., (1995) observed an increase i n whole body C o r t i s o l content i n rainbow trout, i n response to suspension out of water and cold temperatures beginning at 2 weeks post hatch. 60 The v a r i a t i o n between these findings and my own could be due simply to the difference i n water temperature at which the oocytes and embryos were incubated, e s p e c i a l l y i n the case of Barry et al., (1995) who reported using water at 10°C (Pottinger and Mosuwe, (1995) did not report the temperature of t h e i r incubation water), or i t may be the r e s u l t of differences i n the ap p l i c a t i o n of environmental disturbances. I t could be that a repeated disturbance over time, as opposed to a single b r i e f disturbance, allows for greater accumulation of C o r t i s o l within t i s s u e s , and acts to magnify the differences between control and experimental embryos. I t i s d i f f i c u l t to ascertain whether the lack of a measurable C o r t i s o l response i n the steelhead and coho embryos p r i o r to days 6 and 9 post hatch respectively, i s due to a lack of development of the HPI axis, or i s homologous to the SNRP reported i n ra t s , and i s an area for future investigations. In adult and juvenile salmonids plasma C o r t i s o l l e v e l s have been observed to r i s e s i g n i f i c a n t l y a f t e r the ap p l i c a t i o n of a disturbance (<10 min.), and continue to be elevated i f the disturbance i s maintained (Sumpter et al., 1986). A single, b r i e f applied disturbance (1 to 5 min. handling) also e l i c i t s a rapid elevation i n plasma C o r t i s o l that i s seen to peak within a period of 0.5 to 1 hours, before returning to pre-application l e v e l s within 4 to 6 hours (Barry et a l . , 1993; Barton et a l . , 1980; Barton et al., 1986; Biron and Benfey, 1994; Pickering et a l . , 1982) . 61 I observed, beginning at 9 dph i n coho salmon, a s i g n i f i c a n t increase i n whole body C o r t i s o l content i n those embryos receiving a ser i e s of applied disturbances. At 9 dph t h i s increase was seen to be maintained for the 6 hour period a f t e r cessation of the treatment (Fig. 12) . This continuation of elevated C o r t i s o l l e v e l s could be due to a number of factors. I t may be that the embryonic HPI axis i s s t i l l not completely functional, and that C o r t i s o l feedback at the l e v e l of the hypothalamus and/or p i t u i t a r y i s unresponsive, r e s u l t i n g i n continued release of corticotrophin releasing factor (CRF), ACTH, or both. I t may also be possible that the metabolic clearance rate of C o r t i s o l at t h i s point i n development i s less e f f i c i e n t , and that C o r t i s o l i s simply not being cleared from tissues as quickly. At 16 dph whole body C o r t i s o l content i n the coho embryos was seen to decline over the 6 hour period a f t e r cessation of the treatment, and at 8 hours was not s i g n i f i c a n t l y d i f f e r e n t from pre-treatment l e v e l s (Fig. 13) . This was also the pattern observed i n whole body C o r t i s o l content of coho embryos receiving the repeated disturbance at 30 dph (Fig. 15). At 23 dph the whole body C o r t i s o l content of those coho embryos receiving the repeated disturbance was elevated above pre-treatment l e v e l s over the duration of the experiment (8 hours). This was also the pattern observed i n the undisturbed coho embryos with the exception of the 4 hour i n t e r v a l (Fig 14). T h i s continued elevation i n whole body C o r t i s o l content observed i n the experimental coho embryos, and i n the seemingly undisturbed coho 62 embryos can be explained by the fa c t that, a f t e r the experiment had commenced, another worker at the f a c i l i t y inadvertently entered the hatchery area and disturbed the f i s h . Even with t h i s disturbance however, the coho embryos i n the treatment groups demonstrated s i g n i f i c a n t l y higher whole body C o r t i s o l contents than those observed i n the control groups over the f i r s t 4 hours of the experiment. This demonstrates that coho embryos at t h i s stage of development are able to express a graded response to s t i m u l i i n terms of C o r t i s o l production, an e f f e c t observed i n juvenile salmonids (Barton et al., 1986). My r e s u l t s suggest that embryonic steelhead and coho salmon have a functional HPI axis by days 6 and 9 post hatch respectively, and that by day 16 post hatch the C o r t i s o l response to an environmental disturbance, over an 8 hour period at l e a s t , i n coho embryos i s s i m i l a r to that of adults and juveniles. CHAPTER 4 Effects of Exogenous Cortisol and/or an Environmental Disturbance on Growth and Survival of Chinook Salmon {Oncorhynchus tshawytscha) Embryos. 64 Introduction: A nearly universal response of te l e o s t s to environmental disturbances or perturbations i s the a c t i v a t i o n of the hypothalamic-pituitary-interrenal (HPI) axis (Sumpter et al., 1993) . This neuroendocrine response to environmental disturbances i n i t i a t e s a hormonal cascade that culminates i n the release of the co r t i c o s t e r o i d , C o r t i s o l , from the i n t e r r e n a l c e l l s into c i r c u l a t i o n (Donaldson, 1981; Bonga, 1993). The elevation of c i r c u l a t i n g C o r t i s o l above baseline l e v e l s i n response to varying environmental s t i m u l i has been studied extensively i n many v a r i e t i e s of f i s h (Barry et al., 1993; Davis and Parker, 1986; Foo and Lam, 1993; Lamers et al., 1992; Thorpe et al., 1987), and i s often used as a diagnostic t o o l to indicate the presence of a "stressed" condition i n salmonids (Barton et al., 1986; Sumpter et al., 1986; Pottinger and Pickering, 1992; Mazur and Iwama, 1993). C o r t i s o l has also been demonstrated to cause hyperglycaemia, and appears to stimulate gluconeogenesis and g l y c o l y s i s from l i p i d and protein sources i n tel e o s t s (Davis et al., 1985; Freeman and Idler, 1973; Leach and Taylor, 1980; Leach and Taylor, 1982; Vijayan et al., 1990; Vijayan et al., 1991), and as such may have an e f f e c t on growth and performance (Schreck, 1990). C o r t i s o l has also been observed to modulate l i p i d metabolism i n salmonids (Sheridan, 1986), and to have important e f f e c t s on immune physiology (Barton and Iwama, 1991). Exposure of salmonids to adverse environmental conditions, and to exogenous C o r t i s o l , has 65 been demonstrated to have deleterious e f f e c t s on immune function (Pickering and Pottinger, 1985; Pickering and Pottinger, 1988; Maule et al., 1989). This can lead to increased mortality and decreased performance i n salmonids under culture conditions, and i s of primary concern to f i s h c u l t u r i s t s . The onset of a functional HPI axis begins early i n the ontogeny of salmonids (Pottinger and Mosuwe, 1994; Barry et al., 1995) , and has been demonstrated to be p a r t i a l l y functional i n steelhead (Oncorhynchus mykiss) and coho salmon (Oncorhynchus kisutch), as early as 6 and 9 days post hatch respectively (this t h e s i s , chpt 3) . Since C o r t i s o l , and secondary p h y s i o l o g i c a l responses to environmental perturbations, have been associated with changes i n growth and impairment of immune function i n salmonids, i t i s int e r e s t i n g to speculate whether the repeated a c t i v a t i o n of t h i s response to environmental s t i m u l i , and the r e s u l t i n g elevation of c i r c u l a t i n g C o r t i s o l , may e f f e c t growth and s u r v i v a l during the early ontogeny of salmonids. The purpose of t h i s investigation was to observe what e f f e c t s repeated disturbance and/or exogenous C o r t i s o l may have on the growth and su r v i v a l of salmonids immediately p r i o r to hatch, and during early development. 66 Materials and Methods: Oocytes from a single adult female, and m i l t from three adult male, chinook salmon (Oncorhynchus tshawytscha) were obtained from the Tenderfoot Ck. Hatchery (Squamish, B r i t i s h Columbia). Oocytes and m i l t were placed i n p l a s t i c bags f i l l e d with oxygen and placed on ice i n a cooler, before being transported to the Department of Fisher i e s and Oceans research f a c i l i t y i n West Vancouver. Oocytes were mixed with m i l t pooled from the three males before water was added to activate the sperm and i n i t i a t e f e r t i l i z a t i o n . Oocytes were then placed i n a single hatchery tray (Heath Techna Corp.) supplied with flowing well water (10 l i t r e s min - 1) at 11.4 ± 0.2° C, i n the on s i t e hatchery. Oocytes were allowed to develop to a point approximately 5 days p r i o r to hatch, and were divided into four equal groups, with each group placed i n a separate hatchery tray. Each group contained seven r e p l i c a t e s of 50 oocytes each. Beginning approximately 5 days p r i o r to hatch, experimental manipulation was i n i t i a t e d and consisted of two treatments, an applied disturbance consisting of a 60 sec. emersion from water (E), or C o r t i s o l immersion (I), applied to i n d i v i d u a l trays i n a 2 X 2 f a c t o r i a l design. One tray received no E and no I, and acted as a control (C) , while the remaining three trays received I and no E, E and no I, or E and I, treatments respectively. Before i n i t i a t i o n of treatment, water flow was interrupted to each of the hatchery trays. The E treatment was applied every 15 minutes over a period of two hours. Since C o r t i s o l q u a n t i f i c a t i o n required the measurement of whole body C o r t i s o l content, as opposed 67 to plasma C o r t i s o l , i t was f e l t that a series of applications of E over time was preferable to a single episode. This would allow for the de novo synthesis of C o r t i s o l from precursors, and f o r the accumulation of C o r t i s o l i n ti s s u e . The I treatment consisted of the addition of 6 mg of exogenous C o r t i s o l (Fisher) suspended i n 5 ml of 95% ethanol to the 10 l i t r e hatchery tray, r e s u l t i n g i n a f i n a l C o r t i s o l concentration of 600 ug/L while keeping the concentration of ethanol at 0.05% ( P i f f e r r e r and Donaldson, 1992). The non-immersed groups received 5 ml of 95% ethanol alone. Afte r two hours water flow was re-established to a l l trays to allow exogenous C o r t i s o l to f l u s h from the I treated embryos. Those trays receiving exogenous C o r t i s o l were placed below the non-immersed groups to avoid the p o s s i b i l i t y of contamination, and the two trays containing the groups receiving the E treatment, and the two that did not, were placed i n separate adjacent hatchery stacks. Water flow to the two stacks was c a r e f u l l y adjusted and monitored to insure that each tray received water at the same temperature, and at the same rate of flow during the course of the experiment. The I and E treatments were applied every 5 days from -1 to 24 days post hatch. At each treatment i n t e r v a l one embryo from each r e p l i c a t e was removed from each group a f t e r the two hour treatment period and k i l l e d by being placed for 15 minutes i n an overdose of anaesthetic (MS222, buffered 1:1 with sodium bicarbonate) at a concentration of 200 mg/L. Embryos were then weighed and measured before being frozen at -50° C for l a t e r assessment of whole body C o r t i s o l content. 68 M o r t a l i t i e s were recorded and removed a f t e r each treatment i n t e r v a l . Tissue Extraction and C o r t i s o l . Q u a n t i f i c a t i o n . Embryo ti s s u e was extracted, and whole body C o r t i s o l content was quantified, as described i n chapter 2. S t a t i s t i c a l Analysis. S i g n i f i c a n t differences among, and s i g n i f i c a n t interactions between, treatments i n regard to whole body C o r t i s o l content, length, and weight at each sampling i n t e r v a l ; and cumulative m o r t a l i t i e s over the 25 day period, were determined by 2 way analysis of variance (ANOVA) (Sigma Stat™, Jandel S c i e n t i f i c ) . The Student-Newman-Keuls t e s t was u t i l i z e d to determine s i g n i f i c a n c e among means. The Levene Median and Kolmogorov-Smirnov te s t s were used to t e s t for equal variance and normality respectively. Data were l o g 1 0 or square root transformed where assumptions of normality and homogeneity of variance were not met. 69 Results: At -1 and 4 dph both groups of embryos rec e i v i n g the I treatment (with and without E) demonstrated mean whole body C o r t i s o l contents that were s i g n i f i c a n t l y elevated over those observed i n embryos that did not receive I (with and without E) (2 way ANOVA, p < 0.05) (Fig. 16). At 9, 14, 19, and 24 dph those embryos receiving both the E and I treatments demonstrated mean whole body C o r t i s o l contents that were s i g n i f i c a n t l y higher than those observed i n the three remaining groups (2 way ANOVA, p < 0.05). During these same sampling i n t e r v a l s those embryos receiving the I, without the E, treatments demonstrated mean whole body C o r t i s o l contents that were s i g n i f i c a n t l y higher than those observed i n the groups receiving the E alone, or neither treatment (C) (2 way ANOVA, p < 0.05). At 14 and 19 dph those embryos that received the E treatment alone demonstrated mean whole body C o r t i s o l contents that were s i g n i f i c a n t l y higher than those observed i n the C embryos (2 way ANOVA, p < 0.05). There was a s i g n i f i c a n t i n t e r a c t i o n between the E and I treatments observed on the mean whole body C o r t i s o l content of the embryos at the 9, 14, 19, and 24 dph sampling periods. (2 way ANOVA, p < 0.05). At 4, 9, 14, 19, and 24 dph those embryos i n the E and I treatment group demonstrated a mean length that was s i g n i f i c a n t l y lower than that observed i n any of the other groups (2 way ANOVA, 70 p < 0.05) (Fig 17). There was no s i g n i f i c a n t differences observed i n the mean length of the remaining three treatment groups at any of the sampling i n t e r v a l s (2 way ANOVA, p > 0.05). There was a s i g n i f i c a n t i n t e r a c t i o n between the E and I treatments observed on the mean length of the embryos at the 4, 9, 19 and 24 dph sampling periods. (2 way ANOVA, p < 0.05). At 14, 19 and 24 dph those embryos i n the E and I group demonstrated a mean weight that was s i g n i f i c a n t l y lower than that observed i n the other three treatment groups (2 way ANOVA, p < 0.05) (Fig 18). At 19 and 24 dph those embryos i n the E group demonstrated a mean body weight that was s i g n i f i c a n t l y higher than that observed i n the E and I group, and s i g n i f i c a n t l y lower than that seen i n the I and C groups (2 way ANOVA, p < 0.05). There was a s i g n i f i c a n t i n t e r a c t i o n between the E and I treatments observed on the mean weight of the embryos at the 9, 14, and 19 dph sampling periods. (2 way ANOVA, p < 0.05) There was a s i g n i f i c a n t l y higher mean mortality rate among those embryos receiving both the E and I treatments (Fig. 19), and there was a s i g n i f i c a n t i n t e r a c t i o n between the E and I treatments observed on the mean mortality rate (2 way ANOVA, p < 0.05) . There was no s i g n i f i c a n t difference i n the mortality rate observed among the other three groups (2 way ANOVA, p < 0.05). 71 Figure 16. Whole body C o r t i s o l content of chinook salmon (Oncorhynchus tshawytscha), embryos receiving either repeated emersion (E), exposure to exogenous C o r t i s o l (I) (600 ug/L for 2 hrs.), both treatments (E+I), or neither treatment (C). Embryos were exposed every 5 days from 1 day before, to 24 days a f t e r , hatch. Points represent mean ± SE (n=7). * denotes a s i g n i f i c a n t difference i n whole body C o r t i s o l content from the control group at that sampling i n t e r v a l (p < 0.05). + denotes a s i g n i f i c a n t differences i n whole body C o r t i s o l content from the next highest group (excluding controls) (p < 0.05). 72 Figure 17. Body length of chinook salmon (Oncorhynchus tshawytscha), embryos receiving either repeated emersion (E) , exposure to exogenous C o r t i s o l (I) (600 ug/L for 2 hrs. ) , both treatments (E+I), or neither treatment (C). Embryos were exposed every 5 days from 1 day before, to 24 days a f t e r , hatch. Points represent mean ± SE (n=7). * denotes a s i g n i f i c a n t difference i n length from the other three groups at that sampling i n t e r v a l (p < 0.05). 73 400 Figure 18. Body weight of chinook salmon (Oncorhynchus tshawytscha), embryos receiving either repeated emersion (E) , exposure to exogenous C o r t i s o l (I) (600 ug/L f o r 2 hr s . ) , both treatments (E+I), or neither treatment (C). Embryos were exposed every 5 days from 1 day before, to 24 days a f t e r , hatch. Points represent mean ± SE (n=7). * denotes a s i g n i f i c a n t difference i n weight from the other three groups at that sampling i n t e r v a l (p < 0.05) . Figure 19. Percent Mortality of chinook salmon (Oncorhynchus tshawytscha), embryos receiving either repeated emersion (E) , exposure to exogenous C o r t i s o l (I) (600 ug/L f o r 2 hrs. ) , both treatments (E+I), or neither treatment (C). Embryos were exposed every 5 days from 1 day before, to 24 days a f t e r , hatch. Points represent mean ± SE (n=7). * denotes a s i g n i f i c a n t difference i n mortality rate from the other three groups (p < 0.05). 75 Discussion: My r e s u l t s indicate that exposure of embryonic salmonids to the combination of an environmental disturbance and exogenous C o r t i s o l has s i g n i f i c a n t e f f e c t s on growth and s u r v i v a l . I t appears that by 9 dph onwards there i s an in t e r a c t i o n between the E and I treatments that r e s u l t s i n increased whole body C o r t i s o l content, an increase greater than would be expected by the sum of the two treatments alone. I t may be that the combination of the two treatments r e s u l t s i n heightened s e n s i t i v i t y of some aspect of the HPI axis and potentiated C o r t i s o l synthesis. I t may also be possible that some aspect of the embryo's response to the applied disturbance altered i t s permeability to exogenous C o r t i s o l . Catecholamines, which increase i n the c i r c u l a t i o n i n response to environmental disturbances, have been associated with increased permeability of ti s s u e to water i n salmonids (Mazeaud et al., 1977). The onset of t h i s i n t e r a c t i o n e f f e c t coincided with the onset of the C o r t i s o l response to emersion observed i n coho salmon (Oncorhynchus kisutch), suggesting that t h i s i n t e r a c t i o n e f f e c t coincides with the onset of a functional HPI axis (this t h e s i s , chpt 3). The s i g n i f i c a n t l y higher mortality rate observed among those embryos receiving both the I and E treatments may be p a r t i a l l y a t t r i b u t a b l e to t h e i r s i g n i f i c a n t l y increased l e v e l s of whole body C o r t i s o l , as compared to the C and sing l y treated groups. Environmental disturbances i n general, and C o r t i s o l i n p a r t i c u l a r , have been demonstrated to suppress the immune response i n 76 salmonids, and as such can make them more vulnerable to inf e c t i o u s agents (for review see Barton and Iwama, 1991; Campbell, 1992). Increased mortality due to disease has been associated with increased i n t e r r e n a l response to environmental disturbance. A t l a n t i c salmon (Salmo salar) selected for t h e i r high C o r t i s o l response, indicated by elevated blood C o r t i s o l l e v e l s i n response to s t i m u l i such as crowding, demonstrated s i g n i f i c a n t l y higher mortality rates when challenged by b a c t e r i a l pathogens than d i d low response f i s h (Fevolden et al., 1993). Overwinter mortality rates of S2 a t l a n t i c salmon parr (2 year fresh water residency) have also been associated with c h r o n i c a l l y high plasma C o r t i s o l t i t e r s and reduced c i r c u l a t i n g lymphocyte numbers (Pickering and Pottinger, 1988). Resistance to pathogens has been observed to decrease i n chinook salmon shortly a f t e r handling (Maule et al., 1989), and acute environmental disturbance has also been demonstrated to decrease c i r c u l a t i n g leukocyte numbers i n coho salmon (Maule and Schreck, 1990), as well as c i r c u l a t i n g numbers of lymphocytes i n rainbow trout (Oncorhynchus mykiss) (Barton et al., 1987; Pottinger and Pickering, 1992). Similar immune disfunction has been observed i n coho salmon treated with exogenous C o r t i s o l , with C o r t i s o l appearing to be d i r e c t l y , but not exclusively, involved i n changes i n leukocyte numbers (Maule and Schreck, 1990). C o r t i s o l administered i n food, or v i a implantation, s i g n i f i c a n t l y r a i sed plasma C o r t i s o l l e v e l s while s i g n i f i c a n t l y reducing c i r c u l a t i n g numbers of lymphocytes,, and increased mortality due to b a c t e r i a l and fungal i n f e c t i o n , i n 77 brown trout (Salmo trutta) (Pickering et al., 1989; Pickering and Pottinger, 1985; Pickering, 1984). I t may have been that the increase i n whole body C o r t i s o l content of the E and I treated embryos caused them to experience a depression of t h e i r immune response, and thus rendered them more susceptible to any infectious agents present. The f a c t that I observed no increase i n mortality among the groups re c e i v i n g the E or I treatment alone suggests that the s i g n i f i c a n t l y higher whole body C o r t i s o l content observed among the E and I embryos on days 9 through 30 post hatch was above a l e v e l at which deleterious e f f e c t s were incurred, and that the other groups remained below t h i s threshold l e v e l . The s i g n i f i c a n t l y lower mean length of those embryos receiving both the E and I treatments at a l l sampling i n t e r v a l s , coupled with the s i g n i f i c a n t l y lower mean weight i n these same embryos observed on the f i n a l three sampling dates, suggests a s i g n i f i c a n t e f f e c t of these combined treatments on the growth of these embryos. Environmental disturbances have been observed to depress growth i n salmonids. Brook trout (Salvelinus fontinalis) reared at pH 4.5 demonstrated stunted growth and increased plasma C o r t i s o l t i t e r s (Tam et al., 1988). Fagerlund et al., (1981) reported that increased f i s h stocking density was associated with decreased growth and increased i n t e r r e n a l c e l l nuclear diameter i n juvenile coho salmon, while Pickering and Stewart (1984) observed a s i m i l a r suppression of growth rate i n c h r o n i c a l l y crowded brown trout. They also observed a s i g n i f i c a n t transient increase i n plasma 78 C o r t i s o l , though they concluded that suppression of growth rate was not mediated by chronic elevation of c o r t i c o s t e r o i d s . This was also the conclusion reached by Vijayan et al. , (1990), who observed decreased growth i n brook trout raised at high stocking density, but saw no accompanying elevation i n plasma C o r t i s o l . Application of exogenous C o r t i s o l however, has been linked to reduced growth rate i n some te l e o s t s . One year o ld rainbow trout fed C o r t i s o l for 10 weeks showed a decrease i n growth and condition factor (Barton et al., 1987). Davis et al., (1985) observed s i g n i f i c a n t l y lower body weight and condition factor among yea r l i n g channel c a t f i s h (Ictalurus punctatus) fed with food containing C o r t i s o l , and suggested t h i s as a possible explanation for the decreased growth of f i s h under conditions that activate the secretion of endogenous C o r t i s o l . My r e s u l t s suggest that application of exogenous C o r t i s o l alone does not a f f e c t growth early i n ontogeny. I did however, see some reduction i n the weight of those embryos subjected to E alone, suggesting that other phy s i o l o g i c a l responses of the embryos, other than the C o r t i s o l response, to an environmental disturbance can s i g n i f i c a n t l y a f f e c t growth. The greatest reduction i n both weight and length was observed among those embryos receiving both the E and I treatments. This suggests that there i s some i n t e r a c t i o n between the two factors that acts to negatively impact on growth, and indeed, a s t a t i s t i c a l l y s i g n i f i c a n t i n t e r a c t i o n e f f e c t was observed on either embryo weight, length, or both, at each sampling i n t e r v a l . I t i s d i f f i c u l t to ascertain whether t h i s reduction i n 79 growth i s due s t r i c t l y to elevated whole body C o r t i s o l content, or whether some other phy s i o l o g i c a l response(s) to emersion are involved. My observation that the E, and not the I, treatment acted to reduce weight seems to indicate the l a t t e r as a more l i k e l y determining factor. I suggest, based on the r e s u l t s of t h i s study, that growth and s u r v i v a l among the Chinook embryos as a whole was much more negatively impacted by the combination of the E and I treatments, than by either of these factors i n i s o l a t i o n . I t may be that the elevated whole body C o r t i s o l content experienced by the embryos i n the E and I group somehow "overloaded" t h e i r capacity to deal p h y s i o l o g i c a l l y with C o r t i s o l , and t h i s resulted i n reduced growth and immunocompetence. I t i s d i f f i c u l t to speculate on the ramifications of exposure of salmonids to the combination of both an environmental disturbance and exogenous C o r t i s o l as previous studies done on te l e o s t s usually involved the a p p l i c a t i o n of either of these treatments i n i s o l a t i o n , and not i n conjunction (Davis et a l . , 1985; Barton et al., 1987). My r e s u l t s suggest that, i n terms of growth and s u r v i v a l , Chinook salmon embryos appear to be r e l a t i v e l y r e s i s t a n t to the e f f e c t s of emersion or elevated whole body C o r t i s o l content, and only begin to exh i b i t negative impacts when severely treated (treatments combined). CHAPTER 5 GENERAL DISCUSSION 81 General Discussion The r e s u l t s of t h i s study suggest that increased c i r c u l a t i n g C o r t i s o l l e v e l s , as a r e s u l t of an environmental disturbance (mechanical a g i t a t i o n ) , i n adult female coho salmon (Oncorhynchus kisutch) during the la t e stages of oogenesis, can lead to an increase i n the C o r t i s o l content of t h e i r oocytes (Chapter 2) . The presence of C o r t i s o l has been demonstrated i n the f r e s h l y ovulated or f e r t i l i z e d oocytes of a number of te l e o s t s , including chum salmon (Oncorhynchus keta), coho salmon, rainbow trout (Oncorhynchus mykiss), T i l a p i a (Oreochromis mossambicus), and Japanese flounder (Paralicthys olivaces) (de Jesus and Hirano, 1992; t h i s thesis, chpt 2; Yeoh et al. , 1993; Rothbard et al. , 1987; de Jesus et al., 1991). The functional s i g n i f i c a n c e of C o r t i s o l during the early ontogeny of tel e o s t s i s undetermined, although there i s some evidence for i t e f f e c t i n g hatching rate i n salmonids (Yeoh, 1993). The current study investigated the f l u x of C o r t i s o l i n developing ooctyes, and how parameters such as hatch, growth and s u r v i v a l were affected by d i f f e r e n t l e v e l s of oocyte C o r t i s o l (Chapter 2). I saw no s i g n i f i c a n t differences between oocytes with normal, and those with a r t i f i c i a l l y elevated C o r t i s o l content, and suggest that the rapid decline i n ooctye C o r t i s o l observed during very early ontogeny ( f i r s t 7 days) to r e l a t i v e l y low l e v e l s (<10 ng/gm) among both control and manipulated oocytes, may preclude the involvement of maternally derived C o r t i s o l i n l a t e r developmental processes. 82 This study also investigated the onset of endogenous C o r t i s o l production, as an ind i c a t i o n of a functional hypothalamic-p i t u i t a r y - i n t e r r e n a l (HPI) axis, i n the early ontogeny of steelhead and coho salmon (Chapter 3), and the e f f e c t s exposure to an environmental disturbance and/or exogenous C o r t i s o l may have on growth and su r v i v a l of chinook salmon (Oncorhynchus tshawytscha) during t h i s same period (Chapter 4). I was able to demonstrate that embryonic steelhead and coho salmon begin to respond to an environmental disturbance with increased synthesis of endogenous C o r t i s o l as early as 6 and 9 days post hatch (dph) respectively. Work by other researchers with rainbow trout has indicated that endogenous production of C o r t i s o l , i n response to s t i m u l i , begins as early as 5 (Pottinger and Mosuwe, 1994) and 2 (Barry et al., 1995) weeks post hatch. The discrepancy between my observations and t h e i r s may be attri b u t e d to a number of factors. An elevation i n the water temperature at which my embyros were cultured (13° vs 10° C) may have resulted i n t h e i r developing at an increased rate, and thus exh i b i t i n g responsiveness to disturbance e a r l i e r . I t i s also possible that my ap p l i c a t i o n of a repeated disturbance over time, as opposed to a single disturbance, may have allowed for the accumulation of C o r t i s o l i n t i s s u e , and provided a more sen s i t i v e measure of t h i s p a r t i c u l a r aspect of the "stress response". The time course of the C o r t i s o l response to environmental disturbance i n coho salmon was si m i l a r to that reported i n other studies involving juvenile and adult salmonids (Barry et al., 1993; 83 Barton et al., 1980; Pickering et al., 1982) by day 16 post hatch. At 9 days post hatch whole body C o r t i s o l remained elevated 6 hours a f t e r the f i n a l emersion application, i n d i c a t i n g that either the HPI axis was not f u l l y functional at t h i s time and synthesis of C o r t i s o l i s s t i l l ongoing, or that the clearance of C o r t i s o l was not as developed or e f f i c i e n t at t h i s early stage i n development. I was able to demonstrate that embryonic chinook salmon exposed to a repeated environmental disturbance coupled with exposure to exogenous C o r t i s o l , during the period when endogenous C o r t i s o l production has been observed to begin i n salmonids, displayed reduced growth and increased mortality. I also observed a s i g n i f i c a n t i n t e r a c t i o n e f f e c t between these two treatments on whole body C o r t i s o l content that coincided with the onset of the C o r t i s o l response to environmental disturbance observed i n coho salmon (Chapter 3) . This resulted i n s i g n i f i c a n t increases i n whole body C o r t i s o l content either by potentiation of endogenous C o r t i s o l production, or by increased permeability to exogenous C o r t i s o l . I suggest that t h i s increase i n whole body C o r t i s o l content was at le a s t i n part responsible f o r the increased mortality observed i n the doubly treated group, as C o r t i s o l has been demonstrated to reduce immunocompetence i n salmonids (Pickering et al., 1989; Pickering and Pottinger, 1985; Maule and Schreck, 1990). The reduced length and weight observed i n those chinook embryos receiving both the emersion and C o r t i s o l immersion treatment was most probably due to factors other than elevated 84 C o r t i s o l content. This i s supported by the fac t that I observed no reduction i n weight or length i n the Chinook embryos receiving exogenous C o r t i s o l alone, but did see some reduction i n weight i n embyros receiving the emersion treatment alone. Reduction i n growth has been observed i n salmonids raised under adverse conditions (high stocking density) without an accompanying r i s e i n plasma C o r t i s o l (Vijayan et al., 1990), and Pickering and Stewart (1984) concluded that growth rate suppression i n c h r o n i c a l l y crowded brown trout was not mediated by chronic elevations of plasma C o r t i s o l . The r e s u l t s of t h i s study suggest that c i r c u l a t i n g l e v e l s of C o r t i s o l i n adult coho salmon during the period of l a t e oogenesis are r e f l e c t e d i n the C o r t i s o l content of t h e i r oocytes, and that r e l a t i v e l y high, or low, oocyte C o r t i s o l content during early ontogeny (immediately post f e r t i l i z a t i o n ) has l i t t l e e f f e c t on subsequent development or s u r v i v a l . 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