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Calcium and calcitonin studies in Pacific salmon, genus Oncorhynchus, and rainbow trout, Salmo gairdneri Watts, Eric George 1973

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172-V-Z^ cl CALCIUM AND CALCITONIN STUDIES IN PACIFIC SALMON, GEIMUS ONCORHYNCHUS, AND RAINBOW TROUT, SALMO GAIRDNERI by ERIC GEORGE WATTS B.Sc. (Hons.), McMaster University, 1968 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in the Department • f Physiology We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA JUNE, 1973 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 representatives., It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of The University of British Columbia Vancouver 8, Canada Date Jfj ft?3 ii ABSTRACT In mammals, calcium homeostasis is under the control of parathyroid hormone and calcitonin. Fish lack parathyroid glands but large amounts of calcitonin are located in the ultimobranchial gland. The objective of this thesis was to examine calcium metabolism and the possible physiological role of calcitonin in rainbow trout, Salmo gai-rdneri, and Pacific salmon, genus •ncorhynchus. Ultimobranchial gland calcitonin concentrations were measured in trout and salmon under a variety of conditions, using the rat bioassay. Assays indicated that calcitonin concentrations in the ultimobranchial glands varied widely and showed no consis tent relationship to plasma calcium and phosphate levels, sex, sexual maturation, environmental calcium concentration or species. The ultimobranchial gland calcitonin concentrations of fingerling trout (age 7-8 months) were lower than adult trout, suggesting a possible relationship between calcitonin and growth. The biological half-life of salmon calcitonin was measured in free-swimming cannulated trout and salmon. Results indicate that the half-life of salmon calcitonin in fish (trout 27.6 min., salmon 48.0 min.) is considerably longer than that found in mammals. The effect of salmon calcitonin on plasma calcium and phosphate was examined in trout and salmon. Salmon calcitonin injection did not cause hypocalcaemia or hypophosphatemia in fingerling trout and uas also ineffective in cannulated adult trout. iii Pdo significant change in plasma electrolytes or urinary electro lyte excretion was observed following infusion of salmon calcitonin into cannulated adult female salmon,, A migration study on the Chilko race of sockeye salmon was carried out to investigate plasma electrolyte and tissue changes as these fish migrate from sea to fresh water. Ionic and total serum calcium were determined and results indicate that the sockeye maintain a relatively constant serum ionic calcium level throughout their spawning migration, indicating effective homeo-static control. Measurements using a sensitive and specific radioimmuno assay, revealed that calcitonin can be detected in the plasma of salmon and that this hormone was continuously secreted under basal conditions. The levels of calcitonin detected in salmon plasma were higher than those found in most mammals. A sex difference in plasma calcitonin levels (females higher than males) was found in sockeye, as well as in the coho and chindok adult salmon. This sex difference appears to be unique to salmonids. Female plasma calcitonin levels were found to rise during the migration and to decrease following spawning« Plasma calcitonin changes Followed a different pattern in the migrating male sockeye. The plasma calcitonin changes were clearly not re lated to plasma calcium and phosphate alterations. In the female sockeye, calcitonin appears to be involved in sexual maturation and spawning. Removal of the gonads from mature female sockeye resulted iv in a marked drop in circulating plasma calcitonin levels. Estrogen injection into these gnnadectomized salmon dramatic ally elevated plasma calcium but did not restore the plasma calcitonin levels. These investigations indicate that- the physiological role of calcitonin in calcium metabolism in fish may be different from that in mammals. V TABLE DF CONTENTS Page ABSTRACT ii LIST OF TABLES • vii LIST OF FIGURES X LIST OF PLATES ' xiii ACKNOWLEDGMENTS xiv PREFACE GENERAL INTRODUCTION 1 GENERAL MATERIALS AND METHODS 7 Experimental AnimalsOperating Procedures 10 Analytical Procedures 21 CHAPTER I ULTIMOBRANCHIAL GLAND CALCITONIN CONCEN TRATIONS 37 IntroductionMaterials and Methods hh Results ^8 Discussion 55 CHAPTER II BIOLOGICAL HALF-LIFE OF SALMON CALCITONIN IN TROUT AND SALMON 6Introduction 5 Materials and Methods 66 Results 68 Discussion 7CHAPTER III PLASMA AND RENAL EFFECTS OF SALMON CALCITONIN 85 Introduction 8Materials and Methods 87 Results 91 Discussion 106 ui Page CHAPTER IU PLASMA CALCITONIN AND TISSUE MINERAL CHANGES IN MIGRATING SALMON 116 Introduction . 116 Materials and Methods 120 Results 129 Discussion 175 CHAPTER V EFFECT OF ESTROGEN ON SERUM IONIC CALCIUM IN TROUT AND GONADECTOMY AND ESTRDGEN ON PLASMA CALCITONIN AND CALCIUM IN SALMON 197 Introduction 19Materials and Methods 198 Results 202 Discussion 211 SUMMARY 22BIBLIOGRAPHY 4 vii LIST OF TABLES Table Page I Physical measurements, plasma electrolytes and calcitonin activities of rainbow trout k9 II Physical measurements, plasma electrolytes and calcitonin activities of caho and chinook salmon 50 III Distribution of calcitonins among salmon species 61 IU Specific biological activities of calcitonins from various species . 62 V Trout physical measurements 69 UI Plasma calcitonin levels and biological half-lives of salmon calcitonin in trout 70 UII Percent calcitonin activity remaining with time 72 VIII Plasma calcium, percent water and haematacrit changes in trout 73 IX Salmon physical measurements Ih X Plasma measurements in twD sockeye salmon 75 XI Effects of salmon calcitonin on plasma electro lytes in fingerling rainbow trout ..... 92 XII Physical.measurements and individual plasma calcium levels of cannulated trout.. 96 XIII Effect of salmon calcitonin on plasma calcium levels in cannulated trout 97 XIV Salmon physical measurements 100 XV Chilko sockeye migration 1971 123 XVI Chilko sockeye migration 1972 125 XVII Coho salmon study: summary of sampling data.. 127 XVIII Physical and plasma measurements - Chilko migration 1971 132 XIX Plasma electrolyte and calcitonin levels -Chilko migration 1971 ... 133 viii Table Page XX Water analysis - Chilko migration 1971 134 XXI Physical parameters, ultimobranchial gland and plasma calcitonin levels in migrating female Chilko sockeye (1971) 137 XXII Physical and plasma measurements - Chilko migration 1972 ..... lkZ XXIII Plasma electrolyte levels - Chilko migration 1972 143 XXIV Ionic and total serum calcium, serum pH and plasma protein changes in migrating Chilko sockeye (1972) Ikk XXV Water temperatures - Chilko migration 1972 146 XXVI Soft tissue mineral changes - Chilko migration 1972 148 XXVII Hard tissue mineral changes - Chilko migration 1972 9 XXVIII Dry weights, phosphate and calcium contents of the premaxilla bone - Chilko migration 1972 160 XXIX Percentage dry weights of tissues 164 XXX Calcium and phosphate concentrations in tissues of average Chilko freshwater arrival sockeye 166 XXXI Physical and. plasma measurements - coho salmon study 168 XXXII Plasma electrolyte and calcitonin levels - coho salmon study 169 XXXIII Physical measurements, plasma calcitonin and electrolyte levels in adult spawning chinook, coho and sockeye salmon 172 XXXIV Plasma and ultimobranchial gland calcitonin concentrations in coho and chinook salmon 174 XXXV Calcium utilized by growing tissues in maturing sockeye salmon 192 XXXVI Physical measurements, plasma and serum electro lytes in control and estrogen-treated trout 203 ix Table Page XXXVII Bane measurements in control and estrogen-treated trout 205 XXXV/III Physical parameters, plasma calcitonin and plasma electrolytes of intact control, Gx control and Gx estrogen sockeye 207 XXXIX Bone measurements in intact control, Gx control and Gx estrogen sockeye 210, X LIST OF FIGURES Figure Page la. Rainbow trout dorsal aortic cannulation......... Ik b. Caudal vein cannulation technique 19 2. Blood and urine sampling technique in salmon.... • 22 3. Plasma calcium and inorganic phosphorus levels in trout and salmon 51 k. Ultimobranchial gland calcitonin concentrations (mU/mg gland) in trout and salmon 53 5. Ultimobranchial gland calcitonin concentrations (U/kg fish) in trout and salmon 5k 6. Biological half-life of salmon calcitonin in rainbow trout.... 71 7. Biological half-life of salmon calcitonin in male sockeye salmon 6 8. Disappearance of salmon calcitonin in trout and salmon 77 9. Plasma calcium changes in fingerling trout-effect of salmon calcitonin. Injection at time • 93 ID. Plasma inorganic phosphorus changes in fingerling trout - effect of salmon calcitonin. Injection at time • 3k 11. Mean plasma calcium changes in adult cannulated trout - effect of salmon calcitonin 98 12. Individual plasma calcium changes in cannulated adult trout - effect of salmon calcitonin 99 13. Plasma electrolyte changes in a sockeye salmon -., effect of salmon calcitonin infusion. Female sockeye V 101 lk. Plasma electrolyte changes in a sockeye salmon -effect of salmon calcitonin infusion. Female sockeye Id. 102 15. Plasma electrolyte changes in a sockeye salmon -effect of salmon calcitonin infusion. Female sockeye Z 103 xi Figure Page 16. Urinary electrolyte excretion and urine flaw in 3 sockeye salmon - effect of salmon calcitonin infusion 105 17. Map of Fraser River and British Columbia 122 18. Plasma calcitonin changes in migrating Chilko sockeye 135 19. Plasma electrolyte changes in migrating Chilko sockeye 138 20. Plasma calcitonin,.plasma calcium and gonad-samatic index changes in migrating Chilko sockeye 139 21. Haematocrit, plasma protein and plasma % water changes in migrating Chilko sockeyB 141 22. Serum ionic and total calcium changes in migrating Chilko sockeye (Chilko Migration 1972) 145 23. Soft tissue calcium changes in migrating Chilko sockeye 150 24. Soft tissue phosphate changes in migrating Chilko sockeye 151 25. Hard tissue calcium changes in migrating Chilko sockeye 4 26. Hard tissue phosphate changes in migrating Chilko sockeye 155 27. Vertebrae mineral content changes in migrating Chilko sockeye 157 28. Scale mineral content changes in migrating Chilko sockeye 158 29. Premaxillae dry weight increases in migrating Chilko sockeye 161 30. Premaxillae calcium content changes in migrating Chilko sockeye 162 31. Premaxillae phosphate content changes in mig rating Chilko sockeye 163 32. Plasma calcium, plasma calcitonin and gonad-somatic index measurements in 3 groups of coho salmon..... 170 xii Figure Page 33. Plasma calcitonin levels in 3 species of salmon 173 3k. Serum ionic and total calcium and plasma inorganic phosphorus levels in immature trout -effect of estrogen 20k 35. Total plasma calcium levels in sockeye - effect of gonadectomy and estrogen replacement......... 208 36. Plasma calcitonin levels in sockeye - effect of gonadectomy and estrogen replacement 209 xiii LIST OF PLATES Plate Page 1 Fish operating table 11 2. Trout dorsal aortic cannulation .. 15 3. Calcium Activity Flow-Thru System 29 4. Infusion pump and electrodes with water jacket... 30 5. Trout ultimobranchial gland 39 6. Salman ultimobranchial gland 40 7. Seawater Chilko sockeye 138. Freshwater arrival Chilko sockeye 130 9. Spawning male Chilko sockeye 131 10. Spawning female Chilko sockeye 1311. Gonadectomized female sockeye (Great Central race) 219 xiv ACKNOWLEDGMENTS This thesis uas accomplished uith the assistance of a number of people. First, I would like to thank my research supervisor, Dr. D. H. Copp, for his enthusiastic guidance in this study. Technical assistance in the laboratory, particularly from Elspeth McGowan, Joan Rogers, Kathy Perry and Frances Newsome, was invaluable. The help and encouragement of Dr. Harold Messer, Dr. Louise Messer, Stephanie Ma and Y. Shami was greatly appreciated. Special thanks are due to Kurt Henze and Ralph Assina for the preparation of the thesis illustrations. I am particularly indebted to Dr. Len Deftos, Department of Medicine, University of California, San Diego, U.S.A., for his collaboration in the measurements of plasma calcitonin and his interest in this study. The co-operation and assistance of Jack McBride, Dr. John Davis and Dr. Gordon Bell, Fisheries Research Board of Canada and Ian Williams, Forrest Scott and Stan Killick of the International Pacific Salmon Commission, made the studies on salmon possible. Working with these gentlemen was truly a pleasure. My typist, Mrs. Mildred Brown has done a remarkable job, not only in the long hours she has spent typing this thesis, but also in the helpful advice she has given. Finally, I wish to thank my wife, Sue, for her help, under standing and love, for without these I could not have made it. Financial assistance in the form of a Studentship from the Medical Research Council of Canada is gratefully acknowledged. XV PREFACE "There they were,11 he said, pointing at the huge fish; "nearly two hundred years old; perfectly healthy; no symptoms of senility; no apparent reason uhy they shouldn't go on for another three or four centuries " He paused and stood for a moment in silence, drumming with his fingers on the glass of the aquarium. Poised between mud and air, the two obese and aged carp hung in their greenish twilight, serenely unaware of him. Dr. Obispo shook his head at them. "The worst experimental animals in the world," he said in a tone of resentment mingled with a certain gloomy pride. "Nobody had a right to talk about technical difficulties who hadn't tried to work with fish. Take the simplest operation; it was a nightmare. Had you ever tried to keep its gills properly wet while it was anaesthetized on the operating table? Or, alternatively, to do your surgery under water? Had you ever set out to determine a fish's basal metabolism, or take an electrocardiograph of its heart action, or measure its excreta? And, if so, did you know how hard it was even to collect them? " "No, you had not," said Dr. Obispo contemptuously. "And until you had, you had no right to complain about anything." After Many A Summer Dies the Swan Aldous Huxley GENERAL INTRODUCTION According to Romer (1962), teleosts are unquestionably the most numerous and versatile of all the vertebrates. This fact was acknowledged by Bern (1967) who, when discussing problems in fish endocrinology remarked, "In their endocrine systems, as in all other aspects of their anatomy and physiology, the fishes reveal a broader range of variation and a longer history of adaptation than do the 'land-living' (tetrapod) vertebrates. It has now been determined that calcium metabolism in teleosts is under endocrine control (Hoar, 1957a; Simmons, 1971; Chan, 1972). The discovery of calcitonin in fish (Copp et al, 1967a) gave rise to the question, "Is calcitonin involved in this hormonal regulation of calcium metabolism?" The purpose of the present thesis was to investigate calcium metabolism in fish and the physiological role of calcitonin in this process. With regard to the endocrine control of calcium homeostasis, fish are unique among the vertebrates in that they lack a para thyroid gland (Fleischmann, 1951; Pickford, 1953; Hoar, 1951, 1957a; Bern, 1967). Most teleosts possess an endocrine gland, the cor puscles of Stannius, which appears to be intimately involved in calcium and other electrolyte homeostasis (Chan, 1969, 1972; Pang, 1971a). The pituitary gland, adrenal cortex, gonads and Bern, H.A. Hormones and Endocrine Glands of Fishes, Science, N.Y. 158: (1967) pg. 455. -2-thyroid gland are also definitely involved in calcium metabolism in fish (Hoar, 1957a; Henderson et al, 1970; Simmons, 1971; Chan, 1972). The importance of the calcium ion to fish uas first re ported by Ringer (1883) uho noted that uhile unfed fish live for ueeks in tapuater, they soon die if placed in distilled uater. Uptake of calcium from the environment occurs at the gills, fins and oral epithelia (Moss, 1965; see Simmons, 1971). This uptake appears to be more efficient in freshuater fish and is facilitated by the presence of phosphate (see Love, 1970). The freshuater fish salves the problems af electrolyte loss and uater influx by absorbing ions from the environment, main taining an impermeable integument (scales, skin, mucous) and excreting a hypo-osmotic urine (Black, 1957; Bentley, 1971). The problems of calcium regulation in seauater teleosts are. quite different from those in freshuater. Marine teleosts drink seauater. This means that electrolytes,. including a significant quantity of calcium, are absorbed by the gastro intestinal tract (Chan ej_ al, 1967; Henderson et al, 1970). Excess salts are then excreted directly from the blood through the gills, retaining uater in the fish (Smith, 1930; see Parry, 1966; Potts, 1968). Again, a dilute urine is farmed and divalent ion excretion occurs. Thus, the environment plays an important role in calcium regulation in fish. For this reason, both the freshuater trout, Salmo qairdneri, and the anadramous Pacific salmon, genus Oncorhynchus, uere studied ta ascertain the effect of environmental -3-water calcium concentration on calcium homeostasis. The osmoregulatory problems of a freshwater or seawater fish are enormous but these same problems encountered by a migrating anadromaus or catadromous fish are even more complex. A major portion of this thesis concerns research done on the migrating sockeye salmon, •ncorhynchus nerka, and its ability to control its internal ionic environment as the external environ ment changes from sea to freshwater. Since many of the hormones which affect calcium metabolism in fish are also involved in migration and sexual maturation, there are numerous endocrine inter-relationships. Many female teleosts develop hypercalcaemia during the breeding season and this condition can be produced by estrogen injection in the labor atory. The pituitary hormones, thyroxine and gonadal steroids appear to be involved in migratory behaviour changes as well as in sexual maturation (Hoar, 1953, 1957b, 1963; LJoodhead and Woodhead, 1965). Environmental factors such as temperature, light or rainfall may serve to initiate, potentiate, and integrate the hormonal activities in the above processes (Hoar, 1965a,b; Henderson et al, 1970). The degree of.involvement of bone in calcium homeostasis in fish has been a subject of controversy for some time (Fleming, 1967; Moss, 1965; Simmons, 1971). Teleost bone exhibits a wide range of histo-morphology, from acellular to cellular bone (Moss, 1961). The degree of calcification of the teleost skeleton appears to be independent of bone type (cellular versus acellular) and histology (Moss and Freilich, 1963). Moss (1961, 1962, 1963, 1965) -k-and others (see Fleming, 1967) have proposed that calcium in the environmental uater is important in calcium homeostasis and that mineral stores in the fish skeleton play only a minor role in this process. In contrast, Urist (1962, 1966) believes that the teleost skeleton is involved in calcium homeostasis and that the bone and tissues of the fish form a "bone-body fluid continuum" uhich acts as a closed-cycle system. Resorption of teleostean bone has seldom been reported but it may be that cellular-boned species, such as the eel and salmon, are able to drau on the bone mineral under certain conditions (Simmons, 1971). Resorption of fish scales, uhich contain substantial amounts of calcium, has been observed in starved fish (Simmons, 1971) and during salmon migration (van Someren, 1937). The importance of the soft tissues, such as muscle and skin, in teleost calcium regulation has been emphasized by several researchers (Norris et_ a_l, 1963; Rosenthal, 1963; Podoliak and Holden, 1965). These tissues serve as important storage depots far exchangeable calcium, the mobilization of uhich, at least in the eel, appears to be under the endocrine control of the pituitary, adrenal cortex, corpuscles of Stannius and the ultimobranchial glands (Chan,1969, 1972; Simmons, 1971). • The dietary supply of calcium, even in marine fish, is generally not as important as direct absorption from uater (Moss, 1962; Berg, 1968, 1970; Simmons, 1971) although food can supply calcium in freshuater fish under certain experimental conditions . (•phel and Judd, 1967). The main excretory routes for calcium are the gills and kidneys, fecal loss of calcium probably being -5-smaller than in mammals (Simmons, 1971). Although the parathyroid gland is absent in fish, they do possess ultimobranchial glands which contain a rich supply of calcitonin (Copp and Parkes, 1968b; Copp et al, 1968b; Copp, 1969a). In fact, calcitonin began phylogenetically in fish (Copp, 1969a; Copp _ al, 1972a; Copp, 1972). The ultimobranchial origin of calcitonin was discovered in 1967 and salmon calcitonin (SCT), the first non-mammalian calcitonin to be characterized, became available in purified form in 1969 (O'Dor e_t a_l, 1969a, b). The amino acid sequence of salmon calcitonin was reported shortly thereafter (IMiall e_t a_l, 1969). Salmon calcitonin has been shown to exert a longer-lasting, more powerful hypacalcaemic effect than mammalian calcitonins when tested in young mammals. This effect is due primarily to the inhibition of bone resorption (Copp, 1970a). When work on the present study commenced, porcine calcitonin had been injected into a few species of teleosts and with variable results (Pang and Pickford, 1967; Louw et al, 1967; Chan et al, 1968). Salmon calcitonin had not been tested in any fish prior to 1969. Therefore, experiments were designed to collect basic information on calcitonin in fish in an attempt to answer the following questions: 1. What is the effect of salmon calcitonin injection on plasma and renal electrolytes in salmonids? 2. How much calcitonin is stored in fish ultimobranchial glands? -6-3. Does the UB gland calcitonin concentration vary uith age, sex or salinity? 4. Uhat is the circulating level of plasma calcitonin in fish and uhat factors govern this level, i.e. migration, sexual maturation, gonadectomy? 5. Is calcitonin involved in calcium and phosphate homeo stasis in fish? 6. Hou do the actions of calcitonin in fish compare to those in mammals? Thus, the investigation of calcium metabolism in fish and the physio logical role of calcitonin uas begun on this broad base. -7-GENERAL MATERIALS AND METHODS In this chapter, these materials and methods uhich were common to all studies are described. Materials and methods specific to individual studies will be outlined in the approp riate chapter. 1. Choice of Experimental Animal The fish was chosen as the experimental animal in this study for several reasons. Phylogenetically, the ultimobranchial (UB) glands first originate in fish. Almost nothing was known about the function of calcitonin in non-mammals until Copp (Copp et al, 1967a, b; Copp and Parkes, 1968a, b) demonstrated that the ultimobranchial gland was a rich source of calcitonin. Salmon and trout were readily available in British Columbia and the surgical techniques for working Dn these fish were well documented. Finally, a very sensitive and specific radioimmunoassay for salmon calcitonin became available and provided an important tool to investigate the physiological changes of calcitonin in these fish. 2. Experimental Animals Several types of fish, obtained from a variety of sources, were studied. The types of fish included rainbow trout (Salmo  qairdneri), echo salmon (Oncorhynchus kisutch), chinook salmon (Oncorhynchus tshawytscha) and sockeye salmon (Oncorhynchus nerka). -8-a) Rainbow Trout Rainbow trout used in this study uere purchased from the Sun Valley Trout Farm, Mission, British Columbia. The trout weighed between 6D and 260 g. The fish were transported to the University of British Columbia in oxygenated 100 gallon tanks and kept in a 25Q gallon self-cleaning, fibreglass, holding tank (Everlast Plastics Co., Vancouver) supplied with fresh running water. Tapwater was filtered and dechlorinated using a filter containing activated charcoal, limestone, oyster shells and sand. Uater temperatures varied seasonally over a range of 4 - 16°C, but remained fairly stable over any one experimental time course. The trout were fed regularly with commercial trout pellets (Purina Trout Chow, Ralston Purina Co.). The light regimen in the fish laboratory was controlled using an Inter-Matic Time Switch (Model T 101). This switch was adjusted regularly to the seasonal light conditions and was kept constant during any one acclimation and experimental period. All fish were acclimated to laboratory conditions for at least one week before being used in any experiments. b) Coho Salmon Mature adult coho salmon were obtained from the Washington State Department of Fisheries at the Samish River holding ponds in Washington, U.S.A. Immature seawater and freshwater coho, were obtained from the Fisheries Research Board of Canada, Vancouver, B. C. -9-c) Chinook Salman Mature adult chinook salmon uere obtained from the Washington State Department of Fisheries at the Deschutes River holding ponds in Olympia, Washington, U.S.A. d) Sockeye Salmon The sockeye salmon uere obtained from two sources, the Chilko Lake race of sockeye and the Great Central Lake race of sockeye. The Chilko sockeye were caught at various stages in their migration during the summers of 1971 (July 23 to September 22) and 1972 (July 21 to September 24). Sexually immature adult sockeye salmon were caught by trap when entering Great Central Lake on Vancouver Island, B.C. in June and July of 1970 and 1971. These fish normally spawn from late September until the end of November in the lake and in creeks feeding the lake. The method of capture and transport to the Vancouver Laboratory, Fisheries Research Board of Canada, have been described (McBride e_t a_L, 1963). The fish were held at the Vancouver Laboratory in large fiberglass holding tanks at seasonal water temperatures and on a natural photoperiod. Fungal infections were treated with salt baths of 3% aqueous sodium chloride and 2-Phenoxyethanol as described by Idler (1961a). A volume of B.5 ml of a 50 mg per ml solution of Terramycin (Phizer, Canada) was injected intramuscularly to control bacterial infection. Prior to an experiment, the sockeye were transported to the Physiology Department fish laboratory as required, using a -10-1:15,000 solution of MS-222 anesthetic (tricaine methanesulfonate, Fraser Medical Supplies Ltd.) and portable air pumps. Unless indicated otherwise, the salmon were not fed in the laboratory. 3. Operating Procedures a) Operating Table and Operating Techniques This study involved the serial sampling of blood from a free-swimming quiet fish, over extended periods of time. For this reason, the dorsal aorta cannulation technique (Smith and Bell, 1964) was employed. An operating table, similar to the one described by Smith and Bell (1967), was constructed and used for all surgical operations (Plate 1, page 11). The fish was first anesthetized in a bucket of a 1:5,000 solution of MS-222 and then placed ventral side up on the operating table. The gills were perfused through the mouth or opercular openings with a 1:15,000 solution of MS-222 (Bell, 1967). The fish was kept moist at all times with wet fish netting. Throughout the procedure, the operating table anesthetic was aerated and the water temperature kept constant by placing plastic bags of ice into the reservoir. Following the operation, the fish was allowed to recover in fresh, aerated running water. The procedures were completed in less than 30 minutes and recovery time was approximately 5 minutes. Complete recovery was indicated by twitching, fin movements, re sumption of normal respiratory movements and swimming efforts. The fish was placed in the appropriate experimental apparatus (aquarium, -11-PLATE 1. Fish Operating Table 1. Trout (upside doun) in position for can nulation 2. Adjustable holding device for fish 3. Rubber hose attachment to direct anes thetic over gills 4. Anesthetic reservoir 5. Uariable-flou pump -12-urine box) immediately following the operation and at least 12 to 2k hours recovery time allowed before use. Since surgical operations can lead to considerable physiological trauma (Houston e_t a_l, 1969), a recovery period of this length is considered necessary. The experimental apparatus was partially covered with black plastic to prevent the fish from being disturbed by the movements of the investigator. b) Dorsal Aortic Cannulation Procedure The dorsal aorta was cannulated at its point of inter section with the second efferent branchial arteries as described by Smith and Bell (1964). The cannula consisted of a 60 cm length of Clay-Adams PE 60 (ID. 0.762 mm) plastic tubing into which a 2-3 cm 21-G (Huber. Point with Closed Bevel-B-D Yale Luer Lok) needle had been inserted. A hole was made mid-dorsally in the tip of the snout of the fish with a 12-G needle. Caution was taken to avoid the olfactory lobes and no injury was evident from this procedure. A 3 cm length of Clay-Adams PE 200 plastic tubing ("sleeve"), heat flared on one end, was passed through this hole in the snout from inside the mouth. This PE 200 sleeve was used to secure the PE 60 cannula in place. The cannula was filled with heparinized (10 USP units Heparin (Ammonium Salt) per ml of saline) Cortland Saline (wolf, 1963) and plugged with a tapered stainless steel pin when not in use. The cannula needle was inserted at the midline junction of the first gill arch in the roof of the mouth at an angle of approximately 15-20°. Successful cannulation was indic ated by bright red blood rushing into the cannula tubing, dis--13-placing the heparinized saline. The cannula uas sutured securely in the roof of the mouth using black silk surgical suture (size ••• Davis & Geek Products) and on the PE 200 sleeve using white surgical cotton thread (Figure 1a, pg.14, and Plate 2, pg. 15). The cannula was flushed with fresh heparinized saline once a day and care was taken to ensure that the needle and cannula were completely filled with heparinized saline. The cannula was allowed to trail freely behind the fish and it did not appear to affect behaviour or swimming ability in any way. c) Blood Sampling Procedure The cannula was gently retrieved from the aquarium with long forceps, dried off with tissue paper, and the plug removed. A heparinized 1 ml syringe was used to withdraw 0.5 ml of the saline-blood mixture which filled the cannula. The dead space of the cannula amounted to D.3 ml (heparin-saline) and so removal of 0.5 ml ensured that the succeeding sample would not be contam inated with heparin -saline. A second heparinized 1 ml syringe was used to withdraw the blood sample. Then the first syringe with the mixture of heparin-saline and blood was immediately returned to the fish and followed by clean heparin-saline so that no blood remained in the cannula. The steel plug was then replaced in the cannula and the cannula was returned to the aquarium so the fish would be free to move. It should be noted that all syringes used in the sampling procedure were plastic (Roehr Monoject) and the dead space of the syringe was filled with concentrated heparin (!••• USP Units = 1 ml Figure 1a. Rainbow trout dorsal aortic cannulation -15-PLATE 2. Trout Dorsal Aortic Cannulation 1. Trout upside doun on operating table 2. Huber needle (21-G) at first gill arch point of entry (tied in with silk thread) 3. PE 2Q.D sleeve tied in place with cotton thread k. PE SO cannula -Il liquid Heparin, Sherwood Medical Industries). Furthermore, in jections were performed slowly and steadily so as to minimize hemolysis and trauma to the fish. If these precautions were taken, the fish always remained perfectly still. In chronic cannulation experiments, where large volumes of blood were taken, the red blood cells were resuspended after plasma collection in an approp riate volume of heparinized-saline (10 U/ml). This mixture was immediately returned to the fish, the cannula was refilled with heparinized saline and plugged. Any significant drop in haema-tocrit due to repeated blood samplings was thus prevented (Hickman, 1968). The blood samples were usually transferred to 6 ml sterile, polystyrene disposable culture tubes and immediately centrifuged for 5 minutes in a standard laboratory clinical centrifuge. Using glass pasteur pipettes, the plasma was separated off into clean culture tubes with caps. The plasma was stored on ice if measure ments were to be made the same day, or frozen on dry ice and stored at -12°C if measurements were to be performed at a later date. Uhen large blood samples (30-50 ml) were taken from salmon, the blood was transferred to 50 ml polycarbonate centrifuge tubes and spun at 1200 x g for 5 minutes on an HIM-S Centrifuge (Inter national Equipment Co. U.S.A.). Injection of substances into the dorsal aorta could be performed immediately fallowing the control bload sample. Care was taken to exclude bubbles from the injectate and to keep the volume injected small (less than 0.3 ml far a 200 g trout). The -17-injectate uas followed first by the heparin-saline and blood mixture and then by a clean O.k ml heparin-saline volume to fill the cannula and needle dead space. The time of injection was calculated from the moment the injectate entered the fish. d) Caudal Vein Sampling Terminal blood samples were obtained from the fish using the caudal vein sampling technique. The fish was usually tapped on the head at the beginning of the procedure and held securely wrapped in netting. To ensure that the sampled blood was well oxygenated, the gills were perfused with aerated water. The trout was laid flat on its side on the operating table and a hepar inized plastic 1 ml syringe with a 21-G lYz inch needle was inserted through the skin of the mid-ventral aspect of the caudal peduncle, approximately 2 cm posterior to the anal fin. The needle was directed forward between the haemal spines into the haemal canal. Using this technique, blood was withdrawn into the syringe from the tail circulation with little injury to the fish. If more than 1 ml of blood was required, the needle was left in place and a second heparinized 1 ml syringe was quickly inserted into the needle. Thus terminal blood samples could be withdrawn quickly and easily from fish in approximately 1 minute. For salmon, the technique was identical, except that a" larger syringe with an 18-G Vfe inch needle was used. In the field, the salmon were restrained by placing them ventral side up in a large V-shaped apparatus constructed of plywood. -18-e) Caudal Vein Cannulation The caudal vein of the salmon was cannulated using the basic technique developed by C. P. Hickman (Hoar and Hickman, 1967). This technique was similar to the caudal vein sampling procedure except that an 18-G 31/z inch thin wall needle (for use with plastic tubing B-D Yale Luer-Lok l\lo. 1295), through which PE 50 Intramedic tubing (Clay-Adams) could be passed, was used. The salmon was first anesthetized, placed on its side on the operating table and perfused with 1:15,000 solution of MS-222. Holding the caudal peduncle in the left hand, the 18-G needle attached to a 2.5 ml plastic syringe containing heparinized-saline, was inserted from the lateral-ventral aspect of the caudal peduncle. The needle was inserted'into the caudal vein, Figure lb, pg. 19, and successful entry was signalled by a flow of blood into the syringe. With the needle held securely in the caudal vein, the syringe was carefully and quickly removed and a 50 cm length of PE 50 cannula (filled with heparinized-saline) was threaded down the needle and into the caudal vein for a distance of 15 cm. Proper insertion of the cannula was indicated by the easy with- . drawal and injection of blood through the PE 50 cannula using a 1 ml syringe and 25-G needle filled with heparinized-saline. The 18-G needle was then extracted from the puncture site, taking care not to dislodge the cannula, and slipped back down the length of the cannula. After filling the cannula with heparinized-saline and pinching it off with the thumb and forefinger, the 1 ml syringe and 25-G needle were detached from the cannula, the 18-G needle was removed and the cannula was plugged with a stainless steel pin. Figure lb. Caudal vein cannulation technique. Diagram from Hoar and Hickman (1967). I -ZD-Pressure uas exerted on the puncture site ta minimize the bleeding and the cannula uas tied securely, 2 cm belau the lateral line, using 2 stitches cf black braided surgical silk. This type of cannulation could only be used on fish Dver 500 g (hence on the salmon in this study) and aluays in volved a small amount of blood loss from the puncture site (about •.5 ml). Blood sampling from the caudal cannula uas similar to that of the dorsal aorta outlined previously. Using the tuo cannulations, a free-suimming, quiet fish could be infused and blood sampled simultaneously. f) Urinary Catheterization The salmon uas anesthetized and placed ventral side up on the operating table. Throughout the procedure, the mouth uas perfused uith a 1:15,••• solution of MS-222. Catheterization uas performed by opening the aperture of the urogenital papilla uith a pair of fine curved forceps and inserting the tip of a rubber pediatric catheter of size French 8 or ID (Ingram and Bell Ltd.) dorsally into the urinary orifice. The catheter uas passed along the urinary duct into the urinary bladder (3-6 cm) and successful catheterization uas indicated by urine flouing from the catheter uhen alloued tD hang beneath the fish. The catheter uas then secured in place by firmly tying off the urinary papilla around the catheter using surgical cotton thread. The urinary aperture uas then closed off around the catheter and over the papilla by a purse string ligature. These procedures anchored the catheter in place and Insured that no leakage -21-•ccurred. The catheter was further secured in position by a long stitch to the caudal peduncle using black surgical silk. The catheterized fish was then quickly transferred to the urine collection box (Figure 2, pg. 22) to recover in fresh running water. The urine collection box used in this study was the same as that described by Smith and Bell (1967). The partitions were adjusted to the size of the fish and the urinary catheter was passed out through a rubber gasket-sealed aperture to a fraction collector (Instrumentation Specialties) for collection of hourly samples. The urine was allowed to flow by gravity and blockage of the catheter rarely occurred. The fish were free-swimming but restricted and urine was collected continuously. The box was con structed of. black acrylic plastic tD shield the fish from outside disturbances and under these conditions, the salmon stayed almost motionless for hours. These precautions were necessary due to the phenomenon of "laboratory diuresis" which normally occurs following any kind of disturbance or handling procedure (Holmes, 1961; Klontz and Smith, 1968; Hammond, 1969; Hurin and Idillford, 1970). Therefore, the fish were allowed to recover after the operation for 15-24 hours before any experiment was conducted. k. Analytical Procedures a) Physical Measurements Total body weights of the trout were measured using a standard laboratory one-arm balance. The salmon were weighed on a larger one-arm balance (Ohaus Scale Corp., cap. 6 kg). A Mettler Figure 2. Blood and urine sampling technique in salmon -23-balance (Type H16, cap. 80g)was used far accurate weighing of chemicals, ash weights and small organs. From the total weight (g) and the weight of both gonads (g), the gonad-somatic index (GSI) was calculated from the fallowing equation: Gonad-Samatic Index = gonad weight (g) x 100 (Q2j) total body wt (g) This "index- of maturity" was used as a measure of the degree af sexual maturation of the fish. Thus, the higher the index, the mare advanced was the degree of sexual maturity (Vladykov, 1951). Fork lengths of individual fish were also recorded. The fork length is the distance (in cm) from the tip of the snout to the fork in the caudal fin. b) Haematocrits Blood far haematacrit estimation was introduced into heparinized capillary tubes (Donlab, Ingram and Bell Ltd.), capped with Critocaps (Sherwood Medical Industries Inc.) and spun in a Micro-Capillary Centrifuge (International Equipment Co. - Model MB) for k minutes. The haematocrits were read immediately on a Critocap Micro-Haematocrit Tube Reader. The plasma was then separ ated from the red cells and used for measurement of percent water or calcium. c) Plasma Water and Total Solids Plasma water and total solids were measured using a TS-Meter (Total Solids Meter, American Optical Instrument Co. Model 10400). Using this instrument and the accompanying conversion tables, -2k-estimates of the total solids percentage composition by weight (TS%), the percent water (,% H^D) by weight (g per 100 g at 20OC) and protein concentration (Cppj g per 10.0 ml at 2Q°C) were de termined on 10 pi of plasma. d) Preparation of Tissues for Electrolyte Analysis i. Drying and Ashing Procedures  Hard Tissues The hard tissues taken far electrolyte analysis included vertebrae, ribs, premaxillae and scales. The tissues were first roughly dissected from the fish and stored frozen at -12°C in air tight plastic bags. The vertebrae, ribs and premaxillae were thawed, freed of soft tissue and samples of approximately equal weight were dried in porcelain crucibles (Coors Labware) to a constant weight (oven temperature 100°C). The dry weight was then calculated and the samples were ashed at 575°C overnight. The samples were again weighed (ash weight). Before drying, the scales were rinsed in deionized water and wiped with tissue paper to remove any mucous or seawater. Soft Tissues Samples of muscle, skin and gonads were dissected and stored similar to the hard tissue. Since a large error due to evaporation was involved with the small samples of muscle and skin, the wet weight for the soft tissue was recorded only for the gonads. The muscle and skin samples were placed in porcelain crucibles and -25-dried in an oven at 100 C to a constant weight. The large gonads were similarly dried to a constant weight in 600 ml pyrex beakers. The dry weight was calculated. Fat-extraction was then carried out on the dried tissues with a 1:1 mixture of absolute ethanol and anhydrous ethyl ether (Analytical Reagent, Mallinckrodt Chemical Works). These samples were further dried to a constant weight at 100°C, and reweighed to obtain the fat-free dry weight (FFDW). The fat-free tissues were then ashed overnight and the ash weight calculated. Due to the large size and biochemical composition of the gonads, it was necessary to ash these samples for several days. ii. Dilutions of Ash The ashed tissue was weighed accurately, dissolved in 6 N hydrochloric acid, and evaporated to near dryness on a hot plate. The sample was then redissolved in 0.1 l\l hydrochloric acid and transferred quantitatively to the appropriate volumetric flask. A further dilution was made using a 0.5% (w/v) lanthanum chloride solution (LaCl^'VH^O) and.analyzed for calcium by atomic absorption spectrophotometry. Phosphate was measured colorimetrically (Alexander, 1968) after making the proper dilution with deionized water. The results were expressed as mg or g of calcium or phos phorus per 100 g dry weight, fat-free dry weight or ash weight. e) Electrolyte Analysis The glassware .used far electrolyte measurements was acid-washed and free of contaminating ions. Small samples (less than 1.0 ml) were dispensed using Oxford Micro-Pipette Samplers (Oxford -26-Labaratories). Lab-Trol and Patho-Trol Chemistry Reference Serums (Dade, Division American Hospital Supply Corp.) uere used in all electro lyte analyses as quality control checks. The simultaneous use of the normal and abnormal controls provided a check of the standards and instrument linearity. i. Calcium Two methods of calcium analysis uere employed in this study. When only small amounts of plasma or solution uere available, calcium uas analyzed fluorometrically using a modification of the Technicon Auto Analyzer Method IM-31 P (IMeusome, 1969). This method could be used to measure extremely small amounts of sample (e.g. 10 pi) and uas found to be both rapid and reproducible. Calcium uas also determined by atomic absorption spectro photometry (Jarrell-Ash, Model 280 Atomsorb) at a uave length of o 4227 A and using 0.5% lanthanum chloride as the diluent. The addition of lanthanum chloride suppressed interference from sulfur and phosphorus (Trudeau and Freier, 1966). The readings uere recorded on a strip chart recorder (Sargent Recorder, Model SR). ii. Magnesium Magnesium uas determined on the same atomic absorption spectrophotometer at a uave length of 2852 8. Samples and standards uere again diluted uith 0.5% lanthanum chloride. iii. Sodium and Potassium Sodium and potassium, in the plasma and urine, uere analyzed -27-by flame photometry (Instrumentation Laboratory Inc., Model 143). A standard lithium solution (15 mEq Li per litre) was used as diluent. Samples were diluted and dispensed with an automatic dilutor (Fisher Dilutor, Model 240). iv'. Inorganic Phosphorus Plasma, urine, and tissue phosphorus were measured using a modification of the Technicon Auto Analyzer l\l-4c Method (Alexander, 1968). This method is based on the formation of phosphomolybdic acid, which is then reduced by stannous chloride-hydrazine. f) Serum Ionic Calciums i. Collection of Blood Samples The blood was sampled by caudal vein puncture using a 12 ml plastic syringe and 18-G V/z inch needle. The syringe, including needle dead space, was previously filled with 2 ml of mineral oil (Nujol, Plough Ltd.) which had been cooled on ice. It should be noted that throughout the entire procedure, the blood and serum were kept anaerobic and on ice. All mineral oil, syringes and test tubes were coaled on ice before use. The blood sample was taken with the syringe in a vertical position i.e. needle down, ta ensure that a layer of oil remained above the blood. After discarding the first few drops of blood, the sample was immediately ejected under the mineral oil in a glass centrifuge tube. The bload was then allowed ta clot for 2 hours (under oil, an ice) and spun for 2 minutes in a clinical centrifuge. To ensure that the serum did.not contact air during -28-transfer, a layer of oil uas first draun up in a pasteur pipette. The serum uas then siphoned into the pipette folloued by a second layer of oil. The sample uas ejected into cooled plastic tubes containing 2 ml of oil, capped and stored on ice until measurement. The temperature of the uater in the fish tank uas carefully noted. ii. Measurement of Serum Ionic Calcium  Equipment Serum ionic calcium activity (Ca++) uas measured potentio-metrically using a Calcium Activity Flou-Thru System (Orion Research Inc., Model 99-20) attached to a digital research pH/mU meter (Corning Scientific Instruments, Digital 112 Model). The pH meter uas connected to a strip chart recorder (Sargent Recorder, Model SRG) as shoun in Plate 3, pg. 29. To minimize external electrical interference, a Faraday cage uas constructed out of galvanized uire mesh (6 mm square) and properly grounded. The strip chart recorder, pH meter, electrode, and syringe pumps uere all grounded to the Faraday cage. Tuo uater jackets uere constructed out of steel (2 mm thickness) to contain both the electrodes and the syringe (Plate k, pg. 30). Directly after determination of serum calcium activity, the serum pH uas measured using an Ultra-Micro pH/Blood Gas Analyzer (Instrumentation Lab. Inc., Model 113-S1) uith a Constant Temper ature Control Module (IL, Model 127). All glassuare used in the procedure uas either acid-uashed or disposable to minimize contamination of specimens uith calcium. Solutions uere prepared using deionized uater. Calcium Activity Flow-Thru System Strip chart recorder Digital pH/mV/ meter Faraday cage Infusion pump and electrode assembly » PLATE k. Infusion Pump and Electrodes uith Uater Jacket 1. Electrode uater jacket (in black) 2. Syringe uater jacket (in black) 3. Inlet and outlet tubings for uater k. Infusion pump -31-Procedure Use Df the Flow-Thru System enabled ionic calcium to be measured an serum samples af 0.2 ml under anaerobic conditions. Calcium standards uere prepared fresh daily by suitable dilution of a stock solution of CaCO^ in 150 mM IMaCl. The standards, corresponding to Orion standards A, B and C, contained 2.00, 4.00 and 8.00 mg/100 ml respectively. These uere checked against reference standards using atomic absorption spectro photometry. Three drops of 0.1 M triethanolamine and 0.006 g of trypsin (Trypsin: 2 x crystalline, salt free, Nutritional Bio chemicals Corps.) uere added to each 10 ml of pure standard. The standards, samples, syringes and electrodes, uere all cooled in running tap water to the same temperature far several hours before measurement. The "B" Standard was run through the system for 20-30 minutes at the beginning of each day to condition the electrodes and remove any accumulated ion exchanger. Separation between the standards was always - 7.5 mV. The standards were repeated several times at the beginning and end of each set of analyses. When the system was stable and the drift was less than - 0.2 mV, the serum samples were introduced. The "B" standard was run between each sample and each serum sample was measured in duplicate. The "C" standard was introduced periodically in order to check the standard curve reproducibility. All serum samples were removed from under the mineral oil using a 1 ml plastic tuberculin syringe and a 26-G Yz inch needle. Care was taken to exclude air bubbles and oil from both the sample and the electrodes. The serum was removed -32-immediately before measurement and a new syringe used for each sample. The standards and samples uere run for at least 3 minutes or until the reading stabilized on the recorder. Immediately following the ionic calcium activity reading, serum pH uas measured using 30 pi of the same sample, at the same temperature. Trial tests indicated that serum samples could be stored anaerobically at <JPC for 3 days uithout any significant change in serum ionic calcium activity. Therefore, all ionic calcium measurements uere performed as soon as possible after sample collection and always uithin the 3 day limit. Total serum calcium uas measured fluorometrically (Neusome, 1969) on duplicate 20 pi samples, directly follouing the ionic calcium activity and pH readings. This alloued the calculation of percent ionic calcium-Percent ionic calcium (.% Ca++) = ionic Ca++(mg/100ml) x IQQ total Ca++(mg/100ml) Calculations The calcium flou-thru electrode developed a potential pro portional to the logarithm of the calcium ion activity in the sample. Potentials became increasingly positive in more concentrated sol utions, and increasingly negative in more dilute solutions. Using 2-cycle, semilogarithmic graph paper, the mean potential developed in each standard (linear axis) uas plotted against the concentration value of the standard (log axis). The calcium concentration of the unknouns uas then determined from this calibration curve. A computer program uas devised on a desk-top computer (Olivetti Underuood, Programma 101 Model) uhich computed the slope -33-•f the standard curve and calculated the ionic and total calcium (in mg per 100 ml) and the percent ionized calcium for each unknown sample. g) Bioassay of Calcitonin in Ultimobranchial Glands i. Rats All calcitonin bioassays were performed on 3-4 week old, 8D g, black hooded male rats of the Long-Evans strain (Blue Spruce Farms, N.Y., U.S.A.). They were housed in metabolic cages (5 per cage) and maintained on a natural photo-period.. The rats were kept on Purina rat chow and tap water a_d libitum for several days before use and starved 24 hours before assay. ii. Collection and Preparation of Ultimobranchial  Glands The fish were sacrificed by severing the spinal cord and the head excised from the body slightly posterior to the operculum. The liver, gonads and esophagous were removed and the transverse septum containing the ultimobranchial gland exposed. Using a fine pair of scissors, the gland was carefully cut out. It was weighed immediately (wet weight), quickly frozen on dry ice and stored at -12DC in capped auto analyzer cups. The UB gland appeared quite distinct in the trout (Plate 5, pg. 39) and just the area around this "moustache-shaped" gland was cut out. However, the salmon UB gland was more diffuse (Plate 6, pg.4Q ) and hence necessitated removal of the majority of the transverse septum. Since the transverse septum of fish contains little fat, it was found unnecessary to fat-extract the ultimobranchial tissue. -3k-The glands uere homogenized using a tissue homogenizer (Tri-R Instruments Inc.) in a vehicle of 0.1 (\l HCl and 0.1% glycine (pH = k.3). Fibrous material uas removed by filtering the homogen ate through a piece of sterile gauze. Care uas taken to keep the gland and homogenate on ice at all times. iii. Bioassay Technique All bioassays uere performed using a modification of the method developed by Kumar e_t al_ (1965). The rats uere starved overnight and injected intravenously via the tail vein uith a dose of 0.3 ml per 80g. body ueight. Five rats uere used for each point and the control group received vehicle alone (0.1N HCl + 0.1% glycine, pH k.3). The injection uas performed under ether anes thesia and the rats uere bled from the tail 60 minutes after the injection. Blood uas collected directly into heparinized capillary tubes (0.2 ml blood) and spun for 10 minutes in a micro-capillary centrifuge. Duplicate plasma calciums uere measured fluorometrically using the Technicon Auto-Analyzer Method (IMeusome, 1969). A rough assay uas first performed to determine the correct dilutions for each gland. Once the dilutions uhich gave the proper curve uere obtained, a fine assay uas performed. The difference betueen the mean plasma calcium level in mg per 100 ml of the control group and the plasma calcium level of each experimental blood sample uas calculated and called the response. A log dose-response curve uas constructed and the amount of calcitonin in m Units/mg uet ueight of gland uas calculated from the standard curve of MRC Research Standard B (Calcitonin, Porcine Thyroid). -35-The concentration of calcitonin per body weight (mU/kg body wt) was also estimated for each gland. A house standard of purified salmon calcitonin (MRC, Mill Hill, England) and checked against the MRC Research Standard B, was injected in assays to ensure the consistency of the rats' hypocalcaemic response. It should be noted that, due to the diffuse nature of the ultimobranchial gland in the salmon and ih e inherent errors of the bioassay, the calcitonin contents of the UB glands represent only an estimate. The method could detect from 2 to 10 mU per 0.3 ml per 80 g rat and had an index of precision ( X) below 0.2. h) Radioimmunoassay of Plasma Calcitonin Plasma calcitonin lev/els were measured using a very sensitive and specific radioimmunoassay for salmon calcitonin developed by Dr. Len Deftos, Endocrine Section, Department of Medicine, University of California, San Diego, U.S.A. (DeftDs e_t a_l, in press). Plasma samples for calcitonin assay were frozen on dry ice immediately after collection and stored at -12°C in sterile polypropylene culture tubes. These samples were then packed in dry ice and shipped to Dr. Deftos by air. All plasma calcitonin measurements reported in this thesis were performed by Dr. Len Deftos. Under optimal conditions, the assay could detect 50-100 pg of calcitonin per millimeter of salmon plasma.. -36-i. Statistical Analysis All measurements uithin each group uere expressed as the mean - standard error about the mean (SE). Group comparisons uere made using the "Student's" t-test calculated uith the aid of a desk-top computer (Olivetti Underwood Co., Programma 101). Probability values were obtained from standard tables. I. ULTIMOBRANCHIAL GLAND CALCITONIN CONCENTRATIONS Introduction The ultimobranchial gland first appears phylogenetically in fish. Van Bemmelen, in. 1885, originally described the gland in elasmobranchs. He named the two small epithelial masses which were found caudal to the last pair of branchial clefts, the "suprapericardial bodies" and considered that they represented a rudimentary seventh pair of branchial pouches. De Meuron (1886) gave a brief account of the suprapericardial body in selachians and amphibians and hamologized the body in these forms with the "accessory thyreoid" of reptiles, birds and mammals. The term "ultimobranchial it body" (ultimobranchialen Harper) was introduced by Greil in 1905 and more correctly describes the embryonic origin and location in most vertebrates. Uatzka reported that the gland was present in all orders of vertebrates except the cyclostomes (Uatzka, 1933). In elasmobranchs, Camp found that the ultimobranchial (UB) gland consisted of large distended vesicles the majority of which intercommunicated (Camp, 1917). Van Bemmelen did not find the suprapericardial bodies in teleosts, but S-upino (1907) described postbranchial bodies in lepto-cephalus lying between the pharynx and the pericardial wall. Giacomini (1909) found them not only in leptocephalus but also in adult Anguilla sp. The ultimobranchial gland has since been des cribed in many species of fish (Giacomini, 1912; Nusbaum-Hilarowicz, 1916; Giacomini, 1936 and Krawarik, 1936). Camp (1917) found that only the left suprapericardial body -38-persisted in the adult selachian. This condition is known to occur in other lower vertebrates. In contrast, the ultimobranchial gland of most teleosts is bilateral and imbedded in the transverse septum between the abdominal cavity and sinus venosus (Krawarik, 1936). In the rainbow trout, it takes the appearance of a small "moustache-shaped" band of white tissue and lies immediately ventro-lateral to the esophagus (Plate 5, pg. 39 ). The salmon UB gland is located in a similar position, except that the gland appears to be much more diffuse (Copp and Parkes, 1968b)(Plate 6,pg. 40). The gland in the dogfish shark, Squalus acanthias, lies imbedded in the connective tissue of the pharyngo-pericardial wall, between the ceratobranchial cartilage laterally and the cardiobranchial cartilage and coracobranchial muscle medially. The ultimobranchial gland has a follicular appearance in sharks (Camp, 1917; Copp, 1969a). A portion of the adult selachian gland was found to be secondarily connected to the pharynx by a true duct, giving the gland the appearance of a cross between an endocrine and an exocrine gland (Camp, 1917). The teleost UB gland normally has a follicular structure (Rasquin and Rosenbloom, 1954; Sehe, I960; Robertson, 1967, 1969; Lopez et_ al, 1968; Copp, 1969a; Deville and Lopez, 1970). Some authors have noted this gland appears as cords of cells (Eggert, 1938; Copp, 1969a;Pang, 1971b;which take on a follicular aspect when the gland becomes hypertrophied. Since the ultimobranchial tissues possess a well-developed vascular and nervous supply, Watzka (1933) believed that the gland, at least in birds and reptiles, might possibly have an endocrine function. Robertson (1969) has demonstrated by electron microscopy, -39-PLATE 5. Trout Ultimobranchial Gland Midline cross-section of a male trout 1 cm posterior to pectoral fins. Triangular-shaped ultimobranchial gland (arrow) lies immediately below esophagus within the transverse septum. -1*0-PLATE 6. Salmon Ultimobranchial Gland Midline cross-section of a seauater female sockeye salmon 1 cm posterior to pectoral fins. Diffuse ultimobranchial gland lies below esophagus within the transverse septum. Kidney tissue lies above the esophagus and liver tissue below. -41-that the UB gland of the rainbow trout consists of epithelial components (columnar and a feu goblet cells) surrounding a simple follicular structure with a ductless central cavity. The presence of an active Golgi apparatus in many cells and the accumulation of membrane-bound cytoplasmic granules, suggested to him a possible endocrine secretory function. The membrane-bound, osmiophilic granules, seen in the teleost UB gland, resemble those seen in the ultimobranchial secretory cell in all jawed vertebrates. Although it had been known for many years that the mammalian ultimobranchial gland cells become incorported into the thyroid gland (Baderstscher, 1918; Kingsbury, 1935a, 1935b; Gorbman, 1947), the function of this curious pharyngeal gland derivative was unknown. In 1932, IMonidez described large epithelial cells with argyrophile granules in the thyroid of the dog. He named them "parafollicular cells" since they lay in the interstitial spaces adjacent to the follicles and were readily distinguished from the follicular epithelium. Godwin (1937) demonstrated that the "para follicular cells" of the thyroid were really of ultimobranchial origin. Foster e_t a_l (1964) hypothesized that the parafollicular or mitochondria-rich cells may be responsible for the secretion of calcitonin. This led to the discovery by Pearse in 1966, that the parafollicular, or "C cells" as he named them, were probably the source of calcitonin. This finding has since been confirmed (Bussolati and Pearse, 1967; Kalina et al, 1970). More recently, it has been shown that the calcitonin-secreting cells are derived from the neural crest (Le Dourain and Le Lievre, 1970, 1972; Pearse and -42-Polak, 1971, 1972). In fish, amphibians, reptiles and birds, the ultimobranchial gland remains separate from the thyroid (Copp, 1967). The first evidence indicating a relationship between the ultimobranchial gland and calcium regulation in fish, was presented by Rasquin and Rosen-bloom (1954). These authors showed that when the Mexican cavefish, Astyanax mexicanus, was raised in the dark, the LIB gland underwent hypertrophy and tissue hyperplasia. Associated with this change were fibrosis and lesions of the skeleton and extensive degeneration and calcification of the kidneys. They postulated that these path ological changes were due to over-secretion of the parathyroid-like ultimobranchial gland. Copp e_t a_l (1967a) were the first to demonstrate in fish, that the ultimobranchial gland did not have a parathyroid function but in contrast, was a rich source of calcitonin. Calcitonin was first extracted from the dogfish shark, Squalus suckleyi and chickens, Gallus domestica (Copp e_t a_l, 1967a, b) and later from the salmon, genus Oncorhynchus (Copp and Parkes, 196Qa, b). The fact that cal citonin was not detectable in the thyroids of these animals provided proof that it was an ultimobranchial rather than a thyroid hormone. The ultimobranchial origin of calcitonin was confirmed by Tauber (1967) in the chicken and by Moseley §__ a_l (1968) in lizards, pigeons and chickens. Calcitonin has since been extracted from the UB glands of the blue shark, Prionace glauca, and horn shark, Heterodontus  francisci (Urist, 1967), the gray cod, Gadus macrocephalus (Copp and Parkes, 1968a), lungfishes, IMeoceratpdus forsteri and Lepidosiren  pardoxa, the killifish, Fundulus heteroclitus and the codfish, Gadus -43-morhua (Pang e_t a_l, 1971), the eel, Anguilla /japonica (Orima e_t al, 1872a), the llngcod, Ophiodon elongatus and the rainbou trout, Salmo  gairdneri (Copp et al, 1972a). Calcitonin activity uas not detect able in extracts of the thyroids of catfish, Ictalurus melas (Louu et al, 1967) or of school sharks, Galeorhinus galeus (Louu e_t al, 1969). The amino acid composition of salmon calcitonin uas deter mined in 1969 by O'Dar e_t a_l and the complete amino acid sequence of salmon calcitonin uas determined by Niall e_t a_l in the same year. Synthesis of the salmon calcitonin molecule by Guttmann e_t a_l (1969) made it the first non-mammalian hormone to be fully characterized. The salmon molecule consists of 32 amino acids but it differs con siderably from that Df porcine, bovine and human calcitonins, the four hormones being homologous in only 9 out of 32 positions. Cur iously, the salmon sequence is more homologous to the human structure (16 out of 32 positions are similar) than it is to either the porcine or the bovine sequence (Niall e_t a_l, 1969). The high specific biological activity of pure salmon calci tonin (5000 MRC U/mg, Q'Dor et_ al, 1969b) is coupled uith its more hypocalcaemic and prolonged action in mammals (Brooks e_t a_l, 1969; Copp e_t a_l, 197D; Guttman e_t al, 1970). In the standard rat bio assay,the biological potency of purified salmon calcitonin is at least 20 times greater than porcine, ovine or bovine calcitonin (O'Dor e_t a_l, 1969b; Keutmann et al, 1972). The high activity of the salmon hormone may be due in part, to its greater stability and is characteristic of the three different forms of salmon calcitonin uhich have been isolated (Keutmann e_t a_l, 1972). Thus, the ultimo branchial glands of fish have been shoun to be a rich source •kk of calcitonin and the study of the function of calcitonin in fish must begin uith an investigation of these glands. Uith SD much information knaun about the salmon calcitonin molecule, it seemed reasonable to characterize the UB gland calcitonin concentrations in salmon and trout. The purpose of this chapter is to present measurements of calcitonin in the ultimobranchial glands of trout and salmon in order to determine the relationship of calcitonin to grouth, sexual maturation and osmoregulation. Materials and Methods  Trout The fish uere all held under laboratory conditions (seasonal photoperiod) and uere acclimated to these conditions at least one ueek before the experiment. Except for the fingerling trout uhich uere fed chopped beef liver, all fish uere fed commercial trout pellets and starved one ueek before sacrifice. Samples uere collect over the period from January 16 to September 11, 197D and the uater temperature and physical measurements (General Materials and Methods pg. 21) uere recorded far each fish. Blood uas taken by caudal vein sampling and plasma calcium and inorganic phosphorus (Pi) uere measured for each fish. In the case of the fingerling trout, blood uas collected from the caudal vein directly into heparinized tubes after severance of the caudal peduncle. The ultimobranchial glands, taken in an identical manner from each fish, uere ueighed and immediately frozen on dry ice. The glands uere then stored at -12°C until bioassay (General Materials and Methods, p. 33). Five groups of trout uere sampled: -1*5-a) Fingerling trout b) Adult immature trout c) Adult mature trout d) Smolting trout e) Sea-water acclimated trout. The fingerling trout uere approximately 7 to 8 months old and their sexes uere indistinguishable at this stage. Since only a small amount of plasma uas available, plasma Pi levels of the finger ling trout uere measured using a micro-phosphorus method (Goldenberg and Fernandez, 1966). The other four groups of trout uere approximately 2-3 years old and the sexes of the individual fish uere recorded. The adult immature trout uere normal, sexually-immature fish and were sampled to obtain control levels of ultimobranchial calci tonin concentrations. A group of sexually-maturing trout uere studied, since it uas noted that the ultimobranchial gland became more distinctly outlined at this stage. The glands uere bioassayed for calcitonin content to determine whether there uas increased UB gland activity during spauning. The UB gland also appeared more active in smolting trout. Smolting is a stage in the life history of a trout or salmon char acterized by morphological, physiological, behavioural and hormonal changes uhich prepare the fish for its seauard migration. The final group of samples uere obtained from trout that had been adapted to sea-uater conditions at the Vancouver Public Aquarium. This group of trout were first held for one week in a 55 gallon tank -46-of fresh running uater, gradually adapted to full strength sea uater (salinity, 26.5-28.4 parts per thousand) over a period of 4 days, and maintained in the running sea-uater for 33 days. It uas not possible to obtain the uet weights of their ultimobranchial glands and since these may have dehydrated somewhat during storage at -12°C, the calcitonin activity of the sea-water acclimated trout may only be meaningful when calculated an the basis of Units per kilogram body weight of fish. The glands were weighed prior to each bioassay test. All trout in this group were sexually immature thus eliminating sexual maturation complications. It should be noted that a problem was encountered in segregating immature from mature trout. Part of the difficulty was due to the fact that the trout mature at different rates and times. By nature, rainbow trout spawn in the spring (Leitritz, 1969), but fish inter-breeding practices have developed trout that spawn in spring and fall. An arbitrary decision was made, therefore, to designate trout with gonad weights under l.D g as immature. Salmon Two species of salmon were obtained from the State of Wash ington, U.S.A., through the co-operation of the Washington State De partment of Fisheries. Both species, captured in freshwater, were in peak spawning condition. The chinook salmon (Oncorhynchus tshawytscha) were obtained an October 21, 1970 from the Deschutes River Holding Ponds near Olympia, Washington, U.S.A. These fish were about 1 mile from the sea (Puget Sound) and had been in freshwater for approximately 2 weeks. The normal spawning season for these chinook salmon extends from -47-the end of September to November 10th (CH. Ellis, Chief Hatchery Management, Washington State Department of Fisheries, personal communication). These fish uere hatchery raised and had an average age of 4 years. The coho salmon (Oncorhynchus kisutch) uere taken on Nov ember 30, 1970 from the Samish River Holding Pond approximately 10 miles from the sea (Puget Sound). The journey from sea to river takes the fish about a day and the spawning season extends from the end of October to the middle of November. The coho were, on the average, 3 years old and had been detained in the holding pond for a period of 2 weeks. Both the chinook and coho salmon were captured by hand-net, stunned by a blow on the skull and immediately blood sampled. Blood was collected from the caudal vein as outlined in General Materials and Methods, pg. 17 ). Measurements on each fish included fork length, total fish weight and gonad weight. The female coho salmon were extremely ripe (sexually mature), making it impassible to obtain an accurate gonad weight. The head of the salmon was excised and the ultimobranchial gland dissected and stored on dry ice. The UB gland weights were recorded upon return to the laboratory. In salmon, the ultimobranchial gland is quite diffuse, so the entire transverse septum was uniformly cut out. The glands were stored at -12DC until assayed and all assays were performed within 2 months of the date of collection. The procedure for collection, homogenization and bioassay of the glands is described in detail in General Materials and Methods page 33 . -48-Results Calcitonin activity and other parameters for the 5 groups of trout and 2 salmon groups are summarized in Tables I and II, pages 49 and 50. Plasma calcium and inorganic phosphorus (mEq/1), and calcitonin activity (mil per mg gland; Units per kg fish) are illustrated in histogram form in Figures 3, 4, 5, pages 51, 53 and 54 respectively. Plasma calcium and inorganic phosphorus levels showed a wide variation among the groups. The highest plasma calcium values for the trout uere recorded for the mature females. Smolt plasma calciums were also somewhat elevated over immature adult trout levels. It is interesting to note that the seawater trout plasma calciums were within the normal range, despite the high environmental calcium concentration. The coho salmon ex hibited the highest plasma calcium values of all the groups and the mean plasma calcium level for the chinook females was slightly higher than that of the mature female trout. The high plasma calcium levels in the salmon were probably a reflection of their advanced stage of gonad development. From Figure 3 it can be seen that the females, far each group display higher plasma calciums than the males, although the difference is only significant in the case of the chinook salmon (p<D.D5). The plasma inorganic phosphorus levels were extremely variable. Individual group plasma Pi measurements also displayed a wide range, as evidenced by the size of the SE bars (Figure 3). Table I . Phyalcal Measurements, Plasma Electrolytes and Calcitonin Activities of Rainbow Trout (Mean - SE) Plasma Calcitonin Activity Fish Group Sex n TotBl Cg) Wt GSI Ca mEq/1 Pi mEq/1 UB Gland Fresh Wt (mg) mU/mg Fresh gland U/gland U/kg fish Fingerling Trout 25 Feb./70 5.5°C m&f 8 12.8 + 1.09 - 4.84 + 0.17 7.66 + o.4o 5.96 + 0.80 91.2 + 21.38 0.5 + 0.09 35.5 + 4.35 Adult Imma m 6 196.6 + 10.00 0.19 + 0.05 4.17 + 0.12 5.00 + 0.18 34.96 + 3.04 833.9 + 124.41 29.3 + 4.87 148.1 + 21.99. ture trout 11 Sept./70 14°C f 7 181..5 + 9.20 0.26 + 0.03 4.50 + 0.19 4.68 + 0.18 35. 24 + 3.17 302.la- "42.35 1.92 sa.tfi 9.97 Adult Mature m 7 236.8 + 15.20 1.55 + 0.28 5.00 + 0.1*9 5.13 0.19 38.80 + 3.81 368.7 + 126.36 15.7 + 6.48 62.3 + 22.55 trout 16 Jan./70 6°C f 7 219.1 + 8.30 6.76 + 2.74 5.65 + 0.24 5.10 + 0.39 47.67 + 2.05 565.1 + 173.40 25.9 + 7.40 114.9 + 29.18 Immature m 5 145.2 + 14.82 0.03 + 0.03 5.38 + 0.26 7.16 + 0.62 31.68 + 3.45 474.8 + 186.35 13.1 + 4.43 92.2 + 29.01 Smolt Trout 18 Mar./70 6°C f 4 144.5 + 5.67 0.1<T + 0.01. 5.51 + 0.25 7.63 + 0.68 27.77 + 2.44 691.7 + 199.22 20.2 + 6.84 145.1 + 53.91 Immature m 2 200.0 + 7.00 0.07 + 0.02 4.55 + 0.25 5.75 + 0.29 75.90 ±26.10 190.9 + 28.34 15.3 + 7.14 75.1 + 33.10 Seauater Trout f 5 207. 4 + 11.90 0.32* + 0.05 4.83 + 0.26 6.31 + 0.47 52.66 ±13.36 327.3 + 72.92 13.9 + 0.63 67.4 + 2.20 2 June/70 10.5°C t-teat probability male vs. female. a. p< 0.005 b. p< 0.01 Table II . Physical Measurements, Plasma Electrolytes and Calcitonin Activities of Coho and Chinook Salmon (Mean - SE) Plasma Calcitonin Activity Fish Group Sex n Total Wt (Kg) GSI Ca mEq/l Pi mEq/l UB Gland Fresh Idt (g) mU/mg Fresh gland U/Gland U/Kg Fish Coho Salmon m 7 5.0 - 0.20 4.72 i 0.10 6.69 ±0.23 8.if. i 0.48 1.84 - 0.09 171.3 i 32.67 317.2 i 66.47 62.7 - 12.59 30 Nov./70 4.5°C f 7 4.5 - 0.21 - 6.90 i 0.50 6.7^ i 0.36 1.3ff ± 0.09 166.7 i 45.72 243.1 - 80.17 52.3 i 16.18 Chinook m 8 8.4 i 0.49 4.63 - 0.70 5.21 i 0.14 5.56 i 0.33 2.48 i 0.26 105.7 - 24.80 241.0 i 45.01 28.2 i 4.51 Salman 21 0ct./70 6.D°C f 5 8.1 - 0.1.7 26.74 I 1.43 5.98 i 0.38 7.04bi 0.48 2.47 i 0.17 203.5 i 86.65 482.3 - 195.71 63.6 - 29.81 t-test probability male vs. female a. p< 0.005 b. p<0.05 I m • -51-Trout 8.00 W 6.00 • E 4.00-2.00 • 0.0 Immature both Sexes Salmon c? 9 0*9 o* 9 o* 9 0* 9 6*9 » 8 4 6 3|76 7 6|7 4 5 5|4 4 2 2| 5 5 77166 8 8|5 5 Finger Mature Seowoter Chinook -ling Immature Smolt Coho Plasma Ca i i Inorganic Phosphorus Figure 3. Plasma calcium and inorganic phosphorus levels in trout and salmon -52-In the trout,, the highest plasma Pi values were recorded for the fingerlings and smolts. The coho males displayed the highest plasma Pi levels of the salmon, while the chinook males were the lowest. In contrast to plasma calciums, plasma Pi levels showed no con sistent sex difference. For example, the coho male mean plasma Pi was significantly higher (p<D.D5) than the female level, whereas in the chinook salmon, the female mean plasma Pi was markedly higher (p<D..rj5) than the male level. In all cases except the mature female trout and the female coho, the mean plasma inorganic phosphorus was higher than the mean plasma calcium level. The data as illustrated in Figures k & 5 on pages 53 and 5k shows that the calcitonin activity of the ultimobranchial glands ex hibited an extremely wide range of values. The individual calcitonin activity variation is indicated by the large SE values for each group of fish. The lowest CT activity was found in the fingerling trout (91.2 - 21.38 mU/mg gland).and the highest activity (833.9 - lZk.kl mU/mg gland) in the immature male trout (Figure 4,pg. 53). The sea-water trout glands contained fairly law CT concentrations. This may in part reflect the fact that the glands weighed slightly more than those of the Dther adult trout. However the wide variation of cal citonin activities in the control group of immature adults, makes a valid comparison difficult. Low levels of calcitonin activity were found in the two groups of salmon. Except for the immature and seawater trout, no significant sex difference in the calcitonin levels of the UB glands was observed. Estimated on a U per kg fish basis (Figure 5fpg. 5k) > "the fingerling trout again exhibit the lowest level of calcitonin activity Figure 4. Ultimobranchial trations (mU/mg gland calcitonin concen-gland) in trout and salmon -5k-Rainbow Trout Salmon 3 £ 40 both Sexes I • 8 <f9 7 7 Fingerling Immature Mature cf 9 d 9 5 4 2 5 Smolt Seawater cf c. 7 7 Coho <s 9 8 5 Chinook Figure 5. Ultimobranchial gland calcitonin concentrations (U/kg fish) in trout and salmon -55-amang all the groups of trout. The chinook male salmon also have low levels of calcitonin activity (U per kg fish). The immature male trout calcitonin activity (U per kg fish) is higher than all other groups uith the exception of the mature females, the smolt females and the smolt males. Discussion Although it gives little information concerning secretion rate, measurement of the calcitonin activity of the ultimobranchial gland provides data an the storage of the hormone. Calcitonin content af the UB gland could be expected to be influenced by many factors such as age, diet, sex,species, secretion rate, bane diseases and other hormones. Thus, Robertson (1968a,b) has noted hyperplasia and cellular hypertrophy of the UB gland in hypercalcaemic frogs. He also presented histological evidence that this uas due to increased production and release of calcitonin. In chickens, Belanger (1971) has described the hypertrophy and hyperplasia of the UB gland paren chymal cells in response to hypercalcaemia and the decrease in secretory activity during prolonged hypacalcaemia. Dther uorkers have confirmed the fact that hypercalcaemia in birds stimulates calcitonin release from the ultimobranchial gland (Ziegler et_ al, 1969; Bates et_ a_l, 1969; Copp et al, 1970; Care and Bates, 1972). Some vertebrates such as the goose, possess relatively small stores of calcitonin (2 U per gram fresh gland ueight) and in contrast to the pig and sheep, must rely on increased biosynthesis in order to increase secretion (Care and Bates, 1972). Finally, calcitonin concentrations have been found to be elevated in the peripheral blood and thyroid gland of -56-humans afflicted uith the condition of medullary carcinoma of the thyroid (Clark e_t al, 1969; Deftas and Potts, 1970; Deftos et al 1971a; Deftos e_t a_l, 1971b). Another factor affecting the calcitonin content of the UB gland, is that the hormone may be stored at the tissue level as an inactive precursor and later converted to the active principle only on the appropriate release stimuli. In order to assess measurements of rat thyroid gland cal citonin content in terms of synthesis and release, Gittes et al (1968) have made the following postulates: 1. A net decrease in calcium lowering activity per gland, represents an excess of release over synthesis of cal citonin. 2. A net increase of calcium lowering activity per gland, represents-a greater synthesis than release of calcitonin. These authors found that persistent hypercalcaemia invariably caused a decrease in the calcitonin content of the rat thyroid glands. Further, they attributed an increased calcitonin content in chronic ally hypocalcaemic rats to a continuous synthesis of the hormone in the absence of any release stimulus. Thus, the interpretation of static UB gland calcitonin content measurements is difficult, and experiments designed to investigate this parameter must be rigidly controlled. To be able to compare different sets of results, the tech nique of extraction and measurement of calcitonin activity should be the same. Parsons and Reynolds (1968) point this out in a statement emphasizing, "the necessity for estimates of biological potency of calcitonins [to] be accompanied by a statement of the assay method and of the standard used." -57-Few ujorkers have investigated the UB gland calcitonin can-tent changes during development. On the premise that calcitonin might be more active in the early stages af growth when there is a high bone turnover rate, Dent et_ a_l (1969) measured the UB gland CT content in developing male chickens (Ghostley strain of non-inbred White Leghorn chickens). These authors found that the calcitonin content of the UB gland increased from 83 mU/mg wet wt gland in the 18 day embryo to a maximum of 408 mU/mg wet wt gland in the 3 day old chick. From this stage until 70 weeks of age, despite considerable variation, there appeared to be no major changes associated with age. Calcitonin content, expressed as U per kg body wt, was shown to decrease with increasing age and no difference was noted between the UB gland CT contents of 70 week old males and females. These authors concluded that calcitonin did not play a major role in the development and maintenance of the bony skeleton of chickens. Although the present study investigated only two ages of fish, the fingerling trout (age 7-8 months) showed considerably lower values of calcitonin content than any of the other 4 groups of trout (age 2-3 years). The calcitonin content (expressed as mU per mg -wet wt gland and U per kg body wt) of the salmon (age 3-5 years) was also very low. However this may be due to the large size of the salmon, the spawning condition, species differences or the method of dissection of the UB gland (see Methods). In contrast to Dent e_t a_l (1969), Wittermgnn e_t al_ (1969) showed that the calcitonin content of the UB glands of 3 week, 3 month and 9 month old female chickens (white Plymouth Rock strain) did not change with age (mU per mg wet wt gland). Calculated as U per kg body wt however, the CT content of the 3 week old chickens -58-uas 50 percent of that found in the 3 month and 9 month old chickens. These conflicting results could be explained by nutrition, sex or species differences. Although it may be superfluous to compare the calcitonin content of fish UB gland uith rat thyroid gland, it has been shoun that 5 and 15 day old rats have significantly louer thyroidal cal citonin contents (mU per mg fresh thyroid) than older age groups (Frankel and Yasumura, 1970). These authors also noted that there uas no significant difference betueen the thyroid gland calcitonin contents in male and female rats of the same age group. They postu lated that the lou levels for immature rats could be attributed to either a lou rate of biosynthesis or a high rate of CT secretion. This explanation could also account for the lou levels of UB gland calcitonin contents of fingerling trout found in the present study. Certainly, the plasma calcium level (the signal for calcitonin re lease in mammals) of the fingerling trout, is not excessively high. The higher plasma calciums in the mature versus immature adult trout, is due to sexual maturation and has been noted in other fish by many others (Miescher, 1897; Hess et_ al, 1928; Pora, 1935, 1936; Booke, 1964; Oguri and Takada, 1967; Urist and Van de Putte, 1967 and Woodhead, 1968). Although the plasma calcium levels in the present study are higher in the mature trout, the UB gland CT content is louer in the mature versus the immature male trout and higher in the mature versus the immature female trout. It is possible that the arbitrary division of the trout into immature and mature groups, could account for the variability of the data. Smolting salmonids are characterized by their silver color ation (Hitching and Falco, 1944) an alteration of body proportions -59-(Hoar, 1939) and the development Df a salinity preference (Bagger-man, I960). The smelting process is also known to be accompanied by an increase in the adrenocortical volume (Qlivereau, 1962) and an elevation of 17-hydroxycorticosteroid plasma levels (Fontaine and Hatey, 1954). Glomerular filtration and urine flow have also been shown to decrease considerably in smolting steelhead trout (Holmes and Stainer, 1966). Thus it can be seen that the physio logical, morphological and behavioural changes occurring in smolting salmonids are extremely complex and appear to prepare the fish for its seaward migration (Hoar, 1951). In the present study, the smolt plasma calcium levels were elevated over those of the immature trout yet were not significantly different from the mature trout levels. Plasma inorganic phosphorus levels were extremely high in both male and female smolts. The smolt ing trout as a group appear to have high UB gland calcitonin contents. These high values are not correlated with either the plasma calcium or Pi levels. The seawater acclimated trout show plasma calcium levels only slightly elevated over those of the immature control trout, while plasma Pi levels were markedly higher. As a group, the sea-water trout have the lowest UB gland CT contents of the adult trout. The low calcitonin contents observed in the seawater trout may re flect the fact that the wet weights of their UB glands were higher than those of the other trout (Table I , pg. 49). However, expressed on a U per kg body weight basis, the seawater trout UB gland CT contents still do not exceed the values for the mature and smolting trout. This finding is quite interesting, in view of the fact that -60-the environmental calcium concentration of the seauater (15 mEq/ litre) uas considerably higher than the freshuater calcium concen tration (less than 0.5 mEq/litre) of the other groups of trout. These results are in marked contrast to those of Orimo et_ a_l (1972a) uho found that UB gland calcitonin contents (U/gland) Df eels, Anguilla japonica, kept in seauater, uere significantly greater than the freshuater controls. This finding uas paralleled by increased plasma calcitonin and serum calcium levels in the seauater eels. Houever, the fact that the eel is a catadromous fish (living in freshuater, spauning in seauater) and the rainbou trout is a euryhaline (living and spauning in freshuater) may account for these contrasting results. It should also be noted that Orimo et_ a_l (1972a) did not report the age and stage of sexual maturation of the eels. The present study is supported by the uork of Pang (1971b) uho found that the ultimobranchial glands of the killifish (Fundulus  heteroclitus) uere more active (histological evidence) in freshuater than in seauater. He also noted that the ultimobranchial body activity uas independent of serum calcium levels, and postulated that the function of the gland might be related to osmoregulation rather than calcium metabolism. The UB gland calcitonin content (mU/mg gland) of the coho salmon uas very similar to the chinook and no sex difference uas detected. The slightly louer values compared to the trout could possibly be due to the fact that the salmon ultimobranchial gland is more diffuse and hence a larger area of salmon transverse septal tissue uas dissected out. This inclusion of excess tissue uould therefore, louer the calcitonin activity when calculated as mU per mg gland. Expressed on a U per kg basis, the salmon UB gland CT -61-contents uere higher (except for the male chinoaks) and approximately the same as the immature female control trout. It is interesting to note that the salmon, which uere a different species, migrating, fasting and extremely sexually mature (note the salmon GSI, Table II, pg. 50) displayed UB gland calcitonin contents that were not very different from those found in the trout. It may be relevant to mention that Keutmann e_fc a_l (1972) have isolated and characterized 3 farms or components of salmon calcitonin. The 3 components, designated calcitonin I, II, III, have been isolated from k species of salmon and their distribution is shown in Table III. Table III. Distribution of Calcitonins Among Salman Species.* Species Calcitonin Component I II III Sockeye (0. nerka) +++ + Chum (0. keta) +++ + Pink (•. qorbuscha) +++ + Coho (0. kisutch) +++ + Data from Keutmann e_t al_ (1972) The specific biological activities of the 3 salmon calcitonin components is compared to the mammalian calcitonins in Table IV, pg. 62. -62-Table IV. Specific Biological Activities of Calcitonins from Various Species.* Preparation Mean Specific Activity MRC Units/mg** Porcine 120 Bovine 60 •vine 70 Human 70 Salman I 2,700 Salman II 2,400 Salman III 600 Data from Keutmann e_t a_l (1972) * All assays carried out on lyophilized preparations of pure hormones using the method of Parsons and Reynolds (1968). It can be seen that only the coho salmon UB gland contains component III which has the lowest specific biological activity (600 U/mg) of the 3 salmon components. Although the salmon were not segregated by sex and trout and chinook salmon have not been examined, these findings have profound implications. As in the case of the trout, the plasma calcium and inorganic phosphorus levels of the salmon appear to bear no consistent re lationship to their UB gland calcitonin contents. Data in the present study confirm the original observations reported by Copp e_t a_l (1967a) that the fish ultimobranchial gland is a rich source of calcitonin. In fact, the fish UB gland cal citonin contents reported in this thesis (range 90 - 830 mU/mg gland, -63-Kumar assay, MRC B Std) are very similar to the values Found in chickens (range 83 - 408 mU/mg gland, Cooper assay, MRC B Std) by Dent et al (1969). The higher values oF UB gland calcitonin contents (mU/mg Fresh gland) For trout and salmon in this study than obtained by Copp et_ a_l (1968b) For chum salmon, grey cod, and dogFish, may be due to species diFFerences, dissection technique, and/or extraction and assay methods. It mould appear that Orimo et_ a_l (1972a,b), cannot claim to have the highest calcitonin activity per kg body weight (Anguilla  japonica, 40 U per kg body weight) since the majority oF the trout and salmon groups in the present study greatly exceeded this value (Figure 5 , pg. 54). The UB gland calitonin content in the adult trout (range 10.7 - 25.9 U/gland) was also signiFicantly higher than those oF the seawater adapted eels (4.3 U/gland) reported by Orimo et al (1972a). The diFFerence may lie in the Fact that Orimo used the Cooper assay while the trout glands were measured by the Kumar assay. It should be noted that Orimo did not report the MRC standard used to evaluate the assay data. In summary, the low UB gland calcitonin contents Found in Fingerling trout, as compared to the adults, may indicate a relation ship between calcitonin and age. The study conFirmed the Fact that the Fish UB gland contains large quantities oF calcitonin. IMo con sistent correlation oF the UB gland calcitonin contents with sex, sexual maturation, smolting, changes in environmental calcium levels or species diFFerences was Found. The wide range oF calcitonin contents Found in birds and mammals (Dent et_ al, 1969; Frankel and -6k-Yasumura, 1970; Copp et_ al_, 1972a), uas also observed in this study on fish. This data therefore, does not provide a firm basis an uhich to outline the physiological role of calcitonin in fish. -65-II. BIOLOGICAL HALF-LIFE DF SALMON CALCITONIN IN TROUT AND SALMON Introduction The Endogenous circulating plasma level of calcitonin depends on the secretion rate from the ultimobranchial gland and the clearance rate from the plasma. In the first chapter, it was demonstrated that the ultimobranchial gland of trout and salmon contains high concentrations of calcitonin and in succeeding chapters, evidence will be given that these fish maintain high circulating plasma levels of calcitonin as well.* A knowledge of the disappearance of the hormone ir_ vivo might explain the high circulating levels and give information on the normal secretion rate of calcitonin. The more powerful and prolonged hypocalcaemic effect of salmon calcitonin (SCT) in mammals has led some workers to inves tigate its biological half-life (T1/2) in mammals (Habener e_t a_l, 1971a,b; 1972a,b; Newsome e_t a_l, 1973). The rate of disappear ance from plasma may account for the rapid response of the animal to calcitonin injection (Copp et a_l, 1968a; Sturtridge and Kumar, 1968; Mills e_t a_l, 1972) and the prompt release of calcitonin in response to hypercalcaemic challenge (Lee et a_l, 1969; Gray and Munson, 1969; Arnaud et al, 197D; Cooper e_t al, 1971; Care and Bates, 1972). The purpose of experiments in this chapter was to determine the biological half-life of salmon calcitonin (a "fish" calcitonin) in trout and salmon. -66-Materials and Methods The biological half-life of salmon calcitonin in trout and salmon uas measured using a modification of the bioassay method of Kumar et_ a_l, 1965 as outlined in General Materials and Methods. This technique uas employed since it avoided the radiation damage and non-specific redistribution Df radioactive label in plasma caused by using labelled hormone. A disadvantage of the bioassay uas that it required rather large blood samples (1.0 ml) and hence made serial sampling Dn small fish difficult. Trout The calcitonin biological half-life experiment uas per formed in the Vancouver Public Aquarium research facilities on May 13, 1970. Eight rainbou trout uere cannulated, placed in darkened bYz gallon aquaria, in running uater (T=8aC) and alloued to recover for 24 hours. The fish had been starved for 5 days previous to the experiment. Purified salmon calcitonin (UBC 5, k.5 U/mg) at a dose of 3.78 Units per 0.25 ml (vehicle 1.0% sodium acetate, 0.1% glycine) uas injected intravenously into each fish at time zero. Davis (1970) has shoun that the circulation time in trout uas 64.1 - 16.4 seconds, therefore injection uas carried out over 30 seconds to facilitate adequate mixing of the hormone in the blood. A further 5 minutes uas alloued before the initial sample to ensure homo geneous distribution of the hormone in the circulation. Bleeding times uere 5, 30, and 65 minutes from injection for the first 2 fish and 5, 30 and 90 minutes far the last 5 fish. One ml of blood -67-uas obtained at each sample point, and centrifuged immediately for 2 minutes. Plasma samples were frozen an dry ice directly following separation and stored at -12°C. The bioassays were completed within 3 weeks of collection. Plasma calcium, hematocrit and percent water were measured for each sample to determine the effect of blood sampling on these parameters. The fish were sacrificed on conclusion of the experiment and all physical measure ments recorded. Salmon Two male sockeye salmon (Great Central Lake race) were cannulated, placed in 50. gallon fibreglass tanks of running water (T=8DC) and allowed to recover for 2k hours. The sockeye were not fed in the laboratory. Purified salmon calcitonin (37.6 U in 0.55 ml vehicle per fish) was injected intravenously at minus 10 minutes. This ten minute interval permitted even distribution of the hormone in the circulation. Four blood samples of 3.0 ml each, were collected at 0, 22, 50 and 79 minutes for fish H and 0, 27, 55 and 91 minutes for fish R. Plasma was separated and stared as previously described. In order to maintain the hematocrit, the red blood cells were re-suspended in the appropriate volume of heparinized Cortland saline and returned to the fish. Plasma sodium, potassium and magnesium, as well as the percent water and haematocrit, were de termined for each sample. Results were plotted directly onto semi-logarithm paper with the plasma calcitonin concentration (mU/ml plasma) on the ordinate and the sample time Dn the abscissa. Results Trout Physical measurements of the trout are presented in Table V, pg. 69. From this data, it is noted that all of the fish uere large, sexually immature trout uith the passible exception of trout C, a male in the early stages of sexual maturity. Since the trout uere quite different in size and the same dose of calcitonin uas given to each, initial plasma calcitonin levels displayed a uide variation (Table Ul, pg. 70). Figure 6, pg. 71, shous the graph of the individual calcitonin disappearance curves used to calculate the half-lives. The mean biological half-life of salmon calcitonin in the trout uas estimated to be 27.6 - 2.90 minutes. The mean percent calcitonin activity remaining uith time uas calculated for each sample, assuming the plasma CT level at 5 min. to be 100 percent (Table VII, pg. 72). Table VIII, pg. 73, shous the plasma calcium, percent uater and haematocrit of each fish at each sample point. The zero sample uas a 0.2 ml blood specimen taken immediately prior to calcitonin injection. -69-Table V. Trout Physical Measurements Fish # Sex Total Wt Fork Length Gonad Wt GSI (g) (cm) (g) 8 f 248.2 28.• 1.1 0.44 C m 215.5 25.5 3.4 1.58 D f 255.0 30.0 0.3 0.12 E f 261.0 30.0 0.7 0.27 F m 260.0 29.0 0.1 0.04 G f 236.0 29.0 1.3 0.55 H m 263.0 29.0 0.1 0.04 n = 7 Mean = 248.4 SD = 15.97 SE = 6.52 7 7 7 28.6 1.0 0.43 1.43 1.07 0.50 0.58 0.43 0.21 -70-Table VI. Plasma Calcitonin Levels and Biological Half-Lives of Salmon Calcitonin in Trout Plasma Calcitonin mU/ml Half-Life Fish # • min. 5 min. 30 min. 65 min. 90 min. Min. B Calcitonin 120.96 60.48 21.17 30.0 C Injection 163.30 99.79 43.85 38.3 D 71.82 42.34 11.34 37.0 E 187.49 77.11 15.12 24.3 F 120.96 45.36 19.66 23.0 G 104.33 38.56 9.98 22.0 H f 102.82 29.48 9.45 18.5 n = 7 7 2 5 7 Mean = 124.53 56.16 32.51 13.11 27.58 SD = 36.15 22.96 11.34 3.83 7.11 SE = 14.76 9.37 11.34 1.91 2.90 Figure 6. Biological half-life of salmon calcitonin in rainbow trout -72-Table VII. Percent Calcitonin Activity Remaining uith Time Sample Time Fish # 5 min. 30 min. 65 min. 90 min. B 100% 50.0% 17.5% C 100 61.2 26.8 D 100 59.0 - 15.8% E 100 51.0 - 8.1 F 100 37.6 - 16.3 G 100 37.0 - 9.8 H 100 28.6 r- 9.2 n = 7 7 2 5 Mean = 100.0 46.3 22.2 11.9 SD = 11.30 4.64 3.48 SE = 4.61 4.64 1.74 -73-Table VIII. Plasma Calcium, Percent Water and Haematocrit Changes in Traut Sample Time Pre-Injection Fish Measurement sample # 0 min. 5 min. 30 min. 65 min. 90 min. B Plasma-Ca mEq/1 4.45 4.60 4.30 5.00 % Water 94.9 95.6 95.7 96.0 Haematccrit 22 26 16' 14 C Plasma-Ca mEq/1 4.90 4.70 4.60 5.40 % Water 93.8 94.2 94.6 95.0 Haematocrit 33 33 26 27 D Plasma-Ca mEq/1 4.75 4.70 4.65 - 4.45 % Water 94.7 - 94.9 - 95.4 Haematocrit 32 34 35 - 20 E Plasma-ca mEq/1 4.75 4.50 4.70 - 4.55 % Water 95.3 95.6 95.5 - 95.9 Haematocrit 26 25 28 - 17 F Plasma-Ca mEq/1 4.70 4.65 4.75 - 4.60 % Water 94.9 95.4 95.2 - 95.5 Haematocrit 28 27 26 - 21 G Plasma-Ca mEq/1 4.65 4.60 4.50 - 4.80 % Water 94.5 94.9 94.9 - 95.5 Haematocrit 24 26 22 - 18 H Plasma-Ca mEq/1 4.50 4.45 4.35 - 4.30 % Water 95.2 95.7 95.7 - 96.1 Haematocrit 26 27 28 - 19 T-test Comparison of 0 min. versus 65 and 90 min. Samples T degrees of freedom p_ Ca mEq/1 0.435 6 NSD % Water 9.66 6 p < .001 Haematocrit 11.1  p< .001 _74-Salmon The physical measurements of the tuo male sockeye salmon are presented in Table IX. Table IX. Salmon Physical Measurements Fish Total Wt Fork Length Gonad Wt GSI # (g) (cm) (g) H 2028 63.5 58 2.86 R 2800 66.0 73 2.61 The sampling intervals and the plasma measurements at each point are shoun in Table X, pg. 75. Figure 7, pg. 76, is a graph illustrating the disappearance curves of CT in the tuo salmon. The biological half-life of salmon calcitonin in the male sockeye salmon uas 46 minutes for salmon H and 50 minutes for salmon R. No significant change in plasma levels of sodium, potassium, or magnesium uere detected due to the injection (Table X). In order to compare the disappearance curves in trout and salmon, the percent of calcitonin activity remaining uas platted against time (Figure 8, pg. 77). The trout appeared to have a slightly faster initial disappearance time than the salmon. Table X. Plasma Measurements in Tuo Male Sockeye Salmon 1 # Sample Time (min) Calcitonin mU/ml plasma Percent Calcitonin Remaining Plasma Percent Water Hct l\)a+ Plasma mEq/l Ions Mg H • 982.8'. 100.00% 94.6 11 153 2.8 1.30 22 899.6 91.53 94.8 11 145 2.5 1.39 50 418.8 42.61 94.9 11 148 2.7 1.47 79 79.4 8.07 95.1 10 153 . 2.8 1.23 R • 831.2 100.00 93.4 28 145 2.6 1.66 27 717.8 86.35 93.6 26 145 2.7 1.39 55 320.9 38.60 93.6 26 147 2.7 1.56 91 86.9 10.45 93.8 26 146 2.8 1.50 Figure 7. Biological half-life of salmon calcitonin in tuo male sockeye salmon T 1 100 Time in Minutes Figure 8. Disappearance of salmon calcitonin in trout and salmon -78-Discussion Polypeptide hormones are known to be rapidly cleared from the blood after intravenous injection into mammals. The half-lives of these hormones are in the order of minutes, for example; T}£ = 8.1 minutes for gastrin in humans (Ganguli et_ a_l, 1971), and Vk = 20 minutes for parathyroid hormone in the cou (Sherwood e_t al, 1968). The biological half-life of any hormone is influenced by many factors, among which are: a) the level of circulating endogenous hormone already present, b) the secretion rate of endogenous hormone, c) the degree of binding of the hormone to plasma proteins, d) the binding of the hormone to receptor sites in the target and other organs, e) the destruction of the hormone in the target and other tissues, f) the inactivation of the hormone by plasma enzymes, g) the renal excretion of the hormone, h) the age, sex, physiological condition and species of the animal used in the test. Lee e_t al (1969) showed there was a rapid turnover of endogenous calcitonin in the rabbit and that the half-life for porcine calcitonin (PCT) in this mammal fallowed first order kinetics. The disappearance of PCT in the pig was shown to follow two expon ential components, the first component had a T1/z = 4-5 minutes and the second component had a T# = 35-4Q minutes (west e_t al_, 1969). -79-The division of the disappearance curve into tuo components has been shown by other workers, the first steep segment representing the distribution and mixing of the hormone in the fluid compart ments and the second, less steep segment the actual rate of in-activation of the hormone (Idest ejt a_l, 1969). Foster e_t a_l (1972a) reported that the initial disappear ance of human calcitonin in the dog (T}& = 3 minutes) was largely due to kidney inactivation. This was demonstrated by measuring arterio-venous differences in plasma CT concentration across both the liver and the kidney. Blood samples during a calcitonin infusion, were collected simultaneously from indwelling catheters in the aorta, hepatic and renal veins of anesthetized dogs and the CT concen tration measured by radioimmunoassay. The liver appeared tD remove calcitonin from the circulation only at levels above 90 ng per ml, while the kidneys consistently removed 30% of the arterial level of calcitonin. On removal of the kidneys, the first rapid component of the disappearance curve was abolished and higher levels of plasma CT were measured. The slower' disappearance of salmon calcitonin in nephrectomized versus normal rats, was also shown by Mewsome e_t a_l (1973). Hepatectomy, in one dog, did not affect the dis appearance curve and Foster e_t a_l (1972a) concluded that the liver plays an insignificant role in the inactivation of human calcitonin in the dog. Since only 0.3% of an infused dose of calcitonin was detected in the urine, they reasoned that the rapid phase of the disappearance curve was due to renal uptake and/or destruction and not due to renal excretion. The role of renal excretion in de termining the half-life disappearance curve was also considered -aa-unimpartant by Habener et al (1972a), who found that the metabolic clearance of porcine calcitonin greatly exceeded the GFR in the dog. The binding of the hormone to plasma proteins, besides providing protection from plasma enzymes, uould also preserve it from renal excretion. Foster e_t al_ (1972a) also found that the slow component of the disappearance curve of human calcitonin in the dog had a T/2 = kO minutes and they postulated that it uas due primarily to protein binding since it did not change uith nephrectomy or hepatectomy. This vieu has been supported by Habener et_ al_ (1971a,b; 1972a) uho demonstrated using gel filtration, that the slou component of the disappearance curve uas due to protein-bound calcitonin, uhereas the free calcitonin disappeared rapidly. 125 Injection of I porcine calcitonin into rats shoued that the major site of accumulation of radioactivity uas the liver (de Luise e_t a_, 1970) and these authors concluded that the liver played a role in the early phase of the disappearance curve. Since the accumulation of labelled CT in the liver could be prevented by simultaneous injection of unlabelled CT and since the authors kneu of no knoun effect of calcitonin on the liver, they postulated that the hepatic.uptake may be related to hormone catabolism. It is interesting to note that at ID minutes, 13.9% of the injected 125 dose of I PCT uas found in the liver, 2.6% in the kidney, 13.0% in the skeletal muscle, h.h% in the bone and 6.5% in the blood. Mare recent uork by de Luise et_ a_l (1972) has indicated 125 that, uhereas, an injection of I PCT accumulated mainly in the liver of the rat, both human and salmon calcitonin uere primarily taken up by the kidney. Salmon calcitonin resisted enzymatic -81-breakdown by homogenates of all rat tissues except the kidney. Salmon calcitonin and human calcitonin have also proven to be very stable in ir_ vitro studies. The incubation of SCT in rat plasma at 37°C showed a JVz of approximately 6 hours, whereas incubation of SCT in salmon plasma showed a JVz of 15 hours (O'Dor et al, 1971). Habener et al (1972b) also found that over 30% of initial SCT activity remained after 24 hours Df incubation at 25°C in salmon and human plasmas. In contrast, the porcine, bovine, and ovine calcitonins are much more rapidly inactivated than either the salmon or human calcitonins (TY2 less than 3 hours). In dog plasma at 37°C, PCT showed a JVz of 96 minutes whereas SCT remained stable for Dver 48 hours (Habener et_ al, 1971b). Thus salmon and human calcitonin appear to resist enzymatic inactiv ation j_n vitro more successfully than the other mammalian calcitonins. Further work demonstrating the superior stability of SCT has come from in vivo experiments as well. Habener e_t a_l (1971a), using specific radioimmunoassays in the dog, demonstrated fast and slow components of porcine and salmon calcitonin (PCT 2.5 and 80 min.; SCT 20 and 80 min.). Salman calcitonin, as measured by bioassay, was shown to have an initial half-life af ID - 15 minutes in normal rats, whereas porcine calcitonin showed a very rapid initial disappearance of 2 minutes (IMewsome e_t a_l, 1973). The long half-life of SCT, both in_ vivo and ir_ vitro, may explain the greater biological activity of salmon calcitonin (Habener e_t a_l, 1972a, b). The stability of the salmon hormone is undoubtedly a reflection of some peculiarity of its structure. Comparison of the results of in vitro versus in vivo experiments, -82-would seem to indicate that enzymatic inactivation plays a minor role in the disappearance of calcitonin. The half-life for salmon calcitonin of 27.6 - 2.30 minutes in trout and 48.0 minutes in salmon, indicates that the salmon hormone has a relatively long half-life in fish. Although single injections of hormones do not equilibrate evenly in the fluid com partments of the body and tend to distort the disappearance curves, these results are in substantial agreement uith the half-times found for SCT in mammals. The fact that the second sample uas taken at 30 minutes makes it passible that these results reflect the measurement of the second, slouer component of the disappearance curve. Even if these findings represent the first, rapid component of the curve, the half-life of SCT in fish is still slower than the initial half-life of SCT in the dog (20 min.) found by Habener et al (1971a). These measurements agree quite well with those of Bass (1970) who demonstrated that the disappearance curve of synthetic SCT in rainbow trout showed two components (Tate = 12.5 min. and Tb)& = 59 min.), as measured by radioimmunoassay. Caution must be exercised when comparing the results of bioassay and radioimmunoassay, since loss of immunological activity'may not coincide with loss of biological activity (Lequin e_t a_l, 1969; Cooper et al, 1971). The shape of the disappearance curve for SCT in the salmon is interesting and may reflect the fact that a longer interval was left following injection of the hormone (mixing time: salmon 10 min., trout 5 min.) before collection of the first sample. Con sequently, there may have been a more homogeneous concentration -83-of the hormone in the fluid compartments than uas observed in the trout. The longer half-life of SCT in the salmon compared to the trout, could be due to many factors. Since the salmon uere in the spauning condition, the level of plasma binding proteins may have been higher than in the trout. This increased protein binding of the hormone in the plasma uould prolong its half-life. In the sexually mature skate, Raja radiata, Fletcher et_ a_l (1969) have shoun that the sex hormone-binding-protein binds the approp riate steroids quite strongly. They demonstrated that the meta bolic clearance rates (MCR) of testosterone from skate plasma uere considerably louer than those reported for humans. Furthermore, the metabolic clearance rates for the females uere consistently higher than far the males. On the other hand, a significant increase in the Cortisol MCR uas observed in the sexual maturation of the sockeye salmon (Donaldson and Fagerlund, 1968, 1970, 1972). This greater MCR uas correlated uith an increased apparent volume of Cortisol dis tribution. Thus, they concluded that the elevated Cortisol levels in maturing and spauning salmon uere not due to a lou MCR but to a rise in Cortisol secretion. The half-life of Cortisol, houever, did increase during maturation and spauning. It appears that the situation in the spauning salmon is quite complex and experiments on the MCR and secretion rate of calcitonin in immature-and mature salmon may help to explain the prolonged half-life in this fish. The fact that fish are poiki-lothermic animals means that they have a louer basal metabolic rate -84-th an mammals, and this undoubtedly contributes to a more prolonged clearance rate. Nothing has been done on the distribution of labelled SCT in fish so the role played by the kidney, liver and other organs in the removal and inactivation of circulating calcitonin is not known. It may be significant to note that although Habener e_t al_ (1971b) claim that the prolonged half-life of salmon calcitonin may explain its increased potency in mammals, the long half-life of SCT in the fish was not accompanied by a hypocalcaemic effect. It would be informative from a structure-function-stability point of view to examine the plasma electrolyte effects and biological half-lives of the mammalian calcitonins in fish. -85-III. PLASMA AND RENAL EFFECTS DF SALMON CALCITONIN Introduction In mammals, calcitonin has been shown to exert a rapid hypocalcaemic and hypophosphatemic response. The evidence is con clusive, from both in_ vitro and in_ vivo studies that the primary target organ for calcitonin is bone. The reduction of plasma calcium and phosphate is achieved through an inhibition of bone resorption (Copp, 1969a, b; Copp, 1970a; Behrens and Grinnan, 1969; Rasmussen and Pechet, 1970. A more marked hypocalcaemic re sponse has been demonstrated in young developing animals (Copp and Kuczerpa, 1967; Phillippo and Hinde, 1968; Sturtridge and Kumar, 1968; Sorenson e_t al, 1970) and this effect is likely due to the higher rate of bone turnover associated uith periods of rapid growth (Frankel and Yasumura, 1970; Copp, 1970a). Further evidence to support the fact that the hypocalcaemic action of calcitonin is mediated by its effect on bone, came from experi ments which demonstrated that this action could be produced in nephrectomized and eviscerated rats (Webster and Frazer, 1967; Copp, 1970a). Salmon calcitonin, the first non-mammalian calcitonin to be characterized, has been shown to possess an extremely high specific biological activity (0'Dor et_ al_, 1969a; 0'Dor et_ al_, 1969b; Keutmann e_t a_l, 1970; Keutmann e_t a_l, 1972) and to exert an extremely potent and long-lasting hypocalcaemic effect in a variety of mammals (Copp et al, 1970; Brooks et al, 1969; Singer et al, 1970; Galante et al, -86-1971; Barlet et al, 1971; Bar let, 1972). Minkin e_t al (1971) have shown salmon calcitonin (k mU/ml) to be more effective than greater concentrations of mammalian calcitonins in preventing calcium release from newborn mouse calvaria. Calcitonin has been shown to exert a variable effect on renal electrolyte excretion in mammals (Hirsch and Munson, 1969; Copp, 1970a; Foster e_t a_l, 1972b). In general, porcine calcitonin in the rat increases the excretion of phosphate, calcium, sodium and potassium and decreases the excretion of magnesium. Salman calcitonin, in addition to causing hypercalciuria (large doses), hyperphasphaturia and marked hypomagnesuria in rats, has been shown to be one of the most patent natriuretic agents known (Aldred e_t a_l, 1970; Keeler e_t al, 1970). These results have recently been confirmed using synthetic salmon calcitonin in the rat (Williams e_t a_l, 1972). Long term (96 hour) infusion of synthetic SCT into male lambs resulted in signifi cant increases in urinary excretion of calcium, inorganic phosphorus and sodium and a marked depression of Mg+* excretion (Barlet,1972)» Up until 1968, only three workers had reported on the effect of injection of mammalian calcitonin into fish with inconsistent results (Pang and Pickford, 1967; Louw e_t a_l, 1967; Chan e_t al, 1968). Following the discovery Df the ultimobranchial origin of calcitonin in 1967 (Copp et a_l, 1967a; Copp e_t a_l, 1968b), salmon calcitonin became available in purified farm. Since this hormone had not been tested in fish, a study of the plasma and renal electrolyte effects of salmon calcitonin in rainbow trout and salmon was performed. -87-Materials and Methods  Trout Purified salmon calcitonin uas injected into 2 groups of trout, fingerlings and cannulated adult trout. In all experiments, calcitonin uias weighed out the day of the experiment and the activity confirmed by bioassay. (i) Fingerling trout Since calcitonin had been shown to be more effective in young mammals, fingerling trout (age 7-8 months) were used to determine the effect of salmon calcitonin an plasma calcium and inorganic phosphorus levels. A group of 150 fingerling trout were tagged behind the dorsal fin with a small length of coloured thread and randomly divided into three groups Df 50 fish. All fish were weighed (in a beaker of water) and measured during the tagging procedure, taking care to return them to the water as quickly as possible. A 3-week period prior to the experiment was then allowed for recovery and for acclimation to laboratory conditions. Food consisted of daily rations of finely-chopped beef liver and fish that were not actively feeding were re moved. Before the experiment, the fish were starved for 2 days. The experimental procedure was as follows: Salmon calcitonin (62.5 mU in 0.1 ml vehicle per fish) was injected intraperitoneally (gills immersed under water) into the fish in Group I at time zero. Group II received vehicle alone (0.1 ml of vehicle, 1.0 percent sodium acetate + 0.1 percent glycine, pH = 4.3). Group III, the control group, was not injected. Samples were taken at -88-intervals of lYz, 3Vz, Hz and 2bVz hours after injection. The fish uere caught by dip net and quickly dried uith tissue paper. Blood samples (D.5 ml per fish) uere collected from the caudal vein directly into heparinized capillary tubes, after severance of the caudal peduncle. Using this technique, blood could be collected in 20 seconds uithout anesthetic. From 5 to 10 fish uere sampled and ueighed at each time period. Plasma calcium uas measured fluorometrically (IMeusome, 1969) and plasma inorganic phosphorus uas determined colorimetrically using the micro-method of Goldenberg and Fernandez (1966). (ii) Cannulated trout Tuenty-four adult immature trout (mean total ut = 205 - 7.3 g) uere divided into k groups of 6 fish each. Each trout uas cannulated and held separately in 51£ gallon darkened aquaria (uater temperature 8°C) as outlined in General Materials and Methods (pg. 12). The trout uere starved 2 ueeks prior to the experiment. The four groups of trout consisted of a control group (no injection), a vehicle group (0.1 ml vehicle per 100 gT ) and tuo calcitonin groups (CT^ = 125 mU per 0.1 ml vehicle per 100 g fish; CT^ = 500 mU per 0.1 ml vehicle per 100 g fish). Injection and sampling procedures have been previously described (General Materials and Methods, pg. 13 ). Three control blood samples (0.15 ml each) uere collected at -2, -1 and 0 hours. The trout uere injected intra venously at time zero and post-injection samples taken at +1, +3, +5 and + 2k hours. Haematocrit, percent uater and plasma calcium uere measured at each sample point. On completion of the experiment, -89-th e fish uere sacrificed and physical measurements recorded. Salmon The effect of salmon calcitonin infusion on plasma electro lyte levels and urine electrolyte excretion was tested in sockeye salmon. Techniques used for cannulation and catheterization were outlined in General Materials and Methods, pg. 12 - 21. The urine box used to restrain the salmon in these experiments is illus trated in Figure 2, pg. 22. Three sockeye salmon (Great Central race) were cannulated, their urinary bladders were catheterized and they were allowed to recover for 2k hours before the experiment. All 3 fish were sexually-ripe females. The experimental procedure was as follows. Salmon calcitonin (2.0 Units/100 g fish in 1.0 ml) was infused (Harvard Apparatus, Infusion Withdrawal Pump) into the 3 fish for a period of 30 min. The infusate was kept cool by surrounding the syringe with a plastic bag filled with ice. Two pre-injection blood samples (1.2 ml) were taken and post-injection samples were collected at lYz, 3Yz and 5Vz hours. On the following day, vehicle (vol. = 1.0 ml) was infused for the same time period (30 minutes) and blood samples collected at the same time intervals. Hourly urine samples were collected by fraction collector in preweighed 15 ml tubes and the urine volume (by weight) recorded. The samples were immediately frozen on dry ice and stored at -12DC. Haematocrit, percent water, and plasma electrolytes were measured for each fish. Urinary calcium, magnesium, phosphorus, potassium, and sodium were also determined. On termination of the -90-experiment, the fish mere sacrificed and physical measure ments recorded. The experiments uere conducted December 5 - 18, 1970 and the water temperature during this period ranged from 6.0 - 6.5DC. -91-Resulta Trout (i) Fingerling trout Results of intraperitoneal injection of salmon calcitonin into fingerling trout are presented in Table XI,pg. 92 . Graphs illustrating the plasma calcium and inorganic phosphorus changes are found on Figure 9 pg. 93,and Figure 10,pg.94 respectively. It is apparent from the data that plasma electrolyte levels displayed a wide range of values. In fact, although the plasma calcium and Pi levels of the vehicle group did not differ signifi cantly from the calcitonin-injected group at any of the sample times, the control plasma calcium and Pi levels were elevated well above the other two groups at 3Yz and 7]k hours. . Control plasma electrolyte levels differed significantly from those of the vehicle group (Ca 3)4 hr. p< 0.025; Pi 3# hr. p< 0.001 and Tk hr. p< 0.D05). By 25}£ hr. the control levels had returned to normal. A comparison of the vehicle versus calcitonin-injected groups does not reveal any significant hypocalcaemic or hypophosphatemic effect of calcitonin. (ii) Cannulated trout Physical measurements and individual plasma calcium values of the h groups of trout are tabulated in Table XII,pg. 96. Mean total body weight of the 24 trout used in this experiment was 205.0 - 7.3 g. Evidence that these fish were sexually immature is demonstrated by the low GSI values. Each group contained approximately equal numbers -92-Table XI. Effects cf Salman Calcitonin on Plasma Electrolytes in Fingerling Rainbow Trout. Sample Time (After Injection) Group Total wt (g)+ Mean - SE Plasma Ions (mg per IPG ml) Total Calcium Mean ± SE (n) Inorganic Phosphorus Mean + SE (n) Vfe hour Control 6 Vehicle 8 Calcitonin 8 10.6 - 1.50 10.0 - 1.75 12.0 - 1.30 9.4 0.39 (5) 11.8 0.25 (3) 9.8 0.27 (8) 11.7 0.65 (6) 9.0 0.35 (8) 12.4 0.52 (8) yk hour Control 10 12.7 - 0.62 10.9 0.21 (9) 15.3 0.36 (10) Vehicle 10 12.0 - 1.07 9.9 0.31 (10) 10.9 0.50 (10) Calcitonin 10 12.2 - 0.62 9.7 0.29 (9) 12.1 0.67 (9) Th hour Control 10 Vehicle 10 Calcitonin 10 11.2 - 0.82 12.9 - 0.97 12.2 - 0.99 10.9 0.38 (10) 15.0 0.48 (8) 10.0 0.34 (10) 12.3 0.65 (8) 10.3 0.27 (10) 12.0 0.90 (5) 25Vz hour Control 5 Vehicle 10 Calcitonin 10 12.5 - 2.15 11.2 - 1.16 10.2 - 0.84 9.7 0.43 (4) 9.5 0.28 (10) 9.7 0.28 (10) 12.7 0.69 (5) 12.3 0.40 (10) 12.9 0.50 (8) Figure 9. Plasma calcium changes in fingerling trout - effect of salmon calcitonin. Injection at time •. i I Figure 10. Plasma inorganic phosphorus changes in fingerling trout -effect of salmon calcitonin. Injection at time 0 i •F-I -95-•f males and females (non-spawning trout are difficult to sex). The data, as presented in Table XII was difficult to analyze statistically due to the variation in individual plasma calciums and was therefore, recalculated (Table XIII, pg. 97). The 3 pre-injection plasma calcium levels for each fish were averaged (Table XIII, column 2). This mean was then arbitrarily adjusted to 10.• mg per 100 ml. Column 5 contains the number added to each individual mean plasma calcium level to equal 10.0 mg per 100 ml. This number was then added to each actual sample time plasma calcium (Table XIII) for the appropriate fish. Calculated in this manner, it is possible to obtain an average of the plasma calcium levels for each group and to compare the results at each time period. The data of Table XIII is presented graphically in Figure 11, pg. 98. Figure 12, pg. 99, illustrates the actual plasma calcium changes in 3 fish taken from the control, vehicle and calcitonin groups. Haematocrits decreased from an average of 25 to 18 percent and plasma percent water increased from 94.8 to 95.2 percent over the 24 hour period experiment. Salmon Physical measurements of the 3 female salmon used in this experiment are given in Table XIV, pg. 100. The mean total weight was 1137 - 59.3 g. All 3 salmon were very sexually mature. Results of calcitonin and vehicle infusions on the plasma electrolyte levels of each fish are shown in Figures 13, 14, 15, pages 101, 102, 103, respectively. (Mo consistent effect of calcitonin was demonstrated on any of the plasma electrolytes at lYz, 3$ and bVz hours Table XII. Physical Measurements end Individual Plasma Calcium Levels of Cannulated Trout. Plasma Calcium (mg per 100 ml.) C(g) (Cm) -2 hr -1 hr 0 • + 1 hr + 3 hr + 5 hr + 2d hr Control 1 M 10d.5 0.1.8 22.3 9.5 9.2 . 9.d 9.3 8.9 8.6 8.6 2 H 207.5 D.05 27.0 8.1 8.2 7.8 7.9 7.5 7.3 7.9 3 M 218.0 0.18 26.6 8.8 8.2 8.6 8.7 8.1. 8.5 8.2 It F 238.0 0.21 28.9 9.0 8.5 8.9 8.6 8.6 8.6 8.1 5 F 2<.9.0 0.1.0 28.7 8.3 8.1 8.1. 8.3 8.3 8.5 8.1. 6 M 209.0 0.05 28.2 8.8 8.3 8.0 8.1. 8.2 8.5 8.6 n - 6 mean » 201..0 a.23 27.0 8.8 8.1. 8.5 8.5 8.3 8.3 8.3 SD - 1.7.1 0.16 2.2d 0.d6 Q.36 0.52 O.dZ 0.d2 0.d5 0.2d SE = 21.0 0.07 1.00 0.20 O.ld 0.22 Q.17 0.17 0.20 0.10 Vehicle 1 H 215.0 0.05 26.7 8.0 8.1 8.0 7.8 7.9 7.5 7.5 2 F 20d.5 0.10 28.0 7.9 7.d 7.5 7.5 7.5 7.5 7.3 3 F 232.5 0.3d 28.0 7.6 7.d 7.6 7.5 7.8 7.9 7.9 d F 220.0 D.d5 26.8 8.2 7.8 8.5 8.0 8.0 8.2 6.2 5 F 196.0 0.10 26.6 8.3 7.7 7.2 7.2 7.2 7.1 8.1 6 M 13d.0 D.15 23.3 8.1 8.0 B.l 8.1 7.8 7.9 fl.d n = 6 mean = 200.0 0.20 26.6 8.0 7.7 7.8 7.7 7.7 7.7 7.9 SD = 31.8 O.ld 1.57 0.23 0.26 0.d2 0.30 0.26 0.3d 0.38 SE = Id.2 0.06 0.70 0.10 0.10 0.17 0.10 0.10 O.ld 0.17 Calcitonin 1 M 228.0 0.09 28.7 8.5 8.7 8.3 8.2 8.6 8.2 8.1 ,„ „.,__ 2 M 2d8.0 O.Od 29.0 9.2 9.0 9.0 9.0 9.5 8.5 8.8 1Z5 mll/100 g 3 M . 20((>0 Q>1D 26.2 6.8 6.6 6.7 6.d 7.1 6.1 6.9 rlafl d F 218.0 0.23 27.3 8.0 7.8 7.5 7.7 8.0 7.d 7.a 5 F 210.0 0.38 28.3 8.3 7.6 8.d 7.8 8.d B.l 8.3 6 M 226.0 D.Od 28.6 9.1 8.B 9.2 8.9 9.1 9.d 8.9 n = 6 mean = 220.0 0.15 28.0 8.3' 8.1 8.2 8.0 8.5 8.0 8.1 SD = Id.2 0.12 0.96 0.80 0.33 0.85 0.86 0.76 1.01 0.56 SE ° 6.3 0.05 0.d2 0.36 0'.37 0.37 0.38 0.33 O.dd 0.28 Calcitonin 1 F 205.0 0.3d 26.7 • 7.d 8.0 B.d 7.8 7.9 7.2 7.3 . 2 F 16d.O 0.06 25.3 8.2 7.9 7.7 7.6 7.5 7.d 7.S mU/lUU g. 3 p 215.5 Q.32 2S.0 a.9 8.9 B.d 8.7 8.5 6.5 8.5 • 80 d M 238.0 O.Od 29.3 8.d 8.6 8.d 8.8 S.d 6.6 8.8 5 M ld9.0 0.07 2d.9 7.3 7.3 6.9 7.3 7.2 7.d 7.8 6 M 182.5 0.05 26.5 5.8 5.7 5.7 5.8 5.7 5.7 5.3 n = 6 mean = 192.0 0.15 26.8 7.7 7.7 7.6 7.7 7.5 7.5 7.7 SD = 30.d 0.13 1.50 1.00 1.03 1.00 0.99 0.93 0.95 0.95 SE = 13.6 0.05 0.67 0.d5 0.d5 O.dd D.d3 O.dl ' 0.d2 0.d2 Table XIII. Effect of Salmon Calcitonin on Plaema Calcium Levels in Cannulated Trout. Mean Control Plasma Plasma Calcium* (mg/100 ml) NumbBr Bdded Calcium Zero Plasma Calcium Change from Zero Time „ ... Time** (mg/100 ml) Group tt mean SD SE to mean mV. i-a  Control 1 9.3 - 0.00 0.00 0.7 10.0 10.0 9.S 9.3 9.3 2 9.0 0.1<t 0.10 2.0 9.9 9.5 9.3 9.9 3 B.G 0.21. 0.11. 1.1. 10.1 9.8 9.9 9.5 •% S.9 0.22 0.10 1.1 9.7 9.7 9.7 9.2 5 8.1% 0.17 0.10 I.S 9.9 9.9 10.1 10.0 6 8.1. 0.31 0.22 1.6 10.0 9.8 10.1 10.2 n = 6 6 6 6 6 mean - 8.60 . 9.93 9.72 9.73 9.70 SD = 0.1.1 0.12 0.13 0.33 0.36 SE » 0.17 0.05 0.05 0.15 0.16 Vehicle 1 a.o i 0.00 0.00 2.0 10.0 9.8 9.9 9.5 9.5 2 7.7 0.21. 0.11. 2.3 9.8 9.B 9.8 9.6 3 7.7 0.20 0.10 2.3 9.B 10.1 10.2 10.2 It 8.2 0.26 0.11. 1.8 9.B 9.8 10.0 10.0 5 7.7 0.1.8 0.31. 2.3 9.5 9.5 9.1. 10.1. 6 8.1 0.00 0.00 1.9 10.0 9.7 9.8 10.3 n = 6 6 6 6 6 mean = 7.90 9.78 9.80 9.78 10.00 SD = 0.20 0.11. 0.18 0.27 0.3U SE = 0.00 0.06 0.08 0.12 0.15 Calcitonin 1 8.6 t 0.22 0.10 1.4 10.0 9.6 10.0 9.6 9.5 125 mU/100 g 2 9.1 0.00 0.00 0.9 9.9 10.1. 9.1. 9.7 fish 3 6.7 0.00 0.00 3.3 9.7 10.1. 9.1. 10.2 (. 7.8 0.17 0.10 2.2 9.9 10.2 9.6 1C.0 5 8.2 0.31 0.17 1.8 9.6 10.2 9.9 10.1 6 9.1 0.11. 0.00 0.9 9.8 10.0 10.3 9.8 n = 6 6 6 6 6 mean « 8.25 9. 75 10.20 9.70 g.aa SD =. 0.83 0.12 0.16 0.32 0.21. SE = 0.36 0.05 0.07 CU 0.10 Calcitonin 1 7.9 i 0.1.0 0.28 2.1 10.0 9.9 10.0 9.3 9.9 500 mU/100 g 2 7.9 0.17 0.10 2.1 9.7 9.6 9.5 9.7 fish 3 8.7 0.22 0.11. 1.3 10.0 9.8 9.8 9.8 1. 8.5 0.00 0.00 1.5 10.3 9.9 10.1 10.3 5 7.2 0.17 0.10 2.8 10.1 10.10 10.2 10.6 6 5.7 0.00 0.00 1..3 10.1 10.10 10.0 10.1 n B 6 6 6 6 6 mean = 7.65 10.02 9.B8 9.82 10.07 SD = 0.99 0.18 0.11. 0.32 0.31 SE = 0.1.3 0.08 0.06 D.ll. 0.11. VO -0 mean of 3 control plasma calcium levels (-2, -1, 0 hour samples) I 'mean plasma calcium (column 2) of each fish taken as 10.0 (mg/100 ml) re 11. Mean plasma calcium changes in adult cannulated trout effect of salmon calcitonin. Figure 12. Individual plasma calcium changes in cannulated adult trout -effect of salmon calcitonin. ^ IX) i -100-Table XIV Salmon Physical Measurements. Salmon Sex Total lilt. (g) Gonad Lit. (g) GSI Fork Length (cm) V U Z female female female 1255 1085 1070 205 250 265 16.33 23.04 24.77 50.2 46.6 47.0 n = mean = SD = SE = 3 1137. 83.8 59.3 3 240. 25.4 18.0 3 21.38 3.63 2.57 3 47.9 1.60 1.13 Figure 13. Plasma electrolyte changes in a sockeye salmon - effect of salmon calcitonin infusion. Female sockeye V. i o i o o 5.01 4.0 \ 3.0-UJ E — 2.0 r 1.0-co 150 1 14 5 o 140 -135 1 Colclum _j _^ Inorganic Phosphorus Time in Hours Salmon CT Infusion 2units/IOOgm in I ml 30 min. Vehicle Infusion Figure 14. Plasma electrolyte changes in a sockeye salmon - effect Df salmon calcitonin infusion. Female sockeye U. o 1 Salmon CT Infusion Vehicle Infusion 2 units/IOO gm in I ml 30 min. Figure 15. Plasma electrolyte changes in a sockeye salmon - effect of salmon calcitonin infusion. Female sockeye Z. • UJ i -104-post-injection. Plasma magnesiums uere particularly stable uhile plasma sodiums shcued uider fluctuations. The effect of calcitonin infusion on renal electrolyte ex cretion and urine flou rates of the 3 salmon are summarized in Figure 16, pg.105. Both vehicle and calcitonin infusions appeared to cause a slight diuresis but the infusion procedure itself may have caused "laboratory diuresis" (Forster and Berglund, 1956). The control period electrolyte excretion and urine flou rates displayed little variation from the calcitonin and vehicle infusion experiments. Calcitonin infusion caused a slightly greater increase in sodium output compared to the vehicle infusion (not significantly different) but in both cases the sodium output uas back to control values uithin 3 hours. Magnesium output also increased slightly due to the in fusion of both calcitonin and vehicle. Calcium excretion uas least affected. No evidence of phosphaturia due to the calcitonin infusion uas observed. Urinary potassium output (not shoun) uas very stable. Urine flous of the 3 salmon uere remarkably similar to control collections ranging from approximately 3.D - 4.5 ml per hour. -105 Figure 16. Urinary electrolyte excretion and urine flow in 3 sockeye salmon - effect of salmon calcitonin infusion. -106-Discussion Plasma Effects of Salman Calcitonin Injection of calcitonin into fish has led to inconsistent results. The first report, published by Pang and Pickford (1967), revealed that intravenous and intraperitoneal injections of partially purified hog calcitonin (2 - k units per gram fish) produced no change in the serum calcium levels of male killifish, Fundulus  heteroclitus, at 1, 2 and 4 hours post-injection. The hormone uas also ineffective in intact and hypophysectomized fish maintained in freshuater and seauater. Louu e_t al_ (1967) reported a hypocalcaemic and hypophosphatemia effect of partially purified porcine calcitonin in the catfish, Ictaluras melas, at 60 minutes post-injection. These results, hDuever, have since been retracted (Kenny, 1972) since the crude thyroid extracts used in the above study uere found to be contaminated uith histamine and other unidentified pharmacologically active substances. Chan et a_l (1968) demonstrated a hypocalcaemic and hyper phosphatemia effect uith intravenous injection of partially purified porcine calcitonin (10 mU/100 g and 50 mU/100 g ) into intact, immature freshuater European eels (Anguilla anguilla L.). The response lasted several hours depending on the dose and the maximal peak response of the 50 mU/100 g dose occurred at 6 hours. The hyperphosphatemia caused by calcitonin is interesting in vieu of the fact that in mammals, calcitonin louers plasma phosphate as a con sequence of the inhibition of bone resorption. Injection of porcine calcitonin (50 mU/100 g ) into -107-stanniectamized eels (their corpuscles of Stannius had been removed one week prior to the experiment) had no effect on plasma calcium levels, while a slight hyperphosphatemia occurred. Removal of the corpuscles of Stannius in the eel is known to significantly elevate plasma and muscle calcium levels and to lower plasma inorganic phosphorus one week after the operation (Chan e_t al, 1967; Chan, 1972). The changes in calcium returned to normal within k to 6 weeks, possibly due to increased calcitonin secretion from the ultimobranchial gland (Chan, 1969; Henderson e_t a_l, 197D). The lack of effect of calcitonin in stanniectomized eels might be related to the almost total disappearance of osteoclasts observed 4 weeks after corpuscle removal (Lopez, 1970a). It may also be that the eel was insensitive to exogenous calcitonin administration since the receptor sites were fully saturated from the high plasma levels of calcitonin (Chan, 1972). Chan (1969) also demonstrated that ultimobranchialectomy of the Asian eel, Anguilla japonica, resulted in a small but significant increase of plasma calcium while plasma phosphate re mained unchanged (samples collected 4 weeks after operation). More recent work (Chan, 1972) has shown that ultimobranchialectomy in Anguilla japonica resulted in a slight drop (P<0„05) in plasma total calcium (haemadilution) at 4 weeks while plasma ionic calcium was unaltered (2 weeks post-operation). Thyroidectomy of rats has no effect on plasma calcium levels (Talmage ejt a_l, 1965; Sturtridge and Kumar, 1967; Cooper e_t al 1970; Sorensen, 1970 ). Recently, Milhaud et al (1972) have shown that thyroidectomy of rats will raise plasma calcium and phosphate levels only if the operation is performed during the dark night -loa-period when they uere feeding. Pang (1971b) obtained a hypocalcaemic and hyperphosphatemia response following injection of salmon calcitonin into the fresh water American eel, Anguilla rostrata. However, the same experi ment, repeated on seawater-adapted eels, produced no effect. The hyperphosphatemia effect found by Pang and Chan has also been observed in the heart-lung preparation (bone-and kidney-free) of the dog by Stahl e_t a_l (1968). This suggests an extra-skeletal and extra-renal mechanism of action for calcitonin. Ma (1972) produced a marked hypocalcaemic and hypakalaemic response in the Asian eel, Anguilla japdnica, an injection of salmon calcitonin at a dose as law as 100 mU/100 g body weight. This response was elicited only after pre-treatment of the eels with L-thyroxine (10 /jg/100 g body weight) and was time-and dose-dependent. A slight but significant elevation of serum inorganic phosphorus was also noted. On the other hand, Pang (1971b) has shown that injections of salmon calcitonin, codfish ultimobranchial extract and porcine calcitonin had no significant effect on any of the serum electrolytes tested in the killifish, Fundulus heteroclitus, in various experiments. Injection Df shark ultimobranchial extracts into the blue shark, Prionace glauca, and the horn shark, Heterodontus francisci, failed to elicit a hypocalcaemic response (Urist, 1967). Porcine cal citonin (10-20 U/kg, IP) extract produced no significant changes in serum calcium or inorganic phosphorus levels in the lazy shark, Pbroderma africanum (Louw e_t al, 1969). Copp et_ a_l (1970) were also unable to detect any change in plasma calcium levels in dogfish -109-sharks, Squalus suckleyi, injected intravenously with 10 units of salman calcitonin. In contrast to the above results, Orimo et_ a_l (1972a) claim that injection of eel calcitonin (5 MRC Units) caused a significant increase in serum calcium in freshwater Anguilla  japonica. These authors also detected a significant hyponatremia and hypochlaremia (p<0.05) following injection of eel calcitonin into a similar group of freshwater-adapted eels. Recently Urist e_t al (1972), reported that injection of purified and synthetic salmon calcitonin (500 MRC U/kg) into the female South American lungfish, Lepsidosiren paradoxa, had no effect on plasma calcium at 1 and k hours post-injection. Porcine calcitonin, at doses of kk, 88 and 176 MRC U/kg, also failed to suppress the plasma calcium level 1 hour post injection. These authors also reported that the skeleton of Lepidosiren, which con tained perichondral bone, apatite mineral, and osteocytes but no osteoclasts, was unresponsive to vitamin D, parathyroid extract and calcitonin. The variability of the fingerling trout control group plasma electrolyte levels in the present study, was probably due to stress. The absence of an effect of salmon calcitonin on the fingerling trout plasma calcium and inorganic phosphorus levels may indicate that in young, growing fish, bone turnover is not as rapid as in mammals. In fact, some authors (Moss, 1962; IMorris ejb a_l, 1963; Nelson, 1967; Simmons e_t al, 1970; Simmons, 1971) have shown that fish bone has a very low rate of turnover. Unlike mammals, fish are able to obtain adequate amounts -110-•f calcium and phosphorus directly from the uater via their gills, oral epithelia and fins (Simmons, 1971). The transport of calcium across the gills also appears to be more efficient in freshuater fish. These points are mentioned to suggest that the action of calcitonin in fish may not be on bone but on other target sites such as the gill.. Certainly the type of bone (acellular versus cellular) found in fish does not appear to influence the hypo calcaemic response to calcitonin since the eel, salmon and trout all have cellular bone uhereas the killifish has acellular bane. The results obtained on the fingerling trout uere con firmed by Pang (1971b) uho did not detect any hypocalcaemic effect in juvenile channel catfish, Ictaluras melas, due to injection of salmon or porcine calcitonin. Sex differences would not appear to explain the negative effect of salmon calcitonin (SCT) in the trout since the hormone was equally ineffective in males and females (Table XII, pg. 96). This contrasts with the results of Hinde and Phillippo (1967) who observed a marked difference in the response of rats to calcitonin injection, the males of a given age being more sensitive than the females. The original report on the negative effect of salmon cal citonin (125 and 500 mU/100 g fish) on plasma calcium levels in the trout (Uatts et a_l, 1970) was supported by Pang (1971b) who was also unable to produce a hypocalcaemic response by injection of salmon calcitonin into freshwater coho salmon, Oncorhynchus  kisutch. Pang (1971b), however, does not report the dosages of calcitonin used in any of his experiments and this undoubtedly is -111-a critical factor. With the sockeye salmon in the present study, the effect of salmon calcitonin infusion might have been obscured by the hormonal changes of sexual maturation. Other endocrine factors such as thyroxine and the corpuscles of Stannius may have compen sated for the infusion of calcitonin. One explanation for the negative result of salmon calcitonin (SCT) in the salmon would appear to reside, in the finding that spawning female salmon have elevated levels of plasma calcitonin (Chapter IV). Measurement of the circulating level of plasma calcitonin at 24 hours post-infusion of vehicle revealed calcitonin levels of 6775 - 1042 pg/ml plasma and 14,700 - 1731 pg/ml plasma for salmon V and W, respectively. The high circulating calcitonin levels in these salmon may indicate that the receptor sites for this hormone were already saturated. Infusion of SCT therefore, might better be tested in male or gonadectomized salmon but un fortunately these fish were not available for this study. It would be interesting to test the hypocalcaemic effect Df SCT in ultimobranchialectomized trout since Talmage and Kennedy (1969) have demonstrated that thyroidectomized rats were more sensitive than intact rats to calcitonin treatment. The effect of different levels of calcium in the food and water on the response Df trout to calcitonin has not been fully investigated. It may be significant that a hypocalcaemic response to calcitonin injection has been demonstrated only in the eel. This may be explained by a species difference or by the fact that the eel is an extraordinarily stable experimental fish. Wttnile the salmon is an•anadromous fish (lives in seawater, spawns in fresh--112-water), the eel is catadromous (lives in freshwater, spawns in sea water) and thus their osmoregulatory problems at similar stages in the life-cycle are quite different. Since Dacke (Chan, 1972, discussion) was unable to confirm Chan's hypocalcaemic effect using salmon and porcine CT in eels, physiological condition and sexual development may be crucial factors. Renal Effects of Salmon Calcitonin Since no effect on plasma electrolyte levels was observed, it is not too surprising to find that infusion of salmon calcitonin did not alter renal electrolyte excretion. It is possible that the dose required to produce renal effects in salmon might be extremely large, owing to the fact that spawning salmon have such high circulating calcitonin levels. However, the dose used in the present study (2 U/100 g body wt) was the same as that which produced a 2- to 3-fold increase in urine volume and a 3- to 5-fDld increase in sodium extretion in saline-loaded rats (Keeler ejt a_l, 1970; Aldred et al, 1970). Aldred et_ al (1970) demonstrated that urine volume, sodium, phosphorus and magnesium excretion levels showed significant changes only at the 3rd hour after treatment. In this study, however, urinary parameters returned to the normal range at approx imately the 3rd hour following infusion. The renal effects of calcitonin in mammals seems dependent upon the species and dose of calcitonin and the experimental animal. For example, Williams et al (1972) have shown that although synthetic salmon calcitonin causes a profound natriuresis in rats, -113-no effect on sodium excretion uas observed uith synthetic human calcitonin. These same authors shoued that SCT produced no effect on phosphate excretion uhereas other uarkers have noted a phosphaturia under similar circumstances (Heeler et_ a_l, 1970; Aldred et al, 1970). It is possible that the urinary effects of SCT in the salmon uere extremely rapid and obscured by the hourly collection of urine samples. Salaka et_ a_l (1971) have shoun this to be true in rabbits uhere intra-aortic injections of porcine calcitonin caused' an immediate increase in urine flou uithin 3 minutes of injection. Tuo other studies on the renal effects of calcitonin an fish have yielded negative results. Goncharevskaya e_t al (1971) reported that bovine calcitonin intramuscular injection (150 units/ 100 g body ut ) had no effect on serum calcium and inorganic phos phorus levels in the sea scorpion, Myoxocephalus scorpius (L.). There uas also no urinary diuresis or change in urinary bladder ionic composition. Hayslett e_t a_l (1972) observed no change in the fractional excretion of calcium or urea, the GFR or urine volume on injection of salmon calcitonin (4.4 U/kg body ut ) into the elasmobranch, Squalus acanthias. A slight decrease in fractional excretion of sodium and potassium uas noted but no hypocalcaemic effect uas observed. Uith regard to the natriuretic effect of salmon calcitonin in mammals, it is of interest to note that purified dogfish cal citonin like the salmon, hormone, is extremely natriuretic in the rat (Maclntyre et al, 1972). Chan (1972) reported that ultimobranchialectomy of Anguilla -114-japonica caused an increase in calcium excretion uhich returned to normal levels in 4 ueeks. IMo change in urine flou rate uas observed. Since the major osmoregulatory problem in freshuater fish is the conservation of electrolytes and excretion of uater, it uould be physiologically inexpedient to excrete electolytes in freshuater. It might prove informative to test the effect of salmon calcitonin in a seauater salmon. Besides the positive hypocalcaemic effect of calcitonin in eels, Lopez et_ a_l (1971) have provided evidence of a passible role af calcitonin in rainbou trout. Porcine calcitonin injection (50 mU every 2 days for 3 ueeks) into immature trout, prevented bone demineralization caused by calcium-free uater and thyroxine. These authors believe therefore, that bone is the target organ for calcitonin and that the ultimobranchial gland, by secreting calcitonin, plays an important rale in calcium homeostasis in fish. Lopez and Deville (1972) have also investigated the effect of salmon calcitonin on vertebral bone morphology and ultimobranchial activity in the mature female eel, Anguilla anguilla L. Immature silver eels uere made experimentally mature by intraperitoneal injection of carp pituitary extract (1 mg per 100 g body ueight, tuice per ueek until maturation). Development of the gonads in sexual maturation is accompanied by a marked hypercalcaemia. Tuo groups of these mature eels uere submitted to prolonged treatment uith salmon calcitonin. The first group, after receiving pituitary injections for B ueeks, uere injected uith SCT (3DQ mU per body ueight, IP, daily for 43 days). The second group, uhich had also received the pituitary injections and had spauned, uere injected -115-uith the same dose of SCT daily far 11 days. Salmon calcitonin, in the first group, did not alter the hypercalcaemia but prevented halastasic demineralization (reduc tion of mineralization of intercellular substance without histological modifications of the organic matrix). Osteoclastic and osteolytic resorption were reduced and the UB gland was still highly stimulated. In the second group, SCT reduced the hypercal caemia by 50 percent and decreased the osteoclastic and osteolytic resorption. The UB gland appeared inactive. These authors conclude that calcitonin in the eel acts on bone to prevent bone resorption as it does in mammals. They attribute the negative effect of calcitonin in preventing the hypercalcaemia to the reduced responsiveness to calcitonin caused by gonadal steroids (Sorensen and Hindberg, 1971). This explanation could apply equally well to the present study. In summary, although salmon calcitonin has negligible effects on plasma and renal electrolytes in trout and salmon, other target organs such as the gill, bone and gut must be investigated before the role of calcitonin in these fish can be elucidated. -116-IV„ PLASMA CALCITONIN AIMD TISSUE MINERAL CHANGES IN MIGRATING SALMON Introduction There are five main species of Pacific salmon under the genus Oncorhynchus: Scientific name Common name Oncorhynchus nerka (LJalbaum) - sockeye, red Oncorhynchus kisutch (Ualbaum) - coho, silver Oncorhynchus tshawytscha (Ualbaum) - chinook, spring, king Oncorhynchus gorbuscha ( Ualbaum) - pink, humpback Oncorhynchus keta (Ualbaum) - chum, keta, dog As mentioned previously, salmon are anadromous fish, which means that they live most of their lives in the sea, but return to fresh water to spawn. The five species all have different life-histories, morphology, sizes, behaviour, feeding and spawning habits. This chapter will deal mainly with the sockeye, chinook and coho salmon. Pacific salmon begin their life cycle in freshwater. Females dig a nest or "redd" in gravel beds of freshwater streams, rivers or lakes and deposit up to 6000 eggs, the amount dependent on the species and size of the individual fish. The eggs are fertil ized by the male, covered with gravel by the female, and remain under the gravel throughout the winter. On completion of the spawning act, both male and female Pacific salmon die within a few days or weeks. The eggs develop through the sac-like alevin stage and emerge from the gravel in the spring as fry. Depending on the species -117-and environmental conditions, the subsequent period of freshuater residence varies from a feu days to several years. All pink and cltoum salmon migrate to the sea directly follouing their emergence from the gravel. Other species, such as the sockeye, descend or ascend the tributary from the spauning grounds to the nursery lake uhere they spend one or tuo years. Here, the fry develop into smalts at uhich stage they migrate to the sea. Chinook salmon migrate uithin the first year uhile mast caho spend one year in freshwater before their seauard migration. The timing of the doun-stream migration also depends on temperature, food availability and fish size (Ricker, 1966). On reaching the sea, the smalts or fry may spend several days in the estuarine uaters af the river mouth. They then move out into the offshore pelagic environment uhere their distribution covers most of the North Pacific Dcean and the Bering Sea (Jackson, 1963). The time spent at sea varies according to species and even uithin a species. Coho and pink salmon stay in seauater for one and tuo years respectively. Fraser River sockeye spend tuo Dr three years in the ocean uhile the chinook salmon commonly ocean feed for four years. Under the influence of a homing instinct, the adult salmon approach inshore uaters, heading in the direction of their natal stream. This shoreuard migration occurs at a characteristic time for each species. For example, maturing Fraser River sockeye appear in coastal uaters from May through October. The majority of these sockeye enter the Strait of Georgia via the Strait of Juan de Fuca uith usually less than 10 percent entering through Johnstone Strait -118-(Ricker, 1966). The salmon delay off the mouth of the Fraser for varying periods of time (Killick, 1955). Generally, the earliest Fraser River runs go furthest upstream. The gonads begin to mature some time before the shore ward migration and are often well-developed before the fish enter their home stream (Hanamura, 1966; Ishida, 1966; Vladykov, 1962). At this time, the salmon also cease feeding (Greene, 1904). The journey up the river is particularly arduous,often involving long distances and swift currents. Some species, such as the coho and chum, generally spawn in coastal streams and hence have very short migrations. After reaching the particular spawning grounds, spawning occurs on a predictable date plus or minus a few days. In general, spawning of sockeye tends to coincide with water temperature. Sockeye in British Columbia spawn at temperatures of 3 - 7° C. Other environmental factors such as light, water level, etc. also undoubtedly influence the time of spawning. The adults spawn and then die, and the life cycle is repeated. During their freshwater migration, Pacific salmon undergo complex physiological changes and are subjected to a variety of internal and environmental stresses. These include: 1. osmoregulatory stress on movement from sea to freshwater 2. exhaustion, from the often long, arduous migration 3. sexual maturation and development of secondary sexual characteristics k. starvation, often for many months, due to cessation of feeding 5. diseases, bacterial and fungal infections, parasites -119-6. uater temperature changes. The dramatic development of the gonads and the immense problems of osmoregulation encountered by the salmon during the spawning migration, have led many researchers to investigate the endocrine changes involved in these processes. Sexual maturation and development of secondary sex characteristics in volves extensive tissue reorganization in a relatively short period of time and under fasting conditions. This chapter presents re sults of experiments designed to investigate the role of calcitonin in osmoregulation and/or sexual maturation and spawning, in the migrating salmon. -120-Materials and Methods Three species of Pacific salmon, sockeye, coho and chinook, uere investigated at various stages in their spauning migrations. The method of collection and storage of samples uas outlined previously in General Materials and Methods. This chapter is divided into three sections. In Section A, experimental material is presented on the migration of the Chilko race of sockeye uhich uas studied during the summers of 1971 and 1972. In Section B, material collected on coho salmon under various conditions is outlined. Section C is a summary of the data from 3 species of spauning salmon: sockeye,coho and chinook. A. Migration of Chilko Sockeye Salmon Chilko Migration 1971 The purpose of this study uas to examine the changes in plasma calcitonin and electrolyte levels in migrating sockeye salmon. The Chilko race of sockeye uere chosen for several reasons. Extensive biochemical and physiological studies had already been performed on migrating sockeye salmon thus providing a good basis for further investigation (Idler and Clemens, 1959; MacLeod e_t al, 1958; Idler and Tsuyuki, 1958; Idler and Bitners, 1958; Idler e_t a_l, 1960). The International Pacific Salmon Fisheries Com mission (Salman Commission) supplied the necessary manpouer and facilities to collect sockeyefrom this particular race both from the sea and on the spauning grounds. By means of scale analysis, -121-the age and race of the seawater sockeye were identified by the Salman Commission and only Chilko sockeye were included in the study. This identification was an important consideration. By examining one discrete race of sockeye, the salmon were essentially at the same stage of sexual development when sampled at any one particular point in their migration. Also, sockeye salmon of the same age group are relatively uniform in body weight and length. All salmon were captured and blood sampled within 5 minutes of capture. In the sea, salmon were caught by reef net and on the spawning grounds by beach seining. Migrating salmon were sampled at 3 points along their migratory route. Phase I, seawater sockeye were sampled at Lummi Island, Washington State, U.S.A. Phase II, freshwater arrival sockeye were sampled at Chilko Lake, British Columbia, Canada. Phase III, spawning sockeye were sampled on the spawning grounds of Chilko River at the outlet from Chilko Lake. Table XV, pg.123 , presents a summary of the sampling times, dates and locations. Figure 17, pg.122,shows the location of the 3 sampling points and the route travelled by the migrating salmon. Physical measurements, plasma electrolytes (calcium, inorganic phosphorus, sodium, potassium), plasma percent water, plasma protein and haematocrit were measured for each fish. Plasma calcitonin was measured by radioimmunoassay an each individual sample. Procedures used for the above analyses were described in General Materials and Methods. The scientists with the Salmon Commission divided the ure 17. Map of Fraser River and British Columbia. Chilko sockeye migratory route indicated by dark line and locations of the 3 sampling points are shown by numbers. -123-spauning female sockeye into 3 groups: • percent spawned (ripe but egg case intact), 50 percent spawned (half of the eggs re maining) and 100 percent spawned or spawned out females (almost no eggs remaining). The sexually mature female Chilko sockeye contains approximately 3000 eggs. The male spawning sockeye were impossible to classify as to degree of spawning and hence were sampled as one group. Table XV. Chilko Sockeye Migration 1971 Phase Sampling Date(s) Uater Temperature Location Number of Salman I Seawater (beginning migration) July 23 & 24/71 August 6/71 11.5°C 1D.5DC Lummi Island (seawater) 45 m 44 f II Freshwater August 17/71 Arrival August 26 & 27/71 (to Chilko Lake) 14.4°C 13.9aC Chilko Lake (freshwater) 26 m 34 f III Spawning September 22/71 (spawning S.9DC Chilko River (freshwater) 15 m 12 f (096 sp) ID f (50% sp) ID f (1DD% sp) grounds Chilko River) -124-Chilko Migration 1972 The purpose of the second migration study uas to inves tigate the serum ionic cylcium changes in migrating salmon in an attempt to correlate these uith plasma calcitonin changes. The calcium and phosphorus contents of vertebrae, premaxillae, scales, muscle, gonads and skin uere also analyzed. Scales, skin, and muscle samples uere taken from the dorsal aspect at the right side of the fish, 1 cm behind the posterior edge of the operculum. Scales uere individually removed from the 1 inch square skin sample and rinsed 3 times in deionized uater before drying. The vertebrae uere also obtained from a position 1 cm behind the posterior edge of the operculum. The premaxillae uere collected from the right side of the jau only and the teeth, uhich had solidly fused to the premaxillae in the spauning fish, uere removed as completely as possible. Both gonads uere taken from each fish for analysis. Methods of analysis uere outlined previously in General Materials and Methods. Due to phosphate interference, it uas found necessary to employ a 1.0. percent lanthanum chloride solution in the analysis of calcium of the soft tissues uhereas a 0.5 percent lanthanum chloride solution uas suitable for the hard tissues. Table XVI, pg.125, gives a summary of the dates and loc ations of sampling points of the 1972 Chilko Migration. Physical measurements, plasma calcium, inorganic phosphorus, sodium, potassium, percent uater, plasma protein and haematocrit uere measured for each fish. The serum ionic calcium uas measured in the laboratory at a temperature close to that of the uater in uhich the fish uere originally captured. Serum pH uas determined on -125-Table XVI. Chilko Sockeye Migration 1972 Phase Sampling Date Location Number of Salmon I Seauater July 21/72 Lummi Island 10 m (beginning (seauater) 10 f migration) II Freshuater August 28/72 Chilko Lake 10 m Arrival (freshuater) 10 f (to Chilko Lake) III Spauning Sept. 24/72 (spauning grounds) the freshuater arrival and spauning samples immediately fallouing the ionic calcium measurements. The female spauning sockeye uere divided into unspauned females (ripe but egg case intact) and spauned out females (almost no eggs remaining). The spauning males uere in various stages of sexual maturation but uere generally very ripe. It should be noted that the location and method of capture of the fish and the handling of the samples, uere the same for the 1971 and 1972 migrations. Chilko River (freshuater) 10 m 10 f (0% sp) 10 f (100% sp) -126-B. Plasma Calcitonin Levels in Coho Salmon: Effect of Sexual Maturation and Environmental Salinity The purpose Df this study uas to investigate the plasma calcitonin levels in coho salmon at different stages of develop ment and in different environments. Table XVII, pg.127>summarizes the experimental conditions of the 3 groups of coho salmon. The first group has been described previously in Chapter I, and con sisted of freshuater spauning adult male and female coho salmon. The second group uere sexually immature coho salmon uhich had spent their entire lives in freshuater. The third group uere young, very sexually immature salmon (grilse) uhich had been in seauater for 7 months. It should be noted that the adult spauning coho salmon in Group I had not been feeding for at least 1 month pior to sacrifice since in nature they cease to feed upon entry into fresh uater. The freshuater immature coho in Group II uere fed trout pellets once ueekly and starved 6 days prior to sacrifice. The coho grilse in Group III uere grouing rapidly and being fed 3 times daily uith a frozen meat diet consisting of canned salmon, beef liver and horse heart. These fish had been fed 4 hours prior to sampling. Physical and electrolyte measurements uere determed for each fish as outlined previously. Plasma calcitonin levels uere again measured using the salmon calcitonin radioimmunoassay. Table XVII. Coho Salmon Study: Summary of Sampling Data Group Sampling Date Location Number of Salmon History I Adults (ripe, spawning) November 30/70 Samish Hatchery Washington, U.S.A, (from river) Freshwater (temp. 4.4°C) 15 m 15 f Wild fish - migrated from seawater to freshwater (Samish River) Age 2 -3 years II Freshwater (immature) July 28/71 U.B.C.Physiology (fish laboratory) Freshwater (temp. 10DC) k m 11 f Hatchery-raised in freshwater Age 3 years III Grilse (very immature) December 10/71 Fisheries Research Board, West Vancouver (outside tank) Seawater for 7 months (salinity range 28-30 ppt, temp. 9 °C) 5 m 5 f Hatchery-raised in freshwater until smolt stage when adapted to sea water Age 1 year -128-C. Plasma Calcitonin Levels in Spauning Adult Sockeye, Coho and Chinook Salmon This section is a summary of the plasma calcitonin levels obtained from spauning sockeye, coho and chinook salmon. Details on the dates and locations of the collection of these salmon are found in Chapter I and in Chapter IU, Sections A and B. Plasma electrolytes and physical measurements uere obtained for each of the three species. Ultimobranchial gland calcitonin con centrations uere measured for several coho and chinook salmon. Blood sampling and handling techniques uere previously described in General Materials and Methods. -129-Results A. Migration of Chilko Sockeye Salmon Chilko Migration 1971 The dramatic changes in morphology of the migrating Chilko sockeye salmon are illustrated in Plates 7 and 8, pg.130,and Plates 9 and 10, pg. 131. The seawater sockeye (Plate 7) have olive-green backs, silver sides and white bellies. The sexes at this stage are indistinguishable upon external examination. Plate 8 shows the sockeye 3-4 weeks later on arrival to Chilko Lake. They have lost the silver colour from their sides and now have a reddish appearance. The secondary sexual characteristics such as the hooked snout and hump back in the male are beginning to develop. In the spawning condition, after approximately 2 months in fresh water, the sexes are clearly distinguishable by the secondary sexual characteristics in the male (Plates 9 and 10). Both the male and female have brilliant crimson backs, black bellies and green heads and tails. The male has developed a cartilaginous hump and a hooked snout. The anterior teeth are much larger than those of the female and are now firmly attached to the jaw bones. The spawn ing male is generally larger in size than his mate, for the body shape of the female changes little from the seawater condition. Table XVIII, pg.132,presents physical parameters and plasma measurements for the male and female sockeye at the 3 stages of the migration. Plasma electrolyte and calcitonin levels in these same fish are shown in Table XIX, pg. 133 . -130-Plate 8. Freshuater arrival Chilko sockeye (Male above, female below). Plate ID. Spawning female Chilku sockeye Table XVIII. Physical and Plasma Measurements - Chilkc Migration 1971 Parameter Sex Seawater Arrival Chilko Lake Spawning Mean i SE (n) Mean i' SE (n) Mean i SE (n) Total Weight (g) m f 2534 2506 52.4 67.5 (45) (44) 2175 1923 103.9 40.9 (26) (34) 0% 50% 100% 2780 2175 1944 1788 130.1 82.2 115.0 63.8 (14) (12) (10) (10) Fork Length (cm) m 59.2 0.3 (45) 59.8 0.8 (26) 61.5 0.8 (14) f 59.0 0.4 (44) 57.6 0.3 (33) 0% 5D% 100% 58.6 57.3 57.3 0.4 0.9 0.7 (12) (10) (10) Gonad/Somatic m 3.07 0.14 (45) 3.06 0.16 (26) 2.64 0.19 (14) Index f 4.01 0.13 (44) 10.14 0.26 (34) 0% 50% 100% 14.81 8.45 1.13 0.31 1.31 0.10 (12) ( 9) (10) Haematocrit m 51 0.7 (44) 40 1.5 (26) 36 3.4 (15) (vols 90 f 49 0.8 (44) 40 0.7 (34) 0% 50% 100% 41 41 36 3.4 3.8 1.9 (12) (10) (10) Plasma Protein Cg/IDQ ml) m f 8.1 8.7 0.20 0.27 (44) (42) 5.4 6.6 0.19 0.12 (26) (34) 0% 50% 100% 2.9 4.2 4.2 2.0 0.36 0.53 0.61 0.39 (15) (12) (10) (10) Plasma m 90.6 0.19 (44) 93.1 0.19 (26) . 95.5 0.34 (15) % H20 (g/lDOg) f 90.0 0.26 (42) 92.0 0.11 (34) 0% 50% 100% 94.3 94.2 96.4 0.55 0.57 0.37 (12) (10) (10) Table XIX. Plasma Electrolyte and Calcitonin Levels - ChilkD Migration 1971 Plasma Measurement Sex Seauater Arrival Chilko Lake Spauning Mean i SE (n) Mean i SE (n) Mean + SE (n) Calcium m 6.9 0.12 (MO 5.6 0. 11 (26) 4.1 0 .24 (15) mEq/l f 9.2 0.29 (MO 8.7 0. 18 (34) 0% 7.3 0 .84 (12) 50% 7.0 0 .81 (10) 100% 4.0 0 .51 (10) Phosphate m 7.6 0.24 (MO. 7.1 0. 19 (26) 5.8 0 .29 (15). mEq/l f 7.B 0.27 (MO 6.8 0. 20 (34) 0% 6.7 0 .52 (12) 50% 6.2 0 .50 (10) 100% 4.9 0 .23 (10) Sodium m 164 1.1 (43) 151 1. 5 (26) 140 2 .9 (15) mEq/l f 159 0.8 (38) 151 0. 7 (34) 0% 50% 133 143 6 6 .1 .1 (12) (10) 100% 130 3 .6 (10) Potassium m • .7 0.08 (43) 1.4 0. 92 (26) 1.2 0 .38 (15) mEq/l f •.a 0.10 (38) 1.9 0. 40 (34) 0% 50% 0.9 0.6 0 0 .26 .14 (12) (10) 100% 0.9 0 .17 (10) Plasma m 117 34 (45) 12 12 (25) 141 29 (14) Calcitonin pg/ml f 545 136 (44) 687 112 (34) 0% 1649 240 (12) 50% 709 330 ( 5) 100% 306 105 ( 9) I t-1 UJ I -134-LJater samples, callected at the same location and time as the collection of the Chilko sockeye, uere analysed for calcium, sodium and potassium concentration (Table XX, pg.134). According to Reid (1961), phosphorus is a trace element in seauater uhere its concentration ranges from 0.0001 to 0.01 rng/lDQ ml, depending upon many factors. Freshuater contains 0.001 to 0.003 mg/100 ml, uhile even phosphate "rich" freshuater contains less than 0.03 mg/100 ml. Table XX. Uater Analysis - Chilko Migration 1971 Date Location Depth . (feet) Calcium July 23, 1971 Lummi Island 15-20 (seauater) Seauater Ions (mEq/l) Sodium Potassium 15.6 350 7.65 August 6,1971 1-5-20 16.9 391 B.50 September 21, ChilkD River 1971 Spauning Grounds (freshuater) 0.17 Plasma calcitonin changes in the sockeye at the 3 stages of migration are illustrated in Figure 18, pg. 135 . It is readily apparent that the females maintained higher circulating levels of calcitonin than the males, at all stages of the migration. The CT levels of the females increased significantly from sea to freshuater up to the 0 percent spauning stage, falling off pre cipitously after spauning. The male CT levels decreased to 12 1800 r-1500 E X 2l200 o ^ 900 o O o | 600 o CL 300 Plasma Calcitonin 34 44 44 T 25 Seawater Arrival Chilko Lake 14 1 50 Spawning WW Male •Z] Female 0 -I % Spawning 50 V Condition 100 J of Female Figure 18. Plasma calcitonin changes in migrating Chilko sockeye. Ul U*l i -136-extremely lou levels on arrival to Chilko Lake and then increased at spawning. In order to relate the plasma calcitonin concentrations of the sockeye with ultimobranchial gland calcitonin concen trations, the UB gland calcitonin contents of 6 seawater females were compared with those of 6 0% spawning females. Table XXI, pg.137 summarizes the physical measurements, UB gland calcitonin contents and plasma CT"levels in these 2 groups of female sockeye. The data show that there were significant increases in the GSI (p< 0.001) and plasma calcitonin levels (p< 0.001). Uhile the UB gland calcitonin content increased from 35.81 to 55.63 Units per gland, the increase was not statistically significant due to the variation in the data. Plasma electrolyte changes in the sockeye throughout the migration are illustrated in Figure 19, pg.138 . The plasma sodium, phosphate and calcium levels decreased gradually through out the migration in both sexes. The total plasma calciums in the females were significantly (p< 0.001) higher than the males at all stages in the migration except the spawned out females. Plasma potassium levels rose on arrival to Chilko Lake and fell with spawning. Figure 20, pg. 139 ,shows the plasma calcium, plasma cal citonin and GSI changes throughout the migration. It can be seen that as spawning time approaches, the female gonads grow rapidly and that on spawning as the eggs are shed, the gonad-somatic index falls off. The GSI of the 0 percent spawning females was 269.3 percent higher than the seawater females. A decrease in the Table XXI. Physical Parameters, Ultimobranchial Gland and Plasma Calcitonin Levels in Migrating Female Chilko Sockeye (1971) Group Location and Total Lilt Calcitonin Plasma Calcitonin Level Date Fish # (g) GSI Content „, U/gland pg/ml mU/ml Seauater Females Lummi Island Seauater August 6/71 94 96 1DD 101 103 105 3019 3043 2099 2626 2541 2028 4.79 3.57 3.50 3.24 3.25 4.87 5.5 72.4 68.5 23.9 21.0 23.6 178 379 0 0 0 1090 0.89 1.90 0 0 0 5.45 n = mean = SD = SE = 2559.33 396.70 177.41 6 3.87 0.69 0.31 a 6 35.81 25.28 11.30 6 274.50 389.72 174.29 a 6 1.37 1.95 0.87 a Spauning Chilko River 69 2391 14.88 34.9 2591 12.96 Females Spauning Grounds 70 2604 14.80 55.0 1466 7.33 (0%) 71 2455 13.01 55.7 1406 7.0September 22/71 72 2044 15.24 40.9 2089 10.45 76 1947 16.05 71.0 1290 6.477 1769 13.96 75.7 1433 7.17 n = 6666 6 6 mean = 2201.66 14.66 55.53 1712.50 8.57 SD = 299.66 0.96 14.64 469.90 2.35 SE = 134.01 0.43 6.54 210.14 1.0t-test probability seauater vs. spauning a. p<0.001 * Plasma CT biological activity based on salmon CT specific biological activity of 5000 MRC U/mg. -138 o co E o 0. LU £ £ 8.0 o — 0. o E o o B 44 44 |piosma Phosphate I 2S 34 12 -tl 10 0 50 H Male Female 0 196 Spawning 50 > Condition IOOI of Females Sea Water Fresh Water Spawning Fresh Arrival Water Figure 19. Plasma electrolyte changes in migrating ChilkD sockeye. -139 o 10 E Gonad/Somatic Index 44 111 Plasma Calcium e 6 E -44 -fl 45 • B 'z 10 ito l°0 1500 1 \ Sl200 Plasma Calcitonin <»<* Li Se<"""ef ChMKoTa'ke Spawnin, Male CZ3 Female O i % S[ 50 Ct 100 ) ot % Spawning Condition Females igure 20. Plasma calcitonin, plasma calcium and gonad-somatic index changes in migrating Chilko sockeye. -140-male GSI of 14.0 percent from seawater to spawning, is evidence vthat some males had partially spawned before capture. Changes in plasma percent water, plasma protein and haematocrit are illustrated in Figure 21, pg. 141 . The data in dicate that the plasma percent water increased throughout the migration with the males having higher readings than the females, except for the spawned out females. Plasma protein levels in both sexes decreased during the migration. The females had con sistently higher levels than the males except for the spawned out females. The haematocrits of both males and females decreased throughout the migration. Chilko Migration 1972 c Physical measurements, haematocrit, plasma protein and percent water for the sockeye sampled in.the 1972 Chilko sockeye migration study are presented in Table XXII, pg. 142 . Plasma electrolyte levels are shown in Table XXIII, pg. 143 . There was close agreement between the data for the 1971 and 1972 migrations. Serum total calcium, serum ionic calcium, percent ionic calcium, plasma protein and serum pH measurements are presented in Table XXIV/, pg„ 144 . Serum pH of the seawater sockeye was not measured. Changes in serum total and ionic calcium are illustrated in Figure 22, pg. 145 . As stated previously, serum ionic calcium levels were measured at a water temperature close to that in which the fish were originally captured. Table XXV/, pg. 146 , shows temperature readings both on location at the time of capture and of the ion electrode water jacket in the laboratory. 97.0 96.0 95.0 o o \ 940 o» w Q> 93.0 O 3 9 2.0 a £ o 91 .0 a. 90.0 85.5 10.0 E O 8.0 O N O) 6.0 e 4.0 2.0 0.0 Percent Water 142 Plasma Protein 34 15 T rh 12 10 50 12 10 15 | I [*1 10 m •HE 50 DH Male • Female O 50 100 . % Spawning 1 Condition of Females * o > O o CT E (U o I Sea Water Freshwater Spawning on Arrival Freshwater at Spawning Grounds Figure 21. Haematocrit, plasma protein and plasma percent water changes in migrating Chilko sockeye. Table XXII. Physical and Plasma Measurements - Chilko Migration 1972 Parameter Sex Seawater Mean SE (n) Arrival Chilko Lake Mean SE (n) Mean Spawning + SE (n) Total Weight (g) m f 2672 2433 192.8 94.5 (10) (12) 2667 2011 114.5 81.9 (11) (10) 2743 0% 2152 100% 1853 118.3 37.8 93.6 (10) (10) (10) Fork Length (cm) m f 60.7 59.4 1.8 0.8 ( 6) (12) 63.3 58.6 0.6 (11) 61.9 0.6 (10) 0% 58.5 100% 59.0 0.7 0.3 0.5 (10) (10) (10) Gonad/Somatic m Index f 2.42 3.98 0.22 0.24 (10) (11) 2.77 9.90 0.14 ( 9) 0.75 ( 9) 0% 100% 1.79 15.36 D.17 0.30 (10) ( 9) Haematocrit (vols %) m f 50 48 2.7 2.1 ( 9) (10) 37 40 1.0 1.2 (10) (10) 0% 1D0% 36 40 40 1.5 1.7 4.2 (10) (10) (10) Plasma Protein (g/100 ml) m f 7.6 8.6 0.26 0.35 ( 8) (11) 5-1 6.9 0.17 (10) 0.17 (10) 0% 100% 3.1 4.8 2.1 0.43 (10) 0.30 0.28 (10) (10) Plasma % m 91.2 0.22 ( 8) 93.4 0.14 (10) 95.3 0.42 (10) H„0 f 90.1 0.33 (11) 91.7 0.17 (10) 0% 93.7 0.30 (10) (g/lOOg) 1QD% 9S'3 D'26 (1Q) Table XXIII. Plasma Electrolyte Levels - Chilko Migration 1972 Seawater Arrival Chilko Lake Spawning Electrolyte . Sex Mean i SE (n) Mean - SE (n) Mean - SE (n) Serum Total Calcium mEq/1 m f 7.3 11.2 0.17 0.36 (10) (10) 5.7 12.7 0.14 0.30 (11) (10) 0% 100% 4.9 8.2 4.5 0.33 0.32 0.29 (10) (11) ( 9) Plasma Phosphate m ld.l 0.48 (10) 7.0 0.26 (10) 6.1 0.17 (10) mEq/1 f ID.5 0.37 (11) 7.5 0.14 (10) 0% 100% 6.3 5.8 0.48 0.44 (10) (10) Plasma Sodium m 165 1.9 (10) 138 8.5 (10) 140 6.3 (10) mEq/1 f 163 2.0 (11) 127 8.3 (10) 0% 100% 151 133 3.4 8.0 (10) (9) Plasma Potassium m 0.4 0.06 (10) 2.3 0.23 (10) 1.1 0.23 (10) mEq/1 f 0.5 0.04 (11) . 1.8 0.23 (ID) 0% 100% 1.3 1.3 0.24 0.26 (10) ( 9) Tabla XXIV. Ionic and Total Serum Calcium, Serum pH and Plasma Protein Changes in Migrating Chilko Sockaye (1972) Seauater Arrival Chilko Lake Spauning Sex Total Ionic % Plasma Total Ionic % Plasma Serum Total Ionic % Plasma Serum Serum Serum Ionic Protein Serum Serum Ionic Protein pH Serum Serum Ionic Protein pH Ca Ca Ca g/100 Ca Ce Ca g/100 ml Ca Ca Ca g/100 ml mEq/i mEq/1 ml mEq/l mEq/1 mEq/1 mEq/i rn n _ 10 e 10 10 c S 11 - 11 11 _ 10 c 11 10 8 8 10 10 mean B 7.25C 3.06 42.2C 7.6a 5.65c 3.02 53.1.C 5.1c 7.329 1..87 2.75 59.1. 3.1 7.515 SD m 0.51 0.30 2.07 0.70 0.50 0.18 2.56 0.51 0.069 0.99 0.31. 7.29 1.33 0.130 SE o 0.17 0.09 0.68 0.26 0.11. 0.05 0.61 0.17 0.022 D.33 0.13 2.75 0.1.3 0.031 f n m 10 10 10 11 10 10 10 10 10 056 , • 11 11 11 10 11 mean - 11.23 3.16 28.2 6.6 12.73 3.17 25.0 6.9 7.282 8.23 3.01. 37.1. 1..8 7.389 SD m 1.08 0.32 2.31. 1.11. 0.91. 0.17 1.73 0.56 0.080 1.02 0.16 1..96 0.91. 0.11.1 SE m 0.36 0.10 0.76 0.36 0.30 0.05 D.58 0.17 0.027 0.32 0.01. 1.56 0.30 0.GU - - 100% 9 7 7 ID 10 (..1.7 2.1.5 57.9 2.1 7.551. 0.83 0.33 1..66 0.85 0.211. 0.29 0.13 1.90 0.28 0.C70 Total 20 18 is 20 21 6.5«.b 2.B1 1.5. l.b 3.5 7.1. £8 Spauning 2.09 0.38 11.11. 1.61. 0.197 0.1.8 0.09 2.70 0.37 0.031 Females t-test probability male vs. female a. p< 0.050 b. p< 0.010 c. p< 0.001 Note: pH uas not measured on seauater serum samples. -F" -F" 12.0 -i 10.0 -8.0-N cr LU t= 6.0-E •D 2. 4.0 -o O E 2 2.0-<o CO 0.0 J 10 I 10 10 Sea Water Fresh Water Arrival 10 \ Total Serum Calcium Serum Ionic Calcium II II cr* 0%9 I00%9 * Spawning Figure 22. Serum ionic and total calcium changes in migrating Chilko sockeye (Chilko Migration 1972). Note constancy of ionic serum calcium levels. -p-ui i -146-Table XXV. Uater Temperatures - Chilko Migration 1972 Temperature of Uater On Location Ion electrode (surface uater jacket temperature). July 21, 1972 Lummi Island 14.5°C 12°C (seauater) August 28, 1972 Chilko Lake (freshuater) 14.4°C 13.5°C September 24, 1972 Chilko River (freshuater) 10.6°C 12.5°C It can be seen from the data that although serum total calcium levels markedly changed throughout the migration, the serum ionic calcium levels remained relatively constant. The spaun ing males shoued a slight but significant (p<Q.D5) decline in ionic calcium from the arrival level. Female serum ionic calcium remained stable until spauning uhen there uas a significant de crease (p<0.DDl) to the 100 percent spauned level. Since the total serum calcium fell dramatically and the ionic serum calcium remained fairly constant, there uas a marked increase in percent ionic calcium from seauater to spauning in both sexes. Although the female total calciums uere significantly higher than those of the male (excepting the spauned out females), the Collection Location Date -147-ionic calcium levels of bath sexes uere very similar. It is emphasized that the serum ionic calcium levels uere the most stable of any of the plasma electrolytes measured and that these levels were maintained despite a marked increase in plasma percent uater and decrease in haematocrit during the migration. There uas a marked decline in plasma protein throughout the migration and a rise in serum pH from arrival to spauning in both males and females. Calcium and phosphate changes in the soft tissues (skin, muscle, gonads) and hard tissues (vertebrae, scales, premaxillae) of the migrating Chilko sockeye are presented in Table XXVI, pg. 148, and Table XXVII, pg. 149, respectively. These same results are illustrated in histogram form in Figures 23, 24, pages 150, 151 , (soft tissues) and Figures 25, 26, pages 154, 155, (hard tissues). The soft tissue mineral contents are presented as mg calcium or phosphate per lOOg fat-free dry ueight (FFDLd). The hard tissue mineral contents are presented as g calcium or phosphate per lOOg ash ueight. Only the premaxillae calcium and phosphate contents are expressed as gCa and PO^ per lOOg dry ueight since ash ueights for these bones uere not available. As illustrated in Figure 23, the skin had the greatest concentration of calcium (range 76-192 mgCa/lOOg FFDW) of the soft tissues. The female gonads also contained large amounts of calcium (150-175 mgCa/lOOg FFDLd) and had 6 to 17 times more calcium than the male gonads. The latter possessed the louest amounts of calcium (10-26 mgCa/lOOg FFDW) of the soft tissues. Measurements for the muscles ranged from 22 to 68 mgCa/lOOg FFDW. In comparing Table XXVI. Soft Tissue Mineral Changes - Chilko Migration 1972 ARRIVAL CHILKO LAKE SPAUNING Tissue Sex (n) % Ash Ut. FF Dry Ut Xean - SE mo PO^ mq Ca lOOg FF Dry lilt. lOOg FF Dry Ut Mean - SE [Mean - SE % Ash Ut FF Dry Ut. Mean - SE . rag PO^ 100 FF Dry Ut Mean i SE mq Ca lOOg FF Dry Ut Mean ± SE i i % Ash Ut 1 mq PO, nq Ca FF Dry Ut | lOOg FF Dry Ut lOOg FF Dry Lit Mean - SE ;Mean - SE Mean t SE Gonads m 9 T 7 14.49 0.71 2667.5 121..63! 9.9 0.61. 3.92C 0.15 805.1° 44.49;i61.8C 6.03 13.90 0.26 4.02° 0.17 3551.0 73.9 | 13.1 0.86 949.3C 28.3 ! 175.DC 8.22 ! 16.46 O.BSj3661.8 140.2 26.4 3.63 0% 3.95G 0.20 ; 856.0G 45.4 150.1C 6.82 Muscles m 7 f 9 7 5.56 0.33 857.7 79.21.! 48.1 5.61 1 5.07 0.26 908.2 1.0.98: 68.3a 7.57 I | 4.61, 0.45 lD6U'5 37-1U\ 22-\ X-B1 5.59 0.19 UQ7-7 21-67! 31-9 U98 1 | 5.05 D.23.1D08.6 38.07 43.4- 3.05 0* 5.83 0.27.1069.5 42.77 33.3 3.65 10054 5.36 0.49 1075.9 90.38 31.2 2.52 Skin ra S f a 9 1.67 O.H. 1,22.1, 21..71:11.1.7 27.30 1.63 0.22 1.23.7 59.93 176.3 36.19 i ! i !2.82 0.08 2.80 0.13 598.5 17.17 549.8 61.52 166.7 24.21 192.7 52.45 547.0 30.05 78.0.. 10.57 0* - - ' ! 547.2 16.70 75.9 6.10 10056 - 528.6 14.65 110.0 12.SO i t-test probability male va. female a. p< 0.05 b. p< 0.005 c. p < 0.001 Tabla XXVII. Hard TIBBUB Mineral Changea - Chilko Migration 1972 ARRIVAL CHILKO LAKE SPAWNING Tissue Sax (n) % Ash Ult Dry Dt' Mean t SE 9 P0fc 100 g Ash Mean SE q Ca 100 g Ash Mean - SE 96 Aah tilt Dry Ut Mean - SE gPO,, lOQg Ash Mean i SE 0 CB 100 g Ash Mean - SE % Ash tilt I g PO^ j q Ca Dry Wt | lOOg Ash j lOOg Ash Mean i SEJMean . i SE|Mean - SE Scales m 7 f 6 9 36.01 0.75 34.018 0.3S 17.65 0.25 17.65 0.20 36.22 0.53 35.64 ^ 0.42 26.79 0.94 26.43 0.67 19.51 0.29 19.62 0.48 32.65 0.64 32.81 0.31 23.53 D.63 0% 24.77 0.68 100% 24.87 0.86 20.29 0.19^ 32.03 0.49 20.36 0.40'32.94 0.39 19.59 0.47!32.14 0.79 i Vertebrae ra 9 f 9 9 31..17 0.33 18.69 0.34 34.07 0.40Jl8.81 0.29 i 1 ! 35.58 0.53 36.19 0.23 38.15 0.87! 18.51 0.91 39.75 0.52; 18.18 0.06 37.11 0.39 36.21 0.48 41.65 0.52 0% 42.54 0.26 100% 45.06 0.64 18.21 0.D5 38.08 0.17 18.04 0.09 ! 36.81 0.62 18.12 0.04 ; 36.10 0.58 i t-test probability male us. female a. p< 0.05 Chilko Migration 1972 -150 <$ 9 d* 9 o* 90% Seawater Chi|ko ^ Spawn,ng Figure 23. Soft tissue calcium changes in migrating Chilko sockeye. -151 Chilko Migration 1972 Muscle Skin o g v 450001 2 a. 4000.0-6 • n i Gonads cf 9 & 9 c* 90% c „,„ Arrival _ Seawa,er Chilko Lake Spawn,n9 Figure 2k. Soft tissue phosphate changes in migrating Chilko sockeye. -152-the 3 soft tissues, the skin showed the greatest variation in calcium content (as indicated by the SE bars) while the muscles showed the least. In the females, the muscle calcium content decreased significantly (p< Q.uul) from a high of 68 mgCa/lQOg FFDW in the sea to 32 mgCa/lOQg FFDU on arrival, at which level it remained stable. The male muscle calcium also fell significantly (p< •.••5) from 48 mgCa/lQDg FFDU in the sea to 23 mgCa/lDOg FFDU on arrival. Spawning males however, had a significantly (p<Q.u01) higher level than the arrival males as the muscle calcium content again rose to the seawater level. In comparing the muscle calcium content for both sexes, the females had significantly higher levels in the seawater and arrival groups while the males had significantly higher measurements in the spawning group. The calcium content of the skin for both male and female sockeye increased slightly on arrival to Chilko Lake. The female skin contained slightly mare calcium but the difference was not statistically significant. In both sexes, the skin calcium content decreased (male p< 0.005, female p<D.Q25) from arrival to spawning. Spawned out females had significantly (p<0.05) higher skin calcium contents than the 0% spawning females. As expected, the gonads Df the female sockeye contained significantly (p< •.••!) greater amounts of calcium than the males at all stages of the migration. The female level rose slightly from sea to arrival then fell (p<D.D5) with spawning. In contrast, the male gonad calcium content increased significantly both from sea to arrival (p<0.01) and from arrival to spawning (p<0.DD5). -153-Ldith regard to soft tissue phosphate (Figure 24), the male gonads contained the greatest concentration (2867 - 3862 mg P0^/100g FFDLd), the skin contained the least (422 - 547 mg PO^/lOOg FFDLd) and the muscles contained intermediate amounts (858 - 100 mg P0^/100g FFDLJ). Only in the case of the gonads uas there a sex difference in the soft tissue phosphate contents. The muscle phosphate level in the male increased slightly (p< 0.05) from sea to arrival and remained constant from arrival to spauning. The female level also rose (p< 0.001) from sea to arrival, remaining stable throughout spauning. The male skin phosphate content increased (p< 0.001) from sea to arrival. The spauning level uas not significantly different from the latter. The female skin phosphate content did not change significantly from sea to spauning. Muscle and skin phosphate contents of both male and female sockeye falloued the same basic pattern throughout the migration. The male gonads exhibited a dramatic increase (p < 0.001) in phosphate content from the sea to arrival uith a further slight increase on spauning. The testes contained 3-4 times more phosphate than the ovaries. Gonad phosphate content in the female increased (p<0.05) from sea to arrival and then returned to the seauater level in the 0 percent spauned female. In the hard tissues, it should be noted that uhile the vertebrae increased in mineral content (% ash/dry ueight) through out the migration, the scales demineralized. The mineral content of the male vertebrae increased (p< 0.001) from 34.17% in the sea to 41.65% at spauning. The female vertebrae increased (p< 0.001) Chilko Migration 1972 38. 0- Scales 34.0 • 4 30.0 O O \ o o o> 38.0-1 34.0 30.0 Vertebrae cf 9 Seawater d* 9 Arrival Chilko Lake cf (J0% 9100% Spawning Figure 25. Hard tissue calcium changes in migrating Chilkc sockeye. -155-Chilko Migration 1972 21.0 i 19.0 • 17.0-< O o 15.0 • a. Scales Vertebrae 19.0-17.0-15.0- cr 9 Seawater cf 9 Arrival Chilko Lake iii 1*1 Cf £O%CMOO% Spawning Figure 26. Hard tissue phosphate changes in migrating Chilko sockeye. -156-from the seauater value of 34.07% to 46.06% in the spawned out females. In the male, the scale mineral content decreased. (p< 0.001) from 36.01% in the sea to 23.53% at spawning while the female level decreased (p< 0.001) from 34.01% in the sea to 24.87% in the spawned out females. Thus the scales of the male sockeye lost more mineral throughout the migration than did those of the female, whereas the female vertebrae mineralized to a greater extent than the males. This relationship is shown in Figures 27 and 28, pages 157 and 158 . As seen in Figure 25, the calcium content of the male vertebrae increased from sea to arrival (p<0.05) and further from arrival to spawning (p< 0.05). In the female, there was no significant change throughout the migration. A comparison of the vertebrae calcium content for both sexes reveals significantly (p<0.05) higher calcium levels for the males than for the females in the spawning condition. As well as losing mineral, the calcium content of the scales of both male and female sockeye decreased markedly during the freshwater migration. In the male, scale calcium decreased from sea to arrival (p< 0.005) with a further decrease at spawning. The female level also decreased significantly (p< 0.001) after the first stage of the migration but did not change with spawning. As shown in Figure 26, the vertebrae phosphate content of bath sexes decreased slightly with migration but the decrease was significant only between the female seawater and spawning vertebrae (p<0.05). In contrast, the male and female scale phosphate content -157 i Chilko Sockeye Migration 1972 Cf 9 Seawater cf 9 Arrival Chilko Lake Cf C)0% Q ioo% Spawning Figure 27. Vertebrae mineral content changes in migrating Chilko sockeye. Chilko Sockeye Migration 1972 ft ft cf 9 Seawater cf 9 Arrival Chilko Lake cf 90% 9 ioo% Spawning lire 28. Scale mineral content changes in migrating Chilko sockeye. -159-showed a marked increase through the migratory stages. In the male, this increase uas significant from sea to arrival (p< 0.001) and from arrival to spawning (p<0.05). The female scale phosphate content also increased significantly (p< •.•Ol) from sea to arrival but there was little change with spawning. Tchernavin (1937; 1938a; 1938b) has documented the phenom enal growth in the jaw bones of the spawning Atlantic salmon. This "breeding growth" as Tchernavin termed it, is particularly evident in the premaxillae (Uladykov, 1962). In males, the premaxilla practically doubles in length from the seawater to the breeding stage. The increase in size is due not only to the growth of the bone itself, but also to its fusion to an ossified plate which develops as a support base for the large breeding teeth (Tchernavin, 1938a). Table XXV/III, pg. 160 , summarizes the dry weights, calcium and phosphate contents (g/lOOg dry weight) of single premaxillary bones taken from Chilko sockeye at each stage of the migration. Figure 29, pg. 161, illustrates the changes in dry weight of the premaxillae. The marked increase in weight for the spawning male reflects the extensive snout and jaw growth. Calcium and phosphate content of the premaxillae are illustrated in Figures 30 and 31, pages 162 and 163 . The spawning male had significantly (p<0.005) more calcium and phosphate (g/lOOg dry wt.) than the spawning female. In order to determine the actual amounts of tissue calcium and phosphate per fish, the tissues of two freshwater arrival Chilko sockeye salmon (male and female) were dissected out, dried Table XXVIII. Ory Weights, Phosphate and Calcium Contents of thB Premaxilla Bona -Chilko Sockeye Migration (1972) SEAWATER ' ARRIVAL CHILKO LAKE . SPAWNING Sax Ory Ut Mean - SE(n) gCa Dry Wt Mean ± SE(n) gCa Dry Wt Mean - SE (n) gpo,, gCa lOOg Dry Wt Mean i S£(n) lOOg Dry Wt Mean i SE(n) lOOg Dry Wt Mean - SE (n) lOOg Dry Ut Mean - SE(n) lOOg Dry Ut Mean i SE (n) lOOg Ory Ut Mean t SE(n) IB f 54.10 10.29(5) 37.92 8.10(7) 4.75 0.32(5) 4.30 0.13(5) 7.37 0.15(5) 8.41 0.46(5) 75.49b 7.38(11) 40.34 2.36(10) 6.63 0.32(11) 6.01 0.32(9) 12.19 0.56(11) 11.40 0.62(10) 213.51b 33.52(10) 0% 56.11 6.90(10) 100% 75.34 7.10 (8) 8.82a 0.32(10) 7.60 0.17(10) 7.50 0.41 (8) 16.073 0.56(10) 13.72 0.35(10) 14.14 0.76 (3) Total sp.f 64.66 5.35(18) 7.55 0.20(18) 13.91 0.33(16) t-teet probability male vs. femala a. p< 0.005 b. p< 0.001 cn o ure 29. Premaxillae dry weight increases i migrating Chilko sockeye. -162-Chilko Migration 1972 170 n Arrival Chilko Lake Spawning Figure 30. Premaxillae calcium content changes in migrating Chilko sockeye. Chilko Migration 1972 10.0 i 8.0 -ft r-, JZ .? 6.0 v Q o> O 2 4.0 \ o a. 2.0 0.0 cf 9 Seawater cf 9 Arrival Chilko Lake cf 90% 9100% Spawning Figure 31. Premaxillae phosphate content changes in migrating Chilko sockeye. -164-and ujaighed. The percent dry ueight organ (g)/total body uet ueight fish (g) uas calculated (Table XXIX, pg. 164). Data from a human cadaver uas inserted in the table for comparison. Table XXIX. Percentage Dry weights of Tissues % dry ut /total body uet ut Total Body Wet Wt (Kg) Skin Muscle Gonads Scales Vertebrae Total Skele ton ** male sockeye 2.540 1.693 24.399 0.5B1 0.079 0.736 2.669 female sockeye 2.526 1.742 21.770 3.486 0.071 0.732 2.514 human* 70.55 2.758 6.464 - 10.119 * Data taken from Mitchell e_t a_l, 1945. ** Total skeleton in sockeye includes vertebrae, ribs, tail, fin bones, gill apparatus and skull bones. It is apparent from the table that the percent dry ueight/total body uet weight for the skin is slightly higher in the human than in the fish. The sockeye have a much greater percent of muscle and a smaller percent of bone than the human. The higher percentage weight of the human skeleton compared to the salmon skeleton, probably reflects the supportative function of the human skeleton (Broun, 1957). From the above dissection data and that given in Tables -165-XXVI and XXVII, pages 148, and 149 , it uas possible to calculate the absolute amount of calcium and phosphate in each tissue of a male and female freshuater arrival Chilko sockeye (Table XXX, pg. 166). The absolute ueight of the fish as uell as the in dividual tissue weights changed during the migration. For example, it has been shoun that salmon muscle and viscera ueights decrease during migration while there are obvious increases in the absolute ueights of the gonads and skeleton (Greene, 1926; Idler and Tsuyuki, 1958; Idler and Bitners, 1958). Therefore, the calcul ations would be different for seawater and spawning salmon. As expected, the major storage of calcium for the sockeye occurred in the hard tissues whereas the soft tissues contained slightly more phosphate. In the human, body calcium amounts to 1.5 - 1.6% body weight (Mitchell e_t al, 1945; Copp, 1970b) whereas in the sockeye, body calcium (total skeleton plus the 3 soft tissues measured) was approximately 0.42 - 0.44% body weight. The percent calcium and phosphate in the human skeleton is 1.58% and 0.72% respectively (Mitchell et_ al, 1945). Corresponding percentages in the sockeye skeleton were 0.40 - 0.43% for calcium and 0.21 - 0.23% for phosphate. In summary, because of its great bulk, the muscle tissue contained the largest stare af calcium and phosphate of the 3 soft tissues examined. The female gonads contained 60 times more calcium and slightly more phosphate than the male gonads. Due to their very low weight, the scales contributed little to the total storage of calcium and phosphate in the hard tissue. The scales did, however, contain mare calcium than any of the soft tissues measured. -166-Table XXX. Calcium and Phosphate Content in Tissues of Average Chilko Freshwater Arrival Sockeye* Tissue Calcium (g) per tissue per fish male female Phosphate (g) per tissue per fish male female Soft Tissues Skin Muscle Gonads 0.075 0.148 • .002 • .•68 • .140 0.122 0.270 6.927 0.550 0.193 4.850 0.664 Total Soft Tissue** 0.225 0.330 7.747 5.7Q7 Hard Tissues Scales Vertebrae Premaxilla • .173 2.159 • .•19 0.158 2.121 • .••9 • .103 1.077 0.010 0.095 1.067 0.005 Total Hard Tissue*** 11.383 8.112 6.086 4.318 •Mean total wet weight: FUA male = 2667g, FLdA female = 2011g **Total soft tissue includes only skin, muscle and gonads. **Total hard tissue includes all bones plus scales. -167-B. Plasma Calcitonin Levels in Coho Salman: Effect of Sexual Maturation and Environmental Salinity Physical parameters and plasma measurements of the 3 groups of CDho salmon are presented in Table XXXI, pg. 168 , and their respective electrolyte and calcitonin levels are shown in Table XXXII, pg. 169 . The GSI, plasma calcitonin and plasma calcium levels for the 3 groups of coho are illustrated in Figure 32, pg. 170 . As shown by the ganad-somatic index the adult coho were very sexually mature while both the coho grilse and freshwater coho were sexually immature. Plasma calcium levels were lowest in the immature freshwater coho and highest in the spawning adult coho. The coho grilse, even though they were living in seawater had lower plasma calcium levels (calcium level = 17.6 mEq/litre) than the freshwater adult spawning coho. Although plasma calciums in the females are slightly higher than in the males in each of the 3 groups, the differences are not statistically significant. Plasma calcitonin levels for the adult spawning coho were markedly higher than the other two groups while the lowest levels were measured in the freshwater immature coho. A sex difference was noted only in the case of the adult spawning coho where the females had significantly higher (p<0.001) plasma calcitonin levels than the males. The mean plasma calcitonin level.of 4 coho "jacks" (2 year old, sexually ripe males, mean total wt. = 409 - 107g) was 2,393 - 754 pg/ml and was thus higher than the level found in the adult spawning coho (1,070 - 294 pg/ml). Table XXXI. Physical and Plasma Measurements - Coho Salman Study Parameter Grilse Seauater Immature Freshuater Spauning Adults Freshuater Sex Mean i SE (n) Mean i SE (n) Mean - SE (n) Total Weight m • .19 • .•21 (5) • .46 • .134 Ck) 5.17 • .23 (15) (Kg) f • .18 • .••6 (5) • .50 • .•58 (ID) 4.36a • .22 (15) Fork Length m 25.3 l.D (5) 35.1 2.7 Ck) 77.8 • .9 (15) (cm) f 25.7 • .3 (5) 36.7 1.5 (10) 72.5b 1.1 (15) Gonad/Somatic m • .•3 0.00 (5) • .5 • .21 Ck) 4.7 • .•• (12) Index f 0.2SC • .•4 (5) • .9 • .14 (10) >15.0 Haematocrit m 44 3.8 (5) . 19 2.5 Ck) 47 1.4 (15) (vols %) f 45 3.8 (5) 12a 1.3 (10) 46 1.9 (15) Plasma Protein m 5.7 • .68 (5) 3.6 • .31 Ck) 6.4 0.23 (15) (g/100 ml) f E>.k 1.29 (5) 3.6 • .ID (10) 6.5 0.38 (15) Plasma m 92.8 • .6 (5) 95.0 • .3 Ck) 92.1 0.2 (15) % H20 f 92.2 1.2 (5) 94.8 • .1 (10) 92.1 0.4 (15) (g/lOQg) t-test probability male us. female a. p<0.05 b. p< 0.010 c. p< 0.001 Table XXXII. Plasma Electrolyte and Calcitonin Levels - Coho Salman Study Plasma Measurement Sex Grilse Seauater Mean - SE . (n) Immature Freshuater Mean - SE (n) Spauning Adults Freshuater Mean - SE (n) Calcium m . 5.7 0.18 (5) 4.4 0 .11 ( 4) 6.9 0 .21 (15) mEq/1 f 6.1 0.13 (5) 4.6 0 .09 (10) 8.0 . 0 .83 (15) Phosphate m 6.2 0.68 (4) 6.0 0 .53 (4) 8.6 0 .37 (15) mEq/1 f 6.2 0.14 (5) . 5.7 0 .17 (10) 7.7 0 .42 (15) Sodium m 145 1 .6 (4) 167 1 .1 (15) mEq/1 f — 146 1 .4 (ID) 163a 1 .5 (15) Potassium m 3.8 0 .18 (4) 3.2 • .27 (15) mEq/1 f — 4.3 0 .17 (10) 3.0 0 .30 (15) Magnesium m 2.02 0 .06 (11) mEq/1 f — 1.79b 0 .03 (15) Calcitonin m 814 169 (5) 292 239 (4) 1,070 294 (13) pg/ml f 1195 394 (5) 272 158 (10) 12,081C 1305 (15) t-test probability male vs. female a. p<D.05 b. p< 0.010 c. p < 0.001 -170 <0 0 14000 12000 10000 8000 o 6000 O 2 000 0 Gonad/Somatic index I Plasma Calcitonin" it. •I • T . •i Male • Female 5 4.0 o a e CO O a. 2.0 -0.0 immature Spawning Adult Fresh Wafer Fresh Water ure 32. Plasma calcium, plasma calcitonin and gonad-somatic index measurements in 3 groups of coho salmon. -171-C. Plasma Calcitonin Levels in Spawning Adult Sockeye, Coho and Chinook Salmon This, section summarizes the plasma calcitonin levels for 3 species of Pacific salmon, sockeye, coho and chinook. It should be noted that the sockeye are the Chilko spauning sockeye salmon (•% spauning females, spauning males) from the 1971 migration study (Chapter IV, Section A). The coho are the freshuater spauning adults described in Section B of the present chapter. A description of the chinook salmon uas given in Chapter r. Physical measurements, plasma calcitonin and electrolyte levels for the 3 groups are presented in Table XXXIII, pg. 172 . The GSI indicates that all 3 species uere very sexually mature. Plasma calcitonin levels for the 3 salmon groups are illustrated in Figure 33, pg. 173 . The coho and chinook levels uere significantly higher (males p<D.DD5 and females p<Q.0Dl) than those measured for the sockeye. TableXXXIV, pg. 174, summarizes the individual fish ueights, ultimobranchial gland calcitonin content and plasma calcitonin level in coho and chinook salmon. Plasma calcitonin is presented in picograms per ml plasma and in mU per ml plasma (assuming a biological activity of 5000 U per mg for pure salmon calcitonin). It can be seen from the table that although the coho and chinook females had significantly higher plasma CT levels than the males, the UB gland, calcitonin content did not reflect this sex difference. There uas no significant difference betueen the LIB gland calcitonin contents of the coho and chinook males or females. Table XXXIII. Physical Measurements, Plasma Calcitonin and Electrolyte Levels in Adult Spawning CHINOOK, COHO AMD SOCKEYE SALMON Weight Gonad/Samatic Plasma % H„0 Plasma Plasma Electrolytes mEq/1 Species Sex Kg Index g/lOOg Calcitonin pg/ml Calcium Phosphate Sodium Potassium Mean - SECn) Mean * SE(n) Mean - SE(n) MBBn - SE(n) MB an - SE(n) MB an i SE(n) Mean - SE(n) Mean - SE(n) Chinook m 7.85 •.33(15) 4.6 0.44(15) 93.3 0.26(15) 2067 600(15) ' 5.3 0.17(15) 5.9 0.31(15) 161 1.3(15) 1.1 0.29(15) f 8.83a 0.33(11.) 25. lc . 0.87(13) 93.5 0.29(14) 13154° 2362(14) 5.Sa 0.17(14) 6.9a 0.21(14) 166a 1.1(14) 1.2 0.29(14) Coho m 5.17 0.23(15) 4.7 0.00(12) 92.1 0.2(15) 1070 294(13) 6.9 0.21(15) 8.6 0.37(15) 167 1.1(15) 3.2 0.27(15) f (..36s 0.22(15) *15.0C - (15) 92.1 0.4(15) 12081° 1305(15) 8.0 0.83(15) 7.7 0.42(15) 163a 1.5(15) 3.Q 0.32(15) Sockeye m 2.78 0.13(14) 2.6 0.19(14) 95.5 0.34(15) 141 29(14) 4.1 0.24(15) 5.8 0.29(15) 140 2.9(15) 1.2 0.32(15) f 2.18° 0.08(12) 14.8C 0.31(12) 94.3 0.55(12) 1649° 240(12) 7.3° 0.84(12) 6.7 0.52(12) 133 6.1(12) 0.9 0.26(12) t-teBt probability male va. female B. p< 0.05 b. p<0.01 c. p< 0.001 'estimate I (-• O ro I 18000 Chinook 2 15 000 E N ~ 12000 c o o 9 000 o O o £ w 6000 o 0-3000 Coho Sockeye o i-n Mean S.E. = 14 ' 141 •±29 •• Male • Female Note: All Salmon are Spawning Adults in Fresh Water 12 1649 + 240 13 1070 294 15 12081 ± 1305 15 2067 ±600 14 13154 ±2362 ure 33. Plasma calcitonin levels in 3 species of salmon. Note higher female plasma CT level in each species. Table XXXIV. Plasma and Ultimobranchial Gland Calcitonin Concentrations in Coho and Chinook Salmon UB Gland Plasma Calcitonin Species Sex ' Fish Total Calcitonin Level ft Ut Content Kg MRC U/gland pg/ml mU/ml* Coho m 19 4.32 112.9 2,575 12.88 20 5.68 191.1 292 1.46 21 5.46 557.1 180 0.90 22 5.00 517.3 739 3.70 24 5.46 313.2 0 0 26 4.55 375.7 - -28 4.55 153.2 880 4.40 n = 7 7 6 6 mean 5.00 317.21 778° 3.89° SO = 0.50 162.82 860 4.30 SE 0.20 66.47 385 1.92 f 1 4.55 594.0 16.8D0 84.00 2 4.09 84.8 5,575 27.88 5 4.09 66.7 8,725 43.63 ' 6 4.32 181.6 13,650 68.25 a • 5.46 494.7 11.15D 55.75 11 5.00 106.2 13,150 65.75 12 3.86 173.8 7,375 36.88 n _ 7 7 7 7 mean = 4.46 243.11 10,918 54.59 SD 0.53 196.37 3,645 18.23 SE — 0.21 80.17 1,488 7.44 Chinook m 2 7.73 233.7 1,166 5.83 5 10.68 202.8 1,433 7.17 6 a.18 166.5 2,816 14.08 7 6.59 64.1 3,033 15.17 a 6.91 192.6 1,166 5.83 9 - 9.00 442.3 1,433 7.17 ID 9.77 417.6 1,475 7.38 11 7.91 208.1 1,350 6.75 n _ B 8 8 8 „ Mean B.35 240.96 1,734° 8.67a SO 1.31 119.10 698 3.49 SE = 0.49 45.01 264 1.32 f ia 8.55 277.6 4,333 21.67 20 8.82 556.1 5,800 29.00 21 9.09 258.9 36,333 181.67 23 6.77 1209.9 18,166 90.83 24 7.14 108.9 10,166 50.83 n = 5 5 5 5 mean = 8.07 482.28 14,960 74.80 SD = 0.94 391.42 11,721 58.61 SE = 0.47 195.71 5,860 29.30 t-test probability male vs. female a. p< 0.025 b. p< 0.001 *Plasma CT biological activity based on salmon CT specific biological activity of 5000 MRC U/mg. -175-Discussicin A. Chilko Sockeye Migration Plasma The decrease in plasma electrolytes which occurred through out the Chilko migration may partially be explained by hemodilution (note the increase in plasma percent water). Relatively high doses of Cortisol, corticosterone, aldo sterone, deoxycorticosterone and cortisone cause decreases in plasma sodium levels in freshwater fish (Henderson e_t a_l, 1970). Therefore, it is passible that the changes in plasma sodium throughout the migration might be related to the high adreno-corticasteraid levels found in both male and female salmon during and after spawning and death (Idler e_t a_l, 1959; Hane and Robertson, 1959; Robertson et al, 1961; Schmidt and Idler, 1962; Fagerlund, 1967; Henderson et al, 1970). The fall in plasma sodium concentrations in the sockeye is consistent with a study done by Greene (1904), who found that the freezing point depression of chinook serum rose from -0.762°C in the sea to -0.612°C at spawning. Fontaine and Koch (1950) reported the freezing point depression of the serum of Atlantic salmon rose _ during the freshwater migration from -0.75aC to -0.66aC at spawning. The very low plasma potassiums in the Chilko sockeye have also been found in chinook salmon (Urist and van de Putte, 1967) -176-and may be related to starvation. Further evidence of the im portance of starvation in explaining the electrolyte changes in migrating salmon comes from Love e_t a_l (1968). They demonstrated a fall in plasma sodium, potassium and muscle potassium in starved immature cod, Gadus morhua, uhile muscle sodium rose. On feeding, this trend uas reversed. Plasma potassium, uhich rose in the freshuater arrival Chilko sockeye, may not reflect the increased Cortisol and corti-costerone levels at spauning since high doses of corticosteroids cause the plasma potassium level of freshuater fish to fall or remain constant (Henderson Et al, 1970). It is passible that the decline in plasma sodium levels is related to a decreased release of prolactin from the pituitary since some evidence has shoun that this gland degenerates in spauning salmon (Robertson and Uexler, 1957, 1960). Subseguent uork (van Overbeeke and McBride, 1967) has revealed that the de generative changes in the sockeye pituitary, uith sexual maturation and spauning, are only moderate. McKeoun and van Overbeeke (1969), also uorking on the Chilko race of sockeye, detected no change in the granulation af the prolactin cells during the migration or subsequent spauning, uhereas the granule density of the ACTH cells gradually increased throughout the latter part of the migration. It has been reported that during their spauning migration in freshuater, flesh sodium concentration and uater content of the sockeye salmon increases, uhile the flesh potassium concen tration decreases (Vinogradov, 1953; MacLeod et_ al^, 1958; Tomlinsan e_t al, 1967). These results agree uith our observations on plasma -177-sodium, potassium and percent uater during the sockeye salmon migration. The higher flesh water content and plasma water in the males may be related to the higher level of 11-ketotestosterone found in spawning male sockeye (Idler e_t a_l, 1961b). Another factor contributing to the decrease in plasma electrolytes in the migrating salmon has been reported by Miles (1971). Using migrating coho salmon, he measured an increase in the glomerular filtration rate from 1.48 ml/(kg x hr) in seawater to 9.06 ml/(kg x hr) in freshwater. Urine flow increased from 0.406 ml/(kg x hr) in the seawater to 4.65 ml/(kg x hr) in fresh water. Urinary excretion rates for sodium, potassium and calcium also increased from the sea to freshwater. The excretion of electrolytes, therefore, increases in freshwater despite the fact that there is no replacement of these ions from the diet. There does not appear to be any consistent relationship between the female plasma calcitonin and electrolyte changes. Female plasma CT levels increased from sea to spawning, falling off after the eggs were shed, whereas the plasma calcium, sodium and phosphate levels declined steadily throughout the migration. Likewise, the male plasma calcitonin changes did not correlate with these electro lyte changes. The decrease in haematocrit observed during the sockeye migration may reflect a decreased production of red blood cells due to the degeneration of the hemopaetic tissues (Robertson and Ldexler, I960). A fall in haematocrit has also been noted in starving fish (Lave, 1970). The decrease observed in the present study may be exaggerated since the gonads are developing rapidly -178-while the fish abstains from food. The fall in plasma protein concentration during the sock eye migration has been reported by other workers (Jonas and MacLeod, I960; Robertson et al, 1961; Qureshi et al, 1971). An explanation may derive from the fishes starvation, the uptake of protein into the developing gonads and/or a decreased plasma protein production due to the degeneration of the liver in the spawning salmon (Robertson and Ulexler, I960; McBride e_t al, 1965; Love, 1970). The plasma and muscle protein depletion in the migrating salmon may be enhanced by protein catabolism caused by the excess glucocorticoids (Robertson e_t a_l, 1961). The dramatic decline in plasma protein in both male and female sockeye appears greater than might be accounted for by blood dilution. Higher plasma protein levels in the female sockeye are likely produced by the liver under the action of estrogen in the sexually maturing female fish (Bailey, 1957; Urist and Schjiede, 1961; Ho and Van-stone, 1961; Phillips et_ ajL, 1964; Holmes and Donaldson, 1969; Love, 1970; McBride and van Overbeeke, 1971; Takashima et al, 1972). The higher total plasma calcium found in the female salmon as compared to the male has been reported by other researchers (van Someren, 1937; Idler and Tsuyuki, 1958; Ho and Vanstone, 1961) and may be explained by the action of the female sex hormones. The decrease in total plasma calcium during the freshwater mig ration was also noticed in the sockeye salmon by Idler and Tsuyuki (1958) and in the Atlantic salmon by Fontaine et_ a_l (1969). These last authors noted that the fall in plasma calcium (25 - 35%) of the adult salmon (both sexes combined) during their freshwater -179-migration could not be accounted for solely by hemodilution. They speculated that the drop in plasma calcium could be caused by calcitonin since the UB gland appeared to be particularly active (histologically) in the spawning salmon. Lopez (1969) has suggested that the corpuscles of Stannius may also be involved in lowering plasma calcium since these glands are very active in spawning Atlantic salmon, especially males. The particularly steep decline in plasma calcium after spawning has also been observed by Ldoodhead and Uoodhead (1965) in the female cod. A general drop in the electrolytes of the post-spawned salmon has been previously reported (Hoar, 1957b) Parry, 1961; Love, 1970). At least some of these electrolytes are used in the production of celomic fluid, which appears rather suddenly just at the time of spawning (Greene, 1904). The electro lyte composition (mEq/litre) of the celomic fluid in the Atlantic salmon has been measured as IMa 151, K 3.2, Ca 7.1, Mg 2.6, Cl 116, HPO^ 4.D and HC03 13.4 (Hayes et al, 1946). It is interesting to note that whereas a significant de crease in total serum calcium occurred from seawater tD the spawning condition, the ionic calcium remained quite constant. This un doubtedly reflects the physiological importance of the calcium ion in muscle contraction, nerve conduction, membrane permeability, etc. (Copp, 1972 ). The decline in serum ionic calcium in both spawning sexes may be partially explained by the rise in serum pH since the binding of calcium to serum proteins increases with increasing pH (Moore, 1969). It has been demonstrated that a rise in pH of 0.1 units is accompanied by a corresponding decrease in -180-ionized calcium of 0.1 mEq/l (Moore, 1969; Seamonds et_ al_, 1972). In the present study, the decrease in ionic calcium level from freshuater arrival to spauning uas slightly greater than could be accounted for by the accompanying increase in serum pH. The breakdoun in control of ionic calcium in the spauned salmon is not surprising in vieu of the fact that the fish die uithin 10 days of spauning. Moore (1969) measured the mean serum ionic calcium level of 18 normal human subjects. He obtained a measurement of 2.33 - 0.006 mEq/l (temp. 25°C; pH 7.42 - 0.005). This is louer than most of the serum ionic calcium readings in the present study (range sockeye serum ionic calcium = 2.45 - 3.17 mEq/l) except for a feu of the spauned out female sockeye. The total serum calciums of bath male and female seauater and freshuater arrival sockeye (Table XXIV., pg. 144) are also higher (range 5.65 - 12.73 mEq/l) than the human level of 5.08 mEq/l reported by Moore. The partition of calcium in the body fluids is influenced by many physical and chemical factors including body temperature, pH, concentration of serum protein, concentration of citrate and other organic complexes, ionic strength of the solution, plasma uater and other conditions (Urist, 1963; Chan and Chester Jones, 1968; Moore, 1969). A comprehensive study of the factors involved uould be necessary to clearly understand the serum ionic calcium changes in the migrating.salmon. Chan (1972) reported the normal plasma ionic calcium of freshuater Anguilla japonica to be 3.24 - 0.24 mEq/l (using the specific calcium ion electrode). The normal plasma ionic calcium -181-level (Murexide method) of yellou and silver freshuater Anguilla  anguilla uas 2.76 - D.02 mEq/1 (Chan and Chester Jones, 1968). The fact that plasma calcitonin changes fallou different patterns in the male and female throughout the sockeye migration, makes it unlikely that calcitonin is involved in the osmoregulatory adaptation changes from seauater to freshuater. In the female, variations in plasma CT are closely related .to GSI changes and hence may be involved in the sexual maturation process.- The in crease in the female plasma CT level from seauater to 0% spauning is remarkable in view of the fact that it occurs concurrently uith a significant hemodilution and decreasing haematocrit. The cause of the increase in plasma CT levels in the female may be related to a rise in production of calcitonin by the ultimobranchial gland (Table XXI, pg. 137) and/or to an increased secretion of calcitonin throughout the migration. The increase in plasma CT levels observed in the female sockeye salmon is supported by the recent uork of Deville and Lopez (197D). These authors reported an increased histological activity of the ultimobranchial gland of the Atlantic salmon, Salmo salar L., during migration and sexual maturation. At spauning, the ultimobranchial cell cytoplasm uhich uas formerly packed uith small, PAS positive granules, became clear and the UB gland hyper-trophied. In the post-spauned salmon, the ultimobranchial gland underuent complete degeneration. Although these authors do not mention any sex difference in their observations, the UB gland histological alterations described could help to explain the plasma calcitonin changes in the female sockeye salmon in the present -182-study. Again, these authors speculate that the decrease in plasma calcium could be attributed to calcitonin secretion. However, this does not explain our results in the spawning female sockeye where the steep fall in plasma calcium is accompanied by a dramatic drop in the plasma CT level. Deville and Lopez (1970) also suggested that the increased calcitonin secretion in the maturing salmon could play a major role in inhibiting bone re sorption in the breeding growth of the salmon skull. Pang (1971b)has also reported that killifish UB glands were more active in freshwater than seawater fish. Results from Chapter I also indicated that the UB calcitonin content of seawater rainbow trout was slightly lower than that of freshwater trout. The situation in the female sockeye appears to be somewhat analogous to Barlet's (1969) findings in milk cows. This author found that the hypocalcemia and hypophosphatemia occurring at calving and in milk fever, were associated with a significant rise of a "calcitonin-like" factor in the plasma. The rise in the plasma CT level exhibited by the sexually maturing female salmon is similar to that shown by free plasma estrogens in the female channel catfish, Ictalurus punctatus (Eleftheriou et_ al, 1966). Cedard et al (1961) have reported a 6-fdd increase in total estrogens in the blood of the spawning Atlantic salmon to a maximum of 7 micrograms/100 ml blood. The estrogen level returned to normal after spawning (the Atlantic salmon does not always die after spawning). The fact that the female sockeye maintained significantly higher plasma calcitonin levels than the males at all stages of -183-the migration is striking. A sex difference in the plasma CT levels of fish has not been previously reported. Deftos e_t a_l (1972b)found plasma calcitonin levels in cows (165 pg/ml) to be significantly (p<0.D5) lower than those in bulls (303 pg/ml) even though total plasma calcium levels were not significantly different. Kenny e_t a_l (1972) have shown that male Japanese quail, 2 - k months of age, had significantly higher plasma CT levels than the females. In this case, the female total plasma calciums were significantly higher than those of the male. Thus, with regard to sex differences in plasma calcitonin levels, the salmon appears to be unique among the vertebrates. The difference in plasma calcitonin levels between the sexes is clearly not related to serum ionic calcium levels, since this parameter showed no sex difference throughout the migration. The only electrolyte that demonstrated a consistent sex difference was total plasma calcium, the females maintaining significantly higher levels than the males except for the spawned out females. This difference was not nearly so evident in the female and male spawning adult chinook and coho salmon (Chapter IV, Section C) al though the females had slightly higher total plasma calciums than the males. While there is a sex difference in plasma calcitonin levels in the maturing salmon, this does not hold for the sex steroids. Indeed, Cedard e_t a_l (1961) have shown that spawning Atlantic male salmon have slightly higher total estrogen levels than the female (not significantly different). Schmidt and Idler (1962) reported that the plasma of both male and female Chilko sockeye captured -184-immediately before spawning, contained high levels of testosterone and 11-ketotestosterone. Testosterone was predominant in the female plasma whereas 11-ketotestosterone was more abundant in the male. Both male and female plasma levels of these steroids de creased after spawning (Schmidt and Idler, 1962). In summary, female sockeye salmon plasma concentrations of testosterone, Cortisol, corticosterone (in mature and post-spawned females) and calcitonin, are higher than in males at all stages of sexual maturation. The plasma calcitonin, plasma electrolyte and tissue electrolyte (especially IMa, H and Ca) changes in the migrating salmon may be most intimately related to the histological changes in the corpuscles of Stannius (Lopez, 1969; Heyl, 1970). In this regard, Heyl (1970) has observed changes in the general architecture and cell types of the corpuscles in the migrating and post-spawned Atlantic salmon which were more closely related to the time spent in freshwater than to gonadal development. It would be informative to examine the simultaneous histological changes of the corpuscles of Stannius and the ultimobranchial gland in the migrating sockeye salmon to reveal the relationship between these two glands. The greater histological activity observed in the spawning male Atlantic salmon corpuscles of Stannius by Lopez (1969), may give some in sight into the observation of the sex difference in plasma calcitonin levels. -185-Tissues It is difficult to assign a role to calcitonin in calcium homeostasis in the migrating, sexually maturing salmon when so many other hormonal changes are occurring at the same time. In fact, the pituitary, thyroid, interrenal, gonads, and corpuscles of Stannius (Hoar, 1953; Robertson et_ a_l, 1961; Hoar, 1963, 1965a, 1965b; Woodhead and woodhead, 1965; Lopez, 1969; Love, 1970; Heyl, 1970) all appear to be involved with fish migration and/or sexual maturation in some way. Add to this the complications of osmoregulation, starvation and death, and the picture becomes exceedingly complex. Nevertheless, an attempt has been made to determine the tissue calcium and phosphate changes in the migrating salmon and the corresponding role of calcitonin. This tissue study was prompted by the suggestion that calcitonin may play a part in the skeletal changes of the breeding salmon, since calcitonin, inhibits bone resorption in mammals in vivo and in_ vitro. Tchernavin (1937) has shown that the alterations in the salmon skull are complicated and involve not only an absolute increase in size of certain banes but also changes in shape. A careful study by this worker (Tchernavin, 1938a, 1938b) revealed that all the tooth-bearing bones of the jaw (dentary, maxilla, premaxilla), the palatines and the vomer grow in size during the freshwater migration whereas the bones forming the gill covers, the branchiostegals and the postarbitals, resorb. The supra-ethmoid grows longer and broader at its anterior end, but is resorbed at its posterior end. -186-Besides these skeletal changes, the salmon lose the teeth they had in the sea ("feeding teeth") and develop an entirely neu set of large "breeding teeth" (Rushton, 1926; Tchernavin, 1937, 1938a). These neu teeth in the spauning male are several times larger than those of the female but the teeth of both sexes become firmly anchored to the jaw bones in the spauning condition. The grouth of the bones and breeding teeth depends on the size of the fish and is invariably greater in the male. This bone and tooth development is quite remarkable considering that it occurs during the freshuater migration uhen the salmon have ceased to feed and in the very short time period Df a feu months! In the Atlantic salmon, which survive spawning and return to the sea, the skull and jaw'bones slowly revert back to their original proportions and sizes. Since the migrating Chilko sockeye in the present study were not feeding, it was important, to determine the source of supply of calcium and phosphate for the growth of the bones and teeth. From Figure 23, pg. 150 , it can be seen that there was a slight decrease in muscle calcium content from seawater to freshwater levels but the total amount of muscle calcium per fish was not large (Table XXX, pg.166 ). The decrease in muscle calcium content during the migration may be related to the increased ACTH and corticosteroid levels (Chan e_t al, 1967; Chan et_ al, 1969; Henderson e_t al, 1970). The corpuscles of Stannius and ultimobranchial gland may also be involved in this mobilization of calcium from soft tissue (Chan, 1969; Chan 1972). Greene (1926) has shown that the chinook salmon lose 51.6% of their total muscle mass by the time spawning is completed, so -187-th e muscle supply af calcium and certainly phosphate might be larger than expected. Chan (1972) has reported that eel muscles contain five times more calcium than tetrapad muscles and that during starvation, the eel obtains food and calcium by digesting its awn muscle. In the sexually maturing salmon, fat and protein from the degenerating muscle are transported to the developing gonads (Greene, 1926; Hoar, 1957b;Idler and Tsuyuki, 1958; McBride et al, I960; Couey, 1965). The skin actually increased in calcium content from sea to. freshuater arrival and then decreased at spauning (skin phosphate content remained fairly constant). The skin could contribute some calcium to the grouing bones but the amount of skin calcium per fish is half that found in the muscle (Table XXX, pg. 166). The sockeye skin contained considerably more calcium (75 - 192 mg Ca/10Dg FFDLd) than rabbit skin (50 - 85 mg Ca/10Dg dry ut) or the skin of dog and man (31 - 59 mg Ca/lDOg dry ut)(Irving, 1957). A further consideration is that the skin of sexually maturing salmon has been shoun to increase in thickness at spauning (Greene, 1926; Robertson and Ldexler, I960). It has since been demonstrated that the increased skin thickness, red coloration and increased size and number of epidermal cells can be produced in salmon by androgen injections, i.e. 11-ketotestosterone and methyltestosterone,, (Idler et al, 1961b;Fagerlund and Donaldson, 1969; McBride and van Overbeeke, 1971; Yamazaki, 1972). These skin changes are important to the salmon since the skin of teleosts is generally considered permeable to uater uhile it is only slightly or not at -188-all permeable to organic substances and ions (van Oosten, 1957). The female gonad calcium and phosphate contents (mg/lQOg FFDLU) remained fairly stable during the migration (Figures 23 and 24, pages 150 and 151). However, the average fat-free dry weight Df the female gonads increased from 31.99 - 2.96g in the sea to 102.58 - 4.08g in the 0% sp awning females. This growth would require approximately 114 mg of calcium and 614 mg of phosphate. It is passible that calcitonin, in conjunction with the female sex steroids plays a role in this development. The male gonads increased from 11.26 - 0.88g in the sea to 15.25 - 1.14g in fresh water and then decreased to 7.97 - 1.36g at spawning. Hence they would not require such large amounts of calcium and phosphate as the females. These increases in gonad calcium and phosphate concentrations may contribute to the fall of plasma calcium and phosphate during the maturation process in the female sockeye. However, from the data it can be seen that both male and female plasma calcium and phosphate decreased approximately the same amount from sea to 096 spawning yet more of these electrolytes were being stored in the female gonad than in the male. The changes in the phosphate content of the gonads and muscle of bath sexes may reflect to same extent changes in the synthesis and storage of nucleic acids. Creelman and Tamlinson (1959) found that migrating sockeye salmon experienced major losses of RNA phosphorus from the flesh, alimentary tract and male gonad, while the gonads of both sexes gained large amounts of DIMA phosphorus. -189-The female sockeye gonad calcium content and % ash/FFDU measured in the present study (150. - 175 mg Ca/lOOg FFDU; 3.92 - 4.02% ash/FFDU) are consistent with the results of Ogino and Yasuda (1962). These workers studied the unfertilized eggs of the rainbow trout, Salmo gairdneri, and found the calcium content to be 182 mg Ca/lOOg dry wt and the % ash/dry weight 3.66%. The teleost scale consists of two-parts: an outer calcified or bony layer with growth-ridges and an inner fibrous or lamellar layer-which is partially calcified (Crichton, 1935; van Oosten, 1957; Harden-Jones, 1968; Brown and Uellings, 1969). The phenomenon of scale resorption in migrating salmon has been reported by many workers (Hutton, 1924; Crichton, 1935; van Someren, 1937; Tchernavin, 1938b; Robertson and Uexler, 1960). Van Someren (1937) found that the resorption process particularly affects the posterior portion of.the scale and may be mediated by "osteoclast-like" cells. Moss (1961), however, has reported that most teleost scales are acellular. It is the outer, calcified layer which is resorbed to a greater extent than the fibrous layer. It was observed that resorption of the scales in the male salmon was frequently more severe than in the female. The scales of spawning salmon are also extremely difficult to remove (descaling in seawater salmon occurs frequently and easily). According to Yamazaki (1972) this phenomenon may be due to testo sterone. Van Someren (1937), in studying the Atlantic salmon, found no correlation between blood calcium level and scale re--190-sorption and suggested that the latter process uas not necessarily related to breeding but to starvation. He noted that the degree of resorption uas proportional to the length of time spent in freshuater, i.e. time of cessation of feeding. Resorption con tinued after spauning and ceased only uhen the fish resumed normal feeding in the sea. Results obtained from the sockeye scales are in substantial agreement uith previous uork on fish scales (van Someren, 1937; van Oosten, 1957; Moss and Freilich, 1963; Broun and Uellings, 1969). The seauater sockeye scales of the present study had mineral contents of 36.01% (male) and 34.01% (female). Broun and LJellings (1969) found that the mineral content of teleost scales varies from 16 to 59%. The calcium (36.22 - 32.14g Ca/lOOg ash) and phosphate (17.65 - 20.36g P0^/100g ash) contents of the sockeye scales uere similar to those of sockeye bone. The present study supports the observation that the male s-almon scales resorb to a greater degree than the female scales since the decrease in mineral content uas slightly more marked in the males (Figure 28, pg. 158). Besides losing mineral, the calcium content (gCa/lOOg ash) of the scales of both sexes declined quite sharply (Figure 25, pg. 154). Associated uith the decline uas a significant rise in phosphate content (g PO^/lOOg ash). This may reflect the fact that there are 2 pools of phosphate in the scales, organic and inorganic. Foerster and Reeve (in Van Someren, 1937), suggested that the calcium resorbed from the scales in maturing salmon is utilized by the grouing bones and teeth. These authors believe -191-that the greater development of secondary sex characteristics in the male (jaw bones, teeth, hump) cause the more extensive scale resorption in the male since an increased supply of Ca and PO^ would be necessary for these developing tissues. This theory, although attractive, can provide only a partial explanation. As previously calculated (Table XXX, pg.166 ), the scales do not contain large amounts of calcium and phosphate since their absolute weight per fish is very small. It is quite likely that the Ca and PO^ lost from the scales remain in the salmon, since the scales are covered by a thin layer of epidermis even during resorption. The calcium could possibly be transported to the skin calcium reservoir and from there contribute to bone and gonad growth or calcium homeostasis. The calcium might also remain in the skin and function to decrease water permeability. This process would account for the fact that the skin Ca and PQ^ content increased from sen to freshwater arrival, the time at which a major decline in scale mineral content occurred. In this connection it has been shown in growing speckled trout, Salvelinus fontinalis, that the calcium content of the skin increases as the scales became larger (Phillips et al, 1953)„ The vertebrae, in contrast to the scales, showed a sig nificant increase in mineral content throughout the migration (Figure 27, pg. 157 ). The vertebral calcium content remained stable in the females and increased in the males (Figure 25, pq.±5k while the vertebrae phosphate content decreased in both sexes (Figure 26, pg.155 ). Thus, it is clear that the vertebrae bones are not supplying Ca and PO, to the growing tissues since they, -192-themselves, are mineralizing. The data in Table XXXV, pg. 192, uere based an actual weights of the growing tissues plus results from the migration study, in order to determine the approximate calcium utilization in the growing tissues. Table XXXV. Calcium Utilized by Growing Tissues in Maturing Sockeye Salman Calcium (mg) Tissue male female Teeth 183 114 Premaxillae 66 18 Vertebrae 132 170 Jaw bones 600 200 Gonads - 114 Total 981 616 Some of the calcium required by the developing tissues could be supplied.by the scales, muscle and skin. As mentioned previously, Tchernavin has shown that while there is growth of the teeth, jaw bones, palatines and vomer, resorption occurs in the gill-covers, branchiostegals, and postorbitals. Tchernavin (1938b) also suggested that the resorbing bones could supply the calcium and phosphate for those bones which were growing. This statement has some basis in fact, since in the freshwater arrival male and female sockeye, the total dry weight of the bones which resorb -193-LjeighEd almost twice as much as the bones which grow. In any case the skeleton, not including the growing bones, could probably supply much of the needed calcium and phosphate. The chicken can mobilize 10 percent of its bone in one day (Simkiss, 1961; Taylor, 1970). If the sockeye salmon could mobilize a similar portion of its skeletal calcium store (Table XXX, pg. 166), it would be able to supply most of the mineral required by the developing tissues. Other workers have shown in fish that the muscle (Chan et al, 1967 ; Chan, 1972) and skin (van Oosten, 1957; Podoliak and Holden, 1965; Fleming, 1967) constitute important storage compart ments for exchangeable calcium. However, Simmons (1971) has pointed out that the skin does not appear to be a major reservoir for calcium in marine fish. Starvation in goldfish and carp has been reported to.be associated with scale resorption (Ichikawa, 1953; Yamada, 1956, 1961). Some of the calcium needed for the growing sockeye tissues could possibly be supplied from the environmental water by absorption through the gills, fins and oral epithelia 45 (see Simmons, 1971 for references). Using Ca , it has been established that calcium ion transport across the gills is mare efficient in freshwater than in seawater fish and the major re positories for this absorbed calcium are the bone and skin. Tracer 45 32 studies, using Ca and P , would reveal the extent to which these ions are absorbed from the water by the sexually maturing salmon. Since calcitonin inhibits bone resorption in mammals.it is cur ious that the mature male salmon, which showed the more extensive scale -194-resorption and bane growth, exhibited lower plasma calcitonin levels than the females. Other endocrine glands, such as the pituitary, thyroid, gonads, corpuscles of Stannius and possibly the adrenal cortex are also likely involved in the skeletal changes in the maturing salmon (Davidson, 1935; Gardner and Pfeiffer, 1943; Hoar, 1957a;Robertson and Uexler, 1962; Love, 1970; Lopez, 1970a, 1970b; Simmons, 1971; Chan, 1972). B. Plasma Calcitonin Levels in Coho Salmon: Effect of Sexual Maturation and Environmental Salinity. As was found with the Chilko sockeye, the highest plasma calcitonin levels were exhibited by the spawning coho females. A sex difference in plasma calcitonin levels (female plasma CT was higher than the male) was clearly evident only in the spawning adults, again indicating a relationship between calcitonin and sexual maturation. The finding of high plasma calcitonin levels in sexually ripe coho "jacks" would appear to indicate that plasma CT is also slightly elevated during male sexual maturation. In contrast to the sockeye, the high coho plasma calcitonin levels were associated with high plasma calcium levels. The very low plasma CT levels found in the freshwater immature coho may partially reflect the high plasma percent water and low haematocrits found in this group. High plasma calcitonin levels in the spawning adult coho do not appear to be correlated with any of the plasma electrolytes measured. -195-C. Plasma Calcitonin Levels in Spawning Adult Sockeye, Coho, and Chinook Salmon. Table XXXIII, pg.172 , shows that in all 3 species of salmon, the female plasma calcitonin levels were significantly (p<•.•01) higher than those of the male. Since the UB gland calcitonin contents of the coho and chinook showed no sex differ ence, the high circulating plasma CT level in the females may be explained by differences in secretary and/or clearance rates of calcitonin in the male and female salmon. The females also had significantly higher total.plasma calciums in the chinook (p<Q.Q5) and sockeye (p<0.01). The higher plasma calcitonin levels found in the chinook and coho compared to the sockeye, could be due to many factors. Since these fish represent 3 separate species under the same genus, Oncorhynchus, there are many morphological, physiological and biochemical variations among them. The different plasma calcitonin levels may reflect different ages, growth rates, size or distances travelled during the freshwater migration. The sockeye travelled approximately 500 miles in freshwater compared with less than 10 miles in the other species. The low plasma electrolyte and cal citonin levels found in the sockeye may partially be explained by the high plasma percent water found in this group. The high plasma water was probably related to the fact that the sockeye had spent more time in freshwater than the other 2 groups. The female sockeye (Table XXI, pg.137) had much lower UB gland calcitonin concentrations than the female chinook or coho salmon (Table XXXIV, pg.i7f+ ), and this would help to account for - 196-the louer plasma CT levels found in the sockeye. Thus, the fact that high female plasma calcitonin levels have been measured in 3 species of spauning salmon suggests that calcitonin is related to sexual maturation, at least in the female. The plasma CT level in the spauning female salmon is much higher than that reported for any mammal, except for some patients uith medullary thyroid carcinoma (Deftos and Potts, 1970; Deftos e_t al, 1972a) and comparable or higher than concentrations found in birds and fish (Copp at al, 1972b; Kenny et al, 1972). Tashjian e_t al (1972) also using the radioimmunoassay, confirmed these observations that fish have higher circulating plasma calcitonin levels than most mammalian species. He found that coho salmon (age 1-3 years) adapted to freshuater, and fed a commercial diet, had higher plasma CT levels than unfed coho adapted to salt-uater for 2 - k months. The data also suggested that freshuater 2 year old female coho (GSI not measured) had higher plasma CT levels than males of the same age. In summary, the role of calcitonin in calcium homeostasis in fish can be expected to be unique among the vertebrates, since they lack parathyroid glands and can obtain calcium from their environment. Results from the Chilko sockeye migration suggest that fish are different from mammals uith respect to calcitonin secretion. In the sheep and pig, hypercalcaemia causes the release of calcitonin (Copp, 1970b; Cooper et al, 1971), uhereas hypo-calcaemia is associated uith high plasma CT levels in the 0% spauning female sockeye. -197-V. EFFECT DF ESTROGEN ON SERUM IONIC CALCIUM IN TROUT AND GONADECTOMY AND ESTROGEN ON PLASMA CALCITONIN AND CALCIUM IN SALMON Introduction Hypercalcaemia has been observed in many female teleosts during the breeding season (Hess at a_l, 1928; Para, 1935, 1936;. van Someren, 1937; Fontaine, 1956; Garrod and Neuall, 1958; Phillips et al, 1964; Booke, 1964; Fleming et al, 1964; Urist and Van de Putte, 1967; Oguri and Takada, 1967; Ldoadhead, 1968; Ldoodhead and Plack, 1968; Fontaine• et al, 1969; Urist et al, 1972). The increase in plasma calcium is associated uith a rise in vitellin, a calcium-binding phosphopratein uhich is produced in the liver Df those louer vertebrates possessing yolky eggs (Urist and Schjeide, 1961; see Simkiss, 1961, 1967). Estrogen injection into both male and female teleosts elevates total plasma calcium (Bailey, 1957; Fleming and Meier, 1961; Urist and Schjeide, 1961; Ha and Vanstone, 1961; Clarke and Fleming, 1963; Fleming e_t al, 1964; Oguri and Takada, 1966, 1967; Chan and Chester Jones, 1968; Uoodhead, 1969a; Urist et al, 1972). Several of these uorkers have noted concomitant increases in serum proteins, phosphorus, lipids and vitamins. These constituents are thought to be mobilized for the developing gonads. The mechanism of action of estrogen in fish and the source of the mobilized calcium has not been clarified. Estrogen appears -198-to affect only the protein-bound fraction of calcium (Bailey, 1957; Urist and Schjeide, 1961; Chan and Chester Jones, 1968; Urist et al, 1972). The results of experiments outlined in Chapter IV indicated a sex difference in plasma calcitonin levels in the salmon and that the plasma CT level in the female increased uith sexual matur ation. These plasma CT changes did not appear tD be correlated uith total plasma calcium or other electrolyte changes. This chapter uill report the effect of gonadectomy and estrogen re placement on plasma calcitonin levels in sockeye salmon. The effect of estrogen on serum ionic and total calcium changes in immature trout uas also investigated. The purpose of these experiments uas to provide further insight into the inter-relationships of calcium metabolism, calcitonin and sexual maturation. Materials and Methods The chapter is divided into tuo sections. In Section A, the effects of estrogen injection on serum ionic and total calcium in sexually immature rainbou trout, Salmo gairdneri, are outlined. As uas noted in Chapter IV, the female salmon exhibited higher plasma CT levels and total calcium levels than the male. It uas important, therefore, to determine uhether estrogen elevated ionic calcium since in mammals increased ionic calcium levels cause the release of calcitonin. Sexually immature trout uere used in these experiments to minimize the influence of endogenous estrogen secretion and its effects on calcium metabolism. Section B outlines the effects of gonadectomy and estrogen -199-rBplacemBnt on plasma calcitonin and total calcium levels of adult sockeye. A. Effect of Estrogen on Serum Ionic and Total Calcium in Immature Rainbow Trout Two groups of ID fish each uere put into separate 50 gallon fibreglass tanks of running water. ThB fish were then acclimated to laboratory conditions for one week. They were fed trout chow pellets daily, except on the day of injsction. The control group recBivBd 0.1 ml cottonseed oil per fish. The experimental group received estradiol cypionate (estradiol-lT^ in the form of the cyclopentyl propionate estsr; Upjohn Pharmaceutical Co.) at a dose of 0.1 mg estradiol/ 0.1 ml cottonseed oil per fish. Injections were performed intraperitoneally once per week for 6 weeks, on fish which were under light anesthetic (2-Phenoxyethanol, 1.0. ml/gal; Eastman Kodak Co.). This experiment was conducted from May 17 to June 28, 1972, during which time the water temperature in the two tanks ranged from 7.5 - 8.5DC. Blood samples were collected from the caudal vein of un-anesthetized fish within 2 minutes of capture. To ensure that the blood was well oxygenated, the gills were perfused with aerated water during the sampling procedure. Other methods of collection and analysis of samples were outlined in General Materials and Methods. Six rib bones were dissected from each of the 20 trout for calcium and phosphate content analysis. Serum ionic calciums were measured at a water temperature of 8.5 - 1.0DC, the water temperature at the time of sacrifice. -2Q0-B. Effect cf Gonadectomy and Estrogen Replacement on Plasma Calcitonin and Electrolyte Levels A group of sexually immature sockeye of the Great Central Lake race uere captured during their spawning migration in June 1971. These fish uere then transported to the Fisheries Research Board Technical Station, Vancouver, B.C. The methods of capture and transportation were those used by McBride et_ a_l (1963). In the laboratory, the fish uere maintained on a natural photoperiod and seasonal water temperatures. In the period from July 15 - 29th, a group of these fish were gonadectomized by Jack McBride, Fisheries Research Board of Canada, using the technique he developed (McBride et_ a_l, 1963). A second group uas left intact and held under similar conditions in the laboratory. The salmon uere divided into 3 experimental groups: (1) Intact controls (normal sockeye) - 5 males, 6 females This group had intact gonads and uere maturing normally. The fish uere not fed during the experiment. In nature, these sockeye spaun from late September until the end of November (McBride e_t _al, 1963). It should be painted out that 4 of the normal males (fish #N8 - Nil) uere sockeye "jacks" but as shoun by their ganad-somatic indices, they uere quite sexually mature. These jacks mature in their 3rd year uhereas the sockeye normally mature in their 4th or 5th year of life (Clemens and Uilby, 1961). -201 (2) Gonadectomized control sockeye - 1 male, 9 females Fish in this group received 1.0 ml intramuscular injections of cottonseed oil once per ueek for 7 ueeks. They uere fed daily throughout the experi mental period. Five females uere sacrificed on February 9, 1972, six months follouing the gonadectomy operation. (3) Gonadectomized estrogen-injected sockeye - 3 males, 3 females These fish received intramuscular injections of estradiol cypionate (1.0 mg estradiol/ml cottonseed oil; Upjohn Pharmaceutical Co.) once a ueek for a period of 7 ueeks. They uere fed on each experimental day. The injection experiment began October 13, 1971, 2-3 month follouing gonadectomy and the fish uere sacrificed on December 1, 1971, one ueek follouing the final injection. All injection and blood sampling operations uere performed on salmon lightly anes thetized in 2-Phenoxyethanol. Water temperatures throughout the injection experiment ranged from 6 - 12°C. Procedures for the collection and analysis of samples uere outlined in General Materials and Methods. Plasma samples uere collected for calcitonin measurement. Ribs uere dissected from each salmon in the December 1, 1971 group far analysis of calcium and phosphate content. It should be noted that approximately the same dosage of -2D2-estradial cypionate uas given to the rainbou trout in Section A and the sockeye salmon. Results A. Effect of Estrogen on Serum Ionic and Total Calcium in Immature Rainbou Trout Physical parameters and plasma electrolyte measurements for the 2 groups of trout are presented in Table XXXVI, pg. 2D3. The total ueights and fork lengths of the estrogen-treated group uere slightly higher than for the controls. The 2 groups of female trout had significantly higher GSI values than the males but both sexes uere very immature. Figure 34, pg. 204, illustrates the serum ionic and total calcium and plasma inorganic phosphorus levels for the immature trout. The male and female electrolyte measurements uere combined since no sex differences uere observed. As can be seen from the data, estrogen injection significantly elevated plasma inorganic phosphorus (p < •.••!) and serum total calcium (p<Q.DDl). Despite a 9-fold increase in serum total calcium, the serum ionic calcium level did not change. . Table XXXVII, pg. 205, presents the percent ash/dry ueight, and calcium and phosphate contents of the rib bones of the control and estrogen-injected trout. There uas no significant difference betueen the 2 groups in any of the 3 parameters. Table XXXVI. Physical Measurements, Plasma and Serum Electrolytes in Control and Estrogen-Treated Trout Group Sex Total Fork GSI Hct Plasma Serum Weight Length Inorganic Ionic Total % Ionic g cm Phosphorus Calcium Calcium Calcium mEq/l mEq/l mEq/l Control m n= 6 6 6 6 5 6 6 6 Cotton mean= 136 24.1 0.08 22 5.52 2.74 4.88 56.23 seed SD= 10.57 0.33 0.06 5.93 0.82 0.17 0.43 1.97 SE= 4.73 0.17 0.03 .2.65 0.41 0.07 0.19 0.88 f n= 4 4 < 4 4 2 4 4 4 rnean= 123 24.3 0.22 24 5.90 2.65 4.73 56.26 SD= 8.46 0.69 0.02 5.12 0.55 0.15 0.30 3.95 SE= 4.88 0.40 0.01 2.95 0.55 0.08 0.17 2.28 Total m&f n= 10 10 10 10 7 10 10 10 Controls mean= 131 24.2 0.14 23 5.63 2.70 . 4.82 56.24 SD= 11.67 0.52 0.08 5.67 0.77 0.17 0.39 2.93 SE= 3.89 0.17 0.03 1.88 0.31 0.05 0.13 0.98 Experi m n= 6 •6 6 6 5 6 6 6 mental mean= 149 24.0 0.12 24 11.68 2.85 36.63 7.90 Estrogen SD= 13.95 0.94 0.02 7.27 0.71 0.17 4.01 1.14 SE= 6.24 0.42 0.01 3.26 '0.36 0.07 1.79 0.51 f n= 4 4 4 4 4 4 4 4 mean= 150 24.6 0.22 25 11.83 2.82 35.38 7.97 SD= 2.74 0.54 0.02 2.12 0.39 0.13 1.35 0.29 SE= 1.58 0.31 0.01 1.22 0.23 0.07 0.78 0.17 Totai m&f n= 10 10 10 10 9 10 10 10 Estrogen mean= 149 a 24.2 0.16 24 11.74° 2.84 36.13° 7.92° SD 10.96 0.85 0.06 5.83 0.60 0.15 3.28 0.90 SE 3.65 0.28 0.02 1.94 0.21 0.04 1.09 0.30 t-test probability: total controls vs. total estrogen a. p< 0.005 b. p< 0.001 -204-40.On £ 30.0-2 o UJ 20.0-o E | 10.0-0.0 Cottonseed n • 10 10 7 Estrogen I Ionic Ca • Total Serum ^ Plasma Phosphorus 10 10 9 Figure 34. Serum ionic and total calcium and plasma inorganic phosphorus levels in immature trout - effect of estrogen. -205-Table XXXVII. Bone Measurements in Control and Estrogen-Treated Trout. % Ash 9 ™k gCa Group n Dry Ut mean - SE lOOg Ash Mean i SE lOOg Ash mean - SE Control Cotton seed a 61.53 0.88 17.91 0.15 34.29 0.52 Experi mental Estrogen 6 63.38 0.36 17.97 0.28 33.91 0.18 -206-B. Effect of Gonadectomy and Estrogen Replacement on Plasma Calcitonin and Electrolyte Levels Physical measurements, plasma calcitonin and plasma electrolyte levels for the 3 groups of salmon are summarized in Table XXXVIII, pg. 2D7. Plasma calcium levels are shown in Figure 35, pg. 208, and the corresponding individual plasma CT levels are illustrated in Figure 36, pg. 209. In the intact control group, the females had significantly higher plasma calcium (p<0.05) and magnesium levels (p<0.05) than the males. These.electrolyte levels uere slightly lower in the gonadectomized controls than in the intact controls. However, the plasma calcium (p <0.001), inorganic phosphorus (p< 0.001) and magnesium (p< 0.001) in the gonadectomized estrogen group were significantly elevated over corresponding . levels for the gonad ectomized controls (sexes combined). Plasma sodium and potassium showed little variation among the 3 groups. The intact control sockeye exhibited the same sex difference in plasma calcitonin levels noted in the migrating Chilko sockeye salmon in Chapter IV. In the normal intact males, plasma CT levels were undetectable. Plasma CT values for the gonadectomized control and gonadectomized estrogen groups were both less than 400 pg/ml. The percent ash/dry weight, and calcium and phosphate con tents of the rib bones for the 3 groups of sockeye are presented in Table XXXIX, pg. 210. The intact control male sockeye had significantly higher percent ash/dry weight (p<0.01) and lower calcium (p<0.05) and phosphate contents (p<0.05) than the intact control fe males. The other groups displayed no large differences in any of the parameters measured. Table XXXVIII. Physical Parameters, Plasma Calcitonin and Plasma Electrolytes af Intact Control, GX Control and GX Estrogen Sockeye Total Plasma Plasma Electrolytes mEq/l Ueight Calcitonin Group Sex g GSI . Hct pg/ml' Ca PD^ Mg IMa Intact m n= 5 5 5 undetect 5 5 5 5 5 Control mean= 757 4.02 33 able 4.55 5.36 1.43 147 2.48 SD 197.90 0.70 5.52 0.33 ' 0.29 0.12 5.49 0.47 December 1,1971 SE 98.95 0.35 2.76 0.16 0.14 0.05 2.75 0.24 f n= 6 6 p 6 6 6 6 6 6 6 mean= 1793C 1S.4SC 36 6603 5.78a 5.23 1.7Qa 150 2.02 SD= 418.50 2.63 3.47 2075.65 1.07 0.78 0.23 2.87 0.75 SE= 187.16 1.18 1.55 928.26 .0.48 ' 0.35 0.10 1.28 0.33 Gonadectomized m n=l 1475 27 400 4.70 4.82 1.56 153 1.90 Control f n= 4 - 4 4 4 3 3 3 December 1,1971 mean= 1594 26 400 4.77 4.46 1.57 152 2.27 SD= 444.19 2.24 0.22 0.10 0.08 2.05 0.17 SE= 256.45 1.29 0.13 0.05 0.05 1.45 0.12 February 9, 1971 f n= 5 _ 5 5 5 5 5 5 mean= 1036 20 400 4.60 3.68 1.26 146 2.64 SD= 130.13 •3.72 0.40 0.19 0.30 8.17 0.15 SE= 65.07 1.86 o.2r 0.09 D.15 4.09 0.07-Gonadectomized m n= 3 3- 3 3 3 3 3 Estroqen mean= 1367 17 400 23.99 10.05 3.78 144 2.60 SD= 455.22 3.09 2.16 0.43 D.21 1.41 0.14 December 1, 1971 SE 321.89 2.19 1.53 0.30 0.14 1.0D 0.10 f n= 3 3 3 3 3 b 3 3 mean= 1425 19 400 30.21 12.32 4.91b 141 2.37 SD= 73.60 2.45 6.14 1.81 0.27 1.25 0.31 SE= 52.04 1.73 4.34 1.28 ' 0.19 0.88 0.22 t-test probability male vs. female a. p< 0.05 b. p<0.01 c. p< 0.001 -208-40.0- Gonadectomized + Cottonseed Oil + Estrogen 30.0-cr LU E E 2 o o O o E CO o 0_ 20.0-10.0-0.0- 789 10II 123456 CMICF3 56 I234SS678 S9I0I Fish Number Figure 35. Total plasma calcium levels in sockeye -effect of gonadectomy and estrogen replacement. Intact Females 9331 7764 4606 Intact Males t 1 undetectable 28*3 7964 563i • Female Male Gonadectomized + Cottonseed Oil + Estradiol 1 (Controls) \[ \ 7 9 II 13 5 CMI CF 5 6 I 3 5 S678 S9 II Fish Number Figure 36. Plasma calcitonin levels in sockeye effect of gonadectomy and estrogen replacement. Table XXXIX. Bone Measurements in Intact Control, GX Control and GX Estrogen Sockeye % Ash gPO, gCa Group Sex Dry Wt lOOg Ash lOOg Ash Intact . m n = 5 5 5 , mean = 68.00 16.64 35.10 LOntro1 SD = 4.57 0.87 1.70 SE = 2.28 0.43 0.85 f n = 5 . 5 , 5 • mean = 59.83° 18.293 37.97 SD = 1.09 1.02 1.39 SE = 0.54 0.51 0.69 Gonadectomized m n = 1 1 1 n„„^ , 60.25 17.54 37.70 December 1, 1971 f n = 4 4 4 mean = 60.51 18..97 37.19 SD = 1.09 1.61 1.04 SE = 0.63 0.93 0.60 Gonadectomized m n = 3 .3 3 c . „„ mean = 60.85 17.89 ' 37.01 Estroo.en SD = 0.90 0.04 0.39 SE = 0.64 0.03 0.27 f n = 3 3 3 mean = "59.92 17.44 36.42 SD = 1.39 0.68 1.03 SE = 0.98 0.48 0.73 t-test probability male us. female a. p<0.05 b. p<0.01 -211-Discussion Total calcium concentration in the serum of vertebrates is present in three distinct fractions: 1. protein-bound calcium 2. complexed calcium bound to anions such as bicarbon ate, phosphate and citrate 3. ionic calcium The complexed and ionic calcium fractions constitute the ultra-filtrable or diffusible fraction and it is the ionic calcium that is the physiologically active form of calcium in the body (see Moore, 1969, 1970; Simkiss, 1967; Chan and Chester Jones, 1968). In mammals, the ionic calcium level is precisely regulated and under the control of calcitonin and parathyroid hormone (Copp 1969c, 1970a). The serum ionic calcium level of the estrogen injected trout in the present study remained quite constant despite a marked elevation in total calcium. These results are in close agreement uith previous uiork on amphibians, reptiles and birds (see Simkiss, 1961, 1967). The effect of estrogen on ultrafiltrable or ionic calcium in teleosts has been investigated in only a feu cases but the general pattern appears to be similar to the above aviviparous vertebrates. Bailey (1957) reported that a single intraperitoneal injection of 0.5 mg estradiol benzoate into gold fish, produced a 10-fold increase in total calcium level an the 20th day post-injection uhereas the ultrafiltrable (mostly ionic) calcium level remained stable. Chan and Chester Janes (1968) shoued -212-that the ionic calcium level of the freshuater European eel, Anguilla anguilla L., remained constant after injection Df estrogen (Premarin 100. microg/100 g per day for 6 days) despite a significant (p<0.01) rise in total calcium. Recently, Urist et al (1972) reported that estradiol valerate injection into male and female lungfish, L_. paradoxa, resulted in a dramatic elevation of total protein and total calcium uith essentially no change in inorganic phosphorus or ultrafiltrable calcium. It is interesting to note that although estrogen injection also causes hypercalcaemia in birds (Riddle and Dotti, 1936; Urist et al, 1958; Taylor, 1970; Simkiss, 1967) amphibians (Urist and Schjeide, 1961; Simkiss, 1961, 1967) and reptiles (Dessauer and Fox, 1959; Urist and Schjeide, 1961; Clarke, 1967; Prosser III and Suzuki, 1968; Simkiss, 1961, 1967), the effect of estrogen on plasma calcium in mammals is more variable and much less con spicuous (Day and Follis, 1941; Gardner and Pfeiffer, 1943; Manunta et_ a_l, 1957; Young et al, 1968; Sorensen and Hindberg, 1971). The serum ionic calcium levels of the trout in this thesis (range 2.39 - 3.03 mEq/1) are uithin the range measured in the migrating Chilko sockeye (males, 2.12 - 3.49 mEq/1; females, 1.84 - 3.83 mEq/1). These results are also similar to the values measured by Chan and Chester Jones (1968) in the European eel under various experimental conditions (plasma ionic calcium range 2.70 - 2.84 mEq/1, Murexide method). The above observations indic ate that teleosts are capable of precisely regulating their ionic -213-calcium levels and provide further evidence to support the con tention that the ionic calcium concentration or activity is one of nature's "physiological constants" (McLean and Hastings, 1935). Estrogen injection significantly elevated the plasma inorganic phosphorus levels in the trout (p< 0.001) and gonad ectomized sockeye (p< 0.001, sexes combined). Bailey (1957) also reported an increase in plasma inorganic phosphorus in goldfish on treatment with estrogen. Ho and Vanstone (1961) showed that intramuscular injections of estradiol monabenzaate (0.2 mg per day for 4 days) into sexually maturing male and female sockeye salmon, caused significant (p< •.•!) increases in both protein and lipid phosphorus as well as total calcium (p<0.01). In contrast, Chan and Chester Jones (1968) did not observe an increase in plasma inorganic phosphorus on injection of estrogen into Anguilla  anguilla L. while Urist e_t a_l (1972) observed a large increase in total phosphorus with little change in inorganic phosphorus. The effect of estrogen on plasma magnesium levels has rarely been investigated. A significant increase in plasma magnesium was observed in the gonadectomized sockeye on treatment with estrogen (p<0.001, sexes combined). Day and Follis (1941) reported a slight (not significant) rise in serum magnesium of young rats treated with estradiol benzoate. The only report an the effect of estrogen on serum magnesium in fish appears to be the work of Oguri and Takada (1967). These authors observed an increase in the serum magnesium levels of goldfish from control levels of 1.63 mEq/1 (males) and 1.50 mEq/1 (females) to 3.5 - 5.6 mEq/1, 8 and 9 days fallowing a single injection of 4.7 mg and -214-7.5 mg of estradiol. In the intact control group of sockeye, there uas no sex difference in the plasma inorganic phosphorus, sodium or potassium levels although the female plasma calciums and magnesiums uere both significantly higher (p<0.05) than the males. In comparing the plasma electrolytes of the intact control females and the gonadectomized control females, it is interesting to note that removal of the gonads, and thus removal of the source of estrogen, resulted in significant declines in plasma calcium (p<0.05), inorganic phosphorus (p<0.01) and magnesium (p<0.05). This decline uas associated uith a decrease in plasma CT levels from a mean of 6603 - 928 pg/ml to less than 400 pg/ml. It is not knoun uhether the decrease in circulating level of plasma CT in the gonadectomized sockeye is related to a reduction in secretory rate or an increased metabolic destruction. The fact that estrogen replacement dramatically increased total plasma calciums but did not restore the plasma CT levels, indicates that the factors governing the circulating level of calcitonin may be quite complex. Resiilts in the male sockeye are even more com plicated since they indicate that the plasma calcitonin level may rise on gonadectomy. Plasma Cortisol has also been shoun to decrease follouing gonadectomy of male and female sockeye (Donaldson and Fagerlund, 1970). Gonadectomy appears to have little effect on plasma calcium levels in rats (Rice et al, 1968; Sorensen and Hindberg, 1971). Castration effectively louers plasma calcium levels in the toad Xenopus but the same operation in dogs produces a marked rise in serum calcium (Gardner and Pfeiffer, 1943). The only report of -215-the effect cf gonadectomy on plasma electrolytes in fish, is that of Pickering and Dockray (1972). These authors showed that gonadectomy of freshwater female lampreys resulted in a signif icant increase in plasma calcium levels. No change occurred in the freshwater male lampreys. The effect of estrogen on plasma calcium, phosphorus and magnesium levels was more marked in the female gonadectomized sockeye than in the male. This observation has been reported by other workers (Ho and Vanstone, 1961; Oguri and Takada, 1967; woodhead, 1969a; Urist e_t al_, 1972). Data from the present study indicate that the more marked elevation of plasma calcium due to estrogen in the females occurred even after removal of the gonads. The hypercalcaemic effect of estrogen in fish does not appear to depend on a source of dietary calcium since most of the experiments reported in the literature were conducted an fasting fish (Bailey, 1957; Ho and Vanstone, 1961; Oguri and Takada, 1966; Chan and Chester Jones, 196B; Woodhead, 1969a). Both the trout and gonadectomized sockeye were fed but examination of the stomach contents of the gonadectomized sockeye at the time of sacrifice revealed that these fish were eating very irregularly. A great deal of literature has been published on the effects of gonadal hormones on vertebrate bone metabolism. The majority of evidence indicates that in many species, estrogen inhibits bone resorption (Day and Follis, 1941; Gardner and Pfeiffer, 1943; Urist et al, 1948; Budy et al, 1952; Linquist et al, I960; Lafferty et al, 1964; Young et al, 1968; Skosey, 1970; Sorensen and Hindberg, 1971). In birds (see Simkiss, 1961, 1967) and -216-mice (Urist et a_l, 1950) estrogen stimulates neu bone formation. The actions of androgens on skeletal metabolism are not so well documented although in some cases, estrogens and androgens act synergistically. The effects of the sex steroids on bone metabolism depends on the species, age of the animal, the hormone(s) used, the dosage and time course and many other factors. This investigation indicated that estrogen had no effect on the percent ash/dry ueight, or the calcium and phosphate con tents Df the rib banes of either the immature trout or the gonad ectomized sDckeye. Thus, although estrogen produced a dramatic increase in total serum calcium and a rise in plasma inorganic phosphorus, the bone mineral content did not change. Fleming et al (1964) pointed out that only a small amount of calcium need be mobilized to obtain a significant hypercalcaemia. It is possible that the methods used in this study uere not sensitive enough to detect these changes in bone mineral. Calcium could also be mobilized from the soft tissues or absorbed via the gills from the environment (Fleming e_t al, 1964; Simmons, 1971). Reports on the effects Df estrogen on fish bone have been feu and variable. Bailey (1957) observed elevated serum calcium levels in estrogen-treated or preovulatory goldfish but found no histological or X-ray evidence of bone deposition. Clark and Fleming (1963) reported that estrogen injection into mature female killifish, Fundulus kansae, elevated total serum calcium but had no detectable effect on bone histology or the bone calcium content as measured by the l/on Hossa technique. Furthermore, Uoodhead -217-1969b) has reported that intramuscular injection of estradiol 11-fi , 3-benzoate (I.D mg/kg, 4 injections on alternate days) into female lesser spatted dogfish, Scyliorhinus canicula, resulted in a significant (p<D.DDl) increase in total plasma calcium from a control level of 10.3 - 0.5(SD) to 11.4 - 0.6(SD) mEq/1. This observation is relevant to the discussion since elasmobranchs possess a cartilaginous skeleton. These findings support the hypothesis that the effects of estrogen on serum and on bone are distinctly separate. In contrast to the above results, some workers have shown that sexual maturation of the female eel, induced by hormones, was accompanied by bone deformation (Boetius e_t al_, 1962; Fontaine e_t _1, 1964). Moreover, Lopez and Martelly-Bagot (1971) showed that injection of carp pituitary extract into female Anguilla  anguilla L. produced sexual maturation accompanied by hyper-calcaemia and hyperphosphatemia. These authors also noticed a marked proliferation of osteoclasts concomitant with significantly enlarged resorption surfaces, increased osteolysis and demineral-ization of the intercellular substance, without histological modification of the organic matrix. Analysis of the bone by X-ray diffraction revealed that the mineral was lost from the amorphorous phase of the bone mineral rather than the crystalline apatite. These skeletal changes were thought to be caused, in part, by the gonadotrophic production of estrogens. Thus, there appears to be species differences in the response of fish bone to estrogens and this may depend on whether the bone structure is cellular or acellular. -218-Only tuo reports an the effects of androgens on calcium metabolism in fish appear in the literature. A single injection of 1.0 mg of testosterone proprionate into goldfish, had no effect on total serum calcium (Bailey, 1957). Recently, Peterson and Shehadeh (1971) observed a dramatic increase in total plasma calcium of the male and female mullet, Mugil cephalus L., follouing intraperitoneal injection of 25.0 mg crystalline methyltestosterone (hormone injected on alternate days for Dne month). Injection of 5.0 mg of partially purified salmon gonad otrophs also elevated the total plasma calcium level in the female mullet. Indirect evidence on the effects of the sex steroids on bone development in salmon has been shoun by McBride and co-uorkers (McBride e_t al, 1963; van Overbeeke and McBride, 1971; McBride and van Overbeeke, 1971). These authors have demonstrated that gonad ectomy of mature salmon not only prolongs their life span beyond the time at uhich they normally uould have spauned and died, but also leads to the arrest of the external secondary sexual character istics such as the snout and teeth development and red skin colour. Plate 11, pg. 219, shaus the 5 gonadectomized control female sockeye sacrificed February 9, 1972. Note the sea-green backs and silver sides uhich give these fish the appearance of sexually immature seauater salmon (Plate 7, pg. 130). Van Overbeeke and McBride (1971) injected 2.50 mg of 11-ketotestosterone and 17«< -methyltestosterone into gonadectomized sockeye tuice ueekly for a period of 7 ueeks. These sockeye, after 11. Gonadectomized female sockeye (Great Central race). IMote similar colouration and body shape to seauater Chilko sockeye (Plate 7). the 7 weeks of androgen treatment, all showed the red spawning colouration as well as hooked snouts and premaxillary teeth. Thus the androgens definitely play a role in skeletal metabolism in the salmon. In summary, in trout and salmon, estrogen appears to influence total calcium without influencing ionic calcium or hard tissue calcium content. Androgens appear to affect skeletal development but have minimal effects on plasma calcium. The role of calcitonin in the above processes remains to be elucidated The factor(s) governing the secretory rate of calcitonin in trout and salmon are under investigation. -221-SUMMARY In the General Introduction, it was stated that the object ive of this thesis uas to investigate calcium metabolism in fish and the passible physiological role of calcitonin in this process. A brief summary of the thesis findings appears belou. Measurement of the ultimobranchial gland calcitonin con tent Df trout and salmon under a variety of conditions displayed great variation. The UB gland CT contents found in the present study are among the highest reported for louer vertebrates and confirm the original observation of Copp in 19G7 that the fish ultimobranchial gland is a rich source of calcitonin. IMo con sistent correlation uas found between the UB gland CT contents and plasma calcium or phosphate, sex, sexual maturation, smolting, changes in environmental calcium levels or species differences. The louer concentrations of calcitonin in fingerling trout may indicate a relationship betueen calcitonin and grouth. The biological half-life for salmon calcitonin (SCT) uas measured in cannulated trout and salmon. The half-life of SCT uas 27.6 minutes in trout and 48.0 minutes in salmon. This is a rather slou disappearance compared to the half-life of SCT in mammals. Salmon calcitonin injection had no effect on plasma calcium levels in fingerling or adult rainbou trout. SCT infusion uas also ineffective in louering plasma calcium and other electrolytes in cannulated adult female sockeye salmon. Renal excretion of calcium, -222-sodium and magnesium, as well as urine flow, uere not significantly altered by SCT infusion into these salmon. Results from cannulated trout and salmon indicated that these fish can regulate plasma calcium and phosphate very effic iently. Data from estrogen-injected trout and migrating salmon, showed that while total plasma calcium changed dramatically, the ionic calcium level remained remarkably constant and well-controlled. Estrogen injection, in addition to causing hypercalcaemia and hyperphosphatemia, significantly elevated plasma magnesium levels in the salmons Evidence of the hormonal status of calcitonin in fish was obtained when calcitonin was detected in the plasma of salmon using the salmon calcitonin radioimmunoassay. As is the case in mammals, calcitonin is continuously secreted under basal conditions. The circulating level of plasma CT in salmon was higher than that found in mammals and comparable to measurements in birds and other fish. A sex difference in the circulating plasma calcitonin levels (females higher than males) was found in three species of adult salmon. This is one of the first reports indicating a sex difference in the circulating level of plasma calcitonin. The higher circulating level of plasma CT in the female may be related to an increased secretory rate since the ultimobranchial glands of male and female salmon contained approximately the same amounts of calcitonin. The cause of the increased secretory rate is not known but it is clearly not related to ionic calcium levels. -223-Plasma calcitonin levels of female salmon increased significantly during migration from sea to freshuater. These plasma CT levels reached maximum values just prior to spawning, after which they fell off precipitously. Plasma CT levels in the male decreased from sea to freshwater and returned almost to their original levels at spawning. The plasma calcitonin changes during the migration do not appear to be related to any of the plasma or tissue calcium and phosphate alterations. The in crease in plasma CT during sexual maturation in the migrating female sockeye and the decrease after spawning, parallels the changes seen in the gonad-somatic index. The high levels of plasma CT in the female decreased following removal of the gonads. Estrogen replacement did not restore the plasma calcitonin levels in the gonadectomized females. These observations have led to the suggestion that calcitonin may be involved in sexual maturation in some way although it is not caused by high estrogen levels. This suggestion is not new since Lewis et a_l (1971) have recently pro posed that calcitonin may play a physiological role in pregnancy and lactation in rats by protecting the skeleton against the osteo lytic action of parathyroid hormone. Many of the questions posed in the General Introduction have been answered through the experiments outlined in this thesis. How ever, new directions of research in the investigation of calcium metabolism and calcitonin in fish have been revealed. These pro cesses appear to be quite different in fish when compared to mammals, indicating that the function of calcitonin during evolution has changed. -224-BIBLIOGRAPHY Aldred, J.P., Kleszynski, R.R. and Bastian, J.U. Effects of acute administration of porcine and salmon calcitonin on urine electrolyte excretion in rats. Proc. Sac. exptl.Biol. 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