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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^  c l  CALCIUM AND CALCITONIN STUDIES IN PACIFIC SALMON, GEIMUS ONCORHYNCHUS, AND RAINBOW TROUT, SALMO GAIRDNERI  by ERIC GEORGE WATTS B.Sc.  (Hons.),  McMaster U n i v e r s i t y ,  1968  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n the  Department  •f Physiology  We accept t h i s required  t h e s i s as conforming to  standard  THE UNIVERSITY OF BRITISH COLUMBIA JUNE,  1973  the  In p r e s e n t i n g  t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements f o r  an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e  and  study.  I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e copying of t h i s t h e s i s f o r s c h o l a r l y purposes may by h i s r e p r e s e n t a t i v e s . ,  be  granted by  permission.  Department of The U n i v e r s i t y of B r i t i s h Vancouver 8, Canada  Date  Jfj  Department or  I t i s understood t h a t copying or p u b l i c a t i o n  of t h i s t h e s i s f o r f i n a n c i a l gain written  the Head of my  Columbia  ft?3  s h a l l not be  allowed without  my  ii  ABSTRACT  In mammals, calcium homeostasis i s 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 consistent relationship to plasma calcium and phosphate levels, sex, sexual maturation, environmental calcium concentration The ultimobranchial gland calcitonin concentrations  or species.  of fingerling  trout (age 7 - 8 months) were lower than adult trout, suggesting a possible relationship between calcitonin and growth. The biological h a l f - l i f e of salmon calcitonin was measured in free-swimming cannulated trout and salmon.  Results indicate  that the h a l f - l i f e of salmon calcitonin in fish (trout 27.6 min., salmon 48.0 min.)  i s 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 i n fingerling trout and uas also ineffective in cannulated adult trout.  iii  Pdo significant change in plasma electrolytes or urinary electrolyte 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 homeostatic control. Measurements using a sensitive and specific radioimmunoassay, 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. salmonids.  This sex difference appears to be unique to  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 related 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 c i r c u l a t i n g plasma c a l c i t o n i n l e v e l s . Estrogen i n j e c t i o n into these gnnadectomized salmon dramatica l l y elevated plasma calcium but did not restore the plasma calcitonin  levels.  These investigations  indicate that- the physiological role  of c a l c i t o n i n in calcium metabolism in f i s h may be different from that in mammals.  V  TABLE DF CONTENTS Page ABSTRACT  i i  LIST OF TABLES  •  X  LIST OF FIGURES LIST OF PLATES  vii  '  ACKNOWLEDGMENTS  xiii xiv  PREFACE GENERAL INTRODUCTION  1  GENERAL MATERIALS AND METHODS  7  CHAPTER I  CHAPTER II  Experimental Animals Operating Procedures Analytical Procedures  7 10 21  ULTIMOBRANCHIAL GLAND CALCITONIN CONCENTRATIONS  37  Introduction Materials and Methods  37 hh  Results Discussion  ^8 55  BIOLOGICAL HALF-LIFE OF SALMON CALCITONIN IN TROUT AND SALMON  65  Introduction  65  Materials and Methods Results Discussion  66 68 78  CHAPTER III PLASMA AND RENAL EFFECTS OF SALMON CALCITONIN  85  Introduction  85  Materials and Methods  87  Results  91  Discussion  106  ui  Page CHAPTER IU  CHAPTER V  PLASMA CALCITONIN AND TISSUE MINERAL CHANGES IN MIGRATING SALMON  116  Introduction  116  .  Materials and Methods  120  Results  129  Discussion  175  EFFECT OF ESTROGEN ON SERUM IONIC CALCIUM IN TROUT AND GONADECTOMY AND ESTRDGEN ON PLASMA CALCITONIN AND CALCIUM IN SALMON  197  Introduction  197  Materials and Methods  198  Results  202  Discussion  211  SUMMARY  221  BIBLIOGRAPHY  224  vii  LIST OF TABLES Table  Page  I  Physical measurements, plasma electrolytes and calcitonin activities of rainbow trout  II  Physical measurements, plasma electrolytes and calcitonin activities of caho and chinook salmon  50  Distribution of calcitonins among salmon species  61  Specific biological activities of calcitonins from various species .  62  V  Trout physical measurements  69  UI  Plasma calcitonin levels and biological halflives of salmon calcitonin in trout  70  UII  Percent calcitonin activity remaining with time  72  III IU  VIII  k9  Plasma calcium, percent water and haematacrit changes in trout  73  IX  Salmon physical measurements  Ih  X XI  Plasma measurements in t w D sockeye salmon Effects of salmon calcitonin on plasma electrolytes in fingerling rainbow trout .....  75  XII  Physical.measurements and individual plasma calcium levels of cannulated trout..  XIII  92 96  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:  127  XVIII  Physical and plasma measurements - Chilko migration 1971 Plasma electrolyte and calcitonin levels Chilko migration 1971 ...  XIX  summary of sampling data..  132 133  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  Hard tissue mineral changes - Chilko migration 1972  149  XXVII 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 Physical and. plasma measurements - coho salmon study  XXXI XXXII  166 168  Plasma electrolyte and calcitonin levels - coho salmon study  169  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  Physical measurements, plasma and serum electrolytes in control and estrogen-treated trout  203  XXXIII  XXXVI  ix  Table  Page  XXXVII  Bane measurements in control and estrogentreated trout  205  XXXV/III  Physical parameters, plasma c a l c i t o n i n and plasma e l e c t r o l y t e s of intact control, Gx control and Gx estrogen sockeye  207  Bone measurements in intact c o n t r o l , Gx control and Gx estrogen sockeye  210,  XXXIX  X  LIST OF FIGURES Figure la. b.  Page Rainbow trout dorsal aortic cannulation.........  Ik  Caudal vein cannulation technique  19  2.  Blood and urine sampling technique in salmon....  3.  Plasma calcium and inorganic phosphorus levels in trout and salmon  51  Ultimobranchial gland calcitonin concentrations (mU/mg gland) in trout and salmon  53  Ultimobranchial gland calcitonin concentrations (U/kg fish) in trout and salmon  5k  Biological h a l f - l i f e of salmon calcitonin in rainbow trout....  71  Biological h a l f - l i f e of salmon calcitonin in male sockeye salmon  76  Disappearance of salmon calcitonin in trout and salmon  77  9.  Plasma calcium changes in fingerling trouteffect of salmon calcitonin. Injection at time •  93  ID.  Plasma inorganic phosphorus changes in fingerling trout - effect of salmon calcitonin. Injection at time •  3k  Mean plasma calcium changes in adult cannulated trout - effect of salmon calcitonin  98  Individual plasma calcium changes in cannulated adult trout - effect of salmon calcitonin  99  k. 5. 6. 7. 8.  11. 12. 13.  lk.  15.  • 22  Plasma electrolyte changes in a sockeye salmon -., effect of salmon calcitonin infusion. Female sockeye V  101  Plasma electrolyte changes in a sockeye salmon effect of salmon calcitonin infusion. Female sockeye Id.  102  Plasma electrolyte changes in a sockeye salmon effect of salmon calcitonin infusion. Female sockeye Z  103  xi  Figure 16.  Page 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  Plasma electrolyte changes in migrating Chilko sockeye  138  Plasma calcitonin,.plasma calcium and gonadsamatic index changes in migrating Chilko sockeye  139  Haematocrit, plasma protein and plasma % water changes in migrating Chilko sockeyB  141  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  154  26.  Hard tissue phosphate changes in migrating Chilko sockeye  155  Vertebrae mineral content changes in migrating Chilko sockeye  157  Scale mineral content changes in migrating Chilko sockeye  158  Premaxillae dry weight increases in migrating Chilko sockeye  161  Premaxillae calcium content changes in migrating Chilko sockeye  162  31.  Premaxillae phosphate content changes in migrating Chilko sockeye  163  32.  Plasma calcium, plasma calcitonin and gonadsomatic index measurements in 3 groups of coho salmon.....  170  19. 20. 21. 22.  27. 28. 29. 30.  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  Total plasma calcium levels in sockeye - effect of gonadectomy and estrogen replacement.........  208  Plasma calcitonin levels in sockeye - effect of gonadectomy and estrogen replacement  209  35. 36.  xiii  LIST OF PLATES  Plate  Page  1  Fish operating table  11  2.  Trout dorsal aortic cannulation  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  130  8.  Freshwater arrival Chilko sockeye  130  9.  Spawning male Chilko sockeye  131  10.  Spawning female Chilko sockeye  131  11.  Gonadectomized female sockeye (Great Central race)  219  ..  15  xiv  ACKNOWLEDGMENTS This thesis uas accomplished uith the assistance of a number of people.  F i r s t , I would l i k e to thank my research supervisor, Dr.  D. H. Copp, for his enthusiastic guidance in this study.  Technical  assistance in the laboratory, p a r t i c u l a r l y 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 p a r t i c u l a r l y indebted to Dr. Len Deftos, Department of Medicine, University of C a l i f o r n i a , San Diego, U . S . A . , for his collaboration i n the measurements of plasma c a l c i t o n i n and his  interest  in this study. The co-operation and assistance of Jack McBride, Dr. John Davis and Dr. Gordon B e l l , Fisheries Research Board of Canada and Ian Williams, Forrest Scott and Stan K i l l i c k of the International P a c i f i c Salmon Commission, made the studies on salmon possible. Working with these gentlemen was truly a pleasure. My t y p i s t , Mrs. Mildred Brown has done a remarkable job, not only in the long hours she has spent typing t h i s thesis,  but also i n  the helpful advice she has given. F i n a l l y , I wish to thank my wife, Sue, for her help, understanding and love, for without these I could not have made i t . 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 f i s h ; "nearly two hundred years o l d ; perfectly healthy; no symptoms of s e n i l i t y ; no apparent reason uhy they shouldn't go on for another three or four centuries  "  He paused and stood for a moment in s i l e n c e , his fingers on the glass of the aquarium.  drumming with  Poised between mud and  a i r , the two obese and aged carp hung in t h e i r greenish t w i l i g h t , serenely unaware of him.  Dr. Obispo shook his head at them. "The  worst experimental animals i n the world," he said in a tone of resentment mingled with a certain gloomy p r i d e .  "Nobody had a  right to talk about technical d i f f i c u l t i e s who hadn't t r i e d to work with f i s h .  Take the simplest operation; i t was a nightmare.  Had you ever t r i e d to keep i t s g i l l s properly wet while i t was anaesthetized on the operating table? your surgery under water?  Or, a l t e r n a t i v e l y , to do  Had you ever set out to determine a f i s h ' s  basal metabolism, or take an electrocardiograph of i t s heart action, or measure i t s excreta? even to c o l l e c t them?  And, i f so,  did you know how hard i t was  "  "No, you had n o t , " said Dr. Obispo contemptuously.  "And  u n t i l 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 v e r s a t i l e of a l l the vertebrates.  This  fact was acknowledged by Bern (1967) who, when discussing problems in fish endocrinology remarked, "In their endocrine systems, as in a l l other aspects of their anatomy and physiology, the fishes reveal a broader range of variation and a longer history of adaptation than do the ' l a n d - l i v i n g ' (tetrapod) vertebrates. It has now been determined that calcium metabolism in teleosts i s under endocrine control (Hoar, 1957a; Simmons, 1971; Chan, 1972).  The discovery of c a l c i t o n i n in f i s h (Copp et a l ,  1967a) gave r i s e to the question,  "Is  c a l c i t o n i n involved in  this hormonal regulation of calcium metabolism?"  The purpose  of the present thesis was to investigate calcium metabolism in f i s h and the physiological role of c a l c i t o n i n in this process. With regard to the endocrine control of calcium homeostasis, f i s h are unique among the vertebrates in that they lack a parathyroid 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, Pang, 1971a).  The p i t u i t a r y gland, adrenal cortex,  Bern, H.A. Hormones and Endocrine Glands of Science, N.Y. 158: (1967) pg. 455.  1972;  gonads and  Fishes,  -2-  thyroid gland are also d e f i n i t e l y involved in calcium metabolism in f i s h (Hoar, 1957a; Henderson et a l , 1970; Simmons, 1971; Chan, 1972). The importance of the calcium ion to f i s h uas f i r s t  re-  ported by Ringer (1883) uho noted that uhile unfed f i s h l i v e for ueeks in tapuater, they soon die i f placed in d i s t i l l e d uater. gills,  Uptake of calcium from the environment occurs at the fins and oral e p i t h e l i a (Moss, 1965; see Simmons, 1971).  This uptake appears to be more e f f i c i e n t  in freshuater  and i s f a c i l i t a t e d by the presence of phosphate The freshuater  (see  f i s h salves the problems af  fish  Love, 1970).  electrolyte  loss and uater influx by absorbing ions from the environment, maintaining an impermeable integument (scales, s k i n , mucous) and excreting a hypo-osmotic urine (Black, 1957; Bentley, 1971). The problems of calcium regulation in seauater teleosts are. quite different drink seauater.  from those in freshuater.  Marine teleosts  This means that e l e c t r o l y t e s , . including a  s i g n i f i c a n t quantity of calcium, are absorbed by the gastroi n t e s t i n a l tract  (Chan ej_ a l , 1967; Henderson et a l , 1970).  Excess  s a l t s are then excreted d i r e c t l y from the blood through the g i l l s , retaining uater in the f i s h (Smith, 1930; see Parry, 1966;  Potts,  1968).  Again, a dilute urine i s farmed and divalent ion excretion  occurs.  Thus, the environment plays an important role in calcium  regulation in f i s h .  For this reason, both the freshuater  trout,  Salmo q a i r d n e r i , and the anadramous P a c i f i c 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 i t s ability to control i t s internal ionic environment as the external environment 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 laboratory.  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 r a i n f a l l 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 i t 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; and Holden, 1965).  Podoliak  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 g i l l s 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 _  a l , 1972a; Copp, 1972).  origin of calcitonin was  discovered in 1967  The ultimobranchial  and salmon calcitonin  (SCT), the f i r s t 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).  to exert a longer-lasting,  Salmon calcitonin has been shown  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; 1968). 1969.  Louw et al, 1967;  Chan et a l ,  Salmon calcitonin had not been tested in any fish prior to 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 c a l c i t o n i n concentration vary uith age,  4.  sex or  salinity?  Uhat i s the c i r c u l a t i n g l e v e l of plasma c a l c i t o n i n in f i s h and uhat factors govern this l e v e l , migration, sexual maturation,  5.  i.e.  gonadectomy?  Is c a l c i t o n i n involved in calcium and phosphate homeos t a s i s in fish?  6.  Hou do the actions of c a l c i t o n i n in f i s h compare to those in mammals?  Thus, the investigation of calcium metabolism in f i s h and the physiol o g i c a l role of c a l c i t o n i n uas begun on this broad base.  -7-  GENERAL MATERIALS AND METHODS  In this chapter, these materials and methods uhich were common to a l l studies are described.  Materials and methods  s p e c i f i c to i n d i v i d u a l studies w i l l be outlined in the appropriate 1.  chapter.  Choice of Experimental Animal The f i s h was chosen as the experimental animal in t h i s  study for several reasons.  Phylogenetically, the ultimobranchial  (UB) glands f i r s t originate in f i s h .  Almost nothing was known  about the function of c a l c i t o n i n in non-mammals u n t i l Copp (Copp et a l , 1967a, b; Copp and Parkes, 1968a, b) demonstrated that the ultimobranchial gland was a r i c h source of c a l c i t o n i n .  Salmon  and trout were readily available in B r i t i s h Columbia and the s u r g i c a l techniques for working Dn these f i s h were well documented. F i n a l l y , a very sensitive and s p e c i f i c radioimmunoassay for salmon c a l c i t o n i n became available and provided an important t o o l to investigate 2.  the physiological changes of c a l c i t o n i n in these f i s h .  Experimental Animals Several types of f i s h , obtained from a variety of  were studied.  sources,  The types of f i s h included rainbow trout (Salmo  q a i r d n e r i ) , echo salmon (Oncorhynchus kisutch), chinook salmon (Oncorhynchus tshawytscha) and sockeye salmon (Oncorhynchus nerka).  -8-  a)  Rainbow Trout  Rainbow trout used in t h i s study uere purchased from the Sun Valley Trout Farm, Mission, B r i t i s h Columbia. weighed between 6D and 260 g.  The trout  The f i s h were transported to the  University of B r i t i s h Columbia in oxygenated 100 gallon tanks and kept in a 25Q gallon s e l f - c l e a n i n g , f i b r e g l a s s , holding tank (Everlast P l a s t i c s C o . , Vancouver) supplied with fresh running water.  Tapwater was f i l t e r e d and dechlorinated using a f i l t e r  containing activated charcoal, limestone, oyster s h e l l s and sand. Uater temperatures varied seasonally  over a range of 4 - 1 6 ° C ,  but remained f a i r l y stable over any one experimental time course. The trout were fed regularly with commercial trout  pellets  (Purina Trout Chow, Ralston Purina C o . ) . The l i g h t regimen in the f i s h laboratory was controlled using an Inter-Matic Time Switch (Model T 101).  This switch was  adjusted regularly to the seasonal l i g h t conditions and was kept constant during any one acclimation and experimental p e r i o d .  All  f i s h were acclimated to laboratory conditions for at least one week before being used i n 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 a i r 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 s e r i a l sampling of blood from a free-swimming quiet f i s h , over extended periods of time.  For this  reason, the dorsal aorta cannulation technique (Smith and B e l l , 1964) was employed. An operating table, similar to the one described by Smith and B e l l (1967), was constructed and used for a l l s u r g i c a l operations (Plate 1, page 11).  The f i s h was f i r s t  anesthetized  in a bucket of a 1:5,000 solution of MS-222 and then placed ventral side up on the operating t a b l e .  The g i l l s were perfused through  the mouth or opercular openings with a 1:15,000 solution of MS-222 ( B e l l , 1967).  The f i s h was kept moist at a l l times with wet  fish netting.  Throughout the procedure, the operating table  anesthetic was aerated and the water temperature kept constant by placing p l a s t i c bags of ice into the r e s e r v o i r . Following the operation, the f i s h 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, f i n movements, resumption of normal respiratory movements and swimming e f f o r t s .  The  f i s h was placed in the appropriate experimental apparatus (aquarium,  -11-  PLATE 1.  Fish Operating Table  1.  Trout (upside doun) in position for cannulation  2.  Adjustable holding device for  3.  Rubber hose attachment to direct anesthetic over g i l l s  4.  Anesthetic  5.  Uariable-flou pump  reservoir  fish  -12-  urine box) immediately following the operation and at least 12 to 2k hours recovery time allowed before use. operations can lead to considerable  Since  physiological trauma (Houston  e_t a_l, 1969), a recovery period of this length is necessary.  surgical  considered  The experimental apparatus was p a r t i a l l y covered with  black p l a s t i c to prevent the f i s h from being disturbed by the movements of the b)  investigator.  Dorsal Aortic Cannulation Procedure  The dorsal aorta was cannulated at i t s point of i n t e r section with the second efferent by Smith and B e l l (1964).  branchial arteries as  described  The cannula consisted of a 60 cm length  of Clay-Adams PE 60 (ID. 0.762 mm) p l a s t i c 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 t i p of the  snout of the f i s h with a 12-G needle. the olfactory  Caution was taken to avoid  lobes and no injury was evident from this  procedure.  A 3 cm length of Clay-Adams PE 200 p l a s t i c tubing ("sleeve"), heat flared on one end, was passed through this hole i n the snout from inside the mouth.  This PE 200 sleeve was used to secure the PE  60 cannula in place.  The cannula was f i l l e d with heparinized  (10 USP units Heparin (Ammonium Salt) per ml of saline) Cortland Saline  (wolf,  1963) and plugged with a tapered stainless s t e e l pin  when not in use.  The cannula needle was inserted at the midline  junction of the f i r s t angle of approximately  g i l l arch in the roof of the mouth at an 15-20°.  Successful cannulation was i n d i c -  ated by bright red blood rushing into the cannula tubing,  dis-  -13-  placing the heparinized s a l i n e .  The cannula uas sutured  securely  in the roof of the mouth using black s i l k 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 f i l l e d with heparinized s a l i n e .  The cannula was  allowed to t r a i l freely behind the f i s h and i t did not appear to affect behaviour or swimming a b i l i t y 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 f i l l e d 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 contaminated with heparin - s a l i n e .  A second heparinized 1 ml syringe  was used to withdraw the blood sample. the mixture of heparin-saline  Then the f i r s t  syringe with  and blood was immediately returned  to the f i s h and followed by clean heparin-saline so that no blood remained in the cannula.  The s t e e l plug was then replaced in the  cannula and the cannula was returned to the aquarium so the f i s h would be free to move. It should be noted that a l l syringes procedure were p l a s t i c  used in the sampling  (Roehr Monoject) and the dead space of the  syringe was f i l l e d with concentrated heparin (!•••  USP Units = 1 ml  Figure  1 a . Rainbow t r o u t d o r s a l a o r t i c  cannulation  -15-  PLATE 2.  Trout Dorsal Aortic Cannulation  1.  Trout upside doun on operating table  2.  Huber needle (21-G) at f i r s t g i l l arch point of entry (tied in with s i l k thread)  3.  PE 2Q.D sleeve tied in place with cotton thread  k.  PE SO cannula  -Il-  l i q u i d Heparin, Sherwood Medical Industries).  Furthermore, i n -  jections were performed slowly and steadily so as to minimize hemolysis and trauma to the f i s h .  If these precautions were taken,  the fish always remained perfectly s t i l l .  In chronic cannulation  experiments, where large volumes of blood were taken, the red blood c e l l s were resuspended after  plasma c o l l e c t i o n in an approp-  riate volume of heparinized-saline (10 U/ml).  This mixture was  immediately returned to the f i s h , the cannula was r e f i l l e d with heparinized saline and plugged.  Any s i g n i f i c a n t drop in haema-  t o c r i t due to repeated blood samplings was thus prevented (Hickman, 1968). The blood samples were usually transferred to 6 ml s t e r i l e , polystyrene disposable culture tubes and immediately centrifuged for 5 minutes in a standard laboratory c l i n i c a l centrifuge.  Using  glass pasteur pipettes, the plasma was separated off into clean culture tubes with caps.  The plasma was stored on ice i f  measure-  ments were to be made the same day, or frozen on dry ice and stored at - 1 2 ° C i f 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 t r o u t ) .  The  -17-  injectate uas followed f i r s t  by the heparin-saline and blood  mixture and then by a clean O.k ml heparin-saline volume to the cannula and needle dead space.  fill  The time of i n j e c t i o n was  calculated from the moment the injectate entered the f i s h . d)  Caudal Vein Sampling  Terminal blood samples were obtained from the f i s h using the caudal vein sampling technique.  The f i s h was usually tapped  on the head at the beginning of the procedure and held securely wrapped in n e t t i n g . oxygenated,  To ensure that the sampled blood was well  the g i l l s were perfused with aerated water.  The  trout was l a i d f l a t on i t s side on the operating table and a heparinized p l a s t i c 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 f i n .  The needle was  directed forward between the haemal spines into the haemal canal. Using this technique, blood was withdrawn into the syringe from the t a i l c i r c u l a t i o n with l i t t l e injury to the f i s h . 1 ml of blood was required, the needle was l e f t  If more than  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 f i s h in approximately 1 minute. For salmon, the technique was i d e n t i c a l , except that a" larger syringe with an 18-G Vfe inch needle was used.  In the f i e l d ,  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 p l a s t i c 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 f i r s t  anesthetized,  placed on i t s side on the  operating table and perfused with 1:15,000 solution of MS-222. Holding the caudal peduncle in the l e f t hand, the 18-G needle attached to a 2.5 ml p l a s t i c syringe containing heparinized-saline, was inserted from the l a t e r a l - v e n t r a l aspect of the caudal peduncle. The needle was i n s e r t e d ' i n t o the caudal vein, and successful syringe.  Figure l b , pg. 19,  entry was signalled by a flow of blood into the  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 ( f i l l e d 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 i n j e c t i o n of blood through the PE 50 cannula using a 1 ml syringe and 25-G needle f i l l e d with heparinized-saline.  The  18-G needle was then extracted from the puncture s i t e , taking care not to dislodge the cannula, and slipped back down the length of the cannula.  After f i l l i n g the cannula with heparinized-saline  and pinching i t 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 s t e e l p i n .  Figure l b . Caudal vein cannulation technique. Diagram from Hoar and Hickman (1967).  I  -ZD-  Pressure and  the  using  uas  exerted  cannula  2  stitches This  Dver  500  volved •.5  g  a  of  on  cf  black  on  the  amount  the  of  sampling  dorsal a  aorta  or  Urinary  The  salmon  operating uith  a  urogenital  The  ID  uas  urine  and  Bell  fish. the  papilla  (Ingram  flouing The  be  used  study)  from  the  caudal  and  cannula  fish  on  line,  uas  be  in-  site  (about  similar  Using  could  fish  aluays  puncture  previously.  quiet  bleeding  silk.  only  this  the  lateral  placed  Throughout  the  procedure,  solution uas  uith  a  the  to  tuo  infused  and  a  fine  rubber Ltd.)  and  The  urinary  catheter  and  over  then  around  the  the  papilla  opening curved  into  the duct  alloued  aperture  forceps of  size  into uas  tD  hang  catheter  using  surgical  catheter  then a in  closed  purse place  string and  urinary  firmly  off  tying  by the off  cotton  around  the  ligature.  Insured  8  orifice.  beneath  by  of  and  indicated  place  by  uas  French  the  in  uas  up  mouth  urinary  catheterization uhen  side  the  the  catheter  urinary  secured  the  aperture  the  anchored  dorsally  catheter  uas  of  pediatric  successful  the  papilla  pair  ventral  MS-222. by  cm)  from  of  performed  passed along  thread.  procedures  the  outlined  uas  catheter  urinary  could  the  and  1:15,•••  of  (3-6  cm b e l a u  surgical  loss  minimize  anesthetized  table.  tip  bladder  ta  Catheterization  the  catheter  from  2  in  blood  site  simultaneously.  f)  inserting  salmon  free-suimming,  Catheterization the  braided  (hence  Blood  perfused  securely,  cannulation  sampled  the  tied  puncture  of  cannulations, blood  the  type  small  ml).  that  uas  on  that  These no  leakage  -21-  •ccurred.  The catheter was further secured in position by a  long stitch to the caudal peduncle using black surgical s i l k . 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 t D 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 t o t a l weight (g) and the weight of both gonads (g),  the gonad-somatic index (GSI) was calculated from the  fallowing equation: Gonad-Samatic Index (Q2j)  =  gonad weight (g) t o t a l body wt (g)  x  100  This "index- of maturity" was used as a measure of the degree af sexual maturation of the f i s h .  Thus, the higher the index, the  mare advanced was the degree of sexual maturity (Vladykov, 1951). Fork lengths of i n d i v i d u a l f i s h were also recorded. fork length i s the distance  The  (in cm) from the t i p of the snout to  the fork in the caudal f i n . b)  Haematocrits  Blood far haematacrit estimation was introduced into heparinized c a p i l l a r y tubes (Donlab, Ingram and B e l l L t d . ) , 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 c e l l s and used for measurement of percent water or calcium. c)  Plasma Water and Total Solids  Plasma water and t o t a l 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 t o t a l solids percentage composition by weight (TS%), the percent water (,% H^D) by weight (g per 1 0 0 g at and  20OC)  protein concentration (Cppj g per 10.0 ml at 2 Q ° C ) were de-  termined on 1 0 pi of plasma. d)  Preparation of Tissues for Electrolyte Analysis i.  Drying and Ashing Procedures  Hard Tissues The vertebrae,  hard tissues taken far e l e c t r o l y t e analysis included r i b s , premaxillae and s c a l e s .  The tissues were f i r s t  roughly dissected from the f i s h and stored frozen at - 1 2 ° C in a i r tight p l a s t i c bags. The  vertebrae, r i b s 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 1 0 0 ° C ) .  The dry weight was then calculated and the  samples were ashed at 5 7 5 ° 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 t i s s u e .  Since a large error due to evaporation  was involved with the small samples of muscle and s k i n , 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 s i m i l a r l y 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 ( A n a l y t i c a l Reagent, Mallinckrodt Chemical Works).  These samples were further dried to a constant weight  at 1 0 0 ° 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,  i t was necessary to ash these samples for several  days. ii.  Dilutions of Ash  The ashed tissue was weighed accurately, dissolved in 6 N hydrochloric a c i d , and evaporated to near dryness on a hot p l a t e . The sample was then redissolved in 0.1 l\l hydrochloric acid and transferred quantitatively to the appropriate volumetric f l a s k . A further d i l u t i o n 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 c o l o r i m e t r i c a l l y  (Alexander, 1968) after making the proper d i l u t i o n 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 e l e c t r o l y t e measurements was acidwashed and free of contaminating ions.  Small samples (less than  1.0 ml) were dispensed using Oxford Micro-Pipette Samplers (Oxford  -26-  Labaratories). Lab-Trol Division lyte of  American  analyses  the  and  as  normal  standards  quality  Chemistry  Supply  control  abnormal  instrument  i. Two  Hospital  and  and  Patho-Trol  Reference  Corp.)  uere  checks.  The  controls  provided  used  Serums in  (Dade,  all  electro-  simultaneous a  check  of  use  the  linearity.  Calcium  methods  of  calcium  analysis  uere  employed  in  this  solution  uere  study. When calcium  uas  Technicon could  be  10 pi)  only  analyzed  Auto used  and  to  uas  amounts  of  plasma  fluorometrically  Analyzer  Method  measure  found  to  uas  also  Calcium photometry  small  IM-31  extremely be  both  (Jarrell-Ash,  Model  using  P  rapid  280  a  by  1969).  amounts  and  available,  modification  (IMeusome,  small  determined  or  of  of  This  sample  the method  (e.g.  reproducible.  atomic  Atomsorb)  absorption at  a  uave  spectro-  length  of  o 4227  A and  addition and  of  using  lanthanum  phosphorus  recorded  on  0.5%  a  strip  spectrophotometer diluted  i i i . Sodium  and  chart  chloride  as  the  diluent.  suppressed interference  Freier,  recorder  1966).  The  (Sargent  from  readings  Recorder,  The sulfur  uere  Model  SR).  Magnesium  Magnesium  again  chloride  (Trudeau  i i .  uere  lanthanum  and  uas at  determined a uave  uith  Sodium  on  length  0.5%  and  potassium,  the of  same  2852  lanthanum  atomic  8.  absorption  Samples  and  standards  chloride.  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 l i t r e ) 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 i s based on the formation of  phosphomolybdic acid, which i s then reduced by stannous chloridehydrazine. 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 f i l l e d with 2 ml of mineral o i l (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. A l l mineral o i l , 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 o i l remained above the blood.  After discarding the f i r s t few drops of blood,  the sample was immediately ejected under the mineral o i l in a glass centrifuge tube.  The bload was then allowed ta clot for 2  hours (under o i l , an ice) and spun for 2 minutes in a c l i n i c a l centrifuge.  To ensure that the serum did.not contact air during  -28-  transfer,  a layer of o i l uas f i r s t  draun up in a pasteur pipette.  The serum uas then siphoned into the pipette folloued by a second layer of o i l .  The sample uas ejected into cooled p l a s t i c  tubes containing 2 ml of o i l , capped and stored on ice u n t i l measurement.  The temperature of the uater in the fish tank uas  carefully noted. ii.  Measurement of Serum Ionic Calcium  Equipment Serum i o n i c calcium a c t i v i t y ( C a + + ) uas measured potentiometrically using a Calcium A c t i v i t y Flou-Thru System (Orion Research Inc., Model 99-20) attached to a d i g i t a l research pH/mU meter (Corning S c i e n t i f i c Instruments,  D i g i t a l 112 Model).  The pH meter  uas connected to a s t r i p chart recorder (Sargent Recorder, Model SRG) as shoun in Plate 3, pg. 29. interference,  To minimize external e l e c t r i c a l  a Faraday cage uas constructed out of galvanized  uire mesh (6 mm square) and properly grounded. recorder, pH meter, electrode, to the Faraday cage. steel  The s t r i p chart  and syringe pumps uere a l l grounded  Tuo uater jackets uere constructed out of  (2 mm thickness) to contain both the electrodes  and the  syringe (Plate k, pg. 30). Directly after  determination of serum calcium a c t i v i t y ,  the serum pH uas measured using an Ultra-Micro pH/Blood Gas Analyzer (Instrumentation Lab. Inc., Model 113-S1) uith a Constant Temperature Control Module (IL, Model 127). A l l 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  S t r i p chart  Flow-Thru System  recorder  Digital  pH/mV/ m e t e r  Faraday  cage  I n f u s i o n pump and e l e c t r o d e a s s e m b l y  »  PLATE k.  Infusion Pump and Electrodes uith Uater Jacket  1.  Electrode uater jacket (in black)  2.  Syringe uater jacket  3.  Inlet and outlet tubings for uater  k.  Infusion pump  (in black)  -31-  Procedure Use D f 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 d i l u t i o n of a stock solution of CaCO^ in 150 mM IMaCl. standards,  The  corresponding to Orion standards A, B and C, contained  2.00, 4.00 and 8.00 mg/100 ml r e s p e c t i v e l y .  These uere checked  against reference standards using atomic absorption spectrophotometry.  Three drops of 0.1 M triethanolamine and 0.006 g of  trypsin (Trypsin: 2 x c r y s t a l l i n e , s a l t free, N u t r i t i o n a l Biochemicals Corps.) uere added to each 10 ml of pure standard. standards,  The  samples, syringes and electrodes, uere a l l 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. standards was always - 7.5 mV.  Separation between the  The standards were repeated several  times at the beginning and end of each set of analyses.  When the  system was stable and the d r i f t 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 p e r i o d i c a l l y in order to check the standard curve r e p r o d u c i b i l i t y .  A l l serum samples were removed from under  the mineral o i l using a 1 ml p l a s t i c tuberculin syringe and a 26-G Yz inch needle.  Care was taken to exclude a i r bubbles and o i l 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 u n t i l the reading s t a b i l i z e d on the recorder.  Immediately  following the i o n i c calcium a c t i v i t y 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 s i g n i f i c a n t  calcium a c t i v i t y .  change in serum ionic  Therefore, a l l i o n i c calcium measurements  performed as soon as possible after  uere  sample c o l l e c t i o n and always  u i t h i n the 3 day l i m i t . Total serum calcium uas measured fluorometrically (Neusome, 1969) on duplicate 20 pi samples, d i r e c t l y follouing the i o n i c calcium a c t i v i t y and pH readings.  This alloued the calculation  of percent i o n i c calciumPercent i o n i c calcium (.% C a + + ) = i o n i c Ca ++ (mg/100ml) t o t a l Ca ++ (mg/100ml)  x  IQQ  Calculations The calcium flou-thru electrode developed a potential proportional to the logarithm of the calcium ion a c t i v i t y in the sample. Potentials became increasingly positive in more concentrated s o l utions, and increasingly negative in more d i l u t e 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 a x i s ) .  The calcium concentration of the  unknouns uas then determined from this c a l i b r a t i o n curve. A computer program uas devised on a desk-top computer ( O l i v e t t i Underuood, Programma 101 Model) uhich computed the slope  -33-  •f  the standard curve and calculated the i o n i c and t o t a l calcium  (in mg per 100 ml) and the percent ionized calcium for each unknown sample. g)  Bioassay of Calcitonin in Ultimobranchial Glands i.  Rats  A l l c a l c i t o n i n bioassays were performed on 3-4 week o l d , 8D g, black hooded male rats of the Long-Evans s t r a i n (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 f i s h were s a c r i f i c e d by severing the spinal cord and the head excised from the body s l i g h t l y posterior to the operculum. The l i v e r , gonads and esophagous were removed and the transverse septum containing the ultimobranchial gland exposed. pair of s c i s s o r s , the gland was carefully cut out.  Using a fine It was weighed  immediately (wet weight), quickly frozen on dry ice and stored at -12 D C in capped auto analyzer cups.  The UB gland appeared quite  d i s t i n c t in the trout (Plate 5, pg. 39) and just the area around this "moustache-shaped"  gland was cut out.  UB gland was more diffuse  However, the salmon  (Plate 6, pg.4Q ) and hence necessitated  removal of the majority of the transverse septum. Since the transverse septum of f i s h contains l i t t l e  fat,  i t was found unnecessary to fat-extract the ultimobranchial t i s s u e .  -3k-  The glands uere homogenized using a tissue homogenizer (Tri-R Instruments (pH = k.3).  Inc.) in a vehicle of 0.1 (\l HCl and 0.1% glycine Fibrous material uas removed by f i l t e r i n g the homogen-  ate through a piece of s t e r i l e  gauze.  Care uas taken to keep  the gland and homogenate on ice at a l l times. iii.  Bioassay Technique  A l l 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 t a i l 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 i n j e c t i o n uas performed under ether anes-  thesia and the rats uere bled from the t a i l 60 minutes after injection.  the  Blood uas collected d i r e c t l y into heparinized c a p i l l a r y  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 f i r s t dilutions for each gland.  performed to determine the correct  Once the d i l u t i o n s uhich gave the proper  curve uere obtained, a fine assay uas performed.  The difference  betueen the mean plasma calcium l e v e l in mg per 100 ml of the control group and the plasma calcium l e v e l of each experimental blood sample uas calculated and called the response.  A log dose-  response curve uas constructed and the amount of c a l c i t o n i n in m Units/mg uet ueight of gland uas calculated from the standard curve of MRC Research Standard B ( C a l c i t o n i n , Porcine Thyroid).  -35-  The concentration of c a l c i t o n i n per body weight (mU/kg body wt) was also estimated for each gland. salmon c a l c i t o n i n (MRC, M i l l H i l l ,  A house standard of p u r i f i e d England) and checked against  the MRC Research Standard B, was injected in assays to ensure the consistency of the r a t s ' hypocalcaemic response. be noted that,  due to the diffuse  It should  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 c a l c i t o n i n lev/els were measured using a very sensitive and s p e c i f i c radioimmunoassay for salmon c a l c i t o n i n developed by Dr. Len Deftos, Endocrine Section, Department of Medicine, University of C a l i f o r n i a , San Diego, U . S . A . (DeftDs e_t a_l, in press). Plasma samples for c a l c i t o n i n assay were frozen on dry ice immediately after c o l l e c t i o n and stored at - 1 2 ° C i n s t e r i l e polypropylene culture tubes.  These samples were then packed  in dry ice and shipped to Dr. Deftos by a i r . A l l plasma c a l c i t o n i n measurements reported in this thesis were performed by Dr. Len Deftos.  Under optimal conditions, the  assay could detect 50-100 pg of c a l c i t o n i n per millimeter of salmon plasma..  -36-  i.  Statistical  Analysis  A l l measurements u i t h i n mean -  standard e r r o r  uere made u s i n g the  about the mean ( S E ) .  Group comparisons  " S t u d e n t ' s " t - t e s t c a l c u l a t e d u i t h the  of a d e s k - t o p computer Probability  each group uere expressed as the  ( O l i v e t t i Underwood C o . , Programma  values were o b t a i n e d from standard  tables.  aid 101).  I. ULTIMOBRANCHIAL GLAND CALCITONIN CONCENTRATIONS Introduction The ultimobranchial gland f i r s t in f i s h .  appears phylogenetically  Van Bemmelen, in. 1885, o r i g i n a l l y described the gland  in elasmobranchs.  He named the two small e p i t h e l i a l masses which  were found caudal to the last pair of branchial c l e f t s , "suprapericardial  the  bodies" and considered that they represented a  rudimentary seventh pair of branchial pouches. a b r i e f account of the suprapericardial  De Meuron (1886) gave  body in selachians and  amphibians and hamologized the body in these forms with the "accessory thyreoid" of r e p t i l e s ,  birds and mammals.  The term "ultimobranchial  it  body" (ultimobranchialen Harper) was introduced by G r e i l in 1905 and more correctly describes the embryonic o r i g i n and location in most vertebrates.  Uatzka reported that the gland was present in a l l orders  of vertebrates except the cyclostomes In elasmobranchs,  (Uatzka,  1933).  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 leptocephalus  lying between the pharynx and the p e r i c a r d i a l w a l l .  Giacomini (1909) found them not only in leptocephalus adult Anguilla sp.  The  but also in  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 l e f t  suprapericardial  body  -38-  persisted in the adult selachian. in other lower vertebrates.  This condition is known to occur  In contrast,  the ultimobranchial gland  of most teleosts i s b i l a t e r a l and imbedded in the transverse septum between the abdominal cavity and sinus venosus (Krawarik, 1936). In the rainbow trout,  i t takes the appearance of a small  "moustache-  shaped" band of white tissue and l i e s immediately ventro-lateral to the esophagus (Plate 5, pg.  39 ).  The salmon UB gland i s located in  a similar p o s i t i o n , 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 l a t e r a l l y and the cardiobranchial cartilage and coracobranchial muscle medially. The ultimobranchial gland has a f o l l i c u l a r 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). normally has a f o l l i c u l a r structure  The teleost UB gland  (Rasquin and Rosenbloom,  1954;  Sehe, I960; Robertson, 1967, 1969; Lopez et_ a l , 1968; Copp, 1969a; Deville and Lopez, 1970). as cords of c e l l s  Some authors have noted this gland appears  (Eggert, 1938; Copp, 1969a;Pang, 1971b;which take  on a f o l l i c u l a r 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 r e p t i l e s ,  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 f i n s .  Triangular-shaped  ultimobranchial gland (arrow) l i e s 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 f i n s .  Diffuse  ultimobranchial gland l i e s below esophagus within the transverse septum.  Kidney tissue l i e s  the esophagus and l i v e r tissue below.  above  -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 c e l l in a l l jawed vertebrates. Although i t 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 i n t e r s t i t i a l spaces adjacent to the f o l l i c l e s 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 a l , 1970).  More recently, i t  has been shown that the calcitonin-secreting cells are derived from the neural crest (Le Dourain and Le Lievre, 1970,  1972;  Pearse and  -42Polak, 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 Rosenbloom (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 and calcification of the kidneys.  degeneration  They postulated that these path-  ological changes were due to over-secretion of the parathyroidlike ultimobranchial gland. Copp e_t a_l (1967a) were the f i r s t 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  f i r s t 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 i t 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 k i l l i f i s h , Fundulus heteroclitus and the codfish, Gadus  -43morhua (Pang e_t a_l, 1971), the e e l , Anguilla /japonica  (Orima e_t a l ,  1872a), the llngcod, Ophiodon elongatus and the rainbou trout, gairdneri (Copp et a l , 1972a).  Salmo  Calcitonin a c t i v i t y uas not detect-  able in extracts of the thyroids of c a t f i s h ,  Ictalurus melas (Louu  et a l , 1967) or of school sharks, Galeorhinus galeus (Louu e_t a l , 1969). The amino acid composition of salmon c a l c i t o n i n uas determined in 1969 by O'Dar e_t a_l and the complete amino acid sequence of salmon c a l c i t o n i n uas determined by N i a l l e_t a_l in the same year. Synthesis of the salmon c a l c i t o n i n molecule by Guttmann e_t a_l (1969) made i t the f i r s t  non-mammalian hormone to be f u l l y  characterized.  The salmon molecule consists of 32 amino acids but i t differs  con-  siderably from that D f porcine, bovine and human c a l c i t o n i n s ,  the  four hormones being homologous in only 9 out of 32 positions.  Cur-  i o u s l y , the salmon sequence i s more homologous to the human structure (16 out of 32 positions  are s i m i l a r ) than i t i s to either the  porcine or the bovine sequence ( N i a l l e_t a_l, 1969). The high s p e c i f i c  b i o l o g i c a l a c t i v i t y of pure salmon c a l c i -  tonin (5000 MRC U/mg, Q'Dor et_ a l , 1969b) i s coupled uith i t s more hypocalcaemic and prolonged action in mammals (Brooks e_t a_l, 1969; Copp e_t a_l, 197D; Guttman e_t a l , 1970).  In the standard rat bio-  assay,the b i o l o g i c a l potency of purified salmon c a l c i t o n i n i s  at  least 20 times greater than porcine, ovine or bovine c a l c i t o n i n (O'Dor e_t a_l, 1969b; Keutmann et a l , 1972).  The high a c t i v i t y of the  salmon hormone may be due i n part, to i t s greater s t a b i l i t y characteristic  of the three different  uhich have been isolated  and i s  forms of salmon c a l c i t o n i n  (Keutmann e_t a_l, 1972).  Thus, the ultimo-  branchial glands of f i s h have been shoun to be a r i c h 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, i t 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 a l l 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, a l l 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 f i s h .  Blood uas taken by caudal vein  sampling and plasma calcium and inorganic phosphorus (Pi) uere measured for each f i s h .  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 t r o u t .  The f i n g e r l i n g 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 fingerl i n g 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 i n d i v i d u a l f i s h uere recorded. The adult immature trout uere normal, sexually-immature f i s h and were sampled to obtain control levels of ultimobranchial c a l c i tonin concentrations. A group of sexually-maturing trout uere studied, since i t uas noted that the ultimobranchial gland became more d i s t i n c t l y outlined at this stage.  The glands uere bioassayed for c a l c i t o n i n content  to determine whether there uas increased UB gland a c t i v i t y during spauning. The UB gland also appeared more active in smolting t r o u t . Smolting i s a stage in the l i f e history of a trout or salmon characterized by morphological, p h y s i o l o g i c a l , behavioural and hormonal changes uhich prepare the f i s h for i t s seauard migration. The f i n a l 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 f i r s t held for one week in a 55 gallon tank  -46-  of fresh running uater,  gradually adapted to f u l l strength sea-  uater ( s a l i n i t y , 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 - 1 2 ° C , the c a l c i t o n i n a c t i v i t y of the sea-water acclimated trout may only be meaningful when calculated an the basis of Units per kilogram body weight of f i s h . bioassay t e s t .  The glands were weighed p r i o r to each  A l l 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 d i f f i c u l t y was due to the fact that the trout  mature at different rates and times.  By nature, rainbow trout spawn  in the spring ( L e i t r i t z , 1969), but f i s h inter-breeding practices have developed trout that spawn in spring and f a l l .  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 Washington, U . S . A . , through the co-operation of the Washington State Department of F i s h e r i e s .  Both species, captured in freshwater,  were in  peak spawning c o n d i t i o n . The chinook salmon (Oncorhynchus tshawytscha) were obtained an October 21, 1970 from the Deschutes River Holding Ponds near Olympia, Washington, U.S.A.  These f i s h 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  -47the end of September to November 10th ( C H . E l l i s ,  Chief Hatchery  Management, Washington State Department of F i s h e r i e s , communication).  personal  These fish uere hatchery raised and had an average  age of 4 years. The coho salmon (Oncorhynchus kisutch) uere taken on November 30, 1970 from the Samish River Holding Pond approximately 10 miles from the sea (Puget Sound).  The journey from sea to r i v e r  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 s k u l l and immediately blood sampled.  Blood  was collected from the caudal vein as outlined in General Materials and Methods, pg. 17 ) .  Measurements on each f i s h included fork  length, t o t a l f i s h weight and gonad weight.  The female coho salmon  were extremely ripe (sexually mature), making i t impassible to obtain an accurate gonad weight.  The head of the salmon was excised and  the ultimobranchial gland dissected and stored on dry i c e .  The UB  gland weights were recorded upon return to the laboratory.  In salmon,  the ultimobranchial gland i s quite diffuse, septum was uniformly cut out.  so the entire transverse  The glands were stored at -12 D C u n t i l  assayed and a l l assays were performed within 2 months of the date of c o l l e c t i o n .  The procedure for c o l l e c t i o n , homogenization and  bioassay of the glands i s described in d e t a i l 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 i s 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 a l l 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 i t 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 .  P h y a l c a l Measurements, Plasma E l e c t r o l y t e s and C a l c i t o n i n A c t i v i t i e s o f Rainbow Trout (Mean - SE)  F i s h Group  Sex  n  Fingerling Trout 25 Feb./70 5.5°C  m&f  8  12.8  Adult Immature trout 11 Sept./70 14°C  m  6  196.6  f  7  181..5  Adult Mature trout 16 Jan./70 6°C  m  7  236.8  f  7  219.1 +  Immature Smolt Trout 18 Mar./70 6°C  m  5  145.2  f  4  144.5  Immature Seauater Trout 2 June/70 10.5°C  m  2  200.0  5  207. 4 + 11.90  f  TotBl Wt Cg) +  +  +  +  + +  +  GSI  1.09  -  10.00  0.19  9.20  0.26  15.20  1.55  8.30  14.82  Ca mEq/1  4.84  + +  0.05  4.17  0.03  4.50  0.28  5.00  6.76 + 2.74  5.65  0.03  5.67  0.1<T  7.00  0.07  +  + +  +  0.03  5.38  0.01.  5.51  0.02  4.55  0.32* + 0.05  t - t e a t p r o b a b i l i t y male v s . female.  +  + +  Plasma Pi mEq/1  0.17  7.66  0.12  5.00  0.19  4.68  0.1*9  5.13  + 0.24  5.10  +  + +  +  0.26 0.25  7.63  0.25  5.75  4.83 + 0.26  a.  7.16  +  + +  +  + +  +  UB Gland Fresh Wt (mg)  o.4o  5.96  0.18  34.96  + +  0.80  +  91.2  +  3.04  833.9  3.17  302.l -  21.38  0.5  124.41  29.3  0.18  35. 24  0.19  38.80  3.81  368.7  0.39  47.67 + 2.05  565.1  0.62  31.68  0.68  27.77 + 2.44  691.7 + 199.22  0.29  75.90 ±26.10  190.9  6.31 + 0.47  p< 0.005  +  Calcitonin Activity mU/mg U/kg f i s h U/gland Fresh gland  b.  +  +  3.45  52.66 ±13.36  p< 0.01  a  474.8  327.3  + +  +  + +  +  +  "42.35  126.36  15.7  173.40  25.9  186.35  13.1  28.34 72.92  + +  +  0.09  35.5  4.87  148.1  13.9  + +  +  1.92  sa.tfi  6.48  62.3  7.40  114.9  4.43  92.2  + 6.84 20.2  15.3  +  7.14 0.63  145.1  75.1 67.4  + +  + +  + +  4.35  21.99. 9.97  22.55 29.18  29.01 53.91  33.10 2.20  Table  II .  P h y s i c a l Measurements, Plasma E l e c t r o l y t e s and C a l c i t o n i n  Activities  of Coho and Chinook Salmon (Mean - SE)  Calcitonin  Plasma F i s h Group  Sex  n  T o t a l Wt (Kg)  GSI  Pi mEq/l  Ca mEq/l  UB Gland Fresh Idt  mU/mg Fresh gland  U/Gland  Activity U/Kg  Fish  (g) Coho Salmon 30 Nov./70 4.5°C  m  7  5.0 - 0.20  f  7  4.5 - 0.21  Chinook Salman 21 0ct./70 6.D°C  m  8  8.4 i 0.49  f  5  8.1 - 0.1.7  ±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  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  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  26.74 I 1.43  5.98 i 0.38  7 . 0 4 i 0.48  2.47 i 0.17  203.5 i 86.65  482.3 - 195.71 63.6 - 29.81  4.72 i 0.10  -  6.69  t - t e s t p r o b a b i l i t y male v s . female  b  a.  p< 0.005  b.  4.51  p<0.05  I  m •  -51-  Salmon  Trout  8.00  W  E  Immature both Sexes  6.00 •  4.00-  2.00 •  0.0  c? » 84  9  0*9  Finger Mature -ling Immature Plasma Ca  Figure 3.  o*  9  6 3 | 7 6 7 6|7 4 5 5|4 4  i  o*  9  2 2| 5 5  0*  9  77166  Seowoter Smolt  6*9 8 8|5 5  Chinook Coho  i Inorganic Phosphorus  Plasma calcium and inorganic phosphorus levels in trout and salmon  -52In the trout,, the highest plasma fingerlings and smolts.  Pi  values were recorded for the  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 consistent sex difference.  For example, the coho male mean plasma Pi  was s i g n i f i c a n t l y higher (p<D.D5) than the female l e v e l ,  whereas  in the chinook salmon, the female mean plasma Pi was markedly higher  (p<D..rj5) than the male l e v e l .  In a l l cases except the mature female  trout and the female coho, the mean plasma inorganic phosphorus was higher than the mean plasma calcium l e v e l . The data as i l l u s t r a t e d in Figures k & 5 on pages 53 and 5k shows that the c a l c i t o n i n a c t i v i t y of the ultimobranchial glands exhibited an extremely wide range of values.  The i n d i v i d u a l c a l c i t o n i n  a c t i v i t y variation i s indicated by the large SE values for each group of f i s h .  The lowest CT a c t i v i t y was found in the f i n g e r l i n g trout  (91.2 - 21.38 mU/mg  gland).and the highest a c t i v i t y  mU/mg gland) in the immature male trout (Figure 4,pg.  (833.9  -  lZk.kl  53).  The sea-  water trout glands contained f a i r l y law CT concentrations.  This may  in part r e f l e c t the fact that the glands weighed s l i g h t l y more than those of the Dther adult t r o u t .  However the wide variation of c a l -  citonin a c t i v i t i e s in the control group of immature adults, makes a v a l i d comparison d i f f i c u l t . Low levels of c a l c i t o n i n a c t i v i t y were found in the two groups of salmon. Except for the immature and seawater t r o u t , no s i g n i f i c a n t sex difference in the c a l c i t o n i n levels of the UB glands was observed. Estimated on a U per kg f i s h basis (Figure 5 f pg. 5k) > "the fingerling trout again exhibit the lowest l e v e l of c a l c i t o n i n a c t i v i t y  Figure 4.  Ultimobranchial gland calcitonin concentrations (mU/mg gland) in trout and salmon  -5k-  Salmon  Rainbow Trout  3  £  40  both Sexes  •  I 8  <f  9  7  7  cf 9 5  4  d 9 2  5  cf c. 7  7  <s 9 8  5  Fingerling Immature Mature Smolt Seawater Coho Chinook  Figure 5.  U l t i m o b r a n c h i a l gland c a l c i t o n i n (U/kg f i s h ) i n t r o u t and salmon  concentrations  -55-  amang a l l the groups of t r o u t .  The chinook male salmon also have  low levels of c a l c i t o n i n a c t i v i t y (U per kg f i s h ) . male trout c a l c i t o n i n a c t i v i t y (U per kg fish)  The immature  i s higher than a l l  other groups uith the exception of the mature females,  the smolt  females and the smolt males.  Discussion Although i t gives l i t t l e information concerning secretion rate, measurement of the c a l c i t o n i n a c t i v i t y 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,  d i e t , sex,species, secretion rate, bane diseases and  other hormones.  Thus, Robertson (1968a,b) has noted hyperplasia  and c e l l u l a r hypertrophy of the UB gland in hypercalcaemic frogs.  He  also presented h i s t o l o g i c a l evidence that t h i s uas due to increased production and release of c a l c i t o n i n .  In chickens, Belanger (1971)  has described the hypertrophy and hyperplasia of the UB gland parenchymal c e l l s in response to hypercalcaemia and the decrease in secretory a c t i v i t y during prolonged hypacalcaemia.  Dther uorkers have confirmed  the fact that hypercalcaemia in birds stimulates  c a l c i t o n i n release  from the ultimobranchial gland (Ziegler et_ a l , 1969; Bates et_ a_l, 1969; Copp et a l , 1970; Care and Bates, 1972).  Some vertebrates such  as the goose, possess r e l a t i v e l y small stores of c a l c i t o n i n (2 U per gram fresh gland ueight) and in contrast to the pig and sheep, must rely on increased biosynthesis and Bates, 1972).  in order to increase secretion  (Care  F i n a l l y , c a l c i t o n i n concentrations have been  found to be elevated in the peripheral blood and thyroid gland of  -56-  humans a f f l i c t e d uith the condition of medullary carcinoma of the thyroid (Clark e_t a l , 1969; Deftas and Potts, 1971a; Deftos e_t a_l, 1971b).  1970; Deftos et a l  Another factor affecting  the c a l c i t o n i n  content of the UB gland, i s that the hormone may be stored at the tissue l e v e l as an inactive precursor and later converted to the active p r i n c i p l e only on the appropriate release s t i m u l i . In order to assess measurements of rat thyroid gland c a l citonin content in terms of synthesis and release, Gittes et a l (1968) have made the following postulates: 1.  A net decrease in calcium lowering a c t i v i t y per gland, represents an excess of release over synthesis of c a l citonin.  2.  A net increase of calcium lowering a c t i v i t y per gland, represents-a greater synthesis than release of c a l c i t o n i n .  These authors found that persistent hypercalcaemia invariably caused a decrease in the c a l c i t o n i n content of the rat thyroid glands. Further, they attributed an increased c a l c i t o n i n content in chronica l l y hypocalcaemic rats to a continuous synthesis of the hormone in the absence of any release stimulus.  Thus, the interpretation of  s t a t i c UB gland c a l c i t o n i n content measurements i s d i f f i c u l t , and experiments designed to investigate this parameter must be r i g i d l y controlled. To be able to compare different  sets of r e s u l t s , the tech-  nique of extraction and measurement of c a l c i t o n i n a c t i v i t y should be the same.  Parsons and Reynolds (1968) point this out in a statement  emphasizing,  "the necessity  calcitonins [to]  for estimates of b i o l o g i c a l potency of  be accompanied by a statement of the assay method  and of the standard  used."  -57Few ujorkers have investigated tent changes during development.  the UB gland c a l c i t o n i n can-  On the premise that c a l c i t o n i n  might be more active in the early stages af growth when there i s a high bone turnover rate, Dent et_ a_l (1969) measured the UB gland CT content in developing male chickens (Ghostley s t r a i n of non-inbred White Leghorn chickens).  These authors found that the c a l c i t o n i n  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 u n t i l 70 weeks of age,  despite  considerable  v a r i a t i o n , 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 c a l c i t o n i n did not play a major role in the development and maintenance of the bony skeleton of chickens. Although the present study investigated the fingerling trout (age 7 - 8  only two ages of f i s h ,  months) showed considerably lower  values of c a l c i t o n i n content than any of the other 4 groups of trout (age 2 - 3  years).  The c a l c i t o n i n content (expressed as mU per mg -  wet wt gland and U per kg body wt) of the salmon (age 3 - 5 was also very low.  years)  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 c a l c i t o n i n 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 c o n f l i c t i n g results could be explained by n u t r i t i o n , sex or species  differences. Although i t may be superfluous  to compare the c a l c i t o n i n  content of fish UB gland uith rat thyroid gland, i t has been shoun that 5 and 15 day old rats have s i g n i f i c a n t l y louer thyroidal c a l citonin contents (mU per mg fresh thyroid) than older age groups (Frankel and Yasumura, 1970). uas no s i g n i f i c a n t  These authors also noted that there  difference betueen the thyroid gland c a l c i t o n i n  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 c a l c i t o n i n contents of f i n g e r l i n g trout found in the present  study.  Certainly, the plasma calcium l e v e l (the signal for c a l c i t o n i n release in mammals) of the f i n g e r l i n g trout, i s not excessively  high.  The higher plasma calciums in the mature versus immature adult trout, i s due to sexual maturation and has been noted in other f i s h by many others  (Miescher, 1897; Hess et_ a l , 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 t r o u t .  It is  possible  that the arbitrary d i v i s i o n of the trout into immature and mature groups,  could account for the v a r i a b i l i t y of the data. Smolting salmonids are characterized by their s i l v e r color-  ation (Hitching and Falco, 1944) an alteration of body proportions  -59-  (Hoar, 1939) and the development D f a s a l i n i t y preference man, I960).  (Bagger-  The smelting process i s also known to be accompanied  by an increase in the adrenocortical volume (Qlivereau, an elevation of 17-hydroxycorticosteroid plasma levels and Hatey, 1954).  1962) and (Fontaine  Glomerular f i l t r a t i o n and urine flow have also  been shown to decrease considerably in smolting steelhead (Holmes and Stainer,  1966).  trout  Thus i t can be seen that the physio-  l o g i c a l , morphological and behavioural changes occurring in smolting salmonids are extremely complex and appear to prepare the f i s h for i t s seaward migration (Hoar, 1951). In the present study, the smolt plasma calcium levels were elevated over those of the immature trout yet were not s i g n i f i c a n t l y different  from the mature trout l e v e l s .  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 c a l c i t o n i n contents. These high values are not correlated with either the plasma calcium or Pi l e v e l s . The seawater acclimated trout show plasma calcium levels only s l i g h t l y 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 t r o u t . The low c a l c i t o n i n contents observed in the seawater trout may ref l e c t the fact that the wet weights of t h e i r 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 s t i l l do not exceed the values for the mature and smolting trout.  This finding is quite i n t e r e s t i n g ,  in view of the fact  that  -60the environmental calcium concentration of the seauater (15 mEq/ l i t r e ) uas considerably higher than the freshuater  calcium concen-  tration (less than 0.5 mEq/litre) of the other groups of t r o u t . These results are in marked contrast to those of Orimo et_ a_l (1972a) uho found that UB gland c a l c i t o n i n contents (U/gland) D f eels, Anguilla japonica, the freshuater  kept in seauater, uere s i g n i f i c a n t l y greater than  controls.  This finding uas p a r a l l e l e d by increased  plasma c a l c i t o n i n and serum calcium levels in the seauater e e l s . Houever, the fact that the eel i s a catadromous f i s h ( l i v i n g i n freshuater,  spauning in seauater) and the rainbou trout i s a  euryhaline ( l i v i n g and spauning in freshuater) contrasting r e s u l t s .  may account for these  It should also be noted that Orimo et_ a_l (1972 a )  did not report the age and stage of sexual maturation of the e e l s . The present study is supported by the uork of Pang (1971b) uho found that the ultimobranchial glands of the k i l l i f i s h heteroclitus) uere more active than in seauater.  (Fundulus  ( h i s t o l o g i c a l evidence) in freshuater  He also noted that the ultimobranchial body  a c t i v i t y uas independent of serum calcium l e v e l s ,  and postulated  that the function of the gland might be related to osmoregulation rather than calcium metabolism. The UB gland c a l c i t o n i n content (mU/mg gland) of the coho salmon uas very similar to the chinook and no sex difference detected.  uas  The s l i g h t l y louer values compared to the trout could  possibly be due to the fact that the salmon ultimobranchial gland i s more diffuse  and hence a larger area of salmon transverse septal  tissue uas dissected out.  This inclusion of excess tissue uould  therefore,  louer the c a l c i t o n i n a c t i v i t y 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 t r o u t .  It i s  interesting  to note that the salmon, which uere a different species, migrating, fasting  and extremely sexually mature (note the salmon GSI, Table I I ,  pg. 50) displayed UB gland c a l c i t o n i n contents that were not very different from those found in the t r o u t . 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 c a l c i t o n i n I,  II,  III,  have been isolated from k species of salmon and t h e i r d i s t r i b u t i o n i s shown in Table III.  Table I I I .  D i s t r i b u t i o n of Calcitonins Among Salman Species.*  Species  Calcitonin Component I  II  Sockeye (0. nerka)  +++  +  Chum (0. keta)  +++  +  Pink (•.  qorbuscha)  +++  +  Coho (0.  kisutch)  +++  III  +  Data from Keutmann e_t al_ (1972) The s p e c i f i c b i o l o g i c a l a c t i v i t i e s  of the 3 salmon c a l c i t o n i n  components is compared to the mammalian c a l c i t o n i n s in Table IV, pg. 62.  -62-  Table I V .  Specific B i o l o g i c a l A c t i v i t i e s of Calcitonins from Various Species.*  Preparation  Mean Specific A c t i v i t y MRC Units/mg** 120  Porcine Bovine  60  •vine  70  Human  70  Salman I  2,700  Salman II  2,400 600  Salman III  Data from Keutmann e_t a_l (1972) *  A l l assays carried out on l y o p h i l i z e d 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 s p e c i f i c b i o l o g i c a l a c t i v i t y (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 t h e i r UB gland c a l c i t o n i n contents. Data in the present study confirm the o r i g i n a l reported by Copp e_t a_l (1967a) i s a r i c h source of c a l c i t o n i n .  observations  that the f i s h ultimobranchial gland In fact, the f i s h UB gland c a l -  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 a l (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 a l (1972a).  The diFFerence may l i e 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 relationship 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 f i s h .  This data therefore,  does not provide a firm  basis an uhich to outline the physiological role of c a l c i t o n i n in f i s h .  -65-  II.  BIOLOGICAL HALF-LIFE DF SALMON CALCITONIN IN TROUT AND SALMON  Introduction The E n d o g e n o u s circulating plasma level of calcitonin depends on the s e c r e t i o n rate from the ultimobranchial gland and the clearance rate from the plasma.  In the f i r s t chapter, i t 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 investigate its biological h a l f - l i f e (T /2) in mammals (Habener e_t a_l, 1  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 a l , 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 h a l f - l i f e 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 i t 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 i t required rather large blood samples (1.0 ml) and hence made serial sampling Dn small fish d i f f i c u l t . Trout The calcitonin biological h a l f - l i f e experiment uas performed in the Vancouver Public Aquarium research f a c i l i t i e s on May 13, 1970.  Eight rainbou trout uere cannulated, placed in  darkened bYz gallon aquaria, in running uater (T=8 C) and alloued a  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 i n i t i a l sample to ensure homogeneous distribution of the hormone in the circulation.  Bleeding  times uere 5, 30, and 65 minutes from injection for the f i r s t 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. completed within 3 weeks of collection.  The bioassays were  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 a l l physical measurements recorded. Salmon Two male sockeye salmon (Great Central Lake race) were cannulated, placed in 50. gallon fibreglass tanks of running water (T=8 C) and allowed to recover for 2k hours. D  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. described.  Plasma was separated and stared as previously  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 determined 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 D n the abscissa.  Results Trout Physical measurements of the trout are presented in Table V, pg. 69. From this data, i t i s noted that a l l 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, i n i t i a l 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 (g)  Fork Length (cm)  Gonad Wt (g)  GSI  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  7 248.4 15.97 6.52  7 28.6  7 1.0  7 0.43  1.07 0.43  0.50 0.21  n = Mean = SD = SE =  1.43 0.58  -70-  Table VI.  Plasma Calcitonin Levels and B i o l o g i c a l of Salmon Calcitonin in Trout  Half-Lives  Plasma Calcitonin mU/ml  Fish #  • min.  5 min.  30 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  102.82  29.48  9.45  18.5  H  n = Mean =  f  7  7  65 min.  Half-Life  2  90 min.  5  Min.  7  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 A c t i v i t y Remaining uith Time  Sample Time Fish #  5 min.  3 0 min.  6 5 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 = Mean =  7  100.0  7  46.3  2  22.2  9 0 min.  5  11.9  SD  =  11.30  4.64  3.48  SE  =  4.61  4.64  1.74  -73Table VIII.  Plasma Calcium, Percent Water and Haematocrit Changes in Traut Sample Time  Fish #  Measurement  B  Plasma-Ca mEq/1  C  D  PreInjection sample 0 min. 4.45  5 min. 4.60  30 min.  65 min.  4.30  5.00  % Water  94.9  95.6  95.7  96.0  Haematccrit  22  26  16'  14  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  Plasma-Ca mEq/1  4.75  4.70  4.65  90 min.  -  4.45  % Water Haematocrit  94.7 32  34  94.9 35  -  95.4 20  E  Plasma-ca mEq/1 % Water Haematocrit  4.75 95.3 26  4.50 95.6 25  4.70 95.5 28  -  4.55 95.9 17  F  Plasma-Ca mEq/1 % Water Haematocrit  4.70 94.9 28  4.65 95.4 27  4.75 95.2 26  -  4.60 95.5 21  G  Plasma-Ca mEq/1 % Water  4.65 94.5  4.60 94.9  4.50 94.9  -  4.80 95.5  Haematocrit  24  26  22  -  18  H  Plasma-Ca mEq/1 % Water Haematocrit  4.50 95.2 26  4.45  4.35  95.7 27  95.7 28  -  4.30  -  96.1 19  T-test Comparison of 0 min. versus 65 and 90 min. Samples T  degrees of freedom  p_ NSD  Ca mEq/1  0.435  6  % Water  9.66  6  p < .001  6  p< .001  Haematocrit  11.1  _74Salmon The physical measurements of the tuo male sockeye salmon are presented in Table IX.  Table IX.  Salmon Physical Measurements  Fish #  Total Wt (g)  Fork Length (cm)  Gonad Wt (g)  GSI  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 h a l f - l i f e 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 i n i t i a l disappearance time than the salmon.  Table X.  1 #  H  R  Plasma Measurements in Tuo Male Sockeye Salmon  Sample Time (min)  •  Calcitonin mU/ml plasma 982.8'.  Plasma Ions mEq/l  Percent Calcitonin Remaining  Plasma Percent Water  Hct  100.00%  94.6  11  153  2.8  1.30  Mg  l\)a  +  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  •  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 .  B i o l o g i c a l h a l f - l i f e of salmon c a l c i t o n i n in tuo male sockeye salmon  T  Time in Minutes  F i g u r e 8.  D i s a p p e a r a n c e o f salmon c a l c i t o n i n i n t r o u t and salmon  1  100  -78-  Discussion Polypeptide  hormones are known to be rapidly cleared from  the blood after intravenous injection into mammals. The halflives 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 a l , 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 f i r s t order kinetics. The disappearance of PCT in the pig was shown to follow two exponential components, the f i r s t component had a T/z = 4-5 minutes and 1  the second component had a T# = 35-4Q minutes (west e_t al_, 1969).  -79-  The d i v i s i o n of the disappearance curve into tuo components has been shown by other workers, the f i r s t  steep segment representing  the d i s t r i b u t i o n and mixing of the hormone in the f l u i d  compart-  ments and the second, less steep segment the actual rate of i n activation of the hormone (Idest ejt a_l, 1969). Foster e_t a_l (1972a)  reported that the i n i t i a l disappear-  ance of human c a l c i t o n i n in the dog (T}& = 3 minutes) was largely due to kidney i n a c t i v a t i o n . venous differences and the kidney.  This was demonstrated by measuring  arterio-  in plasma CT concentration across both the l i v e r  Blood samples during a c a l c i t o n i n infusion, were  collected simultaneously from indwelling catheters in the hepatic and renal veins of anesthetized tration measured by radioimmunoassay.  aorta,  dogs and the CT concenThe l i v e r appeared t D remove  c a l c i t o n i n from the c i r c u l a t i o n only at levels above 90 ng per ml, while the kidneys consistently removed 30% of the a r t e r i a l l e v e l of calcitonin.  On removal of the kidneys, the f i r s t rapid component  of the disappearance curve was abolished and higher levels of plasma CT were measured.  The slower' disappearance of salmon c a l c i t o n i n  in nephrectomized versus normal r a t s , 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 l i v e r plays an i n s i g n i f i c a n t role in the inactivation of human c a l c i t o n i n in the dog.  Since only 0.3% of an infused dose of c a l c i t o n i n 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 h a l f - l i f e disappearance curve was also considered  -aa-  unimpartant by Habener et a l (1972a), who found that the metabolic clearance of porcine c a l c i t o n i n greatly exceeded the GFR in the dog.  The binding of the hormone to plasma proteins,  besides  providing protection from plasma enzymes, uould also preserve from renal excretion.  it  Foster e_t al_ (1972a) also found that the  slow component of the disappearance  curve of human c a l c i t o n i n in  the dog had a T/2 = kO minutes and they postulated that i t uas due primarily to protein binding since i t did not change uith nephrectomy or hepatectomy.  This vieu has been supported by Habener et_ al_  (1971a,b; 1972a) uho demonstrated using gel f i l t r a t i o n , that the slou component of the disappearance  curve uas due to protein-bound  c a l c i t o n i n , uhereas the free c a l c i t o n i n disappeared  rapidly.  125 Injection of  I porcine c a l c i t o n i n into rats shoued  that the major s i t e of accumulation of r a d i o a c t i v i t y uas the l i v e r (de Luise e_t a_, 1970) and these authors concluded that the l i v e r played a role in the early phase of the disappearance  curve.  Since  the accumulation of labelled CT in the l i v e r could be prevented by simultaneous i n j e c t i o n of unlabelled CT and since the authors kneu of no knoun effect  of c a l c i t o n i n on the l i v e r , 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 l i v e r , 2.6% in the kidney, 13.0%  in the s k e l e t a l 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 i n j e c t i o n of  l i v e r of the r a t ,  I PCT accumulated mainly in the  both human and salmon c a l c i t o n i n uere primarily  taken up by the kidney.  Salmon c a l c i t o n i n resisted  enzymatic  -81-  breakdown by homogenates of a l l 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 a l (1972b) also found that over 30%  of i n i t i a l 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 inactivation 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 i n i t i a l h a l f - l i f e af ID - 15 minutes in normal rats, whereas porcine calcitonin showed a very rapid i n i t i a l 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 i s 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 i n a c t i v a t i o n plays a minor role in the disappearance  of c a l c i t o n i n .  The h a l f - l i f e for salmon c a l c i t o n i n of 27.6 - 2.30 minutes in trout and 48.0 minutes in salmon, indicates that the salmon hormone has a r e l a t i v e l y long h a l f - l i f e in f i s h .  Although single  injections of hormones do not equilibrate evenly in the f l u i d compartments of the body and tend to distort the disappearance these results are in substantial found for SCT in mammals.  agreement uith the half-times  The fact that the second sample uas  taken at 30 minutes makes i t passible that these results the measurement of the second, slouer component of the curve.  curves,  Even i f these findings represent the f i r s t ,  reflect disappearance  rapid component  of the curve, the h a l f - l i f e of SCT in f i s h i s s t i l l slower than the i n i t i a l h a l f - l i f e of SCT in the dog (20 min.) found by Habener et a l (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 b i o l o g i c a l a c t i v i t y (Lequin e_t a_l, 1969; Cooper et a l , 1971). The shape of the disappearance  curve for SCT in the salmon  i s interesting and may r e f l e c t the fact that a longer i n t e r v a l was l e f t following i n j e c t i o n of the hormone (mixing time: min., trout 5 min.) before c o l l e c t i o n of the f i r s t  salmon 10  sample.  Con-  sequently, there may have been a more homogeneous concentration  -83-  of  the hormone in the f l u i d compartments than uas observed in  the t r o u t . The  longer h a l f - l i f e of SCT in the salmon compared to the  trout, could be due to many factors.  Since the salmon uere i n  the spauning condition, the l e v e l of plasma binding proteins may have been higher than in the t r o u t . of  This increased protein binding  the hormone in the plasma uould prolong i t s h a l f - l i f e .  the sexually mature skate, Raja radiata,  In  Fletcher et_ a_l (1969)  have shoun that the sex hormone-binding-protein binds the appropr i a t e steroids quite strongly.  They demonstrated that the meta-  b o l i c 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 s i g n i f i c a n t increase in the MCR  Cortisol  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 C o r t i s o l distribution. in  Thus, they concluded that the elevated C o r t i s o l levels  maturing and spauning salmon uere not due to a lou MCR but to a  r i s e in C o r t i s o l s e c r e t i o n .  The h a l f - l i f e of C o r t i s o l , houever, did  increase during maturation and spauning. It appears that the situation i n the spauning salmon is quite complex and experiments on the MCR and secretion rate of c a l c i t o n i n in immature-and mature salmon may help to explain the prolonged h a l f - l i f e in this f i s h .  The fact that f i s h are p o i k i -  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 d i s t r i b u t i o n of labelled SCT in fish so the role played by the kidney, l i v e r and other organs in the removal and i n a c t i v a t i o n of c i r c u l a t i n g c a l c i t o n i n i s not known. It may be s i g n i f i c a n t to note that although Habener e_t al_ (1971b) claim that the prolonged h a l f - l i f e of salmon c a l c i t o n i n may explain i t s increased potency in mammals, the long h a l f - l i f e of SCT in the f i s h was not accompanied by a hypocalcaemic e f f e c t . It would be informative from a s t r u c t u r e - f u n c t i o n - s t a b i l i t y  point  of view to examine the plasma electrolyte effects and b i o l o g i c a l h a l f - l i v e s of the mammalian calcitonins in f i s h .  -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 i t s effect on bone, came from experiments which demonstrated that this action could be produced in nephrectomized and eviscerated rats (Webster and Frazer, 1967; Copp, 1970a). Salmon calcitonin, the f i r s t 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 a l , 1970; Brooks et al, 1969; Singer et al, 1970; Galante et a l ,  -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 a l , 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 s i g n i f i 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 a l , 1968). Following the discovery D f 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 o f the plasma and renal electrolyte effects o f salmon calcitonin in rainbow trout and salmon was performed.  -87-  Materials and Methods Trout Purified salmon c a l c i t o n i n uas injected into 2 groups of trout, fingerlings and cannulated adult t r o u t .  In a l l experiments,  c a l c i t o n i n uias weighed out the day of the experiment and the a c t i v i t y confirmed by bioassay. ( i ) Fingerling trout Since c a l c i t o n i n had been shown to be more effective  in young  mammals, f i n g e r l i n g trout (age 7-8 months) were used to determine the effect  of salmon c a l c i t o n i n an plasma calcium and inorganic  phosphorus  levels.  A group of 150 f i n g e r l i n g trout were tagged behind the dorsal f i n with a small length of coloured thread and randomly divided into three groups D f 50 f i s h .  A l l f i s h 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 f i n e l y -  chopped beef l i v e r and f i s h that were not actively feeding were removed.  Before the experiment, the fish were starved for 2 days. The experimental procedure was as  follows:  Salmon c a l c i t o n i n (62.5 mU in 0.1 ml vehicle per fish) was injected intraperitoneally ( g i l l s I at time zero.  immersed under water) into the f i s h in Group  Group II received vehicle alone (0.1 ml of vehicle,  1.0 percent sodium acetate + 0.1 percent glycine, pH = 4.3). III,  the control group, was not injected.  Group  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. and ueighed at each time period.  From 5 to 10 fish uere sampled Plasma calcium uas measured  fluorometrically (IMeusome, 1969) and plasma inorganic phosphorus uas determined colorimetrically using the micro-method of Goldenberg and Fernandez (1966). ( i i ) 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 g ) and tuo T  calcitonin groups (CT^ = 125 mU per 0.1 ml vehicle per 100 g CT^ = 500 mU per 0.1 ml vehicle per 100 g  fish).  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 lyte  effect  levels  and  of  salmon  urine  calcitonin  electrolyte  salmon.  Techniques  used  outlined  in  General  Materials  box  to  restrain  used  trated  in  Figure  Three their  sexually-ripe The (2.0  hours  bag  filled  taken  and  hours.  g  were  was  with  in  On t h e  kept ice.  following period  the  same  time  intervals.  collector  and  for  these  tested  electro-  in  sockeye  catheterization  pg.  12  -  21.  experiments  Central  is  race)  were  they  were  and  experiment.  at  each  and  ml)  by  was  was  the  were  The  urine  i l l u s -  All  3  cannulated, allowed  fish  to  were  as  infused  3  (Harvard  calcitonin  Apparatus,  a  surrounding  the  syringe  with  a  blood  samples  (1.2  ml)  samples  were  vehicle  at  1.0  lYz,  (vol.  =  blood  samples  and  Hourly  urine  samples  15  tubes  samples  period  collected  minutes)  The  Salmon  for  pre-injection  day,  follows.  fish  preweighed  recorded.  stored  potassium,  (30  in  ml were  ml)  were  and  the  immediately  of  30  min. plastic were  3Yz a n d 5Vz was  infused  collected  at  collected  by  urine frozen  volume on  dry  -12 C. D  Haematocrit, measured  the  into  Two  time  ice  in  (Great  1.0  cool  same  weight)  Methods,  procedure  Pump)  post-injection  (by  and  catheterized  before  the  fraction  and  was  plasma  22.  salmon  fish  Withdrawal  infusate  cannulation  salmon  experimental  The  excretion  on  females.  Units/100  Infusion  pg.  bladders  2k  for  the  sockeye  urinary  recover  2,  for  infusion  percent  fish.  water,  Urinary  sodium were  also  and  plasma  calcium,  electrolytes  magnesium,  determined.  were  phosphorus,  On t e r m i n a t i o n  of  the  for  -90-  experiment,  the f i s h mere s a c r i f i c e d and p h y s i c a l measure-  ments r e c o r d e d .  The experiments uere conducted December 5 -  1970 and the water temperature 6.0  -  6.5 C. D  during t h i s p e r i o d ranged from  18,  -91-  Resulta  Trout  (i)  Fingerling  trout  Results of i n t r a p e r i t o n e a l i n j e c t i o n into  fingerling  illustrating  t r o u t a r e p r e s e n t e d i n T a b l e X I , p g . 92  calcitonin .  Graphs  t h e p l a s m a c a l c i u m and i n o r g a n i c p h o s p h o r u s c h a n g e s  f o u n d on F i g u r e It  o f salmon  9 pg. 93,and  F i g u r e 10,pg.94  respectively.  i s apparent from the data t h a t plasma e l e c t r o l y t e  d i s p l a y e d a wide range o f v a l u e s .  In f a c t ,  levels  a l t h o u g h the plasma  c a l c i u m and P i l e v e l s o f t h e v e h i c l e g r o u p d i d n o t d i f f e r cantly  signifi-  f r o m t h e c a l c i t o n i n - i n j e c t e d g r o u p a t any o f t h e s a m p l e  t h e c o n t r o l p l a s m a c a l c i u m and P i l e v e l s were e l e v a t e d w e l l t h e o t h e r two g r o u p s a t 3Yz and 7]k h o u r s . . C o n t r o l p l a s m a levels differed significantly  from those o f the v e h i c l e  ( C a 3)4 h r . p< 0.025; P i 3# h r . p< 0.001  are  times,  above  electrolyte group  and Tk h r . p < 0 . D 0 5 ) .  By  25}£ h r . t h e c o n t r o l l e v e l s had r e t u r n e d t o n o r m a l . A comparison of the v e h i c l e versus c a l c i t o n i n - i n j e c t e d d o e s n o t r e v e a l any s i g n i f i c a n t effect of  hypocalcaemic or  groups  hypophosphatemic  calcitonin.  (ii)  Cannulated trout  P h y s i c a l m e a s u r e m e n t s and i n d i v i d u a l p l a s m a c a l c i u m of the h groups of t r o u t are t a b u l a t e d  i n Table XII,pg. 96.  body w e i g h t o f t h e 24 t r o u t u s e d i n t h i s e x p e r i m e n t was E v i d e n c e t h a t t h e s e f i s h were s e x u a l l y t h e l o w GSI v a l u e s .  immature  values Mean  205.0 - 7.3  total g.  i s d e m o n s t r a t e d by  E a c h g r o u p c o n t a i n e d a p p r o x i m a t e l y e q u a l numbers  -92-  Table X I .  Effects cf Salman Calcitonin on Plasma Electrolytes in Fingerling Rainbow Trout. Plasma Ions (mg per IPG ml)  Sample Time (After Injection)  Group  Vfe hour  Control  6  10.6 -  1.50  9.4  Vehicle  8  10.0 -  1.75  Calcitonin 8  12.0 -  1.30  Control  10  12.7 - 0.62  Vehicle  10  12.0  Calcitonin 10  12.2  Control  10  11.2 -  Vehicle  10  12.9  yk hour  Th hour  25Vz hour  (n)  Inorganic Phosphorus Mean + SE  (n)  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)  10.9  0.21  (9)  15.3  0.36  (10)  - 1.07  9.9  0.31  (10)  10.9  0.50  (10)  - 0.62  9.7  0.29  (9)  12.1  0.67  (9)  0.82  10.9  0.38  (10)  15.0  0.48  (8)  -  0.97  10.0  0.34  (10)  12.3  0.65  (8)  Calcitonin 10  12.2 -  0.99  10.3  0.27  (10)  12.0  0.90  (5)  Control  5  12.5 -  2.15  9.7  0.43  (4)  12.7  0.69  (5)  Vehicle  10  11.2  -  1.16  9.5  0.28  (10)  12.3  0.40  (10)  10.2 -  0.84  9.7  0.28  (10)  12.9  0.50  (8)  Total wt  (g)  +  Mean - SE  Calcitonin 10  Total Calcium Mean ± SE  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 effect of salmon calcitonin. Injection at time 0  trout i  •FI  -95-  •f males and females (non-spawning trout are d i f f i c u l t to sex). The data, as presented in Table XII was d i f f i c u l t 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, i t i s 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 i s 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. A l l 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) Control  1 2 3 It 5 6  M H M F F M  n 6 mean » SD SE = Vehicle  1 2 3 d 5 6  H F F F F M  n = 6 mean = SD = SE = Calcitonin , „ „.,__ 1Z5 mll/100 g  1 2 3  M  d 5 6  r l a f l  M M . F F M  n = 6 mean = SD = SE ° Calcitonin . mU/lUU g. • 8  0  1 2 3  d 5 6  n = 6 mean = SD = SE =  F F p M M M  (Cm)  -2 hr -1 hr  0 •  + 1 hr  + 3 hr  10d.5 207.5 218.0 238.0 2<.9.0 209.0  0.1.8 D.05 0.18 0.21 0.1.0 0.05  22.3 27.0 26.6 28.9 28.7 28.2  9.5 8.1 8.8 9.0 8.3 8.8  9.2 . 8.2 8.2 8.5 8.1 8.3  9.d 7.8 8.6 8.9 8.1. 8.0  9.3 7.9 8.7 8.6 8.3 8.1.  8.9 7.5  201..0 1.7.1 21.0  a.23 0.16 0.07  27.0 2.2d 1.00  8.8 0.d6 0.20  8.1. Q.36 O.ld  8.5 0.52 0.22  215.0 20d.5 232.5 220.0 196.0 13d.0  0.05 0.10 0.3d D.d5 0.10 D.15  26.7 28.0 28.0 26.8 26.6 23.3  8.0 7.9 7.6 8.2 8.3 8.1  8.1 7.d 7.d 7.8 7.7 8.0  200.0 31.8 Id.2  0.20 O.ld 0.06  26.6 1.57 0.70  8.0 0.23 0.10  7.7 0.26 0.10  228.0 2d8.0  0.09 O.Od  2 0 ( ( > 0  Q  >  1  D  2  28.7 8.5 8.7 29.0 9.2 9.0 6.2 6.8 6.6 27.3 8.0 7.8 28.3 8.3 7.6 28.6 9.1 8.B  218.0 210.0 226.0  0.23 0.38 D.Od  220.0 Id.2 6.3  0.15 0.12 0.05  28.0 0.96 0.d2  205.0 16d.O . 238.0 ld9.0 182.5  0.3d 0.06  26.7 25.3  2 1 5  5  192.0 30.d 13.6  Q  . O.Od 0.07 0.05 3 2  0.15 0.13 0.05  2 S  . 29.3 2d.9 26.5 0  26.8 1.50 0.67  8.3' 0.80 0.36  a  8.1 0.33 0'.37  • 7.d 8.0 8.2 7.9 .9 8.9 8.d 8.6 7.3 7.3 5.8 5.7 7.7 1.00 0.d5  6.7  7.7 1.03 0.d5  B.d  + 5 hr  + 2d hr  8.6 8.3 8.2  8.6 7.3 8.5 8.6 8.5 8.5  8.6 7.9 8.2 8.1 8.1. 8.6  8.5 O.dZ Q.17  8.3 0.d2 0.17  8.3 0.d5 0.20  8.3 0.2d 0.10  8.0 7.5 7.6 8.5 7.2 B.l  7.8 7.5 7.5 8.0 7.2 8.1  7.9 7.5 7.8 8.0 7.2 7.8  7.5 7.5 7.9 8.2 7.1 7.9  7.8 0.d2 0.17  7.7 0.30 0.10  7.7 0.26 0.10  7.7 0.3d O.ld  8.3 9.0 7.5 8.d 9.2  6.d  8.2 9.0 7.7 7.8 8.9  8.1.  7.1  8.6 9.5 8.0 8.d 9.1  6.1  8.2 8.5 7.d B.l 9.d  7.5 7.3 7.9 6.2 8.1 fl.d  7.9 0.38 0.17  6.9  8.1 8.8 7.a 8.3 8.9  8.2 0.85 0.37  8.0 0.86 0.38  8.5 0.76 0.33  8.0 1.01 O.dd  8.1 0.56 0.28  B.d 7.7  7.8 7.6  7.9 7.5  7.2 7.d  7.3 7.S  8.d 6.9 5.7 7.6 1.00 O.dd  8.7  8.8 7.3 5.8 7.7 0.99 D.d3  8.5  S.d 7.2 5.7  6.5  7.5 0.93 O.dl '  6.6 7.d 5.7 7.5 0.95 0.d2  8.5  8.8 7.8 5.3 7.7 0.95 0.d2  Table XIII.  „ tt  Group Control  1 2 3 •%  5 6  1 2 3 It  5 6 n = mean = SD = SE =  1 2 3 1.  5 6 n B mean = SD = SE =  -  0.00 0.1<t 0.21.  B.G S.9  0.22 0.17 0.31  8.1% 8.1.  0.00 0.10 0.11. 0.10 0.10 0.22  to mean 0.7 2.0 1.1. 1.1 I.S 1.6  Plasma Calcium Zero Time** . m V  10.0  a.o i 7.7 7.7 8.2 7.7 8.1  8.6 9.1 6.7 7.8 8.2 9.1  t  7.9 i 7.9 8.7 8.5 7.2 5.7  Plasma Calcium Change from Zero Time (mg/100 ml) i  10.0 9.9 10.1 9.7 9.9 10.0 6 9.93 0.12 0.05  0.00 0.20 0.26  0.00 0.11. 0.10 0.11.  0.1.8  0.31.  0.21.  0.00  0.00  2.0 2.3 2.3 1.8 2.3 1.9  10.0  9.8 9.8 9.B 9.B 9.5 10.0 6 9.78 0.11. 0.06  0.22 0.00 0.00 0.17 0.31 0.11.  0.10 0.00 0.00 0.10 0.17 0.00  1.4 0.9 3.3 2.2 1.8 0.9  10.0  6 8.25 0.83 0.36  n = mean « SD =. SE = Calcitonin 500 mU/100 g fish  9.3  9.0  NumbBr Bdded  6 7.90 0.20 0.00 1 2 3 (. 5 6  Calcitonin 125 mU/100 g fish  Mean Control Plasma Calcium* (mg/100 ml) ... mean SD SE  6 8.60 . 0.1.1 0.17  n = mean SD = SE » Vehicle  Effect of Salmon Calcitonin on Plaema Calcium Levels i n Cannulated Trout.  0.1.0  0.17 0.22 0.00 0.17 0.00  0.28 0.10 0.11.  0.00 0.10 0.00  2.1 2.1 1.3 1.5 2.8  10.0  1..3  6 7.65 0.99 0.1.3  a  9.S 9.5 9.8 9.7 9.9 9.8 6 9.72 0.13 0.05 9.9 9.B 10.1 9.8 9.5 9.7 6 9.80 0.18 0.08  9.3 9.3 9.9 9.7 10.1 10.1 6 9.73 0.33 0.15 9.5 9.8 10.2  10.0  10.1.  10.3  6 9.78 0.27 0.12  6 10.00 0.3U 0.15  9.6  9.5 9.7 10.2 1C.0 10.1 9.8  10.2 10.2 10.0  9.6 9.9 10.3  6 9. 75 0.12 0.05  6 10.20 0.16 0.07  6 9.70 0.32 CU  6 10.02 0.18 0.08  10.0 9.6 9.8 9.9 10.10 10.10 6 9.B8 0.11. 0.06  9.5 9.6 10.2 10.0  9.1.  10.0 10.1.  9.9 9.7 10.0 10.3 10.1 10.1  6 9.70 0.36 0.16  9.8  9.6 9.9 9.7 9.9 9.6 9.8  10.1.  9.3 9.9 9.5 9.2 10.0 10.2  9.1. 9.1.  9.3 9.5 9.8 10.1 10.2 10.0  6 g.aa 0.21.  0.10 9.9 9.7 9.8 10.3 10.6 10.1  6 6 9.82 10.07 0.32 0.31 D.ll. 0.11. VO -0  mean of 3 control plasma calcium levels (-2, -1, 0 hour samples) 'mean plasma calcium (column 2) of each fish taken as 10.0 (mg/100 ml)  I  r e 11.  Mean p l a s m a c a l c i u m c h a n g e s i n a d u l t e f f e c t o f salmon c a l c i t o n i n .  cannulated  trout  F i g u r e 12.  I n d i v i d u a l plasma c a l c i u m changes i n c a n n u l a t e d a d u l t t r o u t e f f e c t o f salmon c a l c i t o n i n .  ^ IX)  i  -100-  Table XIV  Salmon Physical Measurements.  Salmon  Sex  Total lilt. (g)  Gonad Lit. (g)  GSI  Fork Length (cm)  V  female  1255  205  16.33  50.2  U  female  1085  250  23.04  46.6  Z  female  1070  265  24.77  47.0  3 1137. 83.8 59.3  3 240. 25.4 18.0  n = mean = SD = SE =  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  5.01  1  Colclum  4.0 _j \  3.0-  _^ Inorganic Phosphorus  UJ  E  —  2.0  r  1.0-  o  o  co 150 1  14 5  o  140 -  135 Time in Hours Salmon CT Infusion 2units/IOOgm in I ml 3 0 min.  F i g u r e 14.  Vehicle Infusion  Plasma e l e c t r o l y t e changes i n a sockeye salmon - e f f e c t D f salmon c a l c i t o n i n i n f u s i o n . Female sockeye U. o  1  Salmon CT Infusion 2 units/IOO gm in I ml 30 min.  Vehicle Infusion  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 p a r t i c u l a r l y stable uhile  plasma sodiums shcued uider The effect  fluctuations.  of c a l c i t o n i n infusion on renal electrolyte ex-  cretion and urine flou rates of the 3 salmon are summarized in Figure 16, pg.105.  Both vehicle and c a l c i t o n i n infusions  appeared  to cause a s l i g h t diuresis but the infusion procedure i t s e l f may have caused "laboratory d i u r e s i s "  (Forster and Berglund, 1956).  The  control period e l e c t r o l y t e excretion and urine flou rates displayed l i t t l e variation from the c a l c i t o n i n and vehicle infusion  experiments.  Calcitonin infusion caused a s l i g h t l y greater increase in sodium output compared to the vehicle infusion (not s i g n i f i c a n t l y  different)  but in both cases the sodium output uas back to control values u i t h i n 3 hours.  Magnesium output also increased s l i g h t l y due to the i n -  fusion of both c a l c i t o n i n and v e h i c l e . affected.  Calcium excretion uas least  No evidence of phosphaturia due to the c a l c i t o n i n infusion  uas observed.  Urinary potassium output (not shoun) uas very s t a b l e .  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 f i r s t 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 k i l l i f i s h , 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 hyperphosphatemia 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 i s interesting in vieu of the fact that in mammals, calcitonin louers plasma phosphate as a consequence of the inhibition of bone resorption. Injection of porcine calcitonin (50 mU/100 g ) into  -107stanniectamized 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 remained 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 a l 1970; Sorensen, 1970 ). Recently, Milhaud et a l (1972) have shown that thyroidectomy of rats w i l l raise plasma calcium and phosphate levels only i f the operation i s performed during the dark night  -loa-  p e r i o d when t h e y u e r e f e e d i n g . Pang  (1971b) o b t a i n e d a hypocalcaemic and  hyperphosphatemia  response f o l l o w i n g i n j e c t i o n o f salmon c a l c i t o n i n i n t o t h e f r e s h w a t e r A m e r i c a n e e l , A n g u i l l a r o s t r a t a . H o w e v e r , t h e same e x p e r i ment, r e p e a t e d on s e a w a t e r - a d a p t e d e e l s , p r o d u c e d no e f f e c t . hyperphosphatemia  e f f e c t f o u n d by Pang and Chan h a s a l s o  observed i n t h e h e a r t - l u n g p r e p a r a t i o n (bone-and t h e d o g b y S t a h l e_t a_l ( 1 9 6 8 ) .  The  been  kidney-free) of  T h i s s u g g e s t s an e x t r a - s k e l e t a l  and e x t r a - r e n a l mechanism o f a c t i o n f o r c a l c i t o n i n . Ma ( 1 9 7 2 ) p r o d u c e d a m a r k e d h y p o c a l c a e m i c a n d h y p a k a l a e m i c r e s p o n s e i n t h e A s i a n e e l , A n g u i l l a j a p d n i c a , an i n j e c t i o n o f s a l m o n c a l c i t o n i n a t a d o s e a s l a w a s 1 0 0 mU/100 g  body w e i g h t .  T h i s r e s p o n s e was e l i c i t e d o n l y a f t e r p r e - t r e a t m e n t o f t h e e e l s w i t h L - t h y r o x i n e ( 1 0 /jg/100 g dependent. phosphorus  body w e i g h t ) a n d was t i m e - a n d  A s l i g h t b u t s i g n i f i c a n t e l e v a t i o n o f serum  dose-  inorganic  was a l s o n o t e d .  On t h e o t h e r h a n d , P a n g  (1971b) h a s shown t h a t  injections  of salmon c a l c i t o n i n , c o d f i s h u l t i m o b r a n c h i a l e x t r a c t and p o r c i n e c a l c i t o n i n h a d no s i g n i f i c a n t e f f e c t on any o f t h e serum tested i n the k i l l i f i s h ,  electrolytes  Fundulus h e t e r o c l i t u s , i n v a r i o u s experiments.  I n j e c t i o n Df s h a r k u l t i m o b r a n c h i a l e x t r a c t s i n t o t h e b l u e s h a r k , Prionace g l a u c a , and t h e horn shark, Heterodontus f r a n c i s c i , to e l i c i t a hypocalcaemic response ( U r i s t , 1967).  failed  Porcine cal-  c i t o n i n ( 1 0 - 2 0 U / k g , I P ) e x t r a c t p r o d u c e d no s i g n i f i c a n t c h a n g e s i n serum c a l c i u m o r i n o r g a n i c phosphorus Pbroderma  levels i n the lazy shark,  a f r i c a n u m ( L o u w e_t a l , 1 9 6 9 ) .  u n a b l e t o d e t e c t any change  C o p p et_ a_l ( 1 9 7 0 ) w e r e a l s o  i n plasma c a l c i u m l e v e l s i n d o g f i s h  -109-  sharks,  Squalus s u c k l e y i , injected intravenously with 10 units of  salman c a l c i t o n i n . In contrast to the above r e s u l t s , Orimo et_ a_l (1972a) claim that i n j e c t i o n of eel c a l c i t o n i n (5 MRC Units) caused a significant japonica.  increase in serum calcium in freshwater  Anguilla  These authors also detected a s i g n i f i c a n t hyponatremia  and hypochlaremia (p<0.05) following i n j e c t i o n of eel c a l c i t o n i n into a similar group of freshwater-adapted  eels.  Recently Urist e_t a l (1972), reported that i n j e c t i o n of purified and synthetic salmon c a l c i t o n i n (500 MRC U/kg) into the female South American lungfish, Lepsidosiren paradoxa, effect  had no  on plasma calcium at 1 and k hours p o s t - i n j e c t i o n .  Porcine  c a l c i t o n i n , at doses of kk, 88 and 176 MRC U/kg, also f a i l e d to suppress the plasma calcium l e v e l 1 hour post i n j e c t i o n .  These  authors also reported that the skeleton of Lepidosiren, which contained perichondral bone, apatite mineral, and osteocytes but no osteoclasts, was unresponsive to vitamin D, parathyroid extract and calcitonin. The v a r i a b i l i t y of the f i n g e r l i n g trout control group plasma e l e c t r o l y t e levels in the present study, was probably due to s t r e s s .  The absence of an effect  of salmon c a l c i t o n i n on the  fingerling trout plasma calcium and inorganic phosphorus levels may indicate that in young, growing f i s h , bone turnover i s not rapid as i n mammals.  In fact,  some authors  as  (Moss, 1962; IMorris  ejb a_l, 1963; Nelson, 1967; Simmons e_t a l , 1970;  Simmons,  1971)  have shown that f i s h bone has a very low rate of turnover. Unlike mammals, f i s h are able to obtain adequate amounts  -110-  •f  calcium and phosphorus d i r e c t l y from the uater via their g i l l s ,  oral e p i t h e l i a and fins  (Simmons, 1971).  The transport of calcium  across the g i l l s also appears to be more e f f i c i e n t fish.  in freshuater  These points are mentioned to suggest that the action of  c a l c i t o n i n in f i s h may not be on bone but on other target s i t e s such as the g i l l . .  Certainly the type of bone ( a c e l l u l a r  versus  c e l l u l a r ) found in f i s h does not appear to influence the hypocalcaemic response to c a l c i t o n i n since the e e l , salmon and trout a l l have c e l l u l a r bone uhereas the k i l l i f i s h has a c e l l u l a r bane. The results obtained on the f i n g e r l i n g trout uere confirmed by Pang (1971b) uho did not detect any hypocalcaemic in juvenile channel c a t f i s h ,  Ictaluras melas,  effect  due to i n j e c t i o n of  salmon or porcine c a l c i t o n i n . Sex differences effect  would not appear to explain the negative  of salmon c a l c i t o n i n (SCT) i n the trout since the hormone  was equally ineffective  in males and females (Table XII, pg. 96).  This contrasts with the r e s u l t s of Hinde and P h i l l i p p o (1967) who observed a marked difference  in the response of rats to c a l c i t o n i n  i n j e c t i o n , the males of a given age being more sensitive than the females. The o r i g i n a l report on the negative effect  of salmon c a l -  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 i n j e c t i o n of salmon c a l c i t o n i n into freshwater kisutch.  coho salmon, Oncorhynchus  Pang (1971b), however, does not report the dosages of  c a l c i t o n i n used in any of his experiments and this undoubtedly i s  -111-  a critical  factor.  With the sockeye salmon in the present study, the  effect  of salmon c a l c i t o n i n 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 compensated for the infusion of c a l c i t o n i n . One explanation for the negative result of salmon c a l c i t o n i n (SCT) in the salmon would appear to reside, in the finding that spawning female salmon have elevated levels of plasma c a l c i t o n i n (Chapter IV).  Measurement of the c i r c u l a t i n g l e v e l of plasma  c a l c i t o n i n at 24 hours post-infusion of vehicle revealed c a l c i t o n i n levels of 6775 - 1042 pg/ml plasma and 14,700 - 1731 pg/ml plasma for salmon V and W, respectively.  The high c i r c u l a t i n g c a l c i t o n i n  levels in these salmon may indicate that the receptor s i t e s for t h i s hormone were already saturated.  Infusion of SCT therefore,  might better be tested in male or gonadectomized salmon but unfortunately these f i s h were not available for t h i s  study.  It would be interesting to test the hypocalcaemic  effect  D f SCT in ultimobranchialectomized trout since Talmage and Kennedy (1969) have demonstrated that thyroidectomized rats were more sensitive  than intact rats to c a l c i t o n i n treatment.  different  levels of calcium in the food and water on the response D f  trout to c a l c i t o n i n has not been f u l l y  The effect  of  investigated.  It may be s i g n i f i c a n t that a hypocalcaemic response to c a l c i t o n i n i n j e c t i o n has been demonstrated only in the e e l .  This  may be explained by a species difference or by the fact that the eel i s an extraordinarily stable experimental f i s h .  Wttnile the  salmon i s an•anadromous f i s h ( l i v e s in seawater, spawns in fresh-  -112-  water), the e e l i s catadromous  (lives in freshwater,  spawns in sea-  water) and thus their osmoregulatory problems at similar stages in the l i f e - c y c l e are quite d i f f e r e n t .  Since Dacke (Chan, 1972,  discussion) was unable to confirm Chan's hypocalcaemic effect using salmon and porcine CT i n eels, physiological condition and sexual development may be c r u c i a l  factors.  Renal Effects of Salmon Calcitonin Since no effect  on plasma e l e c t r o l y t e levels was observed,  i t i s not too surprising to find that infusion of salmon c a l c i t o n i n did not a l t e r renal e l e c t r o l y t e excretion.  It i s 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 - f D l d  increase  in sodium extretion i n saline-loaded rats (Keeler ejt a_l, 1970; Aldred et al,  1970).  Aldred et_ a l (1970) demonstrated that urine volume, sodium, phosphorus and magnesium excretion levels showed s i g n i f i c a n t changes only at the 3rd hour after  treatment.  In t h i s study,  however, urinary parameters returned to the normal range at approximately the 3rd hour following i n f u s i o n . The renal effects of c a l c i t o n i n in mammals seems dependent upon the species and dose of c a l c i t o n i n and the experimental animal. For example, Williams et a l  (1972) have shown that although  synthetic salmon c a l c i t o n i n causes a profound natriuresis in r a t s ,  -113no 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 a l , 1970). It i s 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 a l (1971)  reported that bovine calcitonin intramuscular injection (150 units/ 100 g body ut ) had no effect on serum calcium and inorganic phosphorus 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, i t i s of interest to note that purified dogfish calcitonin like the salmon, hormone, is extremely natriuretic in the rat (Maclntyre et a l , 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 i s the conservation of electrolytes and excretion of uater,  fish it  uould be physiologically inexpedient to excrete electolytes in freshuater.  It might prove informative to test the effect  of salmon  c a l c i t o n i n in a seauater salmon. Besides the positive hypocalcaemic effect  of c a l c i t o n i n  in eels, Lopez et_ a_l (1971) have provided evidence of a passible role af c a l c i t o n i n in rainbou t r o u t .  Porcine c a l c i t o n i n i n j e c t i o n  (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 c a l c i t o n i n and that the ultimobranchial gland, by secreting c a l c i t o n i n , plays an important rale in calcium homeostasis Lopez and  Deville  in f i s h .  (1972) have also investigated the  effect  of salmon c a l c i t o n i n on vertebral bone morphology and ultimobranchial a c t i v i t y in the mature female e e l , Anguilla anguilla L.  Immature  s i l v e r eels uere made experimentally mature by intraperitoneal injection of carp p i t u i t a r y extract tuice per ueek u n t i l maturation).  (1 mg per 100 g body ueight, Development of the gonads i n  sexual maturation i s accompanied by a marked hypercalcaemia.  Tuo  groups of these mature eels uere submitted to prolonged treatment uith salmon c a l c i t o n i n .  The f i r s t group, after receiving p i t u i t a r y  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 p i t u i t a r y injections and had spauned, uere injected  -115-  uith the same dose of SCT daily far 11 days. Salmon c a l c i t o n i n , in the f i r s t  group, did not a l t e r the  hypercalcaemia but prevented halastasic demineralization (reduction of mineralization of i n t e r c e l l u l a r substance without h i s t o l o g i c a l modifications of the organic matrix).  Osteoclastic  and o s t e o l y t i c resorption were reduced and the UB gland was s t i l l highly stimulated.  In the second group, SCT reduced the hypercal-  caemia by 50 percent and decreased the osteoclastic and o s t e o l y t i c resorption.  The UB gland appeared  inactive.  These authors conclude that c a l c i t o n i n in the eel acts on bone to prevent bone resorption as i t does i n mammals. attribute the negative effect  They  of c a l c i t o n i n in preventing the  hypercalcaemia to the reduced responsiveness to c a l c i t o n i n caused by gonadal steroids (Sorensen and Hindberg, 1971). could apply equally well to the present  This explanation  study.  In summary, although salmon c a l c i t o n i n has negligible on plasma and renal electrolytes organs such as the g i l l ,  effects  in trout and salmon, other target  bone and gut must be investigated  the role of c a l c i t o n i n in these f i s h can be elucidated.  before  -116-  IV„  PLASMA CALCITONIN AIMD TISSUE MINERAL CHANGES IN MIGRATING SALMON  Introduction There are five main species of P a c i f i c salmon under the genus Oncorhynchus: S c i e n t i f i c name  Common name  Oncorhynchus nerka (LJalbaum)  -  sockeye, red  Oncorhynchus kisutch (Ualbaum)  -  coho, s i l v e r  Oncorhynchus tshawytscha (Ualbaum) -  chinook, spring, king  Oncorhynchus gorbuscha ( Ualbaum) -  pink, humpback  Oncorhynchus keta (Ualbaum)  chum, keta, dog  -  As mentioned previously, salmon are anadromous f i s h , which means that they l i v e most of t h e i r l i v e s in the sea, water to spawn.  but return to fresh-  The five species a l l have different  life-histories,  morphology, s i z e s , behaviour, feeding and spawning habits.  This  chapter w i l l deal mainly with the sockeye, chinook and coho salmon. P a c i f i c salmon begin their l i f e cycle i n  freshwater.  Females dig a nest or "redd" in gravel beds of freshwater  streams,  r i v e r s or lakes and deposit up to 6000 eggs, the amount dependent on the species and size of the i n d i v i d u a l f i s h .  The eggs are f e r t i l -  ized by the male, covered with gravel by the female, under the gravel throughout the winter.  and remain  On completion of the spawning  act, both male and female P a c i f i c 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 f r y .  Depending on the species  -117-  and  environmental conditions, the subsequent period of  residence varies from a feu days to several years.  freshuater  A l l pink and  cltoum salmon migrate to the sea d i r e c t l y follouing their emergence from the g r a v e l .  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 u i t h i n the f i r s t 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 a v a i l a b i l i t y and  f i s h size (Ricker, 1966). On reaching the sea,  the smalts or fry may spend several  days in the estuarine uaters af the r i v e r mouth.  They then move  out into the offshore pelagic environment uhere their d i s t r i b u t i o n covers most of the North P a c i f i c Dcean and the Bering Sea 1963).  The time spent at sea varies according to species and even  u i t h i n a species. and  (Jackson,  Coho and pink salmon stay in seauater for one  tuo years r e s p e c t i v e l y .  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 i n s t i n c t , the adult salmon  approach inshore uaters, stream. for  heading in the direction of their natal  This shoreuard migration occurs at a c h a r a c t e r i s t i c time  each species.  For example, maturing Fraser River sockeye appear  in coastal uaters from May through October.  The majority of these  sockeye enter the S t r a i t of Georgia via the S t r a i t 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 ( K i l l i c k , earliest  Fraser River runs go furthest  1955).  Generally, the  upstream.  The gonads begin to mature some time before the shoreward 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 r i v e r i s p a r t i c u l a r l y 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 B r i t i s h Columbia spawn at temperatures of 3 - 7 ° C. Other environmental factors  such as l i g h t , water l e v e l , e t c . also  undoubtedly influence the time of spawning. then die, and the l i f e cycle i s  The adults spawn and  repeated.  During their freshwater migration, P a c i f i c 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 of feeding  5.  diseases, b a c t e r i a l and fungal infections,  cessation 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 i n volves extensive tissue reorganization i n a r e l a t i v e l y short period of time and under fasting conditions.  This chapter presents re-  sults of experiments designed to investigate  the role of c a l c i t o n i n  in osmoregulation and/or sexual maturation and spawning, in the migrating salmon.  -120-  Materials and Methods Three species of P a c i f i c salmon, sockeye,  coho and chinook,  uere investigated at various stages in their spauning migrations. The method of c o l l e c t i o n and storage of samples uas outlined previously in General Materials and Methods. This chapter i s divided into three sections.  In Section  A, experimental material i s 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 i s o u t l i n e d .  Section C i s 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 c a l c i t o n i n and e l e c t r o l y t e levels i n 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 a l , 1958; Idler and Tsuyuki, 1958; e_t a_l, 1960).  Idler and Bitners, 1958;  Idler  The International P a c i f i c Salmon Fisheries Com-  mission (Salman Commission) supplied the necessary manpouer and facilities  to c o l l e c t sockeyefrom t h i s 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 i d e n t i f i e d by the Salman Commission and only Chilko sockeye were included in the study.  This i d e n t i f i c a t i o n 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 r e l a t i v e l y uniform in body weight and length. A l l 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 s e i n i n g . 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 a r r i v a l  sockeye were sampled at Chilko Lake, B r i t i s h 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 t r a v e l l e d by the migrating salmon. Physical measurements,  plasma e l e c t r o l y t e s (calcium,  inorganic phosphorus, sodium, potassium),  plasma percent water,  plasma protein and haematocrit were measured for each f i s h .  Plasma  calcitonin was measured by radioimmunoassay an each i n d i v i d u a l sample.  Procedures used for the above analyses were described  in General Materials and Methods. The s c i e n t i s t s  with the Salmon Commission divided the  ure 17.  Map of Fraser River and B r i t i s h Columbia. Chilko sockeye migratory route indicated by dark l i n e and locations of the 3 sampling points are shown by numbers.  -123-  spauning female sockeye into 3 groups: but egg case i n t a c t ) ,  • percent spawned (ripe  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. were impossible to c l a s s i f y  The male spawning sockeye  as to degree of spawning and hence  were sampled as one group.  Table XV.  Phase  Chilko Sockeye Migration 1971  Sampling Date(s)  Number of Salman  Uater Temperature  Location  July 23 & 24/71 August 6/71  11.5°C 1D.5 D C  Lummi Island (seawater)  45 m 44 f  II Freshwater Arrival (to Chilko Lake)  August 17/71 August 26 & 27/71  14.4°C 13.9 a C  Chilko Lake (freshwater)  26 m 34 f  III Spawning (spawning grounds Chilko River)  September  S.9 D C  Chilko River (freshwater)  I  Seawater (beginning migration)  22/71  15 m 12 f (096 sp) ID f (50% sp) ID f (1DD% sp)  -124-  Chilko Migration 1972 The purpose of the second migration study uas to investigate the serum ionic cylcium changes in migrating salmon in an attempt to correlate these uith plasma c a l c i t o n i n 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 f i s h , 1 cm behind the posterior edge of the operculum.  Scales uere i n d i v i d u a l l y 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 s o l i d l y fused to the premaxillae i n the spauning f i s h , uere removed as completely as possible. analysis.  Methods of analysis uere outlined previously in General  Materials and Methods. necessary  Both gonads uere taken from each f i s h for  Due to phosphate interference, i t uas found  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 t i s s u e s . Table XVI, pg.125, gives a summary of the dates and l o c ations of sampling points of the 1972 Chilko Migration. Physical measurements,  plasma calcium, inorganic phosphorus,  sodium, potassium, percent uater, uere measured for each f i s h .  plasma protein and haematocrit  The serum ionic calcium uas measured  in the laboratory at a temperature close to that of the uater in uhich the fish uere o r i g i n a l l y captured.  Serum pH uas determined on  -125-  Table XVI.  Phase  Chilko Sockeye Migration 1972  Sampling Date  Location  Number of Salmon  I  Seauater (beginning migration)  July 21/72  Lummi Island (seauater)  10 m 10 f  II  Freshuater Arrival (to Chilko Lake)  August 28/72  Chilko Lake (freshuater)  10 m 10 f  Sept. 24/72  Chilko River (freshuater)  10 m 10 f (0% sp) 10 f (100% sp)  III Spauning (spauning grounds)  the freshuater a r r i v a l and spauning samples immediately fallouing the ionic calcium measurements.  The female spauning sockeye uere  divided into unspauned females (ripe but egg case i n t a c t ) and spauned out females (almost no eggs remaining).  The spauning males  uere in various stages of sexual maturation but uere generally very r i p e . It should be noted that the location and method of capture of the f i s h and the handling of the samples, uere the same for the 1971 and 1972 migrations.  -126-  B.  Plasma Calcitonin Levels in Coho Salmon:  Effect of Sexual  Maturation and Environmental S a l i n i t y The purpose D f this study uas to investigate  the plasma  c a l c i t o n i n levels in coho salmon at different stages of development and in different environments.  Table XVII,  pg.127>summarizes  the experimental conditions of the 3 groups of coho salmon. f i r s t group has been described previously in Chapter I, sisted of freshuater  The  and con-  spauning adult male and female coho salmon.  The second group uere sexually immature coho salmon uhich had spent their entire l i v e s in freshuater.  The t h i r d group uere young,  very sexually immature salmon ( g r i l s e ) 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 s a c r i f i c e since in nature they cease to feed upon entry into fresh uater.  The freshuater  immature coho in Group II uere fed  trout p e l l e t s once ueekly and starved 6 days prior to  sacrifice.  The coho g r i l s e in Group III uere grouing rapidly and being fed 3 times daily uith a frozen meat diet consisting of canned salmon, beef l i v e r and horse heart.  These f i s h had been fed 4 hours prior  to sampling. Physical and e l e c t r o l y t e measurements uere determed for each fish as outlined previously.  Plasma c a l c i t o n i n levels uere  again measured using the salmon c a l c i t o n i n radioimmunoassay.  Table XVII.  I  II  Coho Salmon Study:  Group  Sampling Date  Adults (ripe, spawning)  November 30/70  Freshwater (immature)  July 28/71  Summary of Sampling Data  Location Samish Hatchery Washington, U.S.A, (from river) Freshwater (temp. 4.4°C)  U.B.C.Physiology (fish laboratory) Freshwater (temp. 10 C)  Number of Salmon 15 m 15 f  History Wild fish - migrated from seawater to freshwater (Samish River) Age 2 -3 years  k m  11 f  Hatchery-raised in freshwater Age 3 years  D  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 seawater Age 1 year  -128-  C.  Plasma Calcitonin Levels in Spauning Adult Sockeye, Coho and Chinook Salmon This section i s a summary of the plasma c a l c i t o n i n  levels  obtained from spauning sockeye, coho and chinook salmon.  Details on the dates and locations of the c o l l e c t i o n 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 c a l c i t o n i n 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 i l l u s t r a t e d in Plates 7 and 8, pg.130,and Plates 9 and 10, pg. 131.  The seawater sockeye (Plate 7) have o l i v e -  green backs, s i l v e r sides and white b e l l i e s .  The sexes at this  stage are indistinguishable upon external examination. shows the sockeye 3 - 4  Plate 8  weeks later on a r r i v a l to Chilko Lake.  They have lost the s i l v e r colour from their sides and now have a reddish appearance.  The secondary sexual c h a r a c t e r i s t i c s such as  the hooked snout and hump back in the male are beginning to develop. In the spawning condition, after approximately 2 months in freshwater, the sexes are c l e a r l y distinguishable by the secondary sexual c h a r a c t e r i s t i c s in the male (Plates 9 and 10).  Both the male and  female have b r i l l i a n t crimson backs, black b e l l i e s and green heads and t a i l s .  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 i s generally larger i n size than his mate, for the body shape of the female changes l i t t l e from the seawater Table XVIII, pg.132,presents  condition.  physical parameters and plasma  measurements for the male and female sockeye at the 3 stages of the migration.  Plasma e l e c t r o l y t e and c a l c i t o n i n levels in these  same f i s h are shown in Table XIX, pg.  133  .  -130-  Plate 8.  Freshuater a r r i v a l Chilko sockeye (Male above, female below).  Plate ID.  Spawning female Chilku sockeye  Table XVIII.  Parameter  Sex  Physical and Plasma Measurements - Chilkc Migration 1971  Seawater Mean  Total Weight (g)  Fork Length (cm)  i  SE  (n)  Arrival Chilko Lake (n) Mean i' SE  2534  52.4  (45)  2175  103.9  (26)  2780  130.1  (14)  f  2506  67.5  (44)  1923  40.9  (34)  0% 2175 50% 1944 100% 1788  82.2 115.0 63.8  (12) (10) (10)  61.5  0.8  (14)  58.6 57.3 57.3  0.4 0.9 0.7  (12) (10) (10)  2.64  0.19  (14)  14.81 8.45 1.13  0.31 1.31 0.10  (12) ( 9) (10)  36  3.4  (15)  41 41 36  3.4 3.8 1.9  m  59.2  0.3  (45)  59.8  0.8  (26)  f  59.0  0.4  (44)  57.6  0.3  (33)  m  3.07  0.14 (45)  3.06  0.16 (26)  f  4.01  0.13 (44)  10.14  0.26 (34)  Haematocrit (vols 90  m  51  0.7  (44)  40  1.5  (26)  f  49  0.8  (44)  40  0.7  (34)  Plasma Protein Cg/IDQ ml)  m f  8.1 8.7  0.20 (44) 0.27 (42)  5.4  0.19 (26)  6.6  0.12 (34)  Plasma % H0  m  90.6  0.19 (44)  93.1  0.19 (26)  f  90.0  0.26 (42)  92.0  0.11 (34)  (g/lDOg)  (n)  m  Gonad/Somatic Index  2  Spawning Mean i SE  0% 5D% 100% 0% 50% 100% 0% 50% 100% 0% 50% 100% 0% 50% 100%  2.9 4.2 4.2 2.0  0.36 0.53 0.61 0.39  (12) (10) (10) (15) (12) (10) (10)  . 95.5  0.34  (15)  94.3 94.2 96.4  0.55 0.57 0.37  (12) (10) (10)  Table XIX.  Plasma Measurement  Seauater  Sex Mean  Calcium mEq/l  Plasma E l e c t r o l y t e M i g r a t i o n 1971  i  SE  m  6.9  0.12  f  9.2  0.29  and C a l c i t o n i n  Arrival (n)  (MO (MO  Mean  Levels  Chilko  -  ChilkD  Lake  SE  (n)  5.6  0 . 11  (26)  8.7  0 . 18  (34)  i  Spauning + SE  (n)  4.1  0 .24  (15)  7.3 7.0 4.0  0 .84 0 .81 0 .51  (12) (10) (10)  5.8  0 .29  (15).  0% 50% 100%  6.7 6.2 4.9  0 .52 0 .50 0 .23  (12) (10) (10)  0% 50% 100%  140 133 143 130  2 .9 6 .1 6 .1 3 .6  (15) (12) (10) (10)  0% 50% 100%  1.2 0.9 0.6 0.9  0 .38 0 .26 0 .14 0 .17  (15) (12) (10) (10)  Mean  0% 50%  100% Phosphate mEq/l  m  7.6  0.24  (MO.  7.1  0 . 19  (26)  f  7.B  0.27  (MO  6.8  0 . 20  (34)  Sodium  m  1.1  (43)  151  1. 5  (26)  0.8  (38)  151  0.08  (43)  1.4  (38)  1.9  164  mEq/l  f  Potassium mEq/l  m  Plasma Calcitonin pg/ml  m  117  34  f  545  136  f  159  • .7  •.a  0.10  (45) (44)  0. 7  (34)  0 . 92  (26)  0 . 40  (34)  12  12  (25)  141  29  687  112  (34)  0% 1649 50% 709 100% 306  240 330 105  (14) (12) ( 5) ( 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 i s a trace element in seauater uhere i t s 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)  Seauater Ions (mEq/l) Calcium  Sodium  Potassium  July 23, 1971 Lummi Island (seauater)  15-20  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 i s readily  apparent that the females maintained higher circulating levels of calcitonin than the males, at a l l 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 precipitously after spauning.  The male CT levels decreased to  12 Plasma C a l c i t o n i n  1800 r-  1500 E  X 2l200  34  Male  •Z]  Female  0 -I % Spawning 50 V Condition 100 J of Female  o  ^ 900 o  O  WW  44  o | 600 o  CL  300  44  14  T  25 Seawater  Figure 18.  Arrival Chilko Lake  1  50  Spawning  Plasma c a l c i t o n i n changes i n m i g r a t i n g  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 concentrations, 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 s t a t i s t i c a l l y 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 throughout the migration in both sexes.  The total plasma calciums in  the females were significantly (p< 0.001) higher than the males at a l l stages in the migration except the spawned out females. Plasma potassium levels rose on arrival to Chilko Lake and f e l l with spawning. Figure 20, pg. 139 ,shows the plasma calcium, plasma c a l 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 f a l l s o f f . 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 Date  Seauater Females  Lummi Island Seauater August 6/71  n mean SD SE  Total Lilt (g)  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  2559.33 396.70 177.41  6 3.87a 0.69 0.31  6 35.81 25.28 11.30  = = = =  Spauning Females (0%)  n mean SD SE  Fish #  Chilko River Spauning Grounds September 22/71  = = = =  69 70 71 72 76 77 6  2391 2604 2455 2044 1947 1769  GSI  14.88 14.80 13.01 15.24 16.05 13.96  6 6 6 2201.66 14.66 299.66 0.96 134.01 0.43  Calcitonin Content U/gland  Plasma Calcitonin Level „, pg/ml mU/ml 178 379 0 0 0 1090 6 274.50a 389.72 174.29  34.9 55.0 55.7 40.9 71.0 75.7  2591 1466 1406 2089 1290 1433  55.53 14.64 6.54  6 1712.50 469.90 210.14  t-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.  0.89 1.90 0 0 0 5.45 6 1.37 a 1.95 0.87  12.96 7.33 7.03 10.45 6.45 7.17 6 8.57 2.35 1.05  -138  o co E  o  0.  H  £  LU  £ 8.0  44 44  Male  |piosma Phosphate I  o—  S  2  34  Female  12  - t l 10  0.  o E  0  196 Spawning  50 >  Condition  IOOI  of Females  0 50  o o B  S e a Water  Fresh Water Arrival  Figure 19.  Spawning  Fresh  Water  Plasma electrolyte changes i n migrating ChilkD sockeye.  -139  Gonad/Somatic Index  o 10 E  111 44  Plasma Calcium 44  -fl  -  '  45  e  6  E  z  10  ito  •  B l  °0  Plasma Calcitonin  1500  1 \  Male  Sl200  CZ3 O 50 100  Li  Female  i% % S[ Spawning  Ct Condition ot Females  )  <»<*  <"""  Se  igure 20.  ef  ChMKoTa'ke  Spawnin,  Plasma c a l c i t o n i n , plasma calcium and gonad-somatic index changes in migrating Chilko sockeye.  -140male GSI of 14.0 percent from seawater to spawning, is evidence v  that some males had partially spawned before capture. Changes in plasma percent water, plasma protein and haematocrit are illustrated in Figure 21, pg. 141 . dicate  The data i n -  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 . measured.  Serum pH of the seawater sockeye was not  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 15  96.0 95.0  o o \  940  w Q>  93.0  o» O  rh  T  Percent Water  12 10  3  a £ o a.  9 2.0 91 .0 142  90.0  50  85.5 10.0 Plasma Protein E O O N  O)  8.0  34  6.0  12 10  e 4.0  15 |  2.0  m  •HE  0.0  I  DH  Male  •  Female  O % Spawning 50 Condition of 100 . Females 1  [*1  10  50  * o > O  o  CT E  (U  o I  Sea Water  Freshwater on Arrival at Spawning Grounds  Spawning Freshwater  Figure 21. Haematocrit, plasma protein and plasma percent water changes in migrating Chilko sockeye.  Table XXII. Parameter  Total Weight (g) Fork Length  Sex  m f  Mean  SE  2672  192.8  2433 60.7 59.4  (cm)  Gonad/Somatic  m  2.42  Index  f  3.98  Haematocrit  m f  50 48  (n)  Mean  SE  2667  114.5  94.5  (10) (12)  2011  81.9  (11) (10)  1.8 0.8  ( 6) (12)  0.6  (11)  0.6  (10)  0.22 0.24  (10)  2.7 2.1  ( 9) (10)  (11)  2.77 9.90  37 40  1.0 1.2  ( 8)  93.4  0.14 (10)  0.33  (11)  91.7  0.17 (10)  ( 8)  f  8.6  0.35  Plasma %  m  91.2  f  90.1  0% 1D0%  0% Q  D  %  D.17  (10)  0.30  ( 9)  36 40 40  1.5 1.7 4.2  (10) (10) (10)  0.43 (10) 0.30 0.28  '  3  (10) (10)  0.42 (10)  93.7 9 S  (10)  1.79 15.36  95.3 1  (10) (10)  (10) (10)  3.1 4.8 2.1  0% 100%  0.17 (10)  (10)  0.7 0.3 0.5  58.5 59.0  0% 100%  (10) (10)  0.22  0.26  61.9 0% 100%  (n)  SE  118.3 37.8 93.6  0% 2152 100% 1853  0.75 ( 9)  (11)  7.6  2743  0.14 ( 9)  0.17 (10)  m  (g/lOOg)  63.3 58.6  (n)  5-1 6.9  Plasma Protein (g/100 ml)  H„0  Spawning + Mean  Arrival Chilko Lake  Seawater  m f  (vols %)  Physical and Plasma Measurements - Chilko Migration 1972  0.30 (10) D  '  2 6  ( 1 Q )  Table XXIII.  Electrolyte . Serum Total Calcium mEq/1  Sex  Plasma Electrolyte Levels - Chilko Migration 1972  Seawater Mean i  SE  (n)  Arrival Chilko Lake SE (n) Mean -  m  7.3  0.17  (10)  5.7  0.14  f  11.2  0.36  (10)  12.7  0.30  (11) (10)  Plasma Phosphate mEq/1  m  ld.l  0.48  (10)  7.0  0.26  (10)  f  ID.5  0.37  (11)  7.5  0.14  (10)  Plasma Sodium mEq/1  m  1.9 2.0  (10)  138  8.5  (11)  127  8.3  (10) (10)  Plasma Potassium mEq/1  m  0.4  0.06  (10)  2.3  0.23  (10)  f  0.5  0.04  (11)  . 1.8  0.23  (ID)  f  165 163  Spawning Mean 4.9 0% 100%  8.2 4.5  6.1 0% 100%  0% 100%  0.33 0.32 0.29  (n) (10) (11) ( 9)  (10)  0.17 0.48 0.44  (10) (10)  6.3 3.4 8.0  (10) (10) (9)  1.1  0.23  (10)  1.3 1.3  0.24 0.26  (10) ( 9)  6.3 5.8  140 0% 100%  SE  151 133  Tabla XXIV.  Ionic and Total Serum Calcium, Serum pH and Plasma Protein Changes in Migrating Chilko Sockaye (1972)  Seauater Sex  rn  f  Total Serum Ca mEq/i n mean SD SE  _ B m o  -  n m mean SD m SE m  e 7.25 0.51 0.17  1 0  C  10 11.23 1.08 0.36 -  %  Ionic Serum Ca mEq/1  Ionic Ca  10 3.06 0.30 0.09  c 42.2 2.07 0.68  10 3.16 0.32 0.10  Spauning  Arrival Chilko Lake  1 0  C  10 28.2 2.31.  0.76  Plasma Protein g/100 ml  Total Serum Ca mEq/l  S  11 5.65 0.50 0.11.  11 3.02 0.18 0.05  10 12.73  10 3.17 0.17 0.05  7.6  Ionic Serum Ce mEq/1  c  a  0.70 0.26 11 6.6  1.11. 0.36  0.91.  0.30  -  %  Ionic Ca  11 _  53.1.  C  2.56 0.61 10 25.0  1.73 D.58  Plasma Protein g/100 ml  1 0  c  5.1 0.51 0.17 c  10 6.9  0.56 0.17  Serum pH  Total Serum Ca mEq/1  Ionic Serum Ca mEq/i  11 7.329 0.069 0.022  10 1..87 0.99 D.33  8 2.75  10 7.282 0.080 0.027  056 ,• 11 8.23 1.02 0.32 100%  0.31.  0.13 11 3.01.  0.16 0.01.  9  7  (..1.7  2.1.5  0.83 0.29  Total Spauning Females  t-test probability male vs. female  Note:  a.  p< 0.050  b.  p< 0.010  c.  p< 0.001  0.33 0.13  20 6.5«. 2.09  b  0.1.8  18 2.B1 0.38 0.09  %  Ionic Ca  8 59.1.  7.29 2.75 11 37.1.  Plasma Protein g/100 ml  Serum pH  10 3.1 1.33 0.1.3  10 7.515 0.130 0.031  10 1..8  11 7.389  1..96 1.56  0.91.  0.11.1  0.30  0.GU  7 57.9 1..66 1.90  ID 2.1 0.85 0.28  10  is  20 3.5  21  1.5. l. b 11.11.  2.70  1.61.  0.37  7.551. 0.211.  0.C70  7.1. £8  0.197 0.031  pH uas not measured on seauater serum samples.  -F" -F"  12.0 -i  10  Total Serum 10.0 -  Calcium Serum Ionic  8.0-  Calcium  N cr LU  t=  6.0-  E  •D  2. o O  4.0 -  E  2  <o  CO  2.00.0  I 10  J  10  Sea Water  F i g u r e 22.  10  \ II II  cr*  Fresh Water Arrival  0%9  Spawning  I00%9  *  Serum i o n i c and t o t a l calcium changes i n m i g r a t i n g C h i l k o sockeye ( C h i l k o M i g r a t i o n 1972). Note constancy o f i o n i c serum calcium levels. -pui i  -146-  Table XXV. Uater Temperatures - Chilko Migration 1972  Collection Date  Location  Temperature of Uater  July 21, 1972  Lummi Island (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  On Location (surface temperature).  Ion electrode uater jacket  14.5°C  12°C  It can be seen from the data that although serum t o t a l calcium levels markedly changed throughout the migration, the serum ionic calcium levels remained r e l a t i v e l y constant. ing males shoued a s l i g h t but s i g n i f i c a n t ionic calcium from the a r r i v a l l e v e l .  (p<Q.D5)  The spaun-  decline in  Female serum i o n i c calcium  remained stable u n t i l spauning uhen there uas a s i g n i f i c a n t crease (p<0.DDl) to the 100 percent spauned l e v e l .  de-  Since the  t o t a l serum calcium f e l l dramatically and the ionic serum calcium remained f a i r l y constant,  there uas a marked increase  in percent  ionic calcium from seauater to spauning in both sexes. Although the female t o t a l calciums uere s i g n i f i c a n t l y higher than those of the male (excepting the spauned out females), the  -147-  ionic calcium levels of bath sexes uere very s i m i l a r .  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 r i s e in serum pH from a r r i v a l 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, r e s p e c t i v e l y .  These same results  i l l u s t r a t e d in histogram form in Figures 23, 24, pages 150, (soft tissues) and Figures 25, 26, pages  are 151 ,  154, 155, (hard t i s s u e s ) .  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 a v a i l a b l e . As i l l u s t r a t e d 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 l a t t e r possessed the louest amounts of  calcium (10-26 mgCa/lOOg FFDW) of the soft  tissues.  for the muscles ranged from 22 to 68 mgCa/lOOg FFDW.  Measurements In comparing  Table XXVI. Soft Tissue Mineral Changes - Chilko Migration 1972  ARRIVAL CHILKO LAKE  SPAUNING  i Tissue  mo PO^  % Ash Ut.  Sex  lOOg FF Dry lilt.  FF Dry Ut (n) Xean m 9 14.49  Gonads  Muscles  SE Mean 0.71 2667.5  -  SE  [Mean  -  121..63! 9.9  0.15  805.1°  44.49;i61.8  m 7  5.56  0.33  857.7  79.21.! 48.1  9 7  ra f  5.07  0.26  908.2  C  1  1.0.98 68.3 :  SE 0.61.  3.92  C  % Ash Ut  lOOg FF Dry Ut  T 7  f Skin  -  mq Ca  a  6.03  5.61 7.57  FF Dry Ut. Mean 13.90  -  4.61,  0.45  l D 6 U  5.59  0.19  U  21..71:11.1.7  27.30  2.82  0.08  a  1.63  0.22  1.23.7  59.93 176.3  36.19  2.80  0.13  i  lOOg FF Dry Ut  SE  Mean  ±  SE  73.9 | 13.1  8.22  C  ! Q  7  '  -  5  7  3 7  - \  2 2  2 1  -  3 1  1 U  6 7  !  -\ -  9  -  X  U  9  | 598.5  17.17 166.7  24.21  549.8  61.52 192.7  52.45  t-test probability male va. female  a.  p< 0.05  b.  p< 0.005  c.  p < 0.001  FF Dry Ut  |  -  3.95  mq PO,  SE ;Mean  -  O.BSj3661.8 G  nq Ca  lOOg FF Dry Ut  0.20 ; 856.0  G  SE  lOOg FF Dry Lit t  Mean  140.2  26.4  45.4  150.1  SE 3.63  C  6.82  5.05  D.23.1D08.6  38.07  43.4-  3.05  0* 5.83 10054 5.36  0.27.1069.5 0.49 1075.9  42.77 90.38  33.3 31.2  3.65 2.52  30.05  78.0..  B 1  8  1  16.46 0%  i  % Ash Ut Mean  0.86  28.3 ! 175.D  C  1  1,22.1,  !  949.3  0.17  O.H.  i i  i  3551.0  4.02°  1.67  !  Mean  0.26  S  9  mq Ca  100 FF Dry Ut  SE  I  |  .ragPO^  547.0 0* 10056  - - ' -  !  i  547.2 528.6  16.70 75.9 14.65 110.0  10.57 6.10 12.SO  Tabla XXVII.  Hard T I B B U B Mineral Changea - Chilko Migration 1972  ARRIVAL CHILKO LAKE  Tissue  Sax  % Ash  Ult  (n) Mean Scales  t  m 7 36.01 f 6 34.01  8  SE  q Ca  9 P0  fc  Dry Dt'  100 g Mean  Ash SE  100 g Mean  -  96 A a h  tilt  SE  Mean  -  0  gPO,, lOQg A s h  Dry Ut  Ash  SE  0.75 17.65  0.25  36.22  0.53 26.79  0.94  0.3S 17.65  0.20  35.64  ^ 0.42 26.43  0.67  Mean  i  SPAWNING  CB  100 g SE  Mean  -  Ash SE  % A s h tilt  I  Dry Wt  |  Mean  i  g PO^  j  lOOg A s h  SEJMean  . i  j  q  Ca  lOOg A s h  SE|Mean  -  SE  19.51  0.29 32.65  0.64  23.53  D.63 20.29  0.19^ 32.03  0.49  19.62  0.48 32.81  0.31  0% 24.77 100% 24.87  0.68 20.36 0.86 19.59  0.40'32.94 0.47!32.14  0.39 0.79  9  i Vertebrae ra 9  31..17  f 9 34.07 9  0.33  18.69  0.40Jl8.81  0.34  35.58  0.53 38.15  0.87! 18.51  0.91 37.11  0.39  41.65  0.52 18.21  0.D5 38.08  0.29  36.19  0.23 39.75  0.52; 18.18  0.06 36.21  0.48  0% 42.54 100% 45.06  0.26 18.04 0.64 18.12  0.09 36.81 0.04 36.10  i 1  !  ;  i  ! t-test probability male us. female  a. p< 0.05  0.17 0.62 0.58  -150  Chilko  <$ 9 Seawater  Figure 23.  Migration  d* 9 C h i | k o  ^  1972  o* 90% Spawn,ng  Soft tissue calcium changes in migrating Chilko sockeye.  -151  Chilko  Migration  1972  Muscle  Skin  •  o g  n  i  450001  v  2  Gonads  a. 4000.0-  6  cf c S  e  a  w  a  9  & 9  „,„  Arrival Chilko Lake  ,  e  r  c* 90% _ S p a w n , n  9  F i g u r e 2k. S o f t t i s s u e p h o s p h a t e c h a n g e s Chilko sockeye.  i n migrating  -152-  the  3 soft  t i s s u e s , the  calcium content showed t h e  significantly  (p< Q.uul)  from a h i g h of  f r o m 48 m g C a / l Q D g FFDU i n t h e Spawning males however,  to for  arrival  both s e x e s ,  at which l e v e l i t  sea to  the  In  f e m a l e s had s i g n i f i c a n t l y  h i g h e r measurements i n the The c a l c i u m c o n t e n t sockeye i n c r e a s e d s l i g h t l y skin contained s l i g h t l y  spawning of  the s k i n f o r  on a r r i v a l  spawning.  Spawned o u t  to  at  all  stages of the m i g r a t i o n .  from sea to  arrival  then f e l l  t h e male gonad c a l c i u m c o n t e n t to  arrival  (p<0.01)  difference  the  skin  the  was n o t  from a r r i v a l  (p<0.05)  to  higher  females.  female sockeye contained  amounts o f  c a l c i u m than the males  The f e m a l e l e v e l r o s e  (p<D.D5)  with spawning.  increased significantly  and from a r r i v a l  The f e m a l e  calcium  female p<D.Q25)  As e x p e c t e d , t h e g o n a d s D f t h e greater  content  significantly  f e m a l e s had s i g n i f i c a n t l y  (p< • . • • ! )  level  b o t h m a l e and f e m a l e  the  s k i n c a l c i u m c o n t e n t s t h a n t h e 0% s p a w n i n g  significantly  higher  levels in  Chilko Lake.  In b o t h s e x e s ,  d e c r e a s e d ( m a l e p< 0 . 0 0 5 ,  arrival.  group.  mare c a l c i u m b u t  significant.  higher  the m a l e s had  •.••5)  again rose  comparing the muscle c a l c i u m  groups w h i l e  (p<  23 m g C a / l D O g FFDU on (p<Q.u01)  the  remained  significantly  m a l e s as t h e m u s c l e c a l c i u m c o n t e n t  s e a w a t e r and a r r i v a l  statistically  decreased  68 m g C a / l Q O g FFDW i n  had a s i g n i f i c a n t l y  the seawater l e v e l .  content  in  the muscles  f e m a l e s , the muscle c a l c i u m content  The m a l e m u s c l e c a l c i u m a l s o f e l l  than the  variation  ( a s i n d i c a t e d by t h e SE b a r s ) w h i l e  32 m g C a / l O Q g FFDU on a r r i v a l ,  stable.  greatest  least.  In the  sea to  s k i n showed t h e  to  spawning  slightly In  contrast,  both from s e a  (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 l e v e l in the male increased s l i g h t l y (p< 0.05) from sea to a r r i v a l and remained constant from a r r i v a l to spauning.  The female l e v e l also rose (p< 0.001) from sea to  a r r i v a l , remaining stable throughout spauning. The male skin phosphate content increased (p< 0.001) from sea to a r r i v a l . from the l a t t e r .  The spauning l e v e l uas not s i g n i f i c a n t l y different The female skin phosphate content did not change  s i g n i f i c a n t l y 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) i n phosphate content from the sea to a r r i v a l uith a further s l i g h t increase on spauning. than the ovaries.  The testes contained 3-4 times more phosphate  Gonad phosphate content in the female increased  (p<0.05) from sea to a r r i v a l and then returned to the seauater l e v e l in the 0 percent spauned female. In the hard tissues, i t should be noted that uhile the vertebrae increased in mineral content (% ash/dry ueight) throughout 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  38.  0-  Migration 1972  Scales  34.0 •  4 30.0  O O \  Vertebrae  o  o  o> 38.0-1  34.0  30.0  cf  9  Seawater  Figure 25.  d*  9  Arrival Chilko Lake  cf  (J0% 9100%  Spawning  Hard tissue calcium changes in migrating Chilkc sockeye.  -155-  Chilko Migration  21.0  i  1972  Scales  19.0 •  17.0-  < O  o  15.0 •  a.  Vertebrae  19.0-  iii  1*1  17.0-  15.0-  cr  9  Seawater  Figure 26.  cf  9  Arrival Chilko Lake  Cf  £O%CMOO%  Spawning  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 l e v e l 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 a r r i v a l (p<0.05) and further from a r r i v a l to spawning (p< 0.05). significant  In the female,  change throughout the migration.  there was no  A comparison of the  vertebrae calcium content for both sexes reveals s i g n i f i c a n t l y (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 a r r i v a l (p< 0.005) with a further decrease at spawning. The female l e v e l also decreased s i g n i f i c a n t l y first  (p< 0.001) after the  stage of the migration but did not change with spawning. As shown in Figure 26, the vertebrae phosphate content of  bath sexes decreased s l i g h t l y with migration but the decrease was s i g n i f i c a n t  only between the female seawater and spawning  vertebrae ( p < 0 . 0 5 ) . In contrast,  the male and female scale phosphate  content  -157  i  Chilko  Cf  Sockeye  9  Seawater  F i g u r e 27.  Migration 1972  cf  9  Arrival Chilko Lake  Cf  C)0%  Q  ioo%  Spawning  Vertebrae m i n e r a l content changes i n m i g r a t i n g C h i l k o sockeye.  Chilko Sockeye Migration  1972  ft  ft  cf  9  Seawater  lire 28.  cf  9  Arrival Chilko Lake  cf  90% 9 ioo%  Spawning  Scale mineral content changes in migrating Chilko sockeye.  -159-  showed a marked increase through the migratory stages. male, t h i s increase uas s i g n i f i c a n t  from sea to a r r i v a l (p< 0.001)  and from a r r i v a l to spawning ( p < 0 . 0 5 ) . content also increased s i g n i f i c a n t l y  In the  The female scale  phosphate  (p< • . • O l ) from sea to  a r r i v a l but there was l i t t l e change with spawning. Tchernavin (1937; 1938a; 1938b) has documented the phenomenal growth i n the jaw bones of the spawning A t l a n t i c salmon. "breeding growth" as Tchernavin termed i t , in the premaxillae  (Uladykov, 1962).  This  i s p a r t i c u l a r l y evident  In males, the premaxilla  p r a c t i c a l l y doubles in length from the seawater to the breeding stage.  The increase in size i s due not only to the growth of the  bone i t s e l f ,  but also to i t s fusion to an o s s i f i e d  plate which  develops as a support base for the large breeding teeth (Tchernavin, 1938a). Table XXV/III, pg. 160 , summarizes the dry weights, and phosphate contents  (g/lOOg dry weight) of single  calcium  premaxillary  bones taken from Chilko sockeye at each stage of the migration. Figure 29, pg. 161, i l l u s t r a t e s the changes in dry weight of the premaxillae. reflects  The marked increase in weight for the spawning male  the extensive snout and jaw growth.  Calcium and phosphate  content of the premaxillae are i l l u s t r a t e d in Figures 30 and 31, pages 162 and 163 .  The spawning male had s i g n i f i c a n t l y  more calcium and phosphate  (p<0.005)  (g/lOOg dry wt.) than the spawning  female. In order to determine the actual amounts of tissue calcium and phosphate per f i s h , 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  gCa Sax  Ory Ut Mean  lOOg Dry Wt  - SE(n)  lOOg Dry Wt  Mean  i S£(n)  Mean  i SE(n)  gCa Dry Wt Mean  IB  54.10  10.29(5)  4.75  0.32(5)  7.37  0.15(5) 75.49  f  37.92  8.10(7)  4.30  0.13(5)  8.41  0.46(5)  t-teet probability male vs. femala  40.34  ± SE(n)  b  lOOg Dry Wt Mean - SE (n)  lOOg Dry Ut Mean  -  Dry Wt  SE(n)  7.38(11) 6.63  0.32(11)  12.19  0.56(11)  2.36(10) 6.01  0.32(9)  11.40  0.62(10)  gCa  gpo,, Mean  -  lOOg Dry Ut SE (n)  Mean  i  SE (n)  lOOg Ory Ut Mean  t  SE(n)  8.82  0.32(10)  16.07  6.90(10)  7.60  0.17(10)  13.72  75.34  7.10 (8)  7.50  0.41 (8) 14.14  0.76 (3)  64.66  5.35(18)  7.55  0.20(18)  0.33(16)  213.51  33.52(10)  0%  56.11  100%  Total sp.f  b  a  13.91  3  0.56(10) 0.35(10)  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  F i g u r e 30.  Spawning  P r e m a x i l l a e c a l c i u m content changes in migrating Chilko sockeye.  Chilko  Migration  1972  10.0 i  8.0 ft  JZ  .? v  r-,  6.0  Q o> O  2 \  4.0  o a. 2.0  0.0  cf  Seawater  Figure  31.  cf  9  Premaxillae in  cf  9  Arrival Chilko Lake  migrating  Spawning  phosphate Chilko  90% 9100%  content  sockeye.  changes  -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  Total Body Wet Wt (Kg) Skin  % dry ut /total body uet ut Muscle  Gonads  Scales  Vertebrae  Total Skele ton  male sockeye  2.540  1.693  24.399  0.5B1  0.079  0.736  2.669  2.526  1.742  21.770  3.486  0.071  0.732  2.514  2.758  6.464  -  female sockeye human*  70.55  10.119  * Data taken from Mitchell e_t a_l, 1945.  **  Total skeleton in sockeye includes vertebrae, ribs, t a i l , f i n bones, g i l l apparatus and skull bones.  It i s apparent from the table that the percent dry ueight/total body uet weight for the skin i s 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 , i t uas possible to calculate the absolute amount of calcium and phosphate in each tissue of a male and female freshuater pg. 166).  a r r i v a l Chilko sockeye (Table XXX,  The absolute ueight of the f i s h as u e l l as the i n -  dividual tissue weights changed during the migration.  For example,  i t 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 c a l c u l -  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 s l i g h t l y more phosphate.  In the human, body calcium amounts to 1.5 - 1.6%  body weight (Mitchell e_t a l , 1945; Copp, 1970b) whereas i n the sockeye, body calcium ( t o t a l skeleton plus the 3 soft tissues measured) was approximately 0.42 - 0.44% body weight.  The percent  calcium and phosphate in the human skeleton i s 1.58% and 0.72% respectively (Mitchell et_ a l , 1945).  Corresponding percentages i n  the sockeye skeleton were 0.40 - 0.43% for calcium and 0.21 - 0.23% for  phosphate. In summary, because of i t s 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 s l i g h t l y more phosphate than the male gonads.  Due to  their very low weight, the scales contributed l i t t l e to the t o t a l storage of calcium and phosphate in the hard t i s s u e .  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 A r r i v a l Sockeye*  Calcium (g) per tissue per fish  Phosphate (g) per tissue per fish  male  female  male  female  Skin  0.075  • .•68  0.270  0.193  Muscle  0.148  • .140  6.927  4.850  Gonads  • .002  0.122  0.550  0.664  Total Soft Tissue**  0.225  0.330  7.747  5.7Q7  Scales  • .173  0.158  • .103  0.095  Vertebrae  2.159  2.121  1.077  1.067  Premaxilla  • .•19  • .••9  0.010  0.005  Total Hard Tissue***  11.383  8.112  6.086  4.318  Tissue  Soft Tissues  Hard Tissues  •Mean total wet weight:  FUA male = 2667g, FLdA female = 2011g  **Total soft tissue includes only skin, muscle and gonads. **Total hard tissue includes a l l bones plus scales.  -167-  B.  Plasma Calcitonin Levels in Coho Salman:  Effect of Sexual  Maturation and Environmental S a l i n i t y Physical parameters and plasma measurements of the 3 groups of  CDho  salmon are presented in Table XXXI, pg.  168 , and  their respective e l e c t r o l y t e and c a l c i t o n i n levels are shown in Table XXXII, pg. 169 .  The GSI, plasma c a l c i t o n i n and plasma  calcium levels for the 3 groups of coho are i l l u s t r a t e d in Figure 32, pg.  170 . As shown by the ganad-somatic  index the adult coho were  very sexually mature while both the coho g r i l s e 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 g r i l s e ,  even though they were l i v i n g in seawater  had lower plasma calcium levels (calcium l e v e l = 17.6 mEq/litre) than the freshwater adult spawning coho. in the females  Although plasma calciums  are s l i g h t l y higher than i n the males in each of the  3 groups, the differences  are not s t a t i s t i c a l l y  significant.  Plasma c a l c i t o n i n levels for the adult spawning coho were markedly higher than the other two groups while the lowest were measured in the freshwater immature coho.  A sex  levels  difference  was noted only in the case of the adult spawning coho where the females had s i g n i f i c a n t l y higher (p<0.001) plasma c a l c i t o n i n levels than the males.  The mean plasma c a l c i t o n i n l e v e l . o f 4  coho "jacks" (2 year o l d , sexually ripe males, mean t o t a l wt. = 409 - 107g) was 2,393 - 754 pg/ml and was thus higher than the l e v e l found in the adult spawning coho (1,070 - 294 pg/ml).  Table XXXI.  Physical and Plasma Measurements - Coho Salman Study Grilse Seauater  Parameter Sex  Mean  Total Weight (Kg)  m f  • .19 • .18  Fork Length  m  25.3  (cm)  f  25.7  Gonad/Somatic  i  SE  Immature Freshuater (n)  • .•21 (5) • .••6 (5)  SE  (n)  • .46  • .134  Ck)  • .50  • .•58 (ID)  Mean  -  5.17 4.36  a  SE  (n)  • .23 (15) • .22 (15)  35.1  2.7  Ck)  77.8  (5)  36.7  1.5  (10)  72.5  0.00 • .•4  (5)  • .5  • .21  Ck)  4.7  (5)  • .9  • .14  (10)  2.5  Ck)  47  1.4  (15)  1.3  (10)  46  1.9  (15)  m  • .•3  Index  f  0.2S  Haematocrit  m  44  3.8  (5)  f  45  3.8  (5)  (vols %)  i  (5)  l.D • .3  C  Mean  Spauning Adults Freshuater  . 19 12 a  b  • .9  (15)  1.1  (15)  • . • • (12)  >15.0  Plasma Protein  m  5.7  • .68  (5)  3.6  • .31  Ck)  6.4  (g/100 ml)  f  E>.k  1.29  (5)  3.6  • .ID  (10)  6.5  Plasma  m  92.8  • .6  (5)  95.0  • .3  Ck)  92.1  0.2  (15)  % H0 (g/lOQg)  f  92.2  1.2  (5)  94.8  • .1  (10)  92.1  0.4  (15)  2  t-test probability male us. female  a.  p<0.05  b.  p< 0.010  c.  p< 0.001  0.23 (15) 0.38 (15)  Table XXXII. Plasma Electrolyte and Calcitonin Levels - Coho Salman Study  Plasma Measurement  Grilse Seauater Sex  Mean  Calcium mEq/1  m f  . 5.7 6.1  Phosphate  m  6.2  mEq/1  f  6.2  Sodium  -  SE .(n)  0.11 ( 4) 0.09 (10)  (4)  6.0  0.53  (5)  -  SE (n)  (4)  8.6  0.37 (15)  . 5.7  0.17 (10)  7.7  0.42 (15)  m  145  1.6  (4)  167  mEq/1  f  146  1.4  (ID)  163  Potassium  m  3.8  0.18  (4)  mEq/1  f  4.3  0.17 (10)  Magnesium  m  mEq/1  f  Calcitonin pg/ml  m  814  f  1195  0.68 0.14  4.4 4.6  Mean  0.21 (15) . 0.83 (15)  0.13  (5) (5)  Spauning Adults Freshuater  6.9 8.0  t-test probability male vs. female  0.18  Immature Freshuater Mean - SE (n)  —  —  a  3.2 3.0  169 394  b  a. p<D.05  292  (5)  272  b. p< 0.010  239 158  (4) (10)  1,070 12,081  c. p < 0.001  1.5  (15)  .27 (15)  0.30 (15)  1.79 (5)  (15)  •  2.02 —  1.1  C  0.06 (11) 0.03 (15) 294  (13)  1305  (15)  -170  Gonad/Somatic index  <0  0 14000 12000 10000  I Plasma Calcitonin"  8000 o O  6000  •i  Male  •  Female  2 000 0  5 o  2.0 -  e  0.0  O  a.  ure 32.  •I • T .  4.0  a CO  it.  immature Fresh Wafer  Spawning Adult Fresh Water  Plasma c a l c i u m , plasma c a l c i t o n i n and gonad-somatic i n d e x measurements i n 3 g r o u p s o f coho s a l m o n .  -171-  C.  Plasma Calcitonin Levels in Spawning Adult Sockeye, Coho and Chinook Salmon  This, section summarizes the plasma c a l c i t o n i n levels 3 species of P a c i f i c salmon, sockeye, coho and chinook.  for  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 . measurements,  Physical  plasma c a l c i t o n i n and electrolyte levels for the 3  groups are presented in Table XXXIII, pg. 172 .  The GSI indicates  that a l l 3 species uere very sexually mature. Plasma c a l c i t o n i n levels for the 3 salmon groups i l l u s t r a t e d i n Figure 33, pg. 173 .  are  The coho and chinook levels  uere s i g n i f i c a n t l y higher (males p<D.DD5 and females  p<Q.0Dl)  than those measured for the sockeye. TableXXXIV, pg. 174, summarizes the i n d i v i d u a l f i s h  ueights,  ultimobranchial gland c a l c i t o n i n content and plasma c a l c i t o n i n l e v e l in coho and chinook salmon.  Plasma c a l c i t o n i n i s  presented  in picograms per ml plasma and in mU per ml plasma (assuming a b i o l o g i c a l a c t i v i t y of 5000 U per mg for pure salmon c a l c i t o n i n ) . It can be seen from the table that although the coho and chinook females had s i g n i f i c a n t l y higher plasma CT levels than the males, the UB gland, c a l c i t o n i n content did not r e f l e c t this sex There uas no s i g n i f i c a n t  difference.  difference betueen the LIB gland c a l c i t o n i n  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  Species  Chinook  Coho  Sockeye  Sex  Gonad/Samatic Index  Weight Kg Mean  -  SECn)  Mean  *  m  7.85  f  8.83  m  5.17  f  (..36  0.22(15) *15.0  m  2.78  0.13(14)  2.6  f  2.18°  0.08(12)  14.8  t-teBt  •.33(15) a  0.33(11.) 25. l  0.23(15) s  4.6 c  4.7  SE(n)  Mean  0.44(15)  93.3  probability  male  va.  -  Plasma Calcitonin pg/ml  SE(n)  MBBn  0.26(15)  . 0.87(13)  93.5  0.29(14)  0.00(12)  92.1  0.2(15)  -  2067  SE(n)  i SE(n)  an  0.31(15)  161  6.9  0.21(14)  166  0.17(15)  5.9  0.17(14)  - SE(n)  Mean  Potassium Mean  - SE(n)  1.3(15)  1.1  0.29(15)  1.1(14)  1.2  0.29(14)  1.1(15)  3.2  0.27(15)  1.5(15)  3.Q  0.32(15)  6.9  0.21(15)  8.6  0.37(15)  167  1305(15)  8.0  0.83(15)  7.7  0.42(15)  163  4.1  0.24(15)  5.8  0.29(15)  140  2.9(15)  1.2  0.32(15)  7.3°  0.84(12)  6.7  0.52(12)  133  6.1(12)  0.9  0.26(12)  95.5  0.34(15)  141  29(14)  94.3  0.55(12)  1649°  240(12)  b .  MB  294(13)  0.19(14)  p < 0.05  - SE(n)  1070 12081°  B.  an  13154°  0.4(15)  female  MB  600(15) ' 5.3  92.1  0.31(12)  Plasma Electrolytes mEq/1 Sodium Phosphate  Calcium  2362(14)  - (15)  C  C  Plasma % H„0 g/lOOg  p<0.01  c.  p<  5.S  a  a  a  a  0.001  'estimate  I  (-•  O ro I  18000 Chinook  2  15 000  ~  12000  E N  Coho  c o o o  • •  Male  9 000  •  Female  6000  Note: All Salmon are Spawning Adults in Fresh Water  O  o £ w o 0-  3000  Sockeye  o i= 14 12 n Mean ' 141 1649 S.E. •±29 + 240  13 15 1070 12081 294 ± 1305  15 14 2067 13154 ±600 ±2362  ure 33. Plasma calcitonin levels in 3 species of salmon. Note higher female plasma CT level in each species.  Table XXXIV.  Plasma and U l t i m o b r a n c h i a l Gland C a l c i t o n i n Concentrations i n Coho and Chinook Salmon  Sex '  Species  m  Coho  Fish  19 20 21 22 24 26 28  4.32 5.68 5.46 5.00 5.46 4.55 4.55  112.9 191.1 557.1 517.3 313.2 375.7 153.2  7 5.00 0.50 0.20  7 317.21 162.82 66.47  4.55 4.09 4.09 4.32 5.46 5.00 3.86  594.0 84.8 66.7 181.6 494.7 106.2 173.8  16.8D0 5,575 8,725 13,650 11.15D 13,150 7,375  84.00 27.88 43.63 ' 68.25 55.75 65.75 36.88  7 4.46 0.53 0.21  7 243.11 196.37 80.17  7 10,918 3,645 1,488  7 54.59 18.23 7.44  233.7 202.8 166.5 64.1 192.6 442.3 417.6 208.1  1,166 1,433 2,816 3,033 1,166 1,433 1,475 1,350  5.83 7.17 14.08 15.17 5.83 7.17 7.38 6.75  8 240.96 119.10 45.01  8 1,734° 698 264  1 2 5 6 a 11 12  n _ mean = SD SE —  m  Chinook  2 5 6 7 a 9 ID 11  n _ Mean SO SE = ia  20 21 23 24  n = mean = SD = SE =  •  7.73 10.68 a.18 6.59 6.91 9.00 9.77 7.91 B B.35 1.31 0.49  f  Plasma C a l c i t o n i n Level  ft  n = mean SO = SE f  UB Gland Calcitonin Content MRC U/gland  Total Ut Kg  8.55 8.82 9.09 6.77 7.14 5 8.07 0.94 0.47  277.6 556.1 258.9 1209.9 108.9 5 482.28 391.42 195.71  t - t e s t p r o b a b i l i t y male v s . female  pg/ml  mU/ml*  2,575 292 180 739 0  12.88 1.46 0.90 3.70 0  880  4.40  6 778° 860 385  6 3.89° 4.30 1.92  -  -  8 „ 8.67 3.49 1.32 a  4,333 5,800 36,333 18,166 10,166  21.67 29.00 181.67 90.83 50.83  5 14,960 11,721 5,860  5 74.80 58.61 29.30  a. p< 0.025  b. p< 0.001  *Plasma CT b i o l o g i c a l a c t i v i t y based on salmon CT s p e c i f i c b i o l o g i c a l a c t i v i t y o f 5000 MRC U/mg.  -175-  Discussicin  A.  Chilko Sockeye Migration Plasma The decrease in plasma electrolytes which occurred through-  out the Chilko migration may p a r t i a l l y be explained by hemodilution (note the increase in plasma percent water). Relatively high doses sterone,  deoxycorticosterone  o f  C o r t i s o l ,  corticosterone,  aldo-  and cortisone cause decreases in  plasma sodium levels in freshwater  f i s h (Henderson e_t a_l, 1970).  Therefore, i t is passible that the changes in plasma sodium throughout the migration might be related to the high adrenocorticasteraid and after  levels found in both male and female salmon during  spawning and death (Idler e_t a_l, 1959; Hane and Robertson,  1959; Robertson et a l , 1961; Schmidt and Idler,  1962; Fagerlund,  1967; Henderson et a l , 1970). The f a l l in plasma sodium concentrations in the sockeye i s consistent with a study done by Greene (1904), who found that the freezing point depression of chinook serum rose from - 0 . 7 6 2 ° C in the sea to - 0 . 6 1 2 ° C at spawning.  Fontaine and Koch (1950)  reported the freezing point depression of the serum of A t l a n t i c salmon  rose _  during the freshwater migration from -0.75 a C to  -0.66 a C 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 f a l l 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 f a l l or remain constant (Henderson E t a l , 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 degenerative 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 concentration decreases (Vinogradov, 1953; MacLeod et_ al^, 1958; Tomlinsan e_t a l , 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 freshwater.  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 i s 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 electrolyte 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 f a l l 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 f a l l in plasma protein concentration during the sockeye 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 Vanstone, 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 migration 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 f a l l 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 HC0 13.4 (Hayes et al, 1946). 3  It i s interesting to note that whereas a significant decrease in total serum calcium occurred from seawater t D 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 i s 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 i s 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 i s 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 i s 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-  l e v e l (Murexide method) of yellou and s i l v e r freshuater Anguilla anguilla uas 2.76 - D.02 mEq/1 (Chan and Chester Jones,  1968).  The fact that plasma c a l c i t o n i n changes f a l l o u different patterns in the male and female throughout the sockeye migration, makes i t unlikely that c a l c i t o n i n i s 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 i n -  crease in the female plasma CT l e v e l from seauater to 0% spauning is remarkable in view of the fact that i t occurs concurrently uith a s i g n i f i c a n t hemodilution and decreasing haematocrit.  The  cause of the increase in plasma CT levels in the female may be related to a r i s e in production of c a l c i t o n i n by the ultimobranchial gland (Table XXI, pg. 137) and/or to an increased secretion of c a l c i t o n i n throughout the migration. The increase in plasma CT levels observed in the female sockeye salmon i s supported by the recent uork of Deville and Lopez (197D).  These authors reported an increased h i s t o l o g i c a l  a c t i v i t y of the ultimobranchial gland of the A t l a n t i c salmon, Salmo salar L . , during migration and sexual maturation.  At spauning,  the ultimobranchial c e l l cytoplasm uhich uas formerly packed uith small, PAS positive granules, became clear and the UB gland hypertrophied.  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 h i s t o l o g i c a l alterations described could help to explain the plasma c a l c i t o n i n 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 f a l l 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 resorption in the breeding growth of the salmon skull. Pang (1971b)has also reported that k i l l i f i s h UB glands were more active in freshwater than seawater f i s h .  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 a l (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 a l l 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 i s 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) a l 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 and 11-ketotestosterone.  Testosterone was predominant in the  female plasma whereas 11-ketotestosterone male.  testosterone  was more abundant in the  Both male and female plasma levels of these steroids de-  creased after spawning (Schmidt and Idler, 1962). female sockeye salmon plasma concentrations of Cortisol,  In summary,  testosterone,  corticosterone (in mature and post-spawned females) and  c a l c i t o n i n , are higher than in males at a l l stages of sexual maturation. The plasma c a l c i t o n i n , plasma e l e c t r o l y t e and tissue electrolyte (especially IMa, H and Ca) changes in the migrating salmon may be most intimately related to the h i s t o l o g i c a l changes in the corpuscles of Stannius (Lopez, 1969; Heyl, 1970).  In this  regard, Heyl (1970) has observed changes in the general architecture and c e l l types of the corpuscles in the migrating and post-spawned A t l a n t i c salmon which were more closely related to the time spent in freshwater than to gonadal development.  It would be informative  to examine the simultaneous h i s t o l o g i c a l 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 h i s t o l o g i c a l a c t i v i t y observed in the spawning male A t l a n t i c salmon corpuscles of Stannius by Lopez (1969), may give some i n sight into the observation of the sex difference in plasma c a l c i t o n i n levels.  -185Tissues It i s d i f f i c u l t to assign a role to c a l c i t o n i n in calcium homeostasis in the migrating, sexually maturing salmon when so many other hormonal changes are occurring at the same time. In fact, the p i t u i t a r y , thyroid, i n t e r r e n a l , 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) a l l appear to be involved with fish migration and/or sexual maturation in some way. of osmoregulation,  starvation  exceedingly complex.  Add to this the complications  and death, and the picture becomes  Nevertheless,  an attempt has been made to  determine the tissue calcium and phosphate changes in the migrating salmon and the corresponding role of c a l c i t o n i n . This tissue study was prompted by the suggestion that c a l c i t o n i n may play a part in the skeletal changes of the breeding salmon, since calcitonin, i n h i b i t s bone resorption in mammals in vivo and in_ v i t r o .  Tchernavin (1937) has shown that the alterations  in  the salmon s k u l l are complicated and involve not only an absolute increase i n size of certain banes but also changes in shape.  A  careful study by this worker (Tchernavin, 1938a, 1938b) revealed that a l l the tooth-bearing bones of the jaw (dentary, premaxilla), freshwater  maxilla,  the palatines and the vomer grow i n size during the  migration whereas the bones forming the g i l l  the branchiostegals  and the p o s t a r b i t a l s , resorb.  covers,  The supra-  ethmoid grows longer and broader at i t s anterior end, but is resorbed at i t s 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; 1937,  1938a).  Tchernavin,  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 i t occurs during the freshuater migration uhen the salmon have ceased to feed and in the very short time period D f 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, i t 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 , i t 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 a l , 1967; Chan et_ al, 1969; Henderson e_t a l , 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 a l , 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 a l , 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 i t is only slightly or not at  -188a l l 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 freshwater 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 f a l l of plasma calcium and phosphate during the maturation process in the female sockeye.  However, from the  data i t 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; 1957; Harden-Jones, 1968;  van Oosten,  Brown and Uellings, 1969).  The phenomenon of scale resorption in migrating salmon has been reported by many workers (Hutton, 1924; Someren, 1937;  Crichton, 1935;  van  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 i s 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 d i f f i c u l t to remove (descaling in seawater salmon occurs frequently and easily). According to Yamazaki (1972) this phenomenon may be due to testosterone. Van Someren (1937), in studying the Atlantic salmon, found no correlation between blood calcium level and scale re-  -190-  sorption and suggested that the l a t t e r 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, tinued after  i . e . time of cessation of feeding.  Resorption con-  spauning and ceased only uhen the f i s h resumed normal  feeding in the  sea.  Results obtained from the sockeye scales are in substantial agreement uith previous uork on f i s h scales (van Someren, 1937; van Oosten, 1957; Moss and F r e i l i c h , 1969).  1963;  Broun and U e l l i n g s ,  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 s l i g h t l y 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 s i g n i f i c a n t r i s e in phosphate content (g PO^/lOOg ash). This may r e f l e c t 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 i s u t i l i z e d 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 i t has  been shown in growing speckled trout, Salvelinus fontinalis, that the calcium content of the skin increases as the scales became larger (Phillips et a l , 1953)„ The vertebrae, in contrast to the scales, showed a significant 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, i t i s 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  Teeth  183  114  66  18  Vertebrae  132  170  Jaw bones  600  200  -  114  981  616  Premaxillae  Gonads Total  female  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 i s 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 o f the needed calcium and phosphate.  The chicken can  mobilize 10 percent of i t s bone in one day (Simkiss, 1970).  1961; Taylor,  If the sockeye salmon could mobilize a similar portion  of i t s s k e l e t a l calcium store (Table XXX, pg. 166),  i t would be  able to supply most of the mineral required by the developing tissues. Other workers have shown in f i s h that the muscle (Chan et a l , 1967 ; Chan, 1972) and skin (van Oosten, 1957; Podoliak and Holden, 1965; Fleming, 1967) constitute important storage compartments for exchangeable calcium.  However, Simmons (1971) has pointed  out that the skin does not appear to be a major reservoir for calcium in marine f i s h .  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 g i l l s ,  fins and o r a l e p i t h e l i a 45  (see Simmons, 1971 for references).  Using Ca  , i t has been  established that calcium ion transport across the g i l l s i s mare efficient  in freshwater than in seawater f i s h and the major re-  positories studies,  for this absorbed calcium are the bone and s k i n . 45 32  using Ca  and P  Tracer  , would reveal the extent to which  these ions are absorbed from the water by the sexually maturing salmon. Since c a l c i t o n i n i n h i b i t s bone resorption in mammals.it i s curious that the mature male salmon, which showed the more extensive  scale  -194resorption 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 i s 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 a l l 3 species of  salmon, the female plasma c a l c i t o n i n levels were s i g n i f i c a n t l y ( p < • . • 0 1 ) higher than those of the male.  Since the UB gland  c a l c i t o n i n contents of the coho and chinook showed no sex  differ-  ence, the high c i r c u l a t i n g plasma CT l e v e l in the females may be explained by differences  in secretary  and/or clearance rates of  c a l c i t o n i n in the male and female salmon. s i g n i f i c a n t l y higher total.plasma and sockeye  The females also had  calciums in the chinook  (p<Q.Q5)  (p<0.01).  The higher plasma c a l c i t o n i n levels found in the chinook and coho compared to the sockeye, could be due to many factors. Since these f i s h represent 3 separate species under the same genus, Oncorhynchus, there are many morphological, physiological and biochemical variations among them. levels may r e f l e c t  different  The different plasma c a l c i t o n i n  ages, growth rates, size or distances  travelled during the freshwater migration. approximately 500 miles in freshwater miles in the other species.  The sockeye travelled  compared with less than 10  The low plasma electrolyte and c a l -  citonin levels found in the sockeye may p a r t i a l l y be explained by the high plasma percent water found in t h i s 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 c a l c i t o n i n concentrations than the female chinook or coho salmon (Table XXXIV, pg.i7f+ ), and t h i s would help to account for  - 196-  the louer plasma CT levels found in the sockeye. Thus, the fact that high female plasma c a l c i t o n i n levels have been measured in 3 species of spauning salmon suggests that c a l c i t o n i n i s related to sexual maturation, at least in the female.  The plasma CT l e v e l in the spauning female salmon i s  much higher than that reported for any mammal, except for some patients uith medullary thyroid carcinoma (Deftos and Potts,  1970;  Deftos e_t a l , 1972a) and comparable or higher than concentrations found in birds and f i s h (Copp at a l , 1972b; Kenny et al, 1972). Tashjian e_t a l (1972) also using the radioimmunoassay, confirmed these observations that f i s h have higher c i r c u l a t i n g plasma calcitonin levels than most mammalian species.  He found that coho  salmon (age 1 - 3  and fed a commercial  years) adapted to freshuater,  diet, had higher plasma CT levels than unfed coho adapted to s a l t 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 c a l c i t o n i n in calcium homeostasis in f i s h 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 f i s h are different from mammals uith respect to c a l c i t o n i n secretion.  In the sheep and p i g , hypercalcaemia causes the release  of c a l c i t o n i n (Copp, 1970b; Cooper et a l , 1971), uhereas hypocalcaemia i s 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 a l , 1964; Booke, 1964; Fleming et a l , 1964; Urist and Van de Putte, 1967; Oguri and Takada, 1967; Ldoadhead, 1968; Ldoodhead and Plack, 1968; Fontaine• et a l , 1969; Urist et a l , 1972).  The  increase in plasma calcium is associated uith a rise in v i t e l l i n , a calcium-binding phosphopratein uhich i s 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 a l , 1964; Oguri and Takada, 1966, 1967; Chan and Chester Jones, 1968; Uoodhead, 1969a; Urist et a l , 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 c l a r i f i e d .  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 a l , 1972). The results of experiments outlined in Chapter IV indicated a sex difference in plasma c a l c i t o n i n levels in the salmon and that the plasma CT l e v e l in the female increased uith sexual maturation.  These plasma CT changes did not appear t D be correlated  uith t o t a l plasma calcium or other e l e c t r o l y t e changes. chapter u i l l report the effect  This  of gonadectomy and estrogen re-  placement on plasma c a l c i t o n i n levels in sockeye salmon.  The effect  of estrogen on serum ionic and t o t a l calcium changes in immature trout uas also investigated.  The purpose of these experiments  uas to provide further insight into the i n t e r - r e l a t i o n s h i p s of calcium metabolism, c a l c i t o n i n and sexual maturation. Materials and Methods The chapter i s divided into tuo sections.  In Section A,  the effects of estrogen i n j e c t i o n on serum ionic and t o t a l calcium in sexually immature rainbou trout, Salmo g a i r d n e r i , are o u t l i n e d . As uas noted in Chapter IV, the female salmon exhibited higher plasma CT levels and t o t a l 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 c a l c i t o n i n .  Sexually immature trout uere used in these  experiments to minimize the influence of endogenous  estrogen  secretion and i t s effects on calcium metabolism. Section B outlines the effects of gonadectomy and estrogen  -199-  r B p l a c e m B n t  on plasma c a l c i t o n i n and t o t a l calcium levels of  adult sockeye. A.  Effect of Estrogen on Serum Ionic and Total Calcium in Immature Rainbow Trout Two groups of ID f i s h each  u e r e  fibreglass tanks of running water.  put into separate 50 gallon  ThB f i s h were then acclimated  to laboratory conditions for one week.  They were fed trout chow  p e l l e t s d a i l y , except on the day of i n j s c t i o n . The control group  r e c B i v B d  0.1 ml cottonseed o i l per f i s h .  The experimental group received e s t r a d i o l cypionate ( e s t r a d i o l - l T ^ in the form of the cyclopentyl propionate e s t s r ; Upjohn Pharmaceutical Co.)  at a dose of 0.1 mg e s t r a d i o l / 0.1 ml cottonseed o i l per f i s h .  Injections were performed i n t r a p e r i t o n e a l l y once per week for 6 weeks, on f i s h which were under l i g h t anesthetic (2-Phenoxyethanol, 1.0. ml/gal; Eastman Kodak C o . ) .  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 . 5 D C . Blood samples were collected from the caudal vein of unanesthetized f i s h within 2 minutes of capture.  To ensure that the  blood was well oxygenated, the g i l l s were perfused with aerated water during the sampling procedure. and  Other methods of c o l l e c t i o n  analysis of samples were outlined in General Materials and  Methods. Six r i b 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 - 1.0 D C, 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 f i s h 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 f i s h uere maintained on a natural photoperiod and seasonal water  temperatures.  In the period from July 15 - 29th, a group of these f i s h were gonadectomized by Jack McBride, Fisheries Research Board of Canada, using the technique he developed (McBride et_ a_l, 1963).  A  second group uas l e f t 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 f i s h uere not fed during the experiment.  In nature, these sockeye spaun from late September u n t i l the end of November (McBride e_t _al, 1963). It should be painted out that 4 of the normal males (fish #N8 - N i l ) uere sockeye "jacks" but as shoun by t h e i r ganad-somatic sexually mature.  indices, they uere quite  These jacks mature in their 3rd  year uhereas the sockeye normally mature in t h e i r 4th or 5th year of l i f e (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 o i l once per ueek for 7 ueeks.  They uere fed daily throughout the experi-  mental period.  Five females uere s a c r i f i c e d on  February 9, 1972, six months follouing the gonadectomy operation. (3)  Gonadectomized estrogen-injected sockeye - 3 males, 3 females These f i s h received intramuscular injections of e s t r a d i o l 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 i n j e c t i o n experiment began October 13, 1971, 2 - 3 month follouing gonadectomy and the f i s h uere s a c r i f i c e d on December 1, 1971, one ueek follouing the f i n a l i n j e c t i o n .  A l l i n j e c t i o n and  blood sampling operations uere performed on salmon l i g h t l y anesthetized in 2-Phenoxyethanol.  Water temperatures throughout the  injection experiment ranged from 6 - 1 2 ° C . Procedures for the c o l l e c t i o n and analysis of samples uere outlined in General Materials and Methods. collected for c a l c i t o n i n measurement.  Plasma samples uere  Ribs uere dissected from  each salmon i n the December 1, 1971 group far analysis of calcium and phosphate content. It should be noted that approximately the same dosage of  -2D2-  e s t r a d i a l 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 e l e c t r o l y t e  for  measurements  the 2 groups of trout are presented in Table XXXVI, pg. 2D3.  The t o t a l ueights and fork lengths of the estrogen-treated uere s l i g h t l y higher than for the controls.  group  The 2 groups of female  trout had s i g n i f i c a n t l y higher GSI values than the males but both sexes uere very immature. Figure 34, pg. 204, i l l u s t r a t e s  the serum ionic and t o t a l  calcium and plasma inorganic phosphorus levels for the immature trout.  The male and female e l e c t r o l y t e measurements uere combined  since no sex differences uere observed.  As can be seen from the  data, estrogen i n j e c t i o n s i g n i f i c a n t l y elevated plasma inorganic phosphorus (p < • . • • ! ) and serum t o t a l calcium (p<Q.DDl).  Despite  a 9-fold increase in serum t o t a l calcium, the serum ionic calcium l e v e l did not change. . Table XXXVII, pg. 205, presents the percent ash/dry ueight, and calcium and phosphate contents of the r i b bones of the control and estrogen-injected  trout.  There uas no s i g n i f i c a n t  betueen the 2 groups i n any of the 3 parameters.  difference  Table XXXVI. Group  Physical Measurements, Plasma and Serum Electrolytes in Control and Estrogen-Treated Trout  Sex  Total Weight g  Fork Length cm  GSI  Hct  Plasma Inorganic Phosphorus mEq/l  Serum Ionic Calcium mEq/l  Total Calcium mEq/l  % Ionic Calcium  m  n= mean= SD= SE=  6 136 10.57 4.73  6 24.1 0.33 0.17  6 0.08 0.06 0.03  6 22 5.93 .2.65  5 5.52 0.82 0.41  6 2.74 0.17 0.07  6 4.88 0.43 0.19  6 56.23 1.97 0.88  f  n= rnean= SD= SE=  4 123 8.46 4.88  4 < 24.3 0.69 0.40  4 0.22 0.02 0.01  4 24 5.12 2.95  2 5.90 0.55 0.55  4 2.65 0.15 0.08  4 4.73 0.30 0.17  4 56.26 3.95 2.28  m&f n= Total mean= Controls SD= SE=  10 131 11.67 3.89  10 10 24.2 0.14 0.52 0.08 0.03 0.17  10 23 5.67 1.88  7 5.63 0.77 0.31  10 2.70 0.17 0.05  10 . 4.82 0.39 0.13  10 56.24 2.93 0.98  Experimental Estrogen  n= mean= SD= SE=  6 149 13.95 6.24  •6 24.0 0.94 0.42  6 0.12 0.02 0.01  6 24 7.27 3.26  5 11.68 0.71 '0.36  6 2.85 0.17 0.07  6 36.63 4.01 1.79  6 7.90 1.14 0.51  n= mean= SD= SE= m&f Totai n= Estrogen mean= SD SE  4 150 2.74 1.58 10 149 10.96 3.65  4 24.6 0.54 0.31 10 24.2 0.85 0.28  4 0.22 0.02 0.01 10 0.16 0.06 0.02  4 25 2.12 1.22 10 24 5.83 1.94  4 11.83 0.39 0.23  4 2.82 0.13 0.07 10 2.84 0.15 0.04  4 35.38 1.35 0.78  4 7.97 0.29 0.17  10 36.13° 3.28 1.09  10 7.92° 0.90 0.30  Control Cottonseed  m  f  a  t-test probability:  9 11.74° 0.60 0.21  total controls vs. total estrogen  a. p< 0.005  b. p< 0.001  -204-  40.On  Estrogen  Ionic Ca £  30.0-  • ^  2  Serum Total Plasma Phosphorus  o UJ  20.0-  o E  |  10.0-  Cottonseed  0.0 n •  F i g u r e 34.  10 10 7  I  10 10 9  Serum i o n i c and t o t a l c a l c i u m and plasma i n o r g a n i c phosphorus l e v e l s i n immature trout - e f f e c t of estrogen.  -205-  Table XXXVII.  Bone Measurements in Control and EstrogenTreated Trout.  % Ash Group  n  9 ™  Dry Ut mean  -  gCa  k  lOOg Ash SE  Mean  i  lOOg Ash SE  mean  - SE  Control Cottonseed  a  61.53  0.88 17.91  0.15 34.29  0.52  Experimental 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 c a l c i t o n i n and plasma e l e c t r o l y t e  levels for the 3 groups of salmon are summarized i n Table XXXVIII, pg. 2D7. Plasma calcium levels are shown in Figure 35, pg. 208, and the corresponding individual plasma CT levels are i l l u s t r a t e d i n Figure 36, pg. 209. In the intact control group, the females had s i g n i f i c a n t l y higher plasma calcium (p<0.05) and magnesium levels the males.  (p<0.05) than  These.electrolyte levels uere s l i g h t l y lower i n the  gonadectomized controls than i n the intact controls.  However,  the plasma calcium (p <0.001), inorganic phosphorus (p< 0.001) and magnesium (p< 0.001) i n the gonadectomized estrogen group were s i g n i f i c a n t l y elevated over corresponding . levels for the gonadectomized controls (sexes combined).  Plasma sodium and potassium  showed l i t t l e variation among the 3 groups. The intact control sockeye exhibited the same sex difference in plasma c a l c i t o n i n levels noted i n the migrating Chilko sockeye salmon in Chapter IV. were undetectable.  In the normal intact males, plasma CT levels  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 contents of the r i b bones for the 3 groups of sockeye are presented in Table XXXIX, pg. 210.  The intact control male sockeye had  s i g n i f i c a n t l y higher percent ash/dry weight (p<0.01) and lower calcium (p<0.05) and phosphate contents (p<0.05) than the intact control females.  The other groups displayed no large differences  the parameters measured.  in any of  Table X X X V I I I .  P h y s i c a l P a r a m e t e r s , Plasma C a l c i t o n i n and Plasma E l e c t r o l y t e s a f I n t a c t GX C o n t r o l and GX E s t r o g e n Sockeye  Group  Total Ueight g  Sex  Intact Control  m  n= mean= SD SE  5 757 197.90 98.95  f  n= mean= SD= SE=  6 1793 418.50 187.16  December 1,1971  Gonadectomized Control  Gonadectomized Estroqen  . Hct  5 4.02 0.70 0.35 6 1S.4S 2.63 1.18  p C  PD^  undetect5 able 33 5.52 2.76  5 4.55 0.33 0.16  5 5.36 ' 0.29 0.14  5 1.43 0.12 0.05  6 36 3.47 1.55  6 5.78 1.07 .0.48 '  6 5.23 0.78 0.35  6 1.7Q 0.23 0.10  4.70  4.82  4 4.77 0.22 0.13  n=l  f  n= mean= SD= SE=  4 1594 444.19 256.45  -  4 26 2.24 1.29  n= mean= SD= SE=  5 1036 130.13 65.07  _  5 20 •3.72 1.86  n= mean= SD= SE  3 1367 455.22 321.89  317 3.09 2.19  n= mean= SD= SE=  3 1425 73.60 52.04  3 19 2.45 1.73  f  m  December 1, 1971 f  1475  27  t - t e s t p r o b a b i l i t y male v s . f e m a l e  Plasma E l e c t r o l y t e s mEq/l Ca  m  December 1,1971  F e b r u a r y 9, 1971  C  GSI  Plasma Calcitonin pg/ml'  Control,  a. b. c.  6 6603 2075.65 928.26  400 400  400  400  400  p< 0.05 p<0.01 p< 0.001  Mg  IMa 5 147 5.49 2.75  5 2.48 0.47 0.24  6 150 2.87 1.28  6 2.02 0.75 0.33  1.56  153  1.90  4 4.46 0.10 0.05  3 1.57 0.08 0.05  3 152 2.05 1.45  3 2.27 0.17 0.12  5 4.60 0.40 o.2r  5 3.68 0.19 0.09  5 1.26 0.30 D.15  5 146 8.17 4.09  5 2.64 0.15 0.07-  3 23.99 2.16 1.53  3 10.05 0.43 0.30  3 3.78 D.21 0.14  3 144 1.41 1.0D  3 2.60 0.14 0.10  3 30.21 6.14 4.34  3 12.32 1.81 1.28  3 4.91 0.27 ' 0.19  3 141 1.25 0.88  3 2.37 0.31 0.22  a  a  b b  -208-  Gonadectomized + Cottonseed Oil + Estrogen  40.0-  30.0-  cr  LU  E  20.0-  E  2  o  o O o E CO o  10.0-  0_  0.0-  7 8 9 10II  123456  CMICF3 56  I234SS678  S9I0I  Fish Number  Figure 3 5 . Total plasma calcium levels in sockeye effect of gonadectomy and estrogen replacement.  Intact Females 9331  7764  7964  4606 •  563i  Female  Male  28*3 Gonadectomized + Cottonseed Oil  Intact Males  t  1  1  (Controls)  + Estradiol \[  \  undetectable  7 9 II  13 5 Fish  Figure 36.  CMI CF 5 6 I 3 5 S678 S9 II Number  Plasma c a l c i t o n i n levels in sockeye effect of gonadectomy and estrogen replacement.  Table XXXIX. Bone Measurements in Intact Control, GX Control and GX Estrogen Sockeye  % Ash Group  Sex  Intact . ,  Dry Wt  gPO,  gCa  lOOg Ash  lOOg Ash  m  n mean SD SE  = = = =  5 68.00 4.57 2.28  5 16.64 0.87 0.43  f  n mean SD SE  = = = =  5 . 59.83° 1.09 0.54  5 , 18.29 1.02 0.51  5 • 37.97 1.39 0.69  1 60.25  1 17.54  1 37.70  L O n t r o 1  n =  5 35.10 1.70 0.85 3  Gonadectomized „„^ ,  m  December 1, 1971  f  n mean SD SE  = = = =  4 60.51 1.09 0.63  4 18..97 1.61 0.93  4 37.19 1.04 0.60  Gonadectomized . „„ .  m  n mean SD SE  = = = =  3 60.85 0.90 0.64  .3 17.89 0.04 0.03  3 ' 37.01 0.39 0.27  f  n mean SD SE  = = = =  3 "59.92 1.39 0.98  3 17.44 0.68 0.48  3 36.42 1.03 0.73  n  c E s t r o o  e n  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 bicarbonate, phosphate and citrate  3.  ionic calcium  The complexed and ionic calcium fractions constitute the ultrafiltrable or diffusible fraction and i t 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 goldfish, 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 D f estrogen (Premarin 100. microg/100 g per day for 6 days) despite a significant (p<0.01) rise in total calcium.  Recently, Urist  et a l (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 i s interesting to note that although estrogen injection also causes hypercalcaemia in birds (Riddle and Dotti, 1936; Urist et a l , 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 conspicuous (Day and Follis, 1941; Gardner and Pfeiffer, 1943; Manunta et_ a_l, 1957; Young et a l , 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 contention 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 gonadectomized 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 l i p i d 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 l i t t l e 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 difference  intact  c o n t r o l group of sockeye, there uas no sex  i n the plasma i n o r g a n i c phosphorus, sodium or potassium  l e v e l s although the female both s i g n i f i c a n t l y In  higher  plasma calciums and magnesiums uere ( p < 0 . 0 5 ) than the  males.  comparing the plasma e l e c t r o l y t e s  of the  females and the gonadectomized c o n t r o l females,  it  intact is  interesting  to note t h a t removal of the gonads, and thus removal of source of e s t r o g e n , r e s u l t e d i n s i g n i f i c a n t calcium ( p < 0 . 0 5 ) , (p<0.05).  control  the  d e c l i n e s i n plasma  i n o r g a n i c phosphorus ( p < 0 . 0 1 ) and magnesium  T h i s d e c l i n e uas a s s o c i a t e d u i t h a decrease i n plasma  CT l e v e l s from a mean of 6603 - 928 pg/ml to l e s s than 400 p g / m l . It  i s not knoun uhether  the  decrease i n c i r c u l a t i n g  CT i n the gonadectomized sockeye i s r e l a t e d s e c r e t o r y r a t e or an i n c r e a s e d metabolic t h a t estrogen replacement  dramatically  calciums but d i d not r e s t o r e the f a c t o r s governing the quite  complex.  to a r e d u c t i o n  destruction.  The  fact  indicates  that  l e v e l of c a l c i t o n i n may be  R e s i i l t s i n the male sockeye are even more com-  p l i c a t e d s i n c e they  indicate  may r i s e on gonadectomy.  t h a t the plasma c a l c i t o n i n  (Donaldson and F a g e r l u n d ,  Castration e f f e c t i v e l y  al,  1968;  effect  on plasma c a l c i u m  Sorensen and H i n d b e r g ,  1971).  l o u e r s plasma c a l c i u m l e v e l s i n the  Xenopus but the same o p e r a t i o n serum calcium (Gardner  sockeye  1970).  Gonadectomy appears to have l i t t l e (Rice et  level  Plasma C o r t i s o l has a l s o been shoun to  decrease f o l l o u i n g gonadectomy of male and female  levels in rats  in  i n c r e a s e d t o t a l plasma  the plasma CT l e v e l s ,  circulating  l e v e l of plasma  toad  i n dogs produces a marked r i s e  and P f e i f f e r ,  1943).  The only r e p o r t  in 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 significant 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). indicate  Data from the present study  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 F o l l i s , 1941; Gardner and Pfeiffer, 1943; Urist et a l , 1948; Budy et al, 1952; Linquist et a l , I960; Lafferty et a l , 1964; Young et a l , 1968; Skosey, 1970; Sorensen and Hindberg, 1971).  In birds (see Simkiss, 1961, 1967) a n d  -216-  mice The  (Urist  et  a_l,  actions  of  androgens  documented  although  synergistically. depends  hormone(s)  used,  This the  tents  the  ectomized increase  in  the  detect  rib  the  to  out  these  environment  levels  or  (1963)  killifish,  e_t  the  only  X-ray  no  detectable  bone  al,  as  measured  by  the  l/on  rise  uere  mineral. or  (1957) or  absorbed  on  Hossa  elevated  fish  elevated  injection total  histology technique.  or  Fleming need is  serum the  from  the  been  calcium found  no  and  mature but  calcium  Furthermore,  to  be  Clark  calcium  bone  be  have  serum but  al  possible  also  into  et  enough  bone  deposition.  gonad-  inorganic  gills  goldfish  con-  dramatic  It  the  estrogen  effect  the  a  could  1971).  bone  or  sensitive  Simmons,  observed  no  calcium  via  factors.  phosphate  change.  not  act  other  plasma  of  well  the  had  trout  Calcium  that e s t r o g e n  bone  not  preovulatory of  and  in  so  bone  many  hypercalcaemia.  study  Df  on  produced  amount  small  1964;  and  immature  a  not  animal,  calcium  estrogen  formation.  androgens  estrogen  a  evidence  on  that  the  and  tissues  kansae,  effect  course  the  are  and  the  of  did  effects  reported  age  bone  steroids  content  this  Bailey  Fundulus  or  either  estrogen-treated  histological Fleming  on  variable. in  soft  sex  time  although  in in  (Fleming  the  significant  used  the  Reports and  a  metabolism  indicated  mineral  changes  from  and  neu  estrogens  species,  calcium  that  obtain  methods  mobilized  feu  of  serum  bone  of  ueight,  banes  total  cases,  dosage  ash/dry  pointed  mobilized that  the  stimulates  skeletal  some  sDckeye. T h u s ,  phosphorus, (1964)  on  effects  on  the  estrogen  investigation  percent Df  in  The  metabolism  on  1950)  female had content  Uoodhead  -2171 9 6 9 b ) has r e p o r t e d  11-fi  , 3-benzoate  into  female  resulted  that  (I.D  lesser  mg/kg,  spatted  in a significant  c a l c i u m from a c o n t r o l mEq/1.  intramuscular  4 injections  dogfish,  10.3 - 0.5(SD)  is relevant  to  the  hypothesis  on bone a r e d i s t i n c t l y In  contrast  to  that sexual maturation  that  the  that  1964).  the of  Moreover,  injection  anguilla  L.  of  above r e s u l t s , the  i z a t i o n of  the  modification diffraction phase of  of  extract  surfaces,  into  a c c o m p a n i e d by  in  showed  Anguilla hyper-  also noticed a with  substance, without  of  significantly  histological  A n a l y s i s of  than the  response of  the  t h e bone by X - r a y  from the  crystalline  estrogens.  the  may d e p e n d on w h e t h e r  acellular.  Fontaine  i n c r e a s e d o s t e o l y s i s and d e m i n e r a l -  t h e bone m i n e r a l r a t h e r  t o be s p e c i e s d i f f e r e n c e s  hormones,  (1971)  female  These a u t h o r s  organic matrix.  production  have shown  e_t al_, 1 9 6 2 ;  T h e s e s k e l e t a l c h a n g e s were t h o u g h t t o be c a u s e d ,  and t h i s  findings  some w o r k e r s  r e v e a l e d t h a t t h e m i n e r a l was l o s t  the gonadotrophic  0.6(SD)  e s t r o g e n on serum and  o s t e o c l a s t s concomitant  intercellular the  11.4 -  These  L o p e z and M a r t e l l y - B a g o t  c a l c a e m i a and h y p e r p h o s p h a t e m i a .  enlarged resorption  to  plasma  discussion since  (Boetius  produced s e x u a l maturation  of  total  f e m a l e e e l , i n d u c e d by  carp p i t u i t a r y  marked p r o l i f e r a t i o n  canicula,  skeleton. of  days)  separate.  was a c c o m p a n i e d by bone d e f o r m a t i o n e_t _1,  the  effects  estradiol  on a l t e r n a t e  increase in  elasmobranchs possess a c a r t i l a g i n o u s support  of  Scyliorhinus  (p<D.DDl)  l e v e l of  This observation  injection  apatite.  in part,  Thus, there fish  bone s t r u c t u r e  amorphorous  appears  bone t o is  by  estrogens  cellular  or  -218-  Only tuo reports an the effects of androgens on calcium metabolism in f i s h appear in the l i t e r a t u r e .  A single injection  of 1.0 mg of testosterone proprionate into goldfish, had no effect  on t o t a l serum calcium (Bailey, 1957).  Recently, Peterson  and Shehadeh (1971) observed a dramatic increase in t o t a l plasma calcium of the male and female mullet, Mugil cephalus L., follouing intraperitoneal i n j e c t i o n of 25.0 mg c r y s t a l l i n e methyltestosterone month).  (hormone injected on alternate  days for D n e  Injection of 5.0 mg of p a r t i a l l y p u r i f i e d salmon gonad-  o t r o p h s also elevated the t o t a l plasma calcium l e v e l 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 a l , 1963; van Overbeeke and McBride, 1971; McBride and van Overbeeke, 1971).  These authors have demonstrated that gonad-  ectomy of mature salmon not only prolongs t h e i r l i f e span beyond the time at uhich they normally uould have spauned and died, but also leads to the arrest of the external secondary sexual istics  character-  such as the snout and teeth development and red skin colour.  Plate 11, pg. 219, shaus the 5 gonadectomized control female sockeye s a c r i f i c e d February 9, 1972.  Note the sea-green backs  and s i l v e r sides uhich give these f i s h 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 sockeye tuice ueekly for a period of 7 ueeks.  into gonadectomized 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 o f a n d r o g e n t r e a t m e n t , a l l s h o w e d t h e r e d s p a w n i n g  colouration  as w e l l as hooked s n o u t s and p r e m a x i l l a r y  Thus t h e a n d r o g e n s d e f i n i t e l y p l a y  teeth.  a r o l e i n s k e l e t a l metabolism  i n the salmon. I n summary, i n t r o u t influence  total  and s a l m o n , e s t r o g e n a p p e a r s t o  calcium without influencing  hard t i s s u e calcium  content.  Androgens  i o n i c calcium  appear t o a f f e c t s k e l e t a l  d e v e l o p m e n t b u t h a v e m i n i m a l e f f e c t s on p l a s m a c a l c i u m . r o l e o f c a l c i t o n i n i n t h e a b o v e p r o c e s s e s r e m a i n s t o be The  f a c t o r ( s ) governing the secretory  and salmon a r e under  investigation.  or  rate  The elucidated  of c a l c i t o n i n i n trout  -221-  SUMMARY  In the General Introduction, i t was stated that the objective of this thesis uas to investigate  calcium metabolism in fish  and the passible physiological role of c a l c i t o n i n i n this process. A b r i e f summary of the thesis findings appears belou. Measurement of the ultimobranchial gland c a l c i t o n i n content Df trout and salmon under a variety of conditions displayed great v a r i a t i o n .  The UB gland CT contents found in the present  study are among the highest reported for louer vertebrates and confirm the o r i g i n a l observation of Copp in 19G7 that the f i s h ultimobranchial gland i s a r i c h source of c a l c i t o n i n .  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 c a l c i t o n i n i n f i n g e r l i n g trout may indicate a relationship betueen c a l c i t o n i n and grouth. The b i o l o g i c a l h a l f - l i f e for salmon c a l c i t o n i n (SCT) uas measured in cannulated trout and salmon.  The h a l f - l i f e of SCT  uas 27.6 minutes in trout and 48.0 minutes in salmon. rather slou disappearance  This i s a  compared to the h a l f - l i f e of SCT in  mammals. Salmon c a l c i t o n i n i n j e c t i o n had no effect levels in f i n g e r l i n g or adult rainbou t r o u t . ineffective  on plasma calcium  SCT infusion uas also  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 s i g n i f i c a n t l y altered by SCT infusion into these salmon. Results from cannulated trout and salmon indicated that these fish can regulate plasma calcium and phosphate very e f f i c iently.  Data from estrogen-injected  trout and migrating salmon,  showed that while t o t a l plasma calcium changed dramatically, the ionic calcium l e v e l remained remarkably constant and w e l l - c o n t r o l l e d . Estrogen i n j e c t i o n , in addition to causing hypercalcaemia and hyperphosphatemia, s i g n i f i c a n t l y elevated plasma magnesium levels in the salmons Evidence of the hormonal status of c a l c i t o n i n in fish was obtained when c a l c i t o n i n was detected in the plasma of salmon using the salmon c a l c i t o n i n radioimmunoassay.  As i s the case in  mammals, c a l c i t o n i n i s continuously secreted under basal conditions. The c i r c u l a t i n g l e v e l 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 c i r c u l a t i n g plasma c a l c i t o n i n  levels (females higher than males) was found i n three species of adult salmon. difference  This i s one of the f i r s t  reports indicating a sex  in the c i r c u l a t i n g l e v e l of plasma c a l c i t o n i n .  The higher  c i r c u l a t i n g l e v e l 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 i s not  known but i t i s c l e a r l y not related to i o n i c calcium l e v e l s .  -223-  Plasma c a l c i t o n i n significantly  l e v e l s o f female salmon  increased  during m i g r a t i o n from sea to f r e s h u a t e r .  These  plasma CT l e v e l s reached maximum values j u s t p r i o r t o spawning, a f t e r which they f e l l o f f p r e c i p i t o u s l y . the to  Plasma CT l e v e l s i n  male decreased from sea to freshwater and r e t u r n e d almost t h e i r o r i g i n a l l e v e l s at spawning.  The plasma  calcitonin  changes during the m i g r a t i o n do not appear to be r e l a t e d to any o f the  plasma or t i s s u e calcium and phosphate a l t e r a t i o n s .  The i n -  crease i n plasma CT during s e x u a l m a t u r a t i o n i n the m i g r a t i n g female sockeye and the decrease a f t e r spawning, p a r a l l e l s the changes seen i n the gonad-somatic index.  The high l e v e l s o f plasma  CT i n the female decreased f o l l o w i n g removal o f the gonads. Estrogen replacement d i d not r e s t o r e the plasma c a l c i t o n i n i n the gonadectomized females.  levels  These o b s e r v a t i o n s have l e d to the  suggestion t h a t c a l c i t o n i n may be i n v o l v e d i n s e x u a l m a t u r a t i o n i n some way although i t i s not caused by high estrogen l e v e l s .  This  suggestion i s not new s i n c e Lewis e t a_l (1971) have r e c e n t l y  pro-  posed t h a t c a l c i t o n i n may play a p h y s i o l o g i c a l r o l e i n pregnancy and l a c t a t i o n i n r a t s by p r o t e c t i n g the s k e l e t o n a g a i n s t the o s t e o l y t i c a c t i o n o f p a r a t h y r o i d hormone. Many o f the q u e s t i o n s posed i n the G e n e r a l I n t r o d u c t i o n been answered through the experiments o u t l i n e d i n t h i s t h e s i s .  have How-  ever, new d i r e c t i o n s o f r e s e a r c h i n the i n v e s t i g a t i o n o f calcium metabolism and c a l c i t o n i n i n f i s h  have been r e v e a l e d .  These pro-  cesses appear t o be q u i t e d i f f e r e n t i n f i s h when compared  to mammals,  i n d i c a t i n g t h a t the f u n c t i o n o f c a l c i t o n i n during e v o l u t i o n has changed.  -224-  BIBLIOGRAPHY  Aldred, J . P . , Kleszynski, R.R. and Bastian, J . U . Effects of acute administration of porcine and salmon c a l c i t o n i n on urine electrolyte excretion in r a t s . Proc. Sac. e x p t l . B i o l . Med. 134:1175-1180 (1970). Alexander, E . Inorganic phosphorus methods manual. Hasp., V a n e , B.C. (1968).  Vane. Gen.  Arnaud, C D . , L i t t l e d i k e , T . and Tsaa, H.S. 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