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The effect of methallibure and a constant 12 hours light : 12 hours dark photoperiod on the gonadal maturation… Flynn, Michael Bernard 1973

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THE EFFECT OF METHALLIBURE AND A CONSTANT 12 HOURS LIGHT : 12 HOURS DARK PHOTOPERIOD ON THE GONADAL MATURATION OF PINK SALMON (ONCORHYNCHUS GORBUSCHA) by Michael Bernard Flynn B.Sc, University of British Columbia, 1971 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in the Department of Zoology We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA July, 1973 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f the requirements f o r an advanced degree a t 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 c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y purposes may be g r a n t e d by the Head of my Department or by h i s r e p r e s e n t a t i v e s . I t i s understood t h a t c o p y i n g 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 g a i n s h a l l not be allowed without my w r i t t e n p e r m i s s i o n . Department o f The U n i v e r s i t y o f B r i t i s h Columbia Vancouver 8, Canada i ABSTRACT This study was undertaken to t r y to delay gonadal maturation of pink salmon f o r one year beyond t h e i r normal two year l i f e cycle. This would allow these f i s h to spawn i n years of low or nonexistent escapement and p o s s i b l y increase these "poor" year populations. Three experiments were conducted to investigate the e f f i c a c y of the antigonadotropic drug, methallibure, i n i n h i b i t i n g gonadal maturation i n pink salmon. Gonadoso-matic index, oocyte diameter, and stages of c e l l maturation i n the t e s t i s and oocyte maturation i n the ovary were measured. The f i r s t or p i l o t experiment involved a range of doses of methal-l i b u r e (0.10 mg., 0.32 mg., and 1.0 mg./gm./2wks.) to determine the optimal dose.for subsequent experiments. A l l doses had only a s l i g h t slowing e f f e c t on maturation. This r e s u l t and possible undesirable e f f e c t s of higher doses prompted the dec i s i o n to use the 0.10 mg./gm. dose for subsequent experiments. The second or long-term experiment investigated the ef f e c t s of methallibure and a constant 12 hours light:12 hours dark photoperiod on gonadal maturation of males and females for a period of ten months. Methallibure completely i n h i b i t e d t e s t i c u l a r maturation by preventing the transformation of primary into secondary spermatogonia. Ovarian matura-t i o n , however, was only slowed. The treated ovaries possessed oocytes i n the o i l globule stage while control ovaries had oocytes i n the secondary yolk globule stage. Methallibure had an a n t i t h y r o i d a l e f f e c t under natural photoperiod but not under constant 12L:12D photoperiod or at a i i high dose (1.0 mg./gm). Stress from kidney disease may have been operative i n this e f f e c t . Methallibure also slowed the rate of increase i n body weight. The constant 12L:12D photoperiod slowed gonadal matura-tio n i n both males and females. It i s suggested that a s p e c i f i c day-length and an endogenous rhythm stimulate the i n i t i a t i o n , maintenance, and termination of gonadal maturation and that the seasonal daylength fl u c t u a t i o n s function as a synchronizer. The difference i n e f f e c t of methallibure on males and females may be due. to treatment beginning p r i o r to the s t a r t of t e s t i c u l a r maturation but a f t e r the s t a r t of v i t e l -logenesis. To investigate t h i s p o s s i b i l i t y , methallibure treatment was begun at successive i n t e r v a l s p r i o r to the s t a r t of v i t e l l o g e n e s i s i n the t h i r d or sequential experiment. This treatment had no e f f e c t on ovarian maturation which suggests that the females are less s e n s i t i v e to methal-l i b u r e than are the males. Treatment with a higher dose started early i n j u v e n i l e l i f e may i n h i b i t ovarian maturation. From t h i s study, only the males could be delayed and, therefore, p o s s i b l y spawn i n "poor" years. However, Funk and Donaldson (1972) were able to achieve the same goal by maturing males i n the year of hatching, thus making a three year program i m p r a c t i c a l . The value of a long program would be the delay of ovarian maturation since Funk et al_. . (1973) were unable to advance maturation of females by one year. i i i TABLE OF CONTENTS Page ABSTRACT i LIST OF TABLES i v LIST OF FIGURES v ACKNOWLEDGEMENTS . i x INTRODUCTION 1 MATERIALS AND METHODS 6 RESULTS 18 DISCUSSION 45 SUMMARY 61 BIBLIOGRAPHY 63 LIST OF TABLES The mean gonadosomatic index (GSI) values f o r females of the p i l o t experiment The mean oocyte diameters of the p i l o t experiment The mean percentages of oocytes i n the stages of oocyte maturation of the p i l o t experiment The mean gonadosomatic index (GSI) values for males of the long-term experiment The mean gonadosomatic index (GSI) values f o r females of the long-term experiment The percentages of oocytes i n the stages of oocyte maturation and the percentage of oocytes that are a t r e t i c i n the long-term experiment. The mean oocyte diameters of the long-term experiment The mean body weights of pooled males and females of the long-term experiment The mean thyroid f o l l i c l e e p i t h e l i a l heights of the long-term experiment and the high dose group (1.0 mg/gm.) sampled on June 14/72 The mean percentages of oocytes i n the stages of oocyte maturation of the sequential experiment V LIST OF FIGURES Figure Page 1 The hormone and s t e r o i d pathways of the gonad and thyroid that are influenced by methallibure ... 4 2 Design of the sequential experiment showing the period of methallibure treatment from the s t a r t i n g date to the f i n a l sampling date of Oct. 29/72 f o r each of the four groups ... 10 3 Ovary approximately three months p r i o r to the s t a r t of v i t e l l o g e n e s i s showing oocytes i n the early perinucleolus stage (x60) ... 15 4 Ovary at the s t a r t of v i t e l l o g e n e s i s showing oocytes i n the l a t e perinucleolus stage (uni-form cytoplasm) and early yolk v e s i c l e stage (yolk v e s i c l e s i n the peripheral cytoplasm) (x60) ... 15 5 Oocytes i n the late yolk v e s i c l e stage (x60) ... 15 6 The large central oocyte i s i n the o i l globule stage which i s characterized by large v e s i c l e s surrounding the nucleus (x60) ... 15 7 Oocyte i n the primary yolk globule stage (x48) ... 15 8 Oocyte i n the secondary yolk globule stage (xl9) ... 15 9 T e s t i s approximately s i x months p r i o r to the s t a r t of t e s t i c u l a r maturation (x600) ... 17 10 Test i s at the beginning of maturation exhibi-t i n g secondary spermatogonia (x600) ... 17 11 Testis approaching f i n a l maturity and e x h i b i -t i n g the following se r i e s of c e l l stages: primary spermatocyte (1°SPCY), secondary sper-matocytes (2°SPCY)., spermatid (SPTD), and spermatozoa (SPZ) (x600) ... 17 12 Test i s showing secondary spermatogonia from the constant 12L:12D photoperiod control group of the long-term experiment on June 14 (x600) ... 21 L i s t of Figures (cont 1d) Figure 13 Testi s showing primary spermatogonia from the constant 12L:12D photoperiod treatment group of the same sampling as above (x600) 14 Zero control t e s t i s of the long-term experiment, sampled on November 8/71, showing primary spermatogonia (x600) 15 Te s t i s showing spermatozoa (arrow) from the constant 12L:12D photoperiod control group of the long-term experiment from the August 30 sampling (x600) 16 Testi s showing primary spermatogonia from the constant 12L:12D photoperiod. treatment group at the same sampling as above (x600) 17 Oocytes i n the o i l globule stage from the con-stant 12L:12D photoperiod treatment group of the long-term experiment from the August 30 sampling (xl9) 18 Oocytes i n the secondary yolk globule stage from the constant 12L:12D photoperiod control group from the same samplings as above (xl9) 19 Ovary with oocytes i n the ea r l y perinucleolus stage from the s t a r t of the June 29 group of the sequential experiment (x60) 20 Oocytes i n the late yolk v e s i c l e stage from methallibure treated f i s h of the June 29 group at the end of the sequential experiment (October 29) (x60) 21 Oocytes i n the late yolk v e s i c l e stage from control f i s h of the sequential experiment sampled on October 29 (x60) 22 The mean gonadosomatic index (GSI) values f o r females of the p i l o t experiment (the data are l i s t e d i n Table I) v i i L i s t of Figures (cont'd) Figure Page 23 The mean oocyte diameters of the p i l o t experi-ment i n millimeters (The values are l i s t e d i n Table II) ... 24 24 Histograms of the mean percentages of oocytes i n the stages of oocyte maturation of the p i l o t experiment (the data are presented i n Table III) ... 25 25 The mean gonadosomatic index (GSI) values for males of the long-term experiment (the data are shown i n Table IV) ... 26 26 The mean gonadosomatic index (GSI) values for females of the long-term experiment (the data are l i s t e d i n Table V) . ... 27 27 Histograms of the presence and absence of the c e l l stages of t e s t i c u l a r maturation of the long-term experiment ... 28 28 Histograms of the mean percentages of oocytes i n the stages of oocyte maturation of the long-term experiment (the values are l i s t e d i n Table VI) ... 29 29 The mean oocyte diameters of the long-term experiment, i n millimeters (the values are presented i n Table VII) ... 30 30 Histograms of the mean percentage of oocytes that are a t r e t i c i n the long-term experiment (the data are l i s t e d i n Table VI) ... 31 31 The mean body weights of pooled males and females of the long-term experiment (the data are l i s t e d i n Table VIII) ... 32 32 The mean thyroid f o l l i c l e e p i t h e l i a l heights of the long-term experiment and the high dose group (1.0 mg./gm.) sampled on June 14/72 (the values are presented i n Table IX) ... 33 v i i i L i s t of Figures (cont'd) Figure Page 33 Histograms of the mean percentages of oocytes i n the stages of oocyte maturation of the sequential experiment (the data are shown i n Table X) ... 34 34 The daylengths of the natural photoperiod at 48° l a t i t u d e 56 i x ACKNOWLEDGEMENTS I wish to express my sincere appreciation to my thesis supervi-sor, Dr. E.M. Donaldson, for suggesting the problem and f o r h i s guidance and assistance throughout the study. Appreciation i s also extended to my academic supervisor, Dr. W.S. Hoar, f o r h i s advice during the study. The•technical assistance of Mrs. H. Dye and Mr. S. Shaw i n samp-l i n g and of Messrs. J . Culp, R. Corrigan, and J . Tuerlings i n caring for the f i s h i s g r a t e f u l l y acknowledged. I wish to thank Mrs. K. Kramer for processing and blocking the tissues and, along with Mr. J . McBride, f o r advice on h i s t o l o g i c a l procedure. Miss D. Hards and Mr. K. Khoo also helped i n tiss u e pre-paration. I also wish to thank Dr. T. Evelyn of the Nanaimo B i o l o g i c a l Station f o r the autopsy of diseased f i s h and for i d e n t i f y i n g the pre-sence of kidney disease. Mr. G. Shaw, Veterinary Medical Department, Ayerst Laboratories, generously supplied the methallibure. I further wish to acknowledge Mrs. P. Waldron f o r typing the f i n a l manus c r i p t . This research was completed with the support from a Fisheries Research Board grant. 1 INTRODUCTION The manipulation of environmental factors and the s e l e c t i v e use of pharmacological agents are important techniques used to control re-production i n f i s h . Of the environmental f a c t o r s , photoperiod has a very important influence on reproduction i n f i s h i n h a b i t i n g temperate l a t i t u d e s where seasonal daylength fl u c t u a t i o n s are great (Hazard and Eddy, 1951; Corson, 1955; Henderson, 1963; Wiebe, 1968b; Poston and Livingston, 1971; de Vlaming, 1972). Within the salmonids, photoperiod has been studied most extensively i n the brook trout (Salvelinus  f o n t i n a l i s ) which reproductively mature during the increasing daylengths of spring and decreasing daylengths of f a l l . I f these seasonal changes are accelerated, gonadal maturation and subsequent spawning are advanced (Corson, 1955; Henderson, 1963) suggesting that maturation i s regulated by the seasonal daylength f l u c t u a t i o n s . In pink salmon, females mature during decreasing, increasing, and then decreasing daylengths while males mature l a t e r during increasing and then decreasing daylengths. To investigate the importance of these seasonal changes i n daylength on gonadal maturation of pink salmon, a constant 12 hours light:12 hours dark photoperiod was used throughout maturation. S h i r a i s h i and Fukuda (1966) found that constant short photoperiods stimulated while constant long photoperiods retarded gonadal maturation of four species of salmonids which spawn during the approximately equal l i g h t and dark of f a l l and during the short daylength of winter. The constant equal photoperiod used on the pink salmon which spawn during the equal l i g h t and dark of f a l l , therefore, i s thought to be more appropriate than longer or shorter 2 constant photoperiods. One of the pharmacological agents known to i n h i b i t gonadal matura-t i o n i n t e l e o s t s i s methallibure (I.C.I. 33,828). It i s a non-steroidal d e r i v a t i v e of b i s - t h i o u r e a and acts at the hypothalamic and hypophysial l e v e l to i n h i b i t gonadal function by preventing the synthesis and release of gonadotropins from the p i t u i t a r y (Malven, 1971; Brown and Fawke, 1972; Labhsetwar and Walpole, 1972). By i n h i b i t i n g gonadal maturation i n f i s h , methallibure prevents s t e r o i d production and, consequently, the s t e r o i d dependent secondary sexual c h a r a c t e r i s t i c s and reproductive be-haviour. Methallibure i s e f f e c t i v e i n i n h i b i t i n g gametogenesis i n mammals (Paget et a l . , 1961; G e r r i t s and Johnson, 1965; Hemsworth et a l . , 1968; Boris et_ al_. , 1971), birds (Sykes, 1963, 1964; Steel and Hinde, 1972), r e p t i l e s (Rangneker and Kulkarni, 1969), amphibians (Kanakaraj and Gangadhara, 1967; Rastogi et_ al_., 1972), and f i s h (Hoar et a l . , 1967; Carew, 1968; Martin and Bromage, 1970; Pandey, 1970a, b; B i l l a r d et a l . , 1971; Hyder, 1972). In addition to i t s e f f e c t on the synthesis and release of gonado-t r o p i n , methallibure also has an i n h i b i t o r y e f f e c t on thyrotropin synthe-s i s and release from the p i t u i t a r y . Methallibure acts at the hypo-thalamic and hypophysial l e v e l and r e s u l t s i n suppressing t h y r o i d a l function (Hoar e_t al_., 1967; Rangneker and Kulkarni, 1969; Bourke et a l . , 1971). Methallibure i s chemically s i m i l a r to thiourea and presumably shares with i t the a b i l i t y to competitively bind iodine i n the process of thyroxine synthesis. This would lower the amount of a v a i l a b l e iodine and consequently reduce thyroxine production (Pandey and Leatherland, 1970). The amount of feedback on the hypothalamus and p i t u i t a r y would 3 i n turn, be reduced causing increased thyrotropin release and thyroid stimulation. The combined actions on the hypothalamus and p i t u i t a r y and on the thyroid determines methallibure's e f f e c t upon thyr o i d func-t i o n (Tulloch et a l . , 1963; Walpole, 1965; Hoar et a l . , 1967; Rangneker and Kulkarni, 1969). These s i t e s of action by methallibure are shown i n Figure 1. The p r a c t i c a l importance of i n h i b i t i n g gonadal maturation i s e v i -dent considering the l i f e cycle of pink salmon. Hatching normally occurs during January and February and maximum emergence and downstream migration during A p r i l and May. The f i s h remain at sea u n t i l the late summer of t h e i r second year at which time they migrate i n t o freshwater. Spawning mainly occurs during September and'October (Neave, 1966). This s t r i c t two year l i f e cycle maintains two reproductively i s o l a t e d spawn-ing populations of pink salmon on the coast of B r i t i s h Columbia: one spawning i n even years and one i n odd years. In the northern l a t i t u d e s , escapement i n odd years i s very low while that of even years i s much higher. This d i f f e r e n c e between the odd and even year populations i s less marked i n the mid-latitudes of the coast and completely opposite i n the southern regions (Neave, 1962). In an attempt to increase the es-capement of these Wpobr'i-runs1, transplants from streams with high escape-ment to those with low escapement have been undertaken. However, i n almost a l l these programs, the percent of returning adults has not been s u f f i c i e n t to support a s e l f - s u s t a i n i n g population (Neave, 1965; E l l i s , 1969; Walker and L i s t e r , 1971). The only a l t e r n a t i v e would be to a l t e r the reproductive cycle of pink salmon spawning i n , f o r example, the "good" odd years i n the southern regions of B r i t i s h Columbia, so that 4 Figure 1: The hormone and steroid pathways of the gonad and thyroid that are influenced by methallibure. •*'•:'. steps directly inhibited by methallibure GRF - gonadotropin releasing factor TIF - thyrotropin inhibiting,'factor GTH - gonadotropin TSH - thyrotropin 1 - Baker (1969) 2 - Farner and Follett (1966) 3 - Gorbman (1969) 4 - Peter (1970) 5 - Peter (1971) 6 - Sage and Bromage (1970a,b) PHOTOPERIOD2 feedback RECEPTION & PROCESSING JL negative GRF-1 HYPOTHALAMUS release 4,5 negative :::« PITUITARY GTH J synthesis GTH release positive-' negative1 1.5iodine3 incorporation in thyroxine synthesis feedback -SEX STEROIDS gonad maturation reproductive behaviour secondary sex characteristics THYROXINE effects on metabolism 5 subsequent generations would spawn i n the "poor" even years. Funk and Donaldson (1972) and Funk et_ al_. (1973) used p a r t i a l l y p u r i f i e d salmon (Oncorhynchus tshawytscha) gonadotropin (SG-G100) i n an attempt to advance the gonadal maturation of male and female pink salmon by one year. The males completely matured within 98 days of treatment and thus within the year of t h e i r hatching. The females, however, did not mature u n t i l seven months p r i o r to the normal maturation period and exhibited only a small number of mature oocytes. This would provide the genetic complement from only the males f o r use i n the "poor" year popu-l a t i o n . Since the females seem unable to mature i n as short a period of time as the males, i t would seem reasonable to delay ovarian maturation f o r one year and allow the females to mature normally i n the following two years. This could r e s u l t i n pink salmon becoming sexually mature i n t h e i r t h i r d year of l i f e and allow the genetic complement of the females to be used i n the "poor" year population. From t h i s r a t i o n a l e , i t was decided to investigate the e f f e c t s of the drug methallibure and a constant 12 hours l i g h t : 12 hours dark photo-period on gonadal maturation of male and female pink salmon. This would provide information on the f e a s i b i l i t y of using methallibure, and perhaps a constant photoperiod, to delay gonadal maturation of male and, p a r t i -c u l a r l y , female pink salmon. 6 MATERIALS AND METHODS Three experiments were conducted: a p i l o t experiment comprising three d i f f e r e n t doses of methallibure (Ayerst Laboratories) to determine a dose s u f f i c i e n t to i n h i b i t gonadal maturation i n the subsequent ex-periments; a long-term experiment, i n v e s t i g a t i n g the long-term e f f e c t s of methallibure and a constant 12 hours l i g h t : 12 hours dark photoperiod on gonadal maturation; and a sequential experiment, to determine the ef f e c t of s t a r t i n g methallibure treatment at regular i n t e r v a l s before the s t a r t of ovarian development rather than a f t e r the s t a r t of ovarian development as i n the long-term experiment where i n h i b i t i o n of ovarian maturation was unsuccessful. The pink salmon (Ortcorhynchus gorbuscha) used i n a l l three experiments were obtained from the Fi s h e r i e s Research Board Station i n Nanaimo. Experimental design The f i r s t or p i l o t experiment consisted of four groups: a control and three treatment groups receiving 0.10 mg./gm., 0.32 mg./gm., and 1.0 mg. methallibure/gm. body weight. It was started on September 3, 1971 and sampled on October 3 and f i n a l l y on January 12, 1972. Males were excluded from t h i s experiment since t e s t i c u l a r maturation does not begin u n t i l May or June. The 1.0 mg./gm. group received i n j e c t i o n s of methal-l i b u r e only to the f i r s t sampling date (October 3) a f t e r which i t re-ceived no i n j e c t i o n s . The f i s h were of the same stock as those of the long-term experiment (juveniles which hatched i n January or February, 1971) and, therefore, provide information on the stage of maturation and eff e c t of methallibure treatment p r i o r to the s t a r t of the long-term 7 experiment. The f i s h were maintained i n 144 l i t e r s e l f - c l e a n i n g , f i b e r g l a s s aquaria with flowing sea water and aeration and exposed to a simulated natural photoperiod. As i n a l l experiments, the f i s h were fed three times d a i l y with a frozen mixture of beef heart and l i v e r , canned salmon, pablum, and sodium chloride (Donaldson and McBride, 1967). The second or long-term experiment consisted of four groups: a control and treatment group under a natural photoperiod and a control and treatment group under a constant 12 hours l i g h t : 12 hours dark photoperiod. The experiment was started on November 8, 1971 and each group was sampled on January 15, March 13, June 14, August 24, and f i n a l l y on September 5, 1972. The August and September samples were pooled and c a l l e d the August 30 sampling because of the s i m i l a r i t y i n the parameters measured and the short time diffe r e n c e between the two samplings. The natural photoperiod treatment group terminated on June 14 because of complete mortality due to kidney.disease. Thyroid t i s s u e was removed on the June sampling to determine i f me'thallibure has an a n t i -t h y r o i d a l e f f e c t . An additional group from the same stock of f i s h which had j u s t received one month of treatment with a high dose of methallibure (1.0 mg./gm.) was also sampled to investigate the e f f e c t s of a higher dose on the thyroid. This high dose group had previously received i n -jections of the suspension agent, Tween 80 and d i s t i l l e d water, f o r the duration of the p i l o t experiment which ended on January 12, 1972. The thyroid t i s s u e was f i x e d i n Bouin-Hollande sublime (Kraicer et_ a l . , 1967) and stained with Periodic a c i d - S c h i f f ( C u l l i n g , 1957). E p i t h e l i a l heights from 30 thyroid f o l l i c l e s per f i s h were measured microscopically 8 using an ocular micrometer. The f i s h were maintained i n 700 l i t e r aquaria with flowing sea water and aeration. Two l i g h t proof photo-period enclosures were each illuminated by four, 48 inch daylight fluorescent tubes. The natural daylengths were simulated using an "Astro-clock". The constant 12 hours l i g h t : 12 hours dark photoperiod was maintained from 7:00 AM to 7:00 PM by an "intermatic" time switch. During the l a s t four months of the experiment, the f i s h were kept i n four large ten foot diameter f i b e r g l a s s aquaria with flowing sea water and aeration. The aquaria were lightproofed and the same i l l u m i n a t i o n maintained. The water temperature i n a l l the experiments was not con-t r o l l e d and fluctuated seasonally. High mortality i n a l l four groups from kidney disease prompted the use o f a n t i b a c t e r i a l agents. The drug sulfamerizine was incorporated i n t o the d i e t of a l l the groups at a dose of 50 mg. sulfamerizine/Kgm. fish/day f o r 7 days. During t h i s treatment, the drug "Furanase" (P-7138) was added to the aquaria water of the na-t u r a l photoperiod treatment group (which had the greatest mortality) at approximately 1 ppm. f o r 50 min. A f t e r sulfamerizine treatment, the a n t i b i o t i c erythromycin was incorporated i n the diet of a l l groups at a dose of 100 mg. erythromycin/Kgm. fish/day f o r 21 days. These treatments were started on June 9 and terminated on J u l y 8, 1972. In the t h i r d or sequential experiment, j u v e n i l e pink salmon were taken from a stock population which, a f t e r hatching i n January, 1972, had been maintained at a constant temperature of approximately 12°C. As a r e s u l t the f i s h were larger than pink salmon reared at normally colder temperatures. From t h i s stock population, a group of 12 juvenile 9 f i s h were transferred to 144 l i t e r aquaria every 28 days f o r four months s t a r t i n g on May 31, 1972. Each group was i n j e c t e d with methal-l i b u r e from t h e i r i n i t i a t i o n date to the f i n a l sampling date of October 29, 1972 (Fig. 2). At each of the four i n i t i a t i o n s (May 31, June 29, Ju l y 27, and August 24) a zero control sample was taken. A zero con-t r o l sample was also taken on the f i n a l sampling date (October 29) to determine the extent of ovarian maturation of the stock population. A control group was maintained to determine i f the s o l u t i o n of Tween 80 and d i s t i l l e d water had an e f f e c t on gonadal maturation. Males were excluded from t h i s experiment since t e s t i c u l a r maturation does not be-gin u n t i l the succeeding spring. Injection technique The f i s h were in j e c t e d i i n t r a p e f - i t o n e a l l y once every two weeks with 0.10 mg. methallibure/gm. body weight suspended i n 0.1 ml. of a s o l u t i o n of one drop of Tween 80 i n 20 ml. of d i s t i l l e d water (except for the p i l o t experiment which incorporated a range of doses). Control f i s h were in j e c t e d only with the s o l u t i o n of Tween 80 and d i s t i l l e d water. A 26 gauge, 3/4 inch disposable hypodermic needle and 1 ml. disposable sy-ringe were used. The i n j e c t i o n s i t e f o r most of the p i l o t experiment and the f i r s t four i n j e c t i o n s of the long-term experiment was approximately 5 mm. d o r s a l l y and a n t e r i o r l y from the anus. This can, however, r e s u l t i n an i n t r a i n t e s t i n a l i n j e c t i o n and loss of methallibure. An i n j e c t i o n s i t e approximately 5 mm. d o r s a l l y and a n t e r i o r l y from the base of each p e l v i c f i n (al t e r n a t i n g sides) was used f o r subsequent i n j e c t i o n s since the success of an iMtrapefit'oneal i n j e c t i o n at t h i s s i t e was quite high. 10 Figure 2: Design of the sequential experiment showing the period of methallibure treatment from the starting date to the final sampling date of Oct. 29/72 for each of the four groups. 4-I 3-o on O 2-1- period of methallibure treatment MAY • JUNE 1 JULY ' A U G 1 SEPT 1 OCT 1 1972 11 Fish were anesthetized for approximately 45 min. with 10 ppm. of quinaldine (Locke, 1969) i n the aquaria water f o r most of the p i l o t ex-periment and the f i r s t part of the long-term experiment. This concentra-t i o n and length of exposure, however, re s u l t e d i n m o r t a l i t y which may have been due to excessive exposure and concentration (Marking, 1969). To reduce the exposure during subsequent i n j e c t i o n s , the f i s h were anesthetized for only 10 min. i n a large bucket containing only 5 ppm. of quinaldine. This re s u l t e d i n reduced mortality. Sampling technique A minimum of three males and three females were taken from each group at each sampling. The f i s h were anesthetized i n quinaldine and k i l l e d by severing the s p i n a l cord. Excessive moisture was b l o t t e d from each f i s h and the body weight (measured to the nearest 0.1 gm.), fork length (measured to the nearest m i l l i m e t e r ) , r e l a t i v e amount of methal-l i b u r e i n the coelom, presence of kidney disease, and general appear-ance of the f i s h were recorded. Both gonads were removed, f i x e d , par-t i a l l y dehydrated i n 50 and 70 percent alcohol, and then weighed to the nearest milligram. The large s i z e of the maturing testes and some ovar-ies at the f i n a l two sampling dates of the long-term experiment necessi-tated weighing both testes or ovaries fresh rather than f i x e d . P r i o r to the s t a r t of each experiment or treatment, a zero control sample was taken to determine the i n i t i a l " state of gonadal development. The gonado-somatic index was calculated f o r each f i s h by the formula: 12 = gonad weight i n grams body weight m grams A l l s t a t i s t i c a l comparisons were done with the Student's t - t e s t . H i s t o l o g i c a l procedure and analysis One or both whole gonads were fi x e d i n Bouin's f l u i d f o r approxi-mately 24 hours, dehydrated, and r o u t i n e l y imbedded i n Paraplast (mpt. 56-57°C). The gonads were sectioned at 6 microns and stained with Mayer's haematoxylin and eosin ( C u l l i n g , 1957). Oocyte diameters were measured f o r the p i l o t and long-term experiments micro s c o p i c a l l y using an ocular micrometer. The diameter was calculated according to Braekevelt and McMillan (1967) from the formula: oocyte diameter =. T —T~T- T i z—r= z— J kvgreatest diameter x least diameter where k i s the f a c t o r which converts the ocular micrometer measures of the greatest and least oocyte diameters i n t o millimeters. From the prepared s l i d e s of median sections of ovaries, as many oocytes as pos-s i b l e from each f i s h were chosen for measurement on the basis of being i n the most abundant stages of maturation for that ovary and having the major part of t h e i r nucleus present i n the section. Ovaries with oocytes i n the l a t e r stages of maturation at the August 30 sampling of the long-term experiment were d i f f i c u l t to prepare h i s t o l o g i c a l l y . This neces-s i t a t e d measuring 25 whole oocytes microscopically under low magnification. Ovarian maturation was measured using the combined schemes of Ishida et a l . (1961) and Yamazaki (1965): 13 F i r s t Growth Phase Stage 1: ea r l y perinucleolus (Fig. 3) -haematoxylin p o s i t i v e p a l l i a l layer present Stage 2: i-':^ perivucha^matoxylin p o s i t i v e cytoplasm Stage 2: late-p«rinuc4e&l!us o x v' i " ;;j3itive cytoplasm (Fig. 4) - p a l l i a l layer absent -cytoplasm weakly haematoxylin p o s i t i v e Second Growth Phase Stage 3: early yolk v e s i c l e (Fig. 4) -a layer of yolk v e s i c l e s appear i n the peripheral cytoplasm - f o l l i c l e and thecal c e l l layers are sing l e c e l l e d and squamous Stage 4: l a t e yolk v e s i c l e (Fig. 5) -majority of the cytoplasm contains yolk v e s i c l e s - f o l l i c l e layer i s less squamous -thecal layer i s squamous -zona r a d i a t a i s present but t h i n Stage 5: o i l globule (Fig. 6) - d i s t i n c t vacuoles appear around the nucleus which are usu a l l y larger than yolk v e s i c l e s -yolk v e s i c l e s increase i n s i z e - f o l l i c l e layer i s cuboidal -thecal layer i s squamous -zona r a d i a t a increases i n thickness and now exhibits r a d i a l s t r i a t i o n s 14 Stage 6: primary yolk globule (Fig. 7) -a layer of small haematoxylin p o s i t i v e granules appear i n the peripheral cytoplasm - f o l l i c l e layer remains single c e l l e d -thecal layer i s less squamous and i s u s u a l l y two c e l l layers thick -zona r a d i a t a increases i n thickness Stage 7: secondary yolk globule (Fig. 8) -majority of the cytoplasm i s f i l l e d with yolk globules which increase i n s i z e and number -zona r a d i a t a increases i n thickness and r a d i a l s t r i a t i o n s are pronounced -oocyte growth i s greatest during t h i s stage -th i s was the most advanced stage observed. The extent of oocyte maturation f o r an experimental group was de-termined as the mean percentage of oocytes i n each of these stages. Only oocytes i n which the major part of the nucleus i s present i n the section were recorded. In ovaries with oocytes i n the yolk globule stages, morphological d i s t o r t i o n during routine processing necessitated recording only the most advanced stage of maturation f o r each ovary. The extent of oocyte maturation for these groups was determined as the percentage of ovaries i n each stage. This was only necessary i n the June 14 and August 30, 1972 samplings of the long-term experiment. To overcome t h i s problem of d i s t o r t i o n i n determining the stage of oocyte maturation, a small portion of each ovary from the August 30 sampling was embedded i n acrylamide gel and sectioned at 14-16 microns on a 15 Figure 3: Ovary approximately three months prior to the start of vitellogenesis showing oocytes in the early perinucleolus stage (x60). - the dark pallial layer is evident in the peripheral cytoplasm. Figure 4: Ovary at the start of vitellogenesis showing oocytes in the late perinucleolus stage (uniform cytoplasm) and early yolk vesicle stage (yolk vesicles in the peripheral cytoplasm)(x60). Figure 5: Oocytes in the late yolk vesicle stage (x60). Figure 6: The large central oocyte is in the oil globule stage which is characterized by large vesicles surrounding the nucleus (x60). - the increase in oocyte diameter during this maturation is evident comparing Figures 3-6. Figure 7: Oocyte in the primary yolk globule stage (x48). - this is indicated by the faint dark band of granules amongst the vesicles in the peripheral cytoplasm. Figure 8: Oocyte in the secondary yolk globule stage (xl9). - the majority of the cytoplasm is filled with yolk globules. - the zona radia.ta:l:ayer (dark grey band adjacent to the peripheral cytoplasm) is thick and prominent at this stage. - double imbedded in celloidin and paraffin. 16 cryostat at -20°C (Orlowski et_ al_. , 1971). C e l l o i d i n and p a r a f f i n double imbedding was also t r i e d , but only on a small amount of tis s u e ( C u l l i n g , 1957). T e s t i c u l a r maturation was measured for the long-term experiment as the presence or absence of the following spermatogenic stages: primary spermatogonia, secondary spermatogonia, primary spermatocytes, secondary spermatocytes, spermatids, and spermatozoa. These cellsstages were i d e n t i f i e d h i s t o l o g i c a l l y from Ishida et_ al_. (1961) and are shown i n Figures 9, 10, and 11. 1 7 Figure 9: Testis approximately six months prior to the start of testicular maturation (x600). - the arrow indicates a representative example of primary spermatogonia. - spermatogonia! mitosis is evident at the top middle of the micrograph. Figure 10: Testis at the beginning of maturation exhibiting secondary spermatogonia (arrow indicates a representative example)(x600). - at this stage, lobule formation has just begun. - spermatogonia! mitosis is evident in the lower middle of the micrograph. Figure 11: Testis approaching final maturity and exhibiting the following series of cell stages: primary spermatocytes (1° SPCY), secondary spermatocytes (2° SPCY), spermatids (SPTD), and spermatozoa (SPZ)(x600). 18 RESULTS In the f i r s t or p i l o t experiment, a l l three doses of methallibure reduced the rate of increase i n the GSI of females compared to the con t r o l ; the high dose (1.0 mg./gm.) s i g n i f i c a n t l y (p<0.05) on both the sampling dates of Oct. 3/71 and Jan. 12/72 (Fig. 22, Table I ) . A l l doses reduced the rate of increase i n oocyte diameter also, but not s i g n i f i c a n t l y compared to the control on the Jan. 12/72 sampling (Fig. 23, Table I I ) . Methallibure also had no ef f e c t on the stage of oocyte maturation on the Oct. 3/71 sampling (Fig. 24, Table I I I ) . The 0.32 mg./gm. dose group i s s i m i l a r to the control group which exhibits oocytes mainly i n the late perinucleolus and early yolk v e s i c l e stages while the 0.10 mg./gm. and 1.0 mg./gm. dose groups are more advanced i n also ex-h i b i t i n g oocytes i n the late yolk v e s i c l e stage. On Jan. 12/72, the high dose group (in j e c t i o n s of 1.0 mg./gm. terminating on Oct. 3/71) was less advanced than the other groups, including the co n t r o l , because of the higher proportion of the early stages of maturation and the absence of oocytes i n the o i l globule stage (Fig. 24). In the second or long-term experiment, at the time of the f i n a l pooled sampling (Aug. 30/72), methallibure treatment with a constant 12L:12D photoperiod s i g n i f i c a n t l y reduced the rate of increase of GSI i n males (p<0.01) and females (p<0.05) compared to both controls (Fig. 25, Table IV and Fig. 26, Table V). The s i m i l a r i t y i n the GSI of f e -males i n the natural photoperiod treatment group to both controls on the June 14/72 sampling (Fig. 26) i s due only to the weight of these treatment 19 ovaries since the body weights (Fig. 31) and stages of oocyte matura-t i o n of t h i s treated group (Fig. 28) are s i m i l a r to the constant 12L: 12D photoperiod treatment group. From the June and August samplings, methallibure treatment with natural or constant 12L:12D photoperiod completely i n h i b i t e d t e s t i c u l a r maturation (Fig. 27). In the June sampling i t i s evident that methallibure prevented the transformation of primary spermatogonia into secondary spermatogonia (Figs. 12, 13 and 14) . Both controls of the August sampling exhibited spermatozoa (Fig. 15) . In the females, methallibure with constant 12L:12D photoperiod only slowed the rate of ovarian maturation compared to both controls. At the August sampling, the most advanced stage of t h i s group was o i l globule (Fig. 17) whereas i n the c o n t r o l s . i t was secondary yolk globule (Fig. 28 Table VI and F i g . 18). Methallibure with constant 12L:12D photoperiod also only slowed the rate of increase i n oocyte diameter, but not s i g n i f i c a n t l y compared to both controls (Fig. 29, Table VII). Oocyte a t r e s i a increased throughout the experiment i n both controls and treatments (Fig. 30, Table VI). Methallibure reduced the rate of increase i n body weight of pooled males and females (p<0.01) compared to both controls (Fig. 31, Table V I I I ) . The constant 12L:12D photoperiod alone reduced the rate of increase i n the GSI and body weight of males and females (Fig. 25, Table IV; F i g . 26, Table V; and F i g . 31, Table VIII) and i n the oocyte diameter of females (Fig. 29, Table VII) compared to the natural photoperiod con-t r o l , but only s i g n i f i c a n t l y i n the GSI of males (p<0.01). The constant 12L:12D photoperiod slowed t e s t i c u l a r maturation at the June sampling 20 where secondary spermatogonia were the most advanced stage compared to primary spermatocytes of the natural photoperiod c o n t r o l . In August, both controls exhibited spermatozoa but some f i s h i n the constant 12L: 12D photoperiod control group showed complete i n h i b i t i o n of t e s t i c u l a r maturation (Fig. 27). The constant 12L:12D photoperiod slowed ovarian maturation at the June sampling where the most advanced stage was o i l globule compared to primary yolk globule i n the natural photoperiod con-t r o l group (Fig. 28, Table VI). In August, both controls exhibited the secondary yolk globule stage but two out of three f i s h showed t h i s stage under natural photoperiod while only one out of seven f i s h showed t h i s stage under constant 12L:12D photoperiod. The thyroid f o l l i c l e e p i t h e l i a l heights of the natural photoperiod treatment and constant 12L:12D photoperiod control and treatment are s i g n i f i c a n t l y greater (p<0.01, p<0.01, and p<0.05, respectively) than the natural photoperiod control and high dose group (Fig. 32, Table IX). In the t h i r d or sequential experiment, methallibure had no e f f e c t on ovarian maturation even when administered before and during the very e a r l y stages of maturation (Fig. 33, Table X). Each of the four groups showed a l e v e l of maturation higher than t h e i r respective zero controls and s i m i l a r to the control and zero control sampled on Oct. 29/72 (Figs. 19, 20, and 21). The s i m i l a r i t y i n the extent of ovarian maturation of this control and zero control sampled on Oct. 29 indicates that the sol u t i o n of Tween 80 and d i s t i l l e d water had no e f f e c t on the ovary. Methallibure had no e f f e c t on the rate of increase i n GSI. The treated groups increased at a s i m i l a r rate as the stock population. 21 Figure 12: Testis showing secondary spermatogonia from the constant 12L:12D photoperiod control group of the long-term experiment on June 14 (x600). Figure 13: Testis showing primary spermatogonia from the constant 12L: 12D photoperiod treatment group of the same sampling as above (x600). Figure 14: Zero control testis of the long-term experiment, sampled on November 8/71, showing primary spermatogonia (x600). Figure 15: Testis showing spermatozoa (arrow) from the constant 12L:12D photoperiod control group of the long-term experiment from the August 30 sampling (x600). Figure 16: Testis showing primary spermatogonia from the constant 12L:12D photoperiod treatment group of the same sampling as above (x600). 22 Figure 17: Oocytes in the oil globule stage from the constant 12L:12D photoperiod treatment group of the long-term experiment from the August 30 sampling (xl9). - imbedded in acrylamide gel. Figure 18: Oocytes in the secondary yolk globule stage from the constant 12L:12D photoperiod control group from the same sampling as above (xl9). - the difference between the oocyte diameters of control and treatment fish is evident comparing these oocytes with those of Figure 17. Figure 19: Ovary with oocytes in the early perinucleolus stage from the start of the June 29 group of the sequential experiment (x60). Figure 20: Oocytes in the late yolk vesicle stage from methallibure treated fish of the June 29 group at the end of the sequential experiment (October 29)(x60). Figure 21: Oocytes in the late yolk vesicle stage from control fish of the sequential experiment sampled on October 29 (x60). 23 Figure 22: The mean gonadosomatic index (GSI) values for females of the pilot experiment (the data are listed in Table I). (gzero control * injections of this high dose group were terminated on Oct. 3/71. F E M A L E S S E P T 1 O C " T 1 FTov 1 D T C 1 T A N 1 1971 1972 24 Figure 23: The mean oocyte diameters of the pilot experiment, in millimeters (the values are listed in Table II). ©zero control * injections of this high dose group were terminated on Oct. 3/71. S E P T « o c l • N O V 1 D T C 1 T A N 1 1 9 7 1 1972 25 Figure 24: Histograms of the mean percentages of oocytes in the stages of oocyte maturation of the pilot experiment (the data are presented in Table III). * injections of this high dose group were terminated on Oct. 3/71. 1 - early perinucleolus stage 2 - late perinucleolus stage 3 - early yolk vesicle stage 4 - late yolk vesicle stage 5 - oil globule stage •100% 1 0 0 % — ZERO CONTROL 0- 1 2 SEPT. 3 1 2 3 4 a. 1 2 3 1 2 3 4 1 2 3 4 OCT. 3 1 2 3 4 1 2 3 4 5 1 2 3 4 5 1 9 7 1 1 2 3 4 5 J A N .12 1 9 7 2 l.Omg. •0 r 1 0 0 % 0.32 mg. 1-100 % 0.10 mg. «-0 r i o o % CONTROL ^ 0 26 Figure 25: The mean gonadosomatic index (GSI) values for males of the long-term experiment (the data are shown in Table IV). - closed triangles - natural photoperiod - open triangles - constant 12L:12D photoperiod - solid lines - control - dashed lines - treatment ©zero control — natural photoperiod treatment group - the designation of this group was omitted because of lack of space. This group was terminated on June 14 because of high mortality due to kidney disease. MALES 1971 1972 27 Figure 26: The mean gonadosomatic index (GSI) values for females of the long-term experiment (the data are listed in Table V). NAT. PHOTO. - natural photoperiod CONST. PHOTO. - constant 12L:12D photoperiod ©zero control - the natural photoperiod treatment group terminated on June 14 because of high mortality due to kidney disease. FEM A LES 0 I N O V l D E C ' J A N I FEB ' M A R 1 APR "MAY" JUN> J U L ' A U G ' 1971 1972 28 Figure 27: Histograms of the presence and absence of the cell stages of testicular maturation of the long-term experiment. - the group designations above each histogram on the Jan. 15 sampling also apply to the other three sampling dates. - stippled portions - presence of those cell stages - light portions - absence of those cell stages 1° SPGN - primary spermatogonia 2° SPGN - secondary spermatogonia 1° SPCY - primary spermatocytes 2° SPCY - secondary spermatocytes SPTD - spermatids SPZ - spermatozoa CO < mm .treatment'!; ;;;;| NATURAL P H O T O P F R I Q D control |:':"::::TTj M ii p ii > - — i r ^ — I I — z ir % " £ " X " % o " o ^> ?Z °z D- a. 0 0 <^> (/> </) o o o CN — CN < 00 ZERO C O N T R O L CN rs < treatment!:::::::::::.::| C O N S T A N T 12:12 P H O T O P F R IQD ~ -control FT: ••:••••::! y O £ z * 29 Figure 28: Histograms of the mean percentages of oocytes in the stages of oocyte maturation of the long-term experiment (the values are listed in Table VI). * these histograms represent the percentage of the total number of ovaries in which only the most advanced stage of maturation was recorded because of extensive oocyte distortion in processing. 1 - early perinucleolus stage 2 - late perinucleolus stage 3 - early yolk vesicle stage 4 - late yolk vesicle stage 5 - oil globule stage 6 - primary yolk globule stage 7 - secondary yolk globule stage NATURAL PHOTO. CONSTANT PHOTO. 30 Figure 29: The mean oocyte diameters of the long-term experiment, in millimeters (the values are presented in Table VII). ©zero control - both the controls and the constant 12L:12D photoperiod treatment group were not measured on the June 14 sampling because of oocyte distortion during processing. 3-1 NOV I DEC I JANI FEB IMARI APR I MAY I JUNI JUL » AUG' 1971 1972 31 Figure 30: Histograms of the mean percentage of oocytes that are atretic in the long-term experiment (the data are listed in Table VI). - the group designations above each histogram on the Jan. 15 sampling also apply to the other three sampling dates. o CO ]6 3 < LZZI 3^ " Z J 3 CN O CO < treatmentl CONST. PHOTO., ,' control! I £ treatment[ NAT. PHOTO. Z < control Q oo ZERO CONTROLl ^  K 1 O o z -I 1 1 1 1 1 5€> O O O O O Q oo o -*r CN 2 S31XDOO DI13CJ1V lN3Dil3d 32 Figure 31: The mean body weights of pooled males and females of the long-term experiment (the data are listed in Table VIII). ©zero control 250n 1971 1 9 7 2 33 Figure 32: The mean thyroid follicle epithelial heights of the long-term experiment and the high dose group (1.0 mg./gm.) sampled on June 14/72 (the values are presented in Table IX). - the standard deviation of the means within each group is shown above and below each group mean. NATURAL PHOTOPERIOD CONTROL TREATMENT CONSTANT 12:12 PHOTOPERIOD HIGH CONTROL TREATMENT DOSE 34 Figure 33: Histograms of the mean percentages of oocytes in the stages of oocyte maturation of the sequential experiment (the data are shown in Table X). - the bottom row of zero control histograms represent the percentages of oocytes in the stages of maturation at the start of methallibure treatment for each group. - the zero control histogram on Oct. 29 represents the extent of maturation of the stock population at the end of the experiment. - the top row represents the percentages of oocytes in the stages of maturation of each of the four groups on Oct. 29 after methallibure treatment. The date of the start of methallibure treatment and the respective zero control of each group appear directly below on the bottom row. - the control group appears at the right of the top row. 1 - early perinucleolus stage 2 - late perinucleolus stage 3 - early yolk vesicle stage 4 - late yolk vesicle stage I O O V I CM 2 3 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 SAMPLED O N O C T . 29/72 AFTER METHALLIBURE TREATMENT C O N T R O L lOOVl 0 J M A Y 31 1 2 : Z E R O JUNE 29 1 2 C O N T R O L S JULY 27 1 9 7 2 1 2 3 4 A U G . 24 a . 1 2 3 4 OCT. 29 35 Table I: The mean gonadosomatic index (GSI) values for females of the pilot experiment. * injections of this high dose group were terminated on Oct. 3/71. DATE GROUP NUMBER OF MEAN GSI ± SD FEMALES Sept. 3/71 zero control Oct. 3/71 control 0.10 mg./gm. 0.32 mg./gm. 1.0 mg./gm. Jan. 12/72 control 0.10 mg./gm. 0.32 mg./gm. * 1.0 mg./gm. 0.252 0.051 4 3 2 3 0.386 0.440 0.369 0.315 0.041 0.082 0.059 0.042 3 7 4 3 0.752 0.550 0.592 0.409 0.065 0.187 0.215 0.146 o 36 Table II: The mean oocyte diameters of the pilot experiment. * injections of this high dose group were terminated on Oct. 3/71. DATE GROUP NUMBER OF OOCYTES MEASURED Sept. 3/71 zero control 128 Oct. 3/71 control 220 0.10 mg./gm. 144 0.32 mg./gm. 98 1.0 mg./gm. 163 Jan. 12/72 control 123 0.10 mg./gm. 168 0.32 mg./gm. 170 *1.0 mg./gm. 117 NUMBER OF MEAN DIAMETER ± SD OF THE FEMALES IN MILLIMETERS MEANS 3 0.223 0.024 4 0.299 0.010 3 0.328 0.025 2 0.301 0.006 3 0.306 0.055 3 0.625 0.075 3 0.506 0.087 4 0.510 0.058 3 0.492 0.119 37 Table III: The mean percentages of oocytes in the stages of oocyte maturation of the pilot experiment. * 1 - early perinucleolus stage 2 - late perinucleolus stage 3 - early yolk vesicle stage 4 - late yolk vesicle stage 5 - oil globule stage ** injections of this high dose group were terminated on Oct. 3/71. DATE GROUP NUMBER OF NUMBER OF MEAN PERCENT IN OOCYTES FEMALES * EACH STAGE 1 2 3 4 5 Sept. 3/71 zero control 541 4 12 ,88 Oct. 3/71 control 659 4 18 48 31 ,3 0.10 mg./gm. 383 3 13 26 28 33 0.32 mg./gm. 270 2 15 56 29 1.0 mg./gm. 514 3 30 32 19 18 Jan. 12/72 control 232 3 5 3 6 78 8 0.10 mg./gm. 384 3 10 13 16 41 20 0.32 mg./gm. 425 4 16 15 10 46 13 ** 1.0 mg./gm. 358 3 7 25 23 45 38 Table IV: The mean gonadosomatic index (GSI) values for males of the long-term experiment. DATE GROUP Nov. 8/71 zero control Jan. 15/72 natural photoperiod control natural photoperiod treatment constant photoperiod control constant photoperiod treatment Mar. 13/72 natural photoperiod control natural photoperiod treatment constant photoperiod control constant photoperiod treatment June 14/72 natural photoperiod control natural photoperiod treatment constant photoperiod control constant photoperiod treatment Aug. 30/72 natural photoperiod control (terminated in June) constant photoperiod control constant photoperiod treatment NUMBER OF MEAN GSI ± SD MALES 3 0.040 0.003 5 0.026 0.004 5 0.030 0.011 3 0.033 0.003 6 0.032 0.008 6 0.052 0.006 5 0.030 0.012 3 0.046 0.006 8 0.046 0.009 3 0.134 0.023 3 0.066 0.012 3 0.072 0.004 3 0.059 0.016 5 6.223 1.654 9 2.382 1.898 7 0.052 0.009 39 Table V: The mean gonadosomatic index (GSI) values for females of the long-term experiment. DATE GROUP Nov. 8/71 zero control Jan. 15/72 natural photoperiod control natural photoperiod treatment constant photoperiod control constant photoperiod treatment Mar. 13/72 natural photoperiod control natural photoperiod treatment constant photoperiod control constant photoperiod treatment June 14/72 natural photoperiod control natural photoperiod treatment constant photoperiod control constant photoperiod treatment Aug. 30/72 natural photoperiod control (terminated in June) constant photoperiod control constant photoperiod treatment NUMBER OF MEAN GSI ± SD FEMALES 5 0.446 0.053 3 0.664 0.078 3 0.520 0.229 4 0.526 0.044 2 0.662 0.275 3 0.865 0.109 3 0.905 0.465 5 0.868 0.212 3 0.597 0.172 3 1.198 0.132 3 1.079 0.189 3 1.110 0.271 3 0.685 0.169 8 2.627 2.048 9 1.778 1.031 9 0.775 0.677 40 Table VI: The percentages of oocytes in the stages of oocyte maturation and the percentage of oocytes that are atretic in the long-term experiment. * these groups represent the percentage of the total number of ovaries in which only the most advanced stage of maturation was recorded because of extensive oocyte distortion in processing. ** 1 - early perinucleolus stage 2 - late perinucleolus stage 3 - early yolk vesicle stage 4 - late yolk vesicle stage 5 - oil globule stage 6 - primary yolk globule stage 7 - secondary yolk globule stage DATE GROUP NUMBER OF OOCYTES Nov. 8/71 zero control 710 Jan. 15/72 natural photoperiod control 344 natural photoperiod treatment 384 constant photoperiod control 333 constant photoperiod treatment 310 Mar. 13/72 natural photoperiod control 109 natural photoperiod treatment 136 constant photoperiod control 169 constant photoperiod treatment 112 June 14/72 natural photoperiod control natural photoperiod treatment 137 constant photoperiod control constant photoperiod treatment 203 Aug. 30/72 natural photoperiod control (terminated in June) constant photoperiod control constant photoperiod treatment NUMBER OF MEAN PERCENT IN EACH STAGE PERCENT FEMALES ** 1 2 3 4 5 6 7 ATRETIC 5 12 38 50 0 3 4 15 15 66 1 3 12 16 25 47 7 3 2 13 12 73 10 2 8 12 13 67 0 3 7 3 1 48 41 20 3 8 18 9 31 34 17 3 4 7 9 56 24 13 3 11 12 11 40 26 26 *3 67 33 44 3 3 13 10 49 25 20 *3 100 40 3 16 2 13 47 22 11 *3 33 67 81 *7 57 29 14 50 *5 20 80 59 41 Table VII: The mean oocyte diameters of the long-term experiment. * oocytes from this sampling were measured whole because of the difficulty in processing these large oocytes. ** this group was not included on Figure 29 because oocytes from only one fish could be measured. DATE GROUP NUMBER OF OOCYTES NUMBER OF MEAN DIAMETER ± SD OF THE MEASURED FEMALES IN MILLIMETERS MEANS Nov. 8/71 zero control 193 0.420 0.097 Jan. 15/72 natural photoperiod control natural photoperiod treatment constant photoperiod control constant photoperiod treatment 91 99 90 60 3 3 3 2 0.670 0.591 0.729 0.654 0.022 0.067 0.072 0.012 Mar. 13/72 natural photoperiod control natural photoperiod treatment constant photoperiod control constant photoperiod treatment 36 46 69 59 2 2 3 3 0.741 0.586 0.729 0.589 0.049 0.034 0.117 0.090 June 14/72 natural photoperiod control 0 natural photoperiod treatment 33 constant photoperiod control 0 constant photoperiod treatment **30 0 3 0 1 (excessive distortion) 0.526 0.066 (excessive distortion) 0.472 Aug. 30/72* natural photoperiod control (terminated in June) constant photoperiod control constant photoperiod treatment 146 143 48 6 2 2.749 2.258 1.335 0.869 0.630 0.243 42 Table VIII: The mean body weights of pooled males and females of the long-term experiment. DATE GROUP Nov. 8/71 zero control Jan. 15/72 natural photoperiod control natural photoperiod treatment constant photoperiod control constant photoperiod treatment Mar. 13/72 natural photoperiod control natural photoperiod treatment constant photoperiod control constant photoperiod treatment June 14/72 natural photoperiod control natural photoperiod treatment constant photoperiod control constant photoperiod treatment Aug. 30/72 natural photoperiod control (terminated in June) constant photoperiod control constant photoperiod treatment NUMBER OF MEAN BODY WEIGHT ± SD FISH IN GRAMS 8 52.04 26.02 9 80.92 15.74 8 75.96 14.37 7 92.08 5.94 10 73.27 14.37 9 95.68 18.29 8 73.90 21.25 8 94.51 15.28 11 85.86 26.69 6 138.68 35.85 6 111.85 15.38 6 160.68 26.09 6 104.33 37.30 13 245.65 74.40 18 217.79 52.86 16 144.04 42.84 43 Table IX: The mean thyroid follicle epithelial heights of the long-term experiment and the high dose group (1.0 mg./gm.) sampled on June 14/72. DATE GROUP NUMBER OF THYROID NUMBER OF MEAN HEIGHT ± SD OF THE FOLLICLES MEASURED FISH IN MICRONS MEANS June 14/72 natural photoperiod control 200 6 6 1.3 natural photoperiod treatment 180 6 10 2.1 constant photoperiod control 180 6 9 1.3 constant photoperiod treatment 180 6 8 1.0 high dose 180 6 6 1.5 44 Table X: The mean percentages of oocytes i n the stages of oocyte maturation of the sequential experiment. * 1 - e a r l y p e r i n u c l e o l u s stage 2 - l a t e p e r i n u c l e o l u s stage 3 - e a r l y y o l k v e s i c l e stage 4 - l a t e y o l k v e s i c l e stage DATE GROUP NUMBER OF MEAN PERCENT IN EACH STAGE FEMALES * 1 2 3 4 May 31/72 zero control 3 100 June 29/72 zero control 3 71 27 2 July 27/72 zero control 3 63 37 Aug. 24/72 zero control 3 26 56 11 7 Oct. 29/72 zero control 5 10 51 23 16 Oct. 29/72 control 3 10 63 9 18 Oct. 29/72 sampling of group started on May 31/72 1 8 35 16 41 Oct. 29/72 sampling of group started on June 29/72 4 6 49 27 18 Oct. 29/72 sampling of group started on July 27/72 7 4 45 22 29 Oct. 29/72 sampling of group started on Aug. 24/72 1 6 66 22 6 45 DISCUSSION Methallibure was f i r s t used on f i s h by Hoar et a l . (1967). These authors found that i t completely i n h i b i t e d spermatogenesis i n the sea-perch (Cymatogaster aggregata), three-spined stickleback (Gasterosteus  aculeatus), and g o l d f i s h (Carassius auratus). These r e s u l t s were l a t e r confirmed by Wiebe (1968a, 1969) on the seaperch, Carew (1968) on the three-spined stickleback, and B i l l a r d ejt al_. (1971) on the g o l d f i s h . Other workers also found that methallibure i n h i b i t s spermatogenesis i n the guppy P o e c i l i a r e t i c u l a t a (Pandey and Leatherland, 1970; Martin and Bromage, 1970; B i l l a r d et_ al_., 1970) and T i l a p i a species (Dadzie, unpub-l i s h e d data; Hyder, 1972). From the present study, methallibure also completely i n h i b i t s t e s t i c u l a r maturation i n pink salmon (Figs. 27, 15 and 16). The s i m i l a r i t y of the c e l l s i n the treatment testes of the long-term experiment i n August, 1972 (Fig. 16) with the primary spermato-gonia present i n the zero control testes i n November, 1971 (Fig. 14) indicates that methallibure i n h i b i t s the m i t o t i c transformation of p r i -mary into secondary spermatogonia. This i s also evident comparing the primary spea?mat-ogoriiayofpthe^trgatment testes (Fig. 13) with the secon-dary spermatogonia of the control testes (Fig. 12) on June 14, 1972. B i l l a r d et_ al_. (1971) found that methallibure reduced the numbers of B-spermatogonia, spermatocytes, and spermatids but not A-spermatogonia i n the g o l d f i s h , which further supports the above conclusion assuming that A and B r e f e r to primary and secondary, re s p e c t i v e l y . These e f f e c t s of methallibure are i d e n t i c a l to those observed by Ahsan (1966) on hypophy-sectomized lake chub, Couesius plumbeus. He found that testes of these 46 hypophysectomized f i s h were composed almost e n t i r e l y of primary sperma-togonia and only r a r e l y were secondary spermatogonia observed. This suggests that the transformation of primary to secondary spermatogonia i s gonadotropin dependent and that the action of methallibure, therefore, i s through the i n h i b i t i o n of gonadotropin release. However, most wor-kers state that methallibure prevents the meiotic transformation of secondary spermatogonia into primary spermatocytes (Hoar et a l . , 1967; Martin and Bromage, 1970; Pandey, 1970a). This discrepancy may i n part be due to these researchers not d i s t i n g u i s h i n g between primary and secon-dary spermatogonia. Ahsan (1966) also found that hypophysectomy sup-pressed the m i t o t i c d i v i s i o n o f primary spermatogonia. Replacement therapy with ovine LH, fractionated salmon p i t u i t a r y gonadotropin, or whole salmon p i t u i t a r y extracts restored the m i t o t i c a c t i v i t y to normal suggesting that spermatogonial p r o l i f e r a t i o n , as well as the transforma-t i o n into secondary spermatogonia, i s gonadotropin dependent. Wiebe (1969) found that methallibure also suppressed the m i t o t i c a c t i v i t y of spermato-gonia i n the seaperch and that exogenous ovine LH restored t h i s a c t i v i t y . However, Hoar et_ al_. (1967) and Martin and Bromage (1970) found that methallibure had no e f f e c t on the m i t o t i c a c t i v i t y of spermatogonia. In the present study also, m i t o t i c figures were observed i n the methallibure treated testes of pink salmon. The a c t i v i t y was not q u a n t i f i e d and, thus, the presence of a suppression of mitosis i s not known. The e f f e c t of methallibure on m i t o t i c a c t i v i t y , therefore, remains obscure i n l i g h t of the c o n f l i c t i n g evidence. Methallibure did not completely i n h i b i t gonadal development i n female pink salmon but only slowed i t s rate (Figs. 26, 28, and 29). 47 This i s contradictory to the complete i n h i b i t i o n of ovarian maturation by methallibure i n the g o l d f i s h and three-spined stickleback (Hoar et_ al_. , 1967), seaperch (Hoar et al_. , 1967; Wiebe, 1969), guppy (Pandey, 1970b), and T i l a p i a species (Dadzie, unpublished data; Hyder, 1972). This i n -h i b i t o r y e f f e c t of methallibure i s i d e n t i c a l to that of hypophysectomy since both prevent the onset of v i t e l l o g e n e s i s or the second growth phase of oocytes i n j u v e n i l e guppies (Pandey, 1970b) and stop v i t e l l o g e n e s i s and cause subsequent regression of second growth phase oocytes i n gold-f i s h (Yamazaki, 1965; Hoar et_ al_., 1967). The f a i l u r e of methallibure to i n h i b i t ovarian maturation completely i n the pink salmon may be due to an i n s u f f i c i e n t dose since the 0.10 mg./gm./2 wks. dose was lower than the doses used i n the . literature* (Hoar et^ al_., 1967; B i l l a r d et a l . , 1970) . However, a three and ten f o l d higher dose i n the p i l o t experi-ment also f a i l e d to i n h i b i t ovarian development (Figs. 22, 23, and 24) although the 1.0 mg./gm. dose did s i g n i f i c a n t l y slow the rate of increase i n GSI (Fig. 22). It was t h i s s i m i l a r i t y i n the e f f e c t of a l l three doses (0.10 mg./gm., 0.32 mg./gm., and 1.0 mg./gm.) i n the p i l o t experi-ment on oocyte diameter and stages of oocyte maturation that was import-ant i n deciding upon the lower 0.10 mg./gm. dose f o r the subsequent experiments. It was thought that the lower dose would reduce possible undesirable e f f e c t s of methallibure. This was substantiated i n l a t e r unreported data i n which the high dose (1.0 mg./gm.) caused very high mortality i n younger, j u v e n i l e pink salmon. Its long-term e f f e c t on older f i s h i s unknown; however, t h i s evidence suggests that high doses of methallibure would be disadvantageous i n such studies. 48 One other possible explanation f o r methallibure not completely-i n h i b i t i n g ovarian maturation i n the long-term experiment i s that t r e a t -ment was begun approximately one and one h a l f months a f t e r the onset of oocyte maturation or v i t e l l o g e n e s i s . According to Sokol (1961), gonad maturation i s coincident with the d i f f e r e n t i a t i o n o f the gonadotrops i n the guppy. Pandey (1970a) found that methallibure completely i n h i b i t e d t e s t i c u l a r maturation i n j u v e n i l e guppies but only slowed spermatogenesis i n adults. With these f a c t s , Pandey suggested that methallibure was un-able to i n h i b i t gonadotropin synthesis and release completely once these processes had begun but could prevent t h e i r inception i f treatment was started p r i o r to gonadotrop d i f f e r e n t i a t i o n and gonad maturation. This suggestion i s strengthened by the complete i n h i b i t i o n of t e s t i c u l a r maturation i n the present study i n which methallibure treatment was begun s i x months p r i o r to gonadotrop d i f f e r e n t i a t i o n and the s t a r t of t e s t i s maturation. To determine i f t h i s suggestion holds true f o r ovarian ma-tu r a t i o n i n pink salmon, methallibure treatment was begun before the s t a r t of ovarian development and before gonadotrop d i f f e r e n t i a t i o n i n the sequential experiment. Figure 33 indicates that methallibure had no e f f e c t upon ovarian development. From these data and those of the p i l o t experiment i n which the high dose had l i t t l e e f f e c t , i t seems that i n h i -b i t i o n of ovarian maturation may be dependent upon treatment with a high dose before gonadotrop d i f f e r e n t i a t i o n . The high dose treatment of the p i l o t experiment was started j u s t p r i o r to the beginning of v i t e l -logenesis but because of poor i n j e c t i o n technique i n i t i a l l y , the f i s h were probably not exposed to methallibure u n t i l a f t e r gonadotrop d i f -f e r e n t i a t i o n . This would account f o r i t s lack of e f f e c t . The difference 49 between the e f f e c t s of methallibure on the male and female pink salmon indicates that the females are much less s e n s i t i v e to t h i s drug than are the males. The recorded e f f e c t s of methallibure on thyroid function are very c o n f l i c t i n g . Hoar et_ al_. (1967) found a s l i g h t a n t i t h y r o i d a l e f f e c t or increased e p i t h e l i a l height i n the three-spined stickleback and sea-perch as did Pandey and Leatherland (1970) i n adult guppies. This e f f e c t i n the l a t t e r study was not as great as that of thiourea. The authors of these two studies also found that methallibure had no a n t i t h y r o i d a l e f f e c t on g o l d f i s h and juvenile guppies. Carew (1968), however, found no e f f e c t on the three-spined stickleback nor did Wiebe (1968a) on the seaperch. Wright (1963) and Paget et^ al_. (1961) found no a n t i t h y r o i d a l e f f e c t on the fowl and r a t . On the other hand, Rangneker and Kulkarni (1969) observed a depressing e f f e c t of methallibure on the t h y r o i d a l ac-t i v i t y of the l i z a r d , Calotes v e r s i c o l o r . In the present study, methal-l i b u r e had a d i s t i n c t a n t i t h y r o i d a l e f f e c t under natural photoperiod but f a i l e d to produce t h i s e f f e c t under a constant 12L:12D photoperiod or at a ten times higher dose (Fig. 32). These c o n f l i c t i n g observations may be explained by me'thallibure's e f f e c t on thyroxine synthesis and thyro-t r o p i n . As described i n the introduction, the e f f e c t of methallibure on t h y r o i d a l a c t i v i t y i s dependent upon the i n h i b i t i o n of thyrotropin syn-thesis and release, which would depress thyroid a c t i v i t y , and upon the binding of iodine; which would increase thyroid a c t i v i t y . Bourke et_ al_. (1971) indi c a t e that the r e l a t i v e e f f e c t of methallibure on each of these two actions may change. They suggest that low doses of methallibure exert anciinhibitory e f f e c t on the release of thyrotropin whereas higher doses 50 and increases i n time of exposure exert an i n h i b i t o r y e f f e c t on the synthesis. They found, i n r a t s , that the low doses caused the greatest f a l l s i n serum thyrotropin while the higher doses f a i l e d to depress the serum le v e l s because of a stimulation of thyrotropin release due, presum-ably, to decreased feedback by thyroxine. Therefore, the differences i n the doses used and the method and period of administration of methal-l i b u r e , as well as species d i f f e r e n c e s , may account for the contradictory findings i n the l i t e r a t u r e . The absence of an increase i n thyroid ac-t i v i t y i n the high dose group of the present study i s s u r p r i s i n g consi-dering the f a i l u r e of high doses to depress serum thyrotropin l e v e l s i n the study of Bourke e_t_ al_. (1971) and the increased thyroid weight by high doses found by Paget et^ al_. (1961) . Thyroid h i s t o l o g y i n the l a t t e r study, however, remained normal. At t h i s 1.0 mg./gm. dose i n pink s a l -mon, the e f f e c t of methallibure on the two actions described must, there-fore, be i n balance. The d i f f e r e n c e i n the thyroid a c t i v i t i e s between the methallibure treated groups under natural and constant 12L:12D photo-periods may, i n part, bee due to disease (which w i l l be b r i e f l y discussed l a t e r ) . An e f f e c t of the constant 12 hour daylength on t h i s a c t i v i t y i s doubted because of the findings of Swift (1960), that the d i f f e r e n t day-lengths of the natural photoperiod had no e f f e c t on thyroid a c t i v i t y (measured as the rate of loss of radio-iodine from the gland) and of Wagner (1970), that the thyroid a c t i v i t y (measured as the height of the th y r o i d f o l l i c l e epithelium) under constant daylengths of 8.5, 12, and 16 hours was s i m i l a r to that under natural photoperiod. 51 The e f f e c t of methallibure on body weight, i s , l i k e that on the thyroid, very contradictory i n the l i t e r a t u r e . Many researchers found a decrease i n the body weight of r a t s , including Stratman and F i r s t (1969) and Labhsetwar and Walpole (1972), while Harper (1967) found no e f f e c t . Imai (1972) found a s l i g h t weight loss i n the domestic hen while Steel and Hinde (1972) found no e f f e c t i n the canary. Gangadhara and Ramaiah (1968) observed no body weight loss i n the skipper frog (Rana cyanophlyctis) nor did B i l l a r d ejt al_. (1971) or Pandey and Leather-land (1970) i n the g o l d f i s h and guppy. Dadzie (unpublished data), how-ever, found an increase i n the body weight of T i l a p i a . In the present study, methallibure reduced the rate of increase i n the body weight of pink salmon (Fig. 31) which agrees with the majority of the mammalian l i t e r a t u r e . This e f f e c t may be a d i r e c t r e s u l t of a decrease i n the amount of food ingested since Stratman et a l . (1969) found a suppression of appetite i n hamsters and swine and postulated that the s i t e of methal-l i b u r e ' s action was the hypothalamus. Whether t h i s effected an "appe-t i t e center" or releasing factors c o n t r o l l i n g the p i t u i t a r y somatotrops was not known. Evidence for an e f f e c t of methallibure at the hypothalamic-hypophy-s i a l ' l l e v e l comes mainly from studies i n which the serum or p i t u i t a r y l e v e l s of hormones or t h e i r peripheral e f f e c t were measured upon t r e a t -ment. Malven (1971) found that ovulation i n the guinea p i g was i n h i b i t e d with implants of methallibure i n the hypothalamus, p a r t i c u l a r l y the pre-op t i c area and the arcuate nucleus-median eminence. This strongly suggests a hypothalamic l e v e l of action of methallibure. The observation by Garbers and F i r s t (1968) of the i n h i b i t i o n of milk e j e c t i o n i n swine by 52 methallibure suggests an i n h i b i t i o n of oxytocin release which supports t h i s hypothalamic l e v e l of action. There i s also strong evidence to support a hypophysial l e v e l of action as well. Bourke ejt al_. (1971) found that methallibure i n h i b i t s the release and synthesis of thyrotro-p i n i n the r a t but these authors hypothesize that methallibure has a possible u n i t a r y action at the hypothalamic l e v e l . Labhsetwar and Wal-pole (1972) and Brown and Fawke (1972) found that methallibure i n h i b i t s the synthesis and release of the gonadotropins LH and FSH i n the r a t . Stormshak et^ a l . (1970) suggest that methallibure may i n t e r f e r e with the p i t u i t a r y release of gonadotropins i n g i l t s since the hypothalamic LH r e l e a s i n g f a c t o r was not effected. Studies on p i t u i t a r y c e l l mor-phology and h i s t o l o g i c a l s t a i n i n g properties are valuable i n i n t e r p r e t -ing the e f f e c t s of methallibure on the synthesis and release of hormones. This, however, must be done with caution, since both these a c t i v i t i e s may be involved i n the observed e f f e c t . A f t e r methallibure treatment, Pandey and Leatherland (1970), on the guppy, and Leatherland (1969), on the seaperch, observed greatly decreased a c t i v i t y of the gonadotrops shown by decreased number, s i z e , and granulation; a s l i g h t l y decreased somatotrop a c t i v i t y shown by decreased granulation; and a very s l i g h t l y increased thyrotrop a c t i v i t y shown by increased c e l l number and s i z e . These observations by Pandey and Leatherland (1970) on the gonadotrops and thyrotrops p a r a l l e l c l o s e l y the a c t i v i t y o f the target t i s s u e s . These authors found an i n h i b i t i o n of spermatogenesis and an increase i n thyroid a c t i v i t y but no change i n body weight. Mackay (1971) found a degranulation of the gonadotrops and, to a l e s s e r degree, the thyrotrops with a concomitant i n h i b i t i o n of ovarian maturation i n the bass, 53 P l e c t r o p l i t e s ambiguus. No e f f e c t on the thyroid was observed. Dadzie (unpublished data) and Carew (1968) also found a decrease i n gonadotrop granulation accompanied by an i n h i b i t i o n of gonad development i n T i l a p i a species and the three-spined stickleback. From these studies, i t i s evident that methallibure has an e f f e c t on the gonadotrops, thyro-trops, and somatotrops at the hypothalamic and hypophysial l e v e l . The only evidence f o r a d i r e c t peripheral e f f e c t of methallibure i s given by Rastogi and C h i e f f i (1972) i n the adult green frog, Rana esculenta. They found that methallibure plus a p i t u i t a r y homogenate f a i l e d to stimulate gonadal a c t i v i t y i n pars d i s t a l i s - e c t o m i z e d frogs whereas the p i t u i t a r y homogenate alone was stimulatory. As but one example of the studies contradicting this evidence i s that of B i l l a r d et_ al_. (1971) on the gold-f i s h . Methallibure i n combination with powdered carp p i t u i t a r i e s main-tained normal spermatogenesis. In the discussion on the e f f e c t s of methallibure on the thyroid f o l l i c l e e p i t h e l i a l heights, reference was made to the e f f e c t of disease on thyroid a c t i v i t y . This disease was diagnosed as kidney disease and was very prevalent i n the long-term experiment during the l a t t e r four months of maturation.. The metabolic stress induced by t h i s disease (Wedemeyer and Ross, 1973) may have caused, at least i n part, the increase i n e p i t h e l i a l height i n the natural photoperiod treatment and constant 12L:12D photoperiod control groups (Fig. 32). This kidney disease may also have been involved i n causing the large v a r i a t i o n s i n the GSI, oocyte diameter, and body weight measurements i n the l a t t e r months of development. The large numbers of a t r e t i c oocytes i n control as well as treatment groups during t h i s l a t t e r period (Fig. 30) i s the most suggest-54 ive evidence f o r an e f f e c t of t h i s disease on the reproductive and me-t a b o l i c processes. The e f f e c t of the constant 12L:12D photoperiod on gonadal matura-t i o n i n the long-term experiment was a slowing of the rate of increase i n maturation compared to the rate under the natural photoperiod. This means that gonadal maturation was maintained, although slowed, i n the absence of the seasonal daylength fluctuations of the natural photo-period and that some other c h a r a c t e r i s t i c of photoperiod may be opera-t i v e i n stimulating maturation. One such c h a r a c t e r i s t i c may be the t o t a l number of daylight hours. Baggerman (1957), working on the three-spined stickleback, found that sudden increases i n daylength res u l t e d i n grea-t e r a c c e l e r a t i o n of gonadal maturation than did gradual increases. The author concluded that the t o t a l amount of l i g h t i s more important than gradual changes. Larson (1972) found that a wild population of brook trout matured normally even though they were exposed to a sudden increase i n daylength from near darkness because of natural i c e cover during win-te r and spring. He suggested that the gradual increases i n the natural photoperiod are not necessary f o r gonad maturation and that t h i s sudden increase i n l i g h t , and perhaps temperature also, i n i t i a t e d gonad matura-t i o n . In the present study, the pink salmon may be responding, then, to a c e r t a i n number of hours of exposure to l i g h t which L i c h t (1971) terms a c r i t i c a l daylength. Since the male pink salmon were exposed to the constant 12L:12D photoperiod f o r s i x months p r i o r to the s t a r t of t e s t i s maturation, the 12 hours of l i g h t may be stimulatory i n i n i t i a t i n g sper-matogenesis and, therefore, should approximate the c r i t i c a l daylength. 55 Figure 34 indicates that under a natural photoperiod, the males begin maturing i n May, which establishes the c r i t i c a l daylength between approximately 14.5 and 16 hours. Under t h i s hypothesis, the males should not have begun maturing since the constant 12 hour daylength i s below t h i s normal c r i t i c a l daylength. Li c h t (1971) suggests that the c r i t i c a l or threshold daylength for t e s t i s regression i n the l i z a r d , Anolis  c a r o l i n e n s i s , can s h i f t , i n t h i s case upwards, to account f o r a regres-sive e f f e c t under a constant daylength which i s normally stimulatory under natural photoperiod. This mechanism may p o s s i b l y be operating i n male pink salmon. A decrease i n the threshold daylength to or below 12 hours would i n i t i a t e t e s t i s maturation. Presumably t h i s would also be the case i n the females, however, the constant 12L:12D photoperiod was begun a f t e r the s t a r t of ovarian maturation. The stimulatory e f f e c t of s p e c i f i c daylength on the i n i t i a t i o n and termination of gonad maturation i s evident i n many species of f i s h . Sundararaj and Sehgal (1970a) found that a long photoperiod induced ovarian recrudescence i n the c a t f i s h , Heteropneustes f o s s i l i s . Henderson (1963) found that normal ovarian maturation i s dependent upon long followed by short photoperiod i n the brook trout (Salvelinus f o n t i n a l i s ) and Baggerman (1957) found that the f i r s t and second phases of gonad maturation are stimulated by short and long photoperiod, r e s p e c t i v e l y , i n the three-spined stickleback. Combs et a l . (1959) observed advanced maturation with a short daylength i n the sockeye salmon as did S h i r a i s h i and Fukuda (1966) i n the kokanee (Oncorhynchus nerka), Hon-masu (Oncorhynchus masou), rainbow trout (Salmo  g a i r d n e r i ) , and brook trout (Salvelinus f o n t i n a l i s ) . In a l l these studies, 5 6 Figure 34: The daylengths of the natural photoperiod at 48° latitude (Fawcett, 1973). s a n o H iHonAva 57 the daylengths responsible f o r gonadal stimulation are c h a r a c t e r i s t i c of the stimulatory daylengths i n the natural photoperiod. In the present study, the only s l i g h t slowing of gonadal maturation i n the pink salmon may p a r t l y be a r e s u l t of the constant 12 hours of exposure since t h i s daylength approximates that present i n the natural photoperiod when both males and females are i n the f i n a l stages of gonad maturation (Fig. 34). The i n i t i a t i o n and maintenance of gonad maturation ;l ;n pink salmon under the constant 12L:12D photoperiod may also be the r e s u l t of an en-dogenous rhythm. This i s most e a s i l y investigated by maintaining f i s h i n t o t a l darkness and, thus, i n the absence of any photoperiodic cues. Pyle (1969) and Poston (1971) found that, i n brook trout spawning for the f i r s t time, exposure to t o t a l darkness since the f i n g e r l i n g stage had no e f f e c t on the time of f i n a l maturation. I n i t i a t i o n of gonadal maturation occurred without the short and long daylength sequences i n the brook trout as i t d i d i n the male pink salmon of the present study. Bullough (1940) was probably the f i r s t to suggest an i n t e r n a l rhythm i n f i s h . T otal darkness alone delayed the maturation period of male and female minnows (Phoxirius l a e v i s ) . Bullough also suggested that t h i s rhythm be-comes weaker with time i n darkness upon observing that a si n g l e b l i n d minnow had poorly developed gonads. Sundararaj and Sehgal (1970b) state that the ovarian cycle of the c a t f i s h , Heteropneustes f o s s i l i s , exhibits an endogenous rhythm under constant darkness which i s out of phase with that under natural photoperiod by one month. Poston (1971) and Poston and Livingston (1971) found that constant darkness delayed f i n a l matura-t i o n i n second spawning brook trout as did Pa:ng (1971) i n the sea water 58 adapted k i l l i f i s h , Fundulus h e t e r o c l i t u s . S h i r a i s h i and Fukuda (1966) found that the absence of l i g h t advanced the f i n a l maturation period i n male kokanee, Hon-masu, and rainbow and brook trout but had l i t t l e e f f e c t on the females. These studies i n d i c a t e that, under complete darkness, the endo-genous rhythm maintains gonadal maturation somewhat out of phase with that under the natural photoperiod. Ifaan i n t e r n a l rhythm i s also opera-t i v e i n maintaining maturation of the pink salmon i n the present study, the slowing of maturation observed under the constant 12 hour day-length may be a r e s u l t of this rhythm being out of phase. Unlike the s i t u a t i o n under complete darkness, a constant 12 hour daylength would provide an accurate photoperiodic cue o f exactly 12 hours every day. However, t h i s photoperiod, l i k e complete darkness, provides no cue of seasonal daylength f l u c t u a t i o n s and, therefore, should also show the existence of an endogenous rhythm. This difference i n the timing of the spawning period between the natural photoperiod and one devoid of season-a l cues may be due to a synchronizing function of the seasonal daylength f l u c t u a t i o n s (Sundararaj and Sehgal, 1970b). This function i s supported by studies which have advanced the maturation period by exposure to an accelerated natural photoperiod i n the brook trout (Hoover, 1937; Hoover and Hubbard, 1937; Hazard and Eddy, 1951; Corson, 1955; Henderson, 1963) and the three-spined stickleback (Baggerman, 1957). In a long-term study, Vanstone (unpublished data) retarded gonadal maturation i n pink salmon for one year beyond the normal two year cycle by using a delayed photoperiod. This suggests that the endogenous rhythm i s weak i n pink salmon and can e a s i l y be synchronized with a photoperiodic regime g r e a t l y 59 d i f f e r e n t from the natural photoperiod regime. As a summary of the photoperiodic response: the e f f e c t s of a constant 12 hour daylength and a comparison with the l i t e r a t u r e suggest that the i n i t i a t i o n , maintenance, and termination of gonadal maturation i n pink salmon are stimulated by a s p e c i f i c daylength and an endogenous rhythm and synchronized by the seasonal daylength f l u c t u a t i o n s . This ensures that reproduction occurs at the optimum time to maximize egg and f r y sur-v i v a l (Farner and F o l l e t t , 1966). Another environmental f a c t o r which can be important i n in f l u e n c i n g reproduction i s temperature. Photoperiod and temperature together s t i -mulate gonadal maturation i n the minnow (Bullough, 1939), four-spined stickleback (Apeltes quadracus) (Merriman and Schedl, 1941), three-spined stickleback (Baggerman, 1957), k i l l i f i s h (Fundulus confluentus) (Harrington, 1959), brook trout (Henderson, 1963), seaperch (Wiebe, 1968b), and green sunfish (Lepomis cyanellus) (Kaya and Hasler, 1972). In a b r i e f review, de Vlaming (1972) states that temperature i s important i n the photoperiodic response i n gonadal maturation i n most of the tel e o s t s studied. This evidence suggests that temperature may also be stimula-tory i n pink salmon and, therefore, may have had an influence on gonadal maturation i n those f i s h under the constant 12 hour daylength i n the present study. Vanstone (unpublished data), however, using a delayed photoperiod and n a t u r a l l y f l u c t u a t i n g water temperature, delayed gonadal maturation f o r one year i n pink salmon. This strongly suggests that photoperiod i s the most important f a c t o r and that temperature functions i n only a minor way i n gonadal maturation of pink salmon. The influence of the natural temperature fluctuations on the gonadal maturation of the 60 f i s h under the constant 12 hour daylength i s , therefore, thought to be very small. In the present study, methallibure completely i n h i b i t e d the gonadal maturation of only the male pink salmon. I f these males matured a f t e r withdrawal of methallibure treatment, as the l i t e r a t u r e suggests ( B i l l a r d et_ al_., 1971), then the sperm from these males could be used to f e r t i l i z e eggs from females of high escapement or "good" runs. These eggs could then be transplanted into r i v e r s with a very low or nonexist-ent escapement f o r that year. However, male pink salmon have been sex-u a l l y matured i n only one year using p a r t i a l l y p u r i f i e d salmon (Oncorhyn- chus tshawytscha) gonadotropin (SG-G100) (Funk and Donaldson, 1972). This makes a three year program impractical f o r obtaining mature males. However, as discussed i n the introduction, Funk et a l . (1973) were un-able to mature females within the year of hatching which suggests that the only a l t e r n a t i v e i s to delay the females i n a three year program. Ovarian development was not i n h i b i t e d at the dose of methallibure i n the present study, however, treatment with a higher dose begun ea r l y i n j u -v e n i l e l i f e may be successful i n delaying maturation of female pink s a l -mon for one year beyond t h e i r normal two year l i f e c ycle. This would en-able the genetic complement of the females, as well as that of the males, to be used to populate r i v e r s i n "poor" years. 61 SUMMARY At a dose of 0.10 mg./gm. body weight i n the long-term experiment, methallibure completely i n h i b i t e d spermatogenesis i n pink salmon by preventing the transformation of primary into secondary spermatogonia. Ovarian maturation, however, was slowed but not i n h i b i t e d . Treated ovaries possessed oocytes i n the o i l globule stage while control ovaries had oocytes i n the secondary yolk globule stage. One possible explana-t i o n f o r the difference i n e f f e c t between males and females i s that treatment was begun p r i o r to the s t a r t of spermatogenesis but a f t e r the s t a r t of v i t e l l o g e n e s i s . The sequential experiment investigated the ef f e c t of s t a r t i n g methallibure treatment at successive times p r i o r to the s t a r t of v i t e l l o g e n e s i s . At the 0.10 mg./gm. dose used, however, methallibure had no e f f e c t . Females, therefore, seem less s e n s i t i v e to th i s drug than do the males. In the p i l o t experiment, which was started immediately p r i o r to v i t e l l o g e n e s i s , the highest dose (1.0 mg./gm.), l i k e the dose used i n the experiments above (0.10 mg./gm.), f a i l e d to i n h i b i t ovarian development completely. With these r e s u l t s , i t was suggested that treatment e a r l y i n ju v e n i l e l i f e with a higher dose than that used i n the long-term experiment may i n h i b i t ovarian maturation. The dose i s l i m i -ted to below 1.0 mg./gm. since, i n unreported data, treatment with t h i s dose i n juveniles caused high mortality. Methallibure had an a n t i t h y r o i d a l e f f e c t under natural photoperiod but not under constant 12L:12D photoperiod i n the long-term experiment or at a high dose (1.0 mg./gm.). Kidney disease may, to some degree, be involved i n the e f f e c t under natural photoperiod. 62 Methallibure reduced the rate of increase i n body weight. The constant 12L:12D photoperiod slowed the rate of gonadal matura-t i o n of both males and females. Comparing these r e s u l t s with the l i t e r a -ture suggests that a s p e c i f i c daylength i n the natural photoperiod and an endogenous rhythm stimulate the i n i t i a t i o n , maintenance, and termina-ti o n of gonadal maturation and that the seasonal daylength fl u c t u a t i o n s function as a synchronizer. From t h i s study, only t e s t i c u l a r maturation could be delayed which could allow the male gene complement to be used i n populating a "poor" year, assuming that i n h i b i t i o n of t e s t i c u l a r maturation i s r e v e r s i b l e a f t e r methallibure treatment. 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