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An investigation of the induction of precocious sexual maturity in juvenile pink salmon Oncorhynchus… Funk, James D. 1972

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An Investigation of the Induction of Precocious Sexual Maturity in Juvenile Pink Salmon Oncorhynchus gorbuscha by James D. Funk B. Sc. (Hons.) University of Br i t i s h Columbia, A Thesis submitted in Partial Fulfilment of The Requirements of 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 February, 1972 I n 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 t h e r e q u i r e -ments f o r an advanced degree a t the U n i v e r s i t y o f B r i t i s h C olumbia, I agree t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l -a b l e f o r r e f e r e n c e and st u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y purposes may be g r a n t e d by t h e Head o f my Department o r by h i s r e p r e -s e n t a t i v e s . I t i s u n d e r s t o o n t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department o f Z o o l o g y  The U n i v e r s i t y o f B r i t i s h Columbia Vancouver 8, B.C. Date jfh^d Q-X^ |?73 Abstract This study was undertaken to determine whether the gonads of pink salmon (Oncorhynchus gorbuscha), could be stimu-lated with exogenous hormones to reproductive maturity one year e a r l i e r than normal. This procedure, i f successful, could be used as a method f o r repopulating ' o f f 1 year cycles of pink salmon i n numerous r i v e r s i n B r i t i s h Columbia and Washington. In the Juvenile males, complete sexual maturity was attained by September i n the year of hatching with t h r i c e -weekly treatments of 10.0 micrograms and of 1.0 micrograms salmon (Oncorhynchus tshawytscha) gonadotropin per gram body weight. H i s t o l o g i c a l examination of the precociously mature testes, and comparison with testes from uninjected controls re-pealed that the time of onset of the mitotic d i v i s i o n of sper-matogonia to form the primary spermatocyte, and the process of a c t i v e spermatogenesis were accelerated. At sexual maturity, a scattering of l o c a l i z a t i o n s of A 5 - 3 3 hydroxysteroid dehydro-genase a c t i v i t y was observed which corresponded to the d i s t r i -bution of the i n t e r s t i t i a l c e l l s . A larger stock of pink salmon, i n which i n j e c t i o n s were i n i t i a t e d 83 days l a t e r , de-veloped mature testes i n the same time i n t e r v a l as the normal-sized i n d i v i d u a l s . These gonads were four times larger, however. A small species difference i n the action of the gonadotropin preparation was found when comparing i t s e f f e c t on the G.S.I., rate of induction of sexual maturity, and 3 &-ol dehydrogenase a c t i v i t y of immature Oncorhynchus tshawytscha. i i In the females, the yolk stage was induced f i r s t in fish treated three times a week with 1.0 'yg/gram body weight salmon gonadotropin and 1.5 yg/gram body weight estradiol 1? g for 126 days. Oocytes containing yolk globules did not appear in pink salmon treated, with 1.0 p. g/gram body weight salmon-gonadotropin alone for a'further k-2 days. Estradiol l?s alone, o r in combination with salmon, gonadotropin at. a dosage of 15 ^ g/gram body weight inhibited vitellogenesis. Formation of oocytes 2 mm in diameter required seven and one half months of treatment with 1.0 yg/gram body weight, salmon gonadotropin and 1.5 y g/gram ^ody weight; estradiol 1?3 , and nine months of in-jections with l.Oy g/gram body weight salmon gonadotropin alone. Few large yolky oocytes were developed by any of the treatments. Large numbers of pre-ovulatory corpora atretica were observed _ in a l l of the treated f i s h . L i t t l e histochemically demonstrable A 5-36 hydroxysteroid dehydrogenase activity was present in ovaries from pink or spring salmon juveniles treated for 3 months with various dosages of ' salmon gonadotropin. The significance of the results in relation to the original problem are discussed. Table of Contents Page Abstract . * List of Figures.--List of Tables x Acknowledgements x i i i General Introduction x v Chapter I: The Testis 1 Introduction 1 Methods and Materials ^ Animals ^ Treatments °" Experimental Techniques (i) Injection Procedure ? ( i i ) Autopsy Procedure ^ ( i i i ) Histological Techniques 8 (iv) Analysis of the Histological Results 8 (v) Histochemical Techniques IP (vi) Analysis of the Histochemical Results H Results 12 Spermatogenesis 12 45-3 BHydroxysteroid Dehydrogenase 13 Lipids 1^ I n t e r s t i t i a l Cells 1^ Secondary Sex Characteristics 1^ Discussion 16 Table of Contents Page F i g u r e s 22 T a b l e s 31 Chapter 2 : The Ovary 39 I n t r o d u c t i o n 39 Methods and M a t e r i a l s 46 Animals 46 Treatments 47 Experimental Techniques 4-9 ( i ) , ( i i ) , ( i i i ) - same as i n Chapter 1 ( i v ) A n a l y s i s of the H i s t o l o g i c a l R e s u l t s . . 49 (a) Measurements 49 (b) H i s t o l o g i c a l A n a l y s i s 50 (v) H i s t o c h e m i c a l A n a l y s i s 53 (a) Procedure 53 (b) A n a l y s i s of R e s u l t s 53 R e s u l t s 5^ (a) Measurements 5^ ( i ) Gonadosomatic Index 54 ( i i ) Oocyte Diameter 55 ( i i i ) Oogenesis 57 (b) H i s t o c h e m i s t r y 62 ( i ) A 5-33 H y d r o x y s t e r o i d Dehydrogenase 62 ( i i ) L i p i d s 62 (c) Appearance 63 Table of Contents Page D i s c u s s i o n 65 General Summary 71 F i g u r e s 72 Tables 88 References 100 i i i L i s t of F i g u r e s F a c i n g Page 1. C r o s s - s e c t i o n of a t e s t i s from a zero c o n t r o l pink salmon j u v e n i l e ( 7 . 7 cm long) 22 2. Same s e c t i o n as i n F i g . 1, but a t a h i g h e r m a g n i f i c a t i o n . Example of Stage 1 t e s t i s 22 3 . T e s t i s of a s e x u a l l y mature (Stage 4) pink salmon j u v e n i l e (11.0 cm long) 22 4 . Same s e c t i o n as i n F i g . 3» but a t a hi g h e r m a g n i f i c a t i o n to show d e t a i l of spermatogenesis near the duct 22 5 . E p i t h e l i a l c e l l s l i n i n g the sperm duct 22 6. Regressed t e s t i s (Stage 6) of a j u v e n i l e male pink salmon 22 7. Stage 4 t e s t i s of an u n i n j e c t e d pink salmon (Body weight - 360 grams) 22 8. Same s e c t i o n as i n F i g . 7» but a h i g h e r m a g n i f i -c a t i o n to r e v e a l the d e t a i l s of the spermatogenesis i n the l o b u l e s 22 9 . I n t e r s t i t i a l c e l l s i n the t e s t i s of a p r e c o c i o u s l y mature pink salmon 23 10. Frozen s e c t i o n of the t e s t i s of a p r e c o c i o u s l y mature pink salmon s t a i n e d w i t h Sudan Black B f o r l i p i d s 23 11. H i s t o c h e m i c a l l y demonstrable A5-36 h y d r o x y s t e r o i d dehydrogenase a c t i v i t y i n a f r o z e n s e c t i o n of a t e s t i s from a s e x u a l l y mature pink salmon j u v e n i l e . . 23 i v L i s t of F i g u r e s F a c i n g Page 12. A5-3B h y d r o x y s t e r o i d dehydrogenase a c t i v i t y i n the t e s t i s of a p r e c o c i o u s l y mature s p r i n g salmon 23 13; 3 3-ol dehydrogenase a c t i v i t y i n the t e s t i s of a s e x u a l l y mature g o l d f i s h ( C a r a s s l u s a u r a t u s ) . . 23 14. The mean gonadosomatic index (G.S.I.) of male pink (Oncorhynchus gorbuscha) and s p r i n g (Oncorhynchus tshawytscha) salmon j u v e n i l e s t r e a t e d t h r e e times per week w i t h v a r i o u s dosages of salmon (Oncorhynchus tshawytscha) gonadotropin d u r i n g the summer of I 9 6 9 ^ 15 . The mean gonadosomatic index, (G.S.I.) of u n i n -j e c t e d male pink salmon maintained i n a q u a r i a throughout t h e i r l i f e 25 16 . Stages of t e s t i c u l a r m a t u r i t y of pink salmon j u v e n i l e s i n j e c t e d three times per week wi t h v a r i o u s dosages of salmon (Oncorhynchus  tshawytscha) gonadotropin 26 17. Stages of t e s t i c u l a r m a t u r i t y induced i n s p r i n g salmon j u v e n i l e s t r e a t e d three times per week with v a r i o u s dosages of salmon (Oncorhynchus  tshawytscha) gonadotropin 27 18. Stages of r e p r o d u c t i v e m a t u r i t y of u n i n j e c t e d pink salmon maintained i n a q u a r i a throughout t h e i r l i f e ,. c y c l e 28 V L i s t of Figures. Facing Page 19. The histochemically demonstrable a c t i v i t y of A 5 -3 8 hydroxysteroid dehydrogenase i n the testes of juvenile pink salmon injected three times per week with various dosages of salmon (Oncorhynchus tshawytscha) gonadotropin 29 2 0 . The histochemically demonstrable a c t i v i t y of A 5 - 3 3 hydroxysteroid dehydrogenase i n the testes of juvenile pink salmon injected three times per week with various dosages of salmon (Oncorhynchus tshawytscha)gonadotropin during the summer of 1969 30 2 1 . Ovary of a zero control juvenile spring salmon (5.4 cm long) 72 2 2 . Ovary of a juvenile spring salmon 1 0 . 0 cm i n length injected three times per week with f i s h saline f o r 84 days 72 2 3 . Ovary of a juvenile spring salmon ( 1 1 . 2 cm i n length) injected three times per week with 1 0 . 0 n g of salmon gonadotropin per gram body weight f o r 84 days 72 24. F o l l i c l e layer of an early perinucleolar stage oocyte from the same section as seen i n F i g . 2 3 . . 72 2 5 . The two central oocytes are i n the late perinucleolar stage 72 v i L i s t of F i g u r e s F a c i n g Page.' 2 6 . F o l l i c l e l a y e r of a l a t e p e r i n u c l e o l a r stage oocyte 72 27 . Oocyte i n the y o l k v e s i c l e stage from the ovary of a pink salmon ( 1 3 . 2 cm i n length) i n j e c t e d t h r e e times per week w i t h 1 0 . 0 y g salmon gonado-t r o p i n per gram body weight f o r 96 days 73 28. F o l l i c l e l a y e r s and t h e c a l l a y e r of the oocyte i n F i g . 27 7 3 2 9 . Oocyte i n the y o l k v e s i c l e stage s t a i n e d f o r l i p i d s w i t h Sudan B l a c k B 73 3 0 . H l s t o c h e m i c a l l y demonstrable A 5 -3 g h y d r o x y s t e r o i d dehydrogenase a c t i v i t y i n y o l k v e s i c l e stage oocytes from pink salmon i n j e c t e d three times per week wit h 1 0 . 0 y g salmon gonadotropin per gram body weight f o r 96 days „ 73 3 1 . Primary y o l k stage oocyte (pink salmon) 7 3 3 2 . F o l l i c l e and t h e c a l l a y e r s of the primary y o l k stage oocyte i n F i g . 12 73 3 3 . Primary y o l k stage oocyte s t a i n e d f o r l i p i d s w i t h O i l Red 0 , 73 3^. S e c t i o n of an ovary from a pink salmon j u v e n i l e (1^.6 cm i n length) i n j e c t e d three times per week wi t h 1 .0 y g salmon gonadotropin and 1 .5 P g e s t r a d i o l 17 3 per gram body weight f o r 126 days 7^ v i i L i s t of F i g u r e s F a c i n g Page 35. S e c t i o n of an ovary from a pink salmon j u v e n i l e (14.9 cm i n length) i n j e c t e d t hree times per week wit h 1.0 ig salmon gonadotropin per gram body 74 weight f o r 126 days 36. Frozen s e c t i o n of a post primary y o l k stage oocyte from a pink salmon (16.3 cm i n length) i n j e c t e d t h r e e times per week with 1.0 y g salmon gonado-t r o p i n and 1.5 yg e s t r a d i o l 17 8 per gram body weight 74 f o r 210 days. . , ' 37. F r o z e n s e c t i o n of a post primary y o l k stage oocyte from a pink salmon (17.0 cm i n length) i n j e c t e d t h r e e times per week w i t h 1.0 y g salmon gonadotro-74 p i n per gram body weight f o r 258 days 38. Ovary of a f i s h t r e a t e d i d e n t i c a l l y to that de-s c r i b e d i n F i g . 37. Note the p a u c i t y of oocytes 74 more advanced than the e a r l y p e r i n u c l e o l a r stage. . 39. S e c t i o n of an ovary from a pink salmon t r e a t e d w i t h 1.0 yg salmon gonadotropin per gram body weight f o r 126 days, and 1.0 y g e s t r a d i o l 17 8per 74 gram body weight f o r the l a s t 92 days ' 40. Gonadosomatlc index (G.S.I.) of female pink (Oncorhynchus gorbuscha) and s p r i n g (Oncorhynchus  tshawytscha) t r e a t e d three times per week w i t h v a r i o u s dosages of salmon (Oncorhynchus tshawytscha) gonadotropin d u r i n g the summer of 1969 v i i i L i s t of F i g u r e s F a c i n g Page 41. Gonadosomatic index (G.S.I.) of female pink salmon t r e a t e d three times per week wit h v a r i o u s combinations of salmon (Oncorhynchus  tshawytscha) gonadotropin and e s t r a d i o l 17 6 . . . . . 76 42. Gonadosomatic index (G.S.I.) of e x t r a - o r d i n a r i l y l a r g e female pink salmon t r e a t e d w i t h v a r i o u s dosages of salmon (Oncorhynchus tshawytscha) gonadotropin 77 43. Gonadosomatic index (G.S.I.) of u n i n j e c t e d female pink salmon maintained i n a q u a r i a throughout t h e i r l i f e c y c l e 78 44. Mean oocyte diameter (microns) of j u v e n i l e female pink salmon and s p r i n g salmon i n j e c t e d t h r e e times per week wit h v a r i o u s dosages of salmon (Oncorhynchus  tshawytscha) gonadotropin d u r i n g the summer of 1969 79 45. Mean oocyte diameter (microns) of pink salmon t r e a t e d three times per week with s e v e r a l combina-t i o n s of salmon (Oncorhynchus tshawytscha) gonado-t r o p i n and e s t r a d i o l 17 3 80 46. Mean oocyte diameter of e x t r a - o r d i n a r i l y l a r g e pink salmon o b t a i n e d from Dr. J . R. B r e t t , i n j e c t e d t h r e e times per week wit h combinations of v a r i o u s exogenous hormones: Salmon (Oncorhynchus tshawytscha) gonadotropin, e s t r a d i o l 37 g and progesterone 81 i x L i s t of F i g u r e s F a c i n g Page 4 7 . The mean oocyte diameter of oocytes from u n i n f e c t e d pink salmon maintained i n a q u a r i a throughout t h e i r l i f e c y c l e 82 48. Mean percentages of oocytes which comprised the o v a r i e s of j u v e n i l e pink salmon t r e a t e d w i t h v a r i o u s dosages of salmon gonadotropin . . 83 49. Mean percentages of oocytes which comprise the o v a r i e s of pink salmon t r e a t e d w i t h v a r i o u s combinations of salmon (Oncorhynchus tshawytscha) gonadotropin and e s t r a d i o l 27 B 84 5 0 . Mean percentages of oocytes which comprise the o v a r i e s of u n i n j e c t e d pink salmon maintained i n a q u a r i a throughout t h e i r l i f e 85 5 1 . The h i s t o c h e m i c a l l y demonstrable a c t i v i t y of A5 - 3 6 h y d r o x y s t e r o i d dehydrogenase i n the o v a r i e s of j u v e n i l e pink salmon i n j e c t e d t h r e e times per week w i t h v a r i o u s dosages of salmon (Oncorhynchus tshawytscha) gonadotropin d u r i n g the summer of 1969 . . 86 5 2 . I d e n t i c a l experiment to t h a t i n F i g u r e 51» but with s p r i n g salmon j u v e n i l e s 87 X L i s t of T a b l e s F a c i n g Page 1. Male pink salmon j u v e n i l e s t r e a t e d w i t h v a r i o u s dosages of salmon gonadotropin - mean f i s h l e n g t h , mean f i s h weight, mean f i x e d gonad weight, mean gonadosomatic index (G.S.I.) 31 2. Mean body weight, mean body l e n g t h , mean f i x e d t e s t i s weight, mean gonadosomatic index (G.S.I.) of j u v e n i l e s p r i n g salmon t r e a t e d w i t h v a r i o u s dosages of salmon gonadotropin 32 3 . Mean body weight, mean gonad weight, and mean gonadosomatic index (G.S.I.) of u n i n j e c t e d male pink salmon maintained i n a q u a r i a throughout t h e i r l i f e 33 4. The stage of t e s t i c u l a r maturation induced by v a r i o u s dosages of salmon gonadotropin i n pink salmon j u v e n i l e s 3^ 5 . The t e s t i c u l a r stage of s p r i n g salmon j u v e n i l e s i n j e c t e d w i t h v a r i o u s dosages of salmon gonado-t r o p i n 35 6 . The t e s t i c u l a r stage of u n i n j e c t e d pink salmon main-t a i n e d i n a q u a r i a throughout t h e i r l i f e 36 7. H i s t o c h e m i c a l l y demonstrable A 5-3 g h y d r o x y s t e r o i d dehydrogenase a c t i v i t y i n pink salmon t r e a t e d w i t h v a r i o u s dosages of salmon gonadotropin 37 8. H i s t o c h e m i c a l l y demonstrable A 5-3 g h y d r o x y s t e r o i d dehydrogenase a c t i v i t y i n the t e s t e s of s p r i n g salmon j u v e n i l e s t r e a t e d w i t h v a r i o u s dosages of salmon gonadotropin . 3 8 x i L i s t of T a b l e s F a c i n g Page 9 . Mean body weight, mean f i x e d ovary weight, mean gonadosomatic index (G.S.I.) of j u v e n i l e female pink salmon t r e a t e d w i t h v a r i o u s dosages of salmon gonadotropin . 88 1 0 . Mean body l e n g t h , mean body weight, mean f i x e d ovary weight, and mean gonadosomatic index (G.S.I.) of s p r i n g salmon t r e a t e d w i t h v a r i o u s dosages of salmon gonadotropin 89 11 . Mean body l e n g t h , mean body weight, mean gonad weight, and mean gonadosomatic index (G.S.I.) of female pink salmon t r e a t e d w i t h v a r i o u s dosages of gonadotropin and e s t r a d i o l 17g d u r i n g 1970/71 . . 90 12 . Mean body weight, mean body l e n g t h , mean gonad weight, and mean gonadosomatic index (G.S.I.) of e x t r a - o r d i n a r i l y l a r g e female pink salmon j u v e n i l e s t r e a t e d w i t h v a r i o u s dosages of salmon g o n a d o t r o p i c e s t r a d i o l 17 3 and progesterone .91 1 3 . Mean body weight, mean gonad weight, and mean gonadosomatic index (G.S.I.) of u n i n j e c t e d female pink salmon maintained i n a q u a r i a throughout t h e i r l i f e 92 14. Mean oocyte diameter of pink salmon j u v e n i l e s t r e a t e d w i t h v a r i o u s dosages of salmon gonadotropin 93 1 5 . Mean oocyte diameter of s p r i n g salmon i n j e c t e d w i t h v a r i o u s dosages of salmon gonadotropin 94 x i i L i s t of T a b l e s F a c i n g Page 16. Mean oocyte diameter of pink salmon i n j e c t e d w i t h v a r i o u s dosages of salmon gonadotropin and e s t r a -d i o l 17 6 95 17. Mean oocyte diameter of e x t r a - o r d i n a r i l y l a r g e pink salmon i n j e c t e d w i t h v a r i o u s dosages of salmon gonadotropin, e s t r a d i o l 17 6 t progesterone. . . . 96 18. Mean oocyte diameter of u n i n j e c t e d pink salmon maintained i n a q u a r i a throughout t h e i r l i f e . . . 97 19. The h i s t o c h e m i c a l l y demonstrable A5-38 hydroxy-s t e r o i d dehydrogenase a c t i v i t y i n the o v a r i e s of female pink salmon t r e a t e d w i t h v a r i o u s dosages of salmon gonadotropin. . . 98 20. H i s t o c h e m i c a l l y demonstrable A5-36 h y d r o x y s t e r o i d dehydrogenase a c t i v i t y i n the o v a r i e s of female s p r i n g salmon t r e a t e d w i t h v a r i o u s dosages of salmon gonadotropin 99 x i i i A cknowle dgements Special thanks are extended to my supervisor, Dr. E. M. Donaldson for suggesting the problem, and for his interest? advice, and constant encouragement throughout the course of the study. The author would also like to thank Dr. W. S. Hoar, Dr. A. M, Perks and Dr. P. Ford, for reading the manuscript, and offering constructive criticism , I would like to acknow-ledge the helpful suggestions of Dr. V/. S. Hoar, Mr. J. McBride, Dr. W. Vanstone, Dr. R. Brett, Dr. F. Yamazaki, Dr. N. E. Henderson, Dr. B. I. Sundararaj, and Dr. J. Davis during the course of the study. The technical assistance of Mrs. H. Dye in preparation of salmon pituitary gonadotropin, and of Mrs. K. Kramer in paraffin embedding specimens and preparing slides i s grateful-ly acknowledged. Mr. G. Walton sectioned many of the paraffin blocks and his photographs of whole gonads were useful for demonstrations. Mr. A. Dodimead, of the Pacific Environment Institute generously donated an office for use in the f i n a l writing of the thesis. My appreciation is extended to Mr. J. Culp, Mr. J. Tuerlings, and Mr. R. Corrigan for their help with the day-to-day problems. One of my most valuable experiences was the opportunity to work and interact with the scientists and staff of the Fisheries Research Board, West Vancouver and Vancouver Labora-tories, and of the Pacific Environment Institute. x i v The author would l i k e to thank Mrs. Norma Janssen f o r t y p i n g the rough and f i n a l d r a f t s of the t h e s i s . T h i s i n v e s t i g a t i o n was supported by F i s h e r i e s Research Board P r o j e c t No. 9261, p r o j e c t head Dr. E. M. Donaldson and a F i s h e r i e s Research Board a s s i s t a n t s h i p to the author. General Introduction xv Throughout the geographic range of pink salmon, (Oncorhynchus gorbuscha) there are rivers where there i s a pronounced difference in abundance between the even-and-odd numbered years (Ricker, 1962; Neave, 1 9 6 5 ) . In e,xtreme cases, a watershed may have large numbers of adult salmon spawning at 2-year intervals, and virtually none in the intervening years (Ricker, 1 9 6 2 ) . In the Fraser River-Puget Sound-Howe Sound region, for example, even year fish are virtually absent, whereas the rivers on the Queen Charlotte Islands and Northern B.C. mainland are characterized by a lack of pink salmon spawn-ning in off-numbered years,, (Neave, 1 9 6 2 ) . Both of these sys-tems support large runs in their respective "on" years. If previously empty cycles could be repopulated, there could be a great economic benefit to the fishing industry. In 1965» Neave summarized a l l previous literature con-cerning transplants of pink salmon to rivers where there was no spawning. He noted that no substantial self-sustaining run was known to exist. The most successful transplant on record at present i s the transfer of 1 ,866 adult pinks from Bear Harbour to Sashin Creek (Alaska) in 1 9 6 4 , to reinforce a run which had dwindled below 1 , 0 0 0 . Five thousand seven hundred and sixty-one fis h returned in 1966. Circumstantial evidence indicated that the majority of the returning f i s h were from the original transplant ( E l l i s , 1 9 6 9 ) . A recent publication detailed the results for three generations of pink salmon after transplant of pink salmon eggs to the Qualicum River in both 1963 and xvi 1964. The survivals of transplanted fis h were lower than those normally occurring (Walker and Lister, 1 9 7 1 ) . For a transplant to be successful, the introduced fis h should match as closely as possible the native stock in such characteristics as time of migration and spawning, and in the temperature of the native environment waters (Calaprice, 1969; McNeil e_t a l , 1 9 6 9 ) . It would appear that genetic factors play an important role in determining whether a donor stock w i l l survive in the new system. The r i g i d two-year l i f e cycle of pink salmon prevents the possibility of repopulating a barren cycle directly from fish present in the "on" year stock. Another means of employ-ing the genetic complement of the home stream f i s h would be to manipulate the reproductive cycle of pink salmon such that they spawn exactly one year earlier than normal. It was proposed that a preparation of spring salmon (Oncorhynchus tshawytscha) gonadotropin which had already been proved to reinduce a l l phases of sexual maturity in males and females of hypophysecto-mized goldfish (Carasslus auratus) — Yamazaki and Donaldson (1968) be used to accelerate the development of the gonads of immature pink salmon to sexual maturity by September of the year of hatching. If necessary, combinations with other sex hormones would be employed. A concurrent experiment on the spring salmon could reveal to what extent the gonadotropic hormone was species specific in i t s activity. 1 CHAPTER 1 The Testis Introduction There i s considerable evidence to indicate that p i -t u i t a r y gonadotropin controls gonadal maturation i n male t e l e -osts. Hypophysectomy had been shown to arrest spermatogenesis i n adult Pleuronectes platessa (Barr, 19^3 a), Fundulus  heteroclltus (Lofts, 1 9 6 6 ) , Heteropneustes f o s s l l l s (Sundarapaj and Nayyar, 1 9 6 7 ) , Carrasslus auratus (Yamazaki and Donaldson, 1 9 6 8 ) , Cymatogaster aggregata Gibbons (Wiebe, 1969) and P o e c l l i a  r e t i c u l a t a Peters (Pandey, 1969 a). Pandey ( I 9 6 9 b), working with the guppy ( P o e c l l i a r e t i c u l a t a Peters), successfully re-moved the p i t u i t a r y of a juvenile f i s h . The mitotic d i v i s i o n to form primary spermatocytes from spermatogonia was complete-l y suppressed, thereby precluding any further t e s t i c u l a r matur-ation. Previous research had demonstrated a stimulation of t e s t i c u l a r development i n juvenile salmonids with homoplastic preparations of p i t u i t a r y glands. Active spermatogenesis, and the shedding of motile spermatozoa was brought about i n immature trout (Salmo g a l r d n e r i l ) by administration i n cholesterol p e l l e t s of a preparation from the l y o p h i l i z e d p i t u i t a r i e s of spawning salmon (Oncorhynchus keta and Oncorhynchus tshawytscha) (Robertson and Ri n f r e t , 1957). Two weeks of inje c t i o n s into immature trout (Salmo g a l r d n e r i l ) of extracts from the p i t u i t a r y glands of the spring salmon (Oncorhynchus tshawytscha) acceler-ated the process of spermatogenesis i n the test f i s h (Schmidt 2 et a l , 1 9 6 5 ) . A regime c o n s i s t i n g of t h r e e equal doses per week a d m i n i s t e r e d on a l t e r n a t e days was found to be the optimum sequence f o r i n t r a p e r i t o n e a l i n j e c t i o n . F u r t h e r p u r i f i c a t i o n of t h e , p i t u i t a r y e x t r a c t used by Schmidt et a l . , ( 1 9 6 5 ) , which i n v o l v e d g e l f i l t r a t i o n on Sephadex G-100 c u l -minated i n a p a r t i a l l y p u r i f i e d p r e p a r a t i o n of Oncorhynchus  tshawytscha gonadotropin (SG-G100) d e s c r i b e d by Donaldson and Yamazaki ( I 9 6 8 ) and Donaldson et a l ( 1 9 7 1 ) . F o l l o w i n g hypophy-sectomy and t e s t i c u l a r r e g r e s s i o n , i n j e c t i o n of the aforemen-t i o n e d p r e p a r a t i o n i n t o a d u l t C a r a s s l u s a u r atus (Yamazaki and Donaldson, 1968) and Heteropneustes f o s s l l i s (Sundararaj et a l , , 1971 a) r e s u l t e d i n the complete r e s t o r a t i o n of sexual m a t u r i t y , and i n d u c t i o n of s p e r m i a t i o n . The p o s s i b i l i t y of a c c e l e r a t i o n of the development of the t e s t i s i n immature pink salmon (Oncorhynchus gorbuscha) to s e x u a l m a t u r i t y by September i n the y e a r of h a t c h i n g appeared to be very f e a s i b l e . I t was proposed t h a t s e v e r a l dosages of SG-G100 be i n j e c t e d i n t o the t e s t f i s h a t t h r i c e - w e e k l y i n t e r v a l s f o r a p e r i o d of 98 days. Samples every two weeks would r e v e a l the s u c c e s s i v e stages of gonadal m a t u r i t y . The h i s t o c h e m i c a l l y demonstrable a c t i v i t y of A5-3 6 h y d r o x y s t e r o i d dehydrogenase c o u l d i n d i c a t e the p o t e n t i a l of the t e s t e s f o r producing sex hormones. A s i m i l a r experiment to t h a t d e s c r i b e d above was per-formed u s i n g s p r i n g salmon (Oncorhynchus tshawytscha) f r y . The s p e c i e s s p e c i f i c i t y of the salmon gonadotropin p r e p a r a t i o n c o u l d be determined by a comparison of the r e s u l t s between pink salmon and s p r i n g salmon. 3 A l a r g e r pink salmon c o u l d p o s s i b l y develop a t e s t i s of g r e a t e r s i z e on s t i m u l a t i o n w i t h SG-G100. I t would thus c o n s t i t u t e a s u p e r i o r sperm p r o d u c t i o n u n i t to a s m a l l e r f i s h . Recent r e s e a r c h by B r e t t and Sutherland (1970) had shown t h a t y e a r l i n g sockeye salmon (Onchorhynchus nerka) c o u l d be grown from 22 to 100 g a f t e r two and one-half months of treatment w i t h p r e d e f i n e d optimum environmental and d i e t a r y c o n d i t i o n s . The c o n t r o l f i s h weighed 51 g. a f t e r the t e s t p e r i o d . Dr. B r e t t generously donated a stock of pink salmon j u v e n i l e s whose growth had been s t i m u l a t e d i n a s i m i l a r manner f o r th r e e months. Some i n f o r m a t i o n on the r e l a t i v e r a t e s of sexual m a t u r a t i o n , and r e l a t i v e s i z e s of the r e s u l t a n t t e s t e s c o u l d be obt a i n e d , although i n j e c t i o n of the l a r g e r pink salmon would be i n i t i a t e d l a t e r than i n the no r m a l - s i z e d f i s h . U n i n j e c t e d pink salmon maintained i n a q u a r i a throughout t h e i r l i f e c y c l e were used f o r comparison of the p a t t e r n and r a t e of sexual maturation a t v a r i o u s stages w i t h the f i s h whose r e p r o d u c t i v e m a t u r i t y had been a c c e l e r a t e d w i t h SG-G100. 4 Materials and Methods Animals The aquaria i n which experimental animals were held were supplied with running water ( s a l t or fresh), and were equipped with self- c l e a n i n g systems. The mean d a i l y tempera-ture of the s a l t water supply varied from 8° to 1 1 ° C, depend-ing upon the season. The l i g h t i n g i n the bu i l d i n g housing the animals was regulated to approximate the natural photoperiod. A l l f i s h were fed ad libitum a di e t consisting of beef l i v e r , beef heart, canned salmon, pablum, and sodium chloride. (Donaldson and McBride, 1 9 6 7 ) . Pink salmon f r y (Oncorhynchus gorbuscha) were obtained from the F i s h e r i e s Research Board, Nanaimo, i n mid-April I 9 6 9 . They were transported to a 2 0 0 0 - l i t r e f i b r e g l a s s aquarium at the F i s h e r i e s Research Board, West Vancouver s i t e , where they were kept i n s a l t water. From the o r i g i n a l stock of animals, 500 were subsequently moved into two outdoor 7 0 0 0 - 1 . aquaria. The remainder were divided into four groups of 75* kept i n separate 1 7 0 - 1 . f i b r e g l a s s aquaria and subjected to hormonal treatments as described In the following section. The animals i n the outdoor tanks were held f o r a study of the normal cycle of gonad development i n pink salmon, kept i n aquaria. They were maintained over the period of study by Dr. W. Vanstone and Mr. J . Culp, who used them as controls f o r a concurrent photoperiod study. Random thinning over the f o l -lowing nine-month i n t e r v a l reduced the numbers of the animals 5 to 53 per tank on June 3 0 , 1 9 7 0 . Development of a kidney-disease i n the animals at the end of August 1970 prevented a f i n a l sampling at the time of complete sexual maturity. Spring salmon f r y (Oncorhynchus tshawytscha) which had been hatched i n mid-February were obtained from the hatchery at Big Qualicum River, B.C. on May 8 , 19&9. The 600 f i s h were i n i t i a l l y kept i n a 2 0 0 0 - l i t r e f i b r e g l a s s aquarium supplied with freshwater. Four groups of 75 f i s h were subse-quently transferred to separate 170-litre aquaria and sub-jected to hormonal treatments as described i n the following section. The spring salmon were gradually acclimatized to s a l t water from May 20 to 22. A stock of 80 pink salmon f r y which were much larger than the normal size were obtained from Dr. J . R. Brett on August 2 3 , 1970. At that time they had a mean weight of 42.0 grams, as compared to 15.8 grams f o r the normal-sized pink salmon f r y at the West Vancouver Station. The f i s h were from eggs taken from Sweltzer Creek (near Cultus Lake), and hatched at the Fish e r i e s Research Board, Nanaimo Station. They had been maintained since June 1 at an elevated water temperature ( 1 5 ° C) and a photpperiod which lengthened by 5-minutes each day to 16 hours on September 1. These f i s h had been fed to satiety on a diet consisting of Oregon 116 wet p e l l e t s of an appropriate size (Westgate et a l , 1964). At the West Vancouver Laboratory, they were divided equally among f i v e 170-litre f i b r e g l a s s aquaria. As f a c i l i t i e s were not available to maintain t h e i r former conditions, they were kept on a normal photoperiod, at the temperature of the ambient seawater, and 6 were fed the diet described previously f o r the station. A considerable number of f i s h were subsequently removed because of a disease which induced formation of cataracts i n t h e i r eyes. Two further transfers of the experimental groups were made: to 4 5 0-litre f i b r e g l a s s aquaria, November 22, 1970, and to 2 0 0 0-litre f i b r e g l a s s aquaria, January 29, 1971. Treatments A p a r t i a l l y p u r i f i e d preparation of salmon (Oncorhynchus  tshawytscha) gonadotropin SG-G100 (Donaldson and Yamazaki, 1 9 6 8 ; Donaldson et a l , 1971), was used to induce precocious sexual maturity i n pink salmon and spring salmon males. Three dosages of the salmon gonadotropin preparation dissolved i n f i s h saline (Weibe, 1968) (0 .1 , 1.0, and 10 micrograms per gram body weight) plus a sa l i n e - i n j e c t e d control were studied i n experiments involving seventy-five f r y of each of the two species per treatment. These dosages were referred to i n t h i s thesis as treatment A, B, C, and Co respectively. The f i s h were injected i n t r a p e r i t o n e a l l y three times per week with a volume of 0.05 ml of the test solution. The period of treatment began June 10, 1969 f o r the spring salmon f r y and one week l a t e r f o r the pink salmon f r y . The experiments were terminated on September 1, and September 22, I 9 6 9 f o r the spring and pink salmon respectively. Following a zero control sample of ten f i s h , ten specimens were sampled from each treatment every two weeks. There were a t o t a l of s i x samples f o r each treatment i n the spring salmon experi-ment and seven samples f o r each treatment i n the pink salmon experiment. 7 The pink salmon fry in the outdoor tanks received no injections. Five samples of ten specimens were taken from this stock during their normal l i f e cycle. The last sample was ta-ken August 25, 1970. Twenty extra-large pink salmon juveniles of the stock obtained from the Fisheries Research Board, Nanaimo Laboratory, were used in an experiment designed to obtain information on the effect of size of the animal on the rate of induction of sexual maturity. Ten fish were treated with salmon gonadotro-pin dissolved in fish saline (Wiebe, 1968) at a dosage of 1 .0 micrograms per gram body weight. Ten control animals were in-jected with 0 . 1 0 ml of the test solution three times per week with a 25 g needle for a period of three months, starting September 2 , 1 9 7 0 . A l l of the specimens were sampled November 2 6 , 1 9 7 0 . Experimental Techniques (i) Injection Procedure The f i s h were anesthetized in the aquarium by addition with sti r r i n g of a stock solution of quinaldine (Locke, I 9 6 9 ) to make a total concentration of the aquarium water of 10 ppm. A l l of the f i s h in each tank were then netted and transferred to a basin containing a similar concentration of the anesthetic, and a constant supply of oxygen from an aerator. The fis h could be held safely in this solution for 15 minutes. Removal of the fis h from the basin was quickly followed by intraperi-tioneal injection of the test solution with a 27 g needle, 0 . 5 8 cm a n t e r i o r to the anus. The animals r e v i v e d w i t h i n one min-ute when r e t u r n e d to f r e s h water. ( i i ) Autopsy Procedure F i s h were r a p i d l y removed from a bucket s u p p l i e d with an a e r a t o r , and "damp d r i e d " . Wet weight was measured to the ne a r e s t t e n t h of a gram, and f o r k l e n g t h was measured to the near e s t m i l l i m e t e r . The animals were then k i l l e d by d e c a p i -t a t i o n , and the p e r i t o n e a l c a v i t y was opened. One gonad was weighed to the nea r e s t m i l l i g r a m , f r o z e n on dry i c e (-70° C) and s t o r e d a t -20° C. The othe r gonad was removed and f i x e d i n Bouin's s o l u t i o n . ( i i i ) H i s t o l o g i c a l Techniques F o l l o w i n g f i x a t i o n i n Bouin's s o l u t i o n f o r 24-hours, the t e s t e s were washed, dehydrated and embedded i n P a r a p l a s t -mp 56-57 C ( C u l l i n g , 1957). The specimens were then s e c t i o n e d a t 6 microns, and s t a i n e d w i t h Mayer's haematoxylin and e o s i n . ( i v ) A n a l y s i s of the H i s t o l o g i c a l R e s u l t s (a) Measurements D e s s i c a t i o n of the sm a l l c o n t r o l t e s t e s d u r i n g weighing n e c e s s i t a t e d a comparison of gonad weight based on f i x e d r a t h e r than wet weights. One f i x e d gonad from each f i s h was weighed to the n e a r e s t m i l l i g r a m . The gonadosomatic index was d e t e r -mined as f o l l o v r s : 9 r Q T _ weight of one gonad X 2 - body weight X 1 0 0 (b) H i s t o l o g i c a l A n a l y s i s The stage of sexual m a t u r i t y of the t e s t i s was d e t e r -mined from examination of a s i n g l e median s a g g i t a l s e c t i o n from each specimen. The degree of m a t u r i t y was estimated v i s u a l l y on the b a s i s of the f o l l o w i n g s i x d i s t i n c t stages of t e s t i -c u l a r development: (1) Spermatogonia o n l y a r e prese n t ( F i g s . 1 and 2). (2) Spermatogenesis has been i n i t i a t e d . The predomi-nant c e l l types a r e primary spermatocytes. ( 3 ) Primary spermatocytes, secondary spermatocytes, and spermatids a r e a l l present. ( 4 ) Spermatogenesis i s proceeding a c t i v e l y ; spermato-zoa a r e present i n the l o b u l e lamina. In t h i s stage the t e s t i s reaches i t s maximum s i z e ( F i g s . 7 and 8). (5) The t e s t i s i s now f u n c t i o n a l l y mature. There i s an e x t e n s i v e breakdown of the t e s t i c u l a r morphology, and spermatozoa l i e f r e e i n the sperm duct. Semen can be s t r i p p e d from the animal a t t h i s stage ( F i g s . 3 and 4 ) . (6) The t e s t i s i s completely r e g r e s s e d ( F i g . 6). These stages of t e s t i c u l a r development are s i m i l a r to those employed by Nayar and Sundararaj (1970) i n t h e i r a s s e s s -ment of the r e p r o d u c t i v e m a t u r i t y of the male c a t f i s h , 10 H e t e r o p n e u s t e s f o s s l l l s ( B l o c h ) . The st a g e of f u n c t i o n a l m a t u r i t y c o r r e s p o n d s t o t h a t d e s c r i b e d by Henderson (1962) i n the e a s t e r n brook t r o u t S a l v e l l n u s f o n t i n a l i s - ( M i t c h e l l ) . Graphs were p r e p a r e d showing t h e mean s t a g e o f r e p r o -d u c t i v e m a t u r i t y a t each time o f s a m p l i n g f o r a l l of the p i n k and s p r i n g salmon s t u d i e d , (v) H i s t o c h e m i c a l Techniques The samples from the study on the e f f e c t of salmon g o n a d o t r o p i n on m a t u r a t i o n of t h e t e s t i s were i n v e s t i g a t e d h i s t o c h e m i c a l l y f o r t h e pr e s e n c e of A5-3 8 h y d r o x y s t e r o i d dehydrogenase (3 8-ol dehydrogenase). The f r o z e n t e s t e s s t o r e d a t -20° C were s e c t i o n e d a t 10 m i c r o n s on t h e c r y o s t a t and s t a i n e d u s i n g B a r a ' s (I965) p r o c e d u r e . T h i s method was used s u c c e s s f u l l y t o demonstrate 3 8-ol dehydrogenase a c t i v i t y i n t he g o l d f i s h C a r a s s i u s a u r a t u s L. by Yamazaki and Donaldson (I969). and i s c o n s i d e r e d t o be a s e n s i t i v e a s s a y . D e h y d r o e p i a n d r o s t e r o n e (5-andosten-38-ol-17one; C a l b i o c h e m ) . was the o n l y s u b s t r a t e used.' C o n t r o l s e c t i o n s were i n c u b a t e d : (a) w i t h no s u b s t r a t e , and (b) w i t h reduced- n i c o t i n a m i d e a d e n i n e d i n u c l e o t i d e (NADH; Calbiochem.) t o t e s t f o r d l a p h o r a s e a c t i v i t y . S e c t i o n s o f r a t a d r e n a l and g o l d f i s h ( C a r a s s i u s  a u r a t u s ) t e s t i s were i n c u b a t e d p e r i o d i c a l l y t o check t h e e x p e r i m e n t a l p r o c e d u r e . A t e m p e r a t u r e of 37°C was found t o g i v e t h e b e s t r e s u l t s a f t e r one hour of i n c u b a t i o n , when compared t o tempera-t u r e s of 10, 20, and 4 5 ° C. L i t t l e " a d d i t i o n a l a c t i v i t y was o b s e r v e d when s e c t i o n s were I n c u b a t e d a t 37° C f o r more t h a n 11 one hour. The sections were not washed with acetone (to remove l i p i d s ) following incubation, as t h i s procedure reduces the s e n s i t i v i t y of the assay ( B a i l l i e e% a l , 1966). Frozen testes from selected samples were sectioned on the cryostat at 10 microns and stained with Sudan Black B and O i l Red 0 for l i p i d s . (Chayen et a l , 1969). (vi) Analysis of the Histochemical Results A scale i n which v i s u a l estimation of the degree of a c t i v i t y of A5-3 g hydroxysteroid dehydrogenase i n the mature male gold f i s h (Carassius auratus L.) was registered as 5 was used to quantify the formazan deposits obtained i n the frozen testes. A c t i v i t y of one represented an intense pink reaction with no d i s t i n c t regions of l o c a l i z a t i o n of formazan. A c t i v i t y of 2 was considered to represent a scattering of small deposits. Thereafter, a doubling of a c t i v i t y was recorded as an a d d i t i o n a l stage. Specimens which were intermediate between 2 points were evaluated to the nearest 1/2 stage. 12 Results (a) Spermatogenesis The development of the t e s t i s i n the pink and spring salmon injected with various dosages of salmon gonadotropin was compared with one another, and with the cycle of normal maturation i n uninfected pink salmon kept i n aquaria. The r e s u l t s from the experiment i n which pink salmon were injected with a salmon gonadotropin dosage regime corresponding to Treatment B were also considered. A l l of the f i s h administered salmon gonadotropin showed a stimulation of spermatogenesis. Pink salmon f r y of both treatments A and B were found to undergo the complete cycle of gonad develppment (ending i n t e s t i c u l a r regression, F i g . 6), i n a period of 98 days (Fig. 16). The spring salmon f r y , by comparison, did not reach a stage of functional maturity a f t e r 84 days of treatment (Fig. 17); however, they did have a higher G.S.I, at comparable stages of sexual maturation (Fig. 14). No d i r e c t comparison can be made between these groups and the larger f i s h from the Fis h e r i e s Research Board, Nanaimo Laboratory, be-cause treatments were i n i t i a t e d i n the l a t t e r group 78 days l a t e r than i n the normal-sized pink salmon f r y . The larger pink salmon were sampled a f t e r 84 days of i n j e c t i o n s , and were found to have matured sexually i n the same time i n t e r v a l as required f o r the smaller animals. During the normal cycle of t e s t i c u l a r maturation i n pink salmon a dramatic increase was recorded i n the rate of gonad development and G.S.I, following completion of the meiotic 13 division to form primary spermatocytes from spermatogonia (Figs. 15 and 18) i t was early July, and the f i s h were in their second year of l i f e . A corresponding observation was made on the testis of f i s h in both treatments A and B for the normal-sized pink and spring salmon fry, but the time interval to appearance of spermatozoa was shorter. It would therefore appear that both the time of onset of the mitotic division to form primary spermatocytes, and the process of active spermato-genesis were accelerated in f i s h treated with salmon gonadotro-pin. (b) A 5-3 8 Hydroxysteroid Dehydrogenase Formazan deposits, indicating A 5 - 3 3 hydroxysteroid dehydrogenase activity were recorded in testes from normal-sized pink salmon (treatments A and B); the activity was maxi-mal during the period when spermatogenesis was active and spermatozoa were present (Figs. 11 and 19). The localizations were scattered when compared to the reaction obtained in the testes of the mature goldfish (Fig. 13). A slight activity of 3 3-ol dehydrogenase in the seminiferous tubules was obser-ved. The precociously mature salmon testis had less histo-chemically demonstrable activity than did the pink salmon mentioned above (Figs. 12 and 20). Few deposits of formazan were found. The reaction was more intense in the i n t e r s t i t i a l regions than in the lobules (Fig. 12). Controls lacking dehydroepiandrosterone in the incu-bation medium did give a positive reaction in some instances. The activity was always less than that observed in a corresponding 14 medium with substrate. No reaction was ever detected i n con-t r o l sections incubated i n a medium which lacked NADH. Addition of NADH to the reaction medium resulted i n a l l cases i n the formation of many heavy l o c a l i z a t i o n s of formazan i n the i n t e r s t i t i a l region and within the lobules. Therefore, NADH diaphorase was not l i m i t i n g . (c) Lipids Frozen sections of testes from the pink salmon f r y i n -jected with salmon gonadotropin f o r 42 and 84 days were stained with O i l Red 0 and Sudan Black B. No reaction was obtained with O i l Red 0 whereas the r e s u l t s were po s i t i v e when Sudan Black B was employed. The l i p i d s were l o c a l i z e d i n the i n t e r -s t i t i a l regions (Fig. 10). (d) I n t e r s t i t i a l C e l l s I n t e r s t i t i a l c e l l s were found i n a l l of the experimental groups. At sexual maturity, i n t e r s t i t i a l c e l l s were not abun-dant i n any of the treated pink salmon, or i n the uninfected f i s h maturing at a normal rate. Some concentrations of c e l l s were observed (Fig. 9)* but they were widely scattered. By comparison, the precociously developed spring salmon testes con-tained even fewer i n t e r s t i t i a l c e l l s . This observation correlates d i r e c t l y with previous information that 3 8-ol dehydrogenase a c t i v i t y was correspondingly lower i n the spring salmon. (e) Secondary Sex Characteristics The c h a r a c t e r i s t i c humping of the back and the b r i l l i a n t 15 red c o l o r a t i o n of the s i d e s of the pink salmon approaching sexual m a t u r i t y was not apparent i n the pink salmon i n j e c t e d with salmon gonadotropin. There was, t h e r e f o r e , no means of d i s t i n g u i s h i n g between males and females, or t r e a t e d and con-t r o l f i s h by the appearance of the animals. A s i m i l a r pro-blem was encountered w i t h the i n j e c t e d s p r i n g salmon f r y . The secondary sex c h a r a c t e r i s t i c s were present to a much l e s s e r extent than normal d u r i n g the f i n a l stages of t e s t i c u l a r m a t u r i t y i n the u n i n j e c t e d pink salmon maintained i n a q u a r i a . 16 Discussion The t e s t i s of the salmon gonadotropin-injected pink salmon passed through a l l stages of gonad maturation, including terminal t e s t i c u l a r regression. When the f i s h were functionally-mature, semen could be stripped from the testes by stroking the abdomen i n an antero-posterior d i r e c t i o n . The r e s u l t s indicated that the time of sexual maturity of male pink juveniles could be manipulated to coincide with that of any stock of natural spawn-ing f i s h . The rate of attainment of the maximum G.S.I, showed a dose-response rel a t i o n s h i p with the amount of salmon gonadotro-pin administered. A comparable co r r e l a t i o n was not found for the stage of sexual maturity induced between the two highest dosages. A dosage of 1.0 y g salmon gonadotropin per gram body weight was as e f f e c t i v e as a ten-fold greater quantity of hor-mone i n developing testes i n time to f e r t i l i z e pink salmon ova at the normal time of spawning. A comparison between testes from the treated pink s a l -mon, and those which developed normally revealed the stages of maturation to be accelerated i n the precociously mature f i s h . The time of appearance of primary spermatocytes was advanced by eleven months i n juvenile pink salmon injected with salmon gonadotropin. These re s u l t s agreed with previous research on several representative teleosts by Barr ( I 9 6 3 a) Lofts et a l ( I 9 6 6 ) , Ahsan ( I 9 6 6 ) , Sundararaj and Nayyar ( I 9 6 7 ) , Yamazaki and Donaldson ( 1 9 6 8 ) , and Pandey (1969 a, b). These studies demonstrated that the m i t o t i c d i v i s i o n between spermatogonia 17 and the primary spermatocyte was controlled by p i t u i t a r y hor-mones. The probable reason for the marked difference i n weight between the precociously developed testes and the sex organs from the uninfected f i s h at sexual maturity (0.2 grams v. 31.5 grans respectively) was the e a r l i e r t r a n s i t i o n from spermatogonia to spermatocytes i n the treated f i s h . Further d i v i s i o n s of the spermatogonia would thereby be i n h i b i t e d , and the eventual num-ber of more advanced germinal c e l l s reduced. The progress of active spermatogenesis to the production of mature spermatozoa occurred i n one half the normal time i n the precociously developed pink salmon. This cannot be att r i b u t e d to a d i r e c t e f f e c t of gonadotropin, since i t could have been a manifestation of androgen a c t i v i t y . Androgen i n j e c t i o n r e s u l t -ed i n a stimulation of spermatogenesis i n hypophysectomized adult Lebistes r e t i c u l a t u s (Eversole, 19^1) , Fundulus heteroclltus (Lofts et a l , 1966) Heteropneustes f o s s l l l s (Sundararaj and Nayyar, I 9 6 7 ) , and P b e c l l i a r e t i c u l a t a Peters (Pandey, 1969 a). The re s u l t s are variable, however, because methyl testerone i n the dosages administered did not stimulate spermatogenesis i n the t e s t i s of the methallibure-treated adult Cymatogaster  aggregata Gibbons (Wiebe, 1969) or i n juvenile P o e c i l l a  r e t i c u l a t a Peters (Pandey, 1969 b). Pink salmon which were three times larger than normal-sized specimens on September 2, 1970, developed mature testes a f t e r treatment with salmon gonadotropin i n the same time i n t e r v a l as the smaller animals. However, the gonad i t s e l f was larger (0.9 grams v. 0.2 grams). Direct comparisons cannot 18 be made between these two groups because i n j e c t i o n s were i n i t i a t e d i n the l a r g e r . : f i s h 83 days l a t e r than i n the normal-s i z e d animals. There were d i f f e r e n c e s between the e f f e c t s of the salmon gonadotropin p r e p a r a t i o n on the donor s p e c i e s and on the pink salmon. The maximum G.S.I, of the t e s t s p r i n g salmon f r y was approximately two and one h a l f times l a r g e r than t h a t of pink salmon j u v e n i l e s t r e a t e d i n a corresponding manner. Spermatogenesis was r e l a t i v e l y more advanced In the pink salmon a f t e r 84 days of treatment, however. The r e s u l t s may be p u r e l y q u a n t i t a t i v e because of a s m a l l s p e c i e s d i f f e r -ence i n the response to the hormone. The r a t e of i n d u c t i o n of a c t i v e spermatogenesis and the time of appearance of spermatozoa i n the t r e a t e d j u v e n i l e pink and s p r i n g salmon compared f a v o r a b l y w i t h s i m i l a r r e s e a r c h on ot h e r salmonids. A f t e r two weeks of i n j e c t i o n of e x t r a c t s from Oncorhynchus tshawytscha p i t u i t a r i e s i n t o immature t r o u t (Salmo g a i r d n e r l l ) 1 3 . 5 cm i n l e n g t h , spermatogenesis was a c c e l e r a t e d t o the appearance of primary and secondary sper-matocytes (Schmidt et a l , 1 9 6 5 ) . I n j e c t i o n of f i n g e r l i n g coho salmon (Oncorhynchus k l s u t c h ) f o r t h r e e weeks w i t h p i t u i t a r y homogenate from spawning a d u l t coho s t i m u l a t e d t h e - m i t o t i c d i v i s i o n of the spermatogonia; no spermatocytes c o u l d be found (Chestnut, 1 9 7 0 ) . A d m i n i s t r a t i o n of an e x t r a c t from the p i t u i t a r i e s of spawning salmon (Oncorhynchus ke t a and Oncorhynchus tshawytscha) i n c h o l e s t e r o l p e l l e t s to 6^-month o l d rainbow t r o u t (Salmo g a i r d n e r i i ) r e s u l t e d i n the shedding of spermatozoa two months l a t e r (Robertson and R i n f r e t , 1 9 5 7 ) . 19 No h i s t o l o g y of these t e s t e s was performed. There a r e two areas i n which the androgen s e c r e t i n g c e l l s of the t e s t i s of t e l e o s t s are l o c a t e d : the i n t e r s t i t i a l and lobule-boundary r e g i o n s ( M a r s h a l l , I 9 6 O ; Hoar, 1 9 6 5 ) . There i s c o n s i d e r a b l e v a r i a b i l i t y from one salmonid to another as to the s i t e of the t e s t i c u l a r endocrine t i s s u e . I n t e r s t i t i a l c e l l s were observed with the l i g h t microscope i n the immature t e s t e s of Salmo g a i r d n e r i i (Oota and Yamamoto, 1 9 6 6 ) . These c e l l s a p p a r e n t l y developed i n t o g l a n d u l a r - a p p e a r i n g c e l l s i n the i n t e r s t i t i a l and lobule-boundary r e g i o n s i n mature Salmo  g a i r d n e r i i (Robertson, 1 9 5 8 ) . A s i m i l a r o b s e r v a t i o n was made on Salvelirius f o n t i n a l i s by Henderson, 1962. O'Halloran and I d l e r (1970) s t u d i e d the areas of h i s t o c h e m i c a l l y demonstrable A 5-3 8 h y d r o x y s t e r o i d dehydrogenase a c t i v i t y and the d e p o s i t i o n of c y a n o p h i l l i c l i p i d s i n Salmo s a l a r to r e v e a l p o t e n t i a l s i t e s of s t e r o i d o g e n e s i s . The lobule-boundary c e l l s were d e f i n i t e l y i n d i c a t e d as the s i t e s of endocrine a c t i v i t y . By c o n t r a s t , Chestnut (1970 determined t h a t the i n t e r s t i t i a l c e l l s of mature Oncorhynchus k i s u t c h were the r e g i o n where A 5-3f3 - o l dehydrogenase a c t i v i t y was l o c a l i z e d . In the present study, i n t e r s t i t i a l c e l l s were observed i n s m a l l c l u s t e r s w idely s c a t t e r e d throughout the t e s t e s of a l l mature specimens. The s p r i n g salmon had r e l a t i v e l y fewer n e s t s of i n t e r s t i t i a l c e l l s than d i d the pink salmon. P i t u i t a r y gonadotropin had been found i n s e v e r a l t e l e o s t s to c o n t r o l the A 5 -3 8 h y d r o s t e r o i d dehydrogenase a c t i v i t y i n the t e s t i s . Hypophysectomy of a d u l t C a r a s s l u s 20 auratus caused regression of the i n t e r s t i t i a l c e l l s , and a rapid f a l l i n the a c t i v i t y of 3 g-ol dehydrogenase by 25 days a f t e r the operation, (Yamazaki and Donaldson, 19^9). Replace-ment therapy with salmon gonadotropin restored the normal l e v e l of enzymic a c t i v i t y . Testes from Cymatogaster aggregata Gibbons were incubated " i n v i t r o " with mammalian l u t e i n i z i n g hormone (Wiebe, 1969 a). The amount of histochemically demonstrable A 5-3 8hydroxysteroid dehydrogenase a c t i v i t y increased noticeably i n the test gonads a f t e r four hours of incubation. Evidence of a weak a c t i v i t y of 3g-ol dehydrogenase i n the i n t e r s t i t i a l area following the i n j e c t i o n f o r three weeks of f i n g e r l i n g Oncorhynchus  kisutch with extracts of p i t u i t a r y homogenates from spawning adults was reported by Chestnut (1970). In the present experi-ment, an a c t i v i t y of 3g-ol dehydrogenase of two (on a scale where the reaction i n the mature Carassius auratus t e s t i s was taken as 5) was observed i n the testes of precociously mature pink salmon. Few deposits of formazan could be seen i n the t e s t i s of sexually mature spring salmon juveniles; these data correlated d i r e c t l y with the small number of i n t e r s t i t i a l c e l l s evident. The secondary sex c h a r a c t e r i s t i c s t y p i c a l of spawning adult pink salmon and spring salmon were not manifested i n precociously mature members of these same species. This ob-servation confounded any attempt to re l a t e the apparent increase i n s t e r o i d dehydrogenase a c t i v i t y i n treated f i s h to production of androgen by the t e s t i s . I t i s important to mention that the secondary sex c h a r a c t e r i s t i c s of the uninjected pink salmon 21 maintained i n a q u a r i a throughout t h e i r l i f e c y c l e were much l e s s marked than normal a t sexual m a t u r i t y . Perhaps t h i s e f f e c t was a r e s u l t of the s t r e s s of confinement. A l l of the s e x u a l l y mature specimens s t u d i e d had h y p e r t r o p h i e d e p i t h e l i a l c e l l s i n the duct ( F i g . 5 ) . A sub-stance exuded from them which may have p r o v i d e d n u t r i t i o n f o r the spermatozoa. These c e l l s compared i n t h e i r morphology to those r e p o r t e d by Yamazaki and Donaldson ( 1968) i n hypo-physectomized C a r a s s i u s a u r a t u s i n j e c t e d w i t h salmon gonado-t r o p i n . F o r a separate c o o p e r a t i v e experiment i n the summer of 1970 w i t h F. C. W i t h l e r and R. B. Morley of the F i s h e r i e s Research Board, Nanaimo Lab o r a t o r y , the author developed f o u r t e e n pink salmon males which were s e x u a l l y mature e x a c t l y one year e a r l i e r than normal. These f i s h were t r e a t e d w i t h a dosage regime which corresponded to treatment B of the 1969 experiment. The f e r t i l i z i n g c a p a c i t y of the m i l t from the t e s t f i s h was compared to t h a t of the m i l t from w i l d Lower Babine R i v e r pink males when equal volumes of both were mixed w i t h 100 ova from Lower Babine females. There were no marked d i f f e r e n c e s i n p r o p o r t i o n s of ova f e r t i l i z e d , i n s u r v i v a l to hat c h i n g * or i n numbers of deformed l a r v a e . The spermatozoa were t h e r e f o r e v i a b l e . These r e s u l t s a r e r e p o r t e d i n Donaldson et a l , 1971 . 22 F i g u r e 1: C r o s s - s e c t i o n of a t e s t i s from a zero c o n t r o l pink salmon j u v e n i l e ( 7 . 7 cm l o n g ) . Haematoxylin and e o s i n X 2 5 0 . F i g u r e 2; Same s e c t i o n as i n F i g . 1 , but a t a h i g h e r m a g n i f i c a t t i o n . Example of stage 1 t e s t i s . Haematoxylin and e o s i n X 6 0 0 . F i g u r e 3 : T e s t i s of a s e x u a l l y mature (stage 4) pink salmon j u -v e n i l e ( 1 1 . 0 cm l o n g ) . Haematoxylin and e o s i n X 2 0 . F i g u r e 4: Same s e c t i o n as i n F i g . 3 "but a t a h i g h e r m a g n i f i c a -t i o n to show d e t a i l of spermatogenesis near the duct. Haematoxy-l i n and e o s i n X 2 5 0 . F i g u r e 5 : E p i t h e l i a l c e l l s l i n i n g the sperm duct. Note the s e c r e t i o n of a substance which p o s s i b l y n o u r i s h e s the spermato-zoa. Haematoxylin and e o s i n X 6 0 0 . F i g u r e 6 : Regressed t e s t i s (stage 6) of a j u v e n i l e male pink salmon. Haematoxylin and e o s i n X 300. F i g u r e Stage 4 t e s t i s of an u n i n j e c t e d pink salmon (body weight 360 grams). Haematoxylin and e o s i n X 40. F i g u r e 8 : Same s e c t i o n as i n F i g . 7 . but a t a h i g h e r m a g n i f i c a -t i o n to r e v e a l the d e t a i l s of spermatogenesis i n the l o b u l e s . Haematoxylin and e o s i n X 2 5 0 . 23 F i g u r e 9 : I n t e r s t i t i a l c e l l s i n the t e s t i s of a p r e c o c i o u s l y mature pink salmon (body l e n g t h 1 2 . 3 cm). Haematoxylin and e o s i n X 1 5 0 0 . F i g u r e 1 0 ; F r o z e n s e c t i o n of the t e s t i s of a p r e c o c i o u s l y mature pink salmon s t a i n e d w i t h Sudan B l a c k B f o r l i p i d s , (body l e n g t h 1 2 . 3 cm) X 6 0 0 . F i g u r e 11 ; H i s t o c h e m i c a l l y demonstrable A 5 - 3 8 h y d r o x y s t e r o i d dehydrogenase a c t i v i t y i n a f r o z e n s e c t i o n of a t e s t i s from a s e x u a l l y mature pink salmon j u v e n i l e , (body l e n g t h 1 2 . 3 cm). Dehydroepiandrosterone was used as the s u b s t r a t e , and n i t r o - B . T . was the t e t r a z o l i u m s a l t . D e p o s i t s a re gran u l e s of formazan. X 2 0 0 . F i g u r e 12 : A 5 -3 8 h y d r o x y s t e r o i d dehydrogenase a c t i v i t y i n the t e s t i s of a p r e c o c i o u s l y mature s p r i n g salmon (body l e n g t h 9 . 1 cm). The i n c u b a t i o n medium was i d e n t i c a l to t h a t d e s c r i b e d i n F i g u r e 11 ; X 2 0 0 . F i g u r e 13 : 3 g - o l dehydrogenase a c t i v i t y i n the t e s t i s of a s e x u a l -l y mature g o l d f i s h ( C a r a s s i u s a u r a t u s ) . The i n c u b a t i o n medium was i d e n t i c a l to t h a t d e s c r i b e d f o r F i g u r e 11 ; X 2 0 0 . 2k F i g u r e lk: The mean gonado-somatlc index (G.S.I.) of male pink (Oncorhynchus gorbuscha) and s p r i n g (Oncorhynchus tshawytscha) salmon j u v e n i l e s t r e a t e d t h r i c e weekly w i t h v a r i o u s dosages of salmon (Oncorhynchus tshawytscha) gonadotropin d u r i n g the summer of 19&9. Numbers of f i s h and standard d e v i a t i o n a r e i n Ta b l e I and I I . 4 10 0-i o / / / / MALE SALMON / 8 0 _ | O. tschawytscha o ~ j 10pg/g BW O. gorbuscha — • — - J / / / / i f 25 F i g u r e 15: The mean gonado-somatic index (G.S.I.) of u n i n f e c t e d male pink salmon maintained i n a q u a r i a throughout t h e i r l i f e . D e t a i l e d data i n Table I I I . N°V^ 1 DEC. J JAN. I FEB. I MARCH " A P R I L ' M A Y ' JUNE ' JULY I AUG 1 1 9 6 9 I 1 9 7 0 26 F i g u r e 16: Stages of t e s t i c u l a r m a t u r i t y of pink salmon j u v e n i l e s i n j e c t e d t h r e e times per week with v a r i o u s dosages of salmon (Oncorhynchus tshawytscha) gonadotropin. Values taken from T a b l e IV. 27 F i g u r e 17; Stages of t e s t i c u l a r m a t u r i t y Induced i n s p r i n g salmon j u v e n i l e s t r e a t e d three times weekly with v a r i o u s dosages of salmon (Oncorhynchus tshawytscha) gonadotropin. These v a l u e s taken from Ta b l e V. JUNE I JULY ' AUGUST I SEPT r 28 Figure 18: Stages of reproductive maturity of uninfected male pink salmon maintained in aquaria throughout their l i f e cycle. Data were taken from Table VI. 6H 4H 29 F i g u r e 19: The h i s t o c h e m i c a l l y demonstrable of A 5 -3 8 h y d r o x y s t e r o i d dehydrogenase i n the t e s t e s of j u v e n i l e pink salmon i n j e c t e d t h r i c e weekly wi t h v a r i o u s dosages of salmon (Oncorhynchus  tshawytscha) gonadotropin. The formazan d e p o s i t s were q u a n t i -f i e d by v i s u a l o b s e r v a t i o n . The experiment was conducted d u r i n g the summer of 1969. Values taken from Ta b l e V I I . 30 F i g u r e 2 0 : The h i s t o c h e m i c a l l y . demonstrable a c t i v i t y ofA 5-38 h y d r o x y s t e r o i d dehydrogenase i n the t e s t e s of j u v e n i l e s p r i n g salmon i n j e c t e d three times per week w i t h v a r i o u s dosages of salmon (Oncorhynchus tshawystcha) gonadotropin d u r i n g the summer of I 9 6 9 . The formazan d e p o s i t s were q u a n t i f i e d by v i s u a l o b s e r v a t i o n . These v a l u e s taken from Table V I I I . H 1.5 > I— O < 1.0-liJ > -N 2. LU 0.5 H OA i0 .0^g/g BW IOj jg /gBW O l j j g / g B W O CONTROL JUNE I JULY * AUGUST 1 SEPT r 31 T a b l e 1: Male pink salmon j u v e n i l e s t r e a t e d with v a r i o u s dosages of salmon gonadotropin - mean f i s h l e n g t h , mean f i weight, mean f i x e d gonad weight, mean gonadosomatic index (G.S.I.). y = mean; SD = standard d e v i a t i o n . TABLE I: FIXED BODY BODY TESTIS . G-. S. I-. SAMPLE TREAT- n LENGTH (cm) WEIGHT (g) WEIGHT (;y g) MENT y. S. D. y S. D. y S.D. y S.D, Zero Control Co 5 7.7 0.3 3.8 0.5 17/6/69 1 A 6 8.3 0.7 5.2 1.5 B 4 8.4 0.7 5.5 1.2 30/6/69 C 7 8.1 0.6 4.1 1.0 Co 4 8.3 0.8 4.6 1.0 N 0 D A T A 2 A 5 9 > 0.5 7.1 1.6 B 4 9.1 0.7 6.5 1.5 14/7/69 C 4 9.0 0.-5 6.1 1.2 Co 4 8.8 0.3 5.5 0.7 3 A 3 10.4 0.2 9.7 0 .9 66.0 38.8 1 . 3 2 0.71 B 6 0.4 7.2 1.0 12.6 7.1 0.32* 0.17 28/7/69 C 4 9.9 0.8 8.2 2.2 4.7 ^.5 0.07 0 . 0 5 Co 2 9.5 0.1 6.7 0.2 1.0 0.2 0.03 0.01 4 A 5 10.9 0.9 14.2 2.8 210.8 129.9 2.13 1.71 B 4 10.5 0.6 11.2 2.8 52.2 47.8 0.83 0.55 11/8/69 Co 4 11.1 1.0 11.9 2>A 1.9 0.7 0.03 0.01 5 A 4 12.3 0.5 17.7 2.7 219.5 124.6 2.43 1.02 B 4 11.3 1.4 14.1 5.0 68.4 5^.9 1 .06 1.02 25/8/69 Co 6 12.0 0.4 15.6 1.4 2.5 0.7 0.03 0.01 6 A 2 12.2 0.7 16.8 2.1 91.1 65.3 1.14 1.12 B 2 12.6 0.6 17.8 2.5 223.1 99.2 2.45 0.76 8/9/69 Co 5. 12.5 0.7 17.5 2.6 3.7 2.3 0.04 0.02 7 A 4 12.1 1.3 16.1 4.6 72.1 35.2 0.77 0.13 B 2 12.3 1.5 17.8 7.8 90.0 22.1 1.28 0 . 5 0 22/9/69 Co 4 14.4 1.5 28.5 11.4 4.2 1.0 0.03 0.01 U.I. Co 4 14.4 1.5 28.5 11.4 4.2 1.4 0.03 0.01 32 Table I I : Mean body weight, mean body l e n g t h , mean f i x e d t e s t i s weight, and mean gonadosomatic index (G.S.I.) of j u v e n i l e s p r i n g salmon t r e a t e d with v a r i o u s dosages of salmon gonadotropin, y = mean; S.D. = standard d e v i a t i o n . TABLE I I : FIXED BODY BODY TESTIS G.S.I. SAMPLE TREAT- n LENGTH (cm) WEIGHT (g) WEIGHT (yg) MENT y S. D. y S. D. y S:. D. y S.D. Zero 6 C o n t r o l Co 5.5 0.3 1.8 0.2 IO/ 6 / 6 9 1 A 3 6.0 0.3 2.1 0.3 B 4 6.0 0.7 2.4 0.9 C 5 6.3 0.5 2.9 0.6 Co 5 6.1 1.0 2.4 1.2 2 A 6 6.9 0.3 3.3 0.4 B 3 6.8 0.5 3.3 0.5 C 6 7.1 0.4 3.8 0.7 Co 3 6.7 0.6 3.1 0.9 3 A 6 7.9 0.6 5.2 1.3 B 1 7.2 — 3.6 --C 7.0 0.5 3.^ 1 . 0 Co 7.2 0 . 2 3.6 0.4 A 6 8.1 1 .0 5.8 2.4 73.1 52.7 2.53 1.31 B 6 7.9 0 . 8 5.3 1.5 29.9 28.2 1.13 1.22 C 6 7.2 0 . 7 3.5 0.9 5.2 3.8 0.21 0 .07 Co 6 7.6 0 . 8 4.4 1.6 0.6 0.3 0.02 0.01 5 A 3 9.3 0 . 6 8.8 0.9 186.4 73.3 4 . 3 0 1 .92 B 3 9.1 0 . 6 8.8 1.8 117.9 91.5 2.57 1.88 C 5 8.5 1 .2 7.0 2.8 12.8 7.3 0 .25 0.17 Co 7.3 0 . 7 *.3 1.1 0.9 0.5 0.04 0.02 6 A 5 9 . 1 0 .9 9 . 1 2.5 478.2 228.5 10.00 2.91 B 3 8.8 0.9 8.0 3.1 280.9 116.6 6.39 0.17 C 5 9A 1.5 9.2 4.1 7.6 4.8 0.18 0.09 Co 6 8.6 1.1 7.0 3.1 2.2 1.5 0 . 0 5 0 .03 U.I. Co 5 9.0 1.8 8.9 5.1 ^.7 5.1 0.08 0 . 0 5 33 Table I I I : Mean body weight, mean gonad weight, and mean gonado somatic index (G.S.I.) of u n i n f e c t e d male pink salmon main-t a i n e d i n a q u a r i a throughout t h e i r l i f e c y c l e . v = mean; S.D. = Standard D e v i a t i o n . TABLE I I I : BODY TESTIS SAMPLE DATE n WEIGHT WEIGHT G.S.I. (g) (g) y S.D. y S.D. y S.D. 8 5/H / 6 9 4 9 0 . 8 1 0 . 2 0 . 0 3 0 0 . 0 3 0 . 0 1 9 21/1/70 6 1 5 7 . 5 2 0 . 5 0 . 0 7 0 . 0 2 0.04 0 . 0 1 10 9/3/70 2 1 9 6 . 8 18 .7 0 . 0 9 0 . 0 2 0.04 0 . 0 1 11 3 0 / 6 / 7 0 5 2 3 2 . 6 3 9 . 2 0 . 4 3 0.40 0.18 0.14 12 2 5 / 8 / 7 0 4 3 5 5 . 8 2 5 . 1 3 1 . 5 5 . 8 8 . 8 1 1 .15 3k Table IV: The stage of t e s t i c u l a r dosages of salmon gonadotropin p. = mean; S.D. = Standard maturation induced by v a r i o u s i n p-ihk salmon j u v e n i l e s . D e v i a t i o n g A R T . T C T V : SAMPLE 8c TREATMENT n TESTICULAR STAGE DATE S.D. Zero Control 2 1.0 0 17/6/69 1 A 3 1.0 0 B 2 1.0 0 30/6/69 C 3 1.0 0 Co 3 1.0 0 2 A 3 1.0 0 B 3 1.8 0.4 1V7/69 C 3 1.0 0 Co 3 1.0 0 3 A 3 2.8 0.4 B 3 2 . 6 0.2 28/7/69 C 3 1.4 0.2 Co 1 1.0 0 A 3 4.3 0.6 B 3 4.0 0 . 1 11/8/69 Co 3 1.0 0 5 A 3 4.6 0 . 1 B 3 4.8 0.4 25/8/69 Co 3 1.0 0 6 A 3 4.7 0.3 B 2 *.5 0.7 8/9/69 Co 3 1.0 0 7 A 3 5.8 0.3 B 2 5 .6 0 .6 22/9/69 Co 3 1.0 0 U.I. Co 3 1.0 0 35 Table V: The t e s t i c u l a r stage of s p r i n g salmon j u v e n i l e s i n j e c t e d w i t h v a r i o u s dosages of salmon gonadotropin, u. = mean; S.D. = Standard D e v i a t i o n . TABLE V: SAMPLE & TREATMENT n TESTICULAR STAGE DATE S.D. Zero Control 10/6/69 2 1 . 0 0 1 A 3 1 .0 0 B 3 1 . 0 0 24/6/69 C 2 1 .0 0 Co 3 1 . 0 0 2 A 2 1 .0 0 B 3 1 . 0 0 7/7/69 C 3 1 . 0 0 Co 1 1 .0 0 3 A 3 1 .5 0.1 B 1 1.6 0 21/7/69 C 3 1 .3 0.2 Co 3 1 . 0 0 A 3 2 .5 0 B 3 2.2 0 .3 4 / 8 / 6 9 C 3 2.0 0 Co 3 1 .0 0 5 A 3 4.0 0 I 8 / 8 / 6 9 B 3 3.1 0 . 9 C 3 2 .5 0 . 7 Co 3 1.0 0 6 A 3 4.6 0.2 B 2 4.0 0 1/9/69 C 3 2 .3 0 .6 Co 3 1 . 0 0 U.I. Co 3 1 . 0 0 36 Table VI; The t e s t i c u l a r stage of u n i n f e c t e d pink salmon maintained i n a q u a r i a throughout t h e i r l i f e c y c l e . y= mean; S.D. = Standard D e v i a t i o n . TABLE VI: SAMPLE & DATE 1 1 TESTICULAR y STAGE S.D. 8 5/11/69 4 1 .0 0 9 21/1/70 4 1 .0 0 . 1 10 9/3/70 2 1 .1 0 11 3 0 / 6 / 7 0 4 2 . 0 0.1 12 25/8/70 4 4 . 0 0 3 7 Table V I I : H i s t o c h e m i c a l l y demonstrable A5-3 8 h y d r o x y s t e r o i d dehydrogenase a c t i v i t y i n pink salmon j u v e n i l e s t r e a t e d w i t h v a r i o u s dosages of salmon gonadotropin, y = mean; S.D. = Standard D e v i a t i o n . TABLE V I I : SAMPLE ENZYME A C T I V I T Y & TREATMENT n ( V i s u a l U n i t s ) DATE u S.D. Zero C o n t r o l Co 4 0 0 17/6/69 1 A 4 0 . 1 3 6 . 2 ^ B 4 0 . 4 8 0 . 7 5 3 0 / 6 / 6 9 C 4 0 . 3 8 0 . 7 5 Co 4 0 0 2 A 3 0 . 3 8 0 . 7 5 B 2 0 . 5 0 0 . 7 1 1 4 / 7 / 6 9 C 2 0 0 Co 3 0 0 3 A 3 1 .67 0 . 2 9 B 1 . 2 5 0 . 5 0 2 8 / 7 / 6 9 C 3 1 .67 0 . 2 5 Co 1 0 0 4 A 4 2.13 0 . 6 3 B 4 1 .63 0 . 2 5 11/8/69 Co 2 0 0 A 3 1 .67 0 . 2 9 B 4 1 .50 0 2 5 / 8 / 6 9 Co 4 0 0 6 A 2 1 . 2 5 0 . 3 5 B 2 1 .75 0 . 3 5 8 / 9 / 6 9 Co 4 0 0 7 A 4 1 .13 0 . 2 5 B 2 1 . 0 0 2 2 / 9 / 6 9 Co 4 , 0.13 0 . 2 5 U.I. Co 4 0 . 1 3 0 . 2 5 38 Table V I I I ; H i s t o c h e m i c a l l y demonstrable A 5 - 3 $ h y d r o x y s t e r o i d dehydrogenase a c t i v i t y i n the t e s t e s of s p r i n g salmon j u v e n i l e s t r e a t e d w i t h v a r i o u s dosages of salmon gonadotropin, v = mean; S.D. = Standard D e v i a t i o n . TABLE V I I I : SAMPLE ENZYME ACTIVITY & TREATMENT n .." ( V i s u a l Units);/ DATE u S.D. Z e r o . C o n t r o l 4 0 0 IO/ 6 / 6 9 1 A 3 0 0 B 4 0 0 24/6/69 C 4 0 0 Co 4 0 0 2 A 4 0 0 B 2 0 0 7/7/69 C 4 0 0 Co 3 0 0 3 A 4 0 . 8 8 0.63 B 2 0 . 2 5 0.35 21/7/69 C 0 0 Co 3 0 0 4 A 4 1 . 0 0.41 B 0 . 6 3 0.48 4/8/69 C 0.38 0.48 Co 3 0 0 5 A 1 . 2 5 0 . 3 0 B 3 1.17 0.58 18/8/69 C 0.88 0.48 Co 3 0 0 6 A 1.13 0.4b 1/9/69 B 3 0.83 0.58 C 2 0 . 5 0 0 Co 3 0 0 U.I. Co 3 0 0 CHAPTER 2 The Ovary Introduction The literature reviewed by Hoar (1966) indicates that the development of the ovary in young teleosts is dependent upon the pituitary. After hypophysectomy, the f o l l i c l e s f a i l to develop. Therefore, i t appeared that injection of prepara-tions of pituitary gonadotropin would be a promising method for stimulating the ovaries of immature salmon. Few studies had been done on the effect of homoplastic pituitary extracts on the rate of maturity of the ovary in teleosts. Yamazaki and Donaldson ( I 9 6 8 ) succeeded in reinducing vitellogenesis and ovulation in hypophysectomized female goldfish (Carassius auratus). The f u l l y developed ovaries had been allowed to regress for two months prior to treatment with a partially purified preparation of salmon (Oncorhynchus  tshawytscha) gonadotropin. Their study added to the information obtained by Barr (1963 b). Following hypophysectomy of the pliace (Pleuronectes platessa), i t was found that the remaining oocytes could be stimulated to grow by injecting extracts of pliace pituitary glands. There i s , therefore, evidence to indicate that adult female teleosts may be stimulated to sexual maturity in a short period of time by injecting homoplastic extracts of the pituitary. That the ovary of the immature tele-ost i s relatively more refractory to stimulation can be seen from a study by Schmidt et a l ( 1 9 6 5 ) . Two weeks of injection of extracts of pituitary glands from adult pink salmon 40 (Oncorhynchus tshawytscha) r e s u l t e d In a s m a l l i n c r e a s e i n the weight of the o v a r i e s of Immature t r o u t (Salmo g a l r d n e r i l ) 1 3 . 5 cm i n l e n g t h . T h e r e f o r e , i t appeared t h a t a long-term program of i n j e c t i o n of salmon gonadotropin i n t o immature pink salmon would be necessary for;,the development of mature ova i n the o v a r i e s of these f i s h . There i s l i t t l e i n f o r m a t i o n on the e f f e c t of mammalian gonadotropins on any a s p e c t of oogenesis of v i t e l l o g e n e s i s i n t e l e o s t s ( B a r r , 1 9 6 8 ) . An experiment done by Ahsan and Hoar (1963) does r e v e a l to some extent the p o t e n t i a l of these hor-mones. They found t h a t i n j e c t i o n of LH a l o n e , or i n combination w i t h PSH, s t i m u l a t e d v i t e l l o g e n e s i s and growth of oocytes i n the s t i c k l e b a c k (Gasterosteus a c u l e a t u s ) p h y s i o l o g i c a l l y hypophysectomized w i t h r e s p e c t to gonadotropins. A s l i g h t e f f e c t was o b t a i n e d w i t h FSH, but t h i s was thought to be the r e s u l t of LH contamination. N e i t h e r PMS nor HGG was as e f f e c -t i v e as LH, although both produced some s t i m u l a t i o n of oocyte growth. The mammalian gonadotropins do not appear to o f f e r any s i g n i f i c a n t advantage over the use of homoplastic p r e p a r a t i o n s . T h e ^ p o s s i b i l i t y was r e c o g n i z e d t h a t t h e r e was a hormone not present i n s u b s t a n t i a l q u a n t i t i e s i n the immature f i s h , but which i n the maturing t e l e o s t s y n e r g i z e s with p i t u i t a r y gonadotropin d u r i n g normal development of the ovary. I f such a substance were i n j e c t e d s i m u l taneously w i t h salmon gonadotro-p i n , a f a s t e r r a t e of development of the immature ovary c o u l d be achieved. A survey of such f a c t o r s f o l l o w s . I t was l o g i c a l t o study estrogens as f a c t o r s which might s y n e r g i z e with p i t u i t a r y gonadotropin d u r i n g o v a r i a n 41 maturation. Simpson et a l (1963) found t h a t i n S p y l l o r h l n u s  c a n i c u i u s the q u a n t i t y of e s t r o g e n i n c r e a s e d as the oocytes matured. That estrogens a r e present i n the o v a r i e s of t e l e o s t s can be seen from work done by G a l z i n g a ( 1 9 6 1 ) . He demonstrated t h a t e s t r a d i o l 17 6 was the predominant estrogen i n the o v a r i e s of Cyprlnus c a r p i o and Salmo i r i d e u s . The predominant s p e c i e s of estrogen throughout the l i f e c y c l e of the A t l a n t i c Salmon (Salmo s o l a r L.) i s estrone (Cedard et a l , 1 9 6 1 ) . B o t t i c e l l i and Hisaw (1964) e s t a b l i s h e d t h a t e s t r a d i o l 17B was the only e s t r o g e n i n the o v a r i e s of mature Oncorhynchus nerka; i t was present i n q u a n t i t i e s of 36 micrograms per k i l o g r a m body weight. I t was decided, a f t e r c o n s i d e r a t i o n of t h i s l i t e r a t u r e , t h a t e s t r a d i o l 1? g would be employed i n p h y s i o l o g i c a l dosages. A d m i n i s t r a t i o n of exogenous estrogen has u s u a l l y been r e p o r t e d as having an i n h i b i t i n g or r e g r e s s i v e e f f e c t on the f i s h ovary. Sundararaj and Goswami (1968) d i s c o v e r e d that e s t r a d i o l benzoate i n h i b i t e d v i t e l l o g e n e s i s i n the c a t f i s h (Heteropneustes f o s s l l l s ) . A s i m i l a r o b s e r v a t i o n was recorded by B u l l o u g h (1942) on o e s t r o n e - t r e a t e d o v a r i e s of Phoxlnus  l a e v l s . The development of primary oocytes was i n h i b i t e d , l e a d i n g to t h e i r e v e n t u a l d i s i n t e g r a t i o n . These e f f e c t s can be e x p l a i n e d by t a k i n g i n t o c o n s i d e r a t i o n , the e f f e c t of estrogen on p i t u i t a r y gonadotropin p r o d u c t i o n . The compensatory hypertrophy of the ovary of Heteropneustes f o s s l l l s , f o l l o w i n g u n i l a t e r a l ovariectomy was i n h i b i t e d by treatment w i t h e s t r a d i o l benzoate (Sundararaj and Goswami, 1 9 6 8 ) . Simultaneous observa-t i o n s t h a t t h e r e were fewer and s m a l l e r b a s o p h i l s i n the p i t u i -t a r y glands of c a t f i s h t r e a t e d w i t h e s t r a d i o l benzoate suggested 42 t h a t there was a n e g a t i v e feedback r e l a t i o n s h i p between e s t r o -gen and p i t u i t a r y gonadotropin s i m i l a r to t h a t i n h i g h e r v e r t e -b r a t e s . The i n h i b i t o r y e f f e c t of estrogens on the oocytes of Gambusia sp. were a t t e n u a t e d by the simultaneous i n j e c t i o n of e s t r o g e n and f r o g a n t e r i o r p i t u i t a r y hormones ( I s h i l , 1 9 6 1 ) . C l a v e r t (1958) proposed l a r g e l y on t h e o r e t i c a l grounds t h a t estrogen would m o b i l i z e y o l k p r e c u r s o r s , and thus f a c i l i -t a t e y o l k d e p o s i t i o n i n the f o l l i c l e s . T h i s h y p o t h e s i s was s u b s t a n t i a t e d i n the sockeye salmon (Oncorhynchus nerka) by Ho and Vanstone ( 1 9 6 1 ) . The serum changes a s s o c i a t e d w i t h egg f o r m a t i o n were enhanced by a d m i n i s t r a t i o n of e s t r a d i o l mono-benzoate. In the maturing female f i s h , the t i s s u e which responds to estrogen i s the l i v e r , which produces a calcium phosphoprotein-p h o s p h o l i p i d g l y c o - l i p o - p r o t e i n complex. T h i s substance i s then t r a n s p o r t e d to the ova v i a the bloodstream ( U r i s t , 1964; i n Holmes and Donaldson ( 1 9 6 8 ) . Evidence t h a t estrogen has a d i r e c t e f f e c t on development of the f o l l i c l e s of hypophysectomi-zed immature r a t s has been presented by a number of authors (Pencharz, 1 9 4 0 ; W i l l i a m s , 1944; Simpson e t a l , 1 9 4 1 ; de Wit, 1 9 5 3 ) . Estrogens were found to s t i m u l a t e the m i t o s i s of c e l l s i n the f o l l i c u l a r e p i t h e l i u m , l e a d i n g t o the f o r m a t i o n of a m u l t i - l a y e r e d g r anulosa. Small f o l l i c l e s developed i n t o medium-s i z e d f o l l i c l e s w i t h no antrum. The mechanism by which estrogen s t i m u l a t e s the growth of the oocyte would thus appear to be two-f o l d : m o b i l i z a t i o n of y o l k p r e c u r s o r s , and s t i m u l a t i o n of the p r o l i f e r a t i o n of the f o l l i c u l a r e p i t h e l i u m through which the y o l k p r e c u r s o r s must pass. ^3 Uhere a r e s e v e r a l other hormones which c o u l d be i m p l i -cated i n the development of the o v a r i e s . Among them are andro-gens, progesterone, t h y r o i d hormones, and a d r e n a l s t e r o i d s . The p o t e n t i a l of these substances as s t i m u l a n t s of oocyte ma t u r a t i o n w i l l now be d i s c u s s e d . T e s t o s t e r o n e and 1 1 - k e t o t e s t o s t e r o n e are produced i n o v a r i a n t i s s u e of T l l a p l a aurea ( E c k s t e i n , 1 9 7 0 ) . There i s some evidence, t h e r e f o r e , to i n d i c a t e t h a t androgens are present normally i n the ovary of the t e l e o s t . Ramaswami (1962) found t h a t 5 nig methyl t e s t o s t e r o n e alone d i d not s t i m u l a t e o v u l a t i o n i n the c a t f i s h Heteropneustes f o s s l l i s ( B l o c h ) . 1 . 2 5 mg methyl t e s t o s t e r o n e p l u s a subminimal dose of p i t u i t a r y g l a n d gave a p o s i t i v e r e a c t i o n . There have been no s t u d i e s to suggest t h a t androgen p l u s any o t h e r combination cSf agents i s e f f e c t i v e i n s t i m u l a t i n g the stages of o v a r i a n development p r i o r to o v u l a t i o n . Progesterone (Ramaswami, 1962; Yamazaki, 1965) and a d r e n a l c o r t o c o s t e r o i d s (Ramaswami, 1962; Sundararaj and Goswami, 1966) have been shown to s t i m u l a t e o v u l a t i o n i n the o v a r i e s of some t e l e o s t s . There i s no evidence t h a t these s t e r o i d s a c c e l -e r a t e the p r e v i o u s stages of o v a r i a n development. T h y r o t r o p i n was shown to have a s t i m u l a t o r y e f f e c t on the ovary of the t h r e e s p i n e s t i c k l e b a c k , G a s t e r o s t e p s a c u l a e t u s (Ashan and Hoar, 1 9 6 3 ) . I t was mentioned by the a u t h o r s , how-ever, that t h e i r TSH p r e p a r a t i o n contained p o s s i b l e contamination w i t h gonadotropins. Data c o l l e c t e d from d i f f e r e n t s p e c i e s seem to i n d i c a t e t h a t m i l d hypothyroidism and m i l d h y p e r t h y r o i d i s m a r e compatible w i t h normal r e p r o d u c t i o n , whereas severe cases 44 of d e f i c i e n c y or excess reduces the r e p r o d u c t i v e e f f i c i e n c y i n some s p e c i e s (Van Tienhoven, 1 9 6 8 ) . The p o t e n t i a l of t h y r o i d hormones as s t i m u l a t o r y agents of the ovary i s t a n t a l i z i n g , but does not o f f e r enough promise to be employed i n an i n i t i a l study. From the p r e c e d i n g i n f o r m a t i o n , i t appeared t h a t treatments c o n s i s t i n g of p h y s i o l o g i c a l dosages of salmon gonado-t r o p i n a l o n e , or i n combination w i t h e s t r a d i o l 173 would be most l i k e l y to a c c e l e r a t e the m a t u r i t y of the ovary of the immature pink salmon. I t was a n t i c i p a t e d t h a t f i s h i n j e c t e d w i t h both salmon gonadotropin and estrogen would mature the f a s t e s t . In the present study, the s p r i n g salmon f r y were t r e a t e d w i t h salmon gonadotropin to p r o v i d e a comparison of the a c t i v i t y of the salmon gonadotropin p r e p a r a t i o n between the donor f i s h and the t e s t pink salmon. P r o d u c t i o n of a n t i b o d i e s a g a i n s t gonado-t r o p i n s of f o r e i g n o r i g i n have been r e p o r t e d (Pineda et a l 1 9 6 8 ) . I n f o r m a t i o n can a l s o be obtained on the r e l a t i v e e f f e c t of the gonadotropin p r e p a r a t i o n on a j u v e n i l e salmon which matures s e x u a l l y a f t e r two years (Oncorhynchus gorbuscha), and one which reaches r e p r o d u c t i v e m a t u r i t y a f t e r 4 years (Oncorhynchus  tshawytscha). Inducing sexual m a t u r i t y i n salmonids i n the year of h a t c h i n g poses some s p e c i a l d i f f i c u l t i e s r e l a t e d t o the s i z e of the f i s h . They a r e very s m a l l a t t h i s stage i n t h e i r l i f e , and c o u l d never c o n t a i n a n o r m a l - s i z e d mature overy. One approach to the problem i s to s t i m u l a t e the animals to a l a r g -er s i z e . P r e d e f i n e d environmental and d i e t a r y c o n d i t i o n s were used to grow y e a r l i n g sockeye salmon (Oncorhynchus nerka) from ^5 22 to 110 grams over a two and one h a l f month t e s t p e r i o d ( B r e t t and Sutherland, 1970). The average weight of the c o n t r o l s a t the end of the t e s t p e r i o d was 51 grams. A stock of pink salmon j u v e n i l e s which had been t r e a t e d i n a s i m i l a r manner f o r three months were generously donated by Dr. J . R. B r e t t a t the end of August, 1970. A s i z e - f e c u n d i t y r e l a t i o n s h i p does appear to e x i s t f o r pink salmon. F o e r s t e r and P r i t c h a r d (1941) r e p o r t e d an average i n c r e a s e of 57 eggs f o r each i n c r e a s e of one cm i n l e n g t h , and 472 eggs f o r each i n c r e a s e of 1 k i l o g r a m i n weight f o r M c C l i n t o n Creek (Queen C h a r l o t t e I s l a n d s ) . I t i s a n t i c i p a t e d , t h e r e f o r e , t h a t i n d u c t i o n of p r e c o c i o u s s e x u a l m a t u r i t y i n the s m a l l , im-mature f i s h w i l l be c h a r a c t e r i z e d by the development to f u l l s i z e of a much lower complement of eggs than occurs normally. I t i s proposed t h a t u n i n j e c t e d pink salmon maintained throughout t h e i r l i f e c y c l e i n a q u a r i a be s t u d i e d c o n c u r r e n t l y to p r o v i d e a comparison between o v a r i e s which have developed to m a t u r i t y n a t u r a l l y and those s t i m u l a t e d w i t h hormones. 46 Methods and M a t e r i a l s The females were maintained under the same c o n d i t i o n s as d e s c r i b e d e a r l i e r f o r the males. Experiments on the ovary which were from the same stock as have been d e s c r i b e d p r e v i o u s l y i n chapter 1 , were: (1) Summer 1969; Study of the e f f e c t of v a r i o u s dosages of s p r i n g salmon gonadotropin (SG-G100) on development of the ovary i n pink salmon f r y and s p r i n g salmon f r y . (2) Summer I 9 6 9 to F a l l , 1970; Study of the normal c y c l e of gonad development i n u n i n j e c t e d pink salmon maintained i n a q u a r i a . (3) September 1970 to February, 1 9 7 1 ; Study of the e f f e c t of s p r i n g salmon gonadotropin (SG-G100), and combinations i n -v o l v i n g the a d d i t i o n of e s t r a d i o l 176 , and then p r o g e s t e r -one: - Pink salmon f r y which had been s t i m u l a t e d to a l a r -ger than normal s i z e a t an e a r l i e r stage w i t h h i g h e r water temperatures and a c o n s t a n t l y l e n g t h e n i n g photoperiod were used. The n o r m a l - s i z e d female pink salmon f r y used In e x p e r i -ments i n i t i a t e d i n the summer of 1971 were ob t a i n e d from a d i f f e r e n t source. On A p r i l 24, 1 9 7 0 , 600 pink salmon f r y were obtained from Jones Creek (a t r i b u t a r y of the F r a s e r R i v e r ) d u r i n g t h e i r downstream m i g r a t i o n . At the F i s h e r i e s Research •Laboratory i n West Vancouver they were p l a c e d i n a 500-litre freshwater aquarium which was g r a d u a l l y converted to seawater on A p r i l 2 7 . P r i o r to the s t a r t of i n j e c t i o n s , the f i s h were t r a n s -f e r r e d to 1 7 0 - l i t r e f i b r e g l a s s a q u a r i a . 47 Treatments: The female pink and spring salmon f r y , i n the summer of 1969, were treated with a dosage regime Id e n t i c a l to that received concurrently by the males of both species (Chapter 1). The schedule of sampling was also the same. In a second experiment, started i n the summer of 1970. s i x t y pink salmon f r y were placed i n each of seven oval 1 7 0 -l i t r e f i b r e g l a s s aquaria. These f i s h were anesthetized, and injected i n t r a p e r l t o n e a l l y , three times per week, (by the method described i n Chapter 1), with 0.05 ml of the following substances dissolved i n f i s h saline (Wiebe, I 9 6 8 ) : (1) G: spring salmon gonadotropin, 1.0y g per gram body weight. (2) GE a: spring salmon gonadotropin, 1.0 y g per body weight and 15 y g per gram body weight e s t r a d i o l 178 . (3) GEb: spring salmon gonadotropin, 1.0 yg per gram body weight and 1 .5 yg per gram body weight e s t r a d i o l 17 8 . (4) GEC: spring salmon gonadotropin, 1 .0 yg per gram body ' weight and a f t e r three months of treatment, addition of ! E" 1.5 yg per gram body weight e s t r a d i o l 178 . (5) E a: E s t r a d i o l 17 e - 15 yg per gram body weight. (6) Et,: E s t r a d i o l 178 - 1 .5 vg per gram body weight. (7) Co: 10$ ethanol dissolved i n f i s h saline. The spring salmon gonadotropin (SG-G100) i s described i n Donaldson and Yamazaki, ( 1 9 6 8 ) , and Donaldson et a l ( 1 9 7 1 ) . In a l l of the treatments involving the addition of estrogen, 1 , 3 ,5 . (10) - es t r a t r i e n - 3, 17 6 d i o l ( e s t r a d i o l 17g ) -Mann Research Laboratories) was f i r s t dissolved i n 95% ethanol, and then di l u t e d to a f i n a l concentration of 10% ethanol, with addition of f i s h saline. The r e s u l t i n g f i n e suspension was eas i l y injected through a 27 g needle. The f i s h were sampled at six-week intervals,. At each sampling, specimens were removed u n t i l four females had been found, or ten f i s h had been taken. The zero control sample was taken on June 27, 1970. Injections were continued u n t i l March 10, 1971, when only G, GE C, and Co remained. A l l of the specimens with other treatments had been removed p r i o r to that date. A stock of pink salmon juveniles which were much larger than the normal size (described i n Chapter 1) were studied with ] t the purpose of investigating hormonal induction of precocious sexual maturity i n a larger animal. These f i s h were anestheti-zed, and injected i n t r a p e r i t o n e a l l y three times per week (by the method described i n Chapter 1), with 0.10 ml of the follow-ing treatments dissolved i n f i s h saline (Wiebe, 1968): (1) LG: spring salmon gonadotropin (SG-G100) - 1.0 yg per gram body weight. (2) LGE: spring, salmon gonadotropin 1 (SG-G100) - 1.0 yg per gram body weight and 1.5 y g per'^grarn body weight e s t r a d i o l 17 S. .3 (3) LGEP: spring salmon gonadotropin (SG-G100) - 1.0y g per ' gram body weight, e s t r a d i o l l?g - 1.5 y g per gram body weight, and progesterone - 5.0 $g per gram body weight. (4) LCo: 10$ ethanol dissolved i n f i s h saline. 49 E s t r a d i o l 17 3 and progesterone were f i r s t d i s s o l v e d i n 95$ e t h a n o l , and then d i l u t e d to f i n a l c o n c e n t r a t i o n of 10% e t h a n o l by adding f i s h s a l i n e . The f i n e suspension which r e s u l t e d from t h i s procedure was e a s i l y i n j e c t e d w i t h a 25 g needle. I n j e c t i o n s were s t a r t e d on September 2 , 1970, when the mean body weight of the pink salmon was 42.0 grams (compared t o 15.8 grams f o r the no r m a l - s i z e d f r y ) . Twenty f i s h were a s s i g n e d to each hormonal treatment, and samples were taken a t two s u c c e s s i v e , three-month i n t e r v a l s . There were too few specimens to a l l o w a zero c o n t r o l sample. Ten c o n t r o l a n i -mals were kept throughout the experiment, and were not taken u n t i l the t e r m i n a l sample, February 18, 1971. E i g h t of the LGEP group d i e d November 3 , 19&9, because of a b l o c k i n the water l i n e , and were sampled a t t h i s time. Random m o r t a l i t y , due mainly to removal of d i s e a s e d f i s h , reduced the number of specimens i n each sample. Experimental Techniques; ( i ) , ( i i ) , and ( i i i ) : The f i s h were i n j e c t e d , a u t o p s i e d , and processed h i s t o -l o g i c a l l y as d e s c r i b e d i n Chapter 1. ( i v ) A n a l y s i s of tfoe H i s t o l o g i c a l R e s u l t s (a) Measurements: D e s s i c a t i o n of the s m a l l c o n t r o l o v a r i e s d u r i n g weighing n e c e s s i t a t e d a comparison of gonad weight based on f i x e d , r a t h e r than wet weights, i n the experiments on n o r m a l - s i z e d 50 pink and s p r i n g salmon f r y , d u r i n g the summer of 1969. In the experiments on the nor m a l - s i z e d pink salmon f r y i n 1970/71, the e x t r a - l a r g e s i z e d f i s h i n 1970/71, and i n the study of the normal c y c l e of r e p r o d u c t i v e m a t u r i t y i n the pink salmon kept i n c a p t i v i t y , f r e s h weights of the o v a r i e s were taken. In both cases, one gonad from each f i s h was weighed to the n e a r e s t m i l l i g r a m . The t o t a l gonad weight f o r each animal was obtained by d o u b l i n g the r e s u l t f o r the weighed gonad. The gonadosomatic index was determined as f o l l o w s : t o t a l gonad weight x 100 G.S.I. = BODY WEIGHT The mean oocyte diameter was obtained from an average of the diameter of 30 oocytes per ovary, measured w i t h an o c u l a r micrometer. Only oocytes c o n t a i n i n g the g r e a t e r p a r t of the nucleus were measured. The va l u e recorded f o r o v o i d oocytes was the square r o o t o f the product of the g r e a t e s t diameter m u l t i p l i e d by the l e a s t diameter (as i n B r a e k e v e l t and McMi l l a n , 1967). (b) H i s t o l o g i c a l A n a l y s i s : The composition of each ovary was determined by cou n t i n g a l l of the n u c l e a t e d oocytes i n a median s a g i t t a l s e c t i o n , and ex p r e s s i n g the va l u e f o r each type as a percentage of the t o t a l . T h i s i n f o r m a t i o n was then averaged f o r each treatment and p l o t t e d g r a p h i c a l l y . The c h a r a c t e r i s t i c s f o r each type of oocyte were s i m i l a r to those d e s c r i b e d by Yamazaki (I965) i n h i s study of the ovary of the g o l d f i s h ( C a r a s s i u s auratus L ) . They were as f o l l o w s : 51 1) Early peri'nucleolus stage Several n u c l e o l i are apparent near the nuclear membrane. The cytoplasm has a marked a f f i n i t y f o r haematoxylin; certain b a s o p h i l l i c , clumped, granular portions are referred to as a p a l l i a l layer by Kudo i n studies on Plecoglossus a l t l v e l u s (1969 a) and Pseudorasbosa pimula (1969 b). The cytoplasm and nucleus increase greatly i n size. The oocyte varies from 40 to 250 micra i n diameter. During the course of t h i s stage, the oocytes become surrounded by squamous c e l l s from the i n t e r -s t i t i a l region to form a f o l l i c u l a r layer one c e l l thick. The zona radiata i s not yet apparent (Fig. 21, 22, 23, 24). 2) Late perlnuoleolus stage Nucleoli are s t i l l present at the periphery of the nucleus. The ooplasm has l o s t i t s a f f i n i t y f o r hematoxylin. The p a l l i a l layer has condensed to form a yolk-nucleus at the periphery of the cytoplasm. The oocyte has increased i n size* bat not as markedly as that i n the previous stage. The diameter of the oocytes varied from 180 to 320 micra. There i s a d i v i -sion of the c e l l s from the i n t e r s t i t i a l region to form a second layer. The zona radiata i s vaguely apparent (Fig. 25 and 26). 3) Yolk v e s i c l e stage C o r t i c a l a l v e o l i (or vesicles) appear i n the periphery of the ooplasm as a single layer. The size of the oocytes varies from 260 to 900 micra. As the oocytes increase i n diameter, the yolk v e s i c l e s accumulate toward the nucleus, and contain i n t r a v e s i c u l a r eosinophilic granules. The nucleus 52 becomes i r r e g u l a r i n o u t l i n e , i t s t i l l c o n t a i n s p e r i p h e r a l n u c l e o l i . The f o l l i c u l a r e p i t h e l i u m i s composed of a s i n g l e -l a y e r e d zona g r a n u l o s a of m i t o t i c a l l y d i v i d i n g squamous and ov o i d c e l l s , and an o v e r l y i n g theca of one to s e v e r a l l a y e r s of c e l l s . The zona r a d i a t a i s t h i n a t the be g i n n i n g of t h i s stage and i n c r e a s e s i n t h i c k n e s s and e o s i n a f f i n i t y toward the end ( P i g . 2? and 28). 4) Primary Yolk Stage At the b e g i n n i n g of t h i s stage, o i l g l o b u l e s a r e pr e -sent near the n u c l e u s , and s m a l l y o l k g r a n u l e s a r e present a t the p e r i p h e r y of the ooplasm. The y o l k g r a n u l e s c o a l e s c e to form l a r g e g l o b u l e s , and move throughout the cytoplasm of the oocyte. The zona gr a n u l o s a i s composed of c u b o i d a l c e l l s , w h i l e the o v e r l y i n g l a y e r c o n s i s t s of squamous c e l l s . The t h e c a l l a y e r t h i c k e n s ; the c e l l s which compose i t a r e a l l squamous. The zona r a d i a t a s t a i n s i n t e n s e l y w i t h e o s i n , but r a d i a l s t r i a t i o n s a r e on l y vaguely apparent. The oocytes i n t h i s stage ranged i n diameter from 550 to 1500 miera ( F i g . 31 and 3 2 ) . 5) Post-primary Y o l k Stage T h i s stage c o n s i s t s of a l l those oocytes l a r g e r than 1.0 mm and more advanced than the primary y o l k stage. I t was not p o s s i b l e to cut p a r a f f i n s e c t i o n s of t h i s type of oocyte, as they were too b r i t t l e . C r y o s t a t s e c t i o n s were obtained; they d i d not r e v e a l i n f o r m a t i o n c o n s i s t a n t w i t h t h a t found normally w i t h p a r a f f i n s e c t i o n s . ( F i g . 36 and 37). The largest oocytes in this stage measured 4 mm in diameter. (v) Histochemical Analysis: (a) Procedure The procedure used to demonstrate A.5-3 fLhydroxy steroid dehydrogenase activity was identical to that described in Chapter 1. Only the samples from the study of the effect of various dosages of spring salmon gonadotropin (SG-G100) on the repro-ductive maturity of pink and spring salmon fry in the summer of 1969 were investigated. Frozen ovaries from the experiment in 1970/71 to study the effect of various combinations of spring salmon gonadotropin (SG-G100) and estradiol 17 6. on maturity of the gonad in immature pink salmon were sectioned at 10 microns on the Cryostat, and stained for lipids with O i l Red 0 and Sudan Black B (Chayen e_fc a l , 1969). It was found that the section disintegrated when stained by the "floating through" technique. This problem was overcome with the method quoted in Culling (1957) for attaching frozen sections to gelatinized slides. Frozen sections of ovaries from uninjected pink salmon maintained throughout their l i f e in aquaria were stained as described above. (b) Analysis of Results: Analysis of the formazan deposits obtained in the measurement of A5-3 £ hydroxysteroid dehydrogenase activity in the ovary corresponded exactly to that described in Chapter 1. 5^ Observations were made on the l i p i d s t a i n s . The r e s u l t s are d e s c r i b e d i n the f o l l o w i n g s e c t i o n . RESULTS (a) Measurements; ( i ) Gonadosomatic Index The r e l a t i v e e f f e c t s of i n j e c t i o n of salmon gonado-t r o p i n on the G.S.I, d i f f e r e d markedly f o r the pink and s p r i n g salmon female f r y . Treatment A and B on the pink salmon experiment both had h i g h e r gonadosomatic i n d i c e s than, d i d the c o n t r o l . By comparison, none of the treatments of the s p r i n g salmon showed any r e a l d i f f e r e n c e a t the end of the experiment ( P i g . 40). The r e s u l t s o b t ained f o r treatment B, v the c o n t r o l i n the summer of 1969 were d u p l i c a t e d i n t h e i r q u a l i t a t i v e a s p e c t s (over a s i m i l a r time i n t e r v a l ) i n the experiment on pink salmon i n 1970 ( F i g . 41). In the l a t t e r study, f r e s h weights of the gonads were measured as opposed to the f i x e d weights used i n the experiments i n I969. By the t h i r d s a m p l e , f i s h from treatment G, GE^, and GE C had gonadosomatic i n d i c e s a p p r o x i -mately double those of the c o n t r o l s . At the «nd of the e x p e r i -ment, however, none of these groups were s i g n i f i c a n t l y d i f f e r e n t . The h i g h dosage of e s t r a d i o l 17 (alone, or i n combination w i t h salmon gonadotropin) and the low dosage of e s t r a d i o l 17 6 alone had the e f f e c t of d e p r e s s i n g the G.S.I. The t e r m i n a l sample of the l a r g e r - s i z e d pink salmon j u v e n i l e s was compared to the corresponding treatments over a 55 six-month time i n t e r v a l from pink salmon j u v e n i l e s of a normal s i z e ( F i g . 42). The f i s h i n j e c t e d w i t h salmon gonadotropin and the low dosage of estrogen d i d not d i f f e r s i g n i f i c a n t l y from the c o n t r o l i n e i t h e r experiments. The n o r m a l - s i z e d pink salmon i n j e c t e d w i t h salmon gonadotropin had a G.S.I, approximately f o u r times t h a t of the l a r g e r f i s h . I t i s d i f f i c u l t , however, to make d i r e c t comparisons between the two experiments because i n j e c t i o n s of the l a r g e r f i s h began 69 days l a t e r than the nor-m a l - s i z e d animals. A c o n s t a n t l y r i s i n g gonadosomatic index c h a r a c t e r i z e d the o v a r i a n development of pink salmon maintained i n a q u a r i a throughout t h e i r l i f e ( F i g . 43). From the b e g i n n i n g of J u l y ( i n the year of sexual m a t u r i t y ) to September, the G.S.I, rose a t a r a t e which was not p a r a l l e l e d i n any of the experiments i n v o l v i n g treatment with hormones. On August 2 5 , i t was approximately f i v e times h i g h e r than the maximum ob t a i n e d f o r treatment G. ( i i ) Oocyte Diameter A s i g n i f i c a n t i n c r e a s e i n the mean oocyte diameter was found when salmon gonadotropin was i n j e c t e d i n t o pink salmon f r y f o r 98 days i n both treatments A and B ( F i g . 44). There was no corresponding d i f f e r e n c e between the experimental and c o n t r o l groups i n the s p r i n g salmon f r y t r e a t e d f o r a s i m i l a r time. By the end of the experiment, none of the t r e a t e d s p r i n g salmon contained oocytes l a r g e r i n diameter than the zero con-t r o l o f the pink salmon. 56 The r e s u l t s o b t a i n e d f o r the n o r m a l - s i z e d pink salmon i n j e c t e d w i t h v a r i o u s hormonal treatments over an extended time i n t e r v a l a re complex ( P i g . 45). The h i g h dosage of e s t r o -gen (15 y g per gram body weight) when a d m i n i s t e r e d w i t h or w i t h -out salmon gonadotropin and the low dosage of estrogen a l o n e (1.5y g per gram body weight), depressed the mean oocyte diameter. The other treatments, i n v o l v i n g salmon gonadotropin alone or i n combination with a l o w - l e v e l of e s t r a d i o l 17 6 p e r i o d i c a l l y r ose above the average diameter of the c o n t r o l oocytes; a t the time of the t e r m i n a l sample, however, a l l of the treatments were near o r below the va l u e f©r the c o n t r o l s , R e s u l t s from the stock of the l a r g e r pink salmon a r e s i m i l a r l y d i f f i c u l t t o i n t e r p r e t . The average diameter of the oocytes a c t u a l l y d e c l i n e d between the f i r s t sampling a f t e r 42 days of i n j e c t i o n s , and the t e r m i n a l sample a f t e r 84 days. At the end of the experiment, a l l of the hormonal treatments c o n t a i n e d oocytes which were s m a l l e r i n s i z e than the c o n t r o l s ( F i g . 46). During the normal c y c l e of o v a r i a n development i n pink salmon maintained i n a q u a r i a , change i n oocyte diameter w i t h time was d i r e c t l y l i n e a r u n t i l J u l y , a f t e r which i t rose d r a m a t i c a l l y w i t h the approach of sexual m a t u r i t y ( F i g . 47). The d i f f i c u l t y i n r e s o l v i n g the r e s u l t s o b tained f o r mean oocyte diameter and gonadosomatic index i n f i s h t r e a t e d w i t h hormones, when coupled w i t h o b s e r v a t i o n s of enlargement of some oocytes, n e c e s s i t a t e d an examination of the o v a r i a n composition. The r e s u l t s o f t h i s a n a l y s i s can be seen i n the next s e c t i o n . 57 ( i i i ) Oogenesis The ovary proved to be more r e f r a c t i o n a r y t o hormonal s t i m u l a t i o n than the t e s t i s (Chapter 1 ) . F a i l u r e t o induce o v a r i a n maturation i n the pink or s p r i n g salmon f r y a f t e r 98 and 84 days of treatment r e s p e c t i v e l y , w i t h salmon gonadotro-p i n , l e d to a l a r g e r experiment the next year. The second s e r i e s of experiments were designed to t e s t the e f f e c t of salmon gonadotropin alone, e s t r a d i o l 17 6 a l o n e , and v a r i o u s combinations of salmon gonadotropin and e s t r a d i o l 17 e over an extended time p e r i o d . Comparisons were made w i t h l a r g e r f i s h i n j e c t e d a t a l a t e r time w i t h s i m i l a r dosage regimes, and w i t h pink salmon maintained i n a q u a r i a d u r i n g t h e i r l i f e c y c l e . F i g u r e 48 c o n t a i n s the data on the e f f e c t of v a r i o u s dosages of salmon gonadotropin on development of the oocytes i n immature pink salmon d u r i n g the summer of 1 9 6 9 . An a c c e l e r -a t i o n of the r a t e of c o n v e r s i o n of e a r l y p e r i n u c l e o l a r stage oocytes to l a t e p e r i n u c l e o l a r stage oocytes was seen i n treatments A and B. At the end of 98 days of i n j e c t i o n s , both of the aforementioned treatments contained oocytes more advan-ced by, one stage„ than the c o n t r o l s . Y o l k v e s i c l e f o r m a t i o n had been induced i n the m a j o r i t y of oocytes ( F i g . 2 7 ) , whereas the c o n t r o l o v a r i e s were composed predominantely of oocytes i n the l a t e p e r i n u c l e o l a r stage ( F i g . 2 5 ) . Fourteen days of treatment were r e q u i r e d b e f o r e c o r t i c a l a l v e o l i f i r s t appeared. By comparison, s i m i l a r dosages of the p i t u i t a r y hormone f a i l e d to advance the m a t u r i t y of the oocyte i n the s p r i n g salmon f r y i n j e c t e d f o r 84 days. O v a r i e s from both c o n t r o l and e x p e r i -mental f i s h c o n s t a t e d e n t i r e l y of germinal c e l l s and i n the 58 early;, p e r i n u c l e o l a r stage ( F i g . 22 and 23). From the r e s e a r c h mentioned i n the p r e v i o u s s e c t i o n , i t was concluded t h a t 1.0 yg salmon gonadotropin per gram body weight was as e f f e c t i v e i n a c c e l e r a t i n g o v a r i a n maturation as the h i g h e r dosage of 10 u g per gram body weight. The treatments i n v o l v i n g salmon gonadotropin i n the second l a r g e r =experiment t h e r e f o r e corresponded to Treatment B. The r e l a t i v e e f f e c t i v e n e s s between salmon gonadotropin a l o n e and i n combination w i t h estrogen i n i n c r e a s i n g the r a t e of i n c o r p o r a t i o n of y o l k can be determined from F i g . 49, and by comparison of F i g . 3^ and 35. No primary y o l k stage oocytes were observed i n pink salmon o v a r i e s from treatment G a f t e r 126 days of treatment. In both treatment GE-Q and GE C, however, primary y o l k stage oocytes c o u l d be seen. Not only d i d these oocytes c o n t a i n y o l k g l o b u l e s , but they were a l s o surrounded by the f o l l i c l e l a y e r , t y p i c a l of t h i s stage: a g r a n u l o s a of c u b o i d a l c e l l s , w i t h an o v e r l y i n g theca of one to s e v e r a l l a y e r s . E s t r a d i o l 17 galone, or a t a dosage of 15 y g per gram body weight w i t h salmon gonadotropin s t i m u l a t e d maturation of the oocytes i n i t i a l l y , but by the f o u r t h sample the o v e r a l l e f f e c t was i n h i b i t o r y when compared w i t h c o n t r o l s . Formation of a s m a l l number of l a r g e , y o l k y oocytes i n the f i n a l stages of oocyte maturation was observed i n s e v e r a l of the treatments ( F i g . 49). Oocytes i n the post-primary y o l k stage were f i r s t induced i n treatment GEt, a f t e r 168 days of treatment. Forty-two days l a t e r , t h r e e treatments contained f i s h w i t h o v a r i e s e x h i b i t i n g marked development. Treatment G had one f i s h of three sampled w i t h 50 oocytes (1 . 3 mm i n 59 diameter): Treatment GE_ contained one specimen of three sampled with 30 oocytes ( 2 . 2 mm in diameter); and one of the three fish in Treatment GE D had one full- s i z e d oocyte (4 mm in diameter)— Fig. 3 6 . The number of post-primary yolk stage oocytes in Treatment GE C had declined to six (3 mm in diameter) in one of three specimens taken in the terminal sample. The f i n a l sample also revealed one specimen from Treatment G which had 50 oocytes with a mean diameter of 1 .9 mm (Fig. 3 7 ) . As can be deduced from these results, a high degree of var i a b i l i t y in the compo-sition of the ovary was found within the treatments. For example, another fish from the terminal sample of Treatment G had no oocytes beyond the early perinucleolar stage (Fig. 3 8 ) . The pink salmon which had been stimulated to a larger-than-normal size prior to treatment with hormones, had the same pattern of variab i l i t y within treatments as was mentioned above. The f i n a l sample on February 18, contained one fish from the LGE treatment (corresponds to treatment GE^ ,) which had 8 post-primary yolk stage oocytes (averaging in diameter — 1.8 mm), whereas the other two fish in that treatment had none more developed than the late perinucleolar stage. The percentage of advanced stages in the other treatments in the terminal sample was lower than that of the control. No oocytes more advanced than the yolk vesicle stage were found in the treat-ment in which progesterone was added to gonadotropin and estrogen. Direct comparisons cannot be made between the ovarian development obtained in this experiment and that in the previous study because hormonal treatment of the normal-sized fis h was initiated 69 days earlier than in the larger f i s h . 60 In both of the aforementioned experiments and i n samples from uninfected pink salmon ;v-<, the yolk v e s i c l e stage was found to be susceptible to a t r e s i a . E a r l i e r stages never showed signs of regression. Members of the primary yolk stage were also found to subsequently become a t r e t i c . The apparent contradiction (mentioned i n section (a) of the results) between the low average diameter and G.S.I, of some treatments and v i s u a l observations that a marked accelera-t i o n of oogenesis had occurred i n some of the oocytes can be resolved by a consideration of: ( 1 ) The percentage of regressed oocytes present and (2) the percentage of pre-yolk v e s i c l e stages. Extensive a t r e s i a characterized ovaries containing oocyte stages undergoing yol k - v e s i c l e formation and yolk i n -corporation. Regression of oocytes was induced f i r s t In f i s h injected with e s t r a d i o l 17 3 : the higher dosages had the greatest numbers of a t r e t i c stages. Simultaneous i n j e c t i o n of gonadotropin did appear to moderate the ef f e c t somewhat. By the t h i r d sample, some of the yolk v e s i c l e stages were regres-sing i n Treatment G. The pattern of an increasing percentage of a t r e t i c oocytes f o r eaeh treatment with time was disrupted for most treatments i n the l a t e r samples because many oocytes which had regressed p r i o r to the sample had been completely resorbed. The information presented on percentage ovarian composition takes into consideration only those oocytes which could be counted. Another important fiactor to be considered i n the analysis of the re s u l t s was the high percentage of pre-yolk v e s i c l e stages i n f i s h injected with estrogen when compared 61 to a d e c l i n e i n the number of these c e l l s i n the c o n t r o l s . D e s p i t e the f a c t t h a t oogonia were not counted, e x t e r n a l mani-f e s t a t i o n s of s t i m u l a t e d o o g o n i a l mitoses were found i n a l l samples of f i s h t r e a t e d with e s t r a d i o l 17 g , and i n sample number s i x of Treatment G. Oocytes i n the e a r l i e s t forms of the e a r l i e r p e r i n u c l e o l a r stage were seen to be d e v e l o p i n g from n e s t s of germinal c e l l s ( F i g . 39). The subsequent h i g h numbers of s m a l l oocytes g r e a t l y reduced the mean diameter of the oocytes i n t h a t treatment, and others t r e a t e d w i t h estrogen. The r e s u l t s o b tained i n the pink salmon obtained from Dr. J . R. B r e t t q u a l i t a t i v e l y p a r a l l e l e d those d e s c r i b e d above. Treatment LGPE d i f f e r e d from the o t h e r s i n t h a t t h e r e were fewer e a r l y p e r i n u c l e o l a r stages. A comparison was made of the oocytes i n f i s h i n j e c t e d w i t h hormones, s a l i n e - i n j e c t e d c o n t r o l s and u n i n j e c t e d pink salmon a l l o w e d to develop to m a t u r i t y i n a q u a r i a . I t was decided t h a t the v a r i o u s stages of oocytes were a l i k e i n t h e i r appearance r e g a r d l e s s of the treatment from which they o r i g i n -a t e d . The r a t e of development of the oocytes, however, v a r i e d , There were s i g n i f i c a n t d i f f e r e n c e s to be found between the composition of the ovary of the u n i n j e c t e d pink salmon maintained i n a q u a r i a throughout t h e i r normal l i f e c y c l e ( F i g . 5 0 ) and t h a t of f i s h whose gonad development had been s t i m u l a t -ed with hormones. The most dramatic was the d i f f e r e n c e i n the number of post-primary y o l k stage oocytes developed: s e v e r a l hundred i n the normal f i s h compared to one to s e v e r a l i n some treatments of the i n j e c t e d pink salmon. The post-primary y o l k stage obcytes i n the t e r m i n a l sample of the u n i n j e c t e d f i s h 62 had an average diameter of 2 . 7 mm; they were c o n s i d e r e d to be one month from b e i n g r i p e . There was a complete absence of p r e - y o l k stages by J u l y of the year of sexual m a t u r i t y ; t h e r e -f o r e , the o v e r a l l development of the oocytes comprising the ovary was more synchronous. I t was i n t e r e s t i n g to f i n d t h a t more than h a l f of the oocytes were a t r e t i c by the end of August i n the f i s h not t r e a t e d w i t h hormones. Is t h i s a r e s u l t of the s t r e s s of confinement w i t h i n an aquarium? Only a comparison w i t h o v a r i e s of w i l d pink salmon w i l l p r o v i d e the answer. (b) H i s t o c h e m i s t r y : ( i ) A5-3g H y d r o x y s t e r o i d Dehydrogenase The a c t i v i t y of 3 3 - o l dehydrogenase was s t u d i e d i n pink and s p r i n g salmon f r y i n j e c t e d w i t h v a r i o u s dosages of salmon gonadotropin d u r i n g the summer of 1969 ( F i g . 51 and 5 2 ) . No d i s t i n c t l o c a l i z a t i o n s were found i n any of the o v a r i e s . The a c t i v i t y i n the f o l l i c l e l a y e r ( s ) d i d not d i f f e r from t h a t i n the ooplasm. A pink s t a i n was observed i n pink salmon from Treatments A and B which was darker than t h a t found i n the c o n t r o l by the end of the experiment ( F i g . 3 0 ) . No r e a c t i o n was ever o b t a i n e d i n c o n t r o l o v a r i e s of the s p r i n g salmon f r y , the f i s h t r e a t e d w i t h salmon gonadotropin had only s l i g h t a c t i v i t y . ( i i ) L i p i d s The h i s t o c h e m i c a l l y demonstrable l i p i d s i n the oocytes of o v a r i e s from the pink salmon i n the 1970/71 experiment were s t u d i e d by s t a i n i n g f r o z e n s e c t i o n s w i t h O i l Red 0 and Sudan 63 B l a c k B. The r e a c t i o n s obtained were compared wi t h those i n o v a r i e s from u n i n j e c t e d pink salmon maintained i n a q u a r i a throughout t h e i r l i f e c y c l e . A d i r e c t c o r r e l a t i o n between the stage of development of oocytes, and the s i z e and d i s t r i b u t i o n of the l i p i d s was found, r e g a r d l e s s of the treatment the f i s h has r e c e i v e d . E a r l y p e r i n u c l e o l a r stage oocytes showed no r e a c t i o n w i t h the l i p i d s t a i n s . Late p e r i n u c l e o l a r stage oocytes had s m a l l d r o p l e t s l o c a t e d p r i m a r i l y a t the p e r i p h e r y of the ooplasm. By the y o l k v e s i c l e stage, the p e r i p h e r a l d r o p l e t s had i n c r e a s e d i n s i z e , and some l i p i d c o u l d be seen i n the c e n t r a l r e g i o n of the oocyte ( P i g . 2 9 ) . L i p i d g l o b u l e s l a r g e r than those i n p r e v i o u s stages were d i s t r i b u t e d throughout the ooplasm i n the primary y o l k stage ( F i g . 3 3 ) . Post-primary y o l k stage oocytes were c h a r a c t e r i z e d by a v i r t u a l absence of l i p i d s i n the c e n t r a l r e g i o n s of the ooplasm. Large g l o b u l e s c o u l d be found a t the p e r i p h e r y of these c e l l s . A t r e t i c oocytes s t a i n e d i n t e n s e l y w i t h Sudan Black B. (c) Appearance None of the secondary sex c h a r a c t e r i s t i c s t y p i c a l of the s e x u a l l y mature a d u l t were seen i n f i s h from any of the treatments. Female f i s h were u n d i s t i n g u i s h a b l e from males. The c l o a c a of a l l specimens t r e a t e d w i t h e s t r o g e n showed a marked s w e l l i n g . The body weight of specimens i n j e c t e d w i t h , 1 5 yg e s t r a -d i o l 1 7 gper gram body weight with or without concurrent a d m i n i s t r a t i o n of salmon gonadotropin was g r e a t l y depressed. The f i s h were s i c k , and the m o r t a l i t y r a t e from these treatments 64 was h i g h . Pink salmon i n j e c t e d w i t h 1.5y g of estrogen had s i m i l a r symptoms, but to a l e s s e r extent than those d e s c r i b e d above. The appearance and body weight of treatments i n v o l v i n g salmon gonadotropin a l o n e , and i n combination w i t h the low dosage of e s t r a d i o l 17g was not s i g n i f i c a n t l y d i f f e r e n t from the i n j e c t e d c o n t r o l s a f t e r 210 days of treatment. 65 DISCUSSION Ova r i a n maturation i n j u v e n i l e pink and s p r i n g salmon was s t i m u l a t e d w i t h exogenous hormones f o r v a r y i n g p e r i o d s up to n i n e months. No pr e v i o u s r e s e a r c h over a s i m i l a r time i n t e r v a l has been rec o r d e d i n t e l e o s t s . I t was necessary to e s t a b l i s h the e f f e c t of long-term i n j e c t i o n of gonadotropin w i t h or without e s t r a d i o l 17e because o f the p o t e n t i a l of the method w i t h r e g a r d t o r e p o p u l a t l o n of empty c y c l e s of pink salmon, and because of the p a u c i t y of i n f o r m a t i o n on i n d u c t i o n of pr e c o c i o u s s e x u a l m a t u r i t y i n female t e l e o s t s . E s t r o g e n i n c r e a s e d the number of s m a l l e a r l y p e r i n u c l e o -l a r stage oocytes. C e l l d i v i s i o n had been s t i m u l a t e d i n the germ i n a l c e l l s which g i v e r i s e to oocytes. None of the oocytes i n f i s h t r e a t e d w i t h e s t r a d i o l 178 alone developed beyond the l a t e p e r i n u c l e o l a r stage. E s t r o g e n by i t s e l f , t h e r e f o r e , pre-vented the v i t e l l o g e n l c phase of development by a mechanism which was not determined. Estrogens s t i m u l a t e d m i t o t i c d i v i -s i o n s of the germinal e p i t h e l i u m i n Phoxinus l a e v l s ( Bullough, 1942)f and Heteropneustes f o s s l l l s (Sundararaj and Goswami, 1968). In both cases, more oogonia and primary oocytes r e s u l t -ed from the i n c r e a s e d d i v i s i o n s . The primary oocytes i n Phoxinus l a e v l s subsequently degenerated. E s t r a d i o l benzoate increased the number and s i z e o f p r e - f o l l i c u l a r stages of oocyte i n the hypophysectomized r a t , presumably by i n c r e a s i n g the num-bers of p r e c u r s o r c e l l s (de Wit, 1953). V i v i e n (1939, 19^1) advanced a h y p o t h e s i s t h a t there i s a c r i t i c a l s i z e below which oocytes a r e u n a f f e c t e d by hypophysectomy. Numerous subsequent experiments have v e r i f i e d 66 t h i s statement. P r e - v i t e l l o g e n i c stage oocytes have c o n s i s t e n t -l y been seen to be maintained f o l l o w i n g p i t u i t a r y removal. The q u e s t i o n of whether the growth and development of oocytes l e s s mature than the y o l k v e s i c l e stage i s s t i m u l a t e d by p i t u i t a r y gonadotropin i n t e l e o s t s has had l i t t l e study. Chestnut (1970) noted t h a t i n j e c t i o n of f i n g e r l i n g coho salmon f o r three weeks w i t h p i t u i t a r y homogenates from spawning coho caused the r e s i d e n t oocytes to l o s e t h e i r p e r i p h e r a l b a s o p h i l l i a . In one of f i v e f i s h a f u r t h e r advancement was observed. The speciman had oocytes i n the y o l k v e s i c l e stage. S i m i l a r obser-v a t i o n s were made i n the present study. Conversion of e a r l y p e r i n u c l e o l a r stage oocytes (have p e r i p h e r a l b a s o p h i l l i c sub-stance i n ooplasm — r e f e r r e d t o as the p a l l i a l l a y e r ) to l a t e p e r i n u c l e o l a r stage oocytes ( l a r g e r , no c y t o p l a s m i c b a s o p h i l l i a ) occurred e a r l i e r i n j u v e n i l e pink salmon t r e a t e d w i t h salmon gonadotropin than i n c o n t r o l s . There i s , t h e r e f o r e , a s t i m u l a t i o n of the development of p r e - v i t e l l o g e n i c stages by p i t u i t a r y gonadotropin. Formation of c o r t i c a l a l v e o l i i n t r e a t -ment A and B a f t e r one month of i n j e c t i o n s preceded the normal time of appearance o f the y o l k v e s i c l e stage by f o u r months. The r e s u l t s o b t a i n e d f o r the s p r i n g salmon c o n t r a d i c t those mentioned above. Treatment w i t h homologous gonadotropin had no e f f e c t on the stage or s i z e of the e a r l y p e r i n u c l e o l a r stage oocytes. One e x p l a n a t i o n i s t h a t the e a r l y p e r i n u c l e o l a r stage oocytes i n the s p r i n g salmon were r e l a t i v e l y l e s s mature than those of the pink salmon, and were unable to respond to s t i m u l a t i o n a t t h a t time. The f a c t t h a t the oocytes of the s p r i n g salmon never reached the average diameter of the e a r l y 67 p e r i n u c l e o l a r s tage o o c y t e s i n t h e p i n k salmon z e r o c o n t r o l s u p p o r t s t h i s s t a tement. A l t e r n a t i v e l y , perhaps t h e o o c y t e s o f t h e s p r i n g salmon were l e s s r e s p o n s i v e t o g o n a d o t r o p i n t h a n t h o s e o f t h e p i n k salmon because Oncorhynchus t s h a w y t s c h a n o r m a l l y mature two y e a r s l a t e r t h a n do t h e p i n k s . I t was a n t i c i p a t e d t h a t t h e p i n k salmon t r e a t e d w i t h a c o m b i n a t i o n o f salmon g o n a d o t r o p i n and e s t r o g e n would d e v e l o p y o l k y o o c y t e s more q u i c k l y t h a n t h o s e t r e a t e d w i t h salmon gonad-o t r o p i n a l o n e . The r e s u l t s c o n f i r m e d t h i s p r o p o s a l . Oocytes i n t h e p r i m a r y y o l k s t a g e were f i r s t o b s e r v e d i n p i n k salmon i n j e c t e d w i t h 1 . 5 micrograms e s t r a d i o l 176 p e r gram body w e i g h t ( t h r o u g h o u t , o r a f t e r 84 d a y s ) , p l u s 1.0 micrograms p e r gram body w e i g h t salmon g o n a d o t r o p i n f o r 126 days. By c o m p a r i s o n , t h e f i s h t r e a t e d w i t h g o n a d o t r o p i n o n l y (1.0 micrograms p e r gram body w e i g h t ) d i d n o t have o o c y t e s c o n t a i n i n g y o l k g l o b u l e s f o r 42 days more. The h i g h dosage of e s t r o g e n (15 micrograms) p l u s g o n a d o t r o p i n i n h i b i t e d o v a r i a n m a t u r a t i o n . S t u d i e s of o t h e r v e r t e b r a t e s i n w h i c h p h y s i o l o g i c a l of e s t r o g e n and g o n a d o t r o p i n were i n j e c t e d c o n c u r r e n t l y i n t o t h e f e m a l e showed a s i m i l a r enhancement of development of t h e o v a r y . A s y n e r g i s t i c a c t i o n on t h e development o f t h e o v a r i e s of h ypophysectomized r a t s was f o u n d w i t h t r e a t m e n t s o f d i e t h y l -s t i l b e s t r o l and FSH, (Simpson e t a l , 1941), and d i e t h y s t l l b e s t r o l and HCG ( P e n c h a r z , 19^0; Simpson e t a l , 19^1). The i n c r e a s e i n s i z e o f t h e o v a r y was due p r i m a r i l y t o an enlargement o f t h e f o l l i c l e s . P h i l l i p s and Van Tienhoven (I960) o b s e r v e d t h a t p i n t a i l ducks c a p t u r e d from m i g r a t o r y f l o c k s , and h e l d i n c a p t i v i t y f a i l e d t o show o v a r i a n development. O v a r i a n 68 gonadotropin i n j e c t i o n s , i n combination with d i e t h y l s t i l b o e s t e r o l induced nearly normal f o l l i c u l a r development. Four out of six non-breeding black ducks treated with a combination of chicken ante r i o r p i t u i t a r y extract and d i e t h y l s t i l b o e s t e r o l had large f o l l i c l e s with yellow yolk, whereas none of the birds treated with either hormone alone had yellow yolk ( P h i l l i p s , 1 9 5 9 ) . Gonadotropin extracted from teleost p i t u i t a r i e s was e f f e c t i v e as a stimulant to development of the ovary i n pre-vious research on hypophysectomized adult t e l e o s t s . A l l stages of reproductive maturity, including v i t e l l o g e n e s i s and ovulation were reinduced by replacement therapy with salmon gonadotropin SG-G100 following p i t u i t a r y removal i n Carrassius auratus (Yamazaki and Donaldson, 1 9 6 8 ) , P o e c i l l a r e t i c u l a t a ( L i l e y and Donaldson, I 9 6 9 ) , and Heteropneustes f o s s i l i s (Sundararaj, Anand, and Donaldson, 1971). The gonad of the juvenile salmonid, how-ever, i s more refractory to gonadotropin, as demonstrated by the research of Schmidt et a l , I 9 6 5 . There was a small growth i n the size of ovaries from immature Salmo g a i r d n e r i i injected f o r two weeks with an extract of Oncorhynchus tshawytscha p i t u i -t a r i e s . Oocytes i n f i n g e r l i n g Oncorhynchus kisutch matured slowly when tested f o r three weeks with a homoplastic preparation of p i t u i t a r y glands from spawning adults (Chestnut, 1970 - discussed previously). In the present experiment, six months of i n j e c t i o n s of 1 .0 micrograms per gram body weight salmon gonadotropin plus 1 .5 micrograms per gram body weight e s t r a d i o l 178 were required before the f i n a l stage of oocyte maturation was reached i n one of three specimens f o r both normal-sized and enlarged f i s h . A 69 comparable stage of maturity did not occur u n t i l six weeks l a t e r i n dne pink salmon injected with 1.0 milligram per gram body weight SG-G100 and one treated with salmon gonadotropin and e s t r a d i o l 1? g ( a f t e r three months). A l l of the aforementioned f i s h required 42 more days of treatment before t h e i r oocytes were larger than 2 mm i n diameter. High v a r i a b i l i t y within treatments characterized these samples. In the precociously mature ovaries, some of the devia-tions from the normal pattern of development warrant further discussion. Oocytes i n the f i n a l stage of maturation i n the treated pink salmon were f a r fewer i n number than those at a s i m i l a r stage i n the uninjected controls. There was apparently some mechanism which compensated f o r the f a c t that the test pink salmon were small i n size when precocious ovarian development occurred. A t r e s i a of oocytes undergoing v l t e l l o g e n e s i s was f i r s t observed i n treatments i n which estrogen was injected. Six weeks l a t e r , oocytes i n the yolk v e s i c l e stage were regressing i n the pink salmon administered salmon gonadotropin. The i n i -t i a l c o r r e l a t i o n between the l e v e l of estrogen and the number of a t r e t i c oocytes was l o s t i n l a t e r samples because of resorption of the regressed germinal c e l l s . The data indicated that r e l a t i v e l y few yolky oocytes were allowed to develop to maturity i n any of the f i s h treated with exogenous hormonesi the majority of the remainder of the oocytes undergoing v l t e l l o g e n e s i s were converted into pre-ovulatory corpora 70 a t r e t i c a . More than h a l f of the oocytes had been converted to c o r p o r a a t r e t i c a by sexual m a t u r i t y i n the u n i n j e c t e d pink salmon. T h i s phenomenon i s not unusual, as the ovary of every r e p r o d u c t i v e l y mature f i s h c o n t a i n s r e g r e s s e d oocytes ( B a l l , I960). The stages of f o l l i c u l a r a t r e s i a f o l l o w e d the p a t t e r n e s t a b l i s h e d by B r e t s c h n e i d e r and de Wit (19^7). I t was con-f i n e d to those oocytes i n which v i t e l l o g e n e s i s had begun. A s m a l l amount of a c t i v i t y of A5 -36 h y d r o x y s t e r o i d dehydrogenase was found i n o v a r i e s of pink salmon i n j e c t e d w i t h v a r i o u s dosages -of salmon gonadotropin which c o n t a i n e d oocytes to the y o l k v e s i c l e stage. There were no marked depo-s i t s of formazan. The e a r l y p e r i n u c l e o l a r stage oocytes from the o v a r i e s of the t e s t s p r i n g salmon s t a i n e d a f a i n t pink c o l o r . The r e s u l t s of Chestnut (1970) a r e s i m i l a r to those mentioned above. I n j e c t i o n of f l n g e r l i n g Oncorhynchus k l s u t c h f o r t hree weeks w i t h p i t u i t a r y homogenates from spawning a d u l t s r e s u l t e d i n the f o r m a t i o n of a pink hue i n the o v a r i e s , which was sometimes l o c a l i z e d i n the stroma between the oocytes. The f a i l u r e to observe a marked r e a c t i o n i n the j u v e n i l e pink and s p r i n g salmon c o u l d w e l l be r e l a t e d to the immaturity of the oocytes i n the t e s t f i s h . 71 GENERAL SUMMARY T h i s study has p r o v i d e d evidence t h a t the r e p r o d u c t i v e m a t u r i t y of the male and female pink salmon can be a c c e l e r a t e d . In the case of the male j u v e n i l e , the c y c l e of t e s t i c u l a r m a t u ration can be manipulated with salmon gonadotropin i n j e c t i o n to produce v i a b l e spermatozoa one y e a r e a r l i e r than normal and by the time of spawning of any stock of w i l d pink salmon. The o v a r i e s from females t r e a t e d w i t h salmon gonado-t r o p i n w i t h or without estrogen over an extended time i n t e r v a l c o n t a i n e d a s m a l l number of oocytes i n the f i n a l stage of maturation; f u l l r e p r o d u c t i v e m a t u r i t y s i x months e a r l i e r than normal i s p o s s i b l e . The p r e c o c i o u s l y mature pink salmon male appears to o f f e r the most a p p l i c a b l e s o l u t i o n to the problem of r e p o p u l a t i n g ' o f f 1 year c y c l e s of pink salmon. A f i r s t c r o s s of a r e s i d e n t p r e c o c i o u s male w i t h a t r a n s p l a n t would produce progeny w i t h jQ% of the g e n e t i c complement of the home-stream f i s h . Repeating t h i s procedure w i t h the ova from the o f f s p r i n g would r e s u l t i n a stock w i t h 75%* and then 8 7i% of the gene p o o l of the male r e s i d e n t f i s h . T h i s method would be more convenient than a d i r e c t c r o s s employing m i l t and ova from p r e c o c i o u s l y mature f i s h because: (1) the number of ova developed i s s m a l l and r e q u i r e s months of treatment; (2) an advancement of only s i x months i s p o s s i b l e i n the female, thereby n e c e s s i t a t i n g two c r o s s e s before a t o t a l displacement of one year i s a c h i e v e d . 72 F i g u r e 21: Ovary of a zero c o n t r o l j u v e n i l e s p r i n g salmon (5.^ cm l o n g ) . V i s i b l e oocytes are i n the e a r l y p e r i n u c l e o l a r stage. Haematoxylin and e o s i n . X 150. F i g u r e 22: Ovary of a j u v e n i l e s p r i n g salmon 10.0 cm lo n g i n j e c t e d t h r e e times per week wit h f i s h s a l i n e f o r 84 days. Oocytes a r e a l l i n the e a r l y p e r i n u c l e o l a r stage. Haematoxylin and e o s i n X 150. F i g u r e 23: Ovary of a j u v e n i l e s p r i n g salmon 11.2 cm i n l e n g t h , i n j e c t e d t h r e e times per week w i t h 10.0 ug salmon gonadotropin per gram body weight f o r 84 days. Oocytes a r e a l l i n the e a r l y p e r i n u c l e o l a r stage. Haematoxylin and e o s i n X 150. F i g u r e 24: F o l l i c l e l a y e r of an e a r l y p e r i n u c l e o l a r stage oocyte from the same s e c t i o n as seen i n F i g u r e 23. Haematoxylin and e o s i n , X 1500. F i g u r e 25: The two c o n t r o l oocytes a r e i n the' l a t e p e r i n u c l e o l a r stage. N o t i c e the y o l k nucleus near the f o l l i c l e l a y e r i n the one on the l e f t . A t the p e r i p h e r y , e a r l y p e r i n u c l e o l a r stage oocytes can be seen. The s e c t i o n was taken from the ovary of a pink salmon (13.6 cm i n length) i n j e c t e d t h r e e times per week wit h f i s h s a l i n e f o r 98 days. Haematoxylin and e o s i n , X 150. F i g u r e 26: F o l l i c l e l a y e r of a l a t e p e r i n u c l e o l a r stage oocyte (above) Lower oocyte i s i n the e a r l y p e r i n u c l e o l a r stage. Haematoxyline and e o s i n , X 1500. 73 Figure 27; Oocyte in the yolk vesicle stage from the ovary of a pink salmon (13.2 cm in length) injected three times per week with 10.0 g salmon gonadotropin per gram body weight for 96 days. Haematoxylin and eosin X 150. Figure 28: F o l l i c l e layers and thecal layer of the oocyte in Figure 27. Haematoxylin and eosin X 1500. Figure 29: Oocyte in the yolk vesicle stage stained for lip i d s with Sudan Black B. X 80. Figure 30: Histochemically demonstrable A 5-3f3 hydroxysteroid dehydrogenase activity in yolk vesicle stage oocytes from pink salmon Injected thrice weekly with 10.0 yg salmon gonadotropin per gram body weight for 96 days. The incubation medium contained dehydroepiandrosterone, NAD, and nitro-BT. The granules and stained portions are formazan deposits. X 200. Figure 31: Primary yolk stage oocyte (pink salmon). Haematoxylin and eosin. X 60. Figure 32: F o l l i c l e and thecal layers of the primary yolk stage oocyte in Figure 12. Haematoxylin and eosin. X 600. Figure 33: Primary yolk stage oocyte stained for lipids with O i l Red 0. X 50. 74 Figure 34: Section of an ovary from a pink salmon juvenile (14.6 cm i n length) injected three times per week with 1.0 y g salmon gonado-tr o p i n and 1.5 yg e s t r a d i o l 176 per gram body weight f o r 126 days. Note the number of primary yolk stage oocytes. Haematoxylin and eosin. X 20. Figure 35: Section of an ovary from a pink salmon juvenile (14.9 cm i n length) injected three times per week with 1.0 yg salmon gonado-tropin per gram body weight f o r 126 days. Note that the majority of oocytes are i n the yolk v e s i c l e stage. Haematoxylin and eosin. X 25. Figure 36: Frozen section of a post primary yolk stage oocyte from a pink salmon (16.3 cm i n length) injected three times per week with 1.0 yg salmon gonadotropin and 1.5 yg e s t r a d i o l 178 per gram body weight for 210 days. Haematoxylin and eosin. X 15. Figure 37: Frozen section of a post primary yolk stage oocyte from a pink salmon (17 .0 cm i n length) injected three times per week with 1.0 yg salmon gonadotropin per gram body weight f o r 258 days. Haematoxylin and eosin. X 15. Figure 38: Ovary of a f i s h treated i d e n t i c a l l y to that described i n Figure 37. Note the paucity of oocytes more advanced than the early perinucleolar stage. Haematoxylin and eosin. X 50. Figure 39: Section of an ovary from a pink salmon treated with 1.0 yg salmon gonadotropin per gram body weight f o r 126 days, and l.Oy g e s t r a d i o l 17 B per gram body weight for the l a s t 92 days. Note that precursor germinal c e l l s appear to be a c t i v e l y producing early perinucleolar stage oocytes. Haematoxylin and eosin. X 200. 75 Figure 40; Gonadosomatic index (G.S.I.) of female pink (Oncorhynchus  gorbuscha) and spring (Oncorhynchus tshawytscha) salmon treated three times per week with various dosages of salmon (Oncorhynchus  tshawytscha) gonadotropin during the summer of 1969. Values are averages taken from Table IX and X. n JUNE 1 JULY 1 AUGUST ' SEPL 76 Figure 41: Gonadosomatic index (G.S.I.) of female pink salmon treated three times per week with various combinations of salmon (Oncorhynchus tshawytscha) gonadotropin and e s t r a d i o l 178 . Figures were taken from Table XI. GAE = 1.0 yg salmon gonadotropin and 15.0 yg e s t r a d i o l 173 per gram body weight. GBE = 1.0 yg salmon gonadotropin and 1.5 yg e s t r a d i o l 178 per gram body weight. GCE = 1.0 yg salmon gonadotropin per gram body weight throughout the experiment and 1.5 ng e s t r a d i o l 178 per gram body weight a f t e r 84 days. AE = 15 yg e s t r a d i o l 17 8 per gram body weight. BE = 1.5 yg e s t r a d i o l 178 per gram body weight. Co = Control (injected with f i s h s a l i n e ) . U.I. Co = Uninjected control. 1.201-0.80k -6 SI 0.40k D E C J A N F E B 1 9 7 1 77 Figure 42; Gonadosomatic index (G.S.I.) f o r ex t r a - o r d i n a r i l y large female pink salmon treated three times per week with various dosages of salmon (Oncorhynchus tshawytscha) gonadotropin. Data were taken from Table XII. LG = 1.0 yg salmon gonadotropin per gram body weight. LGE = 1.0 y g salmon gonadotropin plus 1.5 yg e s t r a d i o l 17 Bper gram body weight. LGPE = 1.0 yg salmon gonadotropin, 1.5 Hg e s t r a d i o l 176 and 5 vg progesterone per gram body weight. Co = control (injected with f i s h s a l i n e ) . 0 . 8 0 L G o LGE © L G P E n Co • GSI 0 . 4 0 — 1 1 1 i i S E P T OCT NOV DEC J A N F E B 1970 1971 78 Figure 43; Gonadosomatic Index (G.S.I.) of uninfected female pink salmon maintained i n aquaria throughout t h e i r l i f e cycle. Values are averages taken from Table XIII. 79 Figure 44: Mean oocyte diameter (microns) of juvenile female pink salmon and spring salmon injected three times per week with various dosages of salmon (Oncorhynchus tshawytscha) gonadotro-pin during the summer of 1 9 6 9 . . Values are averages taken from Tables XIV and XV. A = 1 0 . 0 yg salmon gonadotropin per gram body weight. B = 1 .0 y g salmon gonadotropin per gram body weight. C = 0 . 1 yg salmon gonadotropin per gram body weight. Co = control (injected with f i s h s a l i n e ) . U.I. Co = uninjected control. 80 Figure 45: Mean oocyte diameter (microns) of pink salmon treated three times per week with several combinations of salmon (Oncorhynchus tshawytscha) gonadotropin and e s t r a d i o l 17g . . Values are averages taken from Table XVI. G = 1.0 yg salmon gonadotropin per gram body weight. GAE = 1.0 yg salmon gonadotropin plus 15.0 yg es t r a d i o l 17 Bper gram body weight. GBE = 1.0 yg salmon gonadotropin plus 1.5 yg es t r a d i o l 17 Bper gram body weight. GCE = 1.0 yg salmon gonadotropin per gram body weight throughout the experiment plus 1.5 yg es t r a d i o l 17B per gram body weight a f t e r 84 days. AE = 15.0 yg salmon gonadotropin per gram body weight. BE = 1.5 yg salmon gonadotropin per gram body weight. Co = saline injected control. U.I. Co = uninjected control. 81 Figure 46; Mean oocyte diameter of ext r a - o r d i n a r i l y large pink salmon obtained from Dr. J . R. Brett, injected three times per week with combinations of various exogeneous hormones; salmon (Oncorhynchus  tshawytscha) gonadotropin, e s t r a d i o l 176, , and progesterone. The values are averages taken from Table XVII. G 1.0 vig salmon goandotropin per gram body weight. GE 1 .0 ug salmon gonadotropin and 1 .5 e s t r a d i o l 17g per gram body weight. GPE 1 . 0 yg salmon gonadotropin, 1 .5 yg e s t r a d i o l 176 • and 15 yg progesterone per gram body weight. Co control (injected with f i s h s a l i n e ) . 82 Figure 47; The mean oocyte diameter of oocytes from uninjected pink salmon maintained throughout t h e i r l i f e cycle. Values are averages taken from Table XVIII. 83 Figure 48: Mean percentages of oocytes which comprised the ovaries of juvenile pink salmon treated with various dosages of salmon gonadotropin. Each complete bar represents 100$. The number of f i s h per treatment (n) seen i n Table XIV. A = 10.0 yg salmon gonadotropin per gram body weight. B = 1.0 y,g salmon gonadotropin per gram body weight. C = 0.1yg salmon gonadotropin per gram body weight. Co = control (injected with f i s h s a l i n e ) . 1 E A R L Y P E R I N U C L E O L A R 2 L A T E P E R I N U C L E O L A R 3 Y O L K V E S I C L E T R E A T M E N T A B C o S A M P L E D A T E 17/6/69 2 2 1 1 1 2 2 2 2 1 i 1 0 1 2 3 4 5 i 6 7 30/6/69 14/7/69 28/7/69 11/8/69 25/B/69 8/9/69 22/9/69 84 Figure 49« Mean percentages of oocytes which comprise the ovaries of pink salmon treated with various combinations of salmon (Oncorhynchus tshawytscha) gonadotropin and e s t r a d i o l 17/9 . Each complete bar represents 100$. The number of f i s h per sample can be seen i n Table XVI. AE - 15 yiig e s t r a d i o l 17/# per gram body weight BE = 1.5/tg e s t r a d i o l 17/3 per gram body weight GAE = 1.0 fig salmon gonadotropin plus 15.0 jig e s t r a d i o l 17/9 per gram body weight. GBE ~ 1.0 ^ g salmon gonadotropin per gram body weight (throughout) plus l ^ / ' - g e s t r a d i o l 17/5 per gram body weight. GCE = 1 .0jxg salmon gonadotropin per gram body weight throughout the experiment, and 1*5 /ig e s t r a d i o l 17/3 p©r gram body weight a f t e r 84 days. G = 1 . 0 ytig salmon gonadotropin per gram body weight Co = control (injected with f i s h s a l i n e ) . T R E A T M E N T A E 1 E A R L Y P E R I N U C L E O L A R 2 L A T E P E R I N U C L E O L A R 3 Y O L K V E S I C L E * P R I M A R Y Y O L K s L A R G E . H A R D O O C Y T E S * A T R E T I C B E G A E G B E G C E C o S A M P L E 0 I D A T E , 25/6/70 8/8/70 2 18/9/70 31/10/70 15/12/70 5 26/1/71 5.4,3 6 10/3/71 85 Figure 50: Mean percentages of oocyte which comprise the ovaries of uninjected pink salmon maintained i n aquaria throughout t h e i r l i f e . Data was taken from Table XVIII. 1 EARLY PERINUCLEOLAR 2 LATE PERINUCLEOLAR 3 YOLK V E S I C L E * PRIMARY YOLK 5 LARGE, HARD OOCYTES 6 A T R E T I C SAMPLE DATE 6 3 8 5/11/69 6 3 9 21/1/70 10 9/3/70 30/6/70 86 Figure 51 '• The histochemically demonstrable a c t i v i t y of A 5-3B hydroxy-st e r o i d dehydrogenase i n the ovaries of juvenile pink salmon i n -jected three times per week with various dosages of salmon (Oncorhynchus tshawytscha) gonadotropin during the summer of 1 9 6 9 . Data were taken from Table XIV. A 1 0 . 0 yg salmon gonadotropin per gram body weight. B 1 .0 yg salmon gonadotropin per gram body weight. C 0 . 1 yg salmon gonadotropin per gram body weight. Co saline injected control. U.I. Co uninjected control. P I N K S A L M O N O V A R I E S B o — o C O- 9 C o c — ® I _J I J U N E J U L Y A U G S E P T 87 Figure 52: Ide n t i c a l experiment to that i n Figure 19, but with spring salmon juveniles. The number of f i s h per treatment per sample (n) and the standard deviation (SD) can be seen Table XX. S P R I N G S A L M O N O V A R I E S i . o V I S U A L U N I T S OF 0 . 5 ENZYME A C T I V I T Y 0 A B C e e Co ©—& U.I.CO a J U N E JU LY A U G S E P T 88 Table IX: Mean body weight, mean fix e d ovary weight, mean gonado-somatic index (G.S.I.) of juvenile female pink salmon treated with various dosages of salmon gonadotropin, y. = mean; S.D. = standard deviation. TABLE IX: FIXED BODY BODY OVARY SAMPLE TREAT- n LENGTH (cm) WEIGHT (g) WEIGHT (ug) G.S.I. Zero MENT y S.D. y S.D. y S.D. y S.D. Control Co 3 7 .7 0 . 5 3 . 6 1 .0 17/6/69 1 A 4 8 . 1 0 . 8 4 . 4 1 .3 B 4 7 . 3 0 . 2 3 . 0 0 . 3 C 3 8 . 3 0 . 9 4 . 5 1 .7 Co 5 8 . 1 0 . 5 4 . 0 0 . 7 2 A 3 9 . 3 0 . 4 6 . 4 0 . 6 B 4 8 . 9 0 . 8 6 . 0 1 .7 C 4 8 . 8 0 . 5 5 . 3 1 .1 Co 4 9 . 0 0 . 8 6 . 2 2 . 2 3 A 6 1 0 . 0 1 . 0 8 . 9 2 . 6 24 .6 5 . 6 0 . 2 3 0 . 0 3 B 4 9 . 2 0 . 6 6 . 6 1 .3 1 7 . 9 3 . 3 0 . 5 7 0 . 1 1 C 4 1 0 . 0 0 . 6 8 . 0 1 .8 14 .2 4 . 1 0 . 5 9 0 . 0 5 Co 6 1 0 . 0 0 . 5 8 . 2 1 . 4 1 2 . 1 3 . 4 0 . 2 9 0.04 4 A 4 1 0 . 1 0 . 5 9 . 0 0 . 9 2 5 . 5 1 6 . 2 0 . 5 8 0.18 B 1 1 . 0 0 . 6 1 2 . 4 3 . 4 3 9 . 4 6 . 7 0 . 6 6 0.08 Co 1 0 . 4 0 . 8 1 0 . 4 2 . 8 2 1 . 5 7 . 0 0.41 0 . 1 5 5 A 5 1 2 . 2 0 . 8 1 6 . 3 3 . 3 7 3 . 9 1 1 . 4 0 . 9 2 0 . 1 3 B 1 2 . 0 0 . 3 1 5 . 0 1 .3 64 .6 28 .9 0 . 8 6 0.18 Co 1 1 2 . 7 18 .9 2 5 . 8 0 . 2 7 — — 6 A 2 1 2 . 1 1 .0 1 6 . 8 5 . 7 7 3 . 6 4 . 0 0 . 9 4 0.18 B 6 1 2 . 3 1 .2 1 7 . 2 7 . 3 84 . 1 31 .4 1 . 0 1 0.04 Co 1 3 . 7 1 .4 2 2 . 7 6 . 9 ^ 3 . 9 1 3 . 6 0 . 3 8 0 . 0 2 7 A 2 1 3 . 2 0 . 1 1 9 . 8 1.1 7 4 . 9 2 0 . 1 0 . 8 9 0 . 1 5 B 5 1 3 . 8 0 . 7 2 6 . 2 4 . 6 114 .1 1 5 . 2 0 . 9 1 0 . 1 0 Co 5 1 3 . 6 0 . 5 2 2 . 4 3 . 2 5 2 . 1 9 . 5 0 . 3 0 0 . 0 5 U.I. Co 4 1 3 . 4 1 .2 24 .2 6 . 4 3 1 . 2 8 . 6 0.46 0 . 1 3 8 9 Table X; Mean body length, mean body weight, mean fix e d ovary weight, and mean gonadosomatic index (G.S.I.) of spring salmon treated with various dosages of salmon gonadotropin. y = mean; S.D. = standard deviation. TABLE X: SAMPLE TREAT- n Zero MENT Control Co 5 1 0 / 6 / 6 9 1 A 6 B 4 C 4 Co 4 2 A 2 B 6 C 4 Co 6 3 A 2 B 6 C 6 Co 4 A 3 B 1 C 3 Co 4 5 A 6 B 3 C Co 6 A 3 B 3 C 4 Co U.I. Co 5 BODY BODY LENGTH (cm) WEIGHT (g) v S.D. u S.D. 5 . 4 0 . 2 1 .8 0 . 2 FIXED OVARY WEIGHT ( y g) G.S.I, y S.D. u S.D. 5 . 9 0 . 5 2 . 0 0 . 5 6 . 2 0 . 7 2 . 9 1 .2 6 . 2 0 . 3 2 . 5 0 . 2 6 . 1 0 . 1 2 . 2 0 . 2 6 . 9 0 . 1 3A 0 . 1 7 . 3 0 . 7 4 . 1 1.4 6 . 6 0 . 8 3 . 0 0 . 8 6 . 8 0 . 5 2 . 8 1 .0 6 . 1 0 . 6 2 . 2 0 . 5 7 . 5 0 . 7 4 . 2 1 .1 6 . 9 0 . 6 3 . 0 0 . 8 7 . 6 0 . 9 4 . 7 2 . 0 6 . 5 0 . 6 2 . 5 0 . 7 5 . 3 2 . 0 0.42 0 . 0 7 7 . 0 — 4 . 0 — — 4.4 — — 0 . 2 2 — 7 . 8 0 . 3 4 . 1 0 . 3 6 . 1 1.4 0.28 0.10 7 . 6 0 . 9 4 . 7 1.7 4 . 1 1 .0 0.18 0 . 0 5 8 . 9 1 .3 7 . 9 3 . 4 1 0 . 1 4 . 1 0 . 2 7 0 . 0 8 7 . 6 1 .0 5 . 2 2 . 1 7 . 7 0 . 2 0.24 0 . 0 1 9 . 2 0 . 6 8 . 5 1 .2 8 . 0 0 . 7 0 . 1 9 0.04 8 . 0 0.4 5 . 8 0 . 8 6 . 2 1 .3 0 . 2 1 0 . 0 3 11 .2 2 . 0 1 9 . 2 6.4 1 7 . 5 8 . 6 0 . 2 2 0 . 0 3 1 0 . 1 1 .7 1 2 . 6 6 . 2 8 . 5 5 . 2 0.28 0 . 2 2 9 . 3 1 .0 8 . 6 2 . 9 7 .3 4 . 1 0 . 1 7 0.08 1 0 . 0 2 . 0 1 2 . 5 8 . 2 1 0 . 1 2 . 8 0 . 1 3 0 . 0 7 9 . 7 1.1 1 0 . 6 3 . 3 6.4 2 . 1 0.14 0 . 0 5 / / / 90 Table XI; Mean body length, mean body weight, mean gonad weight, and mean gonadosomatic index (G.S.I.) of female pink salmon treated with various dosages of gonadotropin and e s t r a d i o l 17 g during 1970/71, y - mean; S.D. = Standard deviation. TABLE XI: FISH PER SAMPLE SAMPLE & TREAT- BODY n DATE MENT BODY LENGTH (cm) WEIGHT (g) S.D. S.D. GONAD WEIGHT ( g) u S.D. G.S.I. S.D, 3 0 8.0 0.4 5.0 1.0 25/6/70 4 1 G 10.0 0.4 8.3 1.4 0.018 0 0.44 0.10 4 8/8/70 GAE 9.0 0.9 6.2 2.6 0.015 0.010 0.46 0.05 4 GBE 9.7 0.7 8.8 2.1 0.014 — 0.28 0.13 4 25/8/70 Co 10.9 0.3 11.9 1.1 0.011 — 0.20 0.02 4 2 G 12.5 0.6 17.5 2.7 0.039 0.006 0.44 0.15 3 18/9/70 GAE 12.3 0.8 14.4 1.8 0.026 0.024 0.34 0.07 4 GBE 11.9 1.0 16.4 4.7 0.047 0.016 0.58 0.10 4 6/10/70 AE 11.0 0.4 10.3 2.0 0.020 0.021 0.18 0.20 4 BE 12.2 0.7 16.7 3.7 0.034 0.014 0.18 0.05 4 Co 13.5 0.7 22.2 4.9 0.027 0.005 0.26 0.10 4 3 G 14.9 1.0 31.1 7.4 0.013 0.060 0.82 0.40 3 31/10/70 GAE 12.8 0.8 17.2 4.1 0.007 -- 0.08 0.02 4 GBE 14.6 0.7 29.9 4.1 0.120 0.080 0.72 0.66 4 GCE 14.7 1.2 31.1 7.2 0.080 0.050 0.58 0.20 2 17/11/70 Co 16.5 1.6 44.0 7.7 0.080 0.020 0.38 0.08 4 4 G 17.1 1.2 43.4 11.1 0.095 0.080 0.40 0.26 4 15/12/70 GAE 13.0 1.3 18.3 8.2 0.007 0.002 0.08 0.02 3 GBE 14.9 0.7 30.3 2.9 0.063 0.069 0.36 0.38 4 GCE 16.0 0.6 37.6 4.1 0.047 0.053 0.24 0.26 3 29/12/70 BE 13.6 0.9 23.8 3.5 0.017 0.007 0.16 0.06 2 Co 16.9 0.8 44.4 5.7 0.117 0.013 0.54 0.06 4 5 G 17.7 1.9 46.6 11.7 0.256 0.164 1.08 0.74 3 26/1/71 GBE 16.3 0.5 37.2 6.3 0.058 0.042 0.30 0.18 3 GCE 17.0 1.3 45.3 10.7 0.158 0.160 0.30 0.18 2 10/2/71 Co 16.3 1.6 37.3 11.1 0.106 0.050 0.56 0.10 4 U. ,1. Co 21.1 1.5 89.0 21.2 0.306 0.100 0.62 0.08 4 6 G 17.0 1.3 43.7 12.1 0.145 0.254 0.52 0.86 3 10/3/71 GCE 17.1 1.8 44.6 15.4 0.132 0.136 0.56 0.44 3 U.I.Co 20.5 1.8 77.9 23.2 0.247 0.045 0.64 0.08 91 Table XII: Mean body weight, mean body length, mean ovary, weight, and mean gonadosomatic index (G.S.I.) of extra-ordinary large female pink salmon juveniles treated with various dosages of salmon gonadotropin, e s t r a d i o l l? g and progesterone. y = mean; S.D. = standard deviation. TABLE XII: SAMPLE TREAT- n BODY BODY OVARY MENT LENGTH(cm) WEIGHT(g) WEIGHT (g) G.S.I. y S.D. y S.D. y S.D. y S.D. 1 LGPE 2 18.2 0.1 55.9 0.8 0.11 0.05 0.40 0.20 3/11/70 1 LG 3 19.1 0.5 64.2 4.0 0.22 0.07 0.71 0.23 26/11/70 LGE 5 19.3 1.5 69.8 16.1 0.16 0.17 0.50 0.54 2 LG 5 21.0 1.4 81 .1 19.1 0.05 0.02 0.12 0.03 18 /2/71 LGE 3 20.6 2.3 81.9 28.7 0.29 0.41 0.58 0.75 LGPE 2 20.6 1.3 69.2 22.3 0.05 0.04 0.15 0.07 Co 5 21.6 0.9 97.9 11.7 0.27 0.15 0.57 0.36 92 Table XIII; Mean body weight, mean gonad weight, and mean gonado-somatic index (G.S.I.) of uninfected female pink salmon main-tained i n aquaria throughout t h e i r l i f e cycle, y = mean; S.D. = standard deviation. TABLE XIII: BODY OVARY SAMPLE DATE n WEIGHT(g) WEIGHT(g) G.S.I. y -S.D. y S.D. y S.D. 8 5/11/69 6 95.2 9.5 0.46 0.04 0.48 0.05 9 21/1/70 4 121.6 14.6 0.98 0.19 0.80 0.14 10 9/3/70 4 177.3 26.9 1.39 0.21 0.82 0.16 11 30/6/70 4 3^9.3 44.4 4.54 1.71 1.29 0.43 12 25/8/70 6 501.3 210. 9 50.40 44.80 8.24 5.92 93 Table XIV; Mean oocyte diameter of pink salmon juveniles treated with various dosages of salmon gonadotropin, y = mean; S.D. = standard deviation. TABLE XIV: SAMPLE & DATE TREATMENT NUMBER OP SAMPLES MEAN OOCYTE DIAMETER (microns) i S.D. Zero Control 2 134.5 11.5 17/6/69 1 A 3 160.8 10.4 30 /6 /69 B 3 142.8 8.2 C 3 187.2 14.6 Co 3 205.6 23.5 2 A 3 227.6 50.8 B 3 216.7 8.8 14 /7 /69 C 3 205.0 2.8 Co 3 215.5 22.6 3 A 3 274.9 21 .1 B 3'.. 212.7 13.8 28 /7 /69 C 3 257.7 12.9 Co 3 176.1 11.5 4 A 3 273.7 27.5 B 3 265.4 7.8 H / 8 / 6 9 Co 2 193.7 3.5 5 A 3 364.6 28.0 25/8 /69 B 2 322.5 27.3 Co 1 238.7 — 6 A 2 378.0 9.0 8 /9 /69 B 3 356.6 23.0 Co 3 234.4 25.3 7 A 2 416.7 14 .4 B 3 414.3 30.0 22/9/69 Co 3 317.8 29.4 94 Table XV: Mean oocyte diameter of spring salmon injected with various dosages of salmon gonadotropin, y = mean; S.D. = Standard Deviation. TABLE XV: SAMPLE MEAN OOCYTE & TREATMENT NUMBER OF DIAMETER (microns) DATE SAMPLES y S.D Zero Control 2 4 7 . 8 6 . 3 IO/6/69 1 A 2 6 9 . 0 7 . 5 B 2 6 8 . 3 6 . 4 24/6/69 C 2., 7 2 . 1 5 . 2 Co 1 5 4 . 6 2 A 2 7 0 . 6 5 . 4 B 3 7 2 . 6 1 0 . 1 7/7/69 C 2 7 4 . 6 1.1 Co 2 6 0 . 2 9 . 8 3 A 2 7 7 . 9 6 . 8 B 2 81 .5 3 . 1 21/7/69 C 2 6 3 . 8 0 . 9 Co 3 7 4 . 4 6 . 0 4 A 2 7 6 . 2 5 . 7 B 1 8 9 . 6 4 / 8 / 6 9 C 2 8 3 . 0 7.7 Co 3 82 .2 1 .9 5 A 3 1 0 0 . 2 6 . 7 B 2 9 0 . 0 1 3 . 7 I 8 / 8 / 6 9 C 3 1 0 1 . 5 3 . 6 Co 3 8 7 . 0 1 .4 6 A 3 104 .0 1 7 . 9 B 2 1 0 1 . 0 1 0 . 0 1/9/69 C 3 1 0 3 . 6 7 . 5 Co 3 9 5 . 6 9 . 7 U.I. Co 3 9 4 . 0 8 . 3 95 Table XVI: Mean oocyte diameter of pink salmon injected with various dosages of salmon gonadotropin and e s t r a d i o l 17B . v = mean; S.D. = standard deviation. TABLE XVI: SAMPLE MEAN OOCYTE & TREATMENT n DIAMETER (micrc DATE M S.D. Zero Control 25/6/70 2 110.95 15.8 1 G 3 193.6 24.0 8/8/70 GAE 3 162.2 8.7 GBE 3 181.3 4.1 25/8/70 Co 3 182.6 5.8 2 G 3 224 .1 15.5 18/9/70 GAE 3 183.6 80.8 GBE 3 228.7 98.8 6/10/70 AE 3 151.9 41.5 BE 3 205.1 59.1 Co 3 230.3 36.7 3 G 3 418.4 42 .0 31/10/70 GAE 3 170.4 23.4 GBE 3 424.7 58.2 GCE 3 303.0 120.3 17/11/70 Co 2 300.4 28.9 4 G 3 364.7 198.3 15/12/70 GAE 2 53.5 , 75.7 GBE 3 327.5 272.4 GCE 3 149.4 140.2 29/12/70 BE 3 133.8 127.8 Co 2 378.7 70.8 5 G ' 3 723.0 376.5 26/1/71 GBE 3 285.1 184.9 GCE 2 992.7 1085.1 10/2/71 Co 3 382.9 35.4 Co. (U.I. ) 3 560.1 65.2 6 G 3 323.9 347.1 10/3/71 GCE 3 529.2 662.9 Co (U.I.) 3 571.4 73.8 96 Table XVII: Mean oocyte diameter of extra-ordinarily large pink salmon Injected with various dosages of salmon gonadotropin e s t r a d i o l 1?3 , and progesterone. y = mean; S.D. = standard deviation. TABLE XVII: SAMPLE & DATE 1 3/11/70 TREATMENT .LPGE n MEAN OOCYTE DIAMETER(microns) v S.D. 445.2 1.5 1 LG 3 471.7 91.3 26/11/70 . LGE ~ 3 307.8 78.7 2 LG. 3 112.8 33.4 LGE 2 342.7 302.5 18 /2/71 LPGE 2 166.3 72.8 LCo 3 601.1 27.4 97 Table XVIII: Mean oocyte diameter of uninjected pink salmon maintained i n aquaria throughout t h e i r l i f e , y = mean; S.D. = standard deviation. TABLE XVIII: NUMBER OF MEAN OOCYTE SAMPLE DATE SAMPLES DIAMETER (microns) v S.D. 8 5/11/69 4 475.8 40 .1 9 2 1 / 1 /70 4 731.2 27.5 10 9/3/70 4 858.8 60.7 11 30/6/70 4 1196.5 175.0 12 25/8/70 4 2641.4 968.I Table XIX; The histochemically demonstrable A 5 - 3 8 hydroxysteroid dehydrogenase a c t i v i t y i n the ovaries of female pink salmon treated with various dosages of salmon gonadotropin. y = mean S.D. = standard deviation. TABLE XIX: NUMBER OF ENZYME ACTIVITY SAMPLE DATE TREATMENT SAMPLES (visua l units) p S.D. Zero Control 17/6/69 Co 4 0 0 1 30/6/69 A 4 0 . 2 5 0 . 2 9 B 3 0 . 2 9 0.33 C 3 0.17 0 . 2 9 Co 3 0 0 2 14/7/69 A 2 0 . 2 5 0.35 B 4 0.38 0.48 C 2 0 . 2 5 0.48 Co 4 0.5 0 3 28/7/69 A 4 0.13 0 . 2 5 B 2 0 . 2 5 0.48 C 4 0 . 2 5 0 . 2 9 Co 4 0 . 2 5 0 . 5 0 4 11/8/69 A 3 0.67 0 . 2 9 B 4 0 . 2 5 0 . 2 9 Co 4 0 . 5 0 0.41 5 2 5/8/ 6 9 A 4 0 . 2 5 0 . 2 9 B 4 0 . 2 5 0 . 2 9 Co 1 0 . 5 0 0 6 8/ 9 / 6 9 A 2 0.75 0.35 B 4 0 . 5 0 0.41 Co 3 0.17 0 . 2 9 7 22/9/69 A 2 0.75 0.35 B 4 0.88 0 . 2 5 Co 4 - • 0.5© 0.41 U.I. Co 4 0.38 0.48 99 Table XX: Histochemically demonstrable A5-38 hydroxysteroid dehydrogenase a c t i v i t y i n the ovaries of female spring salmon treated with various dosages of salmon gaondotropin. y = mean; S.D. = standard deviation. TABLE XX: NUMBER OP ENZYME ACTIVITY SAMPLE DATE TREATMENT SAMPLES (v i s u a l units) v S.D. Zero Control 10/6/69 Co 4 0 0 1 24/6/69 A 4 0 0 B 4 0 0 C 4 0 0 Co 4 0 0 2 7/7/69 A 1 0 0 B 4 0 0 C 4 0 0 Co 4 0 0 3 21/7/69 A 2 0.50 0.71 B 4 0.25 0.13 C 3 0 0 Co 2 0 0 4 V 8 / 6 9 A 3 0.50 0.50 B 2 0.25 0.35 C 4 0.25 0.29 Co 2 0 0 5 I8/8/69 A 1 0.50 0 B 2 0.25 0.35 C 3 0.33 0.29 Co 4 0 0 6 1/9/69 A 3 0.50 0.50 B 2 0.25 0.35 C 4 0.25 0.29 Co 2 0 0 U.I. Co 3 0.29 0.17 100 References Ashan, S. N. 1966. Effects of gonadotropic hormones on male hypophysectomized lake chub Couesius plumbeus. Can. J. Zool. 44: 703-717. Ashan, S. N. and W. S. Hoar. I963. Some effects of gonado-trop i c hormones on the threespine stickleback (Gasterosteus  aculeatus). Can. J. Zool. 41: 1045-1053. B a i l l i e , A. H., M. M. Ferguson, and D. McHart. 1966. Develop-ments i n steroid histochemistry. Academic Press. London and New York. B a l l , J . N. i960. Reproduction i n female bony f i s h e s . Symp. Zool. Soc. London 1: 105-135. Bara, G. 1965. Histochemical l o c a l i z a t i o n s of A5-3ghydroxy-steroid dehydrogenase i n the ovaries of the teleost f i s h : Scomber Scomber L. Gen. Comp. Endocrinol. 5: 284-296. Barr, W". A, 1963b. The endocrine control of the sexual cycle i n the pl a i c e , Pleuronectes platessa (L). I I . The endo-crine control of oogenesis. Gen. Comp. Endocrinol. 3: 205-201. Barr, W. A. 1963c. The endocrine control of the sexual cycle i n p l a i c e , Pleuronectes platessa (L.) I I I . The endocrine control of spermatogenesis. Gen. Comp. Endocrinol. 3: 216-225. Barr, W. A. I968. Patterns i n ovarian a c t i v i t y . In: Perspectives i n Endocrinology - Hormones i n the l i v e s of lower vertebrates (E.J.W. Barrington and C. Barker Jorgensen, eds.). Academic Press. London and New York. 101 B o t t i c e l l i , C. R., and P. L. Hisaw. 1 9 6 4 . Estrogens and progesterone i n the ovaries of the sockeye salmon (Oncorhynchus nerka). Am Zool. 4 : 297 Braekevelt, C. R. and D. B. McMillan. I 9 6 7 . C y c l i c changes i n the ovary of brook stickleback (Eucalia lnconstans -K i r t l a n d ) . . J . Morph, 123: 373-396. Bretschneider,/L. H. and J. J. de Wit. 19^7. Sexual endocrino-logy of non-mammalian vertebrates. E l s e v i e r . Amsterdam. Brett, J . R. and D. B. Sutherland. cl970. Improvement i n the a r t i f i c i a l rearing of sockeye salmon by environmental . control. F i s h . Res. Bd. Canada. B i o l . Stat. Nanaimo. Ci r c . No. 8 9 : 1 4 pp. Bullough, W. S. 19^2. Gametogenesis and some endocrine factor's a f f e c t i n g i t i n the adult minnow (Phoxinus l a e v i s L.) J . Endocrinol. 3: 211-221. Calaprice, J . R. NI969. Production and genetic fac t o r s i n managed salraonid populations. In: T. G. Northcote (ed). • Symposium on salmon and trout i n streams. H. R. MacMillan Lectures i n Fi s h e r i e s . Univ. of B.C. .pp. 377-388. Cedard, L., M. Fontaine, and T. Nomura. I96I. Sur l a teneur oestrogenes du sang du saumon adulte (Salmo solar L.), en eau douce. Compt. Rend. 252: 2656-2657. Chayen, J.,L. Bitensky, R. G.Butcher and L. W. Poulter. I969. A guide to p r a c t i c a l histochemistry. O l i v e r and Boyd. Edinburgh. Chestnut, C..W. 1970. The p i t u i t a r y gland of coho salmon (Oncorhynchus klsutch Walbaum) and i t s function i n gonad maturation and thyroid a c t i v i t y . Ph. D. Thesis, Simon Fraser University. 102 C l a v e r t , J . 1958. C o n t r i b u t i o n s a l 1 e t u d e de l a v i t e l l o g e n e s e chez l e s v i s e a u x . Phases p h y s i o l o g i g u e s et r6le de l a f o l l i c u l i n e dans l a v i t e l l o g e n e s e . Arch. Anat. Miscrosp. et Morphol. Exper, 47: 47-66. C u l l i n g , C. F.A. 1957. Handbook of h i s t o p a t h o l o g l c a l t e c h n i -que. Butterworth and Co. L t d . London. de Wit, J . C. 1953. The e f f e c t of o e s t r a d i o l monobenzoate on f o l l i c l e s of v a r i o u s s i z e s i n the ovary of the hypo-physectomized r a t . A c t a . E n d o c r i n o l . 12: 123-139. Donaldson, E. M. and J . R. McBride. 1967. The e f f e c t s of hypophysectomy i n the rainbow t r o u t (Salmo g a l r d n e r i l 'Rich.) w i t h s p e c i a l r e f e r e n c e to the p i t u i t a r y i n t e r r e n a l a x i s . Gen. Comp. E n d o c r i n o l . 9: 93-101. Donaldson, E. M., J.D. Funk, F. C. W i t h l e r , and R. B. Morley, 1972. S u c c e s s f u l f e r t i l i z a t i o n of pink salmon (Oncorhynchus  gorbuscha) ova by spermatozoa from gonadotropin i n j e c t e d j u v e n i l e pink salmon. J . F i s h . Res. Bd. Canada V o l . 29 ( i n p r e s s ) . Donaldson, E. M. and F. Yamazaki, I968. P r e p a r a t i o n of gonado-t r o p i c hormone .from salmon p i t u i t a r y glands. Annual Conf. Chem. I n s t , of Canada, 51st. (Abstr.) p. 64. Donaldson, E. M. , .Fv'Yamazaki., H. Dye, and W. W. P h i l l e o . 1971. P r e p a r a t i o n of gonadotropin from salmon (Oncorhynchus  tshawytscha) p i t u i t a r y glands. Submitted f o r p u b l i c a t i o n . E c k s t e i n , B. 1970. M e t a b o l i c pathways of s t e r o i d b i o s y n t h e s i s i n o v a r i a n t i s s u e of a t e l e o s t , T i l a p i a aurea. Gen. Comp. E n d o c r i n o l . 14: 303-312. 103 E l l i s , R. J. 1969. Return and behaviour of adults of the f i r s t f i l i a l generation of transplanted pink salmon, and s u r v i -v a l of t h e i r progeny, Sashin Creek, Baronof Island, Alaska. U.S. Fish. W i l d l i f e Serv. Spec. S c i . Rep. Fish. 589: 1-13. Eversole, W. J . 19^1. The eff e c t s of progeninolone and related steroids on the sexual development i n f i s h (Lebistes r e t i c u l a t u s ) . Endocrinology 28: 6 0 3 - 6 1 0 . Foerster, R. E., and A. L. Pritchard. 1941. Observations on the r e l a t i o n of egg content to t o t a l length and weight in the sockeye salmon (Oncorhynchus nerka) and the pink salmon (Oncorhynchus gorbuscha). Trans. Roy. Soc. Canada 35(V): 51-60. Galzinga, L. 1961. Re. Accad. Naz. 31: 9 2 . Henderson, N. E. 1962. The annual cycle i n the t e s t i s of the eastern brook trout, (Salvelinus f o n t i n a l i s M i t c h e l l ) . Can. J . Zool. 40: 631-641. Ho, F. G., and W. E. Vanstone, I 9 6 I . E f f e c t of e s t r a d i o l mono-benzoate on some serum constituents of maturing sockeye salmon (Oncorhynchus nerka). J . Fi s h . Res. Bd. Canada, 18: 859-864. Hoar, W. S. 1965 . Comparative physiology of hormones and reproduction i n f i s h e s . Ann. Rev. Physiol. 27: 5 1 - 7 0 . Hoar, W. S. 1966. Hormonal a c t i v i t i e s of the pars d i s t a l i s i n cyclostomes, f i s h and amphibia. In: The P i t u i t a r y Gland. (G. W.Harris, and B. T. Donovan, eds). Vol. 1, pp. 242-294. Butterworth, London and Washington, D.C. Holmes, W. N. and E. M. Donaldson. 1969. The body compartments and the d i s t r i b u t i o n of e l e c t r o l y t e s . In: F i s h P h y s i o l o g y . (W. S. Hoar and D. J . R a n d a l l , eds.) V o l . 1, Academic Pr e s s . New York and London. I s h i i , S. 1961. E f f e c t s of some hormones on the g e s t a t i o n of the top minnow. J . Fac. S c i . Univ. Tokyo Sec. IV. 9: 279-290. Kudo, S. 1969a. The r o l e of the y o l k - n u c l e u s i n f i s h oocytes: I: The r e l a t i o n between the f o r m a t i o n of c o r t i c a l a l v e o l i and the y o l k - n u c l a s i n the oocytes of the f i s h , P l e c o g l o s s u s a l t i v e l l s . Z o o l . Mag. 78: 297-304. Kudo, S. 1969b. The r o l e of the y o l k nucleus i n f i s h oocytes.,;> I I : The r e l a t i o n between the f o r m a t i o n of c o r t i c a l a l v e o l i and the y o l k - n u c l e u s i n the oocytes of the f i s h , Pseudo-dorasbora pimula. Z o o l . Mag. 78: 333-339. L i l e y , N. R. and E. M. Donaldson. 1969. The e f f e c t s of salmon p i t u i t a r y gonadotropin on the ovary and sexual behaviour of the female guppy, P o e c i l l a r e t i c u l a t a . Can. J . Z o o l . 4?: 569-573. Locke, D. 0. 1969. Q u i n a l d i n e as an a n e s t h e t i c f o r brook t r o u t , l a k e t r o u t , and a t l a n t i c salmon. U. S. Dept. I n t e r i o r , F i s h and W i l d l i f e Serv., I n v e s t i g a t i o n s i n F i s h C o n t r o l 24: 5 P. L o f t s , B., G.E. P i c k f o r d , and J . W. A t z . I966. E f f e c t s of methyl t e s t o s t e r o n e on t e s t e s of a hypophysectomized cyprinodont f i s h , Fundulus h e t e r o c l i t u s . Gen. Comp. E n d o c r i n o l . 6: 74-88. M a r s h a l l , A. J . i960. Reproduction i n male bony f i s h . Symp. Z o o l . Soc. London 1: 137-151. 105 McNeil, W. J . , S. C. Smedley, and R. J. E l l i s . 1969. Trans-planting adult pink salmon to Sashin Creek, Baronof Alaska, and sur v i v a l of t h e i r progeny, U. S. Fish . W i l d l . Serv. Spec. S c i . Rep. Fish. 5 8 7 : 9 p. Nayyar, S. K., and B. I. Sundararaj. 1970. Seasonal reproduc-t i v e a c t i v i t y i n the testes and seminal v e s i c l e s of the c a t f i s h (Heteropneustes f o s s l l l s Bloch). J . Morphol. 130: 207-226. Neave, F. 1962. The observed fluctuations of pink salmon i n B r i t i s h Columbia. In: N. J. Wilimovsky (ed). Symp. on pink salmon. H. R. MacMillan Lectures i n F i s h e r i e s . Univ. of B.C. pp. 3-14. Neave, F. MS 1965. Transplants of pink salmon. Fish. Res. Bd. Canada MS Rept. 83O: 23 p. O'Halloran, M. J . and D. R. Idler. 1970. I d e n t i f i c a t i o n and d i s t r i b u t i o n of the leydig c e l l homologue i n the t e s t i s of sexually mature a t l a n t i c salmon (Salmo s o l a r ) . Gen. Comp. Endocrinol. 15: 361-364. Oota, I. and K. Yamamoto. 1966. I n t e r s t i t i a l c e l l s i n the immature testes of the rainbow trout. Annotationes Zoologicae Japoneuses 39: 142-148. Pandey S. I 9 6 9 . E f f e c t s of hypophysectomy on the t e s t i s and secondary sex characters of the adult guppy, P o e c i l i a  r e t i c u l a t a peters. Can. J . Zool. 47: 775-781. Pandey, S. 1969. The role of p i t u i t a r y and gonadal hormones i n the d i f f e r e n t i a t i o n of the t e s t i s and secondary sex characters of the juvenile guppy, P o e c i l i a r e t i c u l a t a Peters. B i o l . Reprod. 1: 272-281. 106 Pencharz, R. I. 1940. E f f e c t of estrogens and androgens alone and i n combination with chorionic gonadotropin on the ovary of the hypophysectomized r a t . Science 91: 554-555. P h i l l i p s , R. E. 1959. Endocrine mechanisms of the f a i l u r e of p i n t a i l s (Anas acuta) to reproduce i n c a p t i v i t y . Ph. D. thesis. Cornell University. P h i l l i p s , R. E. and A. Van Tienhoven. I960. Endocrine factors involved i n the f a i l u r e of p i n t a i l ducks (Anus acuta) to reproduce i n c a p t i v i t y . J . Endocrinol. 21: 253-261. Pineda, M. H., L. C. Faulkner, M. L. Hopwood, and D. C. Leuker. 1968. E f f e c t s of immunizing female r a b i t s with bovine l u t e i n i z i n g hormone. Proc. Soc. Exp. B i o l . Med. 128: 743-749. Ramaswami, L. S. 1962. Endocrinology of reproduction i n f i s h and frog. Gen. Comp. Endocrinol. Suppl. 1: 286-299. Ricker, W. E. 1962. Regulation of the abundance of pink salmon populations. In: N. J . Wilimovsky (ed.) Symposium on pink salmon. H. R. MacMillan Lectures i n F i s h e r i e s . Univ. of B. C. pp. 155-201. Robertson, 0. H. 1958. Accelerated development of t e s t i s a f t e r u n i l a t e r a l gonadectomy, with observations on normal t e s t i s of rainbow trout. Fish. W i l d l . Serv. B u l l . 58: 9-30. Robertson, 0. H. and A. P. Rinfret. 1957. Maturation of the i n f a n t i l e t e s t i s i n rainbow trout (Salmo g a i r d n e r i l ) pro-duced by salmon p i t u i t a r y gonadotropins administered i n cholesterol p e l l e t s . Endocrinol. 60: 559-562. Schmidt, P. J . B. S. M i t c h e l l , M. Smith and H. Tsuyuki. 1965. P i t u i t a r y hormones of the p a c i f i c salmon. 1: Response of 107 gonads i n immature trout (Salmo g a i r d n e r i i ) to extracts of p i t u i t a r y glands from adult p a c i f i c salmon (Oncorhynchus) Gen. Comp. Endocrinol. 5: 1 9 7 - 2 0 6 . Simpson, M. E., H. M. Evans, H. L. Fraenkel-Conrat, and C. H. L i . 1 9 4 1 . Synergism of estrogens with p i t u i t a r y gonadotropins i n hypophysectomized rat s . Endocrinol. 28: 37-41. Simpson, T. H. R. S. Wright, and S. V. Hunt. 1 9 6 3 . Sex hormones i n f i s h . Part I I : The oestrogens ; of Scyllorhlnus canicuius. J. Endocrinol. 2 6 : 4 9 9 - 5 0 1 . Sundararaj, B. I. and S. V. Goswami. 1966. E f f e c t s of mammalian hypophysical hormones, placental gonadotropins, gonadal hormones, and adrenal corticosteroids on ovulation and spawning i n hypophysectomized c a t f i s h ; Heteropneustes  f o s s l l l s (Bloch). J . Exp. Zool. 161: 2 8 7 - 2 9 6 . Sundararaj, B.I. and S. K. Nayyar. I 9 6 7 . E f f e c t s of exogenous gonadotropins and gonadal hormones on the testes and seminal v e s i c l e s of the hypophysectomized c a t f i s h , Heteropneustes f o s s i l i s . Gen. Comp. Endocrinol. 8: 4 0 3 - 4 1 6 . Sundararaj, B. I. and S. V. Goswami. 1 9 6 8 . E f f e c t s of estrogen, progesterone, and testosterone on the ovary and p i t u i t a r y of c a t f i s h , Heteropneustes f o s s i l i s (Bloch). J. Exp. Zool. 169: 2 1 1 - 2 2 8 . Sundararaj, B. I., T. C. Anand, and E. M. Donaldson. 1971 . E f f e c t s of P a r t i a l l y P u r i f i e d Salmon P i t u i t a r y Gonadotropin on Ovarian Maintenance, Ovulation and Vlt e l l o g e n e s i s i n the Hypophysectomized Ca t f i s h , Heteropneustes f o s s i l i s (Bloch). In press. 108 Sundararaj, B. I., S. K. Nayyar, T. C. Anand, and E. H. " . .Donaldson, 1 9 7 1 . E f f e c t s of salmon p i t u i t a r y gonado-.. tropin, ovine l u t e i n i z i n g hormone, and testosterone on the testes and seminal v e s i c l e s of hypophysectomized c a t f i s h , V. • Heteropneustes f o s s l l l s (Bloch). In press, van Tlenhoven, A, 1968. Reproductive physiology of verte-brates. W. B. Saunders Company. Philadelphia, Toronto, London. . , ".: Vivie n , J . H. 1939. Relations hypophyso-genitales chez quelques teleosteens et selaciens. Compt. Rend. Soc. B i o l . 131: ".' 1222-1224. V i v i e n , J . H. 1941. Contribution a' 1«etude- de l a physiologie hypophysaire dans ses r e l a t i o n s avec l ' a p p a r e i l g e n i t a l , ; l a thyroide et les corps suprarenaux chez l e s poissons selaciens et teleosteens. Scyliorhlnus canlcula et Goblus paganeleus. B u l l . B i o l . Prance et Belgique 75: * 2 5 7 - 3 0 9 . Walker, C. E. and D. B. L i s t e r . 1971. Results f o r three gen-erations from transfers of pink salmon (Oncorhynchus ; gorbuscha) spawn to the Quallcum River i n I963 and 1964. J . Fish. Res. Bd. Canada 28: 647-654. • :Westgate, J . W., G. B. McKee, and D. K. Law. 1964. Ranklng.of wet ingredients f o r Oregon p e l l e t s . Oregon Fis h . Comm. Res. Br i e f #10: 35-40. Wiebe, J . P. 1968. I n h i b i t i o n of p i t u i t a r y gonadotropic a c t i v i t y ' i n the viviparous seaperch Cymatogaster aggregata Gibbons by a dithiocarbamoylhydrazine deri v a t i v e (I.C.I. 33828) Can. J . Zool. 46: 751-758. 109 Wiebe, J. P. 1969a. Steroid dehydrogenase and steroids In gonads of the viviparous seaperch, Cymatogaster aggregata Gibbons, Gen. Comp. Endocrinol. 12: 256-266. Wiebe, J . P. 1969b. Endocrine controls of spermatogenesis and oogenesis i n the viviparous seaperch, Cymatogaster  aggregata Gibbons. Gen. Comp. Endocrinol. 12: 267-275. Williams, P. C. 1944. Studies of the b i o l o g i c a l a ction of serum gonadotropin 2: Ovarian response a f t e r hypophysectomy and estrogen treatment. J . Endocrinol. 4: 131-136. Yamazaki, F. I965. Endocrinological studies on the reproduction of the female g o l d f i s h , Carasslus auratus L., with s p e c i a l reference to the function of the p i t u i t a r y gland. Mem. Fac. Fish. Hokkaido Univ. 13: 1-64. Yamazaki, F. , and E. M. Donaldson. I968. The ef f e c t s of partially-p u r i f i e d salmon p i t u i t a r y gonadotropin on spermatogenesis, v l t e l l o g e n e s i s and ovulation i n hypophysectomized g o l d f i s h (Carasslus auratus). Gen. Comp. Endocrinol. 11: 292-299. Yamazaki, F., and E. M. Donaldson. 1969. Involvement of gonado-trop i n and steroid hormones i n the spermiation of the gold f i s h (Carasslus auratus). Gen. Comp. Endocrinol. 12: 491-497. 

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