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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 i n Juvenile Pink Salmon Oncorhynchus gorbuscha  by James D. Funk  B. Sc. (Hons.) University of B r i t i s h Columbia, A Thesis submitted i n P a r t i a l Fulfilment of The Requirements of the Degree of Master of Science i n the Department of Zoology  We accept t h i s thesis as conforming to the required  standard.  The University of B r i t i s h Columbia February,  1972  In presenting  this  thesis i n partial  f u l f i l m e n t of the require-  ments f o r an a d v a n c e d d e g r e e a t t h e U n i v e r s i t y Columbia, I agree that  the Library  a b l e f o r r e f e r e n c e and study. for  extensive  copying of t h i s  of B r i t i s h  s h a l l make i t f r e e l y  I f u r t h e r agree that thesis f o r scholarly  avail-  permission purposes  may be g r a n t e d by t h e Head o f my D e p a r t m e n t o r b y h i s r e p r e sentatives. this  I t i s understoon that  thesis for financial  written  gain  s h a l l n o t be a l l o w e d w i t h o u t  permission.  Department o f Zoology The U n i v e r s i t y o f B r i t i s h V a n c o u v e r 8, B.C.  Date  jfh^d  Q-X^ |?73  copying or p u b l i c a t i o n of  Columbia  my  Abstract  T h i s study was undertaken to determine whether the gonads of pink salmon (Oncorhynchus  gorbuscha), c o u l d be stimu-  l a t e d w i t h exogenous hormones to r e p r o d u c t i v e m a t u r i t y one year e a r l i e r than normal.  T h i s procedure, i f s u c c e s s f u l ,  c o u l d be used as a method f o r r e p o p u l a t i n g  ' o f f year c y c l e s 1  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 J u v e n i l e males,  complete  s e x u a l m a t u r i t y was  a t t a i n e d by September i n the year of h a t c h i n g w i t h t h r i c e weekly  treatments of 10.0 micrograms and of 1.0  salmon (Oncorhynchus weight.  micrograms  tshawytscha) gonadotropin per gram body  H i s t o l o g i c a l examination of the p r e c o c i o u s l y mature  t e s t e s , and comparison w i t h t e s t e s from u n i n j e c t e d c o n t r o l s r e p e a l e d t h a t the time of onset of the m i t o t i c d i v i s i o n of spermatogonia  to form the primary spermatocyte, and the process  of a c t i v e spermatogenesis were a c c e l e r a t e d . a s c a t t e r i n g of l o c a l i z a t i o n s of  At s e x u a l m a t u r i t y ,  A 5 - 3 3 h y d r o x y s t e r o i d dehydro-  genase a c t i v i t y was observed which  corresponded t o the d i s t r i -  b u t i o n of the i n t e r s t i t i a l c e l l s .  A l a r g e r 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-  v e l o p e d mature t e s t e s i n the same time i n t e r v a l as the normalsized individuals.  These gonads were f o u r times l a r g e r , however.  A s m a l l s p e c i e s d i f f e r e n c e i n the a c t i o n of the gonadotropin p r e p a r a t i o n was found when comparing  i t s e f f e c t on the G.S.I.,  r a t e of i n d u c t i o n of s e x u a l m a t u r i t y , and 3 &-ol a c t i v i t y of immature Oncorhynchus tshawytscha.  dehydrogenase  ii In the females, the yolk stage was induced f i r s t i n f i s h treated three times a week with 1.0 'yg/gram body weight salmon gonadotropin and 1.5 for 126  days.  yg/gram body weight e s t r a d i o l 1? g  Oocytes containing yolk globules did not appear  in pink salmon treated, with 1.0 p. g/gram body weight salmongonadotropin alone f o r a'further k-2 days. o r  E s t r a d i o l l?s alone,  i n combination with salmon, gonadotropin at. a dosage of  15 ^ g/gram body weight i n h i b i t e d v i t e l l o g e n e s i s . oocytes 2 mm  Formation of  i n 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; e s t r a d i o l 1?3 , and nine months of i n 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 a t r e t i c a were observed  _  i n a l l of the treated f i s h . L i t t l e histochemically demonstrable A 5-36  hydroxysteroid  dehydrogenase a c t i v i t y was present i n ovaries from pink or spring salmon juveniles treated f o r 3 months with various dosages of ' salmon gonadotropin. The significance of the results i n r e l a t i o n to the o r i g i n a l problem are discussed.  Table of Contents Page Abstract  .  *  L i s t of Figures.-L i s t of Tables  x  Acknowledgements  xiii  General Introduction  x  Chapter I:  1  The T e s t i s  v  Introduction  1  Methods and Materials  ^  Animals  ^  Treatments  °"  Experimental Techniques (i)  Injection Procedure  ?  (ii)  Autopsy Procedure  ^  (iii)  H i s t o l o g i c a l Techniques  8  (iv)  Analysis of the H i s t o l o g i c a l Results  8  (v)  Histochemical Techniques  IP  (vi)  Analysis of the Histochemical Results  H  Results  12  Spermatogenesis 45-3 BHydroxysteroid  12 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  Figures  22  Tables  31  Chapter 2  The O v a r y  :  39  Introduction  39  Methods and M a t e r i a l s  46  Animals  46  Treatments  47  Experimental Techniques  4-9  (i), (iv)  (v)  (ii),  (iii)  Analysis  - same a s i n C h a p t e r 1  of the H i s t o l o g i c a l Results  49  (b) H i s t o l o g i c a l A n a l y s i s  50  Histochemical  53  Analysis  Procedure  (b) A n a l y s i s  53 of R e s u l t s  Results  (b)  53 5^  Measurements  5^  (i)  Gonadosomatic  (ii)  Oocyte Diameter  55  (iii)  Oogenesis  57  Index  Histochemistry (i)  (ii) (c)  49  (a) Measurements  (a)  (a)  . .  A 5-33  54  62 Hydroxysteroid  Dehydrogenase  62  Lipids  62  Appearance  63  Table  of  Contents Page  Discussion General  Summary  65 71  Figures  72  Tables  88  References  100  iii  List  of Figures Facing Page  1.  Cross-section of a t e s t i s pink  2.  salmon j u v e n i l e  Testis  22  but a t a higher  of a s e x u a l l y mature (11.0  (Stage  4)  22  pink 22  cm l o n g )  Same s e c t i o n a s i n F i g . 3» b u t t a h i g h e r a  m a g n i f i c a t i o n t o show d e t a i l near  Epithelial  6.  Regressed pink  of  spermatogenesis 22  the duct  5.  7.  control  Example o f S t a g e 1 t e s t i s  salmon j u v e n i l e 4.  a zero  cm l o n g )  Same s e c t i o n a s i n F i g . 1, magnification.  3.  (7.7  from  cells  lining  t h e sperm d u c t  testis  (Stage  6) o f a j u v e n i l e  22 male 22  salmon  Stage 4 t e s t i s  of an u n i n j e c t e d pink  salmon  (Body w e i g h t - 360 grams) 8.  Same s e c t i o n a s i n F i g . 7» b u t a h i g h e r m a g n i f i cation in  9.  to reveal the d e t a i l s  Interstitial cells  Frozen  11.  spermatogenesis 22  i n the t e s t i s  of a p r e c o c i o u s l y 23  salmon  section of the t e s t i s  mature pink for  of the  the lobules  mature pink 10.  22  of a p r e c o c i o u s l y  salmon s t a i n e d w i t h  Sudan B l a c k B 23  lipids  H i s t o c h e m i c a l l y demonstrable dehydrogenase a c t i v i t y testis  A5-36 h y d r o x y s t e r o i d  i n a frozen section  from a s e x u a l l y mature p i n k  of a  salmon j u v e n i l e .  . 23  iv  List  of F i g u r e s Facing Page  A5-3B  12. in  hydroxysteroid  the t e s t i s  dehydrogenase  activity  o f a p r e c o c i o u s l y mature  spring 23  salmon 13;  14.  3  3-ol dehydrogenase  a  s e x u a l l y mature  activity  goldfish  The mean g o n a d o s o m a t i c pink  (Oncorhynchus  (Oncorhynchus  i n the t e s t i s of  (Carasslus auratus).  index  .  (G.S.I.) o f male  gorbuscha) and s p r i n g  t s h a w y t s c h a ) salmon  juveniles  treated  t h r e e t i m e s p e r week w i t h v a r i o u s  dosages  o f salmon  (Oncorhynchus  tshawytscha)  g o n a d o t r o p i n d u r i n g t h e summer o f I 9 6 9 15.  23  The mean g o n a d o s o m a t i c  ^  index, ( G . S . I . ) o f u n i n -  j e c t e d male p i n k salmon m a i n t a i n e d i n a q u a r i a throughout 16.  their  25  life  Stages of t e s t i c u l a r maturity juveniles  injected  v a r i o u s dosages  three  of pink  salmon  t i m e s p e r week w i t h  o f salmon  (Oncorhynchus 26  tshawytscha) gonadotropin 17.  Stages of t e s t i c u l a r m a t u r i t y salmon  juveniles  treated  w i t h v a r i o u s dosages  induced i n spring  t h r e e t i m e s p e r week  o f salmon  (Oncorhynchus 27  tshawytscha) gonadotropin 18.  Stages of r e p r o d u c t i v e maturity salmon m a i n t a i n e d i n a q u a r i a cycle  of u n i n j e c t e d  throughout t h e i r  pink l i f e ,. 28  V  L i s t of F i g u r e s . Facing Page  19.  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 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 e e times per week w i t h v a r i o u s dosages of salmon 29  (Oncorhynchus tshawytscha) gonadotropin 20.  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 A 5-3  3 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  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 21.  30  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 (5.4 cm long)  22.  72  Ovary of a j u v e n i l e s p r i n g salmon 1 0 . 0 cm i n l e n g t h i n j e c t e d three times per week w i t h  fish 72  s a l i n e f o r 84 days 23.  Ovary of a j u v e n i l e s p r i n g salmon ( 1 1 . 2 cm i n length) i n j e c t e d three times per week w i t h 1 0 . 0 n g of salmon gonadotropin per gram body weight 72  f o r 84 days 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 . 2 3 . . 72  25.  The two c e n t r a l oocytes a r e i n the l a t e stage  perinucleolar 72  vi List  of F i g u r e s Facing Page.'  26.  Follicle  layer  of a l a t e  perinucleolar  stage 72  oocyte 27.  Oocyte i n the y o l k v e s i c l e of  a pink  three  salmon  times  (13.2  stage from  cm i n l e n g t h )  f o r 96 d a y s  t r o p i n p e r gram body w e i g h t 28.  Follicle  l a y e r s and t h e c a l  injected  y g s a l m o n gonado-  10.0  p e r week w i t h  the ovary  73  l a y e r of the oocyte  i n F i g . 27 29.  7  Oocyte i n the y o l k v e s i c l e  stage  stained f o r l i p i d s 73  w i t h Sudan B l a c k B 30.  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 from  pink  for  Primary  32.  Follicle  salmon g o n a d o t r o p i n  Primary  and t h e c a l  p e r week w i t h  p e r gram body  weight 73  ( p i n k salmon)  layers  7  of the primary  y o l k stage oocyte  73 stained  forlipids  with  , from  a pink  cm i n l e n g t h ) i n j e c t e d  salmon  f o r 126 d a y s  73  juvenile  three times  1.0 y g salmon g o n a d o t r o p i n and 1.5  17 3 p e r gram body w e i g h t  3  yolk  Red 0  (1^.6  oocytes  i n F i g . 12  S e c t i o n of an ovary  with  stage  „  y o l k stage oocyte  stage oocyte  3^.  three times  96 d a y s  31.  Oil  i n yolk vesicle  salmon i n j e c t e d  y g  10.0  33.  3  p e r week Pg  estradiol 7^  vii  List  of F i g u r e s Facing Page  35.  S e c t i o n of an ovary from (14.9  a p i n k salmon  cm i n l e n g t h ) i n j e c t e d  week w i t h 1.0  juvenile  t h r e e times per  ig salmon g o n a d o t r o p i n  p e r gram body  74  36.  weight  f o r 126 d a y s  Frozen  s e c t i o n of a post primary y o l k stage  from a p i n k salmon  (16.3 cm i n l e n g t h )  oocyte  injected  t h r e e t i m e s p e r week w i t h 1.0 y g s a l m o n gonadot r o p i n a n d 1.5  yg e s t r a d i o l  17 8 p e r gram body  weight  74 for 37.  210 d a y s .  Frozen  .  ,  '  s e c t i o n of a post primary y o l k stage  from a p i n k salmon  (17.0 cm i n l e n g t h )  t h r e e t i m e s p e r week w i t h 1.0 y g s a l m o n  oocyte  injected gonadotro-  74 p i n p e r gram body w e i g h t 38.  Ovary of a f i s h scribed  treated  i n F i g . 37.  f o r 258 d a y s identically  t o that de-  Note t h e p a u c i t y o f o o c y t e s  74 more a d v a n c e d 39.  than the e a r l y  perinucleolar  S e c t i o n o f an ovary from a p i n k salmon with  stage. .  treated  1.0 y g s a l m o n g o n a d o t r o p i n p e r gram body  weight  f o r 126 d a y s ,  a n d 1.0 y g e s t r a d i o l  17 8 p e r  74 gram body w e i g h t 40.  f o r the l a s t  Gonadosomatlc index (Oncorhynchus tshawytscha)  '  (G.S.I.) of f e m a l e  gorbuscha) treated  92 d a y s  and s p r i n g  three times  v a r i o u s d o s a g e s o f salmon  pink  (Oncorhynchus  p e r week w i t h  (Oncorhynchus  g o n a d o t r o p i n d u r i n g t h e summer o f 1969  tshawytscha)  viii List  of F i g u r e s  Facing Page  41.  Gonadosomatic  index  salmon t r e a t e d various  of female  pink  t h r e e t i m e s p e r week w i t h  combinations  tshawytscha)  (G.S.I.)  o f salmon  (Oncorhynchus  g o n a d o t r o p i n and  estradiol  17 6 . . . . . 42.  76  Gonadosomatic i n d e x l a r g e female dosages  (G.S.I.)  of e x t r a - o r d i n a r i l y  p i n k salmon t r e a t e d w i t h v a r i o u s  o f salmon  (Oncorhynchus  tshawytscha)  gonadotropin 43.  Gonadosomatic index pink their  44.  77  salmon m a i n t a i n e d life  of u n i n j e c t e d  female  i n a q u a r i a throughout  cycle  78  Mean o o c y t e d i a m e t e r pink  (G.S.I.)  (microns) of j u v e n i l e  s a l m o n and s p r i n g  salmon i n j e c t e d  p e r week w i t h v a r i o u s d o s a g e s tshawytscha)  female  three times  of salmon  (Oncorhynchus  g o n a d o t r o p i n d u r i n g t h e summer o f  1969 45.  79  Mean o o c y t e d i a m e t e r treated tions  salmon  t h r e e t i m e s p e r week w i t h s e v e r a l  o f salmon  (Oncorhynchus  t r o p i n and e s t r a d i o l 46.  (microns) of pink  17  tshawytscha)  three  salmon o b t a i n e d from  80  gonadotropin,  large  D r . J . R. B r e t t ,  t i m e s p e r week w i t h c o m b i n a t i o n s  exogenous hormones:  gonado-  3  Mean o o c y t e d i a m e t e r o f e x t r a - o r d i n a r i l y pink  combina-  Salmon  injected  of v a r i o u s  (Oncorhynchus  tshawytscha)  e s t r a d i o l 37 g a n d p r o g e s t e r o n e  81  ix List  of F i g u r e s Facing Page  47.  48.  T h e mean o o c y t e  diameter  pink  salmon m a i n t a i n e d  life  cycle  of oocytes from u n i n f e c t e d  i n a q u a r i a throughout  their 82  Mean p e r c e n t a g e s  of oocytes which comprised the  ovaries of juvenile  pink  salmon t r e a t e d  with  v a r i o u s dosages of salmon g o n a d o t r o p i n 49.  Mean p e r c e n t a g e s ovaries of pink  50.  51.  salmon t r e a t e d w i t h v a r i o u s  o f salmon  gonadotropin  a n d e s t r a d i o l 27 B  (Oncorhynchus  of u n i n j e c t e d pink  aquaria  throughout  84  their  salmon m a i n t a i n e d i n 85  life  The h i s t o c h e m i c a l l y demonstrable  of  tshawytscha)  of oocytes which comprise the  ovaries  A5-3  a c t i v i t y of  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  juvenile  pink  salmon i n j e c t e d  three times per  week w i t h v a r i o u s d o s a g e s o f salmon tshawytscha)  gonadotropin  (Oncorhynchus  d u r i n g t h e summer o f  .  1969 52.  Identical with  83  of oocytes which comprise t h e  combinations  Mean p e r c e n t a g e s  . .  experiment  spring  salmon  to that  juveniles  .  86  i n F i g u r e 51» b u t 87  X  List  of T a b l e s Facing Page  1.  Male p i n k  salmon j u v e n i l e s  treated with various  dosages o f salmon g o n a d o t r o p i n mean f i s h  weight,  mean f i x e d  gonadosomatic index 2.  Mean body w e i g h t , t e s t i s weight, of  juvenile  - mean f i s h  gonad w e i g h t ,  mean 31  (G.S.I.)  mean body l e n g t h , mean  mean g o n a d o s o m a t i c i n d e x  spring  length,  fixed (G.S.I.)  salmon t r e a t e d w i t h v a r i o u s 32  dosages of salmon g o n a d o t r o p i n 3.  Mean body w e i g h t ,  mean gonad w e i g h t ,  gonadosomatic index pink  4.  ( G . S . I . ) o f u n i n j e c t e d male  salmon m a i n t a i n e d  their  a n d mean  i n aquaria  throughout 33  life  The s t a g e o f t e s t i c u l a r m a t u r a t i o n  i n d u c e d by  v a r i o u s dosages of salmon g o n a d o t r o p i n  i n pink 3^  salmon j u v e n i l e s 5.  The t e s t i c u l a r  stage of s p r i n g  salmon  juveniles  i n j e c t e d w i t h v a r i o u s d o s a g e s o f s a l m o n gonado35  tropin 6.  The t e s t i c u l a r tained  7.  stage of u n i n j e c t e d pink  i n a q u a r i a throughout  H i s t o c h e m i c a l l y demonstrable dehydrogenase a c t i v i t y  their  salmon main36  life  A 5-3 g h y d r o x y s t e r o i d  i n pink  salmon t r e a t e d  with  v a r i o u s dosages o f salmon g o n a d o t r o p i n 8.  H i s t o c h e m i c a l l y demonstrable dehydrogenase a c t i v i t y  37  A 5-3 g h y d r o x y s t e r o i d  i n the testes of spring  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 o f salmon g o n a d o t r o p i n  . 3 8  xi List  of Tables Facing Page  9.  Mean body w e i g h t , gonadosomatic pink  mean f i x e d  index  ovary weight,  (G.S.I.) o f j u v e n i l e  mean  female  salmon t r e a t e d w i t h v a r i o u s dosages o f  salmon g o n a d o t r o p i n 10.  Mean body l e n g t h , mean body w e i g h t , ovary weight, of  spring  88  . mean  a n d mean g o n a d o s o m a t i c  fixed  index  (G.S.I.)  salmon t r e a t e d w i t h v a r i o u s dosages o f 89  salmon g o n a d o t r o p i n 11.  Mean body l e n g t h , mean body w e i g h t , weight,  a n d mean g o n a d o s o m a t i c  of female  index  Mean body w e i g h t , weight,  (G.S.I.)  p i n k salmon t r e a t e d w i t h v a r i o u s dosages 17g  of g o n a d o t r o p i n and e s t r a d i o l 12.  mean gonad  index  13.  l a r g e female  pink  salmon  gonadosomatic female their 14.  juveniles  gonadotropic  17 3 a n d p r o g e s t e r o n e  Mean body w e i g h t ,  pink  .91  mean gonad w e i g h t ,  index  a n d mean  (G.S.I.) of u n i n j e c t e d  salmon m a i n t a i n e d  i n aquaria  throughout 92  life  Mean o o c y t e d i a m e t e r  of pink  salmon j u v e n i l e s  treated 93  w i t h v a r i o u s dosages o f salmon g o n a d o t r o p i n 15.  90  (G.S.I.) o f  t r e a t e d w i t h v a r i o u s dosages o f salmon estradiol  .  mean body l e n g t h , mean gonad  a n d mean g o n a d o s o m a t i c  extra-ordinarily  d u r i n g 1970/71.  Mean o o c y t e d i a m e t e r  of s p r i n g  salmon i n j e c t e d  v a r i o u s d o s a g e s o f salmon g o n a d o t r o p i n  with 94  xii List  of T a b l e s Facing Page  16.  Mean o o c y t e  diameter  of pink  salmon i n j e c t e d  v a r i o u s d o s a g e s o f salmon g o n a d o t r o p i n diol 17.  with  and e s t r a -  17 6  Mean o o c y t e  95 diameter  of e x t r a - o r d i n a r i l y  large  pink  salmon i n j e c t e d w i t h v a r i o u s d o s a g e s o f s a l m o n gonadotropin, 18.  Mean o o c y t e maintained  19.  diameter  17 6 t p r o g e s t e r o n e .  of u n i n j e c t e d pink  i n a q u a r i a throughout  their  The h i s t o c h e m i c a l l y d e m o n s t r a b l e  A5-38  steroid female of  20.  estradiol  dehydrogenase a c t i v i t y pink  96  salmon life  . . .  97  hydroxy-  i n the ovaries of  salmon t r e a t e d w i t h v a r i o u s dosages  salmon g o n a d o t r o p i n .  . .  H i s t o c h e m i c a l l y demonstrable dehydrogenase a c t i v i t y spring  . . .  98  A5-36 h y d r o x y s t e r o i d  i n the o v a r i e s of female  salmon t r e a t e d w i t h v a r i o u s d o s a g e s o f  salmon g o n a d o t r o p i n  99  xiii A cknowle dgements Special thanks are extended  to my supervisor, Dr. E.  M. Donaldson f o r suggesting the problem, and f o r h i s interest? advice, and constant encouragement throughout the course of the study. The author would also l i k e to thank Dr. W. S. Hoar, Dr. A. M, Perks  and Dr. P. Ford, f o r reading the manuscript,  and o f f e r i n g constructive c r i t i c i s m ,  I would l i k e 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 i n preparation of salmon p i t u i t a r y gonadotropin, and of Mrs. K. Kramer i n p a r a f f i n embedding specimens and preparing slides i s g r a t e f u l l y acknowledged.  Mr. G. Walton sectioned many of the p a r a f f i n  blocks and h i s photographs of whole gonads were useful f o r demonstrations.  Mr. A. Dodimead, of the P a c i f i c Environment  I n s t i t u t e generously donated an o f f i c e f o r use i n the f i n a l writing of the thesis. My appreciation i s extended to Mr. J . Culp, Mr. J . Tuerlings, and Mr. R. Corrigan f o r t h e i r help with the day-today problems. One of my most valuable experiences was  the opportunity  to work and interact with the s c i e n t i s t s and staff of the F i s h e r i e s Research Board, West Vancouver and Vancouver Laborat o r i e s , and of the P a c i f i c Environment I n s t i t u t e .  xiv  The typing  a u t h o r would  the rough and This  Board P r o j e c t Fisheries  like  final  t o t h a n k Mrs.  drafts  i n v e s t i g a t i o n was No.  9261,  project  of the  Norma J a n s s e n f o r  thesis.  s u p p o r t e d by F i s h e r i e s head  Dr.  Research Board a s s i s t a n t s h i p  E . M.  Research  Donaldson  to the author.  and  a  xv General Introduction Throughout the geographic range of pink salmon, (Oncorhynchus gorbuscha) there are r i v e r s where there i s a pronounced difference i n abundance between the even-and-odd numbered years (Ricker, 1 9 6 2 ; Neave, 1 9 6 5 ) . In e,xtreme cases, a watershed may have large numbers of adult salmon spawning at 2-year i n t e r v a l s , and v i r t u a l l y none i n the intervening years (Ricker, 1 9 6 2 ) .  In the Fraser River-Puget Sound-Howe  Sound region, f o r example, even year f i s h are v i r t u a l l y absent, whereas the r i v e r s on the Queen Charlotte Islands and Northern B.C. mainland are characterized by a lack of pink salmon spawnning i n off-numbered years,, (Neave, 1 9 6 2 ) .  Both of these sys-  tems support large runs i n t h e i r respective "on" years. previously empty cycles could be repopulated,  If  there could be  a great economic benefit to the f i s h i n g industry. In 1965» Neave summarized a l l previous l i t e r a t u r e concerning transplants of pink salmon to r i v e r s where there was no spawning.  He noted that no substantial s e l f - s u s t a i n i n g run was  known to e x i s t . present  The most successful transplant on record at  i s the transfer of 1 , 8 6 6 adult pinks from Bear Harbour  to Sashin Creek (Alaska) i n 1 9 6 4 , to reinforce a run which had dwindled below 1 , 0 0 0 .  Five thousand seven hundred and s i x t y -  one f i s h returned i n 1 9 6 6 .  Circumstantial evidence indicated  that the majority of the returning f i s h were from the o r i g i n a l transplant ( E l l i s , 1 9 6 9 ) .  A recent publication detailed the  r e s u l t s f o r three generations  of pink salmon a f t e r transplant  of pink salmon eggs to the Qualicum River i n both 1963 n d a  xvi 1964.  The survivals of transplanted f i s h were lower than those  normally occurring (Walker and L i s t e r , 1 9 7 1 ) . For a transplant to be successful, the introduced f i s h should match as closely as possible the native stock i n such c h a r a c t e r i s t i c s as time of migration and spawning, and i n the temperature of the native environment waters McNeil e_t a l , 1 9 6 9 ) .  (Calaprice, 1 9 6 9 ;  I t would appear that genetic factors play  an important role i n determining whether a donor stock w i l l survive i n the new  system.  The r i g i d two-year l i f e cycle of pink salmon prevents the p o s s i b i l i t y of repopulating a barren cycle d i r e c t l y from f i s h present i n 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 e a r l i e r than normal.  I t was  that a preparation of spring salmon (Oncorhynchus  proposed tshawytscha)  gonadotropin which had already been proved to reinduce a l l phases of sexual maturity i n males and females of mized g o l d f i s h (Carasslus auratus) —  hypophysecto-  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 s p e c i f i c i n i t s a c t i v i t y .  1  CHAPTER 1 The  Testis  Introduction There i s c o n s i d e r a b l e evidence to i n d i c a t e t h a t p i t u i t a r y gonadotropin c o n t r o l s osts.  Hypophysectomy had been shown t o a r r e s t  i n adult Pleuronectes platessa heteroclltus  (Lofts, 1966),  and Nayyar, 1 9 6 7 ) , 1968),  gonadal m a t u r a t i o n i n male t e l e spermatogenesis  19^3 a ) , Fundulus  (Barr,  Heteropneustes f o s s l l l s  Carrasslus  a u r a t u s (Yamazaki and (Wiebe, 1969)  Cymatogaster aggregata Gibbons  r e t i c u l a t a P e t e r s (Pandey, 1969 a ) .  Donaldson, and P o e c l l i a  Pandey ( I 9 6 9 b ) , working  w i t h the guppy ( P o e c l l i a r e t i c u l a t a P e t e r s ) , moved the p i t u i t a r y of a j u v e n i l e f i s h .  successfully re-  The m i t o t i c d i v i s i o n  to form primary spermatocytes from spermatogonia was l y suppressed, thereby p r e c l u d i n g  (Sundarapaj  complete-  any f u r t h e r t e s t i c u l a r matur-  ation. P r e v i o u s r e s e a r c h had demonstrated a s t i m u l a t i o n t e s t i c u l a r development  i n j u v e n i l e salmonids w i t h homoplastic  p r e p a r a t i o n s of p i t u i t a r y g l a n d s . the  Active  shedding of m o t i l e spermatozoa was  trout  of  spermatogenesis, and  brought about i n immature  (Salmo g a l r d n e r i l ) by a d m i n i s t r a t i o n  i n cholesterol  p e l l e t s o f a p r e p a r a t i o n 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  (Robertson and R i n f r e t , 1957). immature t r o u t  k e t a and Oncorhynchus  Two weeks of i n j e c t i o n s  (Salmo g a l r d n e r i l ) of e x t r a c t s  glands of the s p r i n g  salmon  tshawytscha)  (Oncorhynchus  into  from the p i t u i t a r y  tshawytscha)  a t e d the process of spermatogenesis i n the t e s t f i s h  acceler-  (Schmidt  2  et a l , 1 9 6 5 ) .  A regime  week a d m i n i s t e r e d optimum  sequence  purification (1965),  consisting  on a l t e r n a t e  d a y s was  for intraperitoneal  of t h e , p i t u i t a r y  which  of three  extract  purified  f o u n d t o be  injection.  involved gel f i l t r a t i o n  minated i n a p a r t i a l l y  equal doses  per  the  Further  u s e d by S c h m i d t  et  on Sephadex G-100  preparation of  al., cul-  Oncorhynchus  t s h a w y t s c h a g o n a d o t r o p i n (SG-G100) d e s c r i b e d by D o n a l d s o n (I968)  Yamazaki sectomy tioned  Donaldson et a l (1971).  and  and t e s t i c u l a r preparation  into adult  a n d D o n a l d s o n , 1968) et a l , ,  1971  regression,  to  possibility  intervals would  injected  aforemen-  (Yamazaki (Sundararaj of  I t was into  (Oncorhynchus  i n the year of hatching  proposed that  the t e s t  fish at  producing  could  of  thrice-weekly e v e r y two  weeks The  o f A5-3 6 h y d r o x y s t e r o i d  the p o t e n t i a l  of the t e s t e s f o r  s e x hormones.  A similar formed u s i n g species  indicate  appeared  s e v e r a l dosages  Samples  demonstrable a c t i v i t y  of  gorbuscha)  r e v e a l the successive stages of gonadal m a t u r i t y .  dehydrogenase  sexual  o f a c c e l e r a t i o n of the development  f o r a p e r i o d o f 98 d a y s .  histochemically  experiment to that  spring  specificity  salmon  d e s c r i b e d a b o v e was  (Oncorhynchus  salmon and  per-  tshawytscha) f r y .  of t h e salmon g o n a d o t r o p i n p r e p a r a t i o n  c o u l d be d e t e r m i n e d by a c o m p a r i s o n o f t h e r e s u l t s pink  hypophy-  spermiation.  by September  t o be v e r y f e a s i b l e . SG-G100 be  Carasslus auratus  i n immature p i n k s a l m o n  sexual maturity  of the  i n the complete r e s t o r a t i o n  m a t u r i t y , and i n d u c t i o n of  the t e s t i s  injection  and H e t e r o p n e u s t e s f o s s l l i s  a) r e s u l t e d  The  Following  and  spring  salmon.  between  The  3 A l a r g e r pink of  greater  size  salmon c o u l d p o s s i b l y d e v e l o p  on s t i m u l a t i o n w i t h  SG-G100.  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  yearling  sockeye salmon  f r o m 22 t o 100 g a f t e r with The  predefined  generously  thus  to a smaller  fish.  h a d shown t h a t  ( O n c h o r h y n c h u s n e r k a ) c o u l d be grown two a n d o n e - h a l f  optimum e n v i r o n m e n t a l  c o n t r o l f i s h w e i g h e d 51  Brett  testis  I t would  (1970)  R e c e n t r e s e a r c h by B r e t t a n d S u t h e r l a n d  a  g. a f t e r  months o f  treatment  and d i e t a r y c o n d i t i o n s . the test  donated a stock of pink  period.  Dr.  salmon j u v e n i l e s  whose g r o w t h h a d b e e n s t i m u l a t e d i n a s i m i l a r manner f o r t h r e e months.  Some i n f o r m a t i o n o n t h e r e l a t i v e  maturation,  and r e l a t i v e  obtained,  although  initiated  later  Uninjected their rate  life  of the l a r g e r pink  i n the normal-sized  pink  sexual  s i z e s o f t h e r e s u l t a n t t e s t e s c o u l d be  injection  than  rates of  salmon m a i n t a i n e d  s a l m o n w o u l d be  fish. i n aquaria  throughout  c y c l e were u s e d f o r c o m p a r i s o n o f t h e p a t t e r n a n d  of sexual maturation  reproductive maturity  at various  stages  with  had b e e n a c c e l e r a t e d w i t h  the f i s h SG-G100.  whose  4 M a t e r i a l s and Methods  Animals The a q u a r i a i n which experimental animals were h e l d were s u p p l i e d with running water ( s a l t o r f r e s h ) , and were equipped w i t h s e l f - c l e a n i n g systems.  The mean d a i l y  tempera-  t u r e of the s a l t water supply v a r i e d from 8 ° to 1 1 ° C, dependi n g upon the season.  The l i g h t i n g i n the b u i l d i n g housing the  animals was r e g u l a t e d t o approximate All  the n a t u r a l photoperiod.  f i s h were f e d ad l i b i t u m a d i e t c o n s i s t i n g of beef  liver,  beef h e a r t , canned salmon, pablum, and sodium c h l o r i d e . (Donaldson and McBride,  1967).  Pink salmon f r y (Oncorhynchus  gorbuscha)  were o b t a i n e d  from the F i s h e r i e s Research Board, Nanaimo, i n m i d - A p r i l I 9 6 9 . They were t r a n s p o r t e d t o a 2 0 0 0 - l i t r e f i b r e g l a s s aquarium a t 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 o f animals,  500 were subsequently moved i n t o two outdoor 7 0 0 0 - 1 . a q u a r i a . The remainder were d i v i d e d i n t o f o u r groups o f 75* kept i n separate 1 7 0 - 1 .  f i b r e g l a s s a q u a r i a and s u b j e c t e d t o hormonal  treatments as d e s c r i b e d In the f o l l o w i n g  section.  The animals i n the outdoor tanks were h e l d f o r a study of the normal i n aquaria.  c y c l e of gonad development i n pink salmon, kept They were maintained over the p e r i o d of study by  Dr. W. Vanstone  and Mr. J . Culp, who used them as c o n t r o l s f o r  a concurrent p h o t o p e r i o d study.  Random t h i n n i n g 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  o f a kidney-  d i s e a s e i n the animals a t the end o f August 1970 prevented a f i n a l sampling a t the time o f complete sexual m a t u r i t y . S p r i n g salmon f r y (Oncorhynchus tshawytscha) which had been hatched i n mid-February were o b t a i n e d from the hatchery a t B i g Qualicum R i v e r , B.C. on May 8 , 19&9. 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 supplied with freshwater.  The 6 0 0 aquarium  Four groups o f 75 f i s h were subse-  q u e n t l y t r a n s f e r r e d t o separate 1 7 0 - l i t r e a q u a r i a and subj e c t e d t o hormonal treatments as d e s c r i b e d i n the f o l l o w i n g section.  The s p r i n g salmon were g r a d u a l l y a c c l i m a t i z e d to  s a l t water from May 20 to 22. A stock of 80 pink salmon f r y which were much l a r g e r than the normal s i z e were o b t a i n e d from Dr. J . R. B r e t t on August 2 3 , 1970.  At t h a t time they had a mean weight of 42.0  grams, as compared t o 15.8 grams f o r the n o r m a l - s i z e d pink salmon f r y a t the West Vancouver S t a t i o n .  The f i s h were from  eggs taken from Sweltzer Creek (near Cultus Lake), and hatched at  the F i s h e r i e s Research Board, Nanaimo S t a t i o n .  They had  been maintained s i n c e June 1 a t an e l e v a t e d water temperature (15°  C) and a p h o t p p e r i o d which lengthened by 5-minutes each  day to 16 hours on September 1.  These f i s h had been f e d to  s a t i e t y on a d i e t c o n s i s t i n g of Oregon 116 wet p e l l e t s o f an a p p r o p r i a t e s i z e (Westgate et a l , 1964).  A t the West Vancouver  L a b o r a t o r y , they were d i v i d e d e q u a l l y among f i v e f i b r e g l a s s aquaria.  170-litre  As f a c i l i t i e s were n o t a v a i l a b l e t o  m a i n t a i n t h e i r former c o n d i t i o n s , they were kept on a normal photoperiod, a t the temperature of the ambient  seawater, and  6  were f e d the d i e t d e s c r i b e d p r e v i o u s l y f o r the s t a t i o n .  A  c o n s i d e r a b l e number o f f i s h were subsequently removed because of a d i s e a s e which induced f o r m a t i o n of c a t a r a c t s i n t h e i r eyes.  Two  f u r t h e r t r a n s f e r s of the experimental groups were  made: to 4 5 0 - l i t r e f i b r e g l a s s a q u a r i a , November 22, to 2 0 0 0 - l i t r e f i b r e g l a s s a q u a r i a , January 29,  1970,  and  1971.  Treatments 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 salmon (Oncorhynchus tshawytscha) gonadotropin SG-G100 (Donaldson and Yamazaki, 1 9 6 8 ; Donaldson e t a l , 1971), was used to induce p r e c o c i o u s sexual m a t u r i t y i n pink salmon and s p r i n g salmon males.  Three dosages  of the salmon gonadotropin p r e p a r a t i o n d i s s o l v e d i n f i s h (Weibe, 1968)  (0.1,  1.0,  saline  and 10 micrograms per gram body weight)  p l u s a s a l i n e - i n j e c t e d c o n t r o l were s t u d i e d i n experiments i n v o l v i n g s e v e n t y - f i v e f r y of each of the two s p e c i e s per treatment.  These dosages were r e f e r r e d to i n t h i s t h e s i s as  treatment A, B, C, and Co r e s p e c t i v e l y .  The f i s h were i n j e c t e d  i n t r a p e r i t o n e a l l y three times per week w i t h a volume of 0.05 of the t e s t s o l u t i o n .  The p e r i o d of treatment began June  ml  10,  1969 f o r the s p r i n g salmon f r y and one week l a t e r f o r the pink salmon f r y . September 22,  The experiments were terminated on September 1,  and  I 9 6 9 f o r the s p r i n g and pink salmon r e s p e c t i v e l y .  F o l l o w i n g a zero c o n t r o l sample of t e n f i s h , t e n 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 s p r i n g salmon e x p e r i ment and seven samples f o r each treatment i n the pink salmon experiment.  7 The pink salmon f r y i n the outdoor tanks received no injections.  Five samples of ten specimens were taken from this  stock during t h e i r normal l i f e cycle. ken August 25,  The l a s t sample was t a -  1970.  Twenty extra-large pink salmon juveniles of the stock obtained from the F i s h e r i e s Research Board, Nanaimo Laboratory, were used i n an experiment designed to obtain information on the effect of size of the animal on the rate of induction of sexual maturity.  Ten f i s h were treated with salmon gonadotro-  pin dissolved i n f i s h saline (Wiebe, 1968) at a dosage of micrograms per gram body weight.  1.0  Ten control animals were i n -  jected with 0 . 1 0 ml of the test solution three times per week with a 25 g needle f o r a period of three months, s t a r t i n g September 2 , 1 9 7 0 . 26,  A l l of the specimens were sampled November  1970.  Experimental Techniques (i)  Injection Procedure The f i s h were anesthetized i n the aquarium by addition  with s t i r r i n g of a stock solution of quinaldine (Locke, I 9 6 9 ) to make a t o t a l concentration of the aquarium water of 10  ppm.  A l l of the f i s h i n 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  could be held safely i n this solution f o r 15 minutes.  fish Removal  of the f i s h from the basin was quickly followed by i n t r a p e r i t i o n e a l i n j e c t i o n of the test solution with a 27 g needle,  0.5  8  cm a n t e r i o r t o t h e a n u s . ute  when r e t u r n e d  (ii)  Autopsy  The a n i m a l s r e v i v e d w i t h i n  to fresh  one m i n -  water.  Procedure  F i s h were r a p i d l y removed f r o m a b u c k e t s u p p l i e d an a e r a t o r ,  a n d "damp d r i e d " .  nearest  tenth  nearest  millimeter.  Wet w e i g h t was m e a s u r e d t o t h e  o f a gram, a n d f o r k l e n g t h  was m e a s u r e d t o t h e  The a n i m a l s were t h e n k i l l e d  t a t i o n , a n d t h e p e r i t o n e a l c a v i t y was opened. weighed and  to the nearest  s t o r e d a t -20° C.  i n Bouin's (iii)  milligram, The o t h e r  frozen gonad  by  decapi-  One gonad  o n d r y i c e (-70°  was C)  was removed a n d f i x e d  solution.  H i s t o l o g i c a l Techniques Following  the  with  fixation  i n B o u i n ' s s o l u t i o n f o r 24-hours,  t e s t e s were washed, d e h y d r a t e d a n d embedded i n P a r a p l a s t -  mp  56-57  at  6 m i c r o n s , and s t a i n e d w i t h Mayer's h a e m a t o x y l i n and e o s i n .  C (Culling,  (iv) Analysis (a)  T h e s p e c i m e n s were t h e n  sectioned  of the H i s t o l o g i c a l Results  Measurements Dessication  necessitated  of the small  control testes during  a c o m p a r i s o n o f gonad  t h a n wet w e i g h t s . to the nearest mined  1957).  One f i x e d gonad  milligram.  as follovrs:  weighing  w e i g h t b a s e d on f i x e d f r o m e a c h f i s h was  The g o n a d o s o m a t i c  i n d e x was  rather  weighed deter-  9  r  (b)  Q T  mined from  o f one g o n a d X 2 body w e i g h t  X  1  0  0  Analysis  stage of sexual maturity of the t e s t i s  was d e t e r -  examination o f a s i n g l e median s a g g i t a l  specimen.  on t h e b a s i s cular  weight  Histological The  each  _ -  The degree  section  o f m a t u r i t y was e s t i m a t e d  of the following  sixdistinct  from  visually  stages of t e s t i -  development: (1)  Spermatogonia  (2)  Spermatogenesis nant  (3)  Primary and  (4)  cell  only are present (Figs. has been i n i t i a t e d .  types a r e primary  spermatocytes,  predomi-  spermatocytes.  secondary  spermatocytes,  i s proceeding a c t i v e l y ;  zoa a r e p r e s e n t i n the l o b u l e stage the t e s t i s  (5)  The  spermatids a r e a l l present.  Spermatogenesis  7  1 a n d 2).  and  lamina.  r e a c h e s i t s maximum  spermatoIn this  size  8).  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 o f t h e t e s t i c u l a r and  spermatozoa  l i e free  (6) These  morphology,  i n t h e sperm d u c t .  can be s t r i p p e d from t h e a n i m a l a t t h i s (Figs.  (Figs.  Semen  stage  3 and 4 ) .  The t e s t i s  i s completely r e g r e s s e d ( F i g . 6).  stages of t e s t i c u l a r  development  t h o s e e m p l o y e d by N a y a r a n d S u n d a r a r a j  (1970)  ment o f t h e r e p r o d u c t i v e m a t u r i t y o f t h e m a l e  are similar to i n their assesscatfish,  10  Heteropneustes f o s s l l l s  The s t a g e o f f u n c t i o n a l  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 H e n d e r s o n (1962) i n  maturity the  (Bloch).  e a s t e r n brook  trout Salvellnus  fontinalis-(Mitchell).  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 o f t h e p i n k and (v)  s p r i n g salmon  studied,  H i s t o c h e m i c a l Techniques The  samples  from t h e study on t h e e f f e c t of  g o n a d o t r o p i n on m a t u r a t i o n o f t h e t e s t i s were  salmon  investigated  h i s t o c h e m i c a l l y f o r t h e p r e s e n c e o f A5-3 8 h y d r o x y s t e r o i d dehydrogenase  (3 8 - o l d e h y d r o g e n a s e ) .  s t o r e d a t -20° and  The f r o z e n  testes  C w e r e s e c t i o n e d a t 10 m i c r o n s o n t h e c r y o s t a t  stained using Bara's  (I965) p r o c e d u r e .  used s u c c e s s f u l l y t o demonstrate  T h i s m e t h o d was  3 8 - o l dehydrogenase  activity  i n t h e 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. b y Y a m a z a k i a n d  Donaldson  (I969). a n d 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 Dehydroepiandrosterone  assay.  (5-andosten-38-ol-17one;  was t h e o n l y s u b s t r a t e u s e d . '  Calbiochem).  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 , a n d ( b ) w i t h r e d u c e d - n i c o t i n a m i d e adenine d i n u c l e o t i d e activity.  (NADH; C a l b i o c h e m . )  to t e s t f o r dlaphorase  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 experimental  procedure.  A t e m p e r a t u r e o f 37°C was f o u n d 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 h o u r o f i n c u b a t i o n , when c o m p a r e d t o t e m p e r a t u r e s o f 10,  20, a n d 4 5 ° C.  Little"additional  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 w e r e 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 s e c t i o n s were not washed w i t h acetone (to  remove l i p i d s ) f o l l o w i n g i n c u b a t i o n , 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 t e s t e s from s e l e c t e d samples were sectioned on the c r y o s t a t at 10 microns and stained w i t h Sudan Black B and O i l Red 0 f o r l i p i d s . (Chayen et a l , 1969). (vi)  A n a l y s i s 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 g o l d f i s h (Carassius auratus L.) was r e g i s t e r e d as 5 was used to quantify the formazan deposits obtained i n the f r o z e n testes.  A c t i v i t y of one represented an intense pink r e a c t i o n  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. of 2 was  Activity  considered to represent a s c a t t e r i n g 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 i n j e c t e d w i t h v a r i o u s dosages of salmon gonadotropin was  compared w i t h one another, and w i t h the c y c l e of normal  m a t u r a t i o n i n u n i n f e c t e d pink salmon kept i n a q u a r i a . r e s u l t s from the experiment  The  i n which pink salmon were i n j e c t e d  w i t h a salmon gonadotropin dosage regime c o r r e s p o n d i n g to Treatment  B were a l s o c o n s i d e r e d .  A l l o f the f i s h a d m i n i s t e r e d salmon gonadotropin showed a s t i m u l a t i o n 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 r e g r e s s i o n , F i g . 6 ) , i n a p e r i o d of 98 days ( F i g . 16). comparison,  The  s p r i n g salmon f r y , by  d i d not reach a stage of f u n c t i o n a l m a t u r i t y a f t e r  84 days of treatment  ( F i g . 17); however, they d i d have a h i g h e r  G.S.I, a t comparable stages of sexual maturation d i r e c t comparison  ( F i g . 14).  No  can be made between these groups and the l a r g e r  f i s h from the F i s h e r i e s Research Board, Nanaimo L a b o r a t o r y , because 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 n o r m a l - s i z e d pink salmon f r y .  The  l a r g e r 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 t o have matured s e x u a l l y i n the same time i n t e r v a l as r e q u i r e d f o r the s m a l l e r animals. During the normal c y c l e of t e s t i c u l a r m a t u r a t i o n i n pink salmon a dramatic i n c r e a s e was  recorded i n the r a t e of gonad  development and G.S.I, f o l l o w i n g completion of the m e i o t i c  13  d i v i s i o n to form primary spermatocytes from  spermatogonia  (Figs. 15 and 18) i t was early July, and the f i s h were i n t h e i r second year of l i f e .  A corresponding observation was  made on the t e s t i s of f i s h i n both treatments A and B f o r the normal-sized pink and spring salmon f r y , but the time i n t e r v a l to appearance of spermatozoa was shorter.  I t would therefore  appear that both the time of onset of the mitotic d i v i s i o n to form primary spermatocytes, and the process of active spermatogenesis were accelerated i n f i s h treated with salmon gonadotropin. (b) A 5-3 8 Hydroxysteroid Dehydrogenase Formazan deposits, i n d i c a t i n g A 5 - 3 3 hydroxysteroid dehydrogenase a c t i v i t y were recorded i n testes from normalsized pink salmon (treatments A and B); the a c t i v i t y was maximal during the period when spermatogenesis was active and spermatozoa were present (Figs. 11 and 19).  The l o c a l i z a t i o n s  were scattered when compared to the reaction obtained i n the testes of the mature g o l d f i s h (Fig. 13).  A slight activity  of 3 3-ol dehydrogenase i n the seminiferous tubules was observed.  The precociously mature salmon t e s t i s had less h i s t o -  chemically demonstrable a c t i v i t y than did the pink salmon mentioned above (Figs. 12 and 20). were found.  Few deposits of formazan  The reaction was more intense i n the i n t e r s t i t i a l  regions than i n the lobules (Fig. 12). Controls lacking dehydroepiandrosterone i n the incubation medium did give a positive reaction i n some instances. The a c t i v i t y was always less than that observed i n a corresponding  14  medium w i t h s u b s t r a t e . trol  No r e a c t i o n was  ever d e t e c t e d i n con-  s e c t i o n s incubated i n a medium which l a c k e d NADH. A d d i t i o n  of NADH to the r e a c t i o n medium r e s u l t e d i n a l l cases i n the f o r m a t i o n 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 r e g i o n and w i t h i n the l o b u l e s . diaphorase was (c)  not  T h e r e f o r e , NADH  limiting.  Lipids F r o z e n s e c t i o n s of t e s t e s from the pink salmon f r y i n -  j e c t e d w i t h salmon gonadotropin f o r 42 and 84 days were s t a i n e d with O i l Red 0 and Sudan B l a c k B.  No r e a c t i o n was  obtained  w i t h O i l Red 0 whereas the r e s u l t s were p o s i t i v e when Sudan B l a c k B was  employed.  The  l i p i d s were l o c a l i z e d i n the  inter-  s t i t i a l regions ( F i g . 10). (d)  Interstitial  Cells  Interstitial groups. dant  c e l l s were found i n a l l of the  experimental  At sexual m a t u r i t y , i n t e r s t i t i a l c e l l s were not abun-  i n any of the t r e a t e d pink salmon, or i n the u n i n f e c t e d  f i s h maturing a t a normal r a t e . were observed comparison,  Some c o n c e n t r a t i o n s of  ( F i g . 9)* but they were widely s c a t t e r e d .  the p r e c o c i o u s l y developed  t a i n e d even fewer i n t e r s t i t i a l c e l l s .  cells By  s p r i n g salmon t e s t e s conThis observation correlates  d i r e c t l y w i t h p r e v i o u s i n f o r m a t i o n t h a t 3 8 - o l dehydrogenase activity (e)  was  c o r r e s p o n d i n g l y lower i n the s p r i n g salmon.  Secondary Sex The  Characteristics  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  coloration  of the s i d e s of the pink  salmon  approaching  s e x u a l m a t u r i t y was n o t a p p a r e n t  i n the pink  with  was, t h e r e f o r e , no means o f  salmon g o n a d o t r o p i n .  There  d i s t i n g u i s h i n g between males and f e m a l e s , trol  fish  secondary extent  with the injected  maturity  spring  A similar  pro-  salmon f r y .  s e x c h a r a c t e r i s t i c s were p r e s e n t t o a much  than normal d u r i n g the f i n a l  injected  o r t r e a t e d and con-  by t h e a p p e a r a n c e o f t h e a n i m a l s .  b l e m was e n c o u n t e r e d  salmon  The  lesser  stages of t e s t i c u l a r  i n t h e u n i n j e c t e d p i n k salmon m a i n t a i n e d  i n aquaria.  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, i n c l u d i n g t e r m i n a l t e s t i c u l a r r e g r e s s i o n . When the f i s h were functionallymature, semen could be s t r i p p e d from the t e s t e s by s t r o k i n g the abdomen i n an a n t e r o - p o s t e r i o r d i r e c t i o n .  The r e s u l t s i n d i c a t e d  that the time of sexual maturity of male pink j u v e n i l e s could be manipulated to c o i n c i d e w i t h that of any stock of n a t u r a l spawning  fish. The r a t e of attainment of the maximum G.S.I, showed a  dose-response r e l a t i o n s h i p w i t h the amount of salmon gonadotropin administered.  A comparable c o r r e l a t i o n was not found f o r  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 t e n - f o l d greater q u a n t i t y of hormone i n developing t e s t e s 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 t e s t e s from the t r e a t e d pink s a l mon,  and those which developed normally revealed the stages of  maturation to be a c c e l e r a t e d i n the p r e c o c i o u s l y mature f i s h . The time of appearance of primary spermatocytes was advanced by eleven months i n j u v e n i l e pink salmon i n j e c t e d w i t h salmon gonadotropin.  These r e s u l t s agreed w i t h previous research on  s e v e r a l r e p r e s e n t a t i v e t e l e o s t s by Barr ( I 9 6 3 a) ( I 9 6 6 ) , Ahsan ( I 9 6 6 ) ,  L o f t s et a l  Sundararaj and Nayyar ( I 9 6 7 ) , Yamazaki  and Donaldson ( 1 9 6 8 ) , and Pandey (1969  a, b).  These s t u d i e s  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 mones.  c o n t r o l l e d by p i t u i t a r y hor-  The probable reason f o r the marked d i f f e r e n c e i n weight  between the p r e c o c i o u s l y developed t e s t e s and the sex organs from the u n i n f e c t e d f i s h a t sexual m a t u r i t y (0.2 grams v. grans r e s p e c t i v e l y ) was  the e a r l i e r t r a n s i t i o n from  to spermatocytes i n the t r e a t e d f i s h . spermatogonia would  31.5  spermatogonia  F u r t h e r d i v i s i o n s of the  thereby be i n h i b i t e d , and the e v e n t u a l num-  ber of more advanced germinal c e l l s reduced. The progress of a c t i v e spermatogenesis to the p r o d u c t i o n of mature spermatozoa  o c c u r r e d i n one h a l f the normal time i n  the p r e c o c i o u s l y developed pink salmon. T h i s cannot be a t t r i b u t e d to a d i r e c t e f f e c t of gonadotropin, s i n c e i t c o u l d have been a m a n i f e s t a t i o n of androgen a c t i v i t y .  Androgen i n j e c t i o n  result-  ed i n a s t i m u l a t i o n of spermatogenesis i n hypophysectomized Lebistes reticulatus ( L o f t s et a l , 1966) Nayyar, I 9 6 7 ) ,  ( E v e r s o l e , 1 9 ^ 1 ) , Fundulus Heteropneustes f o s s l l l s  adult  heteroclltus  (Sundararaj and  and P b e c l l i a r e t i c u l a t a P e t e r s (Pandey, 1969 a ) .  The r e s u l t s a r e v a r i a b l e , however, because methyl t e s t e r o n e i n the dosages a d m i n i s t e r e d d i d not s t i m u l a t e  spermatogenesis  i n the t e s t i s of the m e t h a l l i b u r e - t r e a t e d a d u l t aggregata reticulata  Gibbons  (Wiebe, 1969)  or i n j u v e n i l e  Cymatogaster Poecilla  P e t e r s (Pandey, 1969 b ) .  Pink salmon which were t h r e e times l a r g e r than normals i z e d specimens on September 2, 1970,  developed mature t e s t e s  a f t e r treatment w i t h salmon gonadotropin i n the same time i n t e r v a l as the s m a l l e r animals. was  l a r g e r (0.9 grams v. 0.2  However, the gonad  grams).  itself  D i r e c t comparisons  cannot  18  be made b e t w e e n t h e s e two  groups because  injections  i n t h e l a r g e r . : f i s h 83 d a y s l a t e r  initiated  were  than i n the normal-  sized animals. T h e r e were d i f f e r e n c e s b e t w e e n t h e e f f e c t s s a l m o n g o n a d o t r o p i n p r e p a r a t i o n on t h e d o n o r the p i n k salmon. salmon  f r y was  than that manner. pink may  o f p i n k salmon  purely  and  one h a l f  juveniles treated  S p e r m a t o g e n e s i s was  salmon a f t e r be  s p e c i e s and  The maximum G.S.I, o f t h e t e s t  a p p r o x i m a t e l y two  relatively  of the  spring  times  larger  i n a corresponding  more a d v a n c e d  84 d a y s o f t r e a t m e n t , however.  q u a n t i t a t i v e because of a  on  small  In the  The  results  species  differ-  e n c e i n t h e r e s p o n s e t o t h e hormone. The  rate  of i n d u c t i o n of a c t i v e  time of appearance and  spring  other  salmon  salmonids.  from Oncorhynchus (Salmo g a i r d n e r l l ) accelerated  compared After  two weeks o f i n j e c t i o n  tshawytscha p i t u i t a r i e s 13.5  cm  i n length,  t o the appearance  coho  o f t h e s p e r m a t o g o n i a ; no  (Chestnut,  1970).  pituitaries Oncorhynchus  Administration  o f spawning  salmon  immature  s p e r m a t o z o a two  sper-  the-mitotic  of an e x t r a c t  (Oncorhynchus  resulted  coho  pituitary  s p e r m a t o c y t e s c o u l d be  (Salmo g a i r d n e r i i ) months l a t e r  trout  of f i n g e r l i n g  stimulated  on  was  secondary  found  from the  keta  tshawytscha) i n c h o l e s t e r o l p e l l e t s  rainbow t r o u t  pink  extracts  spermatogenesis  Injection  the  research  ( O n c o r h y n c h u s k l s u t c h ) f o r t h r e e weeks w i t h  division  of  juvenile  of  into  of p r i m a r y and  homogenate f r o m s p a w n i n g a d u l t  old  i n the t r e a t e d  favorably with similar  (Schmidt et a l , 1 9 6 5 ) .  matocytes salmon  of spermatozoa  spermatogenesis and  and t o 6^-month  i n the shedding  ( R o b e r t s o n and R i n f r e t ,  1957).  19  No  histology  o f t h e s e t e s t e s was  T h e r e a r e two cells and  of the t e s t i s  lobule-boundary  There  areas  performed.  i n which the androgen  of t e l e o s t s are l o c a t e d : regions (Marshall,  i s considerable v a r i a b i l i t y  as t o the  site  of the t e s t i c u l a r  from  the i n t e r s t i t i a l and  into  microscope  lobule-boundary  (1970)  A  5-3  studied  of s t e r o i d o g e n e s i s . i n d i c a t e d as the (1970  Chestnut  i n Salmo s a l a r  The  sites  immature  in  Salmo  O'Halloran  and  made  and  demonstrable  the  c e l l s were  of endocrine a c t i v i t y . that  These  deposition  to r e v e a l p o t e n t i a l  lobule-boundary  determined  Interstitial  i n the  the a r e a s o f h i s t o c h e m i c a l l y  lipids  another  A s i m i l a r o b s e r v a t i o n was  8 h y d r o x y s t e r o i d dehydrogenase a c t i v i t y  of c y a n o p h i l l i c  to  r e g i o n s i n mature  on Salvelirius f o n t i n a l i s by H e n d e r s o n , 1 9 6 2 . Idler  salmonid  glandular-appearing c e l l s  (Robertson, 1958).  gairdnerii  one  1965).  Hoar,  ( O o t a a n d Yamamoto, 1 9 6 6 ) .  o f Salmo g a i r d n e r i i  c e l l s apparently developed  I96O;  interstitial  endocrine t i s s u e .  c e l l s were o b s e r v e d w i t h t h e l i g h t testes  the  secreting  By  the i n t e r s t i t i a l  sites  definitely  contrast,  cells  of  m a t u r e O n c o r h y n c h u s k i s u t c h were t h e r e g i o n where A 5-3f3 - o l dehydrogenase a c t i v i t y interstitial scattered spring  was  localized.  c e l l s were o b s e r v e d  throughout  s a l m o n had  than d i d the pink Pituitary  In the present  i n small clusters  study,  widely  the t e s t e s of a l l mature specimens.  relatively  fewer  nests of i n t e r s t i t i a l  The cells  salmon. g o n a d o t r o p i n had b e e n f o u n d the A 5-3  teleosts  to c o n t r o l  activity  i n the t e s t i s .  i n several  8 h y d r o s t e r o i d dehydrogenase  Hypophysectomy o f a d u l t C a r a s s l u s  20 a u r a t u s caused r e g r e s s i o n 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 w i t h salmon gonadotropin r e s t o r e d the normal l e v e l of enzymic a c t i v i t y .  T e s t e s from Cymatogaster aggregata Gibbons  were incubated " i n v i t r o " w i t h mammalian l u t e i n i z i n g hormone (Wiebe, 1969  a).  The amount 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 i n c r e a s e d n o t i c e a b l y i n the t e s t gonads a f t e r f o u r hours of i n c u b a t i o n .  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 f o l l o w i n g 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  k i s u t c h w i t h e x t r a c t s of p i t u i t a r y homogenates from spawning a d u l t s was r e p o r t e d by Chestnut (1970).  In the present  ment, an a c t i v i t y of 3g-ol dehydrogenase of two  experi-  (on a s c a l e  where the r e a c t i o n i n the mature C a r a s s i u s a u r a t u s t e s t i s taken as 5) pink salmon.  was observed i n the t e s t e s of p r e c o c i o u s l y  was  mature  Few d e p o s i t s of formazan could be seen i n the  t e s t i s of s e x u a l l y mature s p r i n g salmon j u v e n i l e s ; these data c o r r e l a t e d 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  cells  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 a d u l t pink salmon and s p r i n g salmon were not manifested i n p r e c o c i o u s l y mature members of these same s p e c i e s .  T h i s ob-  s e r v a t i o n confounded any attempt to r e l a t e the apparent i n c r e a s e i n s t e r o i d dehydrogenase a c t i v i t y i n t r e a t e d f i s h to p r o d u c t i o n of androgen by the t e s t i s .  I t i s important to mention t h a t the  secondary sex c h a r a c t e r i s t i c s of the u n i n j e c t e d pink salmon  21 maintained  i n aquaria  throughout t h e i r  life  c y c l e were much  l e s s marked t h a n n o r m a l a t s e x u a l m a t u r i t y . e f f e c t was a r e s u l t All  Perhaps  this  of the s t r e s s of confinement.  o f t h e s e x u a l l y mature  (Fig. 5).  epithelial  s t a n c e exuded  f r o m them w h i c h may h a v e p r o v i d e d n u t r i t i o n f o r  spermatozoa.  These  cells  t o t h o s e r e p o r t e d by Y a m a z a k i physectomized  i n the duct  s t u d i e d had  hypertrophied  the  cells  specimens  compared  i n their  and Donaldson  sub-  morphology  (1968)  Carassius auratus injected with  A  i n hypo-  salmon  gonado-  tropin. For a separate cooperative  e x p e r i m e n t i n t h e summer o f  1970 w i t h F . C. W i t h l e r a n d R. B. M o r l e y o f t h e F i s h e r i e s R e s e a r c h B o a r d , Nanaimo L a b o r a t o r y , fourteen pink  s a l m o n m a l e s w h i c h were s e x u a l l y m a t u r e  one y e a r e a r l i e r dosage  the author developed  than normal.  exactly  T h e s e f i s h were t r e a t e d w i t h a  r e g i m e w h i c h c o r r e s p o n d e d t o t r e a t m e n t B o f t h e 1969  experiment.  The f e r t i l i z i n g  f i s h was compared  capacity  to that of the milt  of the m i l t from w i l d  from the t e s t  Lower B a b i n e  R i v e r p i n k m a l e s when e q u a l v o l u m e s  o f b o t h were m i x e d  100  T h e r e were no marked  o v a f r o m Lower B a b i n e f e m a l e s .  differences hatching*  i n p r o p o r t i o n s o f ova f e r t i l i z e d ,  o r i n numbers o f d e f o r m e d  were t h e r e f o r e v i a b l e . et a l , 1971.  larvae.  with  i n survival to The s p e r m a t o z o a  These r e s u l t s a r e r e p o r t e d  i n Donaldson  22  1:  Figure  Cross-section of a testis salmon  juvenile  Figure  2;  (7.7  cm l o n g ) .  from a zero c o n t r o l  Haematoxylin and e o s i n X 250.  Same s e c t i o n a s i n F i g . 1 , tion.  Example o f s t a g e 1 t e s t i s .  Figure  3: Testis  venile  (11.0  Figure  4:  but at a higher magnificat Haematoxylin and eosin X 6 0 0 .  o f a s e x u a l l y mature  lin  ( s t a g e 4) p i n k s a l m o n j u -  cm l o n g ) . H a e m a t o x y l i n a n d e o s i n X 2 0 .  Same s e c t i o n a s i n F i g . 3 tion  pink  t o show d e t a i l  "but a t a h i g h e r m a g n i f i c a -  o f s p e r m a t o g e n e s i s n e a r t h e d u c t . Haematoxy-  and e o s i n X 250. 5:  Figure  Epithelial  cells  lining  s e c r e t i o n o f a substance which zoa.  t h e sperm d u c t . possibly  Note t h e  n o u r i s h e s t h e spermato-  Haematoxylin and e o s i n X 6 0 0 .  Figure  6: Regressed t e s t i s  salmon.  ( s t a g e 6) o f a j u v e n i l e male p i n k  H a e m a t o x y l i n a n d e o s i n X 300.  Figure Stage 4 t e s t i s weight  360 g r a m s ) .  Figure  8:  o f an u n i n j e c t e d  (body  H a e m a t o x y l i n a n d e o s i n X 40.  Same s e c t i o n a s i n F i g . 7 . tion to reveal  p i n k salmon  the d e t a i l s  but a t a higher magnifica-  o f spermatogenesis  Haematoxylin and e o s i n X 250.  i n the lobules.  23  Figure  9: Interstitial  mature pink  salmon  cells  i n the t e s t i s  (body l e n g t h  12.3  of a precociously  cm).  Haematoxylin and  e o s i n X 1500. Figure  10; Frozen  pink  salmon  12.3  cm)  Figure  section of the t e s t i s  stained with  of a precociously  Sudan B l a c k  B for lipids,  mature  (body  length  X 600.  11; Histochemically  dehydrogenase  activity  s e x u a l l y mature p i n k  demonstrable A 5-3 8 h y d r o x y s t e r o i d i n a frozen  salmon  section of a t e s t i s  juvenile,  (body l e n g t h  from a  12.3  cm).  D e h y d r o e p i a n d r o s t e r o n e was u s e d a s t h e s u b s t r a t e , a n d n i t r o - B . T . was t h e t e t r a z o l i u m s a l t .  Deposits  are granules  o f formazan.  X 200. Figure  12: A 5-3 8 h y d r o x y s t e r o i d  testis 9.1  The i n c u b a t i o n medium was i d e n t i c a l  i n Figure  11;  (body  i n the length  to that  described  X 200.  13: 3 g - o l dehydrogenase  ly  activity  o f a p r e c o c i o u s l y mature s p r i n g salmon  cm).  Figure  dehydrogenase  mature g o l d f i s h  was i d e n t i c a l  activity  i n the t e s t i s  (Carassius auratus).  to that described  of a  sexual-  T h e i n c u b a t i o n medium  f o r Figure  11;  X 200.  2k  F i g u r e lk:  The mean g o n a d o - s o m a t l c (Oncorhynchus  index  gorbuscha) and s p r i n g  salmon  juveniles  salmon  (Oncorhynchus  summer o f 19&9.  treated  (G.S.I.) o f male  (Oncorhynchus  pink  tshawytscha)  t h r i c e weekly w i t h v a r i o u s dosages o f  tshawytscha) gonadotropin d u r i n g the  Numbers o f f i s h  and standard d e v i a t i o n a r e i n  T a b l e I and I I .  4  10 0-i  o / / / /  MALE SALMON 8  0  _ |  O. tschawytscha O. gorbuscha  / j 10pg/g BW  o ~ — •—-  J  / / / /  i f  25  Figure  15: The  male p i n k Detailed  mean g o n a d o - s o m a t i c i n d e x  salmon m a i n t a i n e d data  i n Table I I I .  (G.S.I.) of u n i n f e c t e d  i n a q u a r i a throughout  their  life.  N°V^  1  DEC.  1969  J  I  JAN.  I  FEB.  I MARCH  " APRIL  '  1970  MAY  '  JUNE  '  JULY  I  AUG  1  26  16:  Figure  Stages injected  t h r e e times  (Oncorhynchus Table  IV.  of t e s t i c u l a r m a t u r i t y of pink  salmon  juveniles  p e r week w i t h v a r i o u s d o s a g e s o f  tshawytscha)  gonadotropin.  Values  taken  salmon from  27  Figure  17; Stages of t e s t i c u l a r maturity  juveniles salmon  treated  three  Induced i n s p r i n g  times weekly w i t h v a r i o u s  (Oncorhynchus tshawytscha) g o n a d o t r o p i n .  taken from Table  V.  salmon  dosages of These  values  JUNE  I  JULY  '  AUGUST  I  SEPT  r  28  Figure 18: Stages of reproductive maturity of uninfected male pink salmon maintained  i n aquaria throughout t h e i r l i f e cycle.  Data were taken from Table VI.  6H  4H  29  Figure  19: The  h i s t o c h e m i c a l l y d e m o n s t r a b l e of A 5-3 8 h y d r o x y s t e r o i d  dehydrogenase i n the t h r i c e weekly w i t h  t e s t e s of  various  tshawytscha) gonadotropin. fied  by  during  visual the  observation.  summer o f 1 9 6 9 .  juvenile  dosages of The The  pink  salmon  salmon  injected  (Oncorhynchus  f o r m a z a n d e p o s i t s were e x p e r i m e n t was  quanti-  conducted  Values taken from Table  VII.  30  Figure 20: The h i s t o c h e m i c a l l y . d e m o n s t r a b l e a c t i v i t y hydroxysteroid  dehydrogenase i n the t e s t e s o f j u v e n i l e s p r i n g  salmon i n j e c t e d t h r e e salmon  visual  t i m e s p e r week w i t h  various  (Oncorhynchus tshawystcha) gonadotropin  summer o f I 9 6 9 .  ofA 5-38  dosages o f  during the  The f o r m a z a n d e p o s i t s were q u a n t i f i e d by  observation.  These v a l u e s  taken from Table  VIII.  H  1.5  > I— O <  i 0 . 0 ^ g / g BW 1.0-  liJ  IOjjg/gBW  >N  2.  Oljjg/gBW  LU 0.5 H  OA  O CONTROL  JUNE  I  JULY  * AUGUST  1  SEPT  r  31  Table  1:  Male  pink  salmon  juveniles  dosages o f salmon g o n a d o t r o p i n weight,  mean f i x e d  (G.S.I.).  y  - mean f i s h  gonad w e i g h t ,  = mean;  SD  treated with  various  l e n g t h , mean f i  mean g o n a d o s o m a t i c  = standard d e v i a t i o n .  index  TABLE I :  SAMPLE Zero Control 17/6/69 1 30/6/69 2 14/7/69 3 28/7/69 4 11/8/69 5 25/8/69 6 8/9/69 7 22/9/69  TREATMENT Co A B C Co A B C Co A B C Co A B Co A B Co A B Co A B Co U.I. Co  n  FIXED BODY BODY TESTIS . G-. S. I-. LENGTH (cm) WEIGHT (g) WEIGHT ( y g) y y S.D. S.D, S. D. S. D. y ;  y.  5  7.7  0.3  6 4 7 4 5 4 4 4 3 6 4 2 5 4 4 4 4 6 2 2 5. 4 2 4 4  8.3 8.4 8.1 8.3 9> 9.1 9.0 8.8 10.4  0.7 0.7 0.6 0.8 0.5 0.7 0.-5 0.3 0.2 0.4 0.8 0.1 0.9 0.6 1.0 0.5 1.4 0.4 0.7 0.6 0.7 1.3 1.5 1.5 1.5  9.9 9.5 10.9 10.5 11.1 12.3 11.3 12.0 12.2 12.6 12.5 12.1 12.3 14.4 14.4  3.8  0.5  5.2 1.5 1.2 5.5 4.1 1.0 4.6 1.0 N 1.6 7.1 6.5 1.5 6.1 1.2 0.7 5.5 66.0 9.7 0 . 9 12.6 7.2 1.0 8.2 2.2 4.7 1.0 6.7 0.2 14.2 2.8 210.8 11.2 2.8 52.2 11.9 2>A 1.9 17.7 2.7 219.5 68.4 14.1 5.0 1.4 15.6 2.5 16.8 2.1 91.1 17.8 223.1 2.5 2.6 3.7 17.5 16.1 4.6 72.1 17.8 7.8 90.0 4.2 28.5 11.4 4.2 28.5 11.4  0  D  38.8 7.1 ^.5 0.2 129.9 47.8 0.7 124.6 5^.9 0.7 65.3 99.2 2.3 35.2 22.1 1.0 1.4  A  T  1.32 0.3 * 0.07 0.03 2.13 0.83 0.03 2.43 1.06 0.03 1.14 2.45 0.04 0.77 1.28 0.03 0.03 2  A  0.71 0.17 0.05 0.01 1.71 0.55 0.01 1.02 1.02 0.01 1.12 0.76 0.02 0.13 0.50 0.01 0.01  32  Table I I : Mean body w e i g h t , mean body  l e n g t h , mean f i x e d  w e i g h t , a n d mean g o n a d o s o m a t i c i n d e x salmon t r e a t e d w i t h v a r i o u s y  = mean;  S.D. = s t a n d a r d  testis  (G.S.I.) of j u v e n i l e  dosages o f salmon deviation.  spring  gonadotropin,  TABLE I I :  SAMPLE Zero Control IO/6/69  1  2  3  5 6  TREATMENT Co A B C Co A B C Co A B C Co A B C Co A B C Co A B C Co U.I. Co  BODY BODY LENGTH (cm) WEIGHT (g) S. D. y y S. D.  n  6  5.5  0.3  1.8  0.2  3 4 5 5 6 3 6 3 6 1  6.0 6.0 6.3 6.1 6.9 6.8 7.1 6.7 7.9 7.2 7.0 7.2 8.1 7.9 7.2 7.6 9.3 9.1 8.5 7.3  0.3 0.7 0.5 1.0 0.3 0.5 0.4 0.6 0.6  0.3 0.9 0.6 1.2 0.4 0.5 0.7 0.9 1.3  0.2 1.0 0.8 0.7 0.8 0.6 0.6 1.2 0.7  2.1 2.4 2.9 2.4 3.3 3.3 3.8 3.1 5.2 3.6 3.^ 3.6 5.8 5.3 3.5 4.4 8.8 8.8 7.0 *.3  0.9 1.5 1.1 1.8  8.0 9.2 7.0 8.9  6 6 6 6 3 3 5 5 3 5 6 5  9.1  8.8 9A  8.6 9.0  —  0.5  0.9  9.1  FIXED TESTIS G.S.I. WEIGHT (yg) S . D. S.D. y y :  --  1.0  0.4 2.4 1.5 0.9 1.6 0.9 1.8 2.8 1.1 2.5 3.1 4.1 3.1 5.1  73.1 29.9  5.2 0.6 186.4 117.9 12.8 0.9 478.2 280.9 7.6 2.2 ^.7  52.7  28.2 3.8 0.3 73.3 91.5 7.3 0.5 228.5 116.6 4.8 1.5 5.1  2.53 1.13 0.21 0.02  4.30  1.31 1.22 0.07  0.01  1.92  1.88 0.17 0.04 0.02 10.00 2.91 6.39 0.17 0.18 0.09 2.57  0.25  0.05  0.08  0.03 0.05  33  Table I I I : Mean body w e i g h t , somatic tained S.D.  index  mean gonad w e i g h t ,  a n d mean  gonado  ( G . S . I . ) o f u n i n f e c t e d male p i n k s a l m o n m a i n -  i n a q u a r i a throughout = Standard Deviation.  their life  cycle.  v  = mean;  TABLE I I I :  SAMPLE  DATE  n  BODY WEIGHT  TESTIS WEIGHT  (g) y  S.D.  G.S.I.  (g) y  S.D.  y  S.D.  8  5/H/69  4  90.8 10.2  0.03  0  0.03  0.01  9  21/1/70  6  157.5 2 0 . 5  0.07  0 . 0 2 0.04  0.01  10  9/3/70  2  196.8 18.7  0.09  0 . 0 2 0.04  0.01  11  30/6/70  5  232.6 3 9 . 2  0.43  0.40 0.18  0.14  12  25/8/70  4  355.8 25.1  31.5  5.8  8.81  1.15  3k  Table  IV: The  stage of  dosages o f salmon p.  = mean;  S.D.  t e s t i c u l a r maturation  gonadotropin i n =  i n d u c e d by v a r i o u s  p-ihk s a l m o n  Standard D e v i a t i o n  juveniles.  gART.TC  T V :  SAMPLE 8c  DATE  TREATMENT  Zero Control 17/6/69 1 30/6/69 2 1V7/69 3 28/7/69  11/8/69 5 25/8/69 6 8/9/69 7 22/9/69  A B C Co A B C Co A B C Co A B Co A B Co A B Co A B Co U.I. Co  n  TESTICULAR STAGE S.D.  2  1.0  0  3 2 3 3  1.0 1.0 1.0 1.0  0 0 0 0  3 3 3 3 3 3 3 1 3 3 3 3  1.0 1.8 1.0 1.0 2.8 2.6 1.4 1.0 4.3 4.0 1.0 4.6 4.8 1.0 4.7 *.5 1.0 5.8 5.6 1.0 1.0  0 0.4 0 0 0.4 0.2 0.2 0 0.6 0.1 0 0.1 0.4 0 0.3 0.7 0 0.3 0.6 0 0  3  3 3 2 3 3 2 3 3  35  Table  V: The  testicular  stage  o f s p r i n g salmon j u v e n i l e s  w i t h v a r i o u s d o s a g e s o f s a l m o n g o n a d o t r o p i n , u. Standard Deviation.  injected  = mean; S.D.  =  TABLE V: SAMPLE &  DATE Zero Control 10/6/69 1 24/6/69 2 7/7/69 3 21/7/69  4/8/69 5 I8/8/69 6 1/9/69  TREATMENT  A B C Co A B C Co A B C Co A B C Co A B C Co A B C Co U.I. Co  n  2 3 3 2 3 2 3 3 1 3 1 3 3 3 3 3 3 3 3 3 3 3 2 3 3 3  TESTICULAR STAGE S.D. 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.5 1.6 1.3 1.0 2.5 2.2 2.0 1.0 4.0 3.1 2.5 1.0 4.6 4.0 2.3 1.0 1.0  0 0 0 0 0 0 0 0 0 0.1 0 0.2 0 0 0.3 0 0 0 0.9 0.7 0 0.2 0 0.6 0 0  36  Table  VI; The  testicular  i n a q u a r i a throughout Deviation.  stage of u n i n f e c t e d pink their  life  cycle.  y=  salmon  mean;  S.D.  maintained =  Standard  TABLE V I :  SAMPLE & DATE  1 1  TESTICULAR STAGE y  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 30/6/70  4  2.0  0.1  12 25/8/70  4  4.0  0  3 7  Table VII: Histochemically dehydrogenase a c t i v i t y various Standard  d e m o n s t r a b l e A5-3 8 h y d r o x y s t e r o i d  i n pink  salmon  juveniles  dosages o f salmon g o n a d o t r o p i n , Deviation.  y =  treated with  mean;  S.D.  =  TABLE V I I : SAMPLE  &  DATE Zero Control 17/6/69 1 30/6/69 2 14/7/69 3 28/7/69 4  11/8/69 25/8/69 6 8/9/69 7 22/9/69  TREATMENT  n  ENZYME  ACTIVITY  (Visual  u  Units) S.D.  Co  4  0  0  A B C Co A B C Co A B C Co A B Co A B Co A B Co A B Co  4 4 4 4  0.13 0.48 0.38 0 0.38 0.50 0 0 1.67 1.25 1.67 0 2.13 1.63 0 1.67 1.50 0 1.25 1.75 0 1.13 1.0  4,  0.13  6.2^ 0.75 0.75 0 0.75 0.71 0 0 0.29 0.50 0.25 0 0.63 0.25 0 0.29 0 0 0.35 0.35 0 0.25 0 0.25  4  0.13  0.25  U.I.  3 2 2 3 3 3 1 4 4  2 3 4 4  2 2 4 4  2 Co  38  Table  VIII; Histochemically  dehydrogenase a c t i v i t y treated with various S.D.  =  Standard  demonstrable A 5-3$hydroxysteroid i n the t e s t e s  of spring  salmon  dosages o f salmon g o n a d o t r o p i n , v  Deviation.  juveniles = mean;  TABLE  VIII:  SAMPLE  TREATMENT & DATE Zero.Control IO/6/69 A 1 B C 24/6/69 Co A 2 B C 7/7/69 Co A 3 B C 21/7/69 Co A 4 B C 4/8/69 Co A 5 B C 18/8/69 Co A 6 B C 1/9/69 Co U.I.  Co  n  4 3 4 4 4 4 2 4 3 4 2  ENZYME ACTIVITY ..." ( V i s u a l U n i t s ) ; / S.D. u 0 0 0 0 0 0 0 0 0 0 0.88 0.25 0 0 1.0 0.63  0 0 0 0 0 0 0 0  3  0 1.25 1.17  0 0.30  3  0  3 4 3  3 2 3 3  0.63 0.35 0 0  0.38  0.41 0.48 0.48  0.88  0.58 0.48  1.13 0.83  0.4b 0.58  0  0  0.50 0  0  0 0  CHAPTER  2  The Ovary Introduction The l i t e r a t u r e reviewed by Hoar (1966) indicates that the development of the ovary i n young teleosts i s dependent upon the p i t u i t a r y . to develop.  A f t e r hypophysectomy, the f o l l i c l e s  fail  Therefore, i t appeared that i n j e c t i o n of prepara-  tions of p i t u i t a r y gonadotropin would be a promising method for  stimulating the ovaries of immature salmon. Few studies had been done on the effect of homoplastic  p i t u i t a r y extracts on the rate of maturity of the ovary i n teleosts.  Yamazaki and Donaldson  ( I 9 6 8 ) succeeded i n reinducing  v i 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 (Carassius auratus).  female  The f u l l y developed ovaries had  been allowed to regress f o r two months p r i o r to treatment with a p a r t i a l l y p u r i f i e d 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 i n j e c t i n g extracts of pliace p i t u i t a r y glands.  There i s , therefore, evidence to  indicate that adult female teleosts may be stimulated to sexual maturity i n a short period of time by i n j e c t i n g homoplastic extracts of the p i t u i t a r y . ost  That the ovary of the immature t e l e -  i s r e l a t i v e l y more refractory to stimulation can be seen  from a study by Schmidt et a l ( 1 9 6 5 ) .  Two weeks of i n j e c t i o n  of extracts of p i t u i t a r y glands from adult pink salmon  40  (Oncorhynchus tshawytscha) weight  i n length.  of  injection  T h e r e f o r e , i t appeared o f salmon g o n a d o t r o p i n  w o u l d be n e c e s s a r y ovaries  of these There  on any  (1963)  does r e v e a l  mones.  They f o u n d  w i t h PSH,  e f f e c t was  i n f o r m a t i o n on  t i v e a s LH,  An  experiment  that  injection  s i g n i f i c a n t advantage over The^possibility  were i n j e c t e d  be a c h i e v e d .  might  was  combination  A  thought  n o r HGG  slight t o be  was  some s t i m u l a t i o n  as of  survey logical  any  of homoplastic preparations. t h e r e was  i n the  immature  synergizes with  o f such f a c t o r s  synergize with p i t u i t a r y  effec-  do n o t a p p e a r t o o f f e r  simultaneously with  to study  the  oocyte  a  hormone  fish,  pituitary  salmon  If  such  gonadotro-  r a t e o f d e v e l o p m e n t o f t h e immature o v a r y A  in  physiologically  recognized that  teleost  hor-  growth o f o o c y t e s  t h i s was  N e i t h e r PMS  in  Hoar  of these  d u r i n g normal development of the ovary.  a faster  It  of v i t e l l o g e n e s i s  done by A h s a n a n d  i n substantial quantities  substance  pin,  but  the use  was  but which i n the maturing  a  salmon  mammalian  o f LH a l o n e , o r i n  mammalian g o n a d o t r o p i n s  gonadotropin  program  i n the  of  with respect to gonadotropins.  although both produced  present  effect  t o some e x t e n t t h e p o t e n t i a l  contamination.  The  long-term  immature p i n k  the  aspect of oogenesis  o b t a i n e d w i t h FSH,  o f LH  growth.  into  (Gasterosteus aculeatus)  hypophysectomized  result  that a  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  stickleback  13.5  (Salmo g a l r d n e r i l )  fish.  (Barr, 1968).  teleosts  s m a l l i n c r e a s e i n the  for;,the development o f mature ova  is little  gonadotropins  not  In a  o f t h e o v a r i e s o f Immature t r o u t  cm  the  resulted  could  follows.  estrogens as gonadotropin  factors  which  during ovarian  41 Simpson e t a l ( 1 9 6 3 )  maturation.  found  that i n Spyllorhlnus  canicuius  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  matured.  That  c a n be that  seen  estrogens  f r o m work done by  estradiol  17 6 was  of  Cyprlnus  of  estrogen throughout  carpio  (Salmo s o l a r L.) and  (1964)  Hisaw  estrogen i n the present It  was  (1961).  Galzinga  Salmo i r i d e u s . the l i f e  i s estrone  cycle  The  He  i n the o v a r i e s  predominant  of the A t l a n t i c  established that e s t r a d i o l  17B  decided, a f t e r  r e p o r t e d as h a v i n g an  inhibiting and  by  laevls. leading be  (1942)  Bullough The  on  (1968)  Goswami  e x p l a i n e d by  taking into  hypertrophy  of the ovary  tions tary  ovariectomy  (Sundararaj  oocytes  was  and  of c a t f i s h  was  inhibited  by  Goswami, 1 9 6 8 ) .  recorded  Phoxlnus  inhibited,  The  can  of  compensatory  f o s s l l l s , following  treatment  with  Simultaneous  estradiol observa-  s m a l l e r b a s o p h i l s i n the  treated with  the  catfish  These e f f e c t s  production.  of Heteropneustes  t h a t t h e r e were f e w e r a n d glands  i n the  c o n s i d e r a t i o n , the e f f e c t  gonadotropin  on  been  discovered that  o b s e r v a t i o n was  eventual disintegration.  pituitary  benzoate  similar  weight. that  usually  oestrone-treated o v a r i e s of  e s t r o g e n on  unilateral  A  development of primary  to t h e i r  i t was  dosages.  or r e g r e s s i v e e f f e c t  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 fossllls).  only  literature,  employed i n p h y s i o l o g i c a l  Sundararaj  (Heteropneustes  the  m i c r o g r a m s p e r k i l o g r a m body  consideration of t h i s  1? g w o u l d be  ovary.  Salmon  o v a r i e s of mature Oncorhynchus nerka; o f 36  species  Botticelli  was  A d m i n i s t r a t i o n o f e x o g e n o u s e s t r o g e n has  fish  teleosts  demonstrated  et a l , 1961).  (Cedard  oocytes  i n the o v a r i e s of  the predominant estrogen  and  in quantities  estradiol  are present  the  e s t r a d i o l benzoate  pitui-  suggested  42  that gen  t h e r e was and  a n e g a t i v e feedback  pituitary  gonadotropin  brates.  The  Gambusia  s p . were a t t e n u a t e d by  e s t r o g e n and  inhibitory  frog anterior  Clavert that  (1958)  similar  substantiated  the  hormones  largely  Vanstone  (1961).  The  benzoate.  In the maturing  e s t r o g e n i s the l i v e r ,  phospholipid  salmon  (Ishil,  female  fish,  Holmes and  direct zed  effect  the t i s s u e which  complex.  a  calcium  1953).  1940;  multi-layered  Evidence  W i l l i a m s , 1944;  that  (Urist,  responds  1964; a  hypophysectomiauthors  Simpson e t a l , 1 9 4 1 ;  to s t i m u l a t e the m i t o s i s  de  Wit,  of  cells  e p i t h e l i u m , l e a d i n g to the formation of a  granulosa.  Small f o l l i c l e s  f o l l i c l e s w i t h no a n t r u m .  The  developed  into  mechanism by w h i c h  m o b i l i z a t i o n o f y o l k p r e c u r s o r s , and  proliferation  egg  e s t r o g e n has  s t i m u l a t e s the growth of the oocyte would thus appear fold:  Ho  phosphoprotein-  b e e n p r e s e n t e d by a number o f  E s t r o g e n s were f o u n d  the f o l l i c u l a r  sized  (1968).  by  T h i s substance i s  on d e v e l o p m e n t o f t h e f o l l i c l e s o f  immature r a t s h a s  (Pencharz,  in  Donaldson  nerka)  was  o f e s t r a d i o l mono-  t h e n t r a n s p o r t e d t o t h e ova v i a t h e b l o o d s t r e a m in  facili-  This hypothesis  which produces  of  grounds  thus  (Oncorhynchus  of  1961).  serum c h a n g e s a s s o c i a t e d w i t h  glyco-lipo-protein  verte-  injection  on t h e o r e t i c a l  f o r m a t i o n were e n h a n c e d by a d m i n i s t r a t i o n  to  i n higher  simultaneous  i n the f o l l i c l e s .  i n the sockeye  estro-  o f e s t r o g e n s on t h e o o c y t e s  pituitary  proposed  to that  e s t r o g e n would m o b i l i z e y o l k p r e c u r s o r s , and  tate yolk deposition  and  effect  r e l a t i o n s h i p between  of the f o l l i c u l a r  y o l k p r e c u r s o r s must  pass.  mediumestrogen  t o be  stimulation  e p i t h e l i u m through which  of  twothe  the  ^3 Uhere a r e cated  i n the  several other  development o f the  gens, progesterone, The  will  now  be  Testosterone ovarian  present found  mg  gland  i n the  produced  teleost.  plus a  i n s t i m u l a t i n g the  are  d i d not  (Bloch).  T h e r e h a v e b e e n no  stages  (1962)  stimulate  subminimal dose o f  other  in  There i s  Ramaswami  Heteropneustes f o s s l l i s  gave a p o s i t i v e r e a c t i o n .  pituitary  studies  to  c o m b i n a t i o n cSf a g e n t s i s of o v a r i a n development  prior  ovulation.  adrenal 1966)  (Ramaswami, 1 9 6 2 ;  cortocosteroids  erate  the  stages  T h y r o t r o p i n was the  o v a r y of  ( A s h a n and  the  ever, t h a t t h e i r TSH with  of o v a r i a n  gonadotropins.  I t was  preparation  with  of  steroids accel-  development. stimulatory  m e n t i o n e d by contained  Data c o l l e c t e d  Goswami,  ovaries  effect  stickleback, Gasterosteps  i n d i c a t e that mild hypothyroidism compatible  that these  shown t o h a v e a  threespine  Hoar, 1 9 6 3 ) .  evidence  and  S u n d a r a r a j and  s t i m u l a t e o v u l a t i o n i n the  T h e r e i s no  previous  Y a m a z a k i , 1965)  (Ramaswami, 1 9 6 2 ;  have b e e n shown t o  some t e l e o s t s .  are  oocyte  (Eckstein, 1970).  ovary of the  catfish  Progesterone  to  steroids.  of  11-ketotestosterone are  and  s u g g e s t t h a t a n d r o g e n p l u s any  to  andro-  discussed.  methyl testosterone  effective  adrenal  stimulants  t h a t 5 nig m e t h y l t e s t o s t e r o n e a l o n e i n the  impli-  Among them a r e  t h e r e f o r e , to i n d i c a t e that androgens  normally  ovulation 1.25  substances as  t i s s u e of T l l a p l a aurea  some e v i d e n c e ,  ovaries.  t h y r o i d hormones, and  p o t e n t i a l of these  maturation  hormones w h i c h c o u l d be  from d i f f e r e n t and  normal r e p r o d u c t i o n ,  mild  aculaetus  the authors,  possible  on  how-  contamination species  seem  hyperthyroidism  whereas severe  cases  44 of  deficiency  in  some s p e c i e s (Van  hormones a s but  or excess  reduces  offer  reproductive  1968).  Tienhoven,  s t i m u l a t o r y agents  does n o t  the  The  potential  of the ovary  enough p r o m i s e t o be  efficiency of  thyroid  i s tantalizing,  employed i n an  initial  study. From t h e p r e c e d i n g i n f o r m a t i o n , i t a p p e a r e d treatments  consisting  tropin alone, likely pink  or i n combination  salmon.  In  of  I t was  anticipated and  the present  study,  tropins  pink  salmon.  ovary  that f i s h  the  two  of the  most  immature  injected with  both  fastest.  s p r i n g s a l m o n f r y were t r e a t e d comparison of the  activity  p r e p a r a t i o n between t h e donor f i s h  o b t a i n e d on  p r e p a r a t i o n on a  sexually after  w o u l d be  years  juvenile  et a l 1968).  (Pineda  the r e l a t i v e  effect  of  the  salmon which matures  (Oncorhynchus gorbuscha), and  reproductive maturity a f t e r 4 years  one  which  (Oncorhynchus  tshawytscha). Inducing  sexual maturity  i n salmonids  h a t c h i n g p o s e s some s p e c i a l d i f f i c u l t i e s of  the f i s h .  and  They a r e v e r y  could never  small at this  i n the year  related  to the  stage  in their  c o n t a i n a n o r m a l - s i z e d mature overy.  approach to the problem i s to s t i m u l a t e the animals er  size.  used  Predefined environmental  t o grow y e a r l i n g  and  P r o d u c t i o n o f a n t i b o d i e s a g a i n s t gonado-  c a n a l s o be  gonadotropin  reaches  of the  of f o r e i g n o r i g i n have been r e p o r t e d  Information  173  estradiol  to provide a  salmon g o n a d o t r o p i n  the t e s t  d o s a g e s o f salmon gonado-  e s t r o g e n would mature the  salmon g o n a d o t r o p i n  the  with  to a c c e l e r a t e the m a t u r i t y  salmon g o n a d o t r o p i n  with  of p h y s i o l o g i c a l  that  sockeye  and  dietary  of  size life,  One to a  larg-  c o n d i t i o n s were  salmon (Oncorhynchus n e r k a )  from  ^5 22  t o 110  grams o v e r a  and  Sutherland,  the  end  of  the  1970). test  two The  end  A pink  472  that  of  F o e r s t e r and 57  A  stock  been t r e a t e d i n a d o n a t e d by  Dr.  controls at of  eggs f o r e a c h i n c r e a s e of  i n d u c t i o n of precocious will  be  o f one  1 kilogram  Brett at  sexual  maturity  c h a r a c t e r i z e d by  the  cm  their  be  to provide  a  cycle i n aquaria  i n the  salmon  studied  comparison between o v a r i e s which have those  for  average  i n length,  eggs t h a n o c c u r s  throughout  n a t u r a l l y and  an  and  McClinton  small,  development t o  pink  life  reported  exist  i n weight f o r  i s proposed that u n i n j e c t e d  to maturity  the  It i s anticipated, therefore,  o f a much l o w e r complement o f It  pink  s i m i l a r manner f o r J . R.  (1941)  Pritchard  (Queen C h a r l o t t e I s l a n d s ) .  mature f i s h size  grams.  (Brett  1970.  eggs f o r e a c h i n c r e a s e  Creek  51  the  period  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 a p p e a r t o  salmon.  increase  h a l f month t e s t  average weight of  months were g e n e r o u s l y  of August,  one  p e r i o d was  s a l m o n j u v e n i l e s w h i c h had three  and  stimulated  with  im-  full  normally. maintained concurrently developed  hormones.  46 Methods a n d M a t e r i a l s The as  females  were m a i n t a i n e d  described earlier Experiments  f o r the  under the  same c o n d i t i o n s  males.  on t h e o v a r y w h i c h were f r o m  a s have b e e n d e s c r i b e d p r e v i o u s l y i n c h a p t e r 1, (1)  Summer 1 9 6 9 ; spring  salmon g o n a d o t r o p i n  the ovary (2)  Study of the e f f e c t  i n pink  same  1970;  stock  were:  of v a r i o u s dosages  (SG-G100) on d e v e l o p m e n t  salmon f r y a n d  Summer I 9 6 9 t o F a l l ,  the  of of  s p r i n g salmon f r y .  Study of the normal c y c l e  gonad d e v e l o p m e n t i n u n i n j e c t e d p i n k  of  salmon m a i n t a i n e d  in  aquaria. (3)  September 1970 spring  salmon g o n a d o t r o p i n  volving one: ger  to February,  1971;  Study of the  (SG-G100), and 176  the a d d i t i o n of e s t r a d i o l  - Pink  s a l m o n f r y w h i c h had  than normal s i z e a t an  temperatures  and  a  effect  of  combinations  , and  then  progester-  been s t i m u l a t e d t o a  earlier  in-  stage with higher  larwater  constantly lengthening photoperiod  were  used. The  normal-sized  ments i n i t i a t e d different obtained  On  April  from Jones Creek  during t h e i r •Laboratory  24,  ferred  27.  salmon f r y used  1970,  downstream m i g r a t i o n .  600  At  pink  to the  to 1 7 0 - l i t r e  start  fibreglass  of the F r a s e r R i v e r ) the F i s h e r i e s  gradually converted of i n j e c t i o n s , aquaria.  a  s a l m o n f r y were  i n West V a n c o u v e r t h e y were p l a c e d i n a  Prior  In e x p e r i -  were o b t a i n e d f r o m  (a t r i b u t a r y  f r e s h w a t e r a q u a r i u m w h i c h was April  pink  summer o f 1971  i n the  source.  female  Research  500-litre t o seawater  on  t h e f i s h were t r a n s -  47  Treatments: The female pink and spring salmon f r y , i n the summer of 1969, were t r e a t e d w i t h a dosage regime I d e n t i c a l to that received concurrently by the males of both species (Chapter 1 ) . The schedule of sampling was a l s o the same. In a second experiment, s t a r t e d i n the summer of 1970. s i x t y pink salmon f r y were placed i n each of seven o v a l 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 a n e s t h e t i z e d , and  i n j e c t e d 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 f o l l o w i n g substances d i s s o l v e d i n f i s h s a l i n e (Wiebe, I 9 6 8 ) : (1)  G:  s p r i n g salmon gonadotropin, 1.0y g per gram body  weight. (2)  GE : spring salmon gonadotropin, 1.0 y g per body weight a  15 y g per gram body weight e s t r a d i o l 178 .  and (3)  GE : b  s p r i n g salmon gonadotropin, 1.0 y g per gram body  weight and 1.5 y g per gram body weight e s t r a d i o l 17 8 . (4)  GE : C  s p r i n g salmon gonadotropin, 1.0 y g per gram body '  weight and a f t e r three months of treatment, a d d i t i o n of ! E" 1.5 yg per gram body weight e s t r a d i o l 178 . (5)  E :  (6)  Et,: E s t r a d i o l 178 - 1.5 vg per gram body weight.  (7)  Co:  a  E s t r a d i o l 17  e  - 15 yg per gram body weight.  10$ ethanol d i s s o l v e d i n f i s h s a l i n e . 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 i n v o l v i n g the a d d i t i o n of estrogen,  1 , 3 , 5 . (10) - e s 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 L a b o r a t o r i e s ) was f i r s t d i s s o l v e d i n 95% ethanol, and then d i l u t e d t o a f i n a l c o n c e n t r a t i o n of 10% ethanol, w i t h a d d i t i o n of f i s h s a l i n e .  The r e s u l t i n g f i n e suspension was  e a s i l y i n j e c t e d through a 27 g needle. The f i s h were sampled a t six-week i n t e r v a l s , .  At each  sampling, specimens were removed u n t i l f o u r females had been found, or t e n f i s h had been taken.  The zero c o n t r o l sample  was taken on June 27, 1970. I n j e c t i o n s were continued u n t i l March 10, 1971, when only G, GE , and Co remained.  A l l of the  C  specimens w i t h other treatments had been removed p r i o r to that date. A stock of pink salmon j u v e n i l e s which were much l a r g e r than the normal s i z e (described i n Chapter 1) were studied w i t h ]  t  the purpose of i n v e s t i g a t i n g hormonal i n d u c t i o n of precocious sexual m a t u r i t y i n a l a r g e r animal.  These f i s h were a n e s t h e t i -  zed, and i n j e c t e d 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 ) , w i t h 0.10 ml of the f o l l o w ing treatments d i s s o l v e d i n f i s h s a l i n e (Wiebe, 1968): (1)  LG:  s p r i n g 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  (3)  S.  .3  LGEP: s p r i n g salmon gonadotropin (SG-G100) - 1.0 g per ' y  gram body weight, e s t r a d i o l l ? g - 1.5 g per gram body y  weight, and progesterone - 5.0 $g per gram body weight. (4)  LCo: 10$ ethanol d i s s o l v e d i n f i s h s a l i n e .  49 Estradiol  17 3 a n d p r o g e s t e r o n e were f i r s t  95$ e t h a n o l , a n d t h e n d i l u t e d e t h a n o l by a d d i n g f i s h  saline.  resulted  from  needle.  I n j e c t i o n s were s t a r t e d  the  this  to f i n a l  mean body w e i g h t t o 15.8  (compared  c o n c e n t r a t i o n o f 10%  The f i n e  p r o c e d u r e was  dissolved i n  easily  suspension  which  i n j e c t e d w i t h a 25 g  on S e p t e m b e r 2 ,  1970,  o f t h e p i n k s a l m o n was 42.0  grams  grams f o r t h e n o r m a l - s i z e d f r y ) .  when  Twenty  f i s h were a s s i g n e d t o e a c h h o r m o n a l t r e a t m e n t , a n d samples t a k e n a t two s u c c e s s i v e , few  specimens  three-month  to allow a zero c o n t r o l  m a l s were k e p t t h r o u g h o u t until  the terminal  LGEP g r o u p water due  line,  mainly  specimens  i n each  Experimental (i),  logically (iv)  19&9,  because  at this  ani-  Eight  of the  of a block i n the  time.  of diseased f i s h ,  Ten c o n t r o l  Random m o r t a l i t y ,  reduced  t h e number o f  sample.  Techniques;  ( i i ) , and The  3,  T h e r e were t o o  a n d were n o t t a k e n  F e b r u a r y 18, 1971.  a n d were sampled to removal  sample.  the experiment,  sample,  d i e d November  intervals.  were  (iii):  f i s h were i n j e c t e d , a u t o p s i e d , a n d p r o c e s s e d as described  Analysis  i n Chapter  histo-  1.  o f tfoe H i s t o l o g i c a l R e s u l t s  (a) M e a s u r e m e n t s : D e s s i c a t i o n of the small c o n t r o l ovaries during weighing n e c e s s i t a t e d a comparison r a t h e r t h a n wet w e i g h t s ,  o f gonad w e i g h t  based  i n the experiments  on f i x e d ,  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 t h e summer o f 1969.  the experiments  on t h e n o r m a l - s i z e d p i n k  the e x t r a - l a r g e  sized  normal in  fish  in  1970/71,  captivity,  f r e s h weights  1970/71,  and i n t h e study  o f t h e o v a r i e s were t a k e n .  each  milligram.  gonad w e i g h t  The t o t a l  doubling the r e s u l t  i n d e x was d e t e r m i n e d  of the  f i s h was w e i g h e d  kept In both  to the nearest  f o r e a c h a n i m a l was o b t a i n e d  f o r t h e w e i g h e d gonad.  The  gonadosomatic  as follows: total  G.S.I. The  salmon f r y i n  c y c l e o f r e p r o d u c t i v e m a t u r i t y i n t h e p i n k salmon  c a s e s , one gonad f r o m  by  In  =  gonad w e i g h t BODY WEIGHT  mean o o c y t e d i a m e t e r was o b t a i n e d  from  x  100  an average  of the  o f 30 o o c y t e s p e r o v a r y , m e a s u r e d w i t h a n o c u l a r  diameter  micrometer.  Only  oocytes  n u c l e u s were m e a s u r e d . was t h e s q u a r e multiplied  containing the greater part  The v a l u e r e c o r d e d f o r o v o i d  of the oocytes  root o f the product of the g r e a t e s t diameter  by t h e l e a s t  diameter  (as i n B r a e k e v e l t and McMillan,  1967). (b) H i s t o l o g i c a l The all  Analysis:  c o m p o s i t i o n o f each  o f t h e n u c l e a t e d o o c y t e s i n a median s a g i t t a l  e x p r e s s i n g t h e v a l u e f o r each This  o v a r y was d e t e r m i n e d  type as a percentage  i n f o r m a t i o n was t h e n a v e r a g e d  graphically. similar  f o r each  t o t h o s e d e s c r i b e d by Y a m a z a k i  the ovary follows:  The c h a r a c t e r i s t i c s  f o r each  of the g o l d f i s h  by c o u n t i n g  s e c t i o n , and of the t o t a l .  treatment and p l o t t e d t y p e o f o o c y t e were  (I965)  i n h i s study o f  (Carassius auratus L ) .  T h e y were a s  51  1) E a r l y peri'nucleolus stage S e v e r a l n u c l e o l i a r e apparent near the n u c l e a r 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, g r a n u l a r p o r t i o n s a r e r e f e r r e d to as a p a l l i a l l a y e r by Kudo i n s t u d i e s on P l e c o g l o s s u s a l t l v e l u s (1969 a) and Pseudorasbosa  pimula  nucleus i n c r e a s e g r e a t l y i n s i z e . to 250 m i c r a i n diameter.  (1969 b ) .  The cytoplasm and  The oocyte v a r i e s from 40  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 r e g i o n t o form a f o l l i c u l a r l a y e r one c e l l t h i c k . zona r a d i a t a i s not y e t apparent  The  ( F i g . 21, 22, 23, 24).  2) Late p e r l n u o l e o l u s stage N u c l e o l i a r e s t i l l present a t the p e r i p h e r y 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 l a y e r has condensed p e r i p h e r y o f the cytoplasm. bat not as markedly  t o form a y o l k - n u c l e u s a t the  The oocyte has i n c r e a s e d i n s i z e *  as t h a t i n the p r e v i o u s stage.  of the oocytes v a r i e d from 180 t o 320 m i c r a .  The diameter  There i s a d i v i -  s i o n of the c e l l s from the i n t e r s t i t i a l r e g i o n t o form a second layer.  The zona r a d i a t a i s vaguely apparent  3) Yolk v e s i c l e  ( F i g . 25 and 26).  stage  Cortical alveoli  ( o r v e s i c l e s ) appear i n the p e r i p h e r y  of the ooplasm as a s i n g l e l a y e r . v a r i e s from 260 t o 900 micra.  The s i z e o f the oocytes  As the oocytes i n c r e a s e i n  diameter, the y o l k v e s i c l e s accumulate  toward the n u c l e u s , and  contain i n t r a v e s i c u l a r e o s i n o p h i l i c granules.  The nucleus  52  becomes i r r e g u l a r nucleoli. layered ovoid of  i n outline,  The f o l l i c u l a r  i t still  e p i t h e l i u m i s composed o f a  zona g r a n u l o s a o f m i t o t i c a l l y  cells,  cells.  contains peripheral  and an o v e r l y i n g  The z o n a r a d i a t a  dividing  single-  squamous a n d  t h e c a o f one t o s e v e r a l l a y e r s  i s t h i n a t the beginning 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 t h e  ( P i g . 2? a n d 2 8 ) .  end  4) P r i m a r y At sent near  Yolk  Stage  the beginning  of this  t h e 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 p r e s e n t a t  the p e r i p h e r y o f t h e ooplasm. form  The y o l k g r a n u l e s  l a r g e g l o b u l e s , a n d move t h r o u g h o u t  oocyte.  The z o n a g r a n u l o s a  the o v e r l y i n g layer The  stage, o i l globules a r e pre-  layer  the cytoplasm  of the  i s composed o f c u b o i d a l c e l l s ,  consists  o f squamous c e l l s .  The  while  thecal  t h i c k e n s ; t h e c e l l s w h i c h compose i t a r e a l l squamous.  zona r a d i a t a  stains  intensely with  s t r i a t i o n s a r e only vaguely stage  coalesce to  ranged  i n diameter  apparent.  from  550  e o s i n , but r a d i a l The o o c y t e s  t o 1500  miera  i n this  ( F i g . 31 a n d  32). 5)  Post-primary This  Yolk  stage  Stage  consists  of a l l those  1.0  mm  not  possible to cut p a r a f f i n  as  a n d more a d v a n c e d t h a n  t h e y were t o o b r i t t l e .  the primary  oocytes  y o l k stage.  sections of t h i s  type  C r y o s t a t s e c t i o n s were  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 with normally  with p a r a f f i n  larger  I t was  of oocyte, obtained;  that  s e c t i o n s . ( F i g . 36 a n d 37).  than  found The  largest oocytes i n t h i s stage measured 4 mm i n diameter. (v)  Histochemical Analysis:  (a)  Procedure The procedure used to demonstrate A.5-3 fLhydroxy steroid  dehydrogenase a c t i v i t y was i d e n t i c a l to that described i n Chapter 1.  Only the samples from the study of the e f f e c t of various  dosages of spring salmon gonadotropin (SG-G100) on the reproductive maturity of pink and spring salmon f r y i n the summer of 1969 were investigated. Frozen ovaries from the experiment i n 1970/71 to study the e f f e c t of various combinations of spring salmon gonadotropin (SG-G100) and e s t r a d i o l 17 6. on maturity of the gonad i n immature pink salmon were sectioned at 10 microns on the Cryostat, and stained f o r l i p i d s with O i l Red 0 and Sudan Black B (Chayen e_fc a l , 1969).  I t was found that the section disintegrated when stained  by the " f l o a t i n g through" technique.  This problem was overcome  with the method quoted i n C u l l i n g (1957) f o r attaching frozen sections to gelatinized s l i d e s .  Frozen sections of ovaries from  uninjected pink salmon maintained throughout their l i f e i n aquaria were stained as described above. (b)  Analysis of Results: Analysis of the formazan deposits obtained i n the  measurement of A5-3 £ hydroxysteroid dehydrogenase a c t i v i t y i n the ovary corresponded exactly to that described i n Chapter 1 .  5^ O b s e r v a t i o n s were made on t h e l i p i d results  are described  i n the following  stains.  The  section.  RESULTS (a)  Measurements;  (i)  Gonadosomatic The  Index  relative effects  o f i n j e c t i o n o f salmon  tropin  on t h e G.S.I, d i f f e r e d m a r k e d l y  salmon  female f r y .  experiment  f o r t h e pink and  A a n d B on t h e p i n k  b o t h had h i g h e r gonadosomatic  control. salmon  Treatment  By c o m p a r i s o n ,  gonado-  indices  spring  salmon than, d i d t h e  none o f t h e t r e a t m e n t s o f t h e s p r i n g  showed a n y r e a l d i f f e r e n c e  a t t h e end o f t h e e x p e r i m e n t  (Pig. 40). The the  results  summer o f 1969 were d u p l i c a t e d  (over a s i m i l a r in  o b t a i n e d f o r t r e a t m e n t B, v t h e c o n t r o l i n  1970  time i n t e r v a l )  ( F i g . 41).  the  experiments  i n I969.  t r e a t m e n t G, GE^, a n d G E mately  i n the experiment  In the l a t t e r  gonads were m e a s u r e d a s o p p o s e d  i n their qualitative aspects  study, f r e s h  on p i n k  weights  salmon  of the  t o t h e f i x e d weights used i n  By t h e t h i r d s a m p l e , f i s h f r o m C  had gonadosomatic  double those of the c o n t r o l s .  indices  approxi-  A t t h e «nd o f the  experi-  ment, however, none o f t h e s e g r o u p s 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  salmon had  of e s t r a d i o l  17 ( a l o n e , o r i n c o m b i n a t i o n w i t h  g o n a d o t r o p i n ) a n d t h e low d o s a g e  of e s t r a d i o l  17 6 a l o n e  t h e e f f e c t o f d e p r e s s i n g t h e G.S.I. The  juveniles  terminal  sample o f t h e l a r g e r - s i z e d  pink  salmon  was compared t o t h e c o r r e s p o n d i n g t r e a t m e n t s o v e r a  55  six-month size  time  i n t e r v a l from  ( F i g . 42).  The f i s h  pink  salmon j u v e n i l e s  injected with  salmon g o n a d o t r o p i n and  t h e low d o s a g e o f e s t r o g e n d i d n o t d i f f e r control  i n either  injected with four  times t h a t  experiments.  mal-sized A  (in  salmon  approximately  It i s difficult,  however,  b e t w e e n t h e two e x p e r i m e n t s b e g a n 69 d a y s l a t e r t h a n  fish  because the nor-  animals. constantly  rising  gonadosomatic index  the o v a r i a n development o f p i n k throughout  h a d a G.S.I,  of the l a r g e r f i s h .  i n j e c t i o n s of the l a r g e r  s i g n i f i c a n t l y from the  The n o r m a l - s i z e d p i n k  salmon g o n a d o t r o p i n  t o make d i r e c t c o m p a r i s o n s  o f a normal  their life  characterized  salmon m a i n t a i n e d  ( F i g . 43).  i n aquaria  From t h e b e g i n n i n g o f J u l y  t h e y e a r o f s e x u a l m a t u r i t y ) t o September,  t h e G.S.I,  rose  at a rate  w h i c h was n o t p a r a l l e l e d i n a n y o f t h e e x p e r i m e n t s  involving  treatment  approximately treatment (ii)  On A u g u s t  f i v e t i m e s h i g h e r t h a n t h e maximum  s i g n i f i c a n t increase  f o r 98 d a y s i n b o t h  time.  i n t h e mean o o c y t e d i a m e t e r  t r e a t m e n t s A a n d B ( F i g . 44).  There  between t h e e x p e r i m e n t a l and  salmon f r y t r e a t e d  By t h e end o f t h e e x p e r i m e n t ,  o f the pink  salmon.  was  salmon  groups i n the s p r i n g  salmon c o n t a i n e d o o c y t e s trol  obtained f o r  into pink  was no c o r r e s p o n d i n g d i f f e r e n c e control  i t was  Diameter  f o u n d when salmon g o n a d o t r o p i n was i n j e c t e d fry  25,  G.  Oocyte A  w i t h hormones.  larger  f o ra  similar  none o f t h e t r e a t e d  i n diameter  than the zero  spring con-  56 The  results  obtained f o r the normal-sized  i n j e c t e d w i t h v a r i o u s hormonal treatments time  i n t e r v a l a r e complex  ( P i g . 45).  over an  pink  salmon  extended  The h i g h d o s a g e o f e s t r o -  gen  ( 1 5 y g p e r gram body w e i g h t ) when a d m i n i s t e r e d w i t h  out  salmon g o n a d o t r o p i n  (1.5y  g p e r gram body w e i g h t ) ,  diameter. alone  or i n combination  oocytes;  a t t h e time  similarly  with a low-level  difficult  actually  salmon  17 6  of the control  the stock of the l a r g e r pink to interpret.  The a v e r a g e  salmon a r e  diameter  sampling  a n d t h e t e r m i n a l sample a f t e r  end o f t h e e x p e r i m e n t ,  a l l o f t h e hormonal  c o n t a i n e d o o c y t e s w h i c h were s m a l l e r i n s i z e (Fig.  gonadotropin  o r below t h e v a l u e f©r t h e c o n t r o l s ,  d e c l i n e d between t h e f i r s t  days o f i n j e c t i o n s ,  oocyte  of e s t r a d i o l  diameter  alone  o f t h e t e r m i n a l s a m p l e , however, a l l o f  were n e a r  R e s u l t s from  t h e mean  involving  r o s e above t h e average  the treatments  oocytes  depressed  The o t h e r t r e a t m e n t s ,  periodically  the  a n d t h e low d o s a g e o f e s t r o g e n  or with-  of the  after  84 d a y s .  42 At  treatments  than  the controls  46). During  t h e normal c y c l e o f o v a r i a n development  salmon m a i n t a i n e d  i n a q u a r i a , change i n o o c y t e  t i m e was d i r e c t l y  linear until July,  dramatically with  the approach of sexual maturity  The mean o o c y t e  difficulty diameter  i n resolving  i n pink  diameter  with  a f t e r which i t rose  the r e s u l t s  and gonadosomatic i n d e x  ( F i g . 47).  obtained f o r  i n fish  treated  w i t h hormones, when c o u p l e d w i t h o b s e r v a t i o n s o f e n l a r g e m e n t of  some o o c y t e s , n e c e s s i t a t e d a n e x a m i n a t i o n  composition. the next  The r e s u l t s  section.  of this analysis  of the ovarian  c a n be s e e n i n  57  (iii)  Oogenesis The  ovary proved  t o be more r e f r a c t i o n a r y  s t i m u l a t i o n than the t e s t i s  (Chapter 1 ) .  o v a r i a n maturation i n the pink or s p r i n g 84 d a y s o f t r e a t m e n t  and pin,  l e d to a l a r g e r  s e r i e s of experiments  extended  were d e s i g n e d t o t e s t  injected at a later pink  induce  salmon  gonadotro-  The  second  the e f f e c t  17 e o v e r  estradiol  salmon m a i n t a i n e d  similar  dosage regimes,  i n aquaria during their  life  F i g u r e 48 c o n t a i n s t h e d a t a on t h e e f f e c t  immature p i n k  ation  of the r a t e  salmon d u r i n g t h e  oocytes to l a t e p e r i n u c l e o l a r  of v a r i o u s  t r e a t m e n t s A a n d B. of  the aforementioned  c e d by, one had  At  o f 98  t h e end  treatments  the l a t e  stage  seen i n  Yolk v e s i c l e  (Fig. 25).  whereas  of oocytes i n  F o u r t e e n days of  t r e a t m e n t 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  first  By  comparison,  hormone  to  advance the m a t u r i t y of the oocyte  injected mental  s i m i l a r dosages of the p i t u i t a r y  f o r 84 d a y s .  fish  i n the s p r i n g  O v a r i e s from both  constated e n t i r e l y  both  formation  (Fig. 27),  o v a r i e s were composed p r e d o m i n a n t e l y stage  acceler-  c o n t a i n e d o o c y t e s more a d v a n -  stage„ t h a n t h e c o n t r o l s .  perinucleolar  An  days of i n j e c t i o n s ,  been i n d u c e d i n the m a j o r i t y o f o o c y t e s  the c o n t r o l  oocytes  perinucleolar  s t a g e o o c y t e s was  with  cycle.  summer o f 1 9 6 9 .  of c o n v e r s i o n of e a r l y  an  fish  and  d o s a g e s o f salmon g o n a d o t r o p i n on d e v e l o p m e n t o f t h e in  of  C o m p a r i s o n s were made w i t h l a r g e r  time w i t h  98  17 6 a l o n e , a n d v a r i o u s  o f salmon g o n a d o t r o p i n and  time p e r i o d .  to  salmon f r y a f t e r  the next year.  salmon g o n a d o t r o p i n a l o n e , e s t r a d i o l combinations  Failure  respectively, with  experiment  t o hormonal  c o n t r o l and  of g e r m i n a l c e l l s and  appeared. failed  salmon f r y experii n the  58 early;, p e r i n u c l e o l a r  stage  23).  ( F i g . 22 a n d  From t h e r e s e a r c h m e n t i o n e d i n t h e p r e v i o u s it  was  weight  concluded was  as  that  1.0  effective  yg  salmon g o n a d o t r o p i n  in accelerating  t h e h i g h e r d o s a g e o f 10  u  g  salmon g o n a d o t r o p i n  therefore  corresponded  The a l o n e and  relative  i n the  to Treatment  ovarian maturation  second  with  by  comparison  35.  and  No  treatment  primary  y o l k stage oocytes  oocytes  c o n t a i n y o l k g l o b u l e s , but  by  the f o l l i c l e  of  cuboidal cells,  layers.  layer,  typical  w i t h an  body w e i g h t  with  the oocytes  initially,  the f i n a l  seen.  of t h i s  overlying  by  of a  s t a g e were f i r s t treatment.  yolk stage  treatment and  only d i d  one  fish  these  granulosa  t h e c a o f one  o r a t a d o s a g e o f 15  to  several  y g per  gram  stimulated maturation  the f o u r t h  126  surrounded  sample t h e  of  overall  controls.  induced  Oocytes  i n treatment  days l a t e r ,  observed  GEt, a f t e r  168  three treatments  o f t h r e e sampled w i t h 50  in  oocytes  (1.3  in  several  i n the post-primary  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 d e v e l o p m e n t . had  oocytes  however,  C  a  and  G after  GE ,  Not  rate  s m a l l number o f l a r g e , y o l k y o o c y t e s  ( F i g . 49).  Forty-two  the  F i g . 49,  from  stage:  s t a g e s o f o o c y t e m a t u r a t i o n was  the treatments  gonadotropin  t h e y were a l s o  salmon g o n a d o t r o p i n but  GE-Q  i n h i b i t o r y when compared w i t h  Formation  of  c o u l d be  E s t r a d i o l 17 g a l o n e ,  e f f e c t was  l a r g e r =experiment  primary  i n p i n k salmon o v a r i e s from In both  treatments  estrogen i n i n c r e a s i n g  i n c o r p o r a t i o n o f y o l k c a n be d e t e r m i n e d o f F i g . 3^  as  B.  of  days of treatment.  The  e f f e c t i v e n e s s b e t w e e n salmon  i n combination  were o b s e r v e d  p e r gram body  p e r gram body w e i g h t .  involving  section,  days  yolk of  contained Treatment mm  in  G  59  diameter): Treatment GE_ contained one specimen of three sampled with 30 oocytes ( 2 . 2 mm i n Treatment GE Fig. 3 6 .  D  i n diameter); and one of the three f i s h  had one f u l l - s i z e d  oocyte (4 mm i n d i a m e t e r ) —  The number of post-primary yolk stage oocytes i n  Treatment GE  C  had declined to six (3 mm i n diameter) i n one of  three specimens taken i n 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 r e s u l t s , a high degree of v a r i a b i l i t y  i n the compo-  s i t i o n of the ovary was found within the treatments.  For example,  another f i s h from the terminal sample of Treatment G had no oocytes beyond the early perinucleolar stage ( F i g . 3 8 ) . The pink salmon which had been stimulated to a largerthan-normal  size p r i o r to treatment with hormones, had the same  pattern of v a r i a b i l i t y within treatments as was mentioned above. The f i n a l sample on February 18, contained one f i s h from the LGE treatment (corresponds to treatment GE^,) which had 8 postprimary yolk stage oocytes (averaging i n diameter —  1.8  mm),  whereas the other two f i s h i n that treatment had none more developed than the late perinucleolar stage. of advanced  The percentage  stages i n the other treatments i n the terminal  sample was lower than that of the control.  No oocytes more  advanced than the yolk v e s i c l e stage were found i n the treatment i n which progesterone was added to gonadotropin and estrogen.  Direct comparisons cannot be made between the  ovarian development obtained i n t h i s experiment and that i n the previous study because hormonal treatment of the normalsized f i s h was i n i t i a t e d fish.  69 days e a r l i e r than i n the larger  60 In both of the aforementioned  experiments  and i n samples  from u n i n f e c t e d pink salmon ;v-<, the y o l k v e s i c l e stage was to be s u s c e p t i b l e to a t r e s i a . of  regression.  E a r l i e r stages never  found  showed s i g n s  Members of the primary y o l k stage were a l s o  found to subsequently become a t r e t i c . The apparent of  c o n t r a d i c t i o n (mentioned  the r e s u l t s ) between the low average  i n section  (a)  diameter and G.S.I, of  some treatments and v i s u a l o b s e r v a t i o n s t h a t a marked a c c e l e r a t i o n of oogenesis had o c c u r r e d i n some of the oocytes can be r e s o l v e d by a c o n s i d e r a t i o n o f : (1)  The percentage  (2)  the percentage of p r e - y o l k v e s i c l e stages.  of r e g r e s s e d oocytes present and  Extensive a t r e s i a characterized ovaries containing oocyte stages undergoing y o l k - v e s i c l e f o r m a t i o n and y o l k i n corporation.  Regression of oocytes was  induced f i r s t  i n j e c t e d w i t h e s t r a d i o l 1 7 3 : the h i g h e r dosages had g r e a t e s t numbers of a t r e t i c stages.  Simultaneous  In f i s h the  i n j e c t i o n of  gonadotropin d i d appear to moderate the e f f e c t somewhat.  By  the t h i r d sample, some of the y o l k v e s i c l e stages were r e g r e s s i n g i n Treatment  G.  The p a t t e r n of an i n c r e a s i n g  percentage  of a t r e t i c oocytes f o r eaeh treatment w i t h time was d i s r u p t e d f o r most treatments i n the l a t e r samples because many oocytes which had r e g r e s s e d p r i o r to the sample had been completely resorbed.  The i n f o r m a t i o n presented on percentage o v a r i a n  composition takes i n t o c o n s i d e r a t i o n o n l y those oocytes which c o u l d be counted.  Another  important  i n the a n a l y s i s of the r e s u l t s was  fiactor  to be c o n s i d e r e d  the h i g h percentage of p r e -  y o l k v e s i c l e stages i n f i s h i n j e c t e d w i t h estrogen when compared  61  to a decline  i n t h e number o f t h e s e  Despite the f a c t festations  of  that  oogonia  treated with  number s i x o f T r e a t m e n t earlier  from  were n o t  G.  perinucleolar  n e s t s of germinal  estradiol Oocytes  counted,  cells  The  reduced  i n that  treatment,  The  results  obtained i n the pink  Treatment fewer  Brett  qualitatively  LGPE d i f f e r e d  early  from  perinucleolar  A comparison  was  decided that  The  50)  and  t h e mean d i a m e t e r  estrogen.  salmon o b t a i n e d  to develop  t h e r e were  c o n t r o l s and  in fish  injected  uninjected pink  to maturity i n aquaria.  It  was  t h e v a r i o u s s t a g e s o f o o c y t e s were a l i k e  were s i g n i f i c a n t  of f i s h  ed w i t h hormones.  in their  from which they  differences  origin-  of the u n i n j e c t e d pink t h e i r normal l i f e  most d r a m a t i c was  compared t o one  treatments  pink  stage obcytes  injected  salmon.  salmon cycle (Fig.  been  developed:  hundred i n the normal f i s h  to several  The  varied, between  stimulat-  the d i f f e r e n c e  number o f p o s t - p r i m a r y y o l k s t a g e o o c y t e s  of the  t o be f o u n d  whose g o n a d d e v e l o p m e n t had  The  from  those d e s c r i b e d above.  made o f t h e o o c y t e s  i n a q u a r i a throughout  that  of  stages.  composition of the ovary  maintained  high  r a t e o f d e v e l o p m e n t o f t h e o o c y t e s , however,  There the  developing  subsequent  the o t h e r s i n t h a t  appearance r e g a r d l e s s of the treatment ated.  forms of  others treated with  paralleled  w i t h hormones, s a l i n e - i n j e c t e d salmon a l l o w e d  ina l l  i n sample  i n the e a r l i e s t  the oocytes  J . R.  e x t e r n a l mani-  17 g , and  ( F i g . 39).  and  controls.  were f o u n d  s t a g e were s e e n t o be  numbers o f s m a l l o o c y t e s g r e a t l y  Dr.  i n the  stimulated oogonial mitoses  samples o f f i s h  the  cells  i n the  several i n some  post-primary  yolk  i n t h e t e r m i n a l sample o f t h e u n i n j e c t e d f i s h  62  an average  one  month f r o m b e i n g r i p e .  pre-yolk fore,  diameter  of 2.7  had  s t a g e s by J u l y  the o v e r a l l  o v a r y was  in  the f i s h  a  complete  I t was  n o t t r e a t e d w i t h hormones.  (b)  Histochemistry:  (i)  A5-3g  by  of  the  to f i n d  t h e end  that  of August  Is t h i s a r e s u l t  w i t h i n an aquarium? p i n k salmon w i l l  Only a  of  the  comparison  p r o v i d e the  o f 3 3 - o l d e h y d r o g e n a s e was  activity  answer.  studied i n pink  s a l m o n f r y i n j e c t e d w i t h v a r i o u s dosages o f  gonadotropin  d u r i n g the  summer o f 1969  ( F i g . 51  distinct  l o c a l i z a t i o n s were f o u n d  i n any  activity  i n the f o l l i c l e  d i d not  the ooplasm.  A pink  Treatments  A and  c o n t r o l by  t h e end  layer(s)  s t a i n was  B w h i c h was  observed  treated with  and  salmon  52).  No  of the o v a r i e s . differ  i n pink  darker than that  of the experiment  ever obtained i n c o n t r o l  the f i s h  be  H y d r o x y s t e r o i d Dehydrogenase  The  was  absence  comprising  interesting  o f t h e o o c y t e s were a t r e t i c  with o v a r i e s of w i l d  spring  was  development o f the o o c y t e s  s t r e s s of confinement  and  There  t h e y were c o n s i d e r e d t o  of the y e a r of s e x u a l m a t u r i t y ; t h e r e -  more s y n c h r o n o u s .  more t h a n h a l f  mm;  salmon g o n a d o t r o p i n  salmon  found  (Fig. 30).  o v a r i e s of the  from  No  spring  had  only  The  that  in  from  i n the reaction salmon f r y , slight  activity. (ii)  Lipids The  of  h i s t o c h e m i c a l l y demonstrable  o v a r i e s from  s t u d i e d by  the pink  staining  lipids  i n the  oocytes  salmon i n the 1970/71 experiment  f r o z e n s e c t i o n s w i t h O i l Red  0 and  were  Sudan  63  B l a c k B.  The  o v a r i e s from throughout  r e a c t i o n s o b t a i n e d were compared w i t h t h o s e i n u n i n j e c t e d p i n k salmon m a i n t a i n e d  their l i f e  cycle.  A direct  s t a g e o f d e v e l o p m e n t o f o o c y t e s , and of  t h e l i p i d s was  has  received.  found,  Early  had  had  By  i n c r e a s e d i n s i z e , and  those  the  s i z e and  stage oocytes  fish no  stage  oocytes the  stage, the p e r i p h e r a l d r o p l e t s  some l i p i d  (Pig. 29).  y o l k stage  the  showed  Late perinucleolar  c o u l d be  Lipid  (Fig. 33).  seen  i n the  globules larger  i n p r e v i o u s s t a g e s were d i s t r i b u t e d  the primary  the  distribution  p r i m a r i l y a t the periphery of  the y o l k v e s i c l e  r e g i o n of the oocyte  in  stains.  small droplets located  ooplasm.  c o r r e l a t i o n between  r e g a r d l e s s of the treatment  perinucleolar  r e a c t i o n w i t h the l i p i d  i n aquaria  throughout  Post-primary  central  than  the  yolk  ooplasm stage  o o c y t e s were c h a r a c t e r i z e d by a v i r t u a l a b s e n c e o f l i p i d s  in  the  be  c e n t r a l r e g i o n s of the ooplasm.  found  a t the  periphery of these  i n t e n s e l y w i t h Sudan B l a c k (c)  cells.  globules could  Atretic  oocytes  stained  B.  Appearance None o f t h e  the  Large  secondary  sex  characteristics  s e x u a l l y m a t u r e a d u l t were s e e n  treatments.  in fish  typical  f r o m any  of  of  the  F e m a l e f i s h were u n d i s t i n g u i s h a b l e f r o m m a l e s .  The  c l o a c a o f a l l s p e c i m e n s t r e a t e d w i t h e s t r o g e n showed a marked swelling. The diol  body w e i g h t  1 7 gper gram body w e i g h t  administration The  o f s p e c i m e n s i n j e c t e d w i t h , 1 5 yg with or without  o f s a l m o n g o n a d o t r o p i n was  f i s h were s i c k ,  and  the m o r t a l i t y  estra-  concurrent  greatly  r a t e from  depressed.  these  treatments  64 was  high.  Pink  salmon i n j e c t e d  similar  symptoms, b u t  above.  The  appearance  to a lesser and  the i n j e c t e d  g of estrogen  extent than those  body w e i g h t  s a l m o n g o n a d o t r o p i n a l o n e , and dosage o f e s t r a d i o l  w i t h 1.5y  of treatments  described involving  i n combination with the  17g was  not  significantly different  controls after  210  days of  treatment.  had  low from  65 DISCUSSION Ovarian maturation was  stimulated with  t o n i n e months.  i n j u v e n i l e pink and s p r i n g  exogenous hormones f o r v a r y i n g p e r i o d s up  No p r e v i o u s r e s e a r c h o v e r a s i m i l a r  i n t e r v a l has been r e c o r d e d i n t e l e o s t s . establish  the effect  or without  salmon  o f long-term  estradiol  17e  time  I t was n e c e s s a r y t o  injection  of gonadotropin  because o f t h e p o t e n t i a l  with  o f t h e 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 o f empty c y c l e s o f p i n k s a l m o n , a n d b e c a u s e o f t h e p a u c i t y o f i n f o r m a t i o n on i n d u c t i o n o f p r e c o c i o u s sexual m a t u r i t y i n female  teleosts.  E s t r o g e n i n c r e a s e d t h e number o f s m a l l e a r l y p e r i n u c l e o lar  stage oocytes.  germinal in  fish  late  c e l l s which g i v e r i s e treated with estradiol  perinucleolar  vented  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 t h e  stage.  the v i t e l l o g e n l c  1942)  f  and  178 a l o n e d e v e l o p e d  Estrogens  fossllls  I n both  ed f r o m  the increased d i v i s i o n s .  c a s e s , more o o g o n i a  subsequently  laevls  t h e hypophysectomized  bers of precursor c e l l s  and primary  The p r i m a r y  degenerated.  a critical  oocytes  result-  oocytes i n  Estradiol  benzoate  stages of oocyte t h e num-  1953).  (1939, 19^1) a d v a n c e d a h y p o t h e s i s t h a t t h e r e  Vivien is  (Bullough,  r a t , p r e s u m a b l y by i n c r e a s i n g (de W i t ,  divi-  ( S u n d a r a r a j a n d Goswami,  i n c r e a s e d t h e number a n d s i z e o f p r e - f o l l i c u l a r in  therefore, pre-  stimulated mitotic  e p i t h e l i u m i n Phoxinus  Heteropneustes  laevls  beyond t h e  p h a s e o f d e v e l o p m e n t by a mechanism  1968).  Phoxinus  None o f t h e o o c y t e s  E s t r o g e n by i t s e l f ,  w h i c h was n o t d e t e r m i n e d . sions of the germinal  to oocytes.  s i z e b e l o w w h i c h o o c y t e s a r e u n a f f e c t e d by  hypophysectomy.  Numerous s u b s e q u e n t  experiments  have  verified  66 this ly  statement.  been seen The  oocytes by  P r e - v i t e l l o g e n i c s t a g e o o c y t e s have  t o be m a i n t a i n e d  p i t u i t a r y removal.  q u e s t i o n o f whether t h e growth and  (1970)  Chestnut  noted  i n t e l e o s t s has  that  had  stimulated  little  study.  t h r e e weeks w i t h p i t u i t a r y homogenates f r o m the r e s i d e n t  oocytes  In  of f i v e  a f u r t h e r a d v a n c e m e n t was  one  speciman had vations  fish  oocytes  late  stage oocytes  perinucleolar  basophillia)  pituitary  occurred e a r l i e r  Similar  Conversion of  peripheral  ( l a r g e r , no  Formation  layer)  to  cytoplasmic  There  i s , therefore,  of c o r t i c a l a l v e o l i  month o f i n j e c t i o n s p r e c e d e d  r e s u l t s obtained f o r the  t h o s e mentioned above. e f f e c t on t h e  stage oocytes. stage oocytes than those  early  b a s o p h i l l i c sub-  o f a p p e a r a n c e o f t h e y o l k v e s i c l e s t a g e by  no  obser-  i n j u v e n i l e p i n k salmon t r e a t e d  i n controls.  gonadotropin.  The  The  of the development of p r e - v i t e l l o g e n i c stages  ment A a n d B a f t e r one time  study.  (have  stage oocytes  than  coho  observed.  r e f e r r e d to as the p a l l i a l  salmon g o n a d o t r o p i n stimulation  spawning  i n the y o l k v e s i c l e stage.  s t a n c e i n ooplasm —  salmon  to lose 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 .  were made i n t h e p r e s e n t  perinucleolar  One i n the  Treatment  stimulation at that  time.  spring  reached  salmon  early  fact that  the average  a  by  in treatnormal  months.  contradict  perinucleolar perinucleolar  s a l m o n were r e l a t i v e l y  The  with  gonadotropin  the e a r l y  were u n a b l e  the  four  w i t h homologous  explanation i s that  o f t h e p i n k s a l m o n , and  salmon n e v e r  spring  stage or s i z e of the  spring  of  is  i n j e c t i o n o f f i n g e r l i n g coho  caused  had  development  l e s s mature than the y o l k v e s i c l e s t a g e  p i t u i t a r y gonadotropin  for  following  consistent-  l e s s mature  to respond  the oocytes diameter  of  of the  to  the early  67 p e r i n u c l e o l a r stage oocytes i n the p i n k salmon z e r o supports t h i s of  statement.  A l t e r n a t i v e l y , perhaps  the s p r i n g salmon were l e s s r e s p o n s i v e t o  control  the  oocytes  gonadotropin  t h a n those o f the p i n k salmon because Oncorhynchus  tshawytscha  n o r m a l l y m a t u r e 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 the 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  estrogen would  develop  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 s a l m o n g o n a d otropin alone. in  The  r e s u l t s confirmed t h i s proposal.  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  i n j e c t e d w i t h 1.5  observed  m i c r o g r a m s e s t r a d i o l 176  ( 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 body w e i g h t  s a l m o n g o n a d o t r o p i n f o r 126  Oocytes  i n pink  salmon  p e r gram b o d y  m i c r o g r a m s p e r gram days.  By  comparison,  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 gram b o d y w e i g h t ) for  weight  per  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  42 d a y s more.  The  h i g h d o s a g e o f e s t r o g e n (15  micrograms)  plus gonadotropin i n h i b i t e d ovarian maturation. 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 which of  e s t r o g e n and  the female ovary. of  g o n a d o t r o p i n were i n j e c t e d  concurrently into  showed a s i m i l a r e n h a n c e m e n t o f d e v e l o p m e n t o f  the  A s y n e r g i s t i c a c t i o n on t h e d e v e l o p m e n t o f t h e o v a r i e s  hypophysectomized  s t i l b e s t r o l a n d FSH, a n d HCG  physiological  r a t s was  found w i t h treatments of  (Simpson  e t a l , 1 9 4 1 ) , and  ( P e n c h a r z , 19^0;  s i z e o f t h e o v a r y was follicles.  due  Simpson e t a l , 19^1).  diethystllbestrol The  increase i n  p r i m a r i l y t o an enlargement  P h i l l i p s a n d Van  Tienhoven  diethyl-  (I960) observed  of  that  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 d e v e l o p m e n t .  Ovarian  the  68  gonadotropin i n j e c t i o n s , i n combination w i t h d i e t h y l s t i l b o e s t e r o l induced n e a r l y normal f o l l i c u l a r development. non-breeding  Four out of s i x  b l a c k ducks t r e a t e d w i t h a combination of c h i c k e n  a n t e r i o r p i t u i t a r y e x t r a c t 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 w i t h y e l l o w y o l k , whereas none of the b i r d s t r e a t e d 1959).  w i t h e i t h e r hormone a l o n e had y e l l o w y o l k ( P h i l l i p s , Gonadotropin  e x t r a c t e d from t e l e o s t p i t u i t a r i e s  was  e f f e c t i v e as a s t i m u l a n t to development of the ovary i n prev i o u s r e s e a r c h on hypophysectomized of  adult teleosts.  A l l stages  r e p r o d u c t i v e m a t u r i t y , i n c l u d i n g v i t e l l o g e n e s i s and  were r e i n d u c e d by replacement  therapy w i t h salmon gonadotropin  SG-G100 f o l l o w i n g p i t u i t a r y removal (Yamazaki and Donaldson, Donaldson,  I969),  and Donaldson,  i n Carrassius auratus  1968), Poecilla reticulata  and Heteropneustes  1971).  ovulation  fossilis  (Liley  (Sundararaj, Anand,  The gonad of the j u v e n i l e salmonid, how-  ever, i s more r e f r a c t o r y t o gonadotropin, as demonstrated the r e s e a r c h of Schmidt et a l , I 9 6 5 .  There was  by  a s m a l l growth  i n the s i z e of o v a r i e s from immature Salmo g a i r d n e r i i for  and  injected  two weeks with an e x t r a c t of Oncorhynchus tshawytscha  taries.  Oocytes  pitui-  i n f i n g e r l i n g Oncorhynchus k i s u t c h matured  slowly when t e s t e d f o r three weeks w i t h a homoplastic p r e p a r a t i o n of p i t u i t a r y glands from spawning a d u l t s (Chestnut, 1970  - discussed previously). In  the present experiment,  s i x months of i n j e c t i o n s of  1.0  micrograms per gram body weight  salmon gonadotropin p l u s  1.5  micrograms per gram body weight  e s t r a d i o l 178  b e f o r e the f i n a l of  stage of oocyte m a t u r a t i o n was  were r e q u i r e d  reached i n one  three specimens f o r both n o r m a l - s i z e d and enlarged f i s h .  A  69 comparable stage of m a t u r i t y d i d not occur u n t i l in  dne pink salmon i n j e c t e d w i t h 1.0  weight  s i x weeks l a t e r  m i l l i g r a m per gram body  SG-G100 and one t r e a t e d w i t h salmon gonadotropin  e s t r a d i o l 1? g ( a f t e r t h r e e months).  A l l o f the  aforementioned  f i s h r e q u i r e d 42 more days of treatment b e f o r e t h e i r were l a r g e r than 2 mm  i n diameter.  treatments c h a r a c t e r i z e d these In  and  oocytes  High v a r i a b i l i t y w i t h i n  samples.  the p r e c o c i o u s l y mature o v a r i e s , some of the d e v i a -  t i o n s from the normal p a t t e r n of development warrant  further  discussion. Oocytes  i n the f i n a l stage of m a t u r a t i o n i n the t r e a t e d  pink salmon were f a r fewer i n number than those a t a stage i n the u n i n j e c t e d c o n t r o l s .  There was  similar  a p p a r e n t l y some  mechanism which compensated f o r the f a c t t h a t the t e s t  pink  salmon were s m a l l i n s i z e when p r e c o c i o u s o v a r i a n 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 observed i n treatments i n which estrogen was  injected.  first Six  weeks l a t e r , oocytes i n the y o l k v e s i c l e stage were r e g r e s s i n g in  the pink salmon a d m i n i s t e r e d salmon gonadotropin.  tial of  The  ini-  c o r r e l a t i o n between the l e v e l of estrogen and the number  a t r e t i c oocytes was  l o s t i n l a t e r samples because of  r e s o r p t i o n of the r e g r e s s e d germinal c e l l s .  The data i n d i c a t e d  t h a t r e l a t i v e l y few y o l k y oocytes were a l l o w e d to develop t o m a t u r i t y i n any of the f i s h t r e a t e d w i t h exogenous hormonesi the m a j o r i t y of the remainder  of the oocytes  undergoing  v l t e l l o g e n e s i s were converted i n t o p r e - o v u l a t o r y corpora  70 atretica. to  More t h a n h a l f  c o r p o r a a t r e t i c a by  salmon.  o f t h e o o c y t e s had  been  converted  sexual maturity i n the u n i n j e c t e d pink  T h i s phenomenon i s n o t  r e p r o d u c t i v e l y mature f i s h  unusual, as  the ovary  of  contains regressed oocytes  every  (Ball,  I960). The established fined  stages of f o l l i c u l a r a t r e s i a by B r e t s c h n e i d e r and  to those A  oocytes  de W i t  found  (19^7).  I t was  i n w h i c h v i t e l l o g e n e s i s had  s m a l l amount o f a c t i v i t y  d e h y d r o g e n a s e was  f o l l o w e d the p a t t e r n  of  A5-36  i n o v a r i e s of pink  sits  to the y o l k v e s i c l e  of formazan.  The  the o v a r i e s of the t e s t color.  The  results  mentioned above. for  salmon  spring  of Chestnut  spring oocytes  (1970)  to observe  i n the t e s t  stroma  a marked r e a c t i o n  s a l m o n c o u l d w e l l be fish.  contained  were no marked  related  depo-  stage oocytes  are  s i m i l a r to  from  pink those  Oncorhynchus  homogenates f r o m  i n t h e f o r m a t i o n o f a p i n k hue i n the  injected  salmon s t a i n e d a f a i n t  I n j e c t i o n of f l n g e r l i n g  sometimes l o c a l i z e d  failure  There  perinucleolar  t h r e e weeks w i t h p i t u i t a r y  resulted was  early  stage.  begun.  hydroxysteroid  w i t h v a r i o u s d o s a g e s -of s a l m o n g o n a d o t r o p i n w h i c h oocytes  con-  klsutch  spawning a d u l t s  i n the o v a r i e s ,  which  between t h e o o c y t e s . i n the  j u v e n i l e pink  to the immaturity  of  The and  the  71  GENERAL SUMMARY This maturity In  the  s t u d y has  provided evidence  o f t h e m a l e and  female  pink  c a s e o f t h e male j u v e n i l e ,  maturation injection  c a n be m a n i p u l a t e d to produce  normal and salmon.  by  The  viable  the time  t r o p i n with or without  c y c l e of  salmon  s p e r m a t o z o a one  females  testicular  gonadotropin  o f s p a w n i n g o f any  o v a r i e s from  the r e p r o d u c t i v e  s a l m o n can be a c c e l e r a t e d .  the  with  that  year  stock of w i l d  treated with  e s t r o g e n over an  contained a  s m a l l number o f o o c y t e s  maturation;  full  earlier  than pink  s a l m o n gonado-  extended  time  i n the f i n a l  interval  stage  of  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 p i n k  salmon m a l e a p p e a r s  offer  t h e most a p p l i c a b l e  solution  to the problem  'off  year  salmon.  A  1  cycles  of p i n k  first  jQ% this  resident  progeny  o f t h e g e n e t i c complement o f t h e home-stream f i s h . procedure  stock with resident direct fish  of r e p o p u l a t i n g  c r o s s of a  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  w i t h t h e ova  75%*  and  fish.  then  87i%  (1)  milt  months i s p o s s i b l e  ova  from  (2)  i n the female,  than  a  p r e c o c i o u s l y mature  t h e number o f o v a d e v e l o p e d  crosses before a t o t a l  Repeating  o f t h e gene p o o l o f t h e m a l e  and  r e q u i r e s months o f t r e a t m e n t ;  with  the o f f s p r i n g would r e s u l t  T h i s method w o u l d be more c o n v e n i e n t  c r o s s employing  because:  from  to  i s small  and  a n advancement o f o n l y s i x thereby n e c e s s i t a t i n g  displacement  o f one  year  two  i s achieved.  in a  72  Figure  21: Ovary of a zero  cm l o n g ) .  Visible  juvenile  spring  oocytes a r e i n the e a r l y  Haematoxylin and e o s i n . Figure  control  perinucleolar  stage.  150.  X  22: Ovary of a j u v e n i l e  spring  s a l m o n 10.0  cm l o n g  t h r e e t i m e s p e r week w i t h f i s h  s a l i n e f o r 84 d a y s .  all  stage.  X  (5.^  salmon  i n the early  perinucleolar  injected  Oocytes a r e  Haematoxylin and  eosin  150.  Figure  23: Ovary of a j u v e n i l e  injected per  spring  10.0  t h r e e t i m e s p e r week w i t h  gram body w e i g h t f o r 84 d a y s .  perinucleolar Figure  stage.  s a l m o n 11.2  Haematoxylin and e o s i n  one  layer  two c o n t r o l  Notice the yolk  of an e a r l y p e r i n u c l e o l a r  c a n be s e e n .  stage  oocyte  H a e m a t o x y l i n and  (13.6  perinucleolar  nucleus near the f o l l i c l e early  layer  perinucleolar  i nthe stage  The s e c t i o n was t a k e n f r o m t h e o v a r y o f a  salmon  fish  s a l i n e f o r 98 d a y s .  cm i n l e n g t h ) i n j e c t e d t h r e e t i m e s p e r week w i t h Haematoxylin and e o s i n ,  X  150.  26: Follicle  (above)  23.  o o c y t e s a r e i n the' l a t e  At the periphery,  pink  Figure  150.  25:  on t h e l e f t .  oocytes  X  1500.  X  The stage.  gonadotropin  Oocytes a r e a l l i n the e a r l y  f r o m t h e same s e c t i o n a s s e e n i n F i g u r e  Figure  u g salmon  24: Follicle  eosin,  cm i n l e n g t h ,  layer  of a late  perinucleolar  stage  Lower o o c y t e i s i n t h e e a r l y p e r i n u c l e o l a r  Haematoxyline and e o s i n ,  X  1500.  oocyte  stage.  73 Figure 27; Oocyte i n the yolk v e s i c l e stage from the ovary of a pink salmon (13.2 cm i n length) injected three times per week with 10.0 days.  g salmon gonadotropin per gram body weight f o r 96  Haematoxylin and eosin X 150.  Figure 28: F o l l i c l e layers and thecal layer of the oocyte i n Figure 27.  Haematoxylin and eosin X 1500.  Figure 29: Oocyte i n the yolk v e s i c l e stage stained f o r l i p i d s with Sudan Black B.  X 80.  Figure 30: Histochemically demonstrable dehydrogenase a c t i v i t y  A 5-3f3  hydroxysteroid  i n yolk v e s i c l e stage oocytes from pink  salmon Injected t h r i c e weekly with 10.0 yg salmon gonadotropin per gram body weight f o r 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 i n Figure 12.  Haematoxylin and eosin.  X 600.  Figure 33: Primary yolk stage oocyte stained f o r l i p i d s with O i l Red 0.  X 50.  74  F i g u r e 34: S e c t i o n of an ovary from a pink salmon j u v e n i l e (14.6 cm i n length) i n j e c t e d three times per week with 1.0 y g salmon gonadot r 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 y o l k stage oocytes. Haematoxylin and e o s i n . X 20. F i g u r e 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 h r e e times per week w i t h 1.0 y g salmon gonadot r o p i n per gram body weight f o r 126 days. Note that the m a j o r i t y of oocytes a r e i n the y o l k v e s i c l e stage. Haematoxylin and e o s i n . X 25. F i g u r e 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 w i t h 1.0 y g salmon gonadotropin and 1.5 y g e s t r a d i o l 178 per gram body weight f o r 210 days. Haematoxylin and e o s i n . X 15. F i g u r e 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 ( 1 7 . 0 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 gonadotropin per gram body weight f o r 258 days. Haematoxylin and e o s i n . X 15. F i g u r e 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 t o t h a t d e s c r i b e d i n F i g u r e 37. Note the p a u c i t y of oocytes more advanced than 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 50. F i g u r e 39: S e c t i o n of an ovary from a pink salmon t r e a t e d 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 f o r the l a s t 92 days. Note t h a t p r e c u r s o r germinal c e l l s appear to be a c t i v e l y producing e a r l y p e r i n u c l e o l a r stage oocytes. Haematoxylin and e o s i n . X 200.  75  F i g u r e 40; Gonadosomatic  index (G.S.I.) of female pink  (Oncorhynchus  gorbuscha) and s p r i n g (Oncorhynchus tshawytscha) salmon 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 tshawytscha) gonadotropin d u r i n g the summer of averages taken from T a b l e IX and X.  1969.  (Oncorhynchus Values a r e  n  JUNE  1  JULY  1  AUGUST '  SEPL  76  F i g u r e 41: Gonadosomatic index (G.S.I.) of female pink salmon t r e a t e d t h r e e times per week with v a r i o u s combinations of salmon (Oncorhynchus  tshawytscha)  gonadotropin and e s t r a d i o l 178 .  F i g u r e s were taken from Table XI. GAE  =  1.0  yg salmon gonadotropin and 15.0  yg  estradiol  173 per gram body weight. GBE  =  1.0  yg salmon gonadotropin and 1.5  yg  estradiol  178 per gram body weight. GCE  =  1.0  yg salmon gonadotropin per gram body weight  throughout  the experiment and 1.5 ng  estradiol  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  Co  =  Control (injected with f i s h  U.I. Co =  yg e s t r a d i o l 178  Uninjected  per gram body weight.  control.  saline).  1.201-  0.80k  -6 SI  0.40k  D  E  C  J  A  N  F 1 9 7 1  E  B  77  Figure  42; Gonadosomatic index (G.S.I.) f o r e x t r a - o r d i n a r i l y  large  female pink salmon t r e a 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.  Data  were taken from Table X I I . LG  =  1.0 yg salmon gonadotropin per gram body weight.  LGE  =  1.0  y  17  Bper gram body weight.  LGPE =  1.0  g  salmon gonadotropin p l u s 1.5  yg e s t r a d i o l  yg salmon gonadotropin, 1.5 Hg e s t r a d i o l  and 5 vg progesterone per gram body weight. Co  =  control (injected with f i s h  saline).  176  LGo LGE © LGPEn  0.80  Co • GSI  0.40  —  SEPT  1  OCT  1  1970  NOV  1  DEC  i  JAN  i  1971  FEB  78  Figure  43; Gonadosomatic Index (G.S.I.) of u n i n f 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 a r e averages taken from Table X I I I .  cycle.  Values  79  F i g u r e 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 w i t h v a r i o u s dosages o f salmon (Oncorhynchus p i n d u r i n g the summer o f 1 9 6 9 . .  tshawytscha)  gonadotro-  Values a r e averages taken from  T a b l e s XIV and XV. A  =  1 0 . 0 y g salmon gonadotropin p e r gram body weight.  B  =  1 . 0 y g salmon gonadotropin p e r gram body weight.  C  =  0 . 1 y g salmon gonadotropin per gram body weight.  Co  =  control (injected with f i s h  U.I. Co =  uninjected  control.  saline).  80  F i g u r e 45: Mean oocyte diameter  (microns) of pink salmon t r e a t e d  t h r e e times p e r week w i t h s e v e r a l combinations o f salmon (Oncorhynchus  tshawytscha)  gonadotropin and e s t r a d i o l 17g  ..  V a l u e s a r e averages taken from T a b l e XVI. G  =  1.0 yg salmon gonadotropin per gram body weight.  GAE  =  1.0  yg salmon gonadotropin p l u s 15.0 yg  e s t r a d i o l 17 Bper gram body weight. GBE  =  1.0 yg salmon gonadotropin p l u s 1.5 yg e s 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  p l u s 1.5 yg  e s t r a d i o l 17B p e r 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  =  U.I. Co =  saline injected  control.  uninjected control.  81  F i g u r e 46; Mean oocyte diameter o f 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 p e r week with combinations o f v a r i o u s exogeneous hormones; tshawytscha)  salmon  (Oncorhynchus  gonadotropin, e s t r a d i o l 176, , and progesterone.  The  v a l u e s a r e averages taken from Table XVII. G  1.0 vig salmon goandotropin per gram body weight.  GE  1.0 17g  GPE  ug salmon gonadotropin and 1 . 5 per gram body weight.  1 . 0 yg salmon gonadotropin, 1 . 5 176  yg e s t r a d i o l  • and 15 yg progesterone p e r gram body  weight. Co  estradiol  control  ( i n j e c t e d with f i s h s a l i n e ) .  82  Figure  47; The mean oocyte diameter of oocytes from u n i n j e c t e d pink  salmon maintained throughout t h e i r l i f e averages taken from Table XVIII.  cycle.  Values a r e  83  Figure  48: Mean percentages of oocytes which comprised  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. of  the o v a r i e s  Each complete  f i s h per treatment A  =  bar r e p r e s e n t s 100$.  (n) seen i n Table  The number  XIV.  10.0 y g salmon gonadotropin per gram body weight.  B  =  1.0 y,g salmon gonadotropin per gram body  C  =  0.1yg  Co  =  c o n t r o l ( i n j e c t e d with f i s h  weight.  salmon gonadotropin per gram body weight. saline).  1  E  A  R  L  2  L  A  T  E  3  Y O L K  T  R  E  A  Y  P P  T  M  E  E  R  V  E  S  E  N  T  R I  I  I N  C  N U  L  U C  C L  L E  E O  O L  L A  A  R  R  E  A  B  2 C o  S  A  M  P  L  E  D  A  T  E  2 1  1 0 17/6/69  1 30/6/69  2  2  2  2  1 2 14/7/69  3  1  28/7/69  4 11/8/69  5  ii  25/B/69  1 6  8/9/69  7 22/9/69  84  Figure  49« Mean percentages o f o o c y t e s which comprise the o v a r i e s  of p i n k salmon t r e a t e d w i t h v a r i o u s combinations o f salmon tshawytscha) g o n a d o t r o p i n and e s t r a d i o l 17/9 .  (Oncorhynchus  Each complete b a r r e p r e s e n t s 100$. sample  The number o f f i s h p e r  can be seen i n T a b l e 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 p e r gram body weight  GAE  =  1.0  fig  salmon g o n a d o t r o p i n p l u s 15.0  jig  e s t r a d i o l 17/9 p e r gram body weight. GBE  ~ 1.0 ^ g salmon g o n a d o t r o p i n p e r gram body weight (throughout) p l u s l ^ / ' - g  estradiol  17/5 per gram body weight. GCE  =  1 . 0 j x g salmon g o n a d o t r o p i n p e r gram body weight throughout the experiment, and 1*5 / i g 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 g o n a d o t r o p i n p e r gram body  weight Co  =  control  ( i n j e c t e d with f i s h  saline).  T  A  R  E  A  T  M  E  N  T  E  1  E  A  R  L  2  L  A  T  E  3  Y  O  L  *  P R  E  G  A  K  I M  V  A  T  R  E  P  E  R  I  N  U  C  L  E  O  L  A  E  R  I  N  U  C  L  E  O  L  A  R  C  Y  T  E  S  I  A R Y  E  T  H I  C  Y A  R  L O  B  C  D  K O  C  E  E  5.4,3  C o  S  A  M  P  L  E  D A T E ,  0  25/6/70  I  8/8/70  2 18/9/70  5 31/10/70  15/12/70  26/1/71  R  E L  E  G  G  P  s L A R G E . *  B  Y  6 10/3/71  O  S  85  Figure  50: Mean percentages of oocyte 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 life.  Data was  taken from T a b l e XVIII.  their  1  EARLY  PERINUCLEOLAR  2 LATE  PERINUCLEOLAR  3 YOLK  VESICLE  *  PRIMARY  YOLK  5 L A R G E , HARD 6  SAMPLE DATE  OOCYTES  ATRETIC  6  6  3  3  8 5/11/69  9 21/1/70  10 9/3/70  30/6/70  86  F i g u r e 51 '• 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 A 5-3B  hydroxy-  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 1969.  tshawytscha)  gonadotropin d u r i n g t h e summer of  Data were taken from Table XIV. A  10.0  yg salmon gonadotropin per gram body  weight. B  1 . 0 yg salmon gonadotropin p e r gram body weight.  C  0.1  yg salmon gonadotropin per gram body  weight. Co  saline injected  U.I. Co  uninjected  control.  control.  PINK  SALMON  OVARIES  B o—o C O- 9 Co —® c  I  JUNE  _J  JULY  I  AUG  SEPT  87  Figure  52: I d e n t i c a l experiment  s p r i n g salmon j u v e n i l e s .  t o t h a t i n F i g u r e 19, but w i t h  The number of f i s h per  per sample (n) and the standard d e v i a t i o n (SD) Table  XX.  treatment  can be  seen  A B C e i.o  SPRING  SALMON  OVARIES  JU LY  AUG  SEPT  e  Co © — & U.I.CO a  VISUAL UNITS OF 0.5 ENZYME ACTIVITY 0  JUNE  88  Table IX: Mean body weight, mean f i x e d ovary weight, mean gonadosomatic index (G.S.I.) o f j u v e n i l e female pink salmon t r e a t e d with v a r i o u s dosages of salmon gonadotropin, y. = =  standard d e v i a t i o n .  mean; S.D.  TABLE IX:  SAMPLE TREATMENT Zero Co Control 17/6/69 1 A B C Co 2 A B C Co A 3 B C Co 4 A B Co A 5 B Co 6 A B Co A 7 B Co U.I. Co  n 3 4 4 3 5 3 4 4 4 6 4 4 6 4 5 1 2 6 2 5 5 4  BODY BODY LENGTH (cm) WEIGHT ( g ) y S.D. S.D. y 3.6 1.0 0.5 7.7 8.1 7.3 8.3 8.1 9.3 8.9 8.8 9.0 10.0 9.2 10.0 10.0 10.1 11.0 10.4 12.2 12.0 12.7 12.1 12.3 13.7 13.2 13.8 13.6 13.4  0.8 0.2 0.9 0.5 0.4 0.8 0.5 0.8 1.0 0.6 0.6 0.5 0.5 0.6 0.8 0.8 0.3 1.0 1.2 1.4 0.1 0.7 0.5 1.2  4.4 3.0 4.5 4.0 6.4 6.0 5.3 6.2 8.9 6.6 8.0 8.2 9.0 12.4 10.4 16.3 15.0 18.9 16.8 17.2 22.7 19.8 26.2 22.4 24.2  1.3 0.3 1.7 0.7 0.6 1.7 1.1 2.2 2.6 1.3 1.8 1.4 0.9 3.4 2.8 3.3 1.3 5.7 7.3 6.9 1.1 4.6 3.2 6.4  FIXED OVARY WEIGHT ( u g ) G.S.I. y y S.D. S.D.  24.6 17.9 14.2 12.1 25.5 39.4 21.5 73.9 64.6 25.8 73.6 84.1 ^3.9 74.9 114.1 52.1 31.2  5.6 3.3 4.1 3.4 16.2 6.7 7.0 11.4 28.9 4. 0 31.4 13.6 20.1 15.2 9.5 8.6  0.23 0.57 0.59 0.29 0.58 0.66 0.41 0.92 0.86 0.27 0.94 1.01 0.38 0.89 0.91 0.30 0.46  0.03 0.11 0.05 0.04 0.18 0.08 0.15 0.13 0.18 —  —  0.18 0.04 0.02 0.15 0.10 0.05 0.13  89  Table X; 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 o f salmon gonadotropin. deviation.  y =  mean; S.D. = standard  TABLE X:  SAMPLE TREATMENT Zero Control Co 10/6/69 A 1 B C Co 2 A B C Co A 3 B C Co 4 A B C Co A 5 B C Co 6 A B C Co U.I. Co  n 5 6 4 4 4 2 6 4 6 2 6 6 3 1 3 4 6 3 3 3 4 5  BODY BODY LENGTH (cm) WEIGHT (g) v S.D. u S.D. 5.4 0.2 1.8 0 . 2 5.9 6.2 6.2 6.1 6.9 7.3 6.6 6.8 6.1 7.5 6.9 7.6 6.5 7.0 7.8 7.6 8.9 7.6 9.2 8.0 11.2 10.1 9.3 10.0 9.7  0.5 0.7 0.3 0.1 0.1 0.7 0.8 0.5 0.6 0.7 0.6 0.9 0.6 —  0.3 0.9 1.3 1.0 0.6 0.4 2.0 1.7 1.0 2.0 1.1  2.0 2.9 2.5 2.2 3A 4.1 3.0 2.8 2.2 4.2 3.0 4.7 2.5 4.0 4.1 4.7 7.9 5.2 8.5 5.8 19.2 12.6 8.6 12.5 10.6  0.5 1.2 0.2 0.2 0.1 1.4 0.8 1.0 0.5 1.1 0.8 2.0 0.7 — —  0.3 1.7 3.4 2.1 1.2 0.8 6.4 6.2 2.9 8.2 3.3  y  FIXED OVARY WEIGHT ( S.D.  5.3 4.4 6.1 4.1 10.1 7.7 8.0 6.2 17.5 8.5 7.3 10.1 6.4  y g  ) G.S.I, u S.D.  2.0  0.42 0.22 0.28 0.18 0.27 0.24 0.19 0.21 0.22 0.28 0.17 0.13 0.14  — —  1.4 1.0 4.1 0.2 0.7 1.3 8.6 5.2 4.1 2.8 2.1  / /  /  0.07 —  0.10 0.05 0.08 0.01 0.04 0.03 0.03 0.22 0.08 0.07 0.05  90  T a b l e XI; 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 17 g d u r i n g 1970/71,  y-  mean; S.D. = Standard  deviation.  TABLE X I : FISH PER SAMPLE SAMPLE & TREATn DATE MENT  BODY BODY LENGTH (cm) WEIGHT (g) S.D. S.D.  GONAD WEIGHT ( ) u S.D. g  8.0  0.4  5.0  1.0  G GAE GBE Co  10.0 9.0 9.7  0.4  8.3 6.2 8.8  1.4 2.6 2.1 1.1  0.018  G GAE GBE AE BE Co  12.5 12.3  2.7 1.8 4.7 2.0 3.7  0.039  4 3 4 4 2  G 3 31/10/70 GAE GBE GCE 17/11/70 Co  14.9  0.013  31.1 44.0  7.4 4.1 4.1 7.2 7.7  4 4 3 4 3 2  4 G 15/12/70 GAE GBE GCE 29/12/70 BE Co  17.1  43.4 18.3  11.1 8.2  0.095  37.6  4.1 3.5 5.7  2.9  0.063  4 3 3 2 4  G GBE GCE 10/2/71 Co U.,1. Co  4 3 3  6 10/3/71  3 4 4 4 4 4 3 4 4 4 4  0 25/6/70  1 8/8/70  25/8/70 2 18/9/70 6/10/70  5 26/1/71  10.9  11.9  11.0 12.2 13.5 12.8 14.6 14.7 16.5  13.0 14.9 16.0 13.6  16.9 17.7 16.3 17.0  0.9  0.7 0.3  11.9  0.6 0.8 1.0 0.4 0.7 0.7  17.5  1.0 0.8 0.7 1.2 1.6  31.1 17.2  1.2 1.3 0.7 0.6  0.9  14.4 16.4 10.3 16.7 22.2  29.9  30.3 23.8  4.9  0.8  44.4  1.9  46.6 37.2 45.3 37.3  89.0  11.7 6.3 10.7 11.1 21.2  43.7 44.6  12.1 15.4  16.3 21.1  0.5 1.3 1.6 1.5  G 17.0 GCE 17.1 U.I.Co 20.5  1.3 1.8 1.8  77.9  23.2  0.015  0.014 0.011 0.026 0.047 0.020 0.034 0.027 0.007 0.120 0.080 0.080  0 0.010 — —  0.006 0.024 0.016 0.021 0.014  0.005 0.060  --  0.080  0.050 0.020  0.007  0.080 0.002  0.047 0.017 0.117  0.007 0.013  0.256 0.058 0.158 0.106  0.069 0.053  0.164 0.042  0.160 0.050  0.306  0.100  0.145 0.132 0.247  0.254 0.136 0.045  G.S.I. S.D,  0.44 0.46 0.28 0.20  0.10  0.44 0.34  0.15  0.18 0.18 0.26  0.05  0.58  0.82 0.08  0.72 0.58 0.38  0.05  0.13 0.02 0.07 0.10 0.20 0.10  0.40 0.02 0.66 0.20 0.08  0.40 0.08 0.36 0.24 0.16 0.54  0.26  1.08  0.74 0.18 0.18 0.10 0.08  0.52 0.56  0.86 0.44 0.08  0.30 0.30 0.56 0.62  0.64  0.02 0.38  0.26 0.06 0.06  91  Table X I I : Mean body weight, mean body l e n g t h , mean ovary, weight, and mean gonadosomatic index (G.S.I.) of e x t r a - o r d i n a r 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 gonadotropin, e s t r a d i o l l ? g and progesterone. standard d e v i a t i o n .  y  =  mean; S.D.  =  TABLE X I I :  SAMPLE  TREATMENT  n  BODY BODY OVARY LENGTH(cm) WEIGHT(g) WEIGHT (g) S.D. S.D. y y y S.D.  1 3/11/70  LGPE  2  18.2  0.1  55.9  0.8  0.11  0.05  0.40  0.20  1 26/11/70  LG LGE  3 5  19.1 19.3  0.5 1.5  64.2 69.8  4.0 16.1  0.22 0.16  0.07 0.17  0.71 0.50  0.23 0.54  2 18/2/71  LG LGE LGPE Co  5 3 2 5  21.0 20.6 20.6 21.6  1.4 2.3 1.3 0.9  81.1 81.9 69.2 97.9  19.1 28.7 22.3 11.7  0.05 0.29 0.05 0.27  0.02 0.41 0.04 0.15  0.12 0.58 0.15 0.57  0.03 0.75 0.07 0.36  G.S.I. S.D. y  92  Table XIII; Mean body weight, mean gonad weight, and mean gonadosomatic index (G.S.I.) of u n i n f e c t e d female pink salmon maint 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 , standard d e v i a t i o n .  y = mean; S.D. =  TABLE X I I I :  SAMPLE  DATE  n  BODY WEIGHT(g) y -S.D.  y  OVARY WEIGHT(g) S.D.  y  G.S.I. 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  T a b l e XIV; Mean oocyte diameter of pink salmon j u v e n i l e s w i t h v a r i o u s dosages o f salmon gonadotropin, standard  deviation.  y  treated  = mean; S.D. =  TABLE XIV: SAMPLE & DATE  TREATMENT  2 14/7/69 3 28/7/69 4  H/8/69 5 25/8/69 6 8/9/69 7 22/9/69  OOCYTE  DIAMETER (microns)  i  2  134.5  11.5  A B C Co  3 3 3 3  160.8 142.8 187.2 205.6  10.4 8.2 14.6 23.5  A B C Co  3 3 3 3  227.6 216.7 205.0 215.5  50.8 8.8 2.8 22.6  A B C Co  3 3'.. 3 3  274.9 212.7 257.7 176.1  21.1 13.8 12.9 11.5  A B Co  3 3 2  273.7 265.4 193.7  27.5 7.8 3.5  A B Co  3 2 1  364.6 322.5 238.7  28.0 27.3  A B Co  2 3 3  378.0 356.6 234.4  9.0 23.0 25.3  A B Co  2 3 3  416.7 414.3 317.8  14.4 30.0 29.4  Zero Control  17/6/69 1 30/6/69  MEAN  NUMBER OP SAMPLES  S.D.  —  94  Table XV: 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 o f salmon gonadotropin, Standard  Deviation.  y = mean; S.D.  =  TABLE XV: SAMPLE & DATE Zero Control IO/6/69 1 24/6/69 2 7/7/69  3 21/7/69 4 4/8/69  5 I8/8/69 6 1/9/69  TREATMENT  MEAN OOCYTE DIAMETER (microns) S.D  NUMBER OF SAMPLES  y  2  47.8  6.3  A B C Co  2 2 2., 1  69.0 68.3 72.1 54.6  7.5 6.4 5.2  A B C Co  2 3 2 2  70.6 72.6 74.6 60.2  5.4 10.1 1.1 9.8  A B C Co  2 2 2 3  77.9 81.5 63.8 74.4  6.8 3.1 0.9 6.0  A B C Co  2 1 2 3  76.2 89.6 83.0 82.2  5.7  A B C Co  3 2 3 3  100.2 90.0 101.5 87.0  6.7 13.7 3.6 1.4  A B C Co U.I. Co  3 2 3 3 3  104.0 101.0 103.6 95.6 94.0  17.9 10.0 7.5 9.7 8.3  7.7 1.9  95  T a b l e XVI: 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 17B . =  standard d e v i a t i o n .  v = mean; S.D.  TABLE XVI: SAMPLE &  DATE Zero Control  TREATMENT  25/6/70 1 8/8/70  25/8/70 2 18/9/70 6/10/70  3 31/10/70 17/11/70 4 15/12/70 29/12/70 5 26/1/71 10/2/71 6 10/3/71  n  MEAN OOCYTE DIAMETER (micrc S.D. M  2  110.95  15.8  G GAE GBE Co  3 3 3 3  193.6 162.2  24.0  181.3 182.6  8.7 4.1 5.8  G GAE GBE AE BE Co  3 3 3 3 3 3  224.1 183.6 228.7  151.9 205.1 230.3  15.5 80.8 98.8  41.5  G GAE GBE GCE Co  3 3 3 3 2  418.4  42.0  G GAE GBE GCE BE Co  3 2 3 3 3 2  364.7 53.5 , 327.5  198.3 75.7 272.4  133.8 378.7  127.8 70.8  170.4  424.7  303.0 300.4  149.4  59.1 36.7  23.4 58.2 120.3 28.9  140.2  G GBE GCE Co Co. (U.I. )  ' 3 3 2 3 3  723.0 285.1 992.7 382.9 560.1  1085.1 35.4 65.2  G GCE Co (U.I.)  3 3 3  323.9 529.2 571.4  347.1 662.9 73.8  376.5 184.9  96  Table XVII: Mean oocyte diameter o f 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 o f salmon gonadotropin e s t r a d i o l 1 ? 3 , and progesterone. deviation.  y  = mean; S.D. = standard  TABLE XVII:  SAMPLE  &  TREATMENT  n v  DATE  1 3/11/70 1 26/11/70 2 18/2/71  .LPGE  MEAN OOCYTE DIAMETER(microns) S.D.  445.2  1.5  LG . LGE ~  3 3  471.7 307.8  91.3 78.7  LG. LGE LPGE LCo  3 2 2 3  112.8 342.7 166.3 601.1  33.4 302.5 72.8 27.4  97  T a b l e XVIII: Mean oocyte diameter maintained S.D.  i n a q u a r i a throughout  = standard d e v i a t i o n .  of u n i n j e c t e d pink salmon their l i f e ,  y  =  mean;  TABLE XVIII:  SAMPLE  DATE  NUMBER OF SAMPLES  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  v  MEAN OOCYTE DIAMETER (microns) S.D.  Table  XIX; The h i s t o c h e m i c a l l y demonstrable  A 5 - 3 8 hydroxysteroid  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. S.D.  = standard d e v i a t i o n .  y = mean  TABLE XIX:  SAMPLE Zero Control  DATE  TREATMENT  NUMBER OF SAMPLES  ENZYME ACTIVITY (visual units) p S.D.  17/6/69  Co  4  0  0  1  30/6/69  A B C Co  4 3 3 3  0.25 0.29 0.17 0  0.29 0.33 0.29 0  2  14/7/69  A B C Co  2 4 2 4  0.25 0.38 0.25 0.5  0.35 0.48 0.48 0  3  28/7/69  A B C Co  4 2 4 4  0.13 0.25 0.25 0.25  0.25 0.48 0.29 0.50  4  11/8/69  A B Co  3 4 4  0.67 0.25 0.50  0.29 0.29 0.41  5  25/8/69  A B Co  4 4 1  0.25 0.25 0.50  0.29 0.29 0  6  8/9/69  A B Co  2 4 3  0.75 0.50 0.17  0.35 0.41 0.29  7  22/9/69  A B Co U.I. Co  2 4 4 - • 4  0.75 0.88 0.5© 0.38  0.35 0.25 0.41 0.48  99  Table  XX: H i s t o c h e m i c a l l y demonstrable A5-38  hydroxysteroid  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 gaondotropin. y = mean; S.D.  = standard  deviation.  TABLE XX:  SAMPLE  DATE  TREATMENT  NUMBER OP SAMPLES  ENZYME ACTIVITY (visual units) S.D. v  Zero  Control 10/6/69  Co  4  0  0  1  24/6/69  A B C Co  4 4 4 4  0 0 0 0  0 0 0 0  2  7/7/69  A B C Co  1 4 4 4  0 0 0 0  0 0 0 0  3  21/7/69  A B C Co  2 4 3 2  0.50 0.25 0 0  0.71 0.13 0 0  4  V8/69  A B C Co  3 2 4 2  0.50 0.25 0.25 0  0.50 0.35 0.29 0  5  I8/8/69  A B C Co  1 2 3 4  0.50 0.25 0.33 0  0 0.35 0.29 0  6  1/9/69  A B C Co U.I.  3 2 4 2 3  0.50 0.25 0.25 0 0.29  0.50 0.35 0.29 0 0.17  Co  100  References Ashan, S. N.  1966.  E f f e c t s of gonadotropic  hormones on male  hypophysectomized lake chub Couesius plumbeus. J . Z o o l . 44:  703-717.  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