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

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THE EFFECT OF METHALLIBURE AND A CONSTANT 12 HOURS LIGHT : 12 HOURS DARK PHOTOPERIOD ON THE GONADAL MATURATION OF PINK SALMON (ONCORHYNCHUS GORBUSCHA)  by Michael Bernard Flynn B.Sc, University of British Columbia, 1971  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE  in the Department of Zoology  We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA July, 1973  In p r e s e n t i n g an the  advanced degree at Library  I further for  this thesis  shall  the  of  this thesis  written  University  of B r i t i s h  permission  s c h o l a r l y p u r p o s e s may his  f u l f i l m e n t of  make i t f r e e l y a v a i l a b l e  agree t h a t  by  in partial  representatives.  be  for f i n a n c i a l gain  permission.  Department  of  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, Canada  the  I t i s understood  Columbia  shall  requirements  Columbia,  for reference  for extensive  g r a n t e d by  the  that  not  and  copying of Head o f my  be  I agree  that  study.  this  thesis  Department  copying or  for  or  publication  allowed without  my  i  ABSTRACT  T h i s study was undertaken  t o t r y t o d e l a y gonadal m a t u r a t i o n o f  p i n k salmon f o r one year beyond t h e i r normal two year l i f e  cycle.  This  would a l l o w these f i s h to spawn i n y e a r s o f low or n o n e x i s t e n t escapement and p o s s i b l y i n c r e a s e these "poor" year p o p u l a t i o n s . were conducted  Three  experiments  t o i n v e s t i g a t e t h e e f f i c a c y o f the a n t i g o n a d o t r o p i c drug,  m e t h a l l i b u r e , i n i n h i b i t i n g gonadal m a t u r a t i o n i n p i n k salmon.  Gonadoso-  matic i n d e x , oocyte diameter, and stages o f c e l l m a t u r a t i o n i n t h e t e s t i s and oocyte m a t u r a t i o n i n the ovary were measured. The libure  first  or p i l o t  experiment  i n v o l v e d a range o f doses  o f methal-  (0.10 mg., 0.32 mg., and 1.0 mg./gm./2wks.) t o determine t h e  o p t i m a l d o s e . f o r subsequent slowing e f f e c t  experiments.  on m a t u r a t i o n .  A l l doses had o n l y a s l i g h t  T h i s r e s u l t and p o s s i b l e u n d e s i r a b l e e f f e c t s  o f h i g h e r doses prompted t h e d e c i s i o n t o use t h e 0.10 mg./gm. dose f o r subsequent The  experiments.  second o r long-term experiment  m e t h a l l i b u r e and a c o n s t a n t 12 hours  i n v e s t i g a t e d the e f f e c t s o f  l i g h t : 1 2 hours dark p h o t o p e r i o d on  gonadal m a t u r a t i o n o f males and females  f o r a p e r i o d o f t e n months.  M e t h a l l i b u r e c o m p l e t e l y i n h i b i t e d t e s t i c u l a r m a t u r a t i o n by p r e v e n t i n g t h e t r a n s f o r m a t i o n o f p r i m a r y i n t o secondary spermatogonia. t i o n , however, was o n l y slowed.  O v a r i a n matura-  The t r e a t e d o v a r i e s p o s s e s s e d oocytes i n  the o i l g l o b u l e s t a g e w h i l e c o n t r o l o v a r i e s had oocytes i n the secondary yolk globule stage.  M e t h a l l i b u r e had an a n t i t h y r o i d a l  e f f e c t under  n a t u r a l p h o t o p e r i o d but not under constant 12L:12D p h o t o p e r i o d o r at a  ii h i g h dose  (1.0 mg./gm).  operative i n this i n body weight.  S t r e s s from k i d n e y d i s e a s e may have been  effect.  M e t h a l l i b u r e a l s o slowed t h e r a t e o f i n c r e a s e  The c o n s t a n t 12L:12D p h o t o p e r i o d slowed  t i o n i n both males and females.  gonadal matura-  I t i s suggested t h a t a s p e c i f i c day-  l e n g t h and an endogenous rhythm s t i m u l a t e the i n i t i a t i o n ,  maintenance,  and t e r m i n a t i o n o f gonadal m a t u r a t i o n and t h a t t h e s e a s o n a l d a y l e n g t h fluctuations  f u n c t i o n as a s y n c h r o n i z e r .  The d i f f e r e n c e i n e f f e c t o f  m e t h a l l i b u r e on males and females may be due. t o treatment b e g i n n i n g p r i o r t o the s t a r t  o f t e s t i c u l a r m a t u r a t i o n but a f t e r t h e s t a r t o f v i t e l -  logenesis. To i n v e s t i g a t e t h i s p o s s i b i l i t y , m e t h a l l i b u r e treatment was begun at s u c c e s s i v e i n t e r v a l s p r i o r t o the s t a r t t h i r d o r s e q u e n t i a l experiment.  o f v i t e l l o g e n e s i s i n the  T h i s treatment had no e f f e c t on o v a r i a n  m a t u r a t i o n which suggests t h a t the females a r e l e s s s e n s i t i v e t o methall i b u r e than a r e the males.  Treatment  w i t h a h i g h e r dose s t a r t e d  early  i n j u v e n i l e l i f e may i n h i b i t o v a r i a n m a t u r a t i o n . From t h i s study, o n l y t h e males c o u l d be d e l a y e d and, t h e r e f o r e , p o s s i b l y spawn i n "poor" y e a r s .  However, Funk and Donaldson  (1972)  were  a b l e t o a c h i e v e the same g o a l by maturing males i n t h e y e a r o f h a t c h i n g , thus making a t h r e e year program i m p r a c t i c a l .  The v a l u e o f a long program  would be the d e l a y o f o v a r i a n m a t u r a t i o n s i n c e Funk unable to advance m a t u r a t i o n o f females by one y e a r .  e t al_. . (1973) were  iii  TABLE OF CONTENTS Page  ABSTRACT  i  LIST OF TABLES  iv  LIST OF FIGURES ACKNOWLEDGEMENTS  v .  i  x  INTRODUCTION  1  MATERIALS AND METHODS  6  RESULTS  18  DISCUSSION  45  SUMMARY  61  BIBLIOGRAPHY  63  LIST OF TABLES  The mean gonadosomatic index (GSI) v a l u e s f o r females o f the p i l o t experiment The mean oocyte diameters o f the p i l o t experiment The mean percentages o f oocytes i n the stages o f oocyte m a t u r a t i o n o f the p i l o t experiment The mean gonadosomatic index (GSI) v a l u e s f o r males o f the long-term experiment The mean gonadosomatic index (GSI) v a l u e s f o r females o f t h e long-term experiment The percentages o f oocytes i n the stages o f oocyte m a t u r a t i o n and the percentage o f oocytes t h a t a r e a t r e t i c i n the long-term experiment. The mean oocyte diameters o f the long-term experiment The mean body weights o f p o o l e d males and females o f t h e long-term experiment The mean t h y r o i d f o l l i c l e e p i t h e l i a l h e i g h t s o f the long-term experiment and the h i g h dose group (1.0 mg/gm.) sampled on June 14/72 The mean percentages o f oocytes i n the stages o f oocyte m a t u r a t i o n o f the s e q u e n t i a l experiment  V  LIST OF FIGURES Figure 1  Page The hormone and s t e r o i d pathways o f the gonad and t h y r o i d t h a t are i n f l u e n c e d by m e t h a l l i b u r e  ...  4  Design o f the s e q u e n t i a l experiment showing the p e r i o d o f m e t h a l l i b u r e treatment from the s t a r t i n g date t o the f i n a l sampling date o f Oct. 29/72 f o r each o f the f o u r groups  ...  10  Ovary a p p r o x i m a t e l y t h r e e months p r i o r t o the s t a r t o f v i t e l l o g e n e s i s showing oocytes i n the e a r l y p e r i n u c l e o l u s stage (x60)  ...  15  Ovary at the s t a r t o f v i t e l l o g e n e s i s showing oocytes i n the l a t e p e r i n u c l e o l u s stage ( u n i form cytoplasm) and e a r l y y o l k v e s i c l e stage (yolk v e s i c l e s i n the p e r i p h e r a l cytoplasm) (x60)  ...  15  5  Oocytes  ...  15  6  The l a r g e c e n t r a l oocyte i s i n the o i l g l o b u l e stage which i s c h a r a c t e r i z e d by l a r g e v e s i c l e s s u r r o u n d i n g the nucleus (x60)  ...  15  7  Oocyte  i n the p r i m a r y y o l k g l o b u l e stage  ...  15  8  Oocyte (xl9)  i n the secondary y o l k g l o b u l e stage ...  15  T e s t i s a p p r o x i m a t e l y s i x months p r i o r t o the s t a r t o f t e s t i c u l a r m a t u r a t i o n (x600)  ...  17  T e s t i s at the b e g i n n i n g o f m a t u r a t i o n t i n g secondary spermatogonia (x600)  ...  17  T e s t i s approaching f i n a l m a t u r i t y and e x h i b i t i n g the f o l l o w i n g s e r i e s o f c e l l s t a g e s : p r i m a r y spermatocyte (1°SPCY), secondary s p e r matocytes (2°SPCY)., spermatid (SPTD), and spermatozoa (SPZ) (x600)  ...  17  T e s t i s showing secondary spermatogonia from the c o n s t a n t 12L:12D p h o t o p e r i o d c o n t r o l group o f the long-term experiment on June 14 (x600)  ...  21  2  3  4  9  10  11  12  i n the l a t e y o l k v e s i c l e stage (x60)  (x48)  exhibi-  List  o f Figures  (cont d) 1  Figure  13  T e s t i s showing p r i m a r y spermatogonia from the constant 12L:12D p h o t o p e r i o d treatment group o f the same sampling as above (x600)  14  Zero c o n t r o l t e s t i s o f the long-term experiment, sampled on November 8/71, showing p r i m a r y spermatogonia (x600)  15  T e s t i s showing spermatozoa (arrow) from t h e c o n s t a n t 12L:12D p h o t o p e r i o d c o n t r o l group o f the long-term experiment from the August 30 sampling (x600)  16  T e s t i s showing p r i m a r y spermatogonia from the c o n s t a n t 12L:12D photoperiod. treatment group at the same sampling as above (x600)  17  Oocytes i n the o i l g l o b u l e stage from t h e cons t a n t 12L:12D p h o t o p e r i o d treatment group o f the long-term experiment from t h e August 30 sampling ( x l 9 )  18  Oocytes i n t h e secondary y o l k g l o b u l e s t a g e from t h e c o n s t a n t 12L:12D p h o t o p e r i o d c o n t r o l group from the same samplings as above ( x l 9 )  19  Ovary w i t h oocytes i n t h e e a r l y p e r i n u c l e o l u s s t a g e from the s t a r t o f t h e June 29 group o f the s e q u e n t i a l experiment (x60)  20  Oocytes i n t h e l a t e y o l k v e s i c l e stage from m e t h a l l i b u r e t r e a t e d f i s h o f the June 29 group at t h e end o f t h e s e q u e n t i a l experiment (October 29) (x60)  21  Oocytes i n the l a t e y o l k v e s i c l e s t a g e from c o n t r o l f i s h o f the s e q u e n t i a l experiment sampled on October 29 (x60)  22  The mean gonadosomatic index (GSI) v a l u e s f o r females o f the p i l o t experiment (the d a t a a r e l i s t e d i n T a b l e I)  vii  List  o f F i g u r e s (cont'd)  Figure  23  24  25  26  27  28  29  30  31  32  Page  The mean oocyte diameters o f the p i l o t e x p e r i ment i n m i l l i m e t e r s (The v a l u e s a r e l i s t e d i n Table II) ...  24  Histograms o f the mean percentages o f oocytes i n the stages o f oocyte m a t u r a t i o n o f the p i l o t experiment (the d a t a are p r e s e n t e d i n T a b l e III) ...  25  The mean gonadosomatic index (GSI) v a l u e s f o r males o f the long-term experiment (the data are shown i n T a b l e IV) ...  26  The mean gonadosomatic index (GSI) v a l u e s f o r females o f the long-term experiment (the d a t a are l i s t e d i n T a b l e V) . ...  27  Histograms o f the presence and absence o f the c e l l stages o f t e s t i c u l a r m a t u r a t i o n o f the long-term experiment ...  28  Histograms o f the mean percentages o f oocytes i n the stages o f oocyte m a t u r a t i o n o f the long-term experiment (the v a l u e s a r e l i s t e d i n T a b l e VI) ...  29  The mean o o c y t e diameters o f the long-term experiment, i n m i l l i m e t e r s (the v a l u e s a r e p r e s e n t e d i n T a b l e VII)  ...  30  Histograms o f t h e mean percentage o f oocytes t h a t are a t r e t i c i n the long-term experiment (the d a t a a r e l i s t e d i n T a b l e VI)  ...  31  The mean body weights o f p o o l e d males and females o f t h e long-term experiment (the d a t a are l i s t e d i n T a b l e V I I I ) ...  32  The mean t h y r o i d f o l l i c l e e p i t h e l i a l h e i g h t s o f the long-term experiment and the h i g h dose group (1.0 mg./gm.) sampled on June 14/72 (the v a l u e s are p r e s e n t e d i n T a b l e IX) ...  33  viii  List  of Figures  (cont'd)  Figure  33  34  Page  Histograms o f the mean percentages o f oocytes i n the stages o f o o c y t e m a t u r a t i o n o f the s e q u e n t i a l experiment (the d a t a are shown i n T a b l e X) ...  34  The d a y l e n g t h s o f the n a t u r a l p h o t o p e r i o d at 48° l a t i t u d e  56  ix  ACKNOWLEDGEMENTS  I wish t o express my s i n c e r e a p p r e c i a t i o n t o my t h e s i s s u p e r v i s o r , Dr. E.M. Donaldson, f o r s u g g e s t i n g  the problem and f o r h i s  guidance and a s s i s t a n c e throughout t h e study. A p p r e c i a t i o n i s a l s o extended t o my academic s u p e r v i s o r , Dr. W.S. Hoar, f o r h i s a d v i c e The•technical ling  d u r i n g the study.  a s s i s t a n c e o f Mrs. H. Dye and Mr. S. Shaw i n samp-  and o f Messrs. J . C u l p , R. C o r r i g a n , and J . T u e r l i n g s i n c a r i n g  f o r t h e f i s h i s g r a t e f u l l y acknowledged. I wish t o thank Mrs. K. Kramer f o r p r o c e s s i n g  and b l o c k i n g t h e  t i s s u e s and, along w i t h Mr. J . McBride, f o r advice procedure.  on h i s t o l o g i c a l  Miss D. Hards and Mr. K. Khoo a l s o h e l p e d  i n t i s s u e pre-  paration. I a l s o wish t o thank Dr. T. E v e l y n S t a t i o n f o r t h e autopsy o f d i s e a s e d sence o f k i d n e y Mr.  f i s h and f o r i d e n t i f y i n g t h e p r e -  disease.  G. Shaw, V e t e r i n a r y M e d i c a l  generously  o f t h e Nanaimo B i o l o g i c a l  Department, A y e r s t  Laboratories,  supplied the methallibure.  I f u r t h e r wish t o acknowledge Mrs. P. Waldron f o r t y p i n g t h e f i n a l manus c r i p t . T h i s r e s e a r c h was completed w i t h t h e support Research Board  grant.  from a F i s h e r i e s  1  INTRODUCTION  The m a n i p u l a t i o n o f environmental  f a c t o r s and the s e l e c t i v e  of p h a r m a c o l o g i c a l agents are important production i n f i s h . v e r y important  techniques used t o c o n t r o l r e -  Of the environmental  f a c t o r s , p h o t o p e r i o d has  i n f l u e n c e on r e p r o d u c t i o n i n f i s h i n h a b i t i n g  l a t i t u d e s where s e a s o n a l d a y l e n g t h f l u c t u a t i o n s are g r e a t Eddy, 1951;  Corson,  L i v i n g s t o n , 1971;  1955;  Henderson, 1963;  de Vlaming,  1972).  (Hazard  s p r i n g and d e c r e a s i n g daylengths o f f a l l .  and  (Salvelinus daylengths  I f these s e a s o n a l changes  are a c c e l e r a t e d , gonadal m a t u r a t i o n and subsequent Henderson, 1963)  and  W i t h i n the salmonids, p h o t o p e r i o d  f o n t i n a l i s ) which r e p r o d u c t i v e l y mature d u r i n g the i n c r e a s i n g  (Corson, 1955;  a  temperate  Wiebe, 1968b; Poston  has been s t u d i e d most e x t e n s i v e l y i n the brook t r o u t  of  use  spawning are advanced  suggesting that maturation i s regulated  by the s e a s o n a l d a y l e n g t h f l u c t u a t i o n s .  In p i n k salmon, females mature  d u r i n g d e c r e a s i n g , i n c r e a s i n g , and then d e c r e a s i n g d a y l e n g t h s w h i l e males mature l a t e r d u r i n g i n c r e a s i n g and then d e c r e a s i n g d a y l e n g t h s . i n v e s t i g a t e the importance  o f t h e s e s e a s o n a l changes i n d a y l e n g t h  gonadal m a t u r a t i o n o f p i n k salmon, a c o n s t a n t 12 hours dark p h o t o p e r i o d was  used throughout  maturation.  light:12  S h i r a i s h i and  To  on  hours Fukuda  (1966) found t h a t c o n s t a n t s h o r t p h o t o p e r i o d s s t i m u l a t e d w h i l e constant long p h o t o p e r i o d s r e t a r d e d gonadal m a t u r a t i o n o f f o u r s p e c i e s o f which spawn d u r i n g the a p p r o x i m a t e l y equal l i g h t d u r i n g the s h o r t d a y l e n g t h o f w i n t e r .  The  and dark o f f a l l  t h e r e f o r e , i s thought  and  constant equal p h o t o p e r i o d  used on the p i n k salmon which spawn d u r i n g the equal l i g h t fall,  salmonids  and dark o f  to be more a p p r o p r i a t e than l o n g e r or s h o r t e r  2  constant One  photoperiods. of the p h a r m a c o l o g i c a l  agents known t o i n h i b i t  tion i n teleosts i s methallibure d e r i v a t i v e o f b i s - t h i o u r e a and l e v e l to i n h i b i t  ( I . C . I . 33,828).  gonadal f u n c t i o n by p r e v e n t i n g  haviour.  et a l . , 1961;  B o r i s et_ al_. ,  1972), r e p t i l e s and  (Rangneker and  Carew, 1968;  et a l . ,  1971;  Rastogi  M a r t i n and  a l s o has  (Sykes,  and h y p o p h y s i a l  function  (Hoar e_t al_.,  1963,  1964;  with  et_ al_.,  1972), and  Bromage, 1970;  fish  The  Hinde,  (Kanakaraj  (Hoar et a l . ,  Pandey, 1970a, b;  Billard  an i n h i b i t o r y e f f e c t  level 1967;  r e l e a s e o f gonadoon t h y r o t r o p i n  synthe-  M e t h a l l i b u r e a c t s at the hypo-  and r e s u l t s i n s u p p r e s s i n g t h y r o i d a l Rangneker and  K u l k a r n i , 1969;  Bourke e t a l . , presumably  i t the a b i l i t y to c o m p e t i t i v e l y b i n d i o d i n e i n the  consequently  1970).  S t e e l and  M e t h a l l i b u r e i s c h e m i c a l l y s i m i l a r to t h i o u r e a and  of thyroxine synthesis. and  be-  Hemsworth et a l . ,  K u l k a r n i , 1969), amphibians  r e l e a s e from the p i t u i t a r y .  thalamic  shares  the  Hyder, 1972).  tropin, methallibure  1971).  maturation  c h a r a c t e r i s t i c s and r e p r o d u c t i v e  In a d d i t i o n t o i t s e f f e c t on the s y n t h e s i s and  s i s and  Fawke,  consequently,  G e r r i t s and Johnson, 1965;  1971), b i r d s  Gangadhara, 1967;  1967;  and,  release  M e t h a l l i b u r e i s e f f e c t i v e i n i n h i b i t i n g gametogenesis i n  mammals (Paget 1968;  Brown and  By i n h i b i t i n g gonadal  steroid production  s t e r o i d dependent secondary s e x u a l  hypophysial  the s y n t h e s i s and  (Malven, 1971;  Labhsetwar and Walpole, 1972).  i n f i s h , m e t h a l l i b u r e prevents  It i s a non-steroidal  a c t s at the hypothalamic and  o f gonadotropins from the p i t u i t a r y 1972;  gonadal matura-  process  T h i s would lower the amount o f a v a i l a b l e i o d i n e  reduce t h y r o x i n e p r o d u c t i o n  (Pandey and  Leatherland,  amount o f feedback on the hypothalamus and p i t u i t a r y would  3  i n t u r n , be reduced stimulation.  c a u s i n g i n c r e a s e d t h y r o t r o p i n r e l e a s e and t h y r o i d  The combined a c t i o n s on t h e hypothalamus and p i t u i t a r y  and on t h e t h y r o i d determines m e t h a l l i b u r e ' s e f f e c t upon t h y r o i d function and  ( T u l l o c h e t a l . , 1963; K u l k a r n i , 1969).  Walpole, 1965; Hoar e t a l . , 1967; Rangneker  These s i t e s o f a c t i o n by m e t h a l l i b u r e a r e shown  i n F i g u r e 1. The p r a c t i c a l importance o f i n h i b i t i n g gonadal m a t u r a t i o n dent c o n s i d e r i n g the l i f e occurs  c y c l e o f p i n k salmon.  d u r i n g J a n u a r y and February  migration during A p r i l  and May.  Hatching  strict ing  occurs  two y e a r l i f e  populations  normally  and maximum emergence and downstream  The f i s h remain at s e a u n t i l the l a t e  summer o f t h e i r second y e a r at which time they migrate Spawning mainly  into  freshwater.  d u r i n g September and'October (Neave, 1966).  c y c l e maintains  two r e p r o d u c t i v e l y i s o l a t e d spawn-  and one i n odd y e a r s .  In the n o r t h e r n  escapement i n odd years i s v e r y low w h i l e t h a t o f even y e a r s  latitudes, i s much  T h i s d i f f e r e n c e between the odd and even y e a r p o p u l a t i o n s i s  l e s s marked i n the m i d - l a t i t u d e s o f the coast and c o m p l e t e l y the southern  regions  (Neave, 1962).  opposite i n  In an attempt t o i n c r e a s e t h e e s -  capement o f these Wpobr'i-runs , t r a n s p l a n t s from streams w i t h h i g h 1  ment t o those with  low escapement have been undertaken.  almost a l l these programs, the p e r c e n t sufficient 1969;  This  o f p i n k salmon on the coast o f B r i t i s h Columbia: one  spawning i n even y e a r s  higher.  i s evi-  t o support  However, i n  o f r e t u r n i n g a d u l t s has n o t been  a self-sustaining population  Walker and L i s t e r , 1971).  escape-  (Neave, 1965; E l l i s ,  The o n l y a l t e r n a t i v e would be t o a l t e r  the r e p r o d u c t i v e c y c l e o f p i n k salmon spawning i n , f o r example, t h e "good" odd years i n the southern  r e g i o n s o f B r i t i s h Columbia, so t h a t  4  Figure 1:  The hormone and steroid pathways of the gonad and thyroid that are influenced by methallibure. •*'•:'. steps directly inhibited by methallibure GRF - gonadotropin releasing factor TIF - thyrotropin inhibiting,'factor GTH - gonadotropin TSH - thyrotropin 1 - Baker (1969) 2 - Farner and Follett (1966) 3 - Gorbman (1969) 4 - Peter (1970) 5 - Peter (1971) 6 - Sage and Bromage (1970a,b)  PHOTOPERIOD2  RECEPTION & PROCESSING JL  HYPOTHALAMUS  negative  release  GRF-1  :::«  GTH J  negative  4,5  positive-  PITUITARY  synthesis  GTH  ' negative1 1.5  release  iodine3 incorporation in thyroxine synthesis  feedback  -SEX  STEROIDS  THYROXINE  gonad maturation effects on metabolism reproductive behaviour secondary sex characteristics  feedback  5  subsequent  g e n e r a t i o n s would spawn i n the "poor" even y e a r s .  Funk and Donaldson  (1972) and Funk et_ al_.  p u r i f i e d salmon (Oncorhynchus tshawytscha) attempt  (1973) used  gonadotropin  partially  (SG-G100) i n an  t o advance the gonadal m a t u r a t i o n o f male and female p i n k salmon  by one y e a r .  The males c o m p l e t e l y matured w i t h i n 98 days o f treatment  and thus w i t h i n the y e a r o f t h e i r h a t c h i n g . not mature u n t i l  The  females, however, d i d  seven months p r i o r t o the normal m a t u r a t i o n p e r i o d and  e x h i b i t e d o n l y a s m a l l number o f mature o o c y t e s .  T h i s would p r o v i d e the  g e n e t i c complement from o n l y the males f o r use i n the "poor" y e a r population.  S i n c e the females  seem unable  to mature i n as s h o r t a p e r i o d o f  time as the males, i t would seem r e a s o n a b l e t o d e l a y o v a r i a n m a t u r a t i o n f o r one y e a r and a l l o w the females two y e a r s .  following  T h i s c o u l d r e s u l t i n p i n k salmon becoming s e x u a l l y mature i n  t h e i r t h i r d year of l i f e t o be used  to mature n o r m a l l y i n the  and a l l o w the g e n e t i c complement o f the  females  i n the "poor" y e a r p o p u l a t i o n .  From t h i s r a t i o n a l e , i t was  d e c i d e d t o i n v e s t i g a t e the e f f e c t s  the drug m e t h a l l i b u r e and a c o n s t a n t 12 hours  l i g h t : 12 hours  p e r i o d on gonadal m a t u r a t i o n o f male and female p i n k salmon.  dark  photo-  T h i s would  p r o v i d e i n f o r m a t i o n on the f e a s i b i l i t y o f u s i n g m e t h a l l i b u r e , and a c o n s t a n t p h o t o p e r i o d , t o d e l a y gonadal m a t u r a t i o n o f male and, c u l a r l y , female p i n k salmon.  of  perhaps parti-  6  MATERIALS AND METHODS  Three experiments were conducted: a p i l o t t h r e e d i f f e r e n t doses o f m e t h a l l i b u r e  experiment c o m p r i s i n g  (Ayerst L a b o r a t o r i e s ) t o determine  a dose s u f f i c i e n t t o i n h i b i t gonadal m a t u r a t i o n i n the subsequent exp e r i m e n t s ; a long-term experiment, i n v e s t i g a t i n g the long-term of  effects  m e t h a l l i b u r e and a c o n s t a n t 12 hours l i g h t : 12 hours dark p h o t o p e r i o d  on gonadal m a t u r a t i o n ; and a s e q u e n t i a l experiment, t o determine t h e e f f e c t o f s t a r t i n g m e t h a l l i b u r e treatment a t r e g u l a r i n t e r v a l s b e f o r e the  start  o f o v a r i a n development  development  r a t h e r than a f t e r the s t a r t o f o v a r i a n  as i n the long-term experiment where i n h i b i t i o n o f o v a r i a n  m a t u r a t i o n was u n s u c c e s s f u l .  The p i n k salmon (Ortcorhynchus gorbuscha)  used i n a l l t h r e e experiments were o b t a i n e d from the F i s h e r i e s  Research  Board S t a t i o n i n Nanaimo.  Experimental design The f i r s t  or pilot  experiment c o n s i s t e d o f f o u r groups: a c o n t r o l  and t h r e e treatment groups r e c e i v i n g 0.10 mg./gm., 0.32 mg./gm., and 1.0 mg. methallibure/gm. body weight. and sampled  I t was s t a r t e d on September 3, 1971  on October 3 and f i n a l l y on January 12, 1972.  Males were  excluded from t h i s experiment s i n c e t e s t i c u l a r m a t u r a t i o n does n o t b e g i n u n t i l May o r June.  The 1.0 mg./gm. group r e c e i v e d i n j e c t i o n s o f methal-  l i b u r e only to the f i r s t c e i v e d no i n j e c t i o n s . long-term experiment  sampling d a t e (October 3) a f t e r which i t r e -  The f i s h were o f the same s t o c k as t h o s e o f the ( j u v e n i l e s which hatched i n January o r February,  1971) and, t h e r e f o r e , p r o v i d e i n f o r m a t i o n on t h e stage o f m a t u r a t i o n and effect  o f m e t h a l l i b u r e treatment p r i o r t o the s t a r t o f the long-term  7  experiment. fiberglass  The f i s h were m a i n t a i n e d i n 144 l i t e r  a q u a r i a w i t h f l o w i n g s e a water and a e r a t i o n and exposed t o  a simulated natural photoperiod. fed  self-cleaning,  As i n a l l experiments, the f i s h were  t h r e e times d a i l y w i t h a f r o z e n mixture o f b e e f h e a r t and  canned salmon, pablum, and sodium c h l o r i d e  liver,  (Donaldson and McBride, 1967).  The second o r long-term experiment c o n s i s t e d o f f o u r groups: a c o n t r o l and treatment group under a n a t u r a l p h o t o p e r i o d and a c o n t r o l and treatment group under a constant 12 hours l i g h t photoperiod. group was  The experiment was  s t a r t e d on November 8, 1971 and each  sampled on J a n u a r y 15, March  f i n a l l y on September  5, 1972.  : 12 hours dark  13, June 14, August 24, and  The August and September  samples were  p o o l e d and c a l l e d the August 30 sampling because o f the s i m i l a r i t y i n the  parameters measured  samplings.  and the s h o r t time d i f f e r e n c e between the two  The n a t u r a l p h o t o p e r i o d treatment group t e r m i n a t e d on June 14  because o f complete m o r t a l i t y due t o k i d n e y . d i s e a s e .  Thyroid tissue  was  removed on the June sampling t o determine i f me'thallibure has an a n t i thyroidal  effect.  An a d d i t i o n a l group from the same s t o c k o f f i s h  which  had j u s t r e c e i v e d one month o f treatment w i t h a h i g h dose o f m e t h a l l i b u r e (1.0 mg./gm.) was  a l s o sampled to i n v e s t i g a t e the e f f e c t s o f a h i g h e r  dose on the t h y r o i d .  T h i s h i g h dose group had p r e v i o u s l y r e c e i v e d i n -  j e c t i o n s o f the s u s p e n s i o n agent, Tween 80 and d i s t i l l e d water, f o r the d u r a t i o n o f the p i l o t t h y r o i d t i s s u e was  experiment which ended on January 12, 1972.  f i x e d i n B o u i n - H o l l a n d e sublime ( K r a i c e r et_ a l . ,  and s t a i n e d w i t h P e r i o d i c a c i d - S c h i f f  ( C u l l i n g , 1957).  h e i g h t s from 30 t h y r o i d f o l l i c l e s p e r f i s h were measured  The 1967)  Epithelial microscopically  8  u s i n g an o c u l a r micrometer.  The f i s h were maintained i n 700  a q u a r i a w i t h f l o w i n g s e a water and a e r a t i o n .  Two l i g h t p r o o f photo-  p e r i o d e n c l o s u r e s were each i l l u m i n a t e d by f o u r , 48 i n c h f l u o r e s c e n t tubes. "Astro-clock".  liter  daylight  The n a t u r a l daylengths were s i m u l a t e d u s i n g an  The constant 12 hours  l i g h t : 12 hours dark p h o t o p e r i o d  was m a i n t a i n e d from 7:00 AM t o 7:00 PM by an " i n t e r m a t i c " time s w i t c h . During the l a s t f o u r months o f the experiment,  the f i s h were kept i n  f o u r l a r g e t e n f o o t diameter f i b e r g l a s s a q u a r i a w i t h f l o w i n g s e a water and a e r a t i o n . maintained. trolled  The a q u a r i a were l i g h t p r o o f e d and the same i l l u m i n a t i o n The water temperature  i n a l l the experiments  and f l u c t u a t e d s e a s o n a l l y .  was not con-  High m o r t a l i t y i n a l l f o u r  groups  from k i d n e y d i s e a s e prompted the use o f a n t i b a c t e r i a l agents.  The drug  s u l f a m e r i z i n e was i n c o r p o r a t e d i n t o the d i e t o f a l l the groups  at a dose  o f 50 mg. sulfamerizine/Kgm.  treatment,  the drug "Furanase"  f i s h / d a y f o r 7 days.  During t h i s  (P-7138) was added t o the a q u a r i a water o f the na-  t u r a l p h o t o p e r i o d treatment group  (which had the g r e a t e s t m o r t a l i t y ) at  a p p r o x i m a t e l y 1 ppm. f o r 50 min.  A f t e r s u l f a m e r i z i n e treatment, the  a n t i b i o t i c e r y t h r o m y c i n was i n c o r p o r a t e d i n the d i e t o f a l l groups dose o f 100 mg. erythromycin/Kgm. f i s h / d a y f o r 21 days.  at a  These treatments  were s t a r t e d on June 9 and t e r m i n a t e d on J u l y 8, 1972. In the t h i r d o r s e q u e n t i a l experiment, taken from a stock p o p u l a t i o n which,  j u v e n i l e p i n k salmon were  a f t e r hatching i n January,  had been m a i n t a i n e d a t a constant temperature  1972,  o f a p p r o x i m a t e l y 12°C.  As a r e s u l t the f i s h were l a r g e r than p i n k salmon r e a r e d a t n o r m a l l y c o l d e r temperatures.  From t h i s stock p o p u l a t i o n , a group o f 12 j u v e n i l e  9  f i s h were t r a n s f e r r e d t o 144 months s t a r t i n g on May  l i t e r a q u a r i a every 28 days f o r f o u r  31, 1972.  Each group was  i n j e c t e d w i t h methal-  l i b u r e from t h e i r i n i t i a t i o n date t o the f i n a l sampling d a t e o f October 29, 1972  ( F i g . 2).  At each o f the f o u r i n i t i a t i o n s  J u l y 27, and August trol  sample was  determine  24) a zero c o n t r o l sample was  a l s o taken on the f i n a l  (May  31, June  taken.  sampling date  29,  A zero con-  (October 29) t o  the e x t e n t o f o v a r i a n m a t u r a t i o n o f the stock p o p u l a t i o n .  A c o n t r o l group was  m a i n t a i n e d t o determine  i f the s o l u t i o n o f Tween 80  and d i s t i l l e d water had an e f f e c t on gonadal m a t u r a t i o n . e x c l u d e d from t h i s experiment  Males were  s i n c e t e s t i c u l a r m a t u r a t i o n does not  be-  g i n u n t i l the s u c c e e d i n g s p r i n g . Injection  0.10  technique  The  f i s h were i n j e c t e d i i n t r a p e f - i t o n e a l l y once every two weeks w i t h  mg.  methallibure/gm. body weight  suspended  i n 0.1  ml. o f a s o l u t i o n  of one drop o f Tween 80 i n 20 ml. o f d i s t i l l e d water (except f o r the pilot  experiment  which i n c o r p o r a t e d a range o f d o s e s ) .  Control f i s h  were i n j e c t e d o n l y w i t h the s o l u t i o n o f Tween 80 and d i s t i l l e d water. 26 gauge, 3/4  i n c h d i s p o s a b l e hypodermic n e e d l e and 1 ml. d i s p o s a b l e sy-  r i n g e were used. the f i r s t 5 mm.  A  The  i n j e c t i o n s i t e f o r most o f the p i l o t  f o u r i n j e c t i o n s o f the long-term experiment  d o r s a l l y and a n t e r i o r l y from the anus.  was  and  approximately  T h i s can, however, r e s u l t  i n an i n t r a i n t e s t i n a l i n j e c t i o n and l o s s o f m e t h a l l i b u r e . s i t e a p p r o x i m a t e l y 5 mm.  experiment  An  injection  d o r s a l l y and a n t e r i o r l y from the base o f each  p e l v i c f i n ( a l t e r n a t i n g s i d e s ) was  used f o r subsequent  injections  the success o f an i M t r a p e f i t ' o n e a l i n j e c t i o n at t h i s s i t e was  since  quite high.  10  Figure 2:  Design of the sequential experiment showing the period of methallibure treatment from the starting date to the final sampling date of Oct. 29/72 for each of the four groups.  4-I  3-  o on O 2-  period  1-  MAY  • JUNE  1  of m e t h a l l i b u r e  JULY  ' 1972  AUG  1  treatment  SEPT  1  OCT  1  11  F i s h were a n e s t h e t i z e d f o r a p p r o x i m a t e l y 45 min. w i t h 10 ppm. quinaldine  (Locke, 1969)  of  i n the a q u a r i a water f o r most o f the p i l o t  periment and the f i r s t p a r t o f the long-term experiment.  This concentra-  t i o n and l e n g t h o f exposure, however, r e s u l t e d i n m o r t a l i t y which have been due t o e x c e s s i v e exposure and c o n c e n t r a t i o n (Marking, To reduce the exposure  d u r i n g subsequent  Sampling  i n j e c t i o n s , the f i s h were  technique  group at each sampling.  and the body weight  (measured  each  The f i s h were a n e s t h e t i z e d i n q u i n a l d i n e  k i l l e d by s e v e r i n g the s p i n a l c o r d .  length  ppm.  T h i s r e s u l t e d i n reduced m o r t a l i t y .  A minimum o f t h r e e males and t h r e e females were taken from  each f i s h  may  1969).  a n e s t h e t i z e d f o r o n l y 10 min. i n a l a r g e bucket c o n t a i n i n g o n l y 5 of quinaldine.  ex-  E x c e s s i v e moisture was  (measured  to the n e a r e s t 0.1  and  blotted gm.),  from  fork  t o the n e a r e s t m i l l i m e t e r ) , r e l a t i v e amount o f methal-  l i b u r e i n the coelom, presence o f k i d n e y d i s e a s e , and g e n e r a l appearance o f the f i s h were r e c o r d e d .  Both gonads were removed, f i x e d , p a r -  t i a l l y dehydrated i n 50 and 70 p e r c e n t a l c o h o l , and then weighed t o the nearest milligram. ies  The  at the f i n a l two  l a r g e s i z e o f the maturing t e s t e s and some ovar-  sampling dates of the long-term experiment  t a t e d weighing both t e s t e s or o v a r i e s f r e s h r a t h e r than f i x e d . the  start  necessiP r i o r to  o f each experiment o r treatment, a zero c o n t r o l sample  taken t o determine the i n i t i a l " s t a t e o f gonadal development. somatic index was  c a l c u l a t e d f o r each f i s h by the f o r m u l a :  The  was gonado-  12  =  All  statistical  gonad weight i n grams body weight m grams  comparisons  H i s t o l o g i c a l procedure and One  were done w i t h the Student's  t-test.  analysis  or both whole gonads were f i x e d i n Bouin's f l u i d f o r a p p r o x i -  mately 24 h o u r s , dehydrated, and r o u t i n e l y imbedded i n P a r a p l a s t 56-57°C).  (mpt.  The gonads were s e c t i o n e d at 6 microns 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 u l l i n g , 1957).  Oocyte diameters were  measured f o r the p i l o t  and long-term experiments m i c r o s c o p i c a l l y u s i n g  an o c u l a r micrometer.  The diameter was  c a l c u l a t e d according to Braekevelt  and M c M i l l a n (1967) from the f o r m u l a :  oocyte diameter =. J  T  T  i  z—r=  k v g r e a t e s t diameter x l e a s t  where k i s the f a c t o r which the  —T~T-  z—  diameter  c o n v e r t s the o c u l a r micrometer measures o f  g r e a t e s t and l e a s t oocyte diameters i n t o m i l l i m e t e r s .  From the  p r e p a r e d s l i d e s o f median s e c t i o n s o f o v a r i e s , as many oocytes as poss i b l e from each f i s h were chosen f o r measurement on the b a s i s o f b e i n g i n the  most abundant stages o f m a t u r a t i o n f o r t h a t ovary and h a v i n g the  major p a r t o f t h e i r n u c l e u s p r e s e n t i n the s e c t i o n . i n the l a t e r stages of m a t u r a t i o n a t the August  O v a r i e s w i t h oocytes  30 sampling o f the l o n g -  term experiment were d i f f i c u l t to p r e p a r e h i s t o l o g i c a l l y . s i t a t e d measuring  25 whole oocytes m i c r o s c o p i c a l l y under  O v a r i a n m a t u r a t i o n was  T h i s neceslow m a g n i f i c a t i o n .  measured u s i n g the combined schemes o f  I s h i d a et a l . (1961) and Yamazaki  (1965):  13  First  Growth Phase  Stage 1:  early (Fig.  perinucleolus 3)  -haematoxylin p o s i t i v e p a l l i a l  layer present  Stage 2:  i-':^ p e r i v u c h a ^ m a t o x y l i n p o s i t i v e cytoplasm  Stage 2:  late-p«rinuc4e&l!us ' oxv  (Fig.  4)  -pallial  i  " ; ; j 3 i t i v e cytoplasm  layer  absent  -cytoplasm weakly h a e m a t o x y l i n p o s i t i v e Second Growth Phase Stage 3:  early yolk v e s i c l e (Fig.  4)  -a l a y e r o f y o l k v e s i c l e s appear i n t h e peripheral -follicle  cytoplasm  and t h e c a l  cell  layers  are s i n g l e  c e l l e d and squamous Stage 4:  late yolk v e s i c l e (Fig.  5)  - m a j o r i t y o f t h e cytoplasm c o n t a i n s y o l k vesicles -follicle -thecal  l a y e r i s l e s s squamous  l a y e r i s squamous  -zona r a d i a t a i s p r e s e n t but t h i n Stage 5:  o i l globule (Fig.  6)  - d i s t i n c t v a c u o l e s appear around the n u c l e u s which are u s u a l l y  l a r g e r than y o l k v e s i c l e s  -yolk v e s i c l e s increase -follicle -thecal  i n size  layer i s cuboidal  l a y e r i s squamous  -zona r a d i a t a i n c r e a s e s  i n t h i c k n e s s and now  exhibits radial striations  14 Stage 6: p r i m a r y y o l k g l o b u l e (Fig.  7)  -a l a y e r o f s m a l l haematoxylin p o s i t i v e appear i n the p e r i p h e r a l -follicle  l a y e r remains  granules  cytoplasm  single  celled  - t h e c a l l a y e r i s l e s s squamous and i s u s u a l l y two  cell  layers  thick  -zona r a d i a t a i n c r e a s e s i n t h i c k n e s s Stage  7:  secondary y o l k g l o b u l e (Fig.  8)  - m a j o r i t y o f the cytoplasm i s f i l l e d w i t h y o l k g l o b u l e s which i n c r e a s e i n s i z e and number -zona r a d i a t a i n c r e a s e s i n t h i c k n e s s and  radial  s t r i a t i o n s are pronounced -oocyte growth i s g r e a t e s t d u r i n g t h i s - t h i s was The  the most advanced stage  stage  observed.  extent o f oocyte m a t u r a t i o n f o r an e x p e r i m e n t a l group was  termined as the mean percentage  de-  o f oocytes i n each o f t h e s e s t a g e s .  Only oocytes i n which the major p a r t o f the n u c l e u s i s p r e s e n t i n the s e c t i o n were r e c o r d e d .  In o v a r i e s w i t h oocytes i n the y o l k g l o b u l e  stages, morphological d i s t o r t i o n during r o u t i n e processing n e c e s s i t a t e d r e c o r d i n g o n l y the most advanced stage o f m a t u r a t i o n f o r each The  extent o f oocyte m a t u r a t i o n f o r these groups was  percentage  o f o v a r i e s i n each s t a g e .  June 14 and August 30, 1972 overcome t h i s problem  samplings  T h i s was  determined  as the  o n l y n e c e s s a r y i n the  o f the long-term experiment.  To  o f d i s t o r t i o n i n d e t e r m i n i n g the s t a g e o f oocyte  m a t u r a t i o n , a s m a l l p o r t i o n o f each ovary from the August 30 was  ovary.  embedded i n a c r y l a m i d e g e l and s e c t i o n e d a t 14-16  microns  sampling on a  15  Figure 3:  Ovary approximately three months prior to the start of vitellogenesis showing oocytes in the early perinucleolus stage (x60). - the dark pallial layer is evident in the peripheral cytoplasm.  Figure 4:  Ovary at the start of vitellogenesis showing oocytes in the late perinucleolus stage (uniform cytoplasm) and early yolk vesicle stage (yolk vesicles in the peripheral cytoplasm)(x60).  Figure 5:  Oocytes in the late yolk vesicle stage (x60).  Figure 6:  The large central oocyte is in the oil globule stage which is characterized by large vesicles surrounding the nucleus (x60). - the increase in oocyte diameter during this maturation is evident comparing Figures 3-6.  Figure 7:  Oocyte in the primary yolk globule stage (x48). - this is indicated by the faint dark band of granules amongst the vesicles in the peripheral cytoplasm.  Figure 8:  Oocyte in the secondary yolk globule stage (xl9). - the majority of the cytoplasm is f i l l e d with yolk globules. - the zona radia.ta:l:ayer (dark grey band adjacent to the peripheral cytoplasm) is thick and prominent at this stage. - double imbedded in celloidin and paraffin.  16  c r y o s t a t at -20°C  (Orlowski et_ al_. , 1971).  double imbedding was (Culling,  C e l l o i d i n and  paraffin  a l s o t r i e d , but o n l y on a s m a l l amount o f t i s s u e  1957).  T e s t i c u l a r m a t u r a t i o n was  measured f o r the long-term experiment  as  the presence o r absence o f the f o l l o w i n g spermatogenic s t a g e s : p r i m a r y spermatogonia, secondary spermatogonia, p r i m a r y spermatocytes, secondary spermatocytes, s p e r m a t i d s , and spermatozoa.  These c e l l s s t a g e s were  i d e n t i f i e d h i s t o l o g i c a l l y from I s h i d a et_ al_.  (1961) and are shown i n  F i g u r e s 9, 10, and  11.  17  Figure 9:  Testis approximately six months prior to the start of testicular maturation (x600). - the arrow indicates a representative example of primary spermatogonia. - spermatogonia! mitosis is evident at the top middle of the micrograph.  Figure 10:  Testis at the beginning of maturation exhibiting secondary spermatogonia (arrow indicates a representative example)(x600). - at this stage, lobule formation has just begun. - spermatogonia! mitosis is evident in the lower middle of the micrograph.  Figure 11:  Testis approaching final maturity and exhibiting the following series of cell stages: primary spermatocytes (1° SPCY), secondary spermatocytes (2° SPCY), spermatids (SPTD), and spermatozoa (SPZ)(x600).  18  RESULTS  In the f i r s t  or p i l o t  experiment,  a l l t h r e e doses o f m e t h a l l i b u r e  reduced the r a t e o f i n c r e a s e i n t h e GSI o f females compared t o the c o n t r o l ; t h e h i g h dose  (1.0 mg./gm.) s i g n i f i c a n t l y  (p<0.05) on both the  sampling dates o f O c t . 3/71 and J a n . 12/72 ( F i g . 22, T a b l e I ) . doses  All  reduced the r a t e o f i n c r e a s e i n oocyte diameter a l s o , but n o t  s i g n i f i c a n t l y compared t o the c o n t r o l on the J a n . 12/72 sampling ( F i g . 23, T a b l e I I ) .  M e t h a l l i b u r e a l s o had no e f f e c t on t h e s t a g e o f oocyte  m a t u r a t i o n on the O c t . 3/71 sampling mg./gm. dose group  ( F i g . 24, T a b l e I I I ) . The 0.32  i s s i m i l a r t o the c o n t r o l group which e x h i b i t s  oocytes  m a i n l y i n the l a t e p e r i n u c l e o l u s and e a r l y y o l k v e s i c l e stages w h i l e the 0.10 mg./gm. and 1.0 mg./gm. dose groups  are more advanced  h i b i t i n g oocytes i n the l a t e y o l k v e s i c l e s t a g e . h i g h dose group l e s s advanced  i n a l s o ex-  On J a n . 12/72, t h e  ( i n j e c t i o n s o f 1.0 mg./gm. t e r m i n a t i n g on O c t . 3/71) was  than t h e o t h e r groups, i n c l u d i n g the c o n t r o l , because o f  the h i g h e r p r o p o r t i o n o f the e a r l y stages o f m a t u r a t i o n and the absence of oocytes i n the o i l g l o b u l e stage  (Fig.  In the second o r long-term experiment, p o o l e d sampling  24). a t t h e time o f the f i n a l  (Aug. 30/72), m e t h a l l i b u r e treatment w i t h a constant  12L:12D p h o t o p e r i o d s i g n i f i c a n t l y reduced t h e r a t e o f i n c r e a s e o f GSI i n males  (p<0.01) and females  (p<0.05) compared t o both c o n t r o l s ( F i g .  25, T a b l e IV and F i g . 26, T a b l e V ) .  The s i m i l a r i t y i n the GSI o f f e -  males i n t h e n a t u r a l p h o t o p e r i o d treatment group t o both c o n t r o l s on t h e June 14/72 sampling  ( F i g . 26) i s due o n l y t o the weight  o f t h e s e treatment  19  o v a r i e s s i n c e the body weights tion of this 12D  ( F i g . 31) and stages o f o o c y t e matura-  t r e a t e d group ( F i g . 28) are s i m i l a r to the c o n s t a n t  p h o t o p e r i o d treatment  m e t h a l l i b u r e treatment  group.  From the June and August  samplings,  w i t h n a t u r a l or c o n s t a n t 12L:12D p h o t o p e r i o d  completely i n h i b i t e d t e s t i c u l a r maturation sampling  12L:  ( F i g . 27).  In the June  i t i s e v i d e n t t h a t m e t h a l l i b u r e p r e v e n t e d the t r a n s f o r m a t i o n  o f p r i m a r y spermatogonia  i n t o secondary  spermatogonia  ( F i g s . 12,  13  14) .  Both c o n t r o l s o f the August sampling  15) .  In the females, m e t h a l l i b u r e w i t h c o n s t a n t 12L:12D p h o t o p e r i o d  o n l y slowed  the r a t e o f o v a r i a n m a t u r a t i o n  At the August sampling, globule  e x h i b i t e d spermatozoa ( F i g .  compared t o both  controls.  the most advanced stage o f t h i s group was o i l  ( F i g . 17) whereas i n the c o n t r o l s . i t was  ( F i g . 28 T a b l e VI and F i g . 18). photoperiod  and  a l s o o n l y slowed  secondary  yolk globule  M e t h a l l i b u r e w i t h c o n s t a n t 12L:12D  the r a t e o f i n c r e a s e i n oocyte  diameter,  but not s i g n i f i c a n t l y compared t o both c o n t r o l s ( F i g . 29, T a b l e V I I ) . Oocyte a t r e s i a i n c r e a s e d throughout treatments  ( F i g . 30, T a b l e V I ) .  the experiment  M e t h a l l i b u r e reduced  i n body weight o f p o o l e d males and females controls The  i n both c o n t r o l s and the r a t e o f i n c r e a s e  (p<0.01) compared t o both  ( F i g . 31, T a b l e V I I I ) . constant  i n the GSI  12L:12D p h o t o p e r i o d alone reduced  and body weight o f males and females  the r a t e o f i n c r e a s e  ( F i g . 25, T a b l e  IV;  F i g . 26, T a b l e V; and F i g . 31, T a b l e V I I I ) and i n the oocyte diameter females  ( F i g . 29, T a b l e VII) compared t o the n a t u r a l p h o t o p e r i o d  t r o l , but o n l y s i g n i f i c a n t l y i n the GSI 12L:12D p h o t o p e r i o d slowed  o f males  t e s t i c u l a r maturation  (p<0.01). a t the June  The  of  conconstant  sampling  20  where secondary primary  spermatogonia  spermatocytes  were the most advanced stage compared t o  o f the n a t u r a l p h o t o p e r i o d c o n t r o l .  In August,  both c o n t r o l s e x h i b i t e d spermatozoa but some f i s h i n the c o n s t a n t 12L: 12D p h o t o p e r i o d c o n t r o l group showed complete i n h i b i t i o n o f t e s t i c u l a r maturation  ( F i g . 27).  The constant  maturation  a t the June sampling  12L:12D p h o t o p e r i o d slowed  ovarian  where the most advanced stage was o i l  g l o b u l e compared t o p r i m a r y y o l k g l o b u l e i n the n a t u r a l p h o t o p e r i o d t r o l group secondary  ( F i g . 28, T a b l e V I ) .  con-  In August, both c o n t r o l s e x h i b i t e d the  y o l k g l o b u l e stage but two out o f t h r e e f i s h showed t h i s  stage  under n a t u r a l p h o t o p e r i o d w h i l e o n l y one out o f seven f i s h showed t h i s stage under c o n s t a n t 12L:12D p h o t o p e r i o d . The treatment  thyroid f o l l i c l e  e p i t h e l i a l h e i g h t s o f the n a t u r a l p h o t o p e r i o d  and c o n s t a n t 12L:12D p h o t o p e r i o d  c o n t r o l and treatment are  s i g n i f i c a n t l y g r e a t e r (p<0.01, p<0.01, and p<0.05, r e s p e c t i v e l y ) than the n a t u r a l p h o t o p e r i o d c o n t r o l and h i g h dose group In the t h i r d o r s e q u e n t i a l experiment,  ( F i g . 32, T a b l e I X ) .  m e t h a l l i b u r e had no  effect  on o v a r i a n m a t u r a t i o n even when a d m i n i s t e r e d b e f o r e and d u r i n g the v e r y e a r l y stages o f m a t u r a t i o n  ( F i g . 33, T a b l e X ) .  Each o f the f o u r groups  showed a l e v e l o f m a t u r a t i o n h i g h e r than t h e i r r e s p e c t i v e zero c o n t r o l s and s i m i l a r t o the c o n t r o l and zero c o n t r o l sampled on O c t . 29/72 19, 20, and 21).  (Figs.  The s i m i l a r i t y i n the extent o f o v a r i a n m a t u r a t i o n o f  t h i s c o n t r o l and zero c o n t r o l sampled on Oct. 29 i n d i c a t e s t h a t the s o l u t i o n o f Tween 80 and d i s t i l l e d water had no e f f e c t on the ovary. M e t h a l l i b u r e had no e f f e c t on the r a t e o f i n c r e a s e i n GSI. groups i n c r e a s e d at a s i m i l a r r a t e as the s t o c k p o p u l a t i o n .  The t r e a t e d  21  Figure 12: Testis showing secondary spermatogonia from the constant 12L:12D photoperiod control group of the long-term experiment on June 14 (x600). Figure 13: Testis showing primary spermatogonia from the constant 12L: 12D photoperiod treatment group of the same sampling as above (x600). Figure 14: Zero control testis of the long-term experiment, sampled on November 8/71, showing primary spermatogonia (x600). Figure 15: Testis showing spermatozoa (arrow) from the constant 12L:12D photoperiod control group of the long-term experiment from the August 30 sampling (x600). Figure 16: Testis showing primary spermatogonia from the constant 12L:12D photoperiod treatment group of the same sampling as above (x600).  22  Figure 17: Oocytes in the oil globule stage from the constant 12L:12D photoperiod treatment group of the long-term experiment from the August 30 sampling (xl9). - imbedded in acrylamide gel. Figure 18: Oocytes in the secondary yolk globule stage from the constant 12L:12D photoperiod control group from the same sampling as above (xl9). - the difference between the oocyte diameters of control and treatment fish is evident comparing these oocytes with those of Figure 17. Figure 19: Ovary with oocytes in the early perinucleolus stage from the start of the June 29 group of the sequential experiment (x60). Figure 20: Oocytes in the late yolk vesicle stage from methallibure treated fish of the June 29 group at the end of the sequential experiment (October 29)(x60). Figure 21: Oocytes in the late yolk vesicle stage from control fish of the sequential experiment sampled on October 29 (x60).  23  Figure 22:  The mean gonadosomatic index (GSI) values for females of the pilot experiment (the data are listed in Table I ) . (gzero control * injections of this high dose group were terminated on Oct. 3/71.  F E M A L E S  SE PT  1  OC"T  1  FTov  1971  1  D T C  1  T A N  1  1972  24  Figure 23:  The mean oocyte diameters of the pilot experiment, in millimeters (the values are listed in Table I I ) . ©zero control * injections of this high dose group were terminated on Oct. 3/71.  S  E  P  T  «  o c l  •  N  1971  O  V  1  D  T  C  1  T  A  N  1972  1  25  Figure 24:  Histograms of the mean percentages of oocytes in the stages of oocyte maturation of the pilot experiment (the data are presented in Table III). * injections of this high dose group were terminated on Oct. 3/71. 1 - early perinucleolus stage 2 - late perinucleolus stage 3 - early yolk vesicle stage 4 - late yolk vesicle stage 5 - o i l globule stage  •100%  l.Omg.  1 2  3  4  1  2  3  •0  4  r  a. 1  100%—  100%  0.32 mg. 2  3  1  2  3  4  5 1-100 %  ZERO CONTROL  0.10 mg. 0-  1  2 1 2  3  4  1  2  3  4  5  «-0 r  ioo% CONTROL  1 2  SEPT. 3  OCT. 3 1971  3  4  1  2  3  JAN.12 1  972  4  5  ^0  26  Figure 25: The mean gonadosomatic index (GSI) values for males of the long-term experiment (the data are shown in Table IV). - closed triangles - natural photoperiod - open triangles - constant 12L:12D photoperiod - solid lines - control - dashed lines - treatment ©zero control — natural photoperiod treatment group - the designation of this group was omitted because of lack of space.  This group was terminated on June 14  because of high mortality due to kidney disease.  MALES  1971  1972  27  Figure 26: The mean gonadosomatic index (GSI) values for females of the long-term experiment (the data are listed in Table V). NAT. PHOTO. - natural photoperiod CONST. PHOTO. - constant 12L:12D photoperiod ©zero control - the natural photoperiod treatment group terminated on June 14 because of high mortality due to kidney disease.  F E M A LES  0  I N O V l D E C ' J A N I FEB ' M A R APR " M A Y " JUN> J U L ' A U G ' 1971 1972 1  28  Figure 27: Histograms of the presence and absence of the cell stages of testicular maturation of the long-term experiment. - the group designations above each histogram on the Jan. 15 sampling also apply to the other three sampling dates. - stippled portions - presence of those cell stages - light portions - absence of those cell stages 1° SPGN - primary spermatogonia 2° SPGN - secondary spermatogonia 1° SPCY - primary spermatocytes 2° SPCY - secondary spermatocytes SPTD - spermatids SPZ - spermatozoa  CO  <  CN  rs  mm <  treatment!:::::::::::.::|  C O N S T A N T 12:12 P H O T O P F R IQD  NATURAL  M  ii p  control |:':"::::TTj  0 0  o CN  " °z% <^>  o —  00  CONTROL  ii > - — i r ^ — I I — z  " ^>£ " ?Z X  -  PHOTOPFRIQD  ZERO  %  ~  control FT: ••:••••::! y .treatment'!; ;;;;| <  O £ z * ir  o " o Da.  (/> o CN  </)  29  Figure 28: Histograms of the mean percentages of oocytes in the stages of oocyte maturation of the long-term experiment (the values are listed in Table VI). * these histograms represent the percentage of the total number of ovaries in which only the most advanced stage of maturation was recorded because of extensive oocyte distortion in processing. 1 - early perinucleolus stage 2 - late perinucleolus stage 3 - early yolk vesicle stage 4 - late yolk vesicle stage 5 - o i l globule stage 6 - primary yolk globule stage 7 - secondary yolk globule stage  NATURAL PHOTO.  CONSTANT PHOTO.  30  Figure 29:  The mean oocyte diameters of the long-term experiment, in millimeters (the values are presented in Table VII). ©zero control - both the controls and the constant 12L:12D photoperiod treatment group were not measured on the June 14 sampling because of oocyte distortion during processing.  3-1  NOV I DEC I JANI FEB I M A R I APR I M A Y I JUNI JUL » A U G ' 1971 1972  31  Figure 30:  Histograms of the mean percentage of oocytes that are atretic in the long-term experiment (the data are listed in Table VI). - the group designations above each histogram on the Jan. 15 sampling also apply to the other three sampling dates.  o  CO  ]6 3 <  LZZI 3^  " Z  3  J  CN  O CO  <  treatmentl CONST. PHOTO., ,' control! I£ Z treatment[ < NAT. PHOTO. control Q  oo  ZERO CONTROLl1 ^ K O o z I 5€> Q 2  1 O  1 O  oo o S31XDOO DI13CJ1V  1 O  -*r lN3Dil3d  1 O  CN  1 O  32  Figure 31:  The mean body weights of pooled males and females of the long-term experiment (the data are listed in Table VIII). ©zero control  250n  1971  1972  33  Figure 32: The mean thyroid f o l l i c l e epithelial heights of the long-term experiment and the high dose group (1.0 mg./gm.) sampled on June 14/72 (the values are presented in Table IX). - the standard deviation of the means within each group is shown above and below each group mean.  NATURAL PHOTOPERIOD CONTROL TREATMENT  CONSTANT 12:12 PHOTOPERIOD CONTROL TREATMENT  HIGH DOSE  34  Figure 33:  Histograms of the mean percentages of oocytes in the stages of oocyte maturation of the sequential experiment (the data are shown in Table X). - the bottom row of zero control histograms represent the percentages of oocytes in the stages of maturation at the start of methallibure treatment for each group. - the zero control histogram on Oct. 29 represents the extent of maturation of the stock population at the end of the experiment. - the top row represents the percentages of oocytes in the stages of maturation of each of the four groups on Oct. 29 after methallibure treatment. The date of the start of methallibure treatment and the respective zero control of each group appear directly below on the bottom row. - the control group appears at the right of the top row. 1 - early perinucleolus stage 2 - late perinucleolus stage 3 - early yolk vesicle stage 4 - late yolk vesicle stage  IOOVI  CM  3  4  1  SAMPLED  ON  2  3  4  O C T . 29/72  1 2  AFTER  3  4  1  2  METHALLIBURE  3  4  1  2  3  TREATMENT CONTROL  lOOVl  0  J  1 2 :  M A Y 31  Z E R O JUNE 29  1  2  C O N T R O L S JULY 27 1972  1 2  3  A U G . 24  4  a1. 2  3 4  OCT. 29  35  Table I:  The mean gonadosomatic index (GSI) values for females of the pilot experiment. * injections of this high dose group were terminated on Oct. 3/71.  DATE  GROUP  NUMBER OF  MEAN GSI ±  SD  FEMALES 0.051  4  0.386  0.041  0.10 mg./gm.  3  0.440  0.082  0.32 mg./gm.  2  0.369  0.059  1.0 mg./gm.  3  0.315  0.042  control  3  0.752  0.065  0.10 mg./gm.  7  0.550  0.187  0.32 mg./gm.  4  0.592  0.215  * 1.0 mg./gm.  3  0.409  0.146  zero control  Oct. 3/71  control  Jan. 12/72  o  0.252  Sept. 3/71  36  Table II: The mean oocyte diameters of the pilot experiment. * injections of this high dose group were terminated on Oct. 3/71.  DATE  GROUP  NUMBER OF OOCYTES MEASURED  NUMBER OF  MEAN DIAMETER  ± SD OF THE  FEMALES  IN MILLIMETERS  MEANS  Sept. 3/71  zero control  128  3  0.223  0.024  Oct. 3/71  control  220  4  0.299  0.010  0.10 mg./gm.  144  3  0.328  0.025  0.32 mg./gm.  98  2  0.301  0.006  1.0 mg./gm.  163  3  0.306  0.055  control  123  3  0.625  0.075  0.10 mg./gm.  168  3  0.506  0.087  0.32 mg./gm.  170  4  0.510  0.058  117  3  0.492  0.119  Jan. 12/72  *1.0 mg./gm.  37  Table III: The mean percentages of oocytes in the stages of oocyte maturation of the pilot experiment. * 1 - early perinucleolus stage 2 - late perinucleolus stage 3 - early yolk vesicle stage 4 - late yolk vesicle stage 5 - o i l globule stage ** injections of this high dose group were terminated on Oct. 3/71.  DATE  GROUP  NUMBER OF  NUMBER OF  OOCYTES  FEMALES  MEAN PERCENT IN EACH STAGE  * 1  2  3  4  5  Sept. 3/71  zero control  541  4  12  ,88  Oct. 3/71  control  659  4  18  48  31  ,3  0.10 mg./gm.  383  3  13  26  28  33  0.32 mg./gm.  270  2  15  56  29  1.0 mg./gm.  514  3  30  32  19  18  control  232  3  5  3  6  78  8  0.10 mg./gm.  384  3  10  13  16  41  20  0.32 mg./gm.  425  4  16  15  10  46  13  ** 1.0 mg./gm.  358  3  7  25  23  45  Jan. 12/72  38  Table IV:  The mean gonadosomatic index (GSI) values for males of the long-term experiment.  DATE  GROUP  NUMBER OF  MEAN GSI  ±  SD  MALES Nov. 8/71  zero control  3  0.040  0.003  Jan. 15/72  natural photoperiod control  5  0.026  0.004  natural photoperiod treatment  5  0.030  0.011  constant photoperiod control  3  0.033  0.003  constant photoperiod treatment  6  0.032  0.008  natural photoperiod control  6  0.052  0.006  natural photoperiod treatment  5  0.030  0.012  constant photoperiod control  3  0.046  0.006  constant photoperiod treatment  8  0.046  0.009  natural photoperiod control  3  0.134  0.023  natural photoperiod treatment  3  0.066  0.012  constant photoperiod control  3  0.072  0.004  constant photoperiod treatment  3  0.059  0.016  natural photoperiod control  5  6.223  1.654  constant photoperiod control  9  2.382  1.898  constant photoperiod treatment  7  0.052  0.009  Mar. 13/72  June 14/72  Aug. 30/72  (terminated in June)  39  Table V:  The mean gonadosomatic index (GSI) values for females of the long-term experiment.  DATE  GROUP  NUMBER OF  MEAN GSI  ±  SD  FEMALES Nov. 8/71  zero control  5  0.446  0.053  Jan. 15/72  natural photoperiod control  3  0.664  0.078  natural photoperiod treatment  3  0.520  0.229  constant photoperiod control  4  0.526  0.044  constant photoperiod treatment  2  0.662  0.275  natural photoperiod control  3  0.865  0.109  natural photoperiod treatment  3  0.905  0.465  constant photoperiod control  5  0.868  0.212  constant photoperiod treatment  3  0.597  0.172  natural photoperiod control  3  1.198  0.132  natural photoperiod treatment  3  1.079  0.189  constant photoperiod control  3  1.110  0.271  constant photoperiod treatment  3  0.685  0.169  natural photoperiod control  8  2.627  2.048  constant photoperiod control  9  1.778  1.031  constant photoperiod treatment  9  0.775  0.677  Mar. 13/72  June 14/72  Aug. 30/72  (terminated in June)  40  Table VI: The percentages of oocytes in the stages of oocyte maturation and the percentage of oocytes that are atretic in the long-term experiment. * these groups represent the percentage of the total number of ovaries in which only the most advanced stage of maturation was recorded because of extensive oocyte distortion in processing. ** 1 - early perinucleolus stage 2 - late perinucleolus stage 3 - early yolk vesicle stage 4 - late yolk vesicle stage 5 - o i l globule stage 6 - primary yolk globule stage 7 - secondary yolk globule stage  DATE  GROUP  NUMBER OF OOCYTES  NUMBER OF FEMALES **  MEAN PERCENT IN EACH STAGE 1  2  3  4  5  6  PERCENT 7  ATRETIC  Nov. 8/71  zero control  710  5  12  38  50  Jan. 15/72  natural photoperiod control  344  3  4  15  15  66  1  natural photoperiod treatment  384  3  12  16  25  47  7  constant photoperiod control  333  3  2  13  12  73  10  constant photoperiod treatment  310  2  8  12  13  67  0  natural photoperiod control  109  3  7  3  1  48  41  20  natural photoperiod treatment  136  3  8  18  9  31  34  17  constant photoperiod control  169  3  4  7  9  56  24  13  constant photoperiod treatment  112  3  11  12  11  40  26  26  Mar. 13/72  June 14/72  *3  natural photoperiod control natural photoperiod treatment  137  constant photoperiod control constant photoperiod treatment Aug. 30/72  0  3  67 3  13  10  49  *3 203  3  16  2  13  47  44  25  20  100  40  22  11  *3  33  constant photoperiod control  *7  57  constant photoperiod treatment  *5  natural photoperiod control  33  67  81  14  50  (terminated in June) 20  80  29  59  41  Table VII: The mean oocyte diameters of the long-term experiment. * oocytes from this sampling were measured whole because of the difficulty in processing these large oocytes. ** this group was not included on Figure 29 because oocytes from only one fish could be measured.  DATE  GROUP  NUMBER OF OOCYTES MEASURED  NUMBER OF FEMALES  IN MILLIMETERS  MEANS  0.420  0.097  Nov. 8/71  zero control  Jan. 15/72  natural photoperiod control  91  3  0.670  0.022  natural photoperiod treatment  99  3  0.591  0.067  constant photoperiod control  90  3  0.729  0.072  constant photoperiod treatment  60  2  0.654  0.012  natural photoperiod control  36  2  0.741  0.049  natural photoperiod treatment  46  2  0.586  0.034  constant photoperiod control  69  3  0.729  0.117  constant photoperiod treatment  59  3  0.589  0.090  0  0  33  3  0  0  **30  1  Mar. 13/72  June 14/72  natural photoperiod control natural photoperiod treatment constant photoperiod control constant photoperiod treatment  Aug. 30/72*  natural photoperiod control  193  MEAN DIAMETER ± SD OF THE  146  (excessive distortion) 0.526  0.066  (excessive distortion) 0.472 2.749  0.869  (terminated in June) constant photoperiod control constant photoperiod treatment  143  6  2.258  0.630  48  2  1.335  0.243  42  Table VIII:  The mean body weights of pooled males and females of the long-term experiment.  DATE  GROUP  NUMBER OF FISH  MEAN BODY WEIGHT  ±  SD  IN GRAMS  Nov. 8/71  zero control  8  52.04  26.02  Jan. 15/72  natural photoperiod control  9  80.92  15.74  natural photoperiod treatment  8  75.96  14.37  constant photoperiod control  7  92.08  5.94  10  73.27  14.37  natural photoperiod control  9  95.68  18.29  natural photoperiod treatment  8  73.90  21.25  constant photoperiod control  8  94.51  15.28  11  85.86  26.69  natural photoperiod control  6  138.68  35.85  natural photoperiod treatment  6  111.85  15.38  constant photoperiod control  6  160.68  26.09  constant photoperiod treatment  6  104.33  37.30  13  245.65  74.40  constant photoperiod control  18  217.79  52.86  constant photoperiod treatment  16  144.04  42.84  constant photoperiod treatment Mar. 13/72  constant photoperiod treatment June 14/72  Aug. 30/72  natural photoperiod control (terminated in June)  43  Table IX: The mean thyroid f o l l i c l e epithelial heights of the long-term experiment and the high dose group (1.0 mg./gm.) sampled on June 14/72.  DATE  GROUP  NUMBER OF THYROID FOLLICLES MEASURED  June 14/72  NUMBER OF FISH  MEAN HEIGHT  ± SD OF THE  IN MICRONS  MEANS  natural photoperiod control  200  6  6  1.3  natural photoperiod treatment  180  6  10  2.1  constant photoperiod control  180  6  9  1.3  constant photoperiod treatment  180  6  8  1.0  high dose  180  6  6  1.5  44  T a b l e X:  The mean percentages o f o o c y t e s i n the s t a g e s o f o o c y t e m a t u r a t i o n o f the s e q u e n t i a l  experiment.  * 1 - e a r l y p e r i n u c l e o l u s stage 2 - l a t e p e r i n u c l e o l u s stage 3 - e a r l y y o l k v e s i c l e stage 4 - l a t e y o l k v e s i c l e stage  DATE  GROUP  NUMBER OF FEMALES  MEAN PERCENT IN EACH STAGE *  1  2  3  2  4  May 31/72  zero control  3  100  June 29/72  zero control  3  71  27  July 27/72  zero control  3  63  37  Aug. 24/72  zero control  3  26  56  11  7  Oct. 29/72  zero control  5  10  51  23  16  Oct. 29/72  control  3  10  63  9  18  Oct. 29/72  sampling of group started on May 31/72  1  8  35  16  41  Oct. 29/72  sampling of group started on June 29/72  4  6  49  27  18  Oct. 29/72  sampling of group started on July 27/72  7  4  45  22  29  Oct. 29/72  sampling of group started on Aug. 24/72  1  6  66  22  6  45  DISCUSSION  M e t h a l l i b u r e was  f i r s t used on f i s h by Hoar et a l . (1967).  authors found t h a t i t c o m p l e t e l y i n h i b i t e d spermatogenesis perch  (Cymatogaster  aggregata), three-spined s t i c k l e b a c k  a c u l e a t u s ) , and g o l d f i s h  (Carassius auratus).  confirmed by Wiebe (1968a,  1969)  These  i n the sea-  (Gasterosteus  These r e s u l t s were  later  on the s e a p e r c h , Carew (1968) on the  t h r e e - s p i n e d s t i c k l e b a c k , and B i l l a r d ejt al_.  (1971) on the g o l d f i s h .  Other workers a l s o found t h a t m e t h a l l i b u r e i n h i b i t s spermatogenesis the guppy P o e c i l i a r e t i c u l a t a Bromage, 1970;  (Pandey and L e a t h e r l a n d , 1970; M a r t i n and  B i l l a r d et_ al_., 1970)  l i s h e d d a t a ; Hyder, 1972).  and T i l a p i a s p e c i e s (Dadzie, unpub-  From the p r e s e n t study, m e t h a l l i b u r e a l s o  c o m p l e t e l y i n h i b i t s t e s t i c u l a r m a t u r a t i o n i n p i n k salmon and 16).  The s i m i l a r i t y o f the c e l l s  long-term experiment gonia present  i n August,  1972  ( F i g s . 27,  i n d i c a t e s that methallibure i n h i b i t s mary i n t o secondary spermatogonia.  15  i n the treatment t e s t e s o f the ( F i g . 16) w i t h the p r i m a r y spermato-  i n the zero c o n t r o l t e s t e s i n November, 1971  ( F i g . 14)  the m i t o t i c t r a n s f o r m a t i o n o f p r i T h i s i s a l s o e v i d e n t comparing  p r i m a r y spea?mat-ogoriiayofpthe^trgatment t e s t e s dary spermatogonia  in  o f the c o n t r o l t e s t e s  the  ( F i g . 13) w i t h the secon-  ( F i g . 12) on June 14,  1972.  B i l l a r d et_ al_.  (1971) found t h a t m e t h a l l i b u r e reduced the numbers o f B-  spermatogonia,  spermatocytes, and spermatids but not A-spermatogonia  in  the g o l d f i s h , which f u r t h e r supports the above c o n c l u s i o n assuming t h a t A and B r e f e r t o p r i m a r y and secondary, r e s p e c t i v e l y .  These e f f e c t s o f  m e t h a l l i b u r e are i d e n t i c a l t o those observed by Ahsan (1966) on hypophys e c t o m i z e d l a k e chub, Couesius plumbeus.  He  found t h a t t e s t e s o f these  46  hypophysectomized  f i s h were composed almost e n t i r e l y o f p r i m a r y sperma-  t o g o n i a and o n l y r a r e l y were secondary spermatogonia  observed.  suggests t h a t the t r a n s f o r m a t i o n o f p r i m a r y t o secondary  This  spermatogonia  i s g o n a d o t r o p i n dependent and t h a t the a c t i o n o f m e t h a l l i b u r e , t h e r e f o r e , is  through the i n h i b i t i o n o f gonadotropin r e l e a s e .  However, most wor-  k e r s s t a t e t h a t m e t h a l l i b u r e p r e v e n t s the m e i o t i c t r a n s f o r m a t i o n o f secondary spermatogonia  i n t o p r i m a r y spermatocytes  M a r t i n and Bromage, 1970;  Pandey, 1970a).  (Hoar et a l . ,  T h i s d i s c r e p a n c y may  1967; i n part  be due t o these r e s e a r c h e r s not d i s t i n g u i s h i n g between p r i m a r y and dary spermatogonia.  Ahsan (1966) a l s o found t h a t hypophysectomy  p r e s s e d the m i t o t i c d i v i s i o n o f p r i m a r y spermatogonia.  secon-  sup-  Replacement  t h e r a p y w i t h ovine LH, f r a c t i o n a t e d salmon p i t u i t a r y g o n a d o t r o p i n , o r whole salmon p i t u i t a r y e x t r a c t s r e s t o r e d the m i t o t i c a c t i v i t y t o normal s u g g e s t i n g t h a t s p e r m a t o g o n i a l p r o l i f e r a t i o n , as w e l l as the t r a n s f o r m a t i o n i n t o secondary spermatogonia,  i s gonadotropin dependent.  Wiebe (1969)  found t h a t m e t h a l l i b u r e a l s o suppressed the m i t o t i c a c t i v i t y o f spermatog o n i a i n the seaperch and t h a t exogenous ovine LH r e s t o r e d t h i s  activity.  However, Hoar et_ al_.  that  (1967) and M a r t i n and Bromage (1970) found  m e t h a l l i b u r e had no e f f e c t on the m i t o t i c a c t i v i t y o f spermatogonia.  In  the p r e s e n t study a l s o , m i t o t i c f i g u r e s were observed i n the m e t h a l l i b u r e t r e a t e d t e s t e s o f p i n k salmon.  The  a c t i v i t y was  not q u a n t i f i e d and,  the presence o f a s u p p r e s s i o n o f m i t o s i s i s not known. m e t h a l l i b u r e on m i t o t i c a c t i v i t y , t h e r e f o r e , remains the c o n f l i c t i n g  The  effect  M e t h a l l i b u r e d i d not c o m p l e t e l y i n h i b i t gonadal development i n female p i n k salmon but o n l y slowed i t s r a t e  of  obscure i n l i g h t  evidence.  ( F i g s . 26, 28, and  29).  thus,  of  47  T h i s i s c o n t r a d i c t o r y t o the complete i n h i b i t i o n o f o v a r i a n m a t u r a t i o n by m e t h a l l i b u r e i n the g o l d f i s h and t h r e e - s p i n e d s t i c k l e b a c k  (Hoar et_ al_. ,  1967), seaperch (Hoar e t al_. , 1967; Wiebe, 1969), guppy (Pandey, and T i l a p i a s p e c i e s  (Dadzie, u n p u b l i s h e d d a t a ; Hyder, 1972).  1970b),  This i n -  h i b i t o r y e f f e c t o f m e t h a l l i b u r e i s i d e n t i c a l t o t h a t o f hypophysectomy s i n c e b o t h p r e v e n t the onset o f v i t e l l o g e n e s i s o r the second growth phase o f oocytes i n j u v e n i l e guppies (Pandey, 1970b) and s t o p v i t e l l o g e n e s i s and cause subsequent r e g r e s s i o n o f second growth phase o o c y t e s i n g o l d fish  (Yamazaki, 1965; Hoar et_ al_., 1967).  The f a i l u r e o f m e t h a l l i b u r e  t o i n h i b i t o v a r i a n m a t u r a t i o n c o m p l e t e l y i n the p i n k salmon may be due t o an i n s u f f i c i e n t dose s i n c e the 0.10 mg./gm./2 wks. than the doses used i n the . l i t e r a t u r e * 1970) .  dose was lower  (Hoar et^ al_., 1967;  B i l l a r d et a l . ,  However, a t h r e e and t e n f o l d h i g h e r dose i n the p i l o t  ment a l s o f a i l e d t o i n h i b i t o v a r i a n development  experi-  ( F i g s . 22, 23, and 24)  although the 1.0 mg./gm. dose d i d s i g n i f i c a n t l y slow the r a t e o f i n c r e a s e i n GSI ( F i g . 22). doses  I t was t h i s s i m i l a r i t y i n the e f f e c t o f a l l  three  (0.10 mg./gm., 0.32 mg./gm., and 1.0 mg./gm.) i n the p i l o t  experi-  ment on o o c y t e diameter and stages o f o o c y t e m a t u r a t i o n t h a t was important i n d e c i d i n g upon the lower 0.10 mg./gm. dose f o r the subsequent experiments.  I t was thought t h a t the lower dose would reduce p o s s i b l e  undesirable e f f e c t s o f methallibure.  T h i s was s u b s t a n t i a t e d i n l a t e r  u n r e p o r t e d d a t a i n which the h i g h dose  (1.0 mg./gm.) caused v e r y h i g h  m o r t a l i t y i n younger, j u v e n i l e p i n k salmon. o l d e r f i s h i s unknown; however, t h i s  I t s long-term e f f e c t on  evidence suggests t h a t h i g h doses  o f m e t h a l l i b u r e would be disadvantageous i n such s t u d i e s .  48  One  o t h e r p o s s i b l e e x p l a n a t i o n f o r m e t h a l l i b u r e not  i n h i b i t i n g o v a r i a n m a t u r a t i o n i n the long-term experiment ment was  begun a p p r o x i m a t e l y one  completelyi s that  treat-  and one h a l f months a f t e r the onset o f  oocyte m a t u r a t i o n o r v i t e l l o g e n e s i s .  A c c o r d i n g t o Sokol  (1961), gonad  m a t u r a t i o n i s c o i n c i d e n t w i t h the d i f f e r e n t i a t i o n o f the gonadotrops the guppy.  Pandey (1970a) found t h a t m e t h a l l i b u r e c o m p l e t e l y  t e s t i c u l a r maturation i n j u v e n i l e in adults.  guppies but o n l y slowed  in  inhibited  spermatogenesis  With t h e s e f a c t s , Pandey suggested t h a t m e t h a l l i b u r e was  un-  a b l e t o i n h i b i t gonadotropin s y n t h e s i s and r e l e a s e c o m p l e t e l y once these p r o c e s s e s had begun but c o u l d p r e v e n t t h e i r i n c e p t i o n i f treatment was  started  prior  to gonadotrop  d i f f e r e n t i a t i o n and gonad m a t u r a t i o n .  T h i s s u g g e s t i o n i s s t r e n g t h e n e d by the complete  i n h i b i t i o n of t e s t i c u l a r  m a t u r a t i o n i n the p r e s e n t study i n which m e t h a l l i b u r e treatment was s i x months p r i o r t o gonadotrop maturation. turation  To determine  d i f f e r e n t i a t i o n and the s t a r t o f t e s t i s  i f t h i s suggestion holds true f o r ovarian  i n p i n k salmon, m e t h a l l i b u r e treatment was  s t a r t o f o v a r i a n development and b e f o r e gonadotrop s e q u e n t i a l experiment.  little  differentiation.  o f the p i l o t experiment  started  was  no  e f f e c t , i t seems t h a t  inhi-  be dependent upon treatment w i t h a  h i g h dose b e f o r e gonadotrop  The h i g h dose  just prior  l o g e n e s i s but because o f poor i n j e c t i o n  treatment  to the b e g i n n i n g o f v i t e l -  technique i n i t i a l l y ,  were p r o b a b l y not exposed t o m e t h a l l i b u r e u n t i l ferentiation.  d i f f e r e n t i a t i o n i n the  From t h e s e d a t a and those o f the p i l o t  i n which the h i g h dose had  b i t i o n o f o v a r i a n m a t u r a t i o n may  ma-  begun b e f o r e the  F i g u r e 33 i n d i c a t e s t h a t m e t h a l l i b u r e had  e f f e c t upon o v a r i a n development. experiment  begun  the  a f t e r gonadotrop  T h i s would account f o r i t s l a c k o f e f f e c t .  The  fish dif-  difference  49  between the e f f e c t s o f m e t h a l l i b u r e  on the male and  female p i n k  salmon i n d i c a t e s t h a t the females are much l e s s s e n s i t i v e to  this  drug than are the males. The  recorded  conflicting.  effects of methallibure  Hoar et_ al_.  as d i d Pandey and  i n the  l a t t e r study was  o f these two  i n the t h r e e - s p i n e d  Leatherland not  (1970) i n a d u l t guppies.  as g r e a t as t h a t o f t h i o u r e a .  j u v e n i l e guppies.  e f f e c t on the t h r e e - s p i n e d  seaperch.  s t i c k l e b a c k and  Wright  (1963) and Paget et^ al_.  e f f e c t of methallibure  t i v i t y o f the  the o t h e r hand, Rangneker and  l i z a r d , Calotes v e r s i c o l o r .  a distinct  the  (1961) found no a n t i t h y r o i d a l  (1969) observed a d e p r e s s i n g  Kulkarni  on the t h y r o i d a l ac-  In the p r e s e n t  study,  methal-  a n t i t h y r o i d a l e f f e c t under n a t u r a l p h o t o p e r i o d  f a i l e d to produce t h i s a t e n times h i g h e r dose  e f f e c t under a c o n s t a n t ( F i g . 32).  be e x p l a i n e d by me'thallibure's As  authors  s t i c k l e b a c k nor d i d Wiebe (1968a) on  On  tropin.  effect  Carew (1968), however, found  e f f e c t on the fowl and r a t .  l i b u r e had  sea-  This  The  or  s t u d i e s a l s o found t h a t m e t h a l l i b u r e had no a n t i t h y r o i d a l  e f f e c t on g o l d f i s h and no  very  (1967) found a s l i g h t a n t i t h y r o i d a l e f f e c t  increased e p i t h e l i a l height perch  on t h y r o i d f u n c t i o n are  12L:12D p h o t o p e r i o d  or at  These c o n f l i c t i n g o b s e r v a t i o n s  e f f e c t on t h y r o x i n e s y n t h e s i s and  but  may  thyro-  d e s c r i b e d i n the i n t r o d u c t i o n , the e f f e c t o f m e t h a l l i b u r e  on  t h y r o i d a l a c t i v i t y i s dependent upon the i n h i b i t i o n o f t h y r o t r o p i n synt h e s i s and r e l e a s e , which would depress t h y r o i d a c t i v i t y , b i n d i n g o f i o d i n e ; which would i n c r e a s e t h y r o i d a c t i v i t y . (1971) i n d i c a t e t h a t the r e l a t i v e e f f e c t of m e t h a l l i b u r e two  a c t i o n s may  change.  and upon the Bourke et_ al_. on each o f  They suggest t h a t low doses o f m e t h a l l i b u r e  a n c i i n h i b i t o r y e f f e c t on the r e l e a s e of t h y r o t r o p i n whereas h i g h e r  these exert  doses  50  and  increases  synthesis. falls  i n time o f exposure e x e r t  an i n h i b i t o r y e f f e c t on  They found, i n r a t s , t h a t the  low  the  doses caused the  i n serum t h y r o t r o p i n w h i l e the h i g h e r doses f a i l e d  greatest  to depress  serum l e v e l s because o f a s t i m u l a t i o n of t h y r o t r o p i n r e l e a s e due, a b l y , t o d e c r e a s e d feedback by t h y r o x i n e . i n the doses used and  the method and  l i b u r e , as w e l l as s p e c i e s f i n d i n g s i n the  Therefore,  The  absence o f an i n c r e a s e  (1971) and  h i g h doses found by Paget et^ al_.  the e f f e c t o f m e t h a l l i b u r e i n balance.  the m e t h a l l i b u r e  The  Thyroid  At t h i s 1.0 on the two  may,  i n p a r t , bee  later).  An  e f f e c t o f the  by  h i s t o l o g y i n the  latter  mg./gm. dose i n p i n k  sal-  must,  there-  d i f f e r e n c e i n the t h y r o i d a c t i v i t i e s between  due  to disease  constant  and  constant  12L:12D photo-  (which w i l l be b r i e f l y  discussed  12 hour d a y l e n g t h on t h i s a c t i v i t y i s  doubted because of the f i n d i n g s o f S w i f t o f the n a t u r a l p h o t o p e r i o d had  no  (1960), t h a t the d i f f e r e n t e f f e c t on t h y r o i d  Wagner (1970), t h a t the t h y r o i d a c t i v i t y t h y r o i d f o l l i c l e epithelium)  (measured as the h e i g h t  under constant  daylengths o f 8.5,  s i m i l a r to t h a t under n a t u r a l p h o t o p e r i o d .  day-  activity  (measured as the r a t e o f l o s s o f r a d i o - i o d i n e from the gland) and  hours was  ac-  t h y r o i d weight  actions described  t r e a t e d groups under n a t u r a l  periods  lengths  i n thyroid  study i s s u r p r i s i n g c o n s i -  the i n c r e a s e d  (1961) .  study, however, remained normal.  f o r e , be  contradictory  f a i l u r e o f h i g h doses to depress serum t h y r o t r o p i n l e v e l s i n  the study o f Bourke e_t_ al_.  mon,  differences  account f o r the  t i v i t y i n the h i g h dose group o f the p r e s e n t d e r i n g the  presum-  p e r i o d of a d m i n i s t r a t i o n o f methal-  d i f f e r e n c e s , may  literature.  the  the  of of  12,  the and  16  51  The e f f e c t o f m e t h a l l i b u r e on body weight, i s , l i k e t h a t on the t h y r o i d , v e r y c o n t r a d i c t o r y i n the l i t e r a t u r e .  Many r e s e a r c h e r s found  a d e c r e a s e i n the body weight o f r a t s , i n c l u d i n g Stratman and (1969) and Labhsetwar and Walpole effect.  Imai  (1972), w h i l e Harper  First  (1967) found no  (1972) found a s l i g h t weight l o s s i n the domestic hen  w h i l e S t e e l and Hinde  (1972) found no e f f e c t i n t h e canary.  Gangadhara  and Ramaiah (1968) observed no body weight l o s s i n the s k i p p e r (Rana c y a n o p h l y c t i s ) nor d i d B i l l a r d ejt al_. land  (1970) i n the g o l d f i s h and guppy.  frog  (1971) or Pandey and L e a t h e r -  Dadzie ( u n p u b l i s h e d d a t a ) , how-  e v e r , found an i n c r e a s e i n the body weight o f T i l a p i a .  In the p r e s e n t  study, m e t h a l l i b u r e reduced the r a t e o f i n c r e a s e i n the body weight o f p i n k salmon  ( F i g . 31) which agrees w i t h the m a j o r i t y o f t h e mammalian  literature.  T h i s e f f e c t may  be a d i r e c t r e s u l t o f a d e c r e a s e i n the  amount o f food i n g e s t e d s i n c e Stratman e t a l . (1969) found a s u p p r e s s i o n of a p p e t i t e i n hamsters and swine and p o s t u l a t e d t h a t the s i t e o f methall i b u r e ' s a c t i o n was  the hypothalamus.  Whether t h i s e f f e c t e d an  t i t e c e n t e r " o r r e l e a s i n g f a c t o r s c o n t r o l l i n g the p i t u i t a r y was  "appe-  somatotrops  not known. Evidence f o r an e f f e c t o f m e t h a l l i b u r e at the hypothalamic-hypophy-  sial'llevel  comes m a i n l y from s t u d i e s i n which the serum o r p i t u i t a r y  l e v e l s o f hormones o r t h e i r p e r i p h e r a l e f f e c t were measured upon t r e a t ment.  Malven  (1971) found t h a t o v u l a t i o n i n the guinea p i g was  inhibited  w i t h i m p l a n t s o f m e t h a l l i b u r e i n the hypothalamus, p a r t i c u l a r l y t h e p r e o p t i c a r e a and the a r c u a t e nucleus-median eminence. a hypothalamic l e v e l o f a c t i o n o f m e t h a l l i b u r e . Garbers and F i r s t  T h i s s t r o n g l y suggests  The o b s e r v a t i o n by  (1968) o f the i n h i b i t i o n o f m i l k e j e c t i o n i n swine by  52  methallibure  suggests an i n h i b i t i o n o f o x y t o c i n r e l e a s e which  t h i s hypothalamic l e v e l of a c t i o n . support  a hypophysial  l e v e l o f a c t i o n as w e l l .  found t h a t m e t h a l l i b u r e pin  i n the r a t but  There i s a l s o s t r o n g  inhibits  these authors  the r e l e a s e and hypothesize  (1972) and  Brown and  the s y n t h e s i s and  t h a t m e t h a l l i b u r e has Labhsetwar and  Fawke (1972) found t h a t m e t h a l l i b u r e  Stormshak et^ a l . (1970) suggest t h a t m e t h a l l i b u r e may the p i t u i t a r y r e l e a s e o f gonadotropins i n g i l t s not e f f e c t e d .  to  synthesis of t h y r o t r o -  r e l e a s e o f the gonadotropins LH and  LH r e l e a s i n g f a c t o r was  evidence  Bourke ejt al_. (1971)  p o s s i b l e u n i t a r y a c t i o n at the hypothalamic l e v e l . pole  supports  FSH  a Wal-  inhibits  i n the r a t .  interfere  s i n c e the  with  hypothalamic  S t u d i e s on p i t u i t a r y c e l l mor-  p h o l o g y and h i s t o l o g i c a l s t a i n i n g p r o p e r t i e s are v a l u a b l e i n i n t e r p r e t ing  the e f f e c t s o f m e t h a l l i b u r e  T h i s , however, must be done w i t h may  on the s y n t h e s i s and  c a u t i o n , s i n c e both these  be i n v o l v e d i n the observed e f f e c t .  Pandey and  Leatherland  the seaperch,  (1970), on the guppy, and  number, s i z e , and  activities  After methallibure  observed g r e a t l y decreased  shown by decreased  r e l e a s e o f hormones.  treatment,  Leatherland  a c t i v i t y o f the  gonadotrops  granulation; a s l i g h t l y  somatotrop a c t i v i t y shown by decreased  g r a n u l a t i o n ; and  (1969), on  decreased  a very  slightly  i n c r e a s e d t h y r o t r o p a c t i v i t y shown by i n c r e a s e d c e l l number and These o b s e r v a t i o n s and  by Pandey and  (1970) on the  gonadotrops  t h y r o t r o p s p a r a l l e l c l o s e l y the a c t i v i t y o f the t a r g e t t i s s u e s .  These authors  found an i n h i b i t i o n o f spermatogenesis and  t h y r o i d a c t i v i t y but no degranulation with  Leatherland  size.  change i n body weight.  o f the gonadotrops and,  an i n c r e a s e i n  Mackay (1971) found a  to a l e s s e r degree, the  a concomitant i n h i b i t i o n o f o v a r i a n m a t u r a t i o n  thyrotrops  i n the b a s s ,  53  P l e c t r o p l i t e s ambiguus. Dadzie  (unpublished  No  e f f e c t on the t h y r o i d was  data) and  observed.  Carew (1968) a l s o found a decrease i n  gonadotrop g r a n u l a t i o n accompanied by an i n h i b i t i o n o f gonad development i n T i l a p i a s p e c i e s and it  the t h r e e - s p i n e d  i s e v i d e n t t h a t m e t h a l l i b u r e has  t r o p s , and  From these  and  level.  f o r a d i r e c t peripheral effect of methallibure  Chieffi  studies,  an e f f e c t on the gonadotrops, t h y r o -  somatotrops at the hypothalamic and h y p o p h y s i a l  o n l y evidence Rastogi  stickleback.  The  i s given  (1972) i n the a d u l t green f r o g , Rana e s c u l e n t a .  by They  found t h a t m e t h a l l i b u r e p l u s a p i t u i t a r y homogenate f a i l e d t o  stimulate  gonadal a c t i v i t y i n p a r s d i s t a l i s - e c t o m i z e d f r o g s whereas the  pituitary  homogenate alone was contradicting this fish.  stimulatory.  evidence  As but  one  example o f the  i s that of B i l l a r d  et_ al_.  studies  (1971) on the  M e t h a l l i b u r e i n combination w i t h powdered carp p i t u i t a r i e s  gold-  main-  t a i n e d normal spermatogenesis. In the d i s c u s s i o n on the e f f e c t s o f m e t h a l l i b u r e on the t h y r o i d f o l l i c l e e p i t h e l i a l h e i g h t s , r e f e r e n c e was on t h y r o i d a c t i v i t y . was  T h i s d i s e a s e was  v e r y p r e v a l e n t i n the  months o f maturation.. (Wedemeyer and  The  Ross, 1973)  i n e p i t h e l i a l height 12L:12D p h o t o p e r i o d  made to the e f f e c t o f  diagnosed as k i d n e y  long-term experiment d u r i n g the metabolic may  s t r e s s induced  i n the n a t u r a l p h o t o p e r i o d c o n t r o l groups  ( F i g . 32).  This kidney  four  increase  constant  disease  l a r g e v a r i a t i o n s i n the  l a t t e r period  and  disease  treatment and  l a r g e numbers o f a t r e t i c oocytes  treatment groups d u r i n g t h i s  latter  by t h i s  o o c y t e diameter, and body weight measurements i n the The  disease  have caused, at l e a s t i n p a r t , the  a l s o have been i n v o l v e d i n c a u s i n g the  development.  disease  may  GSI,  l a t t e r months o f  i n c o n t r o l as w e l l  ( F i g . 30)  as  i s the most suggest-  54  ive  evidence f o r an e f f e c t o f t h i s d i s e a s e on the r e p r o d u c t i v e and me-  t a b o l i c processes. The  e f f e c t o f the constant 12L:12D p h o t o p e r i o d on gonadal  t i o n i n the long-term experiment  matura-  was a s l o w i n g o f the r a t e o f i n c r e a s e  i n m a t u r a t i o n compared t o the r a t e under t h e n a t u r a l p h o t o p e r i o d . means t h a t gonadal m a t u r a t i o n was m a i n t a i n e d , although slowed, absence o f the s e a s o n a l d a y l e n g t h f l u c t u a t i o n s o f the n a t u r a l  This  i n the photo-  p e r i o d and t h a t some o t h e r c h a r a c t e r i s t i c o f p h o t o p e r i o d may be operat i v e i n s t i m u l a t i n g maturation. number o f d a y l i g h t hours.  One such c h a r a c t e r i s t i c may be the t o t a l  Baggerman (1957), working  on t h e t h r e e - s p i n e d  s t i c k l e b a c k , found t h a t sudden i n c r e a s e s i n d a y l e n g t h r e s u l t e d i n g r e a ter  a c c e l e r a t i o n o f gonadal m a t u r a t i o n than d i d gradual i n c r e a s e s .  author concluded t h a t the t o t a l g r a d u a l changes.  Larson  amount o f l i g h t i s more important  The than  (1972) found t h a t a w i l d p o p u l a t i o n o f brook  t r o u t matured n o r m a l l y even though t h e y were exposed t o a sudden i n c r e a s e i n d a y l e n g t h from near darkness because o f n a t u r a l i c e cover d u r i n g winter  and s p r i n g .  He suggested t h a t t h e gradual i n c r e a s e s i n the n a t u r a l  p h o t o p e r i o d are not n e c e s s a r y f o r gonad m a t u r a t i o n and t h a t t h i s i n c r e a s e i n l i g h t , and perhaps tion.  temperature  also, i n i t i a t e d  sudden  gonad matura-  In the p r e s e n t study, the p i n k salmon may be r e s p o n d i n g , then, t o  a c e r t a i n number o f hours o f exposure a critical  daylength.  t o l i g h t which L i c h t  (1971) terms  S i n c e the male p i n k salmon were exposed t o t h e  c o n s t a n t 12L:12D p h o t o p e r i o d f o r s i x months p r i o r t o the s t a r t o f t e s t i s m a t u r a t i o n , the 12 hours matogenesis  o f l i g h t may be s t i m u l a t o r y i n i n i t i a t i n g  and, t h e r e f o r e , should approximate  the c r i t i c a l  sper-  daylength.  55  F i g u r e 34 i n d i c a t e s t h a t under a n a t u r a l p h o t o p e r i o d , the males b e g i n maturing  i n May,  which e s t a b l i s h e s the c r i t i c a l d a y l e n g t h between  a p p r o x i m a t e l y 14.5  and  16 hours.  not have begun maturing  Under t h i s h y p o t h e s i s , the males should  s i n c e the constant 12 hour d a y l e n g t h i s below  t h i s normal c r i t i c a l d a y l e n g t h .  Licht  (1971) suggests t h a t the  or t h r e s h o l d d a y l e n g t h f o r t e s t i s r e g r e s s i o n i n the l i z a r d , c a r o l i n e n s i s , can s h i f t , i n t h i s case  upwards, t o account  critical  Anolis for a regres-  s i v e e f f e c t under a constant d a y l e n g t h which i s n o r m a l l y s t i m u l a t o r y under n a t u r a l p h o t o p e r i o d . male p i n k salmon.  T h i s mechanism may  p o s s i b l y be o p e r a t i n g i n  A decrease i n the t h r e s h o l d daylength t o o r below 12  hours would i n i t i a t e t e s t i s m a t u r a t i o n .  Presumably t h i s would a l s o  the case i n the females, however, the c o n s t a n t 12L:12D p h o t o p e r i o d begun a f t e r t h e s t a r t o f o v a r i a n m a t u r a t i o n .  The  be was  stimulatory effect  of  s p e c i f i c d a y l e n g t h on the i n i t i a t i o n  and t e r m i n a t i o n o f gonad m a t u r a t i o n  i s e v i d e n t i n many s p e c i e s o f f i s h .  Sundararaj  and Sehgal  (1970a) found  t h a t a long p h o t o p e r i o d induced o v a r i a n r e c r u d e s c e n c e i n the Heteropneustes  fossilis.  Henderson  catfish,  (1963) found t h a t normal o v a r i a n  m a t u r a t i o n i s dependent upon long f o l l o w e d by s h o r t p h o t o p e r i o d i n the brook t r o u t first  ( S a l v e l i n u s f o n t i n a l i s ) and Baggerman (1957) found t h a t  the  and second phases o f gonad m a t u r a t i o n are s t i m u l a t e d by s h o r t and  long p h o t o p e r i o d , r e s p e c t i v e l y , i n the t h r e e - s p i n e d s t i c k l e b a c k . et a l . (1959) observed  Combs  advanced m a t u r a t i o n w i t h a s h o r t d a y l e n g t h i n the  sockeye salmon as d i d S h i r a i s h i and  Fukuda (1966) i n the kokanee  (Oncorhynchus n e r k a ) , Hon-masu (Oncorhynchus masou), rainbow t r o u t g a i r d n e r i ) , and brook t r o u t  (Salvelinus f o n t i n a l i s ) .  In a l l these  (Salmo studies,  56  Figure 34:  The daylengths of the natural photoperiod at 48° latitude (Fawcett, 1973).  sanoH  iHonAva  57  the daylengths r e s p o n s i b l e f o r gonadal  s t i m u l a t i o n are  characteristic  o f the s t i m u l a t o r y daylengths i n the n a t u r a l p h o t o p e r i o d . study, the o n l y s l i g h t may  s l o w i n g o f gonadal m a t u r a t i o n i n the p i n k salmon  p a r t l y be a r e s u l t o f the c o n s t a n t 12 hours  d a y l e n g t h approximates males and The  females  In the p r e s e n t  o f exposure  since this  t h a t p r e s e n t i n the n a t u r a l p h o t o p e r i o d when both  are i n the f i n a l  stages o f gonad m a t u r a t i o n  i n i t i a t i o n and maintenance o f gonad m a t u r a t i o n  under the constant 12L:12D p h o t o p e r i o d may  ( F i g . 34).  ;l n p i n k salmon ;  a l s o be the r e s u l t o f an  en-  dogenous rhythm.  T h i s i s most e a s i l y i n v e s t i g a t e d by m a i n t a i n i n g f i s h  i n t o t a l darkness  and, thus, i n the absence o f any p h o t o p e r i o d i c cues.  Pyle first  (1969) and Poston time, exposure  (1971) found t h a t , i n brook t r o u t spawning f o r the  t o t o t a l darkness  e f f e c t on the time o f f i n a l m a t u r a t i o n . o c c u r r e d w i t h o u t the s h o r t and  s i n c e the f i n g e r l i n g stage had I n i t i a t i o n o f gonadal  p r o b a b l y the f i r s t  T o t a l darkness  Bullough  t o suggest an i n t e r n a l rhythm i n f i s h .  alone d e l a y e d the m a t u r a t i o n p e r i o d o f male and  minnows (Phoxirius l a e v i s ) .  maturation  l o n g d a y l e n g t h sequences i n the brook  t r o u t as i t d i d i n the male p i n k salmon o f the p r e s e n t study. (1940) was  B u l l o u g h a l s o suggested  female  t h a t t h i s rhythm be-  comes weaker w i t h time i n darkness upon o b s e r v i n g t h a t a s i n g l e minnow had p o o r l y developed  no  gonads.  Sundararaj  and Sehgal  t h a t the o v a r i a n c y c l e o f the c a t f i s h , Heteropneustes  blind  (1970b) s t a t e  fossilis,  exhibits  an endogenous rhythm under constant darkness which i s out o f phase w i t h t h a t under n a t u r a l p h o t o p e r i o d by one month.  Poston  (1971) and  delayed f i n a l  Poston  and L i v i n g s t o n  (1971) found t h a t c o n s t a n t darkness  matura-  t i o n i n second  spawning brook t r o u t as d i d Pa:ng (1971) i n the sea water  58  adapted  killifish,  Fundulus h e t e r o c l i t u s . S h i r a i s h i and Fukuda (1966)  found t h a t the absence o f l i g h t advanced t h e f i n a l m a t u r a t i o n p e r i o d i n male kokanee, Hon-masu, and rainbow and brook t r o u t but had l i t t l e  effect  on the females. These s t u d i e s i n d i c a t e t h a t , under complete darkness, genous rhythm m a i n t a i n s  gonadal  m a t u r a t i o n somewhat out o f phase w i t h  t h a t under the n a t u r a l p h o t o p e r i o d . t i v e i n maintaining maturation  the endo-  I f a a n i n t e r n a l rhythm i s a l s o  o f the pink salmon i n t h e p r e s e n t  opera-  study,  the s l o w i n g o f m a t u r a t i o n observed under the c o n s t a n t 12 hour dayl e n g t h may be a r e s u l t o f t h i s rhythm b e i n g out o f phase. s i t u a t i o n under complete darkness,  U n l i k e the  a constant 12 hour d a y l e n g t h would  p r o v i d e an a c c u r a t e p h o t o p e r i o d i c cue o f e x a c t l y 12 hours every day. However, t h i s p h o t o p e r i o d , l i k e complete darkness, p r o v i d e s no cue o f s e a s o n a l d a y l e n g t h f l u c t u a t i o n s and, t h e r e f o r e , should a l s o show the e x i s t e n c e o f an endogenous rhythm.  This d i f f e r e n c e i n the timing o f the  spawning p e r i o d between t h e n a t u r a l p h o t o p e r i o d and one d e v o i d o f seasonal  cues may be due t o a s y n c h r o n i z i n g f u n c t i o n o f the s e a s o n a l  fluctuations  (Sundararaj  and S e h g a l , 1970b).  daylength  T h i s f u n c t i o n i s supported  by s t u d i e s which have advanced the m a t u r a t i o n p e r i o d by exposure t o an a c c e l e r a t e d n a t u r a l p h o t o p e r i o d i n the brook t r o u t Hoover and Hubbard, 1937; Hazard 1963)  (Hoover, 1937;  and Eddy, 1951; Corson,  and the t h r e e - s p i n e d s t i c k l e b a c k (Baggerman, 1957).  study, Vanstone  1955; Henderson, In a long-term  (unpublished data) r e t a r d e d gonadal m a t u r a t i o n  i n pink  salmon f o r one y e a r beyond the normal two y e a r c y c l e by u s i n g a delayed photoperiod.  T h i s suggests  t h a t the endogenous rhythm i s weak i n p i n k  salmon and can e a s i l y be s y n c h r o n i z e d w i t h a p h o t o p e r i o d i c regime g r e a t l y  59  d i f f e r e n t from the n a t u r a l p h o t o p e r i o d regime. As a summary o f t h e p h o t o p e r i o d i c response: the e f f e c t s o f a c o n s t a n t 12 hour d a y l e n g t h and a comparison w i t h the l i t e r a t u r e suggest t h a t the i n i t i a t i o n , maintenance, salmon  and t e r m i n a t i o n o f gonadal m a t u r a t i o n i n p i n k  are s t i m u l a t e d by a s p e c i f i c d a y l e n g t h and an endogenous rhythm  and s y n c h r o n i z e d by the s e a s o n a l d a y l e n g t h f l u c t u a t i o n s .  This  ensures  t h a t r e p r o d u c t i o n occurs a t t h e optimum time t o maximize egg and f r y s u r vival  ( F a r n e r and F o l l e t t ,  1966).  Another e n v i r o n m e n t a l f a c t o r which can be important i n i n f l u e n c i n g r e p r o d u c t i o n i s temperature.  P h o t o p e r i o d and temperature t o g e t h e r s t i -  mulate gonadal m a t u r a t i o n i n the minnow stickleback  (Apeltes quadracus)  spined stickleback  (Lepomis  b r i e f r e v i e w , de Vlaming the  four-spined  (Merriman and S c h e d l , 1941),  (Baggerman, 1957), k i l l i f i s h  ( H a r r i n g t o n , 1959), brook t r o u t and green s u n f i s h  ( B u l l o u g h , 1939),  (Fundulus c o n f l u e n t u s )  (Henderson, 1963), seaperch  cyanellus)  three-  (Wiebe,  (Kaya and H a s l e r , 1972).  1968b),  In a  (1972) s t a t e s t h a t temperature i s important i n  p h o t o p e r i o d i c response i n gonadal m a t u r a t i o n i n most o f the t e l e o s t s  studied.  T h i s evidence suggests t h a t temperature may a l s o be s t i m u l a -  t o r y i n p i n k salmon and, t h e r e f o r e , may have had an i n f l u e n c e on gonadal m a t u r a t i o n i n those f i s h under t h e c o n s t a n t 12 hour d a y l e n g t h i n t h e p r e s e n t study.  Vanstone  ( u n p u b l i s h e d d a t a ) , however, u s i n g a d e l a y e d  p h o t o p e r i o d and n a t u r a l l y f l u c t u a t i n g water temperature, d e l a y e d gonadal m a t u r a t i o n f o r one y e a r i n p i n k salmon.  T h i s s t r o n g l y suggests t h a t  p h o t o p e r i o d i s the most important f a c t o r and t h a t temperature i n o n l y a minor way i n gonadal m a t u r a t i o n o f p i n k salmon.  functions  The i n f l u e n c e  o f the n a t u r a l temperature f l u c t u a t i o n s on the gonadal m a t u r a t i o n o f the  60  f i s h under the c o n s t a n t 12 hour d a y l e n g t h i s , t h e r e f o r e , thought t o be very small. In  the p r e s e n t study, m e t h a l l i b u r e c o m p l e t e l y i n h i b i t e d the  m a t u r a t i o n o f o n l y the male p i n k salmon. withdrawal  to  I f these males matured  o f m e t h a l l i b u r e treatment, as the l i t e r a t u r e  ( B i l l a r d et_ al_.,  gonadal after  suggests  1971), then the sperm from these males c o u l d be  f e r t i l i z e eggs from females  used  o f h i g h escapement o r "good" r u n s .  These  eggs c o u l d then be t r a n s p l a n t e d i n t o r i v e r s w i t h a v e r y low o r n o n e x i s t ent escapement f o r t h a t y e a r .  However, male p i n k salmon have been sex-  u a l l y matured i n o n l y one y e a r u s i n g p a r t i a l l y p u r i f i e d salmon chus tshawytscha)  gonadotropin  (SG-G100) (Funk and Donaldson,  (Oncorhyn1972).  T h i s makes a t h r e e y e a r program i m p r a c t i c a l f o r o b t a i n i n g mature males. However, as d i s c u s s e d i n the i n t r o d u c t i o n , Funk et a l . (1973) were una b l e t o mature females w i t h i n the y e a r o f h a t c h i n g which suggests the o n l y a l t e r n a t i v e i s t o d e l a y the females O v a r i a n development was  that  i n a t h r e e y e a r program.  not i n h i b i t e d at the dose o f m e t h a l l i b u r e i n the  p r e s e n t study, however, treatment w i t h a h i g h e r dose begun e a r l y i n j u v e n i l e l i f e may mon  be s u c c e s s f u l i n d e l a y i n g m a t u r a t i o n o f female p i n k  f o r one y e a r beyond t h e i r normal two  year l i f e c y c l e .  sal-  T h i s would  en-  a b l e the g e n e t i c complement o f the females, as w e l l as t h a t o f the males, to  be used t o p o p u l a t e r i v e r s i n "poor" y e a r s .  61  SUMMARY  At a dose o f 0.10 mg./gm. body weight  i n the long-term  m e t h a l l i b u r e c o m p l e t e l y i n h i b i t e d spermatogenesis  experiment,  i n p i n k salmon by  p r e v e n t i n g t h e t r a n s f o r m a t i o n o f p r i m a r y i n t o secondary  spermatogonia.  O v a r i a n m a t u r a t i o n , however, was slowed but n o t i n h i b i t e d .  Treated  o v a r i e s p o s s e s s e d oocytes i n t h e o i l g l o b u l e stage w h i l e c o n t r o l o v a r i e s had oocytes i n t h e secondary y o l k g l o b u l e s t a g e .  One p o s s i b l e  explana-  t i o n f o r t h e d i f f e r e n c e i n e f f e c t between males and females i s t h a t treatment was begun p r i o r t o t h e s t a r t o f spermatogenesis start of vitellogenesis.  The s e q u e n t i a l experiment  but a f t e r the  i n v e s t i g a t e d the  e f f e c t o f s t a r t i n g m e t h a l l i b u r e treatment a t s u c c e s s i v e times p r i o r t o the s t a r t o f v i t e l l o g e n e s i s . m e t h a l l i b u r e had no e f f e c t . t h i s drug than do t h e males.  A t the 0.10 mg./gm. dose used, however, Females,  t h e r e f o r e , seem l e s s s e n s i t i v e t o  In t h e p i l o t  experiment, which was s t a r t e d  immediately p r i o r t o v i t e l l o g e n e s i s , the h i g h e s t dose (1.0 mg./gm.), l i k e the dose used i n the experiments  above (0.10 mg./gm.), f a i l e d t o i n h i b i t  o v a r i a n development c o m p l e t e l y .  With these r e s u l t s , i t was suggested  that  treatment e a r l y i n j u v e n i l e l i f e w i t h a h i g h e r dose than t h a t used i n t h e long-term experiment  may i n h i b i t o v a r i a n m a t u r a t i o n .  The dose i s l i m i -  t e d t o below 1.0 mg./gm. s i n c e , i n u n r e p o r t e d d a t a , treatment w i t h t h i s dose i n j u v e n i l e s caused h i g h m o r t a l i t y . M e t h a l l i b u r e had an a n t i t h y r o i d a l e f f e c t under n a t u r a l p h o t o p e r i o d but not under c o n s t a n t 12L:12D p h o t o p e r i o d i n the long-term experiment o r at a h i g h dose (1.0 mg./gm.).  Kidney d i s e a s e may, t o some degree, be  i n v o l v e d i n t h e e f f e c t under n a t u r a l p h o t o p e r i o d .  62  M e t h a l l i b u r e reduced the r a t e o f i n c r e a s e i n body weight. The  constant 12L:12D p h o t o p e r i o d slowed  t i o n o f both males and females. t u r e suggests  the r a t e o f gonadal  matura-  Comparing these r e s u l t s w i t h the  litera-  t h a t a s p e c i f i c d a y l e n g t h i n the n a t u r a l p h o t o p e r i o d  an endogenous rhythm s t i m u l a t e the i n i t i a t i o n ,  maintenance, and  t i o n o f gonadal m a t u r a t i o n and t h a t the s e a s o n a l d a y l e n g t h  and  termina-  fluctuations  f u n c t i o n as a s y n c h r o n i z e r . From t h i s study, o n l y t e s t i c u l a r m a t u r a t i o n c o u l d be d e l a y e d which c o u l d a l l o w the male gene complement t o be used i n p o p u l a t i n g a  "poor"  y e a r , assuming t h a t i n h i b i t i o n o f t e s t i c u l a r m a t u r a t i o n i s r e v e r s i b l e a f t e r m e t h a l l i b u r e treatment.  However, s i n c e Funk and Donaldson  (1972)  were able t o mature male p i n k salmon i n the y e a r o f h a t c h i n g u s i n g part i a l l y p u r i f i e d salmon g o n a d o t r o p i n , a t h r e e y e a r program t o o b t a i n the same r e s u l t s i s i m p r a c t i c a l .  The v a l u e o f a t h r e e y e a r program would  be t o s u c c e s s f u l l y d e l a y gonadal m a t u r a t i o n o f the females tional  year.  f o r one a d d i -  63  BIBLIOGRAPHY  Ahsan, S.N. 1966. E f f e c t s o f g o n a d o t r o p i c hormones on male hypophysectomized l a k e chub, Couesius plumbeus. Can. J . Z o o l . 44: 703717. 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