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The reproductive physiology of triploid Pacific salmonids Benfey, Tillmann J. 1988

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THE REPRODUCTIVE PHYSIOLOGY OF TRIPLOID PACIFIC SALMONIDS by TILLMANN JOACHIM NIENSTEDT BENFEY  B.Sc. (first class honours), McGill University, 1981 M . S c , Memorial University of Newfoundland, 1984  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY  in  THE FACULTY OF GRADUATE STUDIES (Department of Zoology)  We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITSH COLUMBIA May 1988 ©Tillmann Benfey, 1988  In  presenting  degree at the  this  thesis in  University of  partial  fulfilment  of  the  requirements  for  an advanced  British Columbia, I agree that the Library shall make it  freely available for reference and study. I further agree that permission for extensive copying of  this thesis for  department  or  publication of  by  his  or  scholarly purposes may be granted by the her  Department The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3  DF-fin/ft-n  It  is  understood  that  copying  my or  this thesis for financial gain shall not be allowed without my written  permission.  Date  representatives.  head of  page i i  ABSTRACT  Triploidy was induced in rainbow trout, Salmo gairdneri Richardson, by heat shock (10 min at 26, 28 or 30°C, applied 1 min after fertilization at 10°C) and in pink salmon, Oncorhynchus gorbuscha Walbaum, and coho salmon, 0. kisutch Walb., by hydrostatic pressure shock (1, 2, 3 or 4 min at 69,000 kPa, applied  15 min after  fertilization  at  10.5°C).  Triploid  individuals were  identified by the flow cytometric measurement of DNA content of erythrocytes stained with propidium iodide.  Gonadosomatic index was reduced to a much greater extent in triploid females  than  virtually  males.  no oocytes.  Triploid Triploid  ovaries testes  remained  very  became quite  developed beyond the spermatocyte stage.  small, large,  and contained but  few  cells  Triploid male rainbow trout had  significantly lower spermatocrits than diploids, and their  spermatozoa were  aneuploid.  Growth rates were the same for diploid and triploid rainbow trout, but triploid female pink salmon were smaller than maturing diploid females and diploid and triploid males of the same age. Triploid males of both species developed typical secondary sexual characteristics and had normal endocrine profiles for plasma sex steroids and plasma and pituitary gonadotropin, but their  cycle was delayed by about one month. Triploid females developed no  secondary sexual characteristics and showed no endocrine signs of maturation, even at the level of the pituitary.  page i i i  Vitellogenin synthesis was induced in immature diploid and triploid coho salmon by the weekly injection of 17p-estradiol. Plasma vitellogenin and pituitary  gonadotropin  sham-injected  fish,  levels were significantly whereas  plasma  elevated  gonadotropin  over  levels  levels  were  of  slightly  depressed. There was no significant difference between diploids and triploids for  any of  these  results,  indicating  that  normal  vitellogenesis  is  not  impaired by triploidy per se.  It sterile,  but  is  concluded that  that  only  triploids  triploid  of  females  both  do  not  sexes  are  undergo  genetically  physiological  maturation. Triploid testes develop sufficiently for their steroidogenic cells to become active, which is not the case for triploid ovaries. The occasional cells that pass through the normal meiotic block develop to full maturity in triploid males but not in triploid females, probably due to the absence of the appropriate triploids  of  stimulus  to  initiate  and  maintain  vitellogenesis.  both sexes should make valuable tools for  Although  basic research on  reproductive physiology, only the females will be useful for practical fish culture to avoid the economically detrimental destined for human consumption.  effects of maturation in fish  page iv  TABLE OF CONTENTS ABSTRACT  ii  LIST OF TABLES  v  LIST OF FIGURES  vi  ACKNOWLEDGEMENTS  vii  PREFACE  ix  INTRODUCTION Literature Review: The Physiology of Triploid Fish Research Outline: The Reproductive Physiology of Triploid Pacific Salmonids  13  MATERIALS AND METHODS Fish Radioimmunoassay Procedures Reproductive Endocrinology Spermiation Induced Vitellogenin Production Statistics  16 19 26 27 28 29  RESULTS Gonadal Development (Pink Salmon) Growth Rate and Reproductive Endocrinology (Pink Salmon) Growth Rate and Reproductive Endocrinology (Rainbow Trout) Secondary Sexual Characteristics (Pink Salmon and Rainbow Trout) Spermiation (Rainbow Trout) Induced Vitellogenesis (Coho Salmon)  30 38 41 46 48 51  DISCUSSION  57  REFERENCES  . 68  APPENDICES Appendix 1: Papers describing or utilizing spontaneously-arisen or experimentally-induced triploid fish in species that are normally only dioecious diploids Appendix 2: Papers describing or utilizing spontaneously-arisen or experimentally-induced triploid HYBRID fish of species that are normally only dioecious diploids Appendix 3 [manuscript in review]: An homologous radioimmunoassay for coho salmon (Oncorhynchus kisutch) vitellogenin, with general applicability to other Pacific salmonids  1  97  104  108  page v  LIST OF TABLES Table 1. Growth and endocrine status of diploid and triploid male pink salmon Table 2. Growth and endocrine status of diploid and triploid female pink salmon Table 3. Maturity status of diploid and triploid rainbow trout and reproductive characteristics of mature males  39 40 49  page vi  LIST OF FIGURES Figure 1. Timing of shocks (*) for the production of triploids and tetraploids ( ^ a n d represent haploid maternal and paternal chromosome sets, respectively) Figure 2. Diploid (2n) and triploid (3n) ovaries (top) and testes (bottom) from adult pink salmon (scale in cm) Figure 3. Histological sections of advanced vitellogenic stage oocytes from diploid pink salmon (bar = 500 jjm) Figure 4. Histological sections of pre-vitellogenic stage oocytes (a) and atretic, vitellogenic stage oocytes (b) from diploid pink salmon (bar = 500 jjm) Figure 5. Histological sections of triploid pink salmon ovaries devoid of oocytes (a), or with one late perinucleolar stage oocyte (b) (bar = 500 pm) Figure 6. Histological section of a late perinucleolar stage oocyte from a triploid pink salmon (close-up from Figure 5b, bar = 100 ^im) Figure 7. Histological sections of diploid (a) and triploid (b) pink salmon testes at high magnification (I = primary spermatocytes, II = secondary spermatocytes, St = spermatids, bar = 50 pm) Figure 8. Histological sections of diploid (a) and triploid (b) pink salmon testes at low magnification (light blue = primary and secondary spermatocytes, dark blue = spermatids, bar = 500 pm). . . Figure 9. Growth rates of diploid and triploid rainbow trout ( A and A = 2-year-old diploid and triploid males respectively, O and • = 3-year-old diploid and triplod females, respectively). . . Figure 10. Plasma steroid and gonadotropin levels in diploid and triploid male rainbow trout (ng/ml, A = diploid, A = triploid). . . Figure 11. Plasma steroid and gonadotropin levels in diploid and triploid female rainbow trout (ng/ml, 0= diploid, • = triploid, •fc= ovulated) Figure 12. External appearance of adult diploid (2n) and triploid (3n) rainbow trout (a = males, b = females, scale in cm) Figure 13. Typical DNA content profiles of (a) sperm from a diploid male, (b) sperm from a triploid male, (c) blood from a diploid male, and (d) blood from a triploid male rainbow trout Figure 14. Histological sections of diploid (a) and triploid (b) coho salmon ovaries (bar = 500 pm) Figure 15. Histological sections of early vitellogenic (a) and atretic (b) oocytes from diploid coho salmon.(yg = yolk globule, bar = 500 jum) Figure 16. Histological sections of diploid (a) and triploid (b) coho salmon testes (bar = 50 pm) Figure 17. Change in plasma vitellogenin (Vtg) and gonadotropin (GtH) over 3 weeks, and final values for hepatosomatic index and pituitary GtH content in coho salmon (oand A = diploid and triploid sham-injected, respectively, • and A = diploid and triploid 17B-estradiol treated, respectively)  4 31 32 33 34 .  35  36 .  37  .  43  .  44 45 47 50 53 54 55  56  page vii  ACKNOWLEDGEMENTS  First and foremost, I wish to thank Catherine Clancy, who became an egg-picker extraordinaire  in Newfoundland, followed me across the breadth of  Canada in 1984, encouraged and supported me through seven years of M.Sc. and Ph.D. research and writing, and became my wife on August 8, 1987.  I  also thank my advisory committee:  without whose f a c i l i t i e s possible,  and resources this  and Dave Randall, who maintained  co-supervisors Ed Donaldson, research would not have been  the link  with U.B.C.,  and the  remaining committee members: Fred D i l l , Cas Lindsey, Ray Peterson and George Iwama. Special thanks to Monica Thain for ensuring that all ran smoothly at U.B.C. in spite of my infrequent v i s i t s .  I sincerely thank the staff of the Fish Culture Research Section at the West Vancouver Laboratory: especially Helen Dye, who was a tremendous help and good friend throughout my research, and who showed me that it is possible to survive government bureaucracy; and also Ian Baker, Morva Booth, Andy Lamb, Jack McBride and Igor Solar. I also thank Ted Down, who helped and advised me with  various  aspects  of  my  research,  especially  with  the  protein  radioimmunoassays.  I especially wish to thank John Sumpter of Brunei University, who gave me the impetus to start my research when he visited in 1984, encouragement  and highly  beneficial  criticism  throughout  the  provided  intervening  page v i i i  years, and helped me to understand what it  all meant during another visit in  1987.  I  also thank Gary de Jong of the Cancer Control Agency of British  Columbia for his help and time spent with the flow cytometer; Terry Owen of Helix Biotech Ltd. and John Sumpter for providing materials for the protein radioimmunoassays;  the  Salmonid  Enhancement  Program for  providing  salmon  gametes; and the Natural Sciences and Engineering Research Council, the Quebec Fonds FCAR, the Department  of Fisheries and Oceans, and the University of  British Columbia for providing financial support.  This thesis is dedicated to my parents: Bruno G. Benfey, M.D., who introduced me to science and biology, and Jutta Benfey (geb. Nienstedt), who ensured that I remembered my humanity.  page ix  PREFACE  Common names have generally been used for fish species described in this  thesis;  their  correct  scientific  names and taxonomic classification  (based on Golvan, 1962) appear in Appendices 1 and 2.  Following convention,  the female parent is always given f i r s t when describing hybrids.  - 1 -  INTRODUCTION  Literature Review: The Physiology of Triploid Fish.  Sexually  maturing  fish  undergo  behavioural changes that may reduce their  numerous  physiological  and  value as fish destined for human  consumption. Various methods have therefore been devised and investigated to delay or entirely (Chevassus et  prevent  al.,  1979;  sexual maturation Stanley, 1979,  Refstie,  1982;  Yamazaki,  Shelton,  1986;  Donaldson and Benfey,  Shelton,  1987). One of  triploidy,  1983;  induced triploid  1981;  Bye and  1987;  is the only practical  aquaculture  Donaldson and Hunter,  Lincoln,  the most effective  because the resulting fish  in fish used for  1986;  Pandian of  these  Donaldson,  1986;  and Varadaraj,  1987;  is  are genetically  1982;  the  induction of  sterile.  means by which to  In  fact,  sterilize  large  numbers of fish without the use of potentially harmful chemicals or radiation treatment.  Triploids have three  sets of chromosomes rather  than the normal  (diploid) number of two. Natural populations of triploids have evolved in six genera representing three  orders of f i s h .  Three of  these cases  are well  documented, and have been reviewed by Schultz (1979) and Purdom (1984): these are  in  Poeci1i a and  Poeciliidae)  Poeci1iopsis  (both  order  Cyprinodontiformes,  family  and Carassius (Cypriniformes, Cyprinidae). The remaining three  are recent discoveries in Menidia (Mugi1iformes, Atherinidae) (Echelle et a l . , 1983), Phoxinus (Cyprinidae) Rutilus (Cyprinidae)  (Joswiak et a l . , 1985; Dawley et a l . , 1987) and  (Collares-Pereira, 1987). These fish are always fertile  - 2 -  unisexual females that reproduce by atypical means involving gynogenesis or hybridogenesis, and hence will not be considered further.  Triploid individuals of species that are normally dioecious diploids are occasionally found among wild f i s h , and can readily be produced in species for which it  is possible to manipulate  spawning. A comprehensive l i s t  of  papers describing or utilizing spontaneously-arisen or experimentally-induced triploids appears in Appendices 1 and 2. Virtually all these fish have become triploid  by the retention  of the second polar body, and thus contain two  maternal and one paternal haploid chromosome sets. The notable exceptions are triploid  rainbow trout produced by the breeding of  tetraploid  males with  diploid females (Chourrout et a l . , 1986b; Blanc et a l . , 1987; Chourrout and Nakayama,  1987;  Oliva-Teles  and  Kaushik,  1987a)  or  by  the  dispermic  fertilization of haploid eggs after treating the spermatozoa with polyethylene glycol  (Ueda et  al.,  1986).  In  these cases,  the resultant  fish have one  maternal and two paternal haploid chromosome sets.  Induced interspecific  triploidy  hybrids,  (Chevassus, 1983;  can  although  be it  Krasznai, 1987;  used is  to  increase  the  by no means clear  Neavdal  and Dalpadado,  viability  why this 1987).  of  is so  Increased  viability of triploid hybrids has been demonstrated in salmonids (Capanna et a l . , 1974; Chevassus et a l . , 1983; Scheerer and Thorgaard, 1983; Utter et a l . , 1983;  Arai, 1984;  Ueda et  al.,  1984; Arai, 1986;  Parsons et a l . , 1986; Seeb et a l . , 1986;  Chourrout, 1986a, 1986c;  Scheerer et  al.,  1987), cyprinids  (Vasil'ev et a l . , 1975; Stanley, 1976; Stanley et a l . , 1976; Beck and Biggers, 1982, 1983b; Beck et a l . , 1984)  and tilapia  (Chourrout and Itskovich, 1983).  - 3 -  This increased viability has enabled the use of hybridization as a means to confer such valuable traits as disease resistance (Dorson and Chevassus, 1985, 1986; Parsons et a l . , 1986)  and salinity tolerance (Glebe et a l . , 1986)  salmonid hybrids that are not viable as diploids. Intraspecific may be a useful  way of  decreasing treatment-associated  to  hybridization  mortalities  when  inducing triploidy (Sutterlin et a l . , 1987).  Retention  of  the  second polar  body can easily  be achieved by  interfering with its movement shortly after f e r t i l i z a t i o n .  Vertebrate eggs  always possess three chromosome sets for a short time after  fertilization,  since it is the actual process of fertilization by a haploid spermatozoan that stimulates the haploid second polar body to leave the egg with its haploid pronucleus (Figure 1). Both thermal and hydrostatic pressure shocks are very effective  for  inhibiting movement of the second polar  probably act by different  body in  fish,  but  means (Chourrout, 1986b, 1987). Retention of the  second polar body increases the genomic heterozygosity of triploids (Allendorf and Leary, 1984; Leary et a l . , 1985). Chromosome set manipulation in f i s h , including induced triploidy,  has been the  subject of many recent  reviews  (Allen and Stanley, 1981b; Purdom, 1983; Thorgaard, 1983; Chevassus et a l . , 1984;  Lincoln and Bye, 1984b; Purdom, 1984;  Allen and Wattendorf,  1986;  Chourrout et a l . , 1986a; Purdom, 1986; Thorgaard, 1986; Allen and Wattendorf, 1987; Chevassus, 1987; Chourrout, 1987; Clugston and Shireman, 1987; Nagy, 1987; Thorgaard and Allen, 1987).  Because triploid cells have 50% more DNA than diploid c e l l s ,  their  nuclei and the cells themselves are significantly larger than diploid nuclei  -  Figure 1. Timing  of  tetraploids  shocks  (*)  ( (^) and  4 -  for  the  production  of  represent haploid maternal  chromosome sets, respectively).  triploids  and  and paternal  - 5 -  and c e l l s .  This is likely  a general  feature of all  tissues and organs in  triploid fish (Swarup, 1959a; Small and Benfey, 1987), and has been clearly demonstrated for  the erythrocytes  of many species (coho salmon: Small and  Benfey, 1987; rainbow trout: Lou and Purdom, 1984; Chourrout et a l . , 1986b; Kim et a l . , 1986; Atlantic salmon: Benfey and Sutterlin, 1984c; Benfey et a l . , 1984; Graham et a l . , 1985; ayu: Taniguchi et a l . ,  1985;  grass carp hybrids  with bighead carp: Beck and Biggers, 1983a; common carp: Ueno, 1984;  loach:  Suzuki et a l . , 1985; European catfish: Krasznai et a l . , 1984b; Krasznai and Marian, 1986; channel catfish: Wolters et a l . , 1982a; Chrisman et a l . , 1983; African catfish: Richter et a l . , 1987; threespine stickleback: Swarup, 1959a; tilapia: Don and Avtalion, 1986). Furthermore, the erythrocytes of tetraploids are larger s t i l l  It  (Chourrout et a l . , 1986b).  can be inferred that cell  numbers must be reduced in  triploid  fish to compensate for increased cell size, since, with the exception of gonad size (discussed below),  organ and body size do not change with increased  ploidy. However, this has only been confirmed for triploid erythrocytes, where the increase in cell size is balanced by a decrease in cell numbers, such that there is no effect of triploidy on hematocrit (Barker et a l . , 1983; Benfey and Sutterlin, 1984c; Ueno, 1984; Graham et a l . , 1985; Small and Randall, 1988).  Cellular and total blood hemoglobin concentrations are the same for diploid and triploid  grass carp hybrids with bighead carp (Barker et  al.,  1983), but this is not the case for salmonids. Both cellular and total blood hemoglobin levels  are  lower  for  triploids  in Atlantic  salmon (Benfey and  Sutterlin, 1984c; Graham et a l . , 1985), and the latter is also true for coho  - 6 -  salmon (Small  and Randall, 1988). More detailed analyses by Graham et  (1985) on Atlantic salmon have shown that hemoglobin-oxygen affinity  al.  (p50)  is  the same for the blood of diploids and triploids, but that triploids have lower  blood  pH and hence  both  a lower  hemoglobin-oxygen  loading  ratio  (Hufner's constant) and maximum blood-oxygen carrying capacity than diploids.  In spite of these hematological differences, there is l i t t l e or no apparent effect of triploidy on oxygen consumption rate (Swarup, 1959c; Benfey and Sutterlin, 1984b; Oliva-Teles and Kaushik, 1987a, 1987b), oxygen level at asphixiation (Small  (Benfey  and Sutterlin,  1984b), or critical  swimming velocity  and Randall, 1988). Osmoregulation is also unaffected  in  triploids  (Lincoln and Bye, 1984a; Johnson et a l . , 1986; Quillet et a l . , 1987).  These factors indicate either decrease  in  cell  consequence to triploids  are  numbers  have  associated with  an effect  able to  that the increase in cell  regulate  triploidy  on most physiological their  sufficient degree to overcome potential  are  of  insufficient  processes,  physiological control  size and  or  that  systems to a  problems. The latter is more likely  the case, since there is some evidence that triploids perform  more poorly  than diploids when raised under suboptimal conditions (Lincoln and Bye, 1984b; Johnson et a l . , 1986; Boulanger, 1987; Quillet et a l . , 1987).  Triploids are sterile because their gonial cells (spermatogonia in males and oogonia in females) cannot complete meiosis. This is likely due to the inability of homologous chromosomes to pair as divalents in prophase of the f i r s t meiotic division. However, mitosis is unaffected, since such pairing  - 7 -  of  homologues does not  occur  in  this  type  of  cell  division,  and hence  triploids grow normally and, with one exception (Sutterlin et a l . , 1987), are morphologically similar  to diploids  and have the same developmental  rates  (Swarup, 1959a; Leary et a l . , 1985).  The gonadal development of triploids is retarded to a much greater extent in females than in males. Generally only a very small proportion of gonial cells develop beyond the f i r s t meiotic prophase in triploids of either sex. However,  because of the enormous difference  between the sexes in the  number of pre-meiotic gonial cells normally produced, triploid testes become much larger than triploid ovaries. Furthermore, the small proportion of cells that do pass through the normal meiotic block appear to develop normally in triploid testes, but not in triploid ovaries.  This sexual difference in the degree of gonadal development and fate of post-meiotic cells in triploids has been documented in many species (coho salmon: Johnson et a l . , 1986; rainbow trout: Thorgaard and Gall, 1979; Lincoln and Scott, 1983; Yamazaki, 1983; Chevassus et a l . , 1984; Lincoln and Scott, 1984; Solar et a l . , 1984; Okada, 1985; Solar and Donaldson, 1985; Nakamura et al., 1986;  1987; Atlantic salmon: Benfey and Sutterlin,  1984a; ayu: Ueno et  grass carp: Thompson et a l . , 1987; common carp: Gervai et a l . ,  al., 1980;  Taniguchi et a l . , 1986b; willow gudgeon: Ueno, 1985; rose bitterling: Ueno and Arimoto, 1982;  loach: Suzuki et  al.,  1985;  European catfish: Krasznai and  Marian, 1986; channel catfish: Wolters et a l . , 1982b; Chrisman et a l . ,  1983;  African catfish: Henken et a l . , 1987; Richter et a l . , 1987; tilapia: Shah and Beardmore,  1986;  Penman et  al.,  1987b;  and plaice  and  its  hybrid  with  - 8 -  flounder: Purdom, 1972, 1976; Lincoln, 1981a, 1981b).  Wu et  al.  (1986) have suggested that true triploid  carp are in fact f e r t i l e , sterile the  are  in  fact  overwhelming  and that the presumed triploid  aneuploid. However,  evidence  supporting  this  the  female common  females that are  seems unlikely  virtually  in  complete  light  of  absence  of  post-meiotic oocyte growth in triploid females not only of this species, but of all other normally dioecious diploid species of fish listed in the previous paragraph.  Plasma sex steroid levels were found to be the same in spermiating diploid and triploid male plaice (Lincoln, 1981b) and post-ovulatory diploid and sterile  triploid  female  hybrids between plaice and flounder  1981c). However, only three diploids and three triploids  (Lincoln,  of each sex were  examined.  More detailed studies have revealed that triploid male rainbow trout have normal plasma sex steroid hormone levels (Lincoln and Bye, 1984b; Lincoln and Scott, 1984; Benfey et a l . , 1987), although levels peak about one month later than in diploid males (Benfey et a l . ,  1987). On the other hand, sex  steroid levels remain low or not detectable through the normal pre-spawning period in triploid females (Lincoln and Bye, 1984b; Lincoln and Scott, Benfey et obtained  al., for  1987;  sex  Nakamura et  steroid  levels  al., in  1987).  triploid  Similar pink  results  salmon;  in  1984;  have been addition,  pituitary gonadotropin levels are much higher in maturing diploid females and diploid and triploid males than in triploid females, but plasma levels remain low in all these fish (Benfey et a l . , 1987). Even one year prior to spawning,  - 9 -  diploid female rainbow trout have higher plasma sex steroid and gonadotropin levels than triploid females ever reach (Sumpter et a l . , 1984).  Similar results have been reported for plasma sex steroid levels in triploid hybrids between grass carp and bighead carp when compared to diploids of either parental species (Krasznai et a l . , 1984a), i f one assumes that the triploids  of  unidentifiable  sex  were  females.  available for comparison in this study, so i t  No  diploid  remains unclear whether  results are a reflection of triploid s t e r i l i t y or hybrid  Johnson et levels  between  al.  diploid  hybrids  were these  sterility.  (1986) found no difference in plasma 17p-estradiol and triploid  female  coho salmon at  the  onset  of  maturation in the diploids, but levels were s t i l l very low and near the limits of assay detectabi1ity. diploids.  Triploid  Nevertheless, vitellogenin was detectable only in the  female  salmonids  generally  have  much  lower  plasma  vitellogenin levels than diploid females (Sumpter et a l . , 1984; Benfey et a l . , 1987). This is likely due to the absence of the normal estrogen stimulus from the ovaries on vitellogenin  synthesis by the liver,  since the injection  of  17j3-estradiol results in a normal rate of vitellogenin synthesis in triploid coho salmon (Benfey et a l . , 1987).  When compared with maturing diploid females, triploid females have large fat reserves in the viscera (Thorgaard and Gall, 1979; Chevassus et a l . , 1984; Lincoln and Scott, 1984; Johnson et a l . , 1986; Taniguchi et a l . , 1986b), higher lipid  and lower water content in the flesh (Chevassus et a l . ,  1984;  Lincoln and Bye, 1984b, 1987), and smaller livers (Lincoln and Scott,  1984;  - 10 -  Johnson et  al.,  1986). These traits can all  be attributed  to the lack of  vitellogenin synthesis by the liver and consequent lack of lipid withdrawal from the flesh of triploid females (Lincoln and Scott, 1984). A further result of their s t e r i l i t y  is that triploid females have more consumable flesh than  maturing diploid females of the same size (Lincoln, 1981c; Chrisman et a l . , 1983; Chevassus et a l . , 1984; Lincoln and Bye, 1984b; Lincoln and Scott, 1984; Ueno et a l . , 1986; Henken et a l . , 1987; Lincoln and Bye, 1987; Penman et a l . , 1987b; Richter et a l . , 1987), and their flesh remains fully pigmented and has a firmer texture and better flavour (Lincoln and Bye, 1984b, 1987).  The development  of  secondary sexual  characteristics  in  fish  is  controlled by the gonadal sex steroids (Matty, 1985). Thus, because triploid males have normal sex steroid levels and triploid females do not, generally only the males develop secondary sexual characteristics at the normal time of maturation,  with the external  appearance of mature diploid males  (Swarup,  1959a; Thorgaard and Gall, 1979; Chevassus et a l . , 1984; Lincoln and Scott, 1984; Okada, 1985; Benfey et a l . , 1986; Ueno et a l . , 1986; Benfey et  al.,  1987). However, the absence of secondary sexual characteristics has been noted in triploid 1983)  male channel catfish  and tilapia  (Don  (Wolters  and Avtalion,  et a l . , 1982b; Chrisman et  1986),  suggesting that  al.,  testicular  steroidogenesis is in some way diminished in triploids of these two species. Conversely, triploids of both sexes develop secondary sexual characteristics in  the  willow  gudgeon,  the  only  dioecious  species  of  fish  for  which  substantial ovarian development has been documented in triploid females (Ueno, 1985).  - 11 -  Not only do most triploid males develop typical  secondary sexual  characteristics, but they are able to produce milt, albeit  with a very low  density of spermatozoa (Lincoln, 1981b; Chevassus et a l . , 1984; Lincoln and Scott, 1984; Dawley et a l . , 1985; Okada, 1985; Ueno, 1985; Allen et a l . , 1986; Benfey et a l . , 1986; Penman et a l . , 1987b). These spermatozoa are larger than normal haploid spermatozoa (Lincoln, 1981b; Lincoln and Scott, 1984; Okada, 1985; Ueno, 1985), and are aneuploid due to their having 50% more DNA per cell (Allen et a l . , 1986; Benfey et a l . ,  1986). These aneuploid spermatozoa are  motile and are able to f e r t i l i z e normal eggs, but the resultant embryos are themselves aneuploid (Ueda et a l . , 1987), and generally do not survive beyond hatching (Lincoln, 1981b; Lincoln and Scott, 1984; Dawley et a l . , 1985; Okada, 1985; Ueno, 1985; Nagy, 1987; Penman et a l . , 1987b; Thompson et a l . , 1987). Triploid males will attempt to spawn with normal diploid females i f placed in the appropriate environment (Dawley et a l . , 1985). In rainbow trout, triploid females  have  better  survival  than  diploids  through  the  spawning  and  post-spawning period (Chevassus et a l . , 1984; Lincoln and Bye, 1984a; Quillet et a l . , 1987), which is not the case for triploid males (Benfey et a l . , 1986, 1987; Quillet et a l . , 1987).  Thus, in spite of being genetically sterile, triploid males appear to mature endocrinologically, physiologically and behaviourally. By producing all-female triploids, all these traits attributable to normal maturation can be avoided.  This has been done in rainbow trout by fertilizing  normal eggs  with milt from sex-reversed females, followed by either heat shock (Lincoln and Scott, 1983; Lincoln and Bye, 1987) or hydrostatic pressure shock (Okada, 1985) to induce triploidy. This method has the greatest promise for providing  - 12 -  sterile rainbow trout for  aquaculture (Chevassus et a l . , 1984;  Lincoln and  Bye, 1984b; Bye and Lincoln, 1986; Chourrout et a l . , 1986a; Purdom, 1986), and should be useful for other species as well (Donaldson, 1986; Donaldson and Benfey, 1987; Shelton, 1987).  Triploids have metabolic rates the same or very similar to diploids (Fauconneau et  al.,  1986;  Wiley  and Wike,  1986;  Oliva-Teles and Kaushik,  1987a). However, there appear to be minor differences in protein  utilization  (Oliva-Teles and Kaushik, 1987a; Henken et a l . , 1987; Richter et a l . , 1987), which may necessitate developing different diets for triploids (Henken et a l . , 1987).  Triploids  have the  same ability  to  fix  dietary  canthaxanthin  as  diploids (Choubert and Blanc, 1985). Food conversion rates appear to be the same  for  immature  diploids  and  triploids  (Solar  and  Donaldson,  1985;  Boulanger, 1987; Henken et a l . , 1987), although this was not found to be the case  for  triploid  conversion rates  plaice  than  hybrids  with  flounder,  immature diploid hybrids  which  (Lincoln,  have  lower  1981c).  food  However,  triploids have higher food conversion rates than maturing diploids of the same size and age (Lincoln, 1981c; Wolters et a l . , 1982b; Chrisman et a l . , 1983). There is some indication that triploids have lower food consumption rates than diploids, which may lead to decreased growth rates (Lincoln, 1981c; Wiley and Wike, 1986).  Data on the growth rates of immature-aged triploids are equivocal. Most studies have reported that their  growth rate is the same as that of  immature diploids (Purdom, 1976; Gervai et a l . , 1980; Wolters et a l . , 1982b; Chrisman et a l . , 1983; Benfey and Sutterlin, 1984a; Solar and Donaldson, 1985;  - 13 -  Cassani and Caton, 1986b; Don and Avtalion, 1986; Johnson et a l . , 1986; Kim et al.,  1986; Shah and Beardmore, 1986;  Boulanger, 1987;  Henken et  al.,  1987;  Kowtal, 1987; Lincoln and Bye, 1987; Penman et a l . , 1987b; Richter et 1987), but there have been reports both of inferior  al.,  growth (Lincoln, 1981c;  Utter et a l . , 1983; Chevassus et a l . , 1984; Solar et a l . , 1984; Okada, 1985; Suzuki et  al.,  1985;  Cassani and Caton, 1986b; Chourrout  et  al.,  1986a;  Taniguchi et a l . , 1986b; Blanc et a l . , 1987; Lincoln and Bye, 1987; Penman et a l . , 1987b; Quillet et a l . , 1987) and superior growth (Valenti, 1975; Krasznai and Marian, 1986;  Taniguchi et  al.,  1986b). These variable results may be  attributable to competition between diploids and triploids (Cassani and Caton, 1986b; Lincoln and Bye, 1987; Penman et a l . , 1987b) or to stock differences (Solar et a l . , 1984; Solar and Donaldson, 1985).  Data on the growth rates of mature-aged triploids are, on the other hand, unequivocal. When diploid females begin to mature their decrease,  whereas  triploid  females  continue  to  grow  growth  through  the  rates normal  spawning period (Purdom, 1976; Lincoln, 1981c; Chevassus et a l . , 1984; Lincoln and Bye,  1984a,  Thorgaard, 1986; true of  triploid  1984b;  Suzuki  et  al.,  1985;  Chourrout  et  al.,  1986a;  Lincoln and Bye, 1987; Quillet et a l . , 1987). The same is channel  catfish  of  both  sexes  (Wolters  et  al.,  1982b;  Chrisman et a l . , 1983).  Research Outline:  The  The Reproductive Physiology of Triploid Pacific Salmonids.  preceding  literature  review  showed  that  fundamental differences between diploid and triploid f i s h . is  increased  in  triploids,  with a concomitant  decrease  there  are  two  F i r s t l y , cell size in  cell  numbers.  - 14 -  Secondly,  gonadal  development  is  impaired  dimorphism in the degree of this impairment.  in  triploids,  with  a sexual  These two facts have been well  documented, but their resultant effect on the general physiology of triploids has received l i t t l e attention.  The aim of my research was to examine, in detail, the reproductive physiology  of  triploid  Pacific  salmon and rainbow  trout.  confirming that gonadal development is impaired in triploid  I  began by  pink and coho  salmon in a similar way to that of triploids of other fish species, including rainbow trout.  My goal was then to test the two hypotheses that triploid  males would mature endocrinologically, whereas triploid females would not, but that some aspects of normal maturation could be induced in triploid females by the appropriate replacement therapy with lacking steroids.  These hypotheses were based on the knowledge that triploid males appear to mature physiologically, in spite of meiotic disfunction, whereas triploid females do not.  Sexual maturation is controlled endocrinologically,  with many of the critical  hormones originating from the  gonads.  dimorphism in the gonadal development of triploids may therefore  Sexual influence  and/or result from a sexual dimorphism in reproductive endocrinology. Gonads are the only organs directly affected by problems with meiosis.  Therefore,  any effects of triploidy on other organs involved in sexual maturation may not be the result of triploidy per se, but rather an indication of some effect originating  from  the  gonads.  The  absence of  hormones  possibility, and can be tested by replacement therapy.  is  an obvious  - 15 -  To a certain extent, this research parallels and expands upon work published  by others  in  the  intervening  years  since  it  was  begun  (see  especially Lincoln and Scott, 1984; Allen et a l . , 1986; Johnson et a l . , 1986; and Nakamura et a l . , 1987).  - 16 -  MATERIALS AND METHODS  Fish.  The diploid and triploid rainbow trout (Salmo gairdneri Richardson) used for this research were provided by Igor Solar (Department of Fisheries and Oceans [DFO],  West  Vancouver  Laboratory).  Their  background has been  described by Solar and Donaldson (1985) and Benfey et a l . (1986), but will be expanded upon here. The fish came from a domesticated stock (Spring Valley Trout Farm, Langley, B.C.), that had been reared for one generation pens at the Pacific Biological Station females  and  transported  sperm from separately  4 males,  in sea  (DFO) in Nanaimo, B.C. Eggs from 4  collected  on  January  24,  1984,  on ice to the West Vancouver Laboratory,  were  but were  pooled prior to fertilization that same day.  Triploids were produced by heat shock, using a thermoregulated water bath  (Haake Mess-Technik GmbH u. Co. model F3-C, obtained through Fisher  Scientific, Vancouver, B.C.). Heat shocks of 10 min at 26, 28 or 30°C, applied to separate batches of eggs 1 min after fertilization at 10°C, yielded 23 ± 11, 86 ± 3, and 100% triploids, respectively. These fish were combined with controls and diploid survivors from unsuccessful heat shocks ( i . e . , 10 min at 24°C and 1 min at 34-35°C, also applied 1 min after fertilization at 10°C) at the yearling stage.  Pink salmon (Oncorhynchus gorbuscha Walbaum) gametes were collected by hatchery staff at the DFO Quinsam River Salmonid Enhancement Program (SEP)  - 17 -  hatchery in Campbell River, B.C., on October 9, 1984. kept separated,  Eggs from 4 females,  and pooled sperm from 7 males were transported  plastic bags f i l l e d  on ice in  with 100% oxygen to the West Vancouver Laboratory for  fertilization that same day.  Triploids were produced by hydrostatic pressure shock using a 40 ml standard French pressure cell  (SLM Instruments,  Inc./American Instrument Co.  model FA-073, obtained through Technical Marketing Associates Ltd., Richmond, B.C.) and a Carver laboratory press (13-872, Fisher). With this apparatus a pressure of 69,000 kPa (10,000 psi) could be attained with 4 to 5 strokes (i.e.,  in  4 to  5 sec)  and pressure release was virtually  instantaneous.  Pressure within the cell was not measured directly, but could be calculated from a gauge which displayed the force applied to the c e l l ' s piston. Pressure shocks of 1, 2, 3 or 4 min at 69,000 kPa were applied to separate batches of eggs 15 min after fertilization at 10.5°C. All these groups had to be combined with controls shortly after f i r s t feeding, prior to ploidy determination.  Coho salmon (Oncorhynchus kisutch Walbaum) gametes were collected on November 20, 1984, from fish raised for one generation at the West Vancouver Laboratory, originally of stock from the DF0 Capilano River SEP hatchery in North Vancouver, B.C. Eggs from 3 females and sperm from 10 males were pooled prior  to fertilization that same day. Additional  coho salmon gametes were  collected by hatchery staff at the Capilano River SEP hatchery on November 30, 1984.  Portions of the pooled eggs from 10 females and pooled sperm from an  unspecified number of males were brought to the West Vancouver Laboratory and held on ice in plastic bags f i l l e d  with 100% oxygen for  2 days prior  to  - 18 -  fertilization.  Triploids  were produced  on both  occasions  by  hydrostatic  pressure shocks of 4 min at 69,000 kPa applied 15 min after fertilization at 10.5°C,  using the  equipment  described above. Again,  all  these  fish were  combined shortly after f i r s t feeding, prior to ploidy determination.  All the fish were reared at the West Vancouver Laboratory, using standard techniques for  salmonid culture.  Fertilized  vertically-stacked, flow through incubation trays  eggs were placed in  (Heath Tecna Corp., Kent,  WA), and the larvae were allowed to hatch and develop through yolk absorption before being transferred to 20 L fibreglass tanks. were transferred Freshwater  at  to increasingly larger  10 to  As the fish grew,  they  tanks, ultimately of 3 m diameter.  11°C was supplied from  an on-site  well.  At  the  appropriate development stage, fish were acclimated to salt water from the Burrard  Inlet,  Laboratory.  pumped  in  from  200  m offshore  from  the  Salt water temperatures fluctuated seasonally.  in constant darkness while in the incubation trays; all used a natural photoperiod.  West  Vancouver  Embryos were kept subsequent rearing  The fish were fed a commercially-obtained diet of  appropriate pellet size (Oregon Moist Pellet, Moore-Clark Co. Inc., La Conner, WA).  The ploidy of all fish used for subsequent research was determined from adults by the flow cytometric measurement of erythrocyte  DNA content,  using a Coulter EPICS V System (Coulter Electronics, Inc., Hialeah, FL) at the Cancer Control Agency of British Columbia (Vancouver, B.C.). This system uses an argon-ion laser to cause specifically stained particles in suspension to fluoresce as they pass through the laser beam in a high speed stream. The  - 19 -  intensity  of  fluorescence,  amplified by photomultipliers  proportional  to  DNA content,  and converted to digital  is measured and  form for display and  disc storage (Anonymous, 1982). The determination of ploidy level in nucleated single-cell suspensions is one of the simplest and most basic functions that can be performed with a flow cytometer (G. de Jong, Cancer Control Agency of B.C., pers. comm.), and hence diploid trout or salmon erythroctyes  are now  frequently used as standards during more complicated analyses (e.g.,  Iversen  and Laerum, 1987).  The staining technique used was based on that of Allen (1983), but was greatly simplified. One jil of whole blood was added to 1 ml of staining solution containing 50 mg/1 propidium iodide (P-5264, Sigma Chemical Co., St. Louis,  MO) in  0.1% sodium citrate  (C-7254, Sigma, prepared  water), and immediately mixed on a vortex mixer.  distilled  Cells were stained for a  minimum of 1 hour at 4°C, after which 100 jul of dimethyl Fisher) was added as a cryoprotectant.  in  sulfoxide (D-128,  Samples were stored at -30°C  until  analyzed on the flow cytometer. Fish of known ploidy were then tagged with numbered anchor tags (FD-68B, Floy Tag and Manufacturing, Inc.,  Seattle, WA)  for easy identification.  Radioimmunoassay Procedures.  Plasma sex steroid  levels were measured in diploid and triploid  rainbow trout using the radioimmunoassays described and validated by Van Der Kraak  et  al.  (1984)  20j3-dihydroprogesterone  for  testosterone,  (17,20-P)  and  17p-estradiol by  Dye  et  and al.  17«.-hydroxy(1986)  for  11-ketotestosterone. Prior to their assay, plasma samples were diluted 20-fold  - 20 -  in the steroid assay buffer, incubated at 70°C for 1 hour in covered 10x75 mm borosilicate culture tubes, centrifuged at 1,335 g (2,800 rpm) for 15 min at 4°C, and the supernatant decanted into plastic tubes and stored at -30°C. By denaturing plasma steroid binding proteins in this way, the need for plasma steroid extraction can be eliminated (Scott et a l . , 1982).  The steroid assay buffer dibasic  sodium phosphate  phosphate [S-0751,  Sigma]  [S-0876, in  was a 0.05M phosphate buffer Sigma]  distilled  and  water)  (G-2500, Sigma) and 65 mg/1 sodium azide  1.315  g/1  monobasic sodium  containing 1.0  (S-2002,  (5.75 g/1  Sigma).  g/1  gelatin  The buffer was  heated to 37°C to dissolve the gelatin, and its pH adjusted to 7.6. Standards of  testosterone  (Q-1850,  (T-1500,  Steraloids,  Inc.,  Sigma), Wilton,  173-estradiol  (E-8875,  Sigma),  NH) and 11-ketotestosterone  (lot  17,20-P 2607,  Syndel Laboratories L t d . , Vancouver, B.C.) were i n i t i a l l y dissolved in ethanol at 1 mg/ml, and then serially-diluted at 10-fold intervals to 10 ng/ml in the assay  buffer.  The  actual  standards  used  for  the  assays  were  further  serially-diluted at 2-fold intervals from 10,000 to 78.1 pg/ml.  Steroids radiolabeled with  were obtained from Amersham Canada  Ltd. (Oakville, Ontario) for testosterone (TRK.402), 17[3-estradiol  (TRK.322),  17<* -hydroxyprogesterone  (TRK.676).  (TRK.611)  and  11-ketotestosterone  Radiolabelled 17,20-P was made from the 17<*-hydroxyprogesterone  label by Dr.  J. Stoss, using the method of Scott et a l . (1982). Antibodies to testosterone and 178-estradiol  (ICN  ImmunoBiologicals  61-315  and 61-305,  respectively,  obtained through Miles Laboratories L t d . , Rexdale, Ontario) were prepared by adding 21 ml assay buffer  to 1 vial  of lyophilized antibody. Antibody to  - 21 -  17,20-P was a gift from Dr. A.P. Scott (MAFF Fisheries Laboratory, Lowestoft, England), and was used at a dilution of 1:100 from stock antibody of uncertain concentration  kept  at  the  West  Vancouver  Laboratory.  Antibody  to  11-ketotesterone was a gift from Dr. T.G. Owen (Helix Biotech Ltd., Richmond, B.C.), and was used at a concentration of 1:4,000.  For the radioimmunoassay of testosterone and 178-estradiol duplicate 10x75mm borosilicate culture tubes were set up to contain 200 |il of standard or  plamsa, 200 jjl  activity  in  assay  of  ^-steroid  buffer)  and  (5,000 cpm, diluted 200  jul  of  antibody.  to  the For  appropriate 17,20-P  and  11-ketotestosterone the tubes contained 100 yu 1 of standard or plamsa, 50yjl of ^H-steroid (2,000 cpm) and 50 ^1 of antibody. Non-specific and maximum binding controls were prepared by substituting both standard and antibody with assay buffer in the former case, and standard alone with assay buffer in the latter case.  These tubes were vortexed and left to incubate overnight at room temperature. The following morning the samples were put on ice for at least 15 min,  after  which either  200 yu 1 (testosterone  (17,20-P and 11-ketotestosterone) g/1  dextran  T-70  [Pharmacia  and 17S-estradiol)  of dextran-coated activated  (Canada)  Ltd.,  Dorval,  Quebec]  or 1 ml  charcoal  (0.5  and 5.0  g/1  activated charcoal [C-5260, Sigma] in assay buffer) was added to each tube to bind any remaining unbound steroid. The tubes were vortexed, held on ice for at least 10 min, centrifuged at 1,335 g for 10 min at 4°C, and the supernatant decanted into glass scintillation vials. Ten ml of Scinti Verse II  (So-X-12,  Fisher) was added to each v i a l , they were vigorously shaken, and placed in the  - 22 -  dark at room temperature LKB Wallac  overnight. The next morning they were counted on a  1214 RackBeta liquid  scintillation  counter  (Wallac  Oy, Turku,  Finland) for 5 min each.  Plasma testosterone and 17p-estradiol were measured in diploid and triploid rather  pink salmon using kits with the steroids radiolabeled than  (1102-D  and  1018,  respectively,  with  Radioassay  125j  Systems  Laboratories, Inc., Carson, CA). Plasma samples did not need to be extracted prior  to  their  assay with this  method. Assays were performed  exactly as  outlined in the procedures supplied with the kits. Both assays were validated by demonstrating that salmon plasmas diluted parallel  to the standards, and  that unlabelled hormone added to plasma samples at known concentrations was recovered in the correct amount.  Plasma and pituitary gonadotropin levels were measured using the radioimmunoassay described and validated by Pickering et a l . (1987), but using an immunologically less potent form of purified hormone. This assay measures the so-called "maturational" or "ovulatory" gonadotropin; the significance of this is raised in the Discussion section. gift  from  Dr.  J.P.  Sumpter  (Brunei  The hormone and its antibody were a  University,  Uxbridge,  England).  The  radioimmunoassay for plasma vitellogenin has been described and validated by Benfey et a l . (manuscript in review,  see Appendix 3).  Purified  vitellogenin  and its antibody were a gift from Dr. T.6. Owen (Helix Biotech Ltd., Richmond, B.C.).  Radiolabelled proteins (gonadotropin and vitellogenin) were prepared  - 23 -  by the  'iodogen' method (Salacinski et  al.,  1981). Thirty jil  of iodogen  (T-9018, Sigma) at 40 yjg/ml in dichloromethane was dried onto the bottom of a 1.5 ml plastic micro-centrifuge tube by allowing the solvent to evaporate. A small amount of protein (2.38 yjg of gonadotropin or 5.6 jxq of  vitellogenin)  dissolved in 20 jul of distilled water or protein-free buffer was placed in the bottom of this tube, followed immediately by 6 to 10 yul of sodium-125j  a  t  approximately 100 mCi/ml (IMS.30, Amersham). The amount of 125j added depended on its activity. The protein and sodium-125i  w e r e  mixed once by drawing both  up into a pipette tip and carefully expelling them back into the bottom of the iodogen tube. The reaction was stopped after 10 min by adding 250 /jl of column buffer  (0.05M  phosphate buffer  containing  1.0  g/1  bovine  serum albumin  [A-9647, Sigma] and 8.48 g/1 sodium chloride [S-671, Sigma], with pH adjusted to 7.4 with 1 N hydrochloric acid [S0-A-55, Sigma] or 1 N sodium hydroxide [S0-S-26, Sigma]) to the iodogen tube.  This 0.28 ml was put onto a 13x17 mm Sephadex G25 column. Another 250 /jl of column buffer was added to the iodogen tube, withdrawn, and placed onto the column as well. When the entire  0.53 ml had been drawn into the  column, it was flushed through with column buffer. Ten fractions containing 9 drops each were collected in 10x75 mm plastic culture tubes, beginning when the f i r s t 0.28 ml was added to the column. Ten jul aliquots from each fraction were counted on a LKB Wallac 1272 CliniGamma gamma counter for 10 sec each to determine  which  fraction  contained  the  most  radiolabeled  protein.  All  remaining fractions were discarded.  Further purification of the radio!abelled protein was achieved by  - 24 -  running 10 to 20 million cpm of the peak G25 fraction, diluted to 0.5 ml with column buffer, through a 10x240 mm Sephadex G100 column at 5°C. When this 0.5 ml had all gone into the column, it was flushed with column buffer at a flow rate of 1 drop every 16 to 18 sec. Up to 100 fractions were collected, with each fraction  containing 0.3  to 0.4  ml collected over 4 min. All  these  fractions were counted on the gamma counter to determine which contained the most radiolabel 1 ed protein.  These fractions were pooled and the remaining  fractions discarded.  The  protein  phosphate, 8.77  g/1  assay  buffer  contained  sodium chloride, 10.0 g/1  1.42  g/1  dibasic  sodium  bovine serum albumin, and 65  mg/1 sodium azide in distilled water. The stock solution of gonadotropin standard was at 1 jjg/ml, and was serially-diluted at 10-fold intervals to 10 ng/ml. The standards used for the assays were further  serially-diluted  at  2-fold intervals from 5,000 to 78.1 pg/ml. The stock solution of vitellogenin standard was at 280 yug/ml, and was similarly diluted to 2,800 ng/ml. The standards used for the assays were diluted from 2,800 to 10.9 ng/ml.  The radioimmunoassay for gonadotropin was performed at 4°C over a four  day period,  while  that  for  vitellogenin  was  carried  out  at  room  temperature in two days. Duplicate 10x75 mm plastic culture tubes were set up to contain 100 jul of standard or unextracted plasma and 100 ^il of antibody (at 1:40,000 for normal  rabbit  gonadotropin or serum [NRS]  1:12,000 for  vitellogenin,  [Calbiochem 869019,  Laboratories L t d . , Edmonton, Alberta]  obtained  diluted  1:400  through Terochem  prepared in assay buffer).  were vortexed and briefly centrifuged to ensure that all  in  The tubes  fluid was at  the  - 25 -  bottom of the tubes. The gonadotropin assay was left to incubate for 24 hours prior to the addition of 100 fi ! of 125i_g -  0nac  | tropin 0  (5,000 cpm, diluted to  the appropriate activity in assay buffer); 100 J J I of l25i_ -jtellogenin v  (20,000  cpm) was added immediately after the antibody. Non-specific binding controls had the standard replaced by buffer and the antibody replaced by 1:400 NRS. Maximum binding controls had only the standard replaced by buffer.  All  the tubes were again  assays were incubated (vitellogenin) rabbit  for  vortexed  a further  and briefly  24 hours  prior to the addition of 100 /jl  centrifuged. The  (gonadotropin)  or  6 hours  (1 unit) of goat antibody to  gamma-globulin (GARGG) (Calbiochem 539844, Terochem, prepared by the  addition of 12.5 ml assay buffer to 1 vial of lyophilized GARGG). The tubes were vortexed, centrifuged, and incubated for an additional 24 hours or 18 hours, respectively.  Finally,  500 ^il of buffer  was added, the tubes were  vortexed, centrifuged at 2,086 g (3500 rpm) for 30 min, aspirated, and counted for 60 sec each on the gamma counter.  For the calculation of steroid or protein concentration, counts for all  tubes f i r s t  had the non-specific binding cpm subtracted, and were then  expressed as a percentage of the maximum binding cpm. In this way, values for percentage binding should always f a l l  between 0 and 100%. Standard curves of  percentage  against  steroid or  binding protein  (Y-axis) standards  plotted  (X-axis)  log of  were used to  the  concentration  determine  of  steroid or  protein concentration in the samples. If the percentage binding was less than 20% or greater than 80% ( i . e . , off the linear part of the standard curve), the samples were either diluted in assay buffer and reassayed, or taken to have  - 26 -  less than detectable levels, respectively.  Reproductive Endocrinology.  The  monthly  collection  of  blood  samples  for  the  study  of  reproductive endocrinology began in July, 1985, for all  the pink salmon and  for the male rainbow trout, as they entered their f i r s t  reproductive phase.  Spawning was expected to begin 4 to 6 months later, when the fish were 2 years old. Monthly collection of samples from female rainbow trout began one year later, 4 to 6 months before they were expected to spawn as 3-year-olds. Fish were anesthetized  in  weight  length  and  fork  0.04%  2-phenoxyethanol  measured.  (lot  Blood was  2913,  Syndel)  collected  from  and their the  caudal  vasculature into heparinized vacutainer tubes or syringes, and centrifuged at 1,335 g for 10 min at 4°C to separate the plasma. Plasma samples were stored at -30°C until they were assayed.  All diploid  the pink salmon (11 diploid females, 11 triploid  males,  and 8 triploid  shortly after the f i r s t  males)  developed  sampling, and further  minimize stress on these f i s h .  However,  all  bacterial  females, 10  kidney  disease  sampling was discontinued to the surviving fish  (4 diploid  females, 7 triploid females, 8 diploid males, and 4 triploid males) died in September due to a water failure. These fish had their weight and fork length measured, and their  gonads were removed and weighed for the calculation of  gonadosomatic index  (GSI=[gonad weight/somatic  weight] X 100%). The gonads  were fixed and prepared for histology using standard techniques for  paraffin  embedded tissue (Humason, 1972). Pituitaries were removed, homogenized in  1.0  - 27 -  ml of protein assay buffer, and stored at -30°C.  Sampling of the male rainbow trout  (5 diploids and 5  triploids)  continued through the spawning period to mid-March, by which time they had stopped spermiating. No pituitaries female rainbow trout  were collected from these f i s h . All the  (12 diploids and 11 triploids)  died in mid-December,  again due to a water failure. By this time the diploids were just beginning to spawn  (3  females  had  ovulated).  The  pituitaries  from  these  fish  were  homogenized and stored for later assay.  The following  steroids  and proteins  were measured from plasma  samples: testosterone and gonadotropin for all f i s h , 17B-estradiol for female rainbow  trout  and  11-ketotestosterone salmon. Pituitary  all  pink  salmon,  17,20-P  for male rainbow trout,  for  all  rainbow  and vitellogenin  for  all  trout, pink  gonadotropin content was measured for female rainbow trout  and all pink salmon.  Spermiation.  On January 24, 1986, male rainbow trout developing secondary sexual characteristics (darkening of the skin and development of a hooked jaw) were separated from the remaining fish  and gradually  acclimated  to  freshwater.  Anesthetized fish were checked regularly for spermiation by manual stripping. Spermiating  triploids  could be identified  more easily  if  they were  first  stripped under water, where they produced a cloudy f l u i d . Sperm samples were collected in 10x75 mm borosilicate glass culture tubes and held on ice. Blood samples were collected as described above.  - 28 -  Spermatozoan and erythrocyte DNA content were measured by a more careful  technique  than  for  ploidy  determination,  using  the  same  flow  cytometer. One J J I of blood or sperm was generally stained, but 7-8 yu 1 of sperm from  triploid  males was  needed because of  their  low  spermatocrit.  The  appropriate volume of blood or sperm was mixed with 1.0 ml of chilled 0.5% para-formaldehyde  (Polysciences Chemicals 4018,  obtained through Analychem  Corporation L t d . , Markam, Ontario) and incubated in ice water for 10 min. The samples were then centrifuged at 170 g (1,000 rpm) for 10 min at 4°C, and the supernatant discarded. The cells were resuspended in 1.0 ml of chilled 0.1% Triton X-100 (T-6878, Sigma) and incubated in ice water for a further  3 min.  After a second identical centrifugation the cells were resuspended in 1.0 ml of the staining solution and prepared for storage as described earlier. The diploid DNA content of rainbow trout was assumed to be 5.84 pg/cell (Schmidtke et a l . , 1976).  However, this may be a slight overestimation (Johnson et a l . ,  1987).  For hematocrit  the  calculation  capillary  tubes  of  spermatocrit,  and centrifuged  for  sperm was  collected  10 min  a  in  in  hematocrit  centrifuge. On March 7, 1986, the fish were transferred to 50% seawater, and then to full seawater 3 days later. Those fish that did not survive, as well as two others that died during the freshwater phase, were weighed and had their testes removed and weighed for the calculation of GSI.  Induced Vitellogenin Production.  To induce vitellogenin production, immature diploid and triploid  - 29 -  coho salmon were injected intraperitoneally with 176-estradiol at 1 mg/kg body weight. The 173-estradiol was f i r s t dissolved in ethanol at 12.5 mg/ml, and then a 5-fold dilution was made in peanut oil to give a concentration of 2.5 mg/ml. Thus, for an average fish (0.56 kg), the injection volume was 0.22 ml. Controls were sham-injected with the same solution of ethanol in peanut o i l , but lacking the 176-estradiol. Fish were anesthetized, injected, and bled once a week for three weeks. Plasma samples were prepared as described above. All the fish were bled and killed one week after the third injection, their gonads and  livers  were  removed  and  weighed  for  the  calculation  of  GSI  and  hepatosomatic index (HSI), and their pituitaries were removed and prepared for the assay of gonadotropin content, as described earlier. The gonads were kept for histology. Gonadotropin and vitellogenin levels were measured from the plasma samples using the assays described earlier.  Statistics.  The only statistical analysis that was required throughout this work was single-factor analysis of variance. This analysis was done using 'Minitab' (Ryan et a l . , 1982), a statistical computing package available at the West Vancouver Laboratory. one  standard  Variation about the mean for any data is expressed as ±  deviation.  However,  for  graphical  presentation,  only  the  positive or negative standard deviation is shown to avoid overlapping lines.  - 30 -  RESULTS  Gonadal Development (Pink Salmon).  The ovaries from triploid pink salmon were much smaller than those from diploids when the fish died in September diploids were at  (Figure  2).  Nine of  advanced stages of vitellogenesis at this  time.  the  Their  oocytes were f u l l of yolk globules (Figure 3a), and in many cases these had begun to coalesce into solid yolk masses (Figure 3b). The remaining 2 diploid females were strikingly different: still  at  the  one appeared to be immature, with oocytes  pre-vitellogenic yolk  vesicle stage  (Figure  4a),  while  the  oocytes of the other were atretic (Figure 4b). Five of the 6 triploid females examined histologically had no developing oocytes (Figure 5a); the sixth had a single small oocyte at the late perinucleolar stage (Figure 5b). Although no diploid oocytes at  this  stage were available for  comparison, this  single  oocyte appeared normal when examined at higher magnification (Figure 6).  Triploid pink salmon testes were somewhat smaller and less white than those of diploids in September (Figure 2). were  histologically  similar,  containing  Diploid and triploid testes large  numbers  of  primary  spermatocytes, secondary spermatocytes, and spermatids (Figures 7a and 7b). However, diploid males had a greater proportion of spermatids in their testes; this was most noticeable at  lower magnifications (Figures 8a and 8b). No  spermatozoa were present in any of the testes.  - 31 -  Figure 2.  Diploid  (2n)  and t r i p l o i d  (3n)  ovaries (top)  from adult pink salmon (scale in cm).  and testes  (bottom)  - 32 -  Figure 3.  Histological  sections of  advanced v i t e l l o g e n i c  d i p l o i d pink salmon (bar = 500 urn).  stage oocytes from  - 33 -  Histological atretic,  sections  vitellogenic  (bar = 500  pm).  of  pre-vitel1ogenic  stage  oocytes  (b)  stage  oocytes  from d i p l o i d pink  (a)  and  salmon  - 34 -  Histological  sections  of  triploid  pink  salmon ovaries  devoid  oocytes ( a ) , or with one l a t e perinucleolar stage oocyte (b) 500 p ) .  (bar  - 35 -  -  Figure  7.  Histological testes  at  sections  high  of  36  diploid  magnification  secondary spermatocytes,  -  (I  (a) =  and t r i p l o i d primary  St = s p e r m a t i d s ,  bar  (b)  pink  spermatocytes, = 50 urn).  salmon II  =  - 37 -  Figure 8.  H i s t o l o g i c a l sections of d i p l o i d testes  at  low magnification  (a) and t r i p l o i d (b)  (light  blue = primary  spermatocytes, dark blue = spermatids, bar = 500 um).  pink  salmon  and secondary  - 38 -  Growth Rate and Reproductive Endocrinology (Pink Salmon).  Diploids and triploids could be separated into two distinct groups, based both on their size and their endocrine status (Tables 1 and 2): those that were maturing  (9 of the diploid females and all  triploid males) and those that were immature, sterile  (1 each of the diploid females  abnormal  and all  of the diploid and (i.e.,  atretic), or  of the triploid  females,  respectively). The maturing fish were larger both in weight and length, and had larger  gonads and GSIs. The' maturing diploid females had the highest  plasma testosterone and pituitary gonadotropin levels, followed closely by the maturing diploid and triploid males. These hormones were low or not detectable in  the  immature  178-estradiol  and  abnormal  diploid  and  sterile  triploid  females.  and vitellogenin levels were high only in the maturing diploid  females. Plasma gonadotropin was not detectable in any of the f i s h .  - 39 -  Table 1. Growth and endocrine status of diploid and triploid female pink salmon.  JULY  Maturing  Atretic  Immature  diploids  diploid  diploid  Sterile triploids  Sample size  9  1  1  11  Weight (kg)  0.37 ± 0.07  0.23  0.26  0.26 ± 0.04  Length (cm)  31.6  + 1.7  27.6  27.9  Testosterone (ng/ml)  4.5  ± 2.2  < 0.72  < 0.72  176-estradiol  1.9  ± 1.3  0.11  0.07  0.05 ± 0.01(6)  6339  ± 5794  0.19  0.11 ± 0.02  (ng/ml)  Vitellogenin (}jg/ml) Plasma gonadotropin  < 2.5  10.3 < 2.5  < 2.5  28.9  ± 1.1  < 0.72  < 2.5  (ng/ml) SEPTEMBER  Sample size  2  1  1  7  0.29  0.31 ± 0.11  Weight (kg)  0.69, 0.36  0.20  Length (cm)  34.6, 31.1  27.1  Gonad weight (gm)  42.6, 7.2  GSI (%) Pituitary gonadotropin  6.6,  2.1  7450, 1680  28.4  28.9  ± 1.1  1.13  3.02  0.09 ± 0.04  0.56  1.04  0.03 ± 0.02  1.57  0.38  1.6  ± 2.3  content (ng) (numbers in brackets = reduced sample sizes because levels were not detectable the remaining f i s h ) .  - 40 -  Table 2.  Growth and endocrine status of diploid and triploid male  pink salmon.  Maturing  Maturing  diploids  triploids  Sample size  10  8  Weight (kg)  0.37 + 0.13  0.33 ± 0.07  JULY  Length (cm)  31.3  ± 2.9  31.4  Testosterone (ng/ml)  2.7  + 1.1  2.4  17G-estradiol (ng/ml)  0.13 + 0.02(5)  0.07 + 0.03(4)  Vitellogenin (pg/ml)  0.10 ± 0.04(9)  0.11 ± 0.05(6)  Plasma gonadotropin  < 2.5  ± 2.1 ± 0.6(7)  < 2.5  (ng/ml)  SEPTEMBER  Sample size  8  Weight (kg)  0.46 +  4 0.18  0.44 +  0.1*  Length (cm)  33.4  +  4.0  32.9  +  Gonad weight (gm)  38.4  + 16.9  36.2  + 15.5  GSI {%) Pituitary gonadotropin  8.9  +  1.7  8.8  1538  ±  1051  1473  +  2.8  1.4  + 794  content (ng)  (numbers in brackets = reduced sample sizes because levels were not detectable in the remaining f i s h ) .  - 41 -  Growth Rate and Reproductive Endocrinology (Rainbow Trout).  There was no significant difference in growth rate between diploid and triploid  male rainbow trout  endocrine profiles increased  gradually  (Figure  9),  and both groups had similar  (Figure 10). Testosterone and 11-ketotestosterone from  July  to  November,  at  which  point  levels  testosterone  levelled off and 11-ketotestosterone rose dramatically. Both steroids began to decline in January with the onset of spermiation, at which time 17,20-P levels began to r i s e . There was a slight observed in the triploids,  lag in the increase in androgen levels  with both testosterone  and  11-ketotestosterone  levels being significantly lower than in the diploids in September and again in December (P<0.05), but higher than in the diploids in March (P<0.05 for testosterone only). Plasma gonadotropin levels were low prior to spermiation, but  began to  rise  in  January.  There was  only  a short  peak  of  plasma  gonadotropin in the diploid males, whereas levels continued to rise in the triploid males, and were significantly higher by mid-March (P<0.01).  Growth rates were also the same for diploid and triploid females (Figure 9 ) . The females were considerably larger than the males because they were one year older. Steroid hormone and gonadotropin levels were low or not detectable  in  triploid  females  throughout  the  normal  pre-spawning period  (Figure 11). Diploid females had constantly high 17p-estradiol and increasing testosterone levels until ovulation, at which time levels of these hormones began to drop. There was a tremendous rise in 17,20-P levels in the 3 females that ovulated in December. Plasma gonadotropin levels began to rise in the diploids with the approach of spawning, and by December were significantly  - 42 -  higher than in the triploid females (P<0.01 for the non-ovulated and P<0.001 for the ovulated females).  The ovulated diploids had significantly  higher  plasma 17,20-P and gonadotropin levels in December than those that had not yet ovulated  (P<0.001),  significantly  lower.  but  178-estradiol  Pituitary  and  testosterone  gonadotropin content  levels  was not  were  not  significantly  different between the non-ovulated and ovulated diploids (0.26 ± 0.14 and 0.55 i 0.14 mg, respectively), but was much higher than in triploid females (0.0017 ± 0.0016 mg, P<0.001 in both cases).  - 43 -  1  07  1  08  1  09  1  10  1  11  1  12  1  01  1  02  1  03  MONTH  Figure 9. Growth rates of diploid and triploid  rainbow trout  2- year-old diploid and triploid males respectively, 3 - year-old diploid and triplod females, respectively).  ( A and • = o  and  •  - 45 -  Figure 11.  Plasma steroid  and gonadotropin  levels  in diploid and triploid  female rainbow trout (ng/ml, dashed line denotes limit of assay detectability,  o= diploid, • =  triploid, ^  =  ovulated).  - 46 -  Secondary Sexual Characteristics (Pink Salmon and Rainbow Trout).  Both diploid  and triploid  male  pink  beginning to develop humps when they died in differences  between  ploidies  salmon were yellowing September,  and  with no apparent  in the development of these secondary sexual  characteristics. The maturing diploid females had not yet developed secondary sexual characteristics, and so were indistinguishable from the other females.  There was no difference in secondary sexual characteristics between diploid and triploid  male rainbow trout:  all  developed the  characteristic  hooked jaw, darkening of the skin, and white edges on the ventral fins (Figure 12a).  The  diploid  females  also  developed  normal  secondary  sexual  characteristics (darkening of the skin, white edges on the ventral f i n s , and a protruding  vent),  none of which appeared on the triploid  females  (Figure  12b). The triploid females lost noticeably more scales during handling than any of the maturing f i s h .  - 47 -  Figure  12.  External rainbow  appearance trout  of  (a = m a l e s ,  adult  diploid  b = females,  (2n) s c a l e in  and cm).  triploid  (3  - 48 -  Spermiation (Rainbow Trout).  The proportion of mature males was identical in 2-year-old diploid and triploid  rainbow  trout  (Table  3).  No mature  triploid  observed, whereas 3 diploid females matured at this age. triploid  males were identical  in  external  produced normal amounts of milk-white  females were  Although diploid and  appearance,  only  the diploids  sperm. Only 4 of the triploid males  produced sperm when stripped, and this sperm was very watery. Sperm samples from  these  significantly  4  individuals  higher  had  significantly  spermatozoan DNA content  randomly selected diploids (Table  3).  lower than  spermatocrits  and  sperm samples from 6  The mean DNA content of sperm from  triploid males was 46% greater than that from the diploid males, this being not  significantly  demonstrated that  different all  cell  from  a  50%  types exhibited  increase. discrete  The  flow  DNA content  cytometer profiles  (Figure 13).  Two triploid males died while  in fresh water (on January 28 and  February 25, 1986). In addition, 8 diploid and 3 triploid males died within 5 days of tranfer  back to full  seawater. Although these 5 triploid males were  the same size as the eight diploids, they had significantly lower GSIs (Table 3).  - 49 -  Table 3.  Maturity status of diploid and triploid rainbow trout and  reproductive characteristics of mature males Measure  (sample sizes in parentheses).  Diploids  Triploids  Maturity status Mature males {%) Mature females (%) Immature (%)  a  41.4  (24)  41.3  (19)  5.2  (3)  0.0  (0)  53.4  (31)  58.7  (27)  Reproductive measures  0  Body Weight (g) GSI (%) Spermatocrit  455.3 ± 132.5 *2.83 ± 0.76  {%)  *23.0 ± 9.5  (8) (8)  (6)  489.2 ± 114.7 0.50 + 0.11 1.8 ± 0.6  (5) (5)  (4)  Spermatozoan DNA (pg/cell) a  2.92 ± 0.11  (6)  *4.25 ± 0.09  Four fish lost identification tags and their ploidy was unknown.  (4)  All were  immature. D  Where differences between diploids and triploids are significant (P < 0.01) the larger value is marked with an asterisk.  - 50 -  Figure 13.  Typical DNA content profiles of (a) sperm from a diploid male, (b) sperm from a triploid male, (c) blood from a diploid male, and (d) blood from a triploid male rainbow trout.  - 51 -  Induced Vitellogenesis (Coho Salmon).  Six of the 10 sham-injected diploids were females, with oocytes at the yolk vesicle stage (Figure 14a); 3 of the 7 sham-injected triploids were females,  but  176-estradiol  none had developing oocytes  (Figure  14b).  Seven of  the  10  treated diploids were females; of these, 4 had oocytes at the  yolk vesicle stage (identical to Figure 14a), 2 had early vitellogenic oocytes with some yolk globules (Figure 15a), and 1 had small, atretic oocytes (Figure 15b). Five of the 10 17|3-estradiol treated triploids were females; again, none had developing oocytes (identical to Figure 14b).  With remaining  fish  the  exception  of  one  176-estradiol  were males. There was no apparent  treated  diploid,  histological  the  difference  between diploids and triploids (Figures 16a and 16b, respectively). Most cells were s t i l l at the spermatogonia! stage, but some spermatocytes were present in all the f i s h . The one exceptional diploid appeared to be sterile: it had been positively identified as a diploid by flow cytometry, yet had no developing gonads. Some hormonally-steri1ized diploids had been raised in the same tank as these f i s h , but were separated earlier  on the basis of fin clips. Most  likely this individual was a misclassified hormonally-sterilized f i s h .  The data for this experiment sham-injected  fish,  only  the  diploid  are summarized in Figure 17. Of the females  vitellogenin. These levels remained constant at  had  measurable  3 to 4 jjg/ml  levels  (mean  of  value)  throughout the experiment. Vitellogenin levels were below assay detectabi 1 ity (0.05 ug/ml) for all the sham-injected males and triploid females. There was  - 52 -  no difference  between sexes for any of the remaining results, so males and  females have been combined.  Treatment with 176-estradiol caused a rapid and significant increase in  plasma vitellogenin  vitellogenin  levels  levels.  Within  were significantly  one week of higher  than  the in  first  injection,  sham-injected  fish  (P<0.001), and continued to rise with each subsequent injection. There was no significant  difference  treated diploids  between  and triploids  the at  vitellogenin any given  levels  date.  of  Diploids  176-estradiol treated with  17p-estradiol had a significantly higher HSI than sham-injected diploids by the end of the experiment  (P<0.05); treated triploids also had a higher HSI  than sham-injected triploids, but this difference was not significant.  At any given date, triploids  of  either  there was no difference  sham-injected  fish  or  between diploids and  176-estradiol  treated fish  for  plasma gonadotropin. However, both diploid and triploid 176-estradiol treated fish had lower levels than their respective shams at week 2 (P<0.01 in each case), as well as at week 3 in the case of the triploids  (P<0.05). There was  no change in plasma gonadotropin levels of sham-injected fish over the course of the experiment, whereas 176-estradiol treated fish had lower levels at week 2 than at week 1 (P<0.05 in both cases), and higher levels at week 3 than at week 2 (P<0.05 for diploids and P<0.01 for triploids). There was no difference in pituitary gonadotropin content between diploid and triploid  sham-injected  fish or between diploid and triploid 176-estradiol treated fish at the end of the experiment.  However, 176-estradiol treated fish had significantly higher  pituitary gonadotropin levels (P<0.001).  than their  respective  sham-injected  controls  - 54 -  Figure 15.  Histological  sections of e a r l y v i t e l l o g e n i c  oocytes from d i p l o i d coho salmon (yg = yolk  ym).  (a)  and a t r e t i c  globule,  (b)  bar = 500  -  Figure  16.  Histological testes  (bar  sections = 50  ym).  of  55  -  diploid  (a)  and t r i p l o i d  (b)  coho  salmon  - 56 -  Figure 17.  Change in plasma vitellogenin (Vtg)  and gonadotropin (GtH) over 3  weeks, and final values for hepatosomatic index and pituitary GtH content  in  coho  sham-injected,  salmon  (  respectively,  178-estradiol treated,  o •  and and  respectively).  = diploid  and  triploid  A = diploid  and  triploid  A  - 57 -  DISCUSSION  Nagahama (1983) and Scott (1987) have summarized oocyte development in f i s h . The best light photomicrographs of  the  various  growth in salmonids remain those of Yamamoto et a l . Nagahama (1983). Pre-meiotic mitotic  division.  stages of  oocyte  (1965), reproduced by  oogonia i n i t i a l l y proliferate in the ovary by  These become primary  oocytes when they enter meiosis.  Meiotic development only progresses as far as diplotene of the f i r s t prophase, at which point diplotene,  it  is  arrested  until  chromosome duplication,  final  synapsis  maturation of  the oocytes. By  and crossing-over  have  taken  place.  At this time, oocytes become enclosed in f o l l i c l e s and the primary growth phase begins. Thecal and granulosa cells in these f o l l i c l e s source of  gonadal  sex steroids  in females.  Initially,  the  are the  oocyte nucleus  increases in size and multiple nucleoli appear at its periphery. The oocytes are said to be at the perinucleolar 'Balbiani unknown,  bodies' but  mitochondria,  stage. Ribonucleoprotein particles and  appear in the cytoplasm. The function  may have Golgi  to  do with  bodies,  smooth  the  formation  endoplasmic  of  of  the  organelles  reticulae,  etc.,  latter  is  such as in  the  oocytes.  Oocytes now begin the secondary growth phase, i . e . ,  vitellogenesis,  and are hence referred to as secondary oocytes. The i n i t i a l growth phase is by endogenous vitellogenesis:  yolk  vesicles  appear  and increase  in  size and  number to f i l l the cytoplasm. Ultimately, these yolk vesicles become cortical alveoli  which  release  their  contents  into  the  perivitelline  space  at  - 58 -  fertilization.  Endogenous  vitellogenesis  is  vitellogenesis, which accounts for most of the  followed  by  oocyte's growth. Exogenous  vitellogenesis requires the hepatic synthesis of the protein which is incorporated  exogenous  vitellogenin,  into the oocytes as yolk globules. These eventually  coalesce into a large yolk mass that makes up the bulk of the mature oocyte.  Most of the diploids described here were developing normally. The coho salmon used for the induced vitellogenesis experiment were at advanced stages  of  vesicles.  endogenous Some of  vitellogenesis,  these  were  with  advanced  large  into  estrogen treatment (discussed in more detail  oocytes  exogenous  full  of  yolk  vitellogenesis  by  later). Pink salmon were mostly  at advanced stages of exogenous vitellogenesis with large yolk globule- or yolk mass-filled oocytes. One female pink salmon was clearly immature, with oocytes s t i l l  at the yolk vesicle stage.  salmon normally mature as 2-year-olds.  This was surprising, since pink  However, maturation  is occasionally  delayed in captive pink salmon, possibly due to stress or poor feeding (E.M. Donaldson, pers. comm.). One each of the coho and pink salmon had atretic oocytes,  and obviously would  not  have  matured.  Again,  this  was  likely  indicative of these being captive f i s h . Although no histology was done on the rainbow  trout,  the  fact  that  three of  the  diploids  ovulated  before  the  females all died shows that these fish were developing normally.  Virtually all the triploid females had ovaries apparently devoid of oocytes.  Only one late perinucleolar  triploid  pink  stage oocyte was found  salmon. This appears to be typical  of triploid  in  a single  fish,  i.e.,  ovaries are either entirely devoid of oocytes (Lincoln and Scott, 1983,  1984;  - 59 -  Chevassus et a l . , 1984; Solar et a l . , 1984; Solar and Donaldson, 1985), or, more typically, have a very small number of oocytes that only develop to the perinucleolar  stage  (Thorgaard  and Gall,  1979;  Yamazaki, 1983;  Benfey and  Sutterlin, 1984a; Okada, 1985; Suzuki et a l . , 1985; Krasznai and Marian, 1986; Nakamura et a l . , 1987). Some of these oocytes will occasionally develop as far as the yolk  vesicle  stage  of  endogenous vitellogenesis  (Lincoln,  1981a;  Wolters et a l . , 1982b; Chrisman et a l . , 1983; Johnson et a l . , 1986; Ueno et al.,  1986;  Richter et  al.,  1987). Development to the yolk  globule stage,  indicative of exogenous vitellogenesis with active vitellogenin synthesis by the liver, has only been reported once in triploid fish (Ueno, 1985).  These data indicate that oocyte development in triploids i s , for the most part, blocked at the very earliest  stages of meiosis. The fact  that  triploid cells can undergo normal mitotic division indicates that chromosome duplication at the beginning of meiosis probably functions normally. The next step of meiosis, when homologous chromosomes synapse prior to crossing-over, is likely when triploid cells experience meiotic disfunction. This is prior to the development of the steroidogenic follicular layer around the oocytes. Some oocytes apparently develop beyond this to the perinucleolar stage, and even occasionally begin endogenous vitellogenesis.  Mature oocytes have never been obtained from such triploid f i s h , so it has never been determined what their chromosome content i s . It difficult triploid  is therefore  to predict how they overcome the meiotic block that affects most cells.  One possibility is that they are aneuploid, the result of  random chromosome assortment,  as appears to be the case with spermatozoa  - 60 -  (discussed later). Alternatively, is the case with those few natural on page 1. this  they may be unreduced triploid oocytes, as species of all-female triploids described  Diploid females occasionally produce unreduced diploid oocytes;  is the source of  'spontaneous' triploids.  Presumably triploid  females  should also exhibit such a t r a i t . In fact, both these possibilites are likely true. This certainly appears to be the case with triploid mature eggs are occasionally ovulated.  When fertilized  amphibians, where  with normal  (i.e.,  haploid) spermatozoa, most of these develop into aneuploid embryos with a mean chromosome number between tetraploids  diploid  and triploid,  (Humphrey and Fankhauser, 1949;  This can be explained  by the formation  but some are found to be  Fankhauser and Humphrey, 1950).  of aneuploid  (between haploid and  diploid) or triploid oocytes, respectively.  Testicular development in fish has also been summarized by Nagahama (1983)  and Scott  proliferation  (1987). Testes  initially  of spermatogonia, i . e . ,  division occurs when primary  prior  become quite  large  by  to meiosis. The f i r s t  mitotic meiotic  spermatocytes become secondary spermatocytes,  followed by the second meiotic division to yield spermatids. Spermatids are haploid, and thus represent the final differentiate into  product of meiosis. These spermatids  spermatozoa. Mitotic  proliferation  of  spermatogonia and  subsequent meiotic divisions take place in cysts that are lined with Sertoli cells  and separated  by interstitial  tissue  including  Leydig c e l l s . These  Sertoli and Leydig cells are the sources of gonadal sex steroids in males, and are already present prior to meiosis, in contrast to what is seen in females.  The diploid males used for this research were developing normally  - 61 -  along these lines. Coho salmon were just beginning to develop spermatocytes, but most cells were s t i l l further  at  the  spermatogonia!  stage.  Pink  salmon were  advanced, with virtually no spermatogonia, but many spermatocytes and  spermatids. Again, no histology was done on the rainbow trout, but they  all  spermiated normally.  Triploid  males  developed  large  testes,  but  spermatogenesis was  abnormal compared to the diploids. This was not apparent in the coho salmon, probably because most of the cells were s t i l l pre-meiotic in both diploids and triploids. However, it was clear both from histology of the pink salmon and the  measurement  post-meiotic  of  cells  spermatocrit  from  produced was far  rainbow less than  trout in  that  the  the  number  diploids.  of  As with  triploid females, there is a tremendous reduction in the proportion of cells passing through meiosis in triploid males compared to diploids. The difference is,  however,  that  post-meiotic  cells  in  triploid  testes  develop  to  full  maturity ( i . e . , spermatozoa), which is not the case with post-meiotic cells in triploid ovaries. Furthermore, the actual number of pre-meiotic cells is far greater in testes than in ovaries, which accounts for the great difference in gonad size between triploid males and females.  The spermatozoa produced by triploid rainbow trout were aneuploid, with DNA contents intermediate with  triploid  grass  carp  between haploid and diploid, as is the case  (Allen  et  chromosomes during meiosis in triploid intermediate  al.,  1986).  Random segregation  spermatocytes could account for  of the  DNA content of the resulting spermatozoa. This is supported by  the much wider base in the DNA content profile of spermatozoa from triploid  - 62 -  males compared to that seen in diploid males. It  remains unclear whether  normal bivalents are formed followed by the random segregation of a third set of  univalents,  Triploid  or whether  all  the  chromosomes simply segregate randomly.  amphibians also produce spermatozoa with a chromosome complement  intermediate  between haploid and diploid (Kawamura, 1951a, 1951b; Fankhauser  and Humphrey, 1954), as do triploid Pacific oysters (Allen, 1987).  The diploid rainbow trout and pink salmon were maturing normally, based on what is known of the reproductive endocrinology of these two species (Scott  and  Sumpter,  gonadotropin content  1983;  Dye  was high  levels remained low until  et  al.,  in maturing,  1986).  Pituitary  pre-spawning f i s h ,  "ovulatory" but plasma  spawning. Gonadotropin regulates many aspects of  gonadal development, from the earliest mitotic proliferation of gonial cells through to their  final  maturation  and ovulation  or  spermiation.  However,  plasma "ovulatory" gonadotropin levels are low in maturing, pre-spawning fish due to the inhibition of  its  release from the pituitary  aromatizable androgens, such as 17j3-estradiol  by estrogens and  and testosterone. Plasma levels  of these two steroids, as well as of 11-ketotestosterone, were high throughout the pre-spawning period (178-estradiol  in females only, 11-ketotestosterone in  males only, testosterone in both sexes), but declined as the fish reached spawning.  These  post-meiotic  steroids  gonadal  cells  regulate in  both  the  growth  sexes.  and  The final  development  of  the  maturation-inducing  steroid, 17,20-P, was not detectable until ovulation or spermiation, at which time levels rose dramatically.  The endocrine profiles of triploid males were very similar to those  - 63 -  of the diploids, but there appeared to be a one month lag in the rise and f a l l of plasma gonadotropin and sex steroid levels in the triploids. There was no difference  in  the  development  of  steroid-mediated  secondary  sexual  characteristics between diploid and triploid males. Triploid females showed no signs of  sexual  maturation.  Plasma sex steroid  and plasma and pituitary  gonadotropin levels were low or not detectable throughout the study, and there was no endocrine evidence of any reproductive cycle.  Furthermore,  triploid  of  females  remained  silver,  showing  no  signs  all  secondary  the  sexual  characteristics associated with maturation in salmonids.  Triploid female pink salmon were endocrinologically similar to an immature  diploid female  maturity.  The extremely  that was probably s t i l l low  pituitary  more than  gonadotropin  levels  indicate that even at this level of the brain-pituitary-gonad  one year  from  in  fish  these  axis,  triploid  females remain reproductively inactive. This is probably due to the lack of positive feedback by estrogens from the undeveloped gonad on the pituitary (discussed below). plasma  Thus, there are two entirely  "ovulatory"  gonadotropin  steroid-mediated  inhibition  diploid  and  females  pituitaries  of  of  diploid  levels  its  release  and  triploid  immature diploid and sterile  in  different reasons for the  fish  described  low  here:  from the pituitary in maturing males,  and  triploid  its  lack  females.  It  in  the  would be  interesting to examine the hypothalamic hormone levels of triploid females, to determine at what level reproduction is compromised endocrinologically.  It is important to emphasize the nature of the gonadotropin measured in  this  study,  i.e.,  "ovulatory"  gonadotropin.  It  has  recently  been  - 64 -  demonstrated that there are two distinct gonadotropins in salmon (Kawauchi, 1988).  One corresponds to "ovulatory" gonadotropin, and is released at the  time of  ovulation  vitellogenesis  or  or  radioimmunoassay.  spermiation.  The other,  spermatogenesis,  can  only  which is now  be  released during measured  by  The fact that only the one gonadotropin was measured helps  to explain the low levels of plasma gonadotropin reported  up until  final  maturation in diploid females and diploid and triploid males.  The injection of 178-estradiol identical  for 3 weeks at 1 mg/kg caused an  increase in plasma vitellogenin  level,  hepatosomatic  index,  and  pituitary gonadotropin content in immature diploid and triploid coho salmon. Thus, vitellogenesis itself is not impaired in triploids, but the appropriate stimuli ( i . e . , estrogen-stimulated vitellogenin synthesis by the liver and its gonadotropin-mediated uptake by the oocytes) are lacking. This suggests that the occasional post-meiotic oocytes observed in triploid females do not reach f u l l maturity because of insufficient vitellogenin and gonadotropin production by the liver and pituitary, respectively. This, in turn, is likely due to the greatly diminished number of estrogen-producing f o l l i c l e cells in the triploid ovary. The obtainment of fully mature oocytes from triploids would be of great interest for the production of polyploid lines (Purdom, 1984), and may thus be possible with long-term therapy to induce vitellogenesis. However, long-term estrogen therapy  may not  be suitable  because it  causes the asynchronous  development of oocytes (Lessman and Habibi, 1987).  The -estradiol  significant treated  rise  fish  in  clearly  pituitary  gonadotropin  demonstrates  the  content  positive  in  the  effect  of  - 65 -  estrogens on gonadotropin synthesis and storage in the pituitary of immature fish  (Crim et  al.,  1981;  Peter,  1982;  Kah, 1986).  However,  there was no  increase in plasma gonadotropin levels; in fact, the levels of this hormone in circulation were slightly depressed by estrogen treatment. This is probably a sign that gonadotropin release from the pituitary was inhibited; estrogens have been shown to have such an effect on the pituitary (Peter, 1982; Kah, 1986).  The scale loss observed in the triploid female rainbow trout could have  led to  the  increased  incidence of  infections,  and also would  have  decreased the value of these fish because of their poor appearance. However, no scale loss was observed in other triploid females that were not subjected to this monthly sampling regime, indicating that this is not a general feature of triploid f i s h . Elevated androgen levels associated with maturation cause thickening of the skin in salmonids (e.g., Burton et a l . , 1985; Pottinger and Pickering, females.  1985a;  The  1985b),  increased  which  scale  loss  presumably was  does  likely  not  occur  in  triploid  an indication  of  frequent  handling of all the f i s h , with scale loss being.minimized in the maturing fish due to skin thickening.  Triploids showed no superiority in growth over maturing diploids in this  study,  triploid  and in fact  were growing more slowly in the case of  female  pink salmon. Triploids of a given species were always kept in the  same tank as diploids of the same species, and were thus competing with the diploids throughout their lives. There is some evidence that triploids do not grow as well in competition with diploids as they do when grown on their own  - 66 -  (Cassani and Caton, 1986b; Lincoln and Bye, 1987; Penman et a l . , 1987b), and this may account for the smaller size of the triploid female pink salmon. A more likely  explanation,  however,  is that they were lacking the  anabolic  effect of sex steroids produced by the gonads of maturing fish (Donaldson et al.,  1979), as were the two small diploid females with low plasma steroid  levels. This would explain why triploid males were no smaller than diploid males.  Any growth  advantage of triploids  is generally realized only for  females, and only during the spawning period of the diploids, when growth of the latter decreases or stops entirely (Purdom, 1976; Lincoln, 1981c; Wolters et a l . , 1982b; Chrisman et a l . , 1983; Chevassus et a l . , 1984; Lincoln and Bye, 1984a, 1984b; Suzuki et a l . , 1985; Chourrout et a l . , 1986a; Thorgaard, 1986; Lincoln and Bye, 1987; Quillet et a l . , 1987). All the pink salmon died prior to the diploid spawning period, and the female rainbow trout died just as the diploids were beginning to spawn. It was no evidence of  superior  growth  is therefore in  the  not surprising that there  triploids.  In  fact,  with the  exception of the female triploid pink salmon, the data support the majority of published accounts, in that triploids grew at the same rate as diploids prior to maturation of the latter.  Because triploid males produce aneuploid spermatozoa, they are truly sterile  in spite of the appearance of secondary sexual characteristics and  substantial  testicular  development.  Nonetheless, triploid male salmonids do  not appear to be of any greater benefit for aquaculture than normal diploids, because they s t i l l  undergo all the commercially negative changes associated  - 67 -  with sexual maturation  including, as demonstrated here, change in external  appearance and some post-spawning mortality.  The development of secondary  sexual characteristics alone decreases the market value of such f i s h , and is also indicative of physiological changes which decrease flesh maturity.  quality  at  Furthermore, triploid males of any species may be detrimental  to  natural stocks of fish i f released into the wild in large numbers, because they can be expected to mate with normal females but to produce no viable offspring.  Although ultimately triploid females are sterile because their cells cannot complete meiotic development, the proximate cause of their s t e r i l i t y is that  they  maturation.  exhibit This  none of is  the  the  endocrine changes associated with normal  fundamental  difference  between  male  and female  triploids, since triploid males have normal endocrine profiles and appear to mature  sexually  in  spite  of  their  sterility.  Triploid  females  thus  are  valuable tools for basic research on reproductive physiology, as well as for practical  fish  culture  to  avoid the  economically detrimental  maturation in fish destined for human consumption.  effects  of  - 68 -  REFERENCES  Allen, S.K., Jr. 1983. Flow cytometry: assaying experimental  polyploid fish  and shellfish. Aquaculture 33: 317-328. Allen, S.K., J r . 1987. 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Thorgaard, G.H. and Allen, S.K., J r . 1987. Chromosome manipulation and markers in fishery management. In: Population Genetics and Fishery Management (Ryman, N. and Utter, F.,  eds.),  pp. 319-331. Univ. of Washington  Press, Seattle. Thorgaard, G.H. and Gall, G.A.E.  1979.  Adult triploids  in a rainbow trout  family. Genetics 93: 961-973. Thorgaard, G.H., Jazwin, M.E. and Stier, A.R. 1981. Polyploidy induced by heat shock in rainbow trout. Trans. Am. Fish. Soc. 110: 546-550. Thorgaard, G.H., Rabinovitch, P.S., Shen, M.W., Gall, G.A.E., Propp, J . and Utter,  F.M.  1982.  Triploid  rainbow  trout  identified  by  flow  cytometry. Aquaculture 29: 305-309. Thorgaard, G.H., Scheerer, P.D. inheritance  in  and Parsons, J . E . 1985.  gynogenetic  rainbow  trout:  transfer. Theor. Appl. Genet. 71: 119-121.  Residual  implications  paternal for  gene  - 94 -  Ueda, T . , Kobayashi, M. and Sato, R. 1986. Triploid rainbow trouts induced by polyethylene glycol. Proc. Japan Acad. 62B: 161-164. Ueda, T . , Ojima, Y., Kato, T. and Fukuda, Y. 1983. Chromosomal polymorphisms in  the  rainbow  trout  (Salmo  gairdneri).  Proc.  Japan Acad.  59B:  168-171. Ueda, T . , Ojima, Y., Sato, R. and Fukuda, Y. 1984. Triploid hybrids between female rainbow trout and male brook trout. Bull. Jap. Soc. S c i . Fish. 50: 1331-1336. Ueda, T . , Sawada, M. and Kobayashi, J . 1987. Cytogenetical characteristics of the  embryos between  diploid  female  and triploid  male  in  rainbow  trout. Jap. J . Genet. 62: 461-465. Ueno,  K.  1984.  Induction  of  triploid  carp  and  their  haematological  characteristics. Jap. J . Genet. 59: 585-591. Ueno,  K.  1985.  Sterility  and  secondary  sexual  character  of  Gnathopogon elongathus caerulescens. Can. Trans. Fish.  triploid  Aquat. S c i .  5178: 12 pp. (Trans, of Japanese, Suisan Ikushu 10: 37-41.) Ueno, K. and Arimoto, B. 1982.  Induction of triploids in Rhodeus ocel1atus  ocel1atus by cold shock treatment of fertilized eggs.  Experientia 38:  544-546. Ueno, K.,  Ikenaga,  Y. and Kariya, H. 1986.  Potentiality  of  application of  triploidy to the culture of ayu, Plecoglossus altivelis  temminck et  Schlegel. Jap. J . Genet. 61: 71-77. Utter,  F.M.,  Johnson,  O.W.,  Thorgaard,  G.H.  and Rabinovitch, P.S.  1983.  Measurement and potential applications of induced triploidy in Pacific salmon. Aquaculture 35: 125-135. Valenti,  R.J. 1975.  Induced polyploidy in Tilapia  aurea  (Steindachner)  means of temperature shock treatment. J . Fish Biol. 7: 519-528.  by  - 95 -  Van Der Kraak, G . , Dye, H.M. and Donaldson, E.M. 1984. Effects of LH-RH and Des-GlylO[D-Ala^]LH-RH-ethylamide adult  female  coho  salmon  on plasma sex steroid profiles  (Oncorhynchus  kisutch).  Gen.  in  Comp.  Endocrinol. 55: 36-45. Vasetskii, S.G. 1967. 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CHONDROSTEI Order ACIPENSERIFORMES Family ACIPENSERIDAE Genus Acipenser A. guldenstadti colchicus (Black Sea - Azov Sea sturgeon) Vasetskii (1967): heat A. transmontanus (white sturgeon) Kowtal (1987): heat TELEOSTEI Order CLUPEIFORMES Suborder SALMONOIDEI Family SALMONI DAE Genus Oncorhynchus 0. gorbuscha (pink salmon) Utter et a l . (1983): heat Chernenko (1985): heat Benfey et a l . (1987): [pressure] 0. keta (chum salmon) Arai (1984): pressure Chernenko (1985): colchicine, formalin, heat Arai (1986): pressure Seeb et a l . (1986): heat Benfey et a l . (1988): pressure 0. kisutch (coho salmon) Refstie et a l . (1982): cold Utter et a l . (1983): heat Johnson et a l . (1984): heat Chernenko (1985): heat Johnson et a l . (1986): heat Parsons et a l . (1986): heat Phillips et a l . (1986): heat Seeb et a l . (1986): heat Benfey et a l . (1987): [pressure] Small and Benfey (1987): pressure Small and Randall (1988): pressure 0. nerka (sockeye salmon) Chernenko (1968): spontaneous (18/117) 0. tshawytscha (chinook salmon) Utter et a l . (1983): heat Johnson et a l . (1984): heat Hill et a l . (1985): heat Phillips et a l . (1986): heat Seeb et a l . (1986): heat  - 98 Genus Salmo S. gairdneri (rainbow trout) Lieder (1964): colchicine Cuellar and Uyeno (1972): spontaneous (1/18) Purdom and Lincoln (1973): cold Grammeltvedt (1974): spontaenous (1/34) Refstie et a l . (1977): cytochalain B Thorgaard and Gall (1979): spontaneous (6/11, not random) Chourrout (1980): heat Thorgaard et a l . (1981): heat Chourrout and Quillet (1982): heat Thorgaard et a l . (1982): heat, spontaneous (2/30, not random) Chevassus et a l . (1983): heat Lincoln and Scott (1983): heat Scheerer and Thorgaard (1983): heat Ueda et a l . (1983): spontaneous (2/17) Wright et a l . (1983): spontaneous Yamazaki (1983): pressure Chevassus et a l . (1984): heat Chourrout (1984): pressure Lincoln and Bye (1984a): heat Lincoln and Scott (1984): heat Lou and Purdom (1984): ether, heat, pressure Solar et a l . (1984): heat Sumpter et a l . (1984): [heat] Bolla and Refstie (1985): cytochalasin B Choubert and Blanc (1985): heat Dorson and Chevassus (1985): heat Leary et a l . (1985): heat Okada (1985): pressure Purdom et a l . (1985): heat Solar and Donaldson (1985): heat Thorgaard et a l . (1985): heat Allendorf et a l . (1986): heat Almeida (1986): heat Benfey et a l . (1986): heat Chourrout (1986a): heat Chourrout (1986b): heat Chourrout et a l . (1986b): breeding (2n X 4n), heat, pressure Dorson and Chevassus (1986): heat Fauconneau et a l . (1986): ? Johnstone and Lincoln (1986): heat Kim et a l . (1986): heat Parsons et a l . (1986): heat Phillips et a l . (1986): heat Shelton et a l . (1986): nitrous oxide Ueda et a l . (1986): polyethylene glycol Benfey et a l . (1987): [heat] Blanc et a l . (1987): breeding (2n X 4n), heat Boulanger (1987): pressure Chourrout and Nakayama (1987): breeding (2n X 4n) & (4n X 2n), heat Flajshans and Rab (1987): spontaneous (1/39) Johnstone et a l . (1987): nitrous oxide Lincoln and Bye (1987): heat Nakamura et a l . (1987): heat  - 99 S. gairdneri [cont.] Oliva-Teles and Kaushik (1987a): breeding (2n X 4n), heat Oliva-Teles and Kaushik (1987b): heat Quillet et a l . (1987): heat Sakai et a l . (1987): pressure Ueda et a l . (1987): pressure S. mykiss (Kamchatkan trout) Chernenko (1985): heat S. salar (Atlantic salmon) Lincoln et a l . (1974): cold Refstie et a l . (1977): cytochalasin B Allen and Stanley (1979): cytochalasin B Allen and Stanley (1981a): cytochalasin B Allen (1983): cytochalasin B Holmefjord et a l . (1983): heat unpubl. data cited by Purdom (1983): heat Benfey and Sutterlin (1984a): heat Benfey and Sutterlin (1984b): [heat] Benfey and Sutterlin (1984c): heat Benfey and Sutterlin (1984d): heat, pressure Benfey et a l . (1984): heat Bolla and Refstie (1985): cytochalasin B Graham et a l . (1985): heat Johnstone (1985): heat Fox et a l . (1986): heat Glebe et a l . (1986): heat Johnstone (1987): heat, pressure Small and Benfey (1987): pressure Sutterlin et a l . (1987): heat S. trutta (brown, trout) Scheerer and Thorgaard (1983): heat Arai and Wi1 kins (1987): heat Genus Salvelinus S. alpinus (Arctic char) Grebe et a l . (1986): heat S. fontinalis (brook char) Allen and Stanley (1978): spontaneous Smith and Lemoine (1979): colchicine Lemoine and Smith (1980): cold Scheerer and Thorgaard (1983): heat unpubl. data cited by Boulanger (1987): pressure S. leucomaenis (Japanese char) Arai (1984): pressure  - 100 Family PLECOGLOSSIDAE Genus Plecoglossus P. altivelis (ayu) Taniguchi et a l . (1985): cold Taniguchi et a l . (1986a): cold Taniguchi et a l . (1987): cold Ueno et a l . (1986): cold Family COREGONIDAE Genus Coregonus C. lavaretus (gwyniad) Svardson (1945): cold, spontaneous (1/?) Suborder ESOXOIDEI Family ESOCIDAE Genus Esox E. lucius (northern pike) Lieder (1964): cold Order CYPRINIFORMES Suborder CHARACOIDEI Family CHARACIDAE Genus Astyanax A. schubarti Morelli et a l . (1983): spontaneous (1/21) Family ANOSTOMIDAE Genus Curimata C. modesta Venere and Junior (1985): spontaneous (1/10) Family GYMNOTIDAE Genus Eigenmannia Eigenmannia sp. De Almeida Toledo et a l . (1985): spontaneous (1/6)  - 101 Suborder CYPRINOIDEI Family CYPRINIDAE Genus Aristichthys A. nobilis (bighead carp) Allen and Stanley (1983): spontaneous (1/38) Genus Ctenopharyngodon C. idel 1 a (grass carp) "ATlen and Stanley (1983): spontaneous (1/38) Wattendorf and Anderson (1984): ? (J.M. Malone and Sons) Cassani and Caton (1985): cold, cytochalsin B, heat Allen and Wattendorf (1986): ? (J.M. Malone and Sons) Allen et a l . (1986): ? Burns et a l . (1986): ? Cassani and Caton (1986a): heat, pressure Cassani and Caton (1986b): ? Wattendorf (1986): ? Wiley and Wike (1986): ? (J.M. Malone and Sons) Thompson et a l . (1987): cold, heat Genus Cyprinus C. carpio (carp) Makino and Ozima (1943): cold Ojima and Makino (1978): cold Gervai et a l . (1980): cold Meriwether (1980): colchicine, cold Al-Sabti et a l . (1983; cited by Col 1ares-Pereira, 1987): spontaneous Ueno (1984): cold Taniguchi et a l . (1986b): cold Wu et a l . (1986): cold Genus Gnathopogon G. elongathus caerulescens (willow gudgeon) Ueno (1985): cold Genus Hesperoleucus H. symmetricus ("California roach) Gold and Avise (1976): spontaneous (1/9) Genus Pimephales P. promelas (fathead minnow) Gold (1986): spontaneous (1/15) Genus Rhodeus R. ocel1atus ocel!atus (rose bitterling) Ueno and Arimoto (1982): cold  - 102 Family COBITIDAE Genus Misgurnus M. angui11icaudatus (Japanese common loach) Ojima and Takai (1979): spontaneous (1/80) Suzuki et a l . (1985): cold M. f o s s i l i s (loach) Vasetskii et a l . (1984): pressure Family SILURIDAE Genus Silurus S. glanis (European catfish) Krasznai et a l . (1984b): cold Krasznai and Marian (1986): cold Family AMEIURIDAE Genus Ictalurus I. punctatus (channel catfish) Wolters et a l . (1981): cold Wolters et a l . (1982a): cold Wolters et a l . (1982b): cold Chrisman et a l . (1983): cold Bidwell et a l . (1985): heat Bidwell et a l . (1986): heat Family CLARIIDAE Genus Clarias C. gariepinus (African catfish) Henken et a l . (1987): cold Richter et a l . (1987): cold Order GASTEROSTEI FORMES Family Gasterosteidae Genus Gasterosteus G. aculeatus (threespine stickleback) Swarup (1956): cold, heat Swarup (1959a): cold, heat Swarup (1959b): cold, heat Swarup (1959c): cold, heat Order CYPRINODONTIFORMES Family CYPRINODONTIDAE Genus Oryzi as 0. latipes (medaka) Naruse et a l . (1985): heat 0. melastigma Sriramulu (1962): colchicine  - 103 Order PERCIFORMES Suborder PERCOIDEI Family PERCIDAE Genus Perca P. f l u v i a t i l i s (perch) Lieder (1964): colchicine, cold Family CICHLIDAE Genus Oreochromis [=Tilapia] 0. aureus Valenti (1975): cold, heat Don and Avtalion (1986): heat Penman et a l . (1987a): heat Penman et a l . (1987b): heat 0. mossambicus Pandian and Varadaraj (1987): heat Penman et a l . (1987a): heat Penman et a l . (1987b): heat 0. niloticus Chourrout and Itskovich (1983): heat Shah and Beardmore (1986): heat Penman et a l . (1987a): heat Penman et a l . (1987b): heat Order PLEURONECTIFORMES Suborder PLEURONECTOIDEI Family PARALICHTHYIDAE Genus Paralichthys P. olivaceus (hirame) Tabata et a l . (1986): cold Family PLEURONECTIDAE Genus Liopsetta L. putnami (smooth flounder) unpubl. data cited by Hoornbeek and Burke (1981): cold Genus Pleuronectes P. platessa (plaice) Purdom (1972): cold Purdom et a l . (1976): cold Lincoln (1981a): cold Lincoln (1981b): cold Genus Pseudopleuronectes P. americanus (winterflounder) Hoornbeek and Burke (1981): cold  - 104 Appendix 2: Papers describing or utilizing spontaneouly-arisen or experimentally-induced triploid HYBRID fish of species that are normally only dioecious diploids. TELEOSTEI Order CLUPEIFORMES Suborder SALMONOIDEI Family SALMONIDAE Genus Oncorhynchus 0. gorbuscha (pink salmon) X 0. tshawytscha (chinook salmon) Utter et a l . (1983): heat 0. keta (chum salmon) X 0. kisutch (coho salmon) SeeFTt a l . (1986): heat X 0. tshawytscha (chinook salmon) Seeb and Seeb (1986): heat Seeb et a l . (1986): heat X Salvelinus fontinalis (brook char) Arai (1986): pressure X Salvelinus leucomaenis (Japanese char) Arai (1984): pressure 0. kisutch (coho salmon) X 0. keta (chum salmon) Seeb et a l . (1986): heat X 0. tshawytscha (chinook salmon) Seeb et a l . (1986): heat X Salmo gairdneri (rainbow trout) Parsons et a l . (1986): heat 0. tshawytscha (chinook salmon) X 0. gorbuscha (pink salmon) Utter et a l . (1983): heat X 0. keta (chum salmon) Seeb et a l . (1986): heat X 0. kisutch (coho salmon) Seeb et a l . (1986): heat  - 105 Genus Salmo S. clarki (cutthroat trout) X S. gairdneri (rainbow trout) Rohrer and Thorgaard (1986): heat S. gairdneri (rainbow trout) X Oncorhynchus kisutch (coho salmon) Chevassus et a l . (1983): heat Dorson and Chevassus (1985): heat Dorson and Chevassus (1986): heat Parsons et a l . (1986): heat Quillet et a l . (1987): heat X S. clarki (cutthroat trout) Rohrer (1982; cited by Rohrer and Thorgaard, 1986): heat X S. salar (Atlantic salmon) Purdom et a l . (1985): heat X S. trutta (brown trout) Chevassus et a l . (1983): heat Scheerer and Thorgaard (1983): heat Quillet et a l . (1987): heat X Salvelinus fontinalis (brook char) Capanna et a l . (1974): spontaneous Chevassus et a l . (1983): heat Scheerer and Thorgaard (1983): heat Ueda et a l . (1984): spontaneous Dorson and Chevassus (1986): heat X Thymallus thymallus (grayling) Chourrout (1986a): heat Chourrout (1986c): heat S. salar (Atlantic salmon) X S. trutta (brown trout) Svardson (1945): cold, spontaneous (1/?) Holmefjord et a l . (1983): heat X Salvelinus alpinus (Arctic char) Holmefjord et a l . (1983): heat Glebe et a l . (1986): heat S. trutta (brown trout) X S. gairdneri (rainbow trout) Scheerer and Thorgaard (1983): heat Purdom et a l . (1985): heat X Salvelinus fontinalis (brook char) Scheerer and Thorgaard (1983): heat Scheerer et a l . (1987): heat (S. trutta X C_ salar hybrid) X S^ salar (Atlantic salmon) Johnson and Wright (1986): spontaneous X Salvelinus fontinalis (brook char) Johnson and Wright (1986): spontaneous Genus Salvelinus S. fontinalis (brook char) X Salmo gairdneri (rainbow trout) Scheerer and Thorgaard (1983): heat X Salmo trutta (brown trout) Scheerer and Thorgaard (1983): heat S. leucomaenis (Japanese char) X Oncorhynchus keta (chum salmon) 'Arai (1984): pressure  - 106 Order CYPRINIFORMES Suborder CHARACOIDEI Family CHARACIDAE Genus Colossoma C. macropomum X mitrei De Al meida Toledo et a l . (1987): spontaneous (1/4) Suborder CYPRINOIDEI Family CYPRINIDAE Genus Ctenopharyngodon C. idel 1 a (grass carp) X Aristichthys nobilis (bighead carp) Marian and Krasznai (1978): spontaneous Beck et a l . (1980): spontaneous (Malone, 1979 brood) Cassani (1981): spontaneous (Malone, 1979 brood) Sutton et a l . (1981): spontaneous Beck and Biggers (1982): spontaneous (Malone, 1979 & 1980 broods) Magee and Philipp (1982): spontaneous (Malone, 1979, 1980 & 1981 brood Allen (1983): spontaneous Allen and Stanley (1983): spontaneous (Malone, 1981 brood) Barker et a l . (1983): spontaneous (Malone, 1980 brood) Beck and Biggers (1983a): spontaneous (Malone, 1980 brood) Beck and Biggers (1983b): spontaneous (Malone, ? brood) Beck et a l . (1983): spontaneous (Malone ? brood) Cassani and Caton (1983): spontaneous (Malone, 1979 brood) Shireman et a l . (1983): spontaneous Wattendorf and Shafland (1983; cited by Wattendorf and Anderson, 1984): spontaneous Young et a l . (1983): spontaneous Beck et a l . (1984): spontaneous (Malone, 1980 brood) Cassani et a l . (1984): spontaneous (Malone, 1979, 1980 & 1981 broods) Krasznai et a l . (1984a): spontaneous Bettoli et a l . (1985): spontaneous (Malone, ? brood) Kilambi and Galloway (1985): spontaneous (Malone, ? brood) Wiley and Wike (1986): spontaneous (Malone, ? brood) Wiley et a l . (1986): spontaneous (Malone, 1979 brood) Genus Cyprinus C. carpio (carp) X Ctenopharyngodon idel 1 a (grass carp) Yasil'ev et a l . (1975): spontaneous Stanley (1976): spontaneous Stanley et a l . (1976): spontaneous X Hemiculter eigenmanni Vasil'ev et a l . (1975): spontaneous X Hypophthalmichthys molitrix (silver carp) Bakos et a l . (1978; cited by Krasznai, 1987): spontaneous  - 107 Order PERCIFORMES Suborder PERCOIDEI Family CENTRARCHIDAE Genus Lepomis (L. gibbosus X L. cyanellus hybrid) X L. cyanellus (green sunfish) Dawley et a l . (T985): spontaneous Dawley (1987): spontaneous X L. gibbosus (pumpkinseed) Dawley et a l . (1985): spontaneous X L. macrochirus (bluegill) Dawley (1987): spontaneous Family CICHLIDAE Genus Oreochromis [=Ti1apia] 0. niloticus X 0j_ rendalli Chourrout and Itskovich (1983): heat Order PLEURONECTIFORMES Suborder PLEURONECTOIDEI Genus Pleuronectes P. platessa (plaice) X Platichthys flesus (flounder) Purdom (1972): cold Purdom (1976): cold Lincoln (1981a): cold Lincoln (1981b): cold Lincoln (1981c): cold X (P. platessa X Platichthys flesus hybrid) Purdom~Tl972): cold  - 108 -  Appendix 3 (Manuscript in review for General and Comparative Endocrinology):  An  Homologous  Radioimmunoassay  for  Coho  Salmon  (Oncorhynchus  kisutch)  Vitellogenin, with General Applicability to Other Pacific Salmonids*  TILLMANN J . BENFEY , EDWARD M. DONALDSON, AND TERRANCE G. OWEN 1  1  1  2  Department of Fisheries and Oceans, Biological Sciences Branch, West Vancouver Laboratory, 4160 Marine Drive, West Vancouver, British Columbia V7V 1N6, Canada; and  2  Helix Biotech L t d . , 217-7080 River Road, Vancouver Industrial Park, Richmond, British Columbia V6X 1X5, Canada  *  Reported  in  part  at  the  Third  International  Symposium on Reproductive  Physiology of Fish, St. John's, Newfoundland, August 2-7, 1987  Short t i t l e : Pacific salmonid vitellogenin RIA  - 109 -  ABSTRACT  This paper describes an homologous radioimmunoassay for coho salmon vitellogenin  that  vitellogenin of all  demonstrates  parallel  cross-reactivity  for  plasma  Pacific salmonids tested (chinook, chum, coho, pink and  sockeye salmon, and cutthroat and rainbow trout), but not for Atlantic salmon or a non-salmonid, the sablefish.  Plasma vitellogenin levels in ovulatory  Pacific salmonids were in the hundreds of jug/ml to hundreds of mg/ml range, but were mostly non-detectable in spermiating males of the same species.  - 110 -  INTRODUCTION  Vitellogenin is a protein produced by the liver  in teleosts under  estrogen stimulus, and incorporated into growing oocytes as the yolk proteins lipovitellin  and phosvitin (Wallace  and Selman, 1981;  Scott and Sumpter, 1983a; Wallace, 1985).  Ng and Idler,  1983;  In salmonids, plasma levels of  vitellogenin can reach tens of mg/ml (Scott and Sumpter, 1983a), and can be detected by radioimmunoassay at least two years before spawning (Copeland et a l . , 1986). For this reason, various immunological techniques have been used to sex immature salmonids on the basis of  vitellogenin  production by the  females (Le Bail and Breton, 1981; Gordon et a l . , 1984). To date, three radioimmunoassays for salmonid vitellogenin have been described, all for species of the genus Salmo.  These are for Atlantic salmon,  Salmo salar (So et a l . , 1985), rainbow trout, S^_ gairdneri Copeland et press). yolk  al.,  1986), and brown trout, S_^ trutta  (Sumpter,  1985;  (Norberg and Haux,  in  As well, radioimmunoassays for partially purified Atlantic salmon egg  proteins  (Campbell  and  (Idler  et  al.,  Idler,  1980),  1979) both  and for  having  parallel  conspecific vitellogenins, have been described. demonstrated  that  there  are  clear  rainbow  trout  lipovitellin  displacement  to  their  These radioimmunoassays have  immunological  differences  between  the  vitellogenins of the closely related species and genera of salmonids. Although the rainbow trout radioimmunoassay of Sumpter (1985) has been used to measure plamsa vitellogenin levels in pink salmon, Oncorhynchus gorbuscha (Dye et a l . , 1986),  there  is  a  clear  need  for  the  development  of  homologous  radioimmunoassays for the various genera, i f not species, of salmonids. The  aim  of  the  present  study  was  to  develop  an homologous  - Ill -  radioimmunoassay for coho salmon (0^ kisutch) vitellogenin, and to test the appropriateness of this assay for measuring vitellogenin in other salmonids of the genera Oncorhynchus and Salmo.  MATERIALS AND METHODS  Radioimmunoassay materials. Coho salmon vitellogenin (MW of 390,000; Markert  and Vanstone, 1971)  precipitation  was purified from female coho salmon serum by  with magnesium chloride  and EDTA, followed  by ion-exchange  chromatography (Gordon et a l . , 1984). This material had an optical density of 0.28, and was therefore judged to have a concentration of 280 /jg/ml. Twenty yu"1 (5.6 yjg) were iodinated at  a time,  separation of  vitellogenin  radiolabeled  using the  Sephadex G25 column, followed by further  iodogen method. An initial  from free  iodine was done on a  purification  on a Sephadex G100  column. Antibodies were raised in rabbits using an intramuscular injection of vitellogenin in Freund's complete adjuvant, followed by a subcutaneous booster of Freund's incomplete adjuvant 4-6 weeks later. The rabbits were bled 7-10 days after the booster, and serum was passed over a Protein A column to purify the antibody fraction (Gordon et a l . , 1984). Radioimmunoassay procedure. Triplicate tubes containing 100 yu 1 of standard or plasma, 100 yul radiolabeled vitellogenin (at 20,000 cpm/100 yul) and 100 jul antibody (at 1:12,000 in 1:400 normal rabbit serum) were vortexed and incubated at room temperature for 5-6 hours. goat antibody to rabbit  Next, 100 yul (1 unit) of  gamma-globulin was added, the tubes were vortexed  again, and left to incubate overnight at room temperature. Finally, 500 yul of buffer was added, the tubes were vortexed, centrifuged at 3500 rpm for 30  - 112 -  minutes, aspirated, and then counted for 60 seconds each on a gamma-counter. Based on 22 replicate standard curves, this procedure gave a maximum binding of 30% (± 7% SD) of total binding, with a sensitivity of 50 ng/ml (± 9 ng/ml) to 570 ng/ml (± 82 ng/ml) at 80% and 20% of maximum binding, respectively. Fish. The species and source of fish used are shown in Table 1. Each individual was sexed, either by visual examination of the gonads or on the basis of whether it  produced eggs or sperm at spawning. All fish were  sampled within a few months of spawning, and most were sampled at the actual time of ovulation or spermiation. Males were included only in species of the genus Oncorhynchus. Blood was collected from the caudal sinus into  either  heparinized syringes or vacutainer tubes. Plasma samples were stored in the freezer until they were assayed. Plasma dilutions. To test for parallelism between plasma samples and coho salmon standards, plasma dilutions in the radioimmunoassay buffer (10 mM Na2HP04, 150 mM NaCl, 1 mM NaN3 and 1% BSA) were made by factors of ten for females (neat to 1:10,000,000) or two for males (neat to 1:16). The data were logarithmically  transformed,  to  allow the calculation of slopes by linear  regression for percent binding against either standard concentration or plasma dilution factor.  Parallelism was then determined statistically  by single  factor analysis of variance between slopes of the standards and the plasma dilutions.  RESULTS  Plasma dilutions were made for cutthroat  5 females of each species except  trout, for which only 2 females were available.  Plasma dilutions  - 113 -  were also made for 5 males of each of the 5 Oncorhynchus species. For a given species, plasma dilutions curves always had a similar slope, whether  parallel  to the standard curve or not; the mean curve from each species is shown in Fig. 1.  Plasmas from all 5 species of Oncorhynchus diluted parallel  to the  coho salmon standards (this includes those few males with detectable levels of vitellogenin),  as  did  plasmas  from  cutthroat  and  rainbow  trout.  Plasma  dilutions from Atlantic salmon and sablefish were not parallel to the standard curve (P<0.001). Plasma vitellogenin concentrations were calculated for those species for which parallelism was demonstrated. Most males had undetectable  levels,  and those that were measureable (1 coho salmon and 2 pink salmon) were only in the tens of ng/ml range. This may reflect  non-specific impurities  in  the  vitellogenin preparation from which the antibodies were raised (Gordon et a l . , 1984), or low levels of true vitellogenin. very high plasma vitellogenin  Females, on the other hand, had  concentrations,  in the hundreds of jjg/ml  to  hundreds of mg/ml range (Table 2).  DISCUSSION  This  paper  describes the  first  homologous radioimmunoassay  for  vitellogenin in a Pacific salmon species. The procedure was easily developed using materials  designed for  sexing coho salmon from skin mucus samples  (Gordon et a l . , 1984) and a standard radioimmunoassay protocol. Plasma levels of the antigen were many orders of magnitude higher in mature females than in mature males, reflecting the high levels of vitellogenin required for oocyte growth (Scott and Sumpter, 1983a). High levels of the antigen could be induced  - 114 -  in  immature  male,  178-estradiol  female  and sterile  coho salmon by the  injection  of  (Benfey et a l . , 1988). These two factors clearly support the  identification of the antigen as vitellogenin (Gordon et a l . , 1984). The vitellogenin levels reported here for ovulatory salmonids are in the range of published values for other salmonids (Scott and Sumpter, 1983b; Sumpter, 1985; So et a l . , 1985; Dye et a l . , 1986).  However, our values for  rainbow trout are rather high. The repeated freezing and thawing of plasma samples may cause degredation of vitellogenin to the extent that vitellogenin concentrations become overestimated (Copeland et a l . , 1986; Norberg and Haux, in press).  We kept no record of how many times these samples were frozen and  thawed, but this did occur several times, and may account for the particularly high levels reported here. Plasma concentrations were quite variable between individuals of the same species (hence the high standard deviations reported in Table 2), even though they were presumably all at about the same stage of sexual development.  But regardless of the actual concentration in the plasma,  there is no question that vitellogenin levels were s t i l l  very high in  all  could  be  individuals at ovulation. The  radioimmunoassay described  in  this  paper  easily  sensitized, as discussed by So et a l . (1985) and Copeland et a l . (1986), and used to  study early  events  in  vitellogenesis. The assay also has great  potential for sexing fish that are s t i l l  a long way from spawning, and hence  show no external signs of maturation. This should be possible even for species for which vitellogenin  levels cannot be quantified because of the lack of  parallel displacement to the standards. The  homology  between  immunologically  detectable  forms  of  vitellogenin from the five species of Pacific salmon (Oncorhynchus spp.), and  - 115 -  cutthroat  and rainbow trout  history. Mitochondrial  is  likely  a reflection  of  their  evolutionary  DNA analysis has shown that the genus Salmo can be  divided into two distinct groups: one containing cutthroat  and rainbow trout  (Pacific species), and the other containing Atlantic salmon and brown trout (Atlantic  species) (Gyllensten and Wilson, 1987). Furthermore, rainbow trout  are more closely related to chinook salmon than either  are to brown trout  (Berg and Ferris, 1984; Ferris and Berg, 1987). The evolutionary separation of Pacific from Atlantic  salmonids thus  is  reflected  by the  immunologically  detectable structure of the protein vitellogenin.  ACKNOWLEDGEMENTS  The radioimmunoassay procedure was suggested to us by John Sumpter, and for this and his comments on the manuscript, we are most grateful. wish  to  thank  statistical  Ted  Down for  analysis.  We  his  also  many suggestions,  thank  the  staffs  especially  of  the  Big  We  regarding Qualicum,  Chilliwack and Quinsam River Salmonid Enhancement Program (SEP) Hatcheries and the  Weaver  Creek  SEP Spawning Channel,  and the  Seymour  River  Community  Economic Development Project (CEDP) Hatchery for allowing us to collect blood samples from their broodstock, as well as Bob Roy and Igor Solar for providing additional  plasma  samples.  TJB was  supported  scholarship and a Quebec FCAR graduate fellowship.  by  a NSERC  postgraduate  - 116 -  REFERENCES  Benfey, T . J . , Dye, H.M., and Donaldson, E.M. (1988). Induced vitellogenesis in triploid coho salmon (Oncorhynchus kisutch), and its effect andpituitary gonadotropin. Berg, W.J., and Ferris, S.D.  on plasma  Gen. Comp. Endocrinol., in review. (1984).  Restriction  endonuclease analysis of  salmonid mitochondrial DNA. Can. J . Fish. Aquat. S c i . 41, 1041-1047. Campbell,  CM.,  and  Idler,  D.R.  (1980).  Characterization  of  an  estradiol-induced protein from rainbow trout serum as vitellogenin by the composition and radioimmunological cross reactivity to ovarian yolk fractions. Biol. Reprod. 22, 605-617. Copeland,  P.A.,  Sumpter,  Vitellogenin  J.P.,  Walker,  T.K.,  and  Croft,  M.  (1986).  levels in male and female rainbow trout (Salmo gairdneri  Richardson) at various stages of the reproductive cycle. Comp. Biochem. Physiol. 83B, 487-493. Dye, H.M.,  Sumpter,  J . P . , Fagerlund,  U.H.M.,  and Donaldson, E.M.  (1986).  Changes in reproductive parameters during the spawning migration of pink salmon, Oncorhynchus gorbuscha (Walbaum). J . Fish Biol. 29, 167-176. Ferris, S.D., and Berg, W.J. (1987). The u t i l i t y of mitochondrial DNA in fish genetics and fishery management. Management."  In  "Population Genetics and Fishery  (N. Ryman, and F. Utter, Eds.). Chap. 11. University of  Washington Press, Seattle. Gordon,  M.R.,  Owen,  T.G.,  Ternan,  T.A.,  and  Hildebrand,  L.D.  (1984).  Measurement of a sex-specific protein in skin mucus of premature coho salmon (Oncorhynchus kisutch). Aquaculture 43, 333-339.  - 117 -  Gyllensten, U., and Wilson, A.C. (1987). Mitochondrial "Population Genetics and Fishery Management."  DNA of salmonids. In  (N. Ryman, and F. Utter,  Eds.). Chap. 12. University of Washington Press, Seattle. Idler,  D.R.,  Hwang,  vitellogenin  S.J., in  and  Atlantic  Crim,  L.W.  salmon  (1979). (Salmo  Quantification salar)  plasma  of by  radioimmunoassay. J . Fish. Res. Board Can. 36, 574-578. Le B a i l ,  P.Y.,  puberal  and Breton, salmonid  B.  fish  (1981). Rapid determination by  a  technique  of  of  the sex of  immunoagglutination.  Aquaculture 22, 367-375. Markert,  J.R.,  and  Vanstone,  W.E.  (1971).  Egg proteins  of  coho salmon  (Oncorhynchus kisutch): chromatographic separation and molecular weights of the major proteins in the high density fraction and their presence in salmon plasma. J . Fish. Res. Board Can. 28, 1853-1856. Ng, T . B . ,  and Idler,  D.R.  (1983).  Yolk  formation  and differentiation  in  teleost fishes. In "Fish Physiology, Volume IXA." (Hoar, W.S., Randall, D.J., and Donaldson, E.M., Eds.). Chap. 8. Academic Press, New York. Norberg,  B.,  and Haux,  C.  (in  press).  A homologous radioimmunoassay for  brown trout (Salmo trutta) vitellogenin.  Fish Physiol. Biochem., in  press. Scott, A . P . , and Sumpter, J.P. (1983a). The control of trout reproduction:  basic  and  applied  research  on hormones.  In  "Control  Processes in Fish Physiology." (Rankin, J . C , Pitcher, T . J . , and Duggan, R.T., Eds.). Chap. 11. Croom Helm, London. Scott,  A.P.,  and  reproductive  Sumpter,  J.P.  cycles of autumn-spawning  rainbow trout (Salmo gairdneri 79-85.  (1983b).  A  comparison  of  and winter-spawning  the  female  strains of  Richardson). Gen. Comp. Endocrinol. 52,  - 118 -  So,  Y.P.,  Idler,  D.R.,  and Hwang,  landlocked  Atlantic  salmon  homologous  radioimmunoassay  S.J. (Salmo  and  (1985). salar  Plasma vitellogenin Ouananiche):  immunological  in  isolation,  cross-reactivity  with  vitellogenin from other teleosts. Comp. Biochem. Physiol. 81B, 63-71. Sumpter, J.P. (1985). The purification, radioimmunoassay and plasma levels of vitellogenin  from the  rainbow  trout,  Salmo gairdneri.  In  "Current  Trends in Comparative Endocrinology." (B. Lofts, and W.N. Holmes, Eds.), pp. 355-357. Hong Kong University Press, Hong Kong. Wallace,  R.A.  (1985).  vertebrates.  Vitellogenesis  and oocyte  growth in  nonmammalian  In "Developmental Biology, Volume 1." (L.W. Browder, Ed.),  Chap. 3. Plenum Press, New York. Wallace, R.A., and Selman, K. (1981). Cellular and dynamic aspects of oocyte growth in teleosts. Am. Zool. 21, 325-343.  - 119 -  TABLE 1 SPECIES AND GENERA OF FISH USED  Genus Oncorhynchus chinook salmon (0. tshawytscha)  males  Qualicum River SEP Hatchery (B.C.)  females  DFO Pacific Biological Station (B.C.)  chum salmon (0. keta)  Chilliwack River SEP Hatchery (B.C.)  coho salmon (0. kisutch)  Chilliwack River SEP Hatchery (B.C.)  pink salmon (0. gorbuscha)  Quinsam River SEP Hatchery (B.C.)  sockeye salmon (0. nerka)  Weaver Creek SEP Spawning Channel (B.C.)  Genus Salmo Atlantic salmon (S. salar)  Marine Sciences Research Lab. (Nfld.)  cutthroat trout (S. clarki)  Seymour River CEDP Hatchery (B.C.)  rainbow trout (S. gairdneri)  DFO West Vancouver Laboratory (B.C.)  Genus Anoplopoma sablefish (A. fimbria)  DFO Pacific Biological Station (B.C.)  - 120 -  TABLE 2 VITELLOGENIN LEVELS AT/NEAR OVULATION IN PACIFIC SALMONIDS (IN MG/ML)  chinook salmon  15.7 (+ 2.4, n=5)  chum salmon  0.52 (+ 1.11, n=5)  coho salmon  14.1 (± 18.7, n=5)  pink salmon  0.20 (± 0.20, n=5)  sockeye salmon  0.11 (± 0.12, n=5)  cutthroat trout  2.35 (+ 1.46, n=2)  rainbow trout  131.6 (+ 138.5, n=5)  - 121 -  PLASMA -7  10  [Vtg]  FIG. 1 - Plasma d i l u t i o n (b),  pink  -6  10  coho standards).  -5  10  -4  10  -3  10  FACTOR -2  10  -1  10  o  (ng/ml)  curves f o r v i t e l l o g e n i n  ( c ) , sockeye (d)  cutthroat t r o u t  DILUTION  (g), Atlantic  (Vtg) in Chinook ( a ) , coho  and chum salmon  ( e ) , rainbow  salmon (h) and s a b l e f i s h  (i)  (f)  and  (std =  

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