Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

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

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-UBC_1988_A1 B46.pdf [ 12.56MB ]
Metadata
JSON: 831-1.0097948.json
JSON-LD: 831-1.0097948-ld.json
RDF/XML (Pretty): 831-1.0097948-rdf.xml
RDF/JSON: 831-1.0097948-rdf.json
Turtle: 831-1.0097948-turtle.txt
N-Triples: 831-1.0097948-rdf-ntriples.txt
Original Record: 831-1.0097948-source.json
Full Text
831-1.0097948-fulltext.txt
Citation
831-1.0097948.ris

Full Text

THE REPRODUCTIVE PHYSIOLOGY OF TRIPLOID PACIFIC SALMONIDS by TILLMANN JOACHIM NIENSTEDT BENFEY B.Sc. (first class honours), McGill University, 1981 M.Sc, 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 this thesis in partial fulfilment of the requirements for an advanced degree at the University of 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 scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date DF-fin/ft-n 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 males. Triploid ovaries remained very small, and contained virtually no oocytes. Triploid testes became quite large, but few cells developed beyond the spermatocyte stage. 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 levels were significantly elevated over levels of sham-injected f ish, whereas plasma gonadotropin levels were 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 is concluded that triploids of both sexes are genetically sterile, but that only triploid females do not undergo 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 stimulus to initiate and maintain vitellogenesis. Although triploids of both sexes should make valuable tools for basic research on reproductive physiology, only the females will be useful for practical fish culture to avoid the economically detrimental effects of maturation in fish destined for human consumption. page iv TABLE OF CONTENTS ABSTRACT i i LIST OF TABLES v LIST OF FIGURES vi ACKNOWLEDGEMENTS vii PREFACE ix INTRODUCTION Literature Review: The Physiology of Triploid Fish 1 Research Outline: The Reproductive Physiology of Triploid Pacific Salmonids 13 MATERIALS AND METHODS Fish 16 Radioimmunoassay Procedures 19 Reproductive Endocrinology 26 Spermiation 27 Induced Vitellogenin Production 28 Statistics 29 RESULTS Gonadal Development (Pink Salmon) 30 Growth Rate and Reproductive Endocrinology (Pink Salmon) 38 Growth Rate and Reproductive Endocrinology (Rainbow Trout) 41 Secondary Sexual Characteristics (Pink Salmon and Rainbow Trout) 46 Spermiation (Rainbow Trout) 48 Induced Vitellogenesis (Coho Salmon) 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 97 Appendix 2: Papers describing or utilizing spontaneously-arisen or experimentally-induced triploid HYBRID fish of species that are normally only dioecious diploids 104 Appendix 3 [manuscript in review]: An homologous radioimmunoassay for coho salmon (Oncorhynchus kisutch) vitellogenin, with general applicability to other Pacific salmonids 108 page v LIST OF TABLES Table 1. Growth and endocrine status of diploid and triploid male pink salmon 39 Table 2. Growth and endocrine status of diploid and triploid female pink salmon 40 Table 3. Maturity status of diploid and triploid rainbow trout and reproductive characteristics of mature males 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) 4 Figure 2. Diploid (2n) and triploid (3n) ovaries (top) and testes (bottom) from adult pink salmon (scale in cm) 31 Figure 3. Histological sections of advanced vitellogenic stage oocytes from diploid pink salmon (bar = 500 jjm) 32 Figure 4. Histological sections of pre-vitellogenic stage oocytes (a) and atretic, vitellogenic stage oocytes (b) from diploid pink salmon (bar = 500 jjm) 33 Figure 5. Histological sections of triploid pink salmon ovaries devoid of oocytes (a), or with one late perinucleolar stage oocyte (b) (bar = 500 pm) 34 Figure 6. Histological section of a late perinucleolar stage oocyte from a triploid pink salmon (close-up from Figure 5b, bar = 100 i^m) . 35 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) 36 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). . . . 37 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). . . . 43 Figure 10. Plasma steroid and gonadotropin levels in diploid and triploid male rainbow trout (ng/ml, A = diploid, A = triploid). . . . 44 Figure 11. Plasma steroid and gonadotropin levels in diploid and triploid female rainbow trout (ng/ml, 0= diploid, • = triploid, •fc= ovulated) 45 Figure 12. External appearance of adult diploid (2n) and triploid (3n) rainbow trout (a = males, b = females, scale in cm) 47 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 50 Figure 14. Histological sections of diploid (a) and triploid (b) coho salmon ovaries (bar = 500 pm) 53 Figure 15. Histological sections of early vitellogenic (a) and atretic (b) oocytes from diploid coho salmon.(yg = yolk globule, bar = 500 jum) 54 Figure 16. Histological sections of diploid (a) and triploid (b) coho salmon testes (bar = 50 pm) 55 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) 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: co-supervisors Ed Donaldson, without whose faci l i t ies and resources this research would not have been possible, and Dave Randall, who maintained 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 visits. 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, provided encouragement and highly beneficial criticism throughout the intervening page vi 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 rst when describing hybrids. - 1 -INTRODUCTION Literature Review: The Physiology of Triploid Fish. Sexually maturing fish undergo numerous physiological and behavioural changes that may reduce their value as fish destined for human consumption. Various methods have therefore been devised and investigated to delay or entirely prevent sexual maturation in fish used for aquaculture (Chevassus et a l . , 1979; Stanley, 1979, 1981; Donaldson and Hunter, 1982; Refstie, 1982; Yamazaki, 1983; Bye and Lincoln, 1986; Donaldson, 1986; Shelton, 1986; Donaldson and Benfey, 1987; Pandian and Varadaraj, 1987; Shelton, 1987). One of the most effective of these is the induction of triploidy, because the resulting fish are genetically sterile. In fact, induced triploid is the only practical means by which to 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 ish. Three of these cases are well documented, and have been reviewed by Schultz (1979) and Purdom (1984): these are in Poeci1i a and Poeci1iopsis (both order Cyprinodontiformes, family Poeciliidae) and Carassius (Cypriniformes, Cyprinidae). The remaining three are recent discoveries in Menidia (Mugi1iformes, Atherinidae) (Echelle et a l . , 1983), Phoxinus (Cyprinidae) (Joswiak et a l . , 1985; Dawley et a l . , 1987) and Rutilus (Cyprinidae) (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 ish, and can readily be produced in species for which it is possible to manipulate spawning. A comprehensive l ist 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 a l . , 1986). In these cases, the resultant fish have one maternal and two paternal haploid chromosome sets. Induced triploidy can be used to increase the viability of interspecific hybrids, although it is by no means clear why this is so (Chevassus, 1983; Krasznai, 1987; Neavdal and Dalpadado, 1987). 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 a l . , 1984; Arai, 1986; Chourrout, 1986a, 1986c; Parsons et a l . , 1986; Seeb et a l . , 1986; Scheerer et a l . , 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) to salmonid hybrids that are not viable as diploids. Intraspecific hybridization may be a useful way of decreasing treatment-associated 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 ferti l ization. Vertebrate eggs always possess three chromosome sets for a short time after ferti l ization, 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 body in f ish, but probably act by different 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 ish, 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 cel ls, their nuclei and the cells themselves are significantly larger than diploid nuclei - 4 -Figure 1. Timing of shocks (*) for the production of triploids and tetraploids ( (^) and represent haploid maternal and paternal chromosome sets, respectively). - 5 -and cel ls. 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 (Chourrout et a l . , 1986b). It 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 a l . , 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 a l . (1985) on Atlantic salmon have shown that hemoglobin-oxygen affinity (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 le 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 (Benfey and Sutterlin, 1984b), or crit ical swimming velocity (Small 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 that the increase in cell size and decrease in cell numbers associated with triploidy are of insufficient consequence to have an effect on most physiological processes, or that triploids are able to regulate their physiological control systems to a sufficient degree to overcome potential 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 irst 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 rst 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 a l . , 1987; Atlantic salmon: Benfey and Sutterlin, 1984a; ayu: Ueno et a l . , 1986; grass carp: Thompson et a l . , 1987; common carp: Gervai et a l . , 1980; Taniguchi et a l . , 1986b; willow gudgeon: Ueno, 1985; rose bitterling: Ueno and Arimoto, 1982; loach: Suzuki et a l . , 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; t i lapia: Shah and Beardmore, 1986; Penman et a l . , 1987b; and plaice and its hybrid with - 8 -flounder: Purdom, 1972, 1976; Lincoln, 1981a, 1981b). Wu et al . (1986) have suggested that true triploid female common carp are in fact fert i le , and that the presumed triploid females that are sterile are in fact aneuploid. However, this seems unlikely in light of the overwhelming evidence supporting the virtually complete 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 (Lincoln, 1981c). However, only three diploids and three triploids 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, 1984; Benfey et a l . , 1987; Nakamura et a l . , 1987). Similar results have been obtained for sex steroid levels in triploid pink salmon; in 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. No diploid hybrids were available for comparison in this study, so it remains unclear whether these results are a reflection of triploid steri l i ty or hybrid steri l i ty. Johnson et a l . (1986) found no difference in plasma 17p-estradiol levels between diploid 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. Nevertheless, vitellogenin was detectable only in the diploids. Triploid 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 a l . , 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 steri l i ty 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 a l . , 1987). However, the absence of secondary sexual characteristics has been noted in triploid male channel catfish (Wolters et a l . , 1982b; Chrisman et a l . , 1983) and tilapia (Don and Avtalion, 1986), suggesting that 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 fert i l ize 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 ferti l izing 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 a l . , 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 plaice hybrids with flounder, which have lower food conversion rates than immature diploid hybrids (Lincoln, 1981c). 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 a l . , 1986; Shah and Beardmore, 1986; Boulanger, 1987; Henken et a l . , 1987; Kowtal, 1987; Lincoln and Bye, 1987; Penman et a l . , 1987b; Richter et a l . , 1987), but there have been reports both of inferior growth (Lincoln, 1981c; Utter et a l . , 1983; Chevassus et a l . , 1984; Solar et a l . , 1984; Okada, 1985; Suzuki et a l . , 1985; Cassani and Caton, 1986b; Chourrout et a l . , 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 a l . , 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 growth rates decrease, whereas triploid females continue to grow through the normal spawning period (Purdom, 1976; Lincoln, 1981c; 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). The same is true of triploid channel catfish of both sexes (Wolters et a l . , 1982b; Chrisman et a l . , 1983). Research Outline: The Reproductive Physiology of Triploid Pacific Salmonids. The preceding literature review showed that there are two fundamental differences between diploid and triploid f ish. Firstly, cell size is increased in triploids, with a concomitant decrease in cell numbers. - 14 -Secondly, gonadal development is impaired in triploids, with a sexual dimorphism in the degree of this impairment. These two facts have been well documented, but their resultant effect on the general physiology of triploids has received l i t t le attention. The aim of my research was to examine, in detail, the reproductive physiology of triploid Pacific salmon and rainbow trout. I began by confirming that gonadal development is impaired in triploid 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. Sexual dimorphism in the gonadal development of triploids may therefore 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 is an obvious possibility, and can be tested by replacement therapy. - 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 in sea pens at the Pacific Biological Station (DFO) in Nanaimo, B.C. Eggs from 4 females and sperm from 4 males, collected on January 24, 1984, were transported separately on ice to the West Vancouver Laboratory, 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. Eggs from 4 females, kept separated, and pooled sperm from 7 males were transported on ice in plastic bags f i l led with 100% oxygen to the West Vancouver Laboratory for fertil ization 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 cel 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 rst 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 led with 100% oxygen for 2 days prior to - 18 -ferti l ization. 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 rst feeding, prior to ploidy determination. All the fish were reared at the West Vancouver Laboratory, using standard techniques for salmonid culture. Fertilized eggs were placed in vertically-stacked, flow through incubation trays (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. As the fish grew, they were transferred to increasingly larger tanks, ultimately of 3 m diameter. Freshwater at 10 to 11°C was supplied from an on-site well. At the appropriate development stage, fish were acclimated to salt water from the Burrard Inlet, pumped in from 200 m offshore from the West Vancouver Laboratory. Salt water temperatures fluctuated seasonally. Embryos were kept in constant darkness while in the incubation trays; all subsequent rearing used a natural photoperiod. 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, proportional to DNA content, is measured and amplified by photomultipliers and converted to digital 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 in distil led water), and immediately mixed on a vortex mixer. Cells were stained for a minimum of 1 hour at 4°C, after which 100 jul of dimethyl sulfoxide (D-128, Fisher) was added as a cryoprotectant. 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 a l . (1984) for testosterone, 17p-estradiol and 17«.-hydroxy-20j3-dihydroprogesterone (17,20-P) and by Dye et a l . (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 was a 0.05M phosphate buffer (5.75 g/1 dibasic sodium phosphate [S-0876, Sigma] and 1.315 g/1 monobasic sodium phosphate [S-0751, Sigma] in distil led water) containing 1.0 g/1 gelatin (G-2500, Sigma) and 65 mg/1 sodium azide (S-2002, Sigma). The buffer was heated to 37°C to dissolve the gelatin, and its pH adjusted to 7.6. Standards of testosterone (T-1500, Sigma), 173-estradiol (E-8875, Sigma), 17,20-P (Q-1850, Steraloids, Inc., Wilton, NH) and 11-ketotestosterone (lot 2607, Syndel Laboratories Ltd., Vancouver, B.C.) were init ial ly 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.611) and 11-ketotestosterone (TRK.676). 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 Ltd., 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 of ^-steroid (5,000 cpm, diluted to the appropriate activity in assay buffer) and 200 jul of antibody. For 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 and 17S-estradiol) or 1 ml (17,20-P and 11-ketotestosterone) of dextran-coated activated charcoal (0.5 g/1 dextran T-70 [Pharmacia (Canada) Ltd. , Dorval, Quebec] 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 ia l , they were vigorously shaken, and placed in the - 22 -dark at room temperature overnight. The next morning they were counted on a LKB Wallac 1214 RackBeta liquid scintillation counter (Wallac Oy, Turku, Finland) for 5 min each. Plasma testosterone and 17p-estradiol were measured in diploid and triploid pink salmon using kits with the steroids radiolabeled with 125j rather than (1102-D and 1018, respectively, Radioassay 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 al . (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. The hormone and its antibody were a gift from Dr. J.P. Sumpter (Brunei 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 a l . , 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 distil led water or protein-free buffer was placed in the bottom of this tube, followed immediately by 6 to 10 yul of sodium-125j at 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 rst 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 assay buffer contained 1.42 g/1 dibasic sodium phosphate, 8.77 g/1 sodium chloride, 10.0 g/1 bovine serum albumin, and 65 mg/1 sodium azide in distil led 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 gonadotropin or 1:12,000 for vitellogenin, diluted in 1:400 normal rabbit serum [NRS] [Calbiochem 869019, obtained through Terochem Laboratories Ltd., Edmonton, Alberta] prepared in assay buffer). The tubes were vortexed and briefly centrifuged to ensure that all 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 0 n a c| 0tropin (5,000 cpm, diluted to the appropriate activity in assay buffer); 100 J J I of l25i_v-jtellogenin (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 vortexed and briefly centrifuged. The assays were incubated for a further 24 hours (gonadotropin) or 6 hours (vitellogenin) prior to the addition of 100 /jl (1 unit) of goat antibody to rabbit 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 rst 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 fal l between 0 and 100%. Standard curves of percentage binding (Y-axis) plotted against log of the concentration of steroid or protein standards (X-axis) were used to determine 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 rst 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 0.04% 2-phenoxyethanol (lot 2913, Syndel) and their weight and fork length measured. Blood was collected from 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 the pink salmon (11 diploid females, 11 triploid females, 10 diploid males, and 8 triploid males) developed bacterial kidney disease shortly after the f i rst sampling, and further sampling was discontinued to minimize stress on these f ish. However, all 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 were collected from these f ish. All the female rainbow trout (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 ish, 17B-estradiol for female rainbow trout and all pink salmon, 17,20-P for all rainbow trout, 11-ketotestosterone for male rainbow trout, and vitellogenin for all pink salmon. Pituitary 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 f irst stripped under water, where they produced a cloudy f luid. 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 Ltd., 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 the calculation of spermatocrit, sperm was collected in hematocrit capillary tubes and centrifuged for 10 min in a 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 rst 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. Variation about the mean for any data is expressed as ± one standard 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 (Figure 2). Nine of the diploids were at advanced stages of vitellogenesis at this time. Their oocytes were full 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: one appeared to be immature, with oocytes st i l l at the 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). Diploid and triploid testes were histologically similar, containing 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 ip lo id (3n) ovaries (top) and testes (bottom) from adult pink salmon (scale in cm). - 32 -Figure 3. Histological sections of advanced vitel logenic stage oocytes from diploid pink salmon (bar = 500 urn). - 33 -H i s t o l o g i c a l s ec t i ons of p r e - v i t e l 1 o g e n i c stage oocytes (a) and a t r e t i c , v i t e l l o g e n i c stage oocytes (b) from d i p l o i d pink salmon (bar = 500 pm). - 34 -His to log ica l sections of t r i p l o i d pink salmon ovaries devoid oocytes (a) , or with one late per inucleolar stage oocyte (b) (bar 500 p ) . - 35 -- 36 -F i g u r e 7 . H i s t o l o g i c a l s e c t i o n s o f d i p l o i d (a ) and t r i p l o i d (b) p i n k sa lmon t e s t e s a t h i g h m a g n i f i c a t i o n (I = p r i m a r y s p e r m a t o c y t e s , I I = s e c o n d a r y s p e r m a t o c y t e s , St = s p e r m a t i d s , b a r = 50 urn). - 37 -Figure 8. H is to log ica l sections of d ip lo id (a) and t r i p l o i d (b) pink salmon testes at low magnif ication ( l igh t blue = primary and secondary spermatocytes, dark blue = spermatids, bar = 500 um). - 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 of the diploid and triploid males) and those that were immature, abnormal ( i .e . , atretic), or sterile (1 each of the diploid females and all 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 and abnormal diploid and sterile triploid females. 178-estradiol and vitellogenin levels were high only in the maturing diploid females. Plasma gonadotropin was not detectable in any of the f ish. - 39 -Table 1. Growth and endocrine status of diploid and triploid female pink salmon. Maturing Atretic Immature Sterile JULY diploids diploid diploid 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 28.9 ± 1.1 Testosterone (ng/ml) 4.5 ± 2.2 < 0.72 < 0.72 < 0.72 176-estradiol (ng/ml) 1.9 ± 1.3 0.11 0.07 0.05 ± 0.01(6) Vitellogenin (}jg/ml) 6339 ± 5794 10.3 0.19 0.11 ± 0.02 Plasma gonadotropin < 2.5 < 2.5 < 2.5 < 2.5 (ng/ml) SEPTEMBER Sample size 2 1 1 7 Weight (kg) 0.69, 0.36 0.20 0.29 0.31 ± 0.11 Length (cm) 34.6, 31.1 27.1 28.4 28.9 ± 1.1 Gonad weight (gm) 42.6, 7.2 1.13 3.02 0.09 ± 0.04 GSI (%) 6.6, 2.1 0.56 1.04 0.03 ± 0.02 Pituitary gonadotropin 7450, 1680 1.57 0.38 1.6 ± 2.3 content (ng) (numbers in brackets = reduced sample sizes because levels were not detectable the remaining f ish). - 40 -Table 2. Growth and endocrine status of diploid and triploid male pink salmon. JULY Maturing diploids Maturing triploids Sample size Weight (kg) Length (cm) Testosterone (ng/ml) 17G-estradiol (ng/ml) Vitellogenin (pg/ml) Plasma gonadotropin (ng/ml) 10 0.37 + 0.13 31.3 ± 2.9 2.7 + 1.1 0.13 + 0.02(5) 0.10 ± 0.04(9) < 2.5 8 0.33 ± 0.07 31.4 ± 2.1 2.4 ± 0.6(7) 0.07 + 0.03(4) 0.11 ± 0.05(6) < 2.5 SEPTEMBER Sample size 8 4 Weight (kg) 0.46 + 0.18 0.44 + 0.1* Length (cm) 33.4 + 4.0 32.9 + 2.8 Gonad weight (gm) 38.4 + 16.9 36.2 + 15.5 GSI {%) 8.9 + 1.7 8.8 + 1.4 Pituitary gonadotropin 1538 ± 1051 1473 + 794 content (ng) (numbers in brackets = reduced sample sizes because levels were not detectable in the remaining fish). - 41 -Growth Rate and Reproductive Endocrinology (Rainbow Trout). There was no significant difference in growth rate between diploid and triploid male rainbow trout (Figure 9 ) , and both groups had similar endocrine profiles (Figure 10). Testosterone and 11-ketotestosterone levels increased gradually from July to November, at which point 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 ise. There was a slight lag in the increase in androgen levels observed in the triploids, 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), but 178-estradiol and testosterone levels were not significantly lower. Pituitary gonadotropin content was 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 1 1 1 1 1 1 1 1 07 08 09 10 11 12 01 02 03 MONTH Figure 9. Growth rates of diploid and triploid rainbow trout ( A and • = 2- year-old diploid and triploid males respectively, o and • 3 - year-old diploid and triplod females, respectively). - 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, •= tr iploid, ^ = ovulated). - 46 -Secondary Sexual Characteristics (Pink Salmon and Rainbow Trout). Both diploid and triploid male pink salmon were yellowing and beginning to develop humps when they died in September, with no apparent differences between ploidies 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 ins, 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 ish. - 47 -F i g u r e 12 . E x t e r n a l a p p e a r a n c e o f a d u l t d i p l o i d (2n) and t r i p l o i d (3 r a i n b o w t r o u t (a = m a l e s , b = f e m a l e s , s c a l e i n c m ) . - 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 females were observed, whereas 3 diploid females matured at this age. Although diploid and triploid males were identical in external appearance, only the diploids produced normal amounts of milk-white sperm. Only 4 of the triploid males produced sperm when stripped, and this sperm was very watery. Sperm samples from these 4 individuals had significantly lower spermatocrits and significantly higher spermatozoan DNA content than sperm samples from 6 randomly selected diploids (Table 3). The mean DNA content of sperm from triploid males was 46% greater than that from the diploid males, this being not significantly different from a 50% increase. The flow cytometer demonstrated that all cell types exhibited discrete DNA content 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 (sample sizes in parentheses). Measure Diploids Triploids Maturity status Mature males {%) Mature females (%) Immature (%)a 41.4 (24) 5.2 (3) 53.4 (31) 41.3 (19) 0.0 (0) 58.7 (27) Reproductive measures0 Body Weight (g) GSI (%) Spermatocrit {%) Spermatozoan DNA (pg/cell) 455.3 ± 132.5 (8) *2.83 ± 0.76 (8) *23.0 ± 9.5 (6) 2.92 ± 0.11 (6) 489.2 ± 114.7 (5) 0.50 + 0.11 (5) 1.8 ± 0.6 (4) *4.25 ± 0.09 (4) a Four fish lost identification tags and their ploidy was unknown. 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 none had developing oocytes (Figure 14b). Seven of the 10 176-estradiol 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 the exception of one 176-estradiol treated diploid, the remaining fish were males. There was no apparent histological 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 ish. 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 ish, but were separated earlier on the basis of fin cl ips. Most likely this individual was a misclassified hormonally-sterilized f ish. The data for this experiment are summarized in Figure 17. Of the sham-injected f ish, only the diploid females had measurable levels of vitellogenin. These levels remained constant at 3 to 4 jjg/ml (mean 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 levels. Within one week of the f i rst injection, vitellogenin levels were significantly higher than in sham-injected fish (P<0.001), and continued to rise with each subsequent injection. There was no significant difference between the vitellogenin levels of 176-estradiol treated diploids and triploids at any given date. Diploids 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, there was no difference between diploids and triploids of either sham-injected fish or 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 than their respective sham-injected controls (P<0.001). - 54 -Figure 15. H is to log ica l sections of ear ly v i te l logen ic (a) and a t re t i c (b) oocytes from d ip lo id coho salmon (yg = yolk globule, bar = 500 ym). - 55 -F i g u r e 16 . H i s t o l o g i c a l s e c t i o n s o f d i p l o i d (a) and t r i p l o i d (b) coho sa lmon t e s t e s ( b a r = 50 ym). - 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 salmon ( o and A = diploid and triploid sham-injected, respectively, • and A = diploid and triploid 178-estradiol treated, respectively). - 57 -DISCUSSION Nagahama (1983) and Scott (1987) have summarized oocyte development in f ish. The best light photomicrographs of the various stages of oocyte growth in salmonids remain those of Yamamoto et a l . (1965), reproduced by Nagahama (1983). Pre-meiotic oogonia ini t ia l ly proliferate in the ovary by mitotic division. These become primary oocytes when they enter meiosis. Meiotic development only progresses as far as diplotene of the f i rst prophase, at which point it is arrested until final maturation of the oocytes. By diplotene, chromosome duplication, synapsis and crossing-over have taken place. At this time, oocytes become enclosed in fol l icles and the primary growth phase begins. Thecal and granulosa cells in these fol l icles are the source of gonadal sex steroids in females. Initially, the oocyte nucleus increases in size and multiple nucleoli appear at its periphery. The oocytes are said to be at the perinucleolar stage. Ribonucleoprotein particles and 'Balbiani bodies' appear in the cytoplasm. The function of the latter is unknown, but may have to do with the formation of organelles such as mitochondria, Golgi bodies, smooth endoplasmic reticulae, etc., in the oocytes. Oocytes now begin the secondary growth phase, i .e . , vitellogenesis, and are hence referred to as secondary oocytes. The init ial 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 -ferti l ization. Endogenous vitellogenesis is followed by exogenous vitellogenesis, which accounts for most of the oocyte's growth. Exogenous vitellogenesis requires the hepatic synthesis of the protein vitellogenin, which is incorporated 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 endogenous vitellogenesis, with large oocytes full of yolk vesicles. Some of these were advanced into exogenous vitellogenesis by estrogen treatment (discussed in more detail 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. This was surprising, since pink salmon normally mature as 2-year-olds. 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 ish. 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 stage oocyte was found in a single triploid pink salmon. This appears to be typical of triploid f ish, 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 a l . , 1986; Richter et a l . , 1987). Development to the yolk globule stage, indicative of exogenous vitellogenesis with active vitellogenin synthesis by the l iver, has only been reported once in triploid fish (Ueno, 1985). These data indicate that oocyte development in triploids is , 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 foll icular 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 ish, so it has never been determined what their chromosome content is . It is therefore difficult to predict how they overcome the meiotic block that affects most triploid cel ls. 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, they may be unreduced triploid oocytes, as is the case with those few natural species of all-female triploids described on page 1. Diploid females occasionally produce unreduced diploid oocytes; this is the source of 'spontaneous' triploids. Presumably triploid females should also exhibit such a trait . In fact, both these possibilites are likely true. This certainly appears to be the case with triploid amphibians, where mature eggs are occasionally ovulated. When fertilized with normal ( i .e . , haploid) spermatozoa, most of these develop into aneuploid embryos with a mean chromosome number between diploid and triploid, but some are found to be tetraploids (Humphrey and Fankhauser, 1949; Fankhauser and Humphrey, 1950). This can be explained by the formation of aneuploid (between haploid and diploid) or triploid oocytes, respectively. Testicular development in fish has also been summarized by Nagahama (1983) and Scott (1987). Testes ini t ia l ly become quite large by mitotic proliferation of spermatogonia, i .e . , prior to meiosis. The f irst meiotic division occurs when primary spermatocytes become secondary spermatocytes, followed by the second meiotic division to yield spermatids. Spermatids are haploid, and thus represent the final product of meiosis. These spermatids differentiate into 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 cel ls. 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 at the spermatogonia! stage. Pink salmon were further 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 of spermatocrit from rainbow trout that the number of post-meiotic cells produced was far less than in the diploids. 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 between haploid and diploid, as is the case with triploid grass carp (Allen et a l . , 1986). Random segregation of chromosomes during meiosis in triploid spermatocytes could account for the intermediate 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, or whether all the chromosomes simply segregate randomly. Triploid 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, 1983; Dye et a l . , 1986). Pituitary "ovulatory" gonadotropin content was high in maturing, pre-spawning f ish, but plasma levels remained low until 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 by estrogens and aromatizable androgens, such as 17j3-estradiol 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 steroids regulate the growth and development of the post-meiotic gonadal cells in both sexes. The final 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 fall 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, all the triploid females remained silver, showing no signs of secondary sexual characteristics associated with maturation in salmonids. Triploid female pink salmon were endocrinologically similar to an immature diploid female that was probably s t i l l more than one year from maturity. The extremely low pituitary gonadotropin levels in these fish indicate that even at this level of the brain-pituitary-gonad 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). Thus, there are two entirely different reasons for low plasma "ovulatory" gonadotropin levels in the fish described here: steroid-mediated inhibition of its release from the pituitary in maturing diploid females and diploid and triploid males, and its lack in the pituitaries of immature diploid and sterile triploid females. It 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 or spermiation. The other, which is released during vitellogenesis or spermatogenesis, can only now be measured by radioimmunoassay. 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 for 3 weeks at 1 mg/kg caused an identical 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 full 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 fo l l ic le 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 significant rise in pituitary gonadotropin content in the -estradiol treated fish clearly demonstrates the positive effect of - 65 -estrogens on gonadotropin synthesis and storage in the pituitary of immature fish (Crim et a l . , 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 ish. Elevated androgen levels associated with maturation cause thickening of the skin in salmonids (e.g., Burton et a l . , 1985; Pottinger and Pickering, 1985a; 1985b), which presumably does not occur in triploid females. The increased scale loss was likely an indication of frequent handling of all the f ish, 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, and in fact were growing more slowly in the case of female triploid 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 a l . , 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 is therefore not surprising that there was no evidence of superior growth in the 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 ish, and is also indicative of physiological changes which decrease flesh quality at maturity. Furthermore, triploid males of any species may be detrimental to natural stocks of fish if 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 steri l i ty is that they exhibit none of the endocrine changes associated with normal maturation. This is the fundamental difference between male and female triploids, since triploid males have normal endocrine profiles and appear to mature sexually in spite of their steri l i ty. Triploid females thus are valuable tools for basic research on reproductive physiology, as well as for practical fish culture to avoid the economically detrimental effects of maturation in fish destined for human consumption. - 68 -REFERENCES Allen, S.K., Jr. 1983. Flow cytometry: assaying experimental polyploid fish and shellfish. Aquaculture 33: 317-328. Allen, S.K., Jr. 1987. Gametogenesis in three species of triploid shellfish: My a arenari a, Crassostrea gigas and Crassostrea virginica. In: Selection, Hybridization, and Genetic Engineering in Aquaculture, Vol. II (Tiews, K., ed.), pp. 207-217. Heenemann Verlags. mbH, Berlin. Allen, S.K., Jr. and Stanley, J.G. 1978. Reproductive steri l i ty in polyploid brook trout, Salvelinus fontinalis. Trans. Am. Fish. Soc. 107: 473-478. Allen, S.K., Jr. and Stanley, J.G. 1979. Polyploid mosaics induced by cytochalasin B in landlocked Atlantic salmon Salmo salar. Trans. Am. Fish. Soc. 108: 462-466. Allen, S.K., Jr. and Stanley, J.G. 1981a. Mosaic polyploid Atlantic salmon (Salmo salar) induced by cytochalasin B. Rapp. P.-v. Reun. Cons. int. Explor. Mer 178: 509-510. Allen, S.K., Jr. and Stanley, J.G. 1981b. Polyploidy and gynogenesis in the culture of fish and shellfish. Int. Council Explor. Sea, CM. 1981/F:28, 18 pp. Allen, S.K., Jr. and Stanley, J.G. 1983. Ploidy of hybrid grass carp x bighead carp determined by flow cytometry. Trans. Am. Fish. Soc. 112: 431-435. Allen, S.K., J r . , Thiery, R.G. and Hagstrom, N.T. 1986. Cytological evaluation of the likelihood that triploid grass carp will reproduce. Trans. Am. Fish. Soc. 115: 841-848. - 69 -Allen, S.K., Jr. and Wattendorf, R.J. 1986. A review of the production and quality control of triploid grass carp and progress in implementing 'sterile' triploids as management tools in the U.S. Aquaculture 57: 359 (abstract). Allen, S.K., Jr. and Wattendorf, R.J. 1987. Triploid grass carp: status and management implications. Fisheries 12(4): 20-24. Allendorf, F.W. and Leary, R.F. 1984. Heterozygosity in gynogenetic diploids and triploids estimated by gene-centromere recombination rates. Aquaculture 43: 413-420. Allendorf, F.W., Seeb, J . E . , Knudsen, K.L., Thorgaard, 6.H. and Leary, R.F. 1986. Gene-centromere mapping of 25 loci in rainbow trout. J . Hered. 77: 307-312. Almeida, V. 1986. Induction of triploidy by heat shocks in rainbow trout (Salmo gairdneri R.). Publ. Inst. Zool. "Dr. Augusto Nobre", Fac. Cienc. Porto 193: 7 pp. Anonymous, 1982. The Coulter EPICS V System. Coulter Brochure 4203181B: 16 pp. Coulter Electronics, Inc., Hialeah. Arai, K. 1984. Developmental genetic studies on salmonids: morphogenesis, isozyme phenotypes and chromosomes in hybrid embryos. Mem. Fac. Fish. Hokkaido Univ. 31: 1-94. Arai, K. 1986. Effect of allotriploidization on development of the hybrids between female chum salmon and male brook trout. Bull. Jap. Soc. Sci. Fish. 52: 823-829. Arai, K. and Wilkins, N.P. 1987. Triploidization of brown trout (Salmo trutta) by heat shocks. Aquaculture 64: 97-103. - 70 -Barker, C . J . , Beck, M.L. and Biggers, C.J. 1983. Hematologic and enzymatic analysis of Ctenopharyngodon idel 1 a x Hypophthalmichthys nobilis Fi hybrids. Comp. Biochem. Physiol. 74A: 915-918. Beck, M.L. and Biggers, C.J. 1982. Chromosomal investigation of Ctenopharyngodon idel la x Ari stichthys nobilis hybrids. Experientia 38: 319. Beck, M.L. and Biggers, C.J. 1983a. Erythrocyte measurements of diploid and triploid Ctenopharyngodon i del 1 a x Hypophthalmichthys nobilis hybrids. J . Fish Biol. 22: 497-502. Beck, M.L. and Biggers, C.J. 1983b. Ploidy of hybrids between grass carp and bighead carp determined by morphological analysis. Trans. Am. Fish. Soc. 112: 808-811. Beck, M.L., Biggers, C.J. and Barker, C.J. 1984. Chromosomal and electrophoretic analyses of hybrids between grass carp and bighead carp (Pisces: Cyprinidae). Copeia 1984: 337-342. Beck, M.L., Biggers, C.J. and Dupree, H.K. 1980. Karyological analysis of Ctenopharyngodon idel 1 a, Ari stichthys nobi l i s , and their F\ hybrid. Trans. Am. Fish. Soc. 109: 433-438. Beck, M.L., Biggers, C.J. and Dupree, H.K. 1983. Electrophoretic analysis of protein systems of Ctenopharyngodon idella (Val.), Hypophthalmichthys nobilis (Rich.) and their Fi triploid hybrid. J . Fish Biol. 22: 603-611. Benfey, T . J . , Bosa, P.G., Richardson, N.L. and Donaldson, E.M. 1988. Effectiveness of a commercial-scale pressure shocking device for producing triploid salmonids. Aquae. Engineer.: in press. - 71 -Benfey, T . J . , Solar, I.I., de Jong, G. and Donaldson, E.M. 1986. Flow-cytometric confirmation of aneuploidy in sperm from triploid rainbow trout. Trans. Am. Fish. Soc. 115: 838-840. Benfey, T . J . , Solar, I.I. and Donaldson, E.M. 1987. The reproductive physiology of triploid Pacific salmonids. In: Proc. Third Int. Symp. on the Reproductive Physiology of Fish (Idler, D.R., Crim, L.W. and Walsh, J.M., eds.), p. 128 (abstract). Memorial Univ. of Nfld., St. John's. Benfey, T .J . and Sutterlin, A.M. 1984a. Growth and gonadal development in triploid landlocked Atlantic salmon (Salmo salar). Can. J . Fish. Aquat. Sci. 41: 1387-1392. Benfey, T . J . and Sutterlin, A.M. 1984b. Oxygen utilization by triploid landlocked Atlantic salmon (Salmo salar L.). Aquaculture 42: 69-73. Benfey, T .J . and Sutterlin, A.M. 1984c. The haematology of triploid landlocked Atlantic salmon, Salmo salar L. J . Fish. Biol. 24: 333-338. Benfey, T .J . and Sutterlin, A.M. 1984d. Triploidy induced by heat shock and hydrostatic pressure in landlocked Atlantic salmon (Salmo salar L.). Aquaculture 36: 359-367. Benfey, T . J . , Sutterlin, A.M. and Thompson, R.J. 1984. Use of erythrocyte measurements to identify triploid salmonids. Can. J . Fish. Aquat. Sci. 41: 980-984. Bettoli, P.W., Nei11, W.H. and Kelsch, S.W. 1985. Temperature preference and heat resistance of grass carp, Ctenopharyngodon idel 1 a (Valenciennes), bighead carp, Hypophthalmichthys nobilis (Gray), and their Fi hybrid. J . Fish Biol. 27: 239-247. Bidwell, C.A., Chrisman, C L . and Libey, G.S. 1985. Polyploidy induced by heat shock in channel catfish. Aquaculture 51: 25-32. - 72 -Bidwell, C.A., Chrisman, C.L. and Libey, G.S. 1986. Polyploidy induced by heat shock in channel catfish. Aquaculture 57: 362 (abstract). Blanc, J . -M. , Chourrout, D. and Krieg, F. 1987. Evaluation of juvenile rainbow trout survival and growth in half-sib families from diploid and tetraploid sires. Aquaculture 65: 215-220. Bolla, S. and Refstie, T. 1985. Effect of cytochalasin B on eggs of Atlantic salmon and rainbow trout. Acta Zool. (Stock.) 66: 181-188. Boulanger, Y. 1987. Resultats preliminaires sur la croissance de truites arc-en-ciel steriles (triploidie induite par un choc de pression) en pisciculture. Proc. Ann. Meet. Aquacult. Assoc. Canada 1: 55-58. (In French.) Burns, E.R., Anson, J . F . , Hinson, W.G., Pipkin, J . L . , Kleve, M.G. and Goetz, R.C. 1986. Effect of fixation with formalin on flow cytometric measurement of DNA in nucleated blood cel ls. Aquaculture 55: 149-155. Burton, D., Burton, M.P., Truscott, B. and Idler, D.R. 1985. Epidermal cellular proliferation and differentiation in sexually mature male Salmo salar with androgen levels depressed by o i l . Proc. Roy. Soc. Lond. 225B: 121-128. Bye, V .J . and Lincoln, R.F. 1986. Commercial methods for the control of sexual maturation in rainbow trout (Salmo gairdneri R.). Aquaculture 57: 299-309. Capanna, E . , Cataudella, S. and Volpe, R. 1974. Un ibrido intergenerico tra trota iridea e salmerino di fonte (Salmo gairdneri x Salvelinus  fontinalis). Boll. Pesca Piscic. Idrobiol. 29: 101-106. (In Italian with English summary.) - 73 -Cassani, J.R. 1981. Feeding behaviour of underyearling hybrids of the grass carp, Ctenopharyngodon i del 1 a female and the bighead, Hypophthalmichthys nobi1i s male on selected species of aquatic plants. J . Fish Biol. 18: 127-133. Cassani, J.R. and Caton, W.E. 1983. Feeding behaviour of yearling and older hybrid grass carp. J . Fish Biol. 22: 35-41. Cassani, J.R. and Caton, W.E. 1985. Induced triploidy in grass carp, Ctenopharyngodon idel 1 a Val. Aquaculture 46: 37-44. Cassani, J.R. and Caton, W.E. 1986a. Efficient production of triploid grass carp (Ctenopharyngodon i del la) utilizing hydrostatic pressure. Aquaculture 55: 43-50. Cassani, J.R. and Caton, W.E. 1986b. Growth comparisons of diploid and triploid grass carp under varying conditions. Prog. Fish-Cult. 48: 184-187. Cassani, J.R. , Caton, W.E. and Clark, B. 1984. Morphological comparisons of diploid and triploid hybrid grass carp, Ctenopharyngodon idella female x Hypophthalmichthys nobilis male. J . Fish Biol. 25: 269-278. Chernenko, Ye. V. 1968. Karyotypes of dwarf (residual) and anadromous forms of sockeye salmon [Oncorhynchus nerka (Walb.)] from Lake Dalnee (Kamchatka). Prob. Ichtyol. 8: 668-677. (Trans, of Russian, Vopr. Ikhtiol. 8: 834-846.) Chernenko, E.V. 1985. Induction of triploidy in Pacific salmons (Salmonidae). J . Ichtyol. 25: 124-130. (Trans, of Russian, Vopr. Ikhtiol. 25: 561-567.) Chevassus, B. 1983. Hybridization in f ish. Aquaculture 33: 245-262. - 74 -Chevassus, B. 1987. Caracteristiques et performances des lignees uniparentales et des polyploides chez les poissons d'eau froide. In: Selection, Hybridization, and Genetic Engineering in Aquaculture, Vol. II (Tiews, K., ed.), pp. 145-161. Heenemann Verlags. mbH, Berlin. (In French.) Chevassus, B., Blanc, J.M. and Courrout, D. 1979. Le controle de la reproduction chez les poissons. II. Reproduction differee et ster i l i te. Bull. Fr. Piscic. 274: 32-46. (In French.) Chevassus, B., Guyomard, R., Chourrout, D. and Quillet, E. 1983. Production of viable hybrids in salmonids by triploidization. Genet. Sel. Evol. 15: 519-532. Chevassus, B., Quillet, E. and Chourrout, D. 1984. La production de truites steriles par voie genetique. Piscic. Fr. 78: 10-19. (In French.) Choubert, G. and Blanc, J.-M. 1985. Flesh colour of diploid and triploid rainbow trout (Salmo gairdneri Rich.) fed canthaxanthin. Aquaculture 47: 299-304. Chourrout, D. 1980. Thermal induction of diploid gynogenesis and triploidy in the eggs of the rainbow trout (Salmo gairdneri Richardson). Reprod. Nutr. Develop. 20: 727-733. Chourrout, D. 1984. Pressure-induced retention of second polar body and suppression of f i rst cleavage in rainbow trout: production of al l - tr iploids, al1-tetraploids, and heterozygous and homozygous diploid gynogenetics. Aquaculture 36: 111-126. Chourrout, D. 1986a. Extensive karyological investigations permitting the choice between different methods of gynogenesis and polyploidy induction in rainbow trout. Aquaculture 57: 366 (abstract). - 75 -Chourrout, D. 1986b. Techniques of chromosome manipulation in rainbow trout: a new evaluation with karyology. Theor. Appl. Genet. 72: 627-632. Chourrout, D. 1986c. Use of grayling sperm (Thymallus thymallus) as a marker for the production of gynogenetic rainbow trout (Salmo gairdneri). Theor. Appl. Genet. 72: 633-636. Chourrout, D. 1987. Genetic manipulations in f ish: review of methods. In: Selection, Hybridization, and Genetic Engineering in Aquaculture, Vol. II (Tiews, K., ed.), pp. 111-126. Heenemann Verlags. mbH, Berlin. Chourrout, D., Chevassus, B. and Guyomard, R. 1986a. L'amelioration genetique des poissons. La Recherche 17: 1028-1038. (In French.) Chourrout, D., Chevassus, B., Krieg, F., Happe, A. , Burger, G. and Renard, P. 1986b. Production of second generation triploid and tetraploid rainbow trout by mating tetraploid males and diploid females - potential of tetraploid f ish. Theor. Appl. Genet. 72: 193-206. Chourrout, D. and Itskovich, J . 1983. Three manipulations permitted by artif icial insemination in ti lapia: induced diploid gynogenesis, production of all triploid populations and intergeneric hybridization. In: Int. Symp. on Tilapia in Aquaculture (Fishelson, L. and Yaron, Z. , eds.), pp. 246-255. Tel Aviv Univ., Tel Aviv. Chourrout, D. and Nakayama, I. 1987. Chromosome studies of progenies of tetraploid female rainbow trout. Theor. Appl. Genet. 74: 687-692. Chourrout, D. and Quillet, E. 1982. Induced gynogenesis in the rainbow trout: sex and survival of progenies production of al1-tripioid populations. Theor. Appl. Genet. 63: 201-205. Chrisman, C.L. , Wolters, W.R. and Libey, G.S. 1983. Triploidy in channel catfish. J . World Maricul. Soc. 14: 279-293. - 76 -Clugston, J.P. and Shireman, J.V. 1987. Triploid grass carp for aquatic plant control. U.S. Fish Wildl. Serv., Fish Wildl. Leafl. 8: 3 pp. Collares-Pereira, M.J. 1987. The evolutionary role of hybridization: the example of natural Iberian fish populations. In: Selection, Hybridization, and Genetic Engineering in Aquaculture, Vol. I (Tiews, K., ed.), pp. 83-92. Heenemann Verlags. MbH, Berlin. Crim, L.W., Peter, R.E. and Billard, R. 1981. Onset of gonadotropic hormone accumulation in the immature trout pituitary gland in response to estrogen or aromatizable androgen steroid hormones. Gen. Comp. Endocrinol. 44: 374-381. Cuellar, 0. and Uyeno, T. 1972. Triploidy in rainbow trout. Cytogenetics 11: 508-515. Dawley, R.M. 1987. Hybridization and polyploidy in a community of three sunfish species (Pisces: Centrarchidae). Copeia 1987: 326-335. Dawley, R.M., Graham, J.H. and Schultz, R.J. 1985. Triploid progeny of pumpkinseed x green sunfish hybrids. J . Hered. 76: 251-257. Dawley, R.M., Shultz, R.J. and Goddard, K.A. 1987. Clonal reproduction and polyploidy in unisexual hybrids of Phoxinus eos and Phoxinus neogaeus (Pisces; Cyprinidae). Copeia 1987: 275-283. De Almeida Toledo, L.F. , Foresti, F. and De Almeida Toledo Fo., S. 1985. Spontaneous triploidy and NOR activity in Eigenmannia sp. (Pisces, Sternopygidae) from the Amazon basin. Genetica 66: 85-88. De Almeida-Toledo, L.F. , Foresti, F. , Toledo, S.A., Bernardino, G., Ferrari, W. and Alcantara, R.C.G. 1987. Cytogenetic studies of Colossoma  mitrei, Colossoma macropomum and their interspecific hybrid. In: Selection, Hybridization, and Genetic Engineering in Aquaculture, Vol. I (Tiews, K., ed.), pp. 189-195. Heenemann Verlags. mbH, Berlin. - 77 -Don, J . and Avtalion, R.R. 1986. The induction of triploidy in Oreochromis  aureus by heat shock. Theor. Appl. Genet. 72: 186-192. Donaldson, E.M. 1986. The integrated development and application of controlled reproduction techniques in Pacific salmonid aquaculture. Fish Physiol. Biochem. 2: 9-24. Donaldson, E.M. and Benfey, T .J . 1987. Current status of induced sex manipulation. In: Proc. Third Int. Symp. on the Reproductive Physiology of Fish (Idler, D.R., Crim, L.W. and Walsh, J.M., eds.), pp. 108-119. Memorial Univ. of NfId., St. John's. Donaldson, E.M., Fagerlund, U.H.M., Higgs, D.A. and McBride, J.R. 1979. Hormonal enhancement of growth. In: Fish Physiology, Vol. VIII (Hoar, W.S., Randall, D.J. and Brett, J.R., eds.), pp. 455-597. Academic Press, New York. Donaldson, E.M. and Hunter, G.A. 1982. Sex control in fish with particular reference to salmonids. Can. J . Fish. Aquat. Sci. 39: 99-110. Dorson, M. and Chevassus, B. 1985. Etude de la receptivite d'hybrides triploides truite arc-en-ciel x saumon coho a la necrose pancreatique infectieuse et a la septicemie hemorragique virale. Bull. Fr. Peche Piscic. 296: 29-34. (In French.) Dorson, M. and Chevassus, B. 1986. Susceptibility of two salmonid triploid hybrids (rainbow trout x coho salmon and rainbow trout x brook trout) to infectious pancreatic necrosis and viral haemorrhagic septicaemia viruses. In: EIFAC/FAO Symp. on Selection, Hybridization and Genetic Engineering in Aquaculture of Fish and Shellfish for Consumption and Stocking, Bordeaux (France), 27-30 May, 1986. Report no. EIFAC/86/Symp.E77, 4 pp. - 78 -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. Echelle, A.A., Echelle, A.F. and Crozier, CD . 1983. Evolution of an all-female f ish, Menidia ciarkhubbsi (Atherinidae). Evolution 37: 772-784. Fankhauser, G. and Humphrey, R.R. 1950. Chromosome number and development of progeny of triploid axolotl females mated with diploid males. J . Exp. Zool. 115: 207-249. Fankhauser, G. and Humphrey, R.R. 1954. Chromosome number and development of progeny of triploid axolotl males crossed with diploid females. J . Exp. Zool. 126: 33-58. Fauconneau, B., Kaushik, S.J . and Blanc, J.M. 1986. Utilisation de differents substrats energetiques chez la truite: influence de la ploidie. Diab. Metabol. 12: 111-112 (abstract). (In French.) Flajshans, M. and Rab, P. 1987. A finding of the triploid rainbow trout, Kamloops form (Salmo gairdnerii kamloops). Zivoc. Vyr. 32: 279-282. (In Czech with English summary.) Fox, D.P., Johnstone, R. and Durward, E. 1986. Erythrocyte fusion in heat-shocked Atlantic salmon. J . Fish Biol. 28: 491-499. Gervai, J . , Peter, S. , Nagy, A. , Horvath, L. and Csanyi, V. 1980. Induced triploidy in carp, Cyprinus carpio L. J . Fish Biol. 17: 667-671. - 79 -Glebe, B.D., Delabbio, J . , Lyon, P., Saunders, R.L. and McCormick, S. 1986. Chromosome engineering and hybridization of Arctic char (Salvelinus  alpinus) and Atlantic salmon (Salmo salar) for aquaculture. In: EIFAC/ FAO Symp. on Selection, Hybridization and Genetic Engineering in Aquaculture of Fish and Shellfish for Consumption and Stocking, Bordeaux (France), 27-30 May, 1986. Report no. EIFAC/86/Symp.E35, 14 pp. Gold, J.R. 1986. Spontaneous triploidy in a natural population of the fathead minnow, Pimephales promelas (Pisces: Cyprinidae). Southwest. Nat. 31: 527-529. Gold, J.R. and Avise, J .C. 1976. Spontaneous triploidy in the California roach Hesperoleucus symmetricus (Pisces: Cyprinidae). Cytogenet. Cell Genet. 17: 144-149. Golvan, Y . - J . 1962. Catalogue systematique des noms de genres de poissons actuels. Ann. Parasito. Hum. et Comp. 37(6) (fasc. suppl.): 1-227. Graham, M.S., Fletcher, G.L. and Benfey, T .J . 1985. Effect of triploidy on blood oxygen content of Atlantic salmon. Aquaculture 50: 133-139. Grammeltvedt, A . -F . 1974. A method of obtaining chromosome preparations from rainbow trout (Salmo gairdneri) by leucocyte culture. Norw. J . Zool. 22: 129-134. Henken, A.M., Brunink, A.M. and Richter, C .J .J . 1987. Differences in growth rate and feed utilization between diploid and triploid African catfish, Clarias gariepinus (Burchell 1822). Aquaculture 63: 233-242. H i l l , J.M., Hickerson, A. , Sheldon, D.L. and Warren, K.A. 1985. Production of triploid chinook salmon Oncorhynchus tshawytscha using flow-through heat shock and subsequent, gradual cooling. Proc. 35th Ann. Northwest Fish Culture Workshop, 4-6 Dec, 1984: 20-26. - 80 -Holmefjord, I., Refstie, T. and Hostmark, J . 1983. Preliminary results on induction of triploidy in salmonids using heat shocks. Proc. 33rd Ann. Meet. EAAP, Commission of Animal Genetics, Leningrad (U.S.S.R.), 16-19 Aug., 1982, 9 pp. Hoornbeek, F.K. and Burke, P.M. 1981. Induced chromosome number variation in the winter flounder. J . Hered. 72: 189-192. Humason, G.L. 1972. Animal Tissue Techniques, Third Edition. W.H. Freeman and Co., San Francisco. Humphrey, R.R. and Fankhauser, G. 1949. Three generations of polyploids in ambystomid salamanders. J . Hered. 40: 7-12. Iversen, O.E. and Laerum, O.D. 1987. Trout and salmon erythrocytes and human leukocytes as internal standards for ploidy control in flow cytometry. Cytometry 8: 190-196. Johnson, K.R. and Wright, J .E . 1986. Female brown trout x Atlantic salmon hybrids produce gynogens and triploids when backcrossed to male Atlantic salmon. Aquaculture 57: 345-358. Johnson, O.W., Dickhoff, W.W. and Utter, F.M. 1986. Comparative growth and development of diploid and triploid coho salmon, Oncorhynchus kisutch. Aquaculture 57: 329-336. Johnson, O.W., Rabinovich, P.R. and Utter, F.M. 1984. Comparison of the relability of a Coulter Counter with a flow cytometer in determining ploidy levels in Pacific salmon. Aquaculture 43: 99-103. Johnson, O.W., Utter, F.M. and Rabinovitch, P.S. 1987. Interspecies differences in salmonid cellular DNA identified by flow cytometry. Copeia 1987: 1001-1009. Johnstone, R. 1985. Induction of triploidy in Atlantic salmon by heat shock. Aquaculture 49: 133-139. - 81 -Johnstone, R. 1987. Survival rates and triploidy rates following heat shock in Atlantic salmon ova retained for different intervals in the body cavity after f i rst stripping together with preliminary observations on the use of pressure. In: Selection, Hybridization, and Genetic Engineering in Aquaculture, Vol. II (Tiews, K., ed.), pp. 219-224. Heenemann Verlags. mbH, Berlin. Johnstone, R. and Lincoln, R.F. 1986. Ploidy estimation using erythrocytes from formalin-fixed salmonid fry. Aquaculture 55: 145-148. Johnstone, R., Macdonald, A.G. and Shelton, C.J. 1987. The use of hyperbaric nitrous oxide to induce triploidy in fish eggs. J . Physiol. (Lond.) 384: 38P (abstract). Joswiak, G.R., Stasiak, R.H. and Koop, B.F. 1985. Diploidy and triploidy in the hybrid minnow, Phoxinus eos x Phoxinus neogaeus (Pisces: Cyprinidae). Experientia 41: 505-507. Kah, 0. 1986. Central regulation of reproduction in teleosts. Fish Physiol. Biochem. 2: 25-34. Kawamura, T. 1951a. Reproductive ability of triploid newts with remarks on their offspring. J . Sci. Hiroshima Univ. 12B (Div. 1): 1-10. Kawamura, T. 1951b. The offspring of triploid males of the frog, Rana  nigromaculata. J . Sci. Hiroshima Univ. 12B (Div. 1): 11-20. Kawauchi, H. 1988. The duality of teleost gonadotropins. In: Program and Abstracts, 1st Int. Symp. on Fish Endocrinology, June 12-17, 1988 (Edmonton, Alberta), p. 24 (abstract no. 33). Univ. of Alberta, Edmonton. Kilambi, R.V. and Galloway, M.L. 1985. Temperature preference and tolerance of hybrid carp (female grass carp, Ctenopharyngodon i del 1 a x male bighead, Aristichthys nobilis). Environ. Biol. Fish. 12: 309-314. - 82 -Kim, D.S., Kim, I.-B. and Baik, Y.G. 1986. A report of triploid rainbow trout production in Korea. Bull. Korean Fish. Soc. 19: 575-580. Kowtal, G.V. 1987. Preliminary experiments in induction of polyploidy, gynogenesis and androgenesis in the white sturgeon, Acipenser  transmontanus Richardson. In: Selection, Hybridization, and Genetic Engineering in Aquaculture, Vol. II (Tiews, K., ed.), pp. 317-324. Heenemann Verlags. mbH, Berlin. Krasznai, Z.L. 1987. Interspecific hybridization of warm water f inf ish. In: Selection, Hybridization, and Genetic Engineering in Aquaculture, Vol. II (Tiews, K., ed.), pp. 35-45. Heenemann Verlags. mbH, Berlin. Krasznai, Z. and Marian, T. 1986. Shock-induced triploidy and its effect on growth and gonad development of the European catfish, Silurus glanis L. J . Fish Biol. 29: 519-527. Krasznai, Z . , Marian, T. , Buris, L. and Ditroi, F. 1984a. Production of sterile hybrid grass carp (Ctenopharyngodon idella Val. x Aristichthys  nobilis Rich.) for weed control. Aquae. Hung. (Szarvas) 4: 33-38. Krasznai, Z . , Marian, T. and Kovacs, G. 1984b. Production of triploid European catfish (Silurus glanis L.) by cold shock. Aquae. Hung. (Szarvas) 4: 25-32. Leary, R.F., Allendorf, F.W., Knudsen, K.L. and Thorgaard, G.H. 1985. Heterozygosity and developmental stability in gynogenetic diploid and triploid rainbow trout. Heredity 54: 219-225. Lemoine, H.L., Jr. and Smith, L.T. 1980. Polyploidy induced in brook trout by cold chock. Trans. Am. Fish. Soc. 109: 626-631. Lessman, C A . and Habibi, H.R. 1987. Estradiol-17B silastic implants suppress oocyte development in the brook trout, Salve!inus fontina!is. Gen. Comp. Endocrinol. 67: 311-323. - 83 -Lieder, U. 1964. Polyploidisierungsversuche bei Fischen mittels Temperaturschock und Colchizinbehandlung. Zeit. Fisch. 12: 247-257. (In German with English summary.) Lincoln, R.F. 1981a. Sexual maturation in female triploid plaice, Pleuronectes platessa, and plaice x flounder, Platichthys flesus, hybrids. J . Fish Biol. 19: 499-507. Lincoln, R.F. 1981b. Sexual maturation in triploid male plaice (Pleuronectes  piatessa) and plaice x flounder (Platichthys flesus) hybrids. J . Fish Biol. 19: 415-426. Lincoln, R.F. 1981c. The growth of female diploid and triploid plaice (Pleuronectes piatessa) x flounder (PIatichthys flesus) hybrids over one spawning season. Aquaculture 25: 259-268. Lincoln, R.F., Aulstad, D. and Grammeltvedt, A. 1974. Attempted triploid induction in Atlantic salmon (Salmo salar) using cold shocks. Aquaculture 4: 287-297. Lincoln, R.F. and Bye V.J. 1984a. The growth and survival of triploid rainbow trout (Salmo gairdneri) in sea water over the spawning period. Int. Council Explor. Sea, CM. 1984/F:6, 8 pp. Lincoln, R. and Bye, V. 1984b. Triploid rainbows show commercial potential. Fish Farmer 7(5): 30-32. Lincoln, R.F. and Bye, V.J. 1987. Growth rates of diploid and triploid rainbow trout (Salmo gairdneri R.) over the spawning season. In: Proc. Third Int. Symp. on the Reproductive Physiology of Fish (Idler, D.R., Crim, L.W. and Walsh, J.M., eds.), p. 134 (abstract). Memorial Univ. of NfId. , St. John's. Lincoln, R.F. and Scott, A.P. 1983. Production of all-female triploid rainbow trout. Aquaculture 30: 375-380. - 84 -Lincoln, R.F. and Scott, A.P. 1984. Sexual maturation in triploid rainbow trout, Salmo gairdneri Richardson. J . Fish Biol. 25: 385-392. Lou, Y.D. and Purdom, C.E. 1984. Polyploidy induced by hydrostatic pressure in rainbow trout, Salmo gairdneri Richardson. J . Fish Biol. 25: 345-351. Magee, S.M. and Philipp, D.P. 1982. Biochemical genetic analyses of the grass carp female x bighead carp male ?\ hybrid and the parental species. Trans. Am. Fish-Soc. I l l : 593-602. Makino, S. and Ozima, Y. 1943. Formation of the diploid egg nucleus due to suppression of the second maturation division, induced by refrigeration of fertilized eggs of the carp, Cyprinus carpio. Cytologia 13: 55-60. Marian, T. and Krasznai, Z. 1978. Kariological investigations on Ctenopharyngodon i del 1 a and Hypophthalmichthys nobilis and their cross-breeding. Aquae. Hung. (Szarvas) 1: 44-50. Matty, A .J . 1985. Fish Endocrinology. Croom Helm, London. Meriwether, F., II. 1980. Induction of polyploidy in Israeli carp. Proc. Ann. Conf. S.E. Assoc. Fish & Wildl. Agencies 34: 275-279. Morelli, S. , Bertollo, L.A.C. and Moreira Fo., 0. 1983. Cytogenetic considerations of the genus Astyanax (Pisces, Characidae). II. Occurrence of natural triploidy. Caryologia 36: 245-250. Naevdal, G. and Dalpadado, P. 1987. Intraspecific hybridization - cold water species. In: Selection, Hybridization, and Genetic Engineering in Aquaculture, Vol. II (Tiews, K., ed.), pp. 23-33. Heenemann Verlags. mbH, Berlin. Nagahama, Y. 1983. The functional morphology of teleost gonads. In: Fish Physiology, Vol. IXA (Hoar, W.S., Randall, D.J. and Donaldson, E.M., eds.), pp. 223-275. Academic Press, New York. - 85 -Nagy, A. 1987. Genetic manipulations performed on warm water f ish. In: Selection, Hybridization, and Genetic Engineering in Aquaculture, Vol. II (Tiews, K., ed.), pp. 163-173. Heenemann Verlags. mbH, Berlin. Nakamura, M., Nagahama, Y., Iwahashi, M. and Kojima, M. 1987. Ovarian structure and plasma steroid hormones of triploid female rainbow trout. Can. Trans. Fish. Aquat. Sci. 5334: 6 pp. (Trans, from Japanese, Nippon Suisan Gakkaishi 53: 1105.) Naruse, K., I j i r i , K., Shima, A. and Egami, N. 1985. The production of cloned fish in the medaka (Oryzias latipes). J . Exp. Zool. 236: 335-341. Ojima, Y. and Makino, S. 1978. Triploidy induced by cold shock in fertilized eggs of the carp. A preliminary study. Proc. Japan Acad. 54B: 359-362. Ojima, Y. and Takai, A. 1979. The occurrence of spontaneous polyploid in the Japanese common loach, Misgurnus anguicaudatus. Proc. Japan Acad. 55B: 487-491. Okada, H. 1985. Studies on the art i f icial sex control in rainbow trout, Salmo  gairdneri. Can. Trans. Fish. Aquat. Si . 5329: 105 pp. (Trans, of Japanese, Sci . Rep. Hokkaido Fish Hatchery 40: 1-49). Oliva-Teles, A. and Kaushik, S .J . 1987a. Metabolic utilization of diets by polyploid rainbow trout (Salmo gairdneri). Comp. Biochem. Physiol. 88A: 45-47. Oliva-Teles, A. and Kaushik, S .J . 1987b. Nitrogen and energy metabolism during the early ontogeny of diploid and triploid rainbow trout (Salmo  gairdneri R.). Comp. Biochem. Physiol. 87A: 157-160. Pandian, T .J . and Varadaraj, K. 1987. Techniques to regulate sex ratio and breeding in t i lapia. Curr. Sci . 56: 337-343. - 86 -Parsons, J . E . , Busch, R.A., Thorgaard, G.H. and Scheerer, P.D. 1986. Increased resistance of triploid rainbow trout x coho salmon hybrids to infectious hematopoietic necrosis virus. Aquaculture 57: 337-343. Penman, D.J. , Shah, M.S., Beardmore, J.A. and Skibinski, D.O.F. 1987a. Sex ratios of gynogenetic and triploid t i lapia. In: Selection, Hybridization, and Genetic Engineering in Aquaculture, Vol. II (Tiews, K., ed.), pp. 267-276. Heenemann Verlags. mbH, Berlin. Penman, D.J. , Skibinski, D.O.F. and Beardmore, J.A. 1987b. Survival, growth rate and maturity in triploid t i lapia. In: Selection, Hybridization, and Genetic Engineering in Aquaculture, Vol. II (Tiews, K., ed.), pp. 277-288. Heenemann Verlags. mbH, Berlin. Peter, R.E. 1982. Neuroendocrine control of reproduction in teleosts. Can. J . Fish. Aquat. Sci . 39: 48-55. Phill ips, R.B., Zajicek, K.D., Ihssen, P.E. and Johnson, 0. 1986. Application of silver staining to the identification of triploid fish cel ls. Aquaculture 54: 313-319. Pickering, A.D., Pottinger, T .G. , Carragher, J . and Sumpter, J.P. 1987. The effects of acute and chronic stress on the levels of reproductive hormones in the plasma of mature male brown trout, Salmo trutta L. Gen. Comp. Endocrinol. 68: 249-259. Pottinger, T.G. and Pickering, A.D. 1985a. Changes in skin structure associated with elevated androgen levels in maturing male brown trout, Salmo trutta L. J . Fish Biol. 26: 745-753. Pottinger, T.G. and Pickering, A.D. 1985b. The effects of 11-ketotestosterone and testosterone on the skin structure of brown trout, Salmo trutta L. Gen. Comp. Endocrinol. 59: 335-342. - 87 -Purdom, C.E. 1972. Induced polyploidy in plaice (Pleuronectes piatessa) and its hybrid with the flounder (PIatichthys flesus). Heredity 29: 11-24. Purdom, C.E. 1976. Genetic techniques in flatfish culture. J . Fish. Res. Board Can. 33: 1088-1093. Purdom, C.E. 1983. Genetic engineering by the manipulation of chromosomes. Aquaculture 33: 287-300. Purdom, C.E. 1984. Atypical modes of reproduction in f ish. In: Oxford Reviews of Reproductive Biology, Vol. 6 (Clarke, J.R., ed.), pp. 303-340. Oxford Univ. Press, Oxford. Purdom, C.E. 1986. Genetic techniques for control of sexuality in fish farming. Fish Physiol. Biochem. 2: 3-8. Purdom, C.E. and Lincoln, R.F. 1973. Chromosome manipulation in f ish. In: Genetics and Mutagenesis of Fish (Schroeder, J .H. , ed.), pp. 83-89. Springer-Verlag, New York. Purdom, C.E. , Thompson, D. and Dando, P.R. 1976. Genetic analysis of enzyme polymorphisms in plaice (Pleuronectes platessa). Heredity 37: 193-206. Purdom, C.E. , Thompson, D. and Lou, Y.D. 1985. Genetic engineering in rainbow trout, Salmo gairdnerii Richardson, by the suppression of meiotic and mitotic metaphase. J . Fish Biol. 27: 73-79. Quillet, E. , Chevassus, B. and Krieg, F. 1987. Characterization of auto- and allotriploid salmonids for rearing in seawater cages. In: Selection, Hybridization, and Genetic Engineering in Aquaculture, Vol. II (Tiews, K., ed.), pp. 239-252. Heenemann Verlags. mbH, Berlin. Refstie, T. 1982. Practical application of sex manipulation. In: Proc. Second Int. Symp. on the Reproductive Physiology of Fish (Richter, C .J .J , and Goos, H.J.Th., eds.), pp. 73-77. Cen. Agric. Publ. Doc, Wageningen. - 88 -Refstie, T . , Stoss, J . and Donaldson, E.M. 1982. Production of all female coho salmon (Oncorhynchus kisutch) by diploid gynogenesis using irradiated sperm and cold shock. Aquaculture 29: 67-82. Refstie, T. , Vassvik, V. and Gjedrem, T. 1977. Induction of polyploidy in salmonids by cytochalasin B. Aquaculture 10: 65-74. Richter, C . J . J . , Henken, A.M., Eding, E.H., Van Doesum, J.H. and De Boer, P. 1987. Induction of triploidy by cold-shocking eggs and performance of triploids of the African catfish, CIari as gariepinus (Burchell, 1822). In: Selection, Hybridization, and Genetic Engineering in Aquaculture, Vol. II (Tiews, K., ed.), pp. 225-237. Heenemann Verlags. mbH, Berlin. Rohrer, R.L. and Thorgaard, G.H. 1986. Evaluation of two hybrid trout strains in Henry's Lake, Idaho, and comments on the potential use of sterile triploid hybrids. Nth. Am. J . Fish. Mgmt. 6: 367-371. Ryan, T.A., J r . , Joiner, B.L. and Ryan, B.F. 1982. Minitab Reference Manual. Pennsylvania State Univ., University Park. Salacinski, P.R.P., McLean, C , Sykes, J . E . C . , Clement-Jones, V.V. and Lowry, P.J. 1981. Iodination of proteins, glycoproteins, and peptides using a solid-phase oxidizing agent, l,3,4,6-tetrachloro-3<* ,6oc-diphenyl glycoluril (iodogen). Analyt. Biochem. 117: 136-146. Sakai, D.K., Okada, H., Koide, N. and Tamiya, Y. 1987. Blood type compatibility of lower vertebrates: phylogenetic diversity in blood transfusion between fish species. Develop. Comp. Immunol. 11: 105-115. Scheerer, P.D. and Thorgaard, G.H. 1983. Increased survival in salmonid hybrids by induced triploidy. Can. J . Fish. Aquat. Sci. 40: 2040-2044. - 89 -Scheerer, P.D., Thorgaard, G.H. and Seeb, J .E . 1987. Performance and developmental stability of triploid tiger trout (brown trout female x brook trout male). Trans. Am. Fish. Soc. 116: 92-97. Schmidtke, J . , Schulte, B., Kuhl, P. and Engel, W. 1976. Gene action in fish of tetraploid origin. V. Cellular RNA and protein content and enzyme activities in cyprinid, clupeoid, and salmonoid species. Biochem. Genet. 14: 975-980. Schultz, R.J. 1979. Role of polyploidy in the evolution of fishes. In: Polyploidy: Biological Relevance (Lewis, W.H., ed.), pp. 313-340. Plenum Press, New York. Scott, A.P. 1987. Reproductive endocrinology of f ish. In: Fundamentals of Comparative Vertebrate Endocrinology (Chester-Jones, I., Ingleton, P.M. and Phill ips, J . G . , eds.), pp. 223-256. Plenum Press, New York. Scott, A.P. , Sheldrick, E.L. and Flint, A.P.F. 1982. Measurement of 17oc,206-dihydroxy-4-pregnen-3-one in plasma of trout (Salmo gairdneri Richardson): seasonal changes and response to salmon pituitary extract. Gen. Comp. Endocrinol. 46: 444-451. Scott, A.P. and Sumpter, J.P. 1983. 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.), pp. 200-220. Croom Helm, London. Seeb, J .E . and Seeb, L.W. 1986. Gene mapping of isozyme loci in chum salmon. J . Hered. 77: 399-402. Seeb, J . , Thorgaard, G., Hershberger, W.K. and Utter, F.M. 1986. Survival in diploid and triploid Pacific salmon hybrids. Aquaculture 57: 375 (abstract). - 90 -Shah, M.S. and Beardmore, J.A. 1986. Production of triploid tilapia (Oreochromis niloticus) and their comparative growth performance with normal diploids. In: EIFAC/FAO Symp. on Selection, Hybridization and Genetic Engineering in Aquaculture of Fish and Shellfish for Consumption and Stocking, Bordeaux (France), 27-30 May, 1986, Report no. EIFAC/86/Symp.E22, 14pp. Shelton, C . J . , Macdonald, A.G. and Johnstone, R. 1986. Induction of triploidy in rainbow trout using nitrous oxide. Aquaculture 58: 155-159. Shelton, W.L. 1986. Control of sex in cyprinids for aquaculture. In: Aquaculture of Cyprinids (Billard, R. and Marcel, J . , eds.), pp. 179-194. Inst. Nat. Rech. Agron., Paris. Shelton, W.L. 1987. Genetic manipulations - sex control of exotic fish for stocking. In: Selection, Hybridization, and Genetic Engineering in Aquaculture, Vol. II (Tiews, K. ed.), pp. 175-194. Heenemann Verlags. mbH, Berlin. Shireman, J .V. , Rottmann, R.W. and Aldridge, F .J . 1983. Consumption and growth of hybrid grass carp fed four vegetation diets and trout chow in circular tanks. J . Fish Biol. 22: 685-693. Small, S.A. and Benfey, T .J . 1987. Cell size in triploid salmon. J . Exp. Zool. 241: 339-342. Small, S.A. and Randall, D.J. 1988. The effects of triploidy on the swimming performance of coho salmon (Oncorhynchus kisutch). Can. J . Fish. Aquat. Sc i . : in press. Smith, L.T. and Lemoine, H.L. 1979. Colchicine-induced polyploidy in brook trout. Prog. Fish-Cult. 41: 86-88. - 91 -Solar, I.I. and Donaldson, E.M. 1985. Studies on genetic and hormonal sex control in domesticated rainbow trout. I. The effect of heat shock treatment for induction of triploidy in cultured rainbow trout (Salmo  gairdneri Richardson). Can. Tech. Rep. Fish. Aquat. Sci. 1379: 15 pp. Solar, I.I., Donaldson, E.M. and Hunter, G.A. 1984. Induction of triploidy in rainbow trout (Salmo gairdneri Richardson) by heat shock, and investigation of early growth. Aquaculture 42: 57-67. Sriramulu, V. 1962. Effect of colchicine on the somatic chromosomes of Oryzias  melastigma McClelland. Cellule 63: 367-374. Stanley, J.G. 1976. Production of hybrid, androgenetic, and gynogenetic grass carp and carp. Trans. Am. Fish. Soc. 105: 10-16. Stanley, J.G. 1979. Control of sex in fishes, with special reference to the grass carp. In: Proc. Int. Grass Carp Conference (Shireman, J .V. , ed.), pp. 201-242. Univ. of Florida, Gainesville. Stanley, J.G. 1981. Manipulation of developmental events to produce monosex and sterile f ish. Rapp. P.-v. Reun. Cons. int. Explor. Mer 178: 485-491. Stanley, J .G . , Biggers, C.J. and Schultz, D.E. 1976. Isozymes in androgenetic and gynogenetic white amur, gynogenetic carp, and carp-amur hybrids. J . Hered. 67: 129-134. Sumpter, J . P . , Scott, A.P. , Baynes, S.M. and Witthames, P.R. 1984. Early stages of the reproductive cycle in virgin female rainbow trout (Salmo gairdneri Richardson). Aquaculture 43: 235-242. Sutterlin, A.M., Holder, J . and Benfey, T .J . 1987. Early survival rates and subsequent morphological abnormalities in landlocked, anadromous and hybrid (landlocked x anadromous) diploid and triploid Atlantic salmon. Aquaculture 64: 157-164. - 92 -Sutton, D.L., Stanley, J.G. and Miley, W.W., II. 1981. Grass carp hybridization and observations of a grass carp x bighead hybrid. J . Aquat. Plant Manage. 19: 37-39. Suzuki, R., Nakanishi, T. and Oshiro, T. 1985. Survival, growth and steri l i ty of induced triploids in the cyprinid loach Mi sgurnus anguillicaudatus. Bull. Jap. Soc. Sci. Fish. 51: 889-894. Svardson, G. 1945. Chromosome studies on Salmonidae. Rep. Swed. State Inst. Fresh-water Fish. Res. (Drottningholm) 23: 151 pp. Swarup, H. 1956. Production of heteroploidy in the three-spined stickleback, Gasterosteus aculeatus (L.). Nature 178: 1124-1125. Swarup, H. 1959a. Effect of triploidy on the body size, general organization and cellular structure in Gasterosteus aculeatus (L.). J . Genet. 56: 143-155. Swarup, H. 1959b. Production of triploidy in Gasterosteus aculeatus (L.). J . Genet. 56: 129-142. Swarup, H. 1959c. The oxygen consumption of diploid and triploid Gasterosteus aculeatus (L.). J . Genet. 56: 156-160. Tabata, K., Gorie, S. and Taniguchi, N. 1986. Verification by isozyme gene marker for gynogenetic diploidization and triploidization in hirame, Paralichthys olivacus. Fish Genet. Breed. Sci. 11: 35-41. (In Japanese.) Taniguchi, N., Kijima, A. and Fukai, J . 1987. High heterozygosity at Gpi-1 in gynogenetic diploids and triploids of ayu Plecoglossus alt ivelis. Nippon Suisan Gakkaishi 53: 717-720. Taniguchi, N., Kijima, A. , Fukai, J . and Inada, Y. 1986a. Conditions to induce triploid and gynogenetic diploid in ayu Plecoglossus alt ivelis. Bull. Jap. Soc. Sci . Fish. 52: 49-53. - 93 -Taniguchi, N., Kijima, A. , Tamura, T. , Takegami, K. and Yamasaki, I. 1986b. Color, growth and maturation in ploidy-manipul ated fancy carp. Aquaculture 57: 321-328. Taniguchi, N., Seki, S. , Inada, Y. and Murakami, K. 1985. Induced triploidy in ayu Plecoglossus alt ivel is. Bull. Jap. Soc. Sci . Fish. 51: 503. Thompson, B.Z., Wattendorf, R.J . , Hestand, R.S. and Underwood, J.L. 1987. Triploid grass carp production. Prog. Fish-Cult. 49: 213-217. Thorgaard, G.H. 1983. Chromosome set manipulation and sex control in f ish. In: Fish Physiology, Vol. IXB (Hoar, W.S., Randall, D.J. and Donaldson, E.M., eds.), pp. 405-434. Academic Press, New York. Thorgaard, G.H. 1986. Ploidy manipulation and performance. Aquaculture 57: 57-64. Thorgaard, G.H. and Allen, S.K., Jr. 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. and Parsons, J .E . 1985. Residual paternal inheritance in gynogenetic rainbow trout: implications for gene transfer. Theor. Appl. Genet. 71: 119-121. - 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. Sci. 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 triploid Gnathopogon elongathus caerulescens. Can. Trans. Fish. Aquat. Sci. 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) by means of temperature shock treatment. J . Fish Biol. 7: 519-528. - 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 on plasma sex steroid profiles in adult female coho salmon (Oncorhynchus kisutch). Gen. Comp. Endocrinol. 55: 36-45. Vasetskii, S.G. 1967. Changes in the ploidy of sturgeon larvae induced by heat treatment of eggs at different stages of development. Dokl. Biol. Sci. 172: 23-26. (Trans, of Russian, Dokl. Akad. Nauk SSSR 172: 1234-1237.) Vasetskii, S.G., Betina, M.I. and Kondrat'eva, O.T. 1984. Obtaining triploids by exerting hydrostatic pressure on fertilized eggs of Misgurnus fossi l is . Dokl. Biol. Sci . 279: 667-669. (Trans, of Russian, Dokl. Akad. Nauk SSSR 279: 212-215.) Vasil'ev, V.P., Makeeva, A.P. and Ryabov, I.N. 1975. Triploidy of hybrids of carp with other representatives of the family Cyprinidae. Sov. Genet. 11: 980-985. (Trans, of Russian, Genetika 11(8): 49-56.) Venere, P.C. and Junior, P.M.G. 1985. Natural triploidy and chromosome B in the fish Curimata modesta (Curimatidae, Characiformes). Rev. Brasil. Genet. 8: 681-687. Wattendorf, R.J. 1986. Rapid identification of triploid grass carp with a Coulter Counter and Channelyzer. Prog. Fish-Cult. 48: 125-132. Wattendorf, R.J. and Anderson, R.S. 1984. Hydrilla consumption by triploid grass carp. Proc. Ann. Conf. S.E. Assoc. Fish & Wildl. Agencies 38: 319-326. Wiley, M.J., Pescitel l i , S.M. and Wike, L.D. 1986. The relationship between feeding preferences and consumption rates in grass carp and grass carp x bighead carp hybrids. J . Fish Biol. 29: 507-514. - 96 -Wiley, M.J. and Wike, L.D. 1986. Energy balances of diploid, triploid, and hybrid grass carp. Trans. Am. Fish. Soc. 115: 853-863. Wolters, W.R., Chrisman, C L . and Libey, G.S. 1982a. Erythrocyte nuclear measurements of diploid and triploid channel catfish, Ictalurus punctatus (Rafinesque). J . Fish Biol. 20: 253-258. Wolters, W.R., Libey, G.S. and Chrisman, C L . 1981. Induction of triploidy in channel catfish. Trans. Am. Fish. Soc. 110: 310-312. Wolters, W.R., Libey, G.S. and Chrisman, C L . 1982b. Effect of triploidy on growth and gonad development of channel catfish. Trans. Am. Fish. Soc. I l l : 102-105. Wright, J . E . , Johnson, K.R. and May, B. 1983. Determining gene-centromere distances for isozyme loci in triploid salmonids. Isozyme Bull. 16: 58. Wu, C , Ye, Y. and Chen, R. 1986. Genome manipulation in carp (Cyprinus carpio L.). Aquaculture 54: 57-61. Yamamoto, K., Oota, I., Takano, K. and Ishikawa, T. 1965. Studies on the maturing process of the rainbow trout, Salmo gairdneri irideus. 1. Maturation of the ovary of a one-year old f ish. Bull. Jap. Soc. Sci. Fish. 31: 123-132. Yamazaki, F. 1983. Sex control and manipulation in f ish. Aquaculture 33: 329-354. Young, L.M., Monaghan, J .P . , Jr. and Heidinger, R.C 1983. Food preferences, food intake, and growth of the F\ hybrid of grass carp female x bighead carp male. Trans. Am. Fish. Soc. 112: 661-664. - 97 -Appendix 1: Papers describing or utilizing spontaneously-arisen or experimentally-induced triploid fish in species that are normally only dioecious diploids. 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 al . (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 al . (1984): heat Chernenko (1985): heat Johnson et a l . (1986): heat Parsons et al . (1986): heat Phillips et al . (1986): heat Seeb et al. (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 al . (1983): heat Johnson et al . (1984): heat Hill et al . (1985): heat Phillips et al . (1986): heat Seeb et al . (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 al . (1977): cytochalain B Thorgaard and Gall (1979): spontaneous (6/11, not random) Chourrout (1980): heat Thorgaard et al. (1981): heat Chourrout and Quillet (1982): heat Thorgaard et al . (1982): heat, spontaneous (2/30, not random) Chevassus et al . (1983): heat Lincoln and Scott (1983): heat Scheerer and Thorgaard (1983): heat Ueda et al . (1983): spontaneous (2/17) Wright et al . (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 al . (1984): heat Sumpter et al . (1984): [heat] Bolla and Refstie (1985): cytochalasin B Choubert and Blanc (1985): heat Dorson and Chevassus (1985): heat Leary et al . (1985): heat Okada (1985): pressure Purdom et a l . (1985): heat Solar and Donaldson (1985): heat Thorgaard et a l . (1985): heat Allendorf et al . (1986): heat Almeida (1986): heat Benfey et al . (1986): heat Chourrout (1986a): heat Chourrout (1986b): heat Chourrout et al . (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 al . (1986): heat Phillips et a l . (1986): heat Shelton et al . (1986): nitrous oxide Ueda et a l . (1986): polyethylene glycol Benfey et al . (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 al . (1987): heat - 99 -S. gairdneri [cont.] Oliva-Teles and Kaushik (1987a): breeding (2n X 4n), heat Oliva-Teles and Kaushik (1987b): heat Quillet et al . (1987): heat Sakai et al . (1987): pressure Ueda et al . (1987): pressure S. mykiss (Kamchatkan trout) Chernenko (1985): heat S. salar (Atlantic salmon) Lincoln et al . (1974): cold Refstie et al . (1977): cytochalasin B Allen and Stanley (1979): cytochalasin B Allen and Stanley (1981a): cytochalasin B Allen (1983): cytochalasin B Holmefjord et al . (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 al . (1984): heat Bolla and Refstie (1985): cytochalasin B Graham et al . (1985): heat Johnstone (1985): heat Fox et al . (1986): heat Glebe et al . (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 al . (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 al . (1985): cold Taniguchi et al . (1986a): cold Taniguchi et al . (1987): cold Ueno et al . (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 al . (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 al. (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 al . (1986): ? Burns et al . (1986): ? Cassani and Caton (1986a): heat, pressure Cassani and Caton (1986b): ? Wattendorf (1986): ? Wiley and Wike (1986): ? (J.M. Malone and Sons) Thompson et al . (1987): cold, heat Genus Cyprinus  C. carpio (carp) Makino and Ozima (1943): cold Ojima and Makino (1978): cold Gervai et al . (1980): cold Meriwether (1980): colchicine, cold Al-Sabti et a l . (1983; cited by Col 1ares-Pereira, 1987): spontaneous Ueno (1984): cold Taniguchi et al . (1986b): cold Wu et al . (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. fossi l is (loach) Vasetskii et al . (1984): pressure Family SILURIDAE Genus Silurus S. glanis (European catfish) Krasznai et al . (1984b): cold Krasznai and Marian (1986): cold Family AMEIURIDAE Genus Ictalurus I. punctatus (channel catfish) Wolters et a l . (1981): cold Wolters et al . (1982a): cold Wolters et al . (1982b): cold Chrisman et al. (1983): cold Bidwell et al . (1985): heat Bidwell et al . (1986): heat Family CLARIIDAE Genus Clarias  C. gariepinus (African catfish) Henken et al . (1987): cold Richter et al . (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 luviat i l is (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 al. (1987a): heat Penman et al . (1987b): heat 0. niloticus Chourrout and Itskovich (1983): heat Shah and Beardmore (1986): heat Penman et al . (1987a): heat Penman et al . (1987b): heat Order PLEURONECTIFORMES Suborder PLEURONECTOIDEI Family PARALICHTHYIDAE Genus Paralichthys  P. olivaceus (hirame) Tabata et al. (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 al . (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 al . (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 al . (1986): heat X 0. tshawytscha (chinook salmon) Seeb et al . (1986): heat X Salmo gairdneri (rainbow trout) Parsons et al . (1986): heat 0. tshawytscha (chinook salmon) X 0. gorbuscha (pink salmon) Utter et al . (1983): heat X 0. keta (chum salmon) Seeb et al . (1986): heat X 0. kisutch (coho salmon) Seeb et al . (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 al . (1983): heat Dorson and Chevassus (1985): heat Dorson and Chevassus (1986): heat Parsons et a l . (1986): heat Quillet et al . (1987): heat X S. clarki (cutthroat trout) Rohrer (1982; cited by Rohrer and Thorgaard, 1986): heat X S. salar (Atlantic salmon) Purdom et al . (1985): heat X S. trutta (brown trout) Chevassus et al . (1983): heat Scheerer and Thorgaard (1983): heat Quillet et al . (1987): heat X Salvelinus fontinalis (brook char) Capanna et al. (1974): spontaneous Chevassus et a l . (1983): heat Scheerer and Thorgaard (1983): heat Ueda et al . (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 al . (1983): heat X Salvelinus alpinus (Arctic char) Holmefjord et al . (1983): heat Glebe et al . (1986): heat S. trutta (brown trout) X S. gairdneri (rainbow trout) Scheerer and Thorgaard (1983): heat Purdom et al . (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 al. (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 al . (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 al . (1983): spontaneous Beck et al . (1984): spontaneous (Malone, 1980 brood) Cassani et al . (1984): spontaneous (Malone, 1979, 1980 & 1981 broods) Krasznai et a l . (1984a): spontaneous Bettoli et al . (1985): spontaneous (Malone, ? brood) Kilambi and Galloway (1985): spontaneous (Malone, ? brood) Wiley and Wike (1986): spontaneous (Malone, ? brood) Wiley et al . (1986): spontaneous (Malone, 1979 brood) Genus Cyprinus C. carpio (carp) X Ctenopharyngodon idel 1 a (grass carp) Yasil'ev et al . (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 al . (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 . BENFEY1, EDWARD M. DONALDSON1, AND TERRANCE G. OWEN2 1 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 Ltd., 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 le : Pacific salmonid vitellogenin RIA - 109 -ABSTRACT This paper describes an homologous radioimmunoassay for coho salmon vitellogenin that demonstrates parallel cross-reactivity for plasma vitellogenin of all 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; Ng and Idler, 1983; Scott and Sumpter, 1983a; Wallace, 1985). 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 (Sumpter, 1985; Copeland et a l . , 1986), and brown trout, S_^  trutta (Norberg and Haux, in press). As well, radioimmunoassays for partially purified Atlantic salmon egg yolk proteins (Idler et a l . , 1979) and for rainbow trout lipovitellin (Campbell and Idler, 1980), both having parallel displacement to their conspecific vitellogenins, have been described. These radioimmunoassays have demonstrated that there are clear 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 - I l l -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) was purified from female coho salmon serum by precipitation 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, using the iodogen method. An initial separation of radiolabeled vitellogenin from free iodine was done on a Sephadex G25 column, followed by further 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. Next, 100 yul (1 unit) of goat antibody to rabbit 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 5 females of each species except cutthroat 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. Females, on the other hand, had very high plasma vitellogenin concentrations, in the hundreds of jjg/ml to hundreds of mg/ml range (Table 2). DISCUSSION This paper describes the f irst 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, female and sterile coho salmon by the injection of 178-estradiol (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 individuals at ovulation. The radioimmunoassay described in this paper could easily be sensitized, as discussed by So et a l . (1985) and Copeland et al . (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 is likely a reflection of their evolutionary history. Mitochondrial 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. We wish to thank Ted Down for his many suggestions, especially regarding statistical analysis. We also thank the staffs of the Big 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 by a NSERC postgraduate scholarship and a Quebec FCAR graduate fellowship. - 116 -REFERENCES Benfey, T . J . , Dye, H.M., and Donaldson, E.M. (1988). Induced vitellogenesis in triploid coho salmon (Oncorhynchus kisutch), and its effect on plasma andpituitary gonadotropin. Gen. Comp. Endocrinol., in review. Berg, W.J., and Ferris, S.D. (1984). Restriction endonuclease analysis of salmonid mitochondrial DNA. Can. J . Fish. Aquat. Sci . 41, 1041-1047. Campbell, C M . , 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, J .P . , Walker, T.K., and Croft, M. (1986). Vitellogenin 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 uti l i ty of mitochondrial DNA in fish genetics and fishery management. In "Population Genetics and Fishery Management." (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 DNA of salmonids. In "Population Genetics and Fishery Management." (N. Ryman, and F. Utter, Eds.). Chap. 12. University of Washington Press, Seattle. Idler, D.R., Hwang, S . J . , and Crim, L.W. (1979). Quantification of vitellogenin in Atlantic salmon (Salmo salar) plasma by radioimmunoassay. J . Fish. Res. Board Can. 36, 574-578. Le Bail , P.Y., and Breton, B. (1981). Rapid determination of the sex of puberal salmonid fish by a technique 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 Sumpter, J.P. (1983b). A comparison of the female reproductive cycles of autumn-spawning and winter-spawning strains of rainbow trout (Salmo gairdneri Richardson). Gen. Comp. Endocrinol. 52, 79-85. - 118 -So, Y.P. , Idler, D.R., and Hwang, S.J . (1985). Plasma vitellogenin in landlocked Atlantic salmon (Salmo salar Ouananiche): isolation, homologous radioimmunoassay and immunological 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). Vitellogenesis and oocyte growth in nonmammalian vertebrates. 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 males (0. tshawytscha) females chum salmon (0. keta) coho salmon (0. kisutch) pink salmon (0. gorbuscha) sockeye salmon (0. nerka) Qualicum River SEP Hatchery (B.C.) DFO Pacific Biological Station (B.C.) Chilliwack River SEP Hatchery (B.C.) Chilliwack River SEP Hatchery (B.C.) Quinsam River SEP Hatchery (B.C.) Weaver Creek SEP Spawning Channel (B.C.) Atlantic salmon (S. salar) cutthroat trout (S. clarki) Genus Salmo Marine Sciences Research Lab. (Nfld.) 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 chum salmon coho salmon pink salmon sockeye salmon cutthroat trout rainbow trout 15.7 (+ 2.4, n=5) 0.52 (+ 1.11, n=5) 14.1 (± 18.7, n=5) 0.20 (± 0.20, n=5) 0.11 (± 0.12, n=5) 2.35 (+ 1.46, n=2) 131.6 (+ 138.5, n=5) - 121 -PLASMA DILUTION FACTOR -7 -6 -5 -4 -3 -2 -1 10 10 10 10 10 10 10 o [Vtg] (ng/ml) FIG. 1 - Plasma d i l u t i on curves for v i te l l ogen in (Vtg) in Chinook (a) , coho (b) , pink ( c ) , sockeye (d) and chum salmon (e) , rainbow (f) and cutthroat t rout (g) , A t lan t i c salmon (h) and sab le f ish ( i ) (std = coho standards). 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
https://iiif.library.ubc.ca/presentation/dsp.831.1-0097948/manifest

Comment

Related Items