Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Aspects of the reproductive endocrinology of the Thai carp, Puntius gonionotus (Bleeker) Sukumasavin, Naruepon 1992

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

Item Metadata

Download

Media
831-ubc_1992_spring_sukumasavin_naruepon.pdf [ 5.93MB ]
Metadata
JSON: 831-1.0086398.json
JSON-LD: 831-1.0086398-ld.json
RDF/XML (Pretty): 831-1.0086398-rdf.xml
RDF/JSON: 831-1.0086398-rdf.json
Turtle: 831-1.0086398-turtle.txt
N-Triples: 831-1.0086398-rdf-ntriples.txt
Original Record: 831-1.0086398-source.json
Full Text
831-1.0086398-fulltext.txt
Citation
831-1.0086398.ris

Full Text

ASPECTS OF THE REPRODUCTIVE ENDOCRINOLOGY OF THE THAI CARP,PUNTIUS GONIONOTUS (BLEEKER)byNARUEPON SUKUMASAVINB.Sc. (Fisheries) Kasetsart University, Thailand, 1982A THESIS SUBMITTED IN PARTIAL FULFILLMENT OFTHE REQUIREMENTS FOR THE DEGREE OFMaster of ScienceinTHE FACULTY OF GRADUATE STUDIESDEPARTMENT OF ZOOLOGYWe accept this thesis as conformingto the required standardTHE UNIVERSITY OF BRITISH COLUMBIAApril 1992© Naruepon Sukumasavin, 1992In presenting this thesis in partial fulfilment of the requirements for an advanceddegree at the University of British Columbia, I agree that the Library shall make itfreely available for reference and study. I further agree that permission for extensivecopying of this thesis for scholarly purposes may be granted by the head of mydepartment or by his or her representatives. It is understood that copying orpublication of this thesis for financial gain shall not be allowed without my writtenpermission.(Signature)Department of^7,0()T OGYThe University of British ColumbiaVancouver, CanadaDate^APRIL 1992DE-6 (2/88)ABSTRACTSeveral aspects of the reproductive endocrinology of the Thai carp (Puntius gonionotusBleeker) were investigated. The annual cycle of gonadal development, changes in plasma hormonelevels in male and female fish reared in ponds at Kalasin Freshwater Fisheries Station, Kalasin,Thailand and environmental parameters were observed for a period of 20 months. The interaction of asalmon gonadotropin-releasing hormone analog (D-Argo, Pro 9 NEt-sGnRH; sGnRHA) anddomperidone (Dom) on the induction of gonadotropin (GtH) secretion and spawning in female fishwas investigated. Further, long- and short-term changes in plasma hormone levels during sGnRHAand Dom induced spawning were examined. Moreover, the biological activities of 12 differentmammalian, avian and piscine GnRHs and their analogs were also compared.In females, gonadal recrudescence was highly correlated with the changes in air temperatureand daylength, but spawning coincided with the occurrence of rainfall. Histological analysis of theovary revealed that oocyte development was of the asynchronous type, thus suggesting that the Thaicarp can spawn several times in a spawning season. It was not possible to distinguish pronouncedseasonal changes in plasma hormone levels during this study. Also, there were no correlations betweenseasonal changes in plasma hormone levels and any reproductive parameter.Gonadal development in males was highly correlated with that of females. The structure ofthe testis corresponds to the lobular type. Histological analysis revealed a continuous spermatogeneticactivity. Plasma hormones levels in males exhibited a bimodal pattern of seasonal changes. However,there were no correlations between the changes in plasma reproductive hormones, testiculardevelopment and environmental parameters.Injection of sGnRHA and Dom alone increased plasma GtH levels significantly, but themagnitude of increasing GtH levels was insufficient to induce spawning, except in the case of thehighest dose of domperidone. Administration of various combinations of sGnRHA and Domiiincreased both the plasma GtH level and the number of fish spawning, suggesting that dopamine playsan important role as the gonadotropin release inhibitory factor in this species.Plasma E2 and T levels in females increased significantly following an elevation of GtH duringthe induction period. During the short-term observations, both E2 and T peaked immediately at thetime of ovipositon. Plasma 17a, 20f3 dihydroxy-4-pregnene-3-one was undetectable throughout theinduction period.In males, a significant increase in plasma GtH, T and 11-ketotestosterone (11-KT) wasobserved at the onset of spawning. Both T and 11-KT continued to increase during the process ofspawning, while GtH immediately decreased to a lower level. The changes in plasma 11-KT weresynchronous with the occurrence of spawning, suggesting that 11-KT is probably the major androgenduring the process of spawning in male Thai carp.High concentrations (25 µg/kg) of either native GnRHs or their analogs in combination withDom (25 mg/kg) were equally effective in inducing spawning. At 10 12g/kg, however, sGnRH andchicken GnRH-11, in combination with 10 mg/kg Dom, were found to be the most potent of the nativeGnRHs. GnRHs with a substitution in position 6 with hydrophobic or aromatic D-amino acids possessgreater potencies compared to native forms. Furthermore, [D-Ala 6]-mGnRHA and [D-Trp6]-mGnRHA were found to be the most potent peptides.iiiTABLE OF CONTENTSAbstract^ iiTable of Contents^ ivList of Figures viiList of Tables^ xiAcknowledgement adiCHAPTER 1 General Introduction^ 1CHAPTER 2 Materials and Methods 10A. Experimental Site^ 10B. Experimental Animals and Husbandry^ 10C. Hormone Preparation, Injections and Blood Sampling^ 11D. Histological Analyses^ 12E. Radioimmunoassays 13a. Gonadotropin 131.Chemicals ^ 132.Iodination of carp gonadotropin II (cGtH-II)^133. Radioimmunoassay procedure^ 14b. Steroids^ 151.Chemicals^ 152.Extraction of steroids from plasma samples^ 173.Radioimmunoassay procedure^ 184.Steroid assay validation 19F. Statistics ^ 19CHAPTER 3 Annual Reproductive Cycle of the Thai Carp ^ 20A. Introduction ^ 20B.Materials and Methods^ 20C. Results 21I. Seasonal changes in environmental parameters ^ 23a.Rainfall ^ 23b.Temperature 23c. Daylength 26II. Seasonal changes in reproductive parameters^ 26A. Female ^ 26a.Condition factor^ 26b.Hepatosomatic index 29c. Gonadosomatic index 29B. Male^ 29a. Condition factor^ 29ivb.Hepatosomatic index ^ 32c.Gonadosomatic index 32HI. Seasonal histological changes in gonadal and hepatic tissues^ 32A. Anatomy of the gonads^ 32B. Histology of the ovary 35C. Histology of the testis 44D. Histology of the liver ^ 51IV. Seasonal changes in plasma gonadotropin and steroid hormones^53A. Hormonal changes in female Thai carp^ 53a.Gonadotropin^ 53b.Testosterone 53c. Estradio1-17P 53B. Hormonal changes in male Thai carp^ 59a.Gonadotropin^ 59b.Testosterone 59c. 11-Ketotestosterone 59D. Discussion ^ 64a.Females 64b.Males 69CHAPTER 4 Interaction of sGnRHA and Domperidone on the Induction of Gonadotropin Secretionand Induction of Spawning in the Thai Carp^ 72A. Introduction ^ 72B.Materials and Methods^ 72C. Results 77a.Percent fish spawned 77b.Plasma GtH levels^ 78D. Discussion ^ 83CHAPTER 5 The Effects of sGnRHA and domperidone on plasma gonadotropin and steroidhormones levels during spawning in the male and female Thai carp^ 87A. Introduction^ 87B.Materials and Methods^ 88I. Experiment I. "Long term" changes in plasma hormone levels during induced spawning inmale and female Thai carp^ 88II. Experiment II. "Short term" changes in plasma hormone levels at the time of spawningin male and female Thai carp 88C. Results^ 89Experiment I. ^ 89A. Females 89B.Males 94Experiment II.^ 98A. Females 98B.Males 102D. Discussion ^ 106a.Females 106b.Males 110CHAPTER 6 Biological Activities of GnRHs and Their Analogs in Combination with Domperidoneon the Induction of Gonadotropin Secretion and Spawning in the Thai Carp^114A. Introduction ^ 114B.Materials and Methods^ 115C. Results 119a.Percentage of fish spawned^ 119b.Plasma GtH levels ^ 121D. Discussion ^ 127CHAPTER 7 Summary and Conclusions ^ 130A. Annual Reproductive Cycle of the Thai Carp ^ 1301.Females^ 1302.Males 131B. Interaction of sGnRHA and Domperidone in the Regulation of Gonadotropin Secretion andInduction of Spawning in the Female Thai Carp ^ 132C. Hormonal Changes During sGnRHA and Domperidone Induced Spawning in the Thai Carp ...^ 1331.Females^ 1332.Males 134D. Biological Activities of GnRHs and Their Analogs in Combination with Domperidone on theInduction of Gonadotropin Secretion and Spawning in the Female Thai Carp^135REFERENCES^ 137viLIST OF FIGURESFig. 1. Primary structure of mammalian, avian, and salmon gonadotropin-releasing hormone^9Fig. 2. Annual changes in rainfall at Kalasin during 1987-89 expressed as mm per calendar month ^22Fig. 3. Annual changes in maximum and minimum air temperatures at Kalasin during 1987-89expressed as mean minimum and mean maximum temperatures in each calendar month.^24Fig. 4. Annual changes in daylength at Kalasin during 1987-89 expressed as mean daylength in hour ineach calendar month^ 25Fig. 5. Annual changes in condition factor (CF) in female Thai carp in a rearing pond at KalasinFreshwater Fisheries Station during 1987-89.^ 27Fig. 6. Annual changes in hepatosomatic index (HSI) in female Thai carp in a rearing pond at KalasinFreshwater Fisheries Station during 1987-89^ 28Fig. 7. Annual changes in gonadosomatic index (GSI) in female Thai carp in a rearing pond at KalasinFreshwater Fisheries Station during 1987-89^ 30Fig. 8. Annual changes in condition factor (CF) in male Thai carp in a rearing pond at KalasinFreshwater Fisheries Station during 1987-89^ 31Fig. 9. Annual changes in hepatosomatic index (HSI) in male Thai carp in a rearing pond at KalasinFreshwater Fisheries Station during 1987-89^ 33Fig. 10. Annual changes in gonadosomatic index (GSI) in male Thai carp in a rearing pond at KalasinFreshwater Fisheries Station during 1987-89^ 34Fig. 11.1. Cross-sections of ovaries. (a) Stage I oocyte 160X. (b) Stage II oocyte 160X. (c) Stage IIIoocyte 40X.^ 36Fig. 11.2. Cross-sections of ovaries. (d) Stage IV oocyte, early stage 40X. (dl) Stage IV oocyte, latestage 40X.^ 37Fig. 11.3. Cross-sections of ovaries. (e) Stage V oocyte, germinal vesicle migration stage 40X. (f)Atretic oocyte 40X. (g) Post-ovulatory oocyte 40X^ 38Fig. 12.1. Annual changes in the occurrence of oocytes in different stages in female Thai carp in arearing pond at Kalasin Freshwater Fisheries Station during 1987-89. (a) Stage I oocyte^39Fig. 12.2. Annual changes in the occurrence of oocytes in different stages in female Thai carp in arearing pond at Kalasin Freshwater Fisheries Station during 1987-89. (b) Stage II oocyte. (c)Stage III oocyte. 40vuFig. 12.3. Annual changes in the occurrence of oocytes in different stages in female Thai carp in arearing pond at Kalasin Freshwater Fisheries Station during 1987-89. (d) Stage IV oocyte. (e)Stage V oocyte. 41Fig. 12.4. Annual changes in the occurrence of oocytes in different stages in female Thai carp in arearing pond at Kalasin Freshwater Fisheries Station during 1987-89. (I) Atretic oocyte. (g) Post-ovulatory oocyte^ 42Fig. 13. Cross-sections of testes. (a) Spermatogonia. (b) Primary spermatocytes. (c) Secondaryspermatocytes. (d) Spermatids. (e) Spermatozoa.^ 46Fig. 14.1. Annual changes in the testicular germ cells at different stages in male Thai carp in a rearingpond at Kalasin Freshwater Fisheries Station during 1987-89. (a) Spermatogonia^47Fig. 14.2. Annual changes in the testicular germ cells at different stages in male Thai carp in a rearingpond at Kalasin Freshwater Fisheries Station during 1987-89. (b) Primary spermatocytes. (c)Secondary spermatocytes^ 48Fig. 143. Annual changes in the testicular germ cells at different stages in male Thai carp in a rearingpond at Kalasin Freshwater Fisheries Station during 1987-89. (d) Spermatids. (e) Spermatozoa.49Fig. 15. Annual changes in hepatocyte diameter in female Thai carp in a rearing pond at KalasinFreshwater Fisheries Station during 1987-89^ 52Fig. 16. Annual changes in plasma gonadotropin in female Thai carp in a rearing pond at KalasinFreshwater Fisheries Station during 1987-89^ 55Fig. 17. Annual changes in plasma testosterone in female Thai carp in a rearing pond at KalasinFreshwater Fisheries Station during 1987-89^ 56Fig. 18. Annual changes in plasma estradio1-17P in female Thai carp in a rearing pond at KalasinFreshwater Fisheries Station during 1987-89^ 57Fig. 19. Annual changes in GSI and plasma hormone levels in female Thai carp in a rearing pond atKalasin Freshwater Fisheries Station during 1987-89^ 58Fig. 20. Annual changes in plasma gonadotropin in male Thai carp in a rearing pond at KalasinFreshwater Fisheries Station during 1987-89^ 60Fig. 21. Annual changes in plasma testosterone in male Thai carp in a rearing pond at KalasinFreshwater Fisheries Station during 1987-89^ 61Fig. 22. Annual changes in plasma 11-ketotestosterone in male Thai carp in a rearing pond at KalasinFreshwater Fisheries Station during 1987-89^ 62Fig. 23. Annual changes in GSI and plasma hormone levels in male Thai carp in a rearing pond atKalasin Freshwater Fisheries Station during 1987-89^ 63viiiFig. 24. Effect of various combinations of sGnRHA (µg/kg) and Dom (mg/kg) on the induction ofspawning in female Thai carp during July 1990 at Kalasin Freshwater Fisheries Station.^75Fig. 25. Effect of various combinations of sGnRHA (µg/kg) and Dom (mg/kg) on the induction ofspawning in female Thai carp during May 1991 at Pathumthani Freshwater Fisheries Station. ^76Fig. 26. Effect of various combinations of sGnRHA (µg/kg) and Dom (mg/kg) on the induction ofGtH secretion in female Thai carp at injection time in the 1991 study^ 79Fig. 27. Effect of various combinations of sGnRHA (µg/kg) and Dom (mg/kg) on the induction ofGtH secretion in female Thai carp at 3 hr after injection in the 1991 study.^80Fig. 28. Effect of various combinations of sGnRHA (µg/kg) and Dom (mg/kg) on the induction ofGtH secretion in female Thai carp at 6 hr after injection in the 1991 study.^81Fig. 29. Effect of various combinations of sGnRHA (µg/kg) and Dom (mg/kg) on the induction ofGtH secretion in female Thai carp at 9 hr after injection in the 1991 study.^82Fig. 30. Changes in plasma E2 during sGnRHA (20 µg/kg) and Dom (20 mg/kg) induced spawning infemale Thai carp^ 91Fig. 31. Changes in plasma T during sGnRHA (20 µg/kg) and Dom (20 mg/kg) induced spawning infemale Thai carp 92Fig. 32. Changes in plasma GtH during sGnRHA (20 µg/kg) and Dom (20 mg/kg) induced spawningin female Thai carp^ 93Fig. 34. Changes in plasma 11-KT during spawning in untreated male Thai carp kept with sGnRHAand Dom induced spawning treated females.^ 95Fig. 35. Changes in plasma T during spawning in untreated male Thai carp kept with sGnRHA andDom treated females^ 96Fig. 36. Changes in plasma GtH during spawning in untreated male Thai carp kept with sGnRHA andDom treated females^ 97Fig. 37. Short-term changes in plasma E2 levels in sGnRHA (20 /1g/kg) and Dom (20 mg/kg) inducedspawning in female Thai carp shortly before, during, and after spawning^99Fig. 38. Short-term changes in plasma T levels in sGnRHA (20 µg/kg) and Dom (20 mg/kg) inducedspawning in female Thai carp shortly before, during, and after spawning^100Fig. 39. Short-term changes in plasma GtH levels in sGnRHA (20 µg/kg) and Dom (20 mg/kg)induced spawning in female Thai carp shortly before, during, and after spawning^101Fig. 40. Short-term changes in plasma 11-KT levels in untreated male Thai carp kept with sGnRHAand Dom treated females shortly before, during, and after spawning.^103ixFig. 41. Short-term changes in plasma T levels in untreated male Thai carp kept with sGnRHA andDom treated females shortly before, during, and after spawning^ 104Fig. 42. Short-term changes in plasma GtH levels in untreated male Thai carp kept with sGnRHA andDom treated females shortly before, during, and after spawning^ 105Fig 43. Effect of native mammalian, avian and piscine GnRHs and their analogs (25 µg/kg) incombination with Dom (25 mg/kg) on the induction of spawning in the Thai carp in the 1990study (24-29 July 1990)^ 118Fig. 44. Effect of native mammalian, avian and piscine GnRHs and their analogs (10 µg/kg) incombination with Dom (10 mg/kg) on induction of spawning in the Thai carp in the 1991 study(21-25 May 1991).^ 120Fig. 45. Effects of native mammalian, avian, and piscine GnRHs and their analogs (10 µg/kg) incombination with Dom (10 mg/kg) on the induction of GtH secretion in female Thai carp atinjection time in the 1991 study (21-25 May 1991) 123Fig. 46. Effects of native mammalian, avian, and piscine GnRHs and their analogs (10 µg/kg) incombination with Dom (10 mg/kg) on the induction of GtH secretion in female Thai carp at 3 hrafter injection in the 1991 study (21-25 May 1991).^ 124Fig. 47. Effects of native mammalian, avian, and piscine GnRHs and their analogs (10 µg/kg) incombination with Dom (10 mg/kg) on the induction of GtH secretion in female Thai carp at 6 hrafter injection in the 1991 study (21-25 May 1991).^ 125Fig. 48. Effects of native mammalian, avian, and piscine GnRHs and their analogues (10 µg/kg) incombination with Dom (10 mg/kg) on the induction of GtH secretion in female Thai carp at 9 hrafter injection in the 1991 study (21-25 May 1991).^ 126LIST OF TABLESTable 1Dose combinations of sGnRHA and DomKalasin Freshwater Fisheries Station in July 1990.^ 74Table 2Dose combinations of sGnRHA and DomPathumthani Freshwater Fisheries Station in May 1991^ 74Table 3The primary structure of GnRH and other peptides used in the study^ 117xiACKNOWLEDGEMENTFirst, I wish to thank my research supervisor, Dr. E. M. Donaldson, for his patience,understanding, support and encouragement over the past years. I would also like to express mygratitude to Professor N. R. Liley, my supervisor at UBC, and Professor A. M. Perks, my researchcommittee, for their guidance and assistance.I am also grateful to Mr. Wattana Leelapatra for recognizing my ability to pursue this degreeand for his continuous support.I am sincerely thank Mr. Jack McBride who inspired me the histological techniques, Drs. J. R.Cardwell, G. Van Der Kraak and Mrs. Helen Dye for teaching me the steroid radioimmunoassay, andDr. R. E. Peter and Ms. Carol Nahorniak at the Department of Zoology, University of Alberta forteaching me the gonadotropin radioimmunoassay and allowing me to use the facilities at theDepartment. I also thank Dr. E. McLean for his friendly advice. Additionally, I thank Mr. TharaphanWattanamahart, Mr. Thavee Viputhanumas, and staff of Kalasin Freshwater Fisheries Station andPathumthani Freshwater Fisheries Station, Department of Fisheries, Thailand for assistance duringconducting experiments in ThailandThis study was fmancially supported by CIDA through the Northeast Fisheries Project,Thailand. Thanks are also due to Ms. Deborah Turnbull, Mr. Rolf Schoenert and staff of SRD forassistance during my staying in Canada. Further, this study was made possible by the studies leavepermission from the Department of Fisheries, Thailand.Finally, I extend my sincere gratitude to my family and friends who provided moral supportthroughout the study period.xiiCHAPTER 1GENERAL INTRODUCTIONThe freshwater aquaculture industry is becoming important in Thailand for the provision of aninexpensive and abundant source of protein for human consumption. Since 1980, the number of fishfarms has increased gradually each year, and this has resulted in a shortage in the supply of fry.Though induced spawning by the traditional hypophysation method has been adoptedthroughout Thailand for many years as a major method for fry production, this method still has somelimitations due to the quantity and quality of the pituitary gland supply and method of standardization.In order to have a more reliable supply of fry for the aquaculture industry, it is important to improvethe understanding of the reproductive physiology of cultured species and the relationships between theenvironment, the reproductive hormones, and gonadal development. This information can then beused for the establishment of biotechnologies designed to overcome reproduction related problems inaquaculture.The annual reproductive cycle in teleosts has been intensively studied, and is believed todepend upon changes in environmental factors (Peter and Crim, 1979; Peter, 1981; Crim, 1982). Intemperate fishes, the patterns of gonadal development and spawning have been found to be highlysynchronized with annual cycles in daylength and/or temperature (de Vlaming, 1972; 1974; Lam, 1983).In freshwater tropical fishes, similarly, gonadal development was found to be highly correlated withtemperature and daylength (Lam and Munro, 1987), and final maturation, ovulation, and spawning aremostly triggered by rainfall or water quality changes resulting from rainfall (Munro, 1990).Gonadal development is regulated by hormones within the hypothalamo-pituitary-gonadal axis(Peter, 1983; Donaldson and Hunter, 1983; Idler and Ng, 1983; Fostier et al. 1983). The association ofthe changes in plasma levels of reproductive hormones with gonadal condition has proven to be avaluable tool in the development of an understanding of endocrine control of reproduction in teleosts.1Many studies have investigated the correlations between seasonal changes in plasma levels ofreproductive hormones and gonadal development in a number of fish including salmonids (Crim andIdler, 1978; Lambert et al. 1978; Fostier and Jalabert, 1982; Scott et al. 1980a; 1983; Scott and Sumpter,1983; Whitehead et al. 1983; Ueda et al. 1984; Van Der Kraak et al. 1984; 1985; Young et al. 1983),goldfish, Carassius auratus, (Schreck and Hopwood, 1974; Kagawa et al. 1983; Stacey et al. 1983;Kobayashi et al. 1986a), the white sucker, Catostomus commersoni, (Scott et al. 1984; Stacey et al.1984), catfish, Heteropneustes fossilis, (Lamba et al. 1983), brown bullhead, Ictalurus nebulosus, (Burkeet al. 1984), common carp, Cyprinus carpio, (Yaron and Levavi-Zermonsky, 1986; Galas and Bieniarz,1989; Chang and Chen, 1990), blunt snout bream, Megalobrama amb4rephala, (Weixin et al. 1987), andwalking catfish, Clarias batrachus (Singh and Singh, 1991).The predominant pituitary hormone regulating gonadal function in teleosts is gonadotropin(GtH). Two forms of GtH have been isolated and characterized for teleosts based on theircarbohydrate content or their ability to adsorb to Conconavalin A-Sepharose (Idler and Ng, 1983; VanDer Kraak and Peter, 1987a). The first GtH to be purified was the glycoprotein-rich or maturationalgonadotropin (mGtH). It has a molecular weight of 25,000-40,000 and consists of two subunits(Donaldson, 1973; Peter and Crim, 1979; Idler, 1982; Idler and Ng, 1983) as do the gonadotropins inother vertebrates (Licht et a1. 1977). Maturational GtH is now known to be involved in the processesof steroidogenesis, maturation, and ovulation or spermiation (Idler, 1982; Idler and Ng, 1983; Van DerKraak and Peter, 1987a). Recently, another GtH has been purified from the teleost pituitary gland(Idler and Ng, 1983; Van Der Kraak and Peter, 1987a). This molecule is distinguished from mGtH onthe basis of its lower carbohydrate content and its differences in biological activity. This carbohydrate-poor or vitellogenic gonadotropin (vGtH) participates in vitellogenesis and promotes the uptake of theyolk precursor, vitellogenin, into oocytes (Ng and Idler, 1983; Idler and So, 1987).2More recently, definitive characterization of two pituitary gonadotropins, GtH I and GtH II,has been provided in chum salmon (Kawauchi et al. 1986; 1987; 1989; Itoh et al. 1988, Suzuki et al.1988a; b; c; d), coho salmon (Swanson et al. 1991), grass carp (Yu and Shen, 1989) and common carp(Van Der Kraak et al. 1992). This nomenclature of GtH I and GtH II is based on their relative elutionpositions in anion exchange chromatography. Both GtHs also consist of a and p subunits. The 8subunits have about 30% homology in amino acid sequence (Kawauchi et al. 1989). The a and /3subunits of both GtHs show significant sequence identity to the respective subunits of mammalian FSHand LH (Itoh et al. 1988; Kawauchi et al. 1989). GtH I and GtH II are localized in separate cells in thepituitary and the rate of synthesis of GtH I and GtH II varies during reproductive development (Nozakiet al. 1990a; b). Studies on immunological and biological characteristics reveal that both GtHs aresteroidogenic and are approximately equipotent in inducing estradio1-17P production, but GtH II ismore potent in enhancing the production of 17a20/3-dihydroxyprogesterone and in turn inducingoocyte maturation (Suzuki et al. 1986; Swanson et al. 1989; Van Der Kraak et al. 1992). In rainbowtrout, GtH I has a primary function in stimulating vitellogenin uptake into developing oocytes (Tyler etal. 1991). It is now suggested that GtH II corresponds to a maturational GTH (Kawauchi et a1. 1989;Van Der Kraak et al. 1992). In the following dissertation, the term gonadotropin (GtH) will be takento refer to the maturational gonadotropin (mGtH or GtH II) unless otherwise stated.Recent studies on the reproductive endocrinology of fish have shown that, in the female, GtHcontrols the biosynthesis of gonadal steroids by acting on the ovarian follicle. These steroid hormones,in turn, mediate the processes of gonadal growth, maturation and ovulation (Nagahama, 1987). Inresponse to GtH, theca layers of the oocyte secrete large amounts of aromatizable androgen, mainlytestosterone, which is converted to estradiol-17/3 (E2) by granulosa layers (Nagahama, 1987). E2 thenacts in the liver to stimulate the production of vitellogenin. Finally, vitellogenin is selectively taken up3from the bloodstream by developing oocytes under the influence of vGtH (see Wallace and Selman,1981; Ng and Idler, 1983; Wallace et al. 1987).It is now generally accepted that a preovulatory surge of GtH triggers the processes of fmalmaturation and ovulation in teleosts (Nagahama, 1987). In salmonids which have synchronous oocytedevelopment, a gradual and prolonged rise in plasma GtH has been shown to proceed ovulation inrainbow trout, Salmo gairdneri, (Fostier et al. 1987; Fostier and Jalabert, 1982; Scott et al. 1983), cohosalmon, Oncorhynchus kisutch, (Van Der Kraak et al. 1983; Fitzpatrick et al. 1986), pink salmon, 0.gorbuscha, (Dye et al. 1986) and sockeye salmon, 0. nerka, (Truscott et al. 1986). In cyprinid fisheswhich have asynchronous oocyte development, a rapid surge of plasma GtH was observed prior toovulation in the goldfish, Carassius auratus, (Stacey et al. 1979; Kobayashi et al. 1987), common carp,Cyprinus carpio, (Santos et al. 1986), and bitterling, Acheilognathus rhombea, (Shimizu et al. 1985).The action of GtH in inducing oocyte maturation appears to be dependent on the synthesis ofa maturation-inducing steroid (MIS) (Sundararaj et al. 1985; Nagahama, 1987). In several teleosts, avariety of hydroxylated C21 steroids, such as 17a20P-dihydroxy-4-pregnene-3-one (17,20P-P), havebeen identified as potent maturation inducing steroids (see Jalabert, 1976; Goetz, 1983; Scott andCanario, 1987). In particular, high levels of 17,20P-P have been found during both in in vivo and invitro GtH induced fmal maturation in several teleost species (see Goetz, 1983; Scott and Canario,1987). Furthermore, recent studies of oocyte fmal maturation have revealed that the 17,20/3,21-trihydroxy-4-3-one (17,20P,21-P) trihydroxylated progesterone derivative is also the MIS in severalmarine fishes (Trant et al. 1986; Trant and Thomas, 1989; Patino and Thomas, 1990).Under natural conditions, the surge of GtH in teleosts is considered to occur when bothexogenous and endogenous conditions are optimal (Stacey, 1984). Exogenous factors such astemperature, and to a lesser extent photoperiod, are known to be important for ovulation andspermiation in temperate cyprinids (see Stacey et al. 1979; Peter, 1981; Santos et al. 1986). However,4the endogenous factors required for initiation of the preovulatory GtH surge in tropical cyprinids arenot so clearly understood Many studies have investigated the changes in steroid hormones as anendogenous requisite for the preovulatory GtH surge. In rainbow trout, it has been hypothesized that adecrease in plasma E2 level triggers GtH release ultimately resulting in ovulation (Fostier et al. 1983;Scott et al. 1983). In the goldfish, however, a decline in E2 is thought to signify the completion ofvitellogenesis (Pankhurst and Stacey, 1985; Kobayashi et al. 1987). In these fish the high plasma levelof testosterone observed before ovulation has been suggested to be an important endogenous requisitefor the occurrence of the GtH surge (Kobayashi et al. 1987; 1989).Unlike females, less is known about the reproductive endocrinology of males. The maingonadal steroids are produced by the Leydig cells of the testis under mGtH stimulation (Billard et al.1990). During spermatogenesis in rainbow trout, plasma GtH increases slightly, and later, a peak ofplasma 11-ketotestosterone (11-KT) occurs following that of testosterone (T) (Fostier et al. 1982). Asimilar trend is also found in common carp, but 11-KT, rather than T, is found to be the majorandrogen in in vitro testicular culture of carp (Koldras et al. 1990). Furthermore, 17,201-P has alsobeen found to be the most efficient steroid to induce spermiation in both goldfish and salmonids (Uedaet al. 1984).GtH secretion in the teleost is regulated by gonadotropin-releasing hormone (GnRH) and bydopamine which acts as a gonadotropin release inhibitory factor (GRIF) (Peter et al. 1986; Lin andPeter, 1986). The significance of the GRIF differs between species and may also change along withsexual recrudescence or maturation within the same species. At present, the species in whichdopamine has no inhibitory actions on GtH secretion are the Atlantic croaker, Microgonias undulatus(Copeland and Thomas, 1989). In the species where dopamine plays a minor role in the regulation ofovulatory gonadotropin secretion, injection of GnRH analog alone results in increased GtH andovulation. Species in which this occurs include coho salmon, Oncorhynchus kisutch, (Van Der Kraak et5al. 1986); loach, Paramisgurnus dablyanus, (Lin et al. 1985); and African catfish, Clarias gariepinus, (DeLeeuw et al. 1985). In species that have a strong dopamine inhibitory action, injection of a GnRHanalog alone is generally ineffective in inducing ovulation. This situation is exemplified in goldfish,Carassius auratus, common carp, Cyprinus carpio, (Peter et al. 1986); silver carp, Hypophthalmichthysmolitrix; mud carp, Cirrhinus molitorella; grass carp, Ctenophatyngodon idellus; bighead carp,Aristichthys nobilis; and black carp, Mylopharyngodon piceus, (Peter et al. 1987a).Various types of drugs that block the action of dopamine have been investigated. These drugsare pimozide, a dopamine receptor antagonist (Chang and Peter, 1983), reserpine, a drug which causesgeneral depletion of catecholamine (Lin et al. 1985; 1986), alpha-methyl paratyrosine and carbidopar,drugs which block catecholamine synthesis at steps up to and including the production of dopamine(Peter et al. 1986), and domperidone, a dopamine receptor antagonist (Peter et al. 1987a) and adopamine depletor (Sloley et al. 1990). Of these, pimozide and domperidone are the most potent inpotentiating the action of GnRH analogs on GtH release in goldfish (Peter et al. 1986; Omeljaniuk etal. 1987a).Domperidone is known to be highly specific for dopamine receptors. It binds specifically toreceptors in the pituitary and does not cross the blood-brain barrier in goldfish (Omeljaniuk et al.1987b). Recently, domperidone has been found to cause the depletion of dopamine in the goldfishpituitary (Sloley et al. 1990). Because of the likelihood of fewer undesirable side effects and the factthat it is relatively inexpensive, domperidone has been widely used in studies on the induced ovulationof cultured fish (Peter et al. 1986; Lin and Peter, 1986).More than 2000 forms of GnRH analogs (GnRHA) have been synthesized in an effort toproduce synthetic GnRHA having strong agonistic or antagonistic effects (Karten and Rivier, 1986).Through biochemical studies, it has been demonstrated that mammalian GnRH differs in its aminoacid sequence from those of GnRHs found in bird and fish (Sherwood, 1987) (Fig. 1). In studies on the6activity of various mammalian GnRHs (mGnRH) and salmon GnRH (sGnRH) on goldfish, Peter et al.(1987b) reported that [D-Arg6, Pro9 NEt]-sGnRH (sGnRHA) was the most active analog both in vitroand in vivo. In rainbow trout and landlocked salmon, Crim et al. (1988) demonstrated that all types offish, bird and mammalian GnRH agonists possessed superactive properties in vivo on the fish pituitaryin terms of GtH release. The most active forms were found to be sGnRHA and [D-hArg (Et2) 6 , Pro9NEt]-sGnRH, the mGnRHA, ED-Ala6, Pro9 NEt]-mGnRH, [D-(Na12)6, aza-Gly10]-mGnRH and [D-hArg(Et2)6 , Pro9 NEt]-mGnRH. In the African catfish, comparison of sGnRHA, sGnRH, andmGnRHA determined that the former had the highest activity in vivo, but in vitro, its activity appearedsimilar to [D-Ser(But)6 Pro9 NEt]-mGnRH (Buserelin) (De Leeuw et al. 1988). In coho salmon, VanDer Kraak et al. (1987b) reported similar GtH releasing and ovulation-inducing activity for severalsGnRHA and mGnRHAs.To date, studies on fish reproductive endocrinology have emphasized the temperate or sub-temperate species. Information for tropical fish is not available and is needed for aquaculturedevelopment. Thus, the major objectives of the studies described herein were to investigate theendocrinological aspects of the control mechanisms responsible for the regulation of gonadaldevelopment, gonadotropin secretion, and ovulation in a tropical teleost, the Thai carp, Puntiusgonionotus (Bleeker).The Thai carp is a freshwater cyprinid of great economic importance in Thailand. It iscommonly cultured both in earthen ponds and in paddy fields. In pond culture, the Thai carp oftenmakes up a high proportion of the stocking density in the polyculture of carp, which is commonlypracticed in Thailand (Leelapatra, 1988). Its production from aquaculture increased from 7,311 tons in1985 to 11,145 tons in 1988, and it presently contributes more than 70% of total carp production inThailand (FAO, 1991). The spawning season of the Thai carp ranges from March to August with apeak in May or June and coincides with the occurrence of rainfall (Sipitalliat and Leenanond, 1984).7Induced spawning of the Thai carp has been practiced for many years, mostly with the hypophysationmethod (Sipitakkiat and Leenanond, 1984). Recently, Leelapatra (1988) successfully induced thespawning of the Thai carp using sGnRHA and domperidone therapy. In hatchery conditions, the Thaicarp can be induced to spawn at least 3 times in a spawning period (Sirikul et al. 1986).Like most tropical fish, the pattern of gonadal development and its endocrine control in theThai carp is not clearly understood. Thus, in Chapter 3 of this study, I investigated the role ofenvironmental and hormonal factors in the regulation of gonadal development in the Thai carp.Furthermore, during the appropriate time of the year for induced ovulation of this species observed inChapter 3, I examined the effectiveness of several combinations of salmon gonadotropin-releasinghormone analog (sGnRHA) and domperidone on GtH secretion and ovulation in the Thai carp(Chapter 4). Additionally, the significance of endocrine changes during hormonal induced finalmaturation and spawning was studied, as outlined in Chapter 5. Finally, in Chapter 6, the effectivenessof mammalian, avian, and piscine GnRH analogs in combination with domperidone on GtH releaseand ovulation in the Thai carp was examined8mammalian^pG1u-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2chicken-I^pGlu-His-Trp-Ser-Tyr-Gly-Leu-Gln-Pro-Gly-NH2chicken-II^pGlu-His-Trp-Ser-His-Gly-Trp-Tyr-Pro-Gly-NH2salmon pG1u-His-Trp-Ser-Tyr-Gly-Trp-Leu-Pro-Gly-NH2Fig. 1. Primary structure of mammalian, avian, and salmon gonadotropin-releasing hormone9CHAPTER 2MATERIALS AND METHODSA. EXPERIMENTAL SITEExperiments on the annual reproductive cycle of the Thai carp were carried out at KalasinFreshwater Fisheries Station (Lat. 16 ° 25' Long. 103 ° 31' E), Kalasin, Thailand about 550 kmnortheast of Bangkok. The station is located on the east side of Lam Pao Dam and uses water fromthe Lam Pao reservoir for all aquaculture activities. The air temperature at Kalasin varies from aminimum of 10 0 C in the winter (November to February) to a maximum of 40° C in the summer(March to May). The rainy season extends from April to September, with the highest rainfall beingrecorded during May. A pronounced dry season ranges from November to February every year.Experiments on induced ovulation and the effects of gonadotropin-releasing hormone incombination with domperidone were performed at Kalasin Freshwater Fisheries Station, Kalasin,Thailand in 1990 and at Pathumthani Freshwater Fisheries Station, Pathumthani, Thailand, located 70km northwest of Bangkok in 1991. All fish used in the latter studies were transferred from KalasinFreshwater Fisheries Station approximately 1 month prior to experimentation.B. EXPERIMENTAL ANIMALS AND HUSBANDRYMature (age 1-3 years, weight 200-1000 g) Thai carp, Puntius gonionotus, used in allexperiments were reared as broodstock for fry production in earthen ponds. Stocking densities were200 kg/rai (1600 m2) for females and 400 kg/rai for males. Fish were fed once daily with a 20%protein pellet (9% fishmeal, 15% soybean cake, 25% broken rice, 50% rice bran and 1% vitamins andminerals) at a rate of approximately 3% of body weight. Rearing ponds were also fertilized with 10010kg/rai of dry pig manure once every month. During the pre-spawning season (December to February),pond water was changed regularly i.e., once per month for a period of one week.C. HORMONE PREPARATION, INJECTIONS AND BLOOD SAMPLINGFish were netted from the earthen ponds and transferred into spawning tanks, 2 hr prior toblood sampling and hormone injection. During handling, fish were anesthetized by immersion in 0.05% 2-phenoxyethanol (Sigma Chemical Co.). For sampling during the annual reproductive cycle study,fish were randomly sampled from earthen ponds, whereas, fish for induced ovulation studies wereselected by determining the degree of abdominal swelling and the coloration (reddish) of the genitalpore. Fish were weighed and identified by a color thread tied to the dorsal spine.Mammalian gonadotropin-releasing hormone (mGnRH), mammalian gonadotropin-releasinghormone analog mGnRH-NHEt (mGnRHA), salmon gonadotropin-releasing hormone (sGnRH),Chicken-I gonadotropin-releasing hormone ([G1n 8]-GnRH, cGnRH-I), [D-Trp6]-mGnRH, and [D-Ala6]-mGnRH were purchased from Sigma Chemical Co., St. Louis, U.SA. Des-G1y10 , [D-Ala6,Pro9-NHEt]-mGnRH, des-Gly10, [D-Trp6, Pro9-NHEt]-GnRH, [D-Lys6]-GnRH, des-G1y10, [D-Arg6,Pro9-NHEt]-sGnRH, and des-G1y10, ED-Ala6, Pro9-NHEt]-sGnRH were purchased from BachemInc., California. Chicken-II gonadotropin-releasing hormone (Hiss , Trp7, Tyr8-GnRH, cGnRH-II)was purchased from Peninsula Laboratories, Inc., California, U.S A. Buserelin (D-Ser(But)6-GnRHA;ICI 118630) was a gift from Imperial Chemical Industries, PLC Pharmaceuticals Division, England.Domperidone was purchased from Sigma Chemical Co., St. Louis, U.SA. All gonadotropin-releasinghormones were dissolved in distilled water at the concentration of 1000 µg/10m1. One ml aliquotswere stored in polypropylene tubes and frozen at -20 ° C until use. Domperidone was dissolved in N,N-climethylformamide (Sigma Chemical Co.) at the concentration of 20 mg/ml. Prior to injection, thehormone and domperidone solutions were mixed and the volume of the solution adjusted to 1 ml/kg by11adding distilled water. Hormones were administered by intraperitoneal injection at the base of thepectoral fm, using a 1-ml tuberculin syringe (needle size; 24G). Details of the dosage are provided inthe protocols to the experiments in each chapter.Blood was drawn from the caudal vessels using a 3 or 5 ml heparinized Vacutainer (needle size21G, Terumo, Tokyo, Japan). Blood samples of 1 to 3 ml were held on ice prior to centrifugation at4000 rpm for 8 min to separate blood cells from plasma. Plasma was collected using glass pipettes,transferred to polypropylene vials and then stored at -20 ° C prior to transportation to the WestVancouver Laboratory for further analysis. For transport to Vancouver, samples were placed over dry-ice in a Styrofoam container. All samples were frozen at -40 ° C upon arrival at the West VancouverLaboratory, until assay.Spawning was indicated by the presence of eggs in spawning tanks. Spawned fish wereidentified by the presence of the thinner and flaccid belly resulting from the release of ovulated eggs.D. HISTOLOGICAL ANALYSESWhole gonads and liver were dissected from freshly-killed specimens. After they wereweighed, 1-3 g of either gonads or liver, taken from mid tissue portions, were fixed in Hollande Bouin'ssolution (3:1 w/wt). Histological analysis was carried out after re-fixing the tissues in Bouin's solutionfor 24 hr, following by a rinse in distilled water 3 times within 24 hr. Tissues were then dehydrated in50% ethanol for 1 hr, and preserved in 70% ethanol until analysis. For analysis, tissues weredehydrated in increasing concentrations of ethanol from 70 to 95%, and embedded in Paraplast wax(Fisher Scientific Ltd.). Sections of 5 Am were cut from each block and mounted on microscope slides.The sections were subsequently re-hydrated and stained using Mayer's haematoxyline and eosin(Culling, 1974). Finally, stained sections were dehydrated a second time and cover slips added andsealed with Permount (Fisher Scientific Ltd.).12E. RADIOIMMUNOASSAYSa. GonadotropinPlasma gonadotropin was measured at the Department of Zoology, University of Alberta,Edmonton, Alberta, Canada using a radioimmunoassay described by Peter et al. (1984a). Details ofthe procedure employed are described below.1. ChemicalsThe carp gonadotropin II (cGtH-II) employed was that described in Van Der Kraak et al.(1992). The purified cGtH-II was used for raising antibodies in rabbit (Rabbit Anti-Carpgonadotropin, RAC, first antibody) as described by Peter et al. (1984a), for preparing tracer, and forpreparing standards for RIA.Phosphate buffer stock (0.5 M, 0.84 g potassium dihydrogen phosphate [KH2PO4, BDH,assured grade] and 6.3 g dibasic sodium phosphate [Na2HPO4, S-9763, Sigma] in 100 ml doubledistilled deionized water, pH 7.5]) was prepared fresh for each iodination.The column buffer was a 0.08 M, barbital buffer [5.0 g sodium barbital (B 22-500, Fisher), 3.25g sodium acetate (BDH, assured grade), 0.1 g thimerosal (T-5125, Sigma) and 342 ml 0.1 Nhydrochloric acid in 965.8 ml double distilled deionized water, pH 8.6 1.The assay buffer or diluent was prepared by adding 5 g Bovine serum albumin, BSA (Sigma)into 11 of 0.08 M barbital buffer.2. Iodination of carp gonadotropin II (cGtH-II)cGtH-II was iodinated using a modification of the chloramine T procedure described by Peteret a/. (1984a). In brief, 10 111 (1 mCi) Na 125I (Amersham) was added to a 2 ml polystyrene sample13cup (Fisher Scientific Ltd.) containing 5 I.Lg (25 Al) cGtH-II in 0.05 M phosphate buffer, pH 7.5 (5 mlof 0.5 M phosphate buffer in 45 ml double distilled deionized water). The cGtH-II and Na l25  weremixed once by drawing both up into a pipette tip and carefully expelling them back into the bottom ofthe cup. Further, 40 Al of 0.5 M phosphate buffer and 25 Al chloramine T (10 mg chloramine T[Calbiochem] in 10 ml 0.05 M phosphate buffer, pH 7.5) were then added. The cup was shaken for 90seconds, and the reaction stopped by adding 100 Al sodium metabisulfite (24 mg sodium metabisulfite[J.T. Baker Chemical Co., USA] in 10 ml 0.05 M phosphate buffer, pH 7.5) and 200 ill potassiumiodine (100 mg potassium iodine [J.T. Baker Chemical Co., USA] in 10 ml 0.05 M phosphate buffer,pH 7.5). The entire contents of the cup were transferred onto a 1.1 x 20 cm column containing G-50Sephadex beads which were primed with 2 ml 5% BSA in barbital buffer (1 g BSA in 20 ml barbitalbuffer). A further 200 Al potassium iodine was added into the cup, withdrawn, and also placed ontothe column. When the entire 0.6 ml had been drawn into the column, it was flushed through withcolumn buffer. Fifteen fractions containing 1 ml each were collected and scanned for activities. Thelabelled cGtH-II appeared as the first peak off the column and inorganic 1251 as a second peak. Twohundred Al of 5% BSA in barbital buffer was added to each tube of labelled cGtH-II for preventing theisotopically labelled gonadotropin from sticking to the tubes and also the degradation of the tracer.The labelled GtH was stored at 4 ° C. Under these conditions, the labelled gonadotropin could be usedfor about 2 weeks.3. Radioimmunoassay procedureThe radioimmunoassay for GtH was performed at 4 ° C over a four day period. Duplicate 10 x75 borosilicate glass culture tubes were set up to contain 50 pd of standard (0.16-100 ng/ml) or plasma,200 Al of the first antibody (RAC) at 1:220,000 containing 1:100 normal rabbit serum (NRS, producedat the Department of Zoology, University of Alberta, Edmonton, Alberta) prepared in diluent, and14approximately 15,000 cpm/200 Al of tracer. Non-specific binding controls had the standard replacedby diluent and RAC replaced by 1:100 NRS. Maximum binding controls had only the standardreplaced by diluent, while total count controls contained only 200 1.41 of tracer. After each addition ofsolutions, the tubes were vigorously shaken. The tubes were incubated at 4 ° C for 48 hr, with aninterruption after 24 hr for vigorous shaking. Then 200 Al of the second antibody, Goat Anti-RabbitGlobulin (GAR), at 1:20 in diluent was added into each tube except the total count tubes. The tubeswere vigorously shaken, and further incubated at 4 ° C. About 18-24 hr after incubation, all tubes,except total count tubes, were vigorously shaken and centrifuged at 3000 rpm for 20 min. Aftercentrifugation, the supernatant fluid was decanted onto cotton wool and the tubes were counted for 1min each on an Isomedic Automatic Gamma counter (ICN Biomedicals Inc.).b. Steroids1. ChemicalsDiethyl-ether (BDH, AnalaR grade) and n-heptane (BDH, assured grade) were used in theextraction of steroids from plasma. The steroid assay buffer was a 0.05 M phosphate buffer [5.75 g/1dibasic sodium phosphate (Na2HPO4.2H20, S-0876, Sigma) and 1.315 g/1 mono sodium phosphate(NaH2PO4, S-0751, Sigma) in distilled water] containing 1.0 g/1 gelatin (G-2500, Sigma), to limit non-specific binding, and 65 mg/1 sodium azide (S-2002, Sigma) as a preservative. The buffer was heated to37 ° C to dissolve the gelatin, and its pH adjusted to 7.6.Antibodies to testosterone (ICN ImmunoBiologicals 61-315) and estradiol-17fl (ICNImmunoBiologicals 61-305) were purchased from Miles Laboratories Ltd., Rexdale. Ontario. Anti 11-ketotestosterone was a gift of Dr. D. R. Idler, Memorial University, St. John's Newfoundland.Antibodies to 17,20/3-P were provided from Dr. A. P. Scott (MAFF Fisheries Laboratory, Lowestoft,15England). Cross-reactivity between the antibodies and other steroids was provided by themanufacturer for testosterone and estradiol-17fl. Antibodies against testosterone cross-react with 11-ketotestosterone by 1.5% and with 11 hydroxytestosterone by 2.5% (Miles Laboratories). The crossreaction of 11-ketotestosterone and testosterone was 0.06% at 50% binding level (Cardwell, 1989).Standards of testosterone (T-1500, Sigma), estradiol-17fl (E-8875, Sigma), 17,20fl-P (0-1850,Steraloids, Inc., Wilton, NH) and 11-ketotestosterone (lot 2607, Syndel Laboratories Ltd., Vancouver,B.C.) were initially dissolved in ethanol at 1 mg/ml, and then serially-diluted at 10 fold intervals to 10ng/ml in assay buffer.Steroids radiolabelled with 3H were obtained from Amersham Canada Ltd. (Oakville,Ontario) for testosterone (TRK. 402), estradio1-17/3 (TRK. 322), and 11-ketotestosterone (TRK. 676).Radiolabelled 17,20P-P was prepared according to Scott et al. (1982) and Van Der Kraak et al.(1984). In brief, 40 of [1, 2, 6, 7-3H] 17a-hydroxyprogesterone (Amersham) was placed in a 16x125mm glass tube then dried under a stream of nitrogen. The steroid was redissolved in 500 µl0.05 MTris Buffer pH 7.6 (Sigma Chemical Co.), and mixed with 40 Al of 3a-20P-hydroxysteroiddehydrogenase (Sigma Chemical Co., H 7252) containing 19.2 units/ml (18 Al enzyme + 62 µl Trisbuffer). A further 500 I.41 of 0.05 M Tris containing 2 mg/ml reduced nicotinamide adeninedinucleotide (NADH disodium salt, #481913, Calbiochem, La Jolla, CA) was added to the tube. Themixture was incubated at room temperature (18-22 ° C) for 2 hr, then, 3 ml of diethyl ether (BDH)added. The tube was vortexed for 1 min, and left to separate for 5 min, after which the upper (organic)phase was transferred to a glass scintillation vial (10 ml). Tubes containing the aqueous layer wererinsed with a further 3 ml of diethyl ether, vortexed, allowed to separate, and transferred to a vial. Theether was then evaporated under a stream of nitrogen to dryness, and the vial containing the dry extractwas stored at room temperature overnight. The next morning, 100 Al of diethyl ether was added to thevial, and 15-20 III of the solution was applied in a single spot to one channel of a precoated, prewashed16silicagel plate (LK5DF Thin layer chromatography plate, 250 Am thickness, Whatman), allowed to dry,then repeated to attain a total of 300 Al solution. Fifty Al of 17a0H-[3H] progesterone was applied toan adjacent channel. The plate was developed with a dichloromethane (50 ml): diethyl ether (20 ml)mixture until the solvent front was 2-4 cm from the top of the gel. Then, the plate was allowed to dry ina fumehood (20 min). When dry, the plate was divided into 1 cm fractions, and each section of gel wascarefully scraped into individual 16x125 mm glass tubes. Five hundred Al of distilled water and 5 ml ofdiethyl ether were added, the tubes vortexed for 1 min and left to separate for 5 min The organicfraction was transferred to clean glass tubes. Fifty Al aliquots from each tube were added toscintillation vials and counted. The 17,20/3-P fractions were identified by comparing the activity withthe 17a0H-[31-1] progesterone channel. Once 17,20/3-P was identified, the fraction was re-extracted tomaximize the yield. The ether was then dried, and fmally the dry extract redissolved in absoluteethanol and stored at 4 ° C.2. Extraction of steroids from plasma samplesSteroids were extracted from plasma samples by using diethyl-ether (BDH, AnalaR graded)and n-heptane (BDH assure graded). One or two hundred Al aliquots of plasma were pipetted into25x125 mm borosilicate culture tubes. Approximately 1000 cpm of labelled steroid in 100 Al diluentwere added to the samples, which were then vortexed and left at room temperature for 30 minutes toallow the labelled steroid to equilibrate with endogenous steroids. A similar quantity of labelledsteroid was added to scintillation vials and counted directly as 100% recovery. Four ml diethyl etherand 1 nil n-heptane were added to the sample tubes, vortexed for 15 seconds, left to separate for 1minute, then revortexed for another 15 seconds and finally left to separate for 5 minutes. The tubeswere then frozen in very cool acetone (acetone in dry ice) for 10 seconds. Once the aqueous layer waswell frozen, the organic layer was poured into clean 25x125 mm borosilicate tubes. These tubes were17placed in a warm water bath (37° C), and a light stream of compressed air blown over them to speedevaporation of the solvent. Dried extracts were re-constituted in assay buffer (1:5 or 1:10, v/v),vortexed and incubated in water bath (50 ° C) for 50 minutes. After incubation, the extracts werevortexed, clamped, and left at 4 ° C overnight to redissolve. A 4-500 Al aliquot was then withdrawn andadded to scintillation vials for recovery determinations.3. Radioimmunoassay procedureFor all steroid assays, 10x75 borosilicate glass or plastic tubes were set up to contain 200 Ihl ofextracted plasma or standard (3.9 to 1000 pg/ml for testosterone, estradio1-17/3 and 11-ketotestosterone, 1.9 to 500 pg/ml for 17,20P-P), 200 Al 3H-steroid (3500-4000 cpm for testosterone,1800-2000 cpm for estradio1-17P, 11-ketotestosterone, and 17,20P-P) and 200 Al of antibody (1:80 fortestosterone and estradio1-17P, 1:10000 for 11-ketotestosterone and 17,20P-P). Non-specific andmaximum binding controls were prepared by substituting both standard and antibody with assay buffer.These tubes were vortexed and left to incubate overnight at room temperature. The followingmorning the samples were put in the freezer for 20 minutes, after which 200 Al of dextran-coatedcharcoal [0.5 g/1 dextran T-70 (Pharmacia [Canada] Ltd., Dorval, Quebec) and 5.0 g/1 activatedcharcoal (C-5260, Sigma) in assay buffer] was added to each tube to bind any remaining unboundsteroid. The tubes were vortexed, incubated at 4° C for 12 minutes, centrifuged at 2400 rpm at 4 ° C for12 minutes, and the supernatant fluid decanted into 7 ml plastic scintillation vials (Fisher ScientificLtd.). Four ml of Scinti Verse II (80-X-12, Fisher Scientific Ltd.) was added to each vial, which werethen shaken vigorously, placed in the dark at room temperature for at least 2 hours, following which,they were counted on a LKB Wallac 1214 RackBeta liquid scintillation counter (Wallac Oy, Turku,Finland) for 5 minutes each.184. Steroid assay validationFollowing ether extraction of steroids from plasma, all samples diluted in parallel with thestandard curve. Recovery determinations after ether extraction gave consistent results. All steroidswere extracted with more than 90% efficiency, except for estradio1-17Q (T:93%, 11-KT:93.9%, 17,20P-P: 93%, and E2:83.5%). Following this initial determination, extraction efficiency was assumed to beconstant and was measured only occasionally to make sure that it remained so. All the extractionefficiencies were taken into account in calculating a sample's final concentration.Sensitivity (80% binding) of the assay was approximately 8 pg/ml for T, 25 pg/ml for E2, 30pg/ml for 11-KT, and 7 pg/ml for 17,20P-P. Intraassay (precision) and interassay (accuracy)coefficient of variance were assessed using pooled plasma samples (n = 20). They were 9.1% and11.9% for T, 6.1% and 7.2% for E2, and 11.4% and 6.5% for 11-KT.F. STATISTICSAll data were expressed as mean ± standard error. Analysis of variance and Tukey HSD testwere used to determine differences between means. Log 1° transformation was used to achievehomogeneity of variance. Comparisons between two means or two treatments were analyzed by t-test.Fisher's exact probability test was used to compare the percentage of spawned fish between groups.19CHAPTER 3ANNUAL REPRODUCTIVE CYCLE OF THE THAI CARPA. INTRODUCTIONA full understanding of the processes occurring during the natural reproductive cycle of a fishspecies, and their temporal relationship, is one of the most important prerequisites to theestablishment of biotechnologies designed to overcome reproduction-related problems in aquaculture.At present, there has been no comprehensive description of the annual endocrine changesaccompanying gonadal development and spawning in any tropical fish. This study was conducted toinvestigate the relationship between environmental and hormonal factors and gonadal development inthe female and male Thai carp. The pattern of gonadal development was assessed by changes inreproductive parameters i.e., gonadosomatic index (GSI), hepatosomatic index (HSI), and conditionfactor (CF) and by histological analyses of gonads. Changes in reproductive hormones and the changesin environmental parameters throughout the year were related to ovarian development and the timingof spawning.B. MATERIALS AND METHODSIn this study, 5 adult Thai carp of both sexes were randomly sampled each month over aperiod of 20 months (Dec 1987-July 1989). Data on body weight and total length of each fish wererecorded. Blood samples of 2 ml were taken and centrifuged to obtain plasma. The plasma sampleswere analyzed for gonadotropin (GtH), testosterone (T), estradio1-17/3 (E2), 11-ketotestosterone (11-KT, male only) content by means of specific radioimmunoassays (RIA). After blood sampling, fishwere killed and their gonads and liver removed for determination of GSI (gonad weight x 100/ body20weight), HSI (liver weight x 100/ body weight), CF (body weight x 100/ total length3) and forhistological analysis.For histological analyses, measurements were made with a calibrated eyepiece micrometer.Only those cells which had been sectioned through the nucleus were measured. If the cell wasspherical, the diameter was measured; if oval or irregular, the longest and shortest axes were measuredand the mean taken. For each slide, between 500-600 cells were staged and the results expressed as amonthly mean.Data on rainfall, maximum and minimum air temperature, and daylength were obtained fromthe Kalasin Meteorological office about 3 km from the experimental site (Lat. 16 ° 25' Long. 103 ° 31'E). These data were used to assess any correlation between environmental i.e., air temperature,rainfall, and daylength change and gonadal development.C. RESULTSSpawning occurred in the rearing pond during the rainy season (May-June) following a fewdays of continual rain, and only when male fish were accidentally mixed into the female pond.21o^toM00^0^0^0^0^O^O0 (0 0 10 0 InN^(WW) 11VdNIV7:1LLO0 1—Zv) 0LLcocoOI. Seasonal changes in environmental parametersa.RainfallIn both years, the rainy season began in March (Fig. 2). However, in 1988, rainfall increasedrapidly from March and reached its peak in May (250.2 mm), then slowly decreased to about 50 mm inSeptember and peaked again in October. Similarly, in 1989, the rainy season also started in March.However, instead of gradually increasing, it fell in April, then increased in May and reached its peak inJune. There was no rainfall recorded between November and February of both years.b. TemperatureIn both years, maximum air temperature increased gradually from December and reached itspeak in April (34.8 ° C) (Fig. 3). However, it decreased in May and was maintained at the same leveluntil July, and then slowly decreased reaching its lowest level in November and December in 1988(25.1 ° C).Minimum air temperature showed a similar trend as that observed for maximum airtemperature (Fig. 3). However, instead of peaking in April, it continued to increase and reached itspeak in June (25.6 ° C), then gradually decreased and reached its lowest level in December 1988(15.7 ° C).23 4030200cm 3 0ccm4cccwa.2w 20I-10^  10DJ88FMAMJ J A SONDJ89F MAMJ JMONTHMIN. TEMP --8-- MAX. TEMPFig 3 Annual changes in maximum and minimum air temperatures at Kalasin during 1987-89expressed as mean minimum and mean maximum temperatures in each calendar month.2410^  10DJ88FMAMJJASONDJ89FMAMJJMONTHFig 4 Annual changes in daylength at Kalasin during 1987-89 expressed as mean daylength in hour ineach calendar month.25c. DaylengthDaylength increased gradually from December (Fig. 4) and reached its longest hour in June(13.1 hr). Then, daylength rapidly decreased to its shortest hour in December (11.1 hr).In general, all environmental parameters exhibited seasonal variations. A high correlation wasfound between maximum and minimum air temperature (r = 0.897, P < 0.01) and between minimumair temperature and daylength (r = 0.822, P < 0.01).II. Seasonal changes in reproductive parameters.A. Femalea. Condition factor (Fig. 5)The CF in female Thai carp showed a significant variation during the study period, varyingbetween 1.3 and 1.7%. In general, CF tended to decrease from April 1988 and began to rise again inJune. A similar trend was observed in 1989 when CF values decreased significantly in March andreached lowest levels in April and May (t-Test, P < 0.05).261.81.2DJ88FMAMJJ ASONDJ89FMAMJ JMONTH1.6Ciu.01.4Fig. 5. Annual changes in condition factor (CF) in female Thai carp in a rearing pond at KalasinFreshwater Fisheries Station during 1987-89. Each value represents mean ± SEM. * indicatessignificant difference from previous month as determined by Tukey HSD Test (P < 0.05).270.52.50DJ88FMAMJ J A SONDJ89F MAMJ JMONTH321Fig. 6. Annual changes in hepatosomatic index (HSI) in female Thai carp in a rearing pond at KalasinFreshwater Fisheries Station during 1987-89. Each value represents mean ± SEM.28b. Hepatosomatic index (Fig. 6)HSI gradually decreased from 2.4% in December 1987 and reached its lowest level (1.2%) inMay 1988. HSI remained unchanged until July, then rapidly increased and reached its highest peak(2.4%) in September. There were no significant changes in HSI until January 1989, at which point,HSI gradually decreased, reaching its lowest level by June 1989.c. Gonadosomatic index (Fig. 7)GSI rapidly increased from December 1987 and peaked in May 1988 (18.1%). There were nosignificant changes in GSI between May and July. However, GSI gradually decreased to its lowest levelin August (6.8%) and remained unchanged until December. Similarly, in 1989, GSI values rapidlyincreased in January and peaked in May (19.6%), which indicates an annual cycle for GSI in the Thaicarp.GSI had a high positive correlation with maximum temperature (r = 0.711, P < 0.05). It alsohad a highly negative correlation with HSI (r = -0.75, P < 0.05).B. Malea. Condition factor (Fig. 8)There were no significant changes for CF in the male Thai carp. Mean CF values rangedbetween 1.1-1.6 % throughout the study period. Also, there were no correlations between CF and anyenvironmental parameter.29......^1 5inaae2 02 5100DJ88FMAMJ J A SONDJ89F MAMJ JMONTH5Fig. 7. Annual changes in gonadosomatic index (GSI) in female Thai carp in a rearing pond at KalasinFreshwater Fisheries Station during 1987-89. Each value represents mean ± SEM. • indicatessignificant difference from previous month as determined by Tukey HSD test (P < 0.05).30U.021.510.50DJ88FMAMJJASONDJ89FMAMJJMONTHFig. 8. Annual changes in condition factor (CF) in male Thai carp in a rearing pond at KalasinFreshwater Fisheries Station during 1987-89. Each value represents mean ± SEM.31b. Hepatosomatic index (Fig. 9)HSI decreased from December 1987 to April 1988. Then, it remained almost unchanged untilFebruary 1989, at which point it increased significantly, peaking in March and significantly decreasingto a lower value in April.c. Gonadosomatic index (Fig. 10)GSI increased from February and peaked in the May to July period in 1988, then slowlydecreased to a lower value in December. In 1989, GSI started to increase during January and reachingits peak in the March and May period. Thereafter, GSI decreased significantly to a lower level in June.Unlike CF and HSI, GSI exhibited a highly negative correlation with HSI (r = -0.7).III. Seasonal histological changes in gonadal and hepatic tissuesA. Anatomy of the gonadsThe ovaries of the Thai carp are paired elongated structures lying below air bladder, kidneyand body wall by means of mesovarium. They have a cone-shaped structure: the base projects into theanterior part of the body cavity, while the apex projects towards the tail.The testes are also paired and join anteriorly to form a Y-shaped structure. They aresuspended by mesentaries in the upper section of body cavity. The testes are composed of ill-definedlobules which are separated by thin connective tissue.323 2 . 52110.50DJ88FMAMJJASONDJ89FMAMJJMONTHFig. 9. Annual changes in hepatosomatic index (HSI) in male Thai carp in a rearing pond at KalasinFreshwater Fisheries Station during 1987-89. Each value represents mean ± SEM. * indicatessignificant difference from previous month as determined by Tukey HSD test (P < 0.05).33654Ci3CoCD210DJ88FMAMJ J A SONDJ89F M A M J JMONTHFig. 10. Annual changes in gonadosomatic index (GSI) in male Thai carp in a rearing pond at KalasinFreshwater Fisheries Station during 1987-89. Each value represents mean ± SEM. * indicatessignificant difference from previous month as determined by Tukey HSD test (P < 0.05).34B. Histology of the ovaryOocyte development in the Thai carp can be divided into 5 stages according to the generalclassification of the teleost gonad by West (1990) as follows:a. Stage I: Chromatin nucleolus stage (Fig. 11a)The primary oocytes are characterized by the presence of slightly basophilic cytoplasm. Thenucleus of an oocyte is a large round body with distinct chromosomes in various stages of meioticprophase and exhibits one large centrally located nucleolus. Mean oocyte diameter was 0.058 ± 0.001mm (range from 0.024 to 0.1 mm)Oocytes in the chromatin nucleolus stage were found at every month of the year, and thepercentages of oocytes at this stage are shown in Fig. 12a. The maximum percentage was found inFebruary 1988, after which it decreased to about 15% in March and stayed at this level until the nextspawning season.b. Stage II: Peri-nucleolus stage (Fig. 11b)In oocytes at this stage, several nucleoli were apparent as a ring around the periphery of thenucleus and the cytoplasm exhibited a marked affmity for haematoxylin (basophilic). During this stage,the nucleus enlarged; the chromosomes become less distinct characteristics, and the cytoplasmincreased greatly in volume. At the end of this stage, the cytoplasm lost its affinity for haematoxylin.Mean oocyte diameter at this stage was 0.082 ± 0.001 mm (range from 0.032 to 0.128 mm)Oocytes in the peri-nucleolus stage were also present throughout the year (Fig. 12b). Thepercentage of oocytes at this stage slowly increased in February 1988 (7.1%), peaked in August(45.5%) and maintained this level of frequency until the end of the year. The frequency of peri-nucleolus stage oocytes decreased again in February 1989 which indicated a new cycle of oogenesis.35r^1pFig. 11.1. Cross-sections of ovaries. (a) Stage I oocyte 160X. (b) Stage II oocyte 160X. (c) Stage IIIoocyte 40X.36Fig. 11.2. Cross-sections of ovaries. (d) Stage IV oocyte, early stage 40X. (dl) Stage IV oocyte, latestage 40X.37Fig. 113. Cross-sections of ovaries. (e) Stage V oocyte, germinal vesicle migration stage 40X. (f)Atretic oocyte 40X (g) Post-ovulatory oocyte 40X.388070605040302010a. Stage I oocyte0^DJ88FMAMJ J ASONDJ89FMAMJ JMONTHFig. 12.1. Annual changes in the occurrence of oocytes in different stages in female Thai carp in arearing pond at Kalasin Freshwater Fisheries Station during 1987-89. (a) Stage I oocyte. Eachvalue represents mean ± SEM.398070605040302010807060a- 302010b. Stage II oocyteIrlDJ88FMAMJJASONDJ89FMAMJJMONTHc. Stage III oocyteI 111 T J^0DJ88FMAMJJASONDJ89FMAMJJMONTHFig. 12 2. Annual changes in the occurrence of oocytes in different stages in female Thai carp in arearing pond at Kalasin Freshwater Fisheries Station during 1987-89. (b) Stage II oocyte. (c)Stage HI oocyte. Each value represents mean ± SEM.40CSz 400cca. 30e. Stage V oocyteIIDJ88FMAMJJASONDJ89FMAMJJMONTHDJ88FMAMJJASONDJ89FMAMJJMONTHFig. 123. Annual changes in the occurrence of oocytes in different stages in female Thai carp in arearing pond at Kalasin Freshwater Fisheries Station during 1987-89. (d) Stage N oocyte. (e)Stage V oocyte. Each value represents mean ± SEM.8070605040302010418070605040302010DJ88FMAMJJASONDJ89FMAMJJMONTHDJ88FMAMJJASONDJ89FMAMJJMONTHFig. 12 4. Annual changes in the occurrence of oocytes in different stages in female Thai carp in arearing pond at Kalasin Freshwater Fisheries Station during 1987-89. (f) Atretic oocyte. (g) Post-ovulatory oocyte. Each value represents mean ± SEM.42There was a highly significant negative correlation between stage II oocytes and GSI (r = -0.763, P < 0.05).c.Stage III: Cortical alveoli stage (Fig. 11c)Cortical alveoli or yolk vesicle stage oocytes were characterized by the occurrence of a row ofvacuoles on the periphery of the cytoplasm. There was no pattern in the deposition of yolk vesicleswhich formed randomly. These yolk vesicles also increased in number until they occupied the entirecytoplasm of the oocytes. Nucleoli were still evident in the periphery of the nucleus, but were less welldefined. The cytoplasm was less basophilic at the end of this stage, and the nucleus was stained witheosin. Mean oocyte diameter was 0.143 ± 0.003 mm (range from 0.068 to 0.280 mm)The oocytes at the yolk vesicle stage were found every month during the sampling periods (Fig.12c). The percentage of oocytes at this stage tended to decrease from a peak in February 1988 orMarch 1989 (8.6 or 7.9%) to the lowest levels in June 1988 or July 1989 (4.2 or 5.1%).d. Stage IV: Yolk granule stage (Fig. 11d)This stage was characterized by the presence of yolk droplets associated with a ring of vacuolespresent in stage III oocytes. At the end of this stage, yolk globules were present throughout thecytoplasm, displacing the yolk vesicles to the periphery. The nucleus was still centrally located in theoocyte and contained many nucleoli attached to the nucleus membrane. Mean stage IV oocytediameter was 0354 ± 0.006 mm (range from 0.08 to 054 mm)The percentage of oocytes in stage IV increased from October (2.8%, at the end of spawningseason) and peaked from March to May, (8.7%) then slowly decreased in June (Fig. 12d).e. Stage V: Mature oocyte (Fig. 11e)Most of the oocytes at this stage were filled with yolk, which had coalesced into large globules.The nucleoli had commenced their migration toward the center of the nucleus, away from the nuclearmembrane. Later, the nucleus began to move toward the periphery of the cytoplasm, and all the43nucleoli were concentrated in the center of the nucleus. Finally, the nucleus lost its shape withdisintegration of the nuclear membrane. Mean oocyte diameter for stage V was 0.458 ± 0.003 mm(range from 0320 to 0560 mm)In both years, the frequency of the post-vitellogenic oocytes increased in January (17.5%) andpeaked in May (44.7%) (Fig. 12e). Thereafter, the percentage of stage V oocytes slowly decreased to23.8% in August and maintained this level until January, when it started to increase again.The percentage of stage V oocytes was significantly correlated with GSI (r = 0.9, P < 0.001).f. Post-ovulatory oocytes (Fig. 110Post-ovulatory oocytes were present every month (Fig. 12f), although with a fluctuatingpattern. In 1988, they increased gradually from January (0.1%) and peaked around May (6.1%), afterwhich, they gradually decreased to a lower level in August (2.2%). Similarly, in the 1989 spawningseason, they slowly increased in December 1988 (0.6%) and peaked in June and July (5.4%).There was a highly positive correlation between stage VI oocytes and daylength (r = 0.74).g.Atresia (Fig. 11g)Atretic oocytes are derived from vitellogenic oocytes that failed to undergo maturation andovulation, but underwent degeneration prior to reabsorption. In the Thai carp, atretic oocytes wereobserved every month (Fig. 12g), with the highest percentages being observed during June or July.C. Histology of the testisThe testis of the Thai carp is surrounded by a thin and delicate membrane, the peritoneum.Microscopically, the testis is composed of a complex mass of numerous seminiferous lobules, which areclosely packed together, but separated by the stroma tissue. Each lobule contains several cysts of germcells which may be in various stages of division. Following the descriptions of Lehri (1967), Htun-Han44(1978) and Billard (1986) the spermatogenic cells of the testis of the Thai carp can be divided into 5stages as follows:a. Stage I: Spermatogonia (Fig. 13a)Spermatogonia are generally the largest germ cells, being present either singly or in nests onthe gonadal lamella. Each spermatogonium (0.010-0.020 mm in diameter) was surrounded by a flatsomatic cell which may subsequently develop to form a cyst wall. The cytoplasm stained faintly witheosin, and the nuclei occupied a greater area of the cell.Spermatogonia were found every month of the year (Fig. 14a). In both years, their appearancerapidly decreased from January and reached the lowest level in May, after which they graduallyincreased and peaked in December.There were highly negative correlations between the occurrence of spermatogonia andminimum and maximum temperature (r = -0.74 and -0.76 respectively) and between the occurrence ofspermatogonia and daylength (r = -0.71).45Fig. 13. Cross-sections of testes. (a) Spermatogonia. (b) Primary spermatocytes. (c) Secondaryspermatocytes. (d) Spermatids. (e) Spermatozoa. a,b,c,d, and e 160X.46100908070605040302010a. Spermatogonia-7-0DJ88FMAMJ J ASONDJ89FMAMJ JMONTHFig. 14.1. Annual changes in the testicular germ cells at different stages in male Thai carp in a rearingpond at Kalasin Freshwater Fisheries Station during 1987-89. (a) Spermatogonia. Each valuerepresents mean ± SEM.471009080706050403020100D 88FMAMJJASONDJ89FMAMJJMONTH100 ^c. Secondary spermatocytes80706050403020100DJ88FMAMJJASONDJ89FMAMJJMONTH90Fig. 14.2. Annual changes in the testicular germ cells at different stages in male Thai carp in a rearingpond at Kalasin Freshwater Fisheries Station during 1987-89. (b) Primary spermatocytes. (c)Secondary spermatocytes. Each value represents mean ± SEM.48100d. Spermatids100DJ88 F MAMJJASONDJ89FMAMJJMONTH100e. Spermatozoa80706050403020100 '^DJ88FMAMJJASONDJ89FMAMJJMONTHFig. 143 Annual changes in the testicular germ cells at different stages in male Thai carp in a rearingpond at Kalasin Freshwater Fisheries Station during 1987-89. (d) Spermatids. (e) Spermatozoa.Each value represents mean ± SEM.90807040605030209049b. Stage II: Primary spermatocytes (Fig. 13b)Primary spermatocytes were distinguished from the spermatogonia by their size (0.008-0.010 indiameter) and appearance. They usually stain more deeply than spermatogonia, and exhibit nucleidensely packed with chromatin or a grouping of the chromatin material at one pole of the nucleus.Primary spermatocytes were found throughout the year (Fig. 14b). In 1988, primaryspermatocytes showed a tendency to increase from February and peaked around October. However,this tendency was not very clear in 1989.c. Stage III: Secondary spermatocytes (Fig. 13c)Secondary spermatocytes were smaller in size (0.006-0.008 mm in diameter) and morenumerous than the primary spermatocytes. They were also more uniform in color than primaryspermatocytes, and their nuclei contained a thick clump of chromatin.Secondary spermatocytes were found throughout the year (Fig. 14c). In 1988, their frequencydecreased from January to the lowest level in July. In 1989, however, the percentage of secondaryspermatocytes decreased in February and tended to increase again in April, then, peaking in July.d. Stage IV: Spermatids (Fig. 13d)Further division of secondary spermatocytes gave rise to spermatids. These cells were small(0.003-0.006 mm in diameter) with a deeply staining clumped mass of chromatin.The percentage of spermatids demonstrated a variable pattern throughout the year (Fig. 14d).In the 1988 spawning season, it rapidly decreased from January to achieve a lower level in February.Thereafter, spermatids gradually increased in March and April. From May to July, whichcorresponded to the peak of the spawning season, the pattern tended to be intermittent. However, thenumber of spermatids started to decrease again from August to a lower level in November. In 1989,the frequency of spermatids tended to increase from December, peaked in June, and then slowlydecreased in July.50e. Stage V: Spermatozoa (Fig. 13e)Spermatozoa were transformed from spermatids without further division, but changed shapeand became tailed. The tails tended to agglutinate within the nests, giving the nests a parachute-shapedappearance.Spermatozoa were present in abundance throughout the year (Fig. 14e). In general, theytended to be most numerous during spawning season (February to July) and lowest during August toJanuary. However, the percentage in 1989 appeared lower than observed during 1988.D. Histology of the liverMicroscopically, the liver of the Thai carp was composed of numerous hepatocytes. Thesecells, with a roundish centrally located nucleus, stained weakly with haematoxylin and eosin. Thediameter of the hepatocytes ranged from 0.054 to 0.093 mm (mean = 0.071 ± 0.001), and variedseasonally (Fig. 15). In general, the diameter of hepatocytes tended to decrease significantly fromJanuary (0.079 mm) to reach their smallest size in April to May, and then slowly increased, and peakedaround July to September.A high positive correlation between hepatocyte diameter and HSI (r = 0.7) was observed.51E'E 0.08ccwI—wM '0 07a0.060.090 . 10.05 1 " " 'DJ88FMAMJJ A SONDJ89F MAMJ JMONTHFig. 15. Annual changes in hepatocyte diameter in female Thai carp in a rearing pond at KalasinFreshwater Fisheries Station during 1987-89. Each value represents the mean ± SEM.52IV. Seasonal changes in plasma gonadotropin and steroid hormonesA. Hormonal changes in female Thai carpa. Gonadotropin (GtH)Plasma levels of gonadotropin decreased from December 1987 (2.21 ng/ml) to attain lowestlevels in July 1988 (1.13 ng/ml) (Fig. 16). GtH then gradually increased and peaked in September (3.29ng/ml). The levels tended to remain high until they dropped again in June 1989 (0.61 ng/ml).b. Testosterone (T)Plasma testosterone levels were low (< 1 ng/ml) throughout the year (Fig. 17). T increasedsignificantly from December 1987 to January 1988 (t-test, P < 0.05), then decreased slightly inFebruary. A slightly increased T level was observed in July which corresponded to the spawningseason. A similar trend was also found in 1989, when the level of T rapidly increased from November(0.104 ng/ml) to December (0.59 ng/ml)(P < 0.01) and fell in January 1989 (0.17 ng/ml). However,there were almost no changes in T levels during spawning period.c. Estradiol-17fl (E2)Plasma E2 levels were high, when compared to T, throughout the year (2-10 ng/ml) (Fig. 18).In 1987-88, E2 significantly increased from December (2.2 ng/ml) to January (9.3 ng/ml) (P < 0.01),then decreased rapidly to about 3 ng/ml in February. A slight increase in the level of E/2 was observedagain in June (5.12 ng/ml). A similar trend was also found in 1988-89, when E2 exhibited a rapidincrease in December (6.38 ng/ml), and a slow decline in January (4.58 ng/ml). Further, a rapidlyincrease in the level of E2 was also observed in March (8.83 ng/ml).53In general, plasma levels of GtH, T, and E2 in female carp showed a variable pattern ofseasonal change (Fig. 18). GtH tended to be high during the postspawning period and decreasedduring the spawning season. T and E2 displayed a similar pattern. Both peaked during thepreparatory period (January and December) and showed a small surge during spawning.544310DJ88FMAMJ J ASONDJ89FMAMJ JMONTHFig. 16. Annual changes in plasma gonadotropin in female Thai carp in a rearing pond at KalasinFreshwater Fisheries Station during 1987-89. Each value represents the mean ± SEM.55i0 . 8I— 0 . 40 . 2D..188F M A M J J A S ON D..189F M A M J JMONTHFig. 17. Annual changes in plasma testosterone in female Thai carp in a rearing pond at KalasinFreshwater Fisheries Station during 1987-89. Each value represents the mean ± SEM. *indicates significant difference from previous month as determined by Tukey HSD test (P < 0.05).561614121 0420DJ88FMAMJJASONDJ89FMAMJJMONTHFig. 18 Annual changes in plasma estradiol-17ft in female Thai carp in a rearing pond at KalasinFreshwater Fisheries Station during 1987-89. Each value represents the mean ± SEM. •indicates significant difference from previous month as determined by Tukey HSD test (P < 0.05).570 "^ 0DJ88FMAMJJASONDJ89FMAMJJMONTHGtH(ng/m1)^—4-- GSI(%)--- Testosterone(ng/mI)^-9-- Estradlol(ng/m1)Fig. 19 Annual changes in GSI and plasma hormone levels in female Thai carp in a rearing pond atKalasin Freshwater Fisheries Station during 1987-89.58B. Hormonal changes in male Thai carpa. Gonadotropin (GtH)Plasma GtH levels increased in January 1988 and immediately decreased in February (Fig.20). GtH levels started to increase again in April and peaked in June, after which it graduallydecreased in August. In September, GtH levels slowly increased again and showed another peak inOctober, then rapidly decreased in November and maintained this level throughout the rest of the year.In 1989, however, there were almost no changes in plasma GtH levels.b. Testosterone (T)Plasma levels of testosterone in the male Thai carp significantly increased from December1987 and peaked in January 1988 (Fig. 21). Then, T gradually decreased from February to a lower levelin April. The levels of T then increased and reached another peak in July after which T rapidlydecreased during August. T then rapidly increased in September, remained high until October, beforedecreasing in November to its lowest levels where T remained for the rest of the year. In 1989,similarly, peaks of T, though lower than those of 1988, were recorded in February and May.c. 11-Ketotestosterone (11-KT)11-KT showed similar patterns to those of T. 11-KT increased significantly from Decemberand peaked in January (Fig. 22). The levels of 11-KT then significantly decreased from February to alower level in April, and gradually increased and peaked again in July.598620DJ88F M A M J J A S O N DJ89F M A M J JMONTHFig. 20. Annual changes in plasma gonadotropin in male Thai carp in a rearing pond at KalasinFreshwater Fisheries Station during 1987-89. Each value represents the mean ± SEM.604310DJ88FMAMJ J ASONDJ89FMAMJ JMONTHFig. 21. Annual changes in plasma testosterone in male Thai carp in a rearing pond at KalasinFreshwater Fisheries Station during 1987-89. Each value represents the mean ± SEM. •indicates significant difference from previous month as determined by Tukey HSD test (P < 0.05).61210DJ88FMAMJ J ASONDJ89FMAMJ JMONTHFig. 22. Annual changes in plasma 11-ketotestosterone in male Thai carp in a rearing pond at KalasinFreshwater Fisheries Station during 1987-89. Each value represents the mean ± SEM. *indicates significant difference from previous month as determined by Tukey HSD test (P < 0.05).62E0^ 0DJ88FMAMJJASONDJ89FMAMJJMONTHGSI^Testoterone^11-KT^GtHFig. 23. Annual changes in GSI and plasma hormone levels in male Thai carp in a rearing pond atKalasin Freshwater Fisheries Station during 1987-89.63Concentrations of 11-KT rapidly declined in August before increasing in September. Thelevels of 11-KT then slowly decreased to its lowest level in November which was maintained throughDecember Similarly, in 1989, 11-KT gradually increased from January and peaked in May. 11-KTlevel then rapidly declined in June and increased again in July.11-KT levels exhibited a highly significant positive correlation with T (r = 0.9, P < 0.001). Ingeneral, plasma GtH, T, and 11-KT in the male Thai carp exhibited variable patterns of changes asfound in female (Fig. 23). However, the concentrations of these hormones were generally higher thanthose of the female.D. DISCUSSIONa. FemalesThe present results demonstrate the reproductive cycle of the female Thai carp reared inponds at Kalasin Freshwater Fisheries Station, Kalasin, Thailand. Gonadal recrudescence, in terms ofa gradual increase in GSI, was highly correlated with the sequential changes of environmental factorssuch as air temperatures and daylength. Furthermore, the decline in GSI which indicated the period ofspawning in this species was observed to coincide with the occurrence of rainfall. Similarly, in the wild,spawning of Thai carp was observed to occur during the rainy season in Thailand, i.e., from May to July(Sipitakkiat and Leenanond, 1984). Similar patterns of reproduction have been reported in severaltropical fish such as the Indian catfish, Heteropneustes fossilis, (Lamba et al. 1983) and the walkingcatfish, Clarias batrachhus, (Singh and Singh, 1991).Histological analysis of the ovary in the Thai carp demonstrated the presence of oocytes ofvarious stages, without pronounced clutches, throughout the year. This pattern of oocyte developmentcorresponds to the asynchronous type of oocyte development which is usually observed in species with64protracted spawning seasons and multiple spawns per female (de Vlaming, 1983). This is in agreementwith the fmding of Sirikul et al. (1986) that the female Thai carp can be induced to spawn at least 3times in a spawning period.A rapid increased in the percentage of oocytes in the perinucleolar stage which indicated theperiod of oogenesis (de Vlaming, 1983; Selman and Wallace, 1989) was observed during the months ofAugust and January. At this time, the fish demonstrated the lowest GSI and the highest HSI values.There were no correlations between the occurrence of oocytes in the perinucleolar stage and plasmahormone levels. This indicates that the regulation of oogenesis is independent of endocrine control assuggested earlier by de Vlaming (1974).Oocytes in the cortical alveoli and yolk granule stages were observed throughout the year,suggesting continuous vitellogenesis in the Thai carp. However, the occurrence of oocytes in thesestages was low (less than 10%) compared to the oocytes in mature stage (postvitellogenic stage).Vitellogenesis in teleosts is hormone dependent. The synthesis of yolk protein precursor (vitellogenin)by the liver is under the stimulation of estradio1-17/3 (E2) (Aida et al. 1973; Sundararaj and Nath, 1981;Sundararaj et al. 1982) and GtH I (Tyler et al. 1991). Furthermore, the uptake of vitellogenin intooocytes is regulated by GtH I (Tyler et al. 1991). In the present study, a gradual increase in theoccurrence of the postvitellogenic oocytes and GSI coincides with a surge in plasma E2 in both 1988and 1989. Also, this surge in plasma E2 coincides with the decline in hepatocyte diameter and HSI.Thus, these results were in agreement with earlier studies that the yolk precursor or vitellogenin wassynthesized in the liver under the stimulation of E2 (Aida et al. 1973; Sundararaj and Nath, 1981;Sundararaj et al. 1982).Mature oocytes were presented in the Thai carp throughout the year and constituted 18-48%of the ovarian mass. The pattern of their appearance was highly correlated with GSI (r = 0.9). This65supports the value of GSI as a reliable indicator of ovarian activity in cyprinids (Clemens and Reed,1967; Munkittrick and Leatherland, 1984).The occurrence of mature oocytes seemed to be parallel changes in air temperatures anddaylength (r = 0.6-0.7). The maximum percentage of mature oocytes was observed to coincide with theperiod of maximum air temperatures. It has been suggested that temperature may act directly on theovarian steroidogenic enzymes to regulate the reproductive cycle in common carp (Minning and Kime,1984). Although mature oocytes in the Thai carp were observed throughout the year, spawning, interm of decreasing GSI, only occurred during the rainy season. It has been reported in numeroustropical cyprinid species that gonadal development can be completed over a period of 2 months beforethe usual spawning period (Qasim and Qayyum, 1961; Parameswaran et al. 1970; Tsai et al. 1981; Smithand Jiffry, 1986). This allows the fish to be opportunistic in their responses to suitable spawningconditions (Munro, 1990).Rainfall was found to be the most important proximate factor to initiate spawning in manytropical species of freshwater cyprinids (Chaudhuri, 1968; Sinha et al. 1974; Smith and Jiffry, 1986).However, there is no correlation between the onset of spawning and various aspects of rainfall i e ,changes in depth, current, temperature, turbidity, dissolved oxygen, etc. The adaptive significance ofthe coincidence of spawning and rainfall seem to be the increase in living space and food supply whichbecome available at this time (Schwassmann, 1978).Spawning of the Thai carp in rearing ponds usually occurred after the onset of heavy rain andonly when male(s) were accidentally mixed into the female pond (personal observation). Also,spawning in fully mature fish can be induced in specially prepared ponds with flowing water during therainy season in Thailand (Sipitakkiat and Leenanond, 1984) and throughout the year in Malaysia (Tanand Begum, 1985). The longer period of spawning for this species in Malaysia than in northeastThailand is mainly due to the prolonged rainy season in Malaysia.66Pronounced seasonal changes in reproductive hormones have been observed in the goldfish(Kobayashi et al. 1986a). In the current study, however, it was not possible to distinguish pronouncedseasonal changes in plasma reproductive hormones in the female Thai carp. The reason for this mightbe the fact that the fish used in the study are in hatchery conditions. Though they were exposed to thenatural daylength, temperature, and rainfall, the changes in reproductive hormones might not reflectthe true seasonal cycle which usually occurs in wild fish.In the present study, a gradual increase in plasma GtH levels was observed at the beginning ofvitellogenesis which was confirmed by the elevation of GSI. Lower levels of GtH were observed duringthe spawning period. A similar pattern of seasonal changes in plasma GtH was observed in thecommon carp in Israel (Yaron and Levavi-Zermonsky, 1986). In the goldfish, however, plasma GtHshowed a high amplitude of change, and the maximum concentration was observed during and shortlyafter the spawning period (Kobayashi et al. 1986a). The difference between the profile of plasma GtHin the Thai carp and in the goldfish is probably due to the pattern of their gonadal development. In theThai carp which spawn a few times during an extended spawning period (Sirikul et al. 1986), GSI wasmaintained at the highest value for about 4 months. Whereas in the goldfish, a surge in GSI was foundto last for only a few weeks (Kobayashi et al. 1986a).In the present study, there was no correlation between plasma steroid levels and GSI. In thegoldfish, however, changes in plasma steroid levels were highly correlated with the changes in GSI(Kobayashi et al. 1986a). A rapid surge in plasma E2 levels in the Thai carp was observed, in January1988 and in December 1988, to coincide with the initial increase in GSI. A gradual increase in plasmaE2 during ovarian recrudescence has been reported in several teleosts such as rainbow trout (Lambertet al. 1978; Scott et al. 1980a), Indian catfish, Heteropneustes fossilis, (Lamba et al. 1983) and goldfish(Kobayashi et al. 1986a), and has been shown to play a role in promoting hepatic synthesis of yolkprecursor, vitellogenin (Aida et al. 1973; Sundararaj and Nath, 1981). Though only a rapid surge in67plasma E2 was observed in Thai carp, this surge in E2 is probably sufficient to stimulate vitellogenesissince it has been shown in the Indian catfish that a surge in E2 alone was sufficient to maintain a highrate of vitellogenesis (Sundararaj and Nath, 1981).In the Thai carp, plasma T and E2 exhibited a pattern of seasonal changes which is similar tothat demonstrated in the goldfish (Kobayashi et al. 1986a) and the common carp (Yaron and Levavi-Zermonsky, 1986). However, plasma concentration of E2 in the Thai carp was higher than that of Tthroughout the year. The plasma concentration of T has been observed to be higher than that of E2 inmost female teleosts which exhibit synchronous oocyte development or only spawn once in a spawningseason such as rainbow trout (Scott et al. 1980a; Scott and Sumpter, 1983), white sucker (Scott et al.1984), brown bullhead (Burke et al. 1984), blue cod (Pankhurst and Conroy, 1987), channel catfish(MacKenzie et al. 1989) and walking catfish (Singh and Singh, 1991). In contrast, higher plasma E2than T concentrations have been observed in species with continuous vitellogenesis which spawn morethan once in a spawning season such as the common carp (Yaron and Levavi-Zermonsky, 1986; Galasand Bieniarz, 1989) and red sea bream (Matsuyama et al. 1988).At present, the role of T in female teleosts is not understood. However, it has been suggestedthat T serves as precursors for E2 synthesis ( Scott et al. 1980a; Sundararaj et a1.1982; Kagawa et al.1982; 1984). In this study, the parallel changes in plasma T and E2 confirmed with the role of T as aprecursor for E2 synthesis. The lower concentration of T than that of E2 suggests that T is rapidly andeffectively aromatized into E2 in the ovary of the Thai carp. The role of T in oocyte maturation orovulation is still not known, but T has been suggested to be involved in the feedback regulation of GtHsecretion in the goldfish (Trudeau et al. 1991).68b. MalesGonadal development in male Thai carp exhibited a similar pattern to that observed infemales. Seasonal changes in GSI in males was highly correlated with that of females ( r = 0.6, P <0.001), with the maximum GSI values of both sexes being observed in the same month. Because thespawning of the Thai carp coincides with the occurrence of rainfall which is difficult to predict, thissynchronous gonadal development in males and females is important for ensuring spawning success.Since male fish were raised separately from the females, this synchronous gonadal development isprobably not modulated by the presence of the opposite sex as has been shown in Tilapia, Sarotherodonmossambicus (Silverman, 1978a; b).Similar to the situation in females, the GSI in male Thai carp exhibited a highly negativecorrelation with HSI ( r = -0.7). This inverse correlation between GSI and HSI in females indicatedthe role of liver as the site for yolk protein synthesis as mentioned above. In males, however, thisinverse correlation may reflect the role of the liver as a primary energy reserve since the depletion ofenergy reserves, indicated by a declining HSI, during gonadal development has been suggested in themale Atlantic halibut, Hippoglossus hippoglossus (Haug and Gulliksen, 1988).The structure of the testis of the Thai carp corresponds to the lobular type which is found inmost teleosts studied to date (Billard, 1986). Histological analysis of the testis reveals a continuousspermatogenetic activity throughout the years as indicated by the presence of all stages of germ cells inthe testis. A similar pattern of testis development was also observed in the common carp (Gupta, 1975;Billard et al. 1978).In this study, sermatogenesis, indicated by a gradual increase in the occurrence ofspermatogonia, occurred immediately after spawning when GSI started to decrease. In goldfish, theoccurrence of spermatogonia was hormone independent (Billard et al. 1982). Similarly, in the presentstudy, there were no correlations between plasma GtH, T, or 11-KT levels and the occurrence of69spermatogonia. However, the occurrence of spermatogonia exhibited a highly negative correlation withair temperature ( r = -0.75) and the amount of rainfall ( r = -0.71). This suggests the important ofenvironmental cues in the initiation of spermatogenesis in the Thai carp. Environmental factors werefound to be involved in spermatogenesis in several cyprinids. For example, increasing rearingtemperature accelerated the process of spermatogenesis in tench, Tinca tinca (Breton et al. 1980) andin goldfish (Billard, 1986).Spermatocytes, spermatids and spermatozoa were also observed throughout the year withoutpronounced seasonal changes. However, the occurrence of spermatozoa was always found at thehighest proportion in the testis in every sample. This suggests a rapid transformation of germ cellsfrom spermatocytes to spermatids and further to spermatozoa, which indicates the readiness forspawning in males and coincides with the observation that milt can be stripped from the fishthroughout the year in these pond reared fishSpermatogonial division and subsequent meiosis are hormone dependent (Billard et al. 1982).Hypophysectomy and hormone replacement therapy studies in the goldfish have demonstrated that thetransformation of spermatogonia to spermatocytes was GtH-dependent (Billard et al. 1982). Also,qualitative maintenance or restoration of spermatogenesis has been reported in hypophysectomizedgoldfish after androgen treatment (Yamazaki and Donaldson, 1969; Billard, 1974). In this study,however, there were no statistically significant correlations between plasma GtH, T, and 11-KT levelsand the occurrence of spermatocytes, spermatids and spermatozoa. Similar results were observed inrainbow trout (Scott and Sumpter, 1989) and in common carp (Koldras et a1. 1990) where there wereno relationships between plasma T and 11-KT levels and any particular germ cell stage.Plasma GtH, T and 11-KT levels in the male Thai carp exhibited a similar trend of seasonalchanges. However, only plasma levels of T and 11-KT exhibited a highly positive correlation ( r =0.89). Moreover, plasma 11-KT was always higher that plasma T. This indicates that 11-KT is the70main plasma androgen in the Thai carp as has been demonstrated in common carp (Koldras et al.1990).Plasma hormones levels in the male Thai carp demonstrated bimodal seasonal changes. Thefirst peak of plasma GtH, T and 11-KT was observed at the same time in January 1988 when the GSIstarted to increase. This indicates the important of endogenous cues in the initiation of gonadaldevelopment. However, there was no significant correlation between plasma hormone levels and GSIin the present study as has been observed in goldfish (Kobayashi et al. 1986a). The reason for this isprobably due to the fact that the Thai carp has an extended spawning period and can spawn severaltimes within a spawning period (Sirikul et a1. 1986) while the goldfish spawned a few times during ashort spawning period (Kobayashi et al. 1986a).Two peaks of plasma GtH, T and 11-KT levels were observed during the spawning period inthe male Thai carp which was indicated by the decline of GSI. This suggests the involvement of GtHand androgens in the processes of spawning in the male Thai carp. The role of reproductive hormonesduring spawning is discussed in chapter 5.The importance of hormones in spermatogenesis has been demonstrated in several fishspecies. For example, in goldfish, a high levels of GtH was needed to maintain spermatocytes andspermatids (Breton et al. 1973). Also, plasma T and 11-KT were found to be important in the controlof the spermatogenetic process in several teleost species (Billard et al. 1982), but 11-KT was alsoimportant in stimulating development of male secondary sex characteristics as has been observed insockeye salmon (Idler et al. 1961), rainbow trout (Scott et al. 1980b) and white sucker (Scott et al.1984). In the present study, however, there was no clear association between the levels of plasma GtH,T and 11-KT and testicular development. This probably due to the fact that the Thai carp is a tropicalfish which spawns several times during a protracted spawning period. The changes in plasma levels ofthese hormones are probably so rapid that they cannot be detected by a monthly sampling regime.71CHAFFER 4INTERACTION OF SGNRHA AND DOMPERIDONE ON THE INDUCTION OFGONADOTROPIN SECRETION AND INDUCTION OF SPAWNING IN THE THAI CARPA. INTRODUCTIONGonadotropin secretion in teleosts is under the dual control of gonadotropin-releasinghormone (GnRH) and dopamine which acts directly at the pituitary, via specific dopamine type 2receptors (D-2 receptors), as a gonadotropin release inhibitory factor (GRIP) (Peter et al. 1986).Leelapatra (1988) has demonstrated that administration of [D-Arg6 , Pro9-NHEt]-sGnRH (sGnRHA)or [D-Ala6 , Pro9-NHEt]-mGnRH (D-Ala6-mGnRHA) alone fails to induce ovulation in the Thai carp.This contrasts to the situation in which ovulation is reliably induced by hypophysation, or whensGnRHA or D-A1a6-mGnRHA is used in combination with the dopamine antagonist, domperidone(Dom). This suggests the involvement of dopamine in the regulation of GtH secretion in the Thaicarp.In the present study, the interaction of sGnRHA and Dom on the induction of GtH secretionand ovulation in the Thai carp was examined in order to determine the minimum dose of sGnRHA andDom required for inducing ovulation of this species.B. MATERIALS AND METHODSTwo experiments were performed during 1990 and 1991 spawning seasons. In 1990, theexperiment was carried out at Kalasin Freshwater Fisheries Station during the month of July. Onehundred and forty four sexually mature females Thai carp (612.29 ± 20.88 g) were divided into 16different groups. Each group received intraperitoneal injections of various combinations of sGnRHA(0 to 25 µg/kg BW) and Dom (0 to 25 mg/kg BW) (Table 1). After injection, fish in each group72together with the same number of untreated sexually mature males were transferred to a 8 m 2concrete-lined outdoor spawning tank. The tanks were supplied with running water at 29 ± 1 ° C.Blood samples were taken serially at the time of injection, and at 3, 6, and 9 hr post injection, asdescribed in chapter 2. The fish were allowed to spawn naturally, and the numbers of fish whichspawned during the 24 hr period following injection were noted. As a result of a power failure atKalasin Freshwater Fisheries Station, none of the plasma samples could be used for hormonesanalyses.The 1991 experiment was performed at Pathumthani Freshwater Fisheries Station, during themonth of May. Except for differences in dose level, the design of the experiment was similar to the1990 experiment. One hundred and forty four fully mature females Thai carp (393.19 ± 11.51 g BW)were divided into 16 different groups and injected with different combinations of sGnRHA (0 to 10µg/kg BW) and Dom (0 to 10 mg/kg Bw) (Table 2). After injection, fish were transferred to a 6 m 2concrete-lined outdoor spawning tank supplied with running water at a temperature of 31±1 ° C andblood samples were taken serially as mentioned above. Plasma samples were analyzed for GtHcontent at the Department of Zoology, University of Alberta, Edmonton, Alberta.73Table 1: Dose combinations of sGnRHA and Dom: Kalasin Freshwater Fisheries Station in July 1990.sGnRHA (Mg/kg)Dom (mg/kg) 0 1 5 250 0+0 0+1 0+5 0+251 1+0 1+1 1+5 1+255 5+0 5+1 5+5 5+2525 25+0 25+1 25+5 25+25Table 2: Dose combinations of sGnRHA and Dom: Pathumthani Freshwater Fisheries Station in May1991.sGnRHA (µg/kg)Dom (mg/kg) 0 1 5 100 0+0 0+1 0+5 0+101 1+0 1+1 1+5 1+105 5+0 5+1 5+5 5+1010 10+0 10+1 10+5 10+1074100a-0) 8 0 -beco 60 -a.co 40iiae 20aa a25aaa^aa^0sGnRHA (pg/kg)Fig. 24. Effect of various combinations of sGnRHA (µg/kg) and Dom (mg/kg) on the induction ofspawning in female Thai carp during July 1990 at Kalasin Freshwater Fisheries Station. Eachvalue represents the percentage of fish spawned in each treatment. Groups which were similar inpercentage of fish spawned as determined by Fisher's exact probability test (P > 0.05) areidentified by the same superscript, i.e., a, b, and c.75001 go_ab^locdabcadbcdA 4\4)ab AVAM1111131/AC=^rACIIIVAIE111/ e 01 0 0-a 80a)ae 2010^5^1^0sGnRHA (pg/kg)Fig. 25. Effect of various combinations of sGnRHA (µg/kg) and Dom (mg/kg) on the induction ofspawning in female Thai carp during May 1991 at Pathumthani Freshwater Fisheries Station.Each value represents the percentage of fish spawned in each treatment. Groups which weresimilar in percentage of fish spawned as determined by Fisher's exact probability test (P > 0.05)are identified by the same superscript, i.e., a, b, c, and d.76C. RESULTSIn both years, various number of fish in each treatment group began spawning 4 hr afterinjection. Spawning behavior lasted for about 2 hr (6 hr post injection).a. Percent fish spawnedIn the 1990 (Fig. 24), there was no spawning occurred in the control group (0 µg/kg sGnRHA+ 0 mg/kg Dom, 0+0). In groups receiving Dom alone, spawning was observed in only one third ofthe group treated with the highest concentration of Dom used in this study (0+ 25). Spawning occurredin 1 out of 9 fish in each of the groups receiving the highest dose of sGnRHA alone (25+0) or incombination with 1 mg/kg Dom (25+1). The proportion of fish spawning increased with increasingconcentrations of sGnRHA and/or Dom. All fish spawned in the group that received maximum dosesof both sGnRHA and Dom (25 + 25). At each concentration of sGnRHA, the proportion of fishobserved spawning was highest in groups receiving the maximum dose of Dom; however, thedifferences between groups receiving 5 and 25 mg/kg Dom at each dose levels of sGnRHA were notsignificant.In 1991 (Fig. 25), no spawning occurred in the control group (0+0) and in groups receivingDom only. In the four groups receiving sGnRHA alone, 1 out of 9 fish spawned in the group receiving5 µg/kg sGnRHA (5 +0). In groups receiving combinations of sGnRHA and Dom, the proportion offish spawning increased with increasing concentrations of sGnRHA and/or Dom. The maximumnumber spawning (88.89%) was observed in the group which received 10 µg/kg sGnRHA and 5 mg/kgDom. At the same concentration of sGnRHA (1, 5, or 10 µg/kg), groups treated with 5 mg/kg Domdemonstrated an equal or higher, but not significantly different, percentage of spawned fish comparedto those treated with 10 mg/kg Dom.77b. Plasma GtH levelsThere were no significant differences in plasma GtH levels at 0 hr (Fig. 26). GtH levels variedbetween 1.5 and 3.0 ng/ml.In all groups, except the controls, plasma GtH levels increased significantly 3 hr after injectionof any combination of sGnRHA and Dom compared to their corresponding groups at 0 hr (Fig. 27). Ingeneral, GtH increased with increasing concentrations of sGnRHA and/or Dom. The peak mean GtHvalue (656.10 ng/ml) was recorded following delivery of 10 Ag/kg sGnRHA and 5 mg/kg Dom. Thistreatment also resulted in the maximum number of spawning fish. In groups receiving Dom alone,GtH increased significantly following treatment with 10 mg/kg Dom when compared with control fishGtH increased approximately 5 times when the concentration of Dom was increased from 1 to 5mg/kg.By 6 hr after injection, plasma GtH concentrations in each treatment group decreased slightlycompared with 3 hr post injection (Fig. 28). However, all groups, except controls and the groupreceiving 1 mg/kg Dom alone, exhibited significantly higher plasma GtH than at 0 hr. Plasma GtHvaried with increasing dose of sGnRHA and/or Dom, but unlike the situation at 3 hr, all groupsreceiving 10 mg/kg Dom exhibited the highest GtH levels, regardless of the concentration of sGnRHA.Thus, in contrast to 3 hr, maximum GtH levels at 6 hr were observed following treatment with thecombination of 5 µg/kg sGnRHA and 10 mg/kg Dom. The GtH level in the group treated with Domalone at the concentration of 10 mg/kg remained significantly higher than that of controls.78/ z,L^0/5/0 e411111111%4 -32 -a076 ,E 5 -10^5^1^0sGnRHA (.ig/kg)Fig. 26. Effect of various combinations of sGnRHA (µg/kg) and Dom (mg/kg) on the induction ofGtH secretion in female Thai carp at injection time in the 1991 study. Each value represents themean plasma GtH concentration (ng/ml) in each treatment.79NIVIMSSSSMIIIMIAMS,SS \ MSS,S,700600500400300(n 2000. 100010^5^1^0sGnRHA (pg/kg)Fig. 27. Effect of various combinations of sGnRHA (µg/kg) and Dom (mg/kg) on the induction ofGtH secretion in female Thai carp at 3 hr after injection in the 1991 study. Each value representsthe mean plasma GtH concentration (ng/ml) in each treatment. Plasma GtH levels which weresimilar as determined by Tukey HSD test (P > 0.05) are identified by the same superscript, Le, a,b, c, d, and e.1 = significant difference from 0 hr ( P < 0.05).805 300roco 2000 100010^5^1sGnRHA (lig/kg)b d ef 1 ^abcdbcdel^bcdeE0) 500= 400700-"=" 600Fig. 28. Effect of various combinations of sGnRHA (µg/kg) and Dom (mg/kg) on the induction ofGtH secretion in female Thai carp at 6 hr after injection in the 1991 study. Each value representsthe mean plasma GtH concentration (ng/ml) in each treatment. Plasma GtH levels which weresimilar as determined by Tukey HSD test (P > 0.05) are identified by the same superscript, a, b,c, d, e, and f.1 or 2 = significant difference from 0 or 3 hr respectively ( P < 0.05).81IIII.10.9"NV MUMUMMMUUUW10^5^1^0sGnRHA (pg/kg)10 -N5 \0 0O700= 600rn 5004005 300coE 2000_ 1000Fig. 29. Effect of various combinations of sGnRHA (µg/kg) and Dom (mg/kg) on the induction ofGtH secretion in female Thai carp at 9 hr after injection in the 1991 study. Each value representsthe mean plasma GtH concentration (ng/ml) in each treatment. Plasma GtH levels which aresimilar as determined by Tukey HSD test (P > 0.05) are identified by the same superscript, i.e, a,b, c, d, e, and f.1 or 2 = significant difference from 0 or 3 hr respectively ( P < 0.05).82At 9 hr post injection (Fig. 29), plasma GtH levels were lower, but in each treatment groupremained significantly higher than at 0 hr, except in the control and in the group receiving Dom aloneat the concentration of 1 mg/kg. GtH levels varied with concentrations of sGnRHA and/or Dom. Thepeak level was observed in the group treated with the highest concentrations of sGnRHA and Domused in this study. In groups receiving a combination of sGnRHA and Dom, GtH remained 10-200times higher than their respective controls at 0 hr.D. DISCUSSIONThe present results demonstrate that sGnRHA or Dom alone were relatively ineffective ininducing spawning in sexually mature Thai carp. A very low spawning rate (11.11%) was observed onlyin groups treated with 5 µg/kg or 25 µg/kg sGnRHA alone in the 1991 and 1990 experimentrespectively. Also, in groups receiving Dom alone, spawning was observed only in the group treatedwith the highest dosage used in the 1990 study.Either sGnRHA or Dom alone at appropriate concentrations stimulated a modest plasmaGtH release. sGnRHA alone at 5 and 10 µg/kg BW significantly increased plasma GtH at 3 (except10 µg/kg), 6, and 9 (except 5 mg/kg) hr post injection. Similarly, 10 mg/kg of Dom alone significantlyincreased plasma GtH levels at 3 and 6 hr after injection. Nevertheless, the magnitude of increasingGtH levels stimulated by either sGnRHA or Dom alone, at the maximum dosage of 10 µg/kg or 10mg/kg was initially insufficient to initiate the processes of final maturation and ovulation in the Thaicarp. However, in the situation where dopamine receptors are completely blocked after treatment witha high concentration of Dom (25 mg/kg), the production of exogenous GnRH is high enough tostimulate a sufficient surge of the preovulatory GtH and to induce spawning in this species. Theseresults suggest that dopamine serves as a potent gonadotropin release inhibiting factor in the Thai carp83as previously demonstrated in goldfish (Chang and Peter, 1983; Chang et al. 1984), common carp (Linet al. 1988), the Chinese loach (Lin et al. 1988), and the African catfish (De Leeuw et al. 1986).Two classes of GnRH receptors have been found in the goldfish pituitary, one having a high-affinity and low capacity, the other with a low-affinity and high capacity (Habibi et al. 1987). Fromstudies on structure-activity relationships in GnRHs, high affinity GnRH receptors are thought to beinvolved in the control of GtH secretion from the goldfish pituitary (Habibi et al. 1987; 1989). In thepresent study, increasing concentrations of sGnRHA alone were relatively ineffective in stimulatingGtH secretion. This suggests that increasing GnRH binding capacity may be the basis for theincreased responsiveness to GnRH.The dopaminergic inhibition of basal and GnRH-stimulated gonadotropin release in goldfishwas suggested to be the result of a down-regulation of the pituitary GnRH receptors (De Leeuw et al.1989). Injection of Dom caused an increase in capacity of both the high- and low-affinity GnRH-binding sites in the goldfish pituitary in a time- and dose-dependent manner, and resulted in anincreasing responsiveness to GnRH peptides (De Leeuw et al. 1989). Furthermore, Dom was found toact both as a D-2 receptor antagonist and a pituitary dopamine depletor in goldfish (Sloley et al. 1991).These actions of Dom resulted in increasing in plasma GtH concentrations (Omeljaniuk et al. 1987;1989b; Sloley et a/. 1991).In the present study, application of various combinations of sGnRHA and Dom effectivelyincreased both the number of fish spawning and plasma GtH levels in a dose-related manner. Thisresult suggests that Dom potentiates the effect of sGnRHA on the stimulation of the plasma GtHlevels in the Thai carp and confirms the finding of the earlier study of Leelapatra (1988) that dopaminehas GRIF activity in the Thai carp. The potentiation action of Dom and GnRHA has beendemonstrated in several teleost species including coho salmon (Van Der Kraal( et al. 1986), goldfish(Omeljaniuk et al. 1987b), Chinese loach (Peter et al. 1987) and common carp (Lin et a/. 1988). In84goldfish, it has been suggested that the interaction of sGnRHA and Dom on the regulation of GtHsecretion involves changes in the number of pituitary receptors for GnRH and dopamine (Omeljaniuket al. 1989a).Dom at 1 mg/kg, alone or in combination with sGnRHA, was insufficient to mask the effect ofdopamine as a GRIF in the Thai carp. Hence, it caused only a modest increase in plasma GtH at 3 hrand a low percentage of fish spawning. An intermediate concentration of Dom (5 mg/kg), incombination with sGnRHA, was highly effective in stimulating GtH secretion and increasing thenumber of fish that spawned. However, there was no significant difference in plasma GtH levels or thenumbers of fish spawning when higher concentrations of Dom were used (10 or 25 mg/kg in 1990 or1991 respectively). These results suggest that 5 mg/kg is the most effective dosage of Dom inpotentiating the action of sGnRHA. A higher concentration of Dom appeared to prolong the action ofsGnRHA, as indicated by higher concentrations of plasma GtH at 6 and 9 hr post injection. Similarly,at a particular concentration of Dom, application of sGnRHA at 1, 5, or 10 1.1g/kg induced an increasein plasma GtH level at all sampling times. The lack of significance probably resulted from highlyvariable plasma GtH concentrations among fish within the same group.All fish spawned in the group which receiving the combination of the highest dosage of bothsGnRHA and Dom used in the 1990 study. The lower maximum doses of sGnRHA and Dom used inthe 1991 experiment failed to induce spawning in all individuals in the group. This indicates theimportance of sGnRHA in the regulation of GtH secretion and spawning in the Thai carp, since thehighest concentration of Dom used in the 1991 experiment was higher than 5 mg/kg, which is the mosteffective dosage in potentiating the action of sGnRHA. The failure to attain 100% spawning wasprobably due to the lower concentration of sGnRHA used in the 1991 study. Furthermore, the 1991experiment was conducted in May, 2 months earlier than the 1990 experiment which was carried out inJuly. It has been reported in goldfish that the responsiveness to injection of [D-Ala 6]-mGnRHA and85pimozide, a dopamine receptor antagonist, showed a seasonal variation (Sokolowska et al. 1985). Thus,the difference in the timing of the treatment in this study might also affect the responsiveness of thefish.From the economic and practical points of view, however, since there were no significantdifferences in the numbers of fish spawning among groups treated with any combination of 5 to 25µg/kg sGnRHA and 5 to 25 mg/kg Dom, the most economic and appropriate dosage for inducedspawning in the Thai carp is probably 5 µg/kg sGnRHA + 5 mg/kg Dom.86CHAPTER 5THE EFFECTS OF SGNRHA AND DOMPERIDONE ON PLASMA GONADOTROPIN ANDSTEROID HORMONES LEVELS DURING SPAWNING IN THE MALE AND FEMALE THAICARPA. INTRODUCTIONThe pattern of hormonal changes during final maturation and ovulation provides importantbasic knowledge for a thorough understanding of the endocrine control of ovulation in fish Moststudies have investigated the patterns of hormonal changes during final maturation and ovulation intemperate or subtemperate fishes. At the present time, however, such patterns in tropical fish are notclearly understood.From the previous chapter, it is clear that the preovulatory surge in GtH, which leads to fmalmaturation and spawning in the Thai carp can be achieved using sGnRHA and Dom therapy.However, there is no information regarding the changes in reproductive hormones during spawning inthis species. This study was designed to reveal the patterns of hormonal changes during inducedspawning in the male and female Thai carp.Two experiments were carried out at Pathumthani Freshwater Fisheries Station, Pathumthani,Thailand from May 1 to May 15, 1991. The first experiment (Expt. I) concerned the patterns of "longterm" ( < 9 hr) changes in reproductive hormone concentrations during sGnRHA and domperidoneinduced spawning of the Thai carp. The second experiment (Expt. II) examined the "short term"changes in reproductive hormone concentrations immediately before and after the time of spawning.In this chapter, the term sexual behaviour is used to describe any behavioural interactionbetween males and females leading to the union of gametes. Spawning behaviour refers to the patternsby which males and females directly synchronize their behaviour to achieve a coordinated release of87gametes i.e., oviposition and release of milt (Liley and Stacey, 1983). Courtship is that behaviourinvolves in the search for, and attraction and excitation of, a potential sexual partner. The termspermiation is used to refer to the release of spermatozoa from the cysts into the lobule lumen (Billard,1986). On the other hand, the term production of milt is used to describe the formation of thehydrated suspension of mature spermatozoa that can be released during spawning.B. MATERIALS AND METHODSI. Experiment I. "Long term" changes in plasma hormone levels during induced spawning in male andfemale Thai carpTwelve mature females Thai carp (1-2 years of age, average weight 513.33 ± 41.29 g) wereinduced to spawn by a single intraperitoneal injection of 20 µg/kg [D-Arg 6, Pro9-NHEt]-sGnRH(sGnRHA) and 20 mg/kg domperidone. The females, together with 12 mature untreated males(average weight 352.50 ± 28.71 g), were transferred, in pairs, to a spawning tank and allowed to spawnnaturally. The tank was supplied with running water at 30 ± 1 ° C. All fish were blood sampled at thetime of injection and at 3, 6, and 9 hr after injection. Plasma samples were analyzed for GtH, T, E2,11-KT, and 17,20P-P concentrations.H. Experiment II. "Short term" changes in plasma hormone levels at the time of spawning in maleand female Thai carpMature females Thai carp, average weight 555.56 ± 21.78 g, were divided into 7 groups of 9fish each. Each group received a single intraperitoneal injection of 20 Ag/kg sGnRHa and 20 mg/kgdomperidone. Four groups were injected 4 hr before the other three groups. Fish from the first fourgroups, together with the same number of mature untreated males (average weight 173.02 ± 8.04),88were transferred to a spawning tank which was divided into 4 separate units. The other three groups,together with the same number of mature untreated males, were also transferred to a spawning tankwhich also divided into 3 separate units. Both tanks were supplied with running water at a temperatureof 30 ± 1 ° C. Blood samples were taken 30, 60 and 90 min before the predicted time of spawning,during spawning, and 30, 60 and 90 min after the onset of spawning. The plasma samples wereanalyzed for GtH, T, E2, 11-KT, and 17,20/3-P concentration.C. RESULTSExperiment I.Males exhibited spawning behaviour, as described in Liley and Tan (1985), 3.5-4 hours afterplacement with females. Spawning runs were observed between 4-6 hr after injection. This spawningrun was more frequent during the first 30 min after the first run then gradually declined. Thisspawning activity was completed at 6 hr after injection.A. Femalesa. Estradiol-17fl (E2) (Fig. 30)Plasma E2 concentration increased rapidly from 0.45 ng/ml at 0 hr and peaked 3 hr afterinjection at 4.4 ng/ml. Thereafter, E2 gradually decreased to the pre-experiment level at 9 hr.b. Testosterone (T) (Fig. 31)Plasma T increased from 0.33 ng/ml at 0 hr to 1.26 ng/ml at 3 hr, and peaked at 6 hr. Afterspawning, the level rapidly decreased to 0.19 ng/ml at 9 hr.c. Gonadotropin (GtH) (Fig. 32)89Plasma GtH concentration increased significantly from 1.44 ng/ml at 0 hr to 900.56 ng/ml at 3hr after injection. The levels then decreased significantly to 373.89 ng/ml at 6 hr after injection.Between 6 and 9 hr after injection, GtH levels decreased to 151.67 ng/ml.d. 17,2013-PPlasma 17,2013-P levels were virtually undetectable at all times in this study, though thesensitivity of the assay was only 7 pg/ml. A very low level was observed in some samples 3 hr afterinjection (70.67 pg/ml).905rnca-a-, 4a)caEP,3 2ba^ a0^3^6^9^hrovipositionFig. 30. Changes in plasma E2 during sGnRHA (20 µg/kg) and Dom (20 mg/kg) induced spawning infemale Thai carp. Each value represents the mean ± SEM. Plasma hormone levels which weresimilar as determined by Tukey HSD Test (P > 0.05) are identified by the same superscript, i.e.,a, b, and c.91 a\\a2.52E1.5co1cocC00.500 9 hrFig. 31. Changes in plasma T during sGnRHA (20 µg/kg) and Dom (20 mg/kg) induced spawning infemale Thai carp. Each value represents the mean ± SEM. Plasma hormone levels which weresimilar as determined by Tukey HSD Test (P > 0.05) are identified by the same superscript, i.e., aand b.9212501 0 0 0rnco 750a)5 500caEca25000 3 6 9 h r ovipositi o nFig. 32. Changes in plasma GtH during sGnRHA (20 µg/kg) and Dom (20 mg/kg) induced spawningin female Thai carp. Each value represents the mean ± SEM. Plasma hormone levels whichwere similar as determined by Tukey HSD Test (P > 0.05) are identified by the same superscript,i.e., a, b, and c.93B. Malesa. 11-Ketotestosterone (11-KT) (Fig. 34)There were no significant changes in plasma 11-KT levels during the long term observation ofthe spawning activity of the untreated male Thai carp. The level slightly decreased from 0 hr to 3 hrand then gradually increased at 6 hr i.e., during spawning activity. The level then rapidly decreasedagain after spawning i e , at 9 hr.b. Testosterone (T) (Fig. 35)Plasma T levels gradually increased from 1.18 ng/ml at 0 hr and peaked at 3 hr in the presenceof treated females. The level then slowly decreased to the pretreatement level at 9 hr.c. Gonadotropin (GtH) (Fig. 36)Plasma GtH levels in male fish did not show any significant changes during the spawningperiod. GtH varied between 1 and 2 ng/ml at all times.d. 17,20/3-PAs in the female fish, plasma 17,20/3-P levels were barely detectable (50.67 pg/ml, n=3) at 3hr after placement in the same tank as the females.941.2Ern0.8cocu— 0.6co 0.4EU)co0- 0.200 3 6 9 hr s p awn i ngFig. 34. Changes in plasma 11-KT during spawning in untreated male Thai carp kept with sGnRHAand Dom induced spawning treated females. Each value represents the mean ± SEM.959^h ra3 6a b02.5ECo0)Ena) 1.5coco11.0.5spawningFig. 35. Changes in plasma T during spawning in untreated male Thai carp kept with sGnRHA andDom treated females. Each value represents the mean ± SEM. Plasma hormone levels whichwere similar as determined by Tukey HSD Test (P > 0.05) are identified by the same superscript,i.e., a and b.9632.5Eac 2ti(1) 1.5Es0.500 3 6 9 hr spawningFig. 36. Changes in plasma GtH during spawning in untreated male Thai carp kept with sGnRHA andDom treated females. Each value represents the mean ± SEM.97Experiment II.Four groups of fish displayed spawning activity 4 hr after injection. At this point, bloodsamples were immediately taken from one group; the other 3 groups were sampled at 30 min intervals.Spawning activities ended 5 to 6 hr after injection. The other three groups were sampled at 2.5, 3.0,and 3.5 hr after injection. The fish were retained at their tanks for 9 hr in order to identify the spawnedfish. The data represent mean values of all spawned fish in each group.A. Femalesa. Estradiol-17/3 (E2) (Fig. 37)Plasma E2 levels decreased from 2.60 ng/ml at 90 min before spawning to 1.51 ng/ml at 60min before spawning and then gradually increased and peaked at spawning time (4 hr after injection).The levels slowly decreased to about 2 ng/ml at 60 min after spawning and then increased slightly at 90min after spawning.b. Testosterone (T) (Fig 38)There were no significant changes in plasma T levels shortly before and after spawning infemale Thai carp. Levels varied between 1 and 2 ng/ml throughout the study period.98bab^ ababababa^-90^-60^-30 ovipos Hon^30^60^90 min^2:30 3:00 3:30^4:00^4:30 5:00^5:30 hrmin before and after spawning, hr after injectionFig. 37. Short-term changes in plasma E2 levels in sGnRHA (20 1.1g/kg) and Dom (20 mg/kg) inducedspawning in female Thai carp shortly before, during, and after spawning. Each value representsthe mean ± SEM. Plasma hormone levels which were similar as determined by Tukey HSD test(P > 0.05) are identified by the same superscript i.e, a and b.99 1 reS2.5••••••••E..-.c) 2ccoa)>1.5I-cacoco(7.0.5^-90^-60^-30 ovipositIon^30^60^2:30 3:00 3:30^4:00^4:30 5:00min before and after spawing, hr after injection90 min5:30 hrFig. 38. Short-term changes in plasma T levels in sGnRHA (20 µg/kg) and Dom (20 mg/kg) inducedspawning in female Thai carp shortly before, during, and after spawning. Each value representsthe mean ± SEM.10012001000Ecnc 800a)600400cn0_2000 ifi^-9 ^-60^-30 ovIposItIon 30^60^90 min2:30^3:00 3:30^4:00^4:30 5:00^5:30 hrmin before and after spawning, hr after injectionFig. 39. Short-term changes in plasma GtH levels in sGnRHA (20 µg/kg) and Dom (20 mg/kg)induced spawning in female Thai carp shortly before, during, and after spawning. Each valuerepresents the mean ± SEM. Plasma hormone levels which were similar as determined by TukeyHSD test (P > 0.05) are identified by the same superscript i.e, a and b.101c. Gonadotropin (GtH) (Fig. 39)Plasma GtH levels were high (816.75 ng/ml) 90 min before spawning (2.5 hr after injection)and remained high during the prespawning period. GtH decreased slowly during spawning and for aperiod of 30 min after spawning before declining significantly to approximately 220 ng/ml at 60 and 90min after spawning (5 hr after injection).d. 17,20/3-PPlasma 17,20/3-P levels were undetectable throughout the study period.B. Malesa. 11-Ketotestosterone (11-KT) (Fig. 40)Plasma 11-KT decreased from 3.29 ng/ml at 90 min before spawning to 2.13 ng/ml at 60 minbefore spawning. 11-KT remained at this level until 30 min before spawning and then increased rapidlyat spawning time peaking at 30 min after spawning. The concentration then slowly decreased at 90 minafter spawning.b. Testosterone (T) (Fig. 41)Plasma T showed a similar trend to that of 11-KT, but the levels were higher than those of 11-KT. T levels decreased from 12.73 ng/ml at 90 min before spawning to 5.60 ng/ml at 60 min beforespawning. T remained at this level until 30 min before spawning, then rapidly increased at spawningtime to peak at 30 min post spawning activity. T then rapidly decreased at 60 min after spawning andmaintained at this level until 90 min after spawning.102coE-5 4coa)— 3co•- 2bcbcbcabca^-90^-60^-30 spawning 30^60^90 min^2:30 3:00 3:30^4:00^4:30 5:00 5:30 hrmin before and after spawning, hr after injection of femaleFig. 40. Short-term changes in plasma 11-KT levels in untreated male Thai carp kept with sGnRHAand Dom treated females shortly before, during, and after spawning. Each value represents themean ± SEM. Plasma hormone levels which were similar as determined by Tukey HSD test (P >0.05) are identified by the same superscript i.e, a, b and c.103abcbeabaa2 52 0Ern-515coa)10coCacaCI 50^-90^-60^-30 spawning 30^60^90 min^2:30 3:00 3:30^4:00^4:30 5:00^5:30 hrmin before and after spawning, hr After injection of femaleFig. 41. Short-term changes in plasma T levels in untreated male Thai carp kept with sGnRHA andDom treated females shortly before, during, and after spawning. Each value represents the mean± SEM. Plasma hormone levels which were similar as determined by Tukey HSD test (P > 0.05)are identified by the same superscript Le, a, b and c.104- a ba^aaba14cow 86cvco 420-90^-60^-30 spawning +30^+60^+90 min2:30^3:00^3:30^4:00^4:30^5:00^5:30 hrmin before and after spawning, hr after injection of femaleFig. 42. Short-term changes in plasma GtH levels in untreated male Thai carp kept with sGnRHA andDom treated females shortly before, during, and after spawning. Each value represents the mean± SEM. Plasma hormone levels which were similar as determined by Tukey HSD test (P > 0.05)are identified by the same superscript i.e, a and b.105c. Gonadotropin (GtH) (Fig. 42)Plasma GtH levels varied between 2 and 7 ng/ml before spawning occurred. GtH thenincreased significantly to 9.16 ng/ml at spawning time, and declined rapidly to 3.01 ng/ml at 30 minafter spawning and maintained at approximately this levels throughout the observation period.d. 17,20fl-PPlasma 17,20p-P levels were undetectable in all samples.D. DISCUSSIONa. FemalesThe present results confirmed the previous finding that the preovulatory surge in plasma GtHlevels and spawning in the female Thai carp can be stimulated by administration of appropriatequantities of sGnRHA and Dom. In teleost fishes, this surge in plasma GtH has been shown to beimportant for the initiation of fmal maturation and ovulation processes (Nagahama, 1987). In thisstudy, plasma GtH increased significantly 3 hr after injection. Then GtH gradually decreased afterspawning i.e., at 5 hr, but remained high compared with the levels before injection. These high levelsof plasma GtH after spawning were probably due to the residual effect of sGnRHA and Dom.Plasma E2 significantly increased 3 hr after injection and peaked at spawning time. Anincrease in plasma E2 during spawning induced by hormonal or environmental stimulation has beencommonly found in teleosts with asynchronous oocyte development such as goldfish (Kagawa et al.1983; Stacey et al. 1983; Kobayashi et al. 1987; 1988), the bitterling (Shimizu et al. 1985), and commoncarp (Kime and Dolben, 1985; Santos et al. 1986; Levavi-Zermonsky and Yaron, 1986). In goldfish, E2is known to be synthesized by conversion of T, by aromatase enzyme, in the granulosa layers of thevitellogenic oocytes, under the stimulation of GtH (Kagawa et al. 1984). In the Thai carp, vitellogenic106oocytes were present throughout the reproductive cycle (see chapter 3). Thus, the surge in plasma E2observed in this study at 3 hr post injection was most probably due to gonadotropin-stimulation of thevitellogenic oocytes.Studies of hormonal changes during ovulation in teleosts have revealed that E2 tends todecline preceding ovulation in several teleosts including salmonids (Fostier and Jalabert, 1982; Scott etal. 1983; Van Der Kraak et al. 1984; Dye et al. 1986; Liley et al. 1986; Liley and Rouger, 1990) andcyprinids such as common carp (Kime and Dolben, 1985; Santos et al. 1986) and the bitterling (Shimizuet al. 1985). This decline in E2 was suggested to be a result of the shift in the steroid synthesis pathwayin the granulosa layers from the production of E2 to the production of 17,20/3-P (Nagahama, 1987). Insalmonids the decline in E2 is believed to be a trigger for the GtH surge and ovulation (Fostier et al.1983; Scott et al. 1983). However, in goldfish E2 had no effect on the occurrence of the spontaneousGtH surge and subsequent ovulation (Pankhurst and Stacey, 1985).An increase in plasma E2 levels during induction of ovulation by brain lesion technique hasbeen observed in goldfish, but a peak of E2 was observed 1 hr after ovulation (Stacey et al. 1983).Furthermore, in common carp, a significant increase in E2 was observed during induction of ovulationby treatment with GnRH or crude pituitary extract (Weil et al. 1980). On the other hand, there wereno changes in plasma E2 levels observed during induced ovulation with GnRH in Wuchang fish(Megalobrama amblycephala) (Weixin et al. 1986) and in silver carp (Weixin et al. 1988). The functionof a peak in E2 at spawning time in the female Thai carp is unknown, but it may not be involved in theoccurrence of ovulation since E2 has been found to be ineffective in inducing oocyte maturation inmany teleost species (Goetz, 1983; Scott and Canario, 1987).Clear evidence for a shift in the production of E2 to T, which is commonly seen in salmonids(Fostier and Jalabert, 1982; Kagawa et al. 1983; Scott et al. 1983; Van Der Kraak et al. 1984; Dye et al.1986), was also observed during sGnRHA and Dom induced spawning in the Thai carp. A peak of E2107was observed at 3 hr post injection, then E2 significantly decreased at the end of spawning activity (6hr). On the other hand, after injection, plasma T levels gradually increased and peaked at 6 hr afterinjection. However, the short term observations (Expt. II) showed that both T and E2 actually peakedat spawning time. These data suggest that E2 was synthesized during the first part of the inductionunder the stimulation of GtH. Then, when spawning started, the production of Ea decreased, probablydue to the rapidly decreasing plasma GtH. This resulted in an elevation of T. The decrease in E2production most likely due to a decrease in aromatase activity. Although, the mechanism of theinduction or activation of the granulosa cell aromatase system is unknown at the present time, ingoldfish, however, E/2 is known to be synthesized in response to HCG treatment (Kagawa et al. 1984).In goldfish, T was mainly produced by postvitellogenic oocytes under GtH stimulation(Kagawa et al. 1984; Kobayashi et al. 1987). A peak of T just before ovulation was observed in goldfish(Kagawa et al. 1983; Kobayashi et al. 1988) and common carp (Santos et al. 1986; Kime and Dolben1985). The acute preovulatory rise in T in rainbow trout was suggested to be involved in theproduction of 17,20P-P (Jalabert and Fostier, 1984). In goldfish, high plasma T just before ovulation isconsidered to be a physiological cue for the occurrence of the ovulatory GtH surge (Kobayashi et al.1989).In this study the elevation in plasma T probably resulted from the shift in steroid synthesispathway due to the reduction of aromatase activity as previously found in the common carp duringinduced ovulation by hypophysation (Kime and Dolben, 1985). Injection of sGnRHA and Dom in theThai carp caused a rapid increase in GtH which then stimulated the synthesis of T by vitellogenic andpostvitellogenic oocytes as has been shown in goldfish by Kagawa et al. (1984). In several teleosts, ithas been suggested that under GtH stimulation, T was converted to 52 by the aromatase enzyme in thegranulosa layers of the vitellogenic oocytes (Nagahama, 1987). When spawning occurred, GtH rapidlydecreased as well as the aromatase activity. This resulted in a decline in plasma E2 and hence an108increase in plasma T. In the Thai carp, the importance of the shift in steroid synthesis is unknown, butit seems to be a physiological requirement for ovulation.The decrease in plasma T after spawning is probably due to the loss of postvitellogenic oocytesafter spawning which have been shown to be the main producer of T under the stimulation of GtH(Kobayashi et al. 1988).17,20/3-P has been found to be the most potent maturation inducing steroid (MIS) in mostteleosts studied to date (Goetz, 1983; Scott and Canario, 1987). A surge of plasma 17,20/3-P has beenobserved immediately before ovulation in several teleosts such as salmonids (Fostier and Jalabert,1982; Scott and Baynes, 1982; Scott et al. 1982; Wright and Hunt, 1982; Young et al. 1983; Van DerKraak et al. 1984; Dye et al. 1986), African catfish (Lambert and Van den Hurk, 1982), goldfish (Staceyet al. 1983; Kagawa et al. 1983; Peter et al. 1984b; Kobayashi et al. 1987; 1988), the bitterling (Shimizu etal. 1985), white sucker (Scott et al. 1984), and common carp (Santos et al. 1986; Levavi-Zermonsky andYaron 1986; Kime and Bieniarz 1987). Interestingly, in this study, a very low concentration of 17,20/3-P(70.67 pg/ml) was observed only at 3 hr post injection, when the blood was taken at 3 hr intervals.During the short term observations, however, 17,20/3-P was undetectable at all sampling times.Kime and Dolben (1985) failed to detect 17,20/3-P in common carp during ovulation inducedby pituitary extract. However, in a later in vitro study, Kime and Bieniarz (1987) demonstrated that17,20/3-P could only be detected in fish that had received a priming dose of pituitary extract. Thispriming dose of pituitary extract resulted in migration of the germinal vesicle to the periphery (Yaronet al. 1985) which is a pre-requisite for ovarian synthesis of 17,20/3-P (Kime and Bieniarz, 1987).In salmonids a large surge in 17,20/3-P was observed following the shift in steroid synthesisfrom E2 to T (Fostier and Jalabert, 1982; Kagawa et a/. 1983; Scott et al. 1983; Van Der Kraak et al.1984; Dye et al. 1986). In this study, the shift from E2 to T was observed between 3 and 6 hr afterinjection with sGnRHA and Dom, but no 17,20/3-P was detected.109The reason for the failure to detect plasma 17,20/3-P in this study is unknown. All the fishspawned with a high fertilization rate ( > 80% ), indicating that the oocytes had successfully beeninduced to pass through the normal processes of germinal vesicle migration and germinal vesicle breakdown. Hence, the reason for lack of 17,20/3-P is probably different from that in common carp (Kimeand Bieniarz, 1987). Since the shift in steroid synthesis, which was believed to be the basis for 17,20/3-Pproduction (Jalabert and Fostier, 1984; Kobayashi et al. 1987), was observed in this study, the reasonfor non detection of 17,20/3-P was probably due to inadequate sampling time. In goldfish, rapidchanges in 17,20/3-P were observed which suggested the short-term secretion and/or rapid plasmaclearance of this steroid (Kobayashi et al. 1987). In the current study, the peak of 17,20/3-P might haveoccurred so fast that the sampling time interval designed for this experiment could not detect anychanges.Recently, a trihydroxylated progesterone derivative, 17a,20,21-Trihydroxy-4-pregnen-3-one(17,20/3,21-P), has been identified as the MIS in the Atlantic croaker (Trant et al. 1986; Trant andThomas, 1989; Patino and Thomas, 1990). Moreover, 17,20/3,21-P was found to be at least as potent as17,20/3-P in the induction of fmal oocyte maturation in in vitro bioassays in spotted seatrout (Thomasand Trant, 1989), dab (Scott and Canario, 1987), and rainbow trout (Canario and Scott, 1988). Thesesuggest the possibility that 17,20/3-P might not be the MIS in the Thai carp.b. MalesThere were no significant changes in plasma GtH levels during spawning in male Thai carpwhen samples were taken at 3 hr intervals (Expt. I). However, during short term observations inexperiment II, a significant increase in plasma GtH was observed at the onset of spawning. This surgein GtH was followed by a rapid decrease 30 min later. This may be an indication that the GtH surge inmale Thai carp can also be stimulated by exposure to ovulatory females as previously found in goldfish110(Kobayashi et al. 1986c). In the present study, male spawning behaviour was observed shortly beforespawning occurred, consistent with the time of GtH surge. In an earlier study, however, spawningbehavior in male Thai carp treated with homoplastic pituitary extract (PE) was observed at the sametime as PE or prostaglandin PGF2 a treated females, about 3-3.5 hr after treatment and at least 15 minbefore spawning (Liley and Tan, 1985). In goldfish, similarly, sexual behavior was observed to startbefore ovulation around the beginning of the GtH increase (Kobayashi et al. 1986c). Theseobservations suggest that the GtH surge in male might be involved in triggering male sexual behaviourin the Thai carp.In female goldfish, a surge of 17,20/3-P was found to be highly synchronous with the GtHsurge (Kobayashi et al. 1986a), and 17,20/3-P was found to act as a preovulatory pheromone in goldfishwhich exerts a priming effect on the male endocrine system i.e., stimulate GtH secretion and theproduction of milt (Stacey et al. 1987). In the current study, a peak of GtH in females was observedearlier than that of males, though the fish were placed together immediately after injection. Thisresult, together with the very low level of 17,20/3-P observed in female Thai carp, suggests that 17,20/3-P might not act as the preovulatory pheromone in Thai carp.Plasma T increased gradually in male Thai carp after placement with sGnRHA and Domtreated females, when samples were taken at 3 hr intervals. During short-term observations inexperiment II, however, a surge of T was observed at the time of spawning and at the time of the GtHsurge. Similarly, a surge in plasma 11-KT was also observed at this time, though there were nosignificant changes during the 3 hr interval observations. Stimulation of androgen (both T and 11-KT)secretion by elevation of GtH has been reported in rainbow trout (Hunt et al. 1982), common carp(Takashima et al. 1984; Ngamvongchon et al. 1987), and goldfish (Kobayashi et al. 1986b; Wade andVan Der Kraak, 1991). In rainbow trout, a peak of 11-KT was found to coincide with the peak of miltproduction, suggesting the importance of 11-KT in the process of milt production (Scott et al. 1980b;111Fostier et al. 1982; Baynes and Scott, 1985). In this study, the levels of T and 11-KT continued toincrease during the process of spawning, though GtH immediately decreased to a significantly lowerlevel. Thirty minutes after spawning, plasma T rapidly decreased to a lower level, while 11-KTdecreased gradually as well as the occurrence of spawning activity. These data indicate that 11-KT isprobably the main androgen involved in the process of spawning in male Thai carp.As in female fish, very low concentrations of plasma 17,20/3-P (50.67 pg/ml) could be detectedin males only at 3 hr after placement with females, when samples were taken at 3 hr intervals. 17,20/3-P was undetectable during the short-term observations (Expt II). An increase in plasma 17,20/3-Pduring the process of spawning has been observed in several teleost species including salmonids (Scottand Baynes, 1982; Hunt et al. 1982; Ueda et a/. 1983; 1984; Liley et al. 1986), white sucker (Scott et al.1984), goldfish (Kobayashi et al. 1986a; b), the northern pike (Colombo et al. 1987), and common carp(Barry et al. 1990). In most cases, a surge in 17,20P-P was observed following a decline in plasmaYandrogen. This shift from C19 to C21 steroid synthesis was suggested to be typical of seasonallyreproducing male teleosts prior to spawning (Scott et al. 1984), and was found to be mediated by17,20/3-P or a related progestogen (Barry et a1. 1990). However, a shift from C19 to C21 was not themain key for successful spawning in common carp, since some males which had a GtH surge but nosteroidogenic shift can successfully mate with ovulated females (Barry et a1. 1990). The role of 17,20/3-P in male fish is poorly understood (Fostier et al. 1983). It has been suggested that 17,20/3-P may playa role in the control of sperm motility mediated by changes in K + composition of the seminal fluids(Baynes and Scott, 1985).In the current study, no steroidogenic shift was observed, and plasma androgen increasedduring the process of spawning. A possible explanation for these observations is that the surge in GtHin male Thai carp is probably stimulated by a postovulatory pheromone from females (Liley and Tan,1985; Stacey et a1. 1987) which is released immediately before spawning. This increase in GtH112stimulates T production, from the testes, which will gradually be transformed to 11-KT. Both T and11-KT play key roles in the processes of spawning behavior, milt production and milt release andgradually decrease when spawning activity is completed.113CHAPTER 6BIOLOGICAL ACTIVITIES OF GNRHS AND THEIR ANALOGS IN COMBINATION WITHDOMPERIDONE ON THE INDUCTION OF GONADOTROPIN SECRETION AND SPAWNING INTHE THAI CARPA. INTRODUCTIONThe primary structure of a salmon gonadotropin-releasing hormone (sGnRH) differs frommammalian GnRH (mGnRH) in its amino acids at positions 7 and 8 (Sherwood et al. 1983) (Fig. 1).Recently, the sequence of amino acids from chicken GnRHs have been identified to be [G1n 8)-GnRH(cGnRH-I) (King and Millar, 1982a; b) and [His5, Trp7, Tyr81-GnRH (cGnRH-II) (Miyamoto et al.1984). Several teleost species have more than one form of GnRH (Sherwood et al. 1984; King andMillar, 1985). However, the predominant GnRH molecule appears to be sGnRH, although a numberof minor forms exist i.e., cGnRH-I in tilapia (King and Millar, 1985), cGnRH-11 in goldfish (Yu et al.1988), African catfish (Sherwood et al. 1989), and Thai catfish (Ngamvongchon et al. 1991).At present, more than 2000 forms of GnRH analogs have been synthesized (Karten andRivier, 1986). Substitution of the glycinamide residue at position 10 with an ethylamide residue (Pro9-NHEt) (Fujino et al. 1972) and/or substitution of the Gly residue at position 6 with hydrophobic oraromatic D-amino acids in mGnRH (Monahan et al. 1973) has resulted in the production of GnRHanalogs (GnRHA) which are more potent than the native forms. Both modifications are known toincrease resistance to enzymatic degradation and to enhance receptor binding affinities (Nestor, 1984;Conn et al. 1984).From studies of the structure-activity relationships of a number of GnRHs in teleosts,sGnRHA has been reported to be the most potent analog both in vitro and in vivo in goldfish (Peter etal. 1985; 1987b), rainbow trout (Crim et al. 1988), and African catfish (De Leeuw et al. 1988). In the114previous chapters, it was clearly demonstrated that induction of GtH secretion and spawning in theThai carp can be accomplished by injection of sGnRHA and the dopamine antagonist, domperidone.The purpose of the following study was to compare the biological activities of a number of naturalmammalian, avian, and piscine GnRHs and their analogs. In this in vivo study, I examined the potencyof 12 different forms of GnRHs and GnRHAs in combination with domperidone on GtH secretion andspawning induction in the Thai carp.B. MATERIALS AND METHODSTwo experiments were carried out, one at Kalasin Freshwater Fisheries Station between 24and 29 July 1990 and another at Pathumthani Freshwater Fisheries Station between 21 and 25 May1991. In 1990, 117 fully mature females Thai carp (256.48 ± 8.12 g) were randomly assigned to 13treatment groups of 9 fish each. Each group received an intraperitoneal injection of one of 12 differentGnRHs or GnRHAs (25 µg/kg body weight, BW), together with domperidone (25 mg/kg BW) (table3). Control fish were treated with 0.7% saline. Following treatment, each group of females, togetherwith the same number of untreated sexually mature males, were placed in an 8 m 2 outdoor, concrete-lined holding tank. The tanks were supplied with running water at 28±1 ° C. Blood samples weretaken serially at the time of injection, and at 3, 6, and 9 hr after injection as described in chapter 2. Thenumbers of fish which spawned in each group during the 24 hr period following injection wererecorded. As a result of power failure at Kalasin Freshwater Fisheries Station, plasma samples fromthis experiment were not used for GtH analysis.In 1991, 117 sexually mature females Thai carp (380.08 ± 122.40 g) were randomly assigned to13 different treatment groups of 9 fish each. Each group received an intraperitoneal injection of one of12 different GnRHs or GnRHAs (10 µg/kg BW) together with domperidone (10 mg/kg BW) (table3). Control fish were treated with 0.7% saline. Following injection, fish were treated as described115above. Plasma samples were analyzed for GtH by RIA at the Department of Zoology, University ofAlberta, Edmonton, Alberta.116Table 3: The primary structure of GnRH and other peptides used in the studycommon name^ structure1^2^3^4^5^6^7^8^9^10mGnRH^pGlu His Trp Ser Tyr Gly Leu Arg Pro G1yNH2sGnRH Trp LeucGnRH-?^ Leu GlncGnRH-II His^Trp TyrmGnRHA^ NHEtD-A1a6 mGnRH D-Ala^G1yNH2D-A1a6 mGnRHA^ D-Ala NHEtD-Trp6 mGnRH**^D-Trp^G1yNH2D-Trp6 mGnRHA D-Trp NHEtD-Lys6 mGnRH^ D-Lys^G1yNH2Buserelin D-Ser(But) NHEtsGnRHA^ D-Arg Trp Leu^NHEtD-Ala6 sGnRHA D-Ala Trp Leu^NHEt* only in 1991** only in 1990117100806040200°A) Fish spawnedC^eCinRMA^•ClaRli^inGaRM mOnRMA Gaol:IN-II 0-Aloe D-Alall^0-AlaII^1:1-Ly•O^1:1-TrpO^D-Trpe Bu••r•llnmOn1114 menRMA silnRIIA manliti manlIMA menRHTreatmentsFig 43. Effect of native mammalian, avian and piscine GnRHs and their analogs (25 ag/kg) incombination with Dom (25 mg/kg) on the induction of spawning in the Thai carp in the 1990study (2429 July 1990). Each value represents the percentage of fish spawned in each group.Groups with similar percentage of fish spawned as determined by Fisher's exact probability test (P> 0.05) are identified by the same superscript, i.e., a and b.118C. RESULTSSpawning occurred between 4 to 6 hr following the injection. Eggs were transferred toincubators at the time fish were subjected to the third blood sampling.a. Percentage of fish spawnedIn 1990, spawning occurred in all treatment groups except the control fish (Fig. 43). Therewere no significant differences in percentages of fish spawning among groups receiving GnRHs orGnRHAs. The highest spawning success (77.78%) was obtained in groups treated with [D-Trp6]-mGnRH or Buserelin.In 1991 (Fig. 44), there was no fish spawned in either the control group or the cGnRH-Itreated group. The highest spawning success (88.89%) occurred in the groups receiving [D-Ala 6]-mGnRHA and [D-Trp6]-mGnRHA. Treatment with [D-Ala6]-mGnRH, sGnRHA, and Buserelinresulted in a lower spawning success (80-65%). Fewer spawning success (50-20%) was observed ingroups receiving cGnRH-11, sGnRH, [D-Lys 6]-mGnRH, [D-Ala6]-sGnRHA, and mGnRH. Thelowest percentage of spawners (< 20%) was observed in the mGnRHA treated group.119% Fish spawned100402060800C^•GaRMA^sOnlill^mealill^atOnliMA 038111M-1 eGn1.111-11 0-Ala!^0-Alaa^D-A1•0^D-Ly•O^D-Trpa Ouslor•lin^inGn1111^meal:1MA aGnAHA tallnRM onCinliliATreatmentsFig. 44. Effect of native mammalian, avian and piscine GnRHs and their analogs (10 µg/kg) incombination with Dom (10 mg/kg) on induction of spawning in the Thai carp in the 1991 study(21-25 May 1991). Each value represents the percentage of fish spawned in each group. Groupswith similar percentage of fish spawned as determined by Fisher's exact probability test (P > 0.05)are identified by the same superscript, i.e., a, b, c, d, and e.120b. Plasma GtH levelsThere were no significant differences in plasma GtH levels at 0 hr (time of injection) betweengroups. Mean GtH levels varied between 1.4 and 3.05 ng/ml (Fig. 45).By 3 hr post injection (Fig. 46), plasma GtH levels had increased significantly in all groups,except the control fish. GtH levels were highest ( > 900 ng/ml) in groups receiving [D-Ala 6]-mGnRHor [D-Trp6]-mGnRHA. Moderately high GtH levels (600-450 ng/ml) were observed in groupsreceiving p-Ala6FmGnRHA, Buserelin, or sGnRHA, while groups receiving cGnRH-II, or [D-Lys 6]-mGnRH, exhibited lower GtH levels (200 ng/ml), which were not significantly different from either thecontrols or the highest GtH level groups. GtH levels in groups receiving sGnRH, mGnRH, [D-Ala6]-sGnRHA, or cGnRH-I were not significantly higher than those observed in control animals, but GtHlevels in these groups were significantly lower than those of the groups with the highest GtH level.At 6 hr (Fig. 47), all treatment groups, except the mGnRH injected group, maintainedsignificantly higher plasma GtH levels than at 0 hr. However, within each treatment group, plasmaGtH levels tended to decrease from that recorded at 3 hr. Thus, plasma GtH levels remained highestin groups treated with [D-Ala6]-mGnRH, [D-Trp6]-mGnRHA, or Buserelin (700-450 ng/ml). Groupsreceiving [D-Ala6]-mGnRHA or sGnRHA demonstrated intermediate high GtH levels (300-250ng/ml) compared to the control which were not significantly different from the highest GtH levelgroups. Groups receiving cGnRH-11 or [D-Lys6]-mGnRH exhibited GtH levels not significantlydifferent from either the control or the high GtH level groups. There were no significant differences inplasma GtH levels in groups receiving sGnRH, mGnRH, mGnRHA, cGnRH-I or p-Ala 61-sGnRHAwhen compared with controls. However, GtH levels in these groups were significantly lower than thoseof the high GtH levels groups.At 9 hr post injection (Fig. 48), plasma GtH levels in groups receiving [D-Ala 6]-mGnRH, [D-Trp6]-mGnRHA, Buserelin, [D-Ala6]-mGnRHA, sGnRHA, or [D-Lys6]-mGnRHA remained121significantly higher than those at 0 hr. A significant decrease in plasma GtH levels at 3 and 6 hr postinjection was exhibited in groups treated with p-Ala 61-mGnRH, [D-Trp6]-mGnRHA, and Buserelin,but only when compared to 3 hr post injection in [D-Ala6]-mGnRHA treated fish. Plasma GtH levelsremained higher than those of the control animals in groups receiving [D-Ala6]-mGnRH, [D-Trp6]-mGnRHA, [D-Ala6]-mGnRHA, Buserelin, or sGnRHA.122Plasma GtH (ng/ml)12 ^l i tt i titt li ti CoGnill4 AstInAllinGillilimen 1114 AcCin1114- 1cOnF114 - 110-Alad10-Alasp-Alas0-14sID-Trgiellus*WinmOnlili meinliplA sOnFIFIAlmenlili meni114ATreatme nts1086420Fig. 45. Effects of native mammalian, avian, and piscine GnRHs and their analogs (10 µg/kg) incombination with Dom (10 mg/kg) on the induction of GtH secretion in female Thai carp atinjection time in the 1991 study (21-25 May 1991). Each value represents the mean plasma GtH± SEM.123Plasma GtH (ng/ml) ioox12•enliMA •OnR14^manlill nenRHA cOnlIM-1 eGn/114-11 Cs-Mall^D-Allie^D-Alall^D-ly•fl^0-Type 111,••r•linmOnR14 inenRHA •CInFINA IneInFIN mClaRHATreatmentsFig. 46. Effects of native mammalian, avian, and piscine GnRHs and their analogs (10 mg/kg) incombination with Dom (10 mg/kg) on the induction of GtH secretion in female Thai carp at 3 hrafter injection in the 1991 study (21-25 May 1991). Each value represents the mean plasma GtH± SEM. Groups which were similar as determined by Tukey HSD test (P > 0.05) are identifiedby the same superscript, i.e., a, b, and c.1 = significant difference from plasma GtH at injection time.1241bcdeaC^•OnlINA10864201e1e1de 1cde 1abcde1abc 1-U1abcdeI IPlasma GtH (ng/m1) boox12^11^abcd^1ab^a^a-mTi - -mTm- lit -emir•LinFIN^InGaRM^menRMA •OnR14-1 cOn1111-11 1:1-A1111^D-AI./^D-Ala.^C-Ly•ll^D-Trpe ■ko••r•linneln/111 manAHA •OnRMA nenRHA tallaRHATreatmentsFig. 47. Effects of native mammalian, avian, and piscine GnRHs and their analogs (10 µg/kg) incombination with Dom (10 mg/kg) on the induction of GtH secretion in female Thai carp at 6 hrafter injection in the 1991 study (21-25 May 1991). Each value represents the mean plasma GtH± SEM. Groups which were similar as determined by Tukey HSD Test (P > 0.05) are identifiedby the same superscript, i.e., a, b, c, d, and e.1 = significant difference from plasma GtH at injection time.125123 123da12c da1231a b c1111d1b c d111- a^a^a^a^a-L- -.■t■- -L- -aim-1086420Plasma GtH (ng/ml) ioox12C^•OnIIHA^menRli relnliMA^cOnR14-11 D-A1•0^D-Alse^D-Ly•e^D-Ttp6 Ilu••r•IlnaiOnRM menFIMA •CleRHA •OnFIM manill4ATreatmentsFig. 48. Effects of native mammalian, avian, and piscine GnRHs and their analogues (10 µg/kg) incombination with Dom (10 mg/kg) on the induction of GtH secretion in female Thai carp at 9 hrafter injection in the 1991 study (21-25 May 1991). Each value represents the mean plasma GtH± SEM. Groups which were similar as determined by Tukey HSD test (P > 0.05) are identifiedby the same superscript, i.e., a, b, c, and d.1, 2 or 3 = significant difference from plasma GtH at injection time, at 3, or 6 hr after injectionrespectively.126D. DISCUSSIONThe present results demonstrated that a high concentration of either mammalian, avian, andpiscine GnRHs or their analogs (25 µg/kg BW) in combination with Dom (25 mg/kg BW) was equallyeffective in inducing spawning in fully mature female Thai carp. However, cGnRH-II, [D-Ala 6]-mGnRHA, [D-Trp6]-mGnRHA, and Buserelin tended to give higher spawning percentages.At a lower concentration (10 µg/kg BW), the effectiveness of each peptide, in combinationwith 10 mg/kg Dom, can be distinguished in terms of the proportion of fish which spawned andconcentration of plasma GtH.Among the natural forms of mammalian, avian, and piscine GnRHs, given in combination withDom, cGnRH-I failed to induce spawning and stimulate GtH secretion in the Thai carp. mGnRH wasrelatively ineffective, since it induced less than 40% of fish to spawn and stimulated a modest increasein plasma GtH levels at 3 hr post injection. These results are consistent with the finding that mGnRHand cGnRH-I had similar effectiveness in stimulating GtH release in in vivo studies in goldfish (Peteret al. 1985) and the gilthead seabream, Sparus aurata (Zohar et al. 1989).On the other hand, treatment with sGnRH and cGnRH-II induced high percentage of fish tospawn, in both the 1990 and 1991 studies. However, they stimulated only a modest increase in plasmaGtH level, which was not significantly different from the control. This suggests that natural sGnRHand cGnRH-II are active in the Thai carp, and may exist in this species as already been found in severalteleost species such as goldfish (Yu et al. 1988), African catfish (Sherwood et a1. 1989), and Thai catfish(Ngamvongchon et al. 1991).In mammalian GnRH, a substitution of the glycinamide residue at position 10 with ethylamideresidue [Pro9-NHEt] results in an analog with 6 times more potency than the native form of mGnRH(Fujino et al. 1972), probably due to an enhancement of receptor binding affinity (Conn et al. 1984).However, in the Thai carp, this substitution did not effect the potency, since mGnRHA induced a very127low percentage of fish to spawn and induced only a modest increase in GtH similar to those obtainedwith mGnRH and sGnRH. This suggests that the binding affinity for the Thai carp pituitary GnRHreceptors may differ from that of mammals.Substitution of position 6 with hydrophobic or aromatic D-amino acids in mGnRH was foundto be important in either increasing receptor binding affinity and/or an increase hydrophobicity and agreater resistance to enzymatic degradation (Nestor, 1984). Similarly, in this study, mGnRHAs with asubstitution of position 6 with hydrophobic (alanine), aromatic (tryptophan), or tertiary-butyl ether(tertiary-butyl serine) D-amino acids, in combination with Dom, demonstrated greater potencies ininducing spawning and increasing and prolonging plasma GtH concentration in Thai carp compared tothe native form as previously found in goldfish (Peter et al. 1985), rainbow trout, salmon, and winterfounder (Crim et al. 1988). On the other hand, in sGnRH which has been found to be morehydrophobic than mGnRH due to the presence of tryptophan in position 7 (Sherwood et al. 1983),substitution of position 6 with D-Arg lead to the production of a sGnRH analog which is superactive instimulating GtH secretion and ovulation. In several teleosts, including goldfish (Peter et al. 1985;1987b; Sokolowska et a1. 1988), common carp (Lin et a1. 1988) Chinese loach (Lin et a1. 1988), rainbowtrout, salmon, and winter founder (Crim et al. 1988), and African catfish (De Leeuw et al. 1988),sGnRHA was found to be the most potent analog in both in vitro and in vivo studies. In this study, [D-Arg6]-sGnRHA, in combination with Dom, was effective in inducing GtH secretion and spawning inthe Thai carp, however, its potency was lower (difference not significant) than those of P-Ala61-mGnRH, [D-A1a6J-mGnRHA, [D-Trp6]-mGnRHA, [D-Trp6]-mGnRH, and Buserelin at all samplingtimes. Furthermore, increasing hydrophobicity in sGnRHA by substituting D-Arg 6 with a morehydrophobic D-amino acid such as D-Ala6 has not proven to increase potencies in terms of GtHconcentration and percentage of fish spawned. Similar result has been reported in other teleosts (Peteret a1.1985; Crim et a1. 1988). The reason is probably similar to that suggested in the mammal where128increasing hydrophobicity leads to diminished activity of mGnRH (Nestor, 1984), probably due to thefact that a highly hydrophobic molecule tends to bind to lipids and is not readily available for bindingwith receptors (Peter et a1. 1985).[D-Ala6]-mGnRH and [D-Trp6]-mGnRHA were found to be the most potent peptides used inthis study, although their potency was not significantly different from [D-Ala 6]-mGnRHA, sGnRHA,Buserelin and [D-Trp6]-mGnRH in term of increasing percentage of fish spawned and prolongingincreased plasma GtH levels. Interestingly, two mGnRHAs with only a substitution at position 6 ([13-Trp6]-mGnRH and [D-Ala6]-mGnRH in 1990 and [D-Ala6]-mGnRH] in 1991) were equipotent instimulating GtH secretion and spawning in the Thai carp. This confirms the finding that an increase inbinding affinity of mGnRH by substitution of the glycinamide residue at position 10 with ethylamide isnot the important factor in making mGnRH superactive in the Thai carp. This result suggests that inthe Thai carp, as in the goldfish (Peter et al. 1985) and the gilthead seabream (Zohar et al. 1989), therate of enzymatic degradation seems to be important in determining the degree of superactivity, sincesubstitution in position 6 with an appropriate D-amino acid seems to enhance the potency of theanalogs and results in a prolonged increase in plasma GtH.In conclusion, the present study demonstrates that sGnRHA is not the most potent analog interms of GtH secretion and induction of spawning in sexually mature female Thai carp. Its potencyappears to be lower than those of mGnRH analogs with a substitution at position 6 with hydrophobicor aromatic D-amino acids. Substitution of the glycinamide at position 10 with ethylamide was not abasic requirement for producing superactive analogs for use in the Thai carp. This indicates that, inthe Thai carp, resistance to enzyme degradation is the most important factor in producing superactiveanalogs. Furthermore, sGnRH and cGnRH-II were the most potent natural GnRH used in this study.They probably exist as a native GnRH in the Thai carp.129CHAPTER 7SUMMARY AND CONCLUSIONSA. ANNUAL REPRODUCTIVE CYCLE OF THE THAI CARP1. FemalesThe present studies reveal the annual reproductive cycle of the Thai carp, Puntius gonionotus,reared in ponds at Kalasin Freshwater Fisheries Station, Kalasin, Thailand. Since the fish have beencultured under hatchery conditions, the patterns of their gonadal development might not represent thetrue natural cycle which occurs in the wild fish In this study, however, gonadal recrudescence washighly correlated with the sequential changes of environmental factors such as air temperatures anddaylength. Also, the period of spawning was observed to be highly coincident with the occurrence ofrainfall as previously reported in wild fish (Sipitakkiat and Leenanond, 1984).Histological analysis of the ovary revealed that oocyte development in the Thai carp was ofthe asynchronous type. This confirms the finding of Sirikul et al. (1986) that the Thai carp can spawnseveral times in an extended spawning season.It was not possible to distinguish pronounced seasonal changes in plasma reproductivehormones levels in the female Thai carp during this study. A gradual increase in plasma GtH levelswas observed at the beginning of vitellogenesis, which was indicated by the elevation of GSI. However,lower levels of plasma GtH were observed during the spawning period.There were no correlations between the seasonal changes in plasma steroid levels and theseasonal changes in GSI. Plasma E2 and T exhibited a similar pattern of seasonal changes. However,the concentrations of plasma E2 were higher than those of plasma T throughout the year. A peak ofplasma E2 was observed to coincide with the initial increase in GSI. This indicates the involvement of130E2 in the process of vitellogenesis. Furthermore, another peak of plasma E2 was observed during thespawning period. This surge in plasma E2 was probably synthesized by previtellogenic oocytes underthe stimulation of the preovulatory surge in GtH. The parallel changes in plasma T and E2 confirmedthe role of T as a precursor for E2 synthesis. The lower concentrations of T than those of E2 suggestthat T was actively aromatized into E2 in the ovary of the Thai carp.2. MalesSeasonal changes in GSI in male Thai carp were highly correlated with those seen in thefemales. This synchronous gonadal development is probably important for ensuring spawning success.Further, there were no correlations between the male's GSI and environmental factors in thesemonthly sampling regime. This suggests that gonadal development in the male Thai carp is probablycontrolled by endogenous cues.The structure of the testes of the Thai carp corresponds to the lobular type which is commonlyfound in teleosts. Histological analysis of the testes revealed a continuous spermatogenetic activitywhich was indicated by the presence of all germ cell stages throughout the year. However,spermatozoa were always the most frequent cell type in the testes which coincided with the observationthat milt can be stripped from the fish throughout the year. This indicted a prolonged and constantreadiness for spawning which may allow them to be opportunistic in their responses to suitablespawning conditions.Plasma hormones levels in the male Thai carp exhibited a pattern of seasonal changes asobserved in females. However, there were no statistically significant correlations between seasonalchanges in plasma hormone levels and testicular development. Plasma GtH, T and 11-KT establisheda similar pattern of seasonal changes, but only plasma T and 11-KT demonstrated a highly positivecorrelation. Also, the concentration of plasma 11-KT was always higher than that of T.131The first peak of plasma GtH, T and 11-KT was observed at the time when GSI began toincrease. This indicates the importance of these hormones as endogenous factors in the initiation oftesticular development in the male Thai carp. The second peak in plasma hormones levels wasobserved during the spawning period. This suggests the involvement of GtH and androgen in theprocess of spawning.B. INTERACTION OF SGNRHA AND DOMPERIDONE IN THE REGULATION OFGONADOTROPIN SECRETION AND INDUCTION OF SPAWNING IN THE FEMALETHAI CARPThe results from the current study indicate that GtH secretion in the Thai carp is regulated byGnRH and dopamine Application of either synthetic sGnRHA or Dom, a dopamine antagonist, aloneat an appropriate concentration (5-10 µg/kg or 10 mg/kg respectively) significantly increased plasmaGtH level at 3 and 6 hr after injection. However, the magnitude of increasing GtH levels stimulated byeither sGnRHA or Dom alone was insufficient to induce spawning in the female Thai carp.Interestingly, in the 1990 study, application of Dom alone at 25 mg/kg induced 4 out of 9 fishto spawn. This indicated that in the situation where dopamine receptors are completely blocked by theapplication of a dopamine receptors antagonist such as Dom, the level of exogenous GnRH is highenough to stimulate the preovulatory surge in GtH and to induce spawning in this species. Thus, thisresult suggests that under natural conditions dopamine plays an important role, by acting as GRIF, inthe regulation of GtH secretion and spawning in the Thai carp.Application of various combinations of sGnRHA and Dom increased both the plasma GtHlevels and the number of fish spawning in a dose-related manner. This suggests the interaction ofsGnRHA and Dom on the regulation of GtH secretion in the Thai carp. This interaction of sGnRHA132and Dom probably involves changes in the number of pituitary receptors for GnRH and dopamine ashas been suggested in goldfish (Omeljaniuk et al. 1989a).The potentiation effect of Dom on the response to sGnRHA can be observed only when Domwas administered at or higher than 5 mg/kg, the most effective dosage. Application of Dom at aconcentration higher than this dosage did not increase the magnitude of increasing plasma GtH levelsand the number of fish spawning, but appeared to prolong the action of sGnRHA.The lower concentrations of sGnRHA used in the 1991 experiment failed to induced 100%spawning in all treatment groups, though the concentration of Dom was higher than 5 mg/kg. Thissuggests the importance of sGnRHA in the regulation of GtH secretion and increasing the number offish spawning.From the economic and practical points of view, the most effective dosage of sGnRHA andDom for induction of spawning in the female Thai carp is probably 5 µg/kg and 5 mg/kg respectively.Owning to its low cost, a higher concentration of Dom could be administered.C. HORMONAL CHANGES DURING SGNRHA AND DOMPERIDONE INDUCED SPAWNINGIN THE THAI CARP1. FemalesAdministration of sGnRHA in combination with Dom at an appropriate concentration causeda preovulatory surge in GtH levels. This surge in GtH then triggered the processes of fmal maturation,ovulation and spawning in the female Thai carp. GtH decreased significantly after spawning, butremained higher than the pre-treatment levels.Plasma E2 and T levels increased significantly during the induction period, probably due togonadotropin-stimulation of the follicles. A clear shift in the production of E2 to T, which is commonly133seen in salmonids (Fostier and Jalabert, 1982; Kagawa et al. 1983; Scott et al. 1983; Van Der Kraak etal. 1984; Dye et al. 1986), was observed when the fish were sampled at 3 hr intervals. However, a shortterm observation during the spawning period revealed that both E2 and T peaked at spawning time.Both E2 and T gradually decreased after the onset of spawning, following the decline in plasma GtH.Though 17,20/3-P has been shown to be the most potent MIS in a number of teleost species, inthe female Thai carp, plasma 17,20p-P levels were almost undetectable at all sampling times. This isprobably due to the short-term secretion and/or rapid plasma clearance rate of this steroid as has beenshown in goldfish (Kobayashi et al. 1987). Further, it is possible that 17,20fl-P is not the MIS in theThai carp. Recent studies of oocyte final maturation have revealed that the trihydroxylatedprogesterone derivative, 17,20/3,21-P, is the MIS in several fishes (Scott and Canario, 1987; 1988;Thomas and Trant, 1989).2. MalesNo significant changes in plasma GtH levels were observed in untreated male Thai carp duringthe process of spawning when blood was sampled at 3 hr intervals. However, during short termobservations, a significant increase in plasma GtH was seen at the onset of spawning, which rapidlydecreased 30 min later. This suggests that the GtH surge in male Thai carp can be stimulated byexposure to ovulatory females. This surge in plasma GtH is probably involved in triggering sexualbehavior and the process of sperm release in the male Thai carp.Plasma T increased gradually in untreated male Thai carp after placement with preovulatoryfemales. A peak of T was observed immediately at the time of spawning and at the time of GtH surge.There were no significant changes in plasma 11-KT levels when the fish were sampled at 3 hr intervals,but a peak of 11-KT was observed at spawning time during the short term observation study. Thissurge in plasma T and 11-KT is probably stimulated by elevation of GtH. Both T and 11-KT continued134to increase during the process of spawning, while GtH immediately decreased to a lower level. Thissuggests the importance of these androgens in the process of spawning in the male Thai carp.Furthermore, the changes in plasma 11-KT were synchronous with the occurrence of spawning. Thissuggests that 11-KT is probably the main androgen in the process of spawning in male Thai carp.D. BIOLOGICAL ACTIVITIES OF GNRHS AND THEIR ANALOGS IN COMBINATION WITHDOMPERIDONE ON THE INDUCTION OF GONADOTROPIN SECRETION ANDSPAWNING IN THE FEMALE THAI CARPHigh concentrations (25 µg/kg Bw) of either native mammalian, avian, and piscine GnRHsand their analogs, in combination with domperidone (25 mg/kg) were equally effective in inducingspawning in the female Thai carp. At a lower concentration of GnRHs (10 µg/kg Bw) in combinationwith 10 mg/kg Dom, however, cGnRH-I failed to induce spawning and to stimulate GtH secretion,while mGnRH was relatively ineffective. sGnRH and cGnRH-II were found to be the most potent ofthe natural GnRHs in stimulating GtH secretion and inducing spawning in female Thai carp. sGnRHand cGnRH-II have been shown to be present in several species such as goldfish (Yu et al. 1988),African catfish (Sherwood et al. 1989) and Thai catfish (Ngamvongchon et a1. 1991). This suggests thatthey may exist as a native GnRH in the Thai carp.Substitution of the glycinamide at position 10 of the native mGnRH or its analogs with theethylamide residue [Pro9-NHEt], which increases the binding affinity of mGnRH in mammalian assays,did not effect the potency of these GnRHs. This suggests that the binding affinity of the Thai carppituitary GnRH receptor for this analog may differ from that of mammals.Substitution of the glycine residue in position 6 of mGnRH with hydrophobic or aromatic D-amino acids demonstrated greater potencies in inducing spawning and increasing and prolongingplasma GtH concentration in the female Thai carp compared to the native forms. In mammals, this135substitution has been found to be important in either increasing receptor binding affmity and/or anincreasing hydrophobicity and providing greater resistance to enzymatic degradation. This suggeststhat the rate of degradation seems to be important in determining the degree of superactivity of GnRHanalogs in the Thai carp.[D-Ala6]-mGnRHA and [D-Trp6]-mGnRHA were found to be the most potent peptides ofthose tested in this study. However, their potency was not significantly different from sGnRHA, [D-Ser(But)6]-mGnRH or Buserelin, and [D-Trp6]-mGnRH in terms of increasing the percentage of fishspawned and prolonging increased plasma GtH levels.136REFERENCESAida, K., Horose, K., Yokote, M. and Hibiya, T. 1973. Physiological studies on gonadal maturation offishes-II. Histological changes in the liver cells of Ayu following gonadal maturation andestrogen administration. Bull. Japan. Soc. Sci. Fish. 39(11): 1107-1115.Barry, T. P., Santos, A. J. G., Furukawa, K., Aida, K. and Hanyu, I. 1990. Steroid profiles duringspawning in male common carp. Gen. Comp. Endocrinol. 80: 223-231.Baynes, S. M. and Scott, A. P. 1985. Seasonal variations in parameters of milt production and in plasmaconcentration of sex steroids of male rainbow trout (Salmo gairdneri). Gen. Comp.Endocrinol. 57: 150-160.Billard, R. 1974. Testosterone: Effects on the maintenance of spermatogenesis in intact andhypophysectomized goldfish Carassius auratus. I. R. C. S. 2: 1213.Billard, R. 1986. Spermatogenesis and spermatology of some teleost fish species. Reprod. Nutr.Develop. 26(4): 877-920.Billard, R., Breton, B., Fostier, A., Jalabert, B. and Weil, C. 1978. Endocrine control of the teleostreproductive cycle and its relation to external factors: Salmonid and cyprinid models. In P. J.Gaillard and H. H., Boer (eds.). Comparative endocrinology. Elsevier/North-HollandBiomedical Press, New York. pp. 37-48.Billard, R., Fostier, A., Weil, C. and Breton, B. 1982. Endocrine control of spermatogenesis in teleostfish Can. J. Fish. Aquat. Sci. 39: 65-79.Billard, R., le Gac, F. and Loir, M. 1990. Hormonal control of sperm production in teleost fish. In A.Epple, C. G. Scanes and M. H. Stetson (eds.). Progress in comparative endocrinology. Wiley-Liss, Inc., New York. pp. 329-335.Breton, B., Jalabert, B. and Billard, R. 1973. Pituitary and plasma gonadotropin levels andspermatogenesis in the goldfish Carassius auratus after methallibure treatment. J. Endocr. 59:415-420.Breton, B., Horoszewicz, L., Billard, R. and Bieniarz, K. 1980. Temperature and reproduction in tench:Effect of a rise in the temperature regime on gonadotropin level, gametogenesis and spawning.I. Case in the male. Reprod. Nutr. Develop. 20: 105-118.Burke, M. G., Leatherland, J. F. and Sumpter, J. P. 1984. Seasonal changes in serum testosterone, 11-ketotestosterone, and 17P-estradiol levels in the brown bullhead, Ictalurus nebulosus Lesueur.Can. J. Zool. 62: 1195-1199.Canario, A. V. M. and Scott, A. P. 1988. Structure-activity relationships of C21 steroids in an in vitrooocyte maturation bioassay in rainbow trout, Salmo gairdneri. Gen. Comp. Endocrinol. 71:338-348.137Cardwell, J. R. 1989. Behavioural endocrinology in a wild population of the stoplight parrotfish,Sparisoma viride, Scaridae, a protogynous coral reef fish Ph.D. thesis, University of BritishColumbia, Vancouver, B.C., Canada. 129 pp.Chang, C. F. and Chen, M. R. 1990. Fluctuation in sex steroids and sex steroid-binding protein duringthe development and annual cycle of the male common carp, Cyprinus carpio. Comp. Biochem.Physiol. 97A(4): 565-568.Chang, J. P. and Peter, R. E. 1983. Effects of dopamine on gonadotropin release in female goldfish,Carrasius auratus. Neuroendocrinology. 36: 351-357.Chang, J. P., Mackenzie, D. S., Gould, D. R. and Peter, R. E. 1984. Effects of dopamine andnorepinephine on in vitro spontaneous and gonadotropin-releasing hormone-inducedgonadotropin release by dispersed cells or fragments of the goldfish pituitary. Life Sci. 35:2027-2033.Chaudhuri, H. 1968. Breeding and selection of cultivated warm-water fishes in Asia and the Far East-areview. FAO Fish. Rep. 4(44): 30-66.Clemens, H. P. and Reed, C. A. 1967. Long term gonadal growth and maturation of goldfish (Carassiusauratus) with pituitary injections. Copeia. pp. 465-466.Colombo, L., Belvedere, P. C., Simontacchi, C. and Lazzari, M. 1987. Shift from androgen toprogesterone biosynthesis by the testes of the northern pike, Exox lucius L., during transitionfrom spermatogenesis to spermiation. Gen. Comp. Endocrinol. 66: 18.Conn, P. M., Hsueh, A. J. W. and Crowley, F. Jr. 1984. Gonadotropin-releasing hormone: Molecularand cell biology, physiology, and clinical applications. Federation Proceedings. 43: 2351-2361.Copeland, P. and Thomas, P. 1989. Control of gonadotropin release in Atlantic croaker: Evidence forlack of dopaminergic inhibition. Gen. Comp. Endocrinol. 74: 474-483.Crim, L. W. and Idler, D. R. 1978. Plasma gonadotropin, estradiol, and vitellogenin and phosvitin levelsin relation to the seasonal reproductive cycles of female brown trout. Ann. Biol. Anim.Biochem. Biophys. 18: 1001-1005.Crim, L. W. 1982. Environmental modulation of annual and daily rhythms associated with reproductionin teleost fishes. Can. J. Fish. Aquatic. Sci. 39(1): 17-21.Crim, L. W., Peter, R. E. and Van Der Kraak, G. 1987. The use of LHRH analogs in aquaculture. In B.H. Vickery and J. J. Nestor Jr. (eds.). LHRH and its analogs: Contraceptive and therapeuticapplications part 2. MTP Press Limited, Boston. pp. 489-498.Crim, L. W., Nestor Jr., J. J. and Wilson, C. E. 1988. Studies of the biological activity of LHRH analogsin the rainbow trout, landlocked salmon, and winter flounder. Gen. Comp. Endocrinol. 71:372-382.138Culling, C. F. A. 1974. Handbook of histopathological and histochemical techniques ( 3rd edition).Butterworth & Co. Ltd., Toronto. 712 pp.De Leeuw, R., Goos, H. J. T., Richter, C. J. J. and Eding, E. H. 1985. Pimozide modulates theluteinizing hormone-releasing hormone effect on gonadotropin release in the African catfish,Clarias lazera. Gen. Comp. Endocrinol. 58: 120-127.De Leeuw, R., Goos, H. J. T. and Van Oordt, P. G. W. J. 1986. The dopaminergic inhibition of thegonadotropin-releasing hormone-induced gonadotropin release. An in vivo study withfragments and cell suspensions from pituitaries of the African catfish, Clarias gariepinus(Burchell). Gen. Comp. Endocrinol. 63: 171-177.De Leeuw, R., Van't Veer, C., Smit-Van Dijk, W. and Goos, H. J. T. 1988. Binding affinity andbiological activity of gonadotropin-releasing hormone in the African catfish, Clarias gariepinus.Aquaculture. 71: 119-131.De Leeuw, R., Habibi, H. R., Narhorniak, C. S. and Peter, R. E. 1989. Dopaminergic regulation ofpituitary gonadotropin-releasing hormone receptor activity in the goldfish (Cyprinus carpio). J.Endocrinol. 121: 239-247.de Vlaming, V. L. 1972. Environmental control of teleost reproductive cycles: A brief review. J. Fish.Biol. 4: 131-140.de Vlaming, V. L. 1974. Environmental and endocrine control of teleost reproduction. In Control ofsex in fishes. Virginia Polytechnic Institute, Blachsburg, VA. pp. 13-83.de Vlaming, V. L. 1983. Oocyte development patterns and hormonal involvements among teleost. In J.C. Rankin, T.J. Pitcher and R. T. Duggan (eds.). Control processes in fish physiology. NewYork-Toronto, A Wiley-Interscience Publication. pp. 176-199.Donaldson, E. M. 1973. Reproductive endocrinology of fishes. Am. Zool. 13: 909-927.Donaldson, E. M. and Hunter, G. A. 1983. Induced fmal maturation, ovulation and spermiation incultured fish. In W. S. Hoar, D. J. Randall and E. M. Donaldson (eds.). Fish physiology Vol.9B. Academic Press, New York. pp. 351-403.Dye, H. M., Sumpter, J. P., Fagerlund, U. H. M. and Donaldson, E. M. 1986. Changes in reproductiveparameters during the spawning migration of pink salmon, Oncorhynchus gorbuscha(Walbaum). J. Fish Biol. 29: 167-176.FAO, 1991. Aquaculture production (1985-1988). FAO Fisheries Circular No. 815, Revision 2. FAOInformation, Data and Statistic Service, Fisheries Department, FAO. 135 pp.Fitzpatrick, M. S., Van Der Kraak, G. and Schreck, K. 1986. Profiles of plasma sex steroids andgonadotropin in coho salmon, Oncorhynchus kisutch, during final maturation. Gen. Comp.Endocrinol. 62: 437-451.139Fostier, A., Weil, C., Terqui, M., Breton, B. and Jalabert, B. 1978. Plasma estradiol 17/3 andgonadotropin during ovulation in rainbow trout (Salmo gairnderi). Ann. Biol. Anim. Biochem.Biophys. 18: 929-936.Fostier, A. and Jalabert, B. 1982. Physiological basis of practical means to induce ovulation in fish. InH. J. Th. Goos and C. J. J. Richter (eds.). Proceedings of the international symposium onreproductive physiology of fish. Pudoc, Wageningen, the Natherland. pp. 164-173.Fostier, A., Billard, R. and Breton, B. 1982. Plasma 11-oxotestosterone and gonadotropin during thebeginning of spermiation in rainbow trout (Salmo gairdneri R.). Gen. Comp. Endocrinol. 46:428-438.Fostier, A., Jalabert, B., Billard, R., Breton, B. and Zohar, Y. 1983. The gonadal steroids. In W. S.Hoar, D. J. Randall and E. M. Donaldson (eds.). Fish physiology Vol. 9A. Academic Press,New York. pp. 277-372.Fujino, J., Kobayashi, S., Obayashi, M., Yamazaki, S., Nakahama, R., White, W. F. and Rippel, R. H.1972. Structure-activity relationships in the C-terminal part of luteinizing hormone-releasinghormone(LH-RH). Biochem. Biophys. Res. Commun. 49: 863-869.Galas, J. and Bieniarz, K. 1989. Seasonal changes of sex steroids in mature female and male carp(Cyprinus carpio L.). Pol. Arch. Hydrobiol. 36(3): 407-416.Goetz, F. W. 1983. Hormonal control of oocyte final maturation and ovulation in fishes. In W. S. Hoar,D. J. Randall and E. M. Donaldson (eds.). Fish physiology Vol. 9B. Academic Press, NewYork. pp. 117-170.Gupta, S. 1975. The development of carp gonads in warm water aquaria. J. Fish Biol. 7: 775-782.Habibi, H. R., Peter, R. E., Sokolowska, M., Rivier, J. E. and Vale, W. W. 1987. Characterization ofgonadotropin-releasing hormone (GnRH) binding to pituitary receptors in goldfish (Carassiusauratus). Biol. Reprod. 36: 844-853.Habibi, H. R., De Leeuw, R., Nahorniak, C. S., Goos, H. J. T. and Peter, R. E. 1989. Pituitarygonadotropin-releasing hormone (GnRH) receptor activity in goldfish and catfish: Seasonaland gonadal effects. Fish Physiol. Biochem. 7: 109-118.Haug, T. and Gulliksen, B. 1988. Variations in liver and body condition during gonad development ofAtlantic halibut, Hippoglossus hippoglossus (L.). FiskDir. Ski.. Ser. HavUnders. 18: 351-363.Hora, S. S. and Pillay, T. V. R. 1962. Handbook of fish culture in the Indo-Pacific region. FAOFisheries Biology Technical Paper, No. 14. pp. 81-83.Htun-Han, M. 1978. The reproductive biology of the dab Limanda limanda (L.) in the North Sea:Seasonal changes in the testis. J. Fish Biol. 13: 361-367.140Hunt, S. M. V., Simpson, T. H. and Wright, R. S. 1982. Seasonal changes in the levels of 11-oxo-testosterone and testosterone in the serum of male salmon, Salmo salar L., and theirrelationship to growth and maturation. J. Fish. Biol. 20: 105-119.Idler, D. R., Bitners, I. I. and Schmidt, P. J. 1961. 11-ketotestosterone: An androgen for sockeyesalmon. Can. J. Biochem. Physiol. 39: 1737-1742.Idler, D. R. 1982. Some perspectives on fish gonadotropins. In H. J. Th. Goos and C. J. J. Richter(eds.). Proceedings of the international symposium on reproductive physiology of fish. Pudoc,Wageningen, the Natherland. pp. 4-13.Idler, D. R. and Ng, T. B. 1983. Teleost gonadotropins: Isolation, biochemistry, and function. In W. S.Hoar, D. J. Randall and E. M. Donaldson (eds.). Fish physiology Vol. 9A. Academic Press,New York. pp. 187-221.Idler, D. R. and So, P. 1987. Carbohydrate poor gonadotropins. In D. R. Idler, L. W. Crim and J. M.Walsh (eds.). Proceedings of the third international symposium on reproductive physiology offish Memorial University, St. John's, Newfoundland, Canada. pp. 57-60.Itoh, H., Suzuki, K. and Kawauchi, H. 1988. The complete amino acid sequences of fl-subunits of twodistinct chum salmon GTHs. Gen. Comp. Endocrinol. 71: 438-451.Jalabert, B. 1976. In vitro oocyte maturation and ovulation in rainbow trout (Salmo gairdneri), northernpike (Esox lucius) and goldfish (Carassius auratus). J. Fish. Res. Board Can. 33: 974-988.Jalabert, B. and Fostier, A. 1984. The modulatory effect in vitro of estradio1-17P, testosterone orcortisol on the output of 17a-hydroxy-20fl-dihydroprogesterone by rainbow trout (Salmogairdneri) ovarian follicles stimulated by the maturational gonadotropin sGTH. Reprod. Nutr.Dev. 24: 127-136.Kagawa, H., Young, G., Adachi, S. and Nagahama, Y. 1982. Estradiol-17fl production in amagosalmon (Oncorhynchus rhodurus) ovarian follicles: Role of theca and granulosa cells. Gen.Comp. Endocrinol. 47: 440-448.Kagawa, H., Young, G. and Nagahama, Y. 1983. Changes in plasma steroid hormone levels duringgonadal maturation in female goldfish Carassius auratus. Bull. Japan. Soc. Sci. Fish. 49(12):1783-1787.Kagawa, H., Young, G. and Nagahama, Y. 1984. In vitro estradiol-17fl and testosterone production byovarian follicles of the goldfish, Carassius auratus. Gen. Comp. Endocrinol. 54: 139-143.Karten, M. J. and Rivier, J. E. 1986. Gonadotropin-releasing hormone analog design. Structure-function studies toward the development of agonists and antagonists: Rationale andperspective. Endocr. Rev. 7: 44-66.141Kawauchi, H., Suzuki, K, Nagahama, Y., Adachi, S. and Naito, N. 1986. Occurrence of two distinctgonadotropins in chum salmon pituitary. In F. Yoshimaru and A. Gorbman (eds.). ParsDistalis of the pituitary gland: Structure, function and regulation. Elsevier Science PublishersB. V., Amsterdam. pp. 383-390.Kawauchi, H., Suzuki, K, Itoh, H., Swanson, P. and Nagahama, Y. 1987. Duality of salmon pituitarygonadotropins. In E. Ohnishi and Y. Nagahama (eds.). Proceedings of the first congress Asianand Oceania society of comparative endocrinology. Nagoya, Japan. pp. 15-18.Kawauchi, H., Suzuki, K, Itoh, H., Swanson, P., Naito, N., Nagahama, Y., Nozaki, M., Nakai, Y. andItoh, S. 1989. The duality of teleost gonadotropins. Fish Physiol. Biochem. 7: 29-38.Kime, D. E. and Dolben, I. P. 1985. Hormonal changes during induced ovulation of the carp, Cyprinuscarpio. Gen. Comp. Endocrinol. 58: 137-149.Kime, D. and Bieniarz, K 1987. Gonadotropin-induced changes in steroid production by ovaries of thecommon carp Cyprinus carpio L. around the time of ovulation. Fish Physiol. Biochem. 3: 49-52.King, J. A. and Millar, R. P. 1982a. Structure of avian hypothalamic gonadotropin-releasing hormone.S. Afr. J. Sci. 78: 124-125.King, J. A. and Millar, R. P. 1982b. Structure of chicken hypothalamic luteinizing hormone-releasinghormone. I. Structural determination on partially purified material. J. Biol. Chem. 257: 10722-10732.King, J. A. and Millar, R. P. 1985. Multiple molecular forms of gonadotropin-releasing hormone inteleost fish brain. Peptides. 6: 689-694.Kobayashi, M., Aida, K and Hanyu, I. 1986a. Annual changes in plasma levels of gonadotropin andsteroid hormones in goldfish. Bull. Japan. Soc. Sci. Fish. 52(7): 1153-1158.Kobayashi, M., Aida, K and Hanyu, I. 1986b. Effects of HCG on milt amount and plasma levels ofsteroid hormones in male goldfish. Bull. Japan. Soc. Sci. 52(4): 755.Kobayashi, M., Aida, K and Hanyu, I. 1986c. Gonadotropin surge during spawning in male goldfish.Gen. Comp. Endocrinol. 62: 70-79.Kobayashi, M., Aida, K and Hanyu, I. 1987. Hormone changes during ovulation and effects of steroidhormones on plasma gonadotropin levels and ovulation in goldfish. Gen. Comp. Endocrinol.67: 24-32.Kobayashi, M., Aida, K and Hanyu, I. 1988. Hormone changes during the ovulatory cycle in goldfish.Gen. Comp. Endocrinol. 69: 301-307.Kobayashi, M., Aida, K. and Hanyu, I. 1989. Involvement of steroid hormone in the preovulatorygonadotropin surge in female goldfish. Fish Physiol. Biochem. 7: 141-146.142Koldras, M., Bieniarz, K. and Kime, D. E. 1990. Sperm production and steroidogenesis in testes of thecommon carp, Cyprinus carpio L., at different stages of maturation. J. Fish Biol. 37: 635-645.Lam, T. J. 1983. Environmental influences on gonadal activity in fish. In W. S. Hoar, D. J. Randall andE. M. Donaldson (eds.). Fish physiology Vol. 9B. Acdemic Press, New York. pp. 65-116.Lam, T. J. and Munro, A. D. 1987. Environmental control of reproduction in teleost: An overview. InD. R. Idler, L. W. Crim and J. M. Walsh (eds.). Proceedings of the third internationalsymposium on reproductive physiology of fish Memorial University, St. John's, Newfoundland,Canada. pp. 279-288.Lamba, V. J., Goswami, S. V. and Sundararaj, B. I. 1983. Circannual and ciradian variations in plasmalevels of steroids (cortisol, estradiol-17fl, estron, and testosterone) correlated with the annualgonadal cycle in the catfish, Heteropneustes fossilis (Bloch). Gen. Comp. Endocrinol. 50: 205-225.Lambert, J. G. D., Bosman, G. I. C. G. M., van den Hurk, R. and van Oordt, P. G. W. J. 1978. Annualcycle of plasma oestradiol-17fl in the female trout Salmo gairdneri. Ann. Biol. Anim. Biochem.Biophys. 18: 923-927.Lambert, J. G. D. and van den Hurk, R. 1982. Steroidogenesis in the ovaries of the African catfish,Clarias gariepinus, before and after HCG induced ovulation. Proceedings of the internationalsymposium on reproductive physiology of fish Pudoc, Wageningen, the Natherland. pp. 99-102.Leelapatra, W. 1988. Carps culture in Thailand with particular emphasis on induced spawning.Proceedings of the aquaculture international congress and exposition. Vancouver, B.C.Canada. pp. 331-337.Lehri, G. K. 1967. The annual cycle in the testis of the catfish Clarias batrachus L. Acta Anat. 67: 135-154.Levavi-Zermonsky, B. and Yaron, Y. 1986. Changes in gonadotropin and ovarian steroids associationwith oocyte maturation during spawning induction in the carp. Gen. Comp. Endocrinol. 62: 89-98.Licht, P., Farmer, S. W., Muller, S. W., Tsui, H. W. and Crews, D. 1977. Evolution of gonadotropinstructure and function. Rec. Prog. Horm. Res. 33: 169-248.Liley, N. R. and Stacey, N. E. 1983. Hormones, pheromones and reproductive behaviour in fish. In W.S. Hoar, D. J. Randall and E. M. Donaldson (eds.). Fish physiology Vol. 9B. Acdemic Press,New York. pp. 1-49.Liley, N. R. and Tan, E. S. P. 1985. The induction of spawning behaviour in Puntius gonionotus(Bleeker) by treatment with prostaglandin PGF2a . J. Fish Biol. 26: 491-502.143Liley, N. R., Fostier, A., Breton, B. and Tan, E. S. P. 1986a. Endocrine changes associated withspawning behaviour and social stimuli in a wild population of rainbow trout (Salmo gairdneri)II. Females. Gen. Comp. Endocrinol. 62: 157-167.Liley, N. R., Breton, B., Fostier, A. and Tan, E. S. P. 1986b. Endocrine changes associated withspawning behaviour and social stimuli in a wild population of rainbow trout (Salmo gairdneri)I. males. Gen. Comp. Endocrinol. 62: 145-156.Liley, N. R. and Rouger, Y. 1990. Plasma levels of gonadotropin and 17a,20/3-dihydroxy-4-pregnen-3-one in relation to spawning behaviour of rainbow trout, Oncorhynchus mykiss (Walbaum). J.Fish. Biol. 37: 699-711.Lin, H. R., Peng, C., Lu, L. Z., Zhou, X. J., Van Der Kraak, G. and Peter, R. E. 1985 Induction ofovulation in the loach (Parmisgumus dabryanus) using pimozide and [D-Ala°, Pro9-N-ethylamide]-LHRH. Aquaculture. 46: 333-340.Lin, H. R. and Peter, R. E. 1986. Induction of gonadotropin secretion and ovulation in teleosts usingLHRH analogs and catecholaminergic drugs: A review. In J. L. Maclean, L. B. Dizon and L.V. Hosillos (eds.). The First Asian Fisheries Forum. Asian Fisheries Society, Manila,Philippines. pp. 667-670.Lin, H. R., Van Der Kraak G. Zhou, X. J., Liang, J. Y., Peter, R. E., Rivier, E. and Vale, W. W. 1988.Effects of [D-Arg6, T;p, Leu8, Pro9 NEt]-luteinizing hormone-releasing hormone (sGnRH-A) and [D-A1a6, Pro9 NEt]-luteinizing hormone-releasing hormone (LHRH-A), incombination with pimozide or domperidone, on gonadotropin release and ovulation in theChinese loach and common carp. Gen. Com. Endocrinol. 69: 31-40.MacKenzie, D. S., Thomas, P. and Farrar, S. M. 1989. Seasonal changes in thyroid and reproductivesteroid hormones in female channel catfish (ktalurus punctatus) in pond culture. Aquaculture.78: 63-80.Matsuyama, S., Adachi, S., Nagahama, Y. and Matsuura, S. 1988. Diurnal rhythm of oocytedevelopment and plasma steroid hormone levels in the female red sea bream, Pagrus major,during the spawning season. Aquaculture. 73: 357-372.Minning, N. J. and Kime, D. E. 1984. Temperature regulation of ovarian steroid production in thecommon carp, Cyprinus carpio L., in vivo and in vitro. Gen. Comp. Endocrinol. 56: 376-388.Miyamoto, K., Hasegawa, Y., Nomura, M., Igarashi, M., Kangawa, K. and Matsuo, H. 1984.Identification of the second gonadotropin-releasing hormone in chicken hypothalamus:Evidence that gonadotropin secretion is probably controlled by two distinct gonadotropin-releasing hormones in avian species. Proc. NAt1. Acad. Sci. U.S.A. 81: 3874-3878.Monahan, M. W., Amoss, M. S., Anderson, H. A. and Vale, W. W. 1973. Synthetic analogs of thehypothalamic luteinizing hormone releasing factor with increased agonist or antagonistproperties. Biochemistry. 12: 4619-4620.144Munkittrick, K. R. and Leatherland, J. F. 1984. Seasonal changes in the pituitary-gonad axis of feralgoldfish, Carassius auratus L., from Ontario, Canada. J. Fish Biol. 24: 75-90.Munro, A. D. 1990. Tropical freshwater fishes. In A. D. Munro, A. P. Scott and T. J. Lam (eds.).Reproductive seasonality in teleosts: Environmental influences. CRC Press Inc., Boca Raton,Florida. pp. 145-240.Nagahama, Y. 1987. Review: Gonadatropin action on gametogenesis and steroidogenesis in teleostgonads. Zool. Sci. 4: 209-222.Nestor, J. J. Jr. 1984. Development of agonistic LHRH analogs. In B. H. Vickery, J. J. Nestor Jr., andE. S. E. Hafez (eds.). LHRH and its analogs: Contraceptive and therapeutic applications.Boston, MTP Press Limited. pp. 3-10.Ngamvongchon, S., Kok, L. Y. and Takashima, F. 1987. Changes in endocrine profiles and spermiationresponse in carp after LHRH analogue injection. Bull. Jap. Soc. Sci. Fish. 53(2): 229-234.Ngamvongchon, S., Lovejoy, D. A. and Sherwood, N. M. 1991. Characterization of the primarystructure of gonadotropin-releasing hormone in the Thai catfish (Clarias macrocephalus). InA. P. Scott, J. P. Sumpter, D. E. Kime and M. S. Rolfe (eds.). Proceedings of the 4thinternational symposium on the reproductive physiology of fish. University of East Anglia,Norwich, U. K. p. 64.Nozaki, M., Naito, N., Swanson, P., Miyata, K., Nakai, Y., Oota, Y., Suzuki, K. and Kawauchi, H.1990a. Salmonids pituitary gonadotropins. I. Distinct cellular distributions of twogonadotropins, GtH I and GtH II. Gen. Comp. Endocrinol. 77: 348-357.Nozaki, M., Naito, N., Swanson, P., Dickhoff, W. W., Suzuki, K. and Kawauchi, H. 1990b. Salmonidspituitary gonadotropins. II. Ontogeny of GtH I and GtH II cells in the rainbow trout (Salmogairdneri irideus). Gen. Comp. Endocrinol. 77: 358-367.Omeljaniuk, R. J., Habibi, H. R. and Peter, R. E. 1987a. Actions of a GnRH-agonist and a dopamine-antagonist on pituitary GnRH and dopamine receptors in the goldfish. In D. R. Idler, L. W.Crim and J. M. Walsh (eds.). Proceedings of the third international symposium onreproductive physiology of fish. Memorial University, St. John's, Newfoundland, Canada. pp.35.Omeljaniuk, R. J., Shih, S. H. and Peter, R. E. 1987b. In vivo evaluation of dopamine receptor-mediated inhibition of gonadotropin secretion from the pituitary of the goldfish Carassiusauratus. J. Endocrinol. 114: 449-458.Omeljaniuk, R. J., Habibi, H. R. and Peter, R. E. 1989a. Alterations in pituitary GnRH and dopaminereceptors associated with the seasonal variation and regulation of gonadotropin release in thegoldfish (Carassius auratus). Gen. Comp. Endocrinol. 74: 392-399.Omeljaniuk, R. J., Tonon, M. C. and Peter, R. E. 1989b. Dopamine inhibition of gonadotropin andalpha-melanocyte-stimulating hormone release in vivo from the pituitary of the goldfish(Carassius auratus). Gen. Comp. Endocrinol. 74: 451-467.145Pankhurst, N. W. and Stacey, N. E. 1985. The effect of 17/3-estradiol on spontaneous ovulation in thegoldfish, Carassius auratus. Can. J. Zool. 63: 2979-2981.Pankhurst, N. W. and Conroy, A. M. 1987. Seasonal changes in reproductive condition and plasmalevels of sex steroids in blue cod, Parapercis colias (Bloch and Schneider) (Mugiloidae). FishPhysiol. Biochem. 4: 15-26.Parameswaran, S., Selvaraj, C. and Radhakriahnan, S. 1970. Observations on the maturation andbreeding season of carps in Assam. J. Inland Fish. Soc. India. 2: 16-29.Patin, R. and Thomas, P. 1990. Gonadotropin stimulates 17a,20/3,21-trihydroxy-4-pregnen-3-oneproduction from endogenous substrates in Atlantic croaker ovarian follicles undergoing finalmaturation in vitro. Gen. Comp. Endocrinol. 78: 474-478.Peter, R. E. and Crim, L. W. 1979. Reproductive endocrinology of fishes: Gonadal cycle andgonadotropin. Ann. Rev. Physiol. 41: 323-335.Peter, R. E. 1981. Gonadotropin secretion during reproductive cycles in teleosts: Influences ofenvironmental factors. Gen. Comp. Endocrinol. 45: 294-305.Peter, R. E. 1983. The brain and neurohormones in teleost reproduction. In W. S. Hoar, D. J. Randalland E. M. Donaldson (eds.). Fish physiology Vol. 9B. Academic Press, New York. pp.97-135.Peter, R. E., Nahorniak, C. S., Chang, J. P. and Crim, L. W. 1984a. Gonadotropin release from the parsdistalis of goldfish, Carassius auratus, transplanted beside the brain or into the brain ventricles:Additional evidence for gonadotropin release-inhibitory factor. Gen. Comp. Endocrinol. 55:337-346.Peter, R. E., Sokolowska, M., Truscott, B., Walsh, J. and Idler, D. R. 1984b. Secretion of progestergensduring induced ovulation in goldfish. Can. J. Zool. 62: 1946-1949.Peter, R. E., Nahorniak, C. S., Sokolowska, M., Chang, J. P., Rivier, J. E., Vale, W. W., King, J. A. andMillar, R. P. 1985. Structure-activity relationships of mammalian, chicken, and salmongonadotropin-releasing hormones in vivo in goldfish. Gen. Com . Endocrinol. 58: 231-242.Peter, R. E., Chang, J. P., Nahorniak, C. S., Omeljaniuk, R. J., Sokolowska, M., Shih, S. H. and Billard,R. 1986. Interactions of catecholamines and GnRH in regulation of gonadotropin secretion inteleost fish Recent Prog. Horm. Res. 42: 513-548.Peter, R. E., Lin, H R and Van Der Kraak, G. 1987a. Drug/hormone induced breeding of Chineseteleosts. In D. R. Idler, L. W. Crim and J. M. Walsh (eds.). Proceedings of the thirdinternational symposium on reproductive physiology of fish. Memorial University, St.' John,Newfoundland, Canada. pp. 120-123.Peter, R. E., Nahorniak, C. S., Shih, S., King, J. A. and Millar, R. P. 1987b. Activity of position-8-substituted analogs of mammalian gonadotropin-releasing hormones in goldfish. Gen. Corn.Endocrinol. 65: 385-393.146Qasim, S. Z. and Qayyum, A. 1961. Spawning season and breeding frequencies of some freshwaterfishes with special reference to those occurring in the plains of Northern India. Indian J. Fish.8: 24-43.Santos, A. J. G., Furukawa, K., Kobayashi, M., Bando, K., Aida, K. and Hanyu, I. 1986. Plasmagonadotropin and steroid hormone profiles during ovulation in the carp, Cyprinus carpio. Bull.Japan. Soc. Sci. Fish. 52: 1159-1166.Schreck, C. B. and Hopwood, M. L. 1974. Seasonal androgen and estrogen patterns in the goldfish,Carassius auratus. Trans. Am. Fish Soc. 103: 375-378.Schwassmann, H. 0. 1978. Times of annual spawning and reproductive strategies in Amazonian fishes.In J. E. Thorpe (ed.). Rhythymic activity of fishes, Academic Press, New York. pp. 187-200.Scott, A. P., Bye, V. J. and Baynes, S. M. 1980a. Seasonal variation in sex steroids of female rainbowtrout (Salmo gairdneri Richardson). J. Fish Biol. 17: 587-592.Scott, A. P., Bye, V. J., Baynes, S. M. and Springate, J. R. C. 1980b. Seasonal variations in plasmaconcentrations of 11-ketotestosterone and testosterone in male rainbow trout, Salmo gairdneriRichardson. J. Fish Biol. 17: 495-505.Scott, A. P. and Baynes, S. M. 1982. Plasma levels of sex steroids in relation to ovulation andspermiation in rainbow trout (Salmo gairdneri). In H. J. Th. Goos and C. J. J. Richter (eds.).Proceedings of the international symposium on reproductive physiology of fish Pudoc,Wageningen, the Natherland. pp. 103-106.Scott, A. P., Sheldrick, E. and Flint, A. P. 1982. Measurement of 17a,20P-dihydroxy-4-pregene-3-onein plasma of trout (Salmo gairneri Richardson): Seasonal changes and response to salmonpituitary extract. Gen. Comp. Endocrinol. 46: 444-451.Scott, A. P. and Sumpter, J. P. 1983. 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.Scott, A. P., Sumpter, J. P. and Hardiman, P. A. 1983. Hormone changes during ovulation in therainbow trout (Salmo gairdneri). Gen. Comp. Endocrinol. 49: 128-134.Scott, A. P., Mackenzie, D. S. and Stacey, N. E. 1984. Endocrine changes during natural spawning inthe white sucker, Catostomus commersoni. II. Steroid hormones. Gen. Comp. Endocrinol. 56:349-359.Scott, A. P. and Canario, A. V. M. 1987. Status of oocyte maturation-inducing steroids in teleosts. In D.R. Idler, Crim, L. W. and J. M. Walsh (eds.). Proceedings of the third international symposiumon the reproductive physiology of fish. Memorial University, St. John's, Newfoundland,Canada. pp. 224-234.147Scott, A. P. and Sumpter, J. P. 1989. Seasonal variation in testicular germ cell stages and in plasmaconcentrations of sex steroids in male trout (Salmo gairdneri) maturing at 2 years old. Gen.Comp. Endocrinol. 73: 46-58.Selman, K. and Wallace, R. A. 1989. Cellular aspects of oocyte growth in teleosts. Zool. Sci. 6: 211-231.Sherwood, N. M., Eiden, L., Brownstein, M., Spicess, J., Rivier, J. and Vale, W. 1983. Characterizationof a teleost gonadotropin-releasing hormone. Proc. Natl. Acad. Sci. USA. 80: 2794-2798.Sherwood, N. M., Harvey, B., Broownstein, M. J. and Eiden, L. E. 1984. Gonadotropin-releasinghormone (Gn-RH) in striped mullet (Mugil cephalus), milkfish (Chanos chanos), and rainbowtrout (Salmo gairdneri): Comparison with salmon Gn-RH. Gen. Comp. Endocrinol. 55: 174-181.Sherwood, N. 1987. Gonadotropin-releasing hormones in fishes. In D. 0. Norris and R. E. Jones (eds.).Hormones and reproduction in fishes, amphibians, and reptiles. Plenum Press, New York. pp.31-61.Sherwood, N. M., De Leeuw, R. and Goos, H. 1989. A new member of the gonadotropin-releasinghormone family in teleosts: Catfish gonadotropin-releasing hormone. Gen. Com . Endocrinol.75: 427-436.Shimizu, A., Aida, K. and Hanyu, I. 1985. Endocrine profiles during the short reproductive cycle of theautumn-spawning bitterling, Acheilognathus rhombea. Gen. Comp. Endocrinol. 60: 361-371.Silverman, H. I. 1978a. The effects of visual social stimulation upon age at first spawning in the mouthbrooding cichfid fish Sarotherodon (Tilapia) mosambicus (Peters). Anim. Behay. 26: 1120-1125.Silverman, H. I. 1978b. Effect of different levels of sensory contact upon reproductive activity of adultmale and female Sarotherodon mosambicus (Peters); Pisces; Cichidae. Anim. Behay. 26: 1081-1090.Singh, A. and Singh, T. P. 1991. Changes in the plasma steroid hormone levels during gonadalmaturation in the female catfish Clarias batrachus. Zool. Jb. Physiol. 95: 209-220.Sinha, V. R. P., Jhingran, V. G. and Ganapati, S. V. 1974. A review on spawning of the Indian majorcarps. Arch. Hydrobiol. 73: 518-536.Sipitakkiat, P. and Leenanond, Y. 1984. Life history and culturing of pla-tapian khao, Puntiusgonionotus Bleeker. Technical Paper No. 15, National Inland Fisheries Institute, Departmentof Fisheries, Thailand. 51 pp. (in Thai)Sirikul, C. and colleague 1986. The annaul spawning frequency of Puntius gonoinotus (Bleeker).Annual report of Nakornratchasrima Freshwater Fisheries Station, Department of Fisheries,Thailand. (in Thai)148Sloley, B. D., Trudeau, V. L., Dullca, J. G. and Peter, R. E. 1991. Selective depletion of dopamine in thegoldfish pituitary caused by domperidone. Can. J. Physiol. Pharmacol. 69: 776-781.Smith, M. A. K. and Jiffy, F. 1986. Reproductive strategy of Labeo dussmieri and implications ofhydroelectric and irrigation projects on the Mahaweli Ganga, Sri Lanka. In J. A. L. Maclean,L. B. Dizon and L. V. Hosillos (eds.). Proceedings of the First Asian Fisheries Forum. AsianFisheries Society, Manila. pp. 693-6%.Sokolowska, M., Peter, R. E., Nahorniak, C. S. and Chang, J. P. 1985. Seasonal effects of pimozide anddes G1y10 ED-Ala6] LHRH ethylamide on gonadotropin secretion in goldfish. Gen. Comp.Endocrinol. 57: 472-479.Sokolowska, M., Mikkolajczyk, T., Epler, P., Peter, R. E., Piotrowski, W. and Bieniarz, K. 1988. Theeffects of reserpine and LHRH or salmon GnRH analogues on gonadotropin release,ovulation and spermiation in common carp (Cyprinus carpio). Reprod. Nutr. Devlop. 28(4A):889-897.Stacey, N. E., Cook, A. E. and Peter, R. E. 1979. Ovulation surge of gonadotropin in the goldfish,Carassius auratus. Gen. Comp. Endocrinol. 37: 246-249.Stacey, N. E., Peter, R. E. and Cook, A. F. 1983. Changes in plasma concentrations of gonadotropin,17P-estradiol, testosterone, and 17a-hydroxy-20/3-dihydroxyprogesterone during spontaneousand brain lesion induced ovulation in goldfish. Can. J. Zool. 61: 2646-2652.Stacey, N. E. 1984. Control of the timing of ovulation by exogenous and endogenous factors. In G. W.Potts and R. J. Wootton (eds.). Fish reproduction strategies and tactics. Academic Press,London. pp. 207-222.Stacey, N. E., MacKenzie, D. S., Kyle, A. L. and Peter, R. E. 1984. Endocrine changes during naturalspawning in Catostomus commersoni. I. Gonadotropin, growth hormone, and thyroidhormones. Gen. Comp. Endocrinol. 56: 333-348.Stacey, N. E., Sorensen, P. W., Dam, J. G., Van Der Kraak, G. and Hara, T. J. 1987. Teleost sexpheromone: Recent studies on identity and function. In D. R. Idler, L. W. Crim and J. M.Walsh (eds.). Proceedings of the third international symposium on reproductive physiology offish Memorial University, St. John's, Newfoundland, Canada. pp. 150-153.Sundararaj, B. I. and Nath, P. 1981. Steroid-induced synthesis of vitellogenin in the catfish,Heteropneustes fossilis (Bloch). Gen. Comp. Endocrinol. 43: 201-210.Sundararaj, B. I., Goswami, S. V. and Lamba, V. J. 1982. Role of testosterone, estradio1-17/3, andcortisol during vitellogenin synthesis in the catfish, Heteropneustes fossilis (Bloch). Gen. Comp.Endocrinol. 48: 390-397.Sundararaj, B. I., Goswami, S V. and Lamba, V. J. 1985. Oocyte maturation in teleost fishes. In B.Lofts and W. N. Holmes (eds.). Current trends in comparative endocrinology, Vol. 1. HongKong University Press, Hong Kong. pp. 369-372.149Suzuki, K., Kawauchi, H. and Nagahama, Y. 1988a. Isolation and characterization of two distinctgonadotropins from chum salmon pituitary glands. Gen. Comp. Endocrinol. 71: 292-301.Suzuki, K., Kawauchi, H. and Nagahama, Y. 1988b. Isolation and characterization of subunits of twodistinct gonadotropins from chum salmon pituitary glands. Gen. Comp. Endocrinol. 71: 302-306.Suzuki, K., Nagahama, Y. and Kawauchi, H. 1988c. Steroidogenic activities of two distinct salmongonadotropins. Gen. Comp. Endocrinol. 71: 452-458.Suzuki, K., Kanamori, A., Nagahama, Y. and Kawauchi, H. 1988d. Development of salmon GTH I andGTH II radioimmunoassays. Gen. Comp. Endocrinol. 71: 459-467.Swanson, P., Bernard, M., Nozaki, M., Suzuki, K., Kawauchi, H. and Dickhoff, W. W. 1989.Gonadotropins I and II in juvenile coho salmon. Fish Physiol. Biochem. 7: 169-176.Swanson, P., Suzuki, K., Kawauchi, H. and Dickhoff, W. W. 1991. Isolation and characterization of twocoho salmon gonadotropins, GtH I and GtH II. Biol Reprod. 44: 29-38.Takashima, F., Weil, C., Billard, R., Crim, L. W. and Fostier, A. 1984. Stimulation of spermiation byLHRH analogue in carp. Bull. Japan. Soc. Sci. 50(8): 1323-1329.Tan, E. S. P. and Begum, A. Z. 1985. Induced spawning of Puntius gonionotus a Malaysian cyprinid.Proceedings of the second international conference on warm water aquaculture fmfish. Officeof continuing education, Brigham Young University/Hawii Campus, Laie Hawii. pp. 493-506.Thomas, P. and Trant, J. M. 1989. Evidence that 17a,20fl,21-trihydroxy-4-pregnen-3-one is amaturation-inducing steroid in spotted seatrout. Fish. Physiol. Biochem. 7: 185-191.Trant, J. M., Thomas, P. and Shackleton, C. H. L. 1986. Identification of 17a, 20/3, 21-trihydroxy-4-pregnen-3-one as the major ovarian steroid produced by the teleost Micropogonias undulatusduring final oocyte maturation. Steroids. 47: 89-99.Trant, J. M. and Thomas, P. 1989. Changes in ovarian steroidogenesis in vitro associated with finalmaturation of Atlantic croaker oocytes. Gen. Comp. Endocrinol. 75: 405-412.Trudeau, V. L., Peter, R. E. and Sloley, B. D. 1991. Testosterone and estradiol potentiate the serumgonadotropin response to gonadotropin-releasing hormone in goldfish. Biol. Reprod. 44: 951-960.Truscott, B., Idler, D. R., So, Y. P. and Walsh, J. M. 1986. Maturational steroids and gonadotropin inupstream migratory sockeye salmon. Gen. Comp. Endocrinol. 62: 99-110.Tsai, C., Islam, M. N., Karim, M. R. and Rahman, K. U. M. S. 1981. Spawning of major carps in thelower Halda River, Bangladesh. Estuaries. 4(2): 127-138.150Tyler, C. R., Sumpter, J. P., Kawauchi, H. and Swanson, P. 1991. Involvement of gonadotropin in theuptake of vitellogenin into vitellogenic oocytes of the rainbow trout, Oncorhynchus mykiss.Gen. Comp. Endocrinol. 84: 291-299.Ueda, H., Young, G., Crim, L. W., ICambegawa, A. and Nagahama, Y. 1983. 17a,20/3-dihydroxy-4-pregnen-3-one: Plasma levels during sexual maturation and in vitro production by testis ofamago salmon (Oncorhynchus rhodurus) and rainbow trout (Salmo gairdneri). Gen. Comp.Endocrinol. 51: 106-112.Ueda, H., Hiroi, 0., Hara, A., Yamauchi, K. and Nagahama, Y. 1984. Changes in serum concentrationof steroid hormones, thyroxine, and vitellogenin during spawning migration of the chumsalmon, Oncorhynchus keta. Gen. Comp. Endocrinol. 53: 203-211.Van Der Kraak, G., Lin, H. R., Donaldson, E. M., Dye, H. M. and Hunter, G. A. 1983. Effects of LH-RH and des-G1y10 [D-Ala6] LH-RH-ethylamide on plasma gonadotropin levels and oocytematuration in adult female coho salmon (Oncorhynchus kisutch). Gen. Comp. Endocrinol. 49:470-476.Van Der Kgak, G., Dye, H. M. and Donaldson, E. M. 1984. Effects of LH-RH and des-Gly i° [D-Ala ] LH-RH-ethylamide on plasma sex steroid profiles in adult female coho salmon(Oncorhynchus kisutch). Gen. Comp. Endocrinol. 55: 36-45.Van Der Kraak, G., Donaldson, E. M. and Chang, J. P. 1986. Dopamine involvement in the regulationof gonadotropin secretion in coho salmon. Can. J. Zool. 64: 1245-1248.Van Der Kraak, G. and Peter, R. E. 1987a. Conconavalin A separates two forms of maturationalgonadotropin in goldfish. In D. R. Idler, L. W. Crim and J. M. Walsh (eds.). Proceedings ofthe third international symposium on reproductive physiology of fish. Memorial University, St.John's, Newfoundland, Canada. p. 78.Van Der Kraak, G., Donaldson, E. M., Dye, H. M., Hunter, G. A., Rivier, J. E. and Vale, W. W. 1987b.Effects of mammalian and salmon gonadotropin-releasing hormones and analogs on plasmagonadotropin and ovulation in coho salmon (Oncorhynchus kisutch). Can. J. Fish. Aqua. Sci.44: 1930-1935.Van Der Kraak, G., Suzuki, K. and Peter, R. E. 1992. Properties of common carp gonadotropin I andgonadotropin II. Gen. Comp. Endocrinol. 85: 217-229.Wade, M. G. and Van Der Kraak, G. 1991. The control of testicular androgen production in thegoldfish: Effects of activators of different intracellular signalling pathways. Gen. Comp.Endocrinol. 83: 337-344.Wallace, R. A. and Selman, K. 1981. Cellular and dynamic aspects of oocyte growth in teleosts. Am.Zool. 21: 325-343.151Wallace, R. A., Selman, K., Greeley, M. S. J., Begovac, P. C. and Lin, Y.-W. P. 1987. Current status ofoocyte growth. In D. R. Idler, L. W. Crim and J. M. Walsh (eds.). Proceedings of the thirdinternational symposium on reproductive physiology of fish Memorial University, St. John's,Newfoundland, Canada. pp. 167-177.Weil, C., Fosteir, A., Horvath, L., Marlot, S. and Berscenyi, M. 1980. Profiles of plasma gonadotropinand 177-estradiol in the common carp, Cyprinus carpio L. as related to spawning induced byhypophysation or LH-RH treatment. Reprod. Nutr. Develop. 20(4A): 1041-1050.Weixin, Z., Renliang, J., Shijiao, H. and Hongoqi, Z. 1986. Gonadotropin and 17/3-oestradiol changesduring induced spawning and annual reproductive cycle in wuchang fish (Megalobramaamblycephala). In J. L. Maclean, L. B. Dizon and L. V. Hosillos (eds.). Proceedings of the firstAsian fisheries forum. Manila, Philippines, Asian fisheries society. pp. 707-710.Weixin, Z., Yujun, T., Renliang, J. and Chunxiao, K. 1988. Changes of sex steroids during inducedovulation of silver carp (Hypophthalmichthys molitrix). Acta Hydrobiol. Sin. 12(3): 212-218.West, G. 1990. Methods of assessing ovarian development in fishes: A review. Aust. J. Mar. FreshwaterRes. 41: 199-222.Whitehead, C., Bromage, N. R. and Breton, B. 1983. Changes in serum levels of gonadotropin,oestradiol-17fl and vitellogenin during the first and subsequent reproductive cycles of femalerainbow trout. Aquaculture. 43: 317-326.Wright, R. S. and Hunt, S. V. 1982. A radioimmunoassay for 17a,20P-dihydroxy-4-pregnen-3-one: Itsused in measuring changes in serum levels at ovulation in Atlantic salmon (Salmo salar), cohosalmon (Oncorhynchus kisutch) and rainbow trout (Salmo gairdneri). Gen. Comp. Endocrinol.47: 475-482.Yamazaki, F. and Donaldson, E. M. 1969. Involvement of gonadotropin and steroid hormones in thespermiation of the goldfish (Carassius auratus). Gen. Comp. Endocrinol. 12: 491-497.Yaron, Z., Levavi-Zermonsky, B. and Bogomolnaya, A. 1985. GTH and ovarian steroids duringspawning induction in the carp. Fish culture conference., Bacelona, August 26-28th, 1985. p.15A.Yaron, Z. and Levavi-Zermonsky, B. 1986. Fluctuation of gonadotropin and steroids during the annualcycle and spawning of common carp. Fish Physiol. Biochem. 2: 75-86.Young, G., Kagawa, H. and Nagahama, Y. 1983. Evidence for a decrease in aromatase activity in theovarian granulosa cells amago salmon (Oncorhynchus rhodurus) associated with final oocytematuration. Biol. Reprod. 29: 310-315.Yu, J. Y. L. and Shen, S. T. 1989. Isolation of pituitary glycoprotein gonadotropins from grass carp(Ctenopharyngodon idella). Fish Physiol. Biochem. 7: 117-183.152Yu, K. L., Sherwood, N. M. and Peter, R. E. 1988. Differential distribution of two molecular forms ofgonadotropin-releasing hormone in discrete brain areas of goldfish (Carasius auratus).Peptides. 9: 625-630.Zohar, Y., Goren, A., Tosky, M., Pagelson, G., Leibovitz, D. and Koch, Y. 1989. The bioactivity ofgonadotropin releasing hormones and its regulation in the gilthead seabream, Sparus aurata:In vivo and in vitro studies. Fish Physiol. Biochem. 7: 59-67.153

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:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0086398/manifest

Comment

Related Items