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

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ASPECTS OF THE REPRODUCTIVE ENDOCRINOLOGY OF THE THAI CARP, PUNTIUS GONIONOTUS (BLEEKER) by NARUEPON SUKUMASAVIN B.Sc. (Fisheries) Kasetsart University, Thailand, 1982  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF Master of Science in THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF ZOOLOGY  We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA April 1992 © Naruepon Sukumasavin, 1992  In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission.  (Signature)  Department of^7,0()T OGY The University of British Columbia Vancouver, Canada  Date^APRIL 1992  DE-6 (2/88)  ABSTRACT Several aspects of the reproductive endocrinology of the Thai carp (Puntius gonionotus Bleeker) were investigated. The annual cycle of gonadal development, changes in plasma hormone levels 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 a salmon gonadotropin-releasing hormone analog (D-Argo, Pro 9 NEt-sGnRH; sGnRHA) and domperidone (Dom) on the induction of gonadotropin (GtH) secretion and spawning in female fish was investigated. Further, long- and short-term changes in plasma hormone levels during sGnRHA and Dom induced spawning were examined. Moreover, the biological activities of 12 different mammalian, avian and piscine GnRHs and their analogs were also compared. In females, gonadal recrudescence was highly correlated with the changes in air temperature and daylength, but spawning coincided with the occurrence of rainfall. Histological analysis of the ovary revealed that oocyte development was of the asynchronous type, thus suggesting that the Thai carp can spawn several times in a spawning season. It was not possible to distinguish pronounced seasonal changes in plasma hormone levels during this study. Also, there were no correlations between seasonal changes in plasma hormone levels and any reproductive parameter. Gonadal development in males was highly correlated with that of females. The structure of the testis corresponds to the lobular type. Histological analysis revealed a continuous spermatogenetic activity. Plasma hormones levels in males exhibited a bimodal pattern of seasonal changes. However, there were no correlations between the changes in plasma reproductive hormones, testicular development and environmental parameters. Injection of sGnRHA and Dom alone increased plasma GtH levels significantly, but the magnitude of increasing GtH levels was insufficient to induce spawning, except in the case of the highest dose of domperidone. Administration of various combinations of sGnRHA and Dom  ii  increased both the plasma GtH level and the number of fish spawning, suggesting that dopamine plays an 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 during the induction period. During the short-term observations, both E2 and T peaked immediately at the time of ovipositon. Plasma 17a, 20f3 dihydroxy-4-pregnene-3-one was undetectable throughout the induction period. In males, a significant increase in plasma GtH, T and 11-ketotestosterone (11-KT) was observed at the onset of spawning. Both T and 11-KT continued to increase during the process of spawning, while GtH immediately decreased to a lower level. The changes in plasma 11-KT were synchronous with the occurrence of spawning, suggesting that 11-KT is probably the major androgen during the process of spawning in male Thai carp. High concentrations (25 µg/kg) of either native GnRHs or their analogs in combination with Dom (25 mg/kg) were equally effective in inducing spawning. At 10 12g/kg, however, sGnRH and chicken GnRH-11, in combination with 10 mg/kg Dom, were found to be the most potent of the native GnRHs. GnRHs with a substitution in position 6 with hydrophobic or aromatic D-amino acids possess greater potencies compared to native forms. Furthermore, [D-Ala 6]-mGnRHA and [D-Trp 6]mGnRHA were found to be the most potent peptides.  iii  TABLE OF CONTENTS Abstract^  ii  Table of Contents ^  iv  List of Figures ^  vii  List of Tables ^  xi  Acknowledgement ^  adi  CHAPTER 1 General Introduction ^  1  CHAPTER 2 Materials and Methods ^ A. Experimental Site ^ B. Experimental Animals and Husbandry ^ C. Hormone Preparation, Injections and Blood Sampling ^ D. Histological Analyses ^ E. Radioimmunoassays ^ a. Gonadotropin ^ 1.Chemicals ^ 2. Iodination of carp gonadotropin II (cGtH-II) ^ ^ 3. Radioimmunoassay procedure ^ b. Steroids ^ 1.Chemicals ^ 2.Extraction of steroids from plasma samples ^ 3.Radioimmunoassay procedure ^ 4.Steroid assay validation^ F. Statistics ^  13 14 15 15 17 18 19 19  CHAPTER 3 Annual Reproductive Cycle of the Thai Carp ^ A. Introduction ^ B. Materials and Methods ^ C. Results ^ I. Seasonal changes in environmental parameters ^ a.Rainfall ^ b. Temperature ^ c. Daylength ^ II. Seasonal changes in reproductive parameters ^ A. Female ^ a.Condition factor ^ b. Hepatosomatic index ^ c. Gonadosomatic index ^ B. Male ^ a. Condition factor ^  20 20 20 21 23 23 23 26 26 26 26 29 29 29 29  iv  10 10 10 11 12 13 13 13  b. Hepatosomatic index ^ 32 c.Gonadosomatic index ^ 32 HI. Seasonal histological changes in gonadal and hepatic tissues ^ 32 A. Anatomy of the gonads ^ 32 B. Histology of the ovary ^ 35 C. Histology of the testis ^ 44 D. Histology of the liver ^ 51 IV. Seasonal changes in plasma gonadotropin and steroid hormones ^53 A. Hormonal changes in female Thai carp ^ 53 a. Gonadotropin ^ 53 b. Testosterone ^ 53 c. Estradio1-17P ^ 53 B. Hormonal changes in male Thai carp ^ 59 a.Gonadotropin ^ 59 b. Testosterone ^ 59 c. 11-Ketotestosterone ^ 59 D. Discussion ^ 64 a.Females ^ 64 b. Males ^ 69 CHAPTER 4 Interaction of sGnRHA and Domperidone on the Induction of Gonadotropin Secretion and Induction of Spawning in the Thai Carp ^ 72 A. Introduction ^ 72 B. Materials and Methods ^ 72 C. Results ^ 77 a.Percent fish spawned ^ 77 b. Plasma GtH levels ^ 78 D. Discussion ^ 83 CHAPTER 5 The Effects of sGnRHA and domperidone on plasma gonadotropin and steroid hormones levels during spawning in the male and female Thai carp ^ 87 A. Introduction ^ 87 B. Materials and Methods ^ 88 I. Experiment I. "Long term" changes in plasma hormone levels during induced spawning in male and female Thai carp ^ 88 II. Experiment II. "Short term" changes in plasma hormone levels at the time of spawning in male and female Thai carp ^ 88 C. Results^ 89 Experiment I. ^ 89 A. Females ^ 89 B. Males ^ 94 Experiment II. ^ 98 A. Females ^ 98 B. Males ^ 102 D. Discussion ^ 106 a.Females ^ 106 b. Males ^ 110  CHAPTER 6 Biological Activities of GnRHs and Their Analogs in Combination with Domperidone on the Induction of Gonadotropin Secretion and Spawning in the Thai Carp ^114 A. Introduction ^ 114 B. Materials and Methods ^ 115 C. Results ^ 119 a.Percentage of fish spawned ^ 119 b. Plasma GtH levels ^ 121 D. Discussion ^ 127 CHAPTER 7 Summary and Conclusions ^ 130 A. Annual Reproductive Cycle of the Thai Carp ^ 130 1.Females ^ 130 2.Males ^ 131 B. Interaction of sGnRHA and Domperidone in the Regulation of Gonadotropin Secretion and Induction of Spawning in the Female Thai Carp ^ 132 C. Hormonal Changes During sGnRHA and Domperidone Induced Spawning in the Thai Carp ... ^ 133 1.Females ^ 133 2.Males ^ 134 D. Biological Activities of GnRHs and Their Analogs in Combination with Domperidone on the Induction of Gonadotropin Secretion and Spawning in the Female Thai Carp ^135 REFERENCES ^  137  vi  LIST OF FIGURES Fig. 1. Primary structure of mammalian, avian, and salmon gonadotropin-releasing hormone ^9 Fig. 2. Annual changes in rainfall at Kalasin during 1987-89 expressed as mm per calendar month ^22 Fig. 3. Annual changes in maximum and minimum air temperatures at Kalasin during 1987-89 expressed as mean minimum and mean maximum temperatures in each calendar month. ^24 Fig. 4. Annual changes in daylength at Kalasin during 1987-89 expressed as mean daylength in hour in each calendar month ^ 25 Fig. 5. Annual changes in condition factor (CF) in female Thai carp in a rearing pond at Kalasin Freshwater Fisheries Station during 1987-89. ^  27  Fig. 6. Annual changes in hepatosomatic index (HSI) in female Thai carp in a rearing pond at Kalasin Freshwater Fisheries Station during 1987-89 ^ 28 Fig. 7. Annual changes in gonadosomatic index (GSI) in female Thai carp in a rearing pond at Kalasin Freshwater Fisheries Station during 1987-89 ^ 30 Fig. 8. Annual changes in condition factor (CF) in male Thai carp in a rearing pond at Kalasin Freshwater Fisheries Station during 1987-89 ^  31  Fig. 9. Annual changes in hepatosomatic index (HSI) in male Thai carp in a rearing pond at Kalasin Freshwater Fisheries Station during 1987-89 ^ 33 Fig. 10. Annual changes in gonadosomatic index (GSI) in male Thai carp in a rearing pond at Kalasin Freshwater Fisheries Station during 1987-89 ^ 34 Fig. 11.1. Cross-sections of ovaries. (a) Stage I oocyte 160X. (b) Stage II oocyte 160X. (c) Stage III oocyte 40X. ^ 36 Fig. 11.2. Cross-sections of ovaries. (d) Stage IV oocyte, early stage 40X. (dl) Stage IV oocyte, late stage 40X. ^ 37 Fig. 11.3. Cross-sections of ovaries. (e) Stage V oocyte, germinal vesicle migration stage 40X. (f) Atretic oocyte 40X. (g) Post-ovulatory oocyte 40X ^  38  Fig. 12.1. Annual changes in the occurrence of oocytes in different stages in female Thai carp in a rearing pond at Kalasin Freshwater Fisheries Station during 1987-89. (a) Stage I oocyte ^39 Fig. 12.2. Annual changes in the occurrence of oocytes in different stages in female Thai carp in a rearing pond at Kalasin Freshwater Fisheries Station during 1987-89. (b) Stage II oocyte. (c) Stage III oocyte. 40  vu  Fig. 12.3. Annual changes in the occurrence of oocytes in different stages in female Thai carp in a rearing pond at Kalasin Freshwater Fisheries Station during 1987-89. (d) Stage IV oocyte. (e) Stage V oocyte. 41 Fig. 12.4. Annual changes in the occurrence of oocytes in different stages in female Thai carp in a rearing pond at Kalasin Freshwater Fisheries Station during 1987-89. (I) Atretic oocyte. (g) Postovulatory oocyte ^ 42 Fig. 13. Cross-sections of testes. (a) Spermatogonia. (b) Primary spermatocytes. (c) Secondary spermatocytes. (d) Spermatids. (e) Spermatozoa. ^  46  Fig. 14.1. Annual changes in the testicular germ cells at different stages in male Thai carp in a rearing pond at Kalasin Freshwater Fisheries Station during 1987-89. (a) Spermatogonia ^47 Fig. 14.2. Annual changes in the testicular germ cells at different stages in male Thai carp in a rearing pond at Kalasin Freshwater Fisheries Station during 1987-89. (b) Primary spermatocytes. (c) Secondary spermatocytes ^ 48 Fig. 143. Annual changes in the testicular germ cells at different stages in male Thai carp in a rearing pond at Kalasin Freshwater Fisheries Station during 1987-89. (d) Spermatids. (e) Spermatozoa.49 Fig. 15. Annual changes in hepatocyte diameter in female Thai carp in a rearing pond at Kalasin Freshwater Fisheries Station during 1987-89 ^  52  Fig. 16. Annual changes in plasma gonadotropin in female Thai carp in a rearing pond at Kalasin Freshwater Fisheries Station during 1987-89 ^  55  Fig. 17. Annual changes in plasma testosterone in female Thai carp in a rearing pond at Kalasin Freshwater Fisheries Station during 1987-89 ^  56  Fig. 18. Annual changes in plasma estradio1-17P in female Thai carp in a rearing pond at Kalasin Freshwater Fisheries Station during 1987-89 ^  57  Fig. 19. Annual changes in GSI and plasma hormone levels in female Thai carp in a rearing pond at Kalasin Freshwater Fisheries Station during 1987-89 ^ 58 Fig. 20. Annual changes in plasma gonadotropin in male Thai carp in a rearing pond at Kalasin Freshwater Fisheries Station during 1987-89 ^  60  Fig. 21. Annual changes in plasma testosterone in male Thai carp in a rearing pond at Kalasin Freshwater Fisheries Station during 1987-89 ^  61  Fig. 22. Annual changes in plasma 11-ketotestosterone in male Thai carp in a rearing pond at Kalasin Freshwater Fisheries Station during 1987-89 ^ 62 Fig. 23. Annual changes in GSI and plasma hormone levels in male Thai carp in a rearing pond at Kalasin Freshwater Fisheries Station during 1987-89 ^  viii  63  Fig. 24. Effect of various combinations of sGnRHA (µg/kg) and Dom (mg/kg) on the induction of spawning in female Thai carp during July 1990 at Kalasin Freshwater Fisheries Station. ^75 Fig. 25. Effect of various combinations of sGnRHA (µg/kg) and Dom (mg/kg) on the induction of spawning in female Thai carp during May 1991 at Pathumthani Freshwater Fisheries Station. ^76 Fig. 26. Effect of various combinations of sGnRHA (µg/kg) and Dom (mg/kg) on the induction of GtH secretion in female Thai carp at injection time in the 1991 study ^ 79 Fig. 27. Effect of various combinations of sGnRHA (µg/kg) and Dom (mg/kg) on the induction of GtH secretion in female Thai carp at 3 hr after injection in the 1991 study. ^80 Fig. 28. Effect of various combinations of sGnRHA (µg/kg) and Dom (mg/kg) on the induction of GtH secretion in female Thai carp at 6 hr after injection in the 1991 study. ^81 Fig. 29. Effect of various combinations of sGnRHA (µg/kg) and Dom (mg/kg) on the induction of GtH secretion in female Thai carp at 9 hr after injection in the 1991 study. ^82 Fig. 30. Changes in plasma E2 during sGnRHA (20 µg/kg) and Dom (20 mg/kg) induced spawning in female Thai carp^ 91 Fig. 31. Changes in plasma T during sGnRHA (20 µg/kg) and Dom (20 mg/kg) induced spawning in female Thai carp ^ 92 Fig. 32. Changes in plasma GtH during sGnRHA (20 µg/kg) and Dom (20 mg/kg) induced spawning in female Thai carp^ 93 Fig. 34. Changes in plasma 11-KT during spawning in untreated male Thai carp kept with sGnRHA and Dom induced spawning treated females. ^ 95 Fig. 35. Changes in plasma T during spawning in untreated male Thai carp kept with sGnRHA and Dom treated females^ 96 Fig. 36. Changes in plasma GtH during spawning in untreated male Thai carp kept with sGnRHA and Dom treated females^ 97 Fig. 37. Short-term changes in plasma E2 levels in sGnRHA (20 /1g/kg) and Dom (20 mg/kg) induced spawning in female Thai carp shortly before, during, and after spawning ^99 Fig. 38. Short-term changes in plasma T levels in sGnRHA (20 µg/kg) and Dom (20 mg/kg) induced spawning in female Thai carp shortly before, during, and after spawning ^ 100 Fig. 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^101 Fig. 40. Short-term changes in plasma 11-KT levels in untreated male Thai carp kept with sGnRHA and Dom treated females shortly before, during, and after spawning. ^ 103  ix  Fig. 41. Short-term changes in plasma T levels in untreated male Thai carp kept with sGnRHA and Dom treated females shortly before, during, and after spawning ^ 104 Fig. 42. Short-term changes in plasma GtH levels in untreated male Thai carp kept with sGnRHA and Dom treated females shortly before, during, and after spawning ^ 105 Fig 43. Effect of native mammalian, avian and piscine GnRHs and their analogs (25 µg/kg) in combination with Dom (25 mg/kg) on the induction of spawning in the Thai carp in the 1990 study (24-29 July 1990) ^  118  Fig. 44. Effect of native mammalian, avian and piscine GnRHs and their analogs (10 µg/kg) in combination with Dom (10 mg/kg) on induction of spawning in the Thai carp in the 1991 study (21-25 May 1991). ^ 120 Fig. 45. Effects of native mammalian, avian, and piscine GnRHs and their analogs (10 µg/kg) in combination with Dom (10 mg/kg) on the induction of GtH secretion in female Thai carp at injection time in the 1991 study (21-25 May 1991) 123 Fig. 46. Effects of native mammalian, avian, and piscine GnRHs and their analogs (10 µg/kg) in combination with Dom (10 mg/kg) on the induction of GtH secretion in female Thai carp at 3 hr after injection in the 1991 study (21-25 May 1991). ^ 124 Fig. 47. Effects of native mammalian, avian, and piscine GnRHs and their analogs (10 µg/kg) in combination with Dom (10 mg/kg) on the induction of GtH secretion in female Thai carp at 6 hr after injection in the 1991 study (21-25 May 1991). ^ 125 Fig. 48. Effects of native mammalian, avian, and piscine GnRHs and their analogues (10 µg/kg) in combination with Dom (10 mg/kg) on the induction of GtH secretion in female Thai carp at 9 hr after injection in the 1991 study (21-25 May 1991). ^ 126  LIST OF TABLES Table 1 Dose combinations of sGnRHA and Dom Kalasin Freshwater Fisheries Station in July 1990. ^  74  Table 2 Dose combinations of sGnRHA and Dom Pathumthani Freshwater Fisheries Station in May 1991 ^  74  Table 3 The primary structure of GnRH and other peptides used in the study ^  xi  117  ACKNOWLEDGEMENT  First, 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 my gratitude to Professor N. R. Liley, my supervisor at UBC, and Professor A. M. Perks, my research committee, for their guidance and assistance. I am also grateful to Mr. Wattana Leelapatra for recognizing my ability to pursue this degree  and 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, and Dr. R. E. Peter and Ms. Carol Nahorniak at the Department of Zoology, University of Alberta for teaching me the gonadotropin radioimmunoassay and allowing me to use the facilities at the Department. I also thank Dr. E. McLean for his friendly advice. Additionally, I thank Mr. Tharaphan Wattanamahart, Mr. Thavee Viputhanumas, and staff of Kalasin Freshwater Fisheries Station and Pathumthani Freshwater Fisheries Station, Department of Fisheries, Thailand for assistance during conducting experiments in Thailand This 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 for assistance during my staying in Canada. Further, this study was made possible by the studies leave permission from the Department of Fisheries, Thailand. Finally, I extend my sincere gratitude to my family and friends who provided moral support throughout the study period.  xii  CHAPTER 1 GENERAL INTRODUCTION  The freshwater aquaculture industry is becoming important in Thailand for the provision of an inexpensive and abundant source of protein for human consumption. Since 1980, the number of fish farms 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 adopted throughout Thailand for many years as a major method for fry production, this method still has some limitations 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 improve the understanding of the reproductive physiology of cultured species and the relationships between the environment, the reproductive hormones, and gonadal development. This information can then be used for the establishment of biotechnologies designed to overcome reproduction related problems in aquaculture. The annual reproductive cycle in teleosts has been intensively studied, and is believed to depend upon changes in environmental factors (Peter and Crim, 1979; Peter, 1981; Crim, 1982). In temperate fishes, the patterns of gonadal development and spawning have been found to be highly synchronized 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 with temperature and daylength (Lam and Munro, 1987), and final maturation, ovulation, and spawning are mostly 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 of the changes in plasma levels of reproductive hormones with gonadal condition has proven to be a valuable tool in the development of an understanding of endocrine control of reproduction in teleosts.  1  Many studies have investigated the correlations between seasonal changes in plasma levels of reproductive hormones and gonadal development in a number of fish including salmonids (Crim and Idler, 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, (Burke et 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), and walking 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 their carbohydrate content or their ability to adsorb to Conconavalin A-Sepharose (Idler and Ng, 1983; Van Der Kraak and Peter, 1987a). The first GtH to be purified was the glycoprotein-rich or maturational gonadotropin (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 in other vertebrates (Licht et a1. 1977). Maturational GtH is now known to be involved in the processes of steroidogenesis, maturation, and ovulation or spermiation (Idler, 1982; Idler and Ng, 1983; Van Der Kraak 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 on the basis of its lower carbohydrate content and its differences in biological activity. This carbohydratepoor or vitellogenic gonadotropin (vGtH) participates in vitellogenesis and promotes the uptake of the yolk precursor, vitellogenin, into oocytes (Ng and Idler, 1983; Idler and So, 1987).  2  More 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 elution positions in anion exchange chromatography. Both GtHs also consist of a and p subunits. The 8 subunits have about 30% homology in amino acid sequence (Kawauchi et al. 1989). The a and /3 subunits of both GtHs show significant sequence identity to the respective subunits of mammalian FSH and LH (Itoh et al. 1988; Kawauchi et al. 1989). GtH I and GtH II are localized in separate cells in the pituitary and the rate of synthesis of GtH I and GtH II varies during reproductive development (Nozaki et al. 1990a; b). Studies on immunological and biological characteristics reveal that both GtHs are  steroidogenic and are approximately equipotent in inducing estradio1-17P production, but GtH II is more potent in enhancing the production of 17a20/3-dihydroxyprogesterone and in turn inducing oocyte maturation (Suzuki et al. 1986; Swanson et al. 1989; Van Der Kraak et al. 1992). In rainbow trout, GtH I has a primary function in stimulating vitellogenin uptake into developing oocytes (Tyler et al. 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 taken to 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, GtH controls 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). In response to GtH, theca layers of the oocyte secrete large amounts of aromatizable androgen, mainly testosterone, which is converted to estradiol-17/3 (E2) by granulosa layers (Nagahama, 1987). E2 then acts in the liver to stimulate the production of vitellogenin. Finally, vitellogenin is selectively taken up  3  from 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 fmal maturation and ovulation in teleosts (Nagahama, 1987). In salmonids which have synchronous oocyte development, a gradual and prolonged rise in plasma GtH has been shown to proceed ovulation in rainbow trout, Salmo gairdneri, (Fostier et al. 1987; Fostier and Jalabert, 1982; Scott et al. 1983), coho salmon, 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 fishes  which have asynchronous oocyte development, a rapid surge of plasma GtH was observed prior to ovulation 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 of a maturation-inducing steroid (MIS) (Sundararaj et al. 1985; Nagahama, 1987). In several teleosts, a variety of hydroxylated C21 steroids, such as 17a20P-dihydroxy-4-pregnene-3-one (17,20P-P), have been identified as potent maturation inducing steroids (see Jalabert, 1976; Goetz, 1983; Scott and Canario, 1987). In particular, high levels of 17,20P-P have been found during both in in vivo and in vitro 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 17,20/3,21trihydroxy-4-3-one (17,20P,21-P) trihydroxylated progesterone derivative is also the MIS in several marine 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 both exogenous and endogenous conditions are optimal (Stacey, 1984). Exogenous factors such as temperature, and to a lesser extent photoperiod, are known to be important for ovulation and spermiation in temperate cyprinids (see Stacey et al. 1979; Peter, 1981; Santos et al. 1986). However,  4  the endogenous factors required for initiation of the preovulatory GtH surge in tropical cyprinids are not so clearly understood Many studies have investigated the changes in steroid hormones as an endogenous requisite for the preovulatory GtH surge. In rainbow trout, it has been hypothesized that a decrease 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 of vitellogenesis (Pankhurst and Stacey, 1985; Kobayashi et al. 1987). In these fish the high plasma level of testosterone observed before ovulation has been suggested to be an important endogenous requisite for the occurrence of the GtH surge (Kobayashi et al. 1987; 1989). Unlike females, less is known about the reproductive endocrinology of males. The main gonadal 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 of plasma 11-ketotestosterone (11-KT) occurs following that of testosterone (T) (Fostier et al. 1982). A similar trend is also found in common carp, but 11-KT, rather than T, is found to be the major androgen in in vitro testicular culture of carp (Koldras et al. 1990). Furthermore, 17,201-P has also been found to be the most efficient steroid to induce spermiation in both goldfish and salmonids (Ueda et al. 1984).  GtH secretion in the teleost is regulated by gonadotropin-releasing hormone (GnRH) and by dopamine which acts as a gonadotropin release inhibitory factor (GRIF) (Peter et al. 1986; Lin and Peter, 1986). The significance of the GRIF differs between species and may also change along with sexual recrudescence or maturation within the same species. At present, the species in which dopamine 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 of ovulatory gonadotropin secretion, injection of GnRH analog alone results in increased GtH and ovulation. Species in which this occurs include coho salmon, Oncorhynchus kisutch, (Van Der Kraak et  5  al. 1986); loach, Paramisgurnus dablyanus, (Lin et al. 1985); and African catfish, Clarias gariepinus, (De  Leeuw et al. 1985). In species that have a strong dopamine inhibitory action, injection of a GnRH analog 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, Hypophthalmichthys molitrix; 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 drugs are pimozide, a dopamine receptor antagonist (Chang and Peter, 1983), reserpine, a drug which causes general 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 a dopamine depletor (Sloley et al. 1990). Of these, pimozide and domperidone are the most potent in potentiating the action of GnRH analogs on GtH release in goldfish (Peter et al. 1986; Omeljaniuk et al. 1987a).  Domperidone is known to be highly specific for dopamine receptors. It binds specifically to receptors 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 goldfish pituitary (Sloley et al. 1990). Because of the likelihood of fewer undesirable side effects and the fact that it is relatively inexpensive, domperidone has been widely used in studies on the induced ovulation of cultured fish (Peter et al. 1986; Lin and Peter, 1986). More than 2000 forms of GnRH analogs (GnRHA) have been synthesized in an effort to produce 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 amino acid sequence from those of GnRHs found in bird and fish (Sherwood, 1987) (Fig. 1). In studies on the  6  activity of various mammalian GnRHs (mGnRH) and salmon GnRH (sGnRH) on goldfish, Peter et al. (1987b) reported that [D-Arg 6 , Pro9 NEt]-sGnRH (sGnRHA) was the most active analog both in vitro and in vivo. In rainbow trout and landlocked salmon, Crim et al. (1988) demonstrated that all types of fish, bird and mammalian GnRH agonists possessed superactive properties in vivo on the fish pituitary in terms of GtH release. The most active forms were found to be sGnRHA and [D-hArg (Et2) 6 , Pro9 NEt]-sGnRH, the mGnRHA, ED-Ala 6 , Pro9 NEt]-mGnRH, [D-(Na12) 6, aza-Gly 10]-mGnRH and [DhArg(Et2) 6 , Pro9 NEt]-mGnRH. In the African catfish, comparison of sGnRHA, sGnRH, and mGnRHA determined that the former had the highest activity in vivo, but in vitro, its activity appeared similar to [D-Ser(Bu t) 6 Pro 9 NEt]-mGnRH (Buserelin) (De Leeuw et al. 1988). In coho salmon, Van Der Kraak et al. (1987b) reported similar GtH releasing and ovulation-inducing activity for several sGnRHA and mGnRHAs. To date, studies on fish reproductive endocrinology have emphasized the temperate or subtemperate species. Information for tropical fish is not available and is needed for aquaculture development. Thus, the major objectives of the studies described herein were to investigate the endocrinological aspects of the control mechanisms responsible for the regulation of gonadal development, gonadotropin secretion, and ovulation in a tropical teleost, the Thai carp, Puntius gonionotus (Bleeker).  The Thai carp is a freshwater cyprinid of great economic importance in Thailand. It is commonly cultured both in earthen ponds and in paddy fields. In pond culture, the Thai carp often makes up a high proportion of the stocking density in the polyculture of carp, which is commonly practiced in Thailand (Leelapatra, 1988). Its production from aquaculture increased from 7,311 tons in 1985 to 11,145 tons in 1988, and it presently contributes more than 70% of total carp production in Thailand (FAO, 1991). The spawning season of the Thai carp ranges from March to August with a peak in May or June and coincides with the occurrence of rainfall (Sipitalliat and Leenanond, 1984).  7  Induced spawning of the Thai carp has been practiced for many years, mostly with the hypophysation method (Sipitakkiat and Leenanond, 1984). Recently, Leelapatra (1988) successfully induced the spawning of the Thai carp using sGnRHA and domperidone therapy. In hatchery conditions, the Thai carp 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 the Thai carp is not clearly understood. Thus, in Chapter 3 of this study, I investigated the role of environmental 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 in Chapter 3, I examined the effectiveness of several combinations of salmon gonadotropin-releasing hormone analog (sGnRHA) and domperidone on GtH secretion and ovulation in the Thai carp (Chapter 4). Additionally, the significance of endocrine changes during hormonal induced final maturation and spawning was studied, as outlined in Chapter 5. Finally, in Chapter 6, the effectiveness of mammalian, avian, and piscine GnRH analogs in combination with domperidone on GtH release and ovulation in the Thai carp was examined  8  mammalian^pG1u-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2 chicken-I^pGlu-His-Trp-Ser-Tyr-Gly-Leu-Gln-Pro-Gly-NH2 chicken-II^pGlu-His-Trp-Ser-His-Gly-Trp-Tyr-Pro-Gly-NH2 salmon^pG1u-His-Trp-Ser-Tyr-Gly-Trp-Leu-Pro-Gly-NH2  Fig. 1. Primary structure of mammalian, avian, and salmon gonadotropin-releasing hormone  9  CHAPTER 2 MATERIALS AND METHODS  A. EXPERIMENTAL SITE  Experiments on the annual reproductive cycle of the Thai carp were carried out at Kalasin Freshwater Fisheries Station (Lat. 16 ° 25' Long. 103 ° 31' E), Kalasin, Thailand about 550 km northeast of Bangkok. The station is located on the east side of Lam Pao Dam and uses water from the Lam Pao reservoir for all aquaculture activities. The air temperature at Kalasin varies from a minimum 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 being recorded during May. A pronounced dry season ranges from November to February every year. Experiments on induced ovulation and the effects of gonadotropin-releasing hormone in combination with domperidone were performed at Kalasin Freshwater Fisheries Station, Kalasin, Thailand in 1990 and at Pathumthani Freshwater Fisheries Station, Pathumthani, Thailand, located 70 km northwest of Bangkok in 1991. All fish used in the latter studies were transferred from Kalasin Freshwater Fisheries Station approximately 1 month prior to experimentation.  B. EXPERIMENTAL ANIMALS AND HUSBANDRY  Mature (age 1-3 years, weight 200-1000 g) Thai carp, Puntius gonionotus, used in all experiments were reared as broodstock for fry production in earthen ponds. Stocking densities were 200 kg/rai (1600 m 2) 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 and minerals) at a rate of approximately 3% of body weight. Rearing ponds were also fertilized with 100  10  kg/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 SAMPLING  Fish were netted from the earthen ponds and transferred into spawning tanks, 2 hr prior to blood 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 were selected by determining the degree of abdominal swelling and the coloration (reddish) of the genital pore. Fish were weighed and identified by a color thread tied to the dorsal spine. Mammalian gonadotropin-releasing hormone (mGnRH), mammalian gonadotropin-releasing hormone analog mGnRH-NHEt (mGnRHA), salmon gonadotropin-releasing hormone (sGnRH), Chicken-I gonadotropin-releasing hormone ([G1n 8]-GnRH, cGnRH-I), [D-Trp 6]-mGnRH, and [DAla6 ]-mGnRH were purchased from Sigma Chemical Co., St. Louis, U.SA. Des-G1y 10 , [D-Ala6 , Pro9 -NHEt]-mGnRH, des-Gly 10 , [D-Trp6 , Pro9-NHEt]-GnRH, [D-Lys 6]-GnRH, des-G1y 10 , [D-Arg6 , Pro9 -NHEt]-sGnRH, and des-G1y 10 , ED-Ala6, Pro9 -NHEt]-sGnRH were purchased from Bachem Inc., California. Chicken-II gonadotropin-releasing hormone (His s , Trp7 , Tyr8 -GnRH, cGnRH-II) was purchased from Peninsula Laboratories, Inc., California, U.S A. Buserelin (D-Ser(Bu t) 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-releasing hormones were dissolved in distilled water at the concentration of 1000 µg/10m1. One ml aliquots were 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, the hormone and domperidone solutions were mixed and the volume of the solution adjusted to 1 ml/kg by  11  adding distilled water. Hormones were administered by intraperitoneal injection at the base of the pectoral fm, using a 1-ml tuberculin syringe (needle size; 24G). Details of the dosage are provided in the protocols to the experiments in each chapter. Blood was drawn from the caudal vessels using a 3 or 5 ml heparinized Vacutainer (needle size 21G, Terumo, Tokyo, Japan). Blood samples of 1 to 3 ml were held on ice prior to centrifugation at 4000 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 West Vancouver Laboratory for further analysis. For transport to Vancouver, samples were placed over dryice in a Styrofoam container. All samples were frozen at -40 ° C upon arrival at the West Vancouver Laboratory, until assay. Spawning was indicated by the presence of eggs in spawning tanks. Spawned fish were identified by the presence of the thinner and flaccid belly resulting from the release of ovulated eggs.  D. HISTOLOGICAL ANALYSES  Whole gonads and liver were dissected from freshly-killed specimens. After they were weighed, 1-3 g of either gonads or liver, taken from mid tissue portions, were fixed in Hollande Bouin's solution (3:1 w/wt). Histological analysis was carried out after re-fixing the tissues in Bouin's solution for 24 hr, following by a rinse in distilled water 3 times within 24 hr. Tissues were then dehydrated in 50% ethanol for 1 hr, and preserved in 70% ethanol until analysis. For analysis, tissues were dehydrated 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 and sealed with Permount (Fisher Scientific Ltd.).  12  E. RADIOIMMUNOASSAYS  a. Gonadotropin  Plasma gonadotropin was measured at the Department of Zoology, University of Alberta, Edmonton, Alberta, Canada using a radioimmunoassay described by Peter et al. (1984a). Details of the procedure employed are described below.  1. Chemicals  The 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-Carp gonadotropin, RAC, first antibody) as described by Peter et al. (1984a), for preparing tracer, and for preparing 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 double distilled 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), 3.25 g sodium acetate (BDH, assured grade), 0.1 g thimerosal (T-5125, Sigma) and 342 ml 0.1 N hydrochloric 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 Peter et a/. (1984a). In brief, 10 111 (1 mCi) Na 125 I (Amersham) was added to a 2 ml polystyrene sample  13  cup (Fisher Scientific Ltd.) containing 5 I.Lg (25 Al) cGtH-II in 0.05 M phosphate buffer, pH 7.5 (5 ml of 0.5 M phosphate buffer in 45 ml double distilled deionized water). The cGtH-II and Na l25 were mixed once by drawing both up into a pipette tip and carefully expelling them back into the bottom of the 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 90 seconds, 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 potassium iodine (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-50 Sephadex beads which were primed with 2 ml 5% BSA in barbital buffer (1 g BSA in 20 ml barbital buffer). A further 200 Al potassium iodine was added into the cup, withdrawn, and also placed onto the column. When the entire 0.6 ml had been drawn into the column, it was flushed through with column buffer. Fifteen fractions containing 1 ml each were collected and scanned for activities. The labelled cGtH-II appeared as the first peak off the column and inorganic 125 1 as a second peak. Two hundred Al of 5% BSA in barbital buffer was added to each tube of labelled cGtH-II for preventing the isotopically 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 used for about 2 weeks.  3. Radioimmunoassay procedure  The radioimmunoassay for GtH was performed at 4 ° C over a four day period. Duplicate 10 x 75 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, produced at the Department of Zoology, University of Alberta, Edmonton, Alberta) prepared in diluent, and  14  approximately 15,000 cpm/200 Al of tracer. Non-specific binding controls had the standard replaced by diluent and RAC replaced by 1:100 NRS. Maximum binding controls had only the standard replaced by diluent, while total count controls contained only 200 1.41 of tracer. After each addition of solutions, the tubes were vigorously shaken. The tubes were incubated at 4 ° C for 48 hr, with an interruption after 24 hr for vigorous shaking. Then 200 Al of the second antibody, Goat Anti-Rabbit Globulin (GAR), at 1:20 in diluent was added into each tube except the total count tubes. The tubes were 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. After centrifugation, the supernatant fluid was decanted onto cotton wool and the tubes were counted for 1 min each on an Isomedic Automatic Gamma counter (ICN Biomedicals Inc.).  b. Steroids  1. Chemicals  Diethyl-ether (BDH, AnalaR grade) and n-heptane (BDH, assured grade) were used in the extraction of steroids from plasma. The steroid assay buffer was a 0.05 M phosphate buffer [5.75 g/1 dibasic 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 nonspecific binding, and 65 mg/1 sodium azide (S-2002, Sigma) as a preservative. The buffer was heated to 37 ° C to dissolve the gelatin, and its pH adjusted to 7.6. Antibodies to testosterone (ICN ImmunoBiologicals 61-315) and estradiol-17fl (ICN ImmunoBiologicals 61-305) were purchased from Miles Laboratories Ltd., Rexdale. Ontario. Anti 11ketotestosterone 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,  15  England). Cross-reactivity between the antibodies and other steroids was provided by the manufacturer for testosterone and estradiol-17fl. Antibodies against testosterone cross-react with 11ketotestosterone by 1.5% and with 11 hydroxytestosterone by 2.5% (Miles Laboratories). The cross reaction 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 10 ng/ml in assay buffer. Steroids radiolabelled with 3 H 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, 7-3 H] 17a-hydroxyprogesterone (Amersham) was placed in a 16x125 mm glass tube then dried under a stream of nitrogen. The steroid was redissolved in 500 µl0.05 M Tris Buffer pH 7.6 (Sigma Chemical Co.), and mixed with 40 Al of 3a-20P-hydroxysteroid dehydrogenase (Sigma Chemical Co., H 7252) containing 19.2 units/ml (18 Al enzyme + 62 µl Tris buffer). A further 500 I.41 of 0.05 M Tris containing 2 mg/ml reduced nicotinamide adenine dinucleotide (NADH disodium salt, #481913, Calbiochem, La Jolla, CA) was added to the tube. The mixture 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 were rinsed with a further 3 ml of diethyl ether, vortexed, allowed to separate, and transferred to a vial. The ether was then evaporated under a stream of nitrogen to dryness, and the vial containing the dry extract was stored at room temperature overnight. The next morning, 100 Al of diethyl ether was added to the vial, and 15-20 III of the solution was applied in a single spot to one channel of a precoated, prewashed  16  silicagel 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-[ 3 H] progesterone was applied to an 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 in a fumehood (20 min). When dry, the plate was divided into 1 cm fractions, and each section of gel was carefully scraped into individual 16x125 mm glass tubes. Five hundred Al of distilled water and 5 ml of diethyl ether were added, the tubes vortexed for 1 min and left to separate for 5 min The organic fraction was transferred to clean glass tubes. Fifty Al aliquots from each tube were added to scintillation vials and counted. The 17,20/3-P fractions were identified by comparing the activity with the 17a0H [31 1] progesterone channel. Once 17,20/3-P was identified, the fraction was re-extracted to -  -  maximize the yield. The ether was then dried, and fmally the dry extract redissolved in absolute ethanol and stored at 4 ° C.  2. Extraction of steroids from plasma samples  Steroids 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 into 25x125 mm borosilicate culture tubes. Approximately 1000 cpm of labelled steroid in 100 Al diluent were added to the samples, which were then vortexed and left at room temperature for 30 minutes to allow the labelled steroid to equilibrate with endogenous steroids. A similar quantity of labelled steroid was added to scintillation vials and counted directly as 100% recovery. Four ml diethyl ether and 1 nil n-heptane were added to the sample tubes, vortexed for 15 seconds, left to separate for 1 minute, then revortexed for another 15 seconds and finally left to separate for 5 minutes. The tubes were then frozen in very cool acetone (acetone in dry ice) for 10 seconds. Once the aqueous layer was well frozen, the organic layer was poured into clean 25x125 mm borosilicate tubes. These tubes were  17  placed in a warm water bath (37° C), and a light stream of compressed air blown over them to speed evaporation 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 were vortexed, clamped, and left at 4 ° C overnight to redissolve. A 4-500 Al aliquot was then withdrawn and added to scintillation vials for recovery determinations.  3. Radioimmunoassay procedure  For all steroid assays, 10x75 borosilicate glass or plastic tubes were set up to contain 200  Ihl of  extracted plasma or standard (3.9 to 1000 pg/ml for testosterone, estradio1-17/3 and 11ketotestosterone, 1.9 to 500 pg/ml for 17,20P-P), 200 Al 3 H-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 for testosterone and estradio1-17P, 1:10000 for 11-ketotestosterone and 17,20P-P). Non-specific and maximum 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 following morning the samples were put in the freezer for 20 minutes, after which 200 Al of dextran-coated charcoal [0.5 g/1 dextran T-70 (Pharmacia [Canada] Ltd., Dorval, Quebec) and 5.0 g/1 activated charcoal (C-5260, Sigma) in assay buffer] was added to each tube to bind any remaining unbound steroid. The tubes were vortexed, incubated at 4° C for 12 minutes, centrifuged at 2400 rpm at 4 ° C for 12 minutes, and the supernatant fluid decanted into 7 ml plastic scintillation vials (Fisher Scientific Ltd.). Four ml of Scinti Verse II (80-X-12, Fisher Scientific Ltd.) was added to each vial, which were then 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.  18  4. Steroid assay validation Following ether extraction of steroids from plasma, all samples diluted in parallel with the standard curve. Recovery determinations after ether extraction gave consistent results. All steroids were extracted with more than 90% efficiency, except for estradio1-17Q (T:93%, 11-KT:93.9%, 17,20PP: 93%, and E2:83.5%). Following this initial determination, extraction efficiency was assumed to be constant and was measured only occasionally to make sure that it remained so. All the extraction efficiencies 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, 30 pg/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% and 11.9% for T, 6.1% and 7.2% for E2, and 11.4% and 6.5% for 11-KT.  F. STATISTICS  All data were expressed as mean ± standard error. Analysis of variance and Tukey HSD test were used to determine differences between means. Log 1° transformation was used to achieve homogeneity 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.  19  CHAPTER 3 ANNUAL REPRODUCTIVE CYCLE OF THE THAI CARP  A. INTRODUCTION  A full understanding of the processes occurring during the natural reproductive cycle of a fish species, and their temporal relationship, is one of the most important prerequisites to the establishment of biotechnologies designed to overcome reproduction-related problems in aquaculture. At present, there has been no comprehensive description of the annual endocrine changes accompanying gonadal development and spawning in any tropical fish. This study was conducted to investigate the relationship between environmental and hormonal factors and gonadal development in the female and male Thai carp. The pattern of gonadal development was assessed by changes in reproductive parameters i.e., gonadosomatic index (GSI), hepatosomatic index (HSI), and condition factor (CF) and by histological analyses of gonads. Changes in reproductive hormones and the changes in environmental parameters throughout the year were related to ovarian development and the timing of spawning.  B. MATERIALS AND METHODS  In this study, 5 adult Thai carp of both sexes were randomly sampled each month over a period of 20 months (Dec 1987-July 1989). Data on body weight and total length of each fish were recorded. Blood samples of 2 ml were taken and centrifuged to obtain plasma. The plasma samples were analyzed for gonadotropin (GtH), testosterone (T), estradio1-17/3 (E2), 11-ketotestosterone (11KT, male only) content by means of specific radioimmunoassays (RIA). After blood sampling, fish were killed and their gonads and liver removed for determination of GSI (gonad weight x 100/ body  20  weight), HSI (liver weight x 100/ body weight), CF (body weight x 100/ total length 3) and for histological 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 was spherical, the diameter was measured; if oval or irregular, the longest and shortest axes were measured and the mean taken. For each slide, between 500-600 cells were staged and the results expressed as a monthly mean. Data on rainfall, maximum and minimum air temperature, and daylength were obtained from the 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. RESULTS  Spawning occurred in the rearing pond during the rainy season (May-June) following a few days of continual rain, and only when male fish were accidentally mixed into the female pond.  21  o^to  0  M  LL  O  0 1— Z v) 0  LL co co  ^ 0^0^0^0^0^ O O 0^(0^0^10^0^In N^ (WW) 11VdNIV7:1  O  I. Seasonal changes in environmental parameters  a.Rainfall  In both years, the rainy season began in March (Fig. 2). However, in 1988, rainfall increased rapidly from March and reached its peak in May (250.2 mm), then slowly decreased to about 50 mm in September 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 in June. There was no rainfall recorded between November and February of both years.  b. Temperature  In both years, maximum air temperature increased gradually from December and reached its peak in April (34.8 ° C) (Fig. 3). However, it decreased in May and was maintained at the same level until 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 air temperature (Fig. 3). However, instead of peaking in April, it continued to increase and reached its peak in June (25.6 ° C), then gradually decreased and reached its lowest level in December 1988 (15.7 ° C).  23  40  0 cm 3 0 cc m 4c cc w  30  a. 2  w 20 I-  20  10 ^ 10 DJ88FMAMJ J A SONDJ89F MAMJ J MONTH  MIN. TEMP --8-- MAX. TEMP  Fig 3 Annual changes in maximum and minimum air temperatures at Kalasin during 1987-89 expressed as mean minimum and mean maximum temperatures in each calendar month.  24  10 ^ 10 DJ88FMAMJJASONDJ89FMAMJJ MONTH  Fig 4 Annual changes in daylength at Kalasin during 1987-89 expressed as mean daylength in hour in each calendar month.  25  c. Daylength Daylength 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 was found between maximum and minimum air temperature (r = 0.897, P < 0.01) and between minimum air temperature and daylength (r = 0.822, P < 0.01).  II. Seasonal changes in reproductive parameters.  A. Female  a. Condition factor (Fig. 5) The CF in female Thai carp showed a significant variation during the study period, varying between 1.3 and 1.7%. In general, CF tended to decrease from April 1988 and began to rise again in June. A similar trend was observed in 1989 when CF values decreased significantly in March and reached lowest levels in April and May (t-Test, P < 0.05).  26  1.8  Ci  1.6  u.  0  1.4  1.2  DJ88FMAMJJ ASONDJ89FMAMJ J MONTH  Fig. 5. Annual changes in condition factor (CF) in female Thai carp in a rearing pond at Kalasin  Freshwater Fisheries Station during 1987-89. Each value represents mean ± SEM. * indicates significant difference from previous month as determined by Tukey HSD Test (P < 0.05).  27  3  2.5  2  1  0.5  0  DJ88FMAMJ J A SONDJ89F MAMJ J MONTH  Fig. 6. Annual changes in hepatosomatic index (HSI) in female Thai carp in a rearing pond at Kalasin Freshwater Fisheries Station during 1987-89. Each value represents mean ± SEM.  28  b. Hepatosomatic index (Fig. 6) HSI gradually decreased from 2.4% in December 1987 and reached its lowest level (1.2%) in May 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 no significant changes in GSI between May and July. However, GSI gradually decreased to its lowest level in August (6.8%) and remained unchanged until December. Similarly, in 1989, GSI values rapidly increased in January and peaked in May (19.6%), which indicates an annual cycle for GSI in the Thai carp. GSI had a high positive correlation with maximum temperature (r = 0.711, P < 0.05). It also had a highly negative correlation with HSI (r = -0.75, P < 0.05).  B. Male  a. Condition factor (Fig. 8) There were no significant changes for CF in the male Thai carp. Mean CF values ranged between 1.1-1.6 % throughout the study period. Also, there were no correlations between CF and any environmental parameter.  29  25  20  ......^1 5 ae  in a  10  5  0  DJ88FMAMJ J A SONDJ89F MAMJ J MONTH  Fig. 7. Annual changes in gonadosomatic index (GSI) in female Thai carp in a rearing pond at Kalasin Freshwater Fisheries Station during 1987-89. Each value represents mean ± SEM. • indicates significant difference from previous month as determined by Tukey HSD test (P < 0.05).  30  2  1.5  U. 0  1  0.5  0  DJ88FMAMJJASONDJ89FMAMJJ MONTH  Fig. 8. Annual changes in condition factor (CF) in male Thai carp in a rearing pond at Kalasin Freshwater Fisheries Station during 1987-89. Each value represents mean ± SEM.  31  b. Hepatosomatic index (Fig. 9)  HSI decreased from December 1987 to April 1988. Then, it remained almost unchanged until February 1989, at which point it increased significantly, peaking in March and significantly decreasing to 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 slowly decreased to a lower value in December. In 1989, GSI started to increase during January and reaching its 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 tissues  A. Anatomy of the gonads  The ovaries of the Thai carp are paired elongated structures lying below air bladder, kidney and body wall by means of mesovarium. They have a cone-shaped structure: the base projects into the anterior 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 are suspended by mesentaries in the upper section of body cavity. The testes are composed of ill-defined lobules which are separated by thin connective tissue.  32  3 2.5 2  1  1 0.5 0  DJ88FMAMJJASONDJ89FMAMJJ MONTH  Fig. 9. Annual changes in hepatosomatic index (HSI) in male Thai carp in a rearing pond at Kalasin Freshwater Fisheries Station during 1987-89. Each value represents mean ± SEM. * indicates significant difference from previous month as determined by Tukey HSD test (P < 0.05).  33  6  5  4 Ci Co CD  3  2  1  0  DJ88FMAMJ J A SONDJ89F M A M J J MONTH  Fig. 10. Annual changes in gonadosomatic index (GSI) in male Thai carp in a rearing pond at Kalasin Freshwater Fisheries Station during 1987-89. Each value represents mean ± SEM. * indicates significant difference from previous month as determined by Tukey HSD test (P < 0.05).  34  B. Histology of the ovary  Oocyte development in the Thai carp can be divided into 5 stages according to the general classification 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. The nucleus of an oocyte is a large round body with distinct chromosomes in various stages of meiotic prophase and exhibits one large centrally located nucleolus. Mean oocyte diameter was 0.058 ± 0.001 mm (range from 0.024 to 0.1 mm) Oocytes in the chromatin nucleolus stage were found at every month of the year, and the percentages of oocytes at this stage are shown in Fig. 12a. The maximum percentage was found in February 1988, after which it decreased to about 15% in March and stayed at this level until the next spawning 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 the nucleus and the cytoplasm exhibited a marked affmity for haematoxylin (basophilic). During this stage, the nucleus enlarged; the chromosomes become less distinct characteristics, and the cytoplasm increased 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). The percentage 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 perinucleolus stage oocytes decreased again in February 1989 which indicated a new cycle of oogenesis.  35  r^1  p  Fig. 11.1. Cross-sections of ovaries. (a) Stage I oocyte 160X. (b) Stage II oocyte 160X. (c) Stage III oocyte 40X.  36  Fig. 11.2. Cross-sections of ovaries. (d) Stage IV oocyte, early stage 40X. (dl) Stage IV oocyte, late stage 40X.  37  Fig. 113. Cross-sections of ovaries. (e) Stage V oocyte, germinal vesicle migration stage 40X. (f) Atretic oocyte 40X (g) Post-ovulatory oocyte 40X.  38  80 a. Stage I oocyte  70 60 50 40 30 20 10  0 ^ DJ88FMAMJ J ASONDJ89FMAMJ J MONTH  Fig. 12.1. Annual changes in the occurrence of oocytes in different stages in female Thai carp in a rearing pond at Kalasin Freshwater Fisheries Station during 1987-89. (a) Stage I oocyte. Each value represents mean ± SEM.  39  80 b. Stage II oocyte  70 60 50 40 30 20 10 Irl  DJ88FMAMJJASONDJ89FMAMJJ MONTH  80  c. Stage III oocyte  70 60  a- 30 20  10 0  I  111  T^ J  T  DJ88FMAMJJASONDJ89FMAMJJ MONTH  Fig. 12 2. Annual changes in the occurrence of oocytes in different stages in female Thai carp in a rearing pond at Kalasin Freshwater Fisheries Station during 1987-89. (b) Stage II oocyte. (c) Stage HI oocyte. Each value represents mean ± SEM.  40  CS  z 40 0 cc a. 30  DJ88FMAMJJASONDJ89FMAMJJ MONTH  80 e. Stage V oocyte  70 60 50 40  II  30 20  10  DJ88FMAMJJASONDJ89FMAMJJ MONTH  Fig. 123. Annual changes in the occurrence of oocytes in different stages in female Thai carp in a rearing pond at Kalasin Freshwater Fisheries Station during 1987-89. (d) Stage N oocyte. (e) Stage V oocyte. Each value represents mean ± SEM.  41  DJ88FMAMJJASONDJ89FMAMJJ MONTH  80 70 60 50 40 30 20 10  DJ88FMAMJJASONDJ89FMAMJJ MONTH  Fig. 12 4. Annual changes in the occurrence of oocytes in different stages in female Thai carp in a rearing pond at Kalasin Freshwater Fisheries Station during 1987-89. (f) Atretic oocyte. (g) Postovulatory oocyte. Each value represents mean ± SEM.  42  There 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 of vacuoles on the periphery of the cytoplasm. There was no pattern in the deposition of yolk vesicles which formed randomly. These yolk vesicles also increased in number until they occupied the entire cytoplasm of the oocytes. Nucleoli were still evident in the periphery of the nucleus, but were less well defined. The cytoplasm was less basophilic at the end of this stage, and the nucleus was stained with eosin. 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 or  March 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 vacuoles present in stage III oocytes. At the end of this stage, yolk globules were present throughout the cytoplasm, displacing the yolk vesicles to the periphery. The nucleus was still centrally located in the oocyte and contained many nucleoli attached to the nucleus membrane. Mean stage IV oocyte diameter 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 spawning season) 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 nuclear membrane. Later, the nucleus began to move toward the periphery of the cytoplasm, and all the  43  nucleoli were concentrated in the center of the nucleus. Finally, the nucleus lost its shape with disintegration 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%) and peaked in May (44.7%) (Fig. 12e). Thereafter, the percentage of stage V oocytes slowly decreased to 23.8% 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. 110 -  Post-ovulatory oocytes were present every month (Fig. 12f), although with a fluctuating pattern. In 1988, they increased gradually from January (0.1%) and peaked around May (6.1%), after which, they gradually decreased to a lower level in August (2.2%). Similarly, in the 1989 spawning season, 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 and ovulation, but underwent degeneration prior to reabsorption. In the Thai carp, atretic oocytes were observed every month (Fig. 12g), with the highest percentages being observed during June or July.  C. Histology of the testis  The 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 are closely packed together, but separated by the stroma tissue. Each lobule contains several cysts of germ cells which may be in various stages of division. Following the descriptions of Lehri (1967), Htun-Han  44  (1978) and Billard (1986) the spermatogenic cells of the testis of the Thai carp can be divided into 5 stages as follows: a. Stage I: Spermatogonia (Fig. 13a) Spermatogonia are generally the largest germ cells, being present either singly or in nests on the gonadal lamella. Each spermatogonium (0.010-0.020 mm in diameter) was surrounded by a flat somatic cell which may subsequently develop to form a cyst wall. The cytoplasm stained faintly with eosin, and the nuclei occupied a greater area of the cell. Spermatogonia were found every month of the year (Fig. 14a). In both years, their appearance rapidly decreased from January and reached the lowest level in May, after which they gradually increased and peaked in December. There were highly negative correlations between the occurrence of spermatogonia and minimum and maximum temperature (r = -0.74 and -0.76 respectively) and between the occurrence of spermatogonia and daylength (r = -0.71).  45  Fig. 13. Cross-sections of testes. (a) Spermatogonia. (b) Primary spermatocytes. (c) Secondary spermatocytes. (d) Spermatids. (e) Spermatozoa. a,b,c,d, and e 160X.  46  100 a. Spermatogonia  90 80 70 60 50 40 30 20 -7-  10 0  DJ88FMAMJ J ASONDJ89FMAMJ J MONTH  Fig. 14.1. Annual changes in the testicular germ cells at different stages in male Thai carp in a rearing pond at Kalasin Freshwater Fisheries Station during 1987-89. (a) Spermatogonia. Each value represents mean ± SEM.  47  100 90 80 70 60 50 40 30 20 10 0  D 88FMAMJJASONDJ89FMAMJJ MONTH  100 ^ 90  c. Secondary spermatocytes  80 70 60 50 40 30 20 10 0  DJ88FMAMJJASONDJ89FMAMJJ MONTH  Fig. 14.2. Annual changes in the testicular germ cells at different stages in male Thai carp in a rearing pond at Kalasin Freshwater Fisheries Station during 1987-89. (b) Primary spermatocytes. (c) Secondary spermatocytes. Each value represents mean ± SEM.  48  100 d. Spermatids  90 80 70 60 50 40 30 20 10 0  DJ88 F MAMJJASONDJ89FMAMJJ MONTH  100 e. Spermatozoa  90 80 70 60 50 40 30 20 10  0 ^' DJ88FMAMJJASONDJ89FMAMJJ MONTH  Fig. 143 Annual changes in the testicular germ cells at different stages in male Thai carp in a rearing pond at Kalasin Freshwater Fisheries Station during 1987-89. (d) Spermatids. (e) Spermatozoa. Each value represents mean ± SEM.  49  b. Stage II: Primary spermatocytes (Fig. 13b) Primary spermatocytes were distinguished from the spermatogonia by their size (0.008-0.010 in diameter) and appearance. They usually stain more deeply than spermatogonia, and exhibit nuclei densely 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, primary spermatocytes 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 more numerous than the primary spermatocytes. They were also more uniform in color than primary spermatocytes, and their nuclei contained a thick clump of chromatin. Secondary spermatocytes were found throughout the year (Fig. 14c). In 1988, their frequency decreased from January to the lowest level in July. In 1989, however, the percentage of secondary spermatocytes 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, which corresponded to the peak of the spawning season, the pattern tended to be intermittent. However, the number 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 slowly decreased in July.  50  e. Stage V: Spermatozoa (Fig. 13e)  Spermatozoa were transformed from spermatids without further division, but changed shape and became tailed. The tails tended to agglutinate within the nests, giving the nests a parachute-shaped appearance. Spermatozoa were present in abundance throughout the year (Fig. 14e). In general, they tended to be most numerous during spawning season (February to July) and lowest during August to January. However, the percentage in 1989 appeared lower than observed during 1988.  D. Histology of the liver  Microscopically, the liver of the Thai carp was composed of numerous hepatocytes. These cells, with a roundish centrally located nucleus, stained weakly with haematoxylin and eosin. The diameter of the hepatocytes ranged from 0.054 to 0.093 mm (mean = 0.071 ± 0.001), and varied seasonally (Fig. 15). In general, the diameter of hepatocytes tended to decrease significantly from January (0.079 mm) to reach their smallest size in April to May, and then slowly increased, and peaked around July to September. A high positive correlation between hepatocyte diameter and HSI (r = 0.7) was observed.  51  0.1  0.09  E'  E 0.08 cc w I— w M0' 07  a  0.06  0.05  1  " " '  DJ88FMAMJJ A SONDJ89F MAMJ J MONTH  Fig. 15. Annual changes in hepatocyte diameter in female Thai carp in a rearing pond at Kalasin Freshwater Fisheries Station during 1987-89. Each value represents the mean ± SEM.  52  IV. Seasonal changes in plasma gonadotropin and steroid hormones  A. Hormonal changes in female Thai carp  a. Gonadotropin (GtH)  Plasma levels of gonadotropin decreased from December 1987 (2.21 ng/ml) to attain lowest levels in July 1988 (1.13 ng/ml) (Fig. 16). GtH then gradually increased and peaked in September (3.29 ng/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 increased significantly from December 1987 to January 1988 (t-test, P < 0.05), then decreased slightly in February. A slightly increased T level was observed in July which corresponded to the spawning season. 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 observed again in June (5.12 ng/ml). A similar trend was also found in 1988-89, when E2 exhibited a rapid increase in December (6.38 ng/ml), and a slow decline in January (4.58 ng/ml). Further, a rapidly increase in the level of E2 was also observed in March (8.83 ng/ml).  53  In general, plasma levels of GtH, T, and E2 in female carp showed a variable pattern of seasonal change (Fig. 18). GtH tended to be high during the postspawning period and decreased during the spawning season. T and E2 displayed a similar pattern. Both peaked during the preparatory period (January and December) and showed a small surge during spawning.  54  4  3  1  0  DJ88FMAMJ J ASONDJ89FMAMJ J MONTH  Fig. 16. Annual changes in plasma gonadotropin in female Thai carp in a rearing pond at Kalasin Freshwater Fisheries Station during 1987-89. Each value represents the mean ± SEM.  55  0.8  I— 0 . 4  0.2  i  D..188F M A M J J A S ON D..189F M A M J J MONTH  Fig. 17. Annual changes in plasma testosterone in female Thai carp in a rearing pond at Kalasin Freshwater 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).  56  16 14 12  10  4 2 0  DJ88FMAMJJASONDJ89FMAMJJ MONTH  Fig. 18 Annual changes in plasma estradiol-17ft in female Thai carp in a rearing pond at Kalasin Freshwater 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).  57  0  "^  DJ88FMAMJJASONDJ89FMAMJJ  0  MONTH GtH(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 at Kalasin Freshwater Fisheries Station during 1987-89.  58  B. Hormonal changes in male Thai carp  a. 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 gradually  decreased in August. In September, GtH levels slowly increased again and showed another peak in October, 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 December 1987 and peaked in January 1988 (Fig. 21). Then, T gradually decreased from February to a lower level in April. The levels of T then increased and reached another peak in July after which T rapidly decreased during August. T then rapidly increased in September, remained high until October, before decreasing 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 December and peaked in January (Fig. 22). The levels of 11-KT then significantly decreased from February to a lower level in April, and gradually increased and peaked again in July.  59  8  6  2  0  DJ88F M A M J J A S O N DJ89F M A M J J MONTH  Fig. 20. Annual changes in plasma gonadotropin in male Thai carp in a rearing pond at Kalasin Freshwater Fisheries Station during 1987-89. Each value represents the mean ± SEM.  60  4  3  1  0  DJ88FMAMJ J ASONDJ89FMAMJ J MONTH  Fig. 21. Annual changes in plasma testosterone in male Thai carp in a rearing pond at Kalasin Freshwater 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).  61  2 1  0  DJ88FMAMJ J ASONDJ89FMAMJ J MONTH  Fig. 22. Annual changes in plasma 11-ketotestosterone in male Thai carp in a rearing pond at Kalasin Freshwater 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).  62  E  0 ^ 0 DJ88FMAMJJASONDJ89FMAMJJ MONTH  GSI^Testoterone^11-KT^GtH  Fig. 23. Annual changes in GSI and plasma hormone levels in male Thai carp in a rearing pond at  Kalasin Freshwater Fisheries Station during 1987-89.  63  Concentrations of 11-KT rapidly declined in August before increasing in September. The levels of 11-KT then slowly decreased to its lowest level in November which was maintained through December Similarly, in 1989, 11-KT gradually increased from January and peaked in May. 11-KT level 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). In general, plasma GtH, T, and 11-KT in the male Thai carp exhibited variable patterns of changes as found in female (Fig. 23). However, the concentrations of these hormones were generally higher than those of the female.  D. DISCUSSION  a. Females  The present results demonstrate the reproductive cycle of the female Thai carp reared in ponds at Kalasin Freshwater Fisheries Station, Kalasin, Thailand. Gonadal recrudescence, in terms of a gradual increase in GSI, was highly correlated with the sequential changes of environmental factors such as air temperatures and daylength. Furthermore, the decline in GSI which indicated the period of spawning 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 several tropical fish such as the Indian catfish, Heteropneustes fossilis, (Lamba et al. 1983) and the walking catfish, Clarias batrachhus, (Singh and Singh, 1991). Histological analysis of the ovary in the Thai carp demonstrated the presence of oocytes of various stages, without pronounced clutches, throughout the year. This pattern of oocyte development corresponds to the asynchronous type of oocyte development which is usually observed in species with  64  protracted spawning seasons and multiple spawns per female (de Vlaming, 1983). This is in agreement with the fmding of Sirikul et al. (1986) that the female Thai carp can be induced to spawn at least 3 times in a spawning period. A rapid increased in the percentage of oocytes in the perinucleolar stage which indicated the period of oogenesis (de Vlaming, 1983; Selman and Wallace, 1989) was observed during the months of August 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 plasma hormone levels. This indicates that the regulation of oogenesis is independent of endocrine control as suggested 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 these stages 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 into oocytes is regulated by GtH I (Tyler et al. 1991). In the present study, a gradual increase in the occurrence of the postvitellogenic oocytes and GSI coincides with a surge in plasma E2 in both 1988 and 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 was synthesized 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). This  65  supports 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 and daylength (r = 0.6-0.7). The maximum percentage of mature oocytes was observed to coincide with the period of maximum air temperatures. It has been suggested that temperature may act directly on the ovarian 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, in term of decreasing GSI, only occurred during the rainy season. It has been reported in numerous tropical cyprinid species that gonadal development can be completed over a period of 2 months before the usual spawning period (Qasim and Qayyum, 1961; Parameswaran et al. 1970; Tsai et al. 1981; Smith and Jiffry, 1986). This allows the fish to be opportunistic in their responses to suitable spawning conditions (Munro, 1990). Rainfall was found to be the most important proximate factor to initiate spawning in many tropical 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 of the coincidence of spawning and rainfall seem to be the increase in living space and food supply which become available at this time (Schwassmann, 1978). Spawning of the Thai carp in rearing ponds usually occurred after the onset of heavy rain and only 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 the rainy season in Thailand (Sipitakkiat and Leenanond, 1984) and throughout the year in Malaysia (Tan and Begum, 1985). The longer period of spawning for this species in Malaysia than in northeast Thailand is mainly due to the prolonged rainy season in Malaysia.  66  Pronounced 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 pronounced seasonal changes in plasma reproductive hormones in the female Thai carp. The reason for this might be the fact that the fish used in the study are in hatchery conditions. Though they were exposed to the natural daylength, temperature, and rainfall, the changes in reproductive hormones might not reflect the 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 of vitellogenesis which was confirmed by the elevation of GSI. Lower levels of GtH were observed during the spawning period. A similar pattern of seasonal changes in plasma GtH was observed in the common carp in Israel (Yaron and Levavi-Zermonsky, 1986). In the goldfish, however, plasma GtH showed a high amplitude of change, and the maximum concentration was observed during and shortly after the spawning period (Kobayashi et al. 1986a). The difference between the profile of plasma GtH in the Thai carp and in the goldfish is probably due to the pattern of their gonadal development. In the Thai carp which spawn a few times during an extended spawning period (Sirikul et al. 1986), GSI was maintained at the highest value for about 4 months. Whereas in the goldfish, a surge in GSI was found to 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 the goldfish, 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 January 1988 and in December 1988, to coincide with the initial increase in GSI. A gradual increase in plasma E2 during ovarian recrudescence has been reported in several teleosts such as rainbow trout (Lambert et 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 yolk precursor, vitellogenin (Aida et al. 1973; Sundararaj and Nath, 1981). Though only a rapid surge in  67  plasma E2 was observed in Thai carp, this surge in E2 is probably sufficient to stimulate vitellogenesis since it has been shown in the Indian catfish that a surge in E2 alone was sufficient to maintain a high rate of vitellogenesis (Sundararaj and Nath, 1981). In the Thai carp, plasma T and E2 exhibited a pattern of seasonal changes which is similar to that demonstrated in the goldfish (Kobayashi et al. 1986a) and the common carp (Yaron and LevaviZermonsky, 1986). However, plasma concentration of E2 in the Thai carp was higher than that of T throughout the year. The plasma concentration of T has been observed to be higher than that of E2 in most female teleosts which exhibit synchronous oocyte development or only spawn once in a spawning season 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 E2 than T concentrations have been observed in species with continuous vitellogenesis which spawn more than once in a spawning season such as the common carp (Yaron and Levavi-Zermonsky, 1986; Galas and 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 suggested that 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 a precursor for E2 synthesis. The lower concentration of T than that of E2 suggests that T is rapidly and effectively aromatized into E2 in the ovary of the Thai carp. The role of T in oocyte maturation or ovulation is still not known, but T has been suggested to be involved in the feedback regulation of GtH secretion in the goldfish (Trudeau et al. 1991).  68  b. Males Gonadal development in male Thai carp exhibited a similar pattern to that observed in females. 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 the spawning of the Thai carp coincides with the occurrence of rainfall which is difficult to predict, this synchronous 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 is probably not modulated by the presence of the opposite sex as has been shown in Tilapia, Sarotherodon  mossambicus (Silverman, 1978a; b). Similar to the situation in females, the GSI in male Thai carp exhibited a highly negative correlation with HSI ( r = -0.7). This inverse correlation between GSI and HSI in females indicated the role of liver as the site for yolk protein synthesis as mentioned above. In males, however, this inverse correlation may reflect the role of the liver as a primary energy reserve since the depletion of energy reserves, indicated by a declining HSI, during gonadal development has been suggested in the male 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 in most teleosts studied to date (Billard, 1986). Histological analysis of the testis reveals a continuous spermatogenetic activity throughout the years as indicated by the presence of all stages of germ cells in the 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 of spermatogonia, occurred immediately after spawning when GSI started to decrease. In goldfish, the occurrence of spermatogonia was hormone independent (Billard et al. 1982). Similarly, in the present study, there were no correlations between plasma GtH, T, or 11-KT levels and the occurrence of  69  spermatogonia. However, the occurrence of spermatogonia exhibited a highly negative correlation with air temperature ( r = -0.75) and the amount of rainfall ( r = -0.71). This suggests the important of environmental cues in the initiation of spermatogenesis in the Thai carp. Environmental factors were found to be involved in spermatogenesis in several cyprinids. For example, increasing rearing temperature accelerated the process of spermatogenesis in tench, Tinca tinca (Breton et al. 1980) and in goldfish (Billard, 1986). Spermatocytes, spermatids and spermatozoa were also observed throughout the year without pronounced seasonal changes. However, the occurrence of spermatozoa was always found at the highest proportion in the testis in every sample. This suggests a rapid transformation of germ cells from spermatocytes to spermatids and further to spermatozoa, which indicates the readiness for spawning in males and coincides with the observation that milt can be stripped from the fish throughout the year in these pond reared fish Spermatogonial division and subsequent meiosis are hormone dependent (Billard et al. 1982). Hypophysectomy and hormone replacement therapy studies in the goldfish have demonstrated that the transformation of spermatogonia to spermatocytes was GtH-dependent (Billard et al. 1982). Also, qualitative maintenance or restoration of spermatogenesis has been reported in hypophysectomized goldfish 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 levels and the occurrence of spermatocytes, spermatids and spermatozoa. Similar results were observed in rainbow trout (Scott and Sumpter, 1989) and in common carp (Koldras et a1. 1990) where there were no 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 seasonal changes. 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 the  70  main 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. The first peak of plasma GtH, T and 11-KT was observed at the same time in January 1988 when the GSI started to increase. This indicates the important of endogenous cues in the initiation of gonadal development. However, there was no significant correlation between plasma hormone levels and GSI in the present study as has been observed in goldfish (Kobayashi et al. 1986a). The reason for this is probably due to the fact that the Thai carp has an extended spawning period and can spawn several times within a spawning period (Sirikul et a1. 1986) while the goldfish spawned a few times during a short spawning period (Kobayashi et al. 1986a). Two peaks of plasma GtH, T and 11-KT levels were observed during the spawning period in the male Thai carp which was indicated by the decline of GSI. This suggests the involvement of GtH and androgens in the processes of spawning in the male Thai carp. The role of reproductive hormones during spawning is discussed in chapter 5. The importance of hormones in spermatogenesis has been demonstrated in several fish species. For example, in goldfish, a high levels of GtH was needed to maintain spermatocytes and spermatids (Breton et al. 1973). Also, plasma T and 11-KT were found to be important in the control of the spermatogenetic process in several teleost species (Billard et al. 1982), but 11-KT was also important in stimulating development of male secondary sex characteristics as has been observed in sockeye 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 tropical fish which spawns several times during a protracted spawning period. The changes in plasma levels of these hormones are probably so rapid that they cannot be detected by a monthly sampling regime.  71  CHAFFER 4 INTERACTION OF SGNRHA AND DOMPERIDONE ON THE INDUCTION OF GONADOTROPIN SECRETION AND INDUCTION OF SPAWNING IN THE THAI CARP  A. INTRODUCTION  Gonadotropin secretion in teleosts is under the dual control of gonadotropin-releasing hormone (GnRH) and dopamine which acts directly at the pituitary, via specific dopamine type 2 receptors (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 when sGnRHA 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 Thai carp. In the present study, the interaction of sGnRHA and Dom on the induction of GtH secretion and ovulation in the Thai carp was examined in order to determine the minimum dose of sGnRHA and Dom required for inducing ovulation of this species.  B. MATERIALS AND METHODS  Two experiments were performed during 1990 and 1991 spawning seasons. In 1990, the experiment was carried out at Kalasin Freshwater Fisheries Station during the month of July. One hundred and forty four sexually mature females Thai carp (612.29 ± 20.88 g) were divided into 16 different 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 group  72  together with the same number of untreated sexually mature males were transferred to a 8 m 2 concrete-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, as described in chapter 2. The fish were allowed to spawn naturally, and the numbers of fish which spawned during the 24 hr period following injection were noted. As a result of a power failure at Kalasin Freshwater Fisheries Station, none of the plasma samples could be used for hormones analyses. The 1991 experiment was performed at Pathumthani Freshwater Fisheries Station, during the month of May. Except for differences in dose level, the design of the experiment was similar to the 1990 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 10 µg/kg BW) and Dom (0 to 10 mg/kg Bw) (Table 2). After injection, fish were transferred to a 6 m 2 concrete-lined outdoor spawning tank supplied with running water at a temperature of 31±1 ° C and blood samples were taken serially as mentioned above. Plasma samples were analyzed for GtH content at the Department of Zoology, University of Alberta, Edmonton, Alberta.  73  Table 1: Dose combinations of sGnRHA and Dom: Kalasin Freshwater Fisheries Station in July 1990.  sGnRHA (Mg/kg) 0  1  5  25  0  0+0  0+1  0+5  0+25  1  1+0  1+1  1+5  1+25  5  5+0  5+1  5+5  5+25  25  25+0  25+1  25+5  25+25  Dom (mg/kg)  Table 2: Dose combinations of sGnRHA and Dom: Pathumthani Freshwater Fisheries Station in May 1991.  sGnRHA (µg/kg) 0  1  5  10  0  0+0  0+1  0+5  0+10  1  1+0  1+1  1+5  1+10  5  5+0  5+1  5+5  5+10  10  10+0  10+1  10+5  10+10  Dom (mg/kg)  74  100  -0 a) 8 0  be  a  -  co 60 a. co 40  ii  ae 20  a  a a  a  a^a a^0  25  sGnRHA (pg/kg)  Fig. 24. Effect of various combinations of sGnRHA (µg/kg) and Dom (mg/kg) on the induction of  spawning in female Thai carp during July 1990 at Kalasin Freshwater Fisheries Station. Each value represents the percentage of fish spawned in each treatment. Groups which were similar 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, and c.  75  100  cd  -a 80 a)  001 go_ abcadbcd  A AVAM1111131/ AC=^rACIIIVAIE111/ e 0 ab^  ae 20  ab  lo  4\4)  10^5^1^0  sGnRHA (pg/kg)  Fig. 25. Effect of various combinations of sGnRHA (µg/kg) and Dom (mg/kg) on the induction of spawning 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 were similar 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.  76  C. RESULTS  In both years, various number of fish in each treatment group began spawning 4 hr after injection. Spawning behavior lasted for about 2 hr (6 hr post injection).  a. Percent fish spawned In 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 of the group treated with the highest concentration of Dom used in this study (0+ 25). Spawning occurred in 1 out of 9 fish in each of the groups receiving the highest dose of sGnRHA alone (25+0) or in combination with 1 mg/kg Dom (25+1). The proportion of fish spawning increased with increasing concentrations of sGnRHA and/or Dom. All fish spawned in the group that received maximum doses of both sGnRHA and Dom (25 + 25). At each concentration of sGnRHA, the proportion of fish observed spawning was highest in groups receiving the maximum dose of Dom; however, the differences between groups receiving 5 and 25 mg/kg Dom at each dose levels of sGnRHA were not significant. In 1991 (Fig. 25), no spawning occurred in the control group (0+0) and in groups receiving Dom only. In the four groups receiving sGnRHA alone, 1 out of 9 fish spawned in the group receiving 5 µg/kg sGnRHA (5 +0). In groups receiving combinations of sGnRHA and Dom, the proportion of fish spawning increased with increasing concentrations of sGnRHA and/or Dom. The maximum number spawning (88.89%) was observed in the group which received 10 µg/kg sGnRHA and 5 mg/kg Dom. At the same concentration of sGnRHA (1, 5, or 10 µg/kg), groups treated with 5 mg/kg Dom demonstrated an equal or higher, but not significantly different, percentage of spawned fish compared to those treated with 10 mg/kg Dom.  77  b. Plasma GtH levels There were no significant differences in plasma GtH levels at 0 hr (Fig. 26). GtH levels varied between 1.5 and 3.0 ng/ml. In all groups, except the controls, plasma GtH levels increased significantly 3 hr after injection of any combination of sGnRHA and Dom compared to their corresponding groups at 0 hr (Fig. 27). In general, GtH increased with increasing concentrations of sGnRHA and/or Dom. The peak mean GtH value (656.10 ng/ml) was recorded following delivery of 10 Ag/kg sGnRHA and 5 mg/kg Dom. This treatment 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 fish GtH increased approximately 5 times when the concentration of Dom was increased from 1 to 5 mg/kg. By 6 hr after injection, plasma GtH concentrations in each treatment group decreased slightly compared with 3 hr post injection (Fig. 28). However, all groups, except controls and the group receiving 1 mg/kg Dom alone, exhibited significantly higher plasma GtH than at 0 hr. Plasma GtH varied with increasing dose of sGnRHA and/or Dom, but unlike the situation at 3 hr, all groups receiving 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 the combination of 5 µg/kg sGnRHA and 10 mg/kg Dom. The GtH level in the group treated with Dom alone at the concentration of 10 mg/kg remained significantly higher than that of controls.  78  7 6,  E 54 3  a  -  20  / z ,L^0  411111111%  /5  ^ 10^5^1 0 sGnRHA (.ig/kg)  /0  e  Fig. 26. Effect of various combinations of sGnRHA (µg/kg) and Dom (mg/kg) on the induction of GtH secretion in female Thai carp at injection time in the 1991 study. Each value represents the mean plasma GtH concentration (ng/ml) in each treatment.  79  700 600 500 400 300 (n 200  0. 100  NIVIMSSSSMIIIMIAMS,SS \ MSS,S,  0 10^5^1^0  sGnRHA (pg/kg)  Fig. 27. Effect of various combinations of sGnRHA (µg/kg) and Dom (mg/kg) on the induction of GtH secretion in female Thai carp at 3 hr after injection in the 1991 study. Each value represents the mean plasma GtH concentration (ng/ml) in each treatment. Plasma GtH levels which were similar 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).  80  700 -"=" 600  E  0) 500 = 400  5 300 ro  co 200 0 100  b d ef 1 ^abcd bcdel^bcde  0 10^5^1  sGnRHA (lig/kg)  Fig. 28. Effect of various combinations of sGnRHA (µg/kg) and Dom (mg/kg) on the induction of GtH secretion in female Thai carp at 6 hr after injection in the 1991 study. Each value represents the mean plasma GtH concentration (ng/ml) in each treatment. Plasma GtH levels which were similar 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).  81  700 = 600  rn 500 400  5 300 co E 200  0_ 100  IIII. 10.9"  10 -N  5 \  NV MUMUMMMUUUW  0 0  0 10^5^1^0  O  sGnRHA (pg/kg)  Fig. 29. Effect of various combinations of sGnRHA (µg/kg) and Dom (mg/kg) on the induction of GtH secretion in female Thai carp at 9 hr after injection in the 1991 study. Each value represents the mean plasma GtH concentration (ng/ml) in each treatment. Plasma GtH levels which are similar 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).  82  At 9 hr post injection (Fig. 29), plasma GtH levels were lower, but in each treatment group remained significantly higher than at 0 hr, except in the control and in the group receiving Dom alone at the concentration of 1 mg/kg. GtH levels varied with concentrations of sGnRHA and/or Dom. The peak level was observed in the group treated with the highest concentrations of sGnRHA and Dom used in this study. In groups receiving a combination of sGnRHA and Dom, GtH remained 10-200 times higher than their respective controls at 0 hr.  D. DISCUSSION  The present results demonstrate that sGnRHA or Dom alone were relatively ineffective in inducing spawning in sexually mature Thai carp. A very low spawning rate (11.11%) was observed only in groups treated with 5 µg/kg or 25 µg/kg sGnRHA alone in the 1991 and 1990 experiment respectively. Also, in groups receiving Dom alone, spawning was observed only in the group treated with the highest dosage used in the 1990 study. Either sGnRHA or Dom alone at appropriate concentrations stimulated a modest plasma GtH release. sGnRHA alone at 5 and 10 µg/kg BW significantly increased plasma GtH at 3 (except 10 µg/kg), 6, and 9 (except 5 mg/kg) hr post injection. Similarly, 10 mg/kg of Dom alone significantly increased plasma GtH levels at 3 and 6 hr after injection. Nevertheless, the magnitude of increasing GtH levels stimulated by either sGnRHA or Dom alone, at the maximum dosage of 10 µg/kg or 10 mg/kg was initially insufficient to initiate the processes of final maturation and ovulation in the Thai carp. However, in the situation where dopamine receptors are completely blocked after treatment with a high concentration of Dom (25 mg/kg), the production of exogenous GnRH is high enough to stimulate a sufficient surge of the preovulatory GtH and to induce spawning in this species. These results suggest that dopamine serves as a potent gonadotropin release inhibiting factor in the Thai carp  83  as previously demonstrated in goldfish (Chang and Peter, 1983; Chang et al. 1984), common carp (Lin et 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 highaffinity and low capacity, the other with a low-affinity and high capacity (Habibi et al. 1987). From studies on structure-activity relationships in GnRHs, high affinity GnRH receptors are thought to be involved in the control of GtH secretion from the goldfish pituitary (Habibi et al. 1987; 1989). In the present study, increasing concentrations of sGnRHA alone were relatively ineffective in stimulating GtH secretion. This suggests that increasing GnRH binding capacity may be the basis for the increased responsiveness to GnRH. The dopaminergic inhibition of basal and GnRH-stimulated gonadotropin release in goldfish was 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 GnRHbinding sites in the goldfish pituitary in a time- and dose-dependent manner, and resulted in an increasing responsiveness to GnRH peptides (De Leeuw et al. 1989). Furthermore, Dom was found to act 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 effectively increased both the number of fish spawning and plasma GtH levels in a dose-related manner. This result suggests that Dom potentiates the effect of sGnRHA on the stimulation of the plasma GtH levels in the Thai carp and confirms the finding of the earlier study of Leelapatra (1988) that dopamine has GRIF activity in the Thai carp. The potentiation action of Dom and GnRHA has been demonstrated 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). In  84  goldfish, it has been suggested that the interaction of sGnRHA and Dom on the regulation of GtH secretion involves changes in the number of pituitary receptors for GnRH and dopamine (Omeljaniuk et al. 1989a). Dom at 1 mg/kg, alone or in combination with sGnRHA, was insufficient to mask the effect of dopamine as a GRIF in the Thai carp. Hence, it caused only a modest increase in plasma GtH at 3 hr and a low percentage of fish spawning. An intermediate concentration of Dom (5 mg/kg), in combination with sGnRHA, was highly effective in stimulating GtH secretion and increasing the number of fish that spawned. However, there was no significant difference in plasma GtH levels or the numbers of fish spawning when higher concentrations of Dom were used (10 or 25 mg/kg in 1990 or 1991 respectively). These results suggest that 5 mg/kg is the most effective dosage of Dom in potentiating the action of sGnRHA. A higher concentration of Dom appeared to prolong the action of sGnRHA, 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 increase in plasma GtH level at all sampling times. The lack of significance probably resulted from highly variable plasma GtH concentrations among fish within the same group. All fish spawned in the group which receiving the combination of the highest dosage of both sGnRHA and Dom used in the 1990 study. The lower maximum doses of sGnRHA and Dom used in the 1991 experiment failed to induce spawning in all individuals in the group. This indicates the importance of sGnRHA in the regulation of GtH secretion and spawning in the Thai carp, since the highest concentration of Dom used in the 1991 experiment was higher than 5 mg/kg, which is the most effective dosage in potentiating the action of sGnRHA. The failure to attain 100% spawning was probably due to the lower concentration of sGnRHA used in the 1991 study. Furthermore, the 1991 experiment was conducted in May, 2 months earlier than the 1990 experiment which was carried out in July. It has been reported in goldfish that the responsiveness to injection of [D-Ala 6 ]-mGnRHA and  85  pimozide, 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 the fish. From the economic and practical points of view, however, since there were no significant differences in the numbers of fish spawning among groups treated with any combination of 5 to 25 µg/kg sGnRHA and 5 to 25 mg/kg Dom, the most economic and appropriate dosage for induced spawning in the Thai carp is probably 5 µg/kg sGnRHA + 5 mg/kg Dom.  86  CHAPTER 5 THE EFFECTS OF SGNRHA AND DOMPERIDONE ON PLASMA GONADOTROPIN AND STEROID HORMONES LEVELS DURING SPAWNING IN THE MALE AND FEMALE THAI CARP  A. INTRODUCTION  The pattern of hormonal changes during final maturation and ovulation provides important basic knowledge for a thorough understanding of the endocrine control of ovulation in fish Most studies have investigated the patterns of hormonal changes during final maturation and ovulation in temperate or subtemperate fishes. At the present time, however, such patterns in tropical fish are not clearly understood. From the previous chapter, it is clear that the preovulatory surge in GtH, which leads to fmal maturation 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 in this species. This study was designed to reveal the patterns of hormonal changes during induced spawning 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 "long term" ( < 9 hr) changes in reproductive hormone concentrations during sGnRHA and domperidone induced 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 interaction between males and females leading to the union of gametes. Spawning behaviour refers to the patterns by which males and females directly synchronize their behaviour to achieve a coordinated release of  87  gametes i.e., oviposition and release of milt (Liley and Stacey, 1983). Courtship is that behaviour involves in the search for, and attraction and excitation of, a potential sexual partner. The term spermiation 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 the hydrated suspension of mature spermatozoa that can be released during spawning.  B. MATERIALS AND METHODS  I. Experiment I. "Long term" changes in plasma hormone levels during induced spawning in male and female Thai carp  Twelve mature females Thai carp (1-2 years of age, average weight 513.33 ± 41.29 g) were induced 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 spawn naturally. The tank was supplied with running water at 30 ± 1 ° C. All fish were blood sampled at the time 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 male and female Thai carp  Mature females Thai carp, average weight 555.56 ± 21.78 g, were divided into 7 groups of 9 fish each. Each group received a single intraperitoneal injection of 20 Ag/kg sGnRHa and 20 mg/kg domperidone. Four groups were injected 4 hr before the other three groups. Fish from the first four groups, together with the same number of mature untreated males (average weight 173.02 ± 8.04),  88  were 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 tank which also divided into 3 separate units. Both tanks were supplied with running water at a temperature of 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 were analyzed for GtH, T, E2, 11-KT, and 17,20/3-P concentration.  C. RESULTS  Experiment I.  Males exhibited spawning behaviour, as described in Liley and Tan (1985), 3.5-4 hours after placement with females. Spawning runs were observed between 4-6 hr after injection. This spawning run was more frequent during the first 30 min after the first run then gradually declined. This spawning activity was completed at 6 hr after injection.  A. Females  a. Estradiol-17fl (E2) (Fig. 30) Plasma E2 concentration increased rapidly from 0.45 ng/ml at 0 hr and peaked 3 hr after injection 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. After spawning, the level rapidly decreased to 0.19 ng/ml at 9 hr. c. Gonadotropin (GtH) (Fig. 32)  89  Plasma GtH concentration increased significantly from 1.44 ng/ml at 0 hr to 900.56 ng/ml at 3 hr 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-P Plasma 17,2013-P levels were virtually undetectable at all times in this study, though the sensitivity of the assay was only 7 pg/ml. A very low level was observed in some samples 3 hr after injection (70.67 pg/ml).  90  rn  ca  a  - -,  5 4  a) ca  E  P,3 2 b  a  ^  0^3  a  ^ ^ 6 9^hr oviposition  Fig. 30. Changes in plasma E2 during sGnRHA (20 µg/kg) and Dom (20 mg/kg) induced spawning in female Thai carp. 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, b, and c.  91  2.5  2  E co 1.5  co  1  cC  0  0.5  a a  0  \\  9  0  hr  Fig. 31. Changes in plasma T during sGnRHA (20 µg/kg) and Dom (20 mg/kg) induced spawning in female Thai carp. 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.  92  1250  1000 rn co  750  a) 5 500 ca  E ca  250  0  0  6  3 ovipositi  9  hr  on  Fig. 32. Changes in plasma GtH during sGnRHA (20 µg/kg) and Dom (20 mg/kg) induced spawning in female Thai carp. 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, b, and c.  93  B. Males  a. 11-Ketotestosterone (11-KT) (Fig. 34) There were no significant changes in plasma 11-KT levels during the long term observation of the spawning activity of the untreated male Thai carp. The level slightly decreased from 0 hr to 3 hr and then gradually increased at 6 hr i.e., during spawning activity. The level then rapidly decreased again 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 presence of 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 spawning period. GtH varied between 1 and 2 ng/ml at all times. d. 17,20/3-P As in the female fish, plasma 17,20/3-P levels were barely detectable (50.67 pg/ml, n=3) at 3 hr after placement in the same tank as the females.  94  1.2  E rn co cu —  0.8  0.6  co 0.4  E  U)  co  0  -  0.2  0  0  3  6  9  hr  s p awn i ng  Fig. 34. Changes in plasma 11-KT during spawning in untreated male Thai carp kept with sGnRHA and Dom induced spawning treated females. Each value represents the mean ± SEM.  95  2.5  E En Co 0)  ab  a) 1.5 co co  11.  a  0.5  0  3  6  9^h r  spawning  Fig. 35. Changes in plasma T during spawning in untreated male Thai carp kept with sGnRHA and Dom treated females. 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.  96  3  a  2.5  E c 2 ti (1)  1.5  E s 0.5  0  0  3  6  9  hr  spawning  Fig. 36. Changes in plasma GtH during spawning in untreated male Thai carp kept with sGnRHA and  Dom treated females. Each value represents the mean ± SEM.  97  Experiment II. Four groups of fish displayed spawning activity 4 hr after injection. At this point, blood samples 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 spawned fish. The data represent mean values of all spawned fish in each group.  A. Females  a. 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 60 min 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 90 min after spawning. b. Testosterone (T) (Fig 38) There were no significant changes in plasma T levels shortly before and after spawning in female Thai carp. Levels varied between 1 and 2 ng/ml throughout the study period.  98  ^  b ab^  ab ab ab  ab  a  ^-90^-60^-30 ovipos Hon^30^ 60^90 min 2:30^3:00^3:30^4:00^4:30^ 5:00^5:30 hr  min before and after spawning, hr after injection  Fig. 37. Short-term changes in plasma E2 levels in sGnRHA (20 1.1g/kg) and Dom (20 mg/kg) induced  spawning in female Thai carp 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.  99  ^  2.5 ••••••••  E  ..-. c) 2 c co  a)  >1.5  Ica  co  o co  (7.  0.5  S  1  ^-90^-60^-30 ovipositIon^30^60 2:30^3:00^3:30^4:00^4:30^5:00  min before and after spawing, hr after injection  re  90 min 5:30 hr  Fig. 38. Short-term changes in plasma T levels in sGnRHA (20 µg/kg) and Dom (20 mg/kg) induced spawning in female Thai carp shortly before, during, and after spawning. Each value represents the mean ± SEM.  100  1200  1000  E cn c 800 a) 600  400  cn 0_  ifi  200  0  ^-90^-60^-30 ovIposItIon 30^60^90 min ^2:30^3:00^3:30^4:00^4:30^5:00^5:30 hr  min before and after spawning, hr after injection  Fig. 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 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.  101  c. 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 a period of 30 min after spawning before declining significantly to approximately 220 ng/ml at 60 and 90 min after spawning (5 hr after injection). d. 17,20/3-P Plasma 17,20/3-P levels were undetectable throughout the study period.  B. Males  a. 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 min before spawning. 11-KT remained at this level until 30 min before spawning and then increased rapidly at spawning time peaking at 30 min after spawning. The concentration then slowly decreased at 90 min after 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 11KT. T levels decreased from 12.73 ng/ml at 90 min before spawning to 5.60 ng/ml at 60 min before spawning. T remained at this level until 30 min before spawning, then rapidly increased at spawning time to peak at 30 min post spawning activity. T then rapidly decreased at 60 min after spawning and maintained at this level until 90 min after spawning.  102  ^  •-  bc  E co  -5  bc  bc  4  co  a)  —3 abc co  2  a  -90^-60^-30 spawning 30^60^90 min hr min before and after spawning, hr after injection of female  ^2:30^3:00^3:30^4:00^4:30^5:00^5:30  Fig. 40. Short term changes in plasma 11-KT levels in untreated male Thai carp kept with sGnRHA -  and Dom 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, b and c.  103  ^  25  20  rnE  -515  be  abc  co  ab  a) 10 co a  Ca  ca  a  CI 5  0  -90^-60^-30 spawning 30^60^90 min hr min before and after spawning, hr After injection of female  ^2:30^3:00^3:30^4:00^4:30^5:00^5:30  Fig. 41. Short-term changes in plasma T levels in untreated male Thai carp kept with sGnRHA and Dom 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  14  - a b co w  8  ab  6  cv co  4  a^a  a  2 0  -90^-60^-30 spawning +30^+60^+90 min 2:30^3:00^3:30^4:00^4:30^5:00^5:30 hr min before and after spawning, hr after injection of female  Fig. 42. Short-term changes in plasma GtH levels in untreated male Thai carp kept with sGnRHA and  Dom 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.  105  c. Gonadotropin (GtH) (Fig. 42) Plasma GtH levels varied between 2 and 7 ng/ml before spawning occurred. GtH then increased significantly to 9.16 ng/ml at spawning time, and declined rapidly to 3.01 ng/ml at 30 min after spawning and maintained at approximately this levels throughout the observation period. d. 17,20fl-P Plasma 17,20p-P levels were undetectable in all samples.  D. DISCUSSION  a. Females  The present results confirmed the previous finding that the preovulatory surge in plasma GtH levels and spawning in the female Thai carp can be stimulated by administration of appropriate quantities of sGnRHA and Dom. In teleost fishes, this surge in plasma GtH has been shown to be important for the initiation of fmal maturation and ovulation processes (Nagahama, 1987). In this study, plasma GtH increased significantly 3 hr after injection. Then GtH gradually decreased after spawning i.e., at 5 hr, but remained high compared with the levels before injection. These high levels of 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. An increase in plasma E2 during spawning induced by hormonal or environmental stimulation has been commonly 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 common carp (Kime and Dolben, 1985; Santos et al. 1986; Levavi-Zermonsky and Yaron, 1986). In goldfish, E2 is known to be synthesized by conversion of T, by aromatase enzyme, in the granulosa layers of the vitellogenic oocytes, under the stimulation of GtH (Kagawa et al. 1984). In the Thai carp, vitellogenic  106  oocytes were present throughout the reproductive cycle (see chapter 3). Thus, the surge in plasma E2 observed in this study at 3 hr post injection was most probably due to gonadotropin-stimulation of the vitellogenic oocytes. Studies of hormonal changes during ovulation in teleosts have revealed that E2 tends to decline preceding ovulation in several teleosts including salmonids (Fostier and Jalabert, 1982; Scott et al. 1983; Van Der Kraak et al. 1984; Dye et al. 1986; Liley et al. 1986; Liley and Rouger, 1990) and  cyprinids such as common carp (Kime and Dolben, 1985; Santos et al. 1986) and the bitterling (Shimizu et al. 1985). This decline in E2 was suggested to be a result of the shift in the steroid synthesis pathway  in the granulosa layers from the production of E2 to the production of 17,20/3-P (Nagahama, 1987). In salmonids 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 spontaneous GtH surge and subsequent ovulation (Pankhurst and Stacey, 1985). An increase in plasma E2 levels during induction of ovulation by brain lesion technique has been 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 ovulation by treatment with GnRH or crude pituitary extract (Weil et al. 1980). On the other hand, there were no 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 function  of a peak in E2 at spawning time in the female Thai carp is unknown, but it may not be involved in the occurrence of ovulation since E2 has been found to be ineffective in inducing oocyte maturation in many 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 E2  107  was observed at 3 hr post injection, then E2 significantly decreased at the end of spawning activity (6 hr). On the other hand, after injection, plasma T levels gradually increased and peaked at 6 hr after injection. However, the short term observations (Expt. II) showed that both T and E2 actually peaked at spawning time. These data suggest that E2 was synthesized during the first part of the induction under the stimulation of GtH. Then, when spawning started, the production of Ea decreased, probably due to the rapidly decreasing plasma GtH. This resulted in an elevation of T. The decrease in E2 production most likely due to a decrease in aromatase activity. Although, the mechanism of the induction or activation of the granulosa cell aromatase system is unknown at the present time, in goldfish, 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 Dolben 1985). The acute preovulatory rise in T in rainbow trout was suggested to be involved in the production of 17,20P-P (Jalabert and Fostier, 1984). In goldfish, high plasma T just before ovulation is considered 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 synthesis pathway due to the reduction of aromatase activity as previously found in the common carp during induced ovulation by hypophysation (Kime and Dolben, 1985). Injection of sGnRHA and Dom in the Thai carp caused a rapid increase in GtH which then stimulated the synthesis of T by vitellogenic and postvitellogenic oocytes as has been shown in goldfish by Kagawa et al. (1984). In several teleosts, it has been suggested that under GtH stimulation, T was converted to 52 by the aromatase enzyme in the granulosa layers of the vitellogenic oocytes (Nagahama, 1987). When spawning occurred, GtH rapidly decreased as well as the aromatase activity. This resulted in a decline in plasma E2 and hence an  108  increase in plasma T. In the Thai carp, the importance of the shift in steroid synthesis is unknown, but it seems to be a physiological requirement for ovulation. The decrease in plasma T after spawning is probably due to the loss of postvitellogenic oocytes after 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 most teleosts studied to date (Goetz, 1983; Scott and Canario, 1987). A surge of plasma 17,20/3-P has been observed 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 Der Kraak et al. 1984; Dye et al. 1986), African catfish (Lambert and Van den Hurk, 1982), goldfish (Stacey et al. 1983; Kagawa et al. 1983; Peter et al. 1984b; Kobayashi et al. 1987; 1988), the bitterling (Shimizu et al. 1985), white sucker (Scott et al. 1984), and common carp (Santos et al. 1986; Levavi-Zermonsky and Yaron 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 induced by pituitary extract. However, in a later in vitro study, Kime and Bieniarz (1987) demonstrated that 17,20/3-P could only be detected in fish that had received a priming dose of pituitary extract. This priming dose of pituitary extract resulted in migration of the germinal vesicle to the periphery (Yaron et 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 synthesis from 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 after injection with sGnRHA and Dom, but no 17,20/3-P was detected.  109  The reason for the failure to detect plasma 17,20/3-P in this study is unknown. All the fish spawned with a high fertilization rate ( > 80% ), indicating that the oocytes had successfully been induced to pass through the normal processes of germinal vesicle migration and germinal vesicle break down. Hence, the reason for lack of 17,20/3-P is probably different from that in common carp (Kime and Bieniarz, 1987). Since the shift in steroid synthesis, which was believed to be the basis for 17,20/3-P production (Jalabert and Fostier, 1984; Kobayashi et al. 1987), was observed in this study, the reason for non detection of 17,20/3-P was probably due to inadequate sampling time. In goldfish, rapid changes in 17,20/3-P were observed which suggested the short-term secretion and/or rapid plasma clearance of this steroid (Kobayashi et al. 1987). In the current study, the peak of 17,20/3-P might have occurred so fast that the sampling time interval designed for this experiment could not detect any changes. 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 and Thomas, 1989; Patino and Thomas, 1990). Moreover, 17,20/3,21-P was found to be at least as potent as 17,20/3-P in the induction of fmal oocyte maturation in in vitro bioassays in spotted seatrout (Thomas and Trant, 1989), dab (Scott and Canario, 1987), and rainbow trout (Canario and Scott, 1988). These suggest the possibility that 17,20/3-P might not be the MIS in the Thai carp.  b. Males  There were no significant changes in plasma GtH levels during spawning in male Thai carp when samples were taken at 3 hr intervals (Expt. I). However, during short term observations in experiment II, a significant increase in plasma GtH was observed at the onset of spawning. This surge in GtH was followed by a rapid decrease 30 min later. This may be an indication that the GtH surge in male Thai carp can also be stimulated by exposure to ovulatory females as previously found in goldfish  110  (Kobayashi et al. 1986c). In the present study, male spawning behaviour was observed shortly before spawning occurred, consistent with the time of GtH surge. In an earlier study, however, spawning behavior in male Thai carp treated with homoplastic pituitary extract (PE) was observed at the same time as PE or prostaglandin PGF2 a treated females, about 3-3.5 hr after treatment and at least 15 min before spawning (Liley and Tan, 1985). In goldfish, similarly, sexual behavior was observed to start before ovulation around the beginning of the GtH increase (Kobayashi et al. 1986c). These observations suggest that the GtH surge in male might be involved in triggering male sexual behaviour in the Thai carp. In female goldfish, a surge of 17,20/3-P was found to be highly synchronous with the GtH surge (Kobayashi et al. 1986a), and 17,20/3-P was found to act as a preovulatory pheromone in goldfish which exerts a priming effect on the male endocrine system i.e., stimulate GtH secretion and the production of milt (Stacey et al. 1987). In the current study, a peak of GtH in females was observed earlier than that of males, though the fish were placed together immediately after injection. This result, together with the very low level of 17,20/3-P observed in female Thai carp, suggests that 17,20/3P might not act as the preovulatory pheromone in Thai carp. Plasma T increased gradually in male Thai carp after placement with sGnRHA and Dom treated females, when samples were taken at 3 hr intervals. During short-term observations in experiment II, however, a surge of T was observed at the time of spawning and at the time of the GtH surge. Similarly, a surge in plasma 11-KT was also observed at this time, though there were no significant 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 and Van Der Kraak, 1991). In rainbow trout, a peak of 11-KT was found to coincide with the peak of milt production, suggesting the importance of 11-KT in the process of milt production (Scott et al. 1980b;  111  Fostier et al. 1982; Baynes and Scott, 1985). In this study, the levels of T and 11-KT continued to increase during the process of spawning, though GtH immediately decreased to a significantly lower level. Thirty minutes after spawning, plasma T rapidly decreased to a lower level, while 11-KT decreased gradually as well as the occurrence of spawning activity. These data indicate that 11-KT is probably 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 detected in males only at 3 hr after placement with females, when samples were taken at 3 hr intervals. 17,20/3P was undetectable during the short-term observations (Expt II). An increase in plasma 17,20/3-P during the process of spawning has been observed in several teleost species including salmonids (Scott and 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 plasmaY androgen. This shift from C19 to C21 steroid synthesis was suggested to be typical of seasonally reproducing male teleosts prior to spawning (Scott et al. 1984), and was found to be mediated by 17,20/3-P or a related progestogen (Barry et a1. 1990). However, a shift from C19 to C21 was not the main key for successful spawning in common carp, since some males which had a GtH surge but no steroidogenic shift can successfully mate with ovulated females (Barry et a1. 1990). The role of 17,20/3P in male fish is poorly understood (Fostier et al. 1983). It has been suggested that 17,20/3-P may play a 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 increased during the process of spawning. A possible explanation for these observations is that the surge in GtH in 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 GtH  112  stimulates T production, from the testes, which will gradually be transformed to 11-KT. Both T and 11-KT play key roles in the processes of spawning behavior, milt production and milt release and gradually decrease when spawning activity is completed.  113  CHAPTER 6 BIOLOGICAL ACTIVITIES OF GNRHS AND THEIR ANALOGS IN COMBINATION WITH DOMPERIDONE ON THE INDUCTION OF GONADOTROPIN SECRETION AND SPAWNING IN THE THAI CARP  A. INTRODUCTION  The primary structure of a salmon gonadotropin-releasing hormone (sGnRH) differs from mammalian 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 and Millar, 1985). However, the predominant GnRH molecule appears to be sGnRH, although a number of 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 and Rivier, 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 or aromatic D-amino acids in mGnRH (Monahan et al. 1973) has resulted in the production of GnRH analogs (GnRHA) which are more potent than the native forms. Both modifications are known to increase 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 et al. 1985; 1987b), rainbow trout (Crim et al. 1988), and African catfish (De Leeuw et al. 1988). In the  114  previous chapters, it was clearly demonstrated that induction of GtH secretion and spawning in the Thai 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 natural mammalian, avian, and piscine GnRHs and their analogs. In this in vivo study, I examined the potency of 12 different forms of GnRHs and GnRHAs in combination with domperidone on GtH secretion and spawning induction in the Thai carp.  B. MATERIALS AND METHODS  Two experiments were carried out, one at Kalasin Freshwater Fisheries Station between 24 and 29 July 1990 and another at Pathumthani Freshwater Fisheries Station between 21 and 25 May 1991. In 1990, 117 fully mature females Thai carp (256.48 ± 8.12 g) were randomly assigned to 13 treatment groups of 9 fish each. Each group received an intraperitoneal injection of one of 12 different GnRHs or GnRHAs (25 µg/kg body weight, BW), together with domperidone (25 mg/kg BW) (table 3). Control fish were treated with 0.7% saline. Following treatment, each group of females, together with the same number of untreated sexually mature males, were placed in an 8 m 2 outdoor, concretelined holding tank. The tanks were supplied with running water at 28±1 ° C. Blood samples were taken serially at the time of injection, and at 3, 6, and 9 hr after injection as described in chapter 2. The numbers of fish which spawned in each group during the 24 hr period following injection were recorded. As a result of power failure at Kalasin Freshwater Fisheries Station, plasma samples from this experiment were not used for GtH analysis. In 1991, 117 sexually mature females Thai carp (380.08 ± 122.40 g) were randomly assigned to 13 different treatment groups of 9 fish each. Each group received an intraperitoneal injection of one of 12 different GnRHs or GnRHAs (10 µg/kg BW) together with domperidone (10 mg/kg BW) (table 3). Control fish were treated with 0.7% saline. Following injection, fish were treated as described  115  above. Plasma samples were analyzed for GtH by RIA at the Department of Zoology, University of Alberta, Edmonton, Alberta.  116  Table 3: The primary structure of GnRH and other peptides used in the study  common name^  structure  1^2^3^4^5^6^7^8^9^10 mGnRH^pGlu His Trp Ser Tyr Gly Leu Arg Pro G1yNH2 sGnRH^  Trp Leu  cGnRH-?^  Leu Gln  cGnRH-II^  His^Trp Tyr  mGnRHA^  NHEt  D-A1a6 mGnRH^  D-Ala^G1yNH2  D-A1a6 mGnRHA^  D-Ala^NHEt  ** D-Trp6 mGnRH^D-Trp^G1yNH2  D-Trp6 mGnRHA^  D-Trp^NHEt  D-Lys6 mGnRH^  D-Lys^ G1yNH2  Buserelin^  D-Ser(But)^NHEt  sGnRHA^  D-Arg Trp Leu^NHEt  D-Ala6 sGnRHA^  D-Ala Trp Leu^NHEt  * **  only in 1991 only in 1990  117  100  °A) Fish spawned  80  60  40  20  0  C^eCinRMA^•ClaRli^inGaRM mOnRMA Gaol:IN-II 0-Aloe D-Alall^0-AlaII^1:1-Ly•O^1:1-TrpO^D-Trpe Bu••r•lln mOn1114 menRMA silnRIIA manliti manlIMA menRH  Treatments  Fig 43. Effect of native mammalian, avian and piscine GnRHs and their analogs (25 ag/kg) in combination with Dom (25 mg/kg) on the induction of spawning in the Thai carp in the 1990 study (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.  118  C. RESULTS Spawning occurred between 4 to 6 hr following the injection. Eggs were transferred to incubators at the time fish were subjected to the third blood sampling.  a. Percentage of fish spawned In 1990, spawning occurred in all treatment groups except the control fish (Fig. 43). There were no significant differences in percentages of fish spawning among groups receiving GnRHs or GnRHAs. 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-I treated group. The highest spawning success (88.89%) occurred in the groups receiving [D-Ala 6]mGnRHA and [D-Trp6]-mGnRHA. Treatment with [D-Ala 6]-mGnRH, sGnRHA, and Buserelin resulted in a lower spawning success (80-65%). Fewer spawning success (50-20%) was observed in groups receiving cGnRH-11, sGnRH, [D-Lys 6]-mGnRH, [D-Ala6]-sGnRHA, and mGnRH. The lowest percentage of spawners (< 20%) was observed in the mGnRHA treated group.  119  ^  100  % Fish spawned  80  60  40  20  0 C^•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 onCinliliA  Treatments  Fig. 44. Effect of native mammalian, avian and piscine GnRHs and their analogs (10 µg/kg) in combination 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. 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, b, c, d, and e.  120  b. Plasma GtH levels There were no significant differences in plasma GtH levels at 0 hr (time of injection) between groups. 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]-mGnRH or [D-Trp6 ]-mGnRHA. Moderately high GtH levels (600-450 ng/ml) were observed in groups receiving p-Ala6 FmGnRHA, 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 the controls or the highest GtH level groups. GtH levels in groups receiving sGnRH, mGnRH, [D-Ala 6]sGnRHA, or cGnRH-I were not significantly higher than those observed in control animals, but GtH levels 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, maintained significantly higher plasma GtH levels than at 0 hr. However, within each treatment group, plasma GtH levels tended to decrease from that recorded at 3 hr. Thus, plasma GtH levels remained highest in groups treated with [D-Ala 6]-mGnRH, [D-Trp 6 ]-mGnRHA, or Buserelin (700-450 ng/ml). Groups receiving [D-Ala6 ]-mGnRHA or sGnRHA demonstrated intermediate high GtH levels (300-250 ng/ml) compared to the control which were not significantly different from the highest GtH level groups. Groups receiving cGnRH-11 or [D-Lys 6 ]-mGnRH exhibited GtH levels not significantly different from either the control or the high GtH level groups. There were no significant differences in plasma GtH levels in groups receiving sGnRH, mGnRH, mGnRHA, cGnRH-I or p-Ala 61-sGnRHA when compared with controls. However, GtH levels in these groups were significantly lower than those of the high GtH levels groups. At 9 hr post injection (Fig. 48), plasma GtH levels in groups receiving [D-Ala 6]-mGnRH, [DTrp6]-mGnRHA, Buserelin, [D-Ala 6 ]-mGnRHA, sGnRHA, or [D-Lys 6 ]-mGnRHA remained  121  significantly higher than those at 0 hr. A significant decrease in plasma GtH levels at 3 and 6 hr post injection 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 levels remained higher than those of the control animals in groups receiving [D-Ala 6]-mGnRH, [D-Trp6]mGnRHA, [D-Ala6]-mGnRHA, Buserelin, or sGnRHA.  122  Plasma GtH (ng/ml) 12 ^ 10 8 6 4 2 0  l i tt i titt li ti  CoGnill4 AstInAllinGillilimen 1114 AcCin1114- 1cOnF114 - 110-Alad10-Alasp-Alas0-14sID-Trgiellus*Win mOnlili meinliplA sOnFIFIAlmenlili meni114A  Tre atme nts  Fig. 45. Effects of native mammalian, avian, and piscine GnRHs and their analogs (10 µg/kg) in combination with Dom (10 mg/kg) on the induction of GtH secretion in female Thai carp at injection time in the 1991 study (21-25 May 1991). Each value represents the mean plasma GtH ± SEM.  123  Plasma GtH (ng/ml) ioox 12  •enliMA •OnR14^manlill nenRHA cOnlIM-1 eGn/114-11 Cs-Mall ^D-Allie^D-Alall^D-ly•fl^0-Type 111,••r•lin mOnR14 inenRHA •CInFINA IneInFIN mClaRHA  Treatments  Fig. 46. Effects of native mammalian, avian, and piscine GnRHs and their analogs (10 mg/kg) in combination with Dom (10 mg/kg) on the induction of GtH secretion in female Thai carp at 3 hr after 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 identified by the same superscript, i.e., a, b, and c. 1 = significant difference from plasma GtH at injection time.  124  Plasma GtH (ng/m1) boox 12 ^ 10 1 e  8  1 e  1 de  6 1 bcde  4  1 abcde  2  0  1 1^abcd^1 ab^a^a  a -  C^•OnlINA  mTi  -  -  mTm  -  lit  -  emir  I  1 cde  1 abcde 1 abc  U  1  -  I  •LinFIN^InGaRM^menRMA •OnR14-1 cOn1111-11 1:1-A1111^D-AI./^D-Ala.^C-Ly•ll^D-Trpe ■ko••r•lin neln/111 manAHA •OnRMA nenRHA tallaRHA  Treatments  Fig. 47. Effects of native mammalian, avian, and piscine GnRHs and their analogs (10 µg/kg) in combination with Dom (10 mg/kg) on the induction of GtH secretion in female Thai carp at 6 hr after 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 identified by the same superscript, i.e., a, b, c, d, and e. 1 = significant difference from plasma GtH at injection time.  125  Plasma GtH (ng/ml) ioox 12  10 8  6  4 123  d  2  1 bcd  a 0  111  123  12 cd  d 1  ^a^a^a^a  -  a -L-  -.■t■-  a  abc  -aim-  -L-  123  1111  C^•OnIIHA^menRli relnliMA^cOnR14-11 D-A1•0^D-Alse^D-Ly•e^D-Ttp6 Ilu••r•Iln aiOnRM menFIMA •CleRHA •OnFIM manill4A  Treatments  Fig. 48. Effects of native mammalian, avian, and piscine GnRHs and their analogues (10 µg/kg) in combination with Dom (10 mg/kg) on the induction of GtH secretion in female Thai carp at 9 hr after 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 identified by 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 injection respectively.  126  D. DISCUSSION  The present results demonstrated that a high concentration of either mammalian, avian, and piscine GnRHs or their analogs (25 µg/kg BW) in combination with Dom (25 mg/kg BW) was equally effective 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 combination with 10 mg/kg Dom, can be distinguished in terms of the proportion of fish which spawned and concentration of plasma GtH. Among the natural forms of mammalian, avian, and piscine GnRHs, given in combination with Dom, cGnRH-I failed to induce spawning and stimulate GtH secretion in the Thai carp. mGnRH was relatively ineffective, since it induced less than 40% of fish to spawn and stimulated a modest increase in plasma GtH levels at 3 hr post injection. These results are consistent with the finding that mGnRH and cGnRH-I had similar effectiveness in stimulating GtH release in in vivo studies in goldfish (Peter et 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 to spawn, in both the 1990 and 1991 studies. However, they stimulated only a modest increase in plasma GtH level, which was not significantly different from the control. This suggests that natural sGnRH and cGnRH-II are active in the Thai carp, and may exist in this species as already been found in several teleost 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 ethylamide residue [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 very  127  low percentage of fish to spawn and induced only a modest increase in GtH similar to those obtained with mGnRH and sGnRH. This suggests that the binding affinity for the Thai carp pituitary GnRH receptors may differ from that of mammals. Substitution of position 6 with hydrophobic or aromatic D-amino acids in mGnRH was found to be important in either increasing receptor binding affinity and/or an increase hydrophobicity and a greater resistance to enzymatic degradation (Nestor, 1984). Similarly, in this study, mGnRHAs with a substitution 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 in inducing spawning and increasing and prolonging plasma GtH concentration in Thai carp compared to the native form as previously found in goldfish (Peter et al. 1985), rainbow trout, salmon, and winter founder (Crim et al. 1988). On the other hand, in sGnRH which has been found to be more hydrophobic 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 in stimulating 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), rainbow trout, 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, [DArg6 ]-sGnRHA, in combination with Dom, was effective in inducing GtH secretion and spawning in the Thai carp, however, its potency was lower (difference not significant) than those of P-Ala 61mGnRH, [D-A1a6J-mGnRHA, [D-Trp 6]-mGnRHA, [D-Trp 6]-mGnRH, and Buserelin at all sampling times. Furthermore, increasing hydrophobicity in sGnRHA by substituting D-Arg 6 with a more hydrophobic D-amino acid such as D-Ala 6 has not proven to increase potencies in terms of GtH concentration and percentage of fish spawned. Similar result has been reported in other teleosts (Peter et a1.1985; Crim et a1. 1988). The reason is probably similar to that suggested in the mammal where  128  increasing hydrophobicity leads to diminished activity of mGnRH (Nestor, 1984), probably due to the fact that a highly hydrophobic molecule tends to bind to lipids and is not readily available for binding with receptors (Peter et a1. 1985). [D-Ala6 ]-mGnRH and [D-Trp 6 ]-mGnRHA were found to be the most potent peptides used in this study, although their potency was not significantly different from [D-Ala 6 ]-mGnRHA, sGnRHA, Buserelin and [D-Trp 6 ]-mGnRH in term of increasing percentage of fish spawned and prolonging increased plasma GtH levels. Interestingly, two mGnRHAs with only a substitution at position 6 ([13Trp 6]-mGnRH and [D-Ala6 ]-mGnRH in 1990 and [D-Ala6]-mGnRH] in 1991) were equipotent in stimulating GtH secretion and spawning in the Thai carp. This confirms the finding that an increase in binding affinity of mGnRH by substitution of the glycinamide residue at position 10 with ethylamide is not the important factor in making mGnRH superactive in the Thai carp. This result suggests that in the Thai carp, as in the goldfish (Peter et al. 1985) and the gilthead seabream (Zohar et al. 1989), the rate of enzymatic degradation seems to be important in determining the degree of superactivity, since substitution in position 6 with an appropriate D-amino acid seems to enhance the potency of the analogs and results in a prolonged increase in plasma GtH. In conclusion, the present study demonstrates that sGnRHA is not the most potent analog in terms of GtH secretion and induction of spawning in sexually mature female Thai carp. Its potency appears to be lower than those of mGnRH analogs with a substitution at position 6 with hydrophobic or aromatic D-amino acids. Substitution of the glycinamide at position 10 with ethylamide was not a basic requirement for producing superactive analogs for use in the Thai carp. This indicates that, in the Thai carp, resistance to enzyme degradation is the most important factor in producing superactive analogs. 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.  129  CHAPTER 7 SUMMARY AND CONCLUSIONS  A. ANNUAL REPRODUCTIVE CYCLE OF THE THAI CARP  1. Females  The 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 been cultured under hatchery conditions, the patterns of their gonadal development might not represent the true natural cycle which occurs in the wild fish In this study, however, gonadal recrudescence was highly correlated with the sequential changes of environmental factors such as air temperatures and daylength. Also, the period of spawning was observed to be highly coincident with the occurrence of rainfall as previously reported in wild fish (Sipitakkiat and Leenanond, 1984). Histological analysis of the ovary revealed that oocyte development in the Thai carp was of the asynchronous type. This confirms the finding of Sirikul et al. (1986) that the Thai carp can spawn several times in an extended spawning season. It was not possible to distinguish pronounced seasonal changes in plasma reproductive hormones levels in the female Thai carp during this study. A gradual increase in plasma GtH levels was 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 the seasonal 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 of plasma E2 was observed to coincide with the initial increase in GSI. This indicates the involvement of  130  E2 in the process of vitellogenesis. Furthermore, another peak of plasma E2 was observed during the spawning period. This surge in plasma E2 was probably synthesized by previtellogenic oocytes under the stimulation of the preovulatory surge in GtH. The parallel changes in plasma T and E2 confirmed the role of T as a precursor for E2 synthesis. The lower concentrations of T than those of E2 suggest that T was actively aromatized into E2 in the ovary of the Thai carp.  2. Males Seasonal changes in GSI in male Thai carp were highly correlated with those seen in the females. 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 these monthly sampling regime. This suggests that gonadal development in the male Thai carp is probably controlled by endogenous cues. The structure of the testes of the Thai carp corresponds to the lobular type which is commonly found in teleosts. Histological analysis of the testes revealed a continuous spermatogenetic activity which 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 observation that milt can be stripped from the fish throughout the year. This indicted a prolonged and constant readiness for spawning which may allow them to be opportunistic in their responses to suitable spawning conditions. Plasma hormones levels in the male Thai carp exhibited a pattern of seasonal changes as observed in females. However, there were no statistically significant correlations between seasonal changes in plasma hormone levels and testicular development. Plasma GtH, T and 11-KT established a similar pattern of seasonal changes, but only plasma T and 11-KT demonstrated a highly positive correlation. Also, the concentration of plasma 11-KT was always higher than that of T.  131  The first peak of plasma GtH, T and 11-KT was observed at the time when GSI began to increase. This indicates the importance of these hormones as endogenous factors in the initiation of testicular development in the male Thai carp. The second peak in plasma hormones levels was observed during the spawning period. This suggests the involvement of GtH and androgen in the process of spawning.  B. INTERACTION OF SGNRHA AND DOMPERIDONE IN THE REGULATION OF GONADOTROPIN SECRETION AND INDUCTION OF SPAWNING IN THE FEMALE THAI CARP  The results from the current study indicate that GtH secretion in the Thai carp is regulated by GnRH and dopamine Application of either synthetic sGnRHA or Dom, a dopamine antagonist, alone at an appropriate concentration (5-10 µg/kg or 10 mg/kg respectively) significantly increased plasma GtH level at 3 and 6 hr after injection. However, the magnitude of increasing GtH levels stimulated by either 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 fish to spawn. This indicated that in the situation where dopamine receptors are completely blocked by the application of a dopamine receptors antagonist such as Dom, the level of exogenous GnRH is high enough to stimulate the preovulatory surge in GtH and to induce spawning in this species. Thus, this result suggests that under natural conditions dopamine plays an important role, by acting as GRIF, in the regulation of GtH secretion and spawning in the Thai carp. Application of various combinations of sGnRHA and Dom increased both the plasma GtH levels and the number of fish spawning in a dose-related manner. This suggests the interaction of sGnRHA and Dom on the regulation of GtH secretion in the Thai carp. This interaction of sGnRHA  132  and Dom probably involves changes in the number of pituitary receptors for GnRH and dopamine as has been suggested in goldfish (Omeljaniuk et al. 1989a). The potentiation effect of Dom on the response to sGnRHA can be observed only when Dom was administered at or higher than 5 mg/kg, the most effective dosage. Application of Dom at a concentration higher than this dosage did not increase the magnitude of increasing plasma GtH levels and 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. This suggests the importance of sGnRHA in the regulation of GtH secretion and increasing the number of fish spawning. From the economic and practical points of view, the most effective dosage of sGnRHA and Dom 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 SPAWNING IN THE THAI CARP  1. Females Administration of sGnRHA in combination with Dom at an appropriate concentration caused a 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, but remained higher than the pre-treatment levels. Plasma E2 and T levels increased significantly during the induction period, probably due to gonadotropin-stimulation of the follicles. A clear shift in the production of E2 to T, which is commonly  133  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 observed when the fish were sampled at 3 hr intervals. However, a short  term 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, in the female Thai carp, plasma 17,20p-P levels were almost undetectable at all sampling times. This is probably due to the short-term secretion and/or rapid plasma clearance rate of this steroid as has been shown in goldfish (Kobayashi et al. 1987). Further, it is possible that 17,20fl-P is not the MIS in the Thai carp. Recent studies of oocyte final maturation have revealed that the trihydroxylated progesterone derivative, 17,20/3,21-P, is the MIS in several fishes (Scott and Canario, 1987; 1988; Thomas and Trant, 1989).  2. Males No significant changes in plasma GtH levels were observed in untreated male Thai carp during the process of spawning when blood was sampled at 3 hr intervals. However, during short term observations, a significant increase in plasma GtH was seen at the onset of spawning, which rapidly decreased 30 min later. This suggests that the GtH surge in male Thai carp can be stimulated by exposure to ovulatory females. This surge in plasma GtH is probably involved in triggering sexual behavior and the process of sperm release in the male Thai carp. Plasma T increased gradually in untreated male Thai carp after placement with preovulatory females. 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. This surge in plasma T and 11-KT is probably stimulated by elevation of GtH. Both T and 11-KT continued  134  to increase during the process of spawning, while GtH immediately decreased to a lower level. This suggests 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. This suggests 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 WITH DOMPERIDONE ON THE INDUCTION OF GONADOTROPIN SECRETION AND SPAWNING IN THE FEMALE THAI CARP  High concentrations (25 µg/kg Bw) of either native mammalian, avian, and piscine GnRHs and their analogs, in combination with domperidone (25 mg/kg) were equally effective in inducing spawning in the female Thai carp. At a lower concentration of GnRHs (10 µg/kg Bw) in combination with 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 of the natural GnRHs in stimulating GtH secretion and inducing spawning in female Thai carp. sGnRH and 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 that they 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 the ethylamide residue [Pro 9 -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 carp pituitary 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 Damino acids demonstrated greater potencies in inducing spawning and increasing and prolonging plasma GtH concentration in the female Thai carp compared to the native forms. In mammals, this  135  substitution has been found to be important in either increasing receptor binding affmity and/or an increasing hydrophobicity and providing greater resistance to enzymatic degradation. This suggests that the rate of degradation seems to be important in determining the degree of superactivity of GnRH analogs in the Thai carp. [D-Ala6]-mGnRHA and [D-Trp 6]-mGnRHA were found to be the most potent peptides of those tested in this study. However, their potency was not significantly different from sGnRHA, [DSer(But) 6]-mGnRH or Buserelin, and [D-Trp 6]-mGnRH in terms of increasing the percentage of fish spawned and prolonging increased plasma GtH levels.  136  REFERENCES Aida, K., Horose, K., Yokote, M. and Hibiya, T. 1973. Physiological studies on gonadal maturation of fishes-II. Histological changes in the liver cells of Ayu following gonadal maturation and estrogen 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 during spawning 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 plasma concentration 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 and hypophysectomized 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 teleost reproductive cycle and its relation to external factors: Salmonid and cyprinid models. In P. J. Gaillard and H. H., Boer (eds.). Comparative endocrinology. Elsevier/North-Holland Biomedical Press, New York. pp. 37-48. Billard, R., Fostier, A., Weil, C. and Breton, B. 1982. Endocrine control of spermatogenesis in teleost fish 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. WileyLiss, Inc., New York. pp. 329-335. Breton, B., Jalabert, B. and Billard, R. 1973. Pituitary and plasma gonadotropin levels and spermatogenesis 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, 11ketotestosterone, 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 vitro oocyte maturation bioassay in rainbow trout, Salmo gairdneri. Gen. Comp. Endocrinol. 71: 338-348.  137  Cardwell, 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 British Columbia, Vancouver, B.C., Canada. 129 pp. Chang, C. F. and Chen, M. R. 1990. Fluctuation in sex steroids and sex steroid-binding protein during the 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 and norepinephine on in vitro spontaneous and gonadotropin-releasing hormone-induced gonadotropin 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-a review. FAO Fish. Rep. 4(44): 30-66. Clemens, H. P. and Reed, C. A. 1967. Long term gonadal growth and maturation of goldfish (Carassius auratus) with pituitary injections. Copeia. pp. 465-466. Colombo, L., Belvedere, P. C., Simontacchi, C. and Lazzari, M. 1987. Shift from androgen to progesterone biosynthesis by the testes of the northern pike, Exox lucius L., during transition from spermatogenesis to spermiation. Gen. Comp. Endocrinol. 66: 18. Conn, P. M., Hsueh, A. J. W. and Crowley, F. Jr. 1984. Gonadotropin-releasing hormone: Molecular and 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 for lack of dopaminergic inhibition. Gen. Comp. Endocrinol. 74: 474-483. Crim, L. W. and Idler, D. R. 1978. Plasma gonadotropin, estradiol, and vitellogenin and phosvitin levels in 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 reproduction in 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 therapeutic applications 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 analogs in the rainbow trout, landlocked salmon, and winter flounder. Gen. Comp. Endocrinol. 71: 372-382.  138  Culling, 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 the luteinizing 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 the gonadotropin-releasing hormone-induced gonadotropin release. An in vivo study with fragments 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 and biological 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 of pituitary 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 of sex 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. New York-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 in cultured 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 reproductive parameters 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. FAO Information, 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 and gonadotropin in coho salmon, Oncorhynchus kisutch, during final maturation. Gen. Comp. Endocrinol. 62: 437-451.  139  Fostier, A., Weil, C., Terqui, M., Breton, B. and Jalabert, B. 1978. Plasma estradiol 17/3 and gonadotropin 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. 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. 164-173. Fostier, A., Billard, R. and Breton, B. 1982. Plasma 11-oxotestosterone and gonadotropin during the beginning 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-releasing hormone(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, New York. 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 of gonadotropin-releasing hormone (GnRH) binding to pituitary receptors in goldfish (Carassius auratus). Biol. Reprod. 36: 844-853. Habibi, H. R., De Leeuw, R., Nahorniak, C. S., Goos, H. J. T. and Peter, R. E. 1989. Pituitary gonadotropin-releasing hormone (GnRH) receptor activity in goldfish and catfish: Seasonal and gonadal effects. Fish Physiol. Biochem. 7: 109-118. Haug, T. and Gulliksen, B. 1988. Variations in liver and body condition during gonad development of Atlantic 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. FAO Fisheries 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.  140  Hunt, S. M. V., Simpson, T. H. and Wright, R. S. 1982. Seasonal changes in the levels of 11-oxotestosterone and testosterone in the serum of male salmon, Salmo salar L., and their relationship 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 sockeye salmon. 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 of fish 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 two distinct chum salmon GTHs. Gen. Comp. Endocrinol. 71: 438-451. Jalabert, B. 1976. In vitro oocyte maturation and ovulation in rainbow trout (Salmo gairdneri), northern pike (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 or cortisol on the output of 17a-hydroxy-20fl-dihydroprogesterone by rainbow trout (Salmo gairdneri) 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 amago salmon (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 during gonadal 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 by ovarian 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. Structurefunction studies toward the development of agonists and antagonists: Rationale and perspective. Endocr. Rev. 7: 44-66.  141  Kawauchi, H., Suzuki, K, Nagahama, Y., Adachi, S. and Naito, N. 1986. Occurrence of two distinct gonadotropins in chum salmon pituitary. In F. Yoshimaru and A. Gorbman (eds.). Pars Distalis of the pituitary gland: Structure, function and regulation. Elsevier Science Publishers B. V., Amsterdam. pp. 383-390. Kawauchi, H., Suzuki, K, Itoh, H., Swanson, P. and Nagahama, Y. 1987. Duality of salmon pituitary gonadotropins. In E. Ohnishi and Y. Nagahama (eds.). Proceedings of the first congress Asian and 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. and Itoh, 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, Cyprinus carpio. Gen. Comp. Endocrinol. 58: 137-149. Kime, D. and Bieniarz, K 1987. Gonadotropin-induced changes in steroid production by ovaries of the common 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-releasing hormone. I. Structural determination on partially purified material. J. Biol. Chem. 257: 1072210732. King, J. A. and Millar, R. P. 1985. Multiple molecular forms of gonadotropin-releasing hormone in teleost fish brain. Peptides. 6: 689-694. Kobayashi, M., Aida, K and Hanyu, I. 1986a. Annual changes in plasma levels of gonadotropin and steroid 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 of steroid 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 steroid hormones 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 preovulatory gonadotropin surge in female goldfish. Fish Physiol. Biochem. 7: 141-146.  142  Koldras, M., Bieniarz, K. and Kime, D. E. 1990. Sperm production and steroidogenesis in testes of the common 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 and E. 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. In D. R. Idler, L. W. Crim and J. M. Walsh (eds.). Proceedings of the third international symposium 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 plasma levels of steroids (cortisol, estradiol-17fl, estron, and testosterone) correlated with the annual gonadal cycle in the catfish, Heteropneustes fossilis (Bloch). Gen. Comp. Endocrinol. 50: 205225. Lambert, J. G. D., Bosman, G. I. C. G. M., van den Hurk, R. and van Oordt, P. G. W. J. 1978. Annual cycle 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 international symposium on reproductive physiology of fish Pudoc, Wageningen, the Natherland. pp. 99102. 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: 135154. Levavi-Zermonsky, B. and Yaron, Y. 1986. Changes in gonadotropin and ovarian steroids association with oocyte maturation during spawning induction in the carp. Gen. Comp. Endocrinol. 62: 8998. Licht, P., Farmer, S. W., Muller, S. W., Tsui, H. W. and Crews, D. 1977. Evolution of gonadotropin structure 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 PGF2 a . J. Fish Biol. 26: 491-502.  143  Liley, N. R., Fostier, A., Breton, B. and Tan, E. S. P. 1986a. Endocrine changes associated with spawning 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 with spawning 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-3one 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 of ovulation in the loach (Parmisgumus dabryanus) using pimozide and [D-Ala ° , Pro9 -Nethylamide]-LHRH. Aquaculture. 46: 333-340. Lin, H. R. and Peter, R. E. 1986. Induction of gonadotropin secretion and ovulation in teleosts using LHRH 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 (sGnRHA) and [D-A1a6, Pro9 NEt]-luteinizing hormone-releasing hormone (LHRH-A), in combination with pimozide or domperidone, on gonadotropin release and ovulation in the Chinese 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 reproductive steroid 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 oocyte development 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 the common 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 gonadotropinreleasing 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 the hypothalamic luteinizing hormone releasing factor with increased agonist or antagonist properties. Biochemistry. 12: 4619-4620.  144  Munkittrick, K. R. and Leatherland, J. F. 1984. Seasonal changes in the pituitary-gonad axis of feral goldfish, 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 teleost gonads. Zool. Sci. 4: 209-222. Nestor, J. J. Jr. 1984. Development of agonistic LHRH analogs. In B. H. Vickery, J. J. Nestor Jr., and E. 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 spermiation response 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 primary structure of gonadotropin-releasing hormone in the Thai catfish (Clarias macrocephalus). In A. P. Scott, J. P. Sumpter, D. E. Kime and M. S. Rolfe (eds.). Proceedings of the 4th international 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 two gonadotropins, 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. Salmonids pituitary gonadotropins. II. Ontogeny of GtH I and GtH II cells in the rainbow trout (Salmo gairdneri 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 dopamineantagonist 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 on reproductive 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 receptormediated inhibition of gonadotropin secretion from the pituitary of the goldfish Carassius auratus. J. Endocrinol. 114: 449-458. Omeljaniuk, R. J., Habibi, H. R. and Peter, R. E. 1989a. Alterations in pituitary GnRH and dopamine receptors associated with the seasonal variation and regulation of gonadotropin release in the goldfish (Carassius auratus). Gen. Comp. Endocrinol. 74: 392-399. Omeljaniuk, R. J., Tonon, M. C. and Peter, R. E. 1989b. Dopamine inhibition of gonadotropin and alpha-melanocyte-stimulating hormone release in vivo from the pituitary of the goldfish (Carassius auratus). Gen. Comp. Endocrinol. 74: 451-467.  145  Pankhurst, N. W. and Stacey, N. E. 1985. The effect of 17/3-estradiol on spontaneous ovulation in the goldfish, Carassius auratus. Can. J. Zool. 63: 2979-2981. Pankhurst, N. W. and Conroy, A. M. 1987. Seasonal changes in reproductive condition and plasma levels of sex steroids in blue cod, Parapercis colias (Bloch and Schneider) (Mugiloidae). Fish Physiol. Biochem. 4: 15-26. Parameswaran, S., Selvaraj, C. and Radhakriahnan, S. 1970. Observations on the maturation and breeding 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-one production from endogenous substrates in Atlantic croaker ovarian follicles undergoing final maturation in vitro. Gen. Comp. Endocrinol. 78: 474-478. Peter, R. E. and Crim, L. W. 1979. Reproductive endocrinology of fishes: Gonadal cycle and gonadotropin. Ann. Rev. Physiol. 41: 323-335. Peter, R. E. 1981. Gonadotropin secretion during reproductive cycles in teleosts: Influences of environmental factors. Gen. Comp. Endocrinol. 45: 294-305. Peter, R. E. 1983. The brain and neurohormones in teleost reproduction. In W. S. Hoar, D. J. Randall and 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 pars distalis 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 progestergens during 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. and Millar, R. P. 1985. Structure-activity relationships of mammalian, chicken, and salmon gonadotropin-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 in teleost 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 Chinese teleosts. In D. R. Idler, L. W. Crim and J. M. Walsh (eds.). Proceedings of the third international 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-8substituted analogs of mammalian gonadotropin-releasing hormones in goldfish. Gen. Corn. Endocrinol. 65: 385-393.  146  Qasim, S. Z. and Qayyum, A. 1961. Spawning season and breeding frequencies of some freshwater fishes 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. Plasma gonadotropin 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 rainbow trout (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 plasma concentrations of 11-ketotestosterone and testosterone in male rainbow trout, Salmo gairdneri Richardson. J. Fish Biol. 17: 495-505. Scott, A. P. and Baynes, S. M. 1982. Plasma levels of sex steroids in relation to ovulation and spermiation 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-one in plasma of trout (Salmo gairneri Richardson): Seasonal changes and response to salmon pituitary extract. Gen. Comp. Endocrinol. 46: 444-451. Scott, A. P. and Sumpter, J. P. 1983. A comparison of the female reproductive cycles of autumnspawning 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 the rainbow 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 in the 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 symposium on the reproductive physiology of fish. Memorial University, St. John's, Newfoundland, Canada. pp. 224-234.  147  Scott, A. P. and Sumpter, J. P. 1989. Seasonal variation in testicular germ cell stages and in plasma concentrations 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. Characterization of 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-releasing hormone (Gn-RH) in striped mullet (Mugil cephalus), milkfish (Chanos chanos), and rainbow trout (Salmo gairdneri): Comparison with salmon Gn-RH. Gen. Comp. Endocrinol. 55: 174181. 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-releasing hormone 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 the autumn-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 mouth brooding cichfid fish Sarotherodon (Tilapia) mosambicus (Peters). Anim. Behay. 26: 11201125. Silverman, H. I. 1978b. Effect of different levels of sensory contact upon reproductive activity of adult male and female Sarotherodon mosambicus (Peters); Pisces; Cichidae. Anim. Behay. 26: 10811090. Singh, A. and Singh, T. P. 1991. Changes in the plasma steroid hormone levels during gonadal maturation 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 major carps. Arch. Hydrobiol. 73: 518-536. Sipitakkiat, P. and Leenanond, Y. 1984. Life history and culturing of pla-tapian khao, Puntius gonionotus Bleeker. Technical Paper No. 15, National Inland Fisheries Institute, Department of 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)  148  Sloley, B. D., Trudeau, V. L., Dullca, J. G. and Peter, R. E. 1991. Selective depletion of dopamine in the goldfish 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 of hydroelectric 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. Asian Fisheries Society, Manila. pp. 693-6%. Sokolowska, M., Peter, R. E., Nahorniak, C. S. and Chang, J. P. 1985. Seasonal effects of pimozide and des 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. The effects 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 spontaneous and 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 natural spawning in Catostomus commersoni. I. Gonadotropin, growth hormone, and thyroid hormones. 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 sex pheromone: 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 of fish 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, and cortisol 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. Hong Kong University Press, Hong Kong. pp. 369-372.  149  Suzuki, K., Kawauchi, H. and Nagahama, Y. 1988a. Isolation and characterization of two distinct gonadotropins 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 two distinct gonadotropins from chum salmon pituitary glands. Gen. Comp. Endocrinol. 71: 302306. Suzuki, K., Nagahama, Y. and Kawauchi, H. 1988c. Steroidogenic activities of two distinct salmon gonadotropins. Gen. Comp. Endocrinol. 71: 452-458. Suzuki, K., Kanamori, A., Nagahama, Y. and Kawauchi, H. 1988d. Development of salmon GTH I and GTH 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 two coho 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 by LHRH 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. Office of 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 a maturation-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-4pregnen-3-one as the major ovarian steroid produced by the teleost Micropogonias undulatus during final oocyte maturation. Steroids. 47: 89-99. Trant, J. M. and Thomas, P. 1989. Changes in ovarian steroidogenesis in vitro associated with final maturation 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 serum gonadotropin response to gonadotropin-releasing hormone in goldfish. Biol. Reprod. 44: 951960. Truscott, B., Idler, D. R., So, Y. P. and Walsh, J. M. 1986. Maturational steroids and gonadotropin in upstream 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 the lower Halda River, Bangladesh. Estuaries. 4(2): 127-138.  150  Tyler, C. R., Sumpter, J. P., Kawauchi, H. and Swanson, P. 1991. Involvement of gonadotropin in the uptake 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-4pregnen-3-one: Plasma levels during sexual maturation and in vitro production by testis of amago 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 concentration of steroid hormones, thyroxine, and vitellogenin during spawning migration of the chum salmon, 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 LHRH and des-G1y 10 [D-Ala6 ] LH-RH-ethylamide on plasma gonadotropin levels and oocyte maturation 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° [DAla ] 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 regulation of 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 maturational gonadotropin in goldfish. In D. R. Idler, L. W. Crim and J. M. Walsh (eds.). Proceedings of the 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 plasma gonadotropin 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 and gonadotropin II. Gen. Comp. Endocrinol. 85: 217-229. Wade, M. G. and Van Der Kraak, G. 1991. The control of testicular androgen production in the goldfish: 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.  151  Wallace, R. A., Selman, K., Greeley, M. S. J., Begovac, P. C. and Lin, Y.-W. P. 1987. Current status of oocyte growth. In D. R. Idler, L. W. Crim and J. M. Walsh (eds.). Proceedings of the third international 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 gonadotropin and 177-estradiol in the common carp, Cyprinus carpio L. as related to spawning induced by hypophysation 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 changes during induced spawning and annual reproductive cycle in wuchang fish (Megalobrama amblycephala). In J. L. Maclean, L. B. Dizon and L. V. Hosillos (eds.). Proceedings of the first Asian 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 induced ovulation 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. Freshwater Res. 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 female rainbow trout. Aquaculture. 43: 317-326. Wright, R. S. and Hunt, S. V. 1982. A radioimmunoassay for 17a,20P-dihydroxy-4-pregnen-3-one: Its used in measuring changes in serum levels at ovulation in Atlantic salmon (Salmo salar), coho salmon (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 the spermiation of the goldfish (Carassius auratus). Gen. Comp. Endocrinol. 12: 491-497. Yaron, Z., Levavi-Zermonsky, B. and Bogomolnaya, A. 1985. GTH and ovarian steroids during spawning 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 annual cycle 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 the ovarian granulosa cells amago salmon (Oncorhynchus rhodurus) associated with final oocyte maturation. 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.  152  Yu, K. L., Sherwood, N. M. and Peter, R. E. 1988. Differential distribution of two molecular forms of gonadotropin-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 of gonadotropin releasing hormones and its regulation in the gilthead seabream, Sparus aurata: In vivo and in vitro studies. Fish Physiol. Biochem. 7: 59-67.  153  

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