【作者】 温海深；

【导师】 林浩然；

【作者基本信息】 中山大学 ， 水生生物学， 2001， 博士

【摘要】 采用鲤鱼促性腺激素(GtH)放射免疫测定方法(cGtH RIA)和生长激素(GH)放射免疫测定方法(cGH RIA)测定了珠江和辽河流域野生鲇鱼(Silurus asotus)脑垂体和血清中GtH和GH含量的季节变化,研究促性腺素释放激素类似物(LHRH-A)和多巴胺能药物(多巴胺DA及其激动剂APO)对鲇鱼离体脑垂体碎片GtH和GH释放的作用,注射LHRH-A和DA能药物(DA及其D-2型受体拮抗剂DOM)对鲇鱼血清GtH和GH水平的影响,探讨了鲇鱼生殖和生长与性成熟系数(GSI)和肝体指数(HSI)的相互关系,采用LHRH-A+DOM药物组合(Linpe Method)对鲇鱼进行了人工诱导排卵和催产试验,主要研究结果如下: 1.鲇鱼血清和脑垂体GtH和GH水平的季节变化及其与生殖和生长的关系珠江流域雌雄鲇鱼血清GtH水平从3月份逐渐升高,在4月份达到峰值(雌鱼为10.0ng/ml,雄鱼为2.43ng/ml);辽河流域鲇鱼血清GtH水平从1～3月份开始上升,在5月份达到峰值(雌鱼峰值为8.1ng/ml,雄鱼为2.4ng/ml),雌雄鲇鱼血清GtH水平表现了基本相同的变化趋势,但在产卵期雌鱼血清GtH水平显著高于雄鱼。产卵后雌雄鱼血清GtH水平开始逐渐下降,两个流域均在9月份达到最低。在Ⅲ期卵巢发育阶段,两个流域雌鲇鱼血清GtH水平均较低,Ⅳ期卵巢时血清GtH水平达到全年最高值,Ⅴ期时血清GtH水平仍然保持较高,Ⅵ期卵巢时血清GtH水平降到全年最低。在Ⅲ期和Ⅳ期卵巢发育阶段,珠江流域鲇鱼血清GtH水平显著高于辽河流域,而后两个发育期无显著差异。珠江流域雌雄鲇鱼脑垂体GtH含量从3月份开始升高,3～7月份均保持较高水平,7月达到最大值,分别为13.28μg/mg PG(脑垂体)和13.0μg/mg PG;辽河流域鲇鱼脑垂体GtH含量从1月份开始升高,3～7月份保持高水平状态,在3月份达到最大(雌鱼为14.33μg/mg PG,雄鱼为12.37μg/mg PG)。雌雄鱼脑垂体GtH含量表现出基本相同的变化趋势,而且差异不显著。在Ⅲ期、Ⅳ期和Ⅴ期卵巢发育阶段,两个流域雌鲇鱼脑垂GtH含量均保持较高水平,不存在显著差异,但珠江流域显著高于辽河流域,Ⅵ期卵巢时脑垂体GtH含量降到全年最低。珠江和辽河流域鲇鱼在进入生殖季节前血清GtH水平出现明显的峰值,均在9月份达到最低,其中辽河流域峰值出现时间晚于珠江流域,可能与水温、营养和性腺发育状况有关。雌雄鱼血清和脑垂体GtH含量与周年GSI变化一致,雌雄鱼血清和脑垂体GtH水平与HSI之间温海深 中山大学博士学位论文更多还原

【Abstract】 Based on the radioimmunoassay (RIA) for the common carp gonadotropin (GtH) and growth hormone (GH), heterologous RIAs for GtH and GH of feral catfish (Silurus asotus) collected from Peral River and Liaohe River, were developed and used for to assay the seasonal changes of basal GtH and GH contents in the serum and the pituitary of the catfish. Using in vitro pituitary fragments incubation and in vivo intraperitoneal injection techniques, GtH and GH release response of the pituitary and the serum to luteinizing hormone-releasing hormone analogs (des Gly~10[D-Ala~6 ] LHRH ethylamide , LHRH-A) and dopaminergic drugs, including dopamine (DA) and its agonists apomorphine (APO) and D-2 receptor antagonists domperidone (DOM) were investigated. The interrelationships among seasonal variation of GtH and GH and gonadosomatic index (GSI) and hepatic somatic index (HSI), as well as the use of Linpe Method (LHRH-A plus DOM) for inducing ovulation and artificial propagation of the catfish were also explored. The main results obtained could be briefly summarized as follows: 1. Seasonal variations of GtH and GH contents of the serum and pituitary as well as the relationship among GtH and GH and reproductive and growth physiology in the catfish GtH surge of the serum in female and male catfish occurred in April in Pearl River ( 10.0ng/ml in female catfish and 2.43ng/ml in male catfish) and in May in Liaohe River (8.1ng/ml in female catfish and 2.4ng/ml in male catfish). Annual GtH levels of the serum between female and male catfish paralleled each other in two River, but GtH levels of the serum of female catfish were significantly (P<0.05) higher than male during breeding season, with a gradually decreased tendency after ovulation (in July) and the lowest values in September. The serum GtH levels increased gradually in the stage of early ovary development, with the highest values in the stage of late and mature ovary development, and with the lowest values in the stage ofsexually regressed ovary in two River, whereas the serum GtH levels in the stage of early and late ovary development was higher significantly in Pearl River than those in Liaohe River.GtH content of the pituitary remained at very high levels during March to July, with the highest value in March in Liaohe River (14.3ug/mg PG in female catfish and 12.3ug/mg PG in male catfish), and in July in Pearl River (13.2)ig/mg PG in female catfish and 13.0ug/mg PG in male catfish). No differences were found between female and male catfish in GtH content of the pituitary. The pituitary GtH content in the stage of early, late and mature ovary development was higher in Pearl River than those in Liaohe River, with the lowest values in the stage of sexually regressed ovary. The serum GtH surge of the catfish in Liaohe River occurred lately might be because of the effects of the water temperature and the nutrition factors and the recrudescence of the gonad. The interrelationships among reproductive activity and GtH content of the serum and pituitary and GST were analyzed. The results indicated that annual changes of GtH levels of the serum and pituitary parallel to those in GSIs. GtH seasonal changes of the serum and pituitary were interrelated significantly to HSIs in female and male catfish.GH levels of the serum increased gradually from April, with the highest values from June to September (130.6~160.9ng/ml) and annual average value (101.6ng/ml) in female catfish. Seasonal changes of the serum was the same between male and female catfish, whereas annual average content (75.8ng/ml) was lower significantly in male catfish than those in female catfish. There were two basal GH surge of the pituitary in March ( 268.5ng/mg PG in female catfish and 167.8ng/mg PG in male catfish) and July (196.0 ng/mg PG in female catfish and 120.6ng/mg PG in male catfish) respectively, seasonal basal GH levels of the serum and pituitary in female catfish was higher significantly than those in male catfish and maybe caused faster growth in female catfish . GH levels of the serum had increased before ovulation, with GH surge followed by GSI and GtH surge during breeding season and remained a high content after the ovulation. Two GH surges of the pituitary occurred to respectively in pre-ovulation and post-ovulation, these results suggested that seasonal changes of basal GH levels of the serum and pituitary were correlated significantly to reproductive cycle. GH seasonal changes of thepituitary in female catfish were interrelated significantly to HSI.2. The effects of LHRH-A and dopaminergic drugs on GtH and GH secretion of pituitary fragmentGtH release was able to be stimulated after chronic administration of LHRH-A (lOOnM) from pituitary fragments and the response of the pituitary in the stage of sexually regressed ovary was more sensitive than those in the stage of mature ovary ,and the dessensitive to chronic administration of LHRH-A was not be observed in female catfish. The response to LHRH-A from pituitary fragments of different stage of ovary development was the same in male catfish, the results was consistent to African catfish {Clarias gariepinus) and different from eel (Anguilla japonica). GtH release was also able to be stimulated after repetitive pulse administration of LHRH-A from the pituitary fragments in dose-dependent manner in female and male catfish. Perifusion of pituitary fragments with APO caused a decrease in GtH release and the stimulation of GtH release by LHRH-A was completely blocked when APO was administered pior to LHRH-A. The results were agreement with goldfish (Carassius auratus) and African catfish and indicated that DA inhibits the LHRH-A-induced GtH release at the levels of the gonadotropic cells and might be mediated by receptors similar to the mammalian D-2 type.Perifusion of 2 min pulses administration of different concentrations of LHRH-A (10, 100, lOOOnM) failed to stimulate GH release of in vitro pituitary fragments, perifusion of 2 min pulses administration of different concentrations of APO (10,100,1000nM) increased significantly GH release of the pituitary fragments in dose-dependent manner.3. The effects of injection of LHRH-A and dopaminergic drugs on serum GtH levels in the catfishThe serum GtH levels was not observed to increase after injection of LHRH-A (lOug/kg b.w ) alone in female catfish in the stage of early gonadal development, the stage of late gonadal development and the sexually regressed stage (water temperature was 20-25 °C). I.p injection of LHRH -A alone stimulated a small but significant increase in the serum GtH in the stage of mature gonadal development (water temperature was 30°C ) .The sensitive difference in different gonadal development stage might be affected by seasonal quantity and binding affinity of GnRH receptors as well as steroid hormone. The serum GtH levels in female catfish was more sensitive to injection of LHRH-A alone than those in male catfish. Injection of LHRH-A plus dopamine antagonist DOM enhanced significantly GtH levels of theserum in every gonadal development stage and the serum GtH surge occurred in 6hr (water temperature was 30°C)and 12hr (water temperature was 20-25 °C) after injection. The results indicated that GtH secretion in the catfish was under a dual control of gonadotropin-releasing hormone (GnRH) and DA release from the hypothalamus. Basal serum GtH levels didn’t enhanced after injection of DOM alone and didn’t decrease after injection of DA alone, these results suggested that DA can only inhibit GtH release induced by GnRH and not inhibit spontaneous GtH release, and was agreement with in vitro results.The injection of LHRH-A (lOug/kg.b.w) was ineffective to enhance GH levels of the serum, injection of DA (50mg/kg.b.w) potentiated significantly GH levels of the serum, indicated that GnRH might not involved in regulating GH secretion in the catfish. 4. The ovulation and spawning induced by Linpe methodThe rate of ovulation induced by LHRH-A (lOug/kg b.w or 20ug/kg b.w) alone was 25-37.5%, whereas the rate of ovulation induced by LHRH-A (7ug/kg b.w) plus DOM (5mg/kg b.w) was 100% 8-12hr after injection. The main advantages of the Linpe method compared to traditional methods, which the injection of LHRH-A plus Human chorionic goandotropin (HCG) or LHRH-A plus the carp pituitary extraction (CPE), include the following: reduced cost of the synthetic drugs, long stability of the drugs, they can be successfully applied to a great variety of species, high predictability of the time from injection to ovulation, decreased stress on broodstock because only a single injection is needed, and absence of side effects on subsequent reproductive cycles. It was very applicable to induce ovulation of feral catfish without scales using Linpe method. The results of the experiments and field trials in fish farms for cultured catfish was highly successful. 2926 pairs of spawners were induced to spawn by injection of LHRH-A (7ug/kg b.w) plus DOM(5mg/kg b.w), with average spawning rate of 70% (the highest was 80%) , 2.07 XI06 fry were produced and staring economic incoming were obtained.更多还原