Study On Physiological And Endocrine Characteristics Of Reproduction Of Farmed Spotted Halibut And Barfin Flounder

The physiological and endocrine characteristics of reproduction of farmed Spotted halibut and Barfin flounder were investigated and elucidated in this thesis. This thesis aimed at construction of stable technology for artificial breeding of farmed Spotted halibut and Barfin flounder. The contents mainly focused on pituitary structure, gonadal maturation cycle, sex steroids expression pattern, gametes characteristics, structure and function of GnRH system. Multiple and combined methods including histological observation, electron microscopy technique, immunohistochemistry, radioimmunoassay (RIA), molecular cloning, quantitative real time PCR and exogenous hormone induction etc. were employed. Based on results from this study, successful control of gonadal maturation, ovulation and batches of embryos with high quality from barfin flounder were obtained. Results from this study would be helpful to better understand the physiological and endocrine mechanisms underlying the reproductive cycle of spotted halibut and barfin flounder, also would facilitate the artificial control of gonadal maturation and obtain spawnings from broodstocks under farming conditions.The annual ovarian maturation cycle and related serum sex steroids levels variation, the relationships between gonadosomatic index (GSI), condition factor (CF), hepatosomatic index (HSI), sex steroids expression levels and photothermal regulation were investigated and elucidated for farmed Spotted halibut and Barfin flounder. These two species feature asynchronous oocyte development and batch spawnings. In sexually matured females, the oocyte growth could be divided into five phases and ovary maturation cycle was divided into five stages accordingly. GSI, HSI and CF values were all found to increase prior to, or during the peak phase of vitellogenic growth corresponding to the gonad development. Sex steroids levels fluctuated with the gonadal maturation cycle and closely related with photothermal regimes based on expression results and statistical analysis. The effective accumulated temperatures for Spotted halibut and Barfin flounder broodstocks were 1050 and 1180 d·℃respectively. The effects of exogenous hormone (sGnRH and LHRHa) on gonadal development of immatured females (2 year old) were studied. The results showed that the immatured females exhibited weak endocrine response compared with sexually matured broodstocks, which was manifested by the fact that the oocyte could attain the late vitellogenic stage but could not experience hydration and final maturation. Although the serum sex steroids levels increased significantly, the ovulation failed. These results would highlight the physiological and endocrine mechanism of annual reproductive cycle of captive female broodstocks of spotted halibut and barfin flounder, also could serve as useful tools for broodstocks breeding and hormonal induction of spawning of broodstocks.The physiological and endocrine characteristics during annual maturation cycle of male spotted halibut and barfin flounder were elucidated. The spermatogenesis could be divided into five phases and testis maturation cycle was divided into five stages. The hermaphroditism phenomenon was found in one male spotted halibut, but the mechanism remained unclear. The serum steroids expression patterns during testis maturation cycle were also determined. Spotted halibut sperm shared similar structure with that of barfin flounder, and exhibited similar general structure including spherical head, the reduced mid piece with numerous round unmodified small mitochondria, and the typical'9+2'pattern of the flagellum with quite simple structure. These common structure features meant that the spermatozoon of spotted halibut and barfin flounder can be considered to be of primitive type, which is found in most of the advanced fish taxa. There is no acrosome in the sperm. Anterolateral to the nucleus, there is a chromatin-free, electron-lucent area, that we named the anterior pit of the nucleus. Within and in front of the pit, there is a triangular shaped structure and some vesicles of different size that are encapsulated in a layer of membrane were found. With regarding to acrosome and acrosome vestige present in some other euteleosts, we presume the vesicles may represent acrosome vestiges though they may also function to release nuclear material, whilst the riangular shaped structure in front the nucleus may be caused by the improper manipulation. Study on relationship between sperm and environmental factors indicated that when salinity at 25-35, temperature at 8-12 and pH at 7-8, the spotted halibut spermatozoa showed best motility and longest life span. The sperm of spotted halibut was cryopreserved by a simple and convenient protocol. The sperm was cryopreserved in the different extenders. The fertilization rates of this sperm ranged from 59±2.4% to 70±3.0% with the sperm cryopreserved in the extender containing Gly + egg yolk giving the highest fertility, while the hatching rates of eggs fertilized with this sperm were from 11±3.0% to 18±2.6% with the eggs fertilized by the sperm cryopreserved in Gly + egg yolk producing the highest hatching rate. The effects of exogenous hormones including human chorionic gonadotropin (HCG) and salmon gonadotropin releasing hormone analogue (sGnRHa) on milt quality in male barfin flounder were investigated in the present study. Results showed that exogenous hormone can greatly improve the milt quality of males with milt of high viscosity, including improved milt fluidity, sperm motility and sperm life span. Meanwhile, the sex steroids elevated and remained at a relatively higher level after the exogenous hormone treatment, which was in accordance with the sperm quality elevation. The GnRH induction showed better results although there was no significant difference was observed between GnRH and HCG treatment. This study can serve as useful tool in acquiring milt with high quality and be beneficial to artificial insemination in barfin flounder culture. The histological structure and ultrastructure of barfin flounder pituitary was observed. The pituitary belongs to dorsoventral style, consists of two parts: neurohypophysis and adenohysis. Six types of secretory cells were identified in pituitary: Prolactin hormone (PROL) cell, adrenocorticotopic hormone (ACTH) cell, thyrotropic hormone (TSH) cell, growth hormone (GH) cell, gonadotropin hormone (GtH) cell, melanotropic stimulating hormone (MSH) cell.The present study cloned and sequenced the gonadotropin-releasing hormone (GnRH) genes from spotted halibut by isolating their cDNAs. This species expressed three molecular forms of GnRH in the brain: chicken type GnRH-Ⅱ(cGnRH-Ⅱ), seabream type GnRH (sbGnRH) and salmon type GnRH (sGnRH). Each GnRH cDNA encoded a signal peptide (SP), GnRH and a GnRH-associated peptide (GAP), which was connected to GnRH by a Gly–Lys–Arg sequence. Phylogenetic analysis divided multiple molecular forms of GnRHs into three branches: cGnRH-Ⅱbranch, sGnRH branch and the fish specific GnRH branch. The tissue and sex specific expression of these three genes were determined using real time PCR, they all showed the highest expression levels in brain for both sexes. cGnRH-Ⅱwas exclusively detected in brain while sbGnRH had a global expression pattern in all examined tissues for both sexes. sGnRH was detected in pituitary, gonad besides brain tissue in females but not in gonad of males. All these three GnRH genes exhibited sex-specific expression pattern with females expressed higher mRNA levels than that of males. The seasonal changes of GnRH mRNA expression levels in brains of females during ovary maturation cycle were investigated. During ovary maturation cycle, sbGnRH mRNA level increased significantly (P<0.05) since August (vitellogenic stage), peaked in December (prespawning stage) 2009, and then decreased in April 2010 (postspawning stage). On the contrary, neither sGnRH nor cGnRH-ⅡmRNA levels showed significant changes (P>0.05) correlated with ovary maturation cycle in this study. These results will be helpful to better understand the reproductive endocrine mechanism of spotted halibut.The successful control of gonadal maturation, ovulation and batches of embryos with high quality were obtained in barfin flounder by using photothermal control in combination with hormonal induction during the spawning season in 2008. A stable technique for inducing maturation and ovulation of barfin flounder was constructed and applied to practical production of seedlings, which is beneficial for sustainable and healthy development of culture industry of barfin flounder.