【作者】 张晓彦；

【导师】 刘海金；

【作者基本信息】 东北农业大学 ， 动物遗传育种与繁殖， 2009， 硕士

【摘要】 半滑舌鳎(Cynoglossus semilaevis)俗称鳎米、牛舌头,主要分布在我国黄渤海海域,是我国特有的重要名贵经济鱼类。半滑舌鳎雌雄个体差异很大,雌性生长速度比雄性快2～3倍。因此,实现雌性单性化养殖,对于提高半滑舌鳎的养殖产量和经济效益具有重要意义。近年,我国水产科学研究者对半滑舌鳎的生物学特征、人工繁育等方面进行了大量研究,已经突破了人工繁殖难关。但是,关于半滑舌鳎的全雌化诱导和三倍体人工诱导等研究,迄今未见报道。本研究利用不同浓度的17β-雌二醇浸泡半滑舌鳎鱼苗诱导性转化,观察了半滑舌鳎的性分化过程和激素对性分化的影响,分析了激素诱导对半滑舌鳎的性比、生长速度和成活率的影响。本研究还探索了利用雌核发育二倍体的最佳诱导参数诱导三倍体的方法,观察了三倍体的胚胎发育特点,利用流式细胞仪检测了染色体加倍的有效性。各项试验均取得了良好的效果,为半滑舌鳎全雌化和三倍体诱导研究提供了重要的基础数据和有益经验。本研究取得主要试验结果如下:(1)半滑舌鳎属于雌雄异体分化型鱼类,卵巢分化比精巢早。卵巢分化的解剖学标志主要是卵巢腔的形成,最早出现于孵化后65天;精巢分化的解剖学标志主要是输精管和精小叶的形成,出现于孵化后70天。性分化的细胞学标志主要是性原细胞减数分裂。卵原细胞减数分裂开始于孵化后100天后;精原细胞减数分裂则开始于孵化后90天左右。(2)在激素诱导性转化研究中,1μg/L、3μg/L、10μg/L和30μg/L的17β-雌二醇处理组的雌性比率分别为74%、82%、88%和97%,与对照组(26%)相比,处理组的雌性比率显著提高(P＜0.01);乙醇组的雌性比率为36%,与对照组相比差异不显著(P＞0.05)。由此结果可以看出,17β-雌二醇可促使半滑舌鳎卵巢分化,却抑制精巢分化。激素处理组未发现雌雄间性及不育现象。雌二醇对半滑舌鳎的早期生长影响不大,与对照组相比没有显著差异(P＞0.05);雌二醇处理对半滑舌鳎的死亡率没有明显影响(P＞0.05)。(3)对半滑舌鳎雌核发育冷休克处理条件地优化结果表明:受精卵在正常孵化水温22±0.5℃培养时,雌核发育的最佳条件是:受精后3min,用5±0.5℃的海水冷休克处理20min。参照诱导雌核发育二倍体的条件,用冷休克法诱导出半滑舌鳎三倍体,流式细胞仪检测染色体加倍的有效性。(4)对半滑舌鳎三倍体的胚胎发育观察结果表明,普通二倍体胚胎发育最快,雌核发育二倍体次之,单倍体发育最慢,三倍体胚胎发育速度介于雌核发育二倍体和单倍体之间。破膜后的单倍体脊柱弯曲,器官分化不全,畸形率高。三倍体和雌核发育二倍体与普通二倍体半滑舌鳎的发育时序基本相同,除三倍体和雌核发育二倍体各阶段受冷休克影响发育较慢外,发育特征与发育分期没有明显差别。更多还原

【Abstract】 Half-smooth tongue-sole（Cynoglossus semilaevis） females growed bigger and mature later than males,it was reported that females growed 2～3 times faster than males,in other words its growing taked on sexual otherness.So the production of all-female stock would be of significant benefit for aquaculture of half-smooth tongue-sole.In this research,histological methods were used to observe the sex differentiation of half-smooth tongue-sole,providing the scientific foundation for sex reversal.The induction of all-female populations was carried out by marination treatment,using estradiol-17βwith concentration of 1μg/L,3μg/L,10μg/L and 30μg/L.In the period of female induction,it was essential to observer the sex differentiation of the treated groups,the growth and the mortality rate.It was reported that the triploid populations growed faster and bigger than the normal diploid in some teleosts.So the triploid induction of tongue sole（Cynoglossus semilaevis） with cold shock method were made.Embryonic development of haploid,triploid,gynogenetic diploid and normal diploid were observed.This would provide the basic study material for the growth comparison between the triploid and the normal diploid stock.The results showed as follows:（1） Half-smooth tongue-sole belonged to the differentiated species of gonochoristic fish and ovary differentiated earlier than that of spermary.On the 65^th,the gonad was differentiated,the oviposition plate rudiment was formed,then the dorsal edges of these gonads began to extend laterally and they eventually fused to form the ovary;on the 70^th the anlage of the efferent duct was found,which would develop into testis.Oocytes in early meiosis were first observed from the ovary after 100^th dph,while the primary spermatocytes were found on 90^th dph.（2） A chi-squared analysis on sex ratios in each of the control groups showed they were significantly different from 1:1 and they were not significantly different from each other. Furthermore,chi-squared analyses between duplicates of each treatment revealed their sex ratios were not significantly different.And the 1μg/L,3μg/L,10μg/L and 30μg/L treatments yielded 74%,82%,88%and 97%female ratios respectively,it indicated that the endocrine manipulation to half-smooth tongue-sole（Cynoglossus semilaevis） was effective.In the study of hormomal sex reversal of half-smooth tongue-sole,it was showed that 17β-Estradiol could accelerate the ovarian differentiation,but delayed the spermatic differentiation.The ovarian differentiation in the E₂-treatment group was earlier for 5 days than which of the control group;while the spermatic differentiation was later 20 days than which of the control group.This might relate to the effect of inner gonadal estrogen to gonadal differentiation,the outside estrogen might stimulate the gonad, which would increase the production of inner gonadal estrogen.It also revealed that 17β-Estradiol did not show negative influence to early growth and cumulative mortalities.The cumulative mortalities of treatment groups approximated 2%,which were not significantly different from the blank treatment.And there was also no difference of the growh dates between the blank and the treared groups.It was necessary to lucubrate continually whether such fish would be suitable for commercial culture.（3） The parameters of induction of gynogenetic diploids in half-smooth tongue-sole were optimized.It showed that the time of the starting treatment,the temperature of the cold shock and the duration of the cold shock would affect the hatching rate of the gynogenetic diploids. Chromosome diploid was achieved by suppression of the second polar bady release by early cold shocks applied 2～6 min after fertilization at 22±0.5℃with 10～30 min durations.The optimal cold shock was at 5±0.5℃that was applied at 3 min after fertilization and lasted for 20 min.Then the triploid was induced by cold shock treatment with the optimal parameters in the gynogenetic diploid.Then the triploidy was proved.And the impregnation rate and the hatching rate were compared between the normal diploid and the triploid group.（4） The Embryonic development speed of different groups showed:the normal diploid group developed faster than the gynogenetic diploid group,the gynogenetic diploid group developed faster than the triploid group,and the haploid group were slowest.A high frequency of abnormality, particularly body flexion and the organ differentiation were found in the haploid embryos.The embryos of gynogenetic diploid and triploid had basically the same process with the normal diploid embryos.There were no significant differences in embryonic development among gynogenesis diploid,triploid and normal haploid individuals.However,different time of every stage was found in embryonic development among gynogenesis diploid,triploid and haploid individuals.更多还原