【作者】 杨景峰；

【导师】 陈松林；

【作者基本信息】 中国海洋大学 ， 海洋生物学， 2009， 博士

【摘要】 大多数鱼类雌雄个体在生长速度都存在差异,根据鱼类生长速度的雌雄个体差异进行性别控制和单性养殖可以大幅度提高产量和增加养殖效率。以大菱鲆和半滑舌鳎为代表的海水鲆鲽鱼类工厂化养殖业的兴起,掀起了海水养殖的第四次高潮。大多数鲆鲽鱼类雌性生长速度较快,因而进行全雌的鲆鲽类苗种培育对高效的工厂化养殖意义更加巨大。雌核发育技术是一种非常理想的遗传性别控制技术,结合性反转技术可以使鲆鲽鱼类全雌苗种生产成为可能。在本研究中,我们报道了这几种重要海水鱼类的雌核发育诱导方法,并且通过流式细胞仪和微卫星技术对雌核发育群体进行了鉴定,并结合性反转技术进了全雌苗种的生产。实验结果如下:1.大西洋牙鲆是一种重要的经济鱼类。在本研究中,采用紫外线(UV)照射冷冻保存的花鲈精子和大西洋牙鲆精子诱导犬齿牙鲆雌核发育。实验表明:犬齿牙鲆同源精子经80mJ/cm2的紫外线照射可以完全失活,冻存花鲈精子经紫外线照射后也同样具有诱导犬齿牙鲆雌核发育能力。无论同源精子还是异源精子,最佳诱导条件为,在18℃条件下,精子经80mJ/cm2的紫外线照射,在受精后4~5min,将受精卵放在3℃海水中进行冷休克处理,持续时间为45min,同源精子和异源精子二倍体诱导分别为32.66±7.03%和28.00±6.48%。采用形态学、流式细胞仪DNA含量分析和微卫星标记技术对雌核发育鱼苗进行了分析,证明了雌核发育鱼苗为雌核发育二倍体。100尾雌核发育群体中雌性共38尾,占38%,雄性62尾,占62%。按不同方法进行雄性激素处理2700余尾,培育1年后,雄性比例为100%。2.漠斑牙鲆是一种具有较高经济价值的深海比目鱼类。由于漠斑牙鲆雌性生长快,因此通过雌核发育获得漠斑牙鲆雌性群体是一种有效方法。雌核发育实验表明:同源精子只有经紫外线照射遗传失活后才能诱导雌核发育,否则形成三倍体,而花鲈精子也只有经过紫外线照射遗传失活后,才能诱导产生漠斑牙鲆雌核发育二倍体鱼苗,杂交胚可发育,但不能存活至孵化。经实验筛选出诱导漠斑牙鲆雌核发育的最佳条件为:花鲈冷冻精子采用80mJ/cm2的紫外线照射,然后与卵子进行授精,在受精后4~5min,施以65MPa的静水压休克处理6min。采用形态学、流式细胞仪DNA含量分析和微卫星标记技术对雌核发育鱼苗进行了分析,证明了雌核发育鱼苗为雌核发育二倍体。培育1年后的雌核发育群体经鉴定,雌性占42.8%;雄性激素浸泡处和投喂处理后的雄性比例分别达了95.3%和97.5%。3.条斑星鲽是一种大型冷水性经济鱼类。由于条斑星鲽雌性生长快于雄性,在生产上获得全雌群体对条斑星鲽具有重要意义。使用花鲈精子和同源精子诱导条斑星鲽雌核发育,结果显示花鲈精子只有经过紫外线照射遗传失活后,才能诱导产生条斑星鲽雌核发育二倍体鱼苗。经过大量实验筛选出诱导条斑星鲽雌核发育的最佳条件为:花鲈冷冻精子采用80mJ/cm2的紫外线照射,然后与条斑星鲽卵子进行授精,在受精后7~9min,将受精卵放在-1.0~1.5℃海水中进行冷休克处理,持续时间为60~90min,在此条件下,获得的雌核发育二倍体的最高诱导率达40.68%±7.24%。由于不失活精子与条斑星鲽卵形成的杂交胚只能存活到原肠期,而染色体未被成功加倍的胚胎会由于单倍体的致死效应在孵化前后死亡,所以存活的仔鱼全部为条斑星鲽雌核发育二倍体。采用微卫星标记技术对雌核发育鱼苗进行了分析,证明了雌核发育鱼苗为雌核发育二倍体。培育一年后经鉴定,雌核发育群体生别比例为42.7%;高温、激素和投喂激素处理雌核发育群体和正常苗种,只出现了个别雌性个体,雄性比例达95.1%~100%4.半滑舌鳎是新近开发的一种重要的经济海洋鱼类。为了获得半滑舌鳎全雌苗种并阐明其性别机制,本文首先采用冷冻保存的鲈鱼精子作为刺激半滑舌鳎卵子进行雌核发育的有效精子源,建立了精子遗传失活的条件;其次,筛选出有效的冷休克起始时间、适合的冷休克温度和冷休克处理时间。获得了异源冷冻精子-鲈鱼精子诱导产生的半滑舌鳎雌核发育鱼苗。采用常规方法对雌核发育胚胎进行了染色体分析,观察到单倍体染色体数目为21条,雌核发育二倍体染色体数目为42条,观察到含有2条W染色体的个体,表明雌核发育二倍体包含有ZZ和WW两种类型;采用微卫星标记对雌核发育鱼苗进行了分析,证明了雌核发育鱼苗的可靠性;采用半滑舌鳎雌性特异AFLP标记对雌核发育鱼苗进行了遗传性别鉴定,获得了具有雌性特异DNA标记的雌核发育二倍体鱼苗。在10多次雌核发育实验中获得雌核发育二倍体鱼苗6600多尾。在鱼苗长大后鉴定生理性别,其中同源精子诱导组雌性比例为11.5%,异源精子诱导组雌性比例为17.7%,而同批次正常对照组雌性比例45.8±8.2%。雌核发育群体雌性比例明显低于正常组。本研究采用冷冻保存的鲈鱼精子,在多种鲆鲽鱼类建议了人工雌核发育技术和雌核发育群体鉴别技术,获得了雌核发育二倍体鱼苗,部分种类完成性反转,为鲆鲽鱼类全雌育种技术的建立和全雌苗种的生产奠定了基础,提供了技术手段。更多还原

【Abstract】 The majority of marine fishes have a difference at growth rate between female and male. It was conducted by sex control and mono-sex culture to greatly increase yield and improve the efficiency of breeding according to the difference of growth rate. Recently, the rise of factory farming in flatfishes gave birth to the fourth aquaculture wave, especially the culture of tarbot and half-smooth tongue sole. Thus, the development of all-female group is very important to high-efficiency factory farming.Gynogensis technology is a well sex control technology, combined with the sex reversal make the product of all-female group possible. Here we report the methods of gynogenesis on the four important marine fishes and all-female production were attained by sex reversal. At the same time, the flow cytometry and microsatellite analysis were used to identification on the offspring of the gynogenesis.The following results were received:1. Summer flounder, Paralichthys dentatus, is an important commercial and recreational fish. In this study, diploid gynogenesis was induced by activating egg development with UV irradiated summer flounder sperm or sea perch sperm (80 mJ/cm2) for 5 min in seawater, and then subjecting the eggs to coldshock in 3℃seawater for 45 min. The hybrid of could not survive to hatching, thus the offspring was allogynogenesis only. The normal temperature of cultivation to one years later, the total survival is 228 and 100 randomly selected to identify the sex ratio. The result show that the female were 38 while the male were 62. Sex ratio are not comply the presumed 1:1 by the chi-square test. The 2700 individuals were dealt with using different methods of hormone treatment, 30 to 100 fishes from each treatment group randomly selected after one year cultivation and were determined the sex ratio. The results of all treatment groups were male and the male ratio was 100%.2. Southern flounder, Paralichthys lethostigma, is one member of a large family of distinctive benthic flatfishes with a high economic values. Effective methods for induction of diploid gynogenesis of the southern flounder, Paralichthys lethostigma, are needed to initiate monosex culture, which will allow growers to take advantage of the more rapid growth and larger size attained by females. The results of experiments showed that gynogenetic diploid and triploid can be induced by inactivated homologous sperm and noninactivated, respectively; and only gynogenetic diploid were obtained using inactivated heterologous sperm because that hybrid would be died before hatching. Diploid gynogenesis was induced by activating egg development with UV irradiated homologous sperm (80 mJ/cm2) for 4~5 min in seawater, and then then 65MPa pressure treatment for 6min. Flow cytometry and microsatellite DNA analysis were used to demonstrate diploidy. These results indicate that the use of UV irradiated sperm from Lateolabrax japonicas for activation of flounder eggs and pressure shock for polar body retention is an effective method to produce gynogenetic offspring.subjecting the eggs to coldshock in 3℃seawater for 45 min. The offspring of gynogensis were identified by morphologically examination after one year. The result show that the rate of male was 42.8%. After the Androgen immersion and the feeding, the rate of male can reach to 95.3% and 97.5%, respectively.3. Barfin flounder, Verasper moseri is an important economical marine fish. Since females grow faster than males, the production of all-female populations is highly desirable. To test methods for inducing diploid gynogenesis in barfin flounder using homologous sperm, The Cryopreservative sperm of Lateolabrax japonicas was used to active eggs and cool shock was used to prevent extrusion of the second polar body. Four treatments, named for their expected outcome, were employed: hybrid, haploid, triploid, and gynogenetic diploid to prove the ability of Lateolabrax japonicas sperm activing flounder eggs. Diploid gynogenesis was induced by activating egg development with UVirradiated sperm (80 mJ/cm2) for 7~9 min in seawater, and then subjecting the eggs to cold shock in -1~1.5℃seawater for 60–90 min. The hybrid of could not survive to hatching, thus the offspring was allogynogenesis only and the result of microsatellite markers analysis proved this result. This work provides procedures for induction of diploid gynogenesis in barfin flounder using heterologous sperm.4. Half-smooth tongue sole (Cynoglossus semilaevis) is a newly exploited and commercially important cultured marine fish in China. In order to attain the all-female and elucidation the sex mechanism of tongue sole, a set of experiments were carried out for developing artificial gynogenesis technique in the tongue sole. First of all, the effects of different sperms on gynogenesis induction were examined. The sea perch (Lateolabrax japonicus) sperm was considered to be effective sperm in inducing gynogenesis in the tongue sole. Secondly, iradiation conditions for sea perch spermatozoa were developed. Thirdly, the optimal iniation time for cold shock of gynogenetic eggs was determined, the optimal temperature and treatment time were determined. Chromosomes from gynogenetic haploid, diploid were analyzed, WW chromosomes were discovered in some gynogenetic diploid embryos. Microsatellite marker technique was applied to analyze gynogenetic diploid fry. Among the 30 gynogenetic diploid fry, 11 fry contained only one allele, 19 contained two alleles which had same genetype as their mother. Finally, female-specific DNA marker for half-smooth tongue sole was observed in four individuals of ten gynogenetic diploid fry. Thus, a set of techniques for induction of artificial gynogenesis was developed for the first time in half smooth tongue sole, and more than 6600 gynogenetic diploid tongue sole fry were successfully produced by using this technique.In this study, the method of gynogenesis was established by sperm of the sea bass for four marine fishes and the flow cytometry and microsatellite were involved in the identification of offspring of gynogenesis. Moreover, the fishes of sex reversal were also achieved. Therefore it was a foundation for the gynogenesis and all-female production in practice. ?更多还原