利用鱼类性逆转技术创制黄颡鱼XX雄鱼的方法

目前黄颡鱼(Pelteobagrus fulvidraco Richardson)生产上使用的母本非常混杂, 成为影响黄颡鱼产业发展的一个突出问题。建立一个优良性状稳定的全雌家系对于全雄黄颡鱼和杂交黄颡鱼的生产尤为重要, 而将XX雌性黄颡鱼逆转为XX雄性黄颡鱼是创制全雌家系中的关键一步。黄颡鱼性染色体连锁分子标记的开发为鉴定XX雄鱼提供了技术支撑。研究使用不同浓度的17α-甲基睾酮(MT)和芳香化酶抑制剂来曲唑(LZ)处理黄颡鱼鱼苗54d (7—60日龄)。61日龄测量并统计各组鱼的存活率、体长及体重。解剖观察性腺结构, 结合性腺组织切片和性别分子标记分析各组实验鱼中XX鱼性腺发育情况。结果显示: 2种药物对黄颡鱼的存活率影响较小, 与对照组没有显著差异; MT处理的XX性腺为空腔状精小囊结构, 不能逆转为功能性精巢; 适当剂量的LZ可以将XX雌性黄颡鱼性逆转成正常的XX雄鱼, 诱导效果随着剂量的增加而增强。用完全逆转的XX生理雄鱼分别与XX雌鱼和YY雌鱼交配, 能够正常繁殖并具备较好的繁殖能力。研究建立了一种使用芳香化酶抑制剂来曲唑创制黄颡鱼XX雄鱼的方法, 为全雄黄颡鱼的品种改良及新品种的培育奠定了基础。     

高温和皮质醇对黄颡鱼性别分化的影响

研究以性染色体类型已确定且已有性别特异分子标记的黄颡鱼为研究对象, 开展高温与皮质醇诱导黄颡鱼(Tachysurus fulvidraco Richardson)XX个体雄性化组织学进程研究, 以期为环境应激诱导鱼类雄性化提供研究基础。通过对每尾鱼采用性别特异性标记鉴定遗传性别(XX或XY)及组织学鉴定生理型性别, 仅经过24d的处理(12—35日龄), 高温或皮质醇便能诱导XX遗传型个体雄性化。在此过程中, 部分XX遗传型个体卵母细胞受到抑制, 之后发育成带有卵巢腔的精巢结构。62日龄时, XX伪雄鱼性腺较正常XY雄鱼大, XX伪雄鱼体重与正常XY雄鱼相近, 而显著大于未发生性逆转的XX雌鱼。122日龄时, XX伪雄鱼从62日龄带有卵巢腔的精巢结构发育成具有典型的精小叶结构样精巢, 且都具有生理性雄鱼特有的生殖突, 推测这些雄鱼可能具有与正常雄鱼类似的生殖能力。部分XX个体对高温处理不敏感, 没有发生性逆转, 温度处理反而加快了卵巢发育的进程, 这些个体对高温的耐受性和另外一些发生性逆转的个体对温度的敏感性值得进一步研究。     

左炔诺孕酮对XX遗传型黄颡鱼雄性化的影响

为高效诱导功能性XX黄颡鱼(Tachysurus fulvidraco)伪雄鱼,研究以XX遗传型黄颡鱼为实验对象,以12日龄为左炔诺孕酮(Levonorgestrel, LNG)浸泡处理的起始点,设计高(1μg/L)、中(0.1μg/L)和低(0.01μg/L)浓度的LNG处理组,及对照(0)组,于60日龄处理结束。高、中、低浓度LNG处理组和对照组诱导雄性化比例分别为4.0%、25.0%、62.5%和0。观察性腺结构发现, 1和0.1μg/L处理组雄性化的精巢中只有全精巢型,0.01μg/L处理组则包括全精巢型(20.8%)和部分精巢型(41.7%)。幼鱼122日龄精巢组织学观察到大量精子和生精小管互通现象,推测诱导的XX伪雄鱼具有正常繁殖能力。未雄性化的卵巢发育受到LNG抑制, 62日龄卵母细胞数量和122日龄性腺指数分析结果表明,卵巢发育抑制程度与LNG处理浓度呈正相关。与对照组相比, LNG处理对黄颡鱼生长和死亡率都无显著影响。研究表明,在LNG诱导黄颡鱼雄性化研究中,具有雄性化诱导效率较高、使用剂量小和产生的伪雄鱼精巢发育易成熟的优势。研究为优化制备功能性伪雄鱼技术提供了新的...     

从XY雌鱼雌核发育产生YY超雄黄颡鱼

采用激素性逆转结合雌核发育技术,从XY雌鱼产生YY超雄黄颡鱼。与性逆转和后代测交选种技术比较,本文的方法可以缩短两代的育种时间,并提高超雄鱼的产出。通过测交证明,与正常的XY雄鱼一样,YY超雄黄颡鱼是能成活和有生育力的,其后代雄鱼占75.9%—100%,平均90.30%。从29尾雌鱼产生的雌核发育的后代294尾,只有11尾雄鱼,绝大多数是雌鱼;而在12尾YY超雄鱼测交的后代出现0—24.1%雌鱼。从上述结果可以推测,黄颡鱼的性决定体制是雌性配子同型(XX♀/XY♂),但常染色体性修饰基因的影响是比较明显的。本文还对超雄鱼在商业上大量生产全雄黄颡鱼的可行性和应用前景进行简要的讨论。     

黄颡鱼和杂交黄颡鱼“黄优1号”形态及性腺发育的比较

选取相同养殖条件的10月、22月和34月龄的黄颡鱼和新品种杂交黄颡鱼(黄颡鱼P. fulvidraco♀×瓦氏黄颡鱼P. vachelli♂)“黄优1号”进行形态及性腺发育的比较研究。通过形体指标测量发现“黄优1号”生长性能显著优于黄颡鱼; 在22月和34月龄黄颡鱼中, 雄性的体重是雌性的2倍左右, 雄性生长速度显著高于雌性; 而在“黄优1号”中, 两性生长异形现象被显著减弱。基于性腺解剖形态分析发现雌性“黄优1号”卵巢完全退化, 呈细线状结构且没有卵子产生, 故“黄优1号”雌鱼完全不育; 雄性“黄优1号”精巢组织呈现透明状和退化状态。精巢组织切片HE染色分析发现10月龄“黄优1号”的精小囊为空腔状几乎没有精子产生, 22月和34月龄“黄优1号”的精小囊内出现极少量精子。计算机辅助精子分析系统(CASA)分析发现, 相比于黄颡鱼, “黄优1号”精巢中精子量非常少, 有效活力低下; 经过繁殖能力测试, 22月龄“黄优1号”雄鱼不具备繁殖能力。新品种杂交黄颡鱼“黄优1号”在生长性能提高上显现了杂交优势, 具有推动黄颡鱼产业发展的潜力。     

黄颡鱼母本的肠系膜脂肪沉积对繁育性能的影响

为探索肠系膜脂肪堆积对黄颡鱼母本繁育性能的影响, 选择了4个母本群体来评价肠系膜脂肪沉积与黄颡鱼繁育性能的关联: group 1, 野生黄颡鱼群体[肠系膜脂肪指数(0.56±0.17)%]; group 2, 肠系膜脂肪较少的黄颡鱼养殖群体[肠系膜脂肪指数(1.97±0.40) %]; group 3和4, 肠系膜脂肪较多的2个黄颡鱼养殖群体[肠系膜脂肪指数(5.92±1.85)%和(9.62±1.01)%]。研究结果显示肠系膜脂肪体重比与性腺指数总体上呈负相关。人工繁殖结果表明, group 1和group 2的产卵率、受精率和单尾母鱼出苗量无显著性差异, 但显著高于group 3和group 4; group 1和2的苗种畸形率显著低于group 3和4。黄颡鱼雌鱼血清中促黄体生成素(LH)和卵黄蛋白原(VTG)的水平随着肠系膜脂肪沉积量的增大而降低; 相反, 肝脏和卵子中的油脂和糖原含量随着肠系膜脂肪沉积量的增大而升高。研究发现, 肠系膜脂肪过度沉积的黄颡鱼母本的生理指标和繁殖性能明显下降, 生产出来的苗种质量欠佳; 建立完善的亲本培育模式并减少肠系膜脂肪沉积能够显著改善繁殖性能, 文章为提高鱼类苗种质量提供了思路。     

黄颡鱼鱼苗的养殖

黄颡鱼 (Pseudobagrus fulvidraco) ,鱼危科 ,体青黄色 ,大都具有不规则褐色斑纹 ,喜欢生活在静水或江河的缓流中 ,白天趴在水底休息 ,夜晚游到水面上觅食。黄颡鱼的肉质细嫩 ,味道鲜美 ,营养价值高 ,人工养殖大有可为。从天然水域中捕捞鱼苗 ,一是数量有限 ,二是质量没有保证 ,三是天然苗养殖成活率低。人工繁殖是一条解决苗种问题的行之有效的途径 ,也是致富的新门路。1　亲鱼的选择与暂养要人工繁殖黄颡鱼苗种 ,首先需要性成熟的亲鱼 ,黄颡鱼的生长比较慢 ,在 3龄以上才能性成熟 ,雌鱼在南方地区一般 4～ 5月开始产卵 ,北方地区 6月以后…     

黄颡鱼卵水霉病

黄颡鱼是我国重要的淡水名优鱼类之一,近年其养殖发展迅速,已经形成重要的产业[1]。由于黄颡鱼的苗种繁育阶段其受精卵极易患水霉病,严重制约黄颡鱼的苗种规模化生产。开展黄颡鱼受精卵水霉病的病原学、病原生物学特性以及控制方法研究具有较为重要的理论意义与实际应用价值。     

埋植17α—甲基睾酮诱导点带石班鱼性转化技术

论述了埋植17α-思虑在睾酮诱导点带石斑鱼（Epinephelus malabaricus)性转化的方法,包括药物的处理,用量,手术操作等3龄的雌性点带石班鱼经过2次埋植外源激素手术转化为功能性雄鱼,139d后58％可挤出精液,301d后100％可挤出精液,功能性雄鱼用于人工繁殖,亲鱼自然产卵,1999－2000年2年内共孵出存鱼7103万尾,所得仔鱼用鱼苗培育,得到发育正常的幼鱼,认为埋植外源激素诱导点带石斑鱼完成性转化的方法简便可行,能满足规模化人工繁殖的需要。     

三倍体虹鳟性转换及性腺发育的研究

用高温刺激法获得三倍体虹鳟幼鱼。从开始投饵起,分别投喂含有不同浓度17β-雌二醇(0～60μg/μg)的饵料,持续60天,获得了93%～100%的高三倍体雌性率(对照组的雌性率为52%)。三倍体雄鱼可观察到其精子形成,但性腺成熟度较相对的二倍体差;三倍体雌鱼的生殖细胞多停留在偶线期到粗线期,未发现有性成熟个体。实验结果推测,三倍体虹鳟雌鱼的不育现象看来不是由于雌雄基因组的组成差异而致,更可能是由于三组染色体造成对虹鳟卵巢发育更大的阻力。雌二醇处理对三倍体虹鳟幼鱼的生长和成活没有明显的影响。     

Genetic Differentiation and Efficient Sex-specific Marker Development of a Pair of Y- and X-linked Markers in Yellow Catfish

Pf62-Y and Pf62-X is a pair of allelic Y chromosome-linked and X chromosome-linked markers, and have been used to identify YY super-males, XY males and XX females for commercial production of all-male populations in yellow catfish (Pelteobagrus fulvidraco). However, the SCAR primers used previously have only two nucleotide difference, which restricts the wide utility because of nucleotide polymorphism. In this study, a continuous 8102 bp Pf62-Y sequence and a 5362 bp Pf62-X sequence have been cloned by genome walking, and significant genetic differentiation has been revealed between the corresponding X and Y chromosome allele sequences. Moreover, three pairs of primers were designed to efficiently identify YY super-males, XY males and XX females in an artificial breeding population, and to distinguish XY males and XX females in various wild populations. Together, the three new sex-specific genetic markers develop a highly stable and efficient method for genetic sex identification and sex control application in sustainable aquaculture of all-male yellow catfish.     

Isolation of Y- and X-linked SCAR markers in yellow catfish and application in the production of all-male populations

Sex controls have been performed in some farmed fish species because of significant growth differences between females and males. In yellow catfish ( Pelteobagrus fulvidraco ), adult males are three times larger than female adults. In this study, six Y- and X-linked amplified fragment length polymorphism fragments were screened by sex-genotype pool bulked segregant analysis and individual screening. Interestingly, sequence analysis identified two pairs of allelic genes, Pf33 and Pf62 . Furthermore, the cloned flanking sequences revealed several Y- and X-specific polymorphisms, and four Y-linked or X-linked sequence characterized amplified region (SCAR) primer pairs were designed and converted into Y- and X-linked SCAR markers. Consequently, these markers were successfully used to identify genetic sex and YY super-males, and applied to all-male population production. Thus, we developed a novel and simple technique to help commercial production of YY super-males and all-male populations in the yellow catfish.     

Genetic Manipulation of Sex Ratio for the Large-Scale Breeding of YY Super-Male and XY All-Male Yellow Catfish (Pelteobagrus fulvidraco (Richardson))

Yellow catfish has become one of the most important freshwater aquaculture species in China. The mono-sex male yellow catfish has important application value in aquaculture because the male grows generally faster than the sibling females under the same conditions. This study has screened YY super-male and YY physiological female yellow catfish by sex reversal, gynogenesis, and progeny testing, which can help to achieve the large-scale production of YY super-male and XY all-male. From 2008 to 2010, about 123,000 YY super-male were produced, and about 81 million XY all-male fry were produced with 100 % male rate by random sampling. Therefore, these results indicate that YY super-male and YY physiological female yellow catfish can be viable and fertile. We conclude that the mono-sex breeding technique by YY super-male yellow catfish is stable and reliable, which has great potential for application in yellow catfish aquaculture.     

Massive production of all-female diploids and triploids in the crucian carp

In many species of aquaculture importance, all-female and sterile populations possess superior productivity due to faster growth and a relatively homogenous size of individuals. However, the production of all-female and sterile fish in a large scale for aquaculture is a challenge in practice, because treatments necessary for gynogenesis induction usually cause massive embryonic and larval mortality, and the number of induced gynogens is too small for their direct use in aquaculture. Here we report the massive production of all-female triploid crucian carp by combining artificial gynogenesis, sex reversal and diploid-tetraploid hybridization. Previously, we have obtained an allotetraploid carp population (4n = 200) by hybridization between red crucian carp (Carassius auratus red var; ♀) and common carp (Cyprinus carpio; ♂). We induced all-female diploid gynogens of the Japanese crucian carp (Carassius cuvieri; 2n = 100). We also generated male diploid gynogens of the same species treated gynogenetic fry with 17-α-methyltestosterone, leading to the production of sex-revered gynogenetic males. Finally, these males were used to cross with the female diploid Japanese crucian carp gynogens and the allotetraploid females, resulting in the production of fertile all-female diploid Japanese crucian carp (2n=100) and sterile all-female triploid hybrids (3n = 150), respectively. Therefore, diploid crucian carp gynogenetic females and sex-reversed male together with an allotetraploid line provide an opportunity to produce all-female triploid populations in a large scale to meet demands in aquaculture industry.     

17β-雌二醇诱导斑点叉尾鮰雌性化研究

研究以人工配合饲料为17β-雌二醇(17β-estradiol, 17β-E₂)载体, 对斑点叉尾鮰(Ictalurus punctatus)开口仔鱼连续饲喂27d进行雌性性别诱导, 探究斑点叉尾鮰XY雄鱼雌性化的合适17β-E₂剂量。60日龄测量并统计各组试验鱼的生长数据、存活率, 解剖观察性腺结构, 结合遗传性别鉴定结果计算性逆转率, 并通过组织切片分析各组试验鱼中XX和XY雌鱼卵巢发育情况。270日龄检测XY雌鱼和对照组雌鱼的鳃、心脏、肝脏、肾脏、卵巢和肌肉等组织中17β-E₂含量。研究进一步地采用qRT-PCR检测270日龄雌雄性别分化基因foxl2和dmrt1在XX和XY雌鱼卵巢中的表达水平。结果显示: 各组XY雌鱼的比例和卵母细胞数量均随17β-E₂剂量的提高而增加, 且核仁数量也随之增多, 而核质比相应减小; 各组XY雌鱼的体重和体长随着17β-E₂剂量的提高先增加后减小, 但存活率没有显著性变化; 各组XY雌鱼与对照组雌鱼的鳃、心脏、肝脏、肾脏和肌肉组织中17β-E₂含量均无显著性差异, 卵巢中17β-E₂含量与添加剂量呈正相关; 雌性性别分化基因foxl2在XY雌鱼中的表达量显著升高, 雄性性别分化基因dmrt1在XY雌鱼中的表达量显著降低。研究建立了17β-E₂诱导XY雄性斑点叉尾鮰雌性化方法, 为后续全雄斑点叉尾鮰新品种的培育奠定基础。     

Sexual mosaics induced by tetracycline treatment in the Wolbachia-infected adzuki bean borer, Ostrinia scapulalis.

Wolbachia-infected Ostrinia scapulalis (Lepidoptera: Crambidae) females generate all-female or nearly all-female broods. Curing the infection by tetracycline treatment during larval stages results in the generation of all-male broods in the next generation. Here we show that sexually mosaic offspring are produced by Wolbachia-infected females treated with tetracycline at the adult stage. The sexual mosaics had wings that were composed of distinctive female and male sectors. Besides wings, the sexually dimorphic mid tibiae displayed an intermediate morphology in some of the mosaics. Many of the mosaic individuals had an abnormal structure of the external genitalia as well, a combination of the male uncus and the female ovipositor. We assume that Wolbachia has a feminizing effect on O. scapulalis genetic males and, hence, incomplete curing of the Wolbachia infection results in the generation of sexually mosaic individuals.     

The effect of heated effluent on egg production in the estuarine prawn Upogebia Africana (Ortmann)

Egg production of a population of Upogebia africana (Ortmann) in a pond containing water heated by a thermal power station at Knysna, South Africa was compared with that of the population in the adjacent estuary and in two other estuaries. The breeding cycle, proportions of ovigerous females, incubation time for eggs at different temperatures, number of eggs produced, sex ratios, and size-frequency of males and females were all investigated. The number of eggs produced/100 prawns was found to be nearly twice as high in the population exposed to heated effluent as compared with the population in the adjacent estuary. The increase was due to a combination of a change in sex ratio resulting in more females in the population and a greater proportion of larger females in the pond. Since the number of eggs produced/female is positively related to the size of the prawn, large females in warmer water produced more eggs than smaller females in the adjacent estuary. Exposure to heated effluent did not alter the initiation or duration of the breeding season, nor cause precocious maturity; nor did it alter the number of eggs carried per female at various sizes.     

A Single Injection of Hypertrophied Androgenic Gland Cells Produces All-Female Aquaculture

Monosex culture, common in animal husbandry, enables gender-specific management. Here, production of all-female prawns (Macrobrachium rosenbergii) was achieved by a novel biotechnology comprising three steps: (a) A single injection of suspended hypertrophied androgenic gland cells caused fully functional sex reversal of females into “neo-males” bearing the WZ genotype; (b) crossing neo-males with normal females (WZ) yielded genomically validated WW females; and (c) WW females crossed with normal males (ZZ) yielded all-female progeny. This is the first sustainable biotechnology for large-scale all-female crustacean aquaculture. The approach is particularly suited to species in which females are superior to males and offers seedstock protection, thereby ensuring a quality seed supply. Our technology will thus revolutionize not only the structure of the crustacean aquaculture industry but can also be applied to other sectors. Finally, the production of viable and reproducible females lacking the Z chromosome questions its role, with respect to sexuality.