【作者】 张伟；

【导师】 柳凌；

【作者基本信息】 华中农业大学 ， 水产养殖， 2015， 硕士

【摘要】 黄鳝(Monopters albus)隶属合鳃目、合鳃科、黄鳝属,是我国重要的淡水经济鱼类,由于黄鳝存在性逆转现象,使其成为研究鱼类性别转变的模式生物。许多学者从形态学、生理生化及分子生物学等多方面对其进行了研究,但迄今为止,黄鳝性逆转机制仍存在许多盲点,黄鳝规模化养殖及稳定高效的人工繁育技术还有许多难点以待攻克。本研究首次以人工繁育黄鳝为研究对象,对1~4龄各性别(雌性、间性、雄性)进行连续取样,运用组织学、形态学、计算机辅助精子分析(CASA)等技术对其胚胎发育、性腺分化发生、性逆转、配子质量及生长特征做了系统地研究,主要结果如下:(1)对人工繁殖的黄鳝幼苗从出膜到1龄鱼进行了分阶段连续采样,研究了样本的性腺发生、分化过程,以及性腺的位置变化。结果表明:在实验室静水温度26~28℃恒定条件下,仔鳝出膜12h,原始生殖细胞以单细胞的形式出现在肾管区左边;出膜24h,原始生殖细胞迁入生殖原基中,标志着原始性腺形成;出膜72h,原始性腺分化成相连的一对;出膜25d,一对原始性腺外形成结缔组织膜将其包裹,其中一条出现卵巢腔,标志着向卵巢分化,另一条除形态随鱼体增长而增长外,并无明显的生殖细胞特征变化,为形成精巢的原基;随后一直到1龄都可观察到两条生殖腺组织结构上保持着独立,一条形成卵巢,另一条向卵巢内延伸,形成褶。性腺位置起始于生殖孔附近,逐渐向前生长。由于一对性腺合并成一条,位于鱼体的左侧,因而本应在右侧出现性腺的位置上出现了膀胱。膀胱在出膜7d首次观察到,1龄时呈扁平状,到3龄时发育饱满,其上表皮有长短不一的纵褶,内表面积大。(2)对繁殖季节黄鳝的形态进行观察,发现各性别黄鳝泄殖孔前后体高对比存在差异,导致其腹部形态有所不同:雌性泄殖孔前腹部膨大,且体高大于泄殖孔后,泄殖孔后有微微凸起,似“w”形;间性腹部平滑,泄殖孔不内陷,且泄殖孔附近体高无明显差异;雄性泄殖孔后明显凸起,孔后体高大于孔前。腹部形态和泄殖孔情况与性腺发育状态存在一定的关联,可将其作为重要的形态学指标进行繁殖季节黄鳝性别的判别,且此判断方法对亲本无伤害。(3)获得人工养殖黄鳝1+-4+龄群体性别比例,2+龄黄鳝群体中雌性比例(71.4%)比3+、4+龄的比例高。相同年龄的黄鳝间性和雄性个体之间在体长方面没有显著性差异(P﹥0.05),但均显著大于雌性(P﹤0.05)。体重指标在同龄不同性别的黄鳝中有显著的差异(P﹤0.05),间性、雄性体重显著大于雌性(P﹥0.05),2+龄黄鳝间性个体体重达到雌性个体的2倍。对不同年龄组配子质量进行分析,发现2+龄雌鳝的相对怀卵量最大,卵径相对较小。黄鳝精子运动速度较慢,精子活力低,3+龄雄鳝精子在26℃被蒸馏水激活后平均曲线运动速度(VCL)为18.08±3.43μm?s-1,平均直线运动速度(VSL)为9.66±0.94μm?s-1,平均路径运动速度(VAP)为14.61±2.97μm?s-1。(4)实验室26~28℃静水孵化140±4h时,首次出现仔鳝出膜,约占总数的5%;在158±2h时,出膜达到高峰期,约占总数的41%;胚胎发育过程平均用时151h,而自然环境下为140h左右孵化出膜。根据早期性腺分化发育的研究、早期生长特征及摄食行为,将黄鳝出膜1至10d划分为仔鱼期,即以卵黄囊消失、外源性营养建立为界限;出膜10d至30d为稚鱼期,即以卵巢腔形成、性腺分化向雌性结束、体长快速增长期为界限;出膜30d至初次性成熟前为幼鱼期,即以I期卵巢形成、体重快速增长期开始、初次性成熟为界限。(5)1龄人工养殖雌鱼出现异速生长,体重体长关系为WWL=0.0109L2.3195,R2=0.956,推断此现象与鱼体确保卵巢优先发育至成熟,和消耗部分能量资源用于产后性逆转过程有关。(6)基于以上研究,建议在黄鳝人工繁殖生产中挑选2龄雌鳝和3龄及3龄以上雄鳝作为亲鱼。黄鳝苗种的培育应在稚鱼期末期(出膜30d)前于孵化室中培养,在稚鱼期之后转移至培苗网箱中养殖。更多还原

【Abstract】 The ricefield eel(Monopters albus) is a high economic value freshwater fish in China. Since Liu C K et al. found it has the sex reversal phenomenon in 1944, ricefield eel has become the model organism of sex changing research in fish. A large number of scholars have researched it from all kinds of aspects, such as morphology, physiology, biochemistry and molecular biology, however, there are still some problems on sex reversal mechanisms, scale farming and artificial breeding technology of it. This is the first time that the artificial breeding eel is as the research object in this study, and we continuously acquired the eel samples of each gender(female, intersex and male) from 1 to 4 years old. We investigated the embryonic development, Gonadal differentiation and development, Sex reversal, Gamete quality and growth characteristics of each age of the artificial breeding eel using the methods of histology, statistics and CASA. Our major results are as follows:(1) We continuously collected the artificial breeding eel samples from embryo to 1 year old according to the developmental time points and investigated the process of gonadal ontogenesis, differentiation and position changing. The results showed that: under hydrostatic condition in the laboratory(26~28℃), the primordial germ cell(PGC) was first seen at the left of the nephridium area as single cells at 12 h after hatching; at 24 h after hatching, the PGCs moved into the genital primordium, which meant the formation of primordial gonad; at 72 h after hatching, the primordial gonad divided into two symmetrical parts; at 25 d after hatching, the connective tissue membrane appeared to wrap the primordial gonad, meanwhile, there emerged the ovarian cavity at one part of the primordial gonad which marked the differentiation of ovarian cavity; one side of PGCs proliferated rapidly and differentiated into the primary oocytes, leading to squeeze the other side; the part extruded was going to become genital fold which is the primordium of testis.(2) We found that there were some differences on body height around the cloaca among the different gender of ricefield eel, which leaded to various abdominal shapes among them. In female, the abdominal shape was like “W” and the body height in the back of the cloaca was higher than that in front of the cloaca, but not obviously; In intersex, the abdomen was flat and cloaca was not sunk; In male, the body height in the back of the cloaca was obviously higher than that in front of the cloaca. The abdominal shape and cloacal state has a certain association with the gonadal development status, thus we could use the abdominal shape and cloacal state as a crucial morphological index to identify the gender of ricefield eel.(3) In the ricefield eel of the same age: there were no significant differences between the intersex and male on the body length(P > 0.05), however, the body length of the them were all longer than that of female significantly(P < 0.05); the weight of the intersex and male were heavier than that of female as well(p < 0.05). Gamete quality analysis showed that the relative fecundity of 2+ years old female eel was the largest and the egg diameter of it was relatively small. Additionally, the proportion of females in the 2+ years old eel was higher than that in the 3+ or 4+ years old one. As a whole, the sperm motorial speed of the ricefield eel was relatively slow, and the sperm motility was low.(4) Under hydrostatic condition in the laboratory(26~28℃), at 140±4h, the fertilized egg began to hatch, and the hatchability was 5%; at 158±2h, the hatchability reached the peak(41%); the whole process of embryonic development lasted for about 151 h. In this study, we divided the growth stage of ricefield eel which from incubation to young fish into three periods according to the gonadal development, early growth characteristics and feeding behavior: larva was the period from 1 to 10 d after hatching; juvenile was the period from 10 to 30 d after hatching; young fish was the period from 30 d after hatching to first sexual maturity.(5) The one years old artificial breeding eel appeared allometry(WWL = 0.0109L2.3195, R 2 = 0.956). We speculated this phenomenon was a survival strategy to store energy for ovarian development and sex reversal.(6) In summary, it is better to choose 2 years old female eel and 3 or 4 years old male eel as the parent fish for artificial propagation; additionally, the larvae should be cultivated indoor until 30 d after incubation, after that, transfer them to the foster cages for subsequent cultivation.更多还原