【作者】 王珂；

【导师】 康现江；

【作者基本信息】 河北大学 ， 水生生物学， 2008， 硕士

【摘要】 半滑舌鳎(Cynoglossus semilaevis Gunther)隶属于鲽形目舌鳎科舌鳎属,它在我国沿海均有分布,它是一种近海温水性大型底层鱼类,雌性个体大,雄性个体小。栖息于渤海的半滑舌鳎终年不出渤海,具有活动范围小、营养等级低、食物层次较低、个体大、生长快等优良性状。在自然条件下,半滑舌鳎雌、雄个体差异非常大,；雄性个体生殖能力弱,完全成熟精巢,不管是体积还是重量,都只是成熟卵巢的1／200-1／900,因而导致繁殖能力差,这些先天特性在其他的鱼类种群中是罕见的。故其在幼鱼期是很难根据外观形态预测其性别的,因此为提高育种效率和优选率应选择性别早期鉴定的指标,即来自对其内部遗传类型(即基因型)或生理生化的鉴定。1雌、雄半滑舌鳎血液的指标以20尾半滑舌鳎的成鱼为材料,测定了红细胞数、白细胞总数、血红蛋白值、白细胞分类计数、红细胞脆性与红细胞及其核大小等正常血液指标,并统计了性别与血液指标的关系,其中红细胞长径：雄鱼(7.00±0.15 pm)；雌鱼(8.05±0.13μm)、红细胞核长径：雄鱼(3.06±0.30 pm)；雌鱼(3.75±0.12 pm)、短径：雄鱼(2.02±0.32μm)；雌鱼(2.60±0.14 pm)有极显著差异,其它的血液指标在雌、雄中则无显著差异。2雌、雄半滑舌鳎染色体的核型分析本文采用秋水仙素直接取材法对半滑舌鳎的核型进行分析。选取发育12-50 d的幼鱼,在不同浓度的秋水仙素溶液中处理5-6 h,分别剪取冠状幼鳍、鳃丝和身体周围的鳍条及肾组织,通过低渗、固定、解离,得到细胞悬液,冷冻滴片,Giemsa染色。显微镜下观察分散比较良好的中期分裂相并统计染色体的数量,选取较好的分裂相进行显微照相,放大和测量,分析半滑舌鳎的核型。通过对500个清晰的中期分裂相的观察和统计,最终确定：半滑舌鳎的染色体众数为42,核型为2n=42t,臂数NF=42,全部为端部着丝粒染色体。通过观察多次实验的结果,可初步断定半滑舌鳎的性染色体类型属于ZW／ZZ型。3雌、雄半滑舌鳎染色体的带型分析选用片龄在30 d左右的半滑舌鳎的染色体标本放在1×SSC溶液中恒温(35-40℃)加热并用紫外灯照射约15 min后用蒸馏水冲洗,Gimsa染色7-9 min后晾干待镜检,可观察到在复制早期到中期这段时间,半滑舌鳎的性染色体均具有各自特有的复制带。半滑舌鳎W染色体的长度在所有的染色体中占据第一的位置,而Z染色体无论在雌鱼还是雄鱼中均排在第四位。根据实验结果,雌鱼、雄鱼1-3号染色体和Z、W染色体的带型为：雌鱼：W染色体：染色体长臂中部到下部的区域有3条相邻的特征性晚复制区。两端的两条较大,中间的一条较小。1号染色体：染色体短臂距着丝粒最远端可观察到1条特征性晚复制区,长臂在靠近着丝粒处有3条相邻的特征性晚复制区,大小基本相同。2号染色体：3条特征性晚复制区,其中两条靠近长臂的着丝粒处,另一条在距离着丝粒的最远端,大小基本相同。3号染色体：两条特征性晚复制区,一条靠近长臂着丝粒处,另一条在长臂的近中部。位于近中部的特征性复制区要比靠近着丝粒处的大。雄鱼：1号染色体：在靠近长臂着丝粒处有两条特征性晚复制区,近着丝粒的特征性晚复制区大于远端的。2号染色体：整个染色体仅观察到一条特征性晚复制区,位于靠近长臂的近中部。3号染色体：两条特征性晚复制区,均位于长臂中部靠下的位置,大小基本相同。Z染色体：两条特征性晚复制区,均位于染色体长臂的近中部,靠近着丝粒的特征性晚复制区比远离的大。4雌、雄半滑舌鳎激素水平的分析半滑舌鳎性腺早期发育一般划分为Ⅰ期(发育早期)、Ⅱ期(发育前期)、Ⅲ期(发育中期)、Ⅳ期(发育后期)和V期(成熟期)5个时期。半滑舌鳎雄性血清中睾酮在精巢发育期内的变化为：自Ⅲ期开始升高,至Ⅳ期后期(排精前)升至高峰139.925±0.112 ng／mL,至第Ⅱ期下降到最低点8.700±0.120 ng／mL。雌性血清中雌二醇在卵母细胞发育周期中的变化为：自Ⅲ期开始升高,至Ⅳ期后期,排卵前升至高峰30.967±0.256 pg／mL,排卵后,雌二醇浓度骤然下降。雌性血清中孕酮在卵母细胞发育周期中的变化为：自Ⅳ期早期升高,Ⅳ期升至高峰16.556±0.135 pg／mL,高水平维持至V期,排卵结束。更多还原

【Abstract】 Cynoglossus semilaevis belongs to Phylum Chorata, Osteichthyes, Pleurohe Ctifomes, Cynoglossidae, Cynoglossus Buchanan Hamiltou. It distribution the coastal areas of our country. The biggest amount of these areas is Bohai sea, the bigger amount is Huanghai sea and the last is Donghai sea and Nanhai sea. C. semilaevis are inhabiting in the warm water bottom. Individual of female is big. The average length of them is 523 mm, the largest body length is above 800 mm; individual of male is small, the average of them is 280 mm. Habitating Bohai sea of C. semilaevis were not out of that place throughout the year. They have small scope of activitives, low nutritional levels, individual large, grow fast and higher market value of good characters.Under natural conditions, male and female individual differences is very large. Mature females in length and can weight up to more than double the males; individual male reproductive capacity weak, full mature testis, no matter the size or weight, the only mature ovarian 1/200-1/900. As a result, poor reproductive capacity, these inherent characteristics of the fish stocks in the other is rare. Therefore, in their juvenile period is very difficult to predict under the shape of their gender. So breeding to improve efficiency and optimization of sex selection should be the early identification of indicators. That is, from its internal genetic type (that is, genotype) or the identification of physiological and biochemical.1. Blood cators of females and males C. semilaevisThe haemotological indices, including red blood cells (RBC), white blood cells (WBC), hemoglobin content (Hb), erythrocyte osomotic fragility (Eof), differential leukocyte count (DLC), and red cell size, were measured in C. semilaevis with mean body length of 26.86 cm. In long diameter:male(7.00±0.15μm); female(8.05±0.13μm); For the nucleus of erythrocyte, In long diameter:male(3.06±0.30μm); female(3.75±0.12μm); In short diameter:male(2.02±0.32μm); female(2.60±0.14μm). There were a very significant difference. Other indicators in the blood of male and female in no significant difference. 2. The nuclear-chromosome analysis of females and males C. semilaevisThe experimental use of colchicine direct subjects of C. semilaevis nuclear-analysis. Select the incubation time of 12-50 days of juvenile fish, in different concentrations of colchicine solution to deal with 5-6 hours, david coronavirus were Jianqu fin, gill and kidney strip fin around the body, by low permeability, fixed, dissociation, by cell suspension, frozen droplets, Giemsa stain. Were observed scattered relatively good medium-term split of statistics and the number of chromosomes, select a better split of a microscopic camera, zoom and measurement, analysis C. semilaevis half of the nuclear. Chromosome classification criteria for the classification according to Levan. Through the 500 split of a clear medium-term observation and statistics, final:The result shows that there are 42 acrocentric chromosomes in diploid and their karyotype formula is 2n=42t, and there existed heterotypic sex chromosome which belongs to ZW/ZZ type.3. The band type of chromosome of females and males C. semilaevisSelection of the 30-day-old about half of C. semilaevis on chromosome samples put in 1 X SSC (35-40℃). Heated by ultraviolet light and heat exposure after about 15 min rinse with distilled water, Gimsa staining 7-9 minutes after the examination to be dry, copy can be observed in the early to mid-term period, sex chromosome of C. semilaevis are all with their own unique copy.W chromosome of C. semilaevis in all the chromosomes occupy the first position, and Z chromosome in both females in the male are still ranked fourth. According to the experimental results, females, the male chromosome 1-3 and Z, W chromosome for the band: Males:W chromosome:The long arm of chromosome central to the lower part of the region observed a total of three adjacent three characteristics of the area late copy. The two ends of the larger, a small Central. Chromosome 1:The short arm of chromosome from the most remote centromere observed a characteristic late copy, the long arm near the centromere department has three characteristics of the adjacent copy of the evening, they are basically the same as the size of. Chromosome 2:Observed that the characteristics of a total of three copies of late, and around two of the long arm of the centromere, another one from the centromere of the most remote. They are basically the same size. Chromosome 3:Observed that the characteristics of a total of two copies of the late district. A long arm near the centromere, another in the long arm of the recent Central. Located near the central features of the copy area near the centromere than the big. Females:Chromosome 1:In the long arm near the centromere, there are two characteristics of the area late copy. Near the centromere than far off the one. Chromosome 2:Observed on the entire chromosome characteristics of a late copy of the District. It is located near the long arm of the recent Central. Chromosome 3:Observed that the characteristics of a total of two copies of the night, are located on the long arm of the central location. Their size is basically the same. Chromosome Z:Observed that the characteristics of a total of two copies of the late District. The long arm of chromosome are located in the Near Central. One near the centromere copy of the characteristics of the night away from the area than that big.Through the C. semilaevis with a copy of the analysis we can see that the male chromosome W copy of the advanced features of the female with more than one, but chromosome with a copy to significantly more than females. This may be the male half of C. semilaevis as early as the female sex differentiation of a relationship.4. Hormone levels of females and males C. semilaevisSemi-gonadal C. semilaevis early development can be divided into three stages:Gonad of the original stage, gonadal differentiation and gonad differentiation early stage. Generally divided into I period (early developmental stage), II period (pre-development), III period (of the medium-term),Ⅳperiod (of late) and V period (maturity) five times. C. semilaevis half of testosterone in the testes of the changes for the period:Ⅲincreased since the beginning, to the late stageⅣ(Pai fine before) to the peak 139.925±0.112 ng/mL, to stage II dropped to the lowest point 8.700±0.120 ng/mL C. semilaevis serum estradiol in the oocytes of the cycle of change:Ⅲincreased since the beginning, to the late stageⅣ, to the peak before ovulation 30.967±0.256 pg/mL, after ovulation, E2 suddenly dropped. C. semilaevis serum progesterone in the oocytes of the cycle of change:Since the early stageⅣincreased, IV peak period rose to 16.556±0.135 pg/ml, to maintain a high level V period, the end of ovulation.更多还原