论文标题:质膜完整性在中华绒螯蟹（Eriocheir Sinensis）精子质量评价研究中的应用
Studies on Sperm Viability in Sperm Quality Assessment of Chinese Mitten-handed Crab Eriocheir Sinensis
论文作者 马强
论文导师 王群,论文学位 硕士,论文专业 动物学
论文单位 华东师范大学,点击次数 147,论文页数 60页File Size3284k
2005-05-01论文网 http://www.lw23.com/lunwen_820602/ 中华绒螯蟹;精子获得;胰蛋白酶;消化法;机械匀浆法;台盼兰;伊红;质膜完整性;流式细胞仪;精子活力
Eriocheir sinensis; homogenization method; digest method; sperm viability; trypan-blue; esion; fluorometric; ethidium Bromide; flow cytometry;propidium iodide
一、两种获得中华绒螯蟹(Eriocheir sinensis)游离精子方法的比较研究在pH 7. 4的条件下,采用不同的温度、酶浓度和作用时间进行了正交实验研究了胰蛋白酶消化中华绒螯蟹精荚获得游离精子的最佳反应条件。以每克精荚获得的精子数量为判据,发现酶作用时间是主要的影响因子,其次是温度和酶浓度。酶消化的最适反应条件是:温度为37℃,酶浓度为0. 20%,作用时间为5 min。酶消化法与机械匀浆法的比较研究发现,酶消化法从每克精荚中获得的精子量为7. 41×108个,显著高于匀浆法;且精子的死亡率为4. 75%,显著低于匀浆法。此外,用人工诱导顶体反应法进一步检测酶消化法以及机械匀浆法所获得精子的顶体反应能力,发现酶消化法获得精予的顶体反应率为62. 3%,而匀浆法为64. 5%,两者无显著性差异。因此,酶消化获得游离精予的方法明显优于机械匀浆法。二、两种检测中华绒螯蟹(Eriocheir sinensis)精子活力方法的研究本文采用台盼兰和伊红染色法对中华绒螯蟹精子活力进行了评价研究。结果表明两种染色法中死、活精子分别呈现出明显不同的染色特征:活精子无色透明,顶体中央凸起呈圆锥状,光学显微镜下可见到核杯发出的辐射臂,细胞边界清晰可辨;而死亡精子项体着色且中央有一染色较深圆斑,核杯染色不明显,细胞体积变大,辐射臂消失,边界不明显。而两种染色法最适染液浓度和时间是0. 25%的伊红和0. 5%的台盼兰染色15min。然后对两种染色方法的相关性进行了分析。将新鲜精子和处理致死精子以不同的体积混合,配成含有死精子比例为10%-90%的9个浓度梯度,将其视为该样本死精子含量的理论值;用两种染色法检测这些精子样品的死亡率。结果显示:台盼兰法和伊红法测得的结果与理论值显著正相关(P<0. 05) ,而且他们二者之间亦显著正相关(P<0. 05) 。说明这两种染色法均可用于精子活力检测中,其结果具有可比性。三、荧光分光光度计分析中华绒螯蟹精子质膜的完整性
1, Comparison of two methods for acquiring free sperm of Eriocheir sinensisThe purpose of this paper was to compare different methods of acquiring free sperm cells from spermatophores of the Chinese mitten-handed crab (Eriocheir sinensis), and to find a suitable method of acquiring free sperms from spermatophores of Chinese mitten-handed crab under three factors with five levels including a series of temperature (27,32,37,42 and 47 ℃), different concentrations in Trypsinase sotution(0.05%,0.10%,0.15%,0.20% and 0.25%) and duration (5,10,15,20 and 25 min).The orthogonal experiment results of digesting spermatophore showed it would acquire the largest amount of sperms in per gram spermatophore (7.41×10~8/g) by using 0.2% Trypsinase solution at 37℃ and incubating 5 min. Basing on this, we compared the digestion method with homogenization method. The results showed that digestion method was much better than homogenization method in both the amount of sperm and living rate of sperms.2, Assay the sperm viability of Eriocheir sinensis by two supravital staining techniqueTwo dyes: esion and trypan blue were dissolved in Ca2+-free saline water. And by using them the sperm viability of Eriocheir sinensis was evaluated. They appeared remarkable difference between the dead sperms and live sperms, The dead sperm was stained by both of dyes, especially the acrosome; The size of dead sperm was bigger than the lives; The radial arms and border of death were indistinct. Then two experiments were done to determine a fit dye time and concentration of dyes. In 0.1%and 0.5% esion solution, death rate of sperm appeared significantly higher than in 0.25%, 0.125% (P<0.05) . But there was no significant difference between 1% and0.5% trypan blue solutions. And there was also no significant difference among 0.5% , 0.25% esion solution and 1%, 0.5% trypan blue solution (P>0.05) . So (he lit concentrations of dyes were 0.5% 0.25% esion solution and l%> 0.5% trypan blue solution . The death rate of sperm was increased with the time, because dyes did harm to sperms also. In 0.1%and 0.5% esion solution, death rate of sperm showed significant difference among at 30min, 5 min and 15min. In 0.25%and 0.125% esion solution, death rate of sperm showed significant difference between at 30min, 5min and 15min, but there was no significant difference between at 5 and 15 min. In 0.1%and solution, there was no significant difference in sperm death rate among at 5, 15 and 30min. So the best condition was that 0.5% trypan blue or 0.25% esion under staining 15min. The reliability of esion was compared with the trypan blue staining technique. The sperm samples contained known proportions of fresh and killed cells. The results show that the values determined by esion and trypan blue both agreed closely with the theoretical values(R=0.99, PO.05). And the values determined by esion agreed closely with trypan blue method(R=0.98, P<0.05).3, Assessment of Sperm Viability in Eriocheir sinensis by using a Fluorometric Ethidium Bromide Exclusion ProcedureThe ethidium bromide (EtBr) exclusion procedure, a fluorometric method for measuring sperm cell viability, was studied to optimize the use of this technique on Chinese mitten-handed crab (Eriocheir sinensis). The fluorescence intensities of stained fresh and stained tritonX-100-permeabilized samples were corrected, respectively. The nonspecific fluorescence measures of fresh and tritonX-100-permeabilized samples were both unstained. The contribution of the dye and dye+ tritonX-100 was subtracted from the corrected values, then the ratio between the corrected values of fresh and permeabilized cells provided the proportion of damaged cells in the sample. The working cell concentration range giving a constant proportion of damaged cells was set using diluted semen for Chinese mitten-handed crab (8-32 xl06cell/ml). The reliability of the fluorometric method was compared with the traditional eosin (E) staining technique. The intactness of sperm samples containing known proportions of fresh and killed cells was measured in defin
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