伊红、台盼蓝检测河蟹精子存活率的比较

Evaluation of Sperm Viability Rate of Eriocheir sinensis Stained by Eosin and Trypan Blue

对台盼蓝和伊红染色法检测河蟹(Eriocheir sinensis)精子存活率的方法进行了评价研究。结果表明,两种染色法死、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,光镜下辐射臂及细胞边界清晰;死亡精子顶体着色,且中央有一染色较深的圆斑,核杯染色不明显,细胞体积变大,边界模糊。通过不同染色时间和不同染料浓度的比较发现,两种染色法最适染液浓度分别是0·25%的伊红和0·5%的台盼蓝,染色时间均以15min为佳。在此基础上,将新鲜精子和60℃水浴处理致死精子以不同的体积比混合,配成含致死精子比例为10%～90%的9个梯度样品,用伊红和台盼蓝分别测定各样品精子死亡率,并进行相关性分析。结果发现,各样品实测精子死亡率均略高于样品的理论死亡率,同时两种染色法实测值与样品理论值呈显著正相关(P<0·05),两种染色法之间亦呈显著正相关(P<0·05)。上述结果表明,伊红和台盼蓝可用于河蟹精子的活体染色,且两种染色法在对河蟹精子染色中具有一定的稳定性和可比性。

Sperm quality evaluation is an important part in male reproductive biology. In spite of numerous studies on mammalian and fish ,there are few related reports on crustacean, even no reports on Chinese Mitten Crab （Eriocheir sinensis ） . Eosin staining and trypan blue staining are two common methods for detecting cellular viability rate and both are used for evaluation of sperm quality.In this paper, sperm viability rate of E. sinensis was investigated with eosin staining and trypan blue staining. The results showed that there were significant differences of staining characters between dead and live spermatozoas：the live ones were transparent,while the dead ones were stained easily by either dye. We found that 0.25 % eosin solution and 0.5 % trypan blue solution stained the most effectively, and the optimum reaction time was 15 min. We also showed that there was positive correlation between pratical and theoritical values when either of the two dyes was used（ P 〈 0.05） and there was also positive correlation between these two staining methods（ P 〈 0.05）. These results suggested that both eosin staining and trypan blue staining were suitable for evaluation of sperm viability rate of Chinese Mitten Crab.