伊红、PI染色法检测中华绒螯蟹活精子率

Detection of mortality of Eriocheir sinensis sperms stained by eosin and propidium iodide(PI)

以伊红和PI两种染色法,结合光学显微镜和流式细胞仪对中华绒螯蟹(Eriocheir sinensis)精子的存活率进行评价研究。将新鲜精子和60℃水浴处理致死精子以不同比例混合,配成含有死精子比例为0%、20%、40%、60%、80%、100%的6个浓度梯度,将其作为"理论死亡率",分别用上述两种染色法进行检测。结果表明:伊红能有效区分死、活精子,其实测精子死亡率与"理论死亡率"之间具有明显的相关性,相关系数为R=0.98(P<0.01);经PI染色后通过流式细胞仪分析发现,其实测精子死亡率与"理论死亡率"同样具有极好的相关性,相关系数为R=0.99(P<0.01)。对伊红和PI两种染色法进行相关性比较分析,认为PI染色流式细胞仪检测法在检测中华绒螯蟹精子活力方面更具灵敏性,结果更可靠。

The mortality of sperms of Eriocheir sinensis was detected using microscope and flow cytometry （FCM） after eosin or propidium iodide（PI） stain. Those sperms killed at 60℃ （water bath） were mixed with fresh ones so as to obtain six scalar concentrations（0% , 20%, 40%, 60%, 80%, 100% ） （ for dead sperms）, which were referred to as theoretical mortality and detected by eosin and PI stain. Results showed that dead sperms could be discriminated from live ones by eosin stain. Furthermore, correlation did exist between theoretical and practical mortality of sperms with correlation coefficient R = 0. 98 （P 〈0.01 ）. After PI stain and FCM detection, a better correlation indicated with correlation coefficient R = 0. 99（ P 〈 0.01 ）. Therefore, PI stain along with FCM detection is an excellent method to analyze the mortality of sperms of Eriocheir sinensis.