使用荧光染色和流式细胞术检测山羊精子

分别使用PI(碘化丙碇),FITC-PSA(异硫氰酸荧光素络合豌豆凝集素)和RH123(罗丹明)对山羊冷冻精子进行染色,再使用流式细胞仪进行检测、分析。试验发现,对于山羊的冷冻精子,PI染色阴性的为质膜完整的精子;FITC-PSA染色阴性的可能为顶体完整的精子,染色FITC-PSA+和FITC-PSA++,可能分别为顶体反应中的精子和顶体破损精子;RH123+应为线粒体无活性精子,有绿色荧光可能是因为染料残留在细胞膜上导致,而RH123++可能应为线粒体有活性的活精子。不同个体羊的精子对于低温敏感度不一样。不同个体羊之间的冻精顶体完整率、质膜完整率、线粒体有活性的精子比率之间差异显著,据此应选择合适的山羊个体进行精液冷冻。

Detection of goat sperms by fluorescent staining and flow cytometry

In the present study,we used PI,RH123,FITC-PSA staining of goat frozen sperms and detected by using flow cytometry and analysed the flow cytometry data.We found that frozen sperms of the goats with PI negative staining showed the plasma membrane integrity.The sperms of FITC-PSA negative staining might be the acrosome integrity of sperm,while,sperms of FITC-PSA+ and FITC-PSA++ were the ones with acrosome reaction and acrosome damaged,respectively.RH123+ should be no active mitochondria,green fluorescence might be the dye residual in the membrane.RH123++ might be the live sperms with active mitochondria.Goat sperms of different individuals had different sensitivities to the low temperature.Rates of acrosome integrity,plasma membrane integrity and mitochondrial activity of sperms had a significant differences in individual goats.Therefore,we should select right goat individuals for frozen semen.