贝类精子质量检测与评价方法的研究Ⅲ:FDA-PI双荧光复染法检测马氏珠母贝精子的存活率

将马氏珠母贝新鲜精液和精子水浴致死精液配制含有不同精子死亡率(0%、20%、40%、60%、80%)的5浓度梯度检测样品,并作为理论精子死亡率,采用二乙酰荧光素(Fluorescein diacetate,FDA)-碘化丙啶(Propidium iodide,PI)双荧光复染法检测精子存活率和质膜完整性。结果表明,FDA和PI能有效区分活精子和死精子,活精子发出绿色荧光,死精子发出红色荧光。各样品实测精子死亡率和理论死亡率之间呈极显著正相关,相关系数r=0.99(p<0.01)。FDA-PI双荧光复染法检测马氏珠母贝精子存活率,灵敏度高,重复性好,准确有效。

Sperms Viability Rate and Membrane Integrity of Pinctada martensii with a Combination of FDA and PI

The sperm viability rate and sperms membrane integrity were studied by combining Fluorescein diacetate(FDA) with Propidium iodide(PI) under fluorescence microscope.The sperms were stained with FDA and PI,which showed clear difference between the dead and live sperms: the live sperms were green and the dead ones were red under the fluorescent microscopy.Then,these sperms were killed at 60 ℃(water bath) were mixed with fresh ones so as to obtain six rations(for dead sperms) concentrations(0%,20%,40%,60%,80%).A correlation did exist between theoretical and practical mortality of sperms(P<0.01)..In conclusion,the dual association of FDA-PI staining possesses high sensitivity,good repeatability and accuracy.