JC-1 single staining method was usually used to detect the sperm mitochondrial membrane potential (MMP) and it could distinguish the high-MMP and low-MMP sperm accuratly and rapidly, but it could not indentify the the sperm was alive or not. In this study, co-staining method using two fluorescent probs of JC-1 and propidium iodide (propidium iodide, PI) was developed and applied to the goat fresh and post-thaw semen. Flow cytometry and fluorescence microscopy were utilized to detect the function status of these sperms. The results showed that the head of dead sperm fluoresce was red, and the head of high-MMP sperm fluoresce was orange, whereas the tail of low MMP fluoresces was green. The compared results of two detection means were that the fluorescence microscopy could separate high-MMP and low-MMP effectively, but the detection level for the MMP of live sperm was less than ideal results. In contrast, flow cytometry was able to effectively distinguish the level of MMP of live sperm accuratly and rapidly and with higher sensitivity. The two method showed high corelation and the test results for the same treated sample showed a significant positive correlation (r=0.815,r=0.982,P<0.01). It indicated that using flow cytometry combined with fluorescent microscopy detection might reflect the functional status of sperm more effectively.%为准确、全面研究超低温冷冻技术对山羊附睾精子线粒体膜电位的影响效果,本试验采用荧光探针JC-1单标和JC-1/PI双标2种荧光染色方法,通过流式细胞仪和荧光显微镜两种检测手段对精子线粒体膜电位(Mitochondrial membrane potential,MMP)变化进行检测分析,用JC-1单独染色,能准确、快速地区分高、低MMP精子,但不能明确区分精子的存活状态,采用JC-1与碘化丙啶(Propidium iodide,PI)双标法,则能明显判定精子的存活状态.通过对绒山羊附睾精子在冷冻前和解冻后线粒体膜电位检测,结果表明,单标记后高MMP精子尾部呈橙红色,低MMP精子尾部为绿色；双标后死亡精子头部呈红色(PI+).对两种检测手段的比较研究,结果显示,荧光显微镜能够有效地区分出高、低MMP及死精子,但对于活精子中MMP的高低检测效果不理想；流式细胞仪则能够有效地区分活精子MMP的高低,而且结果准确,速度快,敏感性高.试验表明,两种检测方法相关性很高,分析同一处理样品时用JC+%和JC+/PI-%检测结果呈显著正相关(r=0.815,r=0.982,P＜0.01).因此,对于精子线粒体MMP的分析,采用JC-1单染与JC-1/PI双染的方法,利用流式细胞仪与荧光显微镜进行联合检测,能比较全面、准确的反映出精子线粒体的功能状态.
展开▼
