利用流式细胞仪评价不同防冻液对猪冻融精子成活率及凋亡状况的影响  

Effects of Different Protectants on Viability and Apoptosis of Boar Spermatozoa after Freezing-Thawing by FCM

作　　者：李新红[1] 高超[1] 陈翔宇[1] 赵雷[1] 

机构地区：[1]上海交通大学农业与生物学院,上海200240

出　　处：《上海交通大学学报（农业科学版）》2007年第5期468-473,共6页Journal of Shanghai Jiaotong University(Agricultural Science)

基　　金：上海市科委"登山计划"项目(No.064909004);上海交通大学PRP项目(T15211001)资助

摘　　要：人工采取5头优质大白猪精液,利用BTS稀释液和添加氨基—钠—十二烷基硫酸脂(OEP)、十二烷基硫酸钠(SDS)及卵黄所组成的防冻液稀释后,冷冻程控仪制成细管冻精,采用荧光显微镜及流式细胞仪对冷冻复苏后精子质膜完整率和凋亡状况进行测定,目的在于比较不同检测方法评估猪冻融精子活率的差异性,探讨OEP、SDS对猪冻融精子质膜完整率和凋亡状况的影响。结果表明,Annexin-V/PI染色流式细胞术能准确测出猪冻融精子质膜完整率及处于凋亡状态的数量,同Annexin-V/PI染色流式细胞术相比,利用SYBR-14/PI染色荧光显微镜法往往高估冻融精子的死亡率。各处理组间冻融精子质膜完整率及凋亡数量存在一定差别,添加OEP防冻剂的实验组早期凋亡精子的比例显著低于对照组(P<0.05),而正常活性精子的比例(46.5%)显著高于对照组(37.2%)(P<0.05),结果提示:OEP对维持冻融精子质膜完整性、抑制冻融精子凋亡起到较好作用。Semen of five high quality boars was chosen and diluted with protectant of BTS dilution and different treatments of Orvus ES Paste, SDS and egg-yolk. Then the semen samples were frozen in programmable freezer and preserved in straw, then thawed at 39℃. In order to compare the difference of different analytic methods, plasma membrane integrity and apoptosis of the frozen-thawed spermatozoa were analysed by fluorescence microscopy and flow cytometry （FCM）. The effects of OEP and SDS on plasma membrane integrity and apoptosis of the frozen-thawed spermatozoa were discussed also. Results showed： plasma membrane integrity of the frozen-thawed spermatozoa and the number of apoptotic spermatozoa could be detected with Annexin V-PI assay by FCM analyses exactly. Comparing with Annexin V-PI assay by FCM analyses, death rate of the frozen-thawed spermatozoa was evaluated higher when detected with SYBR-14/PI fluorescence microscopy analyses. The plasma membrane integrity of the frozenthawed spermatozoa and the number of apoptotic spermatozoa of each different treatment groups were different. In the group diluted with protectant of OEP, the rate of early apoptotic spermatozoa was lower than the control group distinctly （P〈0.05）, but the rate of normal viability was higher than the control group distinctly （P〈0.05）. The results proved that OEP play a better role on protecting the plasma membrane integrity and restraining the apoptosis of the frozen-thawed spermatozoa.

关 键 词：猪精子 成活率 凋亡 流式细胞仪 

分 类 号：S821.34[农业科学—畜牧学]