单细胞凝胶电泳检测南美白对虾冷冻精子的 DNA 损伤

摘要

The DNA damage of cryopreservation spermatozoa in Litopenaeus vannamei was detected to as-sess the effect of cryopreservation,using a single cell gel electrophoresis assay (SCGE)method.The sperm were pretreated,electrophoresed in the alkaline electrophoresis solution,dyed with GelRed for 10 min,ob-served under fluorescence microscope,counted the proportion of nuclei with tails in about 50 nuclei at ran-dom,and then calculated the comet rate.Various parameters of DNA migration were analyzed and meas-ured by CASP analysis software,and the sperm nuclei were graded according to the length of comet tail and the fluorescence intensity.Compared with the DNA damage states of Litopenaeus vannamei frozen-thawed sperm and the fresh sperm,the result indicated that when the concentration of DMSO went up,the tail DNA,tail length,tail moment and Olive tail moment increased,and the comet rate and damage coeffi-cient increased too.The fresh sperm were basically no damage,the groups of 50 mL/L DMSO were slight-ly damaged,the groups of 100 mL/L and 150 mL/L DMSO were mainly slightly and midly damaged,the groups of 250 mL/L DMSO were more midly and heavily damaged,but totally damage was few in the sperm for this study.Our analysis suggests that the concentration of DMSO is the chief factor that causes the DNA damage in cryopreserved sperm nuclei.%应用单细胞凝胶电泳（SCGE）技术检测南美白对虾冷冻精子的 DNA 损伤状况，以评价南美白对虾精子超低温冷冻保存的效果。对南美白对虾精子细胞进行预处理，在碱性电泳液中使核 DNA 双链解链变性后电泳，GelRed 染色液染色10 min 后，在荧光显微镜下随机观察50个左右的核 DNA，CASP 分析软件分析测量 DNA 迁移的各种参数，并按彗尾长度及荧光强度对 DNA 损伤划分等级。鲜精和各冻精组的SCGE 检测结果显示，随抗冻剂二甲基亚砜（DMSO）浓度的升高，彗星尾部 DNA 含量、尾长、尾距、Olive 尾距增大，彗星率及损伤系数也逐渐增大；鲜精基本无损伤，50 mL/L DMSO 为抗冻剂组基本为轻度损伤，100 mL/L 和150 mL/L DMSO 为抗冻剂组以轻度损伤和中度损伤为主，250 mL/L DMSO 组中度损伤和重度损伤的比例进一步增大，但完全损伤没有出现。分析认为，抗冻剂 DMSO 的浓度是影响冻后精子 DNA 损伤的主要因素。