农业生物技术学报2011，Vol.19Issue(4)：725-733,9.DOI:10.3969/j.issn.1674-7968.2011.04.019
大黄鱼精子的超低温冻存及细胞结构损伤的检测
Sperm Cryopreservation and the Cytoarchitecture Damage Detection of Pseudosciaena crocea
摘要
Abstract
In order to investigate the effect of cryopreservation on spermatozoal structures in Pseudosciaena crocea, we used dimethyl sulfoxide (DMSO) or ethylene glycol (EG) as cryoprotectant, cortland solutions as extender, and two-step cooling procedure to cryopreserve P. Crocea sperm in 0.25 mL straws. And we detected the DNA damage of frozen sperm by single cell gel eletrophoresis (SCGE) and membranous damage by flow cytometry (FCM). The results showed that there were no significant differences between fresh sperm and frozen-thawed sperm in vitality when 5%~20% DMSO or 5%-20% EG were used. The activation rate, moving time and life-span of frozen-thawed sperm were (87.00±2.45) %, (8.99±0.24) min and (13.11 ±0.65) min at 10% DMSO, and (87.50±2.52) %, (8.45±0.48) min and (12.84±0.50) min at 10% EG. A significant drop in sperm vitality was observed at 25% DMSO or 30% EG. SCGE results showed that there were no significant differencesin DNA fragmentation between fresh sperm and frozen-thawed sperm diluted with 5%~20% DMSO or 5%~20% EG, but the DNA damage to frozen-thawed sperm significantly increased at 25% DMSO or 30% EG. There was a positive correlation between the DNA damage extent of frozen-thawed sperm and concentrations of DMSO and EG. FCM analysis suggested that the integrity of mitochondria and membranous structures of frozen-thawed sperm were similar to that of fresh sperm at 5%~20% DMSO or 5%~20% EG, but the proportion of frozen-thawed sperm maintained the integrity of mitochondria and membranous structures was significantly declined at 25% or 30% DMSO or EG. So we conclud that the weakening of sperm vitality and the enhanced damage of DNA, mitochondria and membranous structures in frozen-thawed sperm are mainly induced by the high concentration of cryoprotectant. 10% DMSO or 10% EG can be considered as the best cryoprotectant to ensure the quality of frozen sperm.关键词
大黄鱼/精子/超低温冻存/单细胞凝胶电泳(SCGE)/荧光双染色-流式细胞术(FCM)Key words
Pseudosciaena crocea/Sperm/Cryopreservation/Single cell gel eletrophoresis (SCGE)/Flow cytometry(FCM)引用本文复制引用
姜建湖,闫家强,竺俊全,杨万喜..大黄鱼精子的超低温冻存及细胞结构损伤的检测[J].农业生物技术学报,2011,19(4):725-733,9.基金项目
本研究由宁波市科技计划项目(No.2006C100044,No.2007A31004)、宁波大学人才工程项目(No.BSL2009004)和教育部创新团队项目(No.IRTO734)共同资助 （No.2006C100044,No.2007A31004）
