PDF《黑鲷精子的超低温冻存及 DNA 损伤的 SCGE 检测》

收稿日期:2008-10-06;

接受日期:2009-01-22 基金项目:宁波市科学技术局项目(2006C100044;

2007A31004) ;

教育部创新团队项目(IRTO734) * 通讯作者(Corresponding author) ,E-mail: zhujunquan@nbu.

edu.cn 第一作者简介:叶霆(1984-), 男, 硕士研究生, 从事水产动物遗传育种研究. E-mail: [email protected] 动物学研究2009, Apr. 30(2): 151?157 CN 53-1040/Q ISSN 0254-5853 Zoological Research DOI:10.3724/SP.J.1141.2009.02151 黑鲷精子的超低温冻存及 DNA 损伤的 SCGE 检测 叶霆1 ,竺俊全1, * ,杨万喜2 ,魏平1 ,吴雄飞3 (1. 宁波大学 教育部应用海洋生物技术重点实验室,浙江 宁波 315211;

2. 浙江大学 生命科学学院,浙江 杭州 310012;

3. 宁波市海洋与渔业研究院,浙江 宁波 315012) 摘要:以0.5mL 的麦细管为冻存管和 DMSO 为抗冻剂进行超低温冷冻黑鲷精子,对冻精核 DNA 的损伤情况 进行单细胞凝胶电泳(SCGE)检测,其结果表明,以Cortland 溶液为稀释液,5%、10%、15%及20%DMSO 为 抗冻剂的超低温冻存的黑鲷精子活力、受精率与鲜精无显著差异.其中以 10%DMSO 为抗冻剂的冻存效果最佳, 冻精的激活率、 运动时间、 寿命及受精率分别达 (92.91±1.25) %、 (39.90±2.70) min、 (53.82±2.84) min 及(89.35±1.99) %;

而以 25%及30%DMSO 为抗冻剂时,冻精活力及受精率显著下降.SCGE 检测结果显示,DMSO 浓度为 5%、 10%、15%及20%时,黑鲷冻精与鲜精的彗星率及损伤系数差异不显著;

DMSO 浓度为 25%及30%时,冻精与鲜 精的彗星率及损伤系数差异显著;

冻精的彗星率与抗冻剂 DMSO 浓度成正相关.黑鲷鲜精及冻精核的 DNA 损伤 主要为轻度和中度损伤, 重度损伤比例较低, 完全损伤仅存在于 25%及30%DMSO 为抗冻剂的冻精中, 且比例低. 分析认为,较高浓度的 DMSO 是引起冻精核 DNA 损伤的主要原因. 关键词:黑鲷;

精子;

超低温冻存;

精子活力;

受精率;

DNA 损伤;

单细胞凝胶电泳 中图分类号:Q959.483;

Q492 文献标识码:A 文章编号:0254-5853-(2009)02-0151-07 Sperm Cryopreservation in Sparus macrocephalus and DNA Damage Detection with SCGE YE Ting1 , ZHU Jun-quan1, * , YANG Wan-xi2 , WEI Pin1 , WU Xiong-fei3 (1. Key Laboratory of Applied Marine Biotechnology by the Ministry of Education, Ningbo University, Ningbo 315211, China;

2. College of Life Science, Zhejiang University, Hangzhou 310012, China;

3. Ningbo Academy of Oceanology and Fisheries, Ningbo 315012, China) Abstract: In this paper, DMSO was used as cryoprotectant for cryopreservation of Sparus macrocephalus spermatozoa in 0.5 mL straws. Detection of DNA damage in response to a cryopreservation process in Sparus macrocephalus spermatozoa was also carried out. The results demonstrated that there were no significant differences between frozen-thawed sperm conserved by Cortland solution diluted with 5%, 10%, 15%, 20% DMSO and fresh sperm in motility. The best motility of frozen-thawed sperm were obtained when DMSO concentration was 10%, and the activation rate, moving time, living time and fertilization rate of frozen-thawed sperm were 92.91±1.25%, 39.90±2.70min, 53.82±2.84min and 89.35±1.99% respectively. However, a significant drop in sperm motility and fertilization rate was observed in sperm cryopreserved with 25% and 30% DMSO. The comet rate and damage coefficient of frozen-thawed sperm conserved with 5%, 10%, 15%, 20% DMSO was similar to fresh sperm, but at 25% and 30% were significantly differed to fresh sperm. In fact, there was a positive correlation between comet rate of frozen-thawed sperm and concentration of DMSO in protocol. The majority of sperm with DNA damage within the nucleus were slightly and mildly damaged, while minorities were heavily damaged. Few were totally damaged, and only occurred under the conditions of 25% and 30% DMSO. Our analysis suggests that high concentration of DMSO is the main factor that causes the DNA damage in frozen-thawed sperm nucleus. Key words: Sparus macrocephalus;