玻璃化冷冻对猪GV期卵母细胞及其DNA的影响  

Effect of vitrification on oocytes and its DNA at GV stage of porcine

作　　者：王勇杰 周悦[1] 季珂萌 张润祥 白熙 唐小川 石德顺[1] 王晓丽[1] WANG Yong-jie;ZHOU Yue;JI Ke-meng;ZHANG Run-xiang;BAI Xi;TANG Xiao-chuan;SHI De-shun;WANG Xiao-li(College of Animal Science and Technology,Guangxi Uuniversity,Nanning 530005,China)

机构地区：[1]广西大学动物科学技术学院

出　　处：《中国兽医科学》2019年第8期1048-1053,共6页Chinese Veterinary Science

基　　金：国家自然科学基金项目(31101777,31460644);广西自然科学基金项目(2016JJA130282,2018JJA130056)

摘　　要：为了探索对猪GV期卵母细胞低毒性以及冷冻效果好的最佳冷冻保护液,并对玻璃化冷冻卵母细胞及其DNA损伤进行评价,本研究以猪GV期卵母细胞作为试验材料,应用玻璃化冷冻方法,就目前应用最多的六种冷冻保护液进行筛选,筛选出最合适的冷冻保护液;透射电镜观察冷冻卵母细胞超微结构的变化;采用彗星电泳技术检测玻璃化冷冻对卵母细胞DNA的影响。结果发现,以二甲基亚砜DMSO和乙二醇EG为主要成分的(HM+7.5%DMSO+7.5%EG)、HM+17%DMSO+17%EG+0.4 mol/L Su)冷冻保护液对猪GV期卵母细胞的损伤最小;玻璃化冷冻后卵母细胞透明带、细胞膜损伤,微绒毛消失,线粒体肿胀、基质不均、嵴不明显,胞质内溶解为絮状,有大量空泡;冷冻保护液处理组(未进行玻璃化冷冻)卵母细胞DNA损伤与对照组差异不显著,玻璃化冷冻组卵母细胞DNA损伤与冷冻保护液处理组和对照组均差异显著。结果表明,以DMSO和EG为主要成分的冷冻保护液适合用于对猪GV期卵母细胞玻璃化冷冻,同时也证实了DNA的损伤主要是由玻璃化冷冻过程的机械损伤所致。In order to explore the best cryoprotection solution with low toxicity and good freezing effect on GV oocytes of porcine,and evaluate DNA damage of vitrified frozen oocytes,the GV stage oocytes of porcine were used as experimental materials,and the most suitable cryoprotection solution was selected among the six most commonly used cryoprotection solution by vitrification.Ultrastructural Changes of frozen oocytes were observed by transmission electron microscope.The DNA damage of oocytes by vitrification was detected by comet electrophoresis.The results showed that(HM+7.5%DMSO+7.5%EG) and HM+17%DMSO+17%EG+0.4 mol/L Su) with DMSO and EG as the main components had the least damage to oocytes at GV stage.After vitrification,the zona pellucida and cell membrane of oocytes were damaged,microvilli disappeared,mitochondria swelled,matrix was uneven,cristae were not obvious,intracy to-plasmic dissolution was flocculent,and there were a lot of vacuoles.There was no significant difference in DNA damage of oocytes in GV stage between the treatment group(no vitrification) and the control group,while DNA damage of oocytes in vitrification group was significantly different from that in treatment group and control group.The results indicated that cryoprotection solution with DMSO and EG as the main components were suitable for freezing GV stage oocytes in porcine.It was also confirmed that DNA damage was mainly caused by mechanical damage in freezing process.

关 键 词：玻璃化冷冻 卵母细胞 DNA损伤 

分 类 号：S852.16[农业科学—基础兽医学]