乙二醇联合丙二醇用于猪GV期卵母细胞玻璃化冷冻保存的研究

摘要

本实验以猪GV期卵母细胞为模型,旨在研究乙二醇(EG)联合丙二醇(PROH)作为渗透性冷冻保护剂的冷冻保存方案.将卵母细胞在室温(25℃)和生理温度(39℃)2种冷冻操作条件下,采用2.5％EG+2.5％ PROH直接处理10 min(预平衡方式Ⅰ)或分步以3.75％ EG+ 3.75％ PROH、7.5％ EG+ 7.5％ PROH 2种溶液各处理2.5 min(预平衡方式Ⅱ)进行预平衡,然后Cryotop法玻璃化冷冻保存,解冻后分析其存活、体外成熟及孤雌发育能力.结果表明:在2种温度下,顸平衡方式Ⅰ组卵母细胞解冻后2h的存活率均显著高于预平衡方式Ⅱ组(P＜0.05),但存活卵母细胞成熟培养后,在各冷冻组间的存活率无明显差异(P＞0.05);冷冻组卵母细胞的核成熟也与新鲜卵母细胞无显著性差异(P＞0.05);在相同温度下,预平衡方式Ⅰ组的卵裂率和囊胚发育率均显著高于预平衡方式Ⅱ组(P＜0.05);当采用同一预平衡方式时,39℃的冷冻操作条件所获卵裂率和囊胚发育率显著低于25℃(P＜0.05);此外,各组间囊胚细胞数均无明显差异(P＞0.05).综上所述,EG联合PROH用于猪GV期卵母细胞的Cryotop法玻璃化冷冻保存时,25℃冷冻操作条件下,采用预平衡方式Ⅰ可获得较高的冷冻存活率及囊胚发育率.%This study was conducted to optimize cryopreservation protocols for vitrification of porcine GV oocytes using a combination of ethylene glycol (EG) and propylene glycol (PROH) as permeable cryoprotectants.Under the environmental temperature of 25℃ or 39℃,oocytes were pre-equilibrated with 2.5％ EG + 2.5％ PROH for 10 min (EMⅠ) or with 3.75％ EG + 3.75％ PROH and 7.5％ EG + 7.5％ PROH for 2.5 min respectively (EMⅡ),and then vitrified using Cryotop method.After warming,the oocytes were evaluated by their survival,nuclear maturation and parthenogenetic development.The results showed that at 2 h after warming,the cryosurvival rate of EMI groups was significantly higher (P＜0.05) than that of EMⅡ groups,regardless the environmental temperatures.After in vitro maturation,the percentages of survival and nuclear maturation were similar in all groups.Both cleavage and blastocyst rates were higher (P＜0.05) in EMⅠ groups than in EMⅡ groups in each temperature.When the same pre-equilibration manner was applied,both cleavage and blastocyst rates at 39℃ was significantly lower than that at 25℃ (P＜0.05).Additionally,total cell numbers of blastocysts were not significantly different among all groups.In conclusion,a higher proportion of cryosurvival and blastocyst development can be obtained from Cryotop vitrified porcine GV oocytes pre-equilibrated using 2.5％ EG + 2.5％ PROH for 10 min under 25℃.