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

The Combination of Ethylene Glycol and Propylene Glycol for Vitrification of Porcine GV Oocytes

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,预平衡方式Ⅰ组卵母细胞解冻后2 h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 （EMIl）, 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 EMIl 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℃.