玻璃化冷冻对猪MII期卵母细胞发育能力的影响

Effects of cryoprotectants and vitrification on in vitro development ability of porcine oocytes at MII stage

以MII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7组冷冻保护液配方进行筛选,并且比较3种不同冷冻载体冷冻卵母细胞的效果。结果发现:7组冷冻保护液对MII期卵母细胞有低毒性作用,各组分裂率、囊胚率、囊胚细胞数均低于对照组;综合各组指标,选取3组对猪MII期卵母细胞的发育能力损伤最小的冷冻保护剂,应用GMP管对其冷冻保护效果进行比较,发现第7组冷冻保护液配方EFS不适于MII期卵母细胞的冷冻,其形态正常率、分裂率分别为1.19%、0,与第3组、第6组形态正常率、分裂率(59.48%、53.55%;24.19%、35.02%)差异显著(P<0.05)。综合考虑,选取HM+7.5%(DMSO+EG),HM+17%(DMSO+EG)+0.4 mol/L Su作为冷冻保护剂来比较3种不同冷冻载体冷冻卵母细胞的效果差异,发现半麦管冻融后MII期卵母细胞的分裂率高达53.67%,与OPS法的29.33%及GMP法的35.00%相比差异显著(P<0.05),半麦管法更适合MII期猪卵母细胞的玻璃化冷冻。

Vitrification freezing methods were used to filtrate seven vitrification solutions and compare effects of three carriers on freezing oocytes by using MII oocytes in the present project. The results showed seven groups of cryoprotectants had low toxicity on MII oocytes. The rate of split and blastocyst, the number of btastocyst cells in each group was lower than those in the control group. Three groups of cryoprotectant with smallest damage to MII oocytes development ability was selected to compare vitrification by GMP. The results indicated that EFS was not suitable for MII oocytes vitrification, which the 1.19% normal rate and 0 cleavage rate was significant different （ P〈0. 05 ） from other two groups （59.48%, 53.55% ; 24. 19%, 35.02% ）. HM＋7.5% （ DMSO＋EG）, HM＋17% （DMSO＋EG） ＋0. 4 mol/L Su was selected as the cryoprotectant to compare the effect of three carriers. The data showed that hemi-straw （53.67%） was significant different （P〈0. 05 ） from OPS （29.33%） and GMP （35.00%） in cleavage rate. Together, the hemi-straw was more suitable for MII porcine oocytes vitrification.