两种冷冻方法对牛卵巢组织窦前卵泡活性的影响

Influence of 2 freezing methods on preantral follicular viability of bovine ovarian tissue

目的:探讨两种冷冻方法对牛卵巢组织窦前卵泡活性的影响。方法:取新鲜的牛卵巢组织随机分为新鲜对照组、程序化冷冻组和玻璃化冷冻组,分别加入Liberase DH酶消化,然后挑取窦前卵泡并分类计数,用calcein AM以及ethidium homodimer对窦前卵泡进行荧光染色,鉴定窦前卵泡的活性。结果:3组各级卵泡的数量及分布差异无统计学意义(P=0.765);与新鲜对照组(91%)相比,程序化冷冻组和玻璃化冷冻组窦前卵泡中未受损卵泡所占比例(80%和78%)略有降低(P=0.012)。3组中所占比例占优势的始基卵泡中未受损卵泡率差异无统计学意义(P=0.079);与新鲜对照组相比,两冷冻组初级卵泡中未受损卵泡率有所降低(P<0.05),但两冷冻组间差异无统计学意义(P>0.05)。结论:程序化冷冻法和玻璃化冷冻法均能较好地保存卵巢组织窦前卵泡的活性。

Aim ： To investigate the influence of 2 freezing methods on the preantral follicular viability of bovine ovari- an tissue. Methods： Pieces of fresh bovine ovarian tissue were randomly allocated into fresh control group, program freez- ing group and vitrification freezing group ; ovarian tissue was digested by Liberase DH, then randomly picked preantral folli- cles and counted. Follicular viability was assessed by double fluorescent labelling with calcein AM and ethidium homodimer staining. Results： There were no significant differences in the number or distribution of preantral follicles among the 3 groups （P = 0. 765 ）. Compared with the fresh control group （91% ）, the percentage of undamaged follicles in the preantral follicles was slightly lower in the program freezing group（ 80% ） and vitrification freezing group（78% ） （ P = 0. 012）. There were no significant differences in the rate of undamaged follicles in primordial follicles which were superior in the number （ P = 0. 079 ） ; the rate of undamaged follicles in primary follicles in the 2 freezing groups was slightly lower than that in the fresh control group（ P 〈 0.05）, but the differences between the 2 freezing groups were not significant（P 〉 0. 05 ） . Conclu- sion ： The 2 freezing methods both can better maintain the viability of preantral follicles.