牦牛卵母细胞体外成熟培养及孤雌发育研究

doi:10.13304/j.nykjdb.2022.0244

摘要/Abstract

摘要：

牦牛体外胚胎培养作为辅助生殖策略，能够有效提高其繁殖能力。为研究牦牛卵母细胞体外培养过程中不同的处理方式对孤雌激活胚胎发育质量的影响，10月份采集5~8岁健康母牦牛卵巢，抽吸法收集卵母细胞进行体外成熟诱导，设置卵丘-卵母细胞复合体（cumulus-oocyte complexs，COCs）和裸卵2个试验组；TCM199培养时间分别为24、36 h；以及体外培养12 h的COCs，消化之后再移入TCM199培养12 h）分别进行孤雌激活，在体外进行孤雌胚胎培养，统计卵裂率和囊胚率。结果显示，牦牛COCs的孤雌激活胚胎发育率显著低于裸卵（P<0.05），且后期出现脱卵丘细胞现象；COCs在TCM199中培养24 h的孤雌胚胎卵裂率明显高于培养36 h的处理组（P<0.05）；裸卵再培养之后卵母细胞成熟率低、进行孤雌激活的卵裂率极低，且大部分发生贴壁。以上结果表明，体外培养24 h的COCs经透明质酸酶消化为裸卵的孤雌激活效果最佳，其卵裂率最高，囊胚率最高。研究结果为揭示牦牛卵母细胞孤雌激活前的不同处理方式与孤雌激活胚胎的发育质量提供了依据，可为优化卵母细胞体外培养、提高孤雌激活效果提供参考。

关键词: 牦牛, 卵母细胞, 孤雌激活, 胚胎发育

Abstract:

As an assisted reproduction strategy， yak embryo culture in vitro can effectively improve the reproductive ability. In order to study the effects of different treatment methods on the developmental quality of parthenogenetically activated embryos of yak oocytes in vitro， ovaries of 5~8 years old healthy female yaks were collected in October and oocytes were collected by aspiration for in vitro maturation induction. Different experimental groups （cumulus-oocyte complexs（COCs）and bare eggs； TCM199 was cultured for 24 and 36 h， respectively； and COCs cultured for 24 h， digested and then transferred to TCM199 for 12 h） were set for parthenogenetic activation， and parthenogenetic embryo culture in vitro， and the cleavage rate and blastocyst rate were calculated. The results showed that the development rate of parthenogenetic activated embryos of yak COCs was significantly lower than naked eggs （P<0.05）， and the phenomenon of cumulus shedding appeared in the late stage. Cleavage rate of parthenogenetic embryos cultured in TCM199 for 24 h was significantly higher than that cultured for 36 h （P<0.05）. The cleavage rate of parthenogenetic activated eggs after reculture was very low and most of them were adherent. The results showed that the parthenogenetic activation effect of naked eggs was the best when COCs was digested by hyaluronidase in vitro for 24 h， and the cleavage rate and blastocyst rate were the highest. The results of this experiment provided a basis for revealing the different treatment methods of yak oocytes before parthenogenetic activation and the development quality of parthenogenetic activated embryos， and thus provided a reference for optimizing oocyte culture in vitro and improving the effect of parthenogenetic activation.

Key words: yak, oocyte, parthenogenetic activation, embryonic development

中图分类号:

S823.8⁺5.2

王立斌, 王强龙, 潘阳阳, 赵天, 丁天翊, 崔燕, 余四九. 牦牛卵母细胞体外成熟培养及孤雌发育研究[J]. 中国农业科技导报, 2023, 25(10): 84-90.

Libin WANG, Qianglong WANG, Yangyang PAN, Tian ZHAO, Tianyi DING, Yan CUI, Sijiu YU. In Vitro Maturation Culture and Parthenogenesis of Yak Oocytes[J]. Journal of Agricultural Science and Technology, 2023, 25(10): 84-90.

图/表 6

图1 牦牛卵母细胞A：收集的不同级别卵母细胞；B：优质卵母细胞；C：培养成熟的卵母细胞

Fig. 1 Yak oocytesA： Oocytes of different grades collected； B： Good quality oocyte； C： Cultured mature oocytes

图2 透明质酸酶消化后的卵母细胞A：透明质酸酶消化后的卵母细胞；B：含第一极体的优质卵母细胞

Fig. 2 Oocytes digested by hyaluronidaseA： Oocytes digested by hyaluronidase； B： Good quality oocytes containing the first polar body

图3 孤雌激活之后的卵母细胞不同发育阶段A：含第一极体的卵母细胞（孤雌激活后）；B：2-细胞胚胎；C：4-细胞胚胎；D：8~16细胞胚胎；E：桑椹胚；F：囊胚

Fig. 3 Different developmental stages of oocytes after parthenogenetic activationA： Oocytes containing the first polar body （after parthenogenetic activation）； B： 2-cell embryo； C： 4-cell embryo； D：8~16 cell embryo； E： Morula； F： Blastocysts

表1 COCs和裸卵的孤雌激活效果

Table 1 Parthenogenetic activation effects of COCs and nude eggs

囊胚数

No. of blastocysts

囊胚率

Blastocyst rate/%

表 2 不同培养时间下牦牛卵母细胞的成熟和孤雌激活胚胎发育

Table 2 Maturation and parthenogenetic development of yak oocytes under different culture time

培养时间

Incubation time/h

培养卵子数

No. of oocytes

成熟卵子数

No. of mature oocytes

成熟率

Mature rate/％

卵裂数

No. of cleavage

卵裂率

Cleavage rate/%

囊胚数

No. of blastocysts

囊胚率

Blastocyst rate/%

表3 裸卵再培养对孤雌激活效果的影响

Table 3 Effects of reculture of nude oocytes on parthenogenetic activation

培养卵子数

No. of oocytes

贴壁个数

No. of attached

成熟个数

No. of mature

卵裂数

No. of cleavage

卵裂率

Cleavage rate/%

囊胚数

No. of blastocysts

囊胚率

Blastocyst rate/%

66.67

42.86

60.00

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