卵泡大小和颗粒细胞对山羊卵泡卵母细胞体外成熟和体外受精的影响

Effects of follicles diameter and granulosa cells on in vitro maturation and in vitro fertilization of goat follicular oocytes

用切割法采集卵泡液,收集卵丘-卵母细胞复合体(Cumulus oocytes comlexs,COCs)和自然裸卵,将部分COCs去除卵丘细胞获得机械裸卵,COCs放入体外成熟培养液中培养为成熟卵母细胞,加入获能的精子液,进行体外受精。结果表明:卵母细胞的体外成熟率和卵裂率与卵泡直径密切相关,大卵泡(80.95%,P<0.01)和中等卵泡(75.50%,P<0.05)的卵母细胞成熟率高于小卵泡(50.27%);大卵泡(53.53%)和中等卵泡(47.13%)的卵裂率显著高于小卵泡的32.26%(P<0.05)。COCs、机械裸卵和自然裸卵的体外成熟率分别为75.0%、54.2%和10.5%,差异极显著(P<0.01),卵裂率分别为53.8%、10.8%和0%,差异极显著(P<0.01)。对照组和1×105、1×106个/mL颗粒细胞组卵母细胞体外成熟率分别为68.6%、69.6%和67.8%,无显著差异(P>0.05),但均显著高于1×107个/mL(51.5%,P<0.05)和1×1010个/mL(35.5%,P<0.05)颗粒细胞组,但各组间的体外受精率无显著差异(P>0.05)。结果提示,大卵泡和中卵泡的卵母细胞的体外成熟率和卵裂率显著高于小卵泡,体外成熟培养液中添加高浓度的颗粒细胞能显著抑制卵母细胞的体外成熟。

Follicular fluid including cumulus oocytes comlexs （COCs） and natural naked oocytes was collected by the cutting method. Some COCs were turned into naked oocytes through mechanical removal of cumulus cells. COCs were cultivated into mature oocytes in the mature culture medium in vitro. IVF was carried out by adding the capaciration sperm. The results showed that the IVM rate and cleavage rate of the oocytes were closely related to the follic- ulair diameter,and the IVM rate of the big follicle（80.95% ,P〈0.01） and medium follicle（75.50%, P〈0. 05） was higher than that of the small follicle（50.27% ）, and the cleavage rate of the big follicle（53.53 % ） and medium follicle（47.13 %） was higher than that of the small follicle （32.26 % ,P〈0.05）. The IVM rates of COCs（75.0 %） and denuded oocytes（54.2 %） was significantly higher than those of naked oocytes （10.5 %, P〈0.01 ）, the cleavage rates of COCs（53.8%） was significantly higher than that of denuded oocytes（10.8 %） and naked oocytes（0% ,P〈 0.01 ）. The IVM rates of oocytes cultured in control group,1 ×10^5 cell/mL and 1 ×10^6 eell/mL granulosa ceils were 68.6% .69.6% and 67.8% ,respectively. And the IVM rates of those were significantly higher than 1 ×10^7 cell/mL （51.5 % ,P〈0.05） and 1 ×10^10 cell/mL （35.5 % ,P〈0. 05）granulosa cells groups. But there was no significant difference in cleavage rate of oocytes among all groups. The restilts suggestted that IVM rate and cleavage rate of large follicles and medium follicles were significantly higher than those of small follicles,maturation of oocytes was significantly inhibited by adding high concentrations of granulosa cells to the maturation medium in vitro.