【作者】 李亚东；

【导师】 黄永宏；

【作者基本信息】 扬州大学 ， 敏殖学， 2001， 硕士

【摘要】 本文系统研究了颗粒细胞、卵泡液对山羊卵泡卵母细胞体外成熟和体外受精的影响，主要结果如下： 将卵丘-卵母细胞复合体（COC_s）、机械裸卵（DO）、自然裸卵（NO）在M199体外成熟液中培养24h后，体外成熟率分别为67.68％、45.3％、7.3％，各组间差异显著（p＜0.05）；体外受精24h后，卵裂率分别为14.1％、2.68％、0，各组间差异显著（p＜0.05）。 将部分体外成熟的COC_s进行剥离卵丘细胞处理（试验组）、对照组不进行剥离处理，然后体外受精，试验组和对照组卵裂率分别为14.32％、14.1％，差异不显著（P＞0.05）。 在体外成熟液中分别添加浓度为0.15×10~6个/ml（Ⅰ组）、1.5×10~6个/ml（Ⅱ组）、15×l0~6个/ml（Ⅲ组）、150×10~6个/ml（Ⅳ）的卵丘颗粒细胞和卵泡颗粒细胞悬液，对照组为不添加颗粒细胞的M199液，卵母细胞经体外成熟培养后，对于添加卵丘细胞悬液组，Ⅰ、Ⅱ组的成熟率（60.0％、64.37％）与对照组（65.41％）差异不显著（P＞0.05）；Ⅲ组（50.15％）、Ⅳ（30.4％）与对照组差异显著（p＜0.05）；成熟后体外受精，卵裂率分别为14.2％、14.6％、12.47％、12.56％，对照组为13.4％，各组间差异不显著（P＞0.05）。添加卵泡颗粒细胞结果与卵丘颗粒细胞相类似。 COC_s分别在卵丘颗粒细胞单层、卵丘颗粒细胞条件液和M199培养液（对照组）中培养，体外成熟率分别为66.10％、44.2％和66.28％，条件液组成熟率显著低于与对照组（p＜0.05），单层组与对照组差异不显著（P＞0.05）；体外受精后卵裂率分别为12.7％、13.2％和14.8％，各组间差异不显著（P＞0.05）。 将COC_s放于山羊卵泡液中培养24h后，体外成熟率为8.3％，与对照组（66.42％）相比，差异显著（p＜0.05）；用M199培养液代替GFF，继续 扬州大学硕士学位论文 培养 24h后，COCs体外成熟率为 62．34％，虽较对照组（68．5％）低，但差 异不显著（P＞0．05）；体外受精后卵裂率为14．5％，与对照组门2．49％）相 比，差异不显著（P＞0．05）。 分别用取自于大卵泡（5－6rum）、中等卵泡（35nun）、小卵泡（2－ 3turn）的不同性周期（卵泡期、早期黄体期、功能黄体期、退化黄体期） 的卵泡液培养 COCS，结果表明：对于同一大小的卵泡，卵泡期u）的卵 泡液对 COCS的成熟抑制作用显著…J．05）低于其它各期，早期黄体期 （11）、退化黄体期（IV）的卵泡液对COCs的抑制作用要低于功能黄体期 （m），且差异显著（p＜0．05），但n 组与 W组之间差异不显著 （P＞0．05）；在卵泡期，来源于大、中、小卵泡的卵泡液对 COCS的成熟 抑制作用依次增强，且组间差异显著（P＜0．05），其它同一性周期内，不 同大小卵泡的卵泡液对卵母细胞的体外成熟抑制作用差异不显著 （P＞0．05）。 在体外成熟培养液中添加浓度为 0、5％、l肌、15％的卵泡液，体外成熟 培养24h后，成熟率分别为66．4％、64．7％、64．3％、60．6％，各组间差异不 显著（P＞0．05）；体外受精后，卵裂率为13．57％、14．0％、13．弧、 13．48％，各组间差异不显著（P＞0．05）。更多还原

【Abstract】 The present study was conducted to investigate the effects of granulosa cells\follicular fluid on in vitro maturation and in vitro fertilization of goat follicular oocytes.The main results are as follows:The IVM rates of cumulus-oocyte-complexes (COC5) .. denuded oocytes (Do) and naked oocytes (No) were 67.68%.. 45.3%.. 7.3%, respectively. There was no significant difference among all groups. After the matured oocytes were inseminated in vitro , The 2-cell cleavage rates were 14.1%.. 2.68%.. 0. respectively. The 2-cell cleavage rate of COC5 was significant higher than that of Do and No.The cleavage rates of maturated oocytes with expanded cumulus cells or with complete cumulus denudation were 14.1% and 14.32%, There was no significant difference between two groups.After cultured for 24h in maturation medium with 0.1 Sx I O6viable cells/mi(I).. 1.5x 106 viable cells/ml(ll) .. lSxlO6viable cells/ml(Lll).. 150X 106 viable cells/ml(JV)of cumulus granulosa cells, the maturation rates of COC5 were 60.0%.. 64.37% 50.15% .. 30.4%,respectively.Group TILT and IV were significantly lower than control group (65.4 l%).After IVM, all maturated oocytes from each group were inseminated ,the 2-cell cleavage were 14.2%.. 14.6%.. 12.47%.. 12.56%.. 13.4%(control group) ,respectively..There was no significant difference among all groups. The effect of follicle granulosa cells on IVM and IVF had also been examed and we had the same results as cumulus granulosa cells. There was no significant difference between them.After cultured in monolayer medium of cumulus granulose cells or in inconditioned medium of cumulus granulosa cells for 24h. the maturation rates of oocytes in monolayer medium was significantly higher than that of conditioned3medium(66. 10% vs 44.2%).There was no significant difference between the monolayer group and the control group(66.10% v~ 66.28%).The effect of goat follicle fluid (GFF) on the DIM and IVF of COC5 were examed. Liberated COC5 were cultured either alone in GFF or in M199. After 24b~the maturation rate of oocytes cultured in GFF was significantly lower than those cultured in M199.When the COC5 were transferred to medium without GFF and incubated for an additional 24h,62.34% of COC5 were matured and there was no significant difference in maturation rates between conditioned and control groups.After WF,2-cell cleavage rates were 14.5% and 12.490/o,respectively.there was no significant difference between two groups.GFF was collected at different stages of the estrous cycle from small follicle (2-3mm)~ medium follicle(3-Smxn) and large follicle (5-6mm),then all oocytes were incubated in each groups. After cultured for 24h,the FF from any follicle size at follicle phase had the least DIM inhibition activity. However to the same follicle size, FF from early luteal phase(group I) and late luteal phase(group IV) had significant lower DIM inhibition activity than luteal phase(group 111 ).There was no significant difference between group II and IV.Except at follicle phase,the DIM inhibition activity of (3FF at the same stage did not differ with different follicle size. At follicle phase ,the DIM inhibition activity of GFF significantly decline during the course of follicular development in small medium and large follicle.Added o%~ 5%.~. l0%.~ 15% (3FF to the maturation medium ,the maturation rates were 66.4% 64.7%~ 64.3%.. 60.6%,respectively after incubated for 24h,and there was no significant difference among all groups. After inseminated , 2-cell cleavage rates were 13.57% .. 14.0% 13.4%13 .48%,respectively. There was also no significant difference among all groups.更多还原