【作者】 唐红星；

【导师】 薛立群；

【作者基本信息】 湖南农业大学 ， 基础兽医学， 2001， 硕士

【摘要】 本研究利用屠宰场采集的母猪卵巢，用机械法从卵巢表面直径为3～6mm的卵泡中分离紧密包裹三层以上卵丘细胞的卵丘—卵母细胞复合体（COC），用于猪卵巢卵母钼胞体外成熟、体外受精及早期胚胎（体外受精卵）体外培养的研究。主要结果如下： 1．卵母细胞的体外成熟 采用微滴培养，卵丘—卵母细胞复合体体外培养42～44h后（38.8℃，5％CaO₂，100％相对湿度），卵丘细胞扩展，部分卵母细胞排出第一极体。mTCM199+15％NCS（v／v）和NCSU23+10％PFF（v／v）为基础培养液用于卵母细胞体外成熟培养，在添加15iu／ml的PMSG和HCG时，卵母细胞体外培养成熟率分别为41.2±3.98％和38.28±4.02％；在添加20iu／mlPMSG和HCG时，卵母细胞体外培养成熟率为44.8±5.34％和39.12±1.25％，在添加相同浓度的激素时mTCM199+5％NCS（v／v）和NCSU23+10％PFF（v／v）对卵母细胞成熟率的影响差异不显著（P>0.05）。mTCM199+15iu／ml PMSG—15iu／ml HCG添加浓度为0，10％，15％，20％的血清（NCS）体外培养卵母细胞，卵母细胞成熟率分别为0，37.78±1.92％，43.33±2.72％，43.33±3.85％，添加组与不添加组之间差异极显著（P<0.01）：添加10％与20％组之间差异显著（P<0.05）；添加15％与20％组之间差异不显著（P>0.05）；添加10％与15％组之间差异显著（P<0.05）。在mTCM199+15％NCS（v／v）中共同添加0iu／ml、10iu／ml、15iu／ml、20iu／ml的相同剂量PMSG和HCG进行体外培养时，卵母细胞的成熟率分别为0，35.56±1.92％，41.2±3.98％，46.27±7.36％，添加组与未添加组之间差异极显著（P<0.01），添加15iu／ml组与20iu／ml组之间的差异不显著（P>0.05），添加15iu／ml组与添加10iu／ml组之间差异不显著（P>0.05），添加20iu／ml组与添加10iu／ml组之间差异显著（P<0.05）。添加一定浓度的血清、激素有利于提高卵母细胞体外成熟率。 2．体外受精 将鲜精（或常温保存精液）在mTBM+100ug／ml肝素中运用“上游法”获能40～60min，然后将获能看的精子与成熟卵母细胞在mTBM-50 u g／。1肝素中共孵育6h （3．4aC，5％CO。，100％相对湿度）。受精后 24～28h有部 分受精卵排出第二极体，部分受精卵发生卵裂，总卵裂率为盯．9弧（“～50h观 察统计）。 3．早期胚胎的体外培养 体外受精后，三种培养体系被用于受精卵 的体外培养：1．TCM199＋15％NCS，11．输卵管上皮细胞＋TCM199＋15％NCS共培养系 统，fll．颗粒／卵丘细胞＋TCM199＋15％＼CS共培养系统，培养条件为38．S”C， 5 ％ CO。，100％相对湿度。三种培养体系中早期胚胎发育率分别为 38．49士6．59％；37．19IS．19％，37．85土4．25％（48～50h观察统计），三种培养体系 对早期胚胎发育率的影响差异不显著（P＞0．05）。在1中，胚胎发育到4细胞后便 不再向前发育，在＊和＊中，部分胚胎能越过 4细胞期发育到 8～16细胞期及至 桑檀胚期。桑堪胚发育率分别为（，12．84土6．04％，4．76土6．73％），11组与1组 间差异极显著中＜O．N)，H组与＊I组间差异显著沪＜O．“)，HI组与I组间无显 著差异（P＞0．05）． 总之，在本实验条件下，猪卵巢卵母细胞在体外条件下能够发育成熟，成熟 卵母细胞与体外获能的精子能进行体外受精发生卵裂，发育成胚胎；通过共培养 体系培养，早期胚胎能突破4细胞发育阻滞，发育到桑堪胚。更多还原

【Abstract】 Porcine ovaries were obtained from Changsha Wulipai abattoir.Cumulus-oocyte complexes (COC) with more than three layer’ s compact cumulu cells were mechanically isolate& from 3~-6 mm sized ovarian surface follicles. The COC were cultured in vitro to study the porcine ovarian oocyte’ s in vitro maturation (IVM), in vitro fertilization (lyE) and in vitro culture (IVC) of the early embryos. The results were presented as follows:1.Oocyte’ s in vitro maturation In the experiment, COCs were cultured in 300 microliters droplets (38. 8~C, 5%C02, 100% humidity). After cultured 42-44h, the cumulu cells expansion, some oocytes extrusion the first polar body (PBI). mTCM199-’-lO%NCS(v/v) and NCSU23-4-l0%PFF(v/v) were used to study their effects on oocyte’ s IVM. The results show that, when supplemented with l5iu/ml PMSG and HCG the maturation rate were 41.2±3.%98 and 38.28±4.02% (P>0.03); When supplemented with 2Oiu/ml PMSG and HCG the maturation rate were 44. 8±5. 34% and 39. 12±l.25%(P>0. 05). When supplemented 0, 10%, 13%, 20%(v/v) NCS(New-born cattle serum) into mTCM199-15iu1"ml PMSG--lSiu ml HCG, the maturation rates were 0,37.78±1.92%, 43. 33±2.72% 43 33±3.85%, maturation rates were remarkbabiy different (P<0.0l) between the groups with lO%-----20%(vlv) serum and the group without supplemented serum, the difference of the maturation rate among 10% and 20% or 15% and 10% were significant (P<0. 05), the difference of the maturation rate among 15% and 20% was not significant (P>0. 05) . When supplemented 0, lOiu/ml, lSiu/ml, 2Oiu/ml PMSG and L-JCG into mTCM199~-l5%NCS, the maturation rates were 0, 35.56±1.92%, 41.2±3.98%, 46.27±7 36% the maturation rates between 0 (group) and l0iui’mb- 20iu11m1 (groups) were remarklablv different (P<0. 01); between l5iu/ml and 2Oiu ml or lSiu,"’mi and lOiu/ml were not significantly differen? (p>O.OS> between 2Oiu"ml (group) and lOiu/ml (group) were3significant different(p<0. 05). The results showed that add serum~ PMSG and HCG can improve oocyte’ s maturation rate in vitro.2.In vitro fertilization Fresh sperm were capacitated in medium mTBM+100 ~ g/ml Heparin with the "swim-up" method. After capacitated 40- 60 mm. the sperm and the maturation oocytes were incubated in mTBM+50 ii g/ml Heparin (39. 40C, 5%C02, 100% humidity) for Gb. After 24’-28h some fertifized eggs extrusion the second polar body (PB II) and some fertilized eggs were clevaged. The clevage rate was 37. 95%(48-50h).3.Early embryo’ s in vitro culture After in vitro fertilization, the fertilized eggs were cultured in three systems. I .TCM199+l5%NCS, II. Porcine oviduct epithelial cell monolayer (POEC)+TCM199-r15%NCS, III. granule/cumulus cell monolayer(G/C>TCM199+l5%NCS. The culture condition was 38. 80C, 5%C02, 100% humidity. Their development rates were 38. 49±6. 59% 37 19±5.19%,37.85±4.25%(48 -~ 50h), there were no significant different (P>0. 05) between them. In group I, the embryos were blocked at 4 cell stage, in group II and Ill some embryos can surmount 4 cell stage block and developed to 8-~l6 cell stage and morula. The morula rates were 0 12 84± 6. 04%, 4. 76 ±6. 73%. The difference of the morula rates among group II and I was remarkably (P<O. 01), among group II and III were significant (P<0.0l), among III and I were not signicficant (P>0.05).In sumftiary, under the present culture conditions, the porcine ovarian oocvtes can matured in vitro, fertilized in vitro and developed to early embryos. When the early embryos co-cultured with monolayer porcine oviduct epithelial cells or granual/cumulus cells some of them can surmount the 4 cell stage block and developed to morula更多还原