Rath D, Niemann H, Tao T
Institut für Tierzucht und Tierverhalten, Mariensee, (FAL), 31535 Neustadt, Germany.
Theriogenology. 1995 Sep;44(4):529-38. doi: 10.1016/0093-691x(95)00224-v.
The objective of the present study was to test the ability of porcine follicular fluid (pFF) to improve maturation of porcine cumulus-oocyte-complexes (COC) in vitro and to observe subsequent effects on fertilization and development to late morula/blastocyst stages under in vitro conditions. The COC were incubated in Tissue Culture Medium (TCM) 199, supplemented with 1% fetal calf serum (FCS), 10% pFF collected from immature follicles (2 to 5 mm), with or without addition of 1microg/ml FSH. Control groups were matured in TCM 199 with or without FSH. Follicular aspirates were centrifuged (1700 x g, 5min.) and the supematants were stored at -20 degrees in 1.5-ml Eppendorff cups until used. On 7 experimental days a total of 3849 immature COC was aspirated from follicles ranging from 2 to 5 mm in diameter. A total of 1117 COC was selected for the experiments, and 239 COC were fixed and stained with 1.5% aceto-orcein after 48 h of in vitro maturation at 39 degrees C with 5% CO(2) in humidified air. Germinal vesicle breakdown (GVBD; 91.7%) and development to metaphase II (60.4%) were superior (P <or= 0.05) when 10% pFF and 1microg/ml FSH were present in the maturation medium. Another 878 COC were mechanically denuded and used for in vitro fertilization (IVF). Eighteen hours after IVF, 561 oocytes were fixed and stained with aceto orcein. No diffeences were found among groups at this time point. Another 317 embryos were continuously cultured to morula and blastocyst stages in medium NCSU 23. Cleavage rates differed significantly (P <or= 0.05) 48 h after IVF, and were superior (52.5% vs 32.5% vs 30.7%) when oocytes were matured in maturation medium supplemented with pFF and FSH as compared with maturation rates in TCM 199 without pFF and/or FSH. Once the embryos had cleaved, no further differences were observed in the development to morula and blastocyst stages. These data indicate that GVBD and nuclear matuation to metaphase II are enhanced by a combination of pFF and FSH and that cytoplasmatic maturation is more complete and better synchronized as indicated by the increased cleavage rates.
本研究的目的是测试猪卵泡液（pFF）在体外改善猪卵丘-卵母细胞复合体（COC）成熟的能力，并观察其对体外条件下受精以及发育至桑葚胚/囊胚晚期阶段的后续影响。将COC在添加了1%胎牛血清（FCS）、10%从未成熟卵泡（2至5毫米）收集的pFF的组织培养液（TCM）199中孵育，添加或不添加1微克/毫升促卵泡素（FSH）。对照组在添加或不添加FSH的TCM 199中成熟。卵泡抽吸液离心（1700×g，5分钟），上清液在-20℃下保存在1.5毫升Eppendorf管中备用。在7个实验日，从直径2至5毫米的卵泡中总共吸出3849个未成熟COC。总共挑选1117个COC用于实验，239个COC在39℃、含5%二氧化碳的湿润空气中体外成熟48小时后，用1.5%醋酸洋红固定并染色。当成熟培养液中存在10% pFF和1微克/毫升FSH时，生发泡破裂（GVBD；91.7%）和发育至中期II（60.4%）的比例更高（P≤0.05）。另外878个COC经机械去卵丘后用于体外受精（IVF）。IVF 18小时后，561个卵母细胞用醋酸洋红固定并染色。此时各实验组之间未发现差异。另外317个胚胎在NCSU 23培养液中继续培养至桑葚胚和囊胚阶段。IVF 48小时后的卵裂率有显著差异（P≤0.05），与在不添加pFF和/或FSH的TCM 199中成熟相比，当卵母细胞在添加pFF和FSH的成熟培养液中成熟时，卵裂率更高（52.5%对32.5%对30.7%）。一旦胚胎发生卵裂，在发育至桑葚胚和囊胚阶段未观察到进一步差异。这些数据表明，pFF和FSH共同作用可增强GVBD以及向中期II的核成熟，并且卵裂率增加表明细胞质成熟更完全且同步性更好。
