IN VITRO MATURATION OF PORCINE OOCYTES IN FOLLICULAR FLUID WITH SUBSEQUENT EFFECTS ON FERTILIZATION AND EMBRYO YIELD IN VITRO

摘要

The objective of the present study was to test the ability of porcine follicular fluid (pFF) to improve maturation of porcine cumulus-oocyte-complexes (COG) 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 1 mu g/ml FSH. Control groups were matured in TCM 199 with or without FSH. Follicular aspirates were centrifuged (1700 x g, 5min.) and the supernatants 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% CO2 in humidified air. Germinal vesicle breakdown (GVBD; 91.7%) and development to metaphase Il (60.4%) were superior (P less than or equal to 0.05) when 10% pFF and 1 mu g/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 differences 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 less than or equal to 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 mutation 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.