In Vitro Maturation and Fertilization of Denuded Buffalo Oocytes with Cumulus Cell Monolayers

Buffalo ovaries were collected from a slaughter house and cumulus cells oocytes complexes (COCs) were recovered for preparation of 2-D, 4-D, 6-D and 8-D monolayer. Cumulus cells were stripped off by repeated pipetting into TCM-199 medium supplemented with 10% FCS. Approximately 2x106/ml cumulus cell were transferred in 100 μl drops of culture medium and incubated in humidifi ed atmosphere of 5% CO2 in air at 39oC. The cumulus cell monolayer medium was refreshed after every 48 h. The culture medium was replaced with a maturation medium before transfer of denuded oocytes containing TCM-199, 10% FCS, 5 μg/ml LH, 0.5 μg/ml FSH and 1 μg/ml estradiol 17β. Denuded oocytes with evenly granulated cytoplasm were cultured for 24 h on 2-D, 4-D, 6-D and 8-D old cumulus cell monolayers. After 24 h of incubation on a cumulus cell monolayer, the matured oocytes were fertilized with spermatozoa. The fertilized oocytes were incubated with buffalo oviductal cells. The resultant maturation and fertilization percentage in 6-D were 60.81 and 32.80, respectively, which was higher than 2, 4 and 8 day old cumulus cells monolayers. However, the denuded oocytes with evenly granulated cytoplasm matured on 6-D old cumulus cells monolayer showed equal maturation and fertilization percentage compared with oocytes containing compact oocytes complexes (COCs). Thus it is concluded that the 6-D culture of cumulus cell monolayers can support comparatively higher maturation and fertilization rates of denuded oocytes with evenly granulated cytoplasm than 2-, 4-, and 8day of cumulus cell monolayers.