Negative effect of cumulus cells and cytochalasin on IVM and viability of vitrified immature sheep oocytes

The influence of cumulus cells and cytochalasin B (CCB) on maturing ability of vitrified immature sheep oocytes were evaluated. For this purpose ovaries from an abbatoir were collected and ovine cumulusoocyte complexes (COCs) were cultured in IVM medium (Exp.1). At 0, 3, 6 h of IVM groups of COCs and denuded oocytes (DOs) were vitrified by equilibration with 10% EG + 10% DMSO (30’’), exposition to 20% EG, 20% DMSO, 0.5 m sucrose (20’’), loading on cryotop and plunging into liquid nitrogen. In Exp. 2, at 0 h of culture, a group of COCs were vitrified after incubation with 7.5 lm CCB for 30’. After warming in 1.25 m, 0.62 m, 0.31 m sucrose solution all the oocytes were matured in vitro. Fresh COCs and DOs were cultured as in vitro controls. Oocytes were then fixed and stained with glycerol- Hoechst 33342 to evaluate nuclear maturation. Results indicated that the number of DOs at MII after vitrification at 0 and 3 h of IVM (62.1%; 41.3%) were higher (p < 0.01; chi-square test) than COCs (31.2%, 24.8%). The lowest rate of MII oocytes were recorded after removal of cumulus cells at 6 h of culture (7.4%). higher number of vitrified COCs treated with CCB degenerated after warming compared to control (85.3% vs 5.6%, p < 0.01). In conclusion, vitrification of DOs at 0 h improved MII rates compared to the other culture times but were still lower than in the non-vitrified group (92.1%.) Treatment with CCB affected negatively the viability of vitrified immature ovine oocytes. Abstract