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