Methods for partial denudment of maturated bovine oocytes submitted to vitrification

Abstract

Different methods of partial denudation of maturated bovine oocytes were evaluated
for in vitro embryo production and vitrification procedures, in three experiments. First the effect of
partial denudation by successive pipetting was evaluated. In the second experiment, the denudation
by pipetting was compared with the use of hyaluronidase, and finally, the effect of both methods
was evaluated in regard to the vitrification of oocytes. Oocytes were submitted to treatments after 22
hours of maturation in TCM 199 medium. For the vitrification, they were firstly exposed for 30 seconds
to an equilibrium solution and for 20 seconds to a vitrification solution (20% EG + 20% DMSO +
0.3M SUC), loaded in open pulled straws (OPS) and plunged into liquid nitrogen. The rewarming of
the oocytes suspensions was carried out by plunged them in decreasing sucrose concentrations.
Oocytes of all treatments were submitted to an additional 2 hours maturation period, followed by
fertilization. Presumptive zygotes were cultured in SOFaaci medium. In the first experiment the
denudation resulted in a decreased blastocyst rate (28.7% to 20.5% P<0.05). Both denudation
methods (pipetting or hyaluronidase) provided similar blastocyst rate after culture (17.7% and 21.7%
- Experiment II), as well as after vitrification (6.4% and 8.9% - Experiment III). Though, partial
denudation was more homogeneous with the effect of hyaluronidase We concluded that
hyaluronidase can replace the use of pipette in the partial denudation of bovine oocytes, with identical
viability, being easier to be conduced.