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.