Efeito do bloqueio meiótico sobre a maturação e fertilização de oócitos bovinos

Abstract

Biotechnology of reproduction has been increasingly used in order to improve genetic material as well as germplasm preservation. Provision of viable oocytes in stage suitable for its use is essential to the success of this technique. An alternative would be to prevent resumption of meiosis after removal of oocytes from follicular environment inducing a meiotic blockage with the aim of providing the oocyte more time available so that it could be possible to suffer modifications required to support fertilization and normal embryonic development. Inhibitors, as Rolipram has been used with this objective. These blockers can improve oocyte competence through a better cytoplasmatic maturation. The goal of this project was to evaluate the effect of rolipram during maturation of bovine oocytes on quantity and quality of embryos produced in vitro. Ovaries was collected at slaughter and transported to the laboratory of Animal Reproduction at UAG/UFRPE - LABRAPE. The COC`s were selected and divided into 5 groups: Control 1 (time 0): oocytes fixed and stained immediately after selection to observe after collection status; control: in vitro maturation for 24 hours in the absence of Rolipram; Rolipram in 3 different treatments: blockage for 24 hours in maturation medium containing (100, 150 and 200 μM). After 24 hours, treatment and control groups were placed in maturation medium for over 24 hours to observe the reversal of the blockage. These oocytes were evaluated using an epifluorescent microscope to check maturation stage. After maturation, oocytes from control (within 24 hours of in vitro maturation) and treatments groups with Rolipram were in vitro fertilized 48 hours post fertilization to assess cleavage and blastocyst formation at D7 post fertilization. In addition, part of the oocytes were stored for expression of the following genes: Mater, BMP15 and BAX, to observe the effect of the blocker on expression of these genes in the embryos. As far as gene expression, no difference was seen between control group and treatments. The oocytes evaluated immediately after follicular aspiration 79.00% were in VG, QVGBD, or MI, characterized as immature and 13.40%, was in MII and 7.60%, were considered degenerate or not identified. In different concentrations of T100 and T200 T150 μ M, we observed significant differences in oocytes that have reached the MII phase comparing with control treatments (P = 0.003). As for the embryonic development, differences in the rate of cleavage (P < 0.05) were observed between T150 and T200 when compared to Control 24 hour Group. A difference was seen on blastocyst rate (P < 0.001) among the treatments compared to control group, being T100 μM the concentration which provided less negative effects during pre-maturing of bovine oocytes.