Vitrificação de oócitos bovinos imaturos

Abstract

The development of reproductive biotechnology has been originating new challenges in many areas. In the cryobiology, the current challenge is to obtain an appropriate methodology for bovine oocyte cryopreservation, especially in the immature stage. This study was aimed to the evaluate the in vitro and in vivo development of embryos derived from immature vitrified bovine oocytes, with OPS technology. In addition was evaluate the effect of the pre-treatment with cytochalasin D (CD), and the viability of a cryoprotectant solution composed by ethylene glycol (EG), dimethyl sulfoxide (Me2SO) and sucrose (SUC), in the vitrification of immature bovine oocytes. The oocytes obtained of slaughterhouse bovine ovaries were randomly allocated in three groups: a control group (n=442), a vitrified group (Vitri n=356) and a group treated with 20μg/ml of CD during 10 to 20 minutes and vitrified (CDVitri n=355). For the vitrification procedure, the oocytes were exposed for 30 seconds to an VS50 solution (10% EG + 10% DMSO), followed by 25 seconds exposure to a VS100 solution (20% EG + 20% DMSO + 0.5M of SUC), loaded in groups of 4 to 5 in OPS and plunged in liquid nitrogen. The warming was performed in two steps of 5 minutes, with sucrose gradients (0.26 and 0.16M) solutions. After that, all groups were submitted to the same in vitro maturation, fertilization and culture procedures. The in vitro viability was evaluated by the cleavage and blastocysts rates, and the in vivo viability was evaluated by the transfer of blastocysts derived from vitrified immature oocytes (1 or 2 for each recipient). No differences (P>0.05) were observed in the cleavage and blastocysts rates among the CDVitri (46.4% and 3.5%) and Vitri (49.0% and 6.1%) groups, respectively, and both were lower (p<0.05) than control group (85.1% and 45.9%). A female calf was obtained from CDVitri group (two recipients) and a female and a male calves birth from the Vitri group (five recipients), were obtained. These data indicate that the cytochalasin D pre-treatment do not improve viability of embryos derived from vitrified immature bovine oocytes. The OPS technology with a cryoprotectant solution composed by 20% EG + 20% Me2SO and 0.5M SUC is effective for vitrification of immature oocytes. The embryos obtained from immature oocytes vitrified with this technology are fully competent to develop healthy offspring, even when submitted to a new vitrification procedure at blastocyst stage.