Criotolerância de oócitos suínos maturados in vitro na presença de IGF-I e vitrificados com glutationa reduzida

Abstract

Although oocyte cryopreservation is an important tool for the exchange of genetic material and the in vitro production of embryos, in pigs this biotechnology is still little practiced due to the low resistance of the oocytes to this process. However, studies have shown that insulin-like growth factor I (IGF-I) can regulate protein synthesis during cellular response to cryopreservation, whereas reduced glutathione (GSH) can maintain the stability of the nucleoprotein structure and increase the viability of cryopreserved swine gametes. So, the objective of this study was to evaluate whether in vitro oocyte maturation with IGF-I, and subsequent vitrification with or without GSH, affects quality, expression of genes involved in antioxidant, apoptotic and stress response, and embryonic developmental competence of vitrified porcine oocytes. In Experiment 1, cumulus-oocyte complexes were matured in the absence or presence of IGF-I (100 ng•mL-1) and then vitrified with or without 2.0 mM of GSH. The maturation rate was evaluated prior to vitrification, and oocyte viability, DNA fragmentation and relative abundance of BAX, BCL2-like1 (BCL2L1), heat shock protein 70 (HSPA1A), glutathione peroxidase 1 (GPX1) and superoxide dismutase 1 (SOD1) were evaluated in fresh and vitrified oocytes. In Experiment 2, fresh and vitrified oocytes were fertilized in vitro and the rate of embryonic development was determined. While the addition of IGF-I to the maturation medium had no effect on oocyte maturation (P <0.05), this hormone increase (P> 0.05) the survival rate of vitrified oocytes. Likewise, the use of GSH in the vitrification/warming media also improved (P <0.05) the oocyte viability after rewarming. The associated use of IGF-I during maturation and GSH in vitrification process promoted a highest rate of live cells with integral DNA of IVM vitrified oocytes. The positive effects of IGF-I and GSH on oocyte survival were accompanied by an increase in the relative abundance of HSPA1A and GPX1 transcripts, respectively, and a decrease in BAX gene expression. Despite the differences observed in experiment I, no significant difference (P> 0.05) was found between the vitrified oocyte groups for the parameters of fertilization and embryonic development. In conclusion, supplementing the maturation medium with 100ng•mL-1 of IGF-I and vitrification solutions with 2 mM GSH improves the quality and cryotolerance of in vitro matured vitrified porcine oocytes through a mechanism that involves the expression of BAX, GPX1 and HSPA1A, in addition to increasing the rates of cleavage and blastocyst formation of vitrified oocytes, which presented values similar to fresh oocytes.