Modulation of oxidative stress and lipid content of bovine oocytes and embryos produced in vitro: effects on development potential and cryotolerance

Abstract

Once bovine in vitro produced (IVP) embryos are more susceptible to oxidative damage, the supplementation with antioxidants to in vitro maturation media has shown satisfactory results on embryonic development. Besides the use of antioxidants, the addition of lipogenesis inhibitors and/or lipolysis stimulators also reflects positively on the development, quality and embryonic cryotolerance. To improve the quality, development potential and the success of cryopreservation of bovine embryos, the aim of this study is to promote changes in some steps of production in vitro system to decrease the damages induced by oxidative stress and by the citoplasmatic lipid accumulation. Therefore, oocytes will be maturated in TCM-199 medium supplemented with 0,6 mM of cysteine associated to 100 ¼M of cysteamine and to 100 UI of catalase (C+C+CAT) or without supplementation of antioxidants (Control). Subsequently, oocytes will be fertilized in vitro (IVF) and the presumptive zygotes will be cultured in vitro (IVC) In this last stage the lipolysis stimulators / lipogenesis inhibitors factors will be added to the culture medium. The zygotes will be cultivated in SOF medium supplemented with 100 ¼M of conjugated linoleic acid (CLA), or 2,5 ¼M of Forskolin (FORSK), or CLA associated with Forskolin (CLA+FORSK). The cleavage will be evaluated 48 hours post-insemination (hpi) and the embryonic development at 144 and 168 hpi. Embryos will be analyzed for total lipid content (Nile Red staining) and for the levels of intracellular reactive oxygen species (H2DCFDA staining). Embryos submitted to vitrification process will be heated and cultured in SOF medium for 24 hours, under the same conditions established during the IVC for control group and the rates of embryonic re-expansion (in vitro survive) and hatching will be evaluated at 24 hours IVC rewarming.