In order to improve the results of cryopreservation of in vitro produced (IVP) bovine embryos, this study will be conducted with the main objective of evaluate the impact of supplementation with linolenic acid (ALA), with or without L-carnitine, during in vitro maturation (IVM) on the maturation and oocyte quality, especially in respect to lipid metabolism, and the development and resistance to cryopreservation of produced embryos. Therefore, in a first step will be carried out dose-response experiments to determine the optimal concentrations of ALA (0, 10, 50 or 100 µM) and L-carnitine (0, 1, 5 or 10 mM) to be added to the IVM medium, supplemented with 10% FCS or 0.6% BSA. The effects of ALA and L-carnitine on nuclear and cytoplasmic maturation will be assessed, as well as intracellular lipid accumulation of bovine oocytes, oxidation potential of the culture medium and production of intracellular reactive oxygen species. In a second step, the effect of ALA supplementation, with or without L-carnitine (concentrations defined in the previous step), will be assessed during the IVM culture, on the subsequent in vitro development, quality and intracytoplasmic lipid accumulation, beyond embryo cryotolerance. Therefore, oocytes will be fertilized for 24 hours and the presumptive zygotes in vitro cultured (IVC). The cleavage rate (48 hpi) and embryo development until the blastocyst stage (D7 of the IVC) will be assessed. These blastocysts will be vitrified and subsequently heated for embryo survival evaluation after cryopreservation, 3 hours after in vitro re-cultive. Also in this stage, the regulation of gene expression involved in lipid metabolism (regulation of lipogenesis: SCD1, FAS and SREBP1; regulation of B-oxidation metabolic pathway: CPT1B and CPT2), in oocytes and bovine embryos supplemented with ALA and/or L-carnitine during the IVM culture will be assessed.
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