The in vitro culture system of embryos is crucial to determine its ability to reach the blastocyst stage and, in particular, its viability and success after transfer. Changes in this system are often employed and many of them are based in the addition of carbohydrates and amino acids as supplements of culture medium. However, this supplementation is usually in different concentrations than those observed physiologically, including for commercial embryos production. However, the impact of these supplements has not been fully elucidated and might have an effect beyond energy generation. One of the currently hypotheses in literature is that metabolites generated during energy production exerts direct and indirect control in methylation and demethylation status of cells, critical processes in pre-implantation embryonic development. During embryonic first cleavages occurs the DNA demethylation (actively and passively), which will be responsible for the production of totipotent cells, until 8-16 cells stage. Then, the DNA of these embryos must be re-methylated until, in the blastocyst stage, its blastomeres are classified as pluripotent and differentiated as belonging to inner cell mass or trophectoderm. It is known that the enzymes responsible for DNA demethylation use ±-ketoglutarate, a TCA cycle metabolite, for its activity. Therefore, the hypothesis of this work is that TCA cycle metabolites affect the DNA demethylation and methylation wave in bovine embryos. To confirm this hypothesis, TCA cycle metabolites will be modulated and their effects evaluated in DNA methylation parameters and gene transcription. It is expected that these results are going to contribute to understand the mechanisms that control embryonic DNA demethylation process and the role of metabolism in this event.
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