In vitro production (IVP) of embryos is a reproductive biotechnique widely used for genetic improvement and increase in animal production, and Brazil is responsible for the largest IVP of bovine embryos in the world. Although IVP is successfully used for commercial purposes, the technique still has some limiting factors for further reduction in costs and greater dissemination of its use, which are related with the lower quality e consequent greater sensibility of IVP embryos to cryopreservation. Such reduced cryotolerance of IVP bovine embryos seems to be related with a greater accumulation of cytoplasmic lipids in comparison with in vivo generated embryos. The reasons for such increased lipid stores have not been clarified, but appear to be related with changes in lipid metabolism caused by standard in vitro culture in the presence of fetal calf serum (FCS). Recent studies have shown that culture of IVP embryos with bovine oviduct epithelial cells or extracellular vesicles (EVs) produced by these cells in culture or present in oviductal fluid (OF), or yet with OF and uterine fluid (UF) in sequential culture, result in embryos of better quality and higher cryotolerance. On the other hand, when the FCS usually used in in vitro culture systems is depleted from its EVs (depFCS), embryo cryotolerance is also improved, suggesting that EVs present in FCS may contribute to the lower quality and cryotolerance of IVP embryos. However, possible effects of these EVs on lipid metabolism and its relation with embryo quality have not been determined. EVs are vesicles secreted by several cell types and that contain varied cargo including proteins and RNAs, among other molecules. The cells that uptake these molecules may have their gene expression and metabolic activity affected by them and embryos have been shown to uptake EVs. Therefore, the aim of the present study is to investigate the role of EVs from OF (EVO) and UF (EVU) on lipid metabolism in bovine embryos produced in vitro with their supplementation during in vitro culture (IVC). In Experiment 1, IVP embryos will be cultured for up to 7 days in the following groups: 1) control+ with FCS, 2) control- with depFCS, 3) with EVO from D1-4 followed by EVU from D5-7 (EVO-EVU) and 4) with EVO-EVU in the presence of depFCS (EVO-EVUdep). At the end of culture, embryos will be assessed by fluorescence confocal microscopy for quantification of lipid contents (Nile red staining) and co-localization of PLIN2 (lipid droplet related protein) with lipid droplets. In Experiment 2, the effects of treatments will be analyzed regarding lipid metabolism by determining levels of active HSL (lipolysis enzyme) and of FAS (lipogenesis enzyme) by western blotting. In Experiment 3, embryos from the different experimental groups will be evaluated for transcript abundance of lipid metabolism genes by qPCR. In the last Experiment (4), EVs will be assessed for their transcriptome (mRNAs) related to lipid metabolism by RNAseq, to identify possible candidate genes influencing lipid metabolism of embryos. Selected identified genes will then be assessed in cultured embryos to evaluate their possible uptake.
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