Reference Gene Selection for Gene Expression Analysis During In Vitro Maturation of Alpaca Oocytes

Among the in vitro embryo production (IVP) procedures, the in vitro maturation (IVM) is an essential step for the oocyte to achieve nuclear and cytoplasmic maturity (competence). In this line, supplementation of the IVM medium with resveratrol have been associated with higher oocyte competence in other species. On the other hand, a precise analysis of transcript levels in mammalian gametes and embryos requires the use of reliable reference genes (RGs) for data normalization. However, RGs specific to IVM of alpaca oocytes have yet to be identified, creating a barrier to functional gene studies in this species. Therefore, this study aimed to identify suitable reference genes to proper analyze gene expression patterns during IVM of alpaca oocytes. Four candidate transcripts (Gapdh, Actb, Rplp0 and Ppia) were selected and their stability during IVM with and without exposure to resveratrol was analyzed using RefFinder software (https://github.com/fulxie/RefFinder). The transcript levels of two target genes (Bmp15 and Sirt1) were normalized by the selected reference genes. Our findings indicate that, among the four candidate reference genes, Rplp0 and Gapdh were the most stable transcripts. Additionally, we observed that transcript levels of Bmp15 were differentially expressed after IVM in the presence of resveratrol (p<0,05). We also demonstrated that supplementation with resveratrol modulates the expression patterns of transcripts associated with chromatin remodeling and folliculogenesis. Thus, this is the first report evaluating suitable reference genes for alpaca oocytes, providing valuable tools to better understand oocyte physiology at the gene expression level of South American camelids.