Oxamflatin and ascorbic acid improves developmental competence and quality of buffalo (Bubalis bubalis) cloned embryos

Buffalo cloning is a powerful assisted reproductive tool for multiplying elite buffalo germplasm. However, the live off-spring production efficiency is low due to aberrant epigenetic reprogramming. Aberrant epigenetic marks can be modified by culturing donor cells and/or one cell stage fused embryo or both with epigenetic modifiers alone or in combination. In the present study, we examined the effect of oxamflatin (OxF), ascorbic acid (AA), and their combined (OxF+AA) effect on in vitro developmental competence, quality, and pregnancy establishment rate of buffalo cloned embryos. Oxamflatin is a histone deacetylase inhibitor, whereas ascorbic acid is a hypomethylating agent. To achieve this aim of the study, reconstructs (fused two enucleated ooplasm + donor cell) were cultured for 8 h, i.e., 4 h post-fusion and 4 h post-activation with 1 μM oxamflatin (OxF), 50 μM ascorbic acid (AA), and there combined (OxF+AA) treatment. There was no significant (p<0.05) difference in cleavage rates when reconstructs were treated with oxamflatin (81.34±0.81%), ascorbic acid (82.76±0.51%), combined treatment (82.17±0.54%) compared with control (82.87±0.63%). The blastocyst production rate was significantly higher (p<0.05) in combined treatment OxF+AA (41.64±0.95%) as compared to OxF (34.88±1.22%), AA (38.99±0.69%) and control (30.29±0.77%). The TUNEL assay showed a significantly lower (p<0.05) apoptotic index in combined (OxF+AA) treatment (1.43±0.43) as compared to oxamflatin (3.54±0.46), ascorbic acid (3.24±0.49) and control (5.06±0.48). The cloned embryos were transferred to the synchronized recipient (n=15 to18 buffaloes in each group), and the conception rate was observed better in combined treatment (OxF+AA) (46.66%) than oxamflatin (16.66%) and ascorbic acid (12.50%). At the same time, no pregnancy was reported in the control group. In conclusion, the combined treatment with oxamflatin and ascorbic acid improves the in vitro and in vivo developmental potential in buffalo-cloned embryos, which could probably be due to decreased methylation and increased acetylation of the embryos.