Interspecific SCNT in yak followed by embryo aggregation improves early in vitro developmental parameters without...

The efficiency of interspecific somatic cell nuclear transfer (iSCNT) remains suboptimal, necessitating novel approaches. This study aimed to evaluate the effect of embryo aggregation on the in vitro development of yak clones produced by iSCNT. Zona free cloned embryos were generated using male yak somatic cells and cultured either individually (Y1x, B1x group) or in pairs (Y2x group) in microwells. Aggregation improved the in vitro development of cloned yak embryos, yielding a blastocyst rate of 40.38 % in the Y2x group, which is significantly higher than the 25.34 % and 18.24 % exhibited in Y1x and B1x groups, respectively (P < 0.05), and comparable to the 48.14 % in the IVF control group. Gene expression analysis demonstrated that Y1x blastocysts exhibited significantly lower CDX2 and NANOG levels than those in the Y2x, B1x, and IVF groups. OCT4 levels expression did not differ between groups, and SOX2 levels expression was similar in Y1x, Y2x, and IVF. Y2x embryos showed a higher total cell number than Y1x, though still lower than IVF. In addition, Y2x embryos exhibited fewer CDX2⁺, OCT4⁺, and SOX2⁺ cells compared to B1x and IVF groups. Embryo transfers were performed as a preliminary evaluation of the developmental potential of aggregated embryos. Although early pregnancy signals were detected exclusively in the Y2x group (13 %; 3/25), no embryos were detected at day 42 of gestation. In conclusion, embryo aggregation in yak iSCNT embryos improves selected early in vitro developmental parameters and blastocyst morphology, but does not restore all developmentally relevant gene expression, nor sustain post-implantation development.