19 Tetraploid complementation and embryo aggregation improve the blastocyst rates of yak heterospecific somatic cell nuclear transfer embryos

Heterospecific cloning allows the availability of embryos to study and preserve endangered species. The complementation of a diploid embryo with a tetraploid embryo (TE), capable of differentiating into trophoblast, results in the generation of a single embryo with an increased number of trophectoderm cells. The complementation of an embryo from an endangered species with a TE from the species to be transferred could improve the development of pregnancy to term. Additionally, embryo aggregation, which involves culturing two or more zona-free (ZF) embryos in close contact to obtain a single blastocyst, has been demonstrated to improve embryo production by somatic cell nuclear transfer (SCNT). Therefore, the objective of this study was to investigate the impact of combining embryonic complementation and aggregation on the in vitro development of yak embryos produced by heterospecific SCNT (hSCNT). For this, bovine COCs were recovered by follicular aspiration from slaughterhouse ovaries and in vitro matured in TCM 199, containing 10% fetal bovine serum, 10 µg/mL FSH, 0.3 mM sodium pyruvate, 100 mM cysteamine, and 2% antibiotic-antimycotic for 22 h, at 6.5% CO2 in humidified air and 38.5°C. For SCNT, after denudation, mature oocytes were stripped of the zona pellucida (ZP) using a protease and then enucleated by micromanipulation. Staining was performed with Hoëchst 33342 to observe metaphase II. Bovine enucleated oocytes were attached to yak (Bos grunniens) cells with phytohemagglutinin and electrofused. Reconstituted embryos were activated by using ionomycin and 6-DMAP for 3 h. To produce TE, IVF was performed with 16 × 10⁶ ssp/mL for 5 h. 30 h post-IVF, embryos with two blastomeres were selected, ZP was removed, and then blastomeres were fused with 0.8 Kv/cm. Only tetraploid and diploid cleaved embryos were cultured in a well-of-the-well system in different experimental groups: two hSCNT yak embryos complemented with two bovine tetraploid embryos (YC2x/TE2x); two hSCNT yak embryos without tetraploid embryos (YC2x); two bovine tetraploid embryos (TE2x), and ZP-free IVF embryos (IVF). Blastocyst rates were significantly higher in the YC2x/TE2x group (65.38%, n = 78) compared to YC2x (40.47%, n = 42) and TE2x (30.46%, n = 13) groups. In addition, there were no significant differences in the blastocyst rates between YC2x/TE2x and IVF groups. In conclusion, the use of complementation of tetraploid bovine embryos and the embryo aggregation of hSCNT yak embryos resulted in improved in vitro developmental rates.