4 Tetraploid complementation and embryo aggregation improve development and quality of heterospecific SCNT yak embryos

Heterospecific somatic cell nuclear transfer (hSCNT) makes it possible to recover the genetics of endangered species, but full-term development rates are very low. The embryo aggregation method, which consists of generating a blastocyst from the culture of two or more embryos in intimate contact, improves the quality of the cloned embryos. Complementation of a diploid aggregated embryo with a tetraploid embryo, capable of differentiating into trophoblastic cells, could improve embryo implantation and development within the tetraploid embryo species. Therefore, the objective of this study was to evaluate the combined effect of tetraploid complementation and embryonic aggregation on the development, cell number, and quality of embryos produced by hSCNT. For this, yak clones were produced using cattle oocytes, and oocyte collection and IVM and SCNT procedures were performed as reported (Savy et al. 2021 Cell. Reprogram. 5, 277–289). To produce tetraploid embryos, IVF was performed with 16 × 10⁶ ssp mL⁻¹ for 5 h. After 30 h of IVF, embryos with two blastomeres were selected, the zona pellucida (ZP) was removed, and then the blastomeres were fused with 0.8 kV cm⁻¹. Only tetraploid and diploid cleaved embryos were cultured into one microwell, in different experimental groups: two hSCNT yak embryos complemented with two bovine tetraploid embryos (Y2x+TE2x); two hSCNT yak embryos without tetraploid embryos (Y2x); two SCNT bovine embryos complemented with two bovine tetraploid embryos (B2x+TE2x); two SCNT bovine embryos without tetraploid embryos (B2x); and ZP-free IVF embryos (IVF). Fisher’s exact test was performed for statistical analysis. Embryo expression was assessed by RT-qPCR (SOX2, CDX2, GATA3, TFAP2c, and IFNT) and immunofluorescence (SOX2, OCT4, and CDX2) analysis, followed by t-test statistical analysis. Blastocyst rates were significantly higher in the Y2x+TE2x (59.37%, n = 96) group compared with Y2x (36.62%, n = 71). In addition, there were no significant differences in the blastocyst rates between YC2x+TE2x, B2x+TE2x (56.82%, n = 44), B2x (45.45%, n = 77), and IVF (56.45%, n = 124) groups. Regarding relative expression levels, no significant differences were observed in the expression of GATA3 and TFAP2c genes. The expression of SOX2 and CDX2 was significantly higher in the Y2x+TE2x group than in the Y2X group. It was observed that the expression of interferon tau was lower in the Y2x group than in the B2x group and IVF groups. However, blastocysts of the Y2x+TE2x group showed a significantly higher total cell number compared with Y2x (mean ± SEM: Y2x+TE2x, 181.7 ± 11.59; Y2x, 109 ± 9.95; B2x+T2x, 184.8 ± 21.41; B2x, 165.9 ± 12.38; IVF, 154.1 ± 9.88). Furthermore, Y2x+TE2x blastocysts showed a higher number of CDX2⁺ cells (144.5 ± 12.45) than Y2x (74.71 ± 7.35). SOX2⁺ cells (34.53 ± 5.86) did not present significant differences compared with Y2x (34.21 ± 4.42), B2x+TE2x (50.75 ± 7.11), B2x (44.67 ± 7.68), and IVF (100.8 ± 9.09) groups. In conclusion, the combined use of complementation with bovine tetraploid embryos and embryo aggregation of cloned yak embryos resulted in better in vitro developmental rates and better embryonic quality.