106 Intergeneric hybrid embryo (Bos taurus × Bubalus bubalis) with trophoblast complementation

The generation of hybrid bovines has productive advantages. However, crossbreeding with buffalo is limited by maternal-fetal incompatibility resulting in the inability to obtain offspring. We hypothesise that the complementation of hybrid embryos with bovine tetraploid embryos could enhance maternal-fetal tolerance during embryo implantation since tetraploid embryos are committed to trophoblast cell development. To demonstrate this, zona free (ZF) 2-cell bovine embryos were electrofused with two pulses of 80 mV, with an interval of 30 µs to produce tetraploid embryos (TE4n). Complementation was performed by culture in microwells that allowed stretch contact of ZF hybrid embryos with TE4n (H4n, n = 81). Other experimental groups were ZF bovine embryos complemented with TE4n (B4n, n = 86), noncomplemented ZF hybrids (HZF, n = 109), and ZF bovine embryos (BZF, n = 181) also cultured in microwells as controls. Blastocyst rates were observed on Day 7 of development. As a proof of concept, H4n, B4n, and BZF were transferred to previously synchronized bovines recipient cows. Pregnancy rates were evaluated on Day 60 after embryo transfer. Embryo in vitro developmental rates were compared using two-tailed Fisher’s exact test. Results are shown in Table 1. Different superscript letters indicate statistical significance (Fisher’s exact test, P < 0.05). As shown in Table 1, the development rates of complemented (22.2%) and noncomplemented (21.1%) buffalo-cow hybrids did not show any significant differences (P > 0.05). However, there was a difference (P < 0.05) in the development rates between complemented (47.7%) and noncomplemented (27%) bovine embryos. In a first instance, two H4n were transferred, resulting in one pregnancy. There were three pregnancies out of nine B4n, while two pregnancies were obtained out of 10 BZF. Although the development rate in H4n was not improved, the survival of one of the two transferred H4n embryos suggests that the use of tetraploids may potentially be applied to produce offspring between Bos taurus and Bubalus bubalis. In conclusion, this experiment demonstrated that homospecific trophoblastic complementation significantly improves blastocyst rates in bovine embryos while no comparable effect was observed when embryos from different species were complemented. Further work is required to determine whether maternal-fetal recognition is enhanced by tetraploid complementation.