74 Impact of tetraploid complementation on trophectoderm and inner cell mass in hybrid (Bubalus bubalis × Bos taurus) and bovine embryos

Crossbreeding between cows and buffalos is naturally limited by maternal-fetal interactions. The use of embryo aggregation with tetraploids (4n-cells) enhances embryonic development and viability because tetraploid cells preferentially migrate to the trophoectoderm (TE). The resulting embryos are derived exclusively from the diploid inner cell mass (ICM). We hypothesized that the aggregation of a diploid bovine or cow-buffalo hybrid embryo with a bovine tetraploid embryo could increase the number of TE cells (CDX2+), while maintaining the ICM cell number (SOX2+). To demonstrate this, we used bovine oocytes from a slaughterhouse and fertilized them with bull or buffalo sperm to produce bovine or hybrids embryos, respectively. Two-cell zona-free (ZF) bovine embryos were electrofused with two pulses of 80 mV, with an interval of 30 µs to produce tetraploid embryos (TE4n). TE4n were cultured with ZF cow-buffalo hybrid (H4n, n = 10) or bovine embryos (B4n, n = 17) in microwells that allowed stretch contact. Non-aggregated ZF hybrids (HZF, n = 12), and ZF bovine embryos (BZF, n = 8) were also cultured in microwells as controls. Blastocyst rates were observed on Day 7 of development. Blastocysts were fixed for 20 min in 4% (vol/vol) paraformaldehyde and permeabilized for 15 min with 0.2% (vol/vol) Triton X-100. Nonspecific immunoreactions were blocked by 30-min incubation with 3% (vol/vol) BSA and 0.1% (vol/vol) Tween-20 in DPBS (blocking solution). Incubation with primary antibody against CDX2 and SOX2 was performed overnight at 4°C. Then, embryos were rinsed in a blocking solution for 15 min and incubated with the secondary antibody for 2 h at 39°C. Stained blastocysts were mounted on glass slides with Vectashield (Vector Laboratories), in 70% (vol/vol) glycerol under a coverslip and stored at 4°C for 24 h before microscopic fluorescence evaluation. Embryos were analyzed on an inverted microscope Olympus Disk Spinning Unit-IX83 Spinning Disc Confocal. Statistical analysis was performed using an ANOVA test in GraphPad Prism Software. Results are shown in Table 1 as cell numbers mean ± s.e.m. As shown in Table 1, the HZF group exhibited a significant difference in the number of CDX2+ and SOX2+ cells compared with the BZF, B4n, and H4n groups. There were no significant differences in cell numbers among the BZF, B4n, and H4n groups. This study demonstrates that heterospecific tetraploid complementation significantly improves the number of cells in blastocysts, whereas no comparable effect was observed when embryos from the same species were complemented.