Production of blastocysts from aggregates of buffalo embryonic stem cells and putative tetraploid buffalo embryos

Abstract

This study was aimed at producing tetraploid (4n) buffalo embryos and their aggregates with buffalo embryonic stem (ES) cells. In experiment 1, immature oocytes obtained from slaughterhouse buffalo ovaries were subjected to IVM and IVF after which the blastomeres of 2-cell stage embryos were electrofused using 4 combinations of pulse field strength (0.75 or 1.25 kV/cm) and pulse duration (50 or 99 μsec). Although the differences in fusion (72–82%), cleavage (76–83%) and blastocyst rate (5–16%) among the 4 groups were not significant, the highest blastocyst rate was obtained with 1.25 kV/cm and 99 μsec. Chromosome analysis of electrofused embryos revealed that among the 30 spreads examined, 5 (16.6%) were tetraploid whereas the rest 83.3% were not tetraploid. In experiment 2, following culture of aggregates of buffalo ES cells stained with Hoechst 33342 and putative tetraploid embryos at 8- to 16-cell stage, 1/47(2.1%) aggregates prepared from ES cells and IVF-derived 8- to 16-cell embryos developed to blastocyst stage whereas when 56 aggregates prepared from ES cells and parthenogenesis-derived 8- to 16-cell stage embryos were cultured,16(28.5%) developed to blastocyst stage. These results demonstrated that tetraploid buffalo embryos can be produced by electrofusion and that these embryos can be aggregated with buffalo ES cells to produce blastocysts. Aggregates prepared from parthenogenetically-derived embryos have better developmental competence than those prepared from IVF-derived embryos.