Fusion and development rates of single blastomere pairs of mouse two- and four-cell embryos using the electrofusion...

The present study was undertaken to find suitable conditions for blastomere fusion of mouse two- and four-cell embryos using the electrofusion method to simplify the nuclear transfer procedure. Single blastomeres of ICR and F1 (C57BL/6J x CBA/N) two-cell embryos or ICR four-cell embryos and F1 two-cell embryos were paired and treated with electric stimulus under different fusion conditions. Two hours after electrofusion treatment, the fused blastomere pairs were encapsulated in alginate gel and cultured for 96 hours to observe their developmental potential. When the single blastomere pairs of two-cell embryos were exposed to electric pulses of 1.0, 1.5 and 2.0 kV/cm for 30, 60 and 90 mu sec, high fusion rates were obtained (84.6 to 100%). However, when two-cell blastomere were paired with four-cell blastomere and then treated under the same conditions, the fusion rates (27.5 to 87.5%) were lower than that of single blastomere pairs of two-cell embryos regardless of the duration and strength of the d.c. pulses. The blastocyst developmental rate after in vitro culture of the fused blastomere pairs of two-cell embryos using the above electrofusion conditions was high (81.8 to 100%). Lower blastocyst developmental rates were obtained on the fused blastomere pairs of two- and four-cell embryos (46.4 to 76.2%). Based on the results of this study, a pulse duration of 60 mu sec and a pulse strength of 1.0kV/cm were the most suitable conditions for single blastomere pair fusion of two-cell or two- and four-cell embryos. The study further showed that alginate gel is a good substitute for zonae pellucidae for encapsulating zona-free embryos.