Electrofusion of two-cell bovine embryos for the production of tetraploid blastocysts in vitro

Abstract

Tetraploid bovine blastocysts were produced experimentally by electrofusion of in vitro matured and fertilized, zona-enclosed two-cell embryos (33-35 hr after initiation of sperm-egg incubation) using three fusion protocols. Field strengths of 1.0, 1.4, and 2.4 kV/cm were tested and the rate of fusion, subsequent cleavage, and blastocyst development were measured for each. High rates of fusion (76.5% +/- 2.8%), cleavage (72.5% +/- 7.4%) and blastocyst development (56.1% +/- 6.4%) were achieved with the application of 1. 4 kV/cm as a single 100-microseconds pulse. Embryos were scored 30 and 60 min after stimulation for fusion. No time effect for fusion, cleavage, or blastocyst development was observed. Chromosome preparations of day 7 blastocysts revealed 12.5% of fused embryos were tetraploid. This is a significant increase from that found in nonfused embryos where spontaneous tetraploidy did not occur. An electrical stimulus of 1.0 kV/cm applied as two 50-microseconds pulses produced significantly less one-cell embryos (64.2% +/- 3.0%) compared to 1.4 kV/cm while cleavage (79.9% +/- 3.4) and blastocyst development (44.6% +/- 4.0%) were not different from that for unexposed control embryos (89.5% +/- 2.3% and 57.2% +/- 3.2%, respectively). Embryos fused at 2.4 kV/cm applied as a single 30-microseconds pulse (69.7% +/- 5.7%) showed significantly lower cleavage (72.1% +/- 3.7%) and blastocyst rates (40.2% +/- 4.6%) compared to the unexposed control.