Efficient Production of Bovine Tetraploid Embryos by Electrofusion of In Vitro Produced Two-Cell Stage Embryos

Abstract

Tetraploid complementation has been used to improve the production of cloned animals. The main objective of this study was to improve the efficiency of bovine tetraploid embryo production and the development potential of bovine tetraploid embryos into blastocysts. We assessed early embryonic cleavage timing and established the defined time quantum to collect synchronous 2-cell stage bovine embryos for electrofusion. Different electrofusion protocols were also tested. The second aim was to monitor ploidy transition by fluorescence visualization to shed lights on the nuclear fusion process of the cytoplasmic membrane-fused 2-cell stage bovine embryos. Karyotypes of day 8 blastocysts (day 0: day of electrofusion) were determined by karyotyping analysis. We found that electrofusion of in vitro produced bovine 2-cell stage embryos results in both tetraploid and diploid embryos and that blastocyst formation rates from fused 2-cell stage embryos are affected by the number of electrofusion pulses and the timing of how soon the 2-cell stage embryos are formed post insemination. We identified two distinct nuclear configurations after electrofusion of 2-cell stage embryos, each of which is uniquely related to the formation of tetraploid or diploid embryos. Based on the observation that tetraploid and diploid embryos derived from fused 2-cell stage embryos undergo different timings to become 2-cell stage embryos again, diploid and tetraploid 2-cell embryos can be readily separated after electrofusion. Finally, our study established an experimental protocol for the effective production of bovine tetraploid embryos by electrofusion of 2-cell embryos.