Study On In Vitro Production Of Bovine Tetroploid Embryos By Electrofusion

Tetraploid embryos are used in the production of transgenic cloned animals as tetraploidcomplementation increases the success rate of reproductive cloning. Mouse tetraploid embryoscan be generated from2-cell stage embryos by electrofusion, however, there is no evidence ofthe successful production of bovine tetraploid embryos. Generally, three basic approaches havebeen employed to produce mammalian tetraploid embryos in vitro. The first approach is basedon the transfer of embryonic nuclei into fertilized eggs, which results in the production oftetraploid blastocysts. It is also possible to produce parthenogenetic tetraploid embryos byinhibiting the formation of both first (1stPB) and second polar bodies (2ndPB) with cytochalasinB or other cytokinesis-inhibition drugs during parthenogenetic activation of oocytes. The thirdmethod is to fuse two diploid blastomeres at the2-cell stage embryo with inactivated Sendaivirus, polyethylene glycol or electrofusion. Electrofusion is also safer than other chemical-orvirus-mediated methods, and more convenient and economical than microsurgical injection,which makes it more popular to the production of tetraploid embryos.We assessed early embryonic cleavage timing, and established middle and late timequantum to collect synchronous and good quality2-cell stage bovine embryos for electrofusion.Different electrofusion protocols were also tested. Ploidy transition was monitored byimmunofluorescence visualization of nuclear remodeling in a fused2-cell stage embryos fromcattle. We tried to use colchicum which is a powerful cytoplasmic division inhibitor to inhibitthe fused embryo nuclear remodeling. The results are as follows:1. Electrofusion of in vitro produced bovine2-cell stage embryos results in tetraploid anddiploid embryos, and the tetraploid embryo formation rate depends on the cell cycle stage of the2-cell embryos.2. Blastocyst formation rates from fused2-cell stage embryos are affected by electrofusionparameters and the timing of how soon the2-cell stage embryos are formed post insemination.3. We identified two distinct nuclear remodeling events by immunofluorescent stainingafter electrofusion of2-cell stage embryos, each of which is uniquely related to the formation oftetraploid or diploid embryos.4. Tetraploid and diploid embryos derived from fused2-cell stage embryos go throughdifferent cell cycles; therefore, separation of diploid and tetraploid embryos after electrofusionaccording to the different time intervals of diploid and tetraploid cell cycle is a very effective way to select tetraploid bovine embryos.5. We treated0.05μg/mL demecolcine for6h after fusion significantly improved bovinetetraploid rate which produced by electrofusion.