ABSTRACT
Because tetraploid embryo is used as a base for growth and development of transgenic cells, one of the most important stages in animal biotechnology is to produce tetraploidy by electrofused 2-cell embryo. The aim of this study was to determine the effect of fusion duration on developmental rate of tetraploid embryos. In this experimental study some of the bovine 2-cell embryos were obtained from in vitro matured and fertilized cumulus oocyte complexes 33-35 hr post fertilization as an unexposed control group [UCG]. The remaining 2-cell embryos were exposed to 0.75 kilovolt per centimeter for 80 microsecond, and were transferred to SOF1 medium. Subsequently those embryos fused at 30 and 60 minute post electrofusion were categorized as fused groups [FG[30] and FG[60]] and separated from unfused embryos as exposed control group [ECG]. The developmental rate was compared between UCG, ECG, FG[30], and FG[60] groups and the relation between fusion duration and cleavage and developmental rate was surveyed.The cleavage rate up to 8-cell stage in FG[60] was increased significantly compared to FG[30][p<0.05] while the blastocyst rate has no significant difference between the two groups. The cleavage and developmental rate in UCG was significantly higher than ECG, FG[60] and FG[30]. Chromosomal analysis showed that 76% of embryos were true tetraploid.The fused embryos in FG[60] had more ability to produce embryos up to 8-cell stage than FG[30]. The electrical pulse can decrease the cleavage and developmental ability of embryo
ABSTRACT
The value of embryonic stem-like cells in cloning is obvious. Production of cloned animals can be achieved by introduction of these cells into a tetraploid embryo. Tetraploid embryo is used as a feeder for development of embryonic stem-like cells and can be produced in vitro by electro-fusion of 2-cell embryos. The aim of this study was to assess the effect of voltage alteration and duration on fusion and cleavage rates of bovine tetraploid embryo produced by electrofusion. After in vitro maturation and fertilization of cumulus oocyte complexes, two-cell embryos were categorized into three groups: 1- Fused group [FG]: include two-cell embryos fused by exposure to different voltages [0.5, 0.75, 1, 1.25 and 1.5 kV/cm] and durations [20, 40, 60, 80 and 100 micro s]. 2- Exposed control group [ECG]: two-cell embryos that remained unfused after electrofusion. 3- Unexposed control group [UCG]: two-cell embryos cultured without exposure to any voltage. The embryos from each group were cultured in SOF1. The fusion and cleavage rates were compared in each group. Increase in voltage resulted in significantly higher fusion and lower cleavage rate. The increased duration had no significant effect on fusion rate. The increased duration of high voltages caused decreased cleavage rate significantly, and in low voltage resulted in increased cleavage rate. The cleavage rate in ECG group followed the same as FG group, and they were lower when compared to UCG. Best fusion and cleavage rate was obtained at 0.75-1 kV/cm for 60 micro s duration