Instructions for use Title ELECTROFUSION OF ZONA-FREE MOUSE EMBRYONIC CELLS IN ELECTROLYTES AND THEIR DEVELOPMENT IN VITRO

The influence of increasing the physical electrofusion parameters, direct current (DC) pulse strength, pulse duration, pulse number, alternating current (AC) voltage and alignment time, in electrolytes on the rates of fusion, degeneration and development of zona-free mouse 2-cell embryos were examined. Furthermore, the effects of physiological saline and mannitol as fusion media and various mouse strains were also evaluated. Dulbecco's phosphate-buffered saline (PBS) supplemented with 10% fetal calf serum was used as the main fusion solution. A significant increase in the rate of fusion (P<0.05) was obtained by increasing pulse strength from 30 to 300 V Imm. The embryos fused at the pulse strengths of 30 to 70 V Imm had significantly higher development rates to blastocysts compared with those fused at 100 to 300 V/mm (P<0.05). There were no significant differences in the rates of fusion, degeneration and development to blastocysts when the pulse duration was increased from 30 to 90 psec. Although fusion rates were increased (P<0.05) by increasing the pulse number up to 4, a significant decrease (P < 0.05) in development to blastocysts was observed when the pulse number was 5. Application of AC voltage prior to the DC pulse tended to increase the fusion rate (89.2-93.8%), compared with fusion with the DC pulse only (75.0%). Prolongation of alignment time from 5 to 15 sec had no effect on the fusion rate. Under the optimum conditions (2 pulses of DC of 70 V/mm, 70 psec pulse duration and AC of 5 V/mm for 5 sec), no significant difference was obtained in the fusion and development rates in different mouse strains, nor were fusion and development rates significantly different among PBS, physiological saline and mannitol solutions (P>0.05).