Demi-embryos were successfully produced by bisection of ICR mouse embryos at preimplantation stages. They were microsurgically bisected using a microsnrgical blade attached to a micromanipulator after pretreatment with 0.5% pronase in PBS for two minutes or not. Embryos with softened zona pellucida were more easily bisected and less damaged than intact embryos. The highest success rate in bisection has been achieved by selecting blastocysts(94.1% in success rate with intact blastocysts and 100% in sucess rate with zona softened blastocysts). Demi-embryos without zona pellucida were cultured in D-PBS or M-16 medium at 37℃, 5% CO₂ in air for 72 hours for 2-cell stage embryos, 48 hours for 4-to 8-cell stage embryos, 24 hours for morula stage embryos and 6∼12 honrs for blastocyst stage embryos. For the dn vitro culture of 2-cell stage embryos, 100 ㎛ 2Na-EDTA was added to the media. M-16 medium was better for the itt vitro development of mouse embryos than PBS, and PBS is not considered to be suitable for long-term culture of embryos, especially at early stage of cleavage. In M-16 medium, developing rate of demi-embryos of which pair underwent development to form eublastocysts was 15.8% at 2-cell stage, 16.8% at 4-cell stage, 38% at 8-cell stage, 89.6% at morula stage and 94.4% at blastocyst stage, respectively.
The more rapid and efficient production of demi-embryos and higher viability after bisection can be expected by softening zona pellucida with pronase and by selectng morulae or blastocysts rather than embryos at early stage of cleavage.
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