PRODUCTION OF MONOZYGOTIC TWINS AFTER FREEZING AND THAWING OF BISECTED MOUSE EMBRYOS

We examined the viability of mouse bisected embryos after freezing and thawing and produced monozygotic twin mice from these embryos. Two-ce ll embryos were collected from superovulated mature agouti, Fl hybrid (C57BL/6 X CBA) female mice. For bisection, one blastomere of the embr yo was aspirated with a micropipette and injected into an empty zona p ellucida. After culture for 24 to 28 h to the compacted 4- to 8-cell s tage or 48 to 52 h to the late morula to blastocyst stage, the embryos were slowly frozen (-0.5 to -1.0 degrees C/min), thawed (30 degrees C /min), cultured for 24 h, and then transferred to recipient females. T he bisected embryos without zonae pellucidae had developmental ability in vitro similar to those with zonae pellucidae (88% vs 89%). However , after freezing and thawing at the compacted 4- to 8-cell stage, bise cted embryos with zonae pellucidae had higher viability than those wit hout (60% vs 15%). Zona enclosed, bisected embryos frozen at the compa cted 4- to 8-cell stage were more resistnat to freezing and thawing th an those at the late morula to blastocyte stage (60% vs 23%). After tr ansfer to recipients 26% of the zona enclosed bisected embryos frozen- thawed at the 4- to 8-cell stage developed to living fetuses a day 17. 5 to 18.0 of pregnancy, which was slightly but not significantly lower than that of fresh bisected embryos (48%). On the other hand, only 5% of bisected embryos frozen-thawed at the late morula to blastocyst st age developed to young. The transfer of 15 sets of twin blastocysts as pairs that had been frozen and thawed at the compacted 4- to 8-cell s tage yielded 2 (13%) sets of monozygotic twins. (C) 1998 Academic Pres s.