Effects of Trophectoderm Dissection on Development of Inner Cell Mass of Fresh and Frozen-Thawed Bovine Blastocysts Derived from In-Vitro Fertilization

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A part of trophectoderm cells was dissected microsurgically from bovine blastocysts fertilized in vitro for sex determination by chromosome analysis and its effects on the proliferation of inner cell mass (ICM) of fresh and frozen-thawed embryos were investigated by a differential fluorochrome staining technique. In total, 188 blastocysts were dissected and 85.5% (65/76) of embryos cultured in vitro for 24 h was repaired. Mitotic index (14.9%) and sexing rate (57.1%) of unrepaired embryos after dissection were lower than those (19.9% and 66.0%) of repaired embryos but not significant. Further culture resulted in significant decreases in the repair rates (48 h: 37.5%, 72 h: 32.5%, 96h: 28.6%) but not in embryos co-cultured with cumulus cells monolayer (96 h: 81.8%). The proliferation of ICM cells was inhibited up to 72 h after in-vitro culture and it was promoted thereafter. Survival rates of frozen-thawed embryos, which was repaired after the dissection decreased significantly from 12 h after the initiation of in-vitro co-culture. Total and live ICM cell numbers increased slightly with in-vitro culture but not significant. These results show that the proliferative ability of ICM cells becomes temporarily arrested by the dissection of trophectoderm cells but is resumed by the subsequent in-vitro culture. In addition, it was suggested that the applicable time of transplantation of dissected and frozen-thawed embryo is within 2 h after thawing.