Effect of using slush nitrogen (SN2) on development of microsurgically manipulated vitrified/warmed mouse embryos

BACKGROUND This study evaluated the effect of vitrification using slush nitrogen (SN2) on cryopreservation of micromanipulated mouse embryos. METHODS The zona pellucida of 4-cell embryos was either left intact or dissected or dissected with biopsy of an intact blastomere. In a second study, a blastomere was destroyed and either removed (removed group) or not removed (remained group) prior to vitrification/freezing. The micromanipulated embryos were equilibrated and loaded into an open pulled straw (OPS), and plunged into liquid nitrogen (LN2) or SN2. RESULTS When using LN2 vitrification, recovery and blastocyst formation rates of embryos were lower for zona pellucida-opened and/or blastomere-biopsied embryos compared with zona pellucida-intact embryos. Using SN2 for vitrification resulted in increased survival and development of vitrified/warmed embryos in both the zona pellucida-opened and blastomere-biopsy groups. Similar results were observed when using embryos with a destroyed blastomere either removed or left remaining before vitrification. However, the number of total and apoptotic cells were similar for both LN2 and SN2. In addition, using SN2 increased the rate of intact recovery and blastocyst formation in warmed hemi-8-cell embryos derived from the same embryo. CONCLUSIONS These results suggest that vitrification using SN2 is useful in cryopreservation of micromanipulated embryos obtained from a variety of programs, including assisted hatching, preimplantation genetic diagnosis and nuclear transfer.