Freezing and thawing of eight-cell ınııuse embr)'os Summary: bı this study the ejfect of subzero temperatures on 8-ceU mouse embryos was examined. Fell1ale mice, 8 weeks old, ıvere indueed to superovulate by intraperitoneal injections of gonadotropie hormones. 8-eeU embryos were recovered from the oviduet and the anterior portion of the uterine homs at 68-72 hours af ter the injection of human ehononie gonadatrophilı (HCG). The mouse emhryos were collected in Phosphate Buffer (PBl) and washed

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... several changes of PBI to remove the debris. Before freezing Dimethyl sulfoxide (DMSO) was added in six inerements to samples eonsisted of 10-20 8-eel! mouse embryos, the final eoneentration of DMSO was ı.5 M. The samples were equilibrated for ı5 minutes at room temperature to ensure eomplete permeation of the additive into embryos and af ter that embryos were plaeed in eaeh of several 1Ox100 mm Pyrex test tubes containing 0.2 ml. PBI at room temperature. Tubes cooled to -6°C and seeded with a seeding forceps to induee ice formatian, then slowly eooled (0.3 C / min.) in 1.5 M DMSO to -33 oc before direct transfer to Iiquid nitrogen. For jreezing embryos a mini-freezer unit type R202/ ıO ıR was used. For thawing the tubes were placed straight into a water bath at 25-30°C and agitated. To reduce osmatic shoek embryos were reeovered and washed in three changes of PBI at room temperature. Mouse embryos were transfered to drops of P Bl under paraffin oil. These emhryos were then cultured to assay viability at 37 oC for 48 hours in 5 % CO 2 in air. Survival was assessed by the ability of the frozen-thawed 8-eel! embryos to develope into expanded blastoeysts during eulture. The highest levels of survival