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Effect of cryopreservation on development and ultrastructure of preimplantation embryos from the dasyurid marsupial Sminthopsis crassicaudata

The effect of different cryopreservation methods on the development and ultrastructure of preimplantation embryos of Sminthopsis crassicaudata, a small carnivorous marsupial and member of the family Dasyuridae, was investigated. Females were primed with 1 iu pregnant mares' serum gonadotrophin to induce oestrus and ovulation. Mating generally ensued and, approximately 6 days after priming, embryos were collected and cultured in 5% CO₂ in air at 35°C for 18–22 h in either Dulbecco's modified Eagles medium (DMEM) with high glucose or human tubal fluid medium (HTF), both supplemented with 10% fetal calf serum. Cleavage rates were higher in DMEM than in HTF. One slow and two ultrarapid freezing methods were used. Two out of 12 (17%) embryos cleaved in culture after freezing and thawing using the slow regimen, compared with six of 16 (38%) non-frozen controls. In addition, two of 11 (18%) embryos cleaved in culture following ultrarapid freezing and thawing by one of the two methods, compared to 31 of 41 (76%) non-frozen controls. Most of the embryos appeared morphologically normal under the light microscope after freezing and thawing by the slow regimen, but considerable variation in the degree of ultrastructural damage to the cellular organelles was evident with the transmission electron microscope. The rather low rate of cleavage after freezing and thawing was probably due, at least in part, to ultrastructural damage of the cells.