Ultrastructure of Bovine Embryos Frozen and Thawed by a Two-Step Freezing Method

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Abstract
An ultrastructural evaluation of a rapid tow-step freezing method, by which 6–7-day-old bovine embryos equilibrated in 1.4 M glycerol in Dulbecco’s phosphate-buffered saline were frozen and thawed, was undertaken. In all non-frozen control embryos trophoblastic and embryonic cells formed a spherical structure enclosed by an intact zona pellucida. The spacial arrangement of the cells of the frozen embryos was less regular and the surrounding zona pellucida was damaged in approximately half of the cases. Some embryonic cells had increased electron density and lysosomal content showing reaction sites for acid phosphatase. In all frozen embryos, cytoplasmic defects appearing as non-membrane-bound ‘empty spaces’ were observed more frequently in the trophoblastic cells than in the embryonic cells. Culture of frozen embryos for 24 h revealed that cells appearing non-defective after culture may have the capability of organizing a viable embryonic structure. It was found that the most commonly used freezing method is associated with certain morphological changes. However, no additional cryoinjuries were observed in comparisons with the more complicated freezing procedures using dimethylsulfoxide as cryoprotectant.

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