Effects of freezing of bovine preimplantation embryos derived from oocytes fertilized in vitro on survival of their inner cell mass cells

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Abstract
The morphology of the inner cell mass (ICM) cells and the proportion of dead ICM cells in frozen‐thawed bovine preimplantation embryos were investigated by differential fluorochrome staining. Embryos at the blastocyst stage of development were frozen and thawed by two different techniques (three‐step and one‐step) in two different basic salt solutions (PBS and TCM 199) containing 1.36M glycerol. After thawing and glycerol removal, embryos were co‐cultured in a cumulus cells monolayer in TCM 199 for 48 hr (morula) or 24 hr (blastocysts). Differential cell counts of the ICM and trophectoderm were then done using differential fluorochrome staining. Overall, there was no significant difference in the viability of embryos frozen in the two basic salt solutions. Low proportions of dead ICM cells were observed in embryos frozen at the morula stage in both PBS (19.1%) or TCM 199 (18.0%). However, blastocyst stage embryos frozen by the three‐step technique had a higher (P < 0.05) proportion of dead ICM cells in TCM 199 (37.7%) than in PBS (18.2%). Blastocysts frozen by the one‐step technique had a higher (P < 0.05) proportion of dead ICM cells (42.2%) than those frozen by the three‐step technique (18.2%), regardless of basic salt solutions. Results indicate that freezing and thawing damages ICM cells in morphologically normal embryos and that the degree of damage depended on the basic salt solution and the freezing method.