Survival and apoptosis rates after vitrification in cryotop devices of in vitro-produced calf and cow blastocysts at different developmental stages

Abstract

Two experiments were designed to determine the ability of in vitro-cultured blastocysts at different stages of development to survive the vitrification procedure using cryotop devices. Day 7 and Day 8 embryos were classified as non-expanded, expanded or hatching and/or hatched blastocysts. In the first experiment, we examined the survival rate of vitrified–warmed blastocysts after 3 h incubation in synthetic oviducal fluid (SOF) medium. In the second experiment, vitrified–warmed blastocysts were evaluated using the terminal deoxyribonucleotidyl transferase-mediated dUTP–digoxigenin nick end-labelling (TUNEL) technique to detect nuclei with damaged DNA. In both experiments, results for cow and calf blastocysts were compared. No differences in survival rates were observed after vitrification of Day 8 expanded (52.4%) and hatched (50%) cow blastocysts or Day 8 expanded (54.5%) and hatched (59.4%) calf blastocysts. When embryos were vitrified on Day 7, survival rates of 78.4% and 66.7% were observed after warming expanded and hatched cow blastocysts, respectively, compared with rates of 80% and 76.9%, respectively, for calf blastocysts. Lowest survival rates were recorded for non-expanded blastocysts (26%–54%) compared with the other developmental stages, particularly those vitrified at Day 8 (≤40%). The DNA integrity index obtained after vitrification–warming was comparable to that for control fresh blastocysts, regardless of the length of embryo culture, the developmental stage of the embryo or the source of the oocytes. Our findings suggest that the cryotop vitrification method is particularly useful for the cryopreservation of blastocysts presenting with a high degree of expansion (expanded or hatched blastocysts), particularly when vitrification is performed after 7 days of in vitro embryo culture.