104 SURVIVAL AND APOPTOSIS RATES AFTER VITRIFICATION OF EARLY, EXPANDED, AND HATCHED CALF AND COW BLASTOCYSTS IN VITRO-PRODUCED USING CRYOTOP AS A DEVICE

Abstract

Two experiments were designed to determine the ability of 7 and 8 day in vitro-produced blastocysts to survive to the vitrification procedure. Embryos were classified as early blastocysts, expanded, or hatching/hatched blastocysts. Vitrification was done using cryotop devices as described Du et al. (2007). After warming, blastocysts were incubated for 3 h in SOF medium. In the first experiment, we examined the developmental competence of early blastocysts, expanded blastocysts, and hatching/hatched blastocysts after vitrification using the Cryotop method. In the second experiment, warmed blastocysts that had been vitrified on cryotops were fixed in 4% formaldehyde and incubated with TUNEL staining for detecting DNA damaged nuclei. The percentage of TUNEL positive and negative blastomeres was assessed by confocal microscopy. In all experiments cow and calf blastocysts were compared. When the results according to the developmental stage were analyzed, no differences in the survival rates after vitrification of expanded and hatched blastocysts were observed at Day 8 from cow and calf blastocysts. After warming, survival rates of 52.4 and 50% were noted in the groups of expanded and hatched blastocysts respectively from Day 8 cow blastocysts. Similar results were observed in the groups of expanded (54.5%) and hatched (59.4%) blastocysts from Day 8 calf blastocysts. When embryos were vitrified at Day 7, survival rates of 78.4 and 66.7% were observed after warming expanded and hatched blastocysts from cows. In calves, a significant increase in the survival up to 83.3 and 80% was observed after warming expanded and hatched blastocysts. The lowest survival rates were observed in early blastocysts (from 26 to 51%), particularly in those vitrified at Day 8 (≤40%). Following vitrification, cell death was monitored in blastocysts 3 h after warming by TUNEL labelling of cells with damaged DNA. The TUNEL staining procedure was undertaken on Day 7 calf (n = 23) and cow (n = 25) blastocysts, as well as on Day 8 calf (n = 22) and cow (n = 30) blastocysts. When taking into account the stage of blastocyst development, there was a trend toward higher DNA integrity index after warming of expanded and hatched blastocysts compared with early blastocysts in calf and cow groups. So, cell damage was minimal in those blastocysts vitrified at expanded and hatched stage and rates were comparable with those from control fresh blastocysts. These findings suggest that the Cryotop technique seems to be particularly useful for blastocysts presenting a high degree of expansion (expanded and hatched blastocysts), mainly those blastocysts vitrified and warmed at Day 7.