Influence of storage at 5 degrees C and vitrification on apoptotic changes in equine blastocysts

The aim of the study was to determine the extent of nuclear DNA fragmentation in equine embryos at the blastocyst stage stored at 5 degrees C for 6 h and 24 h or vitrified in straws or in the Rapid-i system. An additional aim was to determine the relationship between the age of fresh and stored embryos and the intensity of apoptosis. In this study, 42 embryos were analyzed by the TUNEL method and allocated to the following groups: fresh (control -14 embryos), stored at 5 degrees C for 6 h (7 embryos), stored at 5 degrees C for 24 h (7 embryos), vitrified in straws (7 embryos), and vitrified in the ultra-fast Rapid-i system (7 embryos). It was found that 36.7% of fresh equine embryos had apoptotic nuclei, and the mean dead cell index (DCI) was 0.3%. This was evident as an increase in DNA fragmentation in equine embryos after vitrification in straws (2.3%) compared to fresh embryos (0.3%) and those vitrified in the Rapid-i system (0.3%). Embryo storage at 5 degrees C did not affect the apoptosis frequency in nuclei regardless of the time of storage. DCI for embryos stored for 6 h was 0.44%, and after 24 h of incubation it was 0.64%. The intensity of apoptotic changes observed in equine embryos depended on their age and size. The apoptosis frequency was negatively correlated with the age and diameter of fresh embryos, embryos stored at 5 degrees C, and embryos vitrified in Rapid-i, whereas for embryos vitrified in straws the correlation was positive.