Evaluation of DNA Damage of Mare Granulosa Cells Before and After Cryopreservation Using a Chromatin Dispersion Test

Abstract

DNA fragmentation of granulosa cells might be related to developmental competence of the equine oocyte. Granulosa cells are commonly stored before DNA fragmentation assessment, but the effect of preservation methods on this parameter remains unexplored. The aim of this study was to evaluate whether or not cryopreservation of granulosa cells affects the DNA damage. Equine oocytes were recovered from postmortem ovaries of five mares. Granulosa cells were washed by centrifugation and then analyzed (control) or stored in cryovials following four different protocols: P1 = directly plunged in liquid nitrogen (LN2) and then stored at −80°C; P2 = LN2/−80°C adding cryoprotectants (7.5% ethylene glycol + 7.5% dimethyl sulfoxide); P3 = −80°C; P4 = −80°C + cryoprotectants. Granulosa cell samples were processed with the prototype D3-Ovoselect, Halotech DNA, Spain), and DNA was visualized under fluorescence microscopy. High, low, and total DNA fragmentation percentages were compared among treatments by analysis of variance. Results were expressed as mean ± standard error. No significant differences (P >.05) were found among treatments and the control group. Therefore, the four conservation protocols could be considered equally efficient for DNA preservation of granulosa cells from mare oocytes. In conclusion, cryopreservation of granulosa cells in any of the four protocols used adequately preserved the DNA for further analysis.