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

This study explores the effects of different preservation methods on the DNA fragmentation of granulosa cells from equine oocytes, finding no significant impact on DNA damage, meaning any of the evaluated methods could be used for granulosa cells cryopreservation effectively.

Objective of the Study

• The research aimed at establishing if cryopreservation, a process of freezing biological samples for future use, affects the DNA integrity of granulosa cells from mare oocytes. These cells are sometimes stored before conducting DNA fragmentation tests, whose results could be influenced by preservation methods, hence the necessity of this study.

Procedure and Methodology

• Equine oocytes, or female reproductive cells, were retrieved postmortem from five mares. After washing these granulosa cells by centrifugation, they were then analysed, constituting the control group.
• Four different preservation protocols were employed. P1 involved directly plunging the cells in liquid nitrogen (LN) and storing at -80°C. P2 also used LN/-80°C but introduced cryoprotectants namely, 7.5% ethylene glycol and 7.5% dimethyl sulfoxide. P3 only used -80°C storage while P4 combined -80°C storage with the use of cryoprotectants.
• The preserved cells were analysed using a prototype D3-Ovoselect, a device used to measure DNA fragmentation. DNA analysis was made possible by fluorescent microscopy.

Results and Findings

• When compared by analysis of variance, no notable differences were observed in terms of high, low, and total DNA fragmentation percentages between the preserved granulosa cells and the control group. This suggests that the cryopreservation did not damage the DNA.
• Statistical comparison revealed a P value greater than .05, which indicates that the differences observed were probable due to random chance rather than being a direct outcome of the different preservation methods.

Conclusion

• The findings provide an evidence that all four preservation procedures are capable of efficiently preserving DNA of equine oocyte’s granulosa cells. Hence, cryopreservation does not compromise the DNA integrity and the frozen cells could be employed for future analysis.