Effect of cryopreservation techniques on proliferation and apoptosis of cultured equine ovarian tissue.

The research investigates the effects of two cryopreservation techniques on equine ovarian tissue, comparing the functionality, integrity, and proliferative ability of the tissue after cryopreservation. It was found that both slow-freezing (SF) and vitrification (VIT) were effective in preserving follicle morphology and ensuring similar tissue functionality after a period of 7 days in in vitro culture.

Objective of the Study

• The study was aimed at examining the effects of slow-freezing (SF) and vitrification (VIT) methods on the equine ovarian tissue after undergoing in vitro culture (IVC) for varying durations.
• Focus was given to the morphological integrity of preantral follicles and their distribution, and to observing cell proliferation and apoptosis factors in the ovarian tissue.

Methodology and Observations

• The number of normal primordial follicles in slow-freezing control, vitrification control, and fresh control groups showed no significant statistical difference.
• After 7 days of IVC, the total percentage of normal preantral follicles in the VIT group was higher compared to the SF group. However, the fresh group outperformed both of these groups.
• There was an insignificant difference in expression levels of EGFR and Ki-67(proliferation markers) amongst fresh, SF and VIT groups both before and after the 7 day culture period.
• Bax (a marker of apoptosis) expression was higher in the VIT group after 7 days of culture compared to the fresh and SF groups, but Bcl-2 levels (another apoptosis marker) were similar across all groups.
• TUNEL signals, which indicate DNA fragmentation, were similar among all groups prior to the IVC. However, the VIT group exhibited a greater number of TUNEL signals after the 7-day culture compared to the fresh group, suggesting more DNA fragmentation.

Conclusion and Implications

• Both SF and VIT were found to be effective in preserving the morphological integrity of follicles compared to the fresh control group. They also offered similar tissue functionality and cell proliferation capability after equine ovarian tissue cryopreservation (OTC) over a 7-day in vitro culture period.
• The research provides critical insights for equine fertility preservation programs using OTC techniques and suggests both SF and VIT can be used reliably for this purpose.