Method for isolating preantral follicles from mare ovaries.

This research explored the use of collagenase treatment to isolate preantral follicles from equine ovaries and assessed the impact it may have on follicular health. The results showed that collagenase can be utilized to isolate these follicles without any harmful impact on their morphology, if used at an optimal concentration and time.

Methodology

• The study started with mare ovaries, which were processed into smaller pieces.
• These pieces were then individually treated with different concentrations of collagenase (1, 3, or 5 mg). Each enzyme concentration was used for different incubation periods, up to 2 hours.
• The treated samples were passed through a series of filters to remove any large clumps and non-target cells.

Analysis and Results

• Following the isolation, the samples were prepared for histological analysis. Light microscopy was used to examine the sections, measure the isolated follicles and oocytes, and assess follicle health.
• The research showed that few follicles were successfully isolated after a short 30-minute incubation, no matter the collagenase concentration used.
• However, when the incubation period was extended to 60 or 90 minutes, more follicles were isolated, particularly at higher collagenase concentration (3 or 5 mg). But, there was no significant difference in the number of follicles isolated between the 3 mg and 5 mg collagenase treatment.
• It was also noted that the quality of the isolated follicles decreased when the incubation time was extended beyond 90 minutes, with no intact follicles observed after 2 hours of incubation.

Conclusion

• The results of the study indicate that collagenase treatment can be useful for the isolation of preantral follicles from mare ovaries without negatively affecting their morphology — provided the enzyme is used at suitable concentration and incubation time.
• Notably, most of the follicles that were successfully isolated ranged between 90-150 microm in diameter.
• However, it’s important to note that even though the morphology of follicles seems unaffected, the study did not conclusively define the implications of this treatment on oocyte quality and follicle viability.