Method for isolating preantral follicles from mare ovaries

The aims of this study were to evaluate the use of collagenase treatment to isolate preantral follicles from mare ovaries and to assess the effect of this treatment on follicular morphology. Intact mare ovaries were chopped into pieces, incubated individually with 1, 3 or 5 mg collagenase (type 1A) ml(-1) in a shaking waterbath at 37 degrees C for up to 2 h and passed through a series of stainless steel filters with pore size 50-300 microm to remove large clumps and stromal cells. The samples were prepared for histological analysis and sections were examined by light microscopy. Isolated follicles and oocytes were measured and the quality of the follicles was assessed by microscopic examination. Very few intact preantral follicles were isolated after 30 min incubation with 1, 3 or 5 mg collagenase ml(-l). After 60 and 90 min incubations, between eight and 71 intact preantral follicles were isolated with 3 or 5 mg collagenase ml(-1), whereas very few were isolated after incubation with 1 mg collagenase ml(-1). The number of intact follicles isolated after incubation with either 3 or 5 mg collagenase ml(-1) was not significantly different. The quality of the isolated follicles decreased with increasing incubation time and no intact follicles were observed after 2 h of incubation. Preantral follicles 60-300 microm in diameter were isolated from ovaries after treatment with either 3 or 5 mg collagenase ml(-1). Most of the follicles isolated were 90-150 microm in diameter. This study indicates that equine preantral follicles can be isolated from equine ovaries using collagenase and that collagenase does not have a deleterious effect on follicle morphology when used at the appropriate concentration for a minimum period. However, oocyte quality and follicle viability remain to be determined.