Large-scale isolation of equine zonae pellucidae

Abstract

A simple and rapid method is described for the large-scale isolation of cumulus cell-free, zona pellucida-intact equine oocytes. Aspiration of palpable antral follicles present on frozen-thawed equine ovaries was accomplished using a constant vacuum source. The resultant follicular fluid, oocytes, and particulate matter were then filtered through a series of nylon screens of alternating mesh openings in combination with sodium citrate-containing buffer to a final volume of approximately 20 ml. This fluid was transferred to scored Petri dishes and a stereomicroscope was used to locate the oocytes for futher processing or storage. The methodology described is inexpensive, time-efficient, and the recovery rate is similar to or better than other methods previously described for equine oocyte recovery. Collected oocytes are adequate for biochemical evaluation of the equine zona pellucida (EZP) as well as sperm-egg binding assays.