Sperm binding capacity and ultrastructure of the zona pellucida of stored canine oocytes

Abstract Sperm binding to the zona pellucida is a prerequisite for fertilization, and tests that evaluate this function have been described for several species. When carrying out such tests in the canine species, ovaries or oocytes have to be stored to obtain a sufficient number of oocytes at the time of testing. In the present study, the sperm binding capacities of salt-stored oocytes and oocytes from deep frozen ovaries were measured and compared with that of fresh oocytes. Two different procedures for washing the sperm-oocyte complexes (gentle and tough) were used before evaluating the number of bound spermatozoa. The total number of oocytes that bound spermatozoa was significantly lower for both salt-stored and deep frozen oocytes compared with fresh oocytes. Significantly fewer spermatozoa bound to stored oocytes than to fresh oocytes (P </= 0.05) irrespective of washing procedure and there was no significant difference between the two methods of storage. Scanning electron microscopy revealed that the zona pellucida of fresh oocytes was sponge-like in appearance with fenestrations surrounded by a meshwork of filaments. In stored oocytes, the meshwork tended to be wider, with larger fenestrations. The thickness of the zona pellucida was 0.3-1.0 microm in salt-stored oocytes and 6-8 microm in oocytes from frozen ovaries, whereas it was 3-6 microm in fresh oocytes. Changes in morphology that occur under the various storage conditions tested might have contributed to the reduced sperm binding capacity of the stored oocytes. Although stored canine oocytes do not bind as many spermatozoa as do fresh oocytes, the former can be used in a zona pellucida binding assay, and the deep freezing of the ovaries appears to be a better storage method than storing oocytes in salt solution.