Zona pellucida-sperm binding assay for equine oocytes

The binding of a spermatozoon to the zona pellucida is the first step in fertilization. The number of spermatozoa bound to a zona pellucida may reflect the functional status of both the oocyte and spermatozoa. The aim of the present study was to determine whether the stage of maturation of the equine oocyte affects the capacity of the zona pellucida to bind with spermatozoa. Cumulus-oocyte complexes (COCs) were collected from the ovaries of mares from abattoirs or were obtained in vivo by ultrasound-guided follicular aspiration. Oocytes were inseminated directly after collection or after 30 h of in vitro maturation (IVM) with semen from the same ejaculate. The results indicate that the immature equine oocytes collected from abattoirs and inseminated directly after collection bound fewer spermatozoa than did mature (metaphase I or II) oocytes inseminated after 30 h of IVM. A similar correlation was observed with the oocytes collected in vivo. The mean number of spermatozoa bound by mature oocytes collected from preovulatory follicles and inseminated directly after collection was 324 +/- 29, whereas the mean number of spermatozoa bound by immature oocytes from nonpreovulatory follicles inseminated without maturation was 222 +/- 26 (P < 0.05). This difference was not observed in the oocytes inseminated after maturation. It is postulated that immature equine oocytes bind fewer spermatozoa than do oocytes matured either in vivo or in vitro.