Cytofluorescent assay to quantify adhesion of equine spermatozoa to oviduct epithelial cells in vitro

To facilitate the study of interactions between equine spermatozoa and homologous oviduct epithelial cells, we developed an assay to count labelled spermatozoa bound to oviduct epithelial cell (OEC) monolayers and used the assay to compare the binding ability of spermatozoa from different stallions. Washed spermatozoa from three stallions were incubated with the fluorochrome Hoechst 33342 (5 μg/ml) for 1 min. Spermatozoa were then layered over confluent monolayers of oviduct epithelial cells in 2 cm² culture wells. Coculture treatments comprised five concentrations of spermatozoa (10⁵, 5 x 10⁵, 10⁶, 2.5 x 10⁶, and 5 x 10⁶ per well). Cocultures were incubated for 30 min before unattached spermatozoa were aspirated in coculture supernatant. Fluorescent videoimages of attached spermatozoa were digitized, and attached spermatozoa were counted by image processing and analysis. Four wells (replicates) of each concentration were allocated within each ejaculate, and ejaculates were blocked by stallion for ANOVA. The total number of spermatozoa bound was not different between replicate wells (P > 0.1). Stallion, ejaculate, concentration, and all higher level interactions influenced total spermatozoa bound (P < 0.00001). Coefficients of variation between replicates were lowest for inseminate concentrations between 10⁶ and 5 x 10⁶ spermatozoa per well. Within the ejaculate, a log linear relationship exists between the number of bound spermatozoa and a spermatozoal concentration of the inseminate between 5 x 10⁵ and 5 x 10⁶ spermatozoa per well. This assay provides a reliable method of determining numbers of spermatozoa bound to somatic cells in vitro. Furthermore, differences exist in the ability of spermatozoa from different stallions to bind OEC monolayers.