Abstract

Survival of sperm within the cow reproductive tract is dependant on the binding of the sperm to the oviductal epithelial cells (OEC) and is a prime determinant of fertility. An assay of sperm survival was set up using the relative binding of sperm to cultured OEC on multi-well plates. A rapid accurate method of counting sperm in the presence of OEC was developed. A precise volume of cell suspension was mixed with a known number of fluorescent micro-beads and the mixture demembranated with detergent, stained with a DNA-binding fluorescence probe and analysed by flow cytometry. Correlations were obtained between flow cytometry and spectrophotometer measurements (R2 = 0.9815, P<0.001) or manual haemocytometer counts (R2 = 0.9995, P<0.001). Sperm was loaded into a cell culture well with confluent monolayers of OEC. After incubation sperm cells remaining in suspension in the well were removed. The difference in the number of sperm in the initial sample and that removed from the well indicated the number of sperm bound to OEC. While, there were large between-well variations for the same sperm sample, the assay was still able to distinguish differences between bulls in the binding ability of their sperm. Because a minimum of 10 wells per treatment are needed to statistically detect the difference, coupled with the high effort and time requirement in preparing the culture plates, the assay is impractical for large-scale experimental assays or for routine semen assessment.

J, Krzyzosiak, RM McDonald, R Vishwanath, and JF Smith

Proceedings of the New Zealand Society of Animal Production, Volume 62, Palmerston North, 330-333, 2002