Epsilon Archive for Student Projects

Abstract

The Annexin-V/propidium iodide (AV/PI) assay detects early membrane changes in spermatozoa and viability using flow cytometry. It is an objective method that evaluates a much larger number of single spermatozoa compared with the usual estimation of motility, the most commonly used method for routine sperm evaluation. In the present study four ejaculates from four stallions were used in a split-sample design to investigate whether and how the number of intact spermatozoa is affected over time and after incubation at different temperatures, either those recorded during shipment of stallion semen for AI (9 or 5°C) or room temperature (20°C). The Annexin-V results did not reflect the motility results that were monitored in parallel. While motility decreased over time, the number of spermatozoa with intact membranes (viable) remained unchanged for at least 21 hours following a short period of initial instability. Sperm motility was better and the percentages of viable spermatozoa were higher in the samples stored at 20° C samples than in the 5°C or 9°C samples. Significant differences between stallions and between ejaculates from the same stallion were also detected. One stallion had very low fertility results but did not differ from the others in terms of either viable or motile spermatozoa. In conclusion, the use of Annexin-V-PI assay combined with flow cytometry is a suitable method, albeit not for routine use, for assessment of early membrane changes in stallion spermatozoa. The method will however not identify all subfertile stallions or predict fertility, but it might, after further studies, be of use as an alternative assay for identifying subfertile stallions.