Fluorescent labelling of boar spermatozoa for quantitative studies on competitive sperm-oviduct binding

In vitro sperm-oviduct binding assays enable assessment of the capacity of spermatozoa to form a 'reservoir' in the oviduct. Competitive approaches, such as experimental set-ups that test multiple males or semen samples simultaneously on the same tissue explants, are desirable because they reduce the likelihood of bias when using material from different females. Therefore, we established a fluorescent labelling technique that allows tagging and storage of spermatozoa before competitive studies of sperm-oviduct binding invitro. Fluorescent markers were tested for reliability and compatibility with parameters of boar spermatozoa viability. The addition of seminal plasma after density gradient centrifugation was essential to counteract centrifugation stress during the labelling procedure. It was demonstrated that sperm tagged with MitoTracker Green FM or MitoTracker Red FM can be successfully used in competitive sperm-oviduct binding studies. The assay was sensitive enough to indicate subtle effects of semen storage temperature on the ability of the spermatozoa to contribute to the female sperm reservoir