Flow cytometry assessment of the acrosomal status in bull and stallion sperm using FITC peanut agglutinin

The acrosome reaction is an essential step in the process of fertilisation. The assessment of acrosomal status and reaction is therefore important for the characterisation of sperm function for cryopreservation, artificial insemination or in vitro fertilisation. The use of flow cytometry as a simple and rapid method was examined using FITC-conjugated peanut agglutinin (Arachis hypogaea, PNA), for staining fresh stallion sperm and frozen stallion and bull sperm. The study involved a comparison with the results of fluorescence microscopy and FCM before and after the acrosome reaction induced by the inonophore A23187. PNA exhibited a strong binding to the outer acrosomal membrane of intact sperm from both species. Acrosome intact sperm exhibited a bright fluorescence over the head region, whereas the acrosome reacted sperm exhibited fluorescence at the equatorial segment only. These typical patterns produced two clearly distinguished peaks in the flow cytometry histogram. The percentage of acrosome intact bull sperm determined from these peaks was highly correlated to the number of intact sperm estimated by microscopy (r = 0.944, p < 0.001). The percentage of spermatozoa without a stained acrosome was examined by counterstaining with PI and was negligible in bull semen. Fresh stallion sperm had 9.07 +/- 1.35% with unlabelled acrosomes which increased so more than 30 percent after cryopreservation. The acrosome stained sperm revealed additional patterns after freezing/thawing not allowing a simple FCM analysis. The results indicate that FCM is suitable as a rapid one parameter test to quantify the spontaneous and induced acrosome reaction in cattle. The method has to be proven for each species in comparative studies.