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 fertilisa tion. The assessment of acrosomal status and reaction is therefore imp ortant for the characterisation of sperm function for cryopreservation , artificial insemination or in vitro fertilisation. The use of flow c ytometry as a simple and rapid method was examined using FITC-conjugat ed peanut agglutinin (Arachis hypogaea, PNA), for staining fresh stall ion sperm and frozen stallion and bull sperm. The study involved a com parison with the results of fluorescence microscopy and FCM before and after the acrosome reaction induced by the inonophore A23187. PNA exh ibited a strong binding to the outer acrosomal membrane of intact sper m from both species. Acrosome intact sperm exhibited a bright fluoresc ence over the head region, whereas the acrosome reacted sperm exhibite d fluorescence at the equatorial segment only. These typical patterns produced two clearly distinguished peaks in the flow cytometry histogr am. The percentage of acrosome intact bull sperm determined from these peaks was highly correlated to the number of intact sperm estimated b y microscopy (r = 0.944, p < 0.001). The percentage of spermatozoa wit hout a stained acrosome was examined by counterstaining with PI and wa s negligible in bull semen. Fresh stallion sperm had 9.07 +/- 1.35% wi th unlabelled acrosomes which increased so more than 30 percent after cryopreservation. The acrosome stained sperm revealed additional patte rns after freezing/thawing not allowing a simple FCM analysis. The res ults indicate that FCM is suitable as a rapid one parameter test to qu antify the spontaneous and induced acrosome reaction in cattle. The me thod has to be proven for each species in comparative studies.