S. Blottner et al., FLOW-CYTOMETRY ASSESSMENT OF THE ACROSOMAL STATUS IN BULL AND STALLION SPERM USING FITC PEANUT AGGLUTININ, Tierarztliche Umschau, 53(7), 1998, pp. 442
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.