Evaluation of the acrosome reaction and viability in buffalo
spermatozoa using two staining methods: the effects of heparin and calcium
ionophore A23187.
Kitiyanant Y, Chaisalee B, Pavasuthipaisit K.
Department of Anatomy, Faculty of Science, Institute of Science and Technology
for Research and Development, Mahidol University, Nakhon Pathom, Thailand. scykt@mahidol.ac.th
In this study, we investigated the effect of heparin and calcium ionophore
A23187 on in vitro induction of buffalo sperm acrosome reaction (AR). Two
methods for detection of the AR and viability were employed. Fluorescein
isothiocyanate-conjugated Arachis hypogea agglutinin (FITC-PNA) was used as a
vital stain in combination with ethidium homodimer-1 (EthD-1) to determine the
acrosome status of viable spermatozoa. In another experiment, trypan blue
replaced EthD-1 to differentiate live and dead spermatozoa having undergone AR.
The results from the two methods were significantly correlated (r > 0.9). Four
different staining patterns were found in both methods. The FITC-PNA intensely
labels the acrosome region of acrosome-intact spermatozoa. EthD-1 and trypan
blue stained red and blue at the post-acrosomal region of dead spermatozoa,
respectively. Spermatozoa incubated with heparin showed a significant increase (
p < 0.05) in the percentage of live acrosome-reacted sperm after 30 min
incubation period. This trend continued and was significantly different over the
entire incubation period when compared with the control group at the same
interval. In the ionophore-treated group, the proportion of changes in live
acrosome-intact and live acrosome-reacted spermatozoa was statistically
significantly different ( p < 0.001) when compared with those treated with
heparin at the same interval. The AR occurred sooner and to a greater extent
when incubated with the ionophore but at 5 h of incubation the percentage of
false acrosomal reaction was significantly higher than those in the control and
heparin-treated groups. The results in this study indicated that the in vitro
induction of AR by heparin and calcium ionophore evaluated by both methods could
be used to assess sperm fertilizing capacity for in vitro fertilization of this
species.