The mean of six counts was calculated and provided as
the actual sperm concentration given in numbers per mL
[28]. The counts and measurements were conducted using
a Nikon Eclipse E600 microscope equipped with a Sony
Exwave HAD camera and Biocom Visual 2000 software. The
spermatocrit was determined using semen collected
into standard microhematocrit tubes (75 mm length and
1.1–1.2 mm inner Ø) and centrifuged at 3000 rpm for
10 minutes [29].
The activity of spermatozoa was measured by using the
computer-assisted sperm motion analysis system. Microscope
stage and chambers were chilled at 15 2.0 C.
Using a 50-Hz video camera (Panasonic wv-BL600) connected
to an S-VHS-video recorder (Panasonic AG-7350),
sperm activity was recorded and later analyzed. The total
magnification was 320-fold. For digital image analysis, a
PC with appropriate hardware and Mika motion analyzer
software were used. One microliter of diluted sperm (2 mL
of semen in 25 mL of a nonactivating solution [300 mOsm/
kg sucrose, pH ¼ 7.4]) was used for each measurement.
Motility was recorded at 15 and 30 seconds after activation.
Only sperm samples showing 80% motility or higher
were used for the experiments [30]. Each sample was
analyzed three times. The number of cells exceeding the
previously established minimum motile speed was used to
determine percent motility, curvilinear velocity, and
straight-line velocity, which were estimated on this motile
fraction.