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Table 1. Software settings of the Hamilton Thorne Ceros 12.3 used in this study
Parameter Value
Frames acquired 30
Frame rate (Hz) 60
Minimum contrast 60
Minimum cell size (pixels) 6
Minimum static contrast 25
Straightness cut-off (%, STR) 75
Average-path velocity cut-off PM (µm/s,VAP) 50
VAP cut-off static cells (µm/s) 20
Cell intensity 100
Static head size 0.55 – 2.04
Static head intensity 0.45 – 1.70
Static elongation 11 - 99
CHAPTER 4.1
Membrane integrity was evaluated using a fluorescent SYBR14-Propidium Iodide (PI) staining
technique (Molecular Probes cat n°: L-7011, Leiden, The Netherlands) based on a previously
described method (Garner and Johnson, 1995). Briefly, 225 µL HEPES-TALP was mixed with 25 µL of
diluted semen and 1.25 µL SYBR14 (1:50 dilution) was added. After 5 min of incubation at 37 °C, 1.25
µL PI was added and incubated for another 5 min. Two hundred cells were evaluated per slide using
a Leica DMR fluorescence microscope and three populations of sperm cells could be identified
(green = living, red = dead and orange = moribund).
Acrosomal status was determined using fluorescent Pisum Sativum Agglutinin (PSA)
conjugated with fluorescein isothiocyanate (FITC) (Sigma-Aldrich cat n°: L 0770, Bornem, Belgium).
The staining was performed in a similar way as described by Rathi et al. (2003). Briefly, 500 µL of
semen was centrifuged for 10 min at 720 × g and the pellet was resuspended with HEPES-TALP. The
semen was centrifuged again for 10 min at 720 × g, the supernatant was removed and the pellet
fixed in 100 µL absolute ethyl alcohol (Vel cat n°: 1115, Haasrode, Belgium) and cooled for 30 min at
4 °C. A drop of 20 µL semen was smeared on a glass slide and 40 µL PSA-FITC (2 mg PSA-FITC diluted
in 2 mL phosphate buffered saline (PBS)) was added. The glass slide was kept at 4 °C for 15 min,
washed 10 times with aqua bidest and allowed to air-dry in the dark. Immediately after drying, two
hundred sperm cells were evaluated per slide. The acrosomal region of the acrosome intact sperm
cells was labeled heavily green, while the acrosome reacted sperm retained only an equatorial band
of label with little or no labeling of the anterior head region.
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