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