view - Department of Reproduction, Obstetrics and Herd Health
view - Department of Reproduction, Obstetrics and Herd Health
view - Department of Reproduction, Obstetrics and Herd Health
Create successful ePaper yourself
Turn your PDF publications into a flip-book with our unique Google optimized e-Paper software.
CHAPTER 4.2<br />
adding 600 µL <strong>of</strong> AO staining solution. The stained samples were analyzed within 3-5 minutes <strong>of</strong> OA<br />
staining using a FACStar Plus flow cytometer (Becton Dickinson, San José, CA, USA) with settings <strong>and</strong><br />
s<strong>of</strong>tware as described by Morrell et al. (2008). The proportion DFI was available for statistical<br />
analysis.<br />
Membrane integrity<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, after thawing in a 37°C water bath for 30s,<br />
the straws were emptied <strong>and</strong> 25 µL <strong>of</strong> semen was mixed with 225 µL HEPES-TALP <strong>and</strong> 1.25 µL<br />
SYBR14 (1:50 dilution) was added. After 5 min <strong>of</strong> incubation at 37 °C, 1.25 µL PI was added <strong>and</strong><br />
incubated for another 5 min at 37°C. Two hundred cells were evaluated per slide using a Leica DMR<br />
fluorescence microscope <strong>and</strong> the proportion <strong>of</strong> membrane intact (MI) sperm cells was calculated<br />
<strong>and</strong> used for statistical analysis.<br />
Acrosomal status<br />
The acrosomal status was determined using fluorescent Pisum Sativum Agglutinin (PSA)<br />
staining conjugated with fluorescein isothiocyanate (FITC) (Sigma-Aldrich cat n°: L 0770, Bornem,<br />
Belgium). The staining was performed in a similar way as described by Rathi et al. (2003). Briefly,<br />
after thawing in a 37°C water bath for 30s, the straws were emptied <strong>and</strong> 400 µL <strong>of</strong> semen was<br />
centrifuged for 10 min at 720 × g after which 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 was<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 in<br />
the dark, washed 10 times with aqua bidest <strong>and</strong> allowed to air-dry in the dark. Immediately after<br />
drying, two hundred sperm cells were evaluated per slide. The acrosomal region <strong>of</strong> the acrosome<br />
intact sperm cells was labeled heavily green, while the acrosome reacted (AR) sperm retained only<br />
an equatorial b<strong>and</strong> <strong>of</strong> staining with little or no labeling <strong>of</strong> the anterior head region. The percentage<br />
<strong>of</strong> AR sperm was used for statistical analysis.<br />
165