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CHAPTER 4.2
adding 600 µL of AO staining solution. The stained samples were analyzed within 3-5 minutes of OA
staining using a FACStar Plus flow cytometer (Becton Dickinson, San José, CA, USA) with settings and
software as described by Morrell et al. (2008). The proportion DFI was available for statistical
analysis.
Membrane integrity
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, after thawing in a 37°C water bath for 30s,
the straws were emptied and 25 µL of semen was mixed with 225 µL HEPES-TALP 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 at 37°C. Two hundred cells were evaluated per slide using a Leica DMR
fluorescence microscope and the proportion of membrane intact (MI) sperm cells was calculated
and used for statistical analysis.
Acrosomal status
The acrosomal status was determined using fluorescent Pisum Sativum Agglutinin (PSA)
staining 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,
after thawing in a 37°C water bath for 30s, the straws were emptied and 400 µL of semen was
centrifuged for 10 min at 720 × g after which 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 was
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 in
the dark, 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 (AR) sperm retained only
an equatorial band of staining with little or no labeling of the anterior head region. The percentage
of AR sperm was used for statistical analysis.
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