view - Department of Reproduction, Obstetrics and Herd Health
view - Department of Reproduction, Obstetrics and Herd Health
view - Department of Reproduction, Obstetrics and Herd Health
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CHAPTER 4.1<br />
142<br />
4.1.3.3. Semen processing<br />
After determination <strong>of</strong> the initial sperm concentration, the semen was diluted to a final<br />
concentration <strong>of</strong> 25 × 10 6 sperm /mL using Gent Extender. Six conical bottom centrifuge tubes <strong>of</strong> 15<br />
mL (Cellstar ® , Greiner bio-one, Germany) were filled with the diluted semen (total <strong>of</strong> 375 × 10 6 sperm<br />
cells per tube) <strong>and</strong> served as the negative control or were subjected to one <strong>of</strong> the five different CPs,<br />
respectively. After centrifugation, 90% <strong>of</strong> the supernatant was aspirated, leaving a sperm pellet with<br />
a volume <strong>of</strong> approximately 1.5 mL.<br />
The concentration in the aspirated supernatant was determined using a Neubauer<br />
haemocytometer. The obtained concentration was multiplied by the volume <strong>of</strong> aspirated<br />
supernatant in order to calculate the total number <strong>of</strong> sperm cells lost per tube when the<br />
supernatant was discarded. The remaining sperm pellet was resuspended in the appropriate diluter<br />
for further processing as cooled or frozen semen.<br />
4.1.3.4. Evaluation <strong>of</strong> sperm characteristics <strong>and</strong> staining procedures<br />
Prior to analysis, an aliquot (500 µL) <strong>of</strong> semen was equilibrated to 37 °C. The morphology <strong>of</strong><br />
sperm cells was examined on eosin-nigrosin stained smears which were prepared as described by<br />
Barth <strong>and</strong> Oko (1989). At least 200 sperm cells were evaluated <strong>and</strong> recorded per slide, individual<br />
morphological abnormalities were noted according to their location (head, midpiece or tail).<br />
Motility was evaluated with a computer-assisted sperm analyzer (CASA) (Hamilton-Thorne<br />
Ceros 12.3). For each analysis, 5 µL <strong>of</strong> diluted semen was mounted on a disposable Leja counting<br />
chamber (Orange Medical, Brussels, Belgium) <strong>and</strong> maintained at 37 °C using a minitherm stage<br />
warmer. Five r<strong>and</strong>omly selected microscopic fields in the center <strong>of</strong> the slide were scanned 5 times<br />
each, obtaining 25 scans for every semen sample. The mean <strong>of</strong> the 5 scans for each microscopic field<br />
was used for the statistical analysis (Rijsselaere et al., 2003). The s<strong>of</strong>tware settings <strong>of</strong> the HTR 12.3,<br />
based on Loomis <strong>and</strong> Graham (2008), are summarized in Table 1.