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