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CHAPTER 3.4
3.4.3. Materials and Methods
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3.4.3.1. Stallions and semen preparation
The semen used in this experiment was frozen in 0.5 mL straws at two European approved AI
centers in Belgium. The straws were concentrated to contain about 300 × 10 6 spermatozoa per mL.
Four straws per stallion, from the same ejaculate, were thawed in a water bath of 38°C for 30s, dried
and pooled in a small cup, and kept at 38°C. An aliquot of 200µL was diluted tenfold with INRA96
(IMV-technologies, L’Aigle, France), a clear semen extender free of debris when visualized
microscopically. The samples, diluted and undiluted, were incubated at 38°C for 10 minutes prior to
analysis.
3.4.3.2. Analysis with the SQA-Ve
The frozen-thawed semen was analyzed after proper incubation without diluting the thawed
semen according to the manufacturer ‘s guidelines. The semen was aspirated into the specifically
designed capillaries, wiped clean and inserted into the device following onscreen instructions. Prior
to analysis, the SQA-Ve preheats the sample once more during 60 seconds after which the analysis
starts automatically. The SQA-Ve does not require parameter settings, the software version used was
1.00.43 for experiment 1, and 1.00.61 for experiment 2.
When total motility (TM) was equal to or lower than 30%, no actual value is being displayed,
resulting in lack of progressive motility (PM) data. However, the number of progressive motile
spermatozoa (PMS) per mL remains available. As soon as TM is lower than 10%, PMS is not reported
either. As such, analysis with SQA-Ve can either result in actual values (complete data), in limited
data (TM≤30%, and actual value for PMS), or no data at all (TM