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 3.3<br />
a camera, the sperm cells are visualized <strong>and</strong> the actual sperm tracks analyzed. The proper sperm cell<br />
recognition is determined by s<strong>of</strong>tware settings. These systems produce accurate <strong>and</strong> highly<br />
repeatable data <strong>of</strong> different semen-motility parameters in different species (Verstegen et al., 2002).<br />
However, h<strong>and</strong>ling procedures <strong>and</strong> parameter settings still differ between laboratories, making it<br />
difficult to compare results (Davis <strong>and</strong> Katz, 1993; Hoogewijs et al., 2009; Verstegen et al., 2002).<br />
112<br />
Unlike CASA systems, the SQA does not require parameter settings, hence reducing a large<br />
source <strong>of</strong> potential bias (H<strong>of</strong>lack et al., 2005), making this a ‘practical’ device which might be a<br />
bridge between subjective light microscopic assessments <strong>and</strong> highly specialized CASA interpretations.<br />
The aim <strong>of</strong> this study was to investigate the agreement <strong>and</strong> repeatability <strong>of</strong> two different<br />
automated semen analyzers, the SQA-Ve <strong>and</strong> CASA, to assess equine semen quality using<br />
conventional subjective light microscopy (LM) as the gold st<strong>and</strong>ard.<br />
3.3.3. Materials <strong>and</strong> methods<br />
3.3.3.1. Stallions <strong>and</strong> semen preparation<br />
For this study, a total <strong>of</strong> 60 ejaculates (six ejaculates per stallion, 5 Shetl<strong>and</strong> ponies <strong>and</strong> 5<br />
Warmblood stallions, respectively) was collected. After collection, semen was filtered through sterile<br />
gauze to remove the gel fraction <strong>and</strong> debris. For 30 ejaculates (3 per stallion), one part <strong>of</strong> the raw<br />
ejaculate, approximately 5 mL <strong>of</strong> fresh semen, was transferred into a sample container <strong>and</strong> placed in<br />
the preheated block heater. The rest <strong>of</strong> the semen was diluted 1:5 with INRA 96 <strong>and</strong> transferred in a<br />
sample container <strong>and</strong> placed in the heating block. The other 30 ejaculates were, after filtration,<br />
processed in the latter way, i.e. no analyses were performed on the undiluted semen.<br />
3.3.3.2. Analysis<br />
Light microscopy<br />
Subjective analysis <strong>of</strong> motility was performed as described by the World <strong>Health</strong> Organization<br />
(WHO, 2010), <strong>and</strong> considered to be the gold st<strong>and</strong>ard for this study. Briefly, a fixed volume <strong>of</strong> 10 µL<br />
<strong>of</strong> preheated (37°C) diluted semen was placed on a preheated clean glass slide <strong>and</strong> covered with a<br />
22 mm × 22 mm coverslip <strong>and</strong> transferred to the warmed (37°C) stage <strong>of</strong> the phase contrast<br />
microscope. At least five microscopic fields were assessed to classify 200 spermatozoa. Individual