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