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3.1.1. Abstract
CHAPTER 3.1
The repeatability of the analysis of frozen-thawed equine semen using a CASA system is high.
However, the settings used to analyze the semen samples influence the results to a large extent.
Total and progressive motility differed on average 7% between two different settings, although
results were highly correlated. We concluded that settings used in different scientific studies and for
semen reports should be described clearly, facilitating comparison of results.
3.1.2. Introduction
The widespread use of frozen-thawed semen in the equine breeding industry creates
opportunities for breeders as well as stallion owners. Semen can be shipped all over the world so
that any given stallion may become available for any given mare. Inseminations with frozen-thawed
semen, however, are less successful compared to inseminations with cooled and especially fresh
semen. In order to optimize pregnancy outcome, frozen-thawed semen should fulfill a few criteria
such as a minimal total number of spermatozoa per dose and minimal progressive motility. E.g., the
insemination dose for conventional artificial insemination (AI) using frozen-thawed semen should at
least contain 240 × 10 6 progressively motile spermatozoa (PMS) (Loomis, 2001). The PM should at
least be 30% (Loomis, 2001) whereas the World Breeding Federation for Sport Horses advocates a
PM of even 35% (WBFSH, 2010).
Computer assisted sperm analysis (CASA) is accepted as an accurate tool for motility analysis.
A number of motility settings for CASA have been described. In this paper we investigated the
repeatability of CASA. Furthermore the influence of two different CASA motility settings on an
number of sperm parameters when analyzing frozen-thawed semen was investigated.
3.1.3. Materials and Methods
The semen used in this experiment was frozen in 0.5 mL straws at two European approved AI
centers in Belgium. Individual straws of different Warmblood and Arabian stallions (n=63) were
thawed in a water bath of 37 - 38°C for 30s, dried and emptied in a small cup. An aliquot of 200 µL
was diluted with INRA96 ® (IMV-technologies, L’Aigle, France), a good quality semen extender free of
debris when visualized microscopically to a final concentration of 35 - 40× 10 6 /mL. The diluted
samples were incubated at 37-38°C for 10 minutes prior to analysis. Each sample was analyzed with
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