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CHAPTER 3.2
depth but not the chamber brand is mentioned (Aurich and Spergser, 2007; Pagl et al., 2006), or only
the volume used (Price et al., 2008; Quintero-Moreno et al., 2003), and sometimes, nothing at all is
mentioned (Love et al., 2005; Macís-García et al., 2009; Ponthier et al., 2009).One might conclude
that objective semen analysis with CASA systems is not at all standardized in horses.
Also the determination of concentration by means of CASA is not indisputable. Especially the
use of capillary filled 20 µm disposable chambers has been subject of debate after discrepancies
with haemocytometer counts have been described (Kuster, 2005). The variation between these two
techniques has been attributed to the Segre-Silberberg (SS) effect, which describes flow dynamics in
capillary filled chambers resulting in a concentration differential for particles in laminar flow. Based
on a mathematical model, corrections can be made (Douglas-Hamilton et al., 2005a).
In the veterinary clinic of the Department of Reproduction, Obstetrics and Herd Health of
Ghent University, we noticed a huge discrepancy when analyzing an equine semen sample from the
same aliquot with both a Makler chamber and a 20 µm Leja chamber. The obtained concentrations
and motility parameters differed so extremely, that it became clinically relevant. For instance, an
analysis using the Leja chamber could lead to rejection of a sample, ejaculate or stallion, whereas
using the Makler chamber would have resulted in a more favourable outcome.
The major aim of this study was to evaluate the difference between a number of counting
chamber types on equine semen motility parameters. Additionally, the differences in concentration
depending on the chamber type used were scrutinized.
3.2.3. Materials and Methods
3.2.3.1. Semen and preparation for analysis
Fifty straws of frozen semen from 50 different stallions, frozen in two European recognized
artificial insemination centres were used in this experiment. Straws were thawed in a 37°C water
bath for 30 sec and subsequently dried and emptied in a 1.5 mL vial (eppendorf). Concentration was
determined using the NucleoCounter SP-100 (ChemoMetec, A/S, Allerød, Denmark) as described
earlier (Hansen et al., 2006; Morrell et al., 2010). Thawed semen was diluted using INRA96 (IMV,
L’Ailgle cedex, France) at 37°C to obtain a final concentration of 25 – 35 × 10 6 sperm /mL based on
the findings of the NucleoCounter, after which the samples were incubated at 37°C for 5 min prior to
analysis. The dilution ratios varied between 1:7 to 1:14.
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