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Fluorescent Activated Cell Sorter
CHAPTER 1.1
A fluorescent activated cell sorter (FACS) has been evaluated to determine the concentration
of boar semen. The FACS showed a very good correlation and the highest repeatability (Hansen et al.,
2006). This is not surprising since this flow cytometric technique uses an internal standard of
fluorescent beads with every analysis.
Haemocytometer
Since the accurate and precise devices (NucleoCounter SP-100 and FACS) are all rather
expensive, it is not useful to propose them as the “gold standard”. Therefore, the haemocytometer is
most likely the best alternative as standard for concentration determination, in concordance with
human andrology. The counting chamber type is known to have a major influence on the outcome of
sperm concentration. In shallow chambers, loaded with capillarity, the SS effect has an influence on
concentration (Douglas-Hamilton et al., 2005a, 2005b). In the Makler chamber (Fig. 2a), a reusable
chamber with a 10 µm depth, the cover glass is placed upon the loaded area, the time interval
between loading and applying the cover glass is a major factor influencing the obtained
concentration (Matson et al., 1999). The Makler chamber has been documented as having a low
precision (Christensen et al., 2005), and the time interval before applying the cover slide might
attribute to this imprecision (Makler, 2000). Different haemocytometers result in different outcomes,
especially when chambers with different depths are used. Therefore, it might be advisable to
recommend the use of a standard counting chamber. In accordance with the WHO, the improved
Neubauer haemocytometer (Fig. 2b) could be the chamber of preference. This chamber was also
described for use in horses (Vanderwall, 2008). Alternatively, a Bürker haemocytometer can be used
as well (Kuisma et al., 2006). Before filling a haemocytometer, correct placement of the cover slip can
be verified by looking for the presence of iridescence (multiple Newton’s rings) between the two
glass surfaces (WHO, 2010). Following a 1:100 dilution (Vanderwall, 2008) of the semen with a
fixative to immobilize the spermatozoa, the well mixed sample is loaded into the chamber. The filled
chamber should be allowed to rest horizontally (>4 min) at room temperature in a humified chamber
to prevent evaporation. The immobilized cells will sediment onto the grid and this will facilitate
counting. When assessing concentration using a haemocytometer, it is advised to fill and count
duplicate chambers to reduce the variation (Freund and Carol, 1964). For assessing concentration
using a haemocytometer the use of cover slips with a thickness of 400 µm is advised (WHO, 2010).
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