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CHAPTER 1.2
0.5 mL straws, and glycerol concentration was not corrected for the different concentrations. Later
on, Clulow et al. (2008) froze equine semen at 40 × 10 6 /mL and 400 × 10 6 /mL, and adjusted this time
the glycerol levels for each group to 4%. Immediately following thawing, the low concentration
yielded a superior motility, however, after incubation for 3h, motility was clearly higher for the
higher concentration. Semen frozen in BotuCrio® showed better motility characteristics for semen
frozen at 100 × 10 6 /mL, and 200 × 10 6 /mL was better than 400 × 10 6 /mL (Nascimento et al., 2008).
In this study, no correction for cryoprotectant concentration was made. In conclusion, the influence
of concentration on post-thaw semen quality is not uniformly elucidated, however, high
concentrations might negatively influence the outcome of cryopreservation. Interactions between
the spermatozoa and the different components of the diluter cannot be excluded and as such might
be determinant for the optimal sperm concentration for cryopreservation.
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Glycerol as cryoprotectant
The first developed freezing media were rich in egg yolk or milk and contained glycerol as
cryoprotective agent (CPA). After some experimental modifications, following media were developed
and frequently used: (a) modified lactose EDTA (Martin et al., 1979), (b) lactose EDTA egg yolk
(Tischner, 1979), and (c) INRA82 with egg yolk and glycerol (Palmer, 1984); the exact composition of
these extenders is presented in table 3. Although glycerol is still the most frequently used
cryoprotectant, little uniformity exists concerning the glycerol concentration which is used. The
optimal glycerol concentration differs depending on the extender composition and might be
influenced by the egg yolk concentration (Ecot et al., 2000). Nevertheless, glycerol has a clear
negative effect on fertility, since not only post-thaw sperm motility is affected (Burns and Reasner,
1995) but it will also influence female fertility. This effect is well known in hens, although the
contraceptive effect has also been documented in equine species (Bedforf et al., 1995; Vidament,
2005), and is most pronounced in asine species (Vidament, 2005). In literature, a wide variety in
glycerol concentration is found in equine freezing extenders, i.e., 2.5% (Palmer, 1984; Vidament et al.,
2000, 2001), 3% (Wilhelm et al., 1996a), 3.5% (Tischner, 1979), 4% (Leipold et al.,1998, Wilhelm et al.,
1996b), 5% (Martin et al., 1979) and 7% (Pace and Sullivan, 1975). It is advised that glycerol
concentration in frozen stallion semen should not exceed 3.5% in order to avoid negative influences
on fertility (Vidament et al., 2005). However, the final glycerol concentration is often not available
since the glycerol concentration is indicated on the freezing extender but not for the processed
semen, where it is variable depending on the dilution ratio used to resuspend the sperm pellet
following centrifugation (Vidament, 2005). The final glycerol concentration can accurately be