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