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CHAPTER 1.2
in flattened aluminum packs, which were frozen either above the surface of liquid nitrogen (Tischner,
1979) or in copper containers that were immersed in liquid nitrogen (Love et al., 1989). These large
volume flat packs (20-25 mL and 10-12 mL) were replaced by large volume straws (4 – 5 mL) (Martin
et al., 1979; Love et al., 1989; Samper and Morris, 1998), which could be used as single insemination
dose straws. The straw volume further decreased to 1.0 mL (Cochran et al., 1983) and 0.5 mL (Loomis
et al, 1983). Using these smaller straws, the temperature was more uniform for the whole sample
during the freezing process and cooling was achieved more rapidly (Loomis et al., 1983). Following
successes in human medicine when freezing semen in cryotube vials, 0.5 mL straws were compared
to cryotubes (3.6 mL of extended semen) for freezing equine semen. However, using these cryotubes,
post-thaw motility was significantly reduced (Kozink et al., 2006). On the other hand, a big
disadvantage associated with French 0.5 mL straws, is the necessity to use multiple straws for one AI
dose. To reduce the risk of making errors when handling frozen 0.5 mL straws, Love et al. (2005b)
investigated the impact of freezing multiple straws in one globlet, in order to reduce post freeze
handling of straws. However, post-thaw semen quality was hampered and the semen quality for
individual straws varied depending on their localization in the globlet during freezing. The use of
smaller 0.25 mL straws (frequently used for freezing bull sperm) for freezing stallion sperm does not
appear to influence post thaw-sperm quality, when compared to 0.5 mL straws (Nascimento et al.,
2008).
Sperm concentration
Generally, the semen is centrifuged after the first dilution in order to have sufficient sperm
numbers per straw. The second dilution is determining for the final sperm concentration at which
straws will be filled and frozen. It has been reported that this sperm concentration during freezing
has an influence on sperm quality of post-thaw samples. As such, sperm concentrations exceeding
400 × 10 6 /mL could negatively influence post-thaw motility (Heitland et al., 1996). However, these
results were not confirmed by Leipold et al. (1998), who found no decreased post-thaw motility
when semen was frozen at 1600 × 10 6 /mL compared to 400 × 10 6 /mL. The major difference in their
experimental setup was the glycerol concentration. In the latter study, adjustment of the final
glycerol concentrations to 4% was done for both groups. On the other hand, the semen was
progressively further diluted in the first study, resulting in lower glycerol concentrations for high
concentrated samples, and vice versa. Clulow et al. (2007) observed a reduced post-thaw motility for
semen diluted to 20 × 10 6 /mL compared to 200 × 10 6 /mL. In this study the confounding factor was
that semen at 20 × 10 6 /mL was frozen in 0.25 mL straws while semen at 200 × 10 6 /mL was frozen in
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