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CHAPTER 1.2
The actual freezing technique i.e. above liquid nitrogen or in a programmable freezer should
not only be decided on from a mere economical point of view. The cooling curve of straws placed at
a fixed distance above liquid nitrogen, has been recorded in order to measure the exact temperature
changes during cryopreservation. The resulting curve has been entered into a programmable freezer
and ejaculates were frozen using a split design. The semen frozen using the programmable freezer
resulted in better post-thaw in vitro semen characteristics, which is likely due to the more uniform
freezing rate (Clulow et al., 2008). However, these findings are in contrast with a previous report
where no differences were noticed between semen frozen above liquid nitrogen and semen frozen in
a programmable freezer (Cristanelli et al., 1985).
The use of alternative CPAs instead of glycerol might influence the optimal cooling and
freezing curve. As such, sperm that was cooled to 5°C for 1h or without cooling, followed by slow
(-20°C/min) or fast (-70°C/min) freezing rates using MF or DMF as CPA, has been compared. Post-
thaw sperm motility was not affected by the freezing rate, however, cooling prior to freezing resulted
in better post-thaw sperm characteristics (Alvarenga et al., 2005). According to the manufacturer, in
order to obtain an optimal freezing process when using BotuCrio®, it is advised to cool the semen to
5°C in 20 min after resuspending the sperm pellet, after which the straws are frozen 6 cm above
liquid nitrogen or at -30°/min in a programmable freezer. The equilibration time using BotuCrio® is
clearly much shorter compared to other extenders. Increasing this equilibration to 60-80 min using
BotuCrio® has a clear negative impact on post-thaw semen quality (Hoogewijs, unpublished data). A
faster freezing rate of -70°C/min, i.e. 4cm above liquid nitrogen, results in lower post-thaw motility
compared to a moderate freezing rate (Terraciano et al., 2008)
Thawing of frozen semen
Semen can be thawed using quite some different protocols (Vidament et al., 2001). In the
protocol which is commonly used, sperm is thawed during 30s in a 37°C water bath. Thawing of
semen in a 75°C water bath for 10s resulted in a slightly higher motility, however, timing is critical
since 15s at 75°C resulted in a zero motility. In Brazilian studies, semen frozen in BotuCrio® is
consistently thawed at 46°C for 20s (Melo et al., 2008; Pinheiro et al., 2008; Pucci et al., 2008; Papa
et al., 2010).
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