view - Department of Reproduction, Obstetrics and Herd Health

CHAPTER 1.2
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Chemically defined extenders in cryopreservation
Two recent studies were performed to evaluate the use of INRA96 as base for freezing
equine semen. As such, the addition of Cryoguard® (Minitube, Verona, Wi, USA) to INRA 96 resulted
in a higher post-thaw semen quality compared to a skimmed milk glucose extender containing
Cryoguard® (Scherzer et al., 2009). On the other hand, lower motility parameters for semen frozen in
INRA96 based freezing extender were found when compared to INRA82 based extender although
membrane integrity was better for semen frozen in INRA96. Fertility trials showed much higher
pregnancy rates for INRA96 frozen semen, clearly demonstrating the value of the INRA 96 diluter for
cryopreservation as well (Pillet et al., 2008a). In an additional study, comparable conflicting in vitro
quality results were found, such as a lower lipid peroxidation but a higher percentage of acrosome
damaged sperm for semen frozen in INRA96 (Pillet et al., 2008b). These studies led to the
development of a ready to use freezing extender for equine semen, INRA-Freeze which is a INRA96
based extender with 2.5% glycerol and 2% sterilized egg yolk plasma added. The egg yolk plasma is
prepared by ultracentrifugation and resulted in comparable in vitro characteristics and in vivo fertility
when compared to whole egg yolk (Pillet et al., 2010).
Cooling and freezing rate
Over the years, a huge variety of cooling and freezing curves has been described. A long term
follow-up study using the same freezing method and the same extenders has been performed.
Initially, semen was processed and frozen as described by Palmer (1984). Briefly, the initial protocol
was as follows. In a first step semen was diluted in INRA82 + 2% egg yolk at 32°C, followed by cooling
and subsequently centrifuging the semen at 4°C. After resuspension of the sperm pellet in INRA82 +
2% egg yolk + 2.5% glycerol at 4°C and semen was left to equilibrate for 1h at 4°C, next straws were
filled and frozen 4 cm above liquid nitrogen (Palmer, 1984). This protocol was refined by changing
every step one by one in order to obtain the best freezing procedure using these diluters (Vidament
et al., 2000, 2001). As such, it was demonstrated that centrifugation is better performed at 22°C
instead of at 4°C and that a moderate cooling rate is preferred to a slow cooling rate. Additionally,
the first cooling from 37°C to 22°C can be done rapidly au bain-marie by plunging the tubes
containing the semen in a 22°C water bath. These results confirm previous reports by Kayser et al.
(1992). The resuspension of the sperm pellet is preferably done at 22°C after which the semen is
allowed to cool and equilibrate for 80 min at 4°C. After filling, the 0.5 mL straws are subsequently
frozen by placing them 4 cm above liquid nitrogen or in a programmable freezer at -60°C/min until
-140°C, there after the straws are plunged and stored in liquid nitrogen.