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