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 />
centrifugation time <strong>and</strong> force. However, this usually results in decreased sperm quality (Loomis,<br />
2006). In 1984, Cochran et al. described a technique where a dense solution was layered below the<br />
extended semen at the bottom <strong>of</strong> a centrifugation tube. This dense solution could serve as a cushion<br />
for the spermatozoa during centrifugation (Cochran et al., 1984). Initially, a glucose-EDTA cushion<br />
was used <strong>and</strong> later replaced by an egg yolk containing extender cushion supplemented with 4%<br />
glycerol (Amann <strong>and</strong> Pickett, 1987). All these initial reports, except Volkman <strong>and</strong> van Zyl (1987),<br />
showed a beneficial effect on sperm motility following centrifugation. Subsequently, a 60% iodixanol<br />
solution was placed beneath extended semen followed by centrifugation for 25 min at 1000 × g. This<br />
cushion prevented sperm from compacting at the bottom <strong>of</strong> the tube despite the high g-force used<br />
<strong>and</strong> resulted in 30% more normal living sperm (Revell et al., 1997). More recently, high sperm yields<br />
following high speed centrifugation without impairing sperm quality were reported using 3.5mL or<br />
5mL <strong>of</strong> cushion fluid in a 50 mL centrifugation tube (Ecot et al., 2005; Knop et al., 2005). A variation<br />
to these techniques has been described in which a small volume (30 µL) <strong>of</strong> cushion solution is placed<br />
at the bottom <strong>of</strong> a special designed nipple centrifugation tube, followed by centrifugation at 400 × g<br />
for 20 min. This technique has slightly lower sperm yields <strong>and</strong> slightly improved in vitro quality<br />
characteristics, <strong>and</strong> has the additional advantage that it does not require aspiration <strong>of</strong> the cushion<br />
following centrifugation (Waite et al., 2008).<br />
34<br />
Alternatives to centrifugation<br />
As alternative for centrifugation to increase the sperm concentration <strong>and</strong> reduce the amount<br />
<strong>of</strong> seminal plasma, fractionated semen collection can be used. Using a Krakow AV or a modified open<br />
ended Missouri AV, it is possible to collect only the sperm rich fraction <strong>of</strong> the ejaculate. An<br />
automated phantom (Equidame®) can also be used. This device provides semen samples with a lower<br />
bacteriological colony count, <strong>and</strong> with a similar motility compared to semen collected using a<br />
Missouri AV (Lindeberg et al., 1999). However, it is important to realize that fractionated collection<br />
using the Equidame® does not correspond completely with the fractionated collection described<br />
above. Separation using the Equidame® is not in concordance with the jets ejaculated by the stallion,<br />
but is performed based on adjusted volume separation in the semen collection cups. Once a cup is<br />
filled with the preset volume, a following cup is filled (Lindeberg et al., 1999). Sperm originating from<br />
sperm rich fractions had higher motility at 12h <strong>and</strong> 24h post collection than sperm from total<br />
ejaculates (Varner et al., 1987). However, seminal plasma from sperm rich fractions was found to be<br />
more deleterious on sperm motility compared to seminal plasma from sperm poor fractions (Akcay<br />
et al., 2006). Indicating that centrifugation might still be advisable, especially for prolonged storage.