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CHAPTER 1.2
centrifugation time and force. However, this usually results in decreased sperm quality (Loomis,
2006). In 1984, Cochran et al. described a technique where a dense solution was layered below the
extended semen at the bottom of a centrifugation tube. This dense solution could serve as a cushion
for the spermatozoa during centrifugation (Cochran et al., 1984). Initially, a glucose-EDTA cushion
was used and later replaced by an egg yolk containing extender cushion supplemented with 4%
glycerol (Amann and Pickett, 1987). All these initial reports, except Volkman and van Zyl (1987),
showed a beneficial effect on sperm motility following centrifugation. Subsequently, a 60% iodixanol
solution was placed beneath extended semen followed by centrifugation for 25 min at 1000 × g. This
cushion prevented sperm from compacting at the bottom of the tube despite the high g-force used
and resulted in 30% more normal living sperm (Revell et al., 1997). More recently, high sperm yields
following high speed centrifugation without impairing sperm quality were reported using 3.5mL or
5mL of cushion fluid in a 50 mL centrifugation tube (Ecot et al., 2005; Knop et al., 2005). A variation
to these techniques has been described in which a small volume (30 µL) of cushion solution is placed
at the bottom of a special designed nipple centrifugation tube, followed by centrifugation at 400 × g
for 20 min. This technique has slightly lower sperm yields and slightly improved in vitro quality
characteristics, and has the additional advantage that it does not require aspiration of the cushion
following centrifugation (Waite et al., 2008).
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Alternatives to centrifugation
As alternative for centrifugation to increase the sperm concentration and reduce the amount
of seminal plasma, fractionated semen collection can be used. Using a Krakow AV or a modified open
ended Missouri AV, it is possible to collect only the sperm rich fraction of the ejaculate. An
automated phantom (Equidame®) can also be used. This device provides semen samples with a lower
bacteriological colony count, and with a similar motility compared to semen collected using a
Missouri AV (Lindeberg et al., 1999). However, it is important to realize that fractionated collection
using the Equidame® does not correspond completely with the fractionated collection described
above. Separation using the Equidame® is not in concordance with the jets ejaculated by the stallion,
but is performed based on adjusted volume separation in the semen collection cups. Once a cup is
filled with the preset volume, a following cup is filled (Lindeberg et al., 1999). Sperm originating from
sperm rich fractions had higher motility at 12h and 24h post collection than sperm from total
ejaculates (Varner et al., 1987). However, seminal plasma from sperm rich fractions was found to be
more deleterious on sperm motility compared to seminal plasma from sperm poor fractions (Akcay
et al., 2006). Indicating that centrifugation might still be advisable, especially for prolonged storage.