view - Department of Reproduction, Obstetrics and Herd Health

CHAPTER 4.1
4.1.2. Introduction
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Numerous studies have demonstrated the beneficial effect of diluting raw semen with an
appropriate extender. As such, the negative influence of seminal plasma (SP) on the preservation of
equine sperm can be reduced. Centrifugation of diluted equine semen and subsequent resuspension
of the sperm pellet in fresh extender can even further reduce the amount of SP in stored samples. A
small proportion of SP in semen improves sperm motility after cooling and storage (Jasko et al.,
1991).
However, centrifugation itself may act like a double edged sword exerting both beneficial
and deleterious effects on sperm motility (Martin et al., 1979). Although the number of sperm cells
can be maximized by increasing centrifugation time and force, physical damage is exerted on the
sperm cells at the same time. The direct negative outcome of centrifugation on semen quality is
demonstrated by the decrease in motility and velocity in contrast to uncentrifuged controls (Jasko et
al., 1991) which indicates that the centrifugation protocol (CP) may play an important role in the
quality of the inseminate. After 24h of cooled storage the harmful effect of centrifugation is no
longer present (Jasko et al., 1991).
A CP of 600 × g for 10 min is commonly used for equine semen (Ecot et al., 2005; Knop et al.,
2005; Vidament et al., 2000; Weiss et al., 2004). In literature, data on the amount of sperm loss after
using this protocol are contradictory and vary from 1.9% (Weiss et al., 2004) to 25% (Ecot et al., 2005;
Knop et al., 2005). Nowadays, it is generally accepted that sperm losses will vary around 25% when
diluted semen is centrifuged at 400-600 × g for 10-15 min (Aurich, 2008; Loomis, 2006).
The importance of an appropriate CP has been clearly demonstrated for human sperm. The
duration of centrifugation was shown to be more important than the centrifugation force for causing
iatrogenic sperm membrane injuries which resulted in an increased formation of reactive oxygen
species (Shekarriz et al., 1995). In boars, similar findings were described: a CP with a high g-force for
a short time (2400 × g for 3 min) was used without detrimental effects on sperm yield compared to
the standard regime (800 × g for 10 min) (Carvajal et al., 2004). Additionally, a positive effect on
semen quality after cryopreservation was detected when using high g-forces for a shorter period of
time (Carvajal et al., 2004). Experiments with equine semen have been performed where semen was
centrifuged at 400 × g for an increasing period of time. Sperm loss was approximately 20%, if
samples were centrifuged for 10 min or longer, while no adverse effects on motility immediately
after centrifugation were present, unless semen was centrifuged for 20 min. However, effects on
preserved by cooling or freezing were not determined (Heitland et al., 1996). A high g-force for a