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CHAPTER 1.2
multiple mounts are required for ejaculation, the collection receptacle should be emptied or
preferably be replaced together with the inner liner (Loomis, 2006).
Sperm concentration and seminal plasma during storage
In general, it is accepted that equine semen needs to be diluted at least 3:1 in order to dilute
the seminal plasma to levels ≤ 25% (Varner et al., 1988). Additionally, for optimal preservation, it is
advised to dilute the semen to a concentration of 25 to 50 × 10 6 /mL (Varner et al., 1987; Jasko et al.,
1991, 1992). If the initial sperm concentration is too low to use an appropriate dilution and maintain
an acceptable concentration at the same time, centrifugation is advised. Standard centrifugation
protocols for equine semen consist of 400× to 600× g-force for 10 to 15 minutes (Loomis, 2006;
Aurich, 2008), and rely on a 75% sperm recovery after aspirating the supernatant and resuspending
the sperm pellet. Prior to centrifugation, semen should be extended at least to a 1:1 ratio. Seminal
plasma acts like a double edge sword on fertility: it is detrimental for stallion spermatozoa during
storage, but at the other hand it shortens the duration of the postbreeding induced endometritis
caused by the inflammatory response triggered by the spermatozoa (Troedsson et al., 2000, 2005).
With most extenders, it is advisable to retain 5 to 20% seminal plasma following centrifugation (Jasko
et al., 1991). However, DNA integrity was maintained best in complete absence of seminal plasma,
whereas DNA damage increased as seminal plasma levels increased (Love et al., 2005a).
The effect of removal of seminal plasma in stallions whose sperm responds poorly to cooling
(to which we will further refer as “poor cooling” stallions) remains a topic of discussion. Brinsko et al.
(2000a) found that partial removal of seminal plasma after centrifugation had a positive effect on
spermatozoal motility following 48 of cooled storage. A more recent study, using more stallions,
demonstrated no effect of seminal plasma removal of “poor cooling” stallions on motility after
cooled storage. Only the percentage of membrane intact spermatozoa was higher for the samples
devoid of seminal plasma (Barrier-Battut et al., 2010).
Centrifugation of sperm
Normally, a 75% sperm recovery rate, obtained after conventional centrifugation (400× to
600× g for 10 to 15 min) is reported (Loomis, 2006; Aurich, 2008). Contradicting results however are
also present in literature, with recovery rates exceeding 98% using the same centrifugation protocol
(Weiss et al., 2004). Losses of about 25% are agreed on, but can be further reduced by increasing
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