view - Department of Reproduction, Obstetrics and Herd Health
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CHAPTER 1.2<br />
36<br />
→ Colloid centrifugation<br />
Centrifugation <strong>of</strong> semen through (one or more) layers <strong>of</strong> colloid can be used to separate<br />
spermatozoa from seminal plasma <strong>and</strong> to select a subpopulation <strong>of</strong> spermatozoa with good motility,<br />
viability <strong>and</strong> chromatin integrity (Pert<strong>of</strong>t, 2000). The cells move to the point in the gradient which<br />
matches their density, i.e. the isopycnic point, during centrifugation (Fig. 10) (Pretlow <strong>and</strong> Pretlow,<br />
1989), rather than actively swimming through the colloid (Mortimer, 2000). After colloid<br />
centrifugation, the sperm pellet is resuspended in fresh extender (washing medium) <strong>and</strong> washed by<br />
centrifugation. Percoll® was the first colloid used for the selection <strong>of</strong> spermatozoa. In 1996, Percoll®<br />
was restricted for non-clinical use only, due to possible toxicity <strong>of</strong> the product (Avery <strong>and</strong> Greve,<br />
1995; Mortimer, 2000). Different colloids have been developed, <strong>and</strong> this has also resulted in species<br />
specific products. Until recently, the major disadvantage <strong>of</strong> colloid centrifugation was the limitation<br />
in volume that could be processed per centrifugation tube, <strong>and</strong> the time consuming practice <strong>of</strong><br />
carefully preparing the different layers <strong>of</strong> the Density Gradient Centrifugation (DGC) (Morrell <strong>and</strong><br />
Rodriguez-Martinez, 2009).<br />
Fig. 10. Schematic presentation <strong>of</strong> spermatozoa following colloid centrifugation with location <strong>of</strong> the<br />
spermatozoa according to their differences in isopycnic point based on their morphology.<br />
Single Layer Centrifugation (SLC) was described as an alternative to DGC for processing<br />
animal semen, resulting in comparable sperm yield <strong>and</strong> similar sperm quality characteristics (Morrell<br />
et al., 2008; Thys et al., 2009). Additionally, SLC can be used to process larger volumes <strong>of</strong> extended<br />
semen (Morrell et al., 2009) per centrifugation tube, allowing for the processing <strong>of</strong> entire stallion<br />
ejaculates.