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CHAPTER 5.2
5.2.2. Selection of spermatozoa
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The possibility to perform sperm selection using a single layer centrifugation (Morrell et al.,
2008) and the adaptation to scale up the volume per centrifugation tube (Morrell et al., 2009)
extends its practical use in equine andrology. This technique was used to select a superior sperm
population prior to cryopreservation and obtained post-thaw samples with increased overall sperm
quality (Chapter 4.2). The sole intention of the cryopreservation protocol we used was to analyze the
effect of cryopreservation of a superior sperm population. It is not surprising to find an overall
increased sperm quality after cryopreservation. Dead, damaged, or spermatozoa with a dysfunctional
motility are very unlikely to survive the freeze-thaw cycle, or to have a sufficient post-thaw quality.
Until recently, it was accepted that reactive oxygen species (ROS) in (human) semen were
almost exclusively produced by leucocytes. This is true for fertile men, although in oligozoospermic
patients the spermatozoa themselves were identified as a second major source of ROS (Aitken et al.,
1992). Equine semen is normally devoid of leucocytes in contrast to human semen, but equine
spermatozoa are equally capable of generating ROS, since damaged spermatozoa or morphological
abnormal spermatozoa generated significantly greater amounts of ROS than live, morphologically
normal spermatozoa (Ball et al., 2001). The presence of ROS impairs in vitro motility of equine
spermatozoa (Baumber et al., 2002) and promotes DNA fragmentation of equine spermatozoa
( Baumber et al., 2003).
We hypothesize that the increased post-thaw sperm quality we have noticed following pre-
freeze SLC selection could perhaps also be partially attributed to reduced levels of ROS. The use of
Androcoll-E might remedy these ROS related problems by working on two levels. First, due to the
selection procedure, the few leucocytes that are present are being held back from the sperm pellet.
Second, fewer abnormal spermatozoa (dead spermatozoa or with excess residual cytoplasm) are
present in the sperm pellet. As such the ROS content in the selected subpopulation of spermatozoa
should be lower than in the “full” sperm pellet, reducing the ROS related sperm damage.
The effect of sperm selection prior to cryopreservation could even be further increased if
stallion-adapted cryopreservation protocols would be used, fitting specific needs . Additionally,
sperm selection using Androcoll-E and subsequent washing of the sperm pellet removes all seminal
plasma. In our study seminal plasma from the same stallion and the same ejaculate was added back
to rule out any possible effect of presence or absence of seminal plasma on the post-thaw results.
Seminal plasma from stallions with poor freezing sperm was shown to play an important role in the
poor freezability (Aurich et al., 1996). The use of seminal plasma of stallions with good freezability
improved the post-thaw quality of stallions with poor freezability (Aurich et al., 1996). The