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Centrifugation technique
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5.2
Centrifugation is one of the common procedures when processing equine semen. Currently,
centrifugation is mandatory when subjecting an equine ejaculate to cryopreservation. Alternative
approaches such as fractionated collection are not as successful (Sieme et al., 2004) or are still in its
infancy (sperm filters – Alvarenga et al., 2010).
The influence of centrifugation on spermatozoa is not completely understood, or at least the
principle(s) by which spermatozoa are influenced due to centrifugation remain(s) unclear. A first
theory is based on alterations in the shape of cells submitted to centrifugation. Using an atomic force
microscope, fitted in a large centrifuge, Van Loon et al. (2009) were able to visualize mouse
osteoblasts during centrifugation. The osteoblasts were markedly reduced in height during
centrifugation at only 3 × g. Although the structure of a spermatozoon is completely different from
an osteoblast, especially because of the dramatic reduction in the cytoplasm of a mature sperm cell,
it is likely that centrifugation at much higher speeds also affect the structure of a sperm cell. Further
refinement of this technique by Van Loon et al. (2009) and research on different cell types is
necessary in order to accurately describe what happens with spermatozoa during centrifugation.
Another explanation could act at the level of the sperm membrane. Like any plasma
membrane, a sperm membrane consists of a liquid phospholipid bilayer (Amann and Pickett, 1987).
Different domains of the plasma membrane all contain different concentrations and distributions of
intramembranous particles, with different, specific functions in the process of fertilization (Flesch
and Gadella, 2010). These domains undergo redistribution following capacitation (Gadella et al.,
1994). Perhaps centrifugation acts on this liquid lipid layer and disturbs as such its function by
rearranging the distribution of the intramembranous particles. Excessive centrifugation forces induce
even more damage, and provoke a compact sperm pellet that is difficult to resuspend (Webb and
Dean, 2009).
In order to identify the effects of centrifugation force on sperm damage, we investigated the
impact of increasing forces (centrifugation protocols from 600 × g to 2400 × g) on stallion semen for
a reduced period of time (5 min) (Chapter 4.1). Additionally, we investigated sperm loss when
aspirating 90% of the supernatant following centrifugation using the different forces. Not all the
spermatozoa were pelleted at the bottom of the tube, but a part remained in suspension in the