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CHAPTER 4.1
Love and coworkers (2005) where increased DNA damage immediately after centrifugation was
reported. These authors showed that complete removal of SP after centrifugation and subsequent
resuspension of the sperm pellet in fresh diluter resulted in better sperm quality (motility and DNA)
compared to samples in which SP was still present after simple dilution of the semen. Therefore the
impact of the centrifugation on DNA integrity needed to be further examined. In our study, no
difference in DNA damage was present immediately after centrifugation which might be explained
by the method used to evaluate DNA integrity. The study of Love et al. (2005) evaluated DNA
integrity using the sperm chromatin structure assay (SCSA). In comparison to the TUNEL assay, with
the SCSA a larger number of sperm cells is analyzed by a flow cytometer based on the colour of the
metachromatic dye acridine orange (Evenson and Wixon, 2006). Therefore it might be advisable to
measure the direct impact of different CPs with flow cytometry (SCSA or TUNEL) to get a more
accurate impression of the possible side effect of centrifugation on DNA integrity.
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Cushioned centrifugation techniques have previously been described as an answer to reduce
sperm damage while maximizing the sperm harvest. A variety of dense solutions acting as a cushion
have been described (Cochran et al., 1984). The frequently used iodixanol was first reported for
stallion sperm cells in 1997 (Revell et al., 1997). Very high recovery rates have been described (Ecot
et al., 2005; Knop et al., 2005) using such a commercially available cushion, when centrifuging at
1000 × g for 20 min. However, the most critical step is carefully removing the cushion material after
centrifugation. Indeed, it is critical to aspirate as much cushion fluid as possible without aspirating
sperm cells from the adjacent band to avoid possible deleterious effects of the iodixanol solution
(Waite et al., 2008). The use of very small amounts (30 µL) of cushion without removal was proven
to be harmless. However, these small volumes require specially designed, custom-made nipple
centrifugation tubes which are not readily available (Waite et al., 2008). Because of these important
drawbacks, cushioned centrifugation is not commonly used in practice. Therefore, the impact of CP
on sperm quality remains an important subject to examine.
In summary, sperm losses can be reduced substantially by centrifuging stallion semen for 5
min at 1800 or 2400 × g without a cushion and without impairing the in vitro sperm function after
cooled or frozen storage. This leads to an increase in the number of AI doses produced per ejaculate.
It can easily result in an additional two AI doses from an average ejaculate of 10 billion sperm.
Nevertheless, the effects of these CPs on DNA damage requires further investigation as well as the
impact on in vivo fertility.