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