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4.1.5. Discussion
CHAPTER 4.1
In this study, we demonstrated that the loss of sperm after centrifugation can be minimized
by using an appropriate protocol without damaging the sperm. Centrifugation protocol had a limited
effect on in vitro sperm characteristics in our study. We specifically showed that the use of the
standard protocol (CP 1) led to an average sperm loss of 22%, resulting in a considerable reduction in
the number of AI doses obtained. If the centrifugation time was decreased while the exerted g-force
was increased up to 1800 × g or even 2400 × g, sperm losses were reduced to 7.4% and 2.1%,
respectively, without any apparent changes in the in vitro sperm quality.
If semen was cooled and stored after centrifugation, only a minimal effect of the used CP
was noticed on total motility as evaluated by CASA. Surprisingly, the motility was reduced in the
group where the lowest centrifugation force was used for the shortest time (600 × g for 5 min). The
sperm pellet probably was very soft which allowed the most motile sperm cells to swim up
immediately after centrifugation had ceased. As a consequence, these highly motile sperm may be
discarded during aspiration of the supernatant. This was confirmed in a preliminary experiment (n=4)
by analyzing the supernatant of semen samples centrifuged according to CP1, 2 and 5 (data not
shown). The average TM and PM in the supernatant was 41.3% and 23.5%, 54.8% and 31.8% and 8.3%
and 4.0% for CP1, 2 and 5, respectively, suggesting that the motility of the discarded sperm cells in
the supernatant is much higher when low centrifugation forces are used. Therefore, application of a
low g-force for a short time leads to important losses of top quality sperm cells and must definitely
be avoided.
Additionally, we found a significant effect on BCF for sperm samples cooled and stored after
centrifugation. The BCF of the sperm subjected to CPs 2 and 4 was reduced compared to the
standard protocol. Moreover, the quality of the frozen-thawed samples after being subjected to one
of the tested CPs did not differ significantly for any of the tested quality parameters except for BCF.
Protocols 2 and 4 yielded the highest BCF values after thawing, which was in contrast with our
observations for cooled preservation. However, the actual impact of BCF on fertility is difficult to
predict since few data are available in literature (Gil et al., 2009). BCF is a parameter indicative for
spermatic vigor. Changes in BCF under capacitating conditions in vitro are thought to be related to
sperm hyperactivation that occurs in vivo and might favor the penetration of oocytes (Gil et al.,
2009).
The results of the experiment 3 showed a decrease of DNA integrity over time, although
centrifugation did not influence DNA integrity. These findings are contradictory with the study of
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