Centrifugation for concentrating sperm
Centrifugation can be performed either with or without cushion solution.
Cushion is an iodixanol solution which allows the use of higher
centrifugation g-forces which reduces sperm damage and improves sperm
recovery rates. After semen is analyzed, it is diluted 1:1 using a semen
extender. After the semen is diluted, it is transferred to 50-mL conical
tubes. If cushion is not being used, the tubes are balanced and
centrifuged at 400 x g for 20 minutes. If cushion is being used, after
the diluted semen is added to the 50-mL conical tubes, the cushion
solution is layered underneath the diluted semen. The amount of cushion
solution used can vary on user preference and stallion but is typically
between 0.5-mL and 3-mL. The layering of cushion is done by first
drawing the cushion solution into syringes and the appropriate volume.
Next, a Tom-cat catheter, pipette, or blunted spinal needle can be used
to deposit the cushion under the diluted semen. Once this is done the
tubes are weighed and balanced and centrifuged at 1000 x g for 20
minutes. It important for the centrifuge to be well balanced as any
vibration can disrupt the sperm pellet and reduce the recovery rate of
sperm. After centrifugation, the supernatant is removed. This can be
done with a pipette or a vacuum aspirator. It is important to remove the
supernatant as soon as possible after centrifugation to maximize the
sperm recovery rate. Next, the samples are combined and resuspended into
one tube and a concentration and volume measured. When using
centrifugation to process semen the reconstituted volume can be much
lower and sperm concentration much higher than using simple dilution
without reducing sperm longevity. Research has found difference in sperm
longevity after cushion centrifugation in sperm undergoing
cooled-storage at 25 x 106 sperm/mL and 250 x
106 sperm/mL (Bliss et al., 2012). There is an
excellent review on processing semen from subfertile stallions (Varner,
2016) which covers cushioned centrifugation techniques in more detail.