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.