Other Semen Processing Techniques
Continuous density gradient centrifugation (also known as single layer colloid) can be a useful technique in some stallions to improve the sperm morphological profile, motility and DNA integrity post processing (Edmond, 2010). One commercially available product is EquiPureTM. It is important to note that this technique does not improve the post processing sperm quality in all stallions, and that a significant number of sperm are lost. Common recovery rates with density gradient centrifugation are from 30% to 40% but can be much lower (Edmond et al., 2012). Thus, it is important to test the effectiveness of this technique on any given stallion prior to shipping semen to mares, by not only looking at the recovery rate but also the morphological profile of sperm after the use of density gradient centrifugation.
Briefly, for this technique semen is extended 1:1 (v/v) with pre-warmed extender. Using either 15-ml or 50-ml conical tubes, the density gradient media (EquiPureTM) is added to the tube first. The volume of EquiPureTM used is typically equal to the amount of extended semen to be added. Next, the extended semen is then added to form a layer on top being careful not to mix the extended semen with the colloid. The tubes are then centrifuged at 400 X g for 20 minutes and afterwards the supernatant removed, and the pellet resuspended. For more details on the procedure the reader is referred to (Bradecamp, 2014a).
Another method to concentrate sperm without the use of centrifugation is through filtration. The recovery rate of sperm using this method was originally reported at 92% and 95%, depending on the size of the pores in the filter (Alvarenga et al., 2010). The sperm recovery rate of the sperm filter is lower compared to cushion centrifugation and differences in motility between the two techniques were negligible, with the authors suggesting this may be a useful technique when centrifugation is unavailable (Roach et al., 2016).
When using the SpermFilter, a petri dish is needed and the semen is extended 2:1 (semen:extender, v/v). The extended semen is added on top of the SpermFilter while it is gently swirled allowing fluid to flow through the filter, collecting sperm on top. Once the sperm has been filtered the membrane is washed with a predetermined volume into a separate petri dish to recover the sperm. For more details please see (Alvarenga & Segabinazzi, 2021).