Stallion sperm selection prior to freezing using a modified colloid swim-up procedure without centrifugation

- Sperm washing (SW) and colloid sperm selection (SLC) involve centrifugation.
- A new colloid-based swim up (SU) technique without centrifugation was compared.
- SU and SLC before freezing selected sperm with better post-thaw DNA integrity than SW.
- The potential benefits of SU in samples lacking seminal plasma should be considered.

The aims of this study were to: 1) develop a new method for stallion sperm selection using a modified swim-up procedure through a colloid and 2) evaluate its impact in good quality ejaculates from bad freezers in comparison to methods involving centrifugation such as single layer centrifugation and sperm washing. Ejaculates were processed before freezing using three different procedures: sperm washing (SW), colloid single layer centrifugation (SLC) and a modified colloid swim-up (SU). After semen processing, sperm recovery rates were measured and sperm were frozen. Post-thaw sperm motility (assessed by computer-assisted sperm analysis), normal forms and plasma membrane integrity (evaluated under bright-field and fluorescence microscopy respectively), and DNA fragmentation (assessed by the Sperm-Halomax kit) were compared between treatments. Sperm recovery rates were similar between SU and SLC but lower than SW. Sperm motility after thawing was lower in SU in comparison to SLC and SW, maybe due to the incomplete removal of seminal plasma before freezing. Sperm DNA fragmentation was lower in SU and SLC selection methods, particularly in SLC selected samples during the first 6 h of incubation. The remaining sperm parameters assessed were similar among treatments. In conclusion, SLC is more suitable than SW and SU to process stallion semen prior to freezing, in particular when sperm DNA damage is suspected. Further studies are needed in order to determine the potential benefits of SU in samples where centrifugation is not necessary, such as epididymal sperm, ejaculate fractioning or post-thaw semen samples.