Epididymal bull sperm selection by Percoll® density-gradient centrifugation prior to conventional or ultra-rapid freezing enhances post-thaw sperm quality

This study evaluated the effectiveness of Percoll® density gradient centrifugation (Percoll-DGC) for selecting bull epididymal sperm prior to conventional slow (CS) or ultra-rapid (UR) freezing and its effects on sperm quality. Fifteen pooled samples from 30 epididymides (2 different samples/pool) of 15 bulls were split into two aliquots assigned to either CS or UR freezing. Samples were either selected using Percoll-DGC (40/80 %) or left non-selected (control), resulting in four pre-freezing treatments: Percoll-CS, Control-CS, Percoll-UR, and Control-UR. The CS freezing used 5 % glycerol, exposing sperm straws to liquid nitrogen (LN₂) vapors, while UR freezing used 100 mM sucrose with direct submersion of 30 μL samples into LN₂. Overall, sperm quality was higher in CS treatments than in UR treatments. Pre-freezing, Percoll-CS improved straight-line velocity (VSL), linearity (LIN), and beat-cross frequency (BCF) compared to Control-CS (P < 0.05). Similarly, Percoll-UR treatment enhanced progressive motility (PSM), velocities, straightness (STR), amplitude of lateral head displacement (ALH), and BCF compared to Control-UR (P < 0.05). Post-thaw, Percoll-CS demonstrated higher kinematic parameters, viability, and acrosome integrity compared to Control-CS (P < 0.05). Meanwhile, Percoll-UR improved viability and acrosome integrity relative to Control-UR (P < 0.05). Notably, both Percoll-UR and Control-UR resulted in lower DNA fragmentation compared to Percoll-CS. In conclusion, Percoll-DGC selection prior to CS freezing significantly improved post-thaw sperm quality, including kinematics, viability, and acrosome integrity. For UR freezing, Percoll-DGC primarily enhanced post-thaw viability and acrosome integrity.