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

Abstract

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 nonselected (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.