Comparative evaluation of Percoll gradient configurations: impact on the structural and functional integrity of cooled porcine spermatozoa

Context Porcine in vitro fertilisation (IVF) efficiency remains limited by sperm quality constraints, including high polyspermy rates and suboptimal gamete preparation. Density gradient centrifugation using Percoll is widely applied to select motile, structurally competent spermatozoa; however, the impact of different gradient configurations on sperm structural and functional integrity in cooled boar semen has not been systematically evaluated. Aims To compare four discontinuous Percoll density gradient configurations (35/70%, 45/90%, 65/70%, and 90/90%) with respect to their effects on motility, kinematics, plasma membrane integrity, and acrosomal status in cooled porcine semen. Methods Semen was collected from 11 Pietrain boars, cooled, and pooled into three experimental replicates. Each replicate was processed through the four Percoll gradient configurations. Sperm motility and kinematic parameters were assessed by computer-assisted semen analysis (CASA), plasma membrane integrity by the hypo-osmotic swelling test (HOST), and acrosomal integrity by confocal microscopy using FITC-conjugated Pisum sativum agglutinin (FITC-PSA). Key results The 35/70% gradient yielded the highest pellet concentration, whereas the 90/90% configuration produced significantly lower sperm recovery. Total motility and velocity parameters (average path velocity (VAP), curvilinear velocity (VCL), straight-line velocity (VSL)) did not differ significantly across gradients, although beat-cross frequency decreased consistently after all Percoll treatments. Plasma membrane integrity was significantly reduced in all gradient-processed groups relative to the control; the 90/90% configuration showed the lowest values. The 35/70% gradient best preserved acrosomal integrity, with intact acrosome rates comparable to unprocessed semen, whereas the 65/70% and 90/90% configurations were associated with increased acrosomal defects and spontaneous acrosome reaction. Conclusions Increasing Percoll gradient density does not improve sperm kinematic parameters but progressively compromises plasma membrane and acrosomal integrity. The 35/70% configuration provided the most favourable balance between sperm recovery and structural preservation. Implications: These findings provide practical guidance for optimising sperm preparation in porcine IVF systems, supporting the use of moderate-density Percoll gradients to maintain structural sperm integrity while ensuring adequate functional selection.