Thigmotactic navigation and functional enrichment of boar sperm in microfluidic channels.

In brief: Successful fertilization in pigs depends on sperm that can navigate the complex geometry of the sow's reproductive tract, yet current sperm preparation methods select only on motility and can damage cells. This study demonstrates that a biomimetic microfluidic device selects boar sperm on thigmotactic competence, yielding functionally superior populations and revealing species-specific differences in angular navigation. Abstract: Efficient sperm selection is crucial for reproductive success in pigs, where artificial insemination is the main method of assisted reproduction. Traditional methods such as density gradient centrifugation (DGC) can enrich motile sperm but may cause mechanical and oxidative stress and do not replicate the natural filters of the sow's reproductive tract. This study assessed a biomimetic microfluidic (MF) device, previously validated in cattle, sheep, and dogs, for enrichment of high-quality spermatozoa from chilled boar ejaculates (n = 18). Unprocessed extended semen (RAW), DGC, and MF samples were evaluated for concentration, computer-assisted sperm analysis kinematics, viability, morphology, acrosome integrity, DNA fragmentation (sperm chromatin structure assay), and thigmotactic navigation. Microfluidic processing yielded a lower concentration (5.73 ± 2.22 × 106/ml) than RAW (65.20 ± 23.33 × 106/ml) and DGC (22.68 ± 10.95 × 106/ml; both p < 0.0001), an ∼11-fold reduction consistent with functional enrichment. Microfluidic sperm showed higher total motility (91.0% vs 77.4% DGC, 67.1% RAW), progressive motility (74.8% vs 59.6% and 33.1%; 2.2-fold over RAW), intact acrosomes (81.4% vs 71.9% and 62.6%), and lower DNA fragmentation (0.77% vs 1.68% and 4.38%; ∼5.7-fold reduction versus RAW; all p < 0.0001). Angular microchannels revealed graded thigmotaxis, with navigation success decreasing from 95.8% at 45° to 87.9% at 90° and 69.4% at 135°, comparable to bull and dog but lower than ram (89.1% at 135°). Biomimetic microfluidics isolates functionally superior boar sperm, offering a pump-free alternative to centrifugation with potential to enhance artificial insemination and in vitro embryo production.