Substrate compliance modulates sperm flagellar beating

Abstract Sperm motility is significantly influenced by the physical environment of the female reproductive tract, where sperm move in proximity to the epithelium (with an estimated modulus of ~ 1–10 kPa) and within a viscoelastic fluid, such as mucus. Conventional semen analyses are conducted on rigid glass surfaces that do not reflect the softness of the tissues that line the female reproductive tract. Here, we investigated how substrate compliance changes bovine sperm motility and flagellar dynamics using polyethylene glycol diacrylate (PEGDA)-based hydrogel substrates with physiological stiffness and compared sperm behavior on these compliant surfaces with that on glass and polydimethylsiloxane (PDMS), both in identical viscoelastic fluids. Using high-speed imaging and automated flagellar tracking to quantify trajectory dynamics and flagellar beating waveforms, we observed that compliant substrates altered flagellar beating, characterized by reduced beat frequency and increased waveform amplitude compared with rigid substrates. We also found that despite substantial differences in flagellar beating across substrates, wave-propagation speed remained similar. Together, these findings demonstrate that the mechanical compliance of the solid environment changes flagellar dynamics, highlighting the need to incorporate substrate mechanical properties into sperm motility research and clinical assessments.