Hydrostatically driven microfluidic sorting of highly motile sperm using multiple parallel microchannels via rheotaxis and thigmotaxis

Efficient and non-invasive sperm selection is critical for improving outcomes in assisted reproduction and livestock breeding. Herein, we present a hydrostatically driven microfluidic device that exploits intrinsic sperm behaviours, namely rheotaxis and thigmotaxis, to selectively isolate highly motile sperm without external actuation. The device features a multiple parallel-channel architecture designed to increase processing capacity through channel parallelisation while maintaining physiologically relevant conditions. The effects of key geometric and hydrodynamic parameters, including channel numbers, channel spacing, reservoir base aspect ratio, and reservoir volume difference, were systematically investigated through experiments and agent-based numerical simulations. Although increasing the number of channels enhanced sperm recovery, the increase was non-linear owing to recirculation effects induced by thigmotaxis. A cross-shaped channel configuration enabled a maximum recovery efficiency of 2.4%, total recovery of 2.3 × 10⁵ sperm in 30 min, and sperm concentration exceeding 2.3 × 10⁶ cells mL^- 1. However, simulations revealed that 53% of the sperm reaching the recovery reservoir re-entered the channels, highlighting the importance of suppressing recirculation for further performance improvement. The study findings provide design guidelines for hydrostatically driven, parallel-channel sperm-sorting devices by demonstrating that increasing the channel numbers alone does not necessarily result in a proportional increase in sperm recovery owing to thigmotaxis-mediated re-entry. The proposed platform enables pump-free and centrifugation-free sperm selection with low equipment requirements owing to its passive operation.