Objective assessment by optical flow of ram semen wave motion in a 100 µm deep chamber

A pre-warmed 100 µm deep glass chamber (Leja, Nieuw-Vennep, The Netherlands) was loaded with raw semen (30 µl) and observed using a phase contrast microscope (10x magnification) equipped with a controlled heating stage (37 °C). A flow of short dark waves on a grey background could be observed. A sequence of 100 successive images (2560x2160 pixels) was recorded at the speed of 100 frames per second using a sCMOS pco.edge 5.5 camera (PCO AG, Kelheim, Germany) monitored using CamWare software. The velocity of the flow in the chamber was assessed using an accurate and fast method derived from optical flow methods, without linearization. A velocity field was obtained using the optical flow method and several criteria were derived.