Flagellar Waveform Analysis in Sperm

The flagellar waveform was analyzed as previously described [30 (link)] with a Nikon Eclipse TE2000-U microscope. Images were collected at 300 Hz respectively by a M3 high speed camera (IDT, Tallahassee, FL, USA) using a 20× objective and the Motion-Studio 64 software (Imaging Solutions, Ehingen, Germany). For sperm beat frequency analysis, 10 µL of the sperm suspension in demembranation/reactivation buffer was transferred to a FluoroDish^TM (World Precision Instruments, Germany) and were allowed to adhere to the bottom during 3 Min incubation. Cells were perfused with different concentrations of ATP, ADP and Mg²⁺ for one minute (Figure 1B). Single sperm were selected for analysis with Image J (1.48V, National Institute of Health, Washington, DC, USA). Flagellar beat frequency was measured with semi-automated Igor Pro^TM software (Wavemetrics, Lake Oswego, OR, USA) like previously described [30 (link),68 (link)].