Measurement of OCR of the spermatozoa was performed using a Seahorse XFp analyzer (Seahorse Bioscience, North Billerica, MA, USA) as previously described [8 (link)] with slight modifications. Mature spermatozoa were collected from the cauda epididymis in flushing holding medium (FHM, non-capacitation medium) [26 (link)]. The spermatozoa (2.5 × 106 per well) were transferred to an 8-well plate that had been coated with concanavalin A (Fujifilm Wako Pure Chemical) at 0.5 mg/ml (20
µl per well), and the plate was centrifuged at 1,200 × g for 2 min. The OCR was measured using a Seahorse XFp Cell Mito Stress Test Kit. The spermatozoa were exposed to
1.5 µM oligomycin for 19.8 min, to 2.0 µM FCCP for 19.8 min, and then to rotenone and antimycin (1.0 µM each) for 19.8 min. Spermatozoa
were collected from each well for measurement of protein content and normalization of OCR values. Basal respiration was the OCR before drug injection. ATP production was calculated as the
difference between the basal OCR and the OCR after oligomycin injection. Proton leak was the OCR after oligomycin injection. Maximal respiration was the OCR after FCCP injection. Spare
capacity is calculated as the difference between OCR after FCCP injection and basal respiration [27 (link)].