Determination of O2 consumption rate

The O₂ consumption rate was determined with a SensorDish^® Reader (SDR) system (PreSens Gmbh; Regensburg, Germany), as described in Blanco-Prieto et al. (2020). For this purpose, 1-ml aliquots were transferred into a specifically-designed Oxodish^® OD24 plates (24 wells/plate). Plates were sealed with Parafilm^® and then placed into the SDR device, which was introduced in an incubator at 38°C. The O₂ consumption was evaluated once a min for 2 h and, when finished, data were exported to an Excel file. The O₂ consumption rate was calculated as the decrease of O₂ concentration in each well during the 2-h incubation period, normalized against the percentage of viable sperm (SYBR14⁺/PI⁻) determined by flow cytometry.

As mentioned in the Experimental design section and to delve further into how irradiation with red light in the presence of antimycin A and/or PKCi affects O₂ consumption, sperm were also incubated with 5 µM oligomycin A, a specific ATPase inhibitor, or 5 µM FCCP, a specific disruptor of the electron chain, as described in Blanco-Prieto et al. (2020). The inclusion of these additional samples allowed us to determine the following parameters (Supplementary Figure S1): 1) non-mitochondrial O₂ consumption (NMC), which corresponded to O₂ consumption recorded in the presence of 1 µM antimycin A; 2) basal respiratory rates (BR), which were the values obtained in the absence of any inhibitor/disruptor; 3) oligomycin-resistant O₂ consumption (OC), which corresponded to samples incubated with 5 µM oligomycin A; 4) Maximal respiration (MR), which corresponded to samples incubated with 5 µM FCCP; 5) H⁺ leakage (HL): OC-NMC; 6) ATP turnover (AT): BR-HL; and 7) Spare respiratory capacity (SRC): MR-BR. Total O₂ consumption rate resulted from the sum of NMC, BR and SRC, and, for all experimental conditions, the percentage of each of these components was calculated.