To begin, prepare the equipment by turning on the high resolution respirometer and connect it to the respirometry software VatLab for data acquisition and analysis. Replace the 70%ethanol in the oxygraph chamber with double distilled water. Using a magnetic stir bar, stir it continuously at 750 RPM in the chamber.
Let it stand for 10 minutes and then aspirate the water. Wash the chamber three times with double distilled water for five minutes each. To calibrate the oxygen sensors, first remove the double distilled water and pipette two milliliters of the cell preparation medium into the chamber.
Place the stoppers leaving an air exchange bubble. To record the oxygen calibration values, adjust the settings such as gain for the sensor, polarization voltage, and data recording interval. Then label the experiment and enter the medium.
Click on layout and select 01 Calibration Experiment GR3 Temp. Stir the medium with the stir bar at 750 RPM for at least 30 minutes at 37 degrees Celsius, and monitor the performance of the sensor membrane. Holding the left mouse button and the Shift key, drag the mouse to select an area where the change in oxygen concentration is stable.
Then click on oxygraph and then O2 Calibration. In air calibration, change the selected mark to the region selected earlier. Then click on calibrate and copy to clipboard.
Stop the recording and save by clicking on oxygraph. Followed by OK Control and then save and disconnect. Next, to perform respiratory assessment, open the chamber and aspirate the medium inside.
Load sperm cells in a final volume of two milliliters of MRM for permeabilized cell analysis. Load the calibration by double clicking on the POS calib box in the bottom corner and opening the calibration performed earlier. Then stop the experiment.
Inject 4.5 microliters of 10 millimolar digitonin and permeabilize the cells for five minutes. Record the respiration of the intact cells for at least five minutes until a stable signal is obtained. Then add the substrates for complex I or complex II.Measure the oxygen consumption until the signal increases and stabilizes.
Next, inject five microliters of 0.5 molar ADP and measure the oxygen consumption until the signal increases and stabilizes. Add one microliter of four milligrams per milliliter Oligomycin, which is an ATP synthetase inhibitor. Measure the oxygen consumption until the signal decreases and stabilizes.
Titrate by adding one microliter of FCCP in successive steps, starting from 0.1 millimolar to one millimolar until a maximum uncoupled respiration rate is reached. Measure the oxygen consumption until the signal increases and stabilizes. Stop injecting the drug when oxygen consumption begins to decrease.
Finally, inject one microliter of one millimolar rotenone for complex I or five millimolar Antimycin A for complex II to discriminate between the mitochondrial and residual oxygen consumption. Measure the oxygen consumption until the signal decreases and stabilizes. To perform data analysis select regions where the oxygen flux per volume correlated is stable after the injection of a substrate or inhibitor, click on marks, then statistics windows, and export the data.
Normalize the data obtained per 1 million sperm cells and subtract the non-mitochondrial oxygen consumption from all the values. Calculate the indices using these equations. Successful recording of intact sperm cells from a semen sample was achieved where the blue line showed the oxygen concentration and the red line represented the oxygen flow per volume correlated.
Lower sperm cell counts resulted in lower baseline oxygen consumption. Further, oxygen consumption curves specifically for the mitochondrial complex I or complex II were obtained using this protocol.
