*2.13. Lipid peroxidation Intensity Analysis*

Mouse spermatozoa were extracted from the cauda epididymis of mature C57BL/6 males and placed in Whitten-Hepes Medium. Sperm were diluted to a concentration of approximately 2 × 10<sup>6</sup>/mL, and subsequently placed with the BODIPY 581/591 C11 (D3861, Invitrogen Molecular Probes, Eugene, OR, USA) to a final concentration of 5 μM for 20 min at 37 ◦C. A subset of sperm were not treated with the BODIPY C11 probe to act as a baseline for innate fluorescence. Sperm were washed twice at 650× *g* for 5 min before being placed with the GSTO inhibitor (100 μM) or DMSO (Control) for 25 min at 37 ◦C. Spermatozoa were washed again and placed in capacitating medium (Whittens-Hepes Medium supplemented with 5mg/mL BSA and 20 mM NaHCO3) to a final concentration of approximately 1–2 million cells/mL and incubated for one hour at 37 ◦C, with agitation every 15 min. Spermatozoa were subsequently washed and placed on a glass slide, covered with a coverslip and sealed with clear nail polish. Three trials were performed for each treatment, and at least 200 cells per treatment were analyzed each trial by confocal microscopy. Images were captured at the Queen's University Cancer Research Institute Imaging Centre, using a Quorum Wave E ffects spinning disc confocal microscope, and analysis was performed using the MetaMorph Imaging software. The intensities of both the red and green fluorescence were acquired for all treatments and controls. For GSTO Inhibited samples, the fluorescent intensity of the inhibitor itself was subtracted from the overall green intensity to ensure it did not impact the overall ratio of green and red fluorescence. The results were analyzed using a t-test with Welch's correction to determine statistical significance.

### *2.14. Cellular Reactive Oxygen Species Levels*

Mouse spermatozoa were extracted from the cauda epididymis of mature CD1 males and placed in Whitten-Hepes Medium. The sperm were subsequently placed with the GSTO inhibitor (100 μM) or vehicle (DMSO) for 25 min at 37 ◦C. Spermatozoa were then diluted to a concentration 5 × 10<sup>6</sup> cells/mL and placed under capacitating conditions through supplementing the buffer with 5 mg/mL BSA and 20 mM NaHCO3. Spermatozoa were left to incubate for 60 min, with agitation every 15 min at 37 ◦C/5% CO2. Reactive oxygen species levels were then probed using the Cellular ROS Assay Kit (ab186029). Samples were subsequently washed and fixed in 2% formaldehyde before being analyzed using flow cytometry. Flow cytometry was performed at the Queen's University Cardiac Pulmonary Unit using the Sony SH800 Cell Sorter and least 10,000 events were recorded for each sample. Three experimental trails were done for each treatment, each using a different mouse, and the data presented include the average mean intensity over the three trials. Statistical analysis was done using a t-test with Welch's correction.
