*2.11. Swine In Vitro Fertilization*

Ovaries were obtained from a local slaughterhouse and aspirated to obtain oocytes from follicles of 3–6 mm in size. Oocyte with uniform ooplasm and compact cumulus cells were selected and in vitro matured at 38.5 ◦C, 5% CO2, for 42 to 44 h. Cumulus cells from matured cumulus-oocyte complexes (COCs) were removed with 0.1% hyaluronidase in TL-HEPES-PVA and washed three times with TL-HEPES-PVA medium. Twenty-five to thirty oocytes were placed into 100 μL drops of the mTBM medium, while sperm were prepared. Boar semen was collected from sperm rich fraction the day before IVF. Sperm cells were incubated with the GSTO inhibitor for 25 min at 38.5 ◦C immediately after being isolated from the semen and before being placed in a short BTS extender (BTS, IMV Technologies, Maple Grove, MN, USA) until used. Mitochondria of the sperm tail were stained with a viable, mitochondrion-specific probe MitoTracker ® Red CMXRos (Molecular Probes, Inc., Eugene, OR, USA) for 10 min at 38 ◦C in a warm incubator before the sperm solution was diluted to a concentration of 1 × 10<sup>6</sup> cells/mL. Co-incubation of the sperm and the oocytes was left for approximately 6 h before oocytes were removed and transferred to 100 μL drops of PZM-3 medium containing 0.4% BSA (A6003; Sigma, Burlington, MA, USA) for additional culture. Three replicates of each experiment were performed using three di fferent ejaculates and presented as the average. Statistical analysis was performed using a t-test with Welch's correction.

### *2.12. Mouse Computer-Aided Sperm Analysis (CASA)*

Spermatozoa were extracted from the cauda epididymis of mature C57BL/6 males and placed in Whittens-Hepes Medium. Sperm were subsequently placed with the GSTO inhibitor (100 μM) or DMSO (Control) for 25 min at 37 ◦C before being washed and diluted with additional Whittens-Hepes medium or capacitating medium (Whittens Hepes supplemented with 5mg/mL BSA and 20 mM NaHCO3) to a final concentration of approximately 1–2 million cells/mL. Sperm were then placed back at 37 ◦C and left to capacitate for one hour. Samples were then gently spun down and resuspended for analysis. Analysis was done by Sperm Vision HR software version 1.01 (Minitube, Ingersoll, ON, Canada). At least 200 cells from each treatment were analyzed in each experimental trial. All cells that were not unequivocally identifiable as sperm were removed from the analysis by the technician. Four replicates were performed, each using a new mouse, and the data are presented as the average of all replicates. Multiple t-tests were performed to determine statistical significance.
