*2.9. Boar In Vitro Capacitation*

Boar spermatozoa were collected from fresh ejaculate and were subsequently centrifuged and washed to remove seminal plasma. Sperm were counted using a hemocytometer (ThermoFisher Scientific, Waltham, MA), and incubated in a modified TL-HEPES medium with either the GSTO Inhibitor or DMSO (Control) for 25 min at 38 ◦C. Sperm cells were then placed in either capacitating (modified TL-HEPES supplemented with 5 mM sodium pyruvate, 11 mM D-glucose, 2 mM CaCl2, 2 mM sodium bicarbonate, and 2% ( *m*/*v*) bovine serum albumin) or non-capacitating medium (modified TL-HEPES medium without supplementation) to a concentration of two million cells/mL and incubated at 38 ◦ C for four hours. Cells were removed every hour, washed, and placed in reducing sample bu ffer, for tyrosine phosphorylation analysis by Western blotting up until the four-hour time point. The results were analyzed using an ANOVA with a post-hoc Tukey test, comparing the mean intensities from three replicates.

### *2.10. Mouse In Vitro Fertilization*

Mouse oocytes were obtained from super-ovulated 6 week old CD1 females. Females were given 10 UI of pregnan<sup>t</sup> mare serum gonadotropin (PMSG) through intraperitoneal (IP) injection, followed 48 h later by 10 UI of human chorionic gonadotropin (hCG), also administered through IP injection. Oviducts were harvested from the sacrificed females 12–13 h after the last hCG injection into Advanced KSOM medium (MR-101-D, Millipore-Sigma), warmed to 37 ◦C. Cumulus-oocyte complexes were extracted from the oviducts into warmed Advanced KSOM medium, washed and rested at 37 ◦C, 5% CO2 under mineral oil until fertilization droplets were prepared, to a maximum of 30 min. Spermatozoa were extracted from the cauda epididymis and into Whitten-Hepes Medium where they were either incubated with the GSTO inhibitor (100 μM) or DMSO for 25 min under mineral oil at 37 ◦C, 5% CO2. Sperm were subsequently washed and placed into EmbryoMax human tubal fluid (HTF, MR-070-D, Millipore-Sigma) to capacitate for a minimum of 1 h. Sperm were then diluted with HTF into a 50 μl droplet with a concentration of approximately 1 × 10<sup>5</sup>/mL sperm and between 20–25 oocytes were added to each droplet. Fertilization droplets were incubated at 37 ◦C, 5% CO2 under mineral oil for 5 h before oocytes were removed, washed and further cultured in 50 μL Advanced KSOM droplets under mineral oil for approximately 8 h. Oocytes were then fixed in 2% formaldehyde, permeabilized in phosphate-bu ffered saline with 0.1% Triton-X-100 (PBS-Tx) and stained with DAPI to allow for the visualization of the sperm head or pronuclear formation, indicative of successful sperm penetration. Oocytes were then mounted onto glass slides using Vectashield mounting Medium (Vector Laboratories) and sealed with clear nail polish. Images were captured at the Queen's University Cancer Research Institute Imaging Centre, using a Quorum Wave E ffects spinning disc confocal microscope. Three replicates of each treatment were performed, all using di fferent mice, and statistical analysis was performed using a t-test with Welch's correction.
