Hydrogen sulfide (H2S) has been shown to play a significant role in oxidative stress across various tissues and cells; however, its role in sperm function remains poorly understood. This study aimed to investigate the protective effect of GYY4137, a slow-releasing H2S compound, on sperm damage induced by H2O2. We assessed the effects of GYY4137 on motility, viability, lipid peroxidation and caspase-3 activity in human spermatozoa in vitro following oxidative damage mediated by H2O2. Spermatozoa from 25 healthy men were selected using a density gradient centrifugation method and cultured in the presence or absence of 10 μM H2O2, followed by incubation with varying concentrations of GYY4137 (0.625–2.5 μM). After 24 h of incubation, sperm motility, viability, lipid peroxidation, and caspase-3 activity were evaluated. The results indicated that H2O2 adversely affected sperm parameters, reducing motility and viability, while increasing oxidative stress, as evidenced by elevated lipid peroxidation and caspase-3 activity. GYY4137 provided dose-dependent protection against H2O2-induced oxidative stress (OS). We concluded that supplementation with GYY4137 may offer antioxidant protection during in vitro sperm preparation for assisted reproductive technology.

The aim of this study was to optimize protocols for electroporation (EP) and polyfection (PLF) using polyethyleneimine (PEI) for pig sperm transfection and to determine which method was the most efficient. For EP standardization, different voltages, amounts and times of electric pulses were tested using propidium iodide (PI) as reporter. For PLF standardization, different concentrations of fluorescein isothiocyanate (FITC)-labelled PEI (PEI/FITC) were incubated with sperm for different periods of time. Flow cytometry was performed to evaluate the best protocol in terms of cell viability, including cytoplasmic membrane, acrosome, chromatin integrities and mitochondrial potential using the FITC probe, PI, acridine orange (AO) and JC1. Transfections with the plasmid pmhyGENIE-5 were carried out under optimum conditions for each procedure (EP: 500 volts, 500 μs and two pulses; PLF: PEI 0.5 mg/ml and incubation time 10 min). Transfection efficacy was assessed by fluorescence in situ hybridization (FISH). A lower transfection rate was observed for sperm in the control group (17.8%) compared with EP (36.7%), with PLF (76.8%) being the most efficient. These results suggest that the EP and PEI could be an efficient and low cost transfection method for swine sperm. Notably, treated cells showed higher plasmatic the membrane damage (PMD) and/or acrosome damage (AD) indexes, therefore the combination of this procedure with biotechniques that facilitate fecundation (i.e. in vitro fertilization or intracytoplasmic sperm injection) or even inclusion of antioxidant or anti-apoptotic drugs to improve spermatozoa viability would be important.