Stallion Semen Incubated with Hydrogen Peroxide Decreased DNA Fragmentation as Measured by the TUNEL Assay

Spermatozoa are highly specialized cells that are sensitive to environmental factors such as temperature and redox state. Hydrogen peroxide (H2O2) is a reactive oxygen species, and its presence is often associated with a decline in sperm function. The aim of this study was to evaluate the effect of H2O2 and heat stress on DNA damage, membrane integrity, and motility of stallion spermatozoa. Spermatozoa from three light-horse stallions were subjected to thermal stress, treatment with H2O2, or a combination of both. Treatments were organized using a 2 × 2 factorial design consisting of heat stress (control, 41°C) and potential oxidative stress (control, 50 μM H2O2) for 1 hour. The experiments were repeated independently on all stallions. Primary motility parameters were measured using a computer-assisted sperm analysis, whereas DNA damage was assessed using the TUNEL assay. To evaluate membrane integrity, an amine reactive dye was utilized. DNA and membrane integrity were simultaneously assessed in the same flow cytometry assay. Sperm incubated at 41°C were observed to have decreased motility (76.2% vs. 66.6%; P < .05) but not progressive motility (39.2% vs. 25.6%), membrane damaged (30.8% vs. 37.4%), or DNA damage (19.7% vs. 17.3%). Interestingly, when compared to control, treatments with H2O2 had decreased DNA damage (19.7% vs. 7.1%; P < .05), but did not affect any other parameter. Although our experiment demonstrated a favorable effect of 50 μM H2O2 on DNA damage, further studies need to be conducted to confirm these findings and to clarify any possible signaling mechanisms.