Background: Infertility affects many couples and has led to in- creased use of assisted reproductive techniques (ART). Sperm processing, a key step in ART, can cause oxidative stress and re- duce sperm quality. Antioxidants like N-acetylcysteine (NAC) may help protect sperm by neutralizing free radicals. This study investigates the effects of NAC on sperm quality, antioxidant defenses, and reactive oxygen species (ROS) levels in normal sperm during one hour of in vitro incubation. Materials and Methods: After collection and complete lique- faction, semen samples from 30 healthy, fertile men were di- vided into two experimental groups. The first group served as the control (no treatment, 1-hour incubation), and the second group was treated with NAC (50 μM NAC, 1-hour incubation). At the end of the incubation period, both groups were evalu- ated for sperm motility and viability, along with levels of key antioxidant enzymes catalase (CAT), glutathione (Glu), and su- peroxide dismutase (SOD), as well as ROS levels. Data were analyzed using the repeated measures analysis method, and sig- nificant differences were considered at the P<0.05 level. Results: In the NAC-treated group, the mean percentages of total and progressive sperm motility, DNA integrity, and the levels of antioxidant enzymes CAT, Glu, andSOD were signifi- cantly higher compared to the control group (P<0.001). Con- versely, the mean percentage of non-progressive sperm motility and ROS levels were significantly lower in the NAC group than in the control group (P<0.001). Conclusion: This study demonstrated that short-term in vitro treatment with NAC significantly improves sperm quality in healthy and fertile men by enhancing motility, DNA integri- ty, and antioxidant enzyme activity, while reducing oxidative stress, highlighting its potential role in assisted reproductive techniques to support sperm function
