1. Introduction
For many years, silver has been used as an antimicrobacterial agent alone and in part with other agents. With the development of nanotechnology, silver nanoparticles (AgNPs) are widely usedin pharmaceutical applications, wound dressings, food products, electronics, cosmetics, and hygiene products. The extensive use of AgNPs has raised concerns for their potential sensitivity to humans and the environment (1-2). AgNPs have been shown to adversely affect multiple organs, including the skin, liver, lungs, brain, vascular system, and reproductive and urinary tracts following inhalation or exposure. AgNP toxicity is related to oxidative stress, which is an imbalance between radical production and the body's antioxidant system (3). Spermatozoa are among the most sensitive cells to oxidative stress, especially reactive oxygen species (ROS) - induced stress, which may lead to lipid peroxidation, protein damage, apoptosis, and ultimately impaired cellular function (4-6). Resveratrol, a natural polyphenol present in grapes, peanuts, and berries, is a potent antioxidant that promotes antioxidant enzyme production such as catalase, superoxide dismutase, and glutathione peroxidase and reduces oxidative stress (7). Resveratrol also affects the control of inflammation, tumor growth, cardiovascular disease, neurological disease, platelet aggregation, and metabolic disorders, and it has previously been reported to protect reproductive and other organs against various oxidative insults, including heavy metals and nanoparticles (8, 9). However, despite extensive use of silver nanoparticles, data on their specific effects on testicular structure, spermatogenesis, and oxidative status are still limited, and the potential protective role of resveratrol against AgNP‑induced testicular toxicity has not been comprehensively characterized. Therefore, the present study was designed to evaluate whether resveratrol, plant –derived with antioxidant properties can mitigate AgNP‑induced oxidative stress, histopathological alterations, and hormonal changes in the testes of adult male mice.

2. Materials and Methods
2.1 Animals
Twenty‑four adult male mice (8–10 weeks old, 40–50 g) were obtained from the animal house of Lorestan University of Medical Sciences, Lorestan, Iran. Animals were housed under standard laboratory conditions (12:12 h light–dark cycle, temperature 20–25°C, and relative humidity 55–60%) with ad libitum access to food and water. All experimental procedures were approved by the Institutional Ethics Committee and complied with international guidelines for the care and use of laboratory animals.
2.2 Experimental groups and study design
The animals were randomly divided into four equal groups (n = 6 per group): The animals were randomly assigned to four groups: (1) the control group, which received normal saline by oral gavage; (2) the AgNP group, which received silver nanoparticles at a dose of 200 mg/kg/day (10); (3) the resveratrol group, which received resveratrol at a dose of 20 mg/kg/day (11); and (4) the AgNP + resveratrol group, which received both AgNPs (200 mg/kg/day) and resveratrol (20 mg/kg/day). All treatments were administered once daily by oral gavage for 35 consecutive days, which covers more than one complete spermatogenesis cycle in mice.
2.3 Silver nanoparticles (AgNPs)
Commercially available silver nanoparticles (Nano Metal Coating Co., Iran) with a nominal particle size of 20–40 nm were used. The choice of this size range was based on previous toxicological studies demonstrating that AgNPs of approximately 20–40 nm process effective tissue penetration and biological activity in vivo. The nanoparticles were provided as a suspension by the manufacturer, and their physicochemical specifications (size, purity, dispersion stability) were confirmed according to the supplier’s certificate of analysis. AgNPs were administered at 200 mg/kg/day, a dose that has been widely used in earlier studies evaluating nanoparticle‑induced reproductive toxicity.
2.4 Resveratrol treatment
Resveratrol (Raha Pharmaceutical Co., Iran) was administered at 20 mg/kg/day via gavage. This dose was selected based on previous reports showing significant antioxidant and cytoprotective effects without overt toxicity. Resveratrol was used as a free compound to allow independent evaluation of its protective effects against AgNP‑induced oxidative and histological alterations. Resveratrol was dissolved each day fresh lyin an appropriate vehicle before administration.
2.5 Sample collection
At the end of the treatment period, mice were weighed and anesthetized using ketamine/xylazine. Blood samples were collected from the heart using a 2mL syringe without anticoagulants, and serum was separated by centrifugation at 3000 rpm for 10 minutes at -70°C. Testes were excised, weighed, and processed for biochemical assays and histopathological evaluation.
2.6 Biochemical measurements
The obtained serum samples were analyzed for biochemical markers, including malondialdehyde (MDA), total antioxidant capacity (TAC), and superoxide dismutase (SOD), using Navand Salamat biochemical kits via spectrophotometry, following the kit instructions. Testosterone levels were measured using an Ideal Diagnostic laboratory kit via the ELISA method. The 8-hydroxy-2′-deoxyguanosine (8-OHdG) factor in homogenized testicular tissue was assessed using the Zell Bio ELISA kit per the manufacturer's protocol.
2.7 Histological evaluation
The right testis and epididymis were weighed, fixed in 10% formaldehyde (pH7.4), sectioned to 4-μm thickness, stained with hematoxylin-eosin, and examined under a light microscope. The diameter of seminiferous tubules and the number of Leydig cells were counted and compared between groups. Additionally, atrophy, spermatogenic cell status, duct atrophy, sperm content within tubules, and cellular changes were evaluated.
2.8 Statistical Analysis
Statistical analyses were performed using SPSS software version 26 with significance set at p< 0.05. Data were expressed as mean ± standard deviation. Normality was assessed using the Shapiro-Wilk test, and variance homogeneity was checked with Levene’s test. Welch’s test was used when variances were unequal. The Kruskal-Wallis test was applied for non-normally distributed variables. Pairwise comparisons were conducted using the Bonferroni or Dunnett test, depending on variance homogeneity.

3. Result
3.1 Testosterone and oxidative stress parameters
The current study demonstrated a significant reduction in testosterone levels in the AgNP-only group compared to the controls (P<0.001). Conversely, testosterone levels were significantly elevated in the group receiving resveratrol alone, relative to the AgNP-only group (P<0.001). Notably, the testosterone levels in the resveratrol-treated group did not differ significantly from those in the control group (P=0.982), with values being nearly comparable. In the group administered both AgNPs and resveratrol, testosterone levels were increased compared to the AgNP-only group, although the increase was not statistically significant (P=0.06) (Table1I).
According to Table I, the AgNP-only group exhibited significantly elevated SOD levels compared to the control group in terms of oxidative stress markers (P=0.001).
The group receiving Resveratrol alone showed no significant difference in SOD levels relative to the control group (P=0.695). In the group administered both AgNPs and resveratrol, SOD levels were reduced compared to the AgNP-only group (P=1.000.); however, this reduction was not statistically significant. Also, no significant differences between the groups were observed in TAC (P=0.051) and MDA (P=0.065) levels. (Table 1) 8-OHdG levels were significantly elevated in the AgNP-only group compared to the control and resveratrol-only groups (P=0.045). The combined treatment group also showed a significant increase in 8-OHdG levels compared to the control (P=0.012), although the elevation was less pronounced than in the AgNP-only group (P=1.000, Table1).