Comprehensive Assessment of Silver Bioaccumulation and DNA Damage Effects in Coturnix coturnix japonica Using Blood, Feather, and Egg Biomarkers

Comprehensive Assessment of Silver Bioaccumulation and DNA Damage Effects in Coturnix coturnix japonica Using Blood, Feather, and Egg Biomarkers | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Comprehensive Assessment of Silver Bioaccumulation and DNA Damage Effects in Coturnix coturnix japonica Using Blood, Feather, and Egg Biomarkers Nudrat Fatima, Shabana Naz, Rifat Ullah Khan, Ala Abudabos, Ankqash Ayyub, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7086690/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract This study examined the effects of Ag-NPs and AgNO₃ on silver accumulation in the blood, feathers, eggshells, and egg contents of Japanese quails, as well as their potential to cause DNA damage. A total of 480 (fourteen-day-old) quails were divided into five groups of 96 birds each, arranged into six replicates of sixteen birds with a sex ratio of one male to three females. The first group served as a control and was fed a basal diet, while the second and third groups received Ag-NPs at doses of 10 mg/kg and 20 mg/kg, respectively. The fourth and fifth groups were given AgNO₃ at the same concentrations. Results showed that the highest silver accumulation occurred in all tissues in quails fed the higher dose of Ag-NPs. The greatest accumulation was observed in the eggshells, likely due to their porous structure, which facilitates metal deposition. Both Ag-NPs (20 mg/kg) and AgNO₃ (10 and 20 mg/kg) induced DNA damage, although the damage was more severe in the groups exposed to AgNO₃. A positive correlation was observed between treatment groups and comet assay parameters indicating increased DNA fragmentation in exposed birds. In conclusion, the study demonstrated that although Ag-NPs resulted in higher silver accumulation, they caused less DNA damage compared to silver nitrate. This suggests that silver nanoparticles may offer a safer alternative for increasing silver levels in poultry feed while minimizing the genotoxic risks associated with ionic silver compounds. Japanese quail Silver nanoparticles Silver Nitrate Genotoxicity Comet assay Figures Figure 1 Figure 2 Figure 3 Introduction Nanotechnology involves the manipulation of materials at the nanometer scale, typically between 1 and 100 nanometers. This innovative field had a significant impact across various industries, including healthcare, agriculture, and the food and nutrition sectors (Khan et al., 2014; Chand et al., 2020; Rawat et al., 2023). Researchers are increasingly exploring the unique properties of nanostructures such as enhanced electrical, magnetic, and optical characteristics which have considerable implications for improving the nutritional value and bioavailability of food ingredients (Khan et al., 2017; Ahmad et al., 2020). A fundamental component of nanotechnology is the use of nanoparticles extremely small particles with a high surface-area-to-volume ratio. This feature allows nanoparticles to interact more efficiently with biological systems, resulting in improved absorption and targeted delivery of nutrients. Among various types of nanoparticles, silver nanoparticles (Ag-NPs) are particularly notable due to their remarkable antibacterial, antiviral, and antifungal properties, which contribute to food preservation and safety enhancement (Nasrollahzadeh et al., 2019). Silver (Ag) exists in both organic and inorganic forms, each with distinct properties and applications. In its inorganic form, it is commonly used as elemental silver or silver salts, while in its organic form, it can form complexes with various organic molecules (Bhattarai et al., 2018). Ag-NPs have become one of the most widely used materials in biomedicine. They are employed in the treatment of infections and cancer, vaccine development, diabetes management, and wound healing. Additionally, they are extensively applied in the development of chemical and biological sensors, owing to their unique optical properties. A prominent trend in Ag-NP synthesis is the adoption of green methods, such as using plant extracts or microorganisms. These methods are considered safer, more cost-effective, and environmentally friendly (Xu et al., 2020). One plant that plays a crucial role in the eco-friendly synthesis of Ag-NPs is Azadirachta indica , commonly known as neem (Gupta et al., 2017). Neem is renowned for its powerful medicinal properties, including antibacterial, antiviral, antifungal, and anti-inflammatory effects, making it a valuable resource for nanoparticle production (Khan et al., 2022; Wylie et al., 2022). Utilizing neem extracts for the synthesis of Ag-NPs offers a more sustainable and biocompatible alternative, providing both health and environmental benefits. However, despite these advantages, concerns remain regarding the potential toxicity of Ag-NPs (Ahmed et al., 2016). Ag-NPs are known to accumulate in specific organs more effectively than silver ions, which can lead to toxic effects in biological systems. This bioaccumulation is particularly concerning due to its potential long-term impacts on both human health and the environment (Tortella et al., 2020). The tendency of Ag-NPs to bioaccumulate has been observed in various organisms, including birds. Their small size and unique physicochemical properties allow them to be easily absorbed and retained in biological tissues (Chaturvedi et al., 2021). Unlike inorganic silver, which typically exists in ionic form (Ag⁺), Ag-NPs can penetrate cellular membranes more efficiently, resulting in greater accumulation in tissues and organs. This is especially concerning for species such as quails, which are frequently used in environmental research to study the effects of pollutants and nanomaterials (Naz et al., 2024). Quails are ideal model organisms for such studies due to their small size, high reproductive rate, and adaptability to various environments. Their widespread use in agricultural and scientific research, along with their importance in food production, makes them valuable subjects for investigating the potential risks of Ag-NP bioaccumulation and its long-term effects on wildlife and human health. Given the expanding use of Ag-NPs in agriculture, medicine, and consumer products, continued research into their safety and environmental impact is essential. This study was designed to compare the effects of Ag-NPs and AgNO₃ on bioaccumulation and DNA damage in quails, thereby contributing to the understanding of nanoparticle toxicity and guiding future regulatory decisions. Materials and Methods Synthesis of Silver Nanoparticles Fresh neem ( Azadirachta indica ) leaves were first thoroughly washed with tap water, followed by distilled water, and then completely dried. Once dried, the leaves were cut into small pieces and ground into a fine paste using a mortar and pestle. A 10 g sample of the paste was then heated in 100 mL of distilled water at 60°C for 30 minutes to extract the bioactive compounds. After cooling, the extract was filtered through Whatman No. 1 filter paper to remove any solid residues (David et al., 2014). A 1 mM silver nitrate (AgNO₃) stock solution was prepared by dissolving silver nitrate in distilled water. Subsequently, 10 mL of the neem leaf extract was added to 200 mL of the Ag-NO₃ solution and gently stirred on a hot plate. The mixture was maintained at 60°C for 30 minutes. A visible color change from yellow to dark red confirmed the successful synthesis of silver nanoparticles (Ag-NPs), indicating the reduction of silver ions and nanoparticle formation (Sahayaraj & Rajesh, 2011). The synthesized Ag-NPs were characterized using a UV-Visible spectrophotometer (Hitachi U-2800), with absorbance measured in the 300-600 nm range to monitor nanoparticle formation (Ahmed et al., 2016). Fourier Transform Infrared (FTIR) spectroscopy (PerkinElmer) was used to identify the functional groups involved in stabilizing the Ag-NPs. Measurements were taken across a spectral range of 400 to 4000 cm⁻¹ with a resolution of 4 cm⁻¹. The FTIR analysis showed absorption bands corresponding to carbonyl groups from proteins, indicating that proteins in the neem extract likely capped and stabilized the Ag-NPs. X-ray diffraction (XRD) analysis was performed using an X’Pert Pro diffractometer with Cu Kα radiation to determine the amorphous structure and size distribution of the nanoparticles. Scanning was conducted at a rate of 10°/min. The XRD patterns confirmed an amorphous structure (Ugwuoke, et al., 2023). Trial Birds and Research Methodology This study was conducted in accordance with the ethical guidelines for the handling and care of laboratory animals established by Government College University Faisalabad (GCUF). A total of 480 (14-day-old) Japanese quails were acquired and acclimatized for 14 days under controlled conditions, which included a temperature range of 20–25°C, 70% humidity, and a 16-hour light/8-hour dark cycle. The quails were housed in wire cages with free access to a commercial basal diet (NRC, 1994, Table 1) and fresh water. The birds were randomly assigned to five experimental groups, each consisting of 96 birds and six replicates (16 birds per replicate). The groups were as follows: the first group (control) received only the basal diet; the second and third groups received Ag-NPs at 10 mg/kg and 20 mg/kg body weight, respectively; and the fourth and fifth groups received AgNO₃ at 10 mg/kg and 20 mg/kg body weight, respectively. Treatments were administered weekly via oral gavage over 65-day period throughout the trial. Blood and feather samples collection At the conclusion of the trial (day 65), 2-3 mL blood samples were collected from the brachial vein of twelve birds per replicate. Six samples were placed in lavender-topped tubes for comet assay analysis, and Six in gold-topped tubes for silver bioaccumulation measurement, following the method described by Khalid et al (2025). All blood samples were stored at -20°C until chemical analysis. The birds were then euthanized, and chest feathers were collected post-dissection. These feathers were selected as representative samples, as they are considered to best reflect environmental exposure Collection of eggs Eggs were collected daily during the quail breeding season, coinciding with the typical morning laying period. To minimize damage, at least 20 eggs per replicate were collected manually. Each egg was labeled with the date and replicate number, then stored in chemically cleaned jars at 4°C to prevent contamination. Prior to processing, eggs were washed with demineralized water, if required. The eggshells were carefully removed, and the contents were transferred to sterile containers via clean glass dishes. These containers were then frozen, preparing the samples for subsequent chemical analysis (Ashkoo et al., 2020). Silver bioaccumulation screening Sample's digestion Blood samples, collected in gold-topped tubes, were allowed to clot. Serum was then separated from red blood cells (RBCs) by centrifuging the coagulated blood at 2000 rpm for 10 minutes, maximizing serum yield. A 1:10 serum dilution was prepared by mixing 1 ml of serum with 10 ml of demineralized water (Naz et al., 2024). Feather samples were cleaned with acetone, followed by three washes with water. After drying in an oven at 60°C for two days, the feathers were fragmented. A 1-g portion of each sample was then digested using a 5 ml hydrogen peroxide (H₂O₂) and 5 ml nitric acid (HNO₃) mixture on a 70°C hot plate. Once digestion was complete, the mixture was cooled to room temperature and filtered through Whatman filter paper No. 1. The filtered solution was then diluted with 25 ml of deionized water (Rutkowska et al., 2018) Eggs were cleaned with acetone and demineralized water to remove surface contaminants. Egg contents were extracted using a toothpick and placed in petri dishes, while eggshells were placed in separate dishes. Samples were dried in an oven to achieve uniform dry matter. Dried eggshells and egg contents were then ground into homogeneous powders. A 0.5-g portion of each powder was mixed with 10 ml of nitric acid (HNO₃) in conical flasks and heated to 140°C until the solution became clear (Khalid et al., 2025). After cooling, the digests were filtered through Whatman filter paper No. 1 and diluted to 25 ml with deionized water in polypropylene flasks. Digested samples were stored at 4°C until silver content analysis via Atomic Absorption Spectrophotometry (Ashkoo et al., 2020). Chemical analysis of samples Silver bioaccumulation in blood, feather, and egg samples was determined using Atomic Absorption Spectrometry (Aurora AI 1200). Silver concentrations were analyzed at a wavelength of 328.1 nm. Calculation of silver concentration Silver concentrations were determined using the formula: Metal concentration = (Spectrophotometric reading × Dilution factor) / Sample weight. A dilution factor of 25 mL/g was applied to solid samples (feathers, eggshells, and egg contents), while a factor of 10 mL/mL was used for serum samples. DNA damage examination To assess DNA damage resulting from silver exposure, the Comet assay, also known as Single-Cell Gel Electrophoresis (SCGE), was conducted. The procedure adhered to the methodology outlined by Singh et al. (1988) (Fig. 1). Image analysis and data acquisition for seven comet parameters—head length, tail length, total comet length, percentage of DNA in the tail and head, tail moment, and olive tail moment—were performed using Casp_1.2.3b1 software. Statistical analysis Data analysis was carried out using IBM SPSS Statistics 25 and Statistics 8.1. One way ANOVA was used to assess silver levels in blood, feathers, eggshells, and egg contents, as well as to evaluate DNA damage in quails exposed to varying concentrations of Ag‑NPs and AgNO 3 . To determine which groups differed significantly, Tukey’s post-hoc test was used. Furthermore, Pearson’s correlation analysis was performed to examine relationships between comet assay parameters and the different treatment groups of C. japonica receiving low and high doses of Ag‑NPs and AgNO 3 . Results Table 2 illustrates that silver concentrations in C. japonica differed significantly ( P < 0.05) depending on the type and dosage of silver administered. The highest silver levels were found in the blood (0.26 μg/g), feathers (0.36 μg/g), eggshells (0.83 μg/g), and egg contents (0.77 μg/g) of quails fed a high dose (20 mg/kg) of Ag-NPs. A moderate increase was also observed with a 10 mg/kg dose of Ag-NPs. In comparison, birds treated with AgNO₃ exhibited intermediate values, while the control group consistently showed the lowest silver accumulation. Significant differences between treatments were marked with superscript letters ( a, b, c, d, e ), indicating statistical variation among groups. Overall, the pattern of silver accumulation in the quail samples treated with different concentrations of Ag-NPs and AgNO₃ followed this order: eggshells > egg contents > feathers > blood. The highest accumulation of silver was observed in the samples from quails supplemented with a high dose of Ag-NPs (20 mg/kg) (Figure 2). The silver concentrations across the treatment groups were ranked as follows: Ag-NPs (20 mg/kg) > Ag-NPs (10 mg/kg) > AgNO₃ (20 mg/kg) > AgNO₃ (10 mg/kg) > control. All comet parameters were analyzed using ANOVA across groups treated with different doses of Ag-NPs and AgNO₃ (Table 3). Significant differences ( P < 0.05) were observed between the mean values of LHead, LTail, LComet, HeadDNA, TailDNA, TM and OTM. A Post-Hoc Tukey test revealed that the highest values for Lhead (37.00 ± 3.46), LTail (10.67 ± 1.15), LComet (39.00 ± 8.71), TM (2.01 ± 0.45) and OTM (2.59 ± 0.48) were found in the group treated with AgNO₃ (20 mg/kg) compared to the other groups. The greatest damage was observed in the AgNO₃ group, while the Ag-NPs groups (10 mg/kg and 20 mg/kg) showed lower values, indicating less damage in these treatments. Additionally, the microscope analysis revealed significant differences, as shown in Figure 1. There was a strong positive correlation between silver exposure (Ag-NPs and AgNO₃) and several comet assay parameters in C. japonica , including Lhead (r = 0.934), Ltail (r = 0.932), Lcomet (r = 0.946), tailDNA (r = 0.626), TM(r = 0.729), and OTM(r = 0.889) (Figure 3). In contrast, HeadDNA content exhibited a marked negative correlation (r = –0.858) with the treatment groups. These findings indicate that silver compounds, particularly AgNO₃, significantly affected DNA integrity. The extent of DNA damage increased with higher silver concentrations, with the most severe effects observed in the groups treated with AgNO₃ Discussion In the present study, the results showed that the mean values of Ag varied significantly in quails treated with different concentrations of Ag-NPs and AgNO₃. Notably, the highest Ag levels were found in the blood of Japanese quails exposed to the highest dose of Ag‑NPs. Because blood reflects overall health and plays a crucial role in transporting nutrients, metabolic waste, and hormones, this elevated level is especially significant (Chand et al., 2018). It indicates enhanced absorption and retention of silver in nanoparticle form likely due to the small size and unique physicochemical properties of Ag‑NPs, which promote their passage across biological membranes. Indeed, Ag‑NP coatings can modulate ion release and membrane interaction, increasing bioavailability compared to ionic forms (Bhattarai et al., 2018). The enhanced bioaccumulation of Ag from Ag-NPs may be attributed to their nanoscale dimensions and high surface area-to-volume ratio, which increase their reactivity and interaction with biological tissues. Ag-NPs can penetrate epithelial barriers through endocytosis or diffusion, allowing them to enter systemic circulation more efficiently than ionic silver (Ag⁺) from AgNO₃ (Khan et al., 2022). Once in the bloodstream, Ag-NPs may bind to plasma proteins or cellular membranes, leading to prolonged circulation time and accumulation in various tissues. Additionally, the slow dissolution of Ag-NPs into Ag⁺ within biological environments may contribute to sustained silver release, thereby enhancing systemic retention and potential biological effects (Behra et al., 2013). Significant differences were observed in the mean Ag concentrations in the feathers of Japanese quails administered varying doses of Ag-NPs and AgNO₃. The highest Ag level was detected in the feathers of birds receiving high dose of Ag-NPs. Once in systemic circulation, Ag-NPs are more likely to persist in the bloodstream and reach peripheral tissues compared to ionic silver from AgNO₃. As feathers develop, they incorporate circulating trace elements, such as silver, into their keratin matrix. The prolonged presence of Ag-NPs in the body increases the likelihood of their deposition in feather tissue during growth, resulting in higher silver concentrations (Ansari et al., 2024). Feathers were selected as a marker for bioaccumulation due to their ability to reflect internal metal exposure over extended periods. Feathers serve as excretory pathways for metals, and their accumulation indicates chronic exposure over time, as opposed to blood, which reflects more recent exposure events (Brown et al., 2016; Roberts & Wilson, 2017). This is consistent with findings from Khalid et al. (2025), who reported selenium accumulation patterns in quail tissues, reinforcing feathers and eggs as reliable bioindicators. Moreover, feathers can be collected non-invasively, enabling repeated sampling without harming the animal. During feather formation, trace elements present in the bloodstream become permanently embedded in the keratin structure, providing a stable and time-integrated record of exposure. The strong binding affinity of metals to keratin further preserves the elemental composition, making feathers an effective tool for long-term monitoring of environmental contaminants in avian species (Borghesi et al., 2016). Our results indicated that increasing dietary concentrations of Ag-NPs and AgNO₃ from 10 to 20 mg/kg feed led to a significant accumulation of silver in both the eggshell and internal contents of Japanese quail eggs. This suggests that silver, particularly in nanoparticle form, can cross biological barriers and be transferred from the hen to the developing egg. Eggshells serve as both a protective barrier and a mineral reservoir, often accumulating metals through maternal deposition during shell formation (Ashkoo et al., 2020). The incorporation of silver into the eggshell may alter its structural integrity, while the presence of silver in the egg contents indicates potential for vertical transmission and raises concerns about embryotoxic effects (Fonseca et al., 2019). The greatest concentration of Ag was detected in the eggshells of quails, compared to the levels found in egg contents, feathers, and blood samples. The eggshell is primarily composed of calcium carbonate crystals, along with a matrix of proteins and other minerals, which are deposited by specialized cells in the shell gland during egg formation (Hincke et al., 2012). This mineralization process allows the eggshell to serve as a reservoir for various minerals and metals circulating in the hen’s bloodstream, including silver. Metals like silver often have a strong affinity for binding with calcium and other mineral components due to similar chemical properties, facilitating their incorporation into the eggshell’s crystalline matrix. This selective deposition means that metals can accumulate in the shell in higher amounts than in other tissues (Rosaiah et al., 2024). Additionally, the eggshell functions as a protective barrier to shield the developing embryo from harmful substances, including toxic metals. By sequestering silver in the shell, the bird’s physiology limits the amount that reaches the internal contents of the egg, such as the yolk and albumen, thereby reducing potential toxicity to the embryo (Wilson, 2017). Recent pathological studies examining the genotoxic effects of Ag-NPs and AgNO₃, confirmed by comet assays, revealed that DNA damage was significantly more pronounced in samples exposed to AgNO₃ compared to those treated with Ag-NPs or control groups. This finding highlights the critical role of silver’s chemical form in its genotoxic potential. AgNO₃ is a soluble salt that rapidly dissolves in biological fluids, releasing silver ions (Ag⁺) almost immediately. These free silver ions are highly reactive and can readily penetrate cells, where they interact directly with vital biomolecules such as DNA (Behra et al., 2013). Inside the cell, silver ions promote the formation of reactive oxygen species (ROS), including free radicals and peroxides. These ROS attack the DNA molecule, causing oxidative damage such as single- and double-strand breaks, base modifications, and crosslinking. The accumulation of such damage compromises the integrity of genetic material and may lead to mutations or cell death. In contrast, Ag-NPs are solid particles that release silver ions more slowly due to their particulate nature and surface chemistry (Croteau et al., 2011). This gradual ion release results in fewer free silver ions being immediately available to interact with DNA, thereby reducing the extent of direct damage. Furthermore, Ag-NPs are often internalized by cells via endocytosis and become sequestered within cellular compartments like lysosomes, limiting their direct contact with the nucleus where DNA is located. As a result, while Ag-NPs can still induce oxidative stress and cellular damage, the severity and immediacy of DNA damage tend to be lower than those caused by silver ions from AgNO₃ (Mahjoubian et al., 2023). This difference in genotoxicity is consistent with the potent nature of silver ions, which can directly bind DNA bases, generate ROS, and cause strand breaks and chromosomal aberrations (Kumar et al., 2021; Zhang et al., 2016). Despite higher silver levels in tissues following Ag-NPs exposure, the DNA damage is comparatively less severe, likely due to the slower ion release and sequestration of nanoparticles within lysosomes or vesicular compartments (Wang & Li, 2023). Additionally, cellular antioxidant defenses may be more effective against the oxidative stress induced by nanoparticles, further mitigating DNA damage. Conclusions The study demonstrated that Ag-NPs led to the highest silver accumulation in quails, particularly within the eggshells, while causing less DNA damage than AgNO₃. Conversely, AgNO 3 exposure resulted in more severe DNA damage at both low and high concentrations. These results indicate that Ag-NPs may be a safer alternative for elevating silver levels in poultry, as they promote greater bioaccumulation with a lower risk of genetic toxicity compared to ionic silver forms. Declarations Author Contribution N.F., S.N., and R.U.K. conceptualized and designed the study. N.F., A.A., M.U., S.A., H.S., and S.S. conducted the experiments and collected samples. S.N. and R.U.K. supervised the research and provided resources. I.A.A. and A.A. assisted with data interpretation and manuscript review. N.F. performed data analysis and wrote the initial draft. S.N. and R.U.K. revised and finalized the manuscript. All authors reviewed and approved the final version of the manuscript. Disclosure statement No potential conflict of interest was reported by author(s) Ethical Approval The Committee on Animal Rights and Welfare, GC University Faisalabad, Pakistan approved this study (GCUF/ERC/460). Data availability Data will be made available from the authors upon reasonable request. Funding We are thankful to the Ongoing Research Funding (ORF-2025-833), King Saud University, Riyadh, Saudi Arabia References Abid, N., Khan, A. M., Shujait, S., Chaudhary, K., Ikram, M., Imran, M., Maqbool, M., 2022. Synthesis of nanomaterials using various top-down and bottom-up approaches, influencing factors, advantages, and disadvantages: A review. Adv. Coll. Int. Sci. 300, 102597. https://doi.org/10.1016/j.cis.2021.102597 Ahmad, Z., Hafeez, A., Ullah, Q., Naz, S., Khan, R. U., 2020. 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Silver nanoparticles: Synthesis, characterization, properties, applications, and therapeutic approaches. Int. J. Mol. Sci. 17 (9), 1534. https://doi.org/10.3390/ijms17091534 Tables Table 1 Composition of Basal Diets Used for Japanese Quails Ingredients Percentage (%) Soyabean oil 0.50 Soyabean meal 21.60 Wheat 4.10 Calcium phosphate (GR) 1.00 Calcium carbonate (GR) 9.50 Cottonseed meal 2.00 Corn 60.50 Phytases 0.015 Sodium chloride 0.30 DL-methionine 0.08 Vitamin premix a 0.15 Mineral premix b 0.035 50% Choline chloride 0.10 Experimental additives 0.12 Total 100.0 Nutrient Level Methionine + Cysteine (%) 0.55 Apparent metabolize energy (AME), Mcal/kg 2.57 Se c (mg/kg) 0.056 Crude protein (%) 15.0 Lysine (%) 0.74 Total phosphorus (%) 0.54 Available phosphorus (%) 0.31 Calcium (%) 3.7 Methionine (%) 0.3 Abbreviations: Guaranteed Reagent (GR) is provided per kilogram of diet, including vitamin A at 12,500 International Units (IU), vitamin D3 at 2,500 IU, vitamin E at 18.75 milligrams (mg), vitamin K3 at 2.65 mg, vitamin B1 at 2 mg, vitamin B2 at 6 mg, vitamin B12 at 0.025 mg, biotin at 0.325 mg, folic acid at 1.25 mg, and niacin at 50 mg. Additionally, trace minerals provided per kilogram of diet include copper (Cu) at 8 mg, iron (Fe) at 80 mg, zinc (Zn) at 80 mg, manganese (Mn) at 60 mg, and iodine (I) at 1.2 mg. Table 2 Comparative Analysis of Silver Levels (Mean ± SD) in Blood, Feathers, Eggshells, and Egg Contents of C. japonica Supplemented with Low and High Doses of Ag-NPs and AgNO 3 Samples Control Ag-NPs (10 mg/kg) Ag-NPs (20 mg/kg) Ag-NO 3 (10 mg/kg) Ag-NO 3 (20 mg/kg) P . Value Blood 0.04 ± 0.01 d 0.19 ± 0.01 b 0.26 ± 0.01 a 0.06 ± 0.02 d 0.16 ± 0.02 c 0.000*** Feathers 0.15 ± 0.00 d 0.20 ± 0.02 b 0.36 ± 0.01 a 0.17 ± 0.00 cd 0.18 ± 0.01 c 0.000*** Egg shell 0.23±0.02 e 0.62±0.04 b 0.83±0.02 a 0.32±0.03 d 0.37±0.04 c 0.001 ٭٭٭ Egg content 0.22 ±0.04 e 0.49±0.07 b 0.77±0.04 a 0.33±0.04 d 0.42±0.04 c 0.001 ٭٭٭ Ag-NPs = Silver Nanoparticles; AgNO₃ = Silver Nitrate. The mean values with distinct superscripts ( a–e ) in a row exhibit substantial variation at ( P < 0.05). Table 3 Comet parameters (Mean ± SD) in the blood of C. japonica supplemented with different doses of Ag-NPs and AgNO₃ Parameters Control Ag-NPs (10 mg/kg) Ag-NPs (20 mg/kg) AgNO 3 (10 mg/kg) AgNO 3 (20 mg/kg) P . value LHead 26.33 ± 2.31 c 27.21 ± 2.31 c 28.33 ± 8.33 c 33.67 ± 4.16 b 37.00 ± 3.46 a 0.000*** LTail 3.00 ± 0.00 b 4.00 ± 0.00 b 5.00 ± 0.00 b 8.00 ± 1.00 a 10.67 ± 1.15 a 0.000*** LComet 30.34 ± 2.30 c 31.34 ± 2.31 c 35.00 ± 3.61 b 38.67 ± 4.16 a 39.00 ± 8.71 a 0.000*** HeadDNA 98.93 ± 0.94 a 95.17 ± 2.82 b 99.38 ± 0.26 a 87.90 ± 7.87 c 80.84 ± 5.44 d 0.000*** TailDNA 7.89±4.00 c 0.44±0.41 d 2.34±2.04 d 31.19±24.37 a 17.68±27.42 b 0.000*** TM 0.03 ± 0.02 b 0.19 ± 0.11 b 0.03 ± 0.01 b 1.00 ± 0.67 ab 2.01 ± 0.45 a 0.013* OTM 0.01 ± 0.11 e 0.55 ± 0.32 c 0.11 ± 0.53 d 1.54 ± 0.89 b 2.59 ± 0.48 a 0.000*** Ag-NPs = Silver Nanoparticles; AgNO₃ = Silver Nitrate; LHead = Length of Head; LTail = Length of Tail; LComet = Length of Comet; TM = Tail Moment; OTM = Olive Tail Moment. The mean values with distinct superscripts (a-c) in a row exhibit significant variations at ( P < 0.05). Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7086690","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":485978390,"identity":"7d25c64b-4301-470e-8e47-835c502b2073","order_by":0,"name":"Nudrat Fatima","email":"","orcid":"","institution":"Government College University Faisalabad","correspondingAuthor":false,"prefix":"","firstName":"Nudrat","middleName":"","lastName":"Fatima","suffix":""},{"id":485978394,"identity":"e2cbed09-e857-41b4-937b-1aea68a71681","order_by":1,"name":"Shabana Naz","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABDElEQVRIiWNgGAWjYDCCAwxsDIwNUM4HIGYHciSAtAFRWhhnAAmeA6RoYeYhRgvf7ePPHvzcYZfHP7v58WebX3aJPQzMB2/zMNwxxqVF8lyOuWHvmeRiiTvHDIxz+5KBWtiSrXkYnpnh0mJwhodNgreNObHhRoJBcm7PgcT9DDxm0jwMh21wa2F/Jvm3rT5x/o30D4ctgVp6GPi/EdDCYCbN23Y4ccONHMNmhh8gLTxsIC04HSZ5BugM2TPHEzfeyClm7G1INu5hZjO2nGPwDKf3+UAOe7ujOnHejfTNH378sZPtYW9+eONNxR3DBlx6UABjG5BgBjv4AFEagOAPnEW0llEwCkbBKBj+AAABvlyeF2KctgAAAABJRU5ErkJggg==","orcid":"","institution":"Government College University Faisalabad","correspondingAuthor":true,"prefix":"","firstName":"Shabana","middleName":"","lastName":"Naz","suffix":""},{"id":485978396,"identity":"66ad3ce2-7252-4bae-9bb5-ba2b66589058","order_by":2,"name":"Rifat Ullah Khan","email":"","orcid":"","institution":"The University of Agriculture","correspondingAuthor":false,"prefix":"","firstName":"Rifat","middleName":"Ullah","lastName":"Khan","suffix":""},{"id":485978399,"identity":"6efe7073-ea91-4cc2-8f02-2a0fc7e1f1a3","order_by":3,"name":"Ala Abudabos","email":"","orcid":"","institution":"Alcorn State University","correspondingAuthor":false,"prefix":"","firstName":"Ala","middleName":"","lastName":"Abudabos","suffix":""},{"id":485978400,"identity":"449c0f50-b21b-4fe4-bbca-374ff993c88f","order_by":4,"name":"Ankqash Ayyub","email":"","orcid":"","institution":"Government College University Faisalabad","correspondingAuthor":false,"prefix":"","firstName":"Ankqash","middleName":"","lastName":"Ayyub","suffix":""},{"id":485978403,"identity":"f7716853-ec2e-408e-b115-64a815edc256","order_by":5,"name":"Muhammad Usama","email":"","orcid":"","institution":"Government College University Faisalabad","correspondingAuthor":false,"prefix":"","firstName":"Muhammad","middleName":"","lastName":"Usama","suffix":""},{"id":485978406,"identity":"d653dbf5-79ab-4866-b950-3b7724510c6d","order_by":6,"name":"Swaira Ashfaq","email":"","orcid":"","institution":"Government College University Faisalabad","correspondingAuthor":false,"prefix":"","firstName":"Swaira","middleName":"","lastName":"Ashfaq","suffix":""},{"id":485978407,"identity":"0c5e71eb-2e59-4205-aa50-3f2000145aa7","order_by":7,"name":"Hifza Shehzadi","email":"","orcid":"","institution":"Government College University Faisalabad","correspondingAuthor":false,"prefix":"","firstName":"Hifza","middleName":"","lastName":"Shehzadi","suffix":""},{"id":485978410,"identity":"3148fce5-2b36-4d80-a8ec-bd60acc23bbc","order_by":8,"name":"Sania Satti","email":"","orcid":"","institution":"Government College University Faisalabad","correspondingAuthor":false,"prefix":"","firstName":"Sania","middleName":"","lastName":"Satti","suffix":""},{"id":485978412,"identity":"f6963356-d516-4a8f-9bab-8990a0532c21","order_by":9,"name":"Ibrahim A. Alhidary","email":"","orcid":"","institution":"King Saud University","correspondingAuthor":false,"prefix":"","firstName":"Ibrahim","middleName":"A.","lastName":"Alhidary","suffix":""}],"badges":[],"createdAt":"2025-07-09 18:38:08","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7086690/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7086690/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":87254332,"identity":"7d6e2b62-6188-451a-84fc-203112ce596d","added_by":"auto","created_at":"2025-07-22 05:36:46","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":379039,"visible":true,"origin":"","legend":"\u003cp\u003eMicroscopic images of \u003cem\u003eC. japonica\u003c/em\u003e blood samples after comet assay (a) subjected to basal diet (control group) (b) processed with Ag‑NPs (10 mg/kg) (c) processed with Ag‑NPs (20 mg/kg) (d) exposed to AgNO₃ (10 mg/kg) (e) exposed to AgNO₃ (20 mg/kg).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7086690/v1/ee28da037e0bbd588e77aadd.png"},{"id":87254331,"identity":"cbe4d6ac-68a8-418c-9025-894fa7e7d7b5","added_by":"auto","created_at":"2025-07-22 05:36:46","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":75175,"visible":true,"origin":"","legend":"\u003cp\u003eConcentration of Silver (μg/g) in the blood, feather and egg shell and egg content samples of \u003cem\u003eC. japonica\u003c/em\u003e supplemented with low and high doses of Ag-NPs and AgNO\u003csub\u003e3\u003c/sub\u003e(Ag-NPs = Silver nanoparticles; AgNO\u003csub\u003e3\u003c/sub\u003e=Silver Nitrate).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7086690/v1/321583000ae74951f01df080.png"},{"id":87254333,"identity":"9032d66e-9b8d-4d59-861a-7174cd003830","added_by":"auto","created_at":"2025-07-22 05:36:46","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":150935,"visible":true,"origin":"","legend":"\u003cp\u003eCorrelation analysis between comet assay parameters and different treatment groups of \u003cem\u003eC. japonica\u003c/em\u003e exposed to low and high levels of Ag-NPs and AgNO₃. (Ag-NPs = Silver nanoparticles; AgNO₃ = Silver nitrate; 1 = Control; 2 = Ag-NPs (10 mg/kg); 3 = Ag-NPs (20 mg/kg); 4 = AgNO₃ (10 mg/kg); 5 = AgNO₃ (20 mg/kg).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7086690/v1/7fd283c11114082754c85c01.png"},{"id":103292938,"identity":"1db14279-e3b5-4e92-b54c-19bb6a54a70f","added_by":"auto","created_at":"2026-02-24 06:42:13","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1576946,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7086690/v1/6343b225-a64a-45c1-a879-318a7edcbcd8.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Comprehensive Assessment of Silver Bioaccumulation and DNA Damage Effects in Coturnix coturnix japonica Using Blood, Feather, and Egg Biomarkers","fulltext":[{"header":"Introduction","content":"\u003cp\u003eNanotechnology involves the manipulation of materials at the nanometer scale, typically between 1 and 100 nanometers. This innovative field had a significant impact across various industries, including healthcare, agriculture, and the food and nutrition sectors (Khan et al., 2014;\u0026nbsp;Chand\u0026nbsp;et al., 2020; Rawat et al., 2023). Researchers are increasingly exploring the unique properties of nanostructures such as enhanced electrical, magnetic, and optical characteristics which have considerable implications for improving the nutritional value and bioavailability of food ingredients (Khan et al., 2017; Ahmad et al., 2020). A fundamental component of nanotechnology is the use of nanoparticles extremely small particles with a high surface-area-to-volume ratio. This feature allows nanoparticles to interact more efficiently with biological systems, resulting in improved absorption and targeted delivery of nutrients. Among various types of nanoparticles, silver nanoparticles (Ag-NPs) are particularly notable due to their remarkable antibacterial, antiviral, and antifungal properties, which contribute to food preservation and safety enhancement (Nasrollahzadeh et al., 2019).\u003c/p\u003e\n\u003cp\u003eSilver (Ag) exists in both organic and inorganic forms, each with distinct properties and applications. In its inorganic form, it is commonly used as elemental silver or silver salts, while in its organic form, it can form complexes with various organic molecules (Bhattarai et al., 2018). Ag-NPs have become one of the most widely used materials in biomedicine. They are employed in the treatment of infections and cancer, vaccine development, diabetes management, and wound healing. Additionally, they are extensively applied in the development of chemical and biological sensors, owing to their unique optical properties.\u003c/p\u003e\n\u003cp\u003eA prominent trend in Ag-NP synthesis is the adoption of green methods, such as using plant extracts or microorganisms. These methods are considered safer, more cost-effective, and environmentally friendly (Xu et al., 2020). One plant that plays a crucial role in the eco-friendly synthesis of Ag-NPs is \u003cem\u003eAzadirachta indica\u003c/em\u003e, commonly known as neem (Gupta et al., 2017). Neem is renowned for its powerful medicinal properties, including antibacterial, antiviral, antifungal, and anti-inflammatory effects, making it a valuable resource for nanoparticle production (Khan et al., 2022; Wylie et al., 2022). Utilizing neem extracts for the synthesis of Ag-NPs offers a more sustainable and biocompatible alternative, providing both health and environmental benefits. However, despite these advantages, concerns remain regarding the potential toxicity of Ag-NPs (Ahmed et al., 2016).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Ag-NPs are known to accumulate in specific organs more effectively than silver ions, which can lead to toxic effects in biological systems. This bioaccumulation is particularly concerning due to its potential long-term impacts on both human health and the environment (Tortella et al., 2020). The tendency of Ag-NPs to bioaccumulate has been observed in various organisms, including birds. Their small size and unique physicochemical properties allow them to be easily absorbed and retained in biological tissues (Chaturvedi et al., 2021). Unlike inorganic silver, which typically exists in ionic form (Ag⁺), Ag-NPs can penetrate cellular membranes more efficiently, resulting in greater accumulation in tissues and organs. This is especially concerning for species such as quails, which are frequently used in environmental research to study the effects of pollutants and nanomaterials (Naz et al., 2024).\u003c/p\u003e\n\u003cp\u003eQuails are ideal model organisms for such studies due to their small size, high reproductive rate, and adaptability to various environments. Their widespread use in agricultural and scientific research, along with their importance in food production, makes them valuable subjects for investigating the potential risks of Ag-NP bioaccumulation and its long-term effects on wildlife and human health. Given the expanding use of Ag-NPs in agriculture, medicine, and consumer products, continued research into their safety and environmental impact is essential. This study was designed to compare the effects of Ag-NPs and AgNO₃ on bioaccumulation and DNA damage in quails, thereby contributing to the understanding of nanoparticle toxicity and guiding future regulatory decisions.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cem\u003eSynthesis of Silver Nanoparticles\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eFresh neem (\u003cem\u003eAzadirachta indica\u003c/em\u003e) leaves were first thoroughly washed with tap water, followed by distilled water, and then completely dried. Once dried, the leaves were cut into small pieces and ground into a fine paste using a mortar and pestle. A 10 g sample of the paste was then heated in 100 mL of distilled water at 60\u0026deg;C for 30 minutes to extract the bioactive compounds. After cooling, the extract was filtered through Whatman No. 1 filter paper to remove any solid residues (David et al., 2014). A 1 mM silver nitrate (AgNO₃) stock solution was prepared by dissolving silver nitrate in distilled water. Subsequently, 10 mL of the neem leaf extract was added to 200 mL of the Ag-NO₃ solution and gently stirred on a hot plate. The mixture was maintained at 60\u0026deg;C for 30 minutes. A visible color change from yellow to dark red confirmed the successful synthesis of silver nanoparticles (Ag-NPs), indicating the reduction of silver ions and nanoparticle formation (Sahayaraj \u0026amp; Rajesh, 2011).\u003c/p\u003e\n\u003cp\u003eThe synthesized Ag-NPs were characterized using a UV-Visible spectrophotometer (Hitachi U-2800), with absorbance measured in the 300-600 nm range to monitor nanoparticle formation (Ahmed et al., 2016). Fourier Transform Infrared (FTIR) spectroscopy (PerkinElmer) was used to identify the functional groups involved in stabilizing the Ag-NPs. Measurements were taken across a spectral range of 400 to 4000 cm⁻\u0026sup1; with a resolution of 4 cm⁻\u0026sup1;. The FTIR analysis showed absorption bands corresponding to carbonyl groups from proteins, indicating that proteins in the neem extract likely capped and stabilized the Ag-NPs. X-ray diffraction (XRD) analysis was performed using an X\u0026rsquo;Pert Pro diffractometer with Cu K\u0026alpha; radiation to determine the amorphous structure and size distribution of the nanoparticles. Scanning was conducted at a rate of 10\u0026deg;/min. The XRD patterns confirmed an amorphous structure (Ugwuoke, et al., 2023).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eTrial Birds and Research Methodology\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThis study was conducted in accordance with the ethical guidelines for the handling and care of laboratory animals established by Government College University Faisalabad (GCUF). A total of 480 (14-day-old) Japanese quails were \u003cstrong\u003eacquired\u003c/strong\u003e and acclimatized for 14 days under controlled conditions, which included a temperature range of 20\u0026ndash;25\u0026deg;C, 70% humidity, and a 16-hour light/8-hour dark cycle. The quails were housed in wire cages with free access to a commercial basal diet (NRC, 1994, Table 1) and fresh water. The birds were randomly assigned to five experimental groups, each consisting of 96 birds and six replicates (16 birds per replicate). The groups were as follows: the first group (control) received only the basal diet; the second and third groups received Ag-NPs at 10 mg/kg and 20 mg/kg body weight, respectively; and the fourth and fifth groups received AgNO₃ at 10 mg/kg and 20 mg/kg body weight, respectively. Treatments were administered weekly via oral gavage over 65-day period throughout the trial.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eBlood and feather samples collection\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAt the conclusion of the trial (day 65), 2-3 mL blood samples were collected from the brachial vein of twelve birds per replicate. Six samples were placed in lavender-topped tubes for comet assay analysis, and Six in gold-topped tubes for silver bioaccumulation measurement, following the method described by Khalid et al (2025). All blood samples were stored at -20\u0026deg;C until chemical analysis. The birds were then euthanized, and chest feathers were collected post-dissection. These feathers were selected as representative samples, as they are considered to best reflect environmental exposure\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u0026nbsp;Collection of eggs\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eEggs were collected daily during the quail breeding season, coinciding with the typical morning laying period. To minimize damage, at least 20 eggs per replicate were collected manually. Each egg was labeled with the date and replicate number, then stored in chemically cleaned jars at 4\u0026deg;C to prevent contamination. Prior to processing, eggs were washed with demineralized water, if required. The eggshells were carefully removed, and the contents were transferred to sterile containers via clean glass dishes. These containers were then frozen, preparing the samples for subsequent chemical analysis (Ashkoo et al., 2020).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eSilver bioaccumulation screening\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eSample\u0026apos;s digestion\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eBlood samples, collected in gold-topped tubes, were allowed to clot. Serum was then separated from red blood cells (RBCs) by centrifuging the coagulated blood at 2000 rpm for 10 minutes, maximizing serum yield. A 1:10 serum dilution was prepared by mixing 1 ml of serum with 10 ml of demineralized water (Naz et al., 2024). Feather samples were cleaned with acetone, followed by three washes with water. After drying in an oven at 60\u0026deg;C for two days, the feathers were fragmented. A 1-g portion of each sample was then digested using a 5 ml hydrogen peroxide (H₂O₂) and 5 ml nitric acid (HNO₃) mixture on a 70\u0026deg;C hot plate. Once digestion was complete, the mixture was cooled to room temperature and filtered through Whatman filter paper No. 1. The filtered solution was then diluted with 25 ml of deionized water (Rutkowska et al., 2018)\u003c/p\u003e\n\u003cp\u003eEggs were cleaned with acetone and demineralized water to remove surface contaminants. Egg contents were extracted using a toothpick and placed in petri dishes, while eggshells were placed in separate dishes. Samples were dried in an oven to achieve uniform dry matter. Dried eggshells and egg contents were then ground into homogeneous powders. A 0.5-g portion of each powder was mixed with 10 ml of nitric acid (HNO₃) in conical flasks and heated to 140\u0026deg;C until the solution became clear (Khalid et al., 2025). After cooling, the digests were filtered through Whatman filter paper No. 1 and diluted to 25 ml with deionized water in polypropylene flasks. Digested samples were stored at 4\u0026deg;C until silver content analysis via Atomic Absorption Spectrophotometry (Ashkoo et al., 2020).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eChemical analysis of samples\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSilver bioaccumulation in blood, feather, and egg samples was determined using Atomic Absorption Spectrometry (Aurora AI 1200). Silver concentrations were analyzed at a wavelength of 328.1 nm.\u003c/p\u003e\n\u003ch4\u003e\u003cem\u003eCalculation of silver concentration\u003c/em\u003e\u003c/h4\u003e\n\u003cp\u003eSilver concentrations were determined using the formula: Metal concentration = (Spectrophotometric reading \u0026times; Dilution factor) / Sample weight. A dilution factor of 25 mL/g was applied to solid samples (feathers, eggshells, and egg contents), while a factor of 10 mL/mL was used for serum samples.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eDNA damage examination\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTo assess DNA damage resulting from silver exposure, the Comet assay, also known as Single-Cell Gel Electrophoresis (SCGE), was conducted. The procedure adhered to the methodology outlined by Singh et al. (1988) (Fig. 1). Image analysis and data acquisition for seven comet parameters\u0026mdash;head length, tail length, total comet length, percentage of DNA in the tail and head, tail moment, and olive tail moment\u0026mdash;were performed using Casp_1.2.3b1 software.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eStatistical analysis\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eData analysis was carried out using IBM SPSS Statistics 25 and Statistics 8.1. One way ANOVA was used to assess silver levels in blood, feathers, eggshells, and egg contents, as well as to evaluate DNA damage in quails exposed to varying concentrations of Ag‑NPs and AgNO\u003csub\u003e3\u003c/sub\u003e. To determine which groups differed significantly, Tukey\u0026rsquo;s post-hoc test was used. Furthermore, Pearson\u0026rsquo;s correlation analysis was performed to examine relationships between comet assay parameters and the different treatment groups of \u003cem\u003eC. japonica\u003c/em\u003e receiving low and high doses of Ag‑NPs and AgNO\u003csub\u003e3\u003c/sub\u003e.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eTable 2 illustrates that silver concentrations in \u003cem\u003eC. japonica\u003c/em\u003e differed significantly (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) depending on the type and dosage of silver administered. The highest silver levels were found in the blood (0.26 \u0026mu;g/g), feathers (0.36 \u0026mu;g/g), eggshells (0.83 \u0026mu;g/g), and egg contents (0.77 \u0026mu;g/g) of quails fed a high dose (20 mg/kg) of Ag-NPs. A moderate increase was also observed with a 10 mg/kg dose of Ag-NPs. In comparison, birds treated with AgNO₃ exhibited intermediate values, while the control group consistently showed the lowest silver accumulation. Significant differences between treatments were marked with superscript letters (\u003csup\u003ea, b, c, d, e\u003c/sup\u003e), indicating statistical variation among groups.\u003c/p\u003e\n\u003cp\u003eOverall, the pattern of silver accumulation in the quail samples treated with different concentrations of Ag-NPs and AgNO₃ followed this order: eggshells \u0026gt; egg contents \u0026gt; feathers \u0026gt; blood. The highest accumulation of silver was observed in the samples from quails supplemented with a high dose of Ag-NPs (20 mg/kg) (Figure 2). The silver concentrations across the treatment groups were ranked as follows: Ag-NPs (20 mg/kg) \u0026gt; Ag-NPs (10 mg/kg) \u0026gt; AgNO₃ (20 mg/kg) \u0026gt; AgNO₃ (10 mg/kg) \u0026gt; control.\u003c/p\u003e\n\u003cp\u003eAll comet parameters were analyzed using ANOVA across groups treated with different doses of Ag-NPs and AgNO₃ (Table 3). Significant differences (\u003cem\u003eP\u0026nbsp;\u003c/em\u003e\u0026lt; 0.05) were observed between the mean values of LHead, LTail, LComet, HeadDNA, TailDNA, TM and OTM. A Post-Hoc Tukey test revealed that the highest values for Lhead (37.00 \u0026plusmn; 3.46), LTail (10.67 \u0026plusmn; 1.15), LComet (39.00 \u0026plusmn; 8.71), TM (2.01 \u0026plusmn; 0.45) and OTM (2.59 \u0026plusmn; 0.48) were found in the group treated with AgNO₃ (20 mg/kg) compared to the other groups. The greatest damage was observed in the AgNO₃ group, while the Ag-NPs groups (10 mg/kg and 20 mg/kg) showed lower values, indicating less damage in these treatments. Additionally, the microscope analysis revealed significant differences, as shown in Figure 1.\u003c/p\u003e\n\u003cp\u003eThere was a strong positive correlation between silver exposure (Ag-NPs and AgNO₃) and several comet assay parameters in \u003cem\u003eC. japonica\u003c/em\u003e, including Lhead (r = 0.934), Ltail (r = 0.932), Lcomet (r = 0.946), tailDNA (r = 0.626), TM(r = 0.729), and OTM(r = 0.889) (Figure 3). In contrast, HeadDNA content exhibited a marked negative correlation (r = \u0026ndash;0.858) with the treatment groups. These findings indicate that silver compounds, particularly AgNO₃, significantly affected DNA integrity. The extent of DNA damage increased with higher silver concentrations, with the most severe effects observed in the groups treated with AgNO₃\u003c/p\u003e"},{"header":"Discussion ","content":"\u003cp\u003eIn the present study, the results showed that the mean values of Ag varied significantly in quails treated with different concentrations of Ag-NPs and AgNO₃. Notably, the highest Ag levels were found in the blood of Japanese quails exposed to the highest dose of Ag‑NPs. Because blood reflects overall health and plays a crucial role in transporting nutrients, metabolic waste, and hormones, this elevated level is especially significant (Chand et al., 2018). It indicates enhanced absorption and retention of silver in nanoparticle form likely due to the small size and unique physicochemical properties of Ag‑NPs, which promote their passage across biological membranes. Indeed, Ag‑NP coatings can modulate ion release and membrane interaction, increasing bioavailability compared to ionic forms (Bhattarai et al., 2018). The enhanced bioaccumulation of Ag from Ag-NPs may be attributed to their nanoscale dimensions and high surface area-to-volume ratio, which increase their reactivity and interaction with biological tissues. Ag-NPs can penetrate epithelial barriers through endocytosis or diffusion, allowing them to enter systemic circulation more efficiently than ionic silver (Ag⁺) from AgNO₃ (Khan et al., 2022). Once in the bloodstream, Ag-NPs may bind to plasma proteins or cellular membranes, leading to prolonged circulation time and accumulation in various tissues. Additionally, the slow dissolution of Ag-NPs into Ag⁺ within biological environments may contribute to sustained silver release, thereby enhancing systemic retention and potential biological effects (Behra et al., 2013).\u003c/p\u003e\n\u003cp\u003eSignificant differences were observed in the mean Ag concentrations in the feathers of Japanese quails administered varying doses of Ag-NPs and AgNO₃. The highest Ag level was detected in the feathers of birds receiving high dose of Ag-NPs. Once in systemic circulation, Ag-NPs are more likely to persist in the bloodstream and reach peripheral tissues compared to ionic silver from AgNO₃. As feathers develop, they incorporate circulating trace elements, such as silver, into their keratin matrix. The prolonged presence of Ag-NPs in the body increases the likelihood of their deposition in feather tissue during growth, resulting in higher silver concentrations (Ansari et al., 2024). Feathers were selected as a marker for bioaccumulation due to their ability to reflect internal metal exposure over extended periods. Feathers serve as excretory pathways for metals, and their accumulation indicates chronic exposure over time, as opposed to blood, which reflects more recent exposure events (Brown et al., 2016; Roberts \u0026amp; Wilson, 2017). This is consistent with findings from Khalid et al. (2025), who reported selenium accumulation patterns in quail tissues, reinforcing feathers and eggs as reliable bioindicators. Moreover, feathers can be collected non-invasively, enabling repeated sampling without harming the animal. During feather formation, trace elements present in the bloodstream become permanently embedded in the keratin structure, providing a stable and time-integrated record of exposure. The strong binding affinity of metals to keratin further preserves the elemental composition, making feathers an effective tool for long-term monitoring of environmental contaminants in avian species (Borghesi et al., 2016).\u003c/p\u003e\n\u003cp\u003eOur results indicated that increasing dietary concentrations of Ag-NPs and AgNO₃ from 10 to 20 mg/kg feed led to a significant accumulation of silver in both the eggshell and internal contents of Japanese quail eggs. This suggests that silver, particularly in nanoparticle form, can cross biological barriers and be transferred from the hen to the developing egg. Eggshells serve as both a protective barrier and a mineral reservoir, often accumulating metals through maternal deposition during shell formation (Ashkoo et al., 2020). The incorporation of silver into the eggshell may alter its structural integrity, while the presence of silver in the egg contents indicates potential for vertical transmission and raises concerns about embryotoxic effects (Fonseca et al., 2019).\u003c/p\u003e\n\u003cp\u003eThe greatest concentration of Ag was detected in the eggshells of quails, compared to the levels found in egg contents, feathers, and blood samples. The eggshell is primarily composed of calcium carbonate crystals, along with a matrix of proteins and other minerals, which are deposited by specialized cells in the shell gland during egg formation (Hincke et al., 2012). This mineralization process allows the eggshell to serve as a reservoir for various minerals and metals circulating in the hen\u0026rsquo;s bloodstream, including silver. Metals like silver often have a strong affinity for binding with calcium and other mineral components due to similar chemical properties, facilitating their incorporation into the eggshell\u0026rsquo;s crystalline matrix. This selective deposition means that metals can accumulate in the shell in higher amounts than in other tissues (Rosaiah et al., 2024). Additionally, the eggshell functions as a protective barrier to shield the developing embryo from harmful substances, including toxic metals. By sequestering silver in the shell, the bird\u0026rsquo;s physiology limits the amount that reaches the internal contents of the egg, such as the yolk and albumen, thereby reducing potential toxicity to the embryo (Wilson, 2017).\u003c/p\u003e\n\u003cp\u003eRecent pathological studies examining the genotoxic effects of Ag-NPs and AgNO₃, confirmed by comet assays, revealed that DNA damage was significantly more pronounced in samples exposed to AgNO₃ compared to those treated with Ag-NPs or control groups. This finding highlights the critical role of silver\u0026rsquo;s chemical form in its genotoxic potential. AgNO₃ is a soluble salt that rapidly dissolves in biological fluids, releasing silver ions (Ag⁺) almost immediately. These free silver ions are highly reactive and can readily penetrate cells, where they interact directly with vital biomolecules such as DNA (Behra et al., 2013). Inside the cell, silver ions promote the formation of reactive oxygen species (ROS), including free radicals and peroxides. These ROS attack the DNA molecule, causing oxidative damage such as single- and double-strand breaks, base modifications, and crosslinking. The accumulation of such damage compromises the integrity of genetic material and may lead to mutations or cell death. In contrast, Ag-NPs are solid particles that release silver ions more slowly due to their particulate nature and surface chemistry (Croteau et al., 2011). This gradual ion release results in fewer free silver ions being immediately available to interact with DNA, thereby reducing the extent of direct damage. Furthermore, Ag-NPs are often internalized by cells via endocytosis and become sequestered within cellular compartments like lysosomes, limiting their direct contact with the nucleus where DNA is located. As a result, while Ag-NPs can still induce oxidative stress and cellular damage, the severity and immediacy of DNA damage tend to be lower than those caused by silver ions from AgNO₃ (Mahjoubian et al., 2023). This difference in genotoxicity is consistent with the potent nature of silver ions, which can directly bind DNA bases, generate ROS, and cause strand breaks and chromosomal aberrations (Kumar et al., 2021; Zhang et al., 2016). Despite higher silver levels in tissues following Ag-NPs exposure, the DNA damage is comparatively less severe, likely due to the slower ion release and sequestration of nanoparticles within lysosomes or vesicular compartments (Wang \u0026amp; Li, 2023). Additionally, cellular antioxidant defenses may be more effective against the oxidative stress induced by nanoparticles, further mitigating DNA damage.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThe study demonstrated that Ag-NPs led to the highest silver accumulation in quails, particularly within the eggshells, while causing less DNA damage than AgNO₃. Conversely, AgNO\u003csub\u003e3\u003c/sub\u003e exposure resulted in more severe DNA damage at both low and high concentrations. These results indicate that Ag-NPs may be a safer alternative for elevating silver levels in poultry, as they promote greater bioaccumulation with a lower risk of genetic toxicity compared to ionic silver forms.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eN.F., S.N., and R.U.K. conceptualized and designed the study. N.F., A.A., M.U., S.A., H.S., and S.S. conducted the experiments and collected samples. S.N. and R.U.K. supervised the research and provided resources. I.A.A. and A.A. assisted with data interpretation and manuscript review. N.F. performed data analysis and wrote the initial draft. S.N. and R.U.K. revised and finalized the manuscript. All authors reviewed and approved the final version of the manuscript.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eDisclosure statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo potential conflict of interest was reported by author(s)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Committee on Animal Rights and Welfare, GC University Faisalabad, Pakistan approved this study (GCUF/ERC/460).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData will be made available from the authors upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe are thankful to the Ongoing Research Funding (ORF-2025-833), King Saud University, Riyadh, Saudi Arabia\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAbid, N., Khan, A. 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S., Moradi-Shoeili, Z., Tyler, C. R., Mansouri, B., 2023. Oxidative stress, genotoxic effects, and other damages caused by chronic exposure to silver nanoparticles (Ag NPs) and zinc oxide nanoparticles (ZnO NPs), and their mixtures in zebrafish (Danio rerio). Toxicol Appl Pharmacol.\u003cem\u003e 472\u003c/em\u003e, 116569.\u003c/li\u003e\n\u003cli\u003eNasrollahzadeh, A., Mokhtari, S., Khomeiri, M., \u0026amp; Saris, P. E., 2022. Antifungal preservation of food by lactic acid bacteria. Foods. \u003cem\u003e11\u003c/em\u003e(3), 395. https://doi.org/10.3390/foods11030395\u003c/li\u003e\n\u003cli\u003eNaz, S., Bibi, G., Nadeem, R., Alhidary, I., Dai, S., Israr, M., Ullah Khan, R., 2024. Evaluation of biological selenium nanoparticles on growth performance, histopathology of vital organs and genotoxicity in Japanese quails (\u003cem\u003eCoturnix coturnix japonica\u003c/em\u003e). Vet. Q. \u003cem\u003e44\u003c/em\u003e(1), 1\u0026ndash;10. https://doi.org/10.1080/01652176.2023.2296127\u003c/li\u003e\n\u003cli\u003eNaz, S., Muazzam, S., Sagheer, A., Tanveer, A., Khan, N. A., Ali, Z., Khan, R. U., 2020. Captivity stress influences the DNA damage of \u003cem\u003ePavo cristatus\u003c/em\u003e under environmental conditions of Faisalabad, Pakistan. Environ. Sci. Pollut. Res. Int.\u003cem\u003e 27\u003c/em\u003e, 5636\u0026ndash;5639. https://doi.org/10.1007/s11356-019-07235-y\u003c/li\u003e\n\u003cli\u003eNaz, S., Raza, N., Alhidary, I., Satti, S., Rafique, A., Batool, S., Khan, R. U., 2024. Evaluation of copper nanoparticles on growth, organs histology and DNA damage in Japanese quails (\u003cem\u003eCoturnix coturnix japonica\u003c/em\u003e). \u003cem\u003eToxin Reviews. 43\u003c/em\u003e(1), 127\u0026ndash;136. https://doi.org/10.1080/15569543.2022.2070619\u003c/li\u003e\n\u003cli\u003eRawat, B., Bist, A. S., Supriyanti, D., Elmanda, V., Sari, S. N., 2023. AI and nanotechnology for healthcare: A survey. Aptisi Transactions on Management. \u003cem\u003e7\u003c/em\u003e(1), 86\u0026ndash;91. https://doi.org/10.34306/atm.v7i1.1234\u003c/li\u003e\n\u003cli\u003eRosaiah, P., Yue, D., Dayanidhi, K., Ramachandran, K., Vadivel, P., Eusuff, N. S., Kim, W. K., 2024. Eggshells \u0026amp; Eggshell Membranes\u0026ndash;A Sustainable Resource for energy storage and energy conversion applications: A critical review. Adv. Colloid Interface Sci. 103144.\u003c/li\u003e\n\u003cli\u003eRutkowska, M., Płotka-Wasylka, J., Lubinska-Szczygeł, M., R\u0026oacute;żańska, A., Możejko-Ciesielska, J., Namieśnik, J., 2018. Birds\u0026rsquo; feathers\u0026ndash;suitable samples for determination of environmental pollutants. TrAC, Trends Anal. Chem. \u003cem\u003e109\u003c/em\u003e, 97\u0026ndash;115. https://doi.org/10.1016/j.trac.2018.09.007\u003c/li\u003e\n\u003cli\u003eSahayaraj, K., Rajesh, S., \u0026amp; Rathi, J. M., 2012. Silver nanoparticles biosynthesis using marine alga padina pavonica (linn.) And its microbicidal activity. \u003cem\u003eDJNB\u003c/em\u003e, \u003cem\u003e7\u003c/em\u003e(4). 1842-3582\u003c/li\u003e\n\u003cli\u003eTortella, G. R., Rubilar, O., Dur\u0026aacute;n, N., Diez, M. C., Mart\u0026iacute;nez, M., Parada, J., Seabra, A. B., 2020. Silver nanoparticles: Toxicity in model organisms as an overview of its hazard for human health and the environment.\u003cem\u003e \u003c/em\u003e J. Hazard. Mater\u003cem\u003e. 390\u003c/em\u003e, 121974. https://doi.org/10.1016/j.jhazmat.2019.121974\u003c/li\u003e\n\u003cli\u003eUgwuoke, K. C., 2023. Effects of green-synthesized silver nanoparticles from Azadirachta indica on growth performance and liver function parameters in male albino rats. Cell Biol. Dev.\u003cem\u003e 7\u003c/em\u003e(1), 1\u0026ndash;10. https://doi.org/10.13057/cellbioldev/v070104\u003c/li\u003e\n\u003cli\u003e\u003cstrong\u003eWang, E. C., Wang, A. Z.,\u003c/strong\u003e 2014. Nanoparticles and their applications in cell and molecular biology Integr Biol. \u003cem\u003e6\u003c/em\u003e(1), 9\u0026ndash;26. https://doi.org/10.1039/C3IB40165K\u003c/li\u003e\n\u003cli\u003eWilson, P. B., 2017. Recent advances in avian egg science: A review. Poult. Sci. J. \u003cem\u003e96\u003c/em\u003e(10), 3747-3754.\u003c/li\u003e\n\u003cli\u003eWylie, M. R., Merrell, D. S., 2022. The antimicrobial potential of the neem tree Azadirachta indica.\u003cem\u003e \u003c/em\u003eFront. Pharmacol. \u003cem\u003e13\u003c/em\u003e, 891535. https://doi.org/10.3389/fphar.2022.891535\u003c/li\u003e\n\u003cli\u003eXu, L., Wang, Y. Y., Huang, J., Chen, C. Y., Wang, Z. X., Xie, H., 2020. Silver nanoparticles: Synthesis, medical applications and biosafety. Theranostics\u003cem\u003e. 10\u003c/em\u003e(20), 8996\u0026ndash;9005. https://doi.org/10.7150/thno.45413\u003c/li\u003e\n\u003cli\u003eZhang, N., Xiao, H., Li, T. K., Nur-E-Kamal, A., Liu, L. F., 2003. DNA damage-mediated apoptosis induced by selenium compounds.\u003cem\u003e \u003c/em\u003eJ Biol Chem\u003cem\u003e. 278\u003c/em\u003e(32), 29532\u0026ndash;29537. https://doi.org/10.1074/jbc.M303404200\u003c/li\u003e\n\u003cli\u003eZhang, X. F., Liu, Z. G., Shen, W., Gurunathan, S., 2016. Silver nanoparticles: Synthesis, characterization, properties, applications, and therapeutic approaches. Int. J. Mol. Sci. \u003cem\u003e17\u003c/em\u003e(9), 1534. https://doi.org/10.3390/ijms17091534\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable 1\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eComposition of Basal Diets Used for Japanese Quails\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 70.9565%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eIngredients\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29.0435%;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePercentage (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 70.9565%;\"\u003e\n \u003cp\u003eSoyabean oil\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29.0435%;\"\u003e\n \u003cp\u003e0.50\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 70.9565%;\"\u003e\n \u003cp\u003eSoyabean meal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29.0435%;\"\u003e\n \u003cp\u003e21.60\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 70.9565%;\"\u003e\n \u003cp\u003eWheat\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29.0435%;\"\u003e\n \u003cp\u003e4.10\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 70.9565%;\"\u003e\n \u003cp\u003eCalcium phosphate (GR)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29.0435%;\"\u003e\n \u003cp\u003e1.00\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 70.9565%;\"\u003e\n \u003cp\u003eCalcium carbonate (GR)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29.0435%;\"\u003e\n \u003cp\u003e9.50\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 70.9565%;\"\u003e\n \u003cp\u003eCottonseed meal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29.0435%;\"\u003e\n \u003cp\u003e2.00\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 70.9565%;\"\u003e\n \u003cp\u003eCorn\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29.0435%;\"\u003e\n \u003cp\u003e60.50\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 70.9565%;\"\u003e\n \u003cp\u003ePhytases\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29.0435%;\"\u003e\n \u003cp\u003e0.015\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 70.9565%;\"\u003e\n \u003cp\u003eSodium chloride\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29.0435%;\"\u003e\n \u003cp\u003e0.30\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 70.9565%;\"\u003e\n \u003cp\u003eDL-methionine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29.0435%;\"\u003e\n \u003cp\u003e0.08\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 70.9565%;\"\u003e\n \u003cp\u003eVitamin premix \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29.0435%;\"\u003e\n \u003cp\u003e0.15\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 70.9565%;\"\u003e\n \u003cp\u003eMineral premix \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29.0435%;\"\u003e\n \u003cp\u003e0.035\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 70.9565%;\"\u003e\n \u003cp\u003e50% Choline chloride\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29.0435%;\"\u003e\n \u003cp\u003e0.10\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 70.9565%;\"\u003e\n \u003cp\u003eExperimental additives\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29.0435%;\"\u003e\n \u003cp\u003e0.12\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 70.9565%;\"\u003e\n \u003cp\u003eTotal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29.0435%;\"\u003e\n \u003cp\u003e100.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 100%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Nutrient Level\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 70.9565%;\"\u003e\n \u003cp\u003eMethionine + Cysteine (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29.0435%;\"\u003e\n \u003cp\u003e0.55\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 70.9565%;\"\u003e\n \u003cp\u003eApparent metabolize energy (AME), Mcal/kg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29.0435%;\"\u003e\n \u003cp\u003e2.57\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 70.9565%;\"\u003e\n \u003cp\u003eSe\u003csup\u003ec\u003c/sup\u003e (mg/kg)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29.0435%;\"\u003e\n \u003cp\u003e0.056\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 70.9565%;\"\u003e\n \u003cp\u003eCrude protein (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29.0435%;\"\u003e\n \u003cp\u003e15.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 70.9565%;\"\u003e\n \u003cp\u003eLysine (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29.0435%;\"\u003e\n \u003cp\u003e0.74\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 70.9565%;\"\u003e\n \u003cp\u003eTotal phosphorus (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29.0435%;\"\u003e\n \u003cp\u003e0.54\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 70.9565%;\"\u003e\n \u003cp\u003eAvailable phosphorus (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29.0435%;\"\u003e\n \u003cp\u003e0.31\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 70.9565%;\"\u003e\n \u003cp\u003eCalcium (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29.0435%;\"\u003e\n \u003cp\u003e3.7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 70.9565%;\"\u003e\n \u003cp\u003eMethionine (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29.0435%;\"\u003e\n \u003cp\u003e0.3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eAbbreviations: Guaranteed Reagent (GR) is provided per kilogram of diet, including vitamin A at 12,500 International Units (IU), vitamin D3 at 2,500 IU, vitamin E at 18.75 milligrams (mg), vitamin K3 at 2.65 mg, vitamin B1 at 2 mg, vitamin B2 at 6 mg, vitamin B12 at 0.025 mg, biotin at 0.325 mg, folic acid at 1.25 mg, and niacin at 50 mg. Additionally, trace minerals provided per kilogram of diet include copper (Cu) at 8 mg, iron (Fe) at 80 mg, zinc (Zn) at 80 mg, manganese (Mn) at 60 mg, and iodine (I) at 1.2 mg.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eComparative Analysis of Silver Levels (Mean \u0026plusmn; SD) in Blood, Feathers, Eggshells, and Egg Contents of \u003cem\u003eC. japonica\u003c/em\u003e Supplemented with Low and High Doses of Ag-NPs and AgNO\u003csub\u003e3\u003c/sub\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" align=\"\" width=\"726\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSamples\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eControl\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAg-NPs (10 mg/kg)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 105px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAg-NPs (20 mg/kg)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 105px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAg-NO\u003csub\u003e3\u0026nbsp;\u003c/sub\u003e(10 mg/kg)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 105px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAg-NO\u003csub\u003e3\u0026nbsp;\u003c/sub\u003e(20 mg/kg)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eP\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e. Value\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBlood\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e0.04 \u0026plusmn; 0.01\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e0.19 \u0026plusmn; 0.01\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 105px;\"\u003e\n \u003cp\u003e0.26 \u0026plusmn; 0.01\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 105px;\"\u003e\n \u003cp\u003e0.06 \u0026plusmn; 0.02\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 105px;\"\u003e\n \u003cp\u003e0.16 \u0026plusmn; 0.02\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e0.000***\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eFeathers\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e0.15 \u0026plusmn; 0.00\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e0.20 \u0026plusmn; 0.02\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 105px;\"\u003e\n \u003cp\u003e0.36 \u0026plusmn; 0.01\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 105px;\"\u003e\n \u003cp\u003e0.17 \u0026plusmn; 0.00\u003csup\u003ecd\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 105px;\"\u003e\n \u003cp\u003e0.18 \u0026plusmn; 0.01\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e0.000***\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eEgg shell\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e0.23\u0026plusmn;0.02\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e0.62\u0026plusmn;0.04\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 105px;\"\u003e\n \u003cp\u003e0.83\u0026plusmn;0.02\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 105px;\"\u003e\n \u003cp\u003e0.32\u0026plusmn;0.03\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 105px;\"\u003e\n \u003cp\u003e0.37\u0026plusmn;0.04\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e0.001\u003cspan dir=\"RTL\"\u003e٭٭٭\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eEgg content\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e0.22 \u0026plusmn;0.04\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e0.49\u0026plusmn;0.07\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 105px;\"\u003e\n \u003cp\u003e0.77\u0026plusmn;0.04\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 105px;\"\u003e\n \u003cp\u003e0.33\u0026plusmn;0.04\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 105px;\"\u003e\n \u003cp\u003e0.42\u0026plusmn;0.04\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e0.001\u003cspan dir=\"RTL\"\u003e٭٭٭\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eAg-NPs = Silver Nanoparticles; AgNO₃ = Silver Nitrate. The mean values with distinct superscripts (\u003csup\u003ea\u0026ndash;e\u003c/sup\u003e) in a row exhibit substantial variation at (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;Comet parameters (Mean \u0026plusmn; SD) in the blood of C. japonica supplemented with different doses of Ag-NPs and AgNO₃\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" align=\"\" width=\"707\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eParameters\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 107px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eControl\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAg-NPs\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(10 mg/kg)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAg-NPs\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(20 mg/kg)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAgNO\u003csub\u003e3\u003c/sub\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(10 mg/kg)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 101px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAgNO\u003csub\u003e3\u003c/sub\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(20 mg/kg)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 101px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eP\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e. value\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003eLHead\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 107px;\"\u003e\n \u003cp\u003e26.33 \u0026plusmn; 2.31\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e27.21 \u0026plusmn; 2.31\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e28.33 \u0026plusmn; 8.33\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e33.67 \u0026plusmn; 4.16\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 101px;\"\u003e\n \u003cp\u003e37.00 \u0026plusmn; 3.46\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 101px;\"\u003e\n \u003cp\u003e0.000***\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003eLTail\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 107px;\"\u003e\n \u003cp\u003e3.00 \u0026plusmn; 0.00\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e4.00 \u0026plusmn; 0.00\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e5.00 \u0026plusmn; 0.00\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e8.00 \u0026plusmn; 1.00\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 101px;\"\u003e\n \u003cp\u003e10.67 \u0026plusmn; 1.15\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 101px;\"\u003e\n \u003cp\u003e0.000***\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003eLComet\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 107px;\"\u003e\n \u003cp\u003e30.34 \u0026plusmn; 2.30\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e31.34 \u0026plusmn; 2.31\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e35.00 \u0026plusmn; 3.61\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e38.67 \u0026plusmn; 4.16\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 101px;\"\u003e\n \u003cp\u003e39.00 \u0026plusmn; 8.71\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 101px;\"\u003e\n \u003cp\u003e0.000***\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003eHeadDNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 107px;\"\u003e\n \u003cp\u003e98.93 \u0026plusmn; 0.94\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e95.17 \u0026plusmn; 2.82\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e99.38 \u0026plusmn; 0.26\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e87.90 \u0026plusmn; 7.87\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 101px;\"\u003e\n \u003cp\u003e80.84 \u0026plusmn; 5.44\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 101px;\"\u003e\n \u003cp\u003e0.000***\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003eTailDNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 107px;\"\u003e\n \u003cp\u003e7.89\u0026plusmn;4.00\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e0.44\u0026plusmn;0.41\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e2.34\u0026plusmn;2.04\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e31.19\u0026plusmn;24.37\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 101px;\"\u003e\n \u003cp\u003e17.68\u0026plusmn;27.42\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 101px;\"\u003e\n \u003cp\u003e0.000***\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003eTM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 107px;\"\u003e\n \u003cp\u003e0.03 \u0026plusmn; 0.02\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e0.19 \u0026plusmn; 0.11\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e0.03 \u0026plusmn; 0.01\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e1.00 \u0026plusmn; 0.67\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 101px;\"\u003e\n \u003cp\u003e2.01 \u0026plusmn; 0.45\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 101px;\"\u003e\n \u003cp\u003e0.013*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003eOTM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 107px;\"\u003e\n \u003cp\u003e0.01 \u0026plusmn; 0.11\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e0.55 \u0026plusmn; 0.32\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e0.11 \u0026plusmn; 0.53\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e1.54 \u0026plusmn; 0.89\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 101px;\"\u003e\n \u003cp\u003e2.59 \u0026plusmn; 0.48\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 101px;\"\u003e\n \u003cp\u003e0.000***\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eAg-NPs = Silver Nanoparticles; AgNO₃ = Silver Nitrate; LHead = Length of Head; LTail = Length of Tail; LComet = Length of Comet; TM = Tail Moment; OTM = Olive Tail Moment. The mean values with distinct superscripts (a-c) in a row exhibit significant variations at (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Japanese quail, Silver nanoparticles, Silver Nitrate, Genotoxicity, Comet assay","lastPublishedDoi":"10.21203/rs.3.rs-7086690/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7086690/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis study examined the effects of Ag-NPs and AgNO₃ on silver accumulation in the blood, feathers, eggshells, and egg contents of Japanese quails, as well as their potential to cause DNA damage. A total of 480 (fourteen-day-old) quails were divided into five groups of 96 birds each, arranged into six replicates of sixteen birds with a sex ratio of one male to three females. The first group served as a control and was fed a basal diet, while the second and third groups received Ag-NPs at doses of 10 mg/kg and 20 mg/kg, respectively. The fourth and fifth groups were given AgNO₃ at the same concentrations. Results showed that the highest silver accumulation occurred in all tissues in quails fed the higher dose of Ag-NPs. The greatest accumulation was observed in the eggshells, likely due to their porous structure, which facilitates metal deposition. Both Ag-NPs (20 mg/kg) and AgNO₃ (10 and 20 mg/kg) induced DNA damage, although the damage was more severe in the groups exposed to AgNO₃. A positive correlation was observed between treatment groups and comet assay parameters indicating increased DNA fragmentation in exposed birds. In conclusion, the study demonstrated that although Ag-NPs resulted in higher silver accumulation, they caused less DNA damage compared to silver nitrate. This suggests that silver nanoparticles may offer a safer alternative for increasing silver levels in poultry feed while minimizing the genotoxic risks associated with ionic silver compounds.\u003c/p\u003e","manuscriptTitle":"Comprehensive Assessment of Silver Bioaccumulation and DNA Damage Effects in Coturnix coturnix japonica Using Blood, Feather, and Egg Biomarkers","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-22 05:36:42","doi":"10.21203/rs.3.rs-7086690/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9cfba641-5a70-4918-9fbc-b8f73549ce42","owner":[],"postedDate":"July 22nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-02-24T06:40:25+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-22 05:36:42","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7086690","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7086690","identity":"rs-7086690","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}