Open Veterinary Journal, (2026), Vol. 16(4): 2003-2013

Research Article

10.5455/OVJ.2026.v16.i4.4

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Application of nano-selenium as a dietary supplement and its effects on antioxidant status and gene expression related to oxidative stress in broiler chickens

Mustafa Hadi Hamid¹, Abdullah Q. Aswad², Ruya Fareed Jasim³and Mohanad Fadhl Hussain Al-Musodi^1*

¹Department of Animal Production, Agriculture College, Kerbala University, Karbala, Iraq

²College of Political Science, University of Anbar, Ramadi, Iraq

³College of Nursing, University of Kirkuk, Kirkuk, Iraq

*Corresponding Author: Mohanad Fadhl Hussain Al-Musodi. Department of Animal Production, Agriculture College, Kerbala University, Karbala, Iraq. Email: Mohanad.fadhl [at] uokerbala.edu.iq

Submitted: 08/12/2025 Revised: 03/03/2026 Accepted: 15/03/2026 Published: 30/04/2026

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ABSTRACT

Background: Antioxidants are important factors in reducing stress in poultry birds. Recently, nanomaterials have been introduced as part of feed additives to enhance the health of birds.

Aim: This study aimed to evaluate the effects of dietary Nano-Se supplementation on broiler chicken growth performance, serum antioxidant status, and hepatic expression of oxidative stress-related genes. In total, 250 1-day-old Ross 308 broiler chicks were reared for up to 42 days.

Methods: Birds were randomly assigned to five treatments (n=50 birds/treatment; 5 replicates of 10 birds each) in a completely randomized design as follows: firsttreatment (T1; basal diet, control), second treatment (T2; basal diet + 0.3 mg/kg organic Se), third treatment (T3; basal diet + 0.3 mg/kg Nano-Se), fourth treatment (T4; basal diet + 0.6 mg/kg Nano-Se), and fifth treatment (T5; basal diet + 0.9 mg/kg Nano-Se).

Results: Growth performance was recorded weekly. On day 42, blood samples were collected for biochemical analysis of antioxidant enzymes, including glutathione peroxidase (GPx), superoxide dismutase (SOD), total antioxidant capacity (TAC), and Malondialdehyde (MDA). Liver tissue was harvested for quantitative real-time Polymerase chain reaction analysis of glutathione peroxidase 1 (GPX1), superoxide dismutase 1 (SOD1), catalase (CAT), nuclear factor 2 (Nrf2), and heme oxygenase-1 (HO-1) gene expression. T4 showed a significant improvement in the final weight and feed conversion ratio. A significant increase in serum GPx activity was observed for T4 compared to T1 at 59.6% (125.3 vs. 78.5 U/ml; p < 0.001), respectively. SOD activity and TAC were both increased by 49.1% (42.5 vs. 28.5 U/ml; p < 0.001), and was 46.2% (2.85 vs. 1.95 mM Trolox equivalents, p < 0.001) in T4 compared with T1. Likewise, MDA concentration was decreased in T4 by 44.4% compared with that in T1 (3.25 vs. 5.85 nmol/ml, p < 0.001), respectively. Additionally, hepatic gene expression analysis revealed a significant upregulation of Nrf2 (3.80-fold), HO-1 (4.20-fold), GPX1 (3.45-fold), SOD1 (3.15-fold), and CAT (2.95-fold) in T4 (p < 0.001). In conclusion, dietary supplementation with 0.6 mg/kg of nano-Se effectively enhanced the Nrf2-mediated antioxidant defense system, reduced oxidative stress, and enhanced growth performance in broiler chicken.

Conclusion: These findings support the use of nano-Se as a superior alternative to conventional selenium sources in the diet and could improve the productive and physiological aspects of broilers.

Keywords: Broiler, Growth performance, Nano-selenium, Nrf2 signaling, Oxidative stress.

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Introduction

The global poultry industry accounts for approximately 38% of meat production worldwide, serving as a critical component of food security (FAO, 2023). Modern broiler chickens, which are genetically selected for rapid growth and superior feed conversion efficiency, exhibit exceptional production performance, but their elevated metabolic rates increase susceptibility to oxidative stress (Zhang et al., 2015; Surai et al., 2019). Oxidative stress, characterized by an imbalance between reactive oxygen species (ROS) production and antioxidant defense capacity, leads to cellular damage, compromised immune function, and reduced productivity (Al-Musodi and Jaafer, 2019; Surai et al., 2019).

Selenium (Se), an essential trace element, plays a pivotal role in antioxidant defense through its incorporation into selenoproteins, particularly glutathione peroxidase (GPx), which catalyzes the reduction of hydrogen peroxide and lipid hydroperoxides (Rayman, 2012; Weaver and Skouta, 2022). Traditional selenium supplementation uses inorganic forms (sodium selenite) or organic forms (selenomethionine), each with inherent limitations, including narrow safety margins and variable bioavailability (Radomska et al., 2021).

Nanotechnology offers innovative solutions to enhance nutrient bioavailability and efficacy. Nano-selenium (Nano-Se), characterized by small particle size (typically 20–100 nm), large surface area-to-volume ratio, and unique physicochemical properties, demonstrates superior bioavailability, enhanced antioxidant capacity, and reduced toxicity compared to conventional selenium sources (Wang et al., 2007; Hu et al., 2012). Recent studies indicate that Nano-Se can be absorbed through multiple pathways, including endocytosis, paracellular transport, and direct membrane penetration, resulting in more efficient cellular uptake (Klasing, 2007).

The nuclear factor 2 (Nrf2) signaling pathway is the principal regulator of antioxidant reactions in cells (Ma, 2013; Tonelli et al., 2018). Under basal conditions, Kelch-like ECH-associated protein 1 (Keap1) sequesters Nrf2 in the cytoplasm. Upon oxidative stress, Nrf2 dissociates from Keap1, translocates to the nucleus, and binds to antioxidant response elements (ARE) in target gene promoters, inducing the expression of antioxidant enzymes, including GPx, superoxide dismutase (SOD), catalase (CAT), and heme oxygenase-1 (HO-1) (Zhao et al., 2017). Emerging evidence suggests that selenium, particularly nano-Se, can modulate Nrf2 pathway activation, although the precise molecular mechanisms in broiler chickens remain incompletely characterized.

Intestinal health is another critical aspect influenced by oxidative stress and selenium supplementation. The gut epithelium functions as a selective barrier that regulates nutrient absorption and prevents pathogen translocation. Intestinal susceptibility to oxidative stress impairs intestinal integrity by interfering with tight junction proteins (zonula occludens-1, occludin, and claudin), leading to increased permeability and systemic inflammation (Cai et al., 2012). Nano-Se supplementation enhances intestinal morphology, upregulates tight junction protein expression, and modulates gut microbiota composition by promoting beneficial bacteria (Lactobacillus, Bifidobacterium) and suppressing pathogens (Escherichia coli, Clostridium perfringens) (Surai, 2002; Klasing, 2007; Boostani et al., 2015).

Despite accumulating evidence supporting the efficacy of nano-Se, systematic dose-response studies comparing nano-Se with conventional selenium sources at various inclusion levels remain limited. Furthermore, a comprehensive investigation of the effects of Nano-Se on Nrf2 pathway gene expression in broiler liver tissue is warranted to elucidate the molecular mechanisms underlying its superior antioxidant efficacy. Therefore, this study aimed to evaluate the effects of different levels of dietary nano-Se supplementation compared with organic selenium on growth performance, oxidative stress biomarkers in serum, and quantification of the hepatic expression of Nrf2 and antioxidant genes [glutathione peroxidase 1 (GPX1), superoxide dismutase 1 (SOD1), CAT, and HO-1] in broiler chickens.

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Materials and Methods

Preparation and characterization of nano-selenium

Nano-selenium particles were synthesized following the technique reported by Zhang et al. (2001) with slight adjustments. A 0.1 M sodium selenite (Na₂ SeO₃, 99% purity, Sigma-Aldrich, USA) solution was prepared by dissolving sodium selenite in deionized water. The reducing agent was ascorbic acid (C₆H₈O₆, analytical grade, Merck, Germany) at a molar ratio of 3:1 (ascorbic acid:selenium). The reaction was carried out at 25^oC and continuously stirred at 400 rpm for 6 hours. Centrifugation of the resulting colloidal suspension was at 10,000 × g for 20 minutes with 4^oC of deionized water and absolute ethanol to remove any remaining reactants. The recrystallized Nano-Se was lyophilized using a freeze dryer (Christ Alpha 2–4 LD Plus, Germany) and kept at 20^oC in amber vials in the presence of nitrogen.

Nano-Se particles were characterized using several analytical methods. Particle morphology and size distribution were determined using transmission electron microscopy (TEM; JEOL JEM-2100, Japan) at 200 kV. The hydrodynamic diameter and polydispersity index were determined by DLS (Malvern Zetasizer Nano ZS, UK). The zeta potential was measured using laser Doppler electrophoresis. XRD (Bruker D8 Advance, Germany) analysis confirmed the amorphous nature of the synthesized Nano-Se. TEM analysis revealed spherical particles with an average diameter of 45 ± 8 nm. The DLS measurements indicated a hydrodynamic diameter of 68 ± 12 nm with a PDI of 0.22, indicating a narrow size distribution. The zeta potential was −28.5 ± 3.2 mV, indicating good colloidal stability.

Experimental design and housing

A total of 250 1-day-old Ross 308 male broiler chicks with an average initial body weight of 45.3 ± 0.2 g were obtained from Al-Najaf Poultry Company, Iraq. The chicks were randomly allocated to five dietary treatment groups using a completely randomized design, with five replicate pens per treatment and 10 birds per replicate (n=50 birds per treatment). The experimental period lasted for 42 days and was divided into the starter (days 1–21) and finisher (days 22–42) phases.

The birds were housed in floor pens (1.5 m × 1.5 m) equipped with wood shavings as bedding material (5 cm depth). Each pen had one tube feeder and one automatic bell drinker, and they had ad libitum access to feed and water during the experiments. The housing facility was environmentally controlled with forced ventilation. The temperature was maintained at 32°C during the first week and gradually reduced by 3°C per week until it reached 24°C on day 21, and then maintained at 22°C–24°C for the finisher period. The relative humidity was maintained at 60%–65%. The lighting program consisted of 23 hours light:1 hour dark for the first 7 days, followed by 18 hours light:6 hours dark for the remainder of the experiment. The light intensity was maintained at 30 lux using LED bulbs.

The standard vaccination program was implemented as follows: Newcastle disease vaccine (B1 strain) via drinking water on days 7 and 21, and infectious bursal disease (Gumboro) vaccine via drinking water on day 14. No antibiotics or coccidiostats were used uring the experimental period.

Dietary treatments

Five experimental diets were developed to meet or exceed the nutritional requirements of Ross 308 broilers as specified by the breeder’s recommendations (Al-Tememe, 2025). The basal diet was developed to be selenium-deficient (<0.05 mg/kg) using low-selenium-content ingredients. The dietary treatments were as follows:

• T1 (Control): Basal diet without selenium supplementation.
• T2 (Organic Se): Basal diet + 0.3 mg/kg selenium as L-Selen methionine (Sel-Plex^®, Alltech Inc., USA).
• T3 (Nano-Se 0.3): Basal diet + 0.3 mg/kg selenium.
• T4 (Nano-Se 0.6): Basal diet + 0.6 mg/kg selenium.
• T5 (Nano-Se 0.9): Basal diet + 0.9 mg/kg selenium.

Nano-Se and organic selenium were thoroughly mixed with a small amount of carrier (wheat bran) before incorporation into the complete feed to ensure uniform distribution. Diets were prepared in mash form and stored in sealed bags at room temperature. Feed samples were analyzed for proximate composition according to the methods of AOAC (2019). Table 1 shows the ingredients and chemical composition of the nutrients of the basal diets.

Table 1. Composition of ingredients and calculated nutrient content of basal diets.

Growth performance measurements

Body weight (BW) was recorded on day 1 and weekly thereafter until day 42 using a digital scale (±1 g precision). Feed consumption was measured weekly by weighing the offered and refused feed. Body weight gain (BWG) was obtained by measuring the difference between final and initial body weights. The ratio of the total body weight gain to total feed intake was calculated as the feed conversion ratio (FCR) of each pen in the replicate. Mortalities were counted on a daily basis, and dead birds were weighed to correct FCR calculations. (Al-Musodi et al., 2025).

Sample collection and processing

On day 42, two birds per replicate (10 birds per treatment) with body weights close to the pen average were selected for blood and tissue sampling following a 12-hour fasting period. The birds were humanely euthanized by cervical dislocation after blood collection. Approximately 5 ml of blood was collected from the brachial vein using sterile syringes and transferred into non-anticoagulant tubes. Blood was left to clot at room temperature for 30 minutes and centrifuged at 3,000 × g for 15 minutes at 4°C. Serum was separated and aliquoted into sterile microcentrifuge tubes and kept at −20°C until biochemical analysis. The abdominal cavity was opened immediately after euthanasia, and liver tissue samples (approximately 1 g) were collected from the left lobe. Liver samples were rinsed with ice-cold phosphate-buffered saline (pH 7.4), blotted dry, flash-frozen in liquid nitrogen, and stored at −80°C until RNA extraction.

Serum biochemical analysis

The activity of serum GPx was measured according to the oxidation of NADPH into NADP+ catalyzed by glutathione reductase using a commercially available assay kit (Item No. 703102, Cayman Chemical, Ann Arbor, MI, USA). The decreasing absorbance rate at 340 nm is proportional to the GPx activity. Superoxide dismutase activity was measured using a superoxide dismutase assay kit (Item No. 706002, Cayman Chemical) utilizing a tetrazolium salt for the detection of superoxide radicals generated by xanthine oxidase and hypoxanthine. The absorbance was measured at 450 nm.

The capacity to inhibit the oxidation of 2,2’-azino-di-(3-ethylbenzthiazoline sulfonate) (ABTS) to ABTS+ by metmyoglobin (to produce ABTS+) is known as total antioxidant capacity (TAC), which was measured using a total antioxidant capacity assay kit (Item No. 709001, Cayman Chemical). The absorbance at 750 nm was recorded, and the data in millimolar Trolox equivalents were used.

The Malondialdehyde (MDA) concentration was measured by the thiobarbituric acid reactive substances assay as described in Placer et al. (1966) (14) with amendments. Briefly, 100 µl of serum was mixed with 500 µl of 20% trichloroacetic acid and 500 µl of 0.67% trichloroacetic acid, and thio barbituric acid and subjected to 30 minutes in a boiling water bath, chilled on ice, and centrifuged at 3,000 × g during 10 minutes. The supernatant absorbance was measured at 532 nm. The MDA concentration was calculated using a standard curve prepared with 1,1,3,3-tetramethoxypropane and expressed as nmol/ml. Biochemical measurements were performed in triplicate using a microplate reader (Synergy H1 Hybrid Multi-Mode Reader, Bio Tek Instruments, Winooski, VT, USA). Each run of the assays used quality control samples. The intra- and inter-assay coefficients of variation were less than 5% and 10%, respectively.

RNA extraction and real-time Polymerase chain reaction (PCR)

Total RNA was extracted from liver tissue (about 30 mg) using the RNeasy Mini Kit (Cat. No. 74104, Qiagen, Hilden, Germany) and on-column DNase digestion (RNase-Free DNase Set, Qiagen) according to the manufacturer’s instructions. The RNA concentration and purity were assessed using a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA). Only samples with A260/A280 ratios between 1.8 and 2.0 and A260/A230 ratios >1.8 were used. RNA integrity was verified using 1% agarose gel electrophoresis.

First-strand cDNA was synthesized from 1 µg total RNA using the High-Capacity cDNA Reverse Transcription Kit (Cat. No. 4368814, Applied Biosystems, Foster City, CA, USA) in a 20 µl reaction volume. The reaction mixture was incubated at 25°C for 10 minutes, 37°C for 120 minutes, and 85°C for 5 minutes. The synthesized cDNA was diluted 1:10 with nuclease-free water and stored at −20°C.

Relative expression of the target genes, including SOD1, GPX1, CAT, Nrf2, and HO-1, was measured by Real-time polymerase chain reaction. Beta-actin (2-actin) was normalized with the help of beta-actin. The primer sequences are listed in Table 2. All primers were designed using Primer3 software, synthesized by Integrated DNA Technologies (IDT, Coralville, IA, USA), and validated by BLAST and melting curve analyses.

Table 2. Primer sequences Primer sequences to be used for quantitative real-time PCR.

Statistical analysis

All data were processed using SPSS version 26.0 (IBM Corp., Armonk, NY, USA). The replicate pen (n=5) was the experimental unit of growth performance data, and the individual bird (n=10) was the experimental unit for biochemical and gene expression data. The data were checked on the test of normality of data through the Shapiro-Wilk test and homogeneity of variance through the Levene test. Analysis of variance was conducted to establish treatment effects on a one-way basis. Duncan’s multiple range test was applied to separate the means when significant differences were found (p ≤ 0.05) (Duncan,1955). Polynomial regression analysis was performed to evaluate dose-response relationships for nano-Se treatments. Statistical significance was declared at p ≤ 0.05, and trends were noted at 0.05 < p ≤ 0.10.

Ethical approval

The experimental procedures were conducted with the permission of the Institutional Animal Care and Use Committee, based on ethical requirements of animal handling. Moreover, the guidelines on the Care and Use of Agricultural Animals in Research and Teaching (FASS, 2020) were strictly followed.

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Results

Growth performance

Table 3 and Figure 1 show the effects of dietary selenium supplementation on growth performance parameters. During the 42 days of the experiment, the dietary treatments had a significant effect on the final BWand BWG (p < 0.001). Birds receiving 0.6 mg/kg nano-Se (T4) exhibited the highest final BW, representing a 7.4% improvement compared with the control group and a 4.5% improvement compared with the organic selenium group (p < 0.05). The T3 and T5 groups also demonstrated significantly higher final BW than control (p < 0.01), although the differences from T4 were not statistically significant. All experimental treatments showed an improvement in BWG compared with the control.

Table 3. Effect of dietary selenium supplementation on growth performance of broiler chickens (1–42 days).

Fig. 1. Effect of dietary selenium supplementation on growth performance of broiler chickens A. Body weight Gain, B. Feed conversion ratio and C. Total feed intake.

All the selenium-treated groups had a greater improvement in FCR than the control group (p < 0.001). The T4 group recorded the best value, representing a 7.6% improvement over the control and a 4.2% improvement over organic selenium. The T3 and T5 groups showed intermediate FCR values, both significantly better than the control (p < 0.001) but not significantly different from the T4 group.

The total feed intake was not significantly different among treatments, ranging from 3,830 g in the control group to 3,879 g in the T4 group, indicating that the improved FCR resulted from enhanced body weight gain rather than reduced feed consumption. Mortality was low across all experimental treatments compared with T1 but did not differ significantly.

Serum antioxidant status

Table 4 and Figures 3 and 4 present the serum antioxidant enzyme activities and oxidative stress biomarkers at day 42. Dietary selenium supplementation significantly influenced all measured parameters (p < 0.001). The level of GPx was significantly higher in the selenium-enriched groups than in the control. The T4 group exhibited the highest GPx activity, representing a 59.6% increase over the control and a 27.2% increase over organic selenium. The T3 and T5 groups showed intermediate GPx activities, which were significantly higher than those of the control and organic selenium.

Table 4. Effects of dietary selenium supplementation on serum antioxidant indices of day 42 broiler chickens.

Fig. 3. Effects of dietary selenium supplementation on antioxidant activity.

Fig. 4. Effects of dietary selenium supplementation on MDA enzymes.

The SOD activity followed a similar pattern. The T4 group achieved the highest SOD activity, representing a 49.1% increase over the control and a 20.7% increase over organic selenium. Nano-Se supplementation at all levels significantly increased SOD activity compared with that of the control and organic selenium.

TAC was significantly elevated in the selenium-treated groups, with T4 and T5 showing the highest values, representing 46.2% and 42.6% increases over the control, respectively. The organic selenium group showed intermediate TAC, which was significantly higher than the control but lower than the T4 and T5 groups.

The MDA concentration, which is a biomarker of lipid peroxidation, was also significantly lower in all the selenium-supplemented groups (p < 0.001). The T4 group exhibited the lowest MDA, representing a 44.4% reduction compared with the control and a 28.6% reduction compared with organic selenium.

Hepatic gene expression

Table 5 and Figure 2 exhibit the relative mRNA expression levels of antioxidant-related genes in liver tissue. Dietary selenium supplementation had a significant impact on the expression of all target genes (p < 0.001). The master regulator Nrf2 showed the most pronounced response, with T4 exhibiting 3.80-fold upregulation compared with the control, followed by T5 (3.50-fold), T3 (2.85-fold), and T2 (2.20-fold). This dose-dependent increase in Nrf2 expression demonstrates Nano-Se’s ability to activate this critical transcription factor. Among the genes examined, HO-1, one of Nrf2’s target genes, showed the highest level of gene expression, reaching 4.20-fold in the T4 group. This robust HO-1 induction reflects the strong activation of the Nrf2-ARE pathway. The organic selenium group showed only a 2.35-fold upregulation, which was significantly lower than that of all the Nano-Se treatments (p < 0.001).

Table 5. Effects of dietary selenium supplementation on the relative mRNA expression of antioxidant-related genes in the liver of broiler chickens on day 42.

Fig. 2. Effects of dietary selenium supplementation on the relative mRNA expression of antioxidant-related genes in the liver of broiler chickens.

The expression of selenoprotein genes (GPX1 and SOD1) was significantly upregulated in all selenium-supplemented groups. The T4 group showed increases of 3.45-fold and 3.15-fold in GPX1 and SOD1 expression, respectively. These increases were significantly higher than those of organic selenium (2.10-fold and 1.85-fold for GPX1 and SOD1, respectively).

CAT expression followed a similar pattern, with T4 showing a 2.95-fold upregulation compared with the control.

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Discussion

Growth performance

This study provides comprehensive evidence that dietary nanoselenium supplementation, particularly at 0.6 mg/kg, significantly enhances growth performance, strengthens antioxidant defense capacity, and activates the Nrf2-mediated transcriptional response in broiler chickens. The superior efficacy of nano-Se compared with organic selenium represents a significant advancement in poultry nutrition with important implications for commercial production (Bami et al., 2022). Dose-response analysis revealed that 0.6 mg/kg represents the optimal Nano-Se supplementation level for broiler chickens. This finding is consistent with those of previous studies reporting optimal responses at 0.3–0.6 mg/kg (Gangadoo et al., 2020).

The 7.4% improvement in final body weight and 7.6% improvement in FCR observed in the 0.6 mg/kg nano-Se group represent substantial economic benefit for commercial broiler production. These improvements can be attributed to multiple synergistic mechanisms. First, Nano-Se has an enhanced antioxidant status, which reduces oxidative damage to cellular components, particularly in rapidly dividing tissues, thereby supporting optimal growth (Hosnedlova et al., 2018). Second, improved intestinal health, as demonstrated in previous studies with Nano-Se supplementation (Boostani et al. 2015), which enhances nutrient absorption efficiency, was also observed. Third, Nano-Se can reduce oxidative stress and support immune function, thereby decreasing energy expenditure on immune responses and redirecting nutrients toward growth (Huang et al., 2003).

The dose-response relationship observed in this study, with optimal performance at 0.6 mg/kg and no additional benefit at 0.9 mg/kg, suggests that selenoprotein synthesis is saturated or regulatory mechanisms are activated under this respective dose in vivo. This has been corroborated by the idea that selenium supplementation conforms to a hormetic dose-response relationship, wherein an intermediate level offers an advantage, but higher levels might provoke some form of adaptive compensatory mechanism that constrains the acquisition of added advantage (Kensler et al., 2007).

The superior performance of nano-Se compared to organic selenium through achieving higher final BW and better FCR can be explained by its unique absorption mechanisms. While organic selenium is primarily absorbed through amino acid transporters in the small intestine, nano-Se can be taken up through multiple pathways, including endocytosis, paracellular transport, and direct membrane penetration (Klasing, 2007). This multimodal absorption results in higher bioavailability and more efficient delivery to target tissues. The improved intestinal health likely contributed to the enhanced growth performance observed in this study, as a healthy gut is essential for efficient nutrient use and reduced energy expenditure on immune responses. Although direct measurements of intestinal health and gut microbiota modulation were not performed in this study, previous studies have established that nano-Se supplementation has a great effect on improving intestinal morphology and barrier function in broilers (Boostani et al., 2015). The gut epithelium serves as a critical interface between the bird and its environment, and its integrity is essential for optimal nutrient absorption and immune function. Oxidative stress impairs the role of the intestinal barrier by altering the tight junction proteins (ZO-1, occludin, and claudin), resulting in increased intestinal permeability and translocation of pathogens and endotoxins (Cai et al., 2012). Nano-Se upregulates tight junction protein expression through Nrf2-dependent mechanisms, as these proteins contain ARE sequences in their promoter regions (36). Additionally, Nano-Se modulates gut microbiota composition by promoting beneficial bacteria (Lactobacillus, Bifidobacterium) and suppressing pathogens (E. coli, C. perfringens) (Surai, 2002; Klasing, 2007). This microbiota modulation may occur through multiple mechanisms: (1) direct antimicrobial effects of nano-Se particles; (2) modulation of the intestinal redox environment, which selectively favors beneficial anaerobic bacteria; and (3) enhancement of host immune responses that control pathogen populations.

Antioxidant status

The marked increase in GPx and SOD enzyme activity in the group treated with 0.6 mg/kg nano-selenium is strong evidence of the enhancement of the primary antioxidant enzyme defense system. These findings are consistent with the observed upregulation of GPX1 and SOD1 gene expression, demonstrating that Nano-Se enhances antioxidant capacity by increasing enzyme synthesis and potentially improving enzyme stability or cofactor availability (Boostani et al., 2015).

The reduction in MDA level provides compelling evidence of reduced lipid peroxidation, indicating that the enhanced antioxidant enzyme activities can be translated into meaningful protection against oxidative damage. This is particularly important in broiler production, where lipid peroxidation not only impairs cellular function but also affects meat quality during storage (Zhang et al., 2001; Gangadoo et al., 2020).

The superior efficacy of Nano-Se compared with organic selenium in enhancing antioxidant enzyme activities can be attributed to several factors. The small particle size and high surface area of Nano-Se facilitate more efficient cellular uptake and intracellular distribution (Yamamoto et al., 2018). Second, nano-Se may have a longer residence time in tissues, providing sustained selenium availability for selenoprotein synthesis. Third, the unique redox properties of Nano-Se may allow it to function both as a selenium source and as a direct antioxidant, scavenging ROS before they cause cellular damage (Weekley et al., 2013). This might also indicate the superior importance and efficacy of Nano-Se in enhancing selenoprotein expression.

The 3.80-fold upregulation of Nrf2 and 4.20-fold upregulation of HO-1 in the Nano-Se 0.6 mg/kg group represent the most significant finding of this study, providing molecular evidence for the mechanism underlying Nano-Se’s superior antioxidant efficacy. The Nrf2-Keap1-ARE pathway is the central controller of antioxidant responses in cells, and its effective stimulation by Nano-Se is the reason why the antioxidant genes of cells are coordinately activated (Ryter et al., 2006). Under normal conditions, Keap1 maintains Nrf2 in the cytoplasm as it is ubiquitinated and degraded by the proteasome to sustain a low steady-state level. Certain cysteine residues on Keap1 (especially Cys151, Cys273, and Cys288) are altered when exposed to oxidative stress or electrophilic compounds, causing the Keap1-Nrf2 interaction to be disturbed (Suzuki et al., 2015). Nano-Se likely activates this pathway through a controlled pro-oxidant effect, generating mild ROS that serve as signaling molecules rather than damaging agents. This hormetic mechanism, in which low-level stress induces adaptive responses, is a well-established phenomenon in biology (Benko et al., 2012).

The superior ability of Nano-Se to activate Nrf2 compared with that of organic selenium may be related to its unique cellular uptake mechanisms and intracellular distribution. While organic selenium is primarily metabolized through the transculturation pathway to generate selenide for selenoprotein synthesis, nano-Se may undergo biotransformation through different pathways involving glutathione and thioredoxin systems (Weaver and Skouta, 2022). This alternative metabolism may generate redox-active intermediates that activate Nrf2 more effectively.

Notably, HO-1 was highly induced (4.20-fold). HO-1 promotes the breakdown of heme to biliverdin, carbon monoxide, and free iron and has strong antioxidant, anti-inflammatory, and cytoprotective activities (Ahmadi et al., 2018). The high HO-1 expression observed in this study suggests that Nano-Se provides protection not only through classical antioxidant enzymes but also through broader cytoprotective mechanisms.

The multi-criteria comparison presents inorganic selenium (red bars), organic selenium (blue bars), and nano-selenium (green bars) across five parameters: bioavailability (95% for nano-selenium), antioxidant capacity (90%), growth performance (85%), safety profile (90%), and cost-effectiveness (70%). Scores are expressed as relative percentages (0%–100%). Nano-selenium demonstrates superior performance in most categories, supporting its potential as a superior alternative to traditional selenium sources.

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Conclusion

The addition of dietary selenium nanoparticles at 0.6 mg/kg significantly enhances growth performance, stimulates antioxidant defense capacity, and activates the master antioxidant defense pathway by robust upregulation of Nrf2 and its downstream targets HO-1, GPX1, SOD1, and CAT. Therefore, Nano-Se shows significantly greater efficacy than organic selenium in all measured parameters, supporting its potential as a superior alternative in nutrition strategies for maximal broiler production.

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Acknowledgments

I would like to thank everyone who helped us to make this work, especially Al-Najaf Poultry Company, Iraq.

Conflict of interest

There is no conflict of interest.

Funding

The research was fully funded at the expense of the researchers named in the research.

Authors’ contributions

All researchers participated in all stages of the research from the beginning of the experiment to the delivery of a copy to the journal.

Data availability

Data are available to everyone.

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