Dietary quercetagetin attenuates H2O2-induced oxidative damage and preserves meat quality in broilers by modulating redox status and Nrf2/ferroptosis signaling pathway

In modern poultry production, oxidative stress has emerged as a pivotal factor compromising the health status and overall performance of broiler. The aim of this study was to investigate the effects of dietary quercetagetin (QG) supplementation on hydrogen peroxide (H₂O₂)-induced oxidative damage in breast muscle of broilers, focusing on growth performance, meat quality, and antioxidant function, and elucidating the underlying mechanisms. Two hundred and forty one-day-old Cobb broilers were randomly divided into three treatment groups: the control group, the H₂O₂ group and the H₂O₂ + QG group. The control and H₂O₂ groups were fed a basal diet, and the QG group was fed a basal diet supplemented with 100 mg/kg QG. The control group was intraperitoneally injected with normal saline, and the other two groups were treated with the same volume of 10% H₂O₂ solution on day 37. The experimental period was 42 days. The results showed that H₂O₂-induced oxidative stress increased the levels of drip loss, cooking loss, reactive oxygen species (ROS), and oxidation products in the breast muscle, and damaged the mitochondrial function. Compared with the control group, the mRNA expressions of glutathione peroxidase (GSH-Px), NAD(P)H quinone dehydrogenase 1 (NQO1), catalase (CAT), nuclear factor erythroid 2-related factor 2 (Nrf2), transferrin receptor protein 1 (TFR1), and ferritin heavy chain 1 (FTH1) in the breast muscle were decreased (p < 0.05). The addition of QG to the diet reduced the levels of ROS and oxidation products (p < 0.05). Meanwhile, the addition of QG to the diet increased the mRNA expressions of Nrf2 and TFR1, showing no significant difference from those of the control group. In conclusion, H₂O₂-induced oxidative stress impairs breast muscle quality, mitochondrial function, and antioxidant capacity in broilers. Dietary QG alleviates oxidative stress and improves meat quality by regulating the Nrf2 signaling pathway and ferroptosis-related mechanisms. This mechanism-based finding supports QG as a safe and effective dietary additive for broiler production, providing a practical solution to enhance animal health, stabilize meat quality, and promote the sustainability of intensive poultry farming.

Graphical abstract

Graphical Abstract. Diagram illustrating the cellular pathway for oxidative stress response. Key elements include the Nrf2-Keap1 complex, ROS generation, and the role of MAPK signaling. Arrows indicate interactions and pathways involving molecules like H2O2, Quercetagetin, GPX4, and FTH1. The image shows how Nrf2 translocates to the nucleus to activate target gene transcription, influencing antioxidants and iron storage.

Introduction

Nowadays, chicken meat has gained widespread popularity due to its high protein and low-fat content (1), making the preservation of meat quality a critical priority in the poultry industry. However, intensive poultry farming practices expose broilers to various stressors, including high temperatures, diseases, physiological challenges, and immune suppression (2, 3). These adverse factors induce oxidative stress in poultry (4). Accumulating evidence has demonstrated that oxidative stress generates excessive reactive oxygen species (ROS), which severely impairs broiler growth performance and meat quality (5, 6), thereby posing a significant threat to the sustainable development of the poultry industry. ROS compromise cellular integrity, increase drip loss in muscle tissues, and disrupt mitochondrial membrane potential, which in turn triggers apoptosis (7–9). This provides a rationale for using hydrogen peroxide (H₂O₂) as a model to induce oxidative damage in broilers. Previous studies have reported that intraperitoneal injection of H₂O₂ successfully induced oxidative stress and impaired meat quality (10, 11). Among various mitigation strategies, nutritional interventions aimed at scavenging excess ROS and enhancing meat quality and antioxidant capacity in broilers have attracted considerable research attention.

Quercetagetin (QG), a 3,3,4,5,6,7-hexahydroxyflavonol extracted from marigold, exhibits stronger free radical scavenging capacity and antioxidant potential compared to quercetin, attributed to its additional hydroxyl group (12, 13). Consequently, QG represents a promising natural feed additive for improving broiler health and meat quality. In our previous study, QG enhanced broiler antioxidant ability, which may through the regulation of nuclear factor E2-related factor 2 (Nrf2) signaling pathway, and reduced malondialdehyde (MDA) levels. And QG also promoted nutrient digestion by enhancing gut structure and morphology (14, 15). Furthermore, QG has been reported to alleviate zearalenone-induced liver oxidative damage in rabbits via the Kelch-like ECH-associated protein 1 (Keap1)-Nrf2-antioxidant response element (ARE) signaling pathway (16). The efficacy and potential mechanisms of QG in alleviating oxidative stress-induced muscle damage in broilers have not been reported. Especially QG’s potential role in modulating Nrf2, ferroptosis or the signaling pathway of mitogen-activated protein kinase (MAPK), which are critical in oxidative damage and cellular stress responses.

Ferroptosis, characterized by mitochondrial shrinkage, membrane rupture, reduction in internal cristae and lipid peroxidation, has been associated with oxidative stress through its dependence on ROS and reduced glutathione peroxidase 4 (GPX4) activity (17, 18). The MAPK signaling pathway is mainly used to transact extracellular stimulus signals into cells and nuclei, and causes a series of physiological and biochemical reactions. The MAPK pathway, which encompasses c-Jun N-terminal kinase (JNK), extracellular regulated protein kinases (ERK), and p38, is of great significance in modulating cellular responses to ROS-induced stress and apoptosis (19). Despite increasing evidence supporting the involvement of ferroptosis and MAPK in oxidative stress, the mechanisms by which dietary QG modulates these pathways in broilers remain poorly understood. Besides, the regulatory effects of dietary quercetagetin on ferroptosis via Nrf2 signaling in broiler muscle have not yet been investigated.

Therefore, in this study, oxidative stress was induced in broilers via H₂O₂ injection to evaluate whether dietary QG supplementation could mitigate H₂O₂-induced oxidative damage and preserve meat quality through modulating apoptosis, ferroptosis, Nrf2 or MAPK signaling pathways. These findings aim to develop a novel dietary strategy for sustainable poultry production under oxidative stress conditions.

Materials and methods

Animals and treatment

Two hundred and forty Cobb broilers at 1 day of age were procured from Hebei Jiuxing Agriculture & Husbandry Co. Ltd. (Baoding, China). All chickens were randomly allocated into three groups. In each group, there are eight replicate cages and 10 birds were placed in each cage. The birds in the control group were provided with a basal diet and an intraperitoneal injection of normal saline (1.0 mL/kg of BW). In the H₂O₂ group, birds were administered a basal diet and injected with the same volume of 10% H₂O₂ (2.96 mmol/kg BW). In the H₂O₂ + QG group, birds were provided a basal diet supplemented with 100 mg/kg QG and injected with an equal volume of H₂O₂. QG (purity exceeding 80%) was obtained from Chenguang Biotechnology Group Co. Ltd. (Handan, China). The formal trial lasted for 42 days, and the injection was performed on day 37. The basal diets for the starter phase (1–21 days) and grower phase (22–42 days) were formulated based on the Cobb 500 broiler, as described in Broiler Performance & Nutrition Supplement (20). The detailed information on the composition and nutrient levels of the basal diet is shown in Table 1. During the experiment, birds were reared and managed according to the description in the Broiler Performance & Nutrition Supplement. The animal experiments were carried out in strict compliance with the ARRIVE guidelines, and the experimental protocols received approval from the Committee for Animal Care and Use at Hebei Agriculture University (Baoding, China, protocol number: 2023142).

Table 1

Table 1. Composition and nutrient levels of the basal diet (as-fed basis, %).

Sample collection and preparation

Following a 12 h fasting period, feed intake and BW of broilers were recorded on the 37th and the 42th days to determine the growth performance. One bird per cage was selected based on proximity to the average BW. Then these birds were euthanized and immediately slaughter thereafter for breast muscle collection.

Meat quality measurement

The determination of the quality of breast muscle was carried out according to the research methods reported in previous studies (21). Briefly, the pH (pH_{45 min} and pH_{24 h}) of breast muscle was determined at three distinct points using an electronic pH meter (Testo-205) and averaged. The meat color, represented by L* (lightness), b* (yellowness) and a* (redness), was determined using a colorimeter (Iwave W R-18). Drip loss was measured by weighing fresh breast meat in bags and suspending them at 4 °C for 24 h. The breast muscle was blotted dry and weighed again. Cooking loss was measured by weighing the breast muscle in bags, cooking them in water for 15 min, and weighing them again after cooling. The cooked samples were evaluated with a digital meat tenderness meter (C-LM3), and shear force values were recorded.

Meat composition determination

The breast muscle samples were lyophilized for further analysis. The contents of moisture (method 935.13), crude protein (method 976.05), ether extract (method 942.05), and ash (method 2003.05) in breast muscle were determined according to the method in Association of Official Analytical Chemists (AOAC) (22). The final data were expressed as fresh weight basis.

Free amino acids analysis

Sample pretreatment for the detection of free amino acids in breast muscle was performed as described by Oh et al. (23) and Dai et al. (24). Samples were analyzed using a high performance liquid chromatography (HPLC) (Thermo Fisher Scientific, United States) instrument. The methods for sample determination and analysis were performed as described by Boz et al. (25).

Measurement of oxidative parameters and ROS

The breast muscle samples were homogenized with normal saline, and then centrifuged to obtain the supernatant for the subsequent determination of antioxidant indices (1). The total protein content of breast muscle was determined using BCA protein kit (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China). The content of total antioxidant capacity (T-AOC, Catalog number: YX-200118C), total superoxide dismutase (T-SOD, Catalog number: YX-191504C), glutathione peroxidase (GSH-Px, Catalog number: YX-071908C), catalase (CAT, Catalog number: YX-030120C), malondialdehyde (MDA, Catalog number: YX-130401C), 8-hydroxy-2′-deoxyguanosine (8-OHdG, Catalog number: YX-081508C), advanced oxidation protein products (AOPPs, Catalog number: YX-011516C), protein carbonyl (Catalog number: YX-160315C), total sulfhydryl (Catalog number: YX-202019C) and ROS (Catalog number: YX-181519C) were measured. The corresponding commercial kits were purchased from the Beijing Borui Changyuan Technology Co., LTD. The assay procedure was performed in accordance with the kit instructions.

Mitochondrial function analysis

The production levels of mitochondrial complexes I (MRCCI, Catalog number: YX-031513C), complexes III (MRCCIII), and mitochondrial O²⁻ (Catalog number: YX-150200C) and the changes of mitochondrial membrane potential in breast muscle tissue were measured by corresponding kits. The corresponding commercial kits were purchased from the same company as described above.

Quantitative real-time PCR assay

Total RNA was extracted with Trizol reagent (TaKaRa, Japan). The purity of the total RNA was evaluated using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, United States). Subsequently, the RNA was subjected to reverse transcription to synthesize cDNA by means of a PrimeScript RT Master Mix kit (TaKaRa, Japan). RT-qPCR was carried out according to the 7500 HT Sequence detection system with SYBR Premix Ex Taq (TaKaRa, Japan).

The primers were synthesized by Tianjin Qingke Biotechnology Co., LTD (Tianjin, China), with sequences provided in Supplementary Table S1. The β-actin gene was served as the internal reference for normalizing the target genes. The relative mRNA expression was computed by the 2^−∆∆Ct method (26).

Statistical analysis

Growth performance was measured with duplicate cages serving as the experimental units. The remaining data were examined using individual broilers as the experimental units. The comprehensive data were analyzed via the one-way ANOVA function in SPSS software (SPSS 26). The disparities between the means of each treatment were appraised using the Tukey’s HSD test within SPSS software. Statistical significance was defined as p < 0.05. Data were presented as means and SEM.

Results

Growth performance

Before H₂O₂ challenge, no differences were observed in broiler growth performance such as average daily gain (ADG), average daily feed intake (ADFI) and feed to gain ratio (F/G) among groups (Table 2). After H₂O₂ challenge, compared with the control group, the body weight gain rate of broilers in the H₂O₂ group was decreased by 34.62%. When compared with the H₂O₂ group, dietary supplementation with QG increased the body weight gain rate of broilers by 27.83%. However, neither of these differences reached statistical significance (p > 0.05).

Table 2

Table 2. Influence of dietary QG on the growth performance of H₂O₂-challenged broilers.

Meat quality measurement

Compared with the control group, the H₂O₂ group exhibited a significantly lower a* value and a significantly higher cooking loss in broiler breast muscle (p < 0.05, Table 3). Dietary supplementation with QG increased the a* value, reduced cooking loss to a certain extent and improved to the level of control group (p > 0.05). Additionally, in the H₂O₂ group, the L* value of broiler breast muscle showed an 8.68% increasing trend (p = 0.061) and the drip loss tended to increase 12.90% (p = 0.057) compared with the control group.

Table 3

Table 3. Effect of dietary QG supplementation on meat quality in breast muscle of H₂O₂-challenged broilers.

Nutritional composition of breast muscle

Among the three groups, no differences were observed in the contents of nutritional components (such as moisture, crude protein, ether extract, and crude ash) in broiler breast muscle (p > 0.05, Table 4).

Table 4

Table 4. Effect of dietary QG supplementation on nutritional composition within breast muscle of H₂O₂₋challenged broilers (fresh sample, %).

Amino acids profile

No disparities were observed in the levels of bitter amino acids, umami amino acids and sweet amino acids (Table 5) of breast muscle across the three groups (p > 0.05).

Table 5

Table 5. Effect of dietary QG supplementation on free amino acids within breast muscle of H₂O₂-challenged broilers (‰).

Antioxidants activity and contents of oxidative products

Compared with the control group, H₂O₂-induced oxidative stress increased the levels of MDA, 8-OHdG, AOPPs and Sulfhydryl in breast (p < 0.05, Table 6), and decreased the activities of SOD, GSH-Px, T-AOC and CAT (p < 0.05). Compared with the H₂O₂ group, broilers in the H₂O₂ + QG group enhanced SOD, T-AOC and CAT activities, reduced levels of MDA, 8-OHdG and AOPPs (p < 0.05).

Table 6

Table 6. Effect of dietary QG supplementation on antioxidant capacity within breast muscle of H₂O₂-challenged broilers.

Generation of ROS

In contrast to the control group, the H₂O₂ challenge led to an elevation in the ROS level by 140.28% in breast muscle (p < 0.05, Figure 1). The supplementation with QG mitigated the adverse effect by decreasing ROS level by 21.43% (p < 0.05) compared with the H₂O₂ group.

Figure 1

Figure 1. ROS production levels were measured after H₂O₂ and QG treatment within breast muscle.

Mitochondrial damage

Compared with the control group, H₂O₂-induced oxidative stress increased MRCC I and MRCC III levels by 56.73 and 55.79%, respectively (p < 0.05, Figure 2). The addition of QG partially mitigated the adverse effects and had no difference with that in control group. The H₂O₂ + QG group displayed a greater O²⁻ concentration (p < 0.05) than the control group. No variation was detected in mitochondrial membrane potential among all groups.

Figure 2

Figure 2. QG alleviated the symptoms of H₂O₂-induced mitochondrial damage in broiler breast muscle. (A) Level of MRCC I production. (B) Level of MRCC III production. (C) Level of mitochondrial membrane potential. (D) Level of O²⁻ concentration.

Antioxidant enzyme mRNA expression

H₂O₂ challenge reduced the GSH-Px mRNA expression by 39.58% (p < 0.05, Figure 3) and reduced CAT mRNA expression by 58.10% (p < 0.05) compared with the control group. The addition of QG in the H₂O₂ + QG group partially mitigated this reduction in GSH-Px and CAT mRNA expression, which increased by 4.98 and 44.46%, respectively, compared to the H₂O₂ group, almost reaching the level of the control group (p > 0.05).

Figure 3

Figure 3. QG attenuated H₂O₂-induced dysregulation of antioxidant—related genes in broiler breast muscle. (A) The expression level of GSH-Px. (B) The expression level of SOD-1. (C) The expression level of CAT.

Apoptosis response

As shown in Figure 4, H₂O₂-induced oxidative stress reduced B-cell lymphoma-2 (Bcl-2) (by 46.15%, p < 0.05) and Bcl-2-associated X protein (Bax) (by 67.03%, p < 0.05) mRNA expression compared with the control group. No disparities were detected in Caspase-3 mRNA expression among the three groups.

Figure 4

Figure 4. QG alleviated the dysregulation of genes related to apoptosis of cells induced by H₂O₂ in broiler breast muscle. (A) The expression level of Caspase-3. (B) The expression level of Bcl-2. (C) The expression level of Bax.

Nrf2, MAPK signaling and ferroptosis

Compared with the control group, H₂O₂-induced oxidative stress reduced Nrf2, ERK, JNK, transferrin receptor protein 1 (TFR1) and ferritin heavy chain 1 (FTH1) mRNA expression (p < 0.05, Figures 5–7). The addition of QG to the diet partly counteracted the negative impacts by enhancing the mRNA expression of Nrf2 and TFR1, attaining the same level as that of the control group (p > 0.05). There was no impact on ERK and JNK mRNA expression in breast muscle between the H₂O₂ and H₂O₂ + QG groups. No disparities were detected in P38 mRNA expression among the three groups.

Figure 5

Figure 5. QG alleviated H₂O₂-induced dysregulation of Nrf2 signaling pathway related gene in broiler breast muscle. (A) The expression level of Nrf2. (B) The expression level of HO-1. (C) The expression level of NQO1.

Figure 6

Figure 6. QG alleviated H₂O₂-induced dysregulation of MAPK signaling pathway related genes in broiler breast muscle. (A) The expression level of ERK. (B) The expression level of P38. (C) The expression level of JNK.

Figure 7

Figure 7. QG alleviated the dysregulation of genes involved in the ferroptosis induced by H₂O₂ in broiler breast muscle. (A) The expression level of TFR1. (B) The expression level of FTH1. (C) The expression level of SLC7A11. (D) The expression level of GPX4.

Discussion

This study demonstrated the potential of QG to alleviate H₂O₂-induced oxidative stress and improve meat quality and antioxidant capacity in broilers. In this study, intraperitoneal injection of H₂O₂ was used to directly increase ROS levels, effectively creating an oxidative stress model, as established in prior research (27). Previous study had confirmed the detrimental effects of oxidative stress on broilers. For example, broilers treated with H₂O₂ exhibited reduced BW and FI and increased F/G (11). Similarly, other oxidative stress inducers, such as lipopolysaccharide (LPS) and diquat, had been demonstrated to have a negative impact on broilers (28–30). It was previously reported that the BW, feed intake of broilers increased linearly from days 1 to 35 with the addition of quercetin (31, 32). However, in this study, dietary QG supplementation did not improve broilers growth performance during the pre-challenge period (day 1–37). Wu et al. (16) also found that adding QG had no influence on the growth performance of rabbits. Previous study showed that H₂O₂ induced oxidative stress decreased the BW change rate of broiler after challenge (11). However, our results showed no significant differences in body weight changes after H₂O₂ induction. This discrepancy could be associated with variations in molecular properties, broiler breed, muscle type, age, or physiological conditions, such as the degree of oxidative stress.

Meat color is a crucial indicator of freshness (12, 33). Oxidative stress promotes glycolysis, leading to excessive production of lactate, which lowers muscle pH (2). Additionally, lipid oxidation in muscle tissue is promoted by ROS, which negatively affects meat color and stability. Secondary metabolites of lipid oxidation, such as MDA, accelerates myoglobin oxidation (11). Protein oxidation also alters the texture and flavor of meat, ultimately impairing meat quality. In this study, H₂O₂ challenge reduced the a* value and increased cooking loss, along with the tendency to increase the drip loss of breast muscle. Dietary supplementation with QG partially improved the meat color and reduced the cooking loss. This suggested that the addition of QG to the diet ameliorated the damage to meat quality caused by oxidative stress to some extent. QG had one more hydroxyl group than traditional quercetin in structure. Previous study showed that the addition of quercetin enhanced the a* value and reduced the b* value of breast in a dose-dependent fashion (12). Wang et al. (6) found that dietary supplementation with radix astragali suppressed lipid oxidative process, elevated a* value and lessened b* value at 24 h. Quercetin, as an external antioxidant, is capable of safeguarding lipids against oxidation and maintaining the stability of myoglobin content. Thus slowing down the process of color change in meat. Lipid peroxidation has the ability to impair the integrity of cell membranes and lead to an increment in drip loss. Dietary quercetin has been shown to promote the loosening of muscle fiber structure, reducing shear force and improving the tenderness of breast muscle (34, 35). Therefore, QG may also enhance the secondary structure and thermal stability of breast muscle proteins, ultimately achieving the goal of improving the tenderness and quality of the meat. In conclusion, QG effectively alleviated the decline in meat quality caused by oxidative stress, reduce cooking loss, and enhance the commercial value of chicken.

In addition, the content and composition of free amino acids are also important indicators affecting meat quality. Amino acids are essential components of proteins. It not only provides important nutritional value to muscle, but also plays an important role in improving muscle flavor and taste (36). The current research revealed that the addition of QG did not exert any influence on the flavor profile of broiler breast muscle. The precise mechanisms by which QG mediates these effects remain unclear and warrant further investigation.

Owing to the swift multiplication of muscle cells and the high degree of unsaturation of muscle lipids, the breast muscle is especially susceptible to oxidative stress (37). Our findings corroborated these observations, as H₂O₂ exposure increased ROS, MDA, 8-OHdG, AOPPs, and sulfhydryl contents, reduced the GSH-Px, and CAT activities. These results suggested that the ROS-induced damage undermines celluar antioxidant system, leading to lipid per-oxidation, protein oxidation, and mitochondrial dysfunction. These findings were in line with the ones obtained from previous investigations (10, 38). We found that dietary QG supplementation may alleviate oxidative damage by scavenging ROS, enhancing antioxidant enzyme activities, and alleviating mitochondrial impairment. Similarly, pterostilbene, a natural dimethylated analog of resveratrol, was shown to alleviate diquat-induced liver injury and oxidative stress in the liver of broilers (28). These findings were consistent with those of previous investigations (39, 40). The antioxidant effect of QG may be ascribed to its structural configuration possessing six hydroxyl groups, which may help to scavenge ROS, superoxide and hydroxyl radicals, as well as activate antioxidant defense system to alleviate oxidative damage (41, 42). Oxidative stress is also recognized to activate apoptosis pathway, leading to irreversible cell death (43). Our results showed that H₂O₂ impaired mitochondrial function. This indicated that the REDOX imbalance in mitochondria may inhibit the mitochondrial electron transport chain, and intracellular anaerobic respiration acts, leading to apoptosis (31, 44, 45).

Nrf2 represents a critical transcription factor in the cellular defense against oxidative stress. Under oxidative stress, Nrf2 disengages from Keap1, translocates to the nucleus, and then attaches to the ARE sequence. This process consequently triggers the transcription of downstream antioxidant genes (46). Additionally, the activation of Nrf2 is also modulated by upstream kinases, including those in the MAPK pathway (e.g., ERK, JNK, p38), which can phosphorylate and promote Nrf2 nuclear translocation (46). However, our study found that H₂O₂-induced oxidative stress led to a significant reduction in Nrf2, NAD(P)H quinone dehydrogenase 1 (NQO1), ERK, JNK mRNA expression, suggesting inhibition of both the Nrf2 and MAPK signal pathways. This may be due to differences in experimental conditions, such as H₂O₂ dose, exposure time, or animal tissues or models. Bao et al. (47) reported that simvastatin affected the expression of Nrf2/MAPK signaling pathway in Gambusia affinis, and exhibited a time- and dose-dependent relationship. These findings underscored the complexity of oxidative stress regulation, which might be affected by either pre- or post-transcriptional control mechanisms. Moreover, MAPKs with sulfhydryl group are susceptible to ROS (48). This highlights the close association with the regulation of Nrf2. Therefore, the decrease in the presentation of MAPK and Nrf2 pathway genes after H₂O₂ challenge may have a connection with ROS accumulation, inhibition of MAPK signaling pathway, and the complex cross-regulatory mechanism between MAPK/Nrf2. We found that dietary supplementation with QG activated the Nrf2 pathway, partially restoring the Nrf2 mRNA expression to levels comparable to the control group. There were no discernible differences in relation to ERK and JNK gene expression. This suggested that QG may enhance cellular antioxidant defenses by activating Nrf2 signal pathway, whereas the involvement of MAPK signaling requires further investigation.

Ferroptosis is caused by disrupted iron homeostasis and enhanced lipid peroxidation. Under stress, cells often increase iron uptake while reducing iron storage, leading to an iron overload (49). Superfluous iron accretion is capable of facilitating the generation of ROS by means of the Fenton reaction, thereby inducing lipid peroxidation. ROS reacts with PUFA in cell membranes, resulting in membrane disruption, mitochondrial damage, and the commencement of ferroptosis. While some flavonoids such as quercetin have been shown to reduce ROS production through the Fenton reaction (50). Chen et al. (51) reported that LPS injection induced broiler intestinal inflammation and may through ferroptosis. Furthermore, the notion that flavonoids modulated the Nrf2/GPX4 axis to suppress ferroptosis is supported by a recent study, which demonstrated that 7,8-dihydroxyflavone (7,8-DHF) alleviated liver injury by promoting Nrf2 nuclear translocation and upregulating GPX4 expression (52). Our study found that dietary QG supplementation partly alleviated oxidative stress by regulating the Nrf2 signal pathway and ferroptosis-ralated genes (Nrf2 and TFR1). This accorded with the results obtained by Chen et al. (53), who documented similar effects that pterostilbene supplementation to some extent enhanced the mRNA expression level of TFH1 than diquat exposure, suggesting that QG may similarly mitigate H₂O₂-induced iron dysregulation. Intriguingly, this protective effect presents an contrast with the mechanism of action of quercetin in cancer cells, which inhibited the Nrf2 pathway to induce ferroptosis (54). This bidirectional regulation underscores the context-dependent nature of flavonoid activity. Furthermore, GSH and GPX4 are crucial components in the cellular antioxidant defense system. GSH plays the role of a reducing agent, whereas GPX4 is the primary peroxidase responsible (55). The synthesis of GSH is closely regulated by solute carrier family 7 member 11 (SLC7A11) (56). The addition of QG diminished the lipid peroxidation triggered by H₂O₂, yet it did not exert a marked influence on the mRNA expression level of SLC7A11 or GPX4. The precise mechanisms underlying QG’s protective effects, particularly in modulating Nrf2 pathways and ferroptosis, remain unclear and warrant further investigation.

Collectively, the promising results of this study highlight the potential of QG as a dietary antioxidant for broilers under oxidative stress. However, the current findings are limited by the reliance on mRNA data. Future research should include dose–response studies under various challenging conditions and protein-level analyses to fully elucidate the underlying mechanisms and optimize practical application. Besides, longer-term trials would be valuable to assess the sustainability of QG’s effects.

Conclusion

In conclusion, H₂O₂-elicited oxidative stress significantly impaired meat quality and mitochondrial function of breast muscle. Dietary supplementation with QG provided a protective efficacy by alleviating oxidative injury and lipid peroxidative effects, enhancing the antioxidant capacity and improving the meat quality of breast muscle. The protective effects of QG is likely mediated through the activation of the Nrf2 signal pathway and modulation of key ferroptosis-related genes. These findings provide a mechanistic basis for using QG as a novel dietary antioxidant and highlight its potential as a practical strategy to improve meat quality and animal health in sustainable poultry production.

Data availability statement

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

Ethics statement

The animal study was approved by Committee for Animal Care and Use of Hebei Agriculture University. The study was conducted in accordance with the local legislation and institutional requirements.

Author contributions

WH: Writing – original draft, Data curation, Methodology. HL: Methodology, Writing – original draft, Data curation. PZ: Investigation, Supervision, Writing – review & editing. SF: Writing – review & editing, Software, Formal analysis. YL: Writing – review & editing, Methodology, Investigation. FW: Writing – review & editing, Formal analysis. SH: Project administration, Writing – review & editing, Supervision, Conceptualization, Funding acquisition.

Funding

The author(s) declare that financial support was received for the research and/or publication of this article. This research received support from the Hebei Natural Science Foundation (Grant No. C2022204218), National Natural Science Foundation of China (Grant No. 32302787) and Research Project on Basic Research Funding for Provincial Universities in Hebei Province (Grant No. KY2024045).

Conflict of interest

PZ was employed by the Chenguang Biotechnology Group Co., Ltd.

The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Generative AI statement

The authors declare that no Gen AI was used in the creation of this manuscript.

Any alternative text (alt text) provided alongside figures in this article has been generated by Frontiers with the support of artificial intelligence and reasonable efforts have been made to ensure accuracy, including review by the authors wherever possible. If you identify any issues, please contact us.

Publisher’s note

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

Supplementary material

The Supplementary material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fvets.2025.1713728/full#supplementary-material

References

1. Lan, R, Wang, Y, Wang, H, and Zhang, J. Dietary chitosan oligosaccharide supplementation improves meat quality by improving antioxidant capacity and fiber characteristics in the thigh muscle of broilers. Antioxidants (Basel). (2024) 13:366. doi: 10.3390/ANTIOX13030366

PubMed Abstract | Crossref Full Text | Google Scholar

2. Xing, T, Gao, F, Tume, RK, Zhou, G, and Xu, X. Stress effects on meat quality: a mechanistic perspective. Compr Rev Food Sci Food Saf. (2019) 18:380–401. doi: 10.1111/1541-4337.12417

PubMed Abstract | Crossref Full Text | Google Scholar

3. Oke, OE, Akosile, OA, Oni, AI, Opowoye, IO, Ishola, CA, Adebiyi, JO, et al. Oxidative stress in poultry production. Poult Sci. (2024) 103:104003. doi: 10.1016/J.PSJ.2024.104003

PubMed Abstract | Crossref Full Text | Google Scholar

4. Sies, H, Berndt, C, and Jones, DP. Oxidative stress. Annu Rev Biochem. (2017) 86:715. doi: 10.1146/annurev-biochem-061516-045037

PubMed Abstract | Crossref Full Text | Google Scholar

5. Zha, P, Wei, L, Liu, W, Chen, Y, and Zhou, Y. Effects of dietary supplementation with chlorogenic acid on growth performance, antioxidant capacity, and hepatic inflammation in broiler chickens subjected to diquat-induced oxidative stress. Poult Sci. (2023) 102:102479. doi: 10.1016/J.PSJ.2023.102479

PubMed Abstract | Crossref Full Text | Google Scholar

6. Wang, Y, Zhou, X, Liu, M, Zang, H, Zhang, R, Yang, H, et al. Quality of chicken breast meat improved by dietary pterostilbene referring to up-regulated antioxidant capacity and enhanced protein structure. Food Chem. (2023) 405:134848. doi: 10.1016/J.FOODCHEM.2022.134848

Crossref Full Text | Google Scholar

7. Davies, MJ. The oxidative environment and protein damage. Biochim Biophys Acta. (2005) 1703:93. doi: 10.1016/j.bbapap.2004.08.007

PubMed Abstract | Crossref Full Text | Google Scholar

8. Lund, MN, Heinonen, M, Baron, CP, and Estévez, M. Protein oxidation in muscle foods: a review. Mol Nutr Food Res. (2011) 55:83. doi: 10.1002/mnfr.201000453

PubMed Abstract | Crossref Full Text | Google Scholar

9. Guo, Y, Balasubramanian, B, Zhao, ZH, and Liu, WC. Marine algal polysaccharides alleviate aflatoxin B1-induced bursa of Fabricius injury by regulating redox and apoptotic signaling pathway in broilers. Poult Sci. (2020) 100:844. doi: 10.1016/j.psj.2020.10.050

Crossref Full Text | Google Scholar

10. Chen, X, Zhang, L, Li, J, Gao, F, and Zhou, G. Hydrogen peroxide-induced change in meat quality of the breast muscle of broilers is mediated by ROS generation, apoptosis, and autophagy in the NF-κB signal pathway. J Agric Food Chem. (2017) 65:3986–94. doi: 10.1021/acs.jafc.7b01267

PubMed Abstract | Crossref Full Text | Google Scholar

11. Yan, Y, Chen, X, Huang, J, Huan, C, and Li, C. HO-induced oxidative stress impairs meat quality by inducing apoptosis and autophagy via ROS/NF-κB signaling pathway in broiler thigh muscle. Poult Sci. (2022) 101:101759. doi: 10.1016/J.PSJ.2022.101759

PubMed Abstract | Crossref Full Text | Google Scholar

12. Deng, C, Zou, H, Wu, Y, Lou, A, Liu, Y, Luo, J, et al. Dietary supplementation with quercetin: an ideal approach for improving meat quality and oxidative stability of broiler chickens. Poult Sci. (2024) 103:103789. doi: 10.1016/J.PSJ.2024.103789

PubMed Abstract | Crossref Full Text | Google Scholar

13. Qi, W, Qi, W, Xiong, D, and Long, M. Quercetin: its antioxidant mechanism, antibacterial properties and potential application in prevention and control of toxipathy. Molecules. (2022) 27:6545. doi: 10.3390/MOLECULES27196545

PubMed Abstract | Crossref Full Text | Google Scholar

14. Wu, F, Wang, H, Li, S, Wei, Z, Han, S, and Chen, B. Effects of dietary supplementation with quercetagetin on nutrient digestibility, intestinal morphology, immunity, and antioxidant capacity of broilers. Front Vet Sci. (2022) 9:1060140. doi: 10.3389/FVETS.2022.1060140

PubMed Abstract | Crossref Full Text | Google Scholar

15. Liang, H, Fan, D, Hu, W, Wu, F, Tan, K, Zhao, P, et al. Effects of quercetagetin on the growth performance, nutrient digestibility, slaughter performance, meat quality, and antioxidant capacity of broiler chickens. Anim Sci J. (2024) 95:e70008. doi: 10.1111/asj.70008

PubMed Abstract | Crossref Full Text | Google Scholar

16. Wu, F, Wang, F, Tang, Z, Yang, X, Liu, Y, Zhao, M, et al. Quercetagetin alleviates zearalenone-induced liver injury in rabbits through Keap1/Nrf2/ARE signaling pathway. Front Pharmacol. (2023) 14:1271384. doi: 10.3389/FPHAR.2023.1271384

PubMed Abstract | Crossref Full Text | Google Scholar

17. Dixon, SJ, Lemberg, KM, Lamprecht, MR, Skouta, R, Zaitsev, EM, Gleason, CE, et al. Ferroptosis: an iron-dependent form of nonapoptotic cell death. Cell. (2012) 149:1060. doi: 10.1016/j.cell.2012.03.042

PubMed Abstract | Crossref Full Text | Google Scholar

18. Yu, Y, Wang, G, Yin, X, Ge, C, and Liao, G. Effects of different cooking methods on free fatty acid profile, water-soluble compounds and flavor compounds in Chinese Piao chicken meat. Food Res Int. (2021) 149:110696. doi: 10.1016/J.FOODRES.2021.110696

PubMed Abstract | Crossref Full Text | Google Scholar

19. Farkhondeh, T, Mehrpour, O, Buhrmann, C, Pourbagher-Shahri, AM, Shakibaei, M, and Samarghandian, S. Organophosphorus compounds and MAPK signaling pathways. Int J Mol Sci. (2020) 21:4258. doi: 10.3390/ijms21124258

PubMed Abstract | Crossref Full Text | Google Scholar

20. Cobb-Vantress. Broiler Performance & Nutritition Supplement Cobb 500. Springdale, AR: Cobb-Vantress Inc. (2018).

Google Scholar

21. Pu, X, Liang, Y, Lian, J, Xu, M, Yong, Y, Zhang, H, et al. Effects of dietary dihydroartemisinin on growth performance, meat quality, and antioxidant capacity in broiler chickens. Poult Sci. (2025) 104:104523. doi: 10.1016/J.PSJ.2024.104523

PubMed Abstract | Crossref Full Text | Google Scholar

22. Animal Feed. Official methods of analysis of association of official analytical chemists. Gaithersburg, MD: AOAC International (2007).

Google Scholar

23. Oh, M, Kim, EK, Jeon, BT, Tang, Y, Kim, MS, Seong, HJ, et al. Chemical compositions, free amino acid contents and antioxidant activities of Hanwoo (Bos taurus coreanae) beef by cut. Meat Sci. (2016) 119:16–21. doi: 10.1016/j.meatsci.2016.04.016

PubMed Abstract | Crossref Full Text | Google Scholar

24. Dai, Z, Feng, M, Feng, C, Zhu, H, Chen, Z, Guo, B, et al. Effects of sex on meat quality traits, amino acid and fatty acid compositions, and plasma metabolome profiles in white king squabs. Poult Sci. (2024) 103:103524. doi: 10.1016/J.PSJ.2024.103524

PubMed Abstract | Crossref Full Text | Google Scholar

25. Boz, MA, Oz, F, Yamak, US, Sarica, M, and Cilavdaroglu, E. The carcass traits, carcass nutrient composition, amino acid, fatty acid, and cholesterol contents of local Turkish goose varieties reared in an extensive production system. Poult Sci. (2019) 98:3067. doi: 10.3382/ps/pez125

PubMed Abstract | Crossref Full Text | Google Scholar

26. Livak, KJ, and Schmittgen, TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2^-ΔΔCT method. Methods. (2001) 25:402. doi: 10.1006/meth.2001.1262

Crossref Full Text | Google Scholar

27. Xing, T, Chen, X, Li, J, Zhang, L, and Gao, F. Dietary taurine attenuates hydrogen peroxide-impaired growth performance and meat quality of broilers via modulating redox status and cell death signaling. J Anim Sci. (2021) 99:skab089. doi: 10.1093/JAS/SKAB089

PubMed Abstract | Crossref Full Text | Google Scholar

28. Chen, Y, Chen, Y, Zhang, H, and Wang, T. Pterostilbene as a protective antioxidant attenuates diquat-induced liver injury and oxidative stress in 21-day-old broiler chickens. Poult Sci. (2020) 99:3158–67. doi: 10.1016/j.psj.2020.01.021

PubMed Abstract | Crossref Full Text | Google Scholar

29. Zheng, XC, Wu, QJ, Song, ZH, Zhang, H, Zhang, JF, Zhang, LL, et al. Effects of Oridonin on growth performance and oxidative stress in broilers challenged with lipopolysaccharide. Poult Sci. (2016) 95:2281–9. doi: 10.3382/ps/pew161

PubMed Abstract | Crossref Full Text | Google Scholar

30. Yang, S, Huo, M, Su, Z, Wang, F, Zhang, Y, Zhong, C, et al. The impact of dietary supplementation of Quercetagetin on growth, antioxidant capacity, and gut microbiota of diquat-challenged broilers. Front Microbiol. (2024) 15:1453145. doi: 10.3389/FMICB.2024.1453145

PubMed Abstract | Crossref Full Text | Google Scholar

31. Zhang, S, and Kim, IH. Effect of quercetin (flavonoid) supplementation on growth performance, meat stability, and immunological response in broiler chickens. Livest Sci. (2020) 242:104286. doi: 10.1016/j.livsci.2020.104286

Crossref Full Text | Google Scholar

32. Abdel-Latif, MA, Elbestawy, AR, El-Far, AH, Noreldin, AE, Emam, M, Baty, RS, et al. Quercetin dietary supplementation advances growth performance, gut microbiota, and intestinal mRNA expression genes in broiler chickens. Animals (Basel). (2021) 11:2302. doi: 10.3390/ANI11082302

PubMed Abstract | Crossref Full Text | Google Scholar

33. Baéza, E, Guillier, L, and Petracci, M. Review: production factors affecting poultry carcass and meat quality attributes. Animal. (2021):1:100331. doi: 10.1016/J.ANIMAL.2021.100331

Crossref Full Text | Google Scholar

34. Goliomytis, M, Tsoureki, D, Simitzis, PE, Charismiadou, MA, Hager-Theodorides, AL, and Deligeorgis, SG. The effects of quercetin dietary supplementation on broiler growth performance, meat quality, and oxidative stability. Poult Sci. (2014) 93:1957–62. doi: 10.3382/ps.2013-03585

PubMed Abstract | Crossref Full Text | Google Scholar

35. Wang, T, Feng, X, Li, L, Luo, J, Liu, X, Zheng, J, et al. Effects of quercetin on tenderness, apoptotic and autophagy signalling in chickens during post-mortem ageing. Food Chem. (2022) 383:132409. doi: 10.1016/J.FOODCHEM.2022.132409

PubMed Abstract | Crossref Full Text | Google Scholar

36. Ma, X, Yu, M, Liu, Z, Deng, D, Cui, Y, Tian, Z, et al. Effect of amino acids and their derivatives on meat quality of finishing pigs. J Food Sci Technol. (2020) 57:404–12. doi: 10.1007/s13197-019-04077-x

PubMed Abstract | Crossref Full Text | Google Scholar

37. Min, B, Nam, KC, Cordray, J, and Ahn, DU. Endogenous factors affecting oxidative stability of beef loin, pork loin, and chicken breast and thigh meats. J Food Sci. (2008) 73:C439. doi: 10.1111/j.1750-3841.2008.00805.x

Crossref Full Text | Google Scholar

38. Chen, Z, Xing, T, Li, J, Zhang, L, Jiang, Y, and Gao, F. Hydrogen peroxide-induced oxidative stress impairs redox status and damages aerobic metabolism of breast muscle in broilers. Poult Sci. (2020) 100:918. doi: 10.1016/J.PSJ.2020.11.029

Crossref Full Text | Google Scholar

39. Jia, H, Zhang, Y, Si, X, Jin, Y, Jiang, D, Dai, Z, et al. Quercetin alleviates oxidative damage by activating nuclear factor erythroid 2-related factor 2 signaling in porcine enterocytes. Nutrients. (2021) 13:375. doi: 10.3390/NU13020375

PubMed Abstract | Crossref Full Text | Google Scholar

40. Mustafa, T, Khan, I, Iqbal, H, Usman, S, Naeem, N, Faizi, S, et al. Rutin and quercetagetin enhance the regeneration potential of young and aging bone marrow-derived mesenchymal stem cells in the rat infarcted myocardium. Mol Cell Biochem. (2022) 478:1759. doi: 10.1007/S11010-022-04628-5

PubMed Abstract | Crossref Full Text | Google Scholar

41. Sun, L, Xu, G, Dong, Y, Li, M, Yang, L, and Lu, W. Quercetin protects against lipopolysaccharide-induced intestinal oxidative stress in broiler chickens through activation of Nrf2 pathway. Molecules. (2020) 25:1053. doi: 10.3390/molecules25051053

PubMed Abstract | Crossref Full Text | Google Scholar

42. Bulut, O, and Yilmaz, MD. Concise synthesis of quercetagetin (3, 3′, 4′, 5, 6, 7-hexahydroxyflavone) with antioxidant and antibacterial activities. Results Chem. (2021) 3:100255. doi: 10.1016/j.rechem.2021.100255

Crossref Full Text | Google Scholar

43. Kannan, K, and Jain, SK. Oxidative stress and apoptosis. Pathophysiology. (2000) 7:153. doi: 10.1016/s0928-4680(00)00053-5

PubMed Abstract | Crossref Full Text | Google Scholar

44. Aon, MA, Cortassa, S, Marbán, E, and O'Rourke, B. Synchronized whole cell oscillations in mitochondrial metabolism triggered by a local release of reactive oxygen species in cardiac myocytes. J Biol Chem. (2003) 278:44735–44. doi: 10.1074/jbc.M302673200

PubMed Abstract | Crossref Full Text | Google Scholar

45. Chen, Y, Zhang, H, Ji, S, Jia, P, Chen, Y, Li, Y, et al. Resveratrol and its derivative pterostilbene attenuate oxidative stress-induced intestinal injury by improving mitochondrial redox homeostasis and function via SIRT1 signaling. Free Radic Biol Med. (2021) 177:1–14. doi: 10.1016/J.FREERADBIOMED.2021.10.011

PubMed Abstract | Crossref Full Text | Google Scholar

46. He, F, Antonucci, L, and Karin, M. NRF2 as a regulator of cell metabolism and inflammation in cancer. Carcinogenesis. (2020) 41:405. doi: 10.1093/carcin/bgaa039

PubMed Abstract | Crossref Full Text | Google Scholar

47. Bao, S, Lin, J, Xie, M, Wang, C, and Nie, X. Simvastatin affects Nrf2/MAPK signaling pathway and hepatic histological structure change in Gambusia affinis. Chemosphere. (2020) 269:128725. doi: 10.1016/j.chemosphere.2020.128725

Crossref Full Text | Google Scholar

48. Wu, MF, Huang, YH, Chiu, LY, Cherng, SH, Sheu, GT, and Yang, TY. Curcumin induces apoptosis of chemoresistant lung cancer cells via ROS-regulated p38 MAPK phosphorylation. Int J Mol Sci. (2022) 23:8248. doi: 10.3390/ijms23158248

PubMed Abstract | Crossref Full Text | Google Scholar

49. Imai, H, Matsuoka, M, Kumagai, T, Sakamoto, T, and Koumura, T. Lipid peroxidation-dependent cell death regulated by GPx4 and ferroptosis. Curr Top Microbiol Immunol. (2017) 403:143–70. doi: 10.1007/82_2016_508

PubMed Abstract | Crossref Full Text | Google Scholar

50. Kim, GN, and Jang, HD. Protective mechanism of quercetin and rutin using glutathione metabolism on H₂O₂-induced oxidative stress in HepG2 cells. Ann N Y Acad Sci. (2009) 1171:530–7. doi: 10.1111/j.1749-6632.2009.04690.x

PubMed Abstract | Crossref Full Text | Google Scholar

51. Chen, Y, Liu, C, and Lin, Y. Involvement of ferroptosis in lipopolysaccharide-induced intestinal inflammation in broilers. Poult Sci. (2025) 104:105512. doi: 10.1016/j.psj.2025.105512

PubMed Abstract | Crossref Full Text | Google Scholar

52. Pei, XT, Mu, R, Liu, SY, Xiong, L, Wang, XL, Yao, Q, et al. 7,8-Dihydroxyflavone protects acetaminophen induced liver injury through activating PI3K/Akt/NRF2/GPX4 mediated ferroptosis suppression. Free Radic Biol Med. (2025) 242:275. doi: 10.1016/J.FREERADBIOMED.2025.10.297

Crossref Full Text | Google Scholar

53. Chen, Y, Zhang, H, Li, Y, and Wang, T. Pterostilbene confers protection against diquat-induced intestinal damage with potential regulation of redox status and ferroptosis in broiler chickens. Oxidative Med Cell Longev. (2023) 2023:8258354. doi: 10.1155/2023/8258354

PubMed Abstract | Crossref Full Text | Google Scholar

54. Ding, LX, Dang, SW, Sun, MJ, Zhou, DZ, Sun, YY, Li, EC, et al. Quercetin induces ferroptosis in gastric cancer cells by targeting SLC1A5 and regulating the p-Camk2/p-DRP1 and NRF2/GPX4 axes. Free Radic Biol Med. (2024) 213:150. doi: 10.1016/J.FREERADBIOMED.2024.01.002

PubMed Abstract | Crossref Full Text | Google Scholar

55. Ursini, F, and Maiorino, M. Lipid peroxidation and ferroptosis: the role of GSH and GPx4. Free Radic Biol Med. (2020) 152:175. doi: 10.1016/j.freeradbiomed.2020.02.027

PubMed Abstract | Crossref Full Text | Google Scholar

56. Gao, W, Zhang, T, and Wu, H. Emerging pathological engagement of ferroptosis in gut diseases. Oxidative Med Cell Longev. (2021) 2021:4246255. doi: 10.1155/2021/4246255

PubMed Abstract | Crossref Full Text | Google Scholar