Ferroptosis bridges early-life oxidative stress and lifetime meat quality defects in broilers

Abstract

This study investigated whether oxidized oil impairs broiler meat quality by triggering ferroptosis and evaluated the synergistic protective effect of composite antioxidants compared with individual antioxidants. The study integrated in vivo broiler trials with in vitro cell models. First, the effects of normal oil and oxidized oil on meat quality were compared through a broiler feeding trial, and the relief effects of BHT (54 g/ton), EQ (90 g/ton), and their complexes BHT+EQ (BE; 18 and 15 g/ton) and BHT+EQ+citric acid blend (BEC; 18, 15, and 9 g/ton) were evaluated using the oxidized oil group as a positive control. Subsequently, an oxidative damage model was constructed using in ovo injection technology, and its muscle satellite cells were isolated to evaluate their potential for proliferation and differentiation, finally focusing on the Nrf2-GPX4 axis to reveal the molecular mechanism by which composite antioxidants alleviate the meat quality deterioration caused by oxidized oil. The results found that, compared with normal oil, oxidized oil significantly impaired the meat quality of broilers, manifested as a decrease pH_24h and redness (a*), while drip loss and cooking loss increased, antioxidant defense capacity was damaged, and myoglobin was unstable (P < 0.05). Antioxidant intervention can significantly alleviate the above negative effects (P < 0.05), among which the protective effect of the BEC group was the most outstanding, superior to the BE group and individual antioxidant treatments. At the cellular level, compared with normal oil, oxidized oil inhibited the abundance of Pax7 and MyHC and induced mitochondrial dysfunction (P < 0.05). The synergized BE and BEC groups effectively cleared reactive oxygen species and inhibited GPX4-mediated ferroptosis by activating the Nrf2-GPX4 signaling axis and upregulating cytoprotective genes such as NQO1 and HO-1, thereby maintaining cellular redox homeostasis. In summary, oxidized oils cause meat quality deterioration by triggering Nrf2-GPX4-mediated ferroptosis, the BEC composite strategy confirmed in this study provides an important theoretical basis for the precise regulation of meat quality and the optimization of antioxidant programs in poultry production.

Graphical abstract

Introduction

In modern intensive poultry production, dietary lipids serve as essential high energy sources to optimize broiler growth and feed efficiency. Nevertheless, their inherent abundance of polyunsaturated fatty acids renders them highly vulnerable to oxidative rancidity, which is a process catalyzed by high temperatures and metallic ions during processing and storage (Chemat et al., 2023). This oxidative degradation generates a cascade of toxic secondary metabolites, including free radicals, hydroperoxides, and reactive aldehydes such as malondialdehyde (MDA) (Zhao et al., 2026). Upon ingestion by broilers, these harmful products trigger systemic oxidative stress that not only significantly impedes growth performance but also leads to a marked impairment of meat quality. Such degradation is typically characterized by a rapid post-mortem decline in muscle pH, increased drip loss, diminished meat redness (a* values), and elevated lipid peroxidation in muscle tissues (Zhang et al., 2011). Therefore, the oxidative instability of dietary lipids has become one of the major limiting factors in optimizing the economic efficiency and product quality of the poultry industry.

To mitigate the adverse effects of lipid oxidation, synthetic antioxidants such as butylated hydroxytoluene (BHT) and ethoxyquin (EQ) are commonly incorporated into poultry diets (Xiao et al., 2024a). However, the prolonged and exclusive reliance on a single antioxidant is frequently constrained by regulatory dosage limits, concerns over potential tissue residues, and a narrow spectrum of radical-scavenging activity (Shahidi et al., 2005; Szerlauth et al., 2023). Consequently, the application of complex antioxidants has gained significant attention in recent years. The efficacy of these formulations stems from the synergistic effects among diverse antioxidative components, enabling a more robust and comprehensive defense against oxidative degradation (Yatheshappa et al., 2025). Previous studies have demonstrated that the combination of BHT and EQ effectively targets multiple stages of the oxidative cascade, thereby enhancing total antioxidant capacity through the multi-site interception of free radicals (Gao et al., 2025; Ren et al., 2025). Furthermore, citric acid serves as a potent metal chelator that can effectively sequester catalytic factors such as ferrous (Fe²⁺) and cupric(Cu²⁺)ions (Lin et al., 2024). By neutralizing these pro-oxidative metals, it suppresses the lipid peroxidative chain reaction at its initiation stage. While BHT and EQ combinations effectively improve the water-holding capacity (WHC) of broiler breast and thigh muscles (Lauridsen et al., 1994), there remains a lack of comprehensive research evaluating the comparative advantages of antioxidant blends over single agents in preventing meat quality deterioration; furthermore, the intricate molecular mechanisms involved have yet to be fully elucidated.

The biological mechanisms by which oxidized oils impair meat quality are complex; conventional wisdom has primarily focused on protein oxidation and cellular apoptosis triggered by oxidative stress (Dragoev, 2024). However, ferroptosis, a recently identified form of regulated cell death, offers a compelling new perspective for unraveling these complex mechanisms (Zheng and Conrad, 2025). Distinct from apoptosis and necrosis, ferroptosis is characterized by the iron-dependent accumulation of lethal lipid peroxides (Bucarey et al., 2026). Recent research emphasizes that muscle oxidative stability is "programmed" during the critical embryonic window, a period when intensive lipid mobilization for avian development renders embryos highly vulnerable to redox imbalances (Alfian et al., 2025; Park and Kim, 2025). Within this sensitive timeframe, oxidative insults can disrupt mitochondrial integrity and trigger ferroptosis in muscle satellite cells, thereby arresting myogenic development and impairing post-mortem quality (Chen et al., 2023; Zeidan et al., 2026). Given that the core pathological hallmark of oxidized oil ingestion is lipid oxidative stress, coupled with the fact that muscle tissue is an abundant reservoir of heme iron (Ru et al., 2025), ferroptosis emerges as a plausible key link through which oxidized lipids compromise muscle cell development and induce tissue injury. Consequently, investigating whether oxidized oils impair broiler muscle quality by triggering ferroptosis in satellite cells provides a novel and robust framework for understanding meat quality regulation.

The Nrf2-GPX4 signaling axis serves as a master regulator in the defense against ferroptosis during muscle development (Ji et al., 2026). Nuclear factor erythroid 2-related factor 2 (Nrf2) is a core transcription factor that regulates antioxidant stress responses during development (Tripathi et al., 2024); its downstream target gene, glutathione peroxidase 4 (GPX4), is the most critical ferroptosis inhibitor known to date, specifically reducing lipid hydroperoxides into harmless alcohols (Xie et al., 2023). Studies have found that oxidative stress may trigger ferroptosis by inhibiting Nrf2 activity and downregulating GPX4 expression, thereby disrupting intracellular redox homeostasis (Ngo and Duennwald, 2022; Nie et al., 2026). Therefore, investigating whether compound antioxidants—particularly formulations containing metal chelators—can block the ferroptosis process by maintaining the stability of the Nrf2-GPX4 signaling axis holds significant theoretical value.

In summary, this study hypothesizes that compound antioxidants can alleviate oxidized oil-mediated ferroptosis in muscle satellite cells by activating the Nrf2-GPX4 signaling axis, thereby improving muscle quality in broilers. To verify this hypothesis, the study validates the overall phenotype through broiler feeding trials, observes muscle development using an in ovo injection model, and further elucidates the underlying molecular mechanisms via in vitro satellite cell experiments. This research aims to provide a scientific basis and technical support for mitigating the hazards of oxidized fats and enhancing the quality of poultry products.

Materials and methods

Animal experimental design and dietary treatments

All animal procedures were approved by the Institutional Animal Care and Use Committee of the Laboratory Animal Center at China Agricultural University (Approval No. AW52704202-1-1).

In experiment 1, a total of 144 one-day-old male Arbor Acres Plus broilers were randomly assigned to two dietary treatments (six replicates of 12 birds each): a control diet containing fresh soybean oil (CON) and a diet containing oxidized soybean oil (OXI). Fresh oil was stored airtight in cool, dark conditions. The oxidized oil was prepared by aerobically exposing refined, antioxidant-free soybean oil in open containers to ambient sunlight (30–40 °C; 1000 Lux) for 60 days. Detailed physicochemical properties and oxidation indices of the oils are provided in Supplementary Table 4.

In experiment 2, a total of 360 one-day-old male Arbor Acres Plus broilers were randomly distributed into five dietary treatments (six replicates of 12 birds each). A basal diet formulated with the oxidized soybean oil served as the positive control (Con). Four experimental diets were prepared by supplementing this basal diet with: (1) 54 g/ton butylated hydroxytoluene (BHT), (2) 90 g/ton ethoxyquin (EQ), (3) a combination of 18 g/ton BHT and 15 g/ton EQ (BE), (4) a mixture of 18 g/ton BHT, 15 g/ton EQ, and 9 g/ton citric acid (BEC).

The 42-day trial provided all diets ad libitum, meeting the nutrient requirements recommended by Aviagen (2022). Dietary compositions for the starter (days 0–21) and grower (days 22–42) phases are detailed in Supplementary Table 1. The starter diet was provided as crumbled pellets and the grower diet as intact pellets. Birds had free access to water via nipple drinkers, with all husbandry practices following Arbor Acres management guidelines (Aviagen, 2022).

Sample collection

On day 42, broilers with body weights close to the pen average were selected, electrically stunned, and exsanguinated. The left breast muscle was immediately excised, snap-frozen in liquid nitrogen, and stored at −80 °C for molecular analysis. An adjacent tissue segment (approx. 2 mm³) was fixed in 4% paraformaldehyde for histological examination. The right breast muscle was harvested at 45 min post-mortem for initial color measurement. About 2.5 cm-thick section was excised from a consistent anatomical site, weighed, placed on a polystyrene tray, and overwrapped with oxygen-permeable PVC film. This sample, along with the remaining right breast muscle, was stored in darkness at 4 °C. Color and drip loss measurements were repeated after 24 h of storage.

Meat quality measurements

Color and pH

Instrumental color measurement and pH of the breast muscle was conducted following a previously described method (Alnagdy et al., 2024).

Drip and Cooking Loss

Drip loss was assessed following Rinwi method (Rinwi et al., 2024). Cooking loss was determined according to the procedure described by Roobab et al. (2024).

Biochemical assays in breast muscle

Lipid oxidation was assessed by measuring thiobarbituric acid reactive substances (TBARS) according to Ismail et al. (2024). Protein oxidation was determined via the 2,4-dinitrophenylhydrazine (DNPH) method for carbonyl group quantification (Bošković Cabrol et al., 2024), while the total sulfhydryl content was measured using Ellman's reagent (DTNB) as described by Wang (Wang, et al., 2025). The relative proportions of myoglobin derivatives—oxymyoglobin (MbO₂), deoxymyoglobin (DMb), and metmyoglobin (MMb)—were analyzed by reflectance spectrophotometry following Piao,et al. (2025). Additionally, the activities of total superoxide dismutase (T-SOD) and total antioxidant capacity (T-AOC) were determined using commercially available assay kits (Wang et al., 2023).

Emulsion preparation and in ovo injection for satellite cell model establishment

In ovo injection and satellite cell model establishment

To establish a model for investigating the embryonic origin of oxidative damage and the efficacy of early interventions, fertilized Arbor Acres Plus broiler eggs (n = 36) were randomly assigned to six treatment groups. On embryonic day 18 (E18), a 100-µL aliquot of sterile emulsion was injected into the amniotic fluid of each egg. Following injection, the entry hole was sealed with sterile paraffin, and the eggs were incubated under standard conditions until hatching. Primary satellite cells were subsequently isolated from the pectoralis major muscle of 1-day-old chicks from each group, as described by Danoviz and Yablonka-Reuveni (2011). Then, the cell pellet was resuspended in growth medium (DMEM/F12 supplemented with 20% fetal bovine serum, 10% horse serum, 2.5 ng/mL bFGF, and 1% penicillin-streptomycin) and seeded into collagen-coated culture flasks. After 24 h of culture at 37 °C in 5% CO₂, the adherent cells were cultured to 80–90% confluence and passaged using 0.25% trypsin-EDTA. Cells between passages 2–4 were used for experiments, with viability assessed by trypan blue exclusion and satellite cell identity confirmed by Pax7 immunocytochemical detection.

Emulsion preparation

Treatment emulsions were prepared by blending the respective oils with specific antioxidants (BHT, EQ, and Citric Acid) according to the experimental group ratios. The experimental treatments consisted of: (1) fresh soybean oil (CON); (2) oxidized soybean oil (OXI); (3) OXI supplemented with BHT (0.18 µg/mL; OXI+BHT); (4) OXI supplemented with EQ (0.66 µg/mL; OXI+EQ); (5) OXI supplemented with a BHT and EQ combination (0.05 µg/mL BHT + 0.06 µg/mL EQ; OXI+BE); and (6) OXI supplemented with a ternary combination of BHT, EQ, and citric acid (0.05 µg/mL BHT + 0.06 µg/mL EQ + 0.03 µg/mL CA; OXI+BEC). These mixtures were subjected to ultrasonic homogenization for 4 h to ensure uniform dispersion and stable emulsification. Prior to amniotic injection, the resulting emulsions were passed through a 0.45-µm polyethersulfone membrane filter (Millipore Sigma, Burlington, MA) to ensure sterility.

Functional assays in satellite cells

Viability

Satellite cells were seeded in 96-well plates at a density of 5 × 10³ cells per well in 100 μL complete growth medium and cultured for 24 h at 37 °C to allow attachment. Cell viability of satellite cells was determined using the Cell Counting Kit-8 (CCK-8) assay, according to the manufacturer's instructions. The absorbance was measured at 450 nm using a microplate reader (BioTek Synergy H1, Winooski, VT).

Proliferation

Satellite cells were seeded in 6-well plates at a density of 1 × 10⁵ cells per well and cultured in complete growth medium for 24 h at 37 °C. Cell proliferation was assessed via 5-ethynyl-2′-deoxyuridine (EdU) incorporation using the Click-iT EdU Alexa Fluor 488 Flow Cytometry Assay Kit (Thermo Fisher Scientific, Waltham, MA), following the manufacturer’s protocols. Flow cytometric analysis of EdU incorporation was conducted as previously described by Andersen et al. (2013).

Immunofluorescence

Satellite cells were seeded in 12-well plates at a density of 5 × 10⁵ cells/well in complete growth medium and incubated at 37 °C for 24 h to facilitate attachment. To characterize cell identity and myogenic differentiation, immunofluorescence staining for Pax7 and Myosin Heavy Chain (MyHC) was performed, respectively, following established protocols (Andersen et al., 2013; Feng et al., 2018). Detailed information on the primary and secondary antibodies used is provided in Supplementary Table S3. Nuclei were visualized by counterstaining with DAPI (Thermo Fisher Scientific, Waltham, MA). Fluorescence images were observed and acquired using an inverted fluorescence microscope (Leica DMi8, Leica Microsystems, Wetzlar, Germany).

Intracellular superoxide anion levels by dihydroethidium (DHE) staining

Intracellular superoxide anion levels in satellite cells were evaluated using dihydroethidium (DHE) staining, following the procedure described by An et al. (2022). Fluorescence images were observed and acquired using an inverted fluorescence microscope (Eclipse Ti2, Nikon, Tokyo, Japan).

Mitochondrial Function

Satellite cells isolated from breast muscle were seeded at a density of 2 × 10⁴ cells/well in 24-well plates and cultured for 24 h. Mitochondrial membrane potential was assessed with the JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolyl-carbocyanine iodide) probe, following the method described by Liu et al., (2024). For fluorescence microscopy, cells were observed under a fluorescence microscope (Nikon Eclipse Ti2, Nikon Corp., Tokyo, Japan).

ATP content

Satellite cells isolated from breast muscle were seeded at 5 × 10⁵ cells/well in 6-well plates and cultured for 24 h. Intracellular ATP levels were determined using a ATP assay kit (S0026, Beyotime, Beijing) according to the manufacturer's instructions. The luminescence was measured using a multimode microplate reader (BioTek Synergy H1, Winooski, VT). The ATP content was calculated based on the standard curve and normalized to the protein concentration determined by the BCA method. Results were expressed as nmol ATP per mg protein.

Molecular analyses

Western blotting

Quantitative analysis of protein expression was performed by western blotting according to the method of Sule et al. (2023). Bands were visualized by ECL and quantified using ImageJ. The antibodies were listed in Supplementary Table 3.

qRT-PCR

Total RNA extraction, cDNA synthesis, and gene expression analysis via qRT-PCR were conducted following previously described methods (Xiao et al., 2024b). The gene sequences were provided in Supplementary Table 2.

Molecular docking

The 3D structure of broiler GPX4 (NP_001333378.1) was predicted using AlphaFold 2. Molecular docking of BHT, EQ, and citric acid (ligand structures from PubChem) with GPX4 was performed using AutoDock Vina.

Treatment protocols of GPX4 intervention

To evaluate the direct effects and protective mechanisms, satellite cells isolated from chicks hatched from eggs injected in ovo with oxidized oil (as described in section 2.5) were subjected to in vitro treatments. For activation GPX4 intervention, the OXI medium was supplemented with the Acetylcysteine.

NCI677397 (10 μM, HY-176557, MedChemExpress, Monmouth Junction, NJ) is a small-molecule compound that selectively induces ferroptosis by directly binding to and inactivating glutathione peroxidase 4 (GPX4). Acetylcysteine (N-acetyl-l-cysteine, NAC, S1623, Selleck, 2 μM), a reactive oxygen species (ROS) inhibitor, was used to counteract the activity of GPX4 inhibitors. All treatments were applied for 24 hours after cells reached 90% confluence, followed by a medium change.

In addition, to specifically investigate the necessity of glutathione peroxidase 4 (GPX4) in the observed protection, a subset of satellite cells was genetically modified to stably knockdown GPX4 (as detailed in Section 2.9) prior to the aforementioned treatments.

shRNA-mediated gene knockdown and assessment of cellular phospholipid hydroperoxides and labile iron pool

Gene knockdown using shRNA in satellite cells

To establish a stable GPX4-knockdown model in satellite cells, a short hairpin RNA (shRNA) targeting the chicken GPX4 gene (NCBI Accession Number: NM_001031215.2) was designed using the BLOCK-iT™ RNAi Designer online tool. The sense strand sequence of the selected shRNA was: 5‘- CCGCTCGAGGAGTTCGACATGTTCAGCAA-3′. A commercially obtained scrambled non-targeting shRNA (shNC) served as a negative control. Oligonucleotides encoding the shRNA were cloned into the pLKO.1-puro lentiviral vector (Addgene plasmid #8453) between the AgeI and EcoRI restriction sites, and all constructed plasmids were verified by Sanger sequencing.

Lentiviral particles were produced by co-transfecting the packaging plasmids (psPAX2 and pMD2.G) with the transfer plasmid (pLKO.1-shGPX4 or pLKO.1-shNC) into HEK-293T cells using polyethylenimine (PEI). The viral supernatant was collected, concentrated, and used to transduce primary broiler satellite cells. Stable cell pools were selected with puromycin (2 µg/mL) for 72 hours. The knockdown efficiency of GPX4 was confirmed at both the mRNA and protein levels by quantitative real-time PCR (qRT-PCR) and Western blot analysis, respectively. This stable GPX4-knockdown cell line was subsequently utilized in functional assays to investigate the pivotal role of GPX4 in regulating ferroptosis and myogenesis.

To definitively establish GPX4-dependency, satellite cells were isolated from chicks hatched from eggs injected in ovo with none additive (NC), oxidized oil (OXI) at Embryonic day 18 (E18). These primary cells, carrying a history of embryonic oxidative insult, were then subjected to in vitro gene knockdown and treatment assays, including the following key groups: None additive (NC), NC + OXI, NC + OXI + BEC, shGPX4, shGPX4 + OXI, shGPX4 + OXI + BEC, shGPX4 + BEC, and Fer-1 + OXI. For in vitro treatments, the BEC combination (0.05 µg/mL BHT + 0.06 µg/mL EQ + 0.03 µg/mL CA) and the ferroptosis inhibitor Ferrostatin-1 (2 μM Fer-1; Cat# HY-100579, MedChemExpress, Monmouth Junction, NJ) were supplemented directly into the culture medium. All concentrations were maintained as final working concentrations for the duration of the experiments.

Detection of cellular phospholipid hydroperoxides (PHOOHs)

Cellular phospholipid hydroperoxides (PHOOHs) were detected using the fluorescent probe Liperfluo (Dojindo Molecular Technologies, Cat# L248, Japan). The satellite cells were incubated with the probe according to the manufacturer’s protocols.

Measurement of the Labile Iron Pool (LIP)

The intracellular labile iron pool (LIP), which comprises redox-active Fe²⁺ ions essential for the execution of ferroptosis, was quantified in satellite cells using the Fe²⁺-sensitive fluorescent probe FerroOrange (Dojindo Molecular Technologies, Kumamoto, Japan). The satellite cells were incubated with the probe according to the manufacturer’s protocols.

Statistical analysis

All experimental data were expressed as mean ± standard deviation (SD) from at least three independent biological replicates. Comparisons among multiple groups were performed by one-way ANOVA followed by Tukey's test, while a student’s t-test was used for two-group comparisons. Statistical significance was set at p < 0.05. Analyses used SPSS 26.0, and graphs were prepared with GraphPad Prism 10.0.

Result

Oxidized oil compromises breast oxidative damage and myoglobin redox deterioration

Compared to the fresh oil group, breast meat in the oxidized oil group exhibited significantly lower pH_45min, pH_24h, redness (a*₂₄h), and yellowness (b*₂₄h) (P < 0.05; Table 1), higher drip loss (p = 0.012) and cooking loss (p = 0.034). Visual assessment revealed a paler appearance in the oxidized oil group (Fig. 1A), which was accompanied by a marked reduction in oxymyoglobin and deoxymyoglobin, concurrent with a significant accumulation of metmyoglobin (Fig. 1B, p < 0.05). Furthermore, the oxidized oil group exhibited significantly reduced T-SOD and T-AOC activities, concurrent with elevated concentrations of TBARS and protein carbonyls and a depletion of total sulfhydryl groups (Fig. 1C–G; P < 0.05). Meanwhile, Metmyoglobin content was strongly and negatively correlated not only with redness (a*₂₄h, r = −0.954; Fig. 1H) but also with yellowness (b*) at 45 min and 24 h post-mortem. In contrast, no meaningful correlation was observed with lightness (L* value).

Table 1.

Effects of oxidized oil on breast muscle quality of 42-day-old broilers.

Meat quality	CON	OXI	p-value
pH45 min	6.12 ± 0.12	5.95 ± 0.12	0.050
pH24 h	5.87 ± 0.14	5.52 ± 0.10	0.005
L*45 min	54.57 ± 1.62	54.32 ± 0.64	0.762
L*24 h	53.17 ± 1.38	53.58 ± 2.32	0.868
a*45min	4.36 ± 1.26	4.16 ± 0.41	0.952
a*24 h	4.20 ± 0.25	2.01 ± 0.12	0.021
b*45 min	−2.41 ± 0.62	−2.97 ± 0.53	0.813
b*24 h	4.82 ± 2.00	3.57 ± 0.73	0.029
Drop loss, %	2.52 ± 0.09	3.89 ± 0.09	0.012
Cooking loss, %	10.37 ± 2.14	25.92 ± 1.59	0.034

Note: CON: Fresh Oil group; OXI: Oxidized oil group. Data represent the trend of changes in oxidized oil compared to the fresh oil group. L*, a*, and b* values correspond to lightness, redness, and yellowness of meat, respectively, measured at 45 min and 24 h post-mortem. Drip loss and cooking loss are expressed as percentage increases relative to the control. n = 6.

Fig. 1.

Oxidized Oil leads to deteriorated meat quality. (A) Visual appearance of breast meat. (B) Oxymyoglobin, Deoxy-myoglobin, and Metmyoglobin content (μmol/mg·protein). (C, D) Total superoxide dismutase (T-SOD) and Total antioxidant capacity (T-AOC) activity (U/mg·protein). (E) TBARS value (mg MDA/kg muscle). (F, G) carbonyl and total sulfhydryl content (nmol/mg· protein). (H) Correlation Between Metmyoglobin Accumulation and Color Parameters. Abbreviations: CON: Fresh Oil group; OXI: Oxidation group. *means p < 0.05, **means p < 0.01, ***means p < 0.001.n = 6.

Antioxidants improve meat quality and physicochemical stability

Meat quality was significantly improved in all antioxidant groups compared with the control (Con) (Table 2; P < 0.05). Both pH_{45 min} and pH_{24 h} were significantly enhanced. pH_{45 min} followed the order of BEC > BE/BHT > EQ, with no significant difference between BE and BHT. pH_{24 h} followed BEC > BE > BHT/EQ, with no difference between BHT and EQ. L*_{45 min} followed the trend of BHT/EQ > BE > BEC, with no significant difference between BEC and BHT. No significant inter-group variation was observed for L*_{24 h}. a*_{45 min} followed the sequence of BEC > EQ > BHT, with no significant differences between BEC and BE, or between BE and EQ. For a*_{24 h}, the order shifted to BE/EQ/BHT > BEC, with no significant differences among the former three. b*_{45 min} was significantly higher in all treated groups than the Con, with no inter-group differences. For b*_{24 h}, BEC, BE, and EQ were significantly superior to BHT, while these three groups were comparable to each other. All treatments outperformed the Con group. Both drip and cooking loss followed an ascending trend of BHT/EQ > BE > BEC, where no significant difference was observed between BHT and EQ for cooking loss.

Table 2.

Effects of antioxidants on breast muscle quality parameters of 42-day-old broilers induced by oxidized oil.

Meat quality	Con	BHT	EQ	BE	BEC	P value
pH45 min	5.60 ± 0.09d	5.82 ± 0.17c	6.09 ± 0.08b	6.02 ± 0.18b	6.35 ± 0.23a	<0.001
pH24 h	5.28 ± 0.06d	5.40 ± 0.14c	5.46 ± 0.08c	5.82 ± 0.09b	6.24 ± 0.08a	<0.001
L*45 min	50.81 ± 0.51c	51.31 ± 1.92c	51.94 ± 1.28c	52.97 ± 1.43b	53.97 ± 1.42a	0.027
L*24 h	52.25 ± 3.21	51.86 ± 2.03	52.76 ± 1.63	51.91 ± 2.96	50.73 ± 2.41	0.895
a*45 min	4.20 ± 0.60c	6.30 ± 0.82b	6. 42 ± 1.42a	6.63 ± 1.31a	6.71 ± 1.02a	<0.001
a*24 h	2.05 ± 0.39d	2.69 ± 0.56c	3.25 ± 0.19b	3.54 ± 0.66ab	3.84 ± 0.76a	<0.001
b*45 min	−2.84 ± 0.62b	−1.56 ± 1.14a	−1.34 ± 0.52a	−0.62 ± 0.72a	−1.27 ± 0.78a	<0.001
b*24 h	3.40 ± 0.84b	5.01 ± 1.36ab	5.17 ± 1.18a	5.24 ± 1.10a	5.78 ± 1.45a	0.030
Drop loss, %	3.88 ± 0.21a	2.58 ± 0.14b	2.34 ± 0.34b	1.62 ± 0.12c	1.31 ± 0.18c	<0.001
Cooking loss, %	25.40 ± 0.24a	23.01 ± 0.96ab	15.35 ± 0.78b	10.35 ± 2.10c	9.68 ± 2.33d	0.005

Note: Data represent the trend in changes for each parameter relative to the control group (oxidized oil without antioxidants). Con: the oxidation group; Abbreviations: BEC (Butylated Hydroxytoluene + Ethoxyquin + Citric acid), BE (Butylated Hydroxytoluene + Ethoxyquin), EQ (Ethoxyquin), and BHT (Butylated Hydroxytoluene) represent different antioxidant treatments. L*, a*, and b* values correspond to lightness, redness, and yellowness of meat, respectively, measured at 45 min and 24 h post-mortem. Drip loss and cooking loss are expressed as percentage increases relative to the control. Different letters (a, b, c) indicate statistically significant differences (p < 0.05). n = 6.

Antioxidants mitigate oxidative damage and myoglobin redox deterioration

Antioxidant supplementation optimized breast meat color in the order of BEC > BE > EQ > BHT (Fig. 2A). Compared with the control, dietary antioxidants resulted in a sequential reduction in TBARS values following BEC > BE > EQ > BHT and carbonyl content following BEC > BE > EQ / BHT, with no significant difference between EQ and BHT groups. Furthermore, the retention of total sulfhydryl groups increased significantly in the following order: BEC > BE > BHT > EQ (Fig. 2B–D; P < 0.05). Regarding antioxidant defenses, the activities of T-SOD and T-AOC were sequentially elevated in the order of EQ / BHT < BE < BEC, with no significant difference observed between the EQ and BHT groups (Fig. 2E–F; P < 0.05). Regarding myoglobin derivatives, the contents of both oxymyoglobin and deoxymyoglobin increased significantly in the order of BEC > BE > EQ > BHT. Specifically, oxymyoglobin levels were comparable between the EQ and BE groups. Conversely, metmyoglobin content decreased sequentially following the hierarchy of BEC > BE / EQ > BHT (Fig. 2G; P < 0.05), with no significant difference observed between the BE and EQ groups.

Fig. 2.

Effects of different antioxidants on color and oxidative stability of broiler breast meat under oxidative stress. (A) Breast Muscle lightness. (B) TBARS values. (C) Carbonyl content (mg MDA/kg muscle). (D) Total sulfhydryl content. (E, F) T-SOD and T-AOC activities (U/mg·protein). (G) Oxymyoglobin content, deoxy-myoglobin, metmyoglobin content (μm/mg·protein). (H) Correlation Between Metmyoglobin Accumulation and Color Parameters. Abbreviations: Con: the oxidation group; BEC (Butylated Hydroxytoluene + Ethoxyquin + Citric acid), BE (Butylated Hydroxytoluene + Ethoxyquin), EQ (Ethoxyquin), and BHT (Butylated Hydroxytoluene) represent different antioxidant treatments. Different letters (a, b, c) indicate statistically significant differences (p < 0.05). n = 6.

Correlation analysis showed that metmyoglobin was strongly positively correlated with drip and cooking loss (r > 0.78), Conversely, redness (a*) and yellowness(b*) were negatively correlated with drop loss (|r| > 0.6). Additionally, pH_24h showed a strong positive relationship with redness (r = 0.790), and drip loss was positively correlated with cooking loss (r = 0.886) (Fig. 2H).

Oxidized oil impairs satellite cell function and myogenic potential

Satellite cells were isolated following in ovo injection during the embryonic stage to elucidate the cellular mechanisms in vitro. Compared to the control group, cell viability was significantly decreased in OXI groups (Fig. 3A). Simultaneously, Pax7 expression and MyHC-labeled myofibers density were significant reduced (Fig. 3B–C; P < 0.05). JC-1 aggregation and intracellular ATP content starkly decreased (Fig. 3D and E; P < 0.05). Furthermore, reactive oxygen species (ROS) and malondialdehyde (MDA) levels were elevated, alongside superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities decreased (Fig. 3F, H–J; P < 0.05). Finally, cleaved Caspase-3 and PARP-1 expression increased, while Bcl-2 levels concomitantly decreased (Fig. 3G; P < 0.05).

Fig. 3.

Impaired chicken muscle development leads to deteriorated meat quality. (A) Cell viability assessed by CCK-8 assay (%). (B) Pax7 expression (400 μm). (C) MyHC-labeled myofiber abundance (400 μm). (D) Mitochondrial membrane potential (JC-1) staining (400 μm). (E) ATP content (nmol/mg· protein). (F) ROS straining (200 μm). (G) Relative expression of apoptosis-related proteins (PARP-1, Cleaved-Caspase-3, and Bcl-2). (H) MDA levels (nmol/mg· protein). (I, J) Activities of the antioxidant enzymes SOD and GSH-Px (U/mmg·protein). Abbreviations: Vehicle: Control group; OXI: Oxidation group. *means p < 0.05, **means p < 0.01, ***means p < 0.001.n = 6.

Antioxidants restore satellite cell viability and mitochondrial homeostasis

Antioxidant interventions restored satellite cell viability and proliferation in a clear time- and composition-dependent manner under OXI stress. While no significant differences occurred at 12 h, a distinct graded efficacy of BEC > BE > EQ > BHT emerged at 24 h and persisted through 36, 48, and 60 h (Fig. 4A; P < 0.05). Under OXI stress, antioxidant interventions consistently optimized satellite cell function following the hierarchy of BEC > BE > EQ > BHT. Specifically, this order was observed in the enhancement of pro-proliferative effects, myofiber expression, and mitochondrial membrane potential, as well as the reduction of ROS levels (Fig. 4B–D, F). Intracellular ATP content followed a similar ascending trend of BEC > BE > EQ / BHT, with no significant difference between EQ and BHT (Fig. 4G). Regarding apoptosis, PARP-1 and Cleaved-Caspase-3 levels decreased sequentially in the order of BEC > BE > BHT/EQ, while Bcl-2 expression increased following the hierarchy of BEC > BE > BHT > EQ (Fig. 4E; p < 0.05).

Fig. 4.

Protective effects of different antioxidants against OXI-induced oxidative stress in cells. (A) Cell viability assessed by CCK8 assay (%). (B) Flow cytometry analysis of the effects of different antioxidants on satellite cells (400 μm). (C) MyHC-labeled myofiber abundance (400 μm). (D) ROS expression (200 μm). (E) PARP-1, Cleaved-Caspase-3, and Bcl-2 proteins relative expression. (F) JC-1 expression (400 μm). (G) ATP content (nmol/mmg·protein). Abbreviations: OXI: Oxidation; BEC (Butylated Hydroxytoluene + Ethoxyquin + Citric acid), BE (Butylated Hydroxytoluene + Ethoxyquin), EQ (Ethoxyquin), and BHT (Butylated Hydroxytoluene) represent different antioxidant treatments. Different letters (a, b, c) indicate statistically significant differences (p < 0.05). n = 6.

Antioxidants activate NRF2 pathway and inhibit muscle ferroptosis

In muscle satellite cells subjected to OXI-induced stress, antioxidant interventions effectively rescued the suppression of the NRF2/ARE pathway. Specifically, the protein expressions of NRF2 and HO-1 increased in the order of BEC > BE > BHT > EQ. In contrast, KEAP1 protein expression followed the sequence of BEC > BE / EQ / BHT, with no significant differences observed among the latter three groups. Furthermore, NQO1 protein levels followed the hierarchy of BEC > BE > EQ / BHT, with no significant differences observed between EQ and BHT groups (Fig. 5A; P < 0.05). Consistent with the protein profiles, the mRNA expressions of NRF2 and NQO1 increased in the order of BEC > BE > EQ > BHT. For HO-1, mRNA levels followed the sequence of BEC > EQ > BHT, although no significant differences were found between BEC and BE, or BE and EQ. Conversely, KEAP1 mRNA expression exhibited a downward trend in the order of BHT > EQ / BE > BEC, with no significant differences observed between EQ and BE groups (Fig.5B; P < 0.05). Additionally, antioxidants significantly upregulated key genes involved in iron homeostasis (FTH1), glutathione synthesis (GCLM), and lipid metabolism (ACSL3, SCD, and PLA2G6) following the hierarchy of BEC > BE > EQ > BHT (Fig. 5C; P < 0.05).

Fig. 5.

Compound Antioxidants Block Ferroptosis by Regulating the NRF2/GPX4 Axis. (A) cf NRF2/ARE pathway components (NRF2, NQO1, KEAP1, HO-1) in satellite cells. (B) Relative mRNA expression levels of NRF2, HO-1, NQO1, and KEAP1 in satellite cells. (C) mRNA expression levels of FTH1, ACSL3, SCD, PLA2G6, and GCLM in satellite cells. (D, E, F) The content of MDA, SOD, GSH-Px in satellite cells (nmccol/mg protein). (G) AlphaFold2 structural modeling of poultry GPX4 identified distinct ligand-binding pockets for BHT, EQ, and citric acid (CA). (H) Dietary antioxidant treatments altered GPX4 protein expression in breast muscle. (I) GPX4 gene expression in breast muscle. (J) GPX4 enzymatic activity in the breast muscle in satellite cells. (K) The labile iron pool (LIP) in cells (scale bar = 50 μm). (L) Phospholipid hydroperoxides (PHOOHs) in satellite cells (scale bar = 100 μm). (M) Immunofluorescence staining of MyHC in satellite cells. (N) Relative protein abundance of MyoD and MyoG in satellite cells. Abbreviations: OXI: Oxidized Oil group; BEC (Butylated Hydroxytoluene + Ethoxyquin + Citric acid), BE (Butylated Hydroxytoluene + Ethoxyquin), EQ (Ethoxyquin), and BHT (Butylated Hydroxytoluene) represent different antioxidant treatments. Different letters (a, b, c) indicate statistically significant differences (p < 0.05). n = 6.

The antioxidant interventions also successfully alleviated oxidative stress and ferroptosis. MDA levels were significantly reduced, with the degree of reduction following the order of BEC > EQ / BE > BHT, with no significant differences observed between EQ and BE groups. Conversely, protein levels of SOD and GSH-Px were elevated in the sequence of BEC > BE / BHT / EQ, with no significant differences observed among the latter three groups (Fig. 5 D–F; P < 0.05). Molecular docking via AlphaFold2 identified three distinct ligand-binding pockets on the poultry GPX4 protein that independently accommodate BHT, EQ, and CA (Fig. 5G). This was paralleled by a graded recovery in GPX4 protein and mRNA levels, following the sequences of BEC > BE > EQ / BHT and BEC > BE > BHT > EQ, respectively (Fig. 5H–I; P < 0.05). In post-hatch muscle, antioxidants restored GPX4 activity in the order of BEC > BE > EQ > BHT, while significantly lowering labile iron pool (LIP) and phospholipid hydroperoxide (PLOOH) levels following the same hierarchy (Fig. 5 J–L). Moreover, immunofluorescence and protein analysis confirmed that the inhibition of ferroptosis effectively rescued the OXI-induced impairment of MyHC, MyoD, and MyoG expression, as well as myotube fusion (Fig. 5 M–N; P < 0.05).

BEC requires GPX4 to suppress lipid peroxidation and ferroptosis

To confirm knockdown efficiency, Western blot and qRT-PCR results showed that GPX4 expression in the shGPX4 group was significantly reduced compared to the vehicle control (P < 0.001), demonstrating the high inhibitory potency of the shRNA (Fig. 6A and B). The accumulation of phospholipid hydroperoxides (PHOOHs) followed a distinct GPX4-dependent response across the experimental cohorts. OXI challenge significantly increased fluorescence intensity in control cells, whereas co-treatment with either BEC or Fer-1 suppressed these levels to baseline. In the shGPX4 line, baseline lipid peroxidation was elevated compared to NC cells, and OXI-induced PHOOHs accumulation was markedly exacerbated. In these GPX4-deficient cells, BEC supplementation failed to reduce PHOOHs levels, with fluorescence intensity in the shGPX4 + OXI + BEC group remaining equivalent to the shGPX4 + OXI cohort (Fig. 6C). Additionally, BEC treatment alone did not lower the elevated initial peroxidation characteristic of the shGPX4 cells. The dynamics of the labile iron pool (LIP) mirrored the fluctuations in phospholipid hydroperoxides across all experimental groups. OXI challenge induced a significant expansion of the LIP in control cells, which was normalized by co-treatment with either BEC or Fer-1. While shGPX4 cells exhibited a slight elevation in basal LIP, OXI insult triggered a severe increase in iron levels in this line. In the absence of GPX4, BEC treatment failed to restore iron homeostasis; the LIP levels in the shGPX4 + OXI + BEC group remained equivalent to those in the unprotected shGPX4 + OXI cohort (Fig. 6D). We further evaluated the modulatory effects of various treatments on GPX4 protein expression to determine the protective efficacy of the BEC system. the GPX4 inhibitor NCI677397 markedly depleted GPX4 levels compared to the Vehicle, validating the experimental system. Under OXI-induced oxidative stress, co-treatment with Acetylcysteine effectively restored GPX4 expression to baseline levels. Notably, the composite antioxidant BEC exhibited superior efficacy, not only neutralizing OXI-induced suppression but also increasing GPX4 protein abundance compared to the Vehicle group(Fig. 6E; p < 0.05).

Fig. 6.

GPX4 is required for BEC to suppress ferroptotic lipid peroxidation and labile iron pool accumulation in satellite cells (n=6). (A) Validation of GPX4 knockdown at the protein level. (B) Validation of GPX4 knockdown at the gene level. (C) The labile iron pool (LIP) in cells (scale bar = 50 μm). (D) Phospholipid hydroperoxides (PHOOHs) in satellite cells (scale bar = 100 μm). (E) Relative protein expression of GPX4. Abbreviations: OXI: Oxidized oil; BEC (Butylated Hydroxytoluene + Ethoxyquin + Citric acid) represent the antioxidant treatments. Different letters (a, b, c) indicate statistically significant differences (p < 0.05). n = 6.

Discussion

In the modern broiler industry, the relentless pursuit of growth efficiency and muscle yield comes at the cost of increased susceptibility to metabolic and oxidative insults induced by accelerated growth rates (Oke et al., 2024). This innate vulnerability implies that even marginal environmental or nutritional stressors can exceed the biological threshold of the antioxidant defense system (Choi et al., 2023). Our findings demonstrate that dietary oxidized oils act as a hazardous component to animal health, initiating a deleterious cascade that spans from embryonic development to significant post-mortem meat quality deterioration. The significant reduction in ultimate pH, coupled with the concomitant loss of redness and substantial increases in drip and cooking losses within the challenged birds, collectively reflects a profound state of myoglobin redox instability and a systemic collapse of myofibrillar structural integrity.

The pathological accumulation of metmyoglobin, which displayed an exceptionally strong negative correlation with redness, confirms that lipid oxidation products directly catalyze the conversion of pigment proteins from their functional ferrous states into their ferric, discolored forms (Zhang et al., 2026). This damage is not a localized post-mortem event; rather, it is programmed during the critical embryonic window through a persistent redox-programming effect. Avian embryos possess an inherent vulnerability to high levels of dietary pro-oxidants due to the rapid turnover of polyunsaturated fatty acids in the yolk (Liu et al., 2021). The impaired myogenic potential, characterized by the downregulation of Pax7 and MyHC, supports the hypothesis that early-life nutritional insults set a pro-oxidative trajectory for muscle tissue, leading to an irreversible loss of quality at market age. This establishes a pathological bridge linking embryonic oxidative stress to persistent myogenic defects throughout the lifespan. Specifically, the capacity of Fer-1 to effectively counteract the depletion of MyHC, MyoD1 and MyoG identifies ferroptosis as a pivotal mediator of the myogenic failure triggered by oxidative challenge. Mechanistically, this process is governed by the collapse of the GPX4-LIP axis, a definitive hallmark of ferroptotic cell death (Yu et al., 2021). As a distinct form of iron-dependent regulated cell death, ferroptosis is governed by the dynamic interplay between intracellular antioxidant defenses and the accumulation of phospholipid hydroperoxides (Zhou et al., 2025). In the present study, the oxidation challenge induced a pathological expansion of the labile iron pool and a concomitant depletion of glutathione peroxidase 4, the primary enzymatic guardian against membrane-bound lipid hydroperoxides. Following shRNA-mediated GPX4 knockdown, BEC treatment failed to activate GPX4, demonstrating that BEC-mediated meat quality preservation is strictly GPX4-dependent. This indicates that meat quality preservation is not merely a consequence of general radical scavenging but is mechanistically contingent upon a functional GPX4 shield. When this enzymatic defense is compromised, the expanded labile iron pool catalyzes the Fenton reaction to generate hydroxyl radicals, initiating a self-amplifying loop of membrane destruction that cripples the structural and metabolic integrity of the muscle tissue.

In evaluating mitigation strategies, a clear hierarchy of efficacy emerged among the treatment groups, where the performance of the BEC group was superior to the BE group, which in turn surpassed the EQ and BHT groups. This performance gradient can be explained through the multi-nodal defense architecture of the BEC formulation. The lipid-soluble components, BHT and Ethoxyquin, exhibit different partition coefficients (Eskin and Robinson, 2000); BHT tends to anchor within the hydrophobic core of the phospholipid bilayer, while Ethoxyquin resides closer to the polar head groups (Braasch-Turi et al., 2022). The staggered spatial distribution of these antioxidants facilitates a more comprehensive interception of lipid peroxy radicals throughout the membrane architecture. Nevertheless, radical scavenging in isolation remains insufficient when the primary catalytic driver, represented by free labile iron, remains unchecked. The integration of citric acid within the BEC formulation introduces a critical iron-chelating dimension, which effectively deactivates the catalytic iron ions necessary for the initiation of the Fenton reaction. This multi-layered synergy explains why the BEC treatment was uniquely capable of maintaining the functional integrity of glutathione peroxidase 4 and normalizing the mitochondrial membrane potential. Such results align closely with the theory of antioxidant synergy in animal nutrition, which posits that complex oxidative challenges necessitate multi-target interventions to preserve cellular homeostasis and metabolic integrity (Corino and Rossi, 2021).

The restoration of mitochondrial function emerged as a vital link between cellular survival and macro-scale meat color stability. Post-mortem mitochondria remain metabolically active for several hours, competing with myoglobin for available oxygen to maintain a low-oxygen environment that favors the stability of bright-red oxymyoglobin (Kiyimba et al., 2021). Oxidation-induced ferroptotic damage to mitochondrial membranes triggers a collapse in oxygen-management efficiency and the release of catalytic iron, thereby accelerating the conversion of myoglobin into its pale-brown metmyoglobin form (Zhu et al., 2025). Consequently, the apoptotic activity mediated by the mitochondrial pathway serves as a sensitive reflection of post-mortem tissue quality. Bcl-2 serves as a critical anti-apoptotic biomarker, and its up-regulation is essential for maintaining cellular homeostasis under redox crisis. Our data demonstrated that the up-regulation of Bcl-2 was accompanied by a concomitant suppression of the pro-apoptotic markers Cleaved-Caspase-3 and PARP-1. In this study, while single antioxidants (BHT or EQ) partially restored Bcl-2 expression and moderately attenuated the cleavage of Caspase-3, the ternary system (BEC) demonstrated a superior synergistic effect. Specifically, the BEC group exhibited the most profound suppression of Cleaved-Caspase-3 and the highest retention of intact PARP-1, suggesting a comprehensive blockade of the apoptotic cascade. This enhancement suggests that the combination of BHT, ethoxyquin, and citric acid provides a more robust defense mechanism against oxidative-stress-induced apoptosis than individual or binary treatments. The significant increase in Bcl-2 levels coupled with the inhibition of the Caspase-3/PARP-1 proteolytic cascade in the BEC group indicates that this specific antioxidant blend can effectively stabilize the mitochondrial membrane potential and suppress programmed cell death pathways in broilers. By shielding the mitochondrial machinery from such programmed degradation, BEC treatment likely preserves the metabolic competition for oxygen, thereby extending the window of myoglobin stability and maintaining the desirable cherry-red appearance of the meat. Consistently, these findings demonstrate that the protective efficacy of BEC was significantly superior to that of EQ or BHT when administered individually, reinforcing the necessity of a multi-targeted antioxidant approach.

Meanwhile, BEC, by safeguarding the mitochondrial-GPX4 axis, effectively maintained high redness levels compared to the oxidation group. This preservation of water-holding capacity is also an electrochemical consequence of preventing protein carbonylation and the loss of total sulfhydryl groups. Lipid peroxidation byproducts like malondialdehyde, which significantly increased in the oxidation group, are known to cross-link with myofibrillar proteins. This oxidative cross-linking collapses the protein lattice, reducing the capillary forces required to entrap water molecules (Jiang et al., 2021). By suppressing malondialdehyde levels, BEC ensures that the physical structure of the myofibrils remains capable of retaining water, resulting in a higher yield of juicy breast meat. Furthermore, the protective efficacy of BEC across these meat quality parameters was significantly superior to that of EQ or BHT when administered individually.

The molecular foundation of this synergy was further elucidated through AlphaFold2 structural modeling of poultry GPX4, which identified distinct ligand-binding pockets for BHT, Ethoxyquin, and Citric Acid. This suggests that the BEC components do not merely function as independent scavengers but may also act as molecular chaperones, stabilizing the GPX4 enzyme and preventing the oxidative modification of key residues, such as the selenocysteine active site. This structural preservation ensures sustained enzymatic turnover under extreme lipid peroxidation. Notably, this direct enzymatic protection is complemented by a broader metabolic fortification of the cellular defensive state, evidenced by the significant upregulation of GCLM and ACSL3 expression. ACSL3, in particular, plays a critical role in 'membrane fortifying' by promoting the incorporation of monounsaturated fatty acids (MUFAs), which are inherently more resistant to peroxidation than polyunsaturated fatty acids (PUFAs) (Lei et al., 2024). Thus, BEC acts as a master regulator that simultaneously safeguards antioxidant enzyme integrity and optimizes membrane lipid composition to achieve robust resistance against ferroptosis.

These systemic effects are orchestrated through the NRF2/ARE signaling pathway. The oxidation challenge was found to significantly suppress a suite of genes responsible for iron sequestration and glutathione synthesis (Inaba et al., 2021). Strikingly, BEC co-treatment triggered an antioxidant priming effect, facilitating the efficient dissociation of NRF2 from its cytosolic repressor, KEAP1. This triggers a comprehensive transcriptional program to restore redox homeostasis, with the efficacy of BEC being significantly superior to that of EQ or BHT when administered individually. The strong correlation between NRF2 activation and the restoration of post-mortem meat quality parameters highlights this pathway as a primary target for precision nutritional interventions. Based on our findings, we speculate that ultimate pH is a critical determinant of meat quality. Specifically, oxidation-induced stress accelerates post-mortem glycolysis, triggering a rapid pH decline toward the isoelectric point of myofibrillar proteins. The BEC group exhibited the superior buffering capacity against this pH drop, which is likely attributable to the preservation of mitochondrial integrity and the maintenance of cellular energy pools during the early post-mortem period.

The industrial implications of these findings are substantial. Historically, the poultry industry has evaluated feed oil quality using simple chemical indices such as peroxide value (Shurson et al., 2015; Zhang et al., 2023). Furthermore, scientific understanding of meat impairment induced by oxidized oils has traditionally remained confined to the assessment of malondialdehyde (MDA) levels and conventional antioxidant enzyme activities (Geng et al., 2023; Macho-González et al., 2020). However, our data suggest that these parameters may underestimate the long-term biological damage. The ferroptotic bridge identified here demonstrates that even embryonic oxidative stress can lead to significant economic losses at slaughter by inducing mitochondrial impairment and the depletion of energy pools, which ultimately compromises post-mortem redox homeostasis. Therefore, we advocate for a shift toward strategic embryonic fortification using synergistic systems like BEC designed to protect the GPX4-NRF2 axis. In conclusion, this research establishes ferroptosis as the definitive pathological bridge connecting early-life nutritional stress to post-mortem meat quality defects in broilers. The bioenergetic crisis and redox collapse initiated by oxidized oils can be effectively neutralized by the ternary antioxidant BEC through a synergistic mechanism of scavenging lipid radicals, chelating catalytic iron, and inducing NRF2-mediated enzymatic defenses. This holistic approach ensures the production of high-quality, stable breast meat that meets both consumer expectations and economic requirements.

Conclusion

This study identifies ferroptosis as the key pathological link between early-life oxidative stress and post-mortem meat defects in broilers, driven by the collapse of the GPX4-LIP axis. Notably, the ternary BEC system demonstrated superior efficacy over individual (BHT, EQ) or binary (BE) treatments by providing a multi-dimensional defense—integrating radical scavenging, iron chelation, and GPX4 structural stabilization. By uniquely activating the NRF2/ARE pathway and safeguarding mitochondrial integrity more effectively than its components, BEC suppresses programmed cell death to preserve myofibrillar stability and meat quality. These findings advocate for embryonic fortification via synergistic nutritional interventions to mitigate the systemic impact of oxidative insults in poultry production.

Data availability

Data associated with this study are available upon reasonable request.

CRediT authorship contribution statement

Xuyang Gao: Writing – review & editing, Formal analysis, Data curation. Caiwei Luo: Software, Investigation. Bo Wang: Supervision, Project administration. Jianmin Yuan: Writing – review & editing, Project administration, Conceptualization.

Disclosures

The authors declare no known competing financial interests or personal relationships that could have influenced the work reported herein.