HIF-2α could be a key regulator of Fe homeostasis in the gut of yellow-feathered broilers

Abstract

This study examines the effects of age and dietary Fe on the duodenal and liver deposition of Fe, Cu, Mn, and Zn, and expression of genes involved in Fe homeostasis in yellow-feathered broilers. A total of 400 1-day-old male broilers were randomly assigned to 5 groups with different dietary Fe supplementation (0, 20, 80, 320, and 1280 mg/kg, from FeSO₄•7H₂O), and samples were collected at two time points (on d 21 and d 63). The basal diet for different growth stages contained 79.60, 72.64, and 61.79 mg/kg Fe, respectively. Each treatment contained 8 cages with 10 birds per cage. The ADG and ADFI at 21 and 63 d were reduced (P ˂ 0.001) in broilers supplemented with 1280 mg/kg Fe compared to others, while FCR was not affected by treatment. Duodenal Cu (P = 0.048) and hepatic Fe (P = 0.015) deposition exhibited a treatment × age interaction, with both the lowest duodenal Cu and the highest hepatic Fe occurring with 1280 mg/kg of dietary Fe on d 63 (P < 0.001). Dietary Fe supplementation decreased duodenal mucosal Mn concentrations (P < 0.001) but did not change duodenal Zn or hepatic Zn. Higher duodenal Zn and Mn, and hepatic Mn, as well as lower hepatic Cu, were detected in birds on d 21 (P < 0.05). There was an interaction of treatment × age on the expression of duodenal divalent metal transporter 1 (DMT1, P < 0.001) and hypoxia-inducible factor 1α (HIF-1α, P = 0.007), with the expression of DMT1 at d 63 in birds supplemented with 0 or 20 mg/kg of Fe and the expression of HIF-1α at d 63 in birds supplemented with Fe higher than 21 d. Both ferroportin 1 (FPN1) and HIF-2α decreased with elevated dietary Fe supplementation (P < 0.05). The expression of HIF-2α remained stable between ages (P = 0.222). In conclusion, excess dietary Fe may trigger broiler Fe homeostasis via HIF-2α with weaker effects in juveniles.

Introduction

Iron is an essential trace element, and its deficiency can lead to conditions such as anemia, while excess Fe adversely affects animal health through various mechanisms (Nemeth and Ganz, 2023). Fast-growing animals exhibit a high demand for Fe. Yellow-feathered broilers, renowned for their superior meat quality, have achieved comparable production levels to white broilers in China, with a recommended dietary Fe concentration of 80 mg/kg (NRC, 1994; MAPRC, 2020). However, the grain-derived feed ingredients have been found to contain considerable Fe, which may derive not only from intrinsic grain components but also from soil contamination introduced during processing (Hansen et al., 2010). The Fe concentrations detected in corn, wheat, soybean meal, limestone, and dicalcium phosphate are around 36mg/kg, 203.2 mg/kg, 185 mg/kg, 2649 mg/kg, and 14,100 mg/kg, respectively (Sullivan et al., 1994; Wu et al., 2025). Recently, it has been reported that the corn-soybean meal diet, containing commercial limestone and dicalcium phosphate, has sufficient Fe to meet their daily needs (Feijo et al., 2024). Not considering the Fe inherent in feed ingredients, supplemental mineral Fe in broiler diets may lead to excessive total Fe content. Our previous studies have demonstrated that growth performance, antioxidant capacity, and intestinal barrier function decline with high Fe supplementation (Chen et al., 2025; Lei et al., 2024), raising animal welfare issues. Therefore, it is critical to study the regulation of dietary Fe absorption.

Animals maintain homeostatic regulation of Fe to counteract excessive Fe intake, and regulation of excessive Fe intake in animals primarily occurs in the small intestine (Hansen et al., 2010). Divalent metal transporter 1 (DMT1) and ferroportin 1 (FPN1) have been extensively demonstrated to participate in Fe absorption and transport, with their expression and degradation being regulated by hepcidin (Vogt et al., 2021; Bergamaschi et al., 2017). However, no homolog of hepcidin has been isolated in poultry to date (Hilton and Lambert, 2008), leaving the mechanism of Fe homeostasis regulation in avian species unclear. Hypoxia-inducible factors (HIFs) are a class of transcription regulators sensitive to oxygen and Fe concentrations (Sanguigno et al., 2023). Given that the intestinal environment is strictly hypoxic, HIFs remain stabilized in the intestine. Studies have demonstrated a synergistic interaction between HIFs and hepcidin in regulating intestinal Fe absorption in mammals (Shah and Xie, 2014; Mastrogiannaki et al., 2013). Nevertheless, in the absence of hepcidin in poultry, whether HIFs serve as regulators of intestinal Fe absorption remains to be elucidated. Additionally, young animals are believed to have an underdeveloped Fe regulatory system, making them more susceptible to Fe overload (Hansen et al., 2010). Our preliminary studies have also observed a more rapid response to Fe doses in younger animals (in terms of mRNA expression of tight junction proteins, 320 mg/kg at d 21 and 1280 mg/kg at d 63) (Chen et al., 2025; Lei et al., 2024). However, the effect of age on the regulatory chain of Fe homeostasis in broilers is unclear.

Studies have demonstrated an interaction between Fe absorption and Cu, Mn, and Zn uptake in the duodenum and jejunum (Han et al., 2017; Hansen et al., 2009; Storey and Greger, 1987). However, research on such elemental interactions has primarily focused on mammals, with limited systematic evidence available for poultry. Furthermore, investigations into avian responses to Fe overload often employ supraphysiological Fe supplementation (Yang et al., 2024; Wang et al., 2023), lacking systematic dose-response studies to assess the effects of graded Fe comprehensively. Therefore, we propose the hypotheses that moderate or high doses of Fe may have positive or negative effects on growth performance and the deposition of Fe, Cu, Zn, and Mn in the tissue, and that age may have a significant or minor effect on the regulation of Fe absorption.

To test the above scientific hypotheses, a two factorial design with 5 dosages of Fe supplementation and 2 sample collection phases was used in this trial to 1) detect the deposition of Fe, Cu, Zn, and Mn in the duodenal mucosa and liver, 2) focus on the relationship between Fe absorption and Cu, Mn, and Zn uptake in different physiological stages of broilers, and 3) innovatively introduce HIFs into the Fe regulation system in poultry, providing a theoretical foundation for the rational utilization of Fe in yellow-feathered broilers and a novel perspective for elucidating the mechanisms of Fe homeostasis in avian species.

Materials and methods

Animals, diets, and experimental design

A total of 400 1-day-old male yellow-feathered broilers were randomly assigned to 5 groups with different dietary Fe supplementation: 0 (the basal diet), 20 (optimal growth performance at 21 d; Lei et al., 2024), 80 (the recommended amount for feeding standards), 320 (apparent damage to the intestinal barrier at 63 d; Chen et al., 2025), and 1,280 mg/kg (obvious damage to the growth performance; Chen et al., 2025), respectively, in the form of FeSO₄•7H₂O. Tissue samples were collected at d 21 and d 63. Each treatment contained 8 replicates with 10 birds per replicated cage. All the birds were housed in a temperature-controlled room with fiberglass feeders and stainless-steel cages coated with plastic. The birds had free access to feed and deionized water, and a lighting regimen meeting the requirements of yellow-feathered broilers was applied during this experiment. The initial temperature set point was 35.0°C when chicks were placed in cages. The room temperature was maintained at about 35°C for one week and then reduced by 3°C each week until the birds were 35 days old. Subsequently, the temperature was maintained at about 22°C until the end of the experiment. The trial lasted 63 days and was divided into 3 scientific phases: 1-21 d, 22-42 d, 43-63 d. The corn-soybean meal basal diet was formulated to meet or exceed the nutritional requirements for yellow-feathered broilers except for Fe (NRC, 1994; MAPRC, 2020). The formula and nutrient concentration of the basal diet are shown in Table 1. The treatment diets were prepared by mixing 0.64% of a corn starch-FeSO₄•7H₂O premix with 99.36% of a base mix. After blending the FeSO₄•7H₂O with corn starch, the resulting mixture provided supplemental Fe at concentrations of 0, 20, 80, 320, or 1280 mg/kg of diet. The analyzed Fe concentrations of different treatments are shown in Table 2.

Table 1.

Composition of the basal diet for broilers (air-dry basis).

Ingredient	d 1-21	d 22-42	d 43-63
Corn (%)	56.83	64.78	67.70
Soybean meal [46%] (%)	35.75	28.45	23.98
Soybean oil (%)	3.00	2.50	4.30
NaCl (%)1	0.30	0.30	0.30
CaCO3 (%)1	1.00	0.92	0.91
CaHPO4 (%)1	1.84	1.70	1.53
Premix (%)2	0.22	0.22	0.22
L-Lys (%)	0.11	0.20	0.20
DL-Met (%)	0.23	0.26	0.20
L-Thr (%)	0.08	0.03	0.02
Corn starch-Fe premix (%)4	0.64	0.64	0.64
Total	100.00	100.00	100.00
Nutrition level
Metabolic energy (MJ/kg)	12.45	12.58	13.18
Crude protein (%)3	21.33	18.66	16.78
Ca (%)3	0.94	0.96	0.80
Total P (%)3	0.70	0.73	0.60
Nonphytate P (%)	0.41	0.37	0.34
Lys (%)	1.18	1.08	0.96
Met (%)	0.53	0.53	0.45
Cys + Met (%)	0.84	0.80	0.72
Thr (%)	0.85	0.70	0.63
Fe (mg/kg) 3	79.60	72.64	61.76
Cu (mg/kg) 3	14.87	12.12	13.81
Mn (mg/kg) 3	93.03	99.69	93.88
Zn (mg/kg) 3	88.62	90.27	92.88

¹Reagent grade.

²The premix provided the following diets per kilogram: Vitamin A 8,400 IU, vitamin D₃ 3,600 IU, vitamin E 13 IU, vitamin K 1.6 mg, thiamine 5.5 mg, vitamin B₂ 6.8 mg, vitamin B₆ 1.0 mg, vitamin B₁₂ 0.01 mg, biotin 0.08 mg, folic acid 0.80 mg, pantothenic acid 10.2 mg, niacin 28.6 mg, choline (50% choline chloride) 1,000 mg, Cu (as reagent grade blue copperas) 8 mg, Zn (as reagent grade zinc sulfate heptahydrate) 60 mg, Mn (as reagent grade manganese sulfate monohydrate) 80 mg, I (as feed grade calcium iodate) 0.35 mg, Se (as feed grade sodium selenite) 0.15 mg.

³Analyzed values, and each value was based on triplicate determinations.

⁴Fe supplements were added in place of equivalent weights of cornstarch in the form of FeSO₄•7H₂O.

Table 2.

Dietary Fe concentrations (air-dry basis)¹.

Age	Fe supplementation (mg/kg)
	0	20	80	320	1280
D 1-21	79.60	97.64	162.54	393.18	1354.47
D 22-42	72.64	91.37	151.21	387.69	1347.89
D 43-63	61.76	79.39	143.65	377.65	1412.34

¹All the values are measured by flame atomic absorption spectrometry (Contr AA 700; Analytik Jena, Germany).

Sample collection

On d 21 and d 63, 1 bird closest to the average body weight from each pen was selected and harvested by cervical dislocation. About 0.5 g of liver tissue was quickly separated and collected from the left side and lower tip portion, respectively. Samples were rinsed with cold phosphate buffer (PBS) and then drained of surface water with filter paper. Finally, they were placed in self-sealing bags and stored in a freezer at a temperature of -20°C for later trace mineral analysis.

The duodenum was separated and rinsed slowly with cold PBS. After the water on the surface was blotted out with filter paper, the mucosal tissues were gently scraped with slides and collected in cryopreservation tubes. Half of the sample was frozen in liquid nitrogen at -196°C and then stored at -80°C for the determination of mRNA expressions of DMT1, FPN1, HIF-1α, and HIF-2α. The other half of the sample was stored at -20°C for duodenal Fe, Cu, Zn, and Mn analysis.

Growth performance

Birds were weighed on d 21 and d 63; the feed intake was recorded from d 1 to d 63. The average daily weight gain (ADG) per cage was calculated by subtracting the initial weight from the final weight and dividing it by the number of feeding days and the number of surviving birds. The average daily feed intake (ADFI) per cage was corrected by subtracting the feed intake of dead birds from the total feed intake and dividing it by the number of feeding days and the number of surviving birds. The feed conversion ratio (FCR) was calculated by dividing the total feed intake by the total body weight gain per cage, including dead birds (Chen et al., 2025).

Measurements of CP, Ca, total P, Fe, Cu, Zn, and Mn concentration

The concentrations of Fe, Cu, Zn, and Mn in the diet and tissues were determined using flame atomic absorption spectrometry (Analytik Jena, Jena, Germany) after wet ashing by microwave digestion with ultrapure-grade nitric acid (Hansen et al., 2010). The metal ion analysis was validated using bovine liver powder (China National Institute of Metrology, Beijing, China) as a standard reference. Crude protein was analyzed using the Kjeldahl method, and the Ca was determined by the potassium permanganate titration method, while the total phosphorus was measured using the spectrophotometric method (AOAC, 2023).

Gene expression analysis

The mRNA expressions of FPN1, DMT1, HIF-1α, and HIF-2α in the duodenal mucosa were analysed. First, total RNA was extracted using a Trizol reagent (Takara, Dalian, China). Then, Nanodrop ND-1000 (Thermo Fisher, Waltham) was used to analyze the purity of RNA (OD 260/280 ≥ 1.8), and the integrity of RNA was determined by gel electrophoresis. Reverse transcription was performed using the PrimeScript™ RT Reagent Kit (TaKaRa, Ltd., Dalian, China). The resulting cDNA was diluted 8-fold and used as a PCR template for real-time qPCR analysis in the CFX Connect™ RealTime PCR Detection System (Bio‑Rad Laboratories, Inc., California). The reaction system had a volume of 25 μL and contained 12.5 μL TB Green Premix Ex Taq II (Takara, Dalian, China), 2 μL cDNA, 1 μL upstream primer, 1 μL downstream primer, and 8.5 μL ddH₂O. Primers were synthesized (Sangon Biotech (Shanghai) Co., Ltd., Shanghai, China), and the sequence of primers is shown in Table 3. In addition, the PCR parameters were as follows: 30 s at 95°C, 40 cycles at 95°C for 5 s, and 60°C for 30 s. All reactions were performed in triplicate, and the expressions of relative mRNA were determined by the 2^−ΔΔCT method, normalized with the level of β-actin.

Table 3.

Primer sequences of real-time fluorescence quantitative PCR.

Gene	GenBank Accession No.	Primer sequences	Size
β-actin	NM_205518.2	F: 5’-CGGTACCAATTACTGGTGTTAGATG-3’	163
		R: 5’-GCCTTCATTCACATCTATCACTGG-3’
FPN1	NM_014585.6	F: 5’-GGAGACTGGGTGGACAAGAACTC-3’	68
		R: 5’-GATGCATTCTGAACAACCAAGGA-3’
DMT1	NM_001249798.2	F: 5’-AGCCGTTCACCACTTATTTCG-3’	129
		R: 5’-GGTCCAAATAGGCGATGCTC-3’
HIF-1α	XM_046917651	F: 5’-CAGCCAGGTGCCGAAGAAGC-3’	118
		R: 5’-ATGGTCAGCCTCATAATGGATGCC-3’
HIF-2α	XM_046913012	F: 5’-ATCAAGTTCCCCCTCAGGAC-3’	174
		R: 5’-TGTTGCAATGCTTGCTCTTC-3’

Statistical analysis

Data analysis was performed by two-way repeated-measures ANOVA using the MIXED procedure of SAS (SAS Inst. Inc., North Carolina), with cage as the experimental unit. The model for all measures included the fixed effects of dietary Fe concentration and age, and dietary Fe concentration × age of sampling day interaction. Outlier tests were performed on all data (3σ rule), and no outliers were removed from the data set. The LSMEANS procedure of SAS was used to calculate treatment means, and the PDlFF option was used to separate means if the difference was significant. A correlation analysis (CORR) procedure examined the simple Pearson correlation coefficients. Significance was determined at P < 0.05. The statistical model was as follows:

$$y_{ijk}=\mu +{\alpha }_i+{\beta }_j+{\left(\alpha \beta \right)}_{ij}+{\epsilon }_{ijk}$$

Where: $y_{ijk}$ = response k in the i^th treatment group and j^th level of the second factor, μ = the overall mean, ${\alpha }_i$ = the main effect of the i^th treatment, ${\beta }_j$ = the main effect of the j^th factor, ${\left(\alpha \beta \right)}_{ij}$ = the interaction effect between the i^th treatment and j^th factor, and ${\epsilon }_{ijk}$ = the random error, assumed to be independently and normally distributed with a mean of 0 and constant variance (ε_ijk ∼ N(0,σ²))

Results

Growth performance

The effects of age and dietary Fe concentration on the growth performance of yellow-feathered broilers are shown in Table 4. The ADG and ADFI at 21 and 63 d were reduced (P ˂ 0.001) in broilers supplemented with 1280 mg/kg Fe compared to the other Fe levels, while FCR was not affected by dietary Fe. As expected, ADG and ADFI, and FCR were greater (P ˂ 0.001) from d 1to 63 than from d 1 to 21.

Table 4.

Effects of age and dietary Fe concentration on growth performance of broilers¹.

Items		ADG (g/d)	ADFI (g/d)	FCR (g/g)
D 1-21	0	22.06	34.62b	1.57
	20	23.04	35.47b	1.54
	80	22.47	35.49b	1.58
	320	22.35	34.72b	1.55
	1280	19.71	30.37a	1.54
D 1-63	0	38.99	82.68d	2.11
	20	38.46	81.45d	2.15
	80	38.04	82.48d	2.14
	320	37.78	80.36d	2.17
	1280	34.75	72.80c	2.11
SEM		0.365	0.913	0.027
Age	D 1-21	21.93a	34.13	1.56a
	D 1-63	37.60b	79.95	2.14b
SEM		0.164	0.410	0.010
Fe	0	30.47bc	58.54	1.84
	20	30.75c	58.46	1.84
	80	30.25bc	58.98	1.86
	320	30.07b	57.54	1.86
	1280	27.23a	51.58	1.83
SEM		0.235	0.547	0.016
P-value	Age	< 0.001	< 0.001	< 0.001
	Fe	< 0.001	< 0.001	0.446
	Age × Fe	0.135	0.018	0.458

¹DG, average daily body weight gain; ADFI, average daily feed intake; FCR, feed conversion ratio.

^a-dMeans with different letters within the same row differ (P < 0.05). Each value represents the mean of 6 replicates (n = 8).

Deposition of Fe, Cu, Zn, and Mn in the duodenal mucosa

The effects of age and dietary Fe supplementation on duodenal Fe, Cu, Zn, and Mn deposition are shown in Table 5. Duodenal Cu concentration showed a treatment × age interaction (P = 0.048). The lowest Cu concentration appeared in the group supplemented with 1280 mg/kg Fe at d 63 (P < 0.001). When supplemented with 80 mg/kg Fe or higher, the duodenal Cu concentrations at d 21 and 63 were obviously decreased (P < 0.001). No interactions were observed in the concentrations of Fe (P = 0.762), Mn (P = 0.849), and Zn (P = 0.984). Duodenal Fe concentrations were independent of age (P = 0.698); however, Zn (P < 0.001) and Mn (P = 0.001) concentrations were affected by age, with birds at d 21 having higher concentrations than those at d 63. Duodenal Fe concentration increased (P ˂ 0.05) when dietary Fe supplementation reached 320 mg/kg Fe and increased (P ˂ 0.05) further with 1280 mg/kg Fe addition. Manganese concentrations decreased (P ˂ 0.05) when 80 mg/kg Fe or higher was supplemented (P < 0.05). However, the concentration of Zn in the duodenal mucosa was not affected by dietary Fe supplementation (P = 0.475).

Table 5.

Effects of age and dietary Fe concentration on deposition of Fe, Cu, Zn, and Mn in the duodenum mucosa (air dry basis).

Items		Duodenum (mg/kg)
		Fe	Cu	Zn	Mn
D 21	0	113.12	9.18ef	142.98	15.67
	20	192.95	8.64cde	139.22	15.78
	80	227.36	8.41bc	139.77	13.36
	320	1071.42	8.08bc	147.15	11.45
	1280	2087.74	7.57b	149.76	9.88
D 63	0	104.87	9.22ef	127.92	14.00
	20	122.84	9.28f	128.13	13.39
	80	265.51	8.57cd	126.74	11.19
	320	1027.22	8.24bc	130.46	10.71
	1280	2166.31	6.88a	133.53	8.30
SEM		73.674	0.242	5.322	0.793
Age	D 21	738.52	8.38	143.78b	13.23b
	D 63	737.35	8.44	129.35a	11.52a
SEM		33.412	0.110	1.830	0.355
Fe	0	108.99a	9.20	135.45	14.84c
	20	157.90a	8.96	133.67	14.59c
	80	246.43a	8.49	133.26	12.28b
	320	1049.32b	8.16	138.81	11.08b
	1280	2127.03c	7.22	141.64	9.09a
SEM		52.827	0.171	3.881	0.561
P-value	Age	0.698	0.546	< 0.001	0.001
	Fe	< 0.001	< 0.001	0.475	< 0.001
	Age × Fe	0.762	0.048	0.984	0.849

^a-dMeans with different letters within the same row differ (P < 0.05). Each value represents the mean of 6 replicates (n = 8).

Deposition of Fe, Cu, Zn, and Mn in the liver

The effects of age and dietary Fe supplementation on the deposition of Fe, Cu, Zn, and Mn in the liver are shown in Table 6. Hepatic Fe deposition exhibited a treatment × age interaction (P = 0.015). At d 21, birds supplemented with 80 mg/kg Fe had higher hepatic Fe deposition than non-supplemented control birds (P < 0.001), while birds at d 63 had greater Fe accumulation at 320 mg/kg supplementation than controls. (P < 0.001). Liver Fe concentrations continued to increase (P ˂ 0.05) with increasing supplemental Fe above 80 mg/kg at 21 d and above 320 mg/kg Fe at 63 d. However, no interaction was detected in hepatic concentrations of other divalent ions (Cu, P = 0.113; Mn, P = 0.680; Zn, P = 0.337). Changes in hepatic concentrations of Cu and Mn were correlated with age, with lower concentrations of Cu in the liver on d 21 than on d 63 (P < 0.001). However, a higher concentration of Mn in the liver was observed on d 21 (P < 0.001). In contrast to the result of the duodenum, the concentrations of Cu (P = 0.306) and Mn (P = 0.492) in the liver were not affected by dietary Fe supplementation. Additionally, the hepatic Zn concentration was not affected by age (P = 0.108) or dietary Fe supplementation (P = 0.228).

Table 6.

Effects of age and dietary Fe concentration on deposition of Fe, Cu, Zn, and Mn in the liver (air dry basis).

Items		Liver (mg/kg)
		Fe	Cu	Zn	Mn
D 21	0	120.30a	7.32	116.87	9.22
	20	165.41ab	7.97	114.30	9.52
	80	200.90b	8.29	107.66	10.31
	320	278.73c	9.10	113.14	8.70
	1280	323.53cd	10.76	137.76	9.45
D 63	0	307.47cd	11.25	131.25	6.60
	20	329.18cd	9.98	123.22	6.85
	80	361.86d	10.57	124.88	6.71
	320	440.00e	10.97	124.45	6.57
	1280	658.63f	10.32	122.49	6.79
SEM		29.393	0.797	7.957	0.495
Age	D 21	217.77	8.68a	117.95	9.44b
	D 63	419.43	10.62b	125.26	6.71a
SEM		13.158	0.358	3.278	0.221
Fe	0	213.88	9.29	124.06	7.91
	20	247.29	8.98	118.76	8.19
	80	281.38	9.43	116.27	8.51
	320	359.37	10.04	118.79	7.64
	1280	491.08	10.54	130.13	8.12
SEM		20.790	0.564	5.065	0.350
P-value	Age	< 0.001	< 0.001	0.108	< 0.001
	Fe	< 0.001	0.306	0.228	0.492
	Age × Fe	0.015	0.113	0.337	0.680

^a-dMeans with different letters within the same row differ (P < 0.05). Each value represents the mean of 6 replicates (n = 8).

Correlation of Fe, Cu, Zn, and Mn in the liver and duodenal mucosa

Correlation analysis (Fig. 1, Fig. 2) was conducted to examine the potential correlations among these divalent trace minerals. Similar results were observed at d 21 and d 63. The results indicated that the duodenal mucosa Fe concentrations were negatively correlated with Cu (d 21, P < 0.001, r = -0.593; d 63, P < 0.001, r = -0.705) and Mn (d 21, P < 0.001, r = -0.665; d 63, P < 0.001, r = -0.645), while positively correlated with liver Fe (d 21, P < 0.001, r = 0.710; d 63, P < 0.001, r = 0.763). Additionally, hepatic Fe concentrations were negatively correlated with Cu (d 21, P < 0.001, r = -0.538; d 63, P < 0.001, r = -0.559) and Mn (d 21, P < 0.001, r = -0.555; d 63, P < 0.001, r = -0.545) concentrations in the duodenal mucosa.

Fig. 1.

The correlation of Fe, Cu, Zn, and Mn in liver and duodenum mucosa of 21 d yellow-feathered broilers¹.

¹ ** P < 0.01, *** P < 0.001.

Fig. 2.

The correlation of Fe, Cu, Zn, and Mn in liver and duodenum mucosa of 63 d yellow-feathered broilers¹.

¹ ** P < 0.01, *** P < 0.001.

The mRNA expression of Fe transport proteins

The effect of age and dietary Fe supplementation on the mRNA expression of Fe transport proteins in the duodenal mucosa is shown in Fig. 3, Fig. 4. An interaction on the mRNA expression of duodenal DMT1 (P < 0.001) was detected. The highest expression of DMT1 was observed in the duodenal mucosa of 63-day-old birds when they were supplemented at 20 mg/kg of Fe (P < 0.001). Both age groups exhibited progressively decreasing DMT1 expression patterns with increasing dietary Fe concentrations (P < 0.001). In addition, the mRNA expression of DMT1 in the duodenal mucosa of 63 d was higher than that of 21 d at the same Fe supplemental concentrations. However, there was no interaction between age and dietary Fe supplementation on the mRNA expression of FPN1 in the duodenal mucosa (P = 0.546). The expression of FPN1 in duodenal mucosa decreased with increasing concentrations of dietary Fe supplementation (P < 0.001). Birds of d 63 showed a higher expression of FPN1 than those at 21 days of age (P < 0.001).

Fig. 3.

Effect of Fe concentration and age on the mRNA expression of DMT1 in the duodenal mucosa.

^a-d Means with different letters within the same row differ (P < 0.05). Each value represents the mean of 6 replicates (n = 8).

Fig. 4.

Effect of Fe concentration and age on the mRNA expression of FPN1 in the duodenal mucosa

^a-d Means with different letters within the same row differ (P < 0.05). Each value represents the mean of 6 replicates (n = 8).

The mRNA expression of hypoxia-inducible factors

The effects of age and dietary Fe concentration on the mRNA expression of HIFs in the duodenal mucosa are illustrated in Fig. 5, Fig. 6. The results indicated that there was an interaction between age and dietary Fe concentration on duodenal mRNA expression of HIF-1α (P = 0.007). The expression of HIF-1α was elevated in 63-day-old birds when dietary Fe supplementation exceeded 20 mg/kg (P = 0.040), but dietary Fe supplementation did not affect duodenal HIF-1α expression at d 21 (P = 0.138). No interaction was detected on duodenal HIF-2α expression (P = 0.906). The expression of HIF-2α in duodenal mucosa decreased with increasing concentrations of dietary Fe supplementation (P = 0.034), but the change was not associated with age (P = 0.222).

Fig. 5.

Effect of Fe concentration and age on the mRNA expression of HIF-1α in the duodenal mucosa.

^a-d Means with different letters within the same row differ (P < 0.05). Each value represents the mean of 8 replicates (n = 8).

Fig. 6.

Effect of Fe concentration and age on the mRNA expression of HIF-2α in the duodenal mucosa.

^a-d Means with different letters within the same row differ (P < 0.05). Each value represents the mean of 8 replicates (n = 8).

Discussion

The results partially support our hypothesis, indicating that high dietary Fe supplementation impaired growth performance and decreased duodenal mucosal Cu and Mn concentrations, while low dietary Fe supplementation showed no positive effect on those indicators. However, duodenal Zn concentrations or the hepatic depositions of metal ions were not affected by dietary Fe supplementations. Notably, younger birds exhibited a weaker transcriptional response to dietary Fe supplementation in genes regulating Fe homeostasis than older ones. To date, the phenomenon of Fe overloading in feed rations has gradually gained an increasing amount of attention. It is well known that feed-grade limestone and dicalcium phosphate contain significant amounts of Fe as impurities, with concentrations as high as 2,410-12,600 mg/kg (Sullivan et al., 1994). Additionally, the grain-derived feed ingredients have been found to contain considerable Fe, which may derive not only from intrinsic grain components but also from soil contamination introduced during processing (Hansen et al., 2010). Some common feedstuffs that contain considerable Fe are corn (36 mg/kg), wheat (203.2 mg/kg), soybean meal (185 mg/kg), limestone (2649 mg/kg), and dicalcium phosphate (14,100 mg/kg) (Sullivan et al., 1994; Wu et al., 2025). Accordingly, our previous studies showed that a corn‑soybean meal basal diet without Fe supplementation provides adequate Fe for yellow‑feathered broilers (Chen et al., 2025; Lei et al., 2024). Not considering the Fe inherent in feed ingredients, supplemental mineral Fe in broiler diets may lead to excessive total Fe content. Short-term or long-term Fe exposure to birds has been confirmed to induce oxidative stress in broilers and impair the function of their intestinal mechanical barrier (Chen et al., 2025; Lei et al., 2024). Additionally, in order to safeguard animal welfare in production, an upper limit of 450 mg/kg has been established by the European Union (EFSA, 2014). However, from the results available in our previous research, this limited amount of Fe (450 mg/kg) has also brought obvious negative effects on birds (Chen et al., 2025).

We observed that high dietary Fe concentration decreased ADG and ADFI in broilers, and the response did not differ across age groups. Han et al. (2022) found that supplementing broilers with 800 mg/kg Fe reduced ADG and increased FCR at d 21, while supplementation at 80 mg/kg induced similar effects at d 42. This demonstrates that the adverse effects of high-dose Fe supplementation are long-term, and the harm from cumulative Fe exposure becomes more pronounced with increasing age. This conclusion is consistent with that of our previous study (Chen et al., 2025). Age of broilers in the current study did not show a difference in response to high doses of Fe, probably because of the relatively large gradient of Fe supplementation in this trial. Body weight gain is affected by multiple factors, among which feed intake exerts a dominant influence. Feed intake is regulated by appetite-modulating hormones produced in the gastrointestinal tract and adipose tissue (Ranft and Hennig, 1991). Since feeding high Fe reduces fat deposition (Lei et al., 2022) and causes oxidative stress that damages intestinal tissues in broilers of different ages (Chen et al., 2025; Lei et al., 2024), excess Fe may disrupt the homeostasis of these feeding-related hormones, ultimately leading to aberrant feed intake. In addition, intestinal damage induced by high Fe, which reduces the digestion and absorption of nutrients, may also be an important cause of reduced BW gain.

Increased duodenal mucosa and liver Fe concentrations were observed with increased dietary Fe supplementation. The more rapid elevation in duodenal mucosa versus liver Fe levels reflects active homeostatic regulation of systemic Fe balance. The duodenum is the primary site of Fe absorption, while the liver is the main Fe storage site (Vogt et al., 2021). Dietary Fe, primarily in the form of Fe²⁺, is absorbed by enterocytes via the DMT1 transporter. Absorption of Fe³⁺ must be reduced to Fe²⁺ by duodenal cytochrome b on the duodenal brush border before absorption. Younger animals exhibit higher Fe absorption efficiency than older animals (Forbes and Reina, 1972). In this study, dietary supplementation of 80 mg/kg Fe significantly increased hepatic Fe accumulation in yellow-feathered broilers on d 21, whereas 320 mg/kg Fe was required to observe significant changes in broilers on d 63. These findings provide indirect evidence for age-dependent variations in animals' regulatory capacity to dietary Fe overload, consistent with findings by Hansen et al. (2010) who reported age-dependent expression of certain genes involved in Fe homeostasis regulation in pigs.

Dietary Fe supplementation reduced duodenal mucosal Cu and Mn concentrations, with mucosal Fe concentrations exhibiting significant negative correlations with both elements. In contrast, mucosal and hepatic Zn concentrations remained unaffected by dietary Fe and showed no correlation with other trace elements. This differential response likely arises from distinct intestinal absorption and transport mechanisms. Current evidence indicates that Fe, Cu, Mn, and Zn absorption requires initial binding to the intestinal mucus layer. Unbound ions may precipitate due to gastrointestinal pH changes, rendering them unavailable for absorption (Einhorn et al., 2024). Competition for shared mucoprotein binding sites likely explains the reduced mucosal Cu and Mn, as well as their negative correlations with Fe. Additionally, competitive inhibition between divalent metals at transporters further elucidates these correlations.

Manganese absorption primarily relies on the Fe transport proteins DMT1 and FPN1. Intestinal epithelial cells exhibit preferential Fe uptake over manganese when both minerals are present (Prajapati et al., 2024), establishing a competitive inhibition mechanism whereby elevated Fe concentrations suppress Mn absorption through shared pathways. This is demonstrated by mouse perfusion studies showing duodenal Fe overload competitively inhibits Mn absorption (Thomson et al., 1971), with analogous findings in bovine models (Hansen et al., 2009) and consistent observations in 42-day-old broilers (Bai et al., 2020). Copper absorption and transport depend on specialized transporters and chaperone proteins (Chen et al., 2022). However, Cu competes with Fe for shared transporters (e.g., DMT1) and enzymes (e.g., duodenal cytochrome b), establishing a negative correlation between duodenal mucosal Cu and Fe concentrations (Einhorn et al., 2024). This competitive inhibition is evidenced by reduced Cu concentrations and induced deficiency in rats following excessive Fe intake (Lee et al., 2021; Doguer et al., 2018). In contrast, Zn maintains stable mucosal concentrations during dietary Fe supplementation, indicating non-competitive absorption. This finding aligns with Zou et al. (2017). Zinc absorption relies on a dedicated transporter system, primarily ZnT-1 in the proximal small intestine, which critically maintains zinc homeostasis (Krebs, 2000). This molecular independence minimizes competition with Fe for transporter binding sites. Furthermore, Zn stimulates mucin secretion (Einhorn et al., 2024), which synergistically enhances ion absorption efficiency. Together, these mechanisms explain why Fe imbalance exerts limited effects on mucosal Zn concentrations.

Notably, while higher Fe concentration reduced duodenal absorption of Cu and Mn, hepatic concentrations remained stable. This aligns with Baker and Halpin (1991), who observed unchanged Mn deposition in chicken liver and bone across varying Fe concentrations. We further found lower hepatic Fe accumulation rates than duodenal mucosa, demonstrating effective homeostatic regulation of Fe in poultry, primarily mediated through intestinal regulation. This conclusion is consistent with that in the pig model in Hansen et al. (2010).

Factor analysis revealed age-dependent differences in duodenal Zn and Mn uptake, with younger broilers exhibiting enhanced uptake. This phenomenon may be attributed to the elevated metabolic demands for metalloenzymes requiring Cu and Mn as cofactors during rapid growth (Zelko et al., 2002). Interestingly, hepatic deposition patterns showed a different relationship, where the accumulation of Zn in liver tissue remained constant. Zinc is well-established as a critical nutrient for animal growth, with Zn-dependent enzymes playing fundamental roles in protein synthesis and skeletal development (Salgueiro et al., 2002). Similarly, Mn is a key component in bone matrix formation and calcification processes (Cotzias, 1958; Gallup and Norris, 1938). Distinct growth priorities at different developmental stages may explain the differential utilization patterns. According to NRC (1994) and MAPRC (2020), 21-day-old broilers prioritize protein accretion over skeletal mineralization, while more mature birds shift toward bone strengthening with reduced protein deposition rates. This physiological transition likely accounts for the observed age-related differences in mineral metabolism. Furthermore, the decreased hepatic Mn deposition in older birds may reflect increased skeletal Mn sequestration, as bone represents the primary Mn storage site in poultry.

Regulation of excessive Fe intake in animals primarily occurs in the intestinal tract (Hansen et al., 2010). Given that the duodenum and proximal jejunum are key sites for Fe absorption and other trace minerals, avian Fe homeostasis is primarily governed by regulating Fe uptake and transport in these intestinal segments. Studies indicate that DMT1 and FPN1 are the primary transporters involved in Fe absorption (Mastrogiannaki et al., 2013). The absorption of Fe²⁺ in the duodenum and proximal jejunum is mediated by DMT1, while the sole transporter responsible for Fe²⁺ efflux from enterocytes into circulation is FPN1 (Vogt et al., 2021). Our results showed age differences between the expression of DMT1 and FPN1 in the duodenal mucosa, with older animals having higher expression of DMT1 and FPN1 than younger ones.

In the past, younger animals were believed to have a higher Fe requirement and absorption efficiency (Forbes and Reina, 1972). However, this requirement is based on a unit weight. Birds on d 63 possessed a higher body weight than those on d 21, and their total Fe requirement to maintain normal physiological needs increased despite a lower Fe requirement per unit body weight. This elevated total demand may consequently lead to enhanced mRNA expression of Fe transport-related proteins. In addition, the expression of DMT1 and FPN1 at both ages was strictly regulated by dietary Fe, confirming systemic Fe homeostasis throughout development. These results are consistent with observations of a previous study, which documented identical downregulation patterns in d 7 Arbor Acres broilers regardless of Fe source (Bai et al., 2021). The consistent suppression of transporters in response to elevated Fe concentrations across ages demonstrates a functional regulatory pathway where Fe concentration serves as the primary signal, preventing excessive absorption through transcriptional control.

In mammals, elevated dietary Fe concentrations increase hepcidin concentration, a peptide hormone secreted by hepatocytes that regulates duodenal Fe absorption, macrophage Fe recycling, and hepatic Fe release (Nemeth and Ganz, 2023). When Fe is sufficient, non-transferrin-bound Fe stimulates hepatocytes to produce hepcidin, which downregulates the mRNA expression of DMT1 and FPN1, as well as ferroxidases (Bergamaschi et al., 2017), thereby limiting intestinal Fe absorption and storage release (Nemeth and Ganz, 2023). Although Fe homeostasis regulation has been confirmed in poultry, no hepcidin homolog has yet been identified (Hilton and Lambert, 2008). While our results confirmed the existence of systemic Fe homeostasis regulation in poultry, the key molecular mediators governing this process remain to be fully characterized.

Age-stratified HIFs responses to dietary Fe reveal developmentally regulated homeostasis in poultry, with distinct mechanistic patterns emerging at different growth stages. These stage-specific modulations indicate incomplete maturation of Fe regulatory pathways in young birds, consistent with prior observations in swine (Hansen et al., 2010). Hypoxia-inducible factors are master transcriptional regulators of cellular adaptation to hypoxia. These heterodimers, composed of α and β subunits, are influenced by oxygen concentration and have been shown to regulate Fe homeostasis in mammals (Mastrogiannaki et al., 2013). Based on their α subunits, HIFs are classified into HIF-1α, HIF-2α, and HIF-3α, with HIF-1α and HIF-2α regulating distinct yet overlapping target genes (Shah and Xie, 2014). Under normoxia, HIF-α is hydroxylated at proline residues within its oxygen-dependent degradation domain (ODD) by prolyl-4-hydroxylase domain enzymes (PHDs), leading to its binding to the tumor suppressor protein pVHL and subsequent degradation (Mastrogiannaki et al., 2013). Hypoxia-inducible factor 1α, sensitive to oxygen concentrations, promotes erythropoietin expression, and its hydroxylation and degradation are inhibited by mitochondrial ROS (Shah and Xie, 2014). While HIF-1α is not essential for Fe absorption (Mastrogiannaki et al., 2009), it can enhance the transcription of the ferroptosis-related gene TfR1, triggering ferroptosis (Sanguigno et al., 2023). Xu et al. (2014) found that FeSO₄ supplementation in rats increased hepatic HIF-1α gene expression, a finding that is broadly consistent with the trends observed in the present study. Based on these findings, we could infer that excessive Fe uptake by intestinal epithelial cells may trigger a cascade involving Fenton reaction-mediated mitochondrial reactive oxygen species accumulation, which subsequently activates HIF-1α-dependent ferroptosis pathways, ultimately leading to epithelial cell shedding as a potential mechanism for excess Fe elimination. Young animals did not show changes in HIF-1α expression, suggesting that their Fe excretory processes may be limited, corroborating the conclusion of Hansen et al. (2010) that the regulation of Fe stabilization in young animals is poorly developed. Hypoxia-inducible factor 2α, sensitive to intracellular Fe and oxygen concentrations, stabilizes in duodenal cells under Fe deficiency and transcriptionally upregulates DMT1 and FPN expression to enhance Fe absorption (Mastrogiannaki et al., 2013). In mice, duodenal HIF-2α expression is regulated by hepatic hepcidin (Lee, 2019) and acts downstream of hepcidin-mediated FPN degradation, with HIF-2α inhibition showing therapeutic potential for Fe overload in rats (Schwartz et al., 2019). Under high Fe conditions, HIF-2α is degraded via the pVHL pathway (Talbot et al., 2014) through PHD activity mediated by FPN (Schwartz et al., 2019), thereby inhibiting further intestinal Fe absorption. Expression of HIF-2α in the duodenal mucosa was affected only by dietary Fe supplementation and not by age, indicating its conserved regulatory role. These findings strongly suggest that HIF-2α may serve as a key regulatory component in avian Fe homeostasis. Based on an integrated analysis of HIFs, DMT1, and FPN1 expression patterns, we suggest that HIF-1α and HIF-2α are jointly involved in the process of Fe homeostasis regulation in poultry, although this function takes some time to develop. In this study, due to the lack of specific antibodies, only mRNA expression of genes involved in Fe homeostasis regulation was determined, and future research should examine the effect of animal age and dietary Fe concentration on expression of proteins involved in Fe metabolism.

In summary, high dietary Fe reduces the duodenal mucosal uptake of Cu and Mn, potentially leading to deficiencies of these trace elements. Upon excessive Fe intake, the expression of Fe absorption and transport proteins, DMT1 and FPN1, may be down-regulated through the HIF-2α-DMT1/FPN1 axis, limiting further Fe absorption. Simultaneously, high Fe levels may activate the ROS-HIF-1α axis in duodenal epithelial cell mitochondria, promoting ferroptosis and shedding of these cells, thereby facilitating the excretion of excess Fe through feces.

Conclusions

Dietary Fe supplementation exceeding 80 mg/kg (152.47 mg Fe /kg total diet) triggers an intrinsic Fe homeostatic mechanism in broilers to prevent excessive Fe accumulation. The HIF‑2α may play a critical regulatory role in this process, and its modulatory effect seems to be weaker in juvenile birds.

CRediT authorship contribution statement

J. Chen: Writing – original draft, Validation, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. K.W. Lei: Writing – original draft, Validation, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. J.W. Spears: Writing – review & editing, Validation, Resources, Project administration, Methodology, Conceptualization. D.P. Li: Writing – review & editing, Resources, Project administration, Methodology, Conceptualization. X. Wang: Writing – review & editing, Resources, Project administration, Methodology, Conceptualization. X. Bai: Writing – review & editing, Resources, Project administration, Methodology, Conceptualization. Y.L. Huang: Writing – review & editing, Visualization, Resources, Project administration, Methodology, Funding acquisition, Formal analysis, Data curation, Conceptualization.

Disclosures

We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, and there is no professional or other personal interest of any nature or kind in any product, service, company, or both that could be construed as influencing the content of this paper.