Melatonin reduces intestinal oxidative damage in broilers with pulmonary hypertension under high-altitude and cold stress

Highlights

• •Melatonin improved intestinal morphology and antioxidant status in PHS-affected broilers at high altitude.
• •Mel-40 increased body weight and lowered RV:TV ratio, indicating cardioprotection.
• •Mel-20 markedly reduced intestinal MDA levels (stronger antioxidant effect).
• •Both doses improved FCR; Mel-40 showed broader jejunum/ileum antioxidant gene upregulation.

Abstract

Pulmonary hypertension syndrome (PHS) in broilers, exacerbated by high-altitude and cold stress, leads to oxidative damage and intestinal dysfunction. Melatonin, with its antioxidant and anti-inflammatory properties, may mitigate these effects. This study examined how melatonin affects intestinal health and oxidative balance in broilers with PHS. We reared 108 broiler chickens at 2100 m altitude under cold stress and divided them into three groups: control, 20 mg/kg melatonin (Mel-20), and 40 mg/kg melatonin (Mel-40). Intestinal morphology, malondialdehyde (MDA) levels, and antioxidant gene expression (SOD1, GPX) were analyzed. Performance metrics and right-to-total ventricular weight ratio (RV:TV) were also assessed. Mel-40 increased body weight (P < 0.05), while both doses improved feed conversion ratio (P < 0.05). The Mel-40 group showed a lower RV:TV compared to the control group (P < 0.05). Mel-20 lowered MDA levels across all intestinal segments (P < 0.05), with large effect sizes in the duodenum (d = 1.41) and jejunum (d = 2.23). Mel-40 upregulated SOD1 in the jejunum and ileum and GPX in the ileum (P < 0.05), whereas Mel-20 increased both SOD1 and GPX expression in the duodenum and jejunum (P < 0.05). Villus height, width, and surface area increased in the duodenum and jejunum for both doses (P < 0.05). Melatonin supplementation improved intestinal health, antioxidant status, and performance in broilers with PHS under high-altitude and cold stress conditions. Optimal melatonin dosing depends on the therapeutic goal: 20 mg/kg for intestinal antioxidant protection versus 40 mg/kg for cardiovascular support.

Graphical abstract

1. Introduction

Pulmonary hypertension syndrome (PHS) represents a significant metabolic disorder in modern poultry production, particularly affecting fast-growing broilers and resulting in substantial economic losses. This condition develops when increased pulmonary arterial pressure leads to right ventricular hypertrophy, ultimately progressing to heart failure and abdominal fluid accumulation (Chen et al., 2025). The pathogenesis of PHS involves complex interactions between genetic predisposition, rapid growth rates, and environmental factors, with oxidative stress playing a central role in disease progression (Hossain & Akter, 2022). The high metabolic demands of rapidly growing broilers create an oxygen supply-demand imbalance, triggering hypoxia-induced pulmonary vasoconstriction that is further exacerbated by dysregulation of vasoactive mediators, including elevated endothelin-1 and angiotensin alongside reduced nitric oxide bioavailability (Ahmadipour et al., 2026; H Hassanpour et al., 2019; Miao et al., 2022).

At the cellular level, PHS is characterized by profound oxidative stress resulting from excessive reactive oxygen species production through multiple pathways, including mitochondrial dysfunction and NADPH oxidase activation (Oke et al., 2024). This oxidative burden overwhelms endogenous antioxidant defenses, leading to widespread cellular damage through lipid peroxidation, protein modification, and DNA injury (Oke et al., 2024). Environmental stressors like cold exposure and high-altitude conditions markedly worsen these pathological mechanisms through different but complementary pathways (Hossain & Akter, 2022). Cold stress elevates metabolic rate and oxygen requirements while diminishing antioxidant defenses, creating a physiological burden that compels birds to boost cardiac output. High-altitude environments compound this stress through chronic oxygen deprivation, triggering adaptive responses like polycythemia that increase blood viscosity and pulmonary vascular resistance (Biswas, 2019). These combined pressures force the cardiovascular system to work harder to maintain oxygenation - cold stress by driving up metabolic demands and altitude by limiting oxygen availability. This double assault significantly stresses an already overtaxed cardiovascular system, dramatically hastening right ventricular hypertrophy development (Ahmadipour et al., 2025; Chen et al., 2025).

The gastrointestinal system emerges as both a victim and contributor in this pathological cascade, with intestinal mucosa being particularly vulnerable to oxidative damage due to its high metabolic activity and constant exposure to luminal oxidants (Li et al., 2024). Cold stress-induced vasoconstriction reduces intestinal perfusion, creating conditions ripe for ischemia-reperfusion injury and subsequent barrier dysfunction. These changes manifest structurally as villus atrophy and crypt hyperplasia, and functionally as tight junction protein disruption and increased permeability, facilitating bacterial translocation and systemic inflammation (Faraji et al., 2020; Li et al., 2024). Within this context, melatonin's pleiotropic actions - including its potent antioxidant properties, anti-inflammatory effects, and ability to enhance mitochondrial function - position it as a promising therapeutic candidate for mitigating both the primary pathology of PHS and its exacerbation by environmental stressors like cold exposure (Bahadoran et al., 2024).

Melatonin, a potent endogenous antioxidant and free radical scavenger, has demonstrated protective effects in various oxidative stress-related conditions. Beyond its role in regulating circadian rhythms, melatonin modulates antioxidant enzyme activity, reduces lipid peroxidation, and enhances cellular repair mechanisms (Ahmad et al., 2023; Calislar et al., 2018). It directly neutralizes reactive oxygen species (ROS), while also stimulating the expression of key antioxidant enzymes such as superoxide dismutase (SOD) and glutathione peroxidase (GPX). Additionally, melatonin enhances mitochondrial function, reduces pro-inflammatory cytokine production, and promotes tissue regeneration (Ahmad et al., 2023). These multifaceted actions make melatonin a promising therapeutic agent for mitigating oxidative damage in metabolic and cardiovascular disorders, including pulmonary hypertension syndrome in poultry (Bahadoran et al., 2024; Fathi et al., 2023). Given the critical role of intestinal health in poultry performance and the potential of melatonin to mitigate oxidative damage, this study aims to investigate the impact of melatonin on intestinal morphology and the oxidant/antioxidant balance in chickens with pulmonary hypertension induced by high altitude and cold stress. Understanding these effects could provide valuable insights into novel therapeutic strategies to improve gut health and reduce the incidence of PHS in commercial poultry production.

2. Materials and methods

2.1. Rearing of birds at high altitudes under cold stress, and treatments

The experiment was conducted at the experimental facility of Shahrekord University, situated at an altitude of 2100 m above sea level in Shahrekord, Iran. This location presents extreme environmental challenges, characterized by a cold, semi-arid continental climate with severe winter temperatures (historic lows near −19 °C), compounded by chronic hypobaric hypoxia due to low atmospheric pressure, aridity with minimal precipitation (∼172 mm/year), and average humidity around 36%. These harsh conditions made the research difficult to execute. Maintaining animal welfare and collecting viable data required intensive environmental controls and constant monitoring. High mortality risks also made it challenging to achieve adequate sample sizes, which is why large-scale studies under such extreme, combined stressors are rare in poultry science.

The study complied with the ethical guidelines established by the Guide for the Care and Use Committee of Shahrekord University. A total of 108 one-day-old male broiler chickens (Ross 308) were randomly allocated to 36 floor pens, each measuring 1.5 m², with 12 birds per pen. Each pen was furnished with a bell drinker and a feed trough. The chicks were initially reared on a commercial broiler diet until five days of age. After an eight-hour fasting period and the removal of runts, the five-day-old chicks were distributed among the pens to ensure equal average body weight per pen. The birds were maintained under progressively decreasing environmental temperatures according to age: 32±1 °C from days 1 to 7, 25±1 °C from days 8 to 14, 20±1 °C from days 15 to 21, and 15±1 °C from days 22 to 42 (Hassanpour et al., 2023). Throughout the trial, the chicks had ad libitum access to feed and water and were subjected to a lighting schedule of 23 h of light followed by 1 h of darkness.

The control diet, formulated based on corn and soybean meal, was designed for three distinct growth phases: starter (1–10 days), grower (11–28 days), and finisher (29–42 days), following our previous study (Gheytaspour et al., 2025). Two experimental diets were prepared by supplementing the control diet with melatonin powder (≥98% purity, verified by thin-layer chromatography and supplied by RazakPharma Co., Tehran, Iran) at concentrations of 20 mg/kg (Mel-20) and 40 mg/kg (Mel-40), respectively.

2.2. Bird performance and sampling

Body weight gain, feed intake, and feed conversion ratio were calculated for the entire experimental period, spanning from day 1 to day 42. At 42 days of age, all birds were subjected to a 6-hour fasting period with unrestricted access to water to ensure empty digestive tracts and reduce variability in intestinal measurements. Subsequently, 10 birds per group were euthanized by decapitation using a sharp blade, following approved ethical protocols to guarantee instantaneous and humane death. Immediately post-euthanasia, the abdominal cavity was opened, and the entire small intestine was carefully excised. The intestinal segments were delineated as follows: the duodenum, extending from the gizzard to the entry of the bile duct; the jejunum, located between the bile duct entry and Meckel’s diverticulum; and the ileum, spanning from the distal end to the ileocecal junction. From each segment, a 2.00 cm section was collected at standardized anatomical landmarks-specifically, the midpoint of the duodenal loop, the midpoint of the jejunum, and a point 5 cm proximal to the ileocecal junction for the ileum. These tissue samples were immediately rinsed with ice-cold phosphate-buffered saline (PBS, pH 7.4) to remove luminal contents and processed for fixation within two minutes of collection to preserve tissue integrity. For molecular analyses, samples were rapidly frozen in liquid nitrogen and stored at –70 °C for subsequent RNA extraction. For histological preservation, samples were fixed in Clark’s solution (comprising 25% acetic acid and 75% ethyl alcohol) for 45 min, followed by transfer to 55% ethyl alcohol for long-term storage (Abaszadeh et al., 2026; Bahadoran et al., 2025).

The heart ventricles were carefully dissected and weighed to determine the right-to-total ventricular weight ratio (RV:TV ratio), which is utilized as a diagnostic indicator of PHS. Cases in which the RV:TV ratio ≥0.29 were classified as indicative of developed PHS (Hassanpour et al., 2023).

2.3. Measurement of intestinal malondialdehyde (MDA)

All reagents utilized in this assay were procured from Sigma-Aldrich (St. Louis, MO, USA). Frozen intestinal tissue segments were homogenized using a sonicator. MDA, a widely recognized biomarker of lipid peroxidation, was quantified in the homogenates employing the thiobarbituric acid reactive substances (TBARS) assay. To precipitate proteins, trichloroacetic acid was added to the samples, followed by centrifugation to separate the supernatant. Subsequently, an equal volume of thiobarbituric acid was added to the supernatants, and the mixtures were incubated in a boiling water bath for 10 min. After cooling, absorbance was measured at 532 nm using a spectrophotometer (Corning 480, USA). The absorbance values obtained from the TBARS assay were then converted to micromolar concentrations (µM) for quantitative analysis (Tohidifar et al., 2023).

2.4. RNA isolation, cDNA synthesis, and quantitative real-time PCR analysis

Total RNA was extracted from frozen samples of three intestinal segments using RNXPlus reagent (Sinaclon Bioscience, Tehran, Iran). Approximately 100 mg of homogenized tissue was lysed in digestion buffer and mixed with chloroform. Following centrifugation, RNA was isolated from the upper aqueous phase, precipitated with isopropanol, washed with 75% ethanol, and resuspended in DEPC-treated water. Residual genomic DNA was removed by DNase treatment (Sinaclon Bioscience), and RNA concentration and purity were assessed spectrophotometrically. Only samples with an A260/A280 ratio above 1.9 were used for complementary DNA (cDNA) synthesis. cDNA was synthesized from total RNA using the First Strand cDNA Synthesis Kit (Pars Toos, Iran). The reverse transcription reaction was briefly heated at 85 °C for 5 s to inactivate reverse transcriptase and denature RNA, then stored at –20 °C.

Quantitative real-time PCR was performed to measure expression levels of SOD1, GPX, and YWHAZ (tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta), with YWHAZ serving as the endogenous reference gene (Hossein Hassanpour et al., 2019). Primers were designed and represented in Table 2. PCR reactions were conducted in triplicate for each sample using the Rotor-Gene Q 6000 system (Qiagen, USA) with RealQ Plus 2x Master Mix Green (Ampliqon, Odense, Denmark). Each 10 µL reaction contained 0.5 µL cDNA, 5 µL Master Mix Green, and 0.5 mM of each primer. Thermal cycling conditions comprised an initial denaturation at 95 °C for 10 min, followed by 45 cycles of 94 °C for 15 s and annealing/extension at 60–62 °C for 10–20 s. Fluorescence data were collected at the end of each cycle.

Table 2.

Details of the primers used for quantitative real time PCR analysis for chickens.

Target	Primers	PCR product	Accession no.
YWHAZ	5′-AGGAGCCGAGCTGTCCAATG-3′ 5′-CTCCAAGATGACCTACGGGCTC-3′	84 bp	NM_001031343.1
SOD1	5′-CACTGCATCATTGGCCGTACCA-3′ 5′-GCTTGCACACGGAAGAGCAAGT-3′	224 bp	NM_205,064.1
GPX	5′-GCTGTTCGCCTTCCTGAGAG-3′ 5′-GTTCCAGGAGACGTCGTTGC-3′	118 bp	NM_001277853.1

YWHAZ, tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta; SOD1, superoxide dismutase 1; GPX, glutathione peroxidase; bp, base pair.

Gene expression levels were normalized to YWHAZ. Data analysis was performed using LinRegPCR software version 2012.0 (Amsterdam, Netherlands) to determine threshold cycle (Ct) values and PCR efficiencies. Relative transcript abundance and fold changes were calculated using the efficiency-corrected Pfaffl method (Dorak, 2007; Hassanpour et al., 2015).

2.5. Measurement of intestinal villus dimensions and surface area

Following fixation and staining with periodic acid–Schiff (PAS) reagent, the intestinal segments were meticulously dissected to isolate the mucosal layer. Villus height and width were quantified using a calibrated Olympus CX23 light microscope equipped with an ocular micrometer at 10× magnification. For each intestinal segment (duodenum, jejunum, and ileum), three intact and properly oriented villi were randomly selected per sample. Villus height was defined as the perpendicular distance from the villus tip to the villus-crypt junction base, explicitly excluding crypt depth, with measurements taken along the central axis of each villus. Villus width was measured at the villus’s broadest point, perpendicular to the height axis. To ensure measurement reliability, three independent readings were recorded per villus, and their mean was calculated. Only villi exhibiting vertical orientation, intact tips, and a visible lamina propria were included to prevent artifacts caused by oblique sectioning. Final measurements were expressed in millimeters (mm) based on the microscope’s calibration scale. Additionally, lamina propria thickness was measured at the villus base. Villus surface area was calculated using the formula:

Villus Surface Area = π × Villus Width (VW) × Villus Length (VL), where VW represents villus width and VL represents villus height (Hassanpour et al., 2016) .

2.6. Statistical analysis

Data are presented as mean ± standard error of the mean (SEM). Normality of data distribution was verified using the Kolmogorov-Smirnov test. Parametric statistical methods were applied to normally distributed variables. Intergroup comparisons were analyzed using one-way analysis of variance (ANOVA). Additionally, effect sizes were calculated using Cohen's d to assess the magnitude of differences between groups. Cohen's d values were interpreted as: negligible (< 0.20), small (0.20–0.49), medium (0.50–0.79), and large (≥ 0.80). Effect sizes were reported with 95% confidence intervals. All statistical procedures were conducted using SPSS version 26.0 (IBM Corp., Armonk, NY, USA), with statistical significance established at P < 0.05.

3. Results

3.1. Effect of melatonin on chicken performance and PHS index

Fig. 1 indicates the effect of two concentrations of melatonin (20 and 40 mg/kg) on broiler chickens' body weight, feed intake, and FCR at high altitude under cold stress. The body weight was only increased in the Mel-40 group compared to the control group (P = 0.021), with no change in the Mel-20 group (P = 0.083). The FCR was decreased in the Mel-20 and Mel-40 groups compared to the control group (P = 0.014 and P = 0.009, respectively), while it did not differ between the two melatonin-treated groups (P = 0.612). The feed intake did not differ significantly among groups (overall ANOVA: P = 0.113). This suggests that melatonin improved metabolic efficiency rather than appetite, with the 40 mg/kg dose providing additional benefits for growth, possibly through enhanced cardiovascular protection and systemic oxygenation.

Fig. 1.

Comparison of body weight, feed intake, and feed conversion rate (FCR) between the control, melatonin-20 mg/kg (Mel-20), and melatonin-40 mg/kg (Mel-40) groups. Data are represented as mean ± SEM. ^a,b significant difference statistically between the groups (P < 0.05).

The RV:TV ratio was decreased in the Mel-40 group (0.25 ± 0.005) compared to the control (0.31 ± 0.014) (P = 0.032), while it did not differ between the Mel-20 (0.27 ± 0.012) and Mel-40 groups (P = 0.152). The reduction in RV:TV ratio in the Mel-40 group indicates that melatonin alleviated pulmonary hypertension severity, likely through mitigating oxidative stress in pulmonary vasculature and reducing right ventricular workload.

3.2. Effect of melatonin on intestinal MDA levels and genes of SOD1/GPX expression

Fig. 2 shows that MDA levels were significantly lower in the Mel-20 group compared to both the control and Mel-40 groups across the duodenum, jejunum, and ileum (duodenum: P = 0.014; jejunum: P = 0.001; ileum: P = 0.003). However, no significant differences in MDA levels were observed between the Mel-40 and control groups in any of the intestinal segments (duodenum: P = 0.228; jejunum: P = 0.063; ileum: P = 0.108). Effect size analysis using Cohen's d further characterized the magnitude of these differences, as shown in Table 3. In the duodenum, the reduction in MDA for control vs Mel-20 showed a large effect size (d = 1.41, P = 0.014), whereas control vs Mel-40 showed a medium effect size (d = 0.63, P = 0.228). In the jejunum, both Mel-20 and Mel-40 showed large effect sizes compared with the control group (d = 2.23, P = 0.001 and d = 1.01, P = 0.063, respectively). In the ileum, control vs Mel-40 showed a large effect (d = 1.86, P = 0.002), whereas control vs Mel-20 showed only a small effect (d = 0.37, P = 0.469). The comparison between Mel-20 and Mel-40 revealed a large effect in the ileum (d = −1.16, P = 0.037), and large effects in the duodenum and jejunum as well (d = −0.87, P = 0.104 and −0.88, P = 0.101 respectively). The 20 mg/kg dose more effectively reduced intestinal lipid peroxidation, while the 40 mg/kg dose showed weaker effects, possibly due to a biphasic dose-response or pro-oxidant activity at higher concentrations.

Fig. 2.

Comparison of malondialdehyde (MDA) levels between the control, melatonin-20 mg/kg (Mel-20), and melatonin-40 mg/kg (Mel-40) groups in different intestinal segments. Data are represented as mean ± SEM. ^a,b significant difference statistically between the groups (P < 0.05).

Table 3.

Cohen's d effect sizes for malondialdehyde levels in intestinal tissues.

Tissue	Comparison	Cohen's d	95% CI	Magnitude	p-value
Duodenum	Control vs Mel-20	1.41	(0.30, 2.52)	Large	0.014
	Control vs Mel-40	0.63	(−0.38, 1.64)	Medium	0.228
	Mel-20 vs Mel-40	−0.87	(−1.90, 0.16)	Large	0.104
Jejunum	Control vs Mel-20	2.23	(0.95, 3.51)	Large	0.001
	Control vs Mel-40	1.01	(−0.04, 2.06)	Large	0.063
	Mel-20 vs Mel-40	−0.88	(−1.91, 0.15)	Large	0.101
Ileum	Control vs Mel-40	1.86	(0.66, 3.06)	Large	0.002
	Control vs Mel-20	0.37	(−0.62, 1.36)	Small	0.469
	Mel-20 vs Mel-40	−1.16	(−2.23, −0.09)	Large	0.037

As shown in Fig. 3, the SOD transcript levels were significantly elevated in the duodenum and jejunum of the Mel-20 group compared to the control group (duodenum: P = 0.024; jejunum: P = 0.007). The Mel-40 group also exhibited increased SOD transcript expression in the jejunum and ileum compared to the control group (jejunum: P = 0.035; ileum: P = 0.004). Effect size analysis using Cohen's d further characterized the magnitude of these differences, as shown in Table 4. In the duodenum, SOD1 expression for control vs Mel-20 showed a large effect size (d = −1.26, P = 0.024), whereas control vs Mel-40 showed a small effect size (d = −0.21, P = 0.679). In the jejunum, both Mel-20 and Mel-40 showed large effect sizes compared with the control group (d = −1.57, P = 0.007 and d = −1.17, P = 0.035, respectively). In the ileum, both Mel-20 and Mel-40 showed large effect sizes compared with the control group (d = −0.86, P = 0.108 and d = −1.70, P = 0.004, respectively). The comparison between Mel-20 and Mel-40 revealed a large effect in the duodenum (d = 0.98, P = 0.071), a negligible effect in the jejunum (d = 0.17, P = 0.741), and a small effect in the ileum (d = −0.32, P = 0.533). The segment-specific upregulation of SOD1 suggests that melatonin preferentially enhances frontline antioxidant defenses in regions most exposed to oxidative insults, with the 20 mg/kg dose being more effective in proximal segments and the 40 mg/kg dose showing greater efficacy in the distal ileum.

Fig. 3.

Comparison of superoxide dismutase (SOD) gene expression between the control, melatonin-20 mg/kg (Mel-20), and melatonin-40 mg/kg (Mel-40) groups in different intestinal segments. Data are represented as mean ± SEM. ^a,b significant difference statistically between the groups (P < 0.05).

Table 4.

Cohen's d effect sizes for superoxide dismutase 1 gene expression in intestinal tissues.

Tissue	Comparison	Cohen's d	95% CI	Magnitude	p-value
Duodenum	Control vs M-20	−1.26	(−2.35, −0.18)	Large	0.024
	Control vs M-40	−0.21	(−1.19, 0.77)	Small	0.679
	M-20 vs M-40	0.98	(−0.07, 2.02)	Large	0.071
Jejunum	Control vs M-20	−1.57	(−2.71, −0.43)	Large	0.007
	Control vs M-40	−1.17	(−2.24, −0.10)	Large	0.035
	M-20 vs M-40	0.17	(−0.81, 1.15)	Negligible	0.741
Ileum	Control vs M-20	−0.86	(−1.89, 0.17)	Large	0.108
	Control vs M-40	−1.70	(−2.86, −0.53)	Large	0.004
	M-20 vs M-40	−0.32	(−1.31, 0.67)	Small	0.533

As shown in Fig. 4, the GPX transcript levels were significantly elevated in the duodenum and jejunum of the Mel-20 group compared to the control group (duodenum: P = 0.045; jejunum: P = 0.041). The Mel-40 also increased GPX transcript expression only in the ileum compared to the control group (P = 0.018). Effect size analysis using Cohen's d further characterized the magnitude of these differences, as shown in Table 5. In the duodenum, GPX expression for control vs Mel-20 showed a large effect size (d = −1.09, P = 0.045), whereas control vs Mel-40 showed a small effect size (d = −0.26, P = 0.606). In the jejunum, both Mel-20 and Mel-40 showed large effect sizes compared with the control group (d = −1.12, P = 0.041 and d = −1.02, P = 0.060, respectively). In the ileum, control vs Mel-40 showed a large effect (d = −1.34, P = 0.018), whereas control vs Mel-20 showed a negligible effect (d = −0.180, P = 0.723). The comparison between Mel-20 and Mel-40 revealed a large effect in the duodenum (d = 0.93, P = 0.083), a medium effect in the jejunum (d = 0.75, P = 0.155), and a large effect in the ileum (d = −1.02, P = 0.059). The parallel pattern of GPX upregulation with SOD1 expression indicates coordinated activation of the antioxidant cascade, where melatonin enhances both superoxide dismutation and peroxide detoxification. The dose-dependent regional differences suggest that optimal melatonin dosing may vary according to the specific intestinal segment targeted for protection.

Fig. 4.

Comparison of glutathione peroxidase (GPX) gene expression between the control, melatonin-20 mg/kg (Mel-20), and melatonin-40 mg/kg (Mel-40) groups in different intestinal segments. Data are represented as mean ± SEM. ^a,b significant difference statistically between the groups (P < 0.05).

Table 5.

Cohen's d effect sizes for glutathione peroxidase gene expression in intestinal tissues.

Tissue	Comparison	Cohen's d	95% CI	Magnitude	p-value
Duodenum	Control vs M-20	−1.09	(−2.15, −0.04)	Large	0.045
	Control vs M-40	−0.26	(−1.24, 0.72)	Small	0.606
	M-20 vs M-40	0.93	(−0.11, 1.97)	Large	0.083
Jejunum	Control vs M-20	−1.12	(−2.19, −0.06)	Large	0.041
	Control vs M-40	−1.02	(−2.07, 0.03)	Large	0.060
	M-20 vs M-40	0.75	(−0.27, 1.77)	Medium	0.155
Ileum	Control vs M-20	−0.18	(−1.16, 0.80)	Negligible	0.723
	Control vs M-40	−1.34	(−2.43, −0.24)	Large	0.018
	M-20 vs M-40	−1.02	(−2.08, 0.03)	Large	0.059

3.3. Effect of melatonin on the intestinal villus dimensions and surface area

Table 1 indicates the effect of melatonin on the villus height, width, lamina propria, and surface area of duodenum, jejunum, and ileum in the experimental groups after 42 days.

Table 1.

Comparison of Morphologic indices of intestinal villi in different groups after 42 days.

Intestinal segment	Group	Villus height (mm)	Villus width (mm)	Lamina propria (mm)	Villus surface area (mm²)
Duodenum	Control	0.45ᵃ	0.14ᵃ	0.16ᵃ	0.19ᵃ
	Mel-20	0.41ᵃ	0.23ᵇ	0.19ᵃᵇ	0.29ᵇ
	Mel-40	0.53ᵇ	0.35ᶜ	0.26ᵇ	0.58ᶜ
	Pooled SEM	0.012	0.008	0.009	0.010
	P-value	0.011	0.009	0.031	0.012
Jejunum	Control	0.24ᵃ	0.18ᵃ	0.17ᵃ	0.13ᵃ
	Mel-20	0.34ᵇ	0.32ᵇ	0.19ᵃ	0.34ᵇ
	Mel-40	0.41ᶜ	0.19ᵃᵇ	0.24ᵇ	0.24ᶜ
	Pooled SEM	0.010	0.006	0.007	0.007
	P-value	0.007	0.018	0.039	0.009
Ileum	Control	0.16ᵃ	0.17	0.16	0.08
	Mel-20	0.24ᵇ	0.12	0.14	0.09
	Mel-40	0.18ᵃ	0.18	0.17	0.10
	Pooled SEM	0.005	0.006	0.004	0.004
	P-value	0.041	0.188	0.545	0.219

Mel-20, melatonin 20 mg/kg; Mel-40, melatonin 40 mg/kg. Data are represented as mean ± SEM. ^a,b,c significant difference statistically between the groups (P < 0.05).

The duodenal and jejunal villus height was significantly increased in the Mel-40 group compared to the control group (P = 0.038). Similarly, the Mel-20 group exhibited greater villus height in both jejunal and ileal segments compared to the control group (jejunum: P = 0.044; ileum: P = 0.015).

The duodenal villus width was only increased in the Mel-40 group compared to the control group (P = 0.047). Similarly, the Mel-20 group exhibited greater villus width only in both jejunal and duodenum segments compared to the control group (jejunum: P = 0.011; duodenum: P = 0.028).

The duodenal and jejunal villus lamina propria was only increased in the Mel-40 group compared to the control group (duodenum: P = 0.042; jejunum: P = 0.037). The Mel-20 group did not exhibit any change in villus lamina propria of three intestinal segments among the experimental groups (duodenum: P = 0.199; jejunum: P = 0.207; ileum: P = 0.102).

Villus surface area was significantly increased in both duodenal and jejunal segments of the Mel-20 and Mel-40 groups compared to control (duodenum: Mel-20 P = 0.031, Mel-40 P = 0.026; jejunum: Mel-20 P = 0.044, Mel-40 P = 0.019), but not in the ileum (overall ANOVA: P = 0.219). These structural enhancements improve intestinal absorptive capacity and likely underlie the improved FCR observed in both treatment groups.

4. Discussion

Melatonin improved intestinal health and performance in PHS-affected broilers. Both doses enhanced FCR without affecting feed intake, confirming metabolic efficiency rather than appetite stimulation. Only Mel-40 increased body weight and reduced RV:TV ratio, suggesting cardioprotection via oxidative stress mitigation (Bahadoran et al., 2024; Gheytaspour et al., 2025), supported by parallel intestinal antioxidant improvements.

Weight gain and FCR improvement dissociated under these stress conditions: only Mel-40 increased body weight (likely via cardiovascular protection), while both doses enhanced FCR through improved gut health compared to stressed controls. The absence of a non-stressed control group means we cannot determine whether these parameters returned to 'normal' levels, only that melatonin mitigated some of the stress-induced decrements (Domínguez-Rodríguez et al., 2021). Our FCR findings align with some literature but contrast with studies reporting linear dose-responses (Al-Jebory et al., 2024). Unchanged feed intake confirms melatonin improved metabolic efficiency via enhanced intestinal absorption and reduced oxidative stress (Cui et al., 2022; Gao et al., 2024). Melatonin demonstrated dose-dependent, non-linear effects: 40 mg/kg favored cardioprotection and growth, while 20 mg/kg showed superior antioxidant efficacy, underscoring the need for condition-specific optimization in poultry production.

Melatonin increased villus height (Mel-40: duodenum/jejunum; Mel-20: jejunum/ileum) and width (Mel-40: duodenum; Mel-20: duodenum/jejunum), correlating with enhanced absorptive capacity (Ravindran & Abdollahi, 2021). Both doses increased villus surface area in duodenum and jejunum (Mel-40 more pronounced), suggesting dose-dependent gut barrier improvement. Increased villus surface area supports enhanced nutrient-enterocyte contact (Kiela & Ghishan, 2016), though segment-specific responses suggest tailored dosing may optimize benefits across the intestinal tract.

However, segment-specific responses (e.g., ileal height only with Mel-20) likely reflect variations in receptor density, oxidative stress, or metabolic demands (Ahmad et al., 2023; Yang et al., 2025). Conflicting reports of minimal morphological changes (Murugesan & Nidamanuri, 2025) likely reflect differences in stress severity, administration timing, or genetic strain, underscoring melatonin's context-dependent efficacy (Samanta, 2022). These structural enhancements improve intestinal absorptive capacity and likely underlie the improved FCR observed in both treatment groups.

Mel-20 reduced MDA levels across all intestinal segments, confirming melatonin's antioxidant properties (Calislar et al., 2018). However, Mel-40 did not consistently lower MDA, contradicting studies that report dose-dependent antioxidant effects (Reiter et al., 2016). The divergent dose-response, antioxidant superiority at 20 mg/kg versus cardioprotection at 40 mg/kg, likely reflects differential tissue partitioning rather than pro-oxidant effects, as SOD1/GPX remained upregulated at 40 mg/kg. At 20 mg/kg, direct scavenging predominates in the intestinal epithelium (MDA reduction); at 40 mg/kg, greater melatonin may partition to cardiovascular tissues via saturable hepatic metabolism, where MT1/MT2 activation reduces pulmonary vascular resistance, accounting for improved body weight and RV:TV ratio exclusively at this dose.

Melatonin upregulated SOD and GPX in a segment-specific manner. Mel-20 increased expression in the duodenum and jejunum, regions most exposed to dietary oxidants, suggesting preferential boosting of frontline defenses where oxidative insults are greatest. In contrast, Mel-40 predominantly affected the ileum, suggesting compensatory mechanisms in distal segments, possibly from saturation of proximal pathways or differential gut metabolism. This spatial patterning aligns with the intestine's graded oxidative microenvironment, where proximal segments face higher radical generation from digestion (Miranda-Bautista et al., 2017).

The differential response also reflects distinct regulatory mechanisms: SOD and GPX are often co-regulated, but their segment-specific induction suggests melatonin modulates the antioxidant cascade differently across intestinal regions. Conflicting literature likely reflects baseline oxidative status: unstressed birds show minimal gene induction, while severe stress (high-altitude + cold) unmasks melatonin's regulatory potential (Zarezadeh et al., 2022).

Effect size analyses confirmed substantial biological impacts: Mel-20 showed large effects on MDA reduction across segments, while SOD1/GPX patterns corroborated segment-specific responses, proximal for 20 mg/kg, ileal for 40 mg/kg. Several large effect sizes with non-significant p-values suggest biologically meaningful trends that may reach significance with larger samples, reinforcing the value of reporting effect sizes alongside p-values.

The dose-dependent divergence, antioxidant superiority at 20 mg/kg versus cardioprotection/growth at 40 mg/kg, requires mechanistic explanation, as pro-oxidant activity is unsupported by our SOD1/GPX upregulation data at 40 mg/kg. We propose that this dichotomy reflects differential tissue partitioning, where higher doses preferentially accumulate in cardiovascular tissues due to saturable hepatic metabolism, while receptor-mediated cardioprotective effects (via MT1/MT2) require higher concentrations than the saturable direct radical-scavenging activity that predominates at 20 mg/kg (Akhzari et al., 2022). Furthermore, segment-specific responses likely reflect variations in melatonin receptor density and local oxidative burden along the intestinal tract, while alternative antioxidant pathways (Nrf2, glutathione system) not measured here may be preferentially activated at higher doses (Cain et al., 2026). These findings suggest that optimal melatonin dosing depends on the therapeutic goal, intestinal protection (20 mg/kg) versus cardioprotection (40 mg/kg), supporting phase-feeding strategies in poultry production.

Overall, this study highlights melatonin's potential to improve gut health and reduce PHS in poultry under environmental stress. However, variable findings across studies underscore its context-dependent actions (dosage, stress, physiology). Future research should optimize supplementation strategies, investigate long-term effects, and explore synergies with other antioxidants to maximize benefits in commercial production.

5. Conclusion

Melatonin supplementation effectively mitigates aspects of PHS in broilers under high-altitude and cold stress by improving gut health, antioxidant defenses, and growth performance relative to untreated stressed birds. As such, these findings demonstrate melatonin's protective effects under stress rather than restoration to 'normal' physiological baselines. The 40 mg/kg dose improved cardiac health and weight gain, while 20 mg/kg showed stronger antioxidant effects, suggesting differential tissue partitioning. Both doses enhanced feed efficiency, though responses varied by gut segment. These findings support target-specific dosing (20 mg/kg for intestinal protection versus 40 mg/kg for cardiovascular support) and warrant further mechanistic studies.

Ethical statement

This experiment received approval from Shahrekord University’s Animal Ethics Committee in Shahrekord, Iran, ensuring all procedures followed strict animal welfare guidelines (approved code: IR.SKU.REC.1400.0007).

Ethical statement

This experiment was approved by the Institutional Animal Care and Use Committee of Shahrekord University (letter No. IR.SKU.REC.1400.0007).

Ethics declaration

This study was conducted in accordance with the following guidelines for animal welfare and/or reporting: This experiment was approved by the Institutional Animal Care and Use Committee of Shahrekord University. This study was approved by the Shahrekord University. (Approval No. IR.SKU.REC.1400.0007)

CRediT authorship contribution statement

Shahab Bahadoran: Writing – review & editing, Validation, Supervision, Software, Resources, Project administration, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. Amir-Hossein Torabi: Visualization, Validation, Investigation, Formal analysis, Data curation, Conceptualization. Hossein Hassanpour: Writing – review & editing, Writing – original draft, Validation, Resources, Methodology, Investigation, Conceptualization.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability

The data generated in this study will be made available online upon acceptance of the manuscript.