High summer temperatures are a critical stressor that significantly impact the poultry industry. When the ambient temperature goes beyond the thermoneutral zone of poultry, heat stress (HS) occurs. Broiler chickens are more susceptible to HS compared with other domestic animals (Smith and Gregory, 2013; Hridoy et al., 2021). In broiler chickens, HS mainly leads to problems such as reduced performance and survival rates, along with metabolic disorders, immunosuppression and deterioration of meat quality (Quinteiro et al., 2010). These issues are mainly caused by oxidative damage induced by HS, which leads to a redox imbalance (He et al., 2020). It has been demonstrated that HS can induce oxidative damage to intestinal epithelial cells trigger inflammatory responses and disrupt the balance of the intestinal microecology (Nanto-Hara et al., 2020). Therefore, preserving the integrity of the intestinal barrier and enhancing its antioxidant capacity are crucial objectives for alleviating HS-induced damage.
       
Oxidative stress may lead to an excessive accumulation of reactive oxygen species (ROS), potentially impairing cellular function through the damage of critical biomolecules, including DNA, proteins and lipids. This damage can eventually result in cellular malfunction and contribute to various pathological conditions (Oke et al., 2024). Major events linked to heat stress involve the excessive production of heat shock proteins (HSPs) (Wang and Edens, 1998). These proteins protect cells by enhancing their tolerance and survival in stressful conditions and are considered key features of the heat shock response (Hidayat et al., 2020).
       
Various approaches have been documented to reduce the negative impacts of heat stress on poultry farming (Sumanu et al., 2022; Ding et al., 2023). Among these, dietary supplements stand out as a simple, rapid and cost-effective solution and recent studies have shown that supplementation with biologically active natural antioxidants is a promising approach to mitigate the detrimental effects of free radicals produced during HS (Humam et al., 2021; Meng et al., 2022).
       
Resveratrol (3,4',5-trihydroxystilbene, RES) is a naturally bioactive polyphenol that exists in various plants, including peanuts, grapes and berries (Meng et al., 2022). It has garnered significant attention for its antioxidant and anti-inflammatory properties (Ammari et al., 2025). Resveratrol exhibits multiple biological activities, such as reducing oxidative stress, regulating immune responses and promoting cell proliferation (Ding et al., 2023; Greeshma et al., 2024). Resveratrol not only inhibits the formation of glutathione disulfide but also maintains glutathione in its active, reduced state, thereby protecting cells from oxidative damage. Furthermore, it modulates inflammatory mediators such as tumor necrosis factor-alpha and attenuates inflammatory reactions triggered by HS (Palacz-Wrobel et al., 2017). Additionally, resveratrol can modulate cellular signal transduction pathways, activating the SIRT1 signaling pathway, enhancing cell energy metabolism, delaying cellular aging and improving the body’s ability to adapt to stress (Ciccone et al., 2022). In the poultry industry, resveratrol can improve production performance and significantly enhance resistance to the adverse effects of HS (Liu et al., 2014; Zhang et al., 2017). Although numerous investigations have verified the benefits of resveratrol, the mechanism by which it affects HS requires further investigation.
       
In this research, we investigated how dietary resveratrol affects antioxidant capacity and intestinal development in chicks experiencing heat stress. The gut transcriptome of heat-stressed chicks in relation to resveratrol was also analyzed in order to uncover the underlying molecular mechanisms. This study enhances the application potential of resveratrol in poultry production, contributing to the sustainable development of the industry.

Birds and management
 
The research protocols and animal handling procedures were reviewed and approved by the Animal Care and Use Committee of Leshan Normal University. A total of 300 one-day-old male Ros308 broiler chickens were obtained and randomly divided into five experimental groups. Each group was subjected to six replicates, with each replicate comprising ten birds. The broilers were provided with a basal diet and had free access to water at all times. This diet was formulated based on the nutritional specification for Ross308 broilers (Table 1). The normal temperature control group (NT) was kept at 25±2^oC for 24 h d-1. The other four groups were exposed to a daytime temperature of 37±2^oC (from 9:00 AM to 5:00 PM.) and 30±2^oC at night throughout the experimental period, this experiment was conducted at the research institute of Leshan Normal University.


 
Experiment design and sample collection
 
Following a 30-day acclimatization phase for all groups, varying doses of resveratrol were administered to the four treatment groups, while the control group (NT) remained untreated. From days 31 to 45, four groups were maintained in a heat-stress temperature cycle and were given a basic diet with added resveratrol (Aladdin, #R107315) in amounts of 0, 200, 400, or 600 mg per kilogram of body weight (HS, HS+Res200, HS+Res400 and HS+Res600).
       
At 46 days of age, the birds were humanely euthanized to collect samples. Blood samples were obtained from the wing veins (1 sample/replicate, 6 replicates/treatment). The bird specimens were subsequently euthanized before collection of intestinal tissue samples (duodenum, jejunum and ileum). Transcriptome sequencing was partially supported by Hangzhou Lianchuan Biotechnology Co., Ltd. The small intestine was partially fixed with a 4% paraformaldehyde solution, while the remaining portion was stored at -80^oC for gene expression analysis.
 
Serum antioxidant assessment
 
The serum total antioxidant capacity (T-AOC), peroxidase (POD), catalase (CAT), superoxide dismutase (SOD) enzyme activities, malonaldehyde (MDA), glutathione peroxidase (GSH-Px) and thioredoxin peroxidase (TPx) levels were measured using purchased kits (Sinobestbio, Shanghai, China). The sample’s T-AOC was quantified in terms of the concentration of standard antioxidant equivalents required to achieve an equivalent change in absorbance (DA). The nitroblue tetrazolium (NBT) photochemical reduction method at 560 nm was employed to determine the activity of superoxide dismutase (SOD). Using the H₂O₂ ultraviolet absorption method at 240 nm, the activity of catalase (CAT) was determined. The peroxidase (POD) activity was determined using the guaiacol oxidation method at a wavelength of 470 nm. At 340 nm, the activity of GSH-Px was measured by quantifying the oxidation rate.
 
Measurements of intestinal morphology
 
The intestinal segments were fixed with 4% paraformaldehyde, embedded in paraffin and stained with hematoxylin and eosin (H and E). The villus height and crypt depth were measured using a digital camera microscope in conjunction with SlideViewer 2.5 software. Ten intact villi and crypts were randomly selected from each sample for analysis. The height of the villi, the depth of the crypts and their ratio (V/C) were calculated.
 
RNA library and RNA-seq
 
Total RNA was isolated from jejunal tissue samples using Trizol reagent (Thermo Fisher, USA). Following RNA isolation, mRNA was fragmented utilizing the RNA Library Prep Kit (NEB, Cat# E6150). The fragmented mRNA was then converted into cDNA through reverse transcription and subsequently used to synthesize the second strand of DNA. Sequencing was performed using the Illumina NovaSeq 6000 platform. Genes with a false discovery rate (FDR) of less than 0.05 were considered to be statistically significant. Moreover, genes showing a minimum absolute fold change of 2 were classified as differentially expressed genes (DEGs). Based on the differentially expressed gene data obtained through sequencing, we carried out functional enrichment analysis utilizing Gene Ontology (GO) and conducted pathway analysis with the Kyoto Encyclopedia of Genes and Genomes (KEGG).
 
GO and KEGG analysis
 
The GO represents an international standard for classifying gene functions. The count of DEGs linked to every GO term was determined and hypergeometric tests were conducted relative to the genomic background to identify significantly enriched terms. KEGG, an integrated public database related to pathways, was utilized. Among the genes exhibiting differential expression, the KEGG database was utilized to detect metabolic and signaling pathways that were significantly enriched.
 
RT-qPCR analysis
 
RT-qPCR was employed to confirm the DEGs detected through RNA-seq. Fifteen DEGs were selected based on the alterations in their expression levels in the treated cells compared to those in the control cells. Primers specific to these genes were designed according to reference UniGene sequences. These primers are listed in Table 2 and were synthesized commercially by Tsingke Biotechnology Corporation (Beijing, China). The relative expression level of the gene was determined by the threshold cycle (Ct) method and the analysis was conducted using the comparative 2^-∆∆Ct formula. An internal control, the β-actin (ACTB) gene, was employed as an internal control to normalize the expression levels of the target genes.


 
Statistical analysis
 
A t-test was performed to assess the influence of temperature environmental factors on NT and HS samples that were not exposed to resveratrol, as well as the effects of resveratrol on HT samples administered with 400 mg/kg of the compound. To investigate the variations between treatments involving different concentrations of resveratrol (0, 200, 400, or 600 mg/kg) within the HT group, Duncan’s multiple range test was utilized. A P-value less than 0.05 is considered to indicate a significant difference between the samples, while a P-value lower than 0.01 indicates a higher level of significance.

Jejunal mucosal mRNA expression
 
To determine the optimal dosage of resveratrol in chick feeding experiments, we employed RT-qPCR analysis to assess the expression levels of HSPB1, HSP70 and HSP90 genes in the jejunal tissue. The results demonstrated that, following 30 days of high-temperature exposure, administering resveratrol at doses of 200, 400, or 600 milligrams per kilogram effectively reduced the relative mRNA expression levels of heat shock proteins in jejunum tissue. Notably, the administration of a 400 mg/kg dose significantly suppressed the expression levels of HSPB1, HSP70 and HSP90 (P<0.05). The addition of 200 mg/kg markedly altered the expression of HSPB1 (P<0.05), while the addition of 600 mg/kg led to a substantial decrease in the expression of both HSPB1 and HSP70 (P<.05) (Fig 1).


 
Antioxidant biomarkers
 
To explore the influence of resveratrol on the overall antioxidant capacity of heat-stressed chicks, several key serum indicators were examined following supplementation with 0 and 400 mg/kg of resveratrol. The results demonstrated that chickens exposed to thermal stress exhibited significantly reduced activities of SOD, T-AOC, CAT, POD and GSH-Px compared with those maintained under normal temperature conditions (P<0.01). Compared with the normal temperature group, the serum MDA level in the HS group was significantly increased (P<0.01). During the heat stress period, supplementing the diet with 400 mg/kg body weight of resveratrol led to a notable decrease in serum MDA levels (P<0.05). Moreover, dietary addition of resveratrol improved the enzymatic activities of SOD, T-AOC, CAT, POD and GSH-Px in the serum of chickens (P<0.05) (Table 3). These findings suggest that resveratrol effectively mitigates lipid peroxidation in chickens under heat stress and promotes the activities of antioxidant enzymes in serum.


 
Changes in intestinal morphology
 
The H and E staining was conducted to examine the development of intestinal villi and crypts. In the HS group, loss or shortening of jejunal villi, degeneration of mucosal epithelial cells and villus atrophy were observed. Under high-temperature conditions, edema of the ileal mucosal villi and shedding of the apical epithelial villi were observed. The duodenal and jejunal villi of chickens under HS condition supplemented with resveratrol also exhibited mild shedding and breakage. However, the intestinal tissue structure was more intact, with less villus damage and neatly arranged cell layers (Fig 2).


       
The results from the paraffin sections clearly indicate that, compared with the NT group, exposure to high temperature leads to a significant reduction in villus height across all segments of the small intestine (P<0.05). In addition to structural damage and shortening of the villi, the ratio of villus height to crypt depth in each intestinal segment was also significantly decreased (P<0.05). Furthermore, a significant increase in crypt depth was observed specifically in the jejunum (P<0.05). However, under heat stress conditions, resveratrol supplementation did not induce significant changes in villus height or crypt depth across intestinal tissues (Table 4).


 
RNA sequencing information
 
To elucidate the mechanisms by which resveratrol regulates intestinal development, eight transcriptome libraries were constructed from jejunal tissue samples obtained from both the blank control group and the 400 mg/kg resveratrol-treated group. This was done to detect differences in gene expression profiles between untreated chickens and those treated with resveratrol. Using high-throughput sequencing technology, we identified a total of 181 differentially expressed genes in the jejunal tissues of chickens exposed to a high-stress environment. The list of these differentially expressed genes can be found in Supplementary File S1. The transcriptome analysis revealed that, following heat stress, 89 genes exhibited increased expression, while 92 genes showed decreased expression in the HS+Res400 group compared to the control HS group (Fig 3A). The x-axis and y-axis were plotted using multiple log2 (fold change) and -log10 (q-value) respectively, visually presenting all the genes in the differential expression analysis (Fig 3B).




       
The differential gene cluster heatmap intuitively displays the gene expression patterns across four samples from each of the two groups. In this heatmap, the samples are represented on the x-axis, whereas the y-axis corresponds to the selected differentially expressed genes (DEGs). Specifically, Fig 3C only includes the top 100 genes with the smallest q-values for heatmap visualization. The results show that the DEGs were clustered into two distinct groups in the heatmap, suggesting that resveratrol supplementation following heat stress significantly altered gene expression in chicken intestinal tissue at the transcriptional level (Fig 3C).
 
Analysis of GO annotation and enrichment in KEGG pathway
 
The results of Gene Ontology (GO) annotation indicated that, in contrast to the control HS group, the administration of resveratrol resulted in differentially expressed genes (DEGs) predominantly associated with signal transduction, G protein-coupled receptor signaling, the NF-κB signaling pathway, transmembrane transport processes and the positive regulation of RNA polymerase II-mediated transcription (Fig 4). A list of all GO terms is provided in Supplementary File S2.




       
KEGG pathway-based analysis provides valuable insights into the functional roles of genes and their interactions. Fig 5 is a KEGG hierarchical bar chart in which the x-axis standing for the number of DEGs in each pathway, the y-axis lists the pathway names and the color indicates the KEGG primary classification (Fig 5A). A list of all KEGG terms is provided in Supplementary File S3. The top 20 KEGG pathways with the lowest P-values were mapped. In this plot, the y-axis represents the names of the pathways and the x-axis illustrates the -log10 transformed P-values obtained from the KEGG pathway enrichment analysis (Fig 5B). The results demonstrate that, following HS, the DEGs in the resveratrol-treated group were primarily enriched in the VEGF and ErbB signaling, the insulin signaling and the Toll-like receptor signaling pathway.




 
Validation of DEGs by RT-qPCR
 
In this study, eight DEGs were selected to corroborate the gene expression profiles obtained from RNA sequencing. The eight verified DEGs included five upregulated genes (GLDC, TJP3, ACE, GYS2 and GRM1) and three down regulated genes (MLN, SRSF2 and TLR3). The RNA sequencing results were validated by RT-qPCR, demonstrating consistency between the two methods and confirming the reliability of the RNA sequencing data (Fig 6).


       
Chronic heat stress exposure might disturb the equilibrium between oxidative stress and antioxidant defense mechanisms. This occurs through the depletion of enzymatic antioxidants and an increase in lipid peroxidation levels (Habashy et al., 2019). The poultry industry, particularly the broiler sector, is highly vulnerable to heat stress due to the birds’ unique physiological structure and elevated metabolic rate. This sensitivity of broilers to HS causes oxidative imbalances, metabolic disorders and immunosuppression (Mackei et al., 2021). The accumulation of ROS can lead to systemic oxidative damage, particularly affecting the intestinal mucosa and inducing muscle proteolysis (Altan et al., 2003).
       
Resveratrol is a plant-derived polyphenolic compound that enhances immune function and exhibits antioxidant properties. It exerts multiple health benefits, including anti-inflammatory and antioxidant effects (Burns et al., 2000; Putics et al., 2008). There are reports indicating that resveratrol can be used as an anti-stress supplement in poultry to reduce tissue damage caused by HS (Ding et al., 2023). In this research, heat-stressed chicks were given resveratrol. The results demonstrate that the addition of resveratrol for 15 days mitigated the adverse effects of HS. Specifically, the expression levels of HSPB1, HSP70 and HSP90 genes were significantly reduced when 400 mg/kg resveratrol was administered. The heat shock protein family (HSPs) are important nonspecific cell protection proteins (Hidayat et al., 2020). Studies have demonstrated that animals subjected to heat stress exhibit significantly elevated mRNA expression levels of HSPs compared to those under normal conditions (Yu et al., 2008; Gu et al., 2012). Polyphenol supplements may play a role in regulating the expression of HSPs. The research conducted by Al-Zghoul et al. (2020) demonstrated that exposure to HS led to elevated levels of HSP70 expression in the intestines of pigs and rats (Al-Zghoul and Saleh, 2020). Liu et al. (2014) observed that under heat stress conditions, the expression levels of HSP70 and HSP90 in the jejunum tissue of Muchuan black bone Chicken were upregulated (Liu et al., 2014). These findings align closely with the outcomes of our current study on the intestinal mucosa of chickens under similar conditions.
       
Excessive reactive oxygen species generated under heat stress can initiate lipid peroxidation reactions, leading to oxidative damage and inflammatory responses in cellular proteins and DNA, ultimately compromising the integrity of intestinal tissues. When cellular oxidative status is disrupted, the activity of key antioxidants such as T-AOC, POD, SOD, CAT, TPX and GSH-Px decreases, while MDA is overproduced (Circu and Aw, 2010; Yang et al., 2010). It has been demonstrated that resveratrol effectively prevents the formation of free radicals induced by HS, lipopoly-saccharide stress and ultraviolet stress, while also inactivating oxidative precursors generated during these stress conditions. Studies have indicated that supplementing the diet with resveratrol can significantly alleviate HS-induced body damage in rats (Das, 2011) and quail (Sahin et al., 2012), while boosting the activities of SOD, CAT and GSH-PX and lowering MDA levels. According to Liu et al. (2014), the inclusion of 400 mg/kg resveratrol in the diet led to a notable enhancement in the serum GPX, SOD and CAT enzyme activities in black-boned chickens on the fifteenth day of exposure to heat stress. Additionally, it increased GSH content while reducing MDA levels (Liu et al., 2014). This study was conducted under conditions where chicks were exposed to prolonged HS. Compared with the normal temperature control group, heat-stressed chicks exhibited significantly reduced serum activities of T-AOC, SOD, CAT, POD and GSH-Px, along with an increased level of MDA. However, under heat stress conditions, supplementation with resveratrol led to a significant recovery in these antioxidant indices and a marked decrease in MDA levels. These findings suggest that resveratrol can effectively mitigate oxidative damage in the body under HS conditions.
       
Heat stress can induce systemic responses in chicks. Maintaining the integrity of intestinal structure and function, which is vital for nutrient absorption, is essential for poultry health (Liu et al., 2022). Burkholder et al. (2008) found that HS can compromise the structure of intestinal villi and disrupt the normal microbiota in poultry (Burkholder et al., 2008). Quinteiro et al. (2012) also demonstrated that HS is capable of causing intestinal damage and triggering acute enteritis in poultry (Quinteiro et al., 2012). The pathological damage induced by HS mainly involves the mucosal layer, manifesting as exfoliation of mucosal epithelial cells and disruption of intestinal villi (Rostagno, 2020). This finding aligns with the outcomes of the current research. Resveratrol was found to markedly enhance the villus height in the duodenum and jejunum, effectively reducing the intestinal mucosal structural damage induced by HS.
       
The regulation of the heat stress response in poultry is a complex process involving multiple genes. To explore the protective mechanism of resveratrol on the gut of chicks under HS, we performed transcriptome sequencing of the jejunal mucosa of chicks fed with resveratrol. Through high-throughput sequencing technology, 181 DEGs were identified, of which 89 were significantly upregulated, including SHC3, GLDC, CKB, GYS2, GRM1, ITPK1, TJP3, LAMB3, ACE, NOXO1 and ND6. In addition, 92 were significantly downregulated, including MHCY15, SRSF2, INA, USP53, ATP11A, MRTFB, CNP3, CNTN2 and GIP. The DEGs were involved in various biological pathways, including developmental processes, response to multicellular stimuli, oxidative homeostasis, neural cell differentiation and development, phosphotransferase activity, nucleic acid binding and transcriptional regulation, as well as immune and extracellular domain responses. These results indicate that resveratrol induced alterations in multiple genes in the chick gut.
       
The results of GO annotation and KEGG pathway analysis show that, in comparison to the blank control group, the DEGs were mainly enriched in pathways including VEGF signaling, ErbB, insulin signaling and Toll-like receptor signaling. The VEGF signaling pathway is alternatively referred to as the vascular endothelial growth factor signaling pathway. The signaling initiated by VEGF/VEGFR2 can cause endothelial cell proliferation and increase vascular permeability, leading to vascular tumors, edema, inflammation and vascular dysfunction (Yang and Cao, 2022, (Apte et al., 2019). This pathway may be related to intestinal inflammation under HS (Scaldaferri et al., 2009). Our sequencing results revealed that genes associated with this pathway were markedly downregulated in the resveratrol-treated group, providing evidence for positive effects of resveratrol on HS-induced intestinal inflammation. The ErbB pathway exerts control over cell proliferation, differentiation and migration through its interaction with several key signaling cascades, including the PI3K/Akt, JAK/STAT and MAPK pathways (Grant et al., 2002). Depending on the specific cellular context, ErbB receptor activation may promote proliferation, motile adhesion, differentiation or even apoptosis (Appert-Collin et al., 2015). The insulin signaling pathway is a biochemical chain of reactions used to transmit information about the insulin hormone to the interior of the cell to regulate metabolic activity and growth (Hotamisligil and Davis, 2016). The insulin signaling pathway is essential for controlling blood glucose levels, fat metabolism and protein synthesis (Saltiel, 2021). Our sequencing data show that genes enriched in this pathway, such as GYS2 and SHC3, were significantly upregulated in the resveratrol-treated group. This indicates that resveratrol can participate in regulating glycogen metabolism under heat stress conditions.
       
In general, the physiological functions and mechanisms of resveratrol are many, including oxidative stress protection, immune system regulation, growth and development support and cell signal transduction regulation. Together, these effects contribute positively to the development, intestinal health and immune system of heat-stressed chicks. However, the specific mechanisms and effects of resveratrol still require further elucidation. Future studies need to clarify its precise pathway of action in chicks as well as determine the most suitable dosage, in order to better harness the potential of resveratrol in animal husbandry.

Heat stress can lead to intestinal epithelial damage and oxidative stress in broilers. Dietary supplementation with 400 mg/kg resveratrol can effectively mitigate the adverse effects of heat stress. Transcriptomic analyses indicate that the protective mechanism of resveratrol involves modulating genes associated with key signaling pathways. In conclusion, resveratrol may serve as an effective nutritional modifier for alleviating heat stress in poultry.

Not applicable.
 
Funding
 
This work was supported by the Natural Science Foundation of Sichuan Province of China [Grant No. 2023NSFSC0231] and the Southern Sichuan local breed chicken Research Center of Sichuan University project [Grant No. 2025DFPZJ002]. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.
 
Data availability statement
 
The data that support this study are available in the article and accompanying online supplementary material.

Disclaimers
 
The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.
 
Informed consent

All experiments involving animals and study protocols were approved by the Animal Care and Use Commission of the College of Life science, Leshan Normal University.

The authors declare that there are no conflicts of interest regarding the publication of this article.