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. 2025 Jul 2;26:631. doi: 10.1186/s12864-025-11832-2

Comparative transcriptomic analysis reveals the important role of hepatic fatty acid metabolism in the acute heat stress response in chickens

Zi Mei 1, Zhenquan Song 1, Haobo Zhou 1, Bin Zheng 1, Yiwei Xiong 2, Zheya Sheng 1,✉, Yanzhang Gong 1,✉
PMCID: PMC12220621  PMID: 40604375

Abstract

Background

Heat stress poses a major challenge to global poultry production, but the molecular mechanisms driving the acute heat stress response in multiple organs of chickens remain poorly understood. The present study aimed to elucidate these mechanisms by establishing an acute heat stress chicken model and analyzing the multi-tissue transcriptome and physiological responses.

Results

Exposure to 36℃ for 6 h induced marked physiological changes, including elevated rectal temperatures, severe multi-organ damage, and disrupted energy metabolism (increased serum glucose [GLU] and decreased triglycerides [TG] and total cholesterol [TCHO]). Comparative transcriptomic analysis of heart, liver, spleen, lung, and kidney tissues revealed tissue-specific differential gene expression, with the liver and heart showing the highest number of differentially expressed genes (DEGs). KEGG enrichment analyses identified lipid metabolism pathways that are key to the multi-tissue acute heat stress response. Weighted gene co-expression network analysis (WGCNA) further identified 58 differentially modularized hub genes (DMHGs), of which 42 were hepatic differentially expressed genes, and most of these DMHGs were significantly enriched for fatty acid metabolic pathways. Fatty acid metabolic pathway-associated DMHGs were significantly correlated with rectal temperature, serum GLU, TG, lactate dehydrogenase (LDH), and aspartate aminotransferase (AST). Functional validation in primary hepatocytes demonstrated that overexpression of FASN attenuated heat stress-induced reductions in triglyceride levels.

Conclusions

The critical role of hepatic fatty acid metabolism in mediating the acute heat stress response in chickens was revealed by a multi-tissue comparative transcriptome, and it was determined that FASN provides actionable insights into improving heat tolerance in poultry through metabolic interventions.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12864-025-11832-2.

Keywords: Heat stress, Chicken, Transcriptomics, Fatty acid metabolism, Fatty acid synthase

Background

Climate change and global warming are escalating threats to agricultural productivity and heat stress has been a serious challenge for livestock, especially poultry industry [1, 2]. Chickens are highly susceptible to heat stress, which affects poultry in the form of physiological and behavioral disorders, including reduced feed intake, growth retardation, decreased egg production, and altered meat quality [3–8]. Heat stress in poultry is generally categorized as acute (27–38 ℃, 1–24 h), moderate (27–38 ℃, up to 7 days) and chronic (27–38 ℃, 7 days or more) heat stress [3, 9, 10]. Acute heat stress causes more severe consequences than chronic stress because the sudden increase in rectal temperature can cause irreversible tissue damage and death [11]. Intensive genetic selection for rapid growth and feed efficiency over decades has inadvertently increased the sensitivity of poultry to high temperatures, thereby exacerbating the adverse effects of heat stress [12].

At the cellular level, heat stress triggers a series of pathological events, including oxidative stress, protein denaturation and metabolic dysregulation [13]. Metabolic regulatory functions, particularly energy and lipid homeostasis, are critical for recovery from heat stress injury in the body [14].A large body of evidence suggests that heat stress leads to overproduction of reactive oxygen species (ROS) and reduced antioxidant properties, which exacerbate oxidative stress, especially in the liver [15, 16]. The liver, as the central hub of lipid metabolism and energy homeostasis, plays a key role in heat stress, but it is also highly susceptible to heat stress-induced injury [17]. The heart, which is critical for maintaining cardiovascular function, also undergoes structural and functional remodeling in response to heat stress, mitochondrial dysfunction and impaired energy metabolism further exacerbate cardiac damage, as evidenced by disrupted adenosine triphosphate (ATP) production and accumulation of reactive oxygen species (ROS) [16, 18, 19]. While the effects of heat stress on individual tissues have been extensively studied, a comprehensive understanding of multi-tissue coordination remains unclear [20–23]. With advances in transcriptomics and bioinformatics, such as weighted gene co-expression network analysis (WGCNA) that identifies co-expressed gene modules and hub genes, this also provides us with a powerful tool to probe tissue-specific and systemic regulatory networks in acute heat stress [6, 14, 24–26].

In this study, we explored the molecular mechanisms regulating acute heat stress in chickens by integrating multi-tissue transcriptomics, physiological phenotyping and functional validation. Multi-tissue transcriptome analysis of chicken heart, liver, spleen, lung and kidney identified 58 differential molecular hub genes, determined the important role of fatty acid metabolism in liver in acute heat stress and validated the functional role of fatty acid synthase (FASN) in primary hepatocytes. Our findings not only deepen the understanding of the biological mechanisms of heat stress in poultry, but also provide actionable targets for breeding heat-tolerant lines and optimizing nutritional interventions.

Methods

Animals and experimental design

20 Xinhua No. 2 layer hens at 28 weeks of age from Hubei Xinhua Ecological Livestock Development Co. Ltd (Xiaogan City, Hubei Province, China) were used in this study. Xinhua layer is a synthetic layer line, which partially originated from several Chinese indigenous breeds. Before the experiment, hens were acclimated for two weeks under standard rearing conditions (23 ℃ ± 1 ℃, 60-70% humidity, 15 h of light/9 h of dark photoperiod).

The thermal challenges were conducted in a climate-controlled room at the experimental chicken farm of Huazhong Agricultural University, where temperatures could be well controlled within ± 0.5 ℃ and humidity ~ 60%. Based on our previous findings in chicken heat-stress models, exposure to an environmental temperature of 36℃ for 6 h induces severe acute heat stress in layer hens [27]. This condition provides a suitable model for examining the impact of severe heat stress on five major internal organs in subsequent studies. Therefore, hens were randomly divided into two groups: a control group (n = 10, 23 ℃ ± 1 ℃) and a heat stress group (n = 10, 6 h of exposure at 36 ℃). Throughout the experiment, hens had free access to a commercial layer feed provided by Xinhua Company, which was also the supplier of the experimental birds. The feed consisted primarily of corn, soybean meal, calcium phosphate, stone powder, sodium chloride, amino acids, amino acid salts and their complexes, as well as a premix of vitamins and vitamin-like compounds. The guaranteed nutrient composition of the feed was as follows: crude protein ≥ 16.5%, crude fiber ≤ 7.0%, crude ash ≤ 15.0%, calcium 3.0–4.5%, total phosphorus ≥ 0.5%, sodium chloride 0.3–0.8%, moisture ≤ 13.0%, methionine ≥ 0.32%.

Phenotypic measurements and sample collection

Immediately after 6 h of treatment, rectal temperature was measured in both groups. Blood samples were collected from the wing vein for physiological analysis. Serum was obtained by centrifugation (2,500 g, 15 min, 4 ℃) and stored at -80 ℃ prior to analysis. Blood gases were assessed using i-STAT CG8+/EG8 + cartridges (Abbott, USA), and serum biochemical indices were measured using an automated chemistry analyzer (BX-4000, Sysmex, Japan). Four individuals per group were randomly selected for tissue collection. Chickens were euthanized by intravenous administration of sodium pentobarbital at a dosage of 150 mg/kg body weight. Prior to injection, chickens were gently restrained to minimize stress. Sodium pentobarbital was administered via the wing vein, quickly resulting in loss of consciousness, followed by respiratory and cardiac arrest. Animals were closely monitored to confirm the absence of vital signs before proceeding. Heart, liver, spleen, lung, and kidney tissues were collected for subsequent analyses. For histopathological analysis, tissues were immediately fixed in 4% paraformaldehyde. For transcriptome sequencing and gene expression validation, tissues were rapidly snap-frozen in liquid nitrogen and stored at -80 ℃ until further use.

Histological analysis

For morphological evaluation, tissue samples from heart, liver, spleen, lungs and kidneys were fixed in 4% paraformaldehyde solution for 24 h at room temperature. After fixation, samples were dehydrated in a graded ethanol series (70%, 80%, 90%, and 100%), washed in xylene, and embedded in paraffin. Paraffin-embedded tissues were sectioned using a microtome (Leica RM2235, Germany) at a thickness of 6 μm. Hematoxylin-eosin staining was performed according to standard protocols. Histologic examination was performed using a light microscope (Olympus BX53, Japan). Images were analyzed for tissue structure, cellular morphology, and any pathological changes using ImageJ software (National Institutes of Health, USA).

Multi-tissue transcriptome profiling

Total RNA was extracted from 40 samples (heart, liver, spleen, lungs, and kidneys) from normal control (NC, n = 4) and heat-stressed (HS, n = 4) chickens using Trizol RNA reagent (Invitrogen, USA) with RNA integrity verified by Agilent 2100 Bioanalyzer (RIN ≥ 7.0). Poly(A)-tailed mRNA was captured using the NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB). Libraries were constructed using the NEBNext® Ultra™ RNA Library Prep Kit for Illumina® according to the library construction method commonly used by NEB [28]. Fragment size distribution (~ 350 bp peak) and concentration (> 2 nM) were assessed via Agilent 2100 Bioanalyzer and Qubit. An Agilent 2100 Bioanalyzer was used to assess the integrity and amount of RNA. Raw reads were quality controlled with FastQC and clean reads were aligned to the chicken reference genome GRCg6a using Hisat2 (v2.0.5) [29]. And our software for QC was fastp (version 0.19.7) with the parameters fastp -g -q 5 -u 50 -n 15 -I 150. Gene counts were obtained using featureCounts (v1.5.0-p3) [30]. Differentially expressed genes (DEGs) between the NC and HS groups were identified using the DESeq2 R package (v1.20.0) [31], with thresholds set at adjusted P-value (Padj) < 0.05 and |log2 fold change| ≥ 1 [32–36].

Gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analyses.

Gene Ontology (GO) and Kyoto Encyclopedia of the Genome (KEGG) pathway enrichment analyses were performed to elucidate the function of DEGs in response to heat stress in different tissues. These analyses were performed using the R package ClusterProfiler (version 3.18.1) [37]. In GO analysis, DEGs were categorized into three major ontologies: biological process (BP), molecular function (MF), and cellular component (CC). Enrichment analyses were performed using the enrichGO function with the organism set to “gallus gallus” and the annotation database as org.Gg.eg.db [38]. KEGG pathway analyses were performed using the enrichKEGG function to identify significantly enriched biological pathways [39]. For both GO and KEGG analyses, hypermetric tests were used to assess the statistical significance of enrichment. Terms and pathways with adjusted p-values less than 0.05 (Benjamini-Hochberg multiple test correction method) were considered significantly enriched. Bubble and bar graphs were generated using ggplot2 to highlight the most significantly enriched terms and pathways.

Weighted gene co-expression network analysis (WGCNA)

Weighted gene co-expression network analysis by the WGCNA software package (version 1.41) in R was performed to find clusters of highly correlated genes [24]. Transcript abundance was quantified using normalized fragments per kilobase per million (FPKM) values, and gene expression matrices were constructed from all 40 samples. To further assess the correlation between co-expressed gene clusters and tissues’ heat-stress status, we assigned nominal values of 1 and 0 to the normal (NS) and heat-stressed (HS) samples, respectively. Module hub genes (MHGs) were identified based on the criteria of gene significance (GS, |GS| > 0.8, P < 0.05) and module membership (MM, |MM| > 0.95, P < 0.05) [40]. This analysis led to the identification of key gene modules and hub genes associated with heat stress responses in multiple tissues.

Real-time quantitative PCR

Gene expression changes were verified by real-time quantitative PCR (qRT-PCR). qRT-PCR reactions were performed on a Bio-Rad CFX-384 instrument (Bio-Rad Laboratories, USA) using 2×SYBR Green Fast qPCR mix (Abconal, China). Each sample was performed in triplicate. The reaction conditions were as follows: initial denaturation at 95 ℃ for 3 min, followed by 40 cycles of 95 ℃ for 10 s and 60 ℃ for 30 s. Melting curve analysis was performed to ensure amplification specificity. Relative mRNA expression levels were calculated using the 2-ΔΔCt method with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the internal reference gene [41]. 5 samples from each of the control and heat stress groups. Results are expressed as fold change relative to the control. The primer sequences are listed in Table S1.

Plasmid construction for in vitro validation

To construct an overexpression vector for in vitro studies, the coding sequence of the chicken FASN gene was amplified by PCR using high-fidelity DNA polymerase. The amplified FASN fragment was then cloned into the pcDNA3.1 expression vector (Invitrogen, USA) using homologous recombination. The resulting plasmid, pcDNA3.1-FASN, was verified by restriction enzyme digestion and Sanger sequencing to ensure correct insertion and orientation of the FASN gene. This construct was subsequently used in transient transfection experiments to investigate the effect of FASN overexpression in chicken cell lines.

Chicken primary hepatocyte culture

Primary hepatocytes were isolated from 14-day-old chicken embryos using a modified two-step collagenase perfusion method. Livers were aseptically removed, minced, and digested with 1 mg/ml type IV collagenase (Sigma-Aldrich, USA) at 37 ℃ for 30 min. The resulting cell suspension was filtered through 200- and 400-mesh sieves to remove tissue debris. Hepatocytes were purified by centrifugation at 50 × g for three consecutive times at 4 ℃ for 5 min each. Isolated cells were resuspended in Dulbecco’s Modified Eagle Medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. Hepatocytes were seeded at a density of 1 × 106 per well in collagen-coated 6-well plates and cultured in a humidified 5% CO2 environment at 37 ℃.

Primary hepatocyte heat stress modeling and gene modulation

To study the cellular response to heat stress, primary chicken liver cells were thermally challenged after 24 h of culture. The medium was changed and the cells were exposed to a temperature of 42 ℃ for 6 h in an incubator to simulate heat stress conditions. The physiological temperature of control cells was 37 ℃. Cells were transfected with FASN overexpression plasmid (pcDNA3.1-FASN) using Lipofectamine 2000 (Invitrogen, USA) according to the optimization protocol for primary hepatocytes provided by the manufacturer.

Statistical analysis

All statistical analyses were performed using IBM SPSS statistical software (version 20.0, IBM Corp., Armonk, NY, USA). Data are expressed as mean ± standard deviation (SD). Comparisons between two groups were made using a two-tailed unpaired Student’s t-test, and comparisons between three or more groups were made using a one-way analysis of variance (ANOVA), with statistical significance for all analyses set at P < 0.05. Graphs of the data were generated using GraphPad Prism software (version 8.0, GraphPad Software, San Diego, CA, USA).

Results

Acute heat stress induces multi-organ damage in chickens

After 6 h of exposure to 36 ℃, blood gas parameters such as pH and Na⁺ increased significantly, indicating severe panting and electrolyte imbalance—classical physiological responses associated with heat stress (Table S2). In addition, the rectal temperatures of heat-stressed chickens were also significantly increased (P < 0.01, Fig. 1A). These results confirmed the successful establishment of the chicken acute heat stress model.

Fig. 1.

Fig. 1

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Effects of acute heat stress on physiological parameters and organ histopathology in chickens. A Rectal temperature. B Representative histopathological images of major organs (heart, liver, spleen, lungs and kidneys) of chickens from control (ambient temperature) and heat-stressed groups (36 ℃, 6 h). C Serum indicators of tissue damage: creatine kinase (CK), lactate dehydrogenase (LDH) and aspartate aminotransferase (AST). D Serum metabolic indicators: glucose (GLU), total protein (TP), triglyceride (TG) and total cholesterol (TCHO). * P < 0.05; ** P < 0.01

Histopathological examination revealed that acute heat stress resulted in damage to multiple organ tissues in chickens: myocardium exhibited nuclear condensation and hyperpigmentation; hepatic sinusoids were dilated; spleen red medulla was severely congested; lung tissues showed marked dilatation of tertiary bronchioles, rupture of alveolar septa, and fusion of adjacent alveoli; and renal tissues showed an increase in intravascular glomerular cells (Fig. 1B). Analysis of serum biochemical parameters showed significant changes in various physiological indices. Tissue damage markers such as creatine kinase (CK), lactate dehydrogenase (LDH) and aspartate aminotransferase (AST) were significantly elevated in heat-stressed chickens, which was consistent with the histopathological findings (Fig. 1C). In addition, heat stress led to alterations in several energetic markers in chickens: increased glucose (GLU) levels and decreased total protein (TP), triglyceride (TG) and total cholesterol (TCHO) concentrations (Fig. 1D).

Transcriptomic changes in multiple tissues due to acute heat stress

To further explore the mechanisms that further probe the regulation of different tissues under acute heat stress, we performed a comparative transcriptome analysis of heart, liver, spleen, lung, and kidney tissues from control and heat-stressed chickens. A total of 40 mRNA libraries were constructed with 4 biological replicates per tissue, generating a total of 273.58 Gb of high-quality RNA-seq data (Table S3). Principal Component Analysis (PCA) showed that all the different tissue types could be effectively differentiated, and in addition the heat stress group was significantly distinguished from the control group samples within each tissue (Fig. 2A). Differential gene expression analysis identified 552, 519, 213, 103, and 27 DEGs in heart, liver, kidney, lung, and spleen tissues, respectively (Fig. 2B). Most DEGs were tissue-specific, suggesting distinct and diverse responses of the five organs to acute heat stress (Fig. 2C).

Fig. 2.

Fig. 2

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Transcriptome analysis of chicken tissues to acute heat stress. A Principal component analysis plot of RNA-seq data from heart, liver, spleen, lung and kidney tissues of control and heat-stressed chickens. B Number of differentially expressed genes (DEGs) in each tissue of control and heat-stressed chickens. C Venn diagram of the overlap of DEGs among different tissues

Enrichment analyses by GO and KEGG were done (Fig. 3A and Table S4). DEGs identified in heart tissue were predominantly enriched for biological processes associated with cellular structure, including “extracellular structure organization” and “extracellular matrix organization”. Meanwhile, degs identified in liver tissue DEGs are mainly enriched in various metabolic processes, such as “cholesterol metabolism process” and “fatty acid metabolism process”. Furthermore, kidney DEGs were predominantly enriched in biosynthetic processes, including lipid biosynthetic and steroid biosynthetic processes. KEGG secondary classification maps showed that the DEGs of heart, liver, spleen, kidney were enriched in a variety of metabolic pathways, where the number of enriched metabolic pathways by DEGs of liver was the largest (Fig. 3B). In addition, DEGs from liver, heart and kidney were co-enriched in lipid metabolic pathways, whereas DEGs from liver, heart and spleen were co-enriched in carbohydrate metabolic pathways. These findings collectively indicate that multiple tissues in chickens collaborate to perform critical metabolic regulatory functions during acute heat stress.

Fig. 3.

Fig. 3

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Functional enrichment analysis of differentially expressed genes in chicken tissues in response to acute heat stress. A Top 10 significantly enriched gene ontology (GO) terms in heart, liver and kidney tissue bioprocesses. B KEGG secondary classification chart in heart, liver, kidney and spleen tissues

Differential modular hub genes regulating acute heat stress

WGCNA identified 11 co-expressed gene modules and their corresponding module hub genes (MHGs). These gene modules were presumed to be primarily associated with tissue type, as suggested by the PCA results of all 40 samples. To further investigate their potential associations with both tissue type and heat stress, we assigned each sample a binary nominal value (1s for heat stress samples and 0s for control samples) and correlated them with the Module Eigengene (ME) values. Our analysis revealed clear tissue-specific correlations between gene modules and sample types: the red and yellow modules were positively correlated with heart samples, the pink and turquoise modules were positively correlated with liver samples, and the black and cyan modules showed positive correlations specifically with lung samples (Cor > 0.8, P < 0.05) (Fig. 4A). By integrating the tissue specific DEGs with 63 MHGs identified from these 6 modules, we identified 58 differentially expressed module hub genes (DMHGs), 42 of which belonged to the pink and turquoise modules and were differentially expressed in the liver. Further, 42 DMHGs associated with liver were mainly enriched in fatty acid metabolism-related pathways, including fatty acid metabolism, elongation, and degradation; 12 DMHGs found in heart showed significant enrichment in the ECM receptor interaction and Focal adhesion pathways; and 4 lung DMHGs were mainly enriched in the Notch signaling pathway (Fig. 4B). Comparative analysis of KEGG results from DEGs and DMHGs revealed that multiple fatty acid-related pathways (Fatty acid metabolism, Fatty acid elongation and Fatty acid degradation) were overlappingly enriched in the liver.

Fig. 4.

Fig. 4

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Weighted gene co-expression network analysis (WGCNA) and functional enrichment of key genes in acute heat-stressed chicken tissues. A Heat map of correlation between co-expressed gene modules and tissue heat stress status. B KEGG pathway enrichment analysis of differential module hub genes (DMHGs). C Heatmap of correlation between DMHGs expression levels (FPKM values) and physiological parameters in liver. *P < 0.05, **P < 0.01. D qPCR validation of RNA-seq results of DMHGs (n = 5 per group). E RT-qPCR analysis of FASN expression in control and heat-stressed primary hepatocytes. F Effect of FASN overexpression on LDH, TG, AST and ALT levels in supernatants of control and heat-stressed primary hepatocyte cultures. Data are expressed as mean ± SEM (n = 3 per group). *P < 0.05, **P < 0.01. Different letters indicate significant differences between treatments (P < 0.05)

To explore the functions of fatty acid pathway-related DMHGs (FASN, hydroxysteroid 17-Beta Dehydrogenase 12 (HSD17B12), elongase of very long chain fatty acids 6 (ELOVL6), alcohol Dehydrogenase 1 C (ADH1C), enoyl-CoA delta isomerase 2 (ECI2)), we correlated their expression levels (quantified as FPKM values) with various physiological parameters obtained before and after the heat stress. In liver, the expression of all fatty acid pathway-associated DMHGs was, as expected, significantly correlated with rectal temperature (Fig. 4C). Further analyses showed that the expression of FASN, HSD17B12 and ELOVL6 also correlated significantly with the metabolic markers TG and GLU, and the tissue damage markers AST, LDH and CK (Fig. 4C). To validate the expression patterns observed in the RNA-seq data, we performed real-time qPCR analysis of DMHGs that were significantly correlated with rectal temperature. The results from five samples per group (control and heat-stress) showed good agreement between RNA-seq and qPCR expression profiles, confirming the reliability of our transcriptome analysis (Fig. 4D and Fig. S1).

Since most of the DMHGs were associated with fatty acid metabolic pathways, and FASN is a key gene for fatty acid synthesis, a chicken embryo primary hepatocyte model was constructed to further investigate the role of FASN in heat stress response. In the hepatocyte heat stress model (42 ℃ for 6 h), RT-qPCR analysis showed a significant down-regulation of FASN expression, consistent with the above transcriptome results in liver tissue (Fig. 4E). Analysis of cell culture supernatants from heat-stressed hepatocytes showed significant increases in AST, ALT and LDH levels and a significant decrease in TG levels, consistent with changes in blood physiological indices (Fig. 4F). In addition, over expression of FASN in heat-stressed hepatocytes restored TG and AST levels in cell supernatants to near-normal levels. Although no significant changes were found in ALT levels, a decreasing trend was noted. LDH levels remained significantly increased due to heat stress and appeared unaffected by FASN expression levels (Fig. 4F). The results suggest that FASN plays an important role in the regulation of lipid metabolism during heat stress, and may also contribute to mitigating the cellular damage caused by heat stress.

Discussion

In intensive poultry farming, particularly in tropical or subtropical regions, ambient temperatures in modern chicken houses equipped with efficient cooling systems can still reach as high as 32℃ during the summer months, whereas older chicken houses with aging equipment may experience temperatures as high as 36 °C. In a previous study, we developed a heat stress model using temperatures of 32℃ and 36℃ and found that 36 °C induced more severe physiological responses compared to 32 °C, in consistent with findings from several other studies [27, 42–44]. In the present study, heat treatment at 36 ℃ for 6 h was used to construct the acute heat model in chickens, aiming to capture more pronounced differences in transcriptomic profiles across five major organs.

In our study, the comparative transcriptome analyses of five organs showed liver’s critical role in chicken’s acute heat stress response. Among the five organs examined, the liver exhibited the most extensive transcriptional changes and metabolic pathway alterations, consistent with its central role in maintaining metabolic homeostasis and known sensitivity to environmental stressors [45]. The number of DEGs in the liver was 519, enriched for key metabolic pathways such as fatty acid degradation, pyruvate metabolism and glycolysis/gluconeogenesis. Notably, enrichment of lipid metabolism pathways was also observed in the heart and kidney. Furthermore, liver susceptibility to heat injury, confirmed by histologic changes and elevated serum markers of injury (AST, LDH), suggests a complex interaction between liver dysfunction and metabolic dysregulation during acute heat stress.

Six co-expressed gene modules by WGCNA analyses were found to be significantly and highly associated with liver, heart and lung tissues. From these modules, 58 differentially expressed module hub genes (DMHGs) were identified, 42 of which were DEGs found in liver. Enrichment analysis of these DMHGs once again highlighted pathways related to fatty acid metabolism, which further emphasize the importance of hepatic fatty acid metabolism in the response of chickens to acute heat stress. Given that KEGG and GO enrichment analyses are hypothesis-generating tools and do not yield definitive conclusions, we further performed correlation analyses and revealed significant associations between genes involved in fatty acid-related pathways — FASN and physiological indicators such as rectal temperatures and TG in blood. Consistent with our findings, several studies have reported alterations in hepatic lipid metabolism genes in broiler chickens under heat stress conditions [14, 17, 46]. Studies have shown that acute heat exposure elevates circulating non-esterified fatty acids, suggesting enhanced lipolysis to meet energy demands [47]. However, chronic heat stress paradoxically suppresses lipolysis and reduces hepatic β-oxidation capacity, likely due to mitochondrial dysfunction and oxidative damage [48]. Our results showed enrichment of both fatty acid biosynthesis and degradation pathways, suggesting a complex remodeling of lipid metabolism under acute heat stress. In poultry, such metabolic changes may have downstream effects on meat quality and egg production, as reduced lipid synthesis alters membrane composition and nutrient partitioning [49]. For example, reduced muscle cell membrane fluidity may affect ion transport and muscle function, leading to pale, soft, and exudative (PSE) meat common in heat-stressed broilers [46, 50]. Similarly, hepatic lipid depletion may limit the availability of yolk precursors such as very low-density lipoprotein (VLDL), thereby reducing egg production in laying hens [3]. These cascading effects emphasize the need for holistic interventions in the metabolism of heat stress.

The interplay between organ damage and metabolic dysregulation under conditions of acute heat stress is complex and multifaceted [51]. As a key tissue for capacity metabolism, heat stress-induced liver injury may exacerbate metabolic disorders, creating a feedback loop that further impairs an animal’s ability to maintain homeostasis in the body [46]. During heat stress, the liver faces a dual challenge: mitigating the overproduction of reactive oxygen species (ROS) and maintaining energy homeostasis [17, 52]. This metabolic reprogramming aligns with observations in heat-stressed mammals showing enhanced glucose production through phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase catalytic (G6PC) activation [53]. In the present study, decreased blood TG levels and increased GLU levels suggest that possibly during heat stress, hepatic gluconeogenesis is prioritized to reduce lipid synthesis to meet energy demands [54]. Notably, transcriptomic analysis revealed significant enrichment of focal adhesion and extracellular matrix receptor interaction pathways in the heart during acute heat stress. These findings corroborate previous reports of heat-induced structural destabilization in cardiac tissues, characterized by cytoskeletal rearrangement and impaired cell-matrix interactions [16, 55].

As FASN is a key enzyme in lipogenesis, correlation analyses and qPCR based on liver samples confirmed its down-regulation reduced lipid synthesis, leading to lower serum TG levels, a phenomenon consistent with previous observations in heat-stressed broilers [34, 56]. To functionally validate the central role of FASN in this process, we established a hepatocyte heat stress model. Given that the physiological incubation temperature for avian cells is approximately 37 °C, we selected 42 °C as the heat stress condition, in line with previous studies [45–47]. Notably, heat treatment at 42 °C for 1–5 h significantly increased LDH release and HSP70 expression in avian hepatocytes, consistent with our own findings [46]. While overexpression of FASN rescued triglyceride depletion under heat stress, it had no effect on LDH levels, suggesting that its role is limited to lipid homeostasis rather than general cytoprotection. This may be due to the role of FASN in maintaining cell membrane integrity and fluidity, which are critical for cellular heat acclimation mechanism [57] The strong correlation between FASN expression and indicators of heat tolerance (rectal temperature, serum TG, and markers of tissue damage) makes FASN a marker-assisted in breeding program. Dietary interventions targeting fatty acid metabolism, such as supplementation with omega-3 fatty acids, can further alleviate heat-induced metabolic disturbance [58, 59]. Omega-3 fatty acids are known for their anti-inflammatory and membrane-stabilizing properties that counteract oxidative damage while maintaining lipid homeostasis [60]. However, excessive lipid accumulation may impair heat dissipation and exacerbate thermal sensitivity [3].

Conclusions

This study reveals the critical role of fatty acid metabolism of liver during acute heat stress (36℃, 6 h) in chickens. Multi-tissue transcriptome analysis identified the liver as a core response organ, with a key role for the fatty acid pathway and the key regulator FASN. Functional validation confirmed that overexpression of FASN restored triglyceride levels under heat stress, highlighting its therapeutic potential. These findings provide a feasible target for improving heat tolerance in poultry through metabolic intervention.

Supplementary Information

Supplementary Material 1. (85.7KB, docx)

Acknowledgements

The authors thank the members of the HZAU Poultry Laboratory for their assistance in material preparation and animal management.

Abbreviations

ROS

Reactive oxygen species

ATP

Adenosine triphosphate

WGCNA

Weighted gene co-expression network analysis

FASN

Fatty acid synthase

GO

Gene ontology

KEGG

Kyoto encyclopedia of genes and genomes

DEG

Differentially expressed gene

BP

Biological process

MF

Molecular function

CC

Cellular component

MHG

Module hub gene

MM

Odule membership

GAPDH

Lyceraldehyde-3-phosphate dehydrogenase

CK

Reatine kinase

LDH

Actate dehydrogenase

AST

Spartate aminotransferase

ALT

Alanine aminotransferase

GLU

Lucose

TP

Otal protein

TG

Riglyceride

TCHO

Otal cholesterol

PCA

Rincipal Component Analysis

HSD17B12

Hydroxysteroid 17-Beta Dehydrogenase 12

ELOVL6

Elongase of very long chain fatty acids 6

ADH1C

Alcohol dehydrogenase 1C

ECI2

Enoyl-CoA delta isomerase 2

VLDL

Very low-density lipoprotein

PEPCK

Phosphoenolpyruvate carboxykinase

G6PC

Glucose-6-phosphatase catalytic

Authors’ contributions

ZM performed the research, curated and analyzed data, wrote the paper; ZQS performed the research and analyzed data; HBZ performed the research and edited the paper; BZ performed the research, edited the paper; YWX performed the research; ZYS designed the research, curated and analyzed data, wrote the paper, secured funding for the research; YZG designedthe research, curated and analyzed data, wrote the paper, secured funding for the research.

Funding

This work was funded by the Science and Technology Innovation 2030 Major Projects (grant number 2023ZD04052), the Science and Technology Innovation 2030 Major Projects (grant number 2023ZD0407106), and the National Key Research and Development Program (grant number 2018YFE128100).

Data availability

Sequence data that support the findings of this study have been deposited in the National Center for Biotechnology Information (NCBI) with the primary accession code PRJNA1222310. The dataset can be accessed via the following link: https://dataview.ncbi.nlm.nih.gov/object/PRJNA1222310?reviewer=u4nvb2stnkfn8j4n8ncplbekqn.

Declarations

Ethics approval and consent to participate

The present study was approved by the Experimental Animal Management and Ethics Committee of Huazhong Agricultural University (Approval No. HZAUCH-2020-0018).

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Contributor Information

Zheya Sheng, Email: zheya.sheng@mail.hzau.edu.cn.

Yanzhang Gong, Email: poultry@mail.hzau.edu.cn.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Material 1. (85.7KB, docx)

Data Availability Statement

Sequence data that support the findings of this study have been deposited in the National Center for Biotechnology Information (NCBI) with the primary accession code PRJNA1222310. The dataset can be accessed via the following link: https://dataview.ncbi.nlm.nih.gov/object/PRJNA1222310?reviewer=u4nvb2stnkfn8j4n8ncplbekqn.

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