Transcriptome profiling analysis of heat-adapted and heat-sensitive breeder hen jejunal mucosal tissues in response to acute heat stress

Transcriptome profiling analysis of heat-adapted and heat-sensitive breeder hen jejunal mucosal tissues in response to acute heat stress | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Transcriptome profiling analysis of heat-adapted and heat-sensitive breeder hen jejunal mucosal tissues in response to acute heat stress Yongcai Zhu, Satoshi Kubota, Phocharapon Pasri, Sitthipong Rakngam, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9368422/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 10 You are reading this latest preprint version Abstract Background Heat stress impairs intestinal integrity in poultry, compromising nutrient digestion and absorption. Nevertheless, the individual variation in heat tolerance suggests distinct molecular responses that are still not well understood. Materials and methods This study investigated the molecular basis of differential heat tolerance by comparing jejunal transcriptomes in heat-adapted ( HA ) and heat-sensitive ( HS ) breeder hens under acute heat stress (36°C for a 6-h). Fifty 28-week-old hens were randomly allocated to the HA and HS groups (25 hens each). After exposure to acute heat stress for 6 hours, the respiratory rate and cloacal temperature were measured, and jejunal mucosal samples were collected for RNA sequencing ( RNA-seq ). Results The results indicated that, under acute heat stress, the respiratory rates and cloacal temperatures of HS hens were significantly higher than those of HA hens ( P = 0.002). RNA-seq analysis identified 284 differentially expressed genes ( DEGs ), with 155 genes upregulated and 129 downregulated in the HS group compared to the HA group. Gene Ontology analysis revealed significant enrichment in 555 GO terms. Kyoto Encyclopedia of Genes and Genomes pathway analysis identified five pathways that were enriched in upregulated DEGs (VEGF signaling pathway, MAPK signaling pathway, steroid biosynthesis, neuroactive ligand-receptor interaction, and cell cycle) and one pathway enriched in downregulated DEGs (cell adhesion molecules). Protein-protein interaction network identified key genes ( PLK1 , CDC7 , CDC20 , HSPA2 , IL6 , SLC22A19A , LBFABP , SLC6A19 , and SLC2A2 ), involved in cell division, immune function, energy and lipid metabolism, as well as organic acid, glucose, and amino acids transport. Conclusions These findings suggest that acute heat stress differentially affects intestinal function in HA and HS hens, potentially through alterations in cell division, immune function, energy and lipid metabolism, and organic acid and glucose transport mechanisms. The identified DEGs and pathways offer valuable insight into the molecular basis of heat stress susceptibility and may inform nutritional or management strategies to enhance poultry resilience. Transcriptome analysis Breeder hen Acute heat stress Jejunal mucosa Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction As global ambient temperatures rise, heat stress has emerged as a prevalent environmental stressor with significant impacts on animal production and health. Among livestock, poultry are susceptible to elevated temperatures due to their limited capacity to regulate heat loss through evaporation [ 1 ]. Heat stress impairs growth, induces oxidative stress and promotes intestinal inflammation, and compromises gut barrier function—resulting in reduced nutrient absorption and immune dysfunction [ 2 , 3 , 4 , 5 ]. Specifically, heat stress-induced reductions in mesenteric blood flow damage the intestinal epithelium and disrupt the transport of essential nutrients such as glucose, amino acids, and lipids [ 6 , 7 , 8 ]. Despite these established physiological effects, the molecular mechanisms underlying intestinal responses to heat stress, particularly in comparing heat-sensitive ( HS ) and heat-adapted ( HA ) breeders, remain insufficiently understood. The intestine, particularly the jejunum, which plays a crucial role in nutrient digestion and absorption, is recognized as the primary target of heat stress [ 9 ]. In response to heat stress, cells activate protective mechanisms, notably by upregulating heat shock proteins ( HSPs ) [ 10 ]. The HSPs are essential for cell survival under stress conditions and maintain cellular homeostasis by preventing protein misfolding and facilitating the removal of damaged proteins [ 11 ]. Among HSPs, HSP70 and HSP90 are the most extensively studied and serve as biomarkers of cellular stress [ 12 ]. Research has shown that heat stress upregulates the expression of HSP70 mRNA in the jejunal mucosa in broilers [ 13 ], activates the intestinal MAPK signaling pathway, and mitigates both structural and oxidative damage to the intestinal mucosa induced by high temperature [ 13 ]. In addition, heat stress regulates genes involved in nutrient absorption and transport [ 14 ]. Studies have demonstrated that heat stress significantly reduces the expression levels of key transport proteins, including glucose transporter 2 ( GLUT-2 ), fatty-acid-binding protein ( FABP ), and cluster of differentiation 36 ( CD36 ) in broilers [ 15 ]. Furthermore, heat stress has been shown to impair immune responses in the small intestine [ 16 ], with heat stress leading to increased expression of interleukin-6 ( IL6 ) and tumor necrosis factor-alpha in the jejunum of broilers [ 17 ]. However, existing studies have focused largely on broilers, with limited data available on breeder hens—a group with distinct physiological demands. Genetic background plays a critical role in modulating heat tolerance in poultry [ 18 ]. Indigenous or slow-growing breeds, such as Thai native chickens (Leung Hang Khao) [ 19 ], often demonstrate greater thermal resilience than fast-growing commercial lines [ 20 , 21 ]. These breed-specific differences offer a unique opportunity to study how transcriptomic variation contributes to heat adaptability. Comparing the transcriptomes of HA and HS birds under identical stress conditions enables us to identify the genes and pathways responsible for differential tolerance, providing a molecular basis for resilience. Although several studies have reported candidate genes linked to heat stress, immune function, and metabolism across breeds [ 22 , 23 ], few have directly compared jejunal transcriptomes of HA and HS breeder hens under acute heat stress. Transcriptomic technology offers valuable insights into the genetic and molecular responses to heat stress by allowing the identification of differentially expressed genes ( DEGs ) and regulatory pathways [ 24 ], and aids in comparing the transcriptome profile among breeds [ 25 ]. In our previous study using RNA-seq, we identified DEGs associated with steroid biosynthesis, terpenoid backbone biosynthesis, steroid hormone biosynthesis, endoplasmic reticulum protein processing, PPAR signaling pathway, and DNA replication in the jejunal mucosa of HS breeder hens under acute heat stress [ 26 ]. However, the specific transcriptomic profiles that differentiate HA from HS breeder hens remain unknown. Therefore, this study aims to compare the transcriptomic profiles of HA and HS breeder hens under acute heat stress, aiming to identify key genetic markers and pathways associated with heat tolerance. These insights will contribute to genetic selection strategies and targeted interventions to improve intestinal resilience and overall heat adaptation in poultry. Materials and methods Ethics statement All experimental protocols were approved by the Animal Care and Use Committee of Suranaree University of Technology ( SUT ), Thailand (Approval Code: SUT-IACUC-012/2020). All animals used in this study were sourced exclusively from the SUT farm (Nakhon Ratchasima, Thailand), which is owned and operated by Suranaree University of Technology. As the animals were institutionally owned by SUT, formal informed consent was obtained from the SUT farm management prior to inclusion in the study. Housing, birds, and sample collection A total of fifty 22-week-old hens from two breeds—HS breeds (SUT breed) and HA breeds (Leuang Hang Kao breed) used in this study were obtained from the SUT farm, with 25 hens per breed. The Leuang Hang Kao breed is a Thai native breed, while the SUT breed represents a synthesized commercial line developed for producing Thai indigenous crossbred chickens. Prior to the start of the experiment, the hens were individually housed in cages with a size of 40 × 45 × 40 cm³ (width × depth × height) and acclimated for 5 weeks in a thermoneutral (23 ± 1°C) room maintained using an air conditioner. All hens were fed 140 g/day of corn-soy basal diets formulated following the guidelines of the National Research Council 1994 [ 27 ] and Aviagen (2021) [ 28 ] recommendations (2,800 kcal of metabolizable energy/kg and 15% crude protein), with water available ad libitum, and were maintained on a 16-hour light cycle daily. At 28 weeks of age, all hens were moved to a heat stress room with a controlled temperature of 36°C and a humidity of 40–70% for 6 hours using a gas heater with thermostat-controlled equipment, as described by [ 29 ]. After the hens were exposed to heat stress for 6 hours, respiration rate and cloacal temperature were measured from 25 HA and 25 HS hens, respectively. Subsequently, 12 breeder hens from each breed were randomly selected and euthanized by cervical dislocation followed by exsanguination (cutting the neck vein). Jejunal mucosal tissues were then collected by dissection, placed into RNA protect tissue tubes (Qiagen, Duesseldorf, Germany), snap-frozen in liquid nitrogen, and stored at − 80°C until further transcriptome and gene validation analyses. Extraction of total RNA from jejunal mucosa samples Total RNA was extracted from 12 jejunal mucosal tissue samples from each breed (HA and HS breeds) using the RNeasy Mini Kit (Qiagen, Hilden, Germany) and subsequently purified with a QIAamp spin column (Qiagen), following the manufacturer's protocol. The RNA concentration was determined using a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), and its quality was assessed through 1% agarose gel electrophoresis, employing 0.5× TAE buffer and an electric current of 100 V for 25 min. The three pooled RNA samples (each pool consisting of four individual jejunal mucosa samples) from each breed were used to construct an RNA-seq library. Capillary electrophoresis using a QIAxcel Connect (Qiagen) system was employed to assess the RNA integrity number (RIN), with RNA samples having a RIN ≥ 7 selected for cDNA library construction. Library construction and data processing The cDNA library construction and RNA-seq were conducted by BGI Co., Ltd. (BGI, Shenzhen, China). Six libraries were sequenced on the DNBSEQ platform. Sequencing data were processed using SOAPnuke Version v1.5.2 [ 30 ] to generate clean reads. The clean reads were then aligned to the chicken reference genome (GCF_000002315.6_GRCg6a) using HISAT2 v2.0.4 [ 31 ], and gene expression levels were calculated using RSEM Version v1.2.8 [ 32 ]. In addition, heatmap clustering, principal component analysis ( PCA ), and Venn diagram creation analysis on Dr. Tom's analysis system, constructed by BGI-Shenzhen, China. Differentially expressed gene screening and functional enrichment Differential gene expression was analyzed using DESeq2 (v1.4.5) [ 33 ]. Differentially expressed genes ( DEGs ) were identified based on a fold-change ( FC ) of ≥ 1 and an adjusted value of P < 0.05. Gene Ontology ( GO ) and Kyoto Encyclopedia of Genes and Genomes ( KEGG ) pathway enrichment analyses were conducted, with GO terms and KEGG pathways having P < 0.05 considered significantly enriched. The interaction networks were constructed using the network analyzer tool Cytoscape software version 3.9.1 [ 34 ]. Validation of DEGs via quantitative polymerase chain reaction To validate the RNA-seq results, six DEGs were selected for further analysis based on their roles in heat stress (heat shock protein family B (small) member 9 [ HSPB9 ] and heat shock protein family A (Hsp70) member 2 [ HSPA2 ]), lipid metabolism (cluster of differentiation 36 [ CD36 ]), intestinal barrier integrity (claudin 15 [ CLDN15 ]), and immunity (recombination activating gene 2 [ RAG2 ] and interleukin 18 binding protein [ IL18BP ]). These genes were analyzed in the jejunal mucosa tissues of HA and HS breeder hens using quantitative polymerase chain reaction ( qPCR ). qPCR-specific primers were designed using NCBI's online primer design software ( https://www.ncbi.nlm.nih.gov/tools/primer-blast/ ) (Table S1 ). For reverse transcription, 2 µg of total RNA from each sample was used with the SuperScript III RNase H-Reverse Transcriptase Kit (Toyobo, Osaka, Japan) and random primers (Promega, Madison, WI, USA), following the manufacturer’s instructions. Real-time qPCR was performed using the QuantiNova SYBR Green PCR Kit (Qiagen, Hilden, Germany). Briefly, the 10 µL reaction mix was prepared containing 5 µL of SYBR Green, 0.4 µL of forward primer, 0.4 µL of reverse primer, 2 µL of cDNA, and 2.2 µL of nuclease-free water. The parameters of PCR cycles included the following phases: initial heat activation at 94°C for 10 min, followed by 40 cycles of denaturation at 95°C for 10 s, annealing at 60°C for 30 s, and final extension at 72°C for 30 s. The expression of the target gene was normalized against glyceraldehyde-3-phosphate dehydrogenase as the reference gene. Gene expression levels were quantified using the 2 −∆∆CT method [ 35 ]. Statistical analysis Data on respiratory rates and cloacal temperatures were analyzed using a t -test in SPSS version 27.0 (SPSS Inc.) [ 36 ]. Statistical significance was determined with a threshold of P < 0.05. Results and discussion Physical response to heat stress between heat-adapted and heat-sensitive breeder hens The respiratory rates and cloacal temperatures in HS breeder hens were significantly higher than those in HA breeder hens under acute heat stress ( P = 0.002) (Table 1 ). Following the 6-hour heat exposure challenge, the average respiratory rates and cloacal temperature of HA breeder hens were 179 times/min and 41.9°C, respectively. In comparison, HS hens exhibited a respiratory rate of 213 times/min and a cloacal temperature of 42.9°C, respectively. Table 1 Respiratory rates and cloacal temperatures between the heat-adapted and heat-sensitive breeder hens under heat stress conditions 1 . Strains Respiratory rate (times/min) Cloacal temperature (°C) Heat-adapted hens 179.0 b 41.9 b Heat-sensitive hens 213.3 a 42.9 a Pooled SEM 5.9 1.0 1 Values are means from 25 breeder hens (n = 25). a,b Values within each column with different superscripts are significantly different ( P < 0.05). The study demonstrates that HS breeder hens exhibit a higher physiological response to acute heat stress than HA hens, as indicated by their higher respiratory rates and cloacal temperatures. These differences underscore the adaptive mechanisms of heat-tolerant hens, which help mitigate the detrimental effects of heat stress [ 37 ]. This highlights the potential benefits of selecting heat-tolerant breeds to improve poultry welfare in hot climates [ 38 ]. Quality of RNA-seq reads A total of six cDNA libraries were constructed and sequenced on the DNBSEQ platform, and an average of 6.35 GB of data was obtained. The RNA-seq data quality metrics are presented in Table 2 . RNA-seq of jejunal samples from HA and HS breeder hens yielded an average of 43.52 million raw reads and 42.30 million clean reads per sample. The sequencing quality was high, with Q20 and Q30 percentages exceeding 97.76% and 93.13%, respectively. GC content across all samples ranged from 46.76% to 47.41%. Alignment of clean reads to the chicken reference genome resulted in mapping rates of 95.38% to 95.82%. Table 2 RNA-sequencing reads and mapping rates in the jejunal mucosa transcriptome of breeder hens between the heat-adapted (HA) and heat-sensitive (HS) groups. Sample 1 Raw Reads (M) Clean Reads (M) Clean Bases (GB) Q20 2 (%) Q30 2 (%) GC content (%) Total Mapping (%) HA1 45.44 44.09 6.61 98.06 94.05 47.14 95.38 HA2 44.15 43.06 6.46 97.76 93.13 46.76 95.82 HA3 45.44 43.85 6.58 97.89 93.68 47.41 95.66 HS1 40.39 39.33 5.93 97.91 93.64 47.01 95.49 HS2 40.78 39.76 5.96 97.84 93.38 47.13 95.55 HS3 44.94 43.73 6.56 97.91 93.65 47.24 95.65 Average 43.52 42.30 6.35 97.90 93.59 47.12 95.59 1 Each sample consists of four individual jejunal mucosa between the heat-adapted (HA) ( n = 12) and heat-sensitive (HS) ( n = 12) breeder hens under heat stress condition. 2 Q20 and Q30 indicate the percentage of bases with a Phred value ≥ 20 and 30, respectively. HA, heat-adapted, HS, heat-sensitive. Differentially expressed genes analysis To identify DEGs in the jejunal mucosa of HA and HS breeder hens under acute heat stress, RNA-seq and subsequent bioinformatic analysis were performed. PCA of jejunal mucosa transcriptomics revealed a clear separation between HA and HS groups, indicating distinct mRNA expression profiles (Fig. 1 A). Hierarchical clustering of DEGs, based on FPKM values, further confirmed this separation, with samples clustering by groups and distinct gene expression patterns observed between HA and HS hens (Fig. 1 B). A total of 15,258 genes were identified across both groups. Among these, 397 genes were uniquely expressed in the HA group, while 347 genes were exclusively expressed in the HS group (Fig. 2 A), suggesting potential breed-specific regulatory mechanisms underlying thermotolerance or susceptibility. Differential expression analysis identified 284 DEGs, with 155 upregulated and 129 downregulated DEGs in HS hens compared to HA hens (Fig. 2 B and Table S2 ). Upregulated genes in HS hens included HSPB9 , RAG2 , HSPA2 , and heat shock protein family B (small) member 1 ( HSPB1 ), while downregulated genes included CLDN15 and liver basic fatty acid binding protein ( LBFABP ). The primary defense against heat stress involves HSPs, which function as intracellular molecular chaperones by binding to misfolded proteins and preventing their aggregation [ 39 , 40 ]. This study reveals the upregulation of several key HSP family members, including HSPA2 (HSP70), HSPB1 (HSP27), and HSPB9 (HSP25). Notably, the expression of HSPA2 (HSP70) was significantly upregulated, with a fold change of 4.4, aligning with previous studies by Kim et al. [ 41 ] and Zhu et al. [ 26 ] in heat-stressed chickens. HSPB9 (HSP25) expression patterns were characterized by an initial low expression followed by a gradual increase over time [ 42 ]. The protein’s role as a molecular chaperone was confirmed through its involvement in cellular homeostasis maintenance and protein denaturation prevention. In HS hens, HSPB1 was also found to be elevated in the jejunal mucosa, where it exerted multiple protective mechanisms, including protein stability maintenance and oxidative stress protection [ 43 ]. The protein’s interaction with cytosolic cytochrome C was observed to regulate apoptotic pathways [ 44 ], while its involvement in lipid clearance was also documented [ 45 ]. Both HSPB1 and HSPB2 were observed to form large multimeric complexes that were involved in preventing protein aggregation and maintaining cytoskeletal integrity during heat stress [ 46 ]. In addition to HSP-related changes, immune response modifications were observed, as evidenced by the upregulation of the RAG2 gene in the jejunal tissue of the HS group relative to the HA group. The gene’s involvement in V(D)J recombination and lymphocyte development has been well established [ 47 ], suggesting its critical role in maintaining immune function under heat-stress conditions. The role of RAG2 in preserving immune function during heat stress likely centers on its essential role in sustaining immune diversity by facilitating the proper development of T and B cells [ 48 ]. Reduced expression of RAG2 during heat stress may impair the immune system’s ability to produce a full range of antigen-specific receptors, thereby limiting the effectiveness of the immune response to infections. By maintaining adequate levels of RAG2 expression, organisms may better preserve their immune competency during stressful conditions. Furthermore, studies suggest that breed-specific variations in RAG2 expression could influence how different breeds respond to thermal stress, with some breeds, like the HA group, potentially exhibiting more robust immune responses under heat stress conditions [ 48 ]. Interestingly, the HA group exhibited less pronounced RAG2 expression changes, indicating potential breed-specific regulatory mechanisms that may confer enhanced stress tolerance. Moreover, an indirect activation of PPAR-γ by RAG2 was observed, which was associated with increased adipogenesis in the jejunum of heat-stressed breeder hens [ 48 ]. These molecular adaptations were more pronounced in HA breeds compared to HS breeds, indicating the development of a more sophisticated cellular protection system in the former group. The observed changes encompassed multiple aspects of cellular function, including protein stability maintenance, metabolic regulation, and immune system functionality. Heat stress has emerged as a critical factor compromising intestinal barrier integrity in poultry production [ 49 ]. The intestinal barrier is maintained by tight junction proteins such as occludin ( OCLN ) and claudin ( CLDN ), which regulate paracellular permeability and are essential for maintaining gut health [ 50 ]. Our transcriptomic analysis revealed that CLDN15 gene expression was significantly downregulated (FC = − 3.7) in the HS group compared to the HA group. CLDN15 serves as a critical tight junction protein that forms a cation-selective channel, facilitating Na + -dependent nutrient transport and maintaining Na + homeostasis [ 51 ]. This Na + gradient is fundamental for various transport processes, including the Na + -dependent uptake of bile acids into enterocytes [ 52 ] and the absorption of essential nutrients such as glucose and amino acids [ 53 ]. Moreover, CLDN15 has been shown to promote the proliferation of intestinal cryptic cells [ 54 ], which are vital for continuous epithelial renewal and barrier maintenance. The heat stress-induced downregulation of CLDN15 observed in HS hens suggests a compromised intestinal barrier function that may significantly impair nutrient absorption and utilization, a molecular adaptation that was notably less pronounced in HA hens. This breed-specific difference indicates the potential value of identifying molecular adaptations in different breeds to heat stress. Heat stress effects extend beyond barrier functions to impact lipid metabolism and transport mechanisms in the intestine [ 14 ]. Fatty acid-binding proteins (FABPs) are crucial facilitators of long-chain fatty acid uptake and transport from intestinal chyme into intestinal epithelial cells, where they support triglyceride synthesis [ 55 ]. FABP expression may facilitate intracellular fatty acid trafficking from uptake to storage or oxidation, or from lipid droplets for secretion [ 56 ]. Our findings demonstrated significant downregulation (FC = − 2.42) of LBFABP in the HS group compared to the HA group, aligning with previous studies reporting decreased FABP expression in heat-stressed chicken intestine [ 57 ]. While LBFABP (also known as FABP10 ) is dominantly expressed in liver tissue [ 58 ], its presence in intestinal tissue plays a vital role in the efflux and transport of various lipids, including cholesterol and bile acids [ 59 ]. The lower LBFABP expression in the HS group suggests less intracellular trafficking of lipids and consequently a decreased capacity for lipid utilization (including storage and oxidation) in the intestine. In contrast, HA hens demonstrated more stable LBFABP expression, suggesting a breed-specific adaptation that supports lipid transport and energy metabolism under heat stress. Gene ontology (GO) annotation analyses of DEGs To elucidate the molecular mechanism underlying heat stress, GO annotation analysis was performed on the DEGs. The analysis categorized the DEGs into three main functional groups: biological processes ( BP ), molecular functions ( MF ), and cellular components ( CC ). The analysis revealed significant enrichment in 555 GO terms ( P < 0.05), comprising 371 BP, 110 MF, and 74 CC (Fig. 3 , Table S3 ). Within the BP category, cell division ( P = 2.35E–05), chromosome segregation ( P = 2.40E–05), cell cycle ( P = 2.55E–04), fructose transmembrane transport ( P = 2.85E–04), and response to heat ( P = 1.12E–03) pathways are included. The MF revealed prominent enrichment in various transport-related activities, especially microtubule binding ( P = 4.11E–06), fructose transmembrane transport ( P = 2.83E–04), short-chain fatty acid transmembrane transport ( P = 2.83E–04), glucose transmembrane transport ( P = 5.61E–03), and neutral amino acid transmembrane transport activity ( P = 7.40E–03), etc. In terms of CC, the DEGs were predominantly enriched in chromosomes, centromeric regions ( P = 9.86E–09), extracellular space ( P = 6.40E–05), brush border membrane ( P = 5.71E–04), plasma membrane ( P = 4.09E–03), and extracellular regions ( P = 1.05E–03), etc. Among these, three GO terms‒response to heat, extracellular space, and extracellular region were identified, which align with the findings of Kim et al. [ 60 ] in their analysis of chronic heat stress responses in hen jejunal mucosa. In addition, the comparison between HS and HA hens highlighted GO terms related to fructose and glucose transmembrane transport, brush-border membrane, and motor activity in the jejunum. The jejunum is the primary site for the absorption of essential nutrients, including amino acids, carbohydrates, and fatty acids [ 61 ]. Efficient nutrient uptake in this region is facilitated by a range of membrane transporters located in the brush-border membrane, which function to regulate nutrient flux and maintain metabolic homeostasis under stress conditions [ 62 ]. Notably, the reduced expression of transport proteins associated with the transmembrane transport of fructose and glucose in the HS hens suggests a potential vulnerability in nutrient absorption, indicating a potential risk of metabolic dysfunction and energy deficits. This pattern was further corroborated by the protein-protein interaction (PPI) analysis in this study. KEGG pathway analyses of DEGs To identify the biological pathways involved in breeder hens under heat stress, the DEGs from both groups were mapped to the KEGG pathway database. KEGG pathway enrichment analysis revealed significant alterations in multiple signaling cascades following acute heat stress exposure. The analysis identified six significantly enriched pathways ( P < 0.05): VEGF signaling pathway ( P = 7.82E–03), MAPK signaling pathway ( P = 0.0208), cell adhesion molecules ( P = 0.0345), steroid biosynthesis ( P = 0.0413), neuroactive ligand-receptor interaction ( P = 0.0430), and cell cycle ( P = 0.0483) (Table 3 ). These enriched pathways suggested a complex cellular response to acute heat stress, involving multiple regulatory mechanisms and cellular processes. Table 3 Significantly enriched Kyoto Encyclopedia of Genes and Genomes pathways in the jejunal mucosa between the heat-adapted (HA) and the heat-sensitive (HS) breeder hens under heat stress 1 . KEGG 2 Pathway Term Count P value Gene Symbols 3 gga04370: VEGF signaling pathway 2 7.82E–03 HSPB1 ↑, PLA2G4EL2 ↑ gga04010: MAPK signaling pathway 3 0.0208 HSPH1 ↑, HSPA2 ↑, PLA2G4EL2 ↑ gga04514: Cell adhesion molecules 2 0.0345 HLA-F10AL4 ↓, CLDN15 ↓ gga00100: Steroid biosynthesis 1 0.0413 LIPML5 ↑ gga04080: Neuroactive ligand-receptor interaction 3 0.0430 TAC1 ↑, HTR1B ↑, RLN3 ↑ gga:04110 Cell cycle 5 0.0483 BUBIB ↑, CDK1 ↑, PLK1 ↑, CDC7 ↑, CAC20 ↑ 1 Pathways were detected from DEGs in the jejunal mucosa between heat-adapted (HA) ( n = 12) and heat-sensitive (HS) ( n = 12) breeder hens under heat stress conditions. 2 KEGG, Kyoto Encyclopedia of Genes and Genomes 3 Up and down arrows indicate the upregulated and downregulated genes in HS hens compared to HA hens, respectively, in the jejunal mucosa between the heat-adapted (HA) and the heat-sensitive (HS) breeder hens under heat stress conditions. KEGG pathway analysis revealed six enriched pathways influenced by acute heat stress in the jejunal mucosal tissues of the HS group compared to the HA group. Notable among these were the vascular endothelial growth factor ( VEGF ) signaling pathways and neuroactive ligand-receptor interaction, which have been previously identified in heat-stressed and immune-stressed (LPS challenge) broilers' jejunum [ 60 , 63 ]. The VEGF signaling pathway is particularly significant in the context of HS chickens, as excessive VEGF activity has been implicated in vascular hyperpermeability and inflammation under heat stress conditions [ 64 ]. The jejunal mucosal damage induced by heat stress can result in intestinal hypoxia, triggering VEGF regulation via hypoxia-inducible factor-1α (HIF-1α) activation [ 65 ]. While VEGF signaling typically promotes angiogenesis to restore oxygen and nutrient delivery to damaged tissues [ 66 ], the HS group upregulated the VEGF pathway, suggesting a maladaptive response that may exacerbate vascular leakage and inflammatory damage. Moreover, the upregulation of neuroactive ligand-receptor interaction pathways in the HS group indicates potential disruptions in neurotransmitter signaling, further contributing to intestinal dysfunction and impaired nutrient absorption. This overactivation of signaling pathways in HS hens may indicate a heightened stress response that compromises intestinal integrity, whereas the HA group exhibited more balanced pathway activity, suggesting a more controlled adaptation to thermal stress. The mitogen-activated protein kinase ( MAPK ) signaling pathway emerged as another significant pathway affected by acute heat stress in the jejunal mucosa, with notable differences observed between HS and HA breeder hens. The activation of MAPK signaling in response to heat stress has been well-documented in broiler jejunal tissue [ 67 ]. This pathway regulates various physiological functions, including oxidative stress responses, inflammation, cell multiplication, apoptosis, and autophagy [ 68 ]. In HS hens, MAPK signaling was notably upregulated, suggesting a heightened oxidative stress and inflammatory response that may contribute to cellular damage and compromised intestinal integrity. Our analysis revealed the upregulation of two HSPs, HSPH1 and HSPA2 , within the MAPK signaling pathway in HS hens compared to HA hens. HSPH1 , a member of the Hsp110 family, shows increased expression in HS hens, aligning with its established role in preventing cell death and promoting cellular survival under heat-stress conditions [ 69 ]. Concurrently, HSPA2 , an HSP70 family member, was significantly upregulated, suggesting its function in mitigating structural and oxidative damage to the intestinal mucosa during heat stress [ 13 ]. Previous research has demonstrated that heat stress upregulates HSP70 expression in chicken jejunal mucosa, concurrently activating the MAPK signaling pathway as a protective mechanism through its chaperone-mediated stabilization of misfolded proteins and interaction with MAPK intermediates [ 12 ]. The upregulation of HSPH1 and HSPA2 in the HS group suggests a potentially adaptive response to heat stress, wherein the activation of these HSPs may be insufficient to counteract thermal damage effectively, thereby exacerbating cellular stress. Protein-protein interaction network analysis of DEGs PPI network analysis for the DEGs identified three distinct networks. The largest network comprised 30 protein-coding genes, with CDK1 , PLK1 , CDC7 , and CDC20 positioned as core nodes. These core proteins showed primary enrichment in the cell cycle pathway within the jejunum (Table 3 and Fig. 4 ). Notably, most proteins in this network were upregulated in HS hens compared to HA hens, as indicated by red nodes, with a few exceptions, such as KIFC3 and CDK18 , shown in blue. The second network contained 13 interaction proteins, including HSPA2 and interleukin-6 ( IL6 ), all of which were exhibited upregulation (red nodes) in HS hens compared to HA hens. The third network consisted of 10 proteins, all of which were downregulated (blue nodes) in HS hens compared to HA hens, with SLC22A13L serving as the core node of the network. PPI network analysis revealed several upregulated cell cycle-related genes in the jejunum of heat-stressed HS hens, including CDK1 , PLK1 , CDC7 , and CDC20 . Cyclin-dependent kinase 1 ( CDK1 ), a key member of the cyclin-dependent kinase family, is a serine/threonine kinase that influences both the Wnt and fibroblast growth factor signaling pathways, thereby affecting cell proliferation [ 70 , 71 ]. In HS hens, the upregulation of CDK1 may represent a compensatory response to heat-induced cellular stress, promoting cell cycle progression despite adverse thermal conditions. Similarly, the upregulation of the polo-like kinase 1 gene ( PLK1 ), another serine/threonine kinase, suggests an adaptive mechanism to maintain cell division and DNA replication under heat stress. PLK1 regulates cell cycle checkpoints, and its overexpression enables cells to bypass arrest, potentially mitigating DNA damage through interactions with cell division cycle 7 ( CDC7 ) [ 72 ]. CDC7 is a critical cell cycle regulator, and its interaction with PLK1 facilitates mitotic exit and diaphase formation [ 73 ]. Notably, in HS hens, CDC7 expression was markedly elevated, consistent with previous findings in heat-stressed Illinois broilers that reported upregulation of PLK1 , CDC7 , and CDC20 [ 74 ]. This conserved upregulation across breeds suggests that these kinases may play pivotal roles in cellular adaptation to heat-induced DNA damage by promoting cell cycle progression and preventing apoptosis. The observed upregulation of cell cycle regulators in HS hens may indicate a heightened cellular effort to counteract heat stress-induced DNA damage, potentially reducing cell cycle arrest and apoptosis. The second largest network identified comprised 13 upregulated genes, with HSPA2 and IL6 emerging as key core nodes related to the immune response. HSPs are intricately linked to immune system functions [ 75 ]. The pro-inflammatory cytokine IL6 plays an important role in innate and acquired immunity [ 76 ]. Multiple studies have indicated increased IL6 expression following heat stress exposure [ 77 ], including elevated levels in the jejunal mucosae of thermal manipulation chicks under chronic heat stress [ 78 ]. The upregulation of IL6 and HSPA2 suggests a complex interplay between inflammatory signaling and cellular protection mechanisms in HS hens. While elevated IL6 levels may indicate an inflammatory response to heat-induced tissue damage, HSPA2 upregulation may serve to mitigate excessive inflammation by stabilizing proteins and inhibiting pro-inflammatory cytokine expression [ 79 ]. The relationship between HSPA2 and IL6 is particularly significant, as both proteins are regulated by heat shock factor ( HSF ). Research has shown that HSF induces both HSP70 and IL-6 expression in heat-stressed chickens, suggesting IL-6 may act as a heat-shock-responsive gene [ 80 ]. The upregulation of IL6 and HSPA2 in HS hens may indicate a dual response to heat stress, characterized by pro-inflammatory signaling coupled with protective HSP-mediated mechanisms. These findings suggest the role of IL6 as a critical mediator of inflammatory responses and its potential role in modulating immune function in HS hens under heat stress conditions. In addition, PPI analysis identified a network of ten downregulated DEGs associated with nutrient transport and metabolism in HS hens, including SLC22A13L , LBFABP , SLC2A2 , and SLC6A19 , which aligns with previous findings of decreased nutrient absorption and transport gene expression in heat-stressed animals [ 15 ], suggesting a potential impairment in nutrient handling in HS hens compared to HA hens. Solute carrier family 22 member 13 ( SLC22A13L ), also known as organic anion transporter 10 (OAT10) [ 81 ], is predominantly expressed in the apical membrane of proximal tubules in the kidneys, where it mediates urate reabsorption through the exchange of organic anions, such as urate, nicotinate, and orotate, for OH- or lactate anions [ 82 ]. Given the role of SLC22A13L in the kidney, low expression of SLC22A13L in the jejunum of HS hens may disrupt the transport of organic anions, impair the exchange of key metabolites (including lactate and nicotinate), and lead to impaired nutrient absorption, metabolic balance, and ion transport, thus causing intestinal barrier dysfunction in HS hens. Further studies are needed to clarify the role of SLC22A13L in the jejunal mucosa in response to heat stress. The glucose transport gene SLC2A2 (GLUT2) plays a role in glucosamine transport necessary for glycosaminoglycan biosynthesis [ 83 ]. Previous studies in broiler jejunum have demonstrated reduced GLUT2 expression under heat stress [ 15 ], suggesting disrupted intestinal glucose transport. Similarly, solute carrier family 6 member 19 ( SLC6A19 ), located in the apical membrane, encodes the B 0 AT protein responsible for high-affinity amino acid transport through electroneutral exchange coupled with the sodium co-transport [ 84 ]. The downregulation of SLC6A19 in HS hens suggests a disruption in amino acid absorption, potentially affecting protein metabolism and nutrient utilization. Validation of DEGs by qPCR We selected DEGs that regulated representative functions or whose expression profiles were significantly altered in response to acute heat stress. We validated the expression levels of four upregulated genes ( HSPB9 , RAG2 , HSPA2 , and IL18BP ) and two downregulated genes ( CLDN15 and CD36 ) in the jejunal mucosa between HA and HS breeder hens for the validation by using qPCR (Fig. 5 ). All genes showed similar expression trends in both the qPCR and RNA-seq. These results demonstrated the reliability and accuracy of our RNA-seq data in this study. Conclusion This study identified 284 DEGs, among these, 155 were upregulated, and 129 were downregulated DEGs in the jejunal mucosa of HS hens compared to HA hens under heat stress using RNA-seq analysis. Ten DEGs associated with HSP, immune response, intestinal barrier integrity, and the transport of lipids, organic acids, glucose, and amino acids, including HSPB9, HSPA2, HSPB1, RAG2, IL6, CLDN15, LBFABP, SLC22A13L, SLC2A2, and SLC6A19. may play key roles in regulating the jejunal mucosa of HS breeder hens under acute heat stress. The identified DEGs are implicated in critical processes related to heat response, cell division, and nutrient transport, indicating potential molecular targets for mitigating thermal stress effects in HS hens. KEGG pathway enrichment analysis revealed that the main biological pathways were related to the VEGF signaling, MAPK signaling, cell adhesion molecules, neuroactive ligand-receptor interaction, and cell cycle regulation. PPI analyses showed that acute heat stress may affect cell cycle progression, immune function, and the transport of organic acids, glucose, and amino acids in the jejunal mucosa of HS breeder hens. The identification of specific genes and pathways in the present study provides valuable insights for future genetic selection strategies and breeding programs aimed at improving heat tolerance in poultry. Declarations Declaration of competing interest The authors declare that they have no competing financial interests or personal relationships that may have influenced the work reported in this study. Consent for publication Not applicable. CRediT authorship contribution statement Yongcai Zhu: Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Software, Project administration, Methodology, Investigation, Formal analysis, Conceptualization. Satoshi Kubota: Validation, Supervision, Software. Phocharapon Pasri: Software, Methodology, Investigation. Sitthipong Rakngam: Methodology, Investigation, Formal analysis. Shenglin Yang: Visualization, Validation, Supervision, Software. Sutisa Khempaka: Supervision, Software, Resources, Funding acquisition, Data curation. Funding Open access funding provided by Suranaree University of Technology. This work was supported by the National Research Council of Thailand (NRCT5-RSA63009-03), Suranaree University of Technology (SUT), Thailand Science Research and Innovation (TSRI), and the National Science, Research, and Innovation Fund (NSRF). Acknowledgments The authors gratefully acknowledge the Suranaree University of Technology for their financial support through the One Research One Graduate (OROG). We would like to thank Phocharapon Pasri for his indispensable work in the implementation of this project in the bird care tasks, and all authors for the preparation of the laboratory work. Transcriptome data availability The transcriptome sequencing data are available through the Gene Expression Omnibus (accession number: GSE294384). References Oluwagbenga EM, Fraley GS. Heat stress and poultry production: a comprehensive review. Poult Sci. 2023;102:103141. Pearce SC, Mani V, Weber TE, Rhoads RP, Patience JF, Baumgard LH, et al. Heat stress and reduced plane of nutrition decrease intestinal integrity and function in pigs. J Anim Sci. 2013;91:5183–93. Varasteh S, Braber S, Akbari P, Garssen J, Fink-Gremmels J. 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OAT10/SLC22A13 acts as a renal urate re-absorber: clinico-genetic and functional analyses with pharmacological impacts. Front Pharmacol. 2022;13:842717. Uldry M, Ibberson M, Hosokawa M, Thorens B. GLUT2 is a high affinity glucosamine transporter. FEBS Lett. 2002;524:199–203. Bröer S. Amino acid transport across mammalian intestinal and renal epithelia. Physiol Rev. 2008;88:249–86. Additional Declarations No competing interests reported. Supplementary Files TableS1PrimerforqPCR.xlsx Table S1 List of primer sequences used for quantitative PCR TableS2.AllDEGsinformation.xlsx Table S2 All differentially expressed genes in the jejunal mucosa between the heat-adapted (HA) ( n =12) and the heat-sensitive (HS) ( n =12) groups under heat stress. TableS3GOterminformation.xlsx Table S3 Gene ontology terms for the differentially expressed genes in the jejunal mucosa between the heat-adapted (HA) (n=12) and the heat-sensitive (HS) (n=12) groups under heat stress. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9368422","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":633506573,"identity":"4d752bd2-24b3-4433-8fea-ab99e2bd8052","order_by":0,"name":"Yongcai Zhu","email":"","orcid":"","institution":"Guizhou Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Yongcai","middleName":"","lastName":"Zhu","suffix":""},{"id":633506574,"identity":"396e733e-5321-4bc7-b4a4-af18cea0c6c2","order_by":1,"name":"Satoshi Kubota","email":"","orcid":"","institution":"Suranaree University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Satoshi","middleName":"","lastName":"Kubota","suffix":""},{"id":633506575,"identity":"539e9bb2-fca2-497b-82b0-bcb796182f51","order_by":2,"name":"Phocharapon Pasri","email":"","orcid":"","institution":"Suranaree University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Phocharapon","middleName":"","lastName":"Pasri","suffix":""},{"id":633506576,"identity":"9175b2fc-e0cd-4b36-8bb3-a14b9dee885c","order_by":3,"name":"Sitthipong Rakngam","email":"","orcid":"","institution":"Suranaree University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Sitthipong","middleName":"","lastName":"Rakngam","suffix":""},{"id":633506577,"identity":"29a1adc9-d262-4f98-8139-bdac8a0e00c6","order_by":4,"name":"Shenglin Yang","email":"","orcid":"","institution":"Guizhou University","correspondingAuthor":false,"prefix":"","firstName":"Shenglin","middleName":"","lastName":"Yang","suffix":""},{"id":633506578,"identity":"9898f3ec-8ef8-4258-ad1f-11d0428714c0","order_by":5,"name":"Sutisa Khempaka","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA1UlEQVRIiWNgGAWjYBACCQkwlcDAjxAiVotkAwNjA4jJQ7QWgwPEapGc3fx0c0FNmrzx+TPmDxhq7BjspRvwa5GWOWZ2e8axHMNtN3IMGxiOJTPwyBzAr0VOIsHsNg9bBeO2GzxALWwHgA5LIKQl/dttnn8V9pv7zwC1/CNCi7REjtlt3racxA0MQIcxthGhRXJGTtntmX1pyTNupBXOSOxL5uG5QUCLxI30bbcLviXb9vcf3vDhwzc7OfYZBLSAADOcBVTMQ1g9ipZRMApGwSgYBdgAABG2QckeyzYZAAAAAElFTkSuQmCC","orcid":"","institution":"Suranaree University of Technology","correspondingAuthor":true,"prefix":"","firstName":"Sutisa","middleName":"","lastName":"Khempaka","suffix":""}],"badges":[],"createdAt":"2026-04-09 12:10:49","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9368422/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9368422/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":108435935,"identity":"2d5f306d-829e-4e3d-b462-0125df16698f","added_by":"auto","created_at":"2026-05-04 15:40:49","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":144741,"visible":true,"origin":"","legend":"\u003cp\u003ePrincipal component analysis and hierarchical clustering of DEGs in jejunal mucosa between the heat-adapted (HA) (\u003cem\u003en\u003c/em\u003e=12) and the heat-sensitive (HS) (\u003cem\u003en\u003c/em\u003e=12) groups under heat stress. (A) Principal component analysis (\u003cstrong\u003ePCA\u003c/strong\u003e) of DEGs in six heat-stressed breeder hens' jejunal tissues. Blue and orange nodes represent individuals from the HA and HS breeder hens, respectively.\u003c/p\u003e\n\u003cp\u003e(B) Heatmap of DEGs. Each row and column corresponds to a DEG and a sample name, respectively. The color scale from red to blue indicates log2 transcription ratios from 2 to −2; red colors represent the overexpressed genes, and the blue colors represent the genes with lower expression levels.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-9368422/v1/3b1d1f688dd25d8e929201b5.png"},{"id":108435936,"identity":"b0ceba1f-7dbe-4964-a94d-d3a203864e2e","added_by":"auto","created_at":"2026-05-04 15:40:49","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":104305,"visible":true,"origin":"","legend":"\u003cp\u003eDifferentially expressed analysis in the jejunal mucosa between the heat-adapted (HA) (\u003cem\u003en\u003c/em\u003e=12) and heat-sensitive (HS) (\u003cem\u003en\u003c/em\u003e=12) groups under heat stress conditions.\u003c/p\u003e\n\u003cp\u003e(A) Venn diagram of DEGs in heat-stressed breeder hens of jejunal tissues. (B) Volcano plot of DEGs in heat-stressed breeder hens of jejunal tissues. Red and green dots represent significantly upregulated and downregulated genes (adjusted\u003cem\u003e P\u003c/em\u003e\u0026lt; 0.05, |log2 FC| ≥ 1) in HS hens, respectively, and gray dots represent insignificant DEGs. The x and y axes of the volcano plots show the log2 fold changes and –log10 p-adj, respectively.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-9368422/v1/8811f40ac64b5c3ad558cfe2.png"},{"id":108435933,"identity":"f041ddd5-65c3-4d09-af6a-c190b9cb71fa","added_by":"auto","created_at":"2026-05-04 15:40:48","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":250551,"visible":true,"origin":"","legend":"\u003cp\u003eFunctional enrichment analysis of identified differentially expressed genes (DEGs). Top 20 enriched Gene Ontology (GO) terms of DEGs in jejunal mucosa between the heat-adapted (HA) (\u003cem\u003en\u003c/em\u003e=12) and heat-sensitive (HS) (\u003cem\u003en\u003c/em\u003e=12) breeder hens under heat stress conditions. The x-axis indicates –log10. The y-axis indicates functional categories. \u003cstrong\u003eA:\u003c/strong\u003e biological process, \u003cstrong\u003eB:\u003c/strong\u003e molecular function, and \u003cstrong\u003eC: \u003c/strong\u003ecellular component, GO, Gene Ontology. The circle size in each term corresponds to the number of genes. The circle's color goes from blue to red, indicating a lower \u003cem\u003eP\u003c/em\u003e value.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-9368422/v1/5c0ee7afbe72b8c54440e655.png"},{"id":108493418,"identity":"b1a67e89-6ce0-4ba5-80f1-db13996ff460","added_by":"auto","created_at":"2026-05-05 10:00:19","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":352419,"visible":true,"origin":"","legend":"\u003cp\u003eProtein-protein interaction network of differentially expressed genes (GEGs) in the jejunal mucosa between heat-adapted (HA) (\u003cem\u003en\u003c/em\u003e = 12) and heat-sensitive (HS) (\u003cem\u003en\u003c/em\u003e = 12) breeder hens under heat stress conditions. The size of the circle represents the values of log2 fold change. Red and blue nodes indicate the upregulated and downregulated genes in HS hens, respectively.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-9368422/v1/efd2c6e6774f8ed3fb170389.png"},{"id":108435941,"identity":"9d7d07b9-8888-46db-bc79-9214af10c020","added_by":"auto","created_at":"2026-05-04 15:40:50","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":31380,"visible":true,"origin":"","legend":"\u003cp\u003eqPCR validation of 6 DEGs identified using RNA-sequencing. The x-axis represents the genes, and the y-axis represents their mRNA expression levels expressed in fold-change values. Expression levels determined via quantitative polymerase chain reaction (qPCR) and RNA-sequencing (RNA-seq) are represented by black and white fill columns, respectively. Expression levels were examined in jejunal mucosae between the heat-adapted (HA) (\u003cem\u003en \u003c/em\u003e= 12) and heat-sensitive (HS) (\u003cem\u003en \u003c/em\u003e= 12) breeder hens under heat stress conditions. \u003cem\u003eHSPB9\u003c/em\u003e, heat shock protein family B (small) member 9; \u003cem\u003eHSPA2\u003c/em\u003e, heat shock protein family A (Hsp70) member 2; \u003cem\u003eCD36\u003c/em\u003e, cluster of differentiation 36; \u003cem\u003eCLND15\u003c/em\u003e, claudin 15; \u003cem\u003eRAG2\u003c/em\u003e, recombination activating gene 2; \u003cem\u003eIL18BP\u003c/em\u003e, interleukin 18 binding protein.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-9368422/v1/6c3408f475d17cd6acd4c12b.png"},{"id":109204473,"identity":"7b44b62a-3670-4ee3-856f-b34f61b7e2d7","added_by":"auto","created_at":"2026-05-13 15:00:22","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1163674,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9368422/v1/5596c3f4-3f75-4324-aa2e-c33a89c15d44.pdf"},{"id":108435932,"identity":"d7df4230-b620-4144-86c1-49bcc480acdc","added_by":"auto","created_at":"2026-05-04 15:40:48","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":10471,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable S1\u003c/strong\u003e List of primer sequences used for quantitative PCR\u003c/p\u003e","description":"","filename":"TableS1PrimerforqPCR.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-9368422/v1/fa2708de0def141e76b5559e.xlsx"},{"id":108435938,"identity":"5fe1d5a3-a14f-4cbd-8fda-b65abdeb15c1","added_by":"auto","created_at":"2026-05-04 15:40:49","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":27360,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable S2\u003c/strong\u003e All differentially expressed genes in the jejunal mucosa between the heat-adapted (HA) (\u003cem\u003en\u003c/em\u003e=12) and the heat-sensitive (HS) (\u003cem\u003en\u003c/em\u003e=12) groups under heat stress.\u003c/p\u003e","description":"","filename":"TableS2.AllDEGsinformation.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-9368422/v1/6ec5ed3cd21cf867109edee1.xlsx"},{"id":108435937,"identity":"9855da56-f88e-4455-9d9f-1d73e3662145","added_by":"auto","created_at":"2026-05-04 15:40:49","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":64537,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable S3\u003c/strong\u003e Gene ontology terms for the differentially expressed genes in the jejunal mucosa between the heat-adapted (HA) (n=12) and the heat-sensitive (HS) (n=12) groups under heat stress.\u003c/p\u003e","description":"","filename":"TableS3GOterminformation.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-9368422/v1/78de5f854463025fee7a8a36.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Transcriptome profiling analysis of heat-adapted and heat-sensitive breeder hen jejunal mucosal tissues in response to acute heat stress","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAs global ambient temperatures rise, heat stress has emerged as a prevalent environmental stressor with significant impacts on animal production and health. Among livestock, poultry are susceptible to elevated temperatures due to their limited capacity to regulate heat loss through evaporation [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Heat stress impairs growth, induces oxidative stress and promotes intestinal inflammation, and compromises gut barrier function\u0026mdash;resulting in reduced nutrient absorption and immune dysfunction [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Specifically, heat stress-induced reductions in mesenteric blood flow damage the intestinal epithelium and disrupt the transport of essential nutrients such as glucose, amino acids, and lipids [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Despite these established physiological effects, the molecular mechanisms underlying intestinal responses to heat stress, particularly in comparing heat-sensitive (\u003cb\u003eHS\u003c/b\u003e) and heat-adapted (\u003cb\u003eHA\u003c/b\u003e) breeders, remain insufficiently understood.\u003c/p\u003e \u003cp\u003eThe intestine, particularly the jejunum, which plays a crucial role in nutrient digestion and absorption, is recognized as the primary target of heat stress [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. In response to heat stress, cells activate protective mechanisms, notably by upregulating heat shock proteins (\u003cb\u003eHSPs\u003c/b\u003e) [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. The HSPs are essential for cell survival under stress conditions and maintain cellular homeostasis by preventing protein misfolding and facilitating the removal of damaged proteins [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Among HSPs, \u003cem\u003eHSP70\u003c/em\u003e and \u003cem\u003eHSP90\u003c/em\u003e are the most extensively studied and serve as biomarkers of cellular stress [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Research has shown that heat stress upregulates the expression of \u003cem\u003eHSP70\u003c/em\u003e mRNA in the jejunal mucosa in broilers [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], activates the intestinal MAPK signaling pathway, and mitigates both structural and oxidative damage to the intestinal mucosa induced by high temperature [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. In addition, heat stress regulates genes involved in nutrient absorption and transport [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Studies have demonstrated that heat stress significantly reduces the expression levels of key transport proteins, including glucose transporter 2 (\u003cb\u003eGLUT-2\u003c/b\u003e), fatty-acid-binding protein (\u003cb\u003eFABP\u003c/b\u003e), and cluster of differentiation 36 (\u003cb\u003eCD36\u003c/b\u003e) in broilers [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Furthermore, heat stress has been shown to impair immune responses in the small intestine [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], with heat stress leading to increased expression of interleukin-6 (\u003cb\u003eIL6\u003c/b\u003e) and tumor necrosis factor-alpha in the jejunum of broilers [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. However, existing studies have focused largely on broilers, with limited data available on breeder hens\u0026mdash;a group with distinct physiological demands.\u003c/p\u003e \u003cp\u003eGenetic background plays a critical role in modulating heat tolerance in poultry [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Indigenous or slow-growing breeds, such as Thai native chickens (Leung Hang Khao) [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], often demonstrate greater thermal resilience than fast-growing commercial lines [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. These breed-specific differences offer a unique opportunity to study how transcriptomic variation contributes to heat adaptability. Comparing the transcriptomes of HA and HS birds under identical stress conditions enables us to identify the genes and pathways responsible for differential tolerance, providing a molecular basis for resilience.\u003c/p\u003e \u003cp\u003eAlthough several studies have reported candidate genes linked to heat stress, immune function, and metabolism across breeds [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], few have directly compared jejunal transcriptomes of HA and HS breeder hens under acute heat stress. Transcriptomic technology offers valuable insights into the genetic and molecular responses to heat stress by allowing the identification of differentially expressed genes (\u003cb\u003eDEGs\u003c/b\u003e) and regulatory pathways [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], and aids in comparing the transcriptome profile among breeds [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. In our previous study using RNA-seq, we identified DEGs associated with steroid biosynthesis, terpenoid backbone biosynthesis, steroid hormone biosynthesis, endoplasmic reticulum protein processing, PPAR signaling pathway, and DNA replication in the jejunal mucosa of HS breeder hens under acute heat stress [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. However, the specific transcriptomic profiles that differentiate HA from HS breeder hens remain unknown. Therefore, this study aims to compare the transcriptomic profiles of HA and HS breeder hens under acute heat stress, aiming to identify key genetic markers and pathways associated with heat tolerance. These insights will contribute to genetic selection strategies and targeted interventions to improve intestinal resilience and overall heat adaptation in poultry.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eEthics statement\u003c/h2\u003e \u003cp\u003eAll experimental protocols were approved by the Animal Care and Use Committee of Suranaree University of Technology (\u003cb\u003eSUT\u003c/b\u003e), Thailand (Approval Code: SUT-IACUC-012/2020). All animals used in this study were sourced exclusively from the SUT farm (Nakhon Ratchasima, Thailand), which is owned and operated by Suranaree University of Technology. As the animals were institutionally owned by SUT, formal informed consent was obtained from the SUT farm management prior to inclusion in the study.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eHousing, birds, and sample collection\u003c/h3\u003e\n\u003cp\u003eA total of fifty 22-week-old hens from two breeds\u0026mdash;HS breeds (SUT breed) and HA breeds (Leuang Hang Kao breed) used in this study were obtained from the SUT farm, with 25 hens per breed. The Leuang Hang Kao breed is a Thai native breed, while the SUT breed represents a synthesized commercial line developed for producing Thai indigenous crossbred chickens. Prior to the start of the experiment, the hens were individually housed in cages with a size of 40 \u0026times; 45 \u0026times; 40 cm\u0026sup3; (width \u0026times; depth \u0026times; height) and acclimated for 5 weeks in a thermoneutral (23\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C) room maintained using an air conditioner. All hens were fed 140 g/day of corn-soy basal diets formulated following the guidelines of the National Research Council 1994 [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] and Aviagen (2021) [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] recommendations (2,800 kcal of metabolizable energy/kg and 15% crude protein), with water available ad libitum, and were maintained on a 16-hour light cycle daily. At 28 weeks of age, all hens were moved to a heat stress room with a controlled temperature of 36\u0026deg;C and a humidity of 40\u0026ndash;70% for 6 hours using a gas heater with thermostat-controlled equipment, as described by [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. After the hens were exposed to heat stress for 6 hours, respiration rate and cloacal temperature were measured from 25 HA and 25 HS hens, respectively. Subsequently, 12 breeder hens from each breed were randomly selected and euthanized by cervical dislocation followed by exsanguination (cutting the neck vein). Jejunal mucosal tissues were then collected by dissection, placed into RNA protect tissue tubes (Qiagen, Duesseldorf, Germany), snap-frozen in liquid nitrogen, and stored at \u0026minus;\u0026thinsp;80\u0026deg;C until further transcriptome and gene validation analyses.\u003c/p\u003e\n\u003ch3\u003eExtraction of total RNA from jejunal mucosa samples\u003c/h3\u003e\n\u003cp\u003eTotal RNA was extracted from 12 jejunal mucosal tissue samples from each breed (HA and HS breeds) using the RNeasy Mini Kit (Qiagen, Hilden, Germany) and subsequently purified with a QIAamp spin column (Qiagen), following the manufacturer's protocol. The RNA concentration was determined using a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), and its quality was assessed through 1% agarose gel electrophoresis, employing 0.5\u0026times; TAE buffer and an electric current of 100 V for 25 min. The three pooled RNA samples (each pool consisting of four individual jejunal mucosa samples) from each breed were used to construct an RNA-seq library. Capillary electrophoresis using a QIAxcel Connect (Qiagen) system was employed to assess the RNA integrity number (RIN), with RNA samples having a RIN\u0026thinsp;\u0026ge;\u0026thinsp;7 selected for cDNA library construction.\u003c/p\u003e\n\u003ch3\u003eLibrary construction and data processing\u003c/h3\u003e\n\u003cp\u003eThe cDNA library construction and RNA-seq were conducted by BGI Co., Ltd. (BGI, Shenzhen, China). Six libraries were sequenced on the DNBSEQ platform. Sequencing data were processed using SOAPnuke Version v1.5.2 [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e] to generate clean reads. The clean reads were then aligned to the chicken reference genome (GCF_000002315.6_GRCg6a) using HISAT2 v2.0.4 [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], and gene expression levels were calculated using RSEM Version v1.2.8 [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. In addition, heatmap clustering, principal component analysis (\u003cb\u003ePCA\u003c/b\u003e), and Venn diagram creation analysis on Dr. Tom's analysis system, constructed by BGI-Shenzhen, China.\u003c/p\u003e\n\u003ch3\u003eDifferentially expressed gene screening and functional enrichment\u003c/h3\u003e\n\u003cp\u003eDifferential gene expression was analyzed using DESeq2 (v1.4.5) [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Differentially expressed genes (\u003cb\u003eDEGs\u003c/b\u003e) were identified based on a fold-change (\u003cb\u003eFC\u003c/b\u003e) of \u0026ge;\u0026thinsp;1 and an adjusted value of \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Gene Ontology (\u003cb\u003eGO\u003c/b\u003e) and Kyoto Encyclopedia of Genes and Genomes (\u003cb\u003eKEGG\u003c/b\u003e) pathway enrichment analyses were conducted, with GO terms and KEGG pathways having \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 considered significantly enriched. The interaction networks were constructed using the network analyzer tool Cytoscape software version 3.9.1 [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eValidation of DEGs via quantitative polymerase chain reaction\u003c/h2\u003e \u003cp\u003eTo validate the RNA-seq results, six DEGs were selected for further analysis based on their roles in heat stress (heat shock protein family B (small) member 9 [\u003cb\u003eHSPB9\u003c/b\u003e] and heat shock protein family A (Hsp70) member 2 [\u003cb\u003eHSPA2\u003c/b\u003e]), lipid metabolism (cluster of differentiation 36 [\u003cb\u003eCD36\u003c/b\u003e]), intestinal barrier integrity (claudin 15 [\u003cb\u003eCLDN15\u003c/b\u003e]), and immunity (recombination activating gene 2 [\u003cb\u003eRAG2\u003c/b\u003e] and interleukin 18 binding protein [\u003cb\u003eIL18BP\u003c/b\u003e]). These genes were analyzed in the jejunal mucosa tissues of HA and HS breeder hens using quantitative polymerase chain reaction (\u003cb\u003eqPCR\u003c/b\u003e). qPCR-specific primers were designed using NCBI's online primer design software (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/tools/primer-blast/\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/tools/primer-blast/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFor reverse transcription, 2 \u0026micro;g of total RNA from each sample was used with the SuperScript III RNase H-Reverse Transcriptase Kit (Toyobo, Osaka, Japan) and random primers (Promega, Madison, WI, USA), following the manufacturer\u0026rsquo;s instructions. Real-time qPCR was performed using the QuantiNova SYBR Green PCR Kit (Qiagen, Hilden, Germany). Briefly, the 10 \u0026micro;L reaction mix was prepared containing 5 \u0026micro;L of SYBR Green, 0.4 \u0026micro;L of forward primer, 0.4 \u0026micro;L of reverse primer, 2 \u0026micro;L of cDNA, and 2.2 \u0026micro;L of nuclease-free water. The parameters of PCR cycles included the following phases: initial heat activation at 94\u0026deg;C for 10 min, followed by 40 cycles of denaturation at 95\u0026deg;C for 10 s, annealing at 60\u0026deg;C for 30 s, and final extension at 72\u0026deg;C for 30 s. The expression of the target gene was normalized against glyceraldehyde-3-phosphate dehydrogenase as the reference gene. Gene expression levels were quantified using the 2\u003csup\u003e\u0026minus;∆∆CT\u003c/sup\u003e method [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eData on respiratory rates and cloacal temperatures were analyzed using a \u003cem\u003et\u003c/em\u003e-test in SPSS version 27.0 (SPSS Inc.) [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Statistical significance was determined with a threshold of \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results and discussion","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003ePhysical response to heat stress between heat-adapted and heat-sensitive breeder hens\u003c/h2\u003e \u003cp\u003eThe respiratory rates and cloacal temperatures in HS breeder hens were significantly higher than those in HA breeder hens under acute heat stress (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.002) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Following the 6-hour heat exposure challenge, the average respiratory rates and cloacal temperature of HA breeder hens were 179 times/min and 41.9\u0026deg;C, respectively. In comparison, HS hens exhibited a respiratory rate of 213 times/min and a cloacal temperature of 42.9\u0026deg;C, respectively.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eRespiratory rates and cloacal temperatures between the heat-adapted and heat-sensitive breeder hens under heat stress conditions\u003csup\u003e1\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStrains\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRespiratory rate (times/min)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCloacal temperature (\u0026deg;C)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHeat-adapted hens\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e179.0\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e41.9\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHeat-sensitive hens\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e213.3\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e42.9\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePooled SEM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"3\"\u003e\u003csup\u003e1\u003c/sup\u003e Values are means from 25 breeder hens (n\u0026thinsp;=\u0026thinsp;25).\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"3\"\u003e\u003csup\u003ea,b\u003c/sup\u003e Values within each column with different superscripts are significantly different (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe study demonstrates that HS breeder hens exhibit a higher physiological response to acute heat stress than HA hens, as indicated by their higher respiratory rates and cloacal temperatures. These differences underscore the adaptive mechanisms of heat-tolerant hens, which help mitigate the detrimental effects of heat stress [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. This highlights the potential benefits of selecting heat-tolerant breeds to improve poultry welfare in hot climates [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eQuality of RNA-seq reads\u003c/h2\u003e \u003cp\u003eA total of six cDNA libraries were constructed and sequenced on the DNBSEQ platform, and an average of 6.35 GB of data was obtained. The RNA-seq data quality metrics are presented in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. RNA-seq of jejunal samples from HA and HS breeder hens yielded an average of 43.52\u0026nbsp;million raw reads and 42.30\u0026nbsp;million clean reads per sample. The sequencing quality was high, with Q20 and Q30 percentages exceeding 97.76% and 93.13%, respectively. GC content across all samples ranged from 46.76% to 47.41%. Alignment of clean reads to the chicken reference genome resulted in mapping rates of 95.38% to 95.82%.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eRNA-sequencing reads and mapping rates in the jejunal mucosa transcriptome of breeder hens between the heat-adapted (HA) and heat-sensitive (HS) groups.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSample\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRaw Reads (M)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eClean Reads (M)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eClean Bases (GB)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eQ20\u003csup\u003e2\u003c/sup\u003e (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eQ30\u003csup\u003e2\u003c/sup\u003e (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eGC content (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eTotal Mapping (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHA1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e45.44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e44.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e6.61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e98.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e94.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e47.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e95.38\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHA2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e44.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e43.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e6.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e97.76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e93.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e46.76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e95.82\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHA3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e45.44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e43.85\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e6.58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e97.89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e93.68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e47.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e95.66\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHS1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e40.39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e39.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e5.93\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e97.91\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e93.64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e47.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e95.49\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHS2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e40.78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e39.76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e5.96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e97.84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e93.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e47.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e95.55\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHS3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e44.94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e43.73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e6.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e97.91\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e93.65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e47.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e95.65\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAverage\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e43.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e42.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e6.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e97.90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e93.59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e47.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e95.59\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"8\"\u003e\u003csup\u003e1\u003c/sup\u003e Each sample consists of four individual jejunal mucosa between the heat-adapted (HA) (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;12) and heat-sensitive (HS) (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;12) breeder hens under heat stress condition.\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"8\"\u003e\u003csup\u003e2\u003c/sup\u003eQ20 and Q30 indicate the percentage of bases with a Phred value\u0026thinsp;\u0026ge;\u0026thinsp;20 and 30, respectively. HA, heat-adapted, HS, heat-sensitive.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eDifferentially expressed genes analysis\u003c/h2\u003e \u003cp\u003eTo identify DEGs in the jejunal mucosa of HA and HS breeder hens under acute heat stress, RNA-seq and subsequent bioinformatic analysis were performed. PCA of jejunal mucosa transcriptomics revealed a clear separation between HA and HS groups, indicating distinct mRNA expression profiles (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Hierarchical clustering of DEGs, based on FPKM values, further confirmed this separation, with samples clustering by groups and distinct gene expression patterns observed between HA and HS hens (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). A total of 15,258 genes were identified across both groups. Among these, 397 genes were uniquely expressed in the HA group, while 347 genes were exclusively expressed in the HS group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), suggesting potential breed-specific regulatory mechanisms underlying thermotolerance or susceptibility. Differential expression analysis identified 284 DEGs, with 155 upregulated and 129 downregulated DEGs in HS hens compared to HA hens (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB and Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). Upregulated genes in HS hens included \u003cem\u003eHSPB9\u003c/em\u003e, \u003cem\u003eRAG2\u003c/em\u003e, \u003cem\u003eHSPA2\u003c/em\u003e, and heat shock protein family B (small) member 1 (\u003cb\u003eHSPB1\u003c/b\u003e), while downregulated genes included \u003cem\u003eCLDN15\u003c/em\u003e and liver basic fatty acid binding protein (\u003cb\u003eLBFABP\u003c/b\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe primary defense against heat stress involves HSPs, which function as intracellular molecular chaperones by binding to misfolded proteins and preventing their aggregation [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. This study reveals the upregulation of several key HSP family members, including \u003cem\u003eHSPA2\u003c/em\u003e (HSP70), \u003cem\u003eHSPB1\u003c/em\u003e (HSP27), and \u003cem\u003eHSPB9\u003c/em\u003e (HSP25). Notably, the expression of \u003cem\u003eHSPA2\u003c/em\u003e (HSP70) was significantly upregulated, with a fold change of 4.4, aligning with previous studies by Kim et al. [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e] and Zhu et al. [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] in heat-stressed chickens. \u003cem\u003eHSPB9\u003c/em\u003e (HSP25) expression patterns were characterized by an initial low expression followed by a gradual increase over time [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. The protein\u0026rsquo;s role as a molecular chaperone was confirmed through its involvement in cellular homeostasis maintenance and protein denaturation prevention. In HS hens, \u003cem\u003eHSPB1\u003c/em\u003e was also found to be elevated in the jejunal mucosa, where it exerted multiple protective mechanisms, including protein stability maintenance and oxidative stress protection [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. The protein\u0026rsquo;s interaction with cytosolic cytochrome C was observed to regulate apoptotic pathways [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e], while its involvement in lipid clearance was also documented [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Both \u003cem\u003eHSPB1\u003c/em\u003e and \u003cem\u003eHSPB2\u003c/em\u003e were observed to form large multimeric complexes that were involved in preventing protein aggregation and maintaining cytoskeletal integrity during heat stress [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. In addition to HSP-related changes, immune response modifications were observed, as evidenced by the upregulation of the \u003cem\u003eRAG2\u003c/em\u003e gene in the jejunal tissue of the HS group relative to the HA group. The gene\u0026rsquo;s involvement in V(D)J recombination and lymphocyte development has been well established [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e], suggesting its critical role in maintaining immune function under heat-stress conditions. The role of \u003cem\u003eRAG2\u003c/em\u003e in preserving immune function during heat stress likely centers on its essential role in sustaining immune diversity by facilitating the proper development of T and B cells [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Reduced expression of \u003cem\u003eRAG2\u003c/em\u003e during heat stress may impair the immune system\u0026rsquo;s ability to produce a full range of antigen-specific receptors, thereby limiting the effectiveness of the immune response to infections. By maintaining adequate levels of \u003cem\u003eRAG2\u003c/em\u003e expression, organisms may better preserve their immune competency during stressful conditions. Furthermore, studies suggest that breed-specific variations in \u003cem\u003eRAG2\u003c/em\u003e expression could influence how different breeds respond to thermal stress, with some breeds, like the HA group, potentially exhibiting more robust immune responses under heat stress conditions [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Interestingly, the HA group exhibited less pronounced RAG2 expression changes, indicating potential breed-specific regulatory mechanisms that may confer enhanced stress tolerance. Moreover, an indirect activation of PPAR-γ by \u003cem\u003eRAG2\u003c/em\u003e was observed, which was associated with increased adipogenesis in the jejunum of heat-stressed breeder hens [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. These molecular adaptations were more pronounced in HA breeds compared to HS breeds, indicating the development of a more sophisticated cellular protection system in the former group. The observed changes encompassed multiple aspects of cellular function, including protein stability maintenance, metabolic regulation, and immune system functionality.\u003c/p\u003e \u003cp\u003eHeat stress has emerged as a critical factor compromising intestinal barrier integrity in poultry production [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. The intestinal barrier is maintained by tight junction proteins such as occludin (\u003cem\u003eOCLN\u003c/em\u003e) and claudin (\u003cem\u003eCLDN\u003c/em\u003e), which regulate paracellular permeability and are essential for maintaining gut health [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Our transcriptomic analysis revealed that \u003cem\u003eCLDN15\u003c/em\u003e gene expression was significantly downregulated (FC\u0026thinsp;=\u0026thinsp;\u0026minus;\u0026thinsp;3.7) in the HS group compared to the HA group. \u003cem\u003eCLDN15\u003c/em\u003e serves as a critical tight junction protein that forms a cation-selective channel, facilitating Na\u003csup\u003e+\u003c/sup\u003e-dependent nutrient transport and maintaining Na\u003csup\u003e+\u003c/sup\u003e homeostasis [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. This Na\u003csup\u003e+\u003c/sup\u003e gradient is fundamental for various transport processes, including the Na\u003csup\u003e+\u003c/sup\u003e-dependent uptake of bile acids into enterocytes [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e] and the absorption of essential nutrients such as glucose and amino acids [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. Moreover, \u003cem\u003eCLDN15\u003c/em\u003e has been shown to promote the proliferation of intestinal cryptic cells [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e], which are vital for continuous epithelial renewal and barrier maintenance. The heat stress-induced downregulation of \u003cem\u003eCLDN15\u003c/em\u003e observed in HS hens suggests a compromised intestinal barrier function that may significantly impair nutrient absorption and utilization, a molecular adaptation that was notably less pronounced in HA hens. This breed-specific difference indicates the potential value of identifying molecular adaptations in different breeds to heat stress.\u003c/p\u003e \u003cp\u003eHeat stress effects extend beyond barrier functions to impact lipid metabolism and transport mechanisms in the intestine [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Fatty acid-binding proteins (FABPs) are crucial facilitators of long-chain fatty acid uptake and transport from intestinal chyme into intestinal epithelial cells, where they support triglyceride synthesis [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. FABP expression may facilitate intracellular fatty acid trafficking from uptake to storage or oxidation, or from lipid droplets for secretion [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. Our findings demonstrated significant downregulation (FC\u0026thinsp;=\u0026thinsp;\u0026minus;\u0026thinsp;2.42) of \u003cem\u003eLBFABP\u003c/em\u003e in the HS group compared to the HA group, aligning with previous studies reporting decreased FABP expression in heat-stressed chicken intestine [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. While \u003cem\u003eLBFABP\u003c/em\u003e (also known as \u003cem\u003eFABP10\u003c/em\u003e) is dominantly expressed in liver tissue [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e], its presence in intestinal tissue plays a vital role in the efflux and transport of various lipids, including cholesterol and bile acids [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. The lower \u003cem\u003eLBFABP\u003c/em\u003e expression in the HS group suggests less intracellular trafficking of lipids and consequently a decreased capacity for lipid utilization (including storage and oxidation) in the intestine. In contrast, HA hens demonstrated more stable \u003cem\u003eLBFABP\u003c/em\u003e expression, suggesting a breed-specific adaptation that supports lipid transport and energy metabolism under heat stress.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eGene ontology (GO) annotation analyses of DEGs\u003c/h2\u003e \u003cp\u003eTo elucidate the molecular mechanism underlying heat stress, GO annotation analysis was performed on the DEGs. The analysis categorized the DEGs into three main functional groups: biological processes (\u003cb\u003eBP\u003c/b\u003e), molecular functions (\u003cb\u003eMF\u003c/b\u003e), and cellular components (\u003cb\u003eCC\u003c/b\u003e). The analysis revealed significant enrichment in 555 GO terms (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), comprising 371 BP, 110 MF, and 74 CC (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e). Within the BP category, cell division (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2.35E\u0026ndash;05), chromosome segregation (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2.40E\u0026ndash;05), cell cycle (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2.55E\u0026ndash;04), fructose transmembrane transport (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2.85E\u0026ndash;04), and response to heat (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1.12E\u0026ndash;03) pathways are included. The MF revealed prominent enrichment in various transport-related activities, especially microtubule binding (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4.11E\u0026ndash;06), fructose transmembrane transport (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2.83E\u0026ndash;04), short-chain fatty acid transmembrane transport (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2.83E\u0026ndash;04), glucose transmembrane transport (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.61E\u0026ndash;03), and neutral amino acid transmembrane transport activity (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.40E\u0026ndash;03), etc. In terms of CC, the DEGs were predominantly enriched in chromosomes, centromeric regions (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;9.86E\u0026ndash;09), extracellular space (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6.40E\u0026ndash;05), brush border membrane (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.71E\u0026ndash;04), plasma membrane (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4.09E\u0026ndash;03), and extracellular regions (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1.05E\u0026ndash;03), etc. Among these, three GO terms‒response to heat, extracellular space, and extracellular region were identified, which align with the findings of Kim et al. [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e] in their analysis of chronic heat stress responses in hen jejunal mucosa. In addition, the comparison between HS and HA hens highlighted GO terms related to fructose and glucose transmembrane transport, brush-border membrane, and motor activity in the jejunum. The jejunum is the primary site for the absorption of essential nutrients, including amino acids, carbohydrates, and fatty acids [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. Efficient nutrient uptake in this region is facilitated by a range of membrane transporters located in the brush-border membrane, which function to regulate nutrient flux and maintain metabolic homeostasis under stress conditions [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]. Notably, the reduced expression of transport proteins associated with the transmembrane transport of fructose and glucose in the HS hens suggests a potential vulnerability in nutrient absorption, indicating a potential risk of metabolic dysfunction and energy deficits. This pattern was further corroborated by the protein-protein interaction (PPI) analysis in this study.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eKEGG pathway analyses of DEGs\u003c/h2\u003e \u003cp\u003eTo identify the biological pathways involved in breeder hens under heat stress, the DEGs from both groups were mapped to the KEGG pathway database. KEGG pathway enrichment analysis revealed significant alterations in multiple signaling cascades following acute heat stress exposure. The analysis identified six significantly enriched pathways (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05): VEGF signaling pathway (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.82E\u0026ndash;03), MAPK signaling pathway (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0208), cell adhesion molecules (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0345), steroid biosynthesis (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0413), neuroactive ligand-receptor interaction (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0430), and cell cycle (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0483) (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). These enriched pathways suggested a complex cellular response to acute heat stress, involving multiple regulatory mechanisms and cellular processes.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSignificantly enriched Kyoto Encyclopedia of Genes and Genomes pathways in the jejunal mucosa between the heat-adapted (HA) and the heat-sensitive (HS) breeder hens under heat stress\u003csup\u003e1\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eKEGG\u003csup\u003e2\u003c/sup\u003e Pathway Term\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCount\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eP value\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGene Symbols\u003csup\u003e3\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003egga04370: VEGF signaling pathway\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.82E\u0026ndash;03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eHSPB1\u003c/em\u003e\u0026uarr;, \u003cem\u003ePLA2G4EL2\u003c/em\u003e\u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003egga04010: MAPK signaling pathway\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.0208\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eHSPH1\u003c/em\u003e\u0026uarr;, \u003cem\u003eHSPA2\u003c/em\u003e\u0026uarr;, \u003cem\u003ePLA2G4EL2\u003c/em\u003e\u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003egga04514: Cell adhesion molecules\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.0345\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eHLA-F10AL4\u003c/em\u003e\u0026darr;, \u003cem\u003eCLDN15\u003c/em\u003e\u0026darr;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003egga00100: Steroid biosynthesis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.0413\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eLIPML5\u003c/em\u003e\u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003egga04080: Neuroactive ligand-receptor interaction\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.0430\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eTAC1\u003c/em\u003e\u0026uarr;, \u003cem\u003eHTR1B\u003c/em\u003e\u0026uarr;, \u003cem\u003eRLN3\u003c/em\u003e\u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003egga:04110 Cell cycle\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.0483\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eBUBIB\u003c/em\u003e\u0026uarr;, \u003cem\u003eCDK1\u003c/em\u003e\u0026uarr;, \u003cem\u003ePLK1\u003c/em\u003e\u0026uarr;, \u003cem\u003eCDC7\u003c/em\u003e\u0026uarr;, \u003cem\u003eCAC20\u003c/em\u003e\u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003e\u003csup\u003e1\u003c/sup\u003e Pathways were detected from DEGs in the jejunal mucosa between heat-adapted (HA) (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;12) and heat-sensitive (HS) (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;12) breeder hens under heat stress conditions.\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003e\u003csup\u003e2\u003c/sup\u003e KEGG, Kyoto Encyclopedia of Genes and Genomes\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003e\u003csup\u003e3\u003c/sup\u003e Up and down arrows indicate the upregulated and downregulated genes in HS hens compared to HA hens, respectively, in the jejunal mucosa between the heat-adapted (HA) and the heat-sensitive (HS) breeder hens under heat stress conditions.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eKEGG pathway analysis revealed six enriched pathways influenced by acute heat stress in the jejunal mucosal tissues of the HS group compared to the HA group. Notable among these were the vascular endothelial growth factor (\u003cb\u003eVEGF\u003c/b\u003e) signaling pathways and neuroactive ligand-receptor interaction, which have been previously identified in heat-stressed and immune-stressed (LPS challenge) broilers' jejunum [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e, \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]. The VEGF signaling pathway is particularly significant in the context of HS chickens, as excessive VEGF activity has been implicated in vascular hyperpermeability and inflammation under heat stress conditions [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]. The jejunal mucosal damage induced by heat stress can result in intestinal hypoxia, triggering VEGF regulation via hypoxia-inducible factor-1α (HIF-1α) activation [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]. While VEGF signaling typically promotes angiogenesis to restore oxygen and nutrient delivery to damaged tissues [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e], the HS group upregulated the VEGF pathway, suggesting a maladaptive response that may exacerbate vascular leakage and inflammatory damage. Moreover, the upregulation of neuroactive ligand-receptor interaction pathways in the HS group indicates potential disruptions in neurotransmitter signaling, further contributing to intestinal dysfunction and impaired nutrient absorption. This overactivation of signaling pathways in HS hens may indicate a heightened stress response that compromises intestinal integrity, whereas the HA group exhibited more balanced pathway activity, suggesting a more controlled adaptation to thermal stress.\u003c/p\u003e \u003cp\u003eThe mitogen-activated protein kinase (\u003cb\u003eMAPK\u003c/b\u003e) signaling pathway emerged as another significant pathway affected by acute heat stress in the jejunal mucosa, with notable differences observed between HS and HA breeder hens. The activation of MAPK signaling in response to heat stress has been well-documented in broiler jejunal tissue [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e]. This pathway regulates various physiological functions, including oxidative stress responses, inflammation, cell multiplication, apoptosis, and autophagy [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e]. In HS hens, MAPK signaling was notably upregulated, suggesting a heightened oxidative stress and inflammatory response that may contribute to cellular damage and compromised intestinal integrity. Our analysis revealed the upregulation of two HSPs, \u003cem\u003eHSPH1\u003c/em\u003e and \u003cem\u003eHSPA2\u003c/em\u003e, within the MAPK signaling pathway in HS hens compared to HA hens. \u003cem\u003eHSPH1\u003c/em\u003e, a member of the Hsp110 family, shows increased expression in HS hens, aligning with its established role in preventing cell death and promoting cellular survival under heat-stress conditions [\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e]. Concurrently, \u003cem\u003eHSPA2\u003c/em\u003e, an HSP70 family member, was significantly upregulated, suggesting its function in mitigating structural and oxidative damage to the intestinal mucosa during heat stress [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Previous research has demonstrated that heat stress upregulates \u003cem\u003eHSP70\u003c/em\u003e expression in chicken jejunal mucosa, concurrently activating the MAPK signaling pathway as a protective mechanism through its chaperone-mediated stabilization of misfolded proteins and interaction with MAPK intermediates [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. The upregulation of \u003cem\u003eHSPH1\u003c/em\u003e and \u003cem\u003eHSPA2\u003c/em\u003e in the HS group suggests a potentially adaptive response to heat stress, wherein the activation of these HSPs may be insufficient to counteract thermal damage effectively, thereby exacerbating cellular stress.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eProtein-protein interaction network analysis of DEGs\u003c/h2\u003e \u003cp\u003ePPI network analysis for the DEGs identified three distinct networks. The largest network comprised 30 protein-coding genes, with \u003cem\u003eCDK1\u003c/em\u003e, \u003cem\u003ePLK1\u003c/em\u003e, \u003cem\u003eCDC7\u003c/em\u003e, and \u003cem\u003eCDC20\u003c/em\u003e positioned as core nodes. These core proteins showed primary enrichment in the cell cycle pathway within the jejunum (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Notably, most proteins in this network were upregulated in HS hens compared to HA hens, as indicated by red nodes, with a few exceptions, such as \u003cem\u003eKIFC3\u003c/em\u003e and \u003cem\u003eCDK18\u003c/em\u003e, shown in blue. The second network contained 13 interaction proteins, including \u003cem\u003eHSPA2\u003c/em\u003e and interleukin-6 (\u003cb\u003eIL6\u003c/b\u003e), all of which were exhibited upregulation (red nodes) in HS hens compared to HA hens. The third network consisted of 10 proteins, all of which were downregulated (blue nodes) in HS hens compared to HA hens, with \u003cem\u003eSLC22A13L\u003c/em\u003e serving as the core node of the network.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ePPI network analysis revealed several upregulated cell cycle-related genes in the jejunum of heat-stressed HS hens, including \u003cem\u003eCDK1\u003c/em\u003e, \u003cem\u003ePLK1\u003c/em\u003e, \u003cem\u003eCDC7\u003c/em\u003e, and \u003cem\u003eCDC20\u003c/em\u003e. Cyclin-dependent kinase 1 (\u003cb\u003eCDK1\u003c/b\u003e), a key member of the cyclin-dependent kinase family, is a serine/threonine kinase that influences both the Wnt and fibroblast growth factor signaling pathways, thereby affecting cell proliferation [\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e, \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e]. In HS hens, the upregulation of \u003cem\u003eCDK1\u003c/em\u003e may represent a compensatory response to heat-induced cellular stress, promoting cell cycle progression despite adverse thermal conditions. Similarly, the upregulation of the polo-like kinase 1 gene (\u003cb\u003ePLK1\u003c/b\u003e), another serine/threonine kinase, suggests an adaptive mechanism to maintain cell division and DNA replication under heat stress. PLK1 regulates cell cycle checkpoints, and its overexpression enables cells to bypass arrest, potentially mitigating DNA damage through interactions with cell division cycle 7 (\u003cb\u003eCDC7\u003c/b\u003e) [\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e]. \u003cem\u003eCDC7\u003c/em\u003e is a critical cell cycle regulator, and its interaction with PLK1 facilitates mitotic exit and diaphase formation [\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e]. Notably, in HS hens, \u003cem\u003eCDC7\u003c/em\u003e expression was markedly elevated, consistent with previous findings in heat-stressed Illinois broilers that reported upregulation of \u003cem\u003ePLK1\u003c/em\u003e, \u003cem\u003eCDC7\u003c/em\u003e, and \u003cem\u003eCDC20\u003c/em\u003e [\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e]. This conserved upregulation across breeds suggests that these kinases may play pivotal roles in cellular adaptation to heat-induced DNA damage by promoting cell cycle progression and preventing apoptosis. The observed upregulation of cell cycle regulators in HS hens may indicate a heightened cellular effort to counteract heat stress-induced DNA damage, potentially reducing cell cycle arrest and apoptosis. The second largest network identified comprised 13 upregulated genes, with \u003cem\u003eHSPA2\u003c/em\u003e and \u003cem\u003eIL6\u003c/em\u003e emerging as key core nodes related to the immune response. HSPs are intricately linked to immune system functions [\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e]. The pro-inflammatory cytokine \u003cem\u003eIL6\u003c/em\u003e plays an important role in innate and acquired immunity [\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e]. Multiple studies have indicated increased \u003cem\u003eIL6\u003c/em\u003e expression following heat stress exposure [\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e], including elevated levels in the jejunal mucosae of thermal manipulation chicks under chronic heat stress [\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e]. The upregulation of \u003cem\u003eIL6\u003c/em\u003e and \u003cem\u003eHSPA2\u003c/em\u003e suggests a complex interplay between inflammatory signaling and cellular protection mechanisms in HS hens. While elevated \u003cem\u003eIL6\u003c/em\u003e levels may indicate an inflammatory response to heat-induced tissue damage, \u003cem\u003eHSPA2\u003c/em\u003e upregulation may serve to mitigate excessive inflammation by stabilizing proteins and inhibiting pro-inflammatory cytokine expression [\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e]. The relationship between \u003cem\u003eHSPA2\u003c/em\u003e and \u003cem\u003eIL6\u003c/em\u003e is particularly significant, as both proteins are regulated by heat shock factor (\u003cb\u003eHSF\u003c/b\u003e). Research has shown that HSF induces both \u003cem\u003eHSP70\u003c/em\u003e and \u003cem\u003eIL-6\u003c/em\u003e expression in heat-stressed chickens, suggesting \u003cem\u003eIL-6\u003c/em\u003e may act as a heat-shock-responsive gene [\u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e]. The upregulation of \u003cem\u003eIL6\u003c/em\u003e and \u003cem\u003eHSPA2\u003c/em\u003e in HS hens may indicate a dual response to heat stress, characterized by pro-inflammatory signaling coupled with protective HSP-mediated mechanisms. These findings suggest the role of \u003cem\u003eIL6\u003c/em\u003e as a critical mediator of inflammatory responses and its potential role in modulating immune function in HS hens under heat stress conditions.\u003c/p\u003e \u003cp\u003eIn addition, PPI analysis identified a network of ten downregulated DEGs associated with nutrient transport and metabolism in HS hens, including \u003cem\u003eSLC22A13L\u003c/em\u003e, \u003cem\u003eLBFABP\u003c/em\u003e, \u003cem\u003eSLC2A2\u003c/em\u003e, and \u003cem\u003eSLC6A19\u003c/em\u003e, which aligns with previous findings of decreased nutrient absorption and transport gene expression in heat-stressed animals [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], suggesting a potential impairment in nutrient handling in HS hens compared to HA hens. Solute carrier family 22 member 13 (\u003cem\u003eSLC22A13L\u003c/em\u003e), also known as organic anion transporter 10 (OAT10) [\u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e], is predominantly expressed in the apical membrane of proximal tubules in the kidneys, where it mediates urate reabsorption through the exchange of organic anions, such as urate, nicotinate, and orotate, for OH- or lactate anions [\u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e]. Given the role of \u003cem\u003eSLC22A13L\u003c/em\u003e in the kidney, low expression of \u003cem\u003eSLC22A13L\u003c/em\u003e in the jejunum of HS hens may disrupt the transport of organic anions, impair the exchange of key metabolites (including lactate and nicotinate), and lead to impaired nutrient absorption, metabolic balance, and ion transport, thus causing intestinal barrier dysfunction in HS hens. Further studies are needed to clarify the role of \u003cem\u003eSLC22A13L\u003c/em\u003e in the jejunal mucosa in response to heat stress. The glucose transport gene \u003cem\u003eSLC2A2\u003c/em\u003e (GLUT2) plays a role in glucosamine transport necessary for glycosaminoglycan biosynthesis [\u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e83\u003c/span\u003e]. Previous studies in broiler jejunum have demonstrated reduced \u003cem\u003eGLUT2\u003c/em\u003e expression under heat stress [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], suggesting disrupted intestinal glucose transport. Similarly, solute carrier family 6 member 19 (\u003cem\u003eSLC6A19\u003c/em\u003e), located in the apical membrane, encodes the B\u003csup\u003e0\u003c/sup\u003eAT protein responsible for high-affinity amino acid transport through electroneutral exchange coupled with the sodium co-transport [\u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e]. The downregulation of \u003cem\u003eSLC6A19\u003c/em\u003e in HS hens suggests a disruption in amino acid absorption, potentially affecting protein metabolism and nutrient utilization.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eValidation of DEGs by qPCR\u003c/h2\u003e \u003cp\u003eWe selected DEGs that regulated representative functions or whose expression profiles were significantly altered in response to acute heat stress. We validated the expression levels of four upregulated genes (\u003cem\u003eHSPB9\u003c/em\u003e, \u003cem\u003eRAG2\u003c/em\u003e, \u003cem\u003eHSPA2\u003c/em\u003e, and \u003cem\u003eIL18BP\u003c/em\u003e) and two downregulated genes (\u003cem\u003eCLDN15\u003c/em\u003e and \u003cem\u003eCD36\u003c/em\u003e) in the jejunal mucosa between HA and HS breeder hens for the validation by using qPCR (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). All genes showed similar expression trends in both the qPCR and RNA-seq.\u0026nbsp;These results demonstrated the reliability and accuracy of our RNA-seq data in this study.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study identified 284 DEGs, among these, 155 were upregulated, and 129 were downregulated DEGs in the jejunal mucosa of HS hens compared to HA hens under heat stress using RNA-seq analysis. Ten DEGs associated with HSP, immune response, intestinal barrier integrity, and the transport of lipids, organic acids, glucose, and amino acids, including HSPB9, HSPA2, HSPB1, RAG2, IL6, CLDN15, LBFABP, SLC22A13L, SLC2A2, and SLC6A19. may play key roles in regulating the jejunal mucosa of HS breeder hens under acute heat stress. The identified DEGs are implicated in critical processes related to heat response, cell division, and nutrient transport, indicating potential molecular targets for mitigating thermal stress effects in HS hens. KEGG pathway enrichment analysis revealed that the main biological pathways were related to the VEGF signaling, MAPK signaling, cell adhesion molecules, neuroactive ligand-receptor interaction, and cell cycle regulation. PPI analyses showed that acute heat stress may affect cell cycle progression, immune function, and the transport of organic acids, glucose, and amino acids in the jejunal mucosa of HS breeder hens. The identification of specific genes and pathways in the present study provides valuable insights for future genetic selection strategies and breeding programs aimed at improving heat tolerance in poultry.\u003c/p\u003e "},{"header":"Declarations","content":"\u003ch2\u003eDeclaration of competing interest\u003c/h2\u003e\n\u003cp\u003eThe authors declare that they have no competing financial interests or personal relationships that may have influenced the work reported in this study.\u003c/p\u003e\n\u003ch2\u003eConsent for publication\u003c/h2\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003ch2\u003eCRediT authorship contribution statement\u003c/h2\u003e\n\u003cp\u003eYongcai Zhu: Writing \u0026ndash; review \u0026amp; editing, Writing \u0026ndash; original draft, Visualization, Validation, Supervision, Software, Project administration, Methodology, Investigation, Formal analysis, Conceptualization. Satoshi Kubota: Validation, Supervision, Software. Phocharapon Pasri: Software, Methodology, Investigation. Sitthipong Rakngam: Methodology, Investigation, Formal analysis. Shenglin Yang: Visualization, Validation, Supervision, Software. Sutisa Khempaka: Supervision, Software, Resources, Funding acquisition, Data curation.\u003c/p\u003e\n\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eOpen access funding provided by Suranaree University of Technology. This work was supported by the National Research Council of Thailand (NRCT5-RSA63009-03), Suranaree University of Technology (SUT), Thailand Science Research and Innovation (TSRI), and the National Science, Research, and Innovation Fund (NSRF).\u003c/p\u003e\n\u003ch2\u003eAcknowledgments\u003c/h2\u003e\n\u003cp\u003eThe authors gratefully acknowledge the Suranaree University of Technology for their financial support through the One Research One Graduate (OROG). We would like to thank Phocharapon Pasri for his indispensable work in the implementation of this project in the bird care tasks, and all authors for the preparation of the laboratory work.\u003c/p\u003e\n\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eTranscriptome data availability\u003c/h2\u003e \u003cp\u003eThe transcriptome sequencing data are available through the Gene Expression Omnibus (accession number: GSE294384).\u003c/p\u003e \u003c/div\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eOluwagbenga EM, Fraley GS. Heat stress and poultry production: a comprehensive review. Poult Sci. 2023;102:103141.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePearce SC, Mani V, Weber TE, Rhoads RP, Patience JF, Baumgard LH, et al. Heat stress and reduced plane of nutrition decrease intestinal integrity and function in pigs. 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Physiol Rev. 2008;88:249\u0026ndash;86.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-veterinary-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [BMC Veterinary Research](http://bmcvetres.biomedcentral.com/)","snPcode":"12917","submissionUrl":"https://submission.nature.com/new-submission/12917/3?","title":"BMC Veterinary Research","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Transcriptome analysis, Breeder hen, Acute heat stress, Jejunal mucosa","lastPublishedDoi":"10.21203/rs.3.rs-9368422/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9368422/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eHeat stress impairs intestinal integrity in poultry, compromising nutrient digestion and absorption. Nevertheless, the individual variation in heat tolerance suggests distinct molecular responses that are still not well understood.\u003c/p\u003e\u003ch2\u003eMaterials and methods\u003c/h2\u003e \u003cp\u003eThis study investigated the molecular basis of differential heat tolerance by comparing jejunal transcriptomes in heat-adapted (\u003cb\u003eHA\u003c/b\u003e) and heat-sensitive (\u003cb\u003eHS\u003c/b\u003e) breeder hens under acute heat stress (36\u0026deg;C for a 6-h). Fifty 28-week-old hens were randomly allocated to the HA and HS groups (25 hens each). After exposure to acute heat stress for 6 hours, the respiratory rate and cloacal temperature were measured, and jejunal mucosal samples were collected for RNA sequencing (\u003cb\u003eRNA-seq\u003c/b\u003e).\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe results indicated that, under acute heat stress, the respiratory rates and cloacal temperatures of HS hens were significantly higher than those of HA hens (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.002). RNA-seq analysis identified 284 differentially expressed genes (\u003cb\u003eDEGs\u003c/b\u003e), with 155 genes upregulated and 129 downregulated in the HS group compared to the HA group. Gene Ontology analysis revealed significant enrichment in 555 GO terms. Kyoto Encyclopedia of Genes and Genomes pathway analysis identified five pathways that were enriched in upregulated DEGs (VEGF signaling pathway, MAPK signaling pathway, steroid biosynthesis, neuroactive ligand-receptor interaction, and cell cycle) and one pathway enriched in downregulated DEGs (cell adhesion molecules). Protein-protein interaction network identified key genes (\u003cem\u003ePLK1\u003c/em\u003e, \u003cem\u003eCDC7\u003c/em\u003e, \u003cem\u003eCDC20\u003c/em\u003e, \u003cem\u003eHSPA2\u003c/em\u003e, \u003cem\u003eIL6\u003c/em\u003e, \u003cem\u003eSLC22A19A\u003c/em\u003e, \u003cem\u003eLBFABP\u003c/em\u003e, \u003cem\u003eSLC6A19\u003c/em\u003e, and \u003cem\u003eSLC2A2\u003c/em\u003e), involved in cell division, immune function, energy and lipid metabolism, as well as organic acid, glucose, and amino acids transport.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThese findings suggest that acute heat stress differentially affects intestinal function in HA and HS hens, potentially through alterations in cell division, immune function, energy and lipid metabolism, and organic acid and glucose transport mechanisms. The identified DEGs and pathways offer valuable insight into the molecular basis of heat stress susceptibility and may inform nutritional or management strategies to enhance poultry resilience.\u003c/p\u003e","manuscriptTitle":"Transcriptome profiling analysis of heat-adapted and heat-sensitive breeder hen jejunal mucosal tissues in response to acute heat stress","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-04 15:40:43","doi":"10.21203/rs.3.rs-9368422/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-05-18T10:07:42+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-11T13:03:52+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-26T14:15:38+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"285072685417600846587056937711227467154","date":"2026-04-25T04:29:36+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"79560768794592332183964101792655972585","date":"2026-04-23T12:32:23+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-23T04:05:50+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-17T04:05:34+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-04-16T16:53:23+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-16T02:59:28+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Veterinary Research","date":"2026-04-16T02:53:44+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-veterinary-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [BMC Veterinary Research](http://bmcvetres.biomedcentral.com/)","snPcode":"12917","submissionUrl":"https://submission.nature.com/new-submission/12917/3?","title":"BMC Veterinary Research","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"efecd810-1845-4559-b208-de8b280510f9","owner":[],"postedDate":"May 4th, 2026","published":true,"recentEditorialEvents":[{"type":"decision","content":"Revision requested","date":"2026-05-18T10:07:42+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-11T13:03:52+00:00","index":33,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[],"tags":[],"updatedAt":"2026-05-18T10:23:10+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-04 15:40:43","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9368422","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9368422","identity":"rs-9368422","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}