Chicken is the second largest type of meat product in China, except for pork. The annual sales of broilers in China can reach 10.5 billion, and the per capita consumption has been increasing year by year. Compared to laying hens, broilers can provide humans with more high-quality dietary protein and meat products (Resnyk et al., 2017). Heat stress is one of the biggest challenges facing global animal husbandry. The increase in ambient temperature and humidity will affect the production of animals in summer and will cause economic losses in severe cases (Salem et al., 2022). Heat stress causes the high mortality rate and severe economic losses estimated at 240 million United States dollars per year in the poultry sector (St-Pierre et al., 2003), representing, for example, about 7% of total HS-caused losses in the French livestock industry in 2003 (Nardone et al., 2010). According to the time of heat stress, it can be divided into acute heat stress (<7 days) and chronic heat stress (≥7 days); According to the temperature of stress treatment, it can be divided into cyclic heat stress and sustained heat stress (Gonzalez-Esquerra and Leeson, 2006). Research has shown that acute heat stress can cause a heat shock response, leading to rapid initiation of heat shock protein synthesis and rapid changes in gene expression, while chronic heat stress can cause larger scale changes (Xie et al., 2014). In addition, Xie et al. (2015) reported that chronic heat stress can lead to tissue damage. Research has found that the cyclic heat stress pattern is more in line with the characteristics of summer temperature and closer to the actual living environment (Al Qaisi et al., 2022). Heat stress can affect intestinal microflora’s activity and stimulate the hypothalamus’s feeding center to reduce its excitability, leading to the decline of broiler growth performance and even death (Wang, 2013). In addition, broilers will respond to changes in the external environment by adjusting their metabolic levels, which can detect changes in gene, metabolite and microorganism levels through behavioral changes and the use of transcriptome, metabolome and 16S rDNA amplicon sequencing technology (Jastrebski et al., 2017).

It has revealed the influence of heat stress on broilers’ cecum by the transcriptome, metabolome and 16S rDNA amplicon technology. Transcriptome sequencing (RNA-Seq) reflects the expression of a specific cell or tissue gene and has the advantage of high sensitivity. RNA-Seq has been successfully applied to study the molecular basis of complex traits, such as feed efficiency and myopathy, and evaluate the molecular response to nutritional therapy and important aspects of immunity and disease resistance (Zampiga et al., 2018). In addition, in terms of heat stress, RNA-Seq analysis was used to identify key genes that respond to the heat stress induced volatilization in laying hens, such as PDK4 and FGA (Wang et al., 2021). Transcriptome analysis identified potential target genes, especially those involved in cell migration and immune signaling responses to heat stress, which can inform future research on heat stress in broilers and could prove useful for improving disease resistance (Monson et al., 2018). Metabolomics is a sequencing technology that studies the body’s physiological changes and pathological characteristics from a dynamic perspective through the qualitative and quantitative determination of metabolites (Nicholson et al., 1999). It can build a direct correlation between metabolites and biological phenotypes, dynamically track and analyze the metabolites of animal bodies, help to analyze the relationship between phenotypes and genetics, environmental and other factors, and provide a basis for the improvement and breeding of economic traits. Metabolomics is widely used to understand the changes in organism metabolism, such as metabolism, sugar, lipid, amino acid metabolism, and meat quality evaluation (Zhang Y. et al., 2023; Zhang F. et al., 2023; Zhang X. et al., 2023). Zampiga et al. (2021) found that chronic heat stress could regulate the changes of metabolites in breast muscle and plasma of broilers by metabolome, and found metabolites that might play an important role in regulating Energy homeostasis of the body. According to metabolomic analysis, heat stress has caused to changes in serum lipid metabolism of broilers. It is determined that heat stress can reduce the content of lysophosphatidylcholine and increase the content of phosphatidylcholine (Guo et al., 2021a). The 16S rDNA amplicon sequencing technology can analyze microbial communities to obtain information on community structure, differences and functions. It is also widely used in the poultry industry, revealing the interaction between microbial communities such as the gut, animal reproduction, growth and development, nutritional health, environmental factors, immunity and disease treatment (Pan and Yu, 2014). Emami et al. (2022) reported that using microbial amplicon analysis, it was found that heat stress can affect the diversity and composition of gut microbiota in broilers, which can provide a basis for developing nutritional strategies to maintain gut microbiota balance and alleviate the negative effects of heat stress on performance and health of broilers. In addition, the multi-omics analysis has shown unique advantages in revealing the underlying molecular regulatory mechanisms. For example, integrated analysis of transcriptome and metabolome was used to determine the critical amino acid metabolic pathway to improve duck eggs (Yan et al., 2023) and lipopolysaccharides could induce the immune stress pathway in broilers (Bi et al., 2022). Hubbard et al. (2019) reported that the combination of RNA-Seq and metabolomic data can identify new changes in gene regulation of broilers affected by heat stress, which reflect changes in pathways that affect metabolite levels. Integrated analysis of metabolome and microbiome has determined that heat stress can increase the relative abundance of harmful microbes in the cecum of broilers and reduce health-related metabolites such as L-malic acid, which can provide a basis for the impact of heat stress on physiological changes and intestinal health in broilers (Liu et al., 2022, 2022a,b).

The intestine is a sensitive part of heat stress and an important organ for nutrient absorption and immune regulation in poultry, making it crucial for poultry production (Deng et al., 2023). The cecum is the broiler gastrointestinal tract’s most diverse area of microbes and various microbes are crucial in improving growth performance and maintaining physical health (Luo et al., 2021). Previous studies mainly explored the effects of heat stress on broiler intestines from a single omics perspective, including transcriptome, metabolomics, and microbiome (Dridi et al., 2022; Kim et al., 2022; Zhang Y. et al., 2023; Zhang F. et al., 2023; Zhang X. et al., 2023). However, very few studies have comprehensively revealed the heat-stress impact on broilers’ guts. Therefore, this experiment conducted chronic heat stress treatment on broilers and analyzed the effects of heat stress on the cecum of broilers at the genetic, metabolic, and microbial levels to provide a theoretical basis for subsequent research.

As an important economic indicator of meat and poultry, the quality and performance of poultry meat often directly determine the level of breeding efficiency in production, and with the development of the economy, people are increasingly favoring chicken with good taste, good meat quality and richer nutrition (Ma et al., 2022). Poultry exposure to heat stress can disrupt thermoregulation and homeostasis, reduce poultry performance, health and welfare, and lead to weight loss or even negative growth, low immunity, and even death (Mujahid et al., 2009; Kim et al., 2021; Sarsour et al., 2022). Growth performance is an important indicator for evaluating whether the production efficiency of poultry meets people’s expectations. The decrease in growth performance caused by heat stress is related to decreased food intake (Peng et al., 2023). The result of this study indicated that heat stress reduced the weight and ADG of broilers but had no effect on ADFI and FCR. The results of the current study are inconsistent with previous studies on ADFI and FCR. Previous studies have found that high-temperature environments reduced broilers’ body weight, ADG, and ADFI (Deng et al., 2023). Sun S. et al. (2023) conducted heat stress treatment on 28-day-old broilers and found a significant reduction in ADFI, ADG, and FCR in 42-day-old broilers. In addition, Yilmaz and Gul (2023) found that heat stress reduced the ADG in 42-day-old broilers, but had no significant effect on the FCR. The above results indicate that this study demonstrates the negative effects of heat stress on broilers, indicating that a heat stress model has been successfully established. This may be because when the temperature recovers, periodic heat stress can lead to birds overeating, thereby weakening the impact of heat stress on ADFI. In addition, the impact of heat stress on the growth performance of broilers not only depends on feed intake, but also includes other factors such as physiological, biochemical, hormonal changes, breeds of broiler, duration of heat stress, and temperature of heat stress treatment (Al-Abdullatif and Azzam, 2023). Research has shown that heat stress can damage growth performance by reducing protein and nutrient digestibility; Insulin growth factor in the Endocrine system is the main regulator of muscle metabolism, participating in all stages of muscle formation and muscle regeneration, which can increase protein synthesis and promote differentiation, while heat stress will affect the secretion of insulin growth factor and thus reduce protein synthesis (Nawaz et al., 2021).

Research shows that transcriptome can help researchers analyze which pathways and genes are activated in response to the stressor (Zhang Y. et al., 2023; Zhang F. et al., 2023; Zhang X. et al., 2023). Compared to other parts of the intestine, the cecum of broilers plays a more important role in host defense (Khan and Chousalkar, 2020). Therefore, this is more important for studying the effects of heat stress. From the transcriptome results, 96 differential genes were identified, 78 differential genes of which were up-regulated and 18 differential genes were down-regulated. PDK4 (pyruvic acid dehydrogenase 4), as a gene with significant differences, plays an important role in regulating energy homeostasis metabolism, glycolysis, and fat decomposition (Honda et al., 2017; Wen et al., 2021; Forteza et al., 2023). In mice with ischemic stroke, it was found that the cecum metabolism was disordered, and the PDK4 related to fatty acid metabolism was up-regulated, which may lead to the reduction of steroid metabolic process activity (Ge et al., 2022). Research has shown that PDK4 was previously identified as a differential gene in multiple chicken heat stress experiments (Wang et al., 2021). In the experiment on high-altitude-stressed Tibetan sheep, glycolysis can increase ATP content by up-regulating the expression of PDK4, providing energy for resisting hypoxia stress (Wen et al., 2021). As is well known, high temperatures can damage protein stability and lead to dysfunctional cell function (Mackei et al., 2021). FKBP10 is a member of the FK113 binding protein gene family and is involved in many functions, including protein folding and repair in response to heat stress, which is necessary to maintain natural peptides and prevent protein aggregation (Akbarzadeh et al., 2018). Our previous research found that heat stress can affect lipid metabolism and increase cholesterol content in broilers (Zhang L. et al., 2022). Studies have found that LBP (lipopolysaccharide binding protein) has been used as an indicator of the impairment of intestinal barrier function, and its level can reflect the degree of intestinal leakage (Vancamelbeke and Vermeire, 2017; Wu et al., 2023). Intestinal barrier dysfunction can further contribute to the occurrence of alcoholic hepatitis by acting on the gut-liver axis, triggering inflammatory cascade reactions, and aggravating liver inflammation and LBP levels (Tilg et al., 2016). Heat stress can lead to an increase in LBP levels, which is consistent with our trend (Wickramasinghe et al., 2023). GO results indicated that differential genes were mainly enriched in immune processes. The KEGG results further indicated that differential genes were mainly enriched in other pathways, such as the NF-kB signaling pathway, calcium signaling pathway, and intestinal immune network for IgA production. These pathways play an important role in the intestinal injury. It is reported that heat stress can result in gut microflora dysbiosis, cause bacterial translocation, and thus induce the production of intestinal endotoxin. These endotoxins can activate TLR4-mediated reactions, including initiation of the NF-κB signaling pathway (Tang et al., 2021). NF-κB is a major transcription factor involved in inflammatory diseases, which can respond to heat-stress stimuli and activate the NF-κB signaling pathway in broilers (Xu et al., 2023), thereby inducing tissue damage (Liu W. C. et al., 2021; Liu Y. R. et al., 2021). NF-κB acts downstream of TLR4 and other immune receptors, increasing the excessive production of IL-6, IL-1β, and TNF-α leads to the occurrence of inflammatory responses in response to heat stress (Vallabhapurapu and Karin, 2009). Liu et al. (2022, 2022a,b) found that chronic heat stress can enhance the NF-κB signaling pathway and promote the occurrence of liver inflammation in broilers. The intestinal immune network for IgA production have been confirmed to play an important role in immunity (Zhang et al., 2018). The intestines are the largest lymphoid tissue, and intestinal immunity can produce many non-inflammatory IgA antibodies, the first line of defense against heat stress. Multiple cytokines (TGF-β, IL-10, IL-4, IL-5 and IL-6) are essential for B cells to differentiate into IgA plasma cells (Nagatake et al., 2019; Yang et al., 2021). IgA primarily functions in the intestinal cavity through secretory immunoglobulin A (SIgA), which maintain intestinal mucosal homeostasis and prevent harmful substances from adhering and entering the intestinal barrier (Lammers et al., 2010). Research has shown that chronic heat stress can damage intestinal immune function by promoting the inflammatory response and reducing IgA secretion (Yang et al., 2019). Heat stress can also affect the calcium signaling pathway, leading to mitochondrial oxidative stress and severe calcium overload, damaging mitochondrial structure and function, and even leading to apoptosis (Coble et al., 2014; Zhang W. et al., 2022; Yao et al., 2023).

Metabolomics research can understand changes in the metabolism of organisms, helping researchers better understand how chicken products are affected by the external environment (Zhang Y. et al., 2023; Zhang F. et al., 2023; Zhang X. et al., 2023). The fermentation products produced by cecal microorganisms have a positive impact on intestinal health, and numerous cecal metabolites play a crucial role in maintaining intestinal barrier function (Liu et al., 2022, 2022a,b). Therefore, this is more important for studying the effects of heat stress on changes in cecal metabolites in broilers. According to the metabolomics results, 619 differential metabolites were identified, with 47 metabolites known structures, of which 13 metabolites were up-regulated and 34 were down-regulated. As one of the most differential metabolites of metabolome, ascorbyl palmitate has the function of clearing reactive oxygen species (ROS) and protecting DNA damage when coping with stress, and plays a key role in protecting cells and cell membranes from oxidative damage (Xiao et al., 2014). In addition, ascorbyl palmitate is believed that increasing the amount of Vitamin C will increase the resistance to oxidative stress, thus increasing the resistance to certain diseases (Nieva-Echevarría et al., 2021). Therefore, the oxidative damage caused by heat stress also reflects the reduction of ascorbyl palmitate content. Zhang Y. et al. (2023), Zhang F. et al. (2023), and Zhang X. et al. (2023) showed that the increase of ADP ribose content can identify damaged DNA and further activate the base excision repair mechanism to participate in heat stress regulation. This also confirms the upregulation of ADP ribose metabolite content in this study. KEGG enrichment results showed that differential metabolites were mainly enriched in the purine metabolism and ABC transporters. Purine is involved in DNA and RNA functions and is essential for cell survival and proliferation (Pedley and Benkovic, 2017). Heat stress will lead to oxidative stress in organisms, and purine metabolism is the basic reaction of oxidative stress and the imbalance between purine remedy and de novo synthesis pathway will lead to the production of ROS (Yu et al., 2021; Tian et al., 2022). Besides, the metabolism of intestinal microorganisms will affect the content change of metabolites. In this study, the differential pathway of microbial function prediction also includes purine metabolism, which also explains why it is enriched in metabolome data. ABC transporter is a transmembrane protein that can transport many molecules across the cell membrane (Yu et al., 2021). As a member of the ABC transporters, ABCB10 deficiency may lead to mitochondrial oxidative damage and ROS production (Liesa et al., 2012). ROS exceeding the tolerance threshold level of the body can damage the body’s antioxidant system, leading to a decrease in antioxidant enzyme activity (Rahman and Rahman, 2012).

Microorganisms living in the gastrointestinal tract can affect poultry’s nutrition, physiology and intestinal development (Shang et al., 2018). Research has shown that heat stress can increase microbial communities’ richness and abundance indicators, increase OUTs (Operational taxonomic units), and thus increase the Chao1, Shannon, Simpson, and ACE indexes (Goel et al., 2022a; Liu et al., 2022, 2022a,b). This is consistent with the results of this study, which found that heat stress increased the number of OTUs and α diversity index, which may be due to an increase in harmful microbial communities. At the phylum level, heat stress increased the relative abundance of Proteobacteria and decreased the relative abundance of Bacteroidota and Firmicutes. At the genus level, heat stress reduced the relative abundance of the Alistipes and increased the relative abundance of the Fusobacterium. LEfSe analysis showed that p_Proteobacteria and p_Fusobacteriota were the differential microbiota under heat stress. At the same time, the relative abundance of g_Alistipes and f_Rikenellaceae was significantly lower than that of the CON. Functional prediction analysis at levle 2 showed that the gut microbiota of AA broilers was mainly involved in carbohydrate metabolism, membrane transport, amino acid metabolism, replication and repair. This is consistent with the Li et al. report’s functional prediction results (Yi et al., 2023). At the level 3 KEGG metabolic pathway showed that fatty acid metabolism was enhanced in the HS. Research has shown that fatty acid metabolism is an important mechanism closely related to energy homeostasis under heat stress (Lim et al., 2022). This is similar to the report of Jastrebski et al. (2017) that heat stress can increase the expression of enzymes related to fat metabolism, thereby improving fatty acid metabolism. In addition, Goel et al. (2022b) research found that heat stress can reduce the number of Bacteroidetes in the ileum of broilers. Bacteroidetes are generally considered to maintain complex and beneficial relationships with the host, including fermenting carbohydrates to produce volatile fatty acids (VFAs) as an energy source for host utilization and are positively correlated with growth performance (Zhu et al., 2021). In addition, Firmicutes and Proteobacteria are related to the fermentation of undigested dietary components. Firmicutes contribute to the production of the polysaccharide and butyrate (Wu et al., 2018). Notably, a high proportion of Proteobacteria indicates intestinal ecological imbalance and is associated with the pathogenesis of many diseases, such as diarrhea, inflammatory bowel disease, and colitis (Wu et al., 2021). Alistipes belonging to the Bacteroidota is considered a relatively new bacterial genus that has protective effects on specific diseases, including liver fibrosis, colitis, cancer immunotherapy and cardiovascular diseases (Parker et al., 2020). It is also a major producer of short chain fatty acids in bacteria (Qi et al., 2019). They have the characteristics of glycolysis and proteolysis and produce acetic acid by producing fibrinolysis, digesting gelatin and fermenting carbohydrates (Zhu et al., 2019). As a member of Fusobacteriota, the increase in abundance of Fusobacterium can act as a pro-inflammatory factor to promote the occurrence of intestinal tumors (Patra and Kar, 2021). In addition, Fusobacterium metabolites may make the tumor microenvironment more comfortable over time by directly promoting tumor cell proliferation, vascular growth or immune cell infiltration (Kostic et al., 2013). Research found that the Rikenellaceae was related to metabolism and gastrointestinal health in the body and a large number of Rikenellaceae had the potential to protect against cardiovascular and metabolic diseases related to visceral fat and were potential biomarkers of healthy aging and longevity (Pin Viso et al., 2021; Tavella et al., 2021; Wang et al., 2022). Therefore, the reduction of the relative abundance of Rikenellaceae may lead to the shortening of cell life, which also confirms that differential genes are enriched in cellular senescence.

There is a strong interdependence between gut microbiota, genes and metabolites. Therefore, multi-omics analysis can help us analyze specific mechanisms of action. Pearson correlation analysis showed that ornithine was negatively correlated with CALB1 and positively correlated with SULT1C3, GSTT1L and ASV2 (g_Lactobacillus). PE was negatively correlated with CALB1 and CHAC1. Studies have shown that amino acids serve as components of synthetic proteins and as important physiological and behavioral regulators, such as regulating stress responses (Chowdhury et al., 2021). Long-term heat stress can reduce the content of most free amino acids (Chowdhury et al., 2021), such as a decrease in ornithine content in the plasma of laying hens exposed to long-term heat stress (Chowdhury et al., 2014). L-ornithine is one of the metabolites in the urea cycle, proline, glutamate, arginine, and polyamine metabolism. It can stimulate the secretion of growth hormone by the pituitary gland and promote the breakdown and metabolism of proteins, sugars and fats (Xiong et al., 2016). Meanwhile, under heat stress conditions, a decrease in ornithine content may weaken the stimulation of glutathione metabolism, thereby reducing the antioxidant capacity of broilers (Xiong et al., 2016). SULT1C3, which has sulfotransferase activity, plays a role in larger biomolecules, including proteins and carbohydrates, and is vital in maintaining tissue structure and cellular signaling (Rondini et al., 2014). GSTT1L is believed to be related to glutathione metabolism in broilers, participating in antioxidant activity, improving heat tolerance and aging (Zhang et al., 2017; Liu et al., 2019). However, research has found that supplementing Lactobacillus probiotics can improve the antioxidant capacity of broilers (Dashti and Zarebavani, 2021). The above results confirm the findings of this study. Therefore, the downregulation of these genes and microorganisms reflects a decrease in antioxidant levels and heat tolerance in broilers. Heat stress is related to the secretion of many hormones (such as estrogen, glucocorticoid, and catecholamine), which may affect the expression of the CALB1 in tissues in different ways (Ebeid et al., 2012). CALB1, as an intracellular transporter protein, can transport Ca²⁺ and act as a Ca²⁺ sensor to prevent increased intracellular Ca²⁺ concentration from causing toxicity (O'Toole, 2011). In addition, CALB1 is a rate-limiting step for epithelial cells to absorb calcium, thus CALB1 expression is highly correlated with intestinal calcium absorption efficiency (Valable et al., 2020). Bahadoran et al. (2018) found that heat stress can increase the expression of the CALB1 in the uterus of laying hens, which helps to increase the resistance of uterine cells to the harmful effects of heat stress. As the main source of phospholipids, PE plays an important role in the integrity of cells and organelle membranes in broilers (Guo et al., 2021b). Research shows that heat stress leads to the decrease of phospholipid level in broilers, which may be due to the damage to cell membrane caused by heat stress by increasing the activity of phospholipase A2 and promoting the decomposition of phospholipids (Guo et al., 2021b). The overexpression of CHAC1, which is induced in response to endoplasmic reticulum stress, will lead to large glutathione consumption (Aquilano et al., 2014; He et al., 2021). In addition, the high expression of CHAC1 in male broilers indicates that the degradation rate of glutathione is higher than average, which may play an important role in oxidative stress (Brothers et al., 2019). This is consistent with our research results. Therefore, the broiler will upregulate the expression of CALB1 and CHAC1 by responding to cell damage caused by heat stress, thereby increasing the body’s resistance. Further research has found that KEGG co-enrichment of differential genes and differential metabolites indicated such as folate biosynthesis, the FoxO signaling pathway, and the mTOR signaling pathway. According to reports, folic acid not only participates in nutrient metabolism but also has free radical scavenging and ROS activity. Dietary supplementation with folic acid can improve the antioxidant performance and immune status of broilers under heat stress, which may be due to the role of folic acid in regulating protein metabolism (Gouda et al., 2020). The enriched ALPL in folate biosynthesis can involve in inflammatory response, bone growth, and bone calcium metabolism (Sharma et al., 2014). High-temperature environments may lead to inflammatory reactions and disrupt bone calcium metabolism in poultry. Therefore, we speculate that upregulation of the ALPL can maintain bone health and reflect the resistance of poultry to high temperatures and pathogens. The mTOR signaling pathway is a key nutrient perception pathway that regulates cell metabolism and lifespan in response to various changes in stress, growth factors, and cell energy levels (Su et al., 2019). A previous study suggested that bovine mammary epithelial cells may resist heat stress damage by enhancing the absorption and metabolism of intracellular amino acids and activating the mTOR signaling pathway (Fu et al., 2021). In addition, heat stress significantly upregulated the expression of mTOR signaling pathway related genes, which is consistent with our research results (Fu et al., 2021). L-arginine, as a metabolite significantly enriched in the mTOR signaling pathway, is essential for maintaining growth, reproduction, and immunity (Murakami et al., 2012). Research has found that supplementing arginine can enhance the development of the small intestine and nutrient absorption (Abdulkarimi et al., 2019). Therefore, the decrease in L-arginine content also reflects that heat stress can cause damage to the body and reduce immune function. The FoxO signaling pathway is involved in various cellular functions, including cell proliferation, apoptosis, autophagy, oxidative stress, and metabolic disorders (Xing et al., 2018). According to reports, under environmental stress conditions, the FoxO signaling pathway in fish is significantly enriched, which is consistent with our research results (Shang et al., 2023; Sun J. et al., 2023). The ROS produced by heat stress can regulate the expression of FoxO at the levels of transcription, protein activation, phosphorylation, and acetylation and overactivation and overexpression of FoxO can lead to the occurrence of various diseases (Liu et al., 2023). In addition, FoxO can also affect the expression of the Bcl-2 protein family, stimulate the expression of death receptor ligands and tumor necrosis factor related apoptosis inducing ligands, and induce cell death through mitochondrial mediated endogenous pathways and death receptor mediated exogenous pathways (Fu and Tindall, 2008).

In summary, our study found that heat stress led to decreased growth performance, intestinal oxidative damage, and antioxidant capacity in broilers, a process related to the complex regulation of genes, metabolites, and microorganisms. This will provide a theoretical reference for the poultry industry to improve the problem of heat stress.

In this study, heat stress reduced the body weight and ADG, altered the expression of purine metabolism, calcium signaling pathway, intestinal immune network for IgA production and FoxO signaling pathway, and increased the expression of cecal microbiota α diversity index, the relative abundance of Proteobacteria and Alistipes decreased the relative abundance of Bacteroidetes and Firmicutes. PICRUSt prediction showed that the cecum microorganisms of AA broilers in HS were mainly enriched in fatty acid metabolism, while those in CON were mainly enriched in the purine metabolism pathway. The multi-omic analysis found that the KEGG co-participating pathway of differential genes, metabolites and microorganisms was the purine metabolism. Pearson correlation analysis showed that ornithine was positively correlated with SULT1C3, GSTT1L and g_ Lactobacillus, and negatively correlated with CALB1. L-arginine and PC were negatively correlated with IFI6 and positively correlated with SULT1C3. PE was negatively correlated with CALB1 and CHAC1, and positively with g_Alistipes.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: NCBI and MetaboLights database - Transcriptome sequencing (PRJNA987758), 16S rDNA sampling sequencing (PRNA972642), LC-MS (MTBLS8078).

The animal study was approved by Shanxi Agricultural University Ethics Committee. The study was conducted in accordance with the local legislation and institutional requirements.

XL, YW, ZW, and LZ contributed to the conception, design, and investigation of the study. YW, ZM, and ZW contributed to the initial statistical analysis and bioinformatics analysis. XL completed the final statistical analysis, bioinformatics analysis, visualization, and wrote the first draft of the manuscript. XL, ZM, YW, HJ, ZW, and LZ contributed to manuscript revision. All authors contributed to the article and approved the submitted version.

This study was supported by the Youth Fund Project on Application of Basic Research Project of Shanxi Province (202103021223135), the Key R&D project of Shanxi Province (201803D221023-2), the Excellent doctors come to Shanxi to reward scientific research projects (SXBYKY20222057), and the Cultivate Scientific Research Excellence Programs of Higher Education Institutions in Shanxi (2021BQ114).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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