The effects of chronic heat stress on the growth performance, digestive and absorbtive-related parameters, and jejunal metabolomics in broilers

The effects of chronic heat stress on the growth performance, digestive and absorbtive-related parameters, and jejunal metabolomics in broilers | 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 Article The effects of chronic heat stress on the growth performance, digestive and absorbtive-related parameters, and jejunal metabolomics in broilers Shiang Sun, Bing Li, Junjun Yuan, Yongjie Xiong, Shaojun He This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6220478/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Heat stress (HS) is one of the major influencing factors limiting the development of poultry farming. To investigate the effect of chronic HS on the intestinal digestion and absorption function, we divided 80 broilers to control group (TN group), chronic HS group (CHS group), and then illustrated the effects using growth performance, gas chromatography-mass spectrometry (GC-MS)-based metabolomics, digestive and absorptive capacity, and apparent digestibility. Broilers in the CHS group were exposed to 12 h/day HS (32 ± 1 ℃) for 14 consecutive days, and the rest of the time per day was maintained at 24 ± 1 ℃, which was the same as that used for the TN group. The jejunum samples were collected at the end of the experiment, and tested for relevant indexes, and the GC-MS technique was applied to obtain two groups of broiler jejunum metabolic profiles. The study showed that HS reduced the average daily feed intake (ADFI), average daily grit (ADG), intestinal digestive enzyme activity, D-xylose and GSH absorption levels, apparent digestibility, and elevated feed conversion ratio (FCR). A total of 370 metabolites in the broiler jejunum were identified, and 43 up-regulated and 22 down-regulated metabolites were screened. Enrichment analysis of metabolic pathways revealed that eight metabolic pathways were significantly altered (mainly related to energy metabolism). These results indicate that changes in cellular metabolism in the jejunum when broilers are subjected to HS, decreased the activity of digestive enzymes in the jejunum, resulting in a decrease in digestibility and consequently affecting growth performance. Biological sciences/Zoology Earth and environmental sciences/Climate sciences broiler jejunum growth performance gas chromatography-mass spectrometry digestive enzyme Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Due to the lack of sweat glands and feather cover, broilers are highly susceptible to heat stress (HS). A study suggested that significant HS can occur when the environmental temperature of broiler feed reaches 32 ℃ [ 1 ] . With the continuing trend of global warming, the HS response of poultry has further increased. Heat stress has become a significant factor limiting the development of the poultry industry, causing substantial economic losses for the livestock industry every year. HS harms various functions of poultry to varying degrees. The main effects are increased body temperature, immunosuppression, oxidative damage and damage to the intestinal mucosa [ 2 ] . This ultimately leads to reduced feed intake and feed conversion and stunted growth. To adapt to the high summer temperatures, broilers will actively regulate multiple metabolic pathways to maintain internal heat balance. The gut is the leading site for the digestion and absorption of nutrients and is also a significant immune barrier against invading pathogenic bacteria [ 3 ] . HS leads to reduced digestive and absorptive capacity of the intestinal tract, damage to intestinal barrier integrity, an imbalance in the structure of the flora and delayed development of immune organs [ 4 ] . There is a critical need to determine the mechanisms of HS responses and their effects on intestinal permeability in poultry. The jejunum is a vital digestive organ in the digestive tract, and metabolic changes in the jejunum can reflect the health status and function of the digestive tract of broilers [ 5 ] . HS impairs broiler intestinal integrity and increases the permeability of the intestine to endotoxins [ 6 ] . However, these data cannot systematically and comprehensively reveal the absorption and metabolic status of essential nutrients in the body as a whole. Furthermore, the differential metabolic markers and the metabolic pathways altered during HS are still unknown. This hinders the idea of mitigating the harm of HS from the perspective of regulating nutrient uptake and metabolism. After genomics, transcriptomics and proteomics, metabolomics has become vital to systems biology. However, studies and the laws concerning the types and quantities of metabolites and their dynamics after an organism is disconcerted [ 7 ] . Using metabolomics technologies to identify specific biomarkers in the development of diseases can provide a basis for early diagnosis of diseases and suggest targeted therapeutic measures [ 8 ] . Detecting the composition of all small molecules in the broiler gut during HS can help further reveal the mechanism of the damage caused by HS in broilers. However, few studies have analyzed the changes in all intestinal metabolites during HS in broiler chickens using a metabolomics approach, and most metabolomic studies have focused on mammals. Therefore, in this study, we used gas chromatography-mass spectrometry (GC-MS) to investigate the metabolic profiles of the jejunum of heat-stressed broilers and to explore the pattern of changes and related regulatory mechanisms involved. This study also aimed to elucidate the relationships between growth performance, digestive and absorptive capacity and apparent digestibility and metabolic changes in heat stressed broilers. Results Growth performance The growth performance of broilers in the TN and CHS groups is shown in Table 2 . Compared with the TN group, broilers in the CHS group had significantly lower ADG, ADFI, and higher FCR ( P < 0.05). Table 2 Effect of chronic HS on broiler growth performance Groups TN CHS SEM P value ADG (g/d) 188.05 168.93 4.18 < 0.001 ADFI (g/d) 729.78 571.30 24.60 < 0.001 FCR (%) 1.81 2.10 0.04 < 0.001 Note: P < 0.001 indicate highly significant differences. ADG, average daily gain; ADFI, average daily feed intake; FCR, feed conversion ratio; TN, control group; CHS, chronic heat stress group. Metabolomic profiling of the samples The GC-MS total ion chromatograms of the samples from the TN and CHS groups are shown in Fig. 1 . The figure shows that the instrument collected strong signals of jejunal metabolites during this test, and the retention time and volume of the generated peaks met the test requirements. The jejunum assay mass spectra were compared with the standard mass spectra of the NIST database, and a total of 370 metabolites with more than 70% match were identified. Multivariate and unidimensional statistics were used to depict metabolomic characteristics by analyzing differences in broiler jejunal metabolites between the TN and CHS groups. Hierarchical clustering Differences in the accumulation patterns of jejunal metabolites between the two groups were analyzed by clustering heatmaps (Fig. 2 ). The heatmap showed that some metabolites were down-regulated in the TN group and up-regulated in the CHS group, suggesting that there may be significantly different metabolic processes between the TN and CHS groups. In addition, eight samples from the two groups were clustered together, indicating high data reliability with good homogeneity between biological replicates. Multivariate statistical analysis Principal component analysis Figure 3 shows the results of the PCA for the two groups of jejunal metabolites. As seen from the figure, the TN and CHS groups were mainly distributed in different quadrants, indicating some inter-group variability between the two groups and that the variability between the test groups was real. Partial least squares-discriminant analysis To eliminate noise information irrelevant to the classification, we built a PLS-DA model based on the PCA model (Fig. 4 ). It shows that 88.0% of the samples (data records) fit the established discriminant mathematical model, and the prediction accuracy of the model was 60.9%. The PLS-DA model score plot showed that the broiler jejunal metabolites in the TN and CHS groups were better separated on the principal component axes, and the differences were true. The robustness of the model was examined using the 200 response ranking method, and the results are shown in Fig. 5 , where R 2 = 0.907 > 0.5 and Q 2 = -0.482 < 0, indicating that this model is stable and reliable. Significantly different metabolites analysis The peak areas of metabolites were normalized by PLS-DA analysis after relative quantification, and 65 differential metabolites were screened according to the results of the relative variable standard analysis of VIP > 1 and the t-test of P 1], and 22 metabolites such as norvaline, L-cystine and glyceric acid were down-regulated (FC < 1). Table 3 Identification of significantly different metabolites of TN group and CHS group Metabolites RT (min) VIP P FC Change Salicylaldehyde 5.187 2.49 0.001 0.40 ↓ Carinitine 5.24 1.49 0.001 1.61 ↑ 2-hydroxypentanoic acid 5.286 2.02 < 0.001 2.33 ↑ Glutaric acid 5.314 2.33 < 0.001 3.24 ↑ 1-hexanol 5.513 2.54 < 0.001 4.05 ↑ Benzylalcohol 5.536 1.89 0.003 2.20 ↑ 2-hydroxypyrazinyl-2-propenoic acid 5.589 2.51 < 0.001 3.94 ↑ Morpholine 5.599 3.19 < 0.001 10.28 ↑ Hexanamide 5.665 3.29 < 0.001 10.73 ↑ Epsilon-caprolactam 5.698 2.56 < 0.001 4.41 ↑ Diethylcarbamic acid 6.474 1.29 < 0.001 1.37 ↑ Enolpyruvate 6.996 1.25 0.029 1.44 ↑ 1,5-anhydroglucitol 7.078 1.45 < 0.001 1.49 ↑ Resorcinol 7.097 3.23 < 0.001 11.56 ↑ 4-aminophenol 7.576 2.68 0.008 0.41 ↓ Glutathione 8.428 1.81 < 0.001 1.89 ↑ Methanolphosphate 8.49 1.30 0.045 0.57 ↓ N-methylalanine 8.81 2.70 0.001 4.84 ↑ Xanthurenic acid 9.312 2.82 < 0.001 5.89 ↑ Leucinic acid 9.51 2.04 0.030 2.97 ↑ Succinic anhydride 9.537 2.19 0.002 0.47 ↓ Delta-tocopherol 9.681 1.50 0.003 0.67 ↓ L-kynurenine 10.346 2.49 0.005 0.43 ↓ Oxamic acid 10.743 2.90 0.001 7.46 ↑ Pentonic acid 10.794 2.18 < 0.001 2.64 ↑ Levoglucosan 10.984 3.94 < 0.001 49.24 ↑ Succinic acid 11.062 1.22 0.007 1.58 ↑ Glyceric acid 11.352 1.58 0.012 0.53 ↓ 2-imidazolidinone 11.376 2.41 < 0.001 3.70 ↑ Tranexamic acid 11.404 3.05 < 0.001 8.67 ↑ 1,2,4-benzenetriol 11.655 4.99 < 0.001 645.13 ↑ Pyrazine, 3,6-dihydro-3,6-dimethyl-2,5-dihydroxy- 11.714 1.84 0.002 0.56 ↓ Erythrose 12.413 1.38 0.001 1.50 ↑ Glucosaminic acid 12.478 1.80 < 0.001 1.93 ↑ Dodecanol 13.583 1.06 < 0.001 0.78 ↓ Biphenyl 13.761 1.33 0.002 1.41 ↑ 4-hydroxyproline 15.496 1.42 0.016 1.54 ↑ L-asparagine 18.553 1.35 0.018 1.86 ↑ D-xylulose 18.753 1.24 0.008 0.71 ↓ D-arabinose 18.78 1.66 0.029 0.44 ↓ Norvaline 19.358 1.94 0.046 0.14 ↓ D-glyceraldehyde 3-phosphate 19.659 1.79 0.016 1.95 ↑ Dihydroxyacetone phosphate 20.126 1.76 0.007 1.80 ↑ L-glutamine 20.742 2.21 < 0.001 2.63 ↑ N-acetyl-d-hexosamine 20.824 1.14 0.021 0.72 ↓ L-cysteine-glycine 21.543 1.85 < 0.001 2.02 ↑ Myristic acid 22.301 1.63 < 0.001 0.58 ↓ Maltitol 22.301 1.66 < 0.001 0.57 ↓ D-tagatose 22.764 1.37 0.017 0.65 ↓ P-hydroxylphenyllactic acid 22.932 1.65 < 0.001 0.60 ↓ Lysopalmitoyl monogalactosylglycerol 23.643 1.84 0.019 2.04 ↑ Ascorbic acid 24.191 1.25 0.028 2.30 ↑ Cellobiose 25.452 1.28 0.029 1.61 ↑ Barbital 26.119 1.54 0.002 1.74 ↑ Erythrotetrofuranose 26.796 1.03 0.043 1.38 ↑ D-ribose 5-phosphate 27.107 1.82 < 0.001 2.14 ↑ D-ribulose 5-phosphate 27.275 2.20 < 0.001 2.78 ↑ Sedoheptulose 27.439 1.50 0.032 0.53 ↓ Altrose 27.518 1.48 0.035 0.53 ↓ Psilocin 28.121 1.67 0.027 2.34 ↑ L-cystine 29.448 1.80 0.019 0.36 ↓ Mannose 6-phosphate 29.71 1.04 0.046 1.49 ↑ Phosphohexonic acid 30.776 1.86 0.012 2.25 ↑ 3,5-dihydroxyphenylglycine 31.822 1.05 0.029 1.50 ↑ Deoxycholic acid 37.194 1.02 0.005 0.77 ↓ Note: RT, retention time; VIP, variable importance in the projection; FC, fold change; FC values greater than 1 mean that metabolite in CHS group was more than that in TN group. Change, ratio of mean peak area of the CHS group to the mean peak area of the TN group; ↑, metabolites with higher concentrations in the CHS group than in the TN group with values > 1; ↓, metabolites with lower concentrations in the CHS group than in the TN group with values < 1. Metabolite set enrichment analysis The enrichment analysis results of the relevant metabolic pathways are shown in Fig. 6 and Fig. 7 , in which the effect of HS on the glycolysis/gluconeogenesis metabolic pathway was highly significant ( P < 0.01), followed by the effects on the fructose and mannose metabolic pathways ( P < 0.01). In addition, metabolic pathway enrichment analysis revealed that lysosomal, amino and nucleotide sugar metabolism, carbon metabolism, ABC transporter protein, amino acid biosynthesis, and tricarboxylic acid cycle (TCA cycle) metabolic pathways were significantly altered during cyclic HS ( P < 0.05). Intestinal digestive enzyme activities Table 4 displays the intestinal digestive enzyme activities of broilers in the TN and CHS groups. In the CHS group, broilers exhibited significantly lower levels of amylase, lipase, and trypsin activities compared to the TN group ( P < 0.05). Table 4 Effect of chronic HS on the activity of intestinal digestive enzymes in broilers Groups TN CHS SEM P value Amylase (U/mgprot) 7.48 5.97 0.3 0.018 Lipase (U/gprot) 231.38 110.82 12.61 0.001 Trypsin (U/mgprot) 973.32 630.7 51.24 0.004 Note: P < 0.05 indicate significant differences. TN, control group; CHS, chronic heat stress group. D-xylose and GSH absorption The levels of D-xylose and GSH absorption in broilers of the TN and CHS groups are shown in Table 5 . HS significantly reduced the levels of D-xylose and GSH absorption in the intestinal tract of broilers compared to the control group ( P < 0.05). Table 5 Effect of chronic HS on intestinal D-Xylose and GSH absorption in broilers Groups TN CHS SEM P value D-xylose (mmol/L) 1.19 0.96 0.10 0.023 GSH (g/L) 4.25 3.30 0.21 0.016 Note: P < 0.05 indicate significant differences. TN, control group; CHS, chronic heat stress group. Apparent digestibility The effect of chronic HS on apparent digestibility in broilers are shown in Table 6 . Compared with the TN group, heat stress significantly decreased the digestibility of dry matter, organic matter, and crude protein in broilers ( P 0.05). Table 6 Effect of chronic HS on the apparent digestibility of nutrients in broilers Groups TN CHS SEM P value Dry matter 73.64 71.20 0.42 0.001 Organic matter 77.65 75.70 0.34 0.001 Crude protein 61.16 56.93 0.82 0.006 Crude fat 76.39 74.88 1.11 0.535 Note: P < 0.05 indicate significant differences. TN, control group; CHS, chronic heat stress group. Discussion HS leads to disruption of physiological functions in poultry in hot environments. Our previous study indicated that broilers exposed to HS (32 ℃) reduced production performance, decreased ADG and increased FCR, the same as the present experiment results [ 9 ] . It may be due to the loss of appetite and decreased feed intake of broilers in hot environments, so the ADG of broilers in the CHS group decreased, and the FCR increased. In addition, poultry have difficulty dissipating heat due to their feathered bodies and lack of sweat glands. When the ambient temperature exceeds the optimal temperature, energy is consumed to maintain the body temperature, reducing the amount of raw materials that can be converted into muscle and fat, leading to a decrease in growth performance. The intestine is greatly affected by HS and is an important link in the occurrence of HS hazards. Studies have shown that when broilers suffer from HS, intestinal villus height decreases crypt depth increases, and intestinal permeability increases, thus inducing inflammation [ 10 ] . This experiment revealed the most significant glycolysis/gluconeogenesis metabolic pathway changes through metabolic pathway enrichment analysis. When the ambient temperature exceeds the optimal temperature, the organism requires energy substances to maintain a relatively stable body temperature. Glycolysis is a stage that all organisms must pass during glucose catabolism [ 11 ] . Glucose can produce ATP during lactate production to provide the energy required by the body to maintain body temperature homeostasis. Enolpyruvate is a common biochemical molecule in biological cells. It is an intermediate product of glycolysis and gluconeogenesis where it can be metabolized to pyruvate to produce ATP, one of the primary sources of intracellular chemical energy [ 12 ] . In this study, the content of enolpyruvate was significantly greater in the intestine of heat-stressed broilers than in that of control broilers, indicating that the glycolysis pathway was activated, the gluconeogenic pathway was blocked, and the cellular demand for energy was increased during HS. Glycolysis and the TCA cycle are the two crucial energy metabolic pathways, and the TCA cycle is a prevalent metabolic pathway in aerobic organisms, distributed in mitochondria [ 13 ] . Both are essential links in the biochemical pathways central to aerobic bioenergy synthesis and cellular metabolism. A previous study showed that HS can cause alterations in the body’s metabolic pathways of the TCA cycle [ 14 ] . In the enriched metabolic pathway, the TCA cycle also changed significantly, with the same results as above. The energy produced by glycolysis was probably not entirely sufficient when the organism was subjected to HS. The organism further processes the pyruvate produced during glycolysis and forms acetyl-CoA through oxidative decarboxylation, which in turn produces carbon dioxide and water, generating a large amount of ATP for consumption by the organism. Succinic acid is a dicarboxylic acid, an intermediate product of the TCA cycle [ 15 ] . The alteration of the TCA cycle in broilers in broilers exposed to HS suggested that the activity of some enzymes involved in the tricarboxylic acid metabolism process may be significantly altered when the body is exposed to HS. Some metabolic intermediates of this cycle, such as succinic acid, cannot enter the cycle normally to participate in metabolism, causing significant changes in the content of these substances in the body. Mannose 6-phosphate is an intermediate product of the fructose and mannose metabolic pathway, and its main metabolic pathway is catalyzed by mannose phosphate isomerase to produce fructose-6-phosphate, which is broken down into the glycolytic pathway. Mannose enters the cell via the glucose transporter protein, which is present as mannose 6-phosphate for further metabolism. Mannose does not affect cellular glucose uptake, but elevated intracellular mannose 6-phosphate inhibits glucose-6-phosphate dehydrogenase, glucose phosphate isomerase and hexokinase, which are involved in glucose metabolism. This in turn, affects glycolysis and the TCA cycle [ 16 ] . Therefore, it was hypothesized that increasing the mannose 6-phosphate in the broiler intestine during HS would inhibit intracellular glucose metabolism in vivo. This is consistent with the findings of Cui et al [ 17 ] . Ribose 5-phosphate is the raw material for the synthesis of RNA and DNA. The content of ribose 5-phosphate in the jejunum significantly increased during HS in broilers. Therefore, it is speculated that the decrease in DNA synthesis during HS may not be related to the content of ribose 5-phosphate, but rather to the loss of control of DNA synthesis from ribose 5-phosphate [ 18 ] . Among the metabolites related to the gluconeogenesis pathway, the levels of D-arabinose and D-xylulose were significantly reduced, indicating that HS caused abnormal energy metabolism in the body. The body needs to accelerate the breakdown of sugars to provide more energy to counteract the stress. ATP-binding cassette (ABC) transporter proteins are a multifunctional superfamily of membrane proteins that play essential roles in the transcellular transport of nutrients. This pathway is exciting because it is involved in the transport of cholesterol and fat [ 19 ] . The high-temperature environment results in significant changes in the basal metabolic rate of broilers and increased deposition of abdominal fat as an additional form of energy storage [ 20 ] . In this experiment, there were significant differences in the indexes related to the metabolism of various lipids such as leucine, myristic acid, valeric acid and glyceric acid in the jejunum of broilers during HS, indicating that lipid metabolism in broilers was disturbed during the occurrence of HS, as reported in the study by Xiong et al [ 21 ] . In this experiment, broilers’ jejunal valeric acid content was significantly increased during HS, probably due to the energy deficiency caused by the decrease of food intake during HS in broilers, which in turn caused the body to mobilize fat from surrounding tissues for energy supply. Valeric acid, as a readily oxidized fatty acid, may be released during lipolysis, thereby increasing levels in the jejunum. Leucine can help burn visceral fat to provide sufficient energy for body tissues [ 22 ] . In order to reach the heat balance in broilers during HS, all parts of the organism work together, so the leucine content is briefly increased to help the organism burn fat for more energy. Glyceric acid is a three-carbon acid formed by the oxidation of glycerol, and its phosphorylation can produce glycerol 3-phosphate, which can further isomerize into sugar or participate in glycolysis [ 23 ] . The glycolysis pathway was activated in broilers under HS, and the phosphorylation of glyceric acid was accelerated to form intermediate products to ensure the operation of the glycolysis pathway. Therefore, the glyceric acid content was significantly lower than that in the control group. Amino acids and urea are the final forms of protein breakdown in vivo. Amino acids can continue to participate in the body’s sugar and lipid metabolism, and urea is excreted in the form of nitrogenous metabolic waste [ 24 ] . In this experiment, the alanine content in the broiler intestine increased significantly during HS, a result also reported in the study by Guo et al [ 25 ] . This may be linked to the involvement of alanine in energy metabolism in the body. The alanine-glucose cycle is an important pathway for it to exert energy metabolism. When HS occurs in broilers, the body must adjust its energy metabolic processes to adapt to the high-temperature environment. A large production of pyruvate accompanies glucose consumption as an energy supply. To maintain a continuous supply of energy, the alanine metabolic pathway is required [ 26 ] . In addition, arginine and proline in the body can be converted to glutamate via the amino acid conversion pathway. Then, glutamate is deaminated under alanine aminotransferase to produce α-ketoglutarate, a critical intermediate in the TCA cycle pathway that provides energy. In this process, alanine is rapidly converted to pyruvate through deamination to facilitate the tricarboxylic acid supply cycle in the organism [ 27 ] . The valine content in the jejunum decreased significantly during the course of this experiment, similar to Jo’s research results [ 28 ] . Valine is a branched-chain amino acid that plays a crucial role in animal growth and metabolism, and is also a sugar-generating amino acid [ 29 ] . When broilers are exposed to HS, energy in the body is heavily depleted. Valine can produce the glucose required for the body’s stable ecological balance through the gluconeogenesis pathway, alleviating the problem of insufficient glucose content in the body during metabolism. The analysis of amino acid metabolism characteristics during HS showed that adding some amino acids with sugar-generating or energy-promoting substances to rations could help prevent and control HS. To better understand the changes in intestinal metabolism induced by HS in broilers, we examined the variations in digestive enzyme activities in the jejunum. The activity levels of intestinal amylase, lipase, and trypsin can effectively indicate the strength of intestinal digestion of major nutrients [ 30 ] . Normally, the activities of various intestinal enzymes tend to stabilize, with some dynamic elevation after eating. It has been shown that HS reduces the activity of digestive enzymes such as amylase and lipase in the gut [ 31 ] . In this experiment, HS resulted in a significant decrease in amylase, lipase, and trypsin activities in the jejunum of broilers, which is consistent with the results of the above study. It may be related to the inactivation of digestive enzyme activity in the organism under high-temperature conditions. Another reason may be that heat stress alters the metabolism and function of jejunal cells, resulting in decreased secretion and activity of digestive enzymes in the jejunum. Furthermore, HS can cause damage to the intestines through oxidative stress and morphological damage, resulting in reduced enzyme activity. This can significantly impact nutrient absorption and metabolism in broilers, leading to a decreased ability to digest sugars, fats, proteins, and other essential nutrients in their diet. Sugars, fat, and protein are crucial nutrients for the growth and development of the body. The digestive capacity of these nutrients affects broiler feed intake, weight gain, weight ratio, and other indicators, which reduces production benefits. Additionally, we conducted measurements of intestinal absorption function in broilers subjected to HS. D-xylose, a pentose sugar that is primarily absorbed in the jejunum and excreted by the kidneys without participating in body metabolism. GSH is a low molecular weight thiol tripeptide that plays an important role in maintaining intracellular redox balance [ 32 ] . D-xylose and GSH absorption tests are widely used to assess intestinal absorption function [ 33 ] . HS can lead to a significant decrease in serum levels of D-xylose and GSH in broilers, which is consistent with the findings of Wu [ 34 ] . It showed that the intestinal absorption function of broilers decreased under HS, and the reason for this phenomenon may be because the intestinal villi of broilers were damaged under HS conditions, and the morphology of intestinal mucosa was damaged, and the absorption capacity was weakened. The reduced levels of digestive enzymes observed in broilers exposed to HS may also impact their intestinal absorptive capacity, and consequently, the absorption of D-xylose and GSH. It is important to note that this statement is based on objective measurements and not subjective evaluations. Next, we examined changes in the apparent digestibility of nutrients in heat-stressed broilers. The apparent digestibility of nutrients is an important indicator for assessing the ability of animals to digest and absorb nutrients in the diet [ 35 ] . Wickramasuriya [ 36 ] reported that HS reduced the apparent digestibility of crude protein and dry matter in broilers. This finding is consistent with the observed reductions in the digestibility of DM, OM, and CP in feeds during the experiment. When broilers are exposed to HS, their bodies allocate a significant amount of blood to the surface to dissipate heat [ 37 ] . This results in reduced blood flow to the digestive tract, leading to a decrease in the apparent digestibility of nutrients. The reduced activity of digestive enzymes in broilers under HS indicates a decrease in intestinal digestive capacity and a corresponding decrease in the apparent digestibility of nutrients in broilers. Furthermore, the reduced apparent digestibility of heat-stressed broilers may also be attributed to reduced intercellular proteins and altered permeability resulting from changes in jejunal metabolism. Decreased dry matter digestibility in broilers during HS reduces glycogen stores. Gluconeogenesis is necessary to maintain blood glucose concentrations. The metabolic pathway enrichment analysis revealed alterations in the glycolysis/gluconeogenesis pathway to support this finding. Conclusions In this experiment, the metabolomic approach technology was applied to study the alteration of small molecule metabolites in the jejunum of broilers during HS. A total of 65 differential metabolites were identified, indicating that the metabolism of intestinal cells in broilers changed during HS. And by testing the intestinal enzyme activity, absorptive capacity, and apparent digestibility of heat-stressed broilers, it was shown that HS may reduce the intestinal digestive and absorptive capacity of broilers by affecting jejunal cell metabolism, which in turn reduces growth performance. In the future, the ability of broilers to cope with HS can be improved by increasing or decreasing some intermediate metabolites in the metabolic pathways. Materials and methods Experimental design, broilers, and diets The experiment was conducted at the Veterinary Hospital of Anhui Science and Technology University, and the experiment was carried out from August to September. In this experiment, 100 day-old broilers, equally divided between male and female, weighing an average of 45 g, were obtained from commercial farms using Ross 308 broilers as test animals. Chicks were fed a basal diet at the start (days 1–21) and growth stages (days 22–42) in accordance with the Ross 308 recommendations (Table 1 ). The broilers were reared to 28 days old: Eighty healthy broilers of similar body weight were randomly divided into two groups (average weight 1350 g, equal male to female ratio), the standard rearing group (TN group) and the hot environment rearing group (CHS group), with four replicates in each group and ten broilers in each replicate, and reared in the same cage. Each group was allowed to eat and drink freely, and 23 hours of light were provided daily. The set temperature of broiler rearing in the control group was 24 ± 1 ℃. The broilers in the CHS group started to warm at 08:00, stabilized at 32 ± 1 ℃ at 09:00, cooled at 21:00, and stabilized at 24 ± 1 ℃ at 22:00 each day. The duration of heat stress was 12 h. The relative humidity in the rearing house was controlled at 60 ± 5% in all cases. During the feeding period, weights were recorded, feed consumption was measured, and the average daily feed intake (ADFI), average daily gain (ADG) and feed conversion ratio (FCR) were calculated for each group of broilers. Table 1 Composition and nutrient levels of basal diets (air-dry basis) % Items Content 1-21d 22-42d Corn 62.00 64.00 Soybean meal 29.10 27.80 Soybean oil 3.00 3.00 Fish meal 3.00 2.50 CaHPO 4 1.60 1.40 NaCl 0.30 0.30 Premix 1 1.00 1.00 Nutrient level ME/(MJ/kg) 2 12.96 13.03 CP 20.50 20.02 EE 5.98 6.06 Crude fiber 2.49 2.44 Ash 3.85 3.80 Lys 1.05 1.02 Met + Cys 0.80 0.78 Ca 1.01 1.00 TP 0.71 0.67 1 Premix is provided per kg of diet: Cu (as copper sulfate) 9 mg, I (as potassium iodide) 0.4 mg, Mn (as manganese sulfate) 66 mg, Fe (as ferrous sulfate) 50 mg, Zn 44 mg, vitamin A 7 000 IU, vitamin B 2 4.5 mg, vitamin B 6 2.5 mg, vitamin B 12 0.6 mg, vitamin D 3 875 IU, vitamin K 3 1 mg, vitamin E 20 IU, D-pantothenic acid 12 mg, nicotinic acid 50 mg. 2 ME was a calculated value. Ethics approval The ethical approval was obtained from the Institutional Animal Care and Use Committee of Anhui Science and Technology University, China (Approval No. 2023005). All methods and procedures were approved by the Anhui Science and Technology University and conducted in accordance with the relevant guidelines formulated by the Ministry of Agriculture of the People’s Republic of China. This study was conducted in accordance with the ARRIVE guidelines ( https://arriveguidelines.org ). Sample collection Two birds on day 42 were randomly selected from each replicate of each group, and a total of eight birds were selected. Broilers were euthanized by intravenous injection of an overdose of sodium pentobarbital, after which they were dissected, intestines were quickly separated, and the contents were removed by flushing with saline. The jejunal samples were placed in lyophilized tubes, snap-frozen in liquid nitrogen and stored in a -80 ℃ freeze for testing. Sample pretreatment A total of 30 mg of jejunal sample was precisely weighed into a centrifuge tube. Added two tiny steel balls, 600 µL of pre-cooled methanol solution and 20 µL of L-2-chloro-phenylalanine (0.3 mg/mL in methanol), and the mixture was held at -80 ℃ for 2 min. After grinding for 2 min, added 120 µL of chloroform and extracted by ultrasound. The resulting extracts were centrifuged at 4 ℃ for 10 min at 12 000 rpm, and 300 µL of the supernatant was loaded into glass sampling vials. Quality control (QC) samples were prepared by mixing well with extracts from two groups of broiler jejunum samples. The samples were volatilized and added with 80 µL of pyridinium methoxamine hydrochloride solution (15 mg/mL), vortex shaken for 2 min and incubated at 37 ℃ for 90 min. After 90 min, 80 µL N,O-bis(trimethylsilyl) trifluoroacetamide derivatization reagent containing 1% trimethylsilyl chloride and 20 µL n-hexane were added to each mixture, and derivatized at 70 ℃ for 60 min. Metabolomic analysis was performed on the sample using a GC-MS instrument. GC-MS analysis This experiment used GC-MS (7890B-5977A, Agilent, USA) for data acquisition. The prepared samples were injected into the GC-MS system in splitless mode and separated by a DB-5MS capillary column for mass spectrometric detection. The instrument ramp-up procedure was as follows: from 60 ℃ to 125 ℃ at 8 ℃/min, from 125 ℃ to 210 ℃ at 5 ℃/min, from 210 ℃ to 270 ℃ at 10 ℃/min, from 270 ℃ to 305 ℃ at 20 ℃/min and finally held at 305 ℃ for 5 minutes. The inlet temperature, electron bombardment ionization source temperature and voltage were set to 260 ℃, 230 ℃ and 70 eV, respectively. The mass scan range was 50–500 (m/z), and the signal acquisition started at 20 spectra/s after 5 min. During the operation of the instrument analysis, one QC sample was inserted out of every six samples analyzed to verify the accuracy and reproducibility of the analytical process of this test. Determination of intestinal digestive enzyme activity For the determination of the sample, approximately 0.5 g of jejunal tissue (excluding intestinal fascia and fat) was collected. The intestinal tissue was homogenized according to the manufacturer’s instructions (Nanjing Jianjian Biological Co., Ltd., China). The supernatant was stored at -20 ℃. This will be used to determine the activity of intestinal amylase, lipase, and trypsin. Absorptive function measurement in the intestine Serum D-xylose activity and GSH uptake were determined using the methods of Chen et al [ 38 ] . On day 42, four broilers were selected per replicate. After 12 h of fasting and water deprivation, two broilers were given a 10% D-xylose solution by gavage at a dose of 1 mL/kg, and the other two broilers were given reduced GSH by gavage at a dose of 1 g/kg. 2 mL of blood was collected from a vein in the tip of the wing 1 h later, and 30 µL of 2% heparin anticoagulant was added. The blood was then centrifuged at 3000 rpm for 10 min at 4 ℃ to prepare the serum. The corresponding indexes were determined using D-xylose and GSH kits purchased from Nanjing Jianchang Bioengineering Institute. Determination of apparent digestibility Fresh manure (100 g) was collected daily during the test period. To fix nitrogen, 10% concentrated sulfuric acid was added, stirred well, and stored at -20 ℃ until analysis. The weight of the feces excreted by each group of broilers was measured and recorded. At the end of the test, the feed and feces of each group were mixed well and dried in an oven to prepare air-dried samples. The samples were ground and passed through a 40 mesh sieve. Then, they were analyzed for dry matter (DM), organic matter (OM), crude protein (CP), and crude ash (Ash) according to the procedures of the AOAC [ 39 ] . Finally, the apparent digestibility of OM, CP and Ash was calculated. Statistical analysis The study analyzed growth performance, digestive enzyme activity, intestinal absorption capacity, and apparent digestibility of broiler using one-way ANOVA with SPSS 19.0. The results are shown as the mean and standard deviation, with P < 0.05 indicating significant differences. The raw GC-MS data were preprocessed by Chroma TOF software (v4.34, LECO Inc., USA). Internal standard peaks and false positive peaks (including derivatization reagent peaks, column loss and noise) were removed, and redundancy and peak merging were performed to analyze the samples to obtain 370 metabolites. The normalized response intensities of the sample mass spectrometry peaks were imported into the SIM CA-P + 14.0 software package, and the overall distribution among the samples, the stability of the analysis process and the overall differences in metabolic profiles among the groups were analyzed by principal component analysis (PCA) and partial least squares-discriminant analysis (PLS-DA) to identify the differentially abundant metabolites among the groups. In the PLS-DA analysis, variables with a variable importance in projection (VIP) greater than 1 were considered differential variables. The experimental analysis model was prevented from overfitting by a 7-cycle interaction validation analysis and a 200-cycle response ranking test analysis. Metabolite identification The metabolites that differed between the TN and CHS groups were screened by a combination of PLS-DA multidimensional analysis and t-test (VIP > 1, P < 0.05). The GC-MS workstation software automatically compared the mass-to-charge ratio and abundance of the characteristic ion fragments of each substance detected in this experiment with the standard metabolites in the National Institute of Standards and Technology (NIST) database, and the standard selection criteria required a match of more than 70%. Metabolic pathway enrichment analysis The ID conversion function of the MBRole database ( http://csbg.cnb.csic.es/mbrole/ ) was used to obtain the substance IDs of the KEGG database ( http://www.genome.jp/kegg/pathway.html ) for the differential metabolites. The two databases’ pathway analysis and enrichment functions were combined to identify the significantly different metabolic pathways exhibited in the jejuna of the TN and CHS groups. The metabolic pathway maps generated from the database and the histogram of metabolic pathway enrichment analysis during HS in broiler chickens were downloaded. Declarations Ethics approval and consent to participate The Animal Care and Use Committee of the Anhui Science and Technology University approved the animal treatment protocol used in this study (Protocol number 2023005). All experimental procedures were carried out following the guidelines mentioned in the ARRIVE guidelines. Data availability statement The data that support the finding of this study are available from corresponding author upon reasonable request. Competing interests The authors declare that they have no competing interests. Funding This study was supported by the Initiation Program of National Natural Science Foundation of China (No. 31702306), the Key Program of Scientific Research of Higher Education Institutions in Anhui Province (No. 2022AH051620), Major projects supported by Department of Education Anhui Province (No. 2023AH040282), and the Support Program for Excellent Young Talents in Universities and Colleges (No. gxyq2022056). Author contributions Y.J. Xiong and S.J. He conceptualized and supervised the study. S.A. Sun and B. Li performed the experiments. J.J. Yuan provided assistance with the animal experiments. Shiang Sun analyzed the data and wrote the paper. S.J. He provided revisions of important content. All authors read and approved the manuscript. Acknowledgments The author would like to thank all teachers and students who dedicated their time to take part in this study. Also, we are grateful to Dr. Abulaiti, Adili for touching up and revising this paper. References Najafi, P. et al. Environmental temperature and stocking density effects on acute phase proteins, heat shock protein 70, circulating corticosterone and performance in broiler chickens. Int. J. Biometeorol. 59 , 1577–1583 (2015). Qaid, M. M. & Al-Garadi, M. A. Protein and Amino Acid Metabolism in Poultry during and after Heat Stress: A Review. Anim. (Basel) . 11 , 1167 (2021). Ibrahim, D. et al. Dietary Eugenol Nanoemulsion Potentiated Performance of Broiler Chickens: Orchestration of Digestive Enzymes, Intestinal Barrier Functions and Cytokines Related Gene Expression With a Consequence of Attenuating the Severity of E. coli O78 Infection. Front. Vet. Sci. 9 , 847580 (2022). Patra, A. K. & Kar, I. Heat stress on microbiota composition, barrier integrity, and nutrient transport in gut, production performance, and its amelioration in farm animals. J. Anim. Sci. Technol. 63 , 211–247 (2021). Bertocchi, M. et al. Exploring Differential Transcriptome between Jejunal and Cecal Tissue of Broiler Chickens. Anim. (Basel) . 9 , 221 (2019). Nanto-Hara, F., Kikusato, M., Ohwada, S. & Toyomizu, M. Heat Stress Directly Affects Intestinal Integrity in Broiler Chickens. J. Poult. Sci. 57 , 284–290 (2020). Qian, W. et al. The roles and mechanisms of gut microbiome and metabolome in patients with cerebral infarction. Front. Cell. Infect. Microbiol. 13 , 1112148 (2023). Monte, A. A., Brocker, C., Nebert, D. W., Gonzalez, F. J. & Thompson, D. C. Vasiliou, V. Improved drug therapy: triangulating phenomics with genomics and metabolomics. Hum. Genomics . 8 , 16 (2014). Sun, S. et al. Effect of dietary supplemental vitamin C and betaine on the growth performance, humoral immunity, immune organ index, and antioxidant status of broilers under heat stress. Trop. Anim. Health Prod. 55 , 96 (2023). Rostagno, M. H. Effects of heat stress on the gut health of poultry. J. Anim. Sci. 98 , skaa090 (2020). Judge, A. & Dodd, M. S. Metabolism Essays Biochem. 64 , 607–647. (2020). Kondo, Y. et al. Phosphoenolpyruvic acid, an intermediary metabolite of glycolysis, as a potential cytoprotectant and anti-oxidant in HeLa cells. Biol. Pharm. Bull. 35 , 606–611 (2012). Xie, Y. F. et al. Investigation of Efficacy Enhancing and Toxicity Reducing Mechanism of Combination of Aconiti Lateralis Radix Praeparata and Paeoniae Radix Alba in Adjuvant-Induced Arthritis Rats by Metabolomics. 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Joint Transcriptome and Metabolome Analysis Prevails the Biological Mechanisms Underlying the Pro-Survival Fight in In Vitro Heat-Stressed Granulosa Cells. Biology (Basel) . 11 , 839 (2022). He, S., Zhao, S., Dai, S., Liu, D. & Bokhari, S. G. Effects of dietary betaine on growth performance, fat deposition and serum lipids in broilers subjected to chronic heat stress. Anim. Sci. J. 86 , 897–903 (2015). Xiong, Y. et al. Gene expressions and metabolomic research on the effects of polyphenols from the involucres of Castanea mollissima Blume on heat-stressed broilers chicks. Poult. Sci. 95 , 1869–1880 (2016). Chen, R. et al. Exploring the biomarkers and therapeutic mechanism of kidney-yang deficiency syndrome treated by You-gui pill using systems pharmacology and serum metabonomics. RSC Adv. 8 , 1098–1115 (2018). Zhao, C. Q. et al. Classification of Gan Dan Shi Re Pattern and Gan Shen Yin Xu Pattern in Patients with Hepatitis B Cirrhosis Using Metabonomics. Evid Based Complement Alternat Med. 2697468. (2018). (2018). Hidayat, M., Prahastuti, S., Yusuf, M. & Hasan, K. Nutrition profile and potency of RGD motif in protein hydrolysate of green peas as an antifibrosis in chronic kidney disease. Iran J Basic Med Sci. 24, 734–743. (2021). (2021). Guo, J. et al. Blood amino acids profile responding to heat stress in dairy cows. Asian-Australas J. Anim. Sci. 31 , 47–53 (2018). McCommis, K. S. et al. Loss of Mitochondrial Pyruvate Carrier 2 in the Liver Leads to Defects in Gluconeogenesis and Compensation via Pyruvate-Alanine Cycling. Cell. Metab. 22 , 682–694 (2015). Rauckhorst, A. J. et al. The mitochondrial pyruvate carrier mediates high fat diet-induced increases in hepatic TCA cycle capacity. Mol. Metab. 6 , 1468–1479 (2017). Jo, J. H. et al. Characterization of Short-Term Heat Stress in Holstein Dairy Cows Using Altered Indicators of Metabolomics, Blood Parameters, Milk MicroRNA-216 and Characteristics. Anim. (Basel) . 11 , 722 (2021). Li, Y. et al. Isolation of a Highly Efficient Antigenic-Protein-Degrading Bacillus amyloliquefaciens and Assessment of Its Safety. Anim. (Basel) . 10 , 1144 (2020). Qian, Y. et al. Effects of dietary pantothenic acid on growth, intestinal function, anti-oxidative status and fatty acids synthesis of juvenile blunt snout bream Megalobrama amblycephala. PLoS One . 10 , e0119518 (2015). An, J. et al. Effects of supplemental different clay minerals in broiler chickens under cyclic heat stress. J. Anim. Sci. Technol. 65 , 113–131 (2023). Efanova, E. et al. Polymorphisms of the GCLC Gene Are Novel Genetic Markers for Susceptibility to Psoriasis Associated with Alcohol Abuse and Cigarette Smoking. Life (Basel) . 13 , 1316 (2023). Kang, P. et al. Effects of oral administration of spermine on the development of small intestine and growth performance of weaned pigs. J. Anim. Vet. Adv. 11 , 2782–2787 (2012). Wu, Q. J. et al. Effect of glutamine on the growth performance, digestive enzyme activity, absorption function, and mRNA expression of intestinal transporters in heat-stressed chickens. Res. Vet. Sci. 134 , 51–57 (2021). Ling, H., Xiao, H., Zhang, Z., He, Y. & Zhang, P. Effects of Macleaya Cordata Extract on Performance, Nutrient Apparent Digestibilities, Milk Composition, and Plasma Metabolites of Dairy Goats. Anim. (Basel) . 13 , 566 (2023). Wickramasuriya, S. S. et al. Differential Effects of Dietary Methionine Isomers on Broilers Challenged with Acute Heat Stress. J. Poult. Sci. 56 , 195–203 (2019). Kim, D. H., Lee, Y. K., Kim, S. H. & Lee, K. W. The Impact of Temperature and Humidity on the Performance and Physiology of Laying Hens. Animals (Basel). 11 , 56. (2020). Chen, Z., Xie, J., Wang, B. & Tang, J. Effect of γ-aminobutyric acid on digestive enzymes, absorption function, and immune function of intestinal mucosa in heat-stressed chicken. Poult. Sci. 93 , 2490–2500 (2014). Aoac. 18th ed. AOAC Int.; Gaithersburg, MD: 2005. Official methods of analysis. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-6220478","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":444298916,"identity":"8b1d1b3d-0c14-4712-9187-b764f97bfc54","order_by":0,"name":"Shiang Sun","email":"","orcid":"","institution":"Anhui Science and Technology University","correspondingAuthor":false,"prefix":"","firstName":"Shiang","middleName":"","lastName":"Sun","suffix":""},{"id":444298917,"identity":"6691474a-740a-4df4-8a87-b43861521245","order_by":1,"name":"Bing Li","email":"","orcid":"","institution":"Anhui Science and Technology University","correspondingAuthor":false,"prefix":"","firstName":"Bing","middleName":"","lastName":"Li","suffix":""},{"id":444298918,"identity":"19f20627-df7c-4463-a735-81807ec03091","order_by":2,"name":"Junjun Yuan","email":"","orcid":"","institution":"Anhui Science and Technology University","correspondingAuthor":false,"prefix":"","firstName":"Junjun","middleName":"","lastName":"Yuan","suffix":""},{"id":444298919,"identity":"b95a8d72-7758-433e-8f22-0fb606974db1","order_by":3,"name":"Yongjie Xiong","email":"","orcid":"","institution":"Anhui Science and Technology University","correspondingAuthor":false,"prefix":"","firstName":"Yongjie","middleName":"","lastName":"Xiong","suffix":""},{"id":444298920,"identity":"0db70b21-9b73-421a-897b-bbdd0a72dcbd","order_by":4,"name":"Shaojun He","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA70lEQVRIiWNgGAWjYDACCQhOYGBvbDiQUCEhx0+8Fp7DBw88OGNhLNlAjBYGkBaJtOSDD9sqEjcQ0iI/u/nhA8u2O3n8DDkGBxLnSTBuYGB++OgGHi0Gd44ZG0i2PSuWbDgD1LJNgtmcgc3YOAefFokEMwnJtsOJGw72gLWwWTbwsEnj0yI/I/0bWMv+wzxALXMkgCQBLQw3cqC2sLElHEhskJAgqMXgRk6xgcS5w4kzzjAfOJBwTMJAspmAX4AO2/hYouxwYv/8h80ff9TU1fezNz98jNdhQMAsgcoloBwEGD8QoWgUjIJRMApGMAAAh2RT1ChFL1QAAAAASUVORK5CYII=","orcid":"","institution":"Anhui Science and Technology University","correspondingAuthor":true,"prefix":"","firstName":"Shaojun","middleName":"","lastName":"He","suffix":""}],"badges":[],"createdAt":"2025-03-13 13:23:29","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6220478/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6220478/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":82160448,"identity":"10224b15-15f4-40b1-886e-73350e15cf37","added_by":"auto","created_at":"2025-05-07 08:33:17","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":119358,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA \u003c/strong\u003eA total ion chromatograms (TICs) of control group from broilers. TN, control group.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB \u003c/strong\u003eA total ion chromatograms (TICs) of heat stress group from broilers. CHS, chronic heat stress group.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6220478/v1/685cd3fdab191b09558424bc.png"},{"id":82162047,"identity":"c7fe7e04-915c-45bd-ab76-9a21084adfdc","added_by":"auto","created_at":"2025-05-07 08:41:17","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":643368,"visible":true,"origin":"","legend":"\u003cp\u003eHierarchical clustering of differential metabolites. TN, control group; CHS, chronic heat stress group.\u003c/p\u003e","description":"","filename":"Fig.2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6220478/v1/ce1a6cf7aa303d47bbede965.jpg"},{"id":82162751,"identity":"e29f389b-2654-4c33-bb0a-9911dc81d1c3","added_by":"auto","created_at":"2025-05-07 08:49:17","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":245785,"visible":true,"origin":"","legend":"\u003cp\u003ePrincipal component analysis (PCA) score scatter plots of jejunal samples. TN, control group; CHS, chronic heat stress group.\u003c/p\u003e","description":"","filename":"Fig.3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6220478/v1/004611b69ecacf6cf442afb4.jpg"},{"id":82159568,"identity":"f428db30-b483-4a17-8785-e45c8f13609e","added_by":"auto","created_at":"2025-05-07 08:25:17","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":232432,"visible":true,"origin":"","legend":"\u003cp\u003ePartial least squares-discriminant analysis (PLS-DA) score scatter plots of jejunal samples. TN, control group; CHS, chronic heat stress group.\u003c/p\u003e","description":"","filename":"Fig.4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6220478/v1/95ea52e6a3b1fe47d4a1fcb8.jpg"},{"id":82162050,"identity":"affdb0d6-78c6-4c7a-bb56-395a9e349472","added_by":"auto","created_at":"2025-05-07 08:41:17","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":249315,"visible":true,"origin":"","legend":"\u003cp\u003eValidation plots of partial least squares-discriminant analysis (PLS-DA) with 200 permutation.\u003c/p\u003e","description":"","filename":"Fig.5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6220478/v1/9ad01f27c514fbe49bbb3060.jpg"},{"id":82160450,"identity":"017d1047-40b5-4b6a-9f17-df8471c835e9","added_by":"auto","created_at":"2025-05-07 08:33:17","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":183308,"visible":true,"origin":"","legend":"\u003cp\u003eMetabolic pathway bubble diagram. ABC, ATP-binding cassette.\u003c/p\u003e","description":"","filename":"Fig.6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6220478/v1/f70a2b411caf1f15a3bc98e5.jpg"},{"id":82160453,"identity":"1db13cfe-84df-4aa9-a039-3c9c82271e0f","added_by":"auto","created_at":"2025-05-07 08:33:17","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":386393,"visible":true,"origin":"","legend":"\u003cp\u003eMetabolic pathway enrichment analysis. Bars that exceed the blue and red dotted lines indicate pathways with P values of \u0026lt; 0.05 and \u0026lt; 0.01, respectively. ABC, ATP-binding cassette.\u003c/p\u003e","description":"","filename":"Fig.7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6220478/v1/0259310aaf869fcdc5b25383.jpg"},{"id":104508411,"identity":"99d5bf3f-56a2-4aa9-aab1-128b7e4d0e10","added_by":"auto","created_at":"2026-03-12 15:26:00","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3351976,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6220478/v1/04c994dd-bd3c-4385-b58b-a170e5bfb34f.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"The effects of chronic heat stress on the growth performance, digestive and absorbtive-related parameters, and jejunal metabolomics in broilers","fulltext":[{"header":"Introduction","content":"\u003cp\u003eDue to the lack of sweat glands and feather cover, broilers are highly susceptible to heat stress (HS). A study suggested that significant HS can occur when the environmental temperature of broiler feed reaches 32 ℃\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. With the continuing trend of global warming, the HS response of poultry has further increased. Heat stress has become a significant factor limiting the development of the poultry industry, causing substantial economic losses for the livestock industry every year. HS harms various functions of poultry to varying degrees. The main effects are increased body temperature, immunosuppression, oxidative damage and damage to the intestinal mucosa\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. This ultimately leads to reduced feed intake and feed conversion and stunted growth. To adapt to the high summer temperatures, broilers will actively regulate multiple metabolic pathways to maintain internal heat balance. The gut is the leading site for the digestion and absorption of nutrients and is also a significant immune barrier against invading pathogenic bacteria\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. HS leads to reduced digestive and absorptive capacity of the intestinal tract, damage to intestinal barrier integrity, an imbalance in the structure of the flora and delayed development of immune organs\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e. There is a critical need to determine the mechanisms of HS responses and their effects on intestinal permeability in poultry.\u003c/p\u003e \u003cp\u003eThe jejunum is a vital digestive organ in the digestive tract, and metabolic changes in the jejunum can reflect the health status and function of the digestive tract of broilers\u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e. HS impairs broiler intestinal integrity and increases the permeability of the intestine to endotoxins\u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. However, these data cannot systematically and comprehensively reveal the absorption and metabolic status of essential nutrients in the body as a whole. Furthermore, the differential metabolic markers and the metabolic pathways altered during HS are still unknown. This hinders the idea of mitigating the harm of HS from the perspective of regulating nutrient uptake and metabolism. After genomics, transcriptomics and proteomics, metabolomics has become vital to systems biology. However, studies and the laws concerning the types and quantities of metabolites and their dynamics after an organism is disconcerted\u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e. Using metabolomics technologies to identify specific biomarkers in the development of diseases can provide a basis for early diagnosis of diseases and suggest targeted therapeutic measures\u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. Detecting the composition of all small molecules in the broiler gut during HS can help further reveal the mechanism of the damage caused by HS in broilers. However, few studies have analyzed the changes in all intestinal metabolites during HS in broiler chickens using a metabolomics approach, and most metabolomic studies have focused on mammals. Therefore, in this study, we used gas chromatography-mass spectrometry (GC-MS) to investigate the metabolic profiles of the jejunum of heat-stressed broilers and to explore the pattern of changes and related regulatory mechanisms involved. This study also aimed to elucidate the relationships between growth performance, digestive and absorptive capacity and apparent digestibility and metabolic changes in heat stressed broilers.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eGrowth performance\u003c/h2\u003e \u003cp\u003eThe growth performance of broilers in the TN and CHS groups is shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e2\u003c/span\u003e. Compared with the TN group, broilers in the CHS group had significantly lower ADG, ADFI, and higher FCR (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\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 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffect of chronic HS on broiler growth performance\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\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 \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGroups\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTN\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCHS\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSEM\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eADG (g/d)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e188.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e168.93\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e4.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eADFI (g/d)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e729.78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e571.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e24.60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFCR (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003eNote: \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001 indicate highly significant differences. ADG, average daily gain; ADFI, average daily feed intake; FCR, feed conversion ratio; TN, control group; CHS, chronic heat stress group.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMetabolomic profiling of the samples\u003c/h3\u003e\n\u003cp\u003eThe GC-MS total ion chromatograms of the samples from the TN and CHS groups are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The figure shows that the instrument collected strong signals of jejunal metabolites during this test, and the retention time and volume of the generated peaks met the test requirements. The jejunum assay mass spectra were compared with the standard mass spectra of the NIST database, and a total of 370 metabolites with more than 70% match were identified. Multivariate and unidimensional statistics were used to depict metabolomic characteristics by analyzing differences in broiler jejunal metabolites between the TN and CHS groups.\u003c/p\u003e \n\u003ch3\u003eHierarchical clustering\u003c/h3\u003e\n\u003cp\u003eDifferences in the accumulation patterns of jejunal metabolites between the two groups were analyzed by clustering heatmaps (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The heatmap showed that some metabolites were down-regulated in the TN group and up-regulated in the CHS group, suggesting that there may be significantly different metabolic processes between the TN and CHS groups. In addition, eight samples from the two groups were clustered together, indicating high data reliability with good homogeneity between biological replicates.\u003c/p\u003e \n\u003ch3\u003eMultivariate statistical analysis\u003c/h3\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003ePrincipal component analysis\u003c/h2\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003e shows the results of the PCA for the two groups of jejunal metabolites. As seen from the figure, the TN and CHS groups were mainly distributed in different quadrants, indicating some inter-group variability between the two groups and that the variability between the test groups was real.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003ePartial least squares-discriminant analysis\u003c/h2\u003e \u003cp\u003eTo eliminate noise information irrelevant to the classification, we built a PLS-DA model based on the PCA model (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003e). It shows that 88.0% of the samples (data records) fit the established discriminant mathematical model, and the prediction accuracy of the model was 60.9%. The PLS-DA model score plot showed that the broiler jejunal metabolites in the TN and CHS groups were better separated on the principal component axes, and the differences were true. The robustness of the model was examined using the 200 response ranking method, and the results are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003e, where R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.907\u0026thinsp;\u0026gt;\u0026thinsp;0.5 and Q\u003csup\u003e2\u003c/sup\u003e = -0.482\u0026thinsp;\u0026lt;\u0026thinsp;0, indicating that this model is stable and reliable.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSignificantly different metabolites analysis\u003c/h3\u003e\n\u003cp\u003eThe peak areas of metabolites were normalized by PLS-DA analysis after relative quantification, and 65 differential metabolites were screened according to the results of the relative variable standard analysis of VIP\u0026thinsp;\u0026gt;\u0026thinsp;1 and the t-test of \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Among them, 43 metabolites such as enolpyruvate, mannose 6-phosphate and succinic acid were up-regulated [Fold change (FC)\u0026thinsp;\u0026gt;\u0026thinsp;1], and 22 metabolites such as norvaline, L-cystine and glyceric acid were down-regulated (FC\u0026thinsp;\u0026lt;\u0026thinsp;1).\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 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eIdentification of significantly different metabolites of TN group and CHS group\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\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=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMetabolites\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRT (min)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eVIP\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eP\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eFC\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eChange\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSalicylaldehyde\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5.187\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026darr;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCarinitine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2-hydroxypentanoic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5.286\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGlutaric acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5.314\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e3.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1-hexanol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5.513\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBenzylalcohol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5.536\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.003\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2.20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2-hydroxypyrazinyl-2-propenoic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5.589\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e3.94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMorpholine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5.599\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e10.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHexanamide\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5.665\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e10.73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEpsilon-caprolactam\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5.698\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDiethylcarbamic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e6.474\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEnolpyruvate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e6.996\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.029\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1,5-anhydroglucitol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e7.078\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eResorcinol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e7.097\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e11.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4-aminophenol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e7.576\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.008\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026darr;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGlutathione\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e8.428\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMethanolphosphate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e8.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.045\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026darr;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eN-methylalanine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e8.81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4.84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eXanthurenic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e9.312\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5.89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLeucinic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e9.51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.030\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2.97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSuccinic anhydride\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e9.537\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.002\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026darr;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDelta-tocopherol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e9.681\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.003\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026darr;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eL-kynurenine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e10.346\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.005\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026darr;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOxamic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e10.743\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e7.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePentonic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e10.794\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2.64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLevoglucosan\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e10.984\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3.94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e49.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSuccinic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e11.062\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.007\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGlyceric acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e11.352\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.012\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026darr;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2-imidazolidinone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e11.376\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e3.70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTranexamic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e11.404\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e8.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1,2,4-benzenetriol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e11.655\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4.99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e645.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePyrazine, 3,6-dihydro-3,6-dimethyl-2,5-dihydroxy-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e11.714\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.002\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026darr;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eErythrose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e12.413\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGlucosaminic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e12.478\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.93\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDodecanol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e13.583\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026darr;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBiphenyl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e13.761\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.002\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4-hydroxyproline\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e15.496\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.016\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eL-asparagine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e18.553\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.018\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eD-xylulose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e18.753\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.008\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026darr;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eD-arabinose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e18.78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.029\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026darr;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNorvaline\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e19.358\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.046\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026darr;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eD-glyceraldehyde 3-phosphate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e19.659\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.79\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.016\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDihydroxyacetone phosphate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e20.126\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.007\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eL-glutamine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e20.742\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2.63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eN-acetyl-d-hexosamine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e20.824\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.021\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026darr;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eL-cysteine-glycine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e21.543\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.85\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMyristic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e22.301\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026darr;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMaltitol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e22.301\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026darr;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eD-tagatose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e22.764\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.017\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026darr;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP-hydroxylphenyllactic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e22.932\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026darr;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLysopalmitoyl monogalactosylglycerol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e23.643\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.019\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAscorbic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e24.191\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.028\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCellobiose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e25.452\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.029\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBarbital\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e26.119\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.002\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eErythrotetrofuranose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e26.796\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.043\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eD-ribose 5-phosphate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e27.107\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eD-ribulose 5-phosphate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e27.275\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2.78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSedoheptulose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e27.439\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.032\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026darr;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAltrose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e27.518\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.035\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026darr;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePsilocin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e28.121\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.027\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eL-cystine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e29.448\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.019\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026darr;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMannose 6-phosphate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e29.71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.046\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePhosphohexonic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e30.776\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.012\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3,5-dihydroxyphenylglycine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e31.822\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.029\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026uarr;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDeoxycholic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e37.194\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.005\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026darr;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003eNote: RT, retention time; VIP, variable importance in the projection; FC, fold change; FC values greater than 1 mean that metabolite in CHS group was more than that in TN group. Change, ratio of mean peak area of the CHS group to the mean peak area of the TN group; \u0026uarr;, metabolites with higher concentrations in the CHS group than in the TN group with values\u0026thinsp;\u0026gt;\u0026thinsp;1; \u0026darr;, metabolites with lower concentrations in the CHS group than in the TN group with values\u0026thinsp;\u0026lt;\u0026thinsp;1.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e\n\u003ch3\u003eMetabolite set enrichment analysis\u003c/h3\u003e\n\u003cp\u003eThe enrichment analysis results of the relevant metabolic pathways are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003e, in which the effect of HS on the glycolysis/gluconeogenesis metabolic pathway was highly significant (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), followed by the effects on the fructose and mannose metabolic pathways (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). In addition, metabolic pathway enrichment analysis revealed that lysosomal, amino and nucleotide sugar metabolism, carbon metabolism, ABC transporter protein, amino acid biosynthesis, and tricarboxylic acid cycle (TCA cycle) metabolic pathways were significantly altered during cyclic HS (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eIntestinal digestive enzyme activities\u003c/h2\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e4\u003c/span\u003e displays the intestinal digestive enzyme activities of broilers in the TN and CHS groups. In the CHS group, broilers exhibited significantly lower levels of amylase, lipase, and trypsin activities compared to the TN group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\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 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffect of chronic HS on the activity of intestinal digestive enzymes in broilers\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\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 \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGroups\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTN\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCHS\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSEM\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmylase (U/mgprot)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e7.48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5.97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.018\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLipase (U/gprot)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e231.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e110.82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e12.61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTrypsin (U/mgprot)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e973.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e630.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e51.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.004\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003eNote: \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 indicate significant differences. TN, control group; CHS, chronic heat stress group.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eD-xylose and GSH absorption\u003c/h2\u003e \u003cp\u003eThe levels of D-xylose and GSH absorption in broilers of the TN and CHS groups are shown in Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e5\u003c/span\u003e. HS significantly reduced the levels of D-xylose and GSH absorption in the intestinal tract of broilers compared to the control group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffect of chronic HS on intestinal D-Xylose and GSH absorption in broilers\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\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 \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGroups\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTN\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCHS\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSEM\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eD-xylose (mmol/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.023\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGSH (g/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.016\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003eNote: \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 indicate significant differences. TN, control group; CHS, chronic heat stress group.\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\u003eApparent digestibility\u003c/h2\u003e \u003cp\u003eThe effect of chronic HS on apparent digestibility in broilers are shown in Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e6\u003c/span\u003e. Compared with the TN group, heat stress significantly decreased the digestibility of dry matter, organic matter, and crude protein in broilers (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). There was no significant change in crude fat (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 6\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffect of chronic HS on the apparent digestibility of nutrients in broilers\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\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 \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGroups\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTN\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCHS\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSEM\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDry matter\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e73.64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e71.20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOrganic matter\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e77.65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e75.70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCrude protein\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e61.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e56.93\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.006\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCrude fat\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e76.39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e74.88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.535\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003eNote: \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 indicate significant differences. TN, control group; CHS, chronic heat stress group.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eHS leads to disruption of physiological functions in poultry in hot environments. Our previous study indicated that broilers exposed to HS (32 ℃) reduced production performance, decreased ADG and increased FCR, the same as the present experiment results\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e. It may be due to the loss of appetite and decreased feed intake of broilers in hot environments, so the ADG of broilers in the CHS group decreased, and the FCR increased. In addition, poultry have difficulty dissipating heat due to their feathered bodies and lack of sweat glands. When the ambient temperature exceeds the optimal temperature, energy is consumed to maintain the body temperature, reducing the amount of raw materials that can be converted into muscle and fat, leading to a decrease in growth performance.\u003c/p\u003e \u003cp\u003eThe intestine is greatly affected by HS and is an important link in the occurrence of HS hazards. Studies have shown that when broilers suffer from HS, intestinal villus height decreases crypt depth increases, and intestinal permeability increases, thus inducing inflammation\u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e. This experiment revealed the most significant glycolysis/gluconeogenesis metabolic pathway changes through metabolic pathway enrichment analysis. When the ambient temperature exceeds the optimal temperature, the organism requires energy substances to maintain a relatively stable body temperature. Glycolysis is a stage that all organisms must pass during glucose catabolism\u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. Glucose can produce ATP during lactate production to provide the energy required by the body to maintain body temperature homeostasis. Enolpyruvate is a common biochemical molecule in biological cells. It is an intermediate product of glycolysis and gluconeogenesis where it can be metabolized to pyruvate to produce ATP, one of the primary sources of intracellular chemical energy\u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e. In this study, the content of enolpyruvate was significantly greater in the intestine of heat-stressed broilers than in that of control broilers, indicating that the glycolysis pathway was activated, the gluconeogenic pathway was blocked, and the cellular demand for energy was increased during HS.\u003c/p\u003e \u003cp\u003eGlycolysis and the TCA cycle are the two crucial energy metabolic pathways, and the TCA cycle is a prevalent metabolic pathway in aerobic organisms, distributed in mitochondria\u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. Both are essential links in the biochemical pathways central to aerobic bioenergy synthesis and cellular metabolism. A previous study showed that HS can cause alterations in the body\u0026rsquo;s metabolic pathways of the TCA cycle\u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. In the enriched metabolic pathway, the TCA cycle also changed significantly, with the same results as above. The energy produced by glycolysis was probably not entirely sufficient when the organism was subjected to HS. The organism further processes the pyruvate produced during glycolysis and forms acetyl-CoA through oxidative decarboxylation, which in turn produces carbon dioxide and water, generating a large amount of ATP for consumption by the organism. Succinic acid is a dicarboxylic acid, an intermediate product of the TCA cycle\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e. The alteration of the TCA cycle in broilers in broilers exposed to HS suggested that the activity of some enzymes involved in the tricarboxylic acid metabolism process may be significantly altered when the body is exposed to HS. Some metabolic intermediates of this cycle, such as succinic acid, cannot enter the cycle normally to participate in metabolism, causing significant changes in the content of these substances in the body.\u003c/p\u003e \u003cp\u003eMannose 6-phosphate is an intermediate product of the fructose and mannose metabolic pathway, and its main metabolic pathway is catalyzed by mannose phosphate isomerase to produce fructose-6-phosphate, which is broken down into the glycolytic pathway. Mannose enters the cell via the glucose transporter protein, which is present as mannose 6-phosphate for further metabolism. Mannose does not affect cellular glucose uptake, but elevated intracellular mannose 6-phosphate inhibits glucose-6-phosphate dehydrogenase, glucose phosphate isomerase and hexokinase, which are involved in glucose metabolism. This in turn, affects glycolysis and the TCA cycle\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. Therefore, it was hypothesized that increasing the mannose 6-phosphate in the broiler intestine during HS would inhibit intracellular glucose metabolism in vivo. This is consistent with the findings of Cui et al\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. Ribose 5-phosphate is the raw material for the synthesis of RNA and DNA. The content of ribose 5-phosphate in the jejunum significantly increased during HS in broilers. Therefore, it is speculated that the decrease in DNA synthesis during HS may not be related to the content of ribose 5-phosphate, but rather to the loss of control of DNA synthesis from ribose 5-phosphate\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e. Among the metabolites related to the gluconeogenesis pathway, the levels of D-arabinose and D-xylulose were significantly reduced, indicating that HS caused abnormal energy metabolism in the body. The body needs to accelerate the breakdown of sugars to provide more energy to counteract the stress.\u003c/p\u003e \u003cp\u003eATP-binding cassette (ABC) transporter proteins are a multifunctional superfamily of membrane proteins that play essential roles in the transcellular transport of nutrients. This pathway is exciting because it is involved in the transport of cholesterol and fat\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. The high-temperature environment results in significant changes in the basal metabolic rate of broilers and increased deposition of abdominal fat as an additional form of energy storage\u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e. In this experiment, there were significant differences in the indexes related to the metabolism of various lipids such as leucine, myristic acid, valeric acid and glyceric acid in the jejunum of broilers during HS, indicating that lipid metabolism in broilers was disturbed during the occurrence of HS, as reported in the study by Xiong et al\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e. In this experiment, broilers\u0026rsquo; jejunal valeric acid content was significantly increased during HS, probably due to the energy deficiency caused by the decrease of food intake during HS in broilers, which in turn caused the body to mobilize fat from surrounding tissues for energy supply. Valeric acid, as a readily oxidized fatty acid, may be released during lipolysis, thereby increasing levels in the jejunum. Leucine can help burn visceral fat to provide sufficient energy for body tissues\u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e. In order to reach the heat balance in broilers during HS, all parts of the organism work together, so the leucine content is briefly increased to help the organism burn fat for more energy. Glyceric acid is a three-carbon acid formed by the oxidation of glycerol, and its phosphorylation can produce glycerol 3-phosphate, which can further isomerize into sugar or participate in glycolysis\u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e. The glycolysis pathway was activated in broilers under HS, and the phosphorylation of glyceric acid was accelerated to form intermediate products to ensure the operation of the glycolysis pathway. Therefore, the glyceric acid content was significantly lower than that in the control group.\u003c/p\u003e \u003cp\u003eAmino acids and urea are the final forms of protein breakdown in vivo. Amino acids can continue to participate in the body\u0026rsquo;s sugar and lipid metabolism, and urea is excreted in the form of nitrogenous metabolic waste\u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e. In this experiment, the alanine content in the broiler intestine increased significantly during HS, a result also reported in the study by Guo et al\u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e. This may be linked to the involvement of alanine in energy metabolism in the body. The alanine-glucose cycle is an important pathway for it to exert energy metabolism. When HS occurs in broilers, the body must adjust its energy metabolic processes to adapt to the high-temperature environment. A large production of pyruvate accompanies glucose consumption as an energy supply. To maintain a continuous supply of energy, the alanine metabolic pathway is required\u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e. In addition, arginine and proline in the body can be converted to glutamate via the amino acid conversion pathway. Then, glutamate is deaminated under alanine aminotransferase to produce α-ketoglutarate, a critical intermediate in the TCA cycle pathway that provides energy. In this process, alanine is rapidly converted to pyruvate through deamination to facilitate the tricarboxylic acid supply cycle in the organism\u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e. The valine content in the jejunum decreased significantly during the course of this experiment, similar to Jo\u0026rsquo;s research results\u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e. Valine is a branched-chain amino acid that plays a crucial role in animal growth and metabolism, and is also a sugar-generating amino acid\u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e. When broilers are exposed to HS, energy in the body is heavily depleted. Valine can produce the glucose required for the body\u0026rsquo;s stable ecological balance through the gluconeogenesis pathway, alleviating the problem of insufficient glucose content in the body during metabolism. The analysis of amino acid metabolism characteristics during HS showed that adding some amino acids with sugar-generating or energy-promoting substances to rations could help prevent and control HS.\u003c/p\u003e \u003cp\u003eTo better understand the changes in intestinal metabolism induced by HS in broilers, we examined the variations in digestive enzyme activities in the jejunum. The activity levels of intestinal amylase, lipase, and trypsin can effectively indicate the strength of intestinal digestion of major nutrients\u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e. Normally, the activities of various intestinal enzymes tend to stabilize, with some dynamic elevation after eating. It has been shown that HS reduces the activity of digestive enzymes such as amylase and lipase in the gut\u003csup\u003e[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e. In this experiment, HS resulted in a significant decrease in amylase, lipase, and trypsin activities in the jejunum of broilers, which is consistent with the results of the above study. It may be related to the inactivation of digestive enzyme activity in the organism under high-temperature conditions. Another reason may be that heat stress alters the metabolism and function of jejunal cells, resulting in decreased secretion and activity of digestive enzymes in the jejunum. Furthermore, HS can cause damage to the intestines through oxidative stress and morphological damage, resulting in reduced enzyme activity. This can significantly impact nutrient absorption and metabolism in broilers, leading to a decreased ability to digest sugars, fats, proteins, and other essential nutrients in their diet. Sugars, fat, and protein are crucial nutrients for the growth and development of the body. The digestive capacity of these nutrients affects broiler feed intake, weight gain, weight ratio, and other indicators, which reduces production benefits.\u003c/p\u003e \u003cp\u003eAdditionally, we conducted measurements of intestinal absorption function in broilers subjected to HS. D-xylose, a pentose sugar that is primarily absorbed in the jejunum and excreted by the kidneys without participating in body metabolism. GSH is a low molecular weight thiol tripeptide that plays an important role in maintaining intracellular redox balance\u003csup\u003e[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e. D-xylose and GSH absorption tests are widely used to assess intestinal absorption function\u003csup\u003e[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e. HS can lead to a significant decrease in serum levels of D-xylose and GSH in broilers, which is consistent with the findings of Wu\u003csup\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/sup\u003e. It showed that the intestinal absorption function of broilers decreased under HS, and the reason for this phenomenon may be because the intestinal villi of broilers were damaged under HS conditions, and the morphology of intestinal mucosa was damaged, and the absorption capacity was weakened. The reduced levels of digestive enzymes observed in broilers exposed to HS may also impact their intestinal absorptive capacity, and consequently, the absorption of D-xylose and GSH. It is important to note that this statement is based on objective measurements and not subjective evaluations.\u003c/p\u003e \u003cp\u003eNext, we examined changes in the apparent digestibility of nutrients in heat-stressed broilers. The apparent digestibility of nutrients is an important indicator for assessing the ability of animals to digest and absorb nutrients in the diet\u003csup\u003e[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/sup\u003e. Wickramasuriya\u003csup\u003e[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]\u003c/sup\u003e reported that HS reduced the apparent digestibility of crude protein and dry matter in broilers. This finding is consistent with the observed reductions in the digestibility of DM, OM, and CP in feeds during the experiment. When broilers are exposed to HS, their bodies allocate a significant amount of blood to the surface to dissipate heat\u003csup\u003e[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]\u003c/sup\u003e. This results in reduced blood flow to the digestive tract, leading to a decrease in the apparent digestibility of nutrients. The reduced activity of digestive enzymes in broilers under HS indicates a decrease in intestinal digestive capacity and a corresponding decrease in the apparent digestibility of nutrients in broilers. Furthermore, the reduced apparent digestibility of heat-stressed broilers may also be attributed to reduced intercellular proteins and altered permeability resulting from changes in jejunal metabolism. Decreased dry matter digestibility in broilers during HS reduces glycogen stores. Gluconeogenesis is necessary to maintain blood glucose concentrations. The metabolic pathway enrichment analysis revealed alterations in the glycolysis/gluconeogenesis pathway to support this finding.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn this experiment, the metabolomic approach technology was applied to study the alteration of small molecule metabolites in the jejunum of broilers during HS. A total of 65 differential metabolites were identified, indicating that the metabolism of intestinal cells in broilers changed during HS. And by testing the intestinal enzyme activity, absorptive capacity, and apparent digestibility of heat-stressed broilers, it was shown that HS may reduce the intestinal digestive and absorptive capacity of broilers by affecting jejunal cell metabolism, which in turn reduces growth performance. In the future, the ability of broilers to cope with HS can be improved by increasing or decreasing some intermediate metabolites in the metabolic pathways.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eExperimental design, broilers, and diets\u003c/h2\u003e \u003cp\u003eThe experiment was conducted at the Veterinary Hospital of Anhui Science and Technology University, and the experiment was carried out from August to September. In this experiment, 100 day-old broilers, equally divided between male and female, weighing an average of 45 g, were obtained from commercial farms using Ross 308 broilers as test animals. Chicks were fed a basal diet at the start (days 1\u0026ndash;21) and growth stages (days 22\u0026ndash;42) in accordance with the Ross 308 recommendations (Table\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The broilers were reared to 28 days old: Eighty healthy broilers of similar body weight were randomly divided into two groups (average weight 1350 g, equal male to female ratio), the standard rearing group (TN group) and the hot environment rearing group (CHS group), with four replicates in each group and ten broilers in each replicate, and reared in the same cage. Each group was allowed to eat and drink freely, and 23 hours of light were provided daily. The set temperature of broiler rearing in the control group was 24\u0026thinsp;\u0026plusmn;\u0026thinsp;1 ℃. The broilers in the CHS group started to warm at 08:00, stabilized at 32\u0026thinsp;\u0026plusmn;\u0026thinsp;1 ℃ at 09:00, cooled at 21:00, and stabilized at 24\u0026thinsp;\u0026plusmn;\u0026thinsp;1 ℃ at 22:00 each day. The duration of heat stress was 12 h. The relative humidity in the rearing house was controlled at 60\u0026thinsp;\u0026plusmn;\u0026thinsp;5% in all cases. During the feeding period, weights were recorded, feed consumption was measured, and the average daily feed intake (ADFI), average daily gain (ADG) and feed conversion ratio (FCR) were calculated for each group of broilers.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab6\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComposition and nutrient levels of basal diets (air-dry basis) %\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\u003eItems\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eContent\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1-21d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e22-42d\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCorn\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e62.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e64.00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSoybean meal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e29.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e27.80\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSoybean oil\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFish meal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.50\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCaHPO\u003csub\u003e4\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.40\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNaCl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.30\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePremix\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNutrient level\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eME/(MJ/kg)\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e12.96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e13.03\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e20.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e20.02\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5.98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.06\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCrude fiber\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.44\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAsh\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.85\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.80\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLys\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.02\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMet\u0026thinsp;+\u0026thinsp;Cys\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.78\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.67\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\u003ePremix is provided per kg of diet: Cu (as copper sulfate) 9 mg, I (as potassium iodide) 0.4 mg, Mn (as manganese sulfate) 66 mg, Fe (as ferrous sulfate) 50 mg, Zn 44 mg, vitamin A 7 000 IU, vitamin B\u003csub\u003e2\u003c/sub\u003e 4.5 mg, vitamin B\u003csub\u003e6\u003c/sub\u003e 2.5 mg, vitamin B\u003csub\u003e12\u003c/sub\u003e 0.6 mg, vitamin D\u003csub\u003e3\u003c/sub\u003e 875 IU, vitamin K\u003csub\u003e3\u003c/sub\u003e 1 mg, vitamin E 20 IU, D-pantothenic acid 12 mg, nicotinic acid 50 mg.\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"3\"\u003e\u003csup\u003e2\u003c/sup\u003eME was a calculated value.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eEthics approval\u003c/h2\u003e \u003cp\u003e The ethical approval was obtained from the Institutional Animal Care and Use Committee of Anhui Science and Technology University, China (Approval No. 2023005). All methods and procedures were approved by the Anhui Science and Technology University and conducted in accordance with the relevant guidelines formulated by the Ministry of Agriculture of the People\u0026rsquo;s Republic of China. This study was conducted in accordance with the ARRIVE guidelines (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://arriveguidelines.org\u003c/span\u003e\u003cspan address=\"https://arriveguidelines.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eSample collection\u003c/h2\u003e \u003cp\u003eTwo birds on day 42 were randomly selected from each replicate of each group, and a total of eight birds were selected. Broilers were euthanized by intravenous injection of an overdose of sodium pentobarbital, after which they were dissected, intestines were quickly separated, and the contents were removed by flushing with saline. The jejunal samples were placed in lyophilized tubes, snap-frozen in liquid nitrogen and stored in a -80 ℃ freeze for testing.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eSample pretreatment\u003c/h2\u003e \u003cp\u003eA total of 30 mg of jejunal sample was precisely weighed into a centrifuge tube. Added two tiny steel balls, 600 \u0026micro;L of pre-cooled methanol solution and 20 \u0026micro;L of L-2-chloro-phenylalanine (0.3 mg/mL in methanol), and the mixture was held at -80 ℃ for 2 min. After grinding for 2 min, added 120 \u0026micro;L of chloroform and extracted by ultrasound. The resulting extracts were centrifuged at 4 ℃ for 10 min at 12 000 rpm, and 300 \u0026micro;L of the supernatant was loaded into glass sampling vials. Quality control (QC) samples were prepared by mixing well with extracts from two groups of broiler jejunum samples. The samples were volatilized and added with 80 \u0026micro;L of pyridinium methoxamine hydrochloride solution (15 mg/mL), vortex shaken for 2 min and incubated at 37 ℃ for 90 min. After 90 min, 80 \u0026micro;L N,O-bis(trimethylsilyl) trifluoroacetamide derivatization reagent containing 1% trimethylsilyl chloride and 20 \u0026micro;L n-hexane were added to each mixture, and derivatized at 70 ℃ for 60 min. Metabolomic analysis was performed on the sample using a GC-MS instrument.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eGC-MS analysis\u003c/h2\u003e \u003cp\u003eThis experiment used GC-MS (7890B-5977A, Agilent, USA) for data acquisition. The prepared samples were injected into the GC-MS system in splitless mode and separated by a DB-5MS capillary column for mass spectrometric detection. The instrument ramp-up procedure was as follows: from 60 ℃ to 125 ℃ at 8 ℃/min, from 125 ℃ to 210 ℃ at 5 ℃/min, from 210 ℃ to 270 ℃ at 10 ℃/min, from 270 ℃ to 305 ℃ at 20 ℃/min and finally held at 305 ℃ for 5 minutes. The inlet temperature, electron bombardment ionization source temperature and voltage were set to 260 ℃, 230 ℃ and 70 eV, respectively. The mass scan range was 50\u0026ndash;500 (m/z), and the signal acquisition started at 20 spectra/s after 5 min. During the operation of the instrument analysis, one QC sample was inserted out of every six samples analyzed to verify the accuracy and reproducibility of the analytical process of this test.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eDetermination of intestinal digestive enzyme activity\u003c/h2\u003e \u003cp\u003eFor the determination of the sample, approximately 0.5 g of jejunal tissue (excluding intestinal fascia and fat) was collected. The intestinal tissue was homogenized according to the manufacturer\u0026rsquo;s instructions (Nanjing Jianjian Biological Co., Ltd., China). The supernatant was stored at -20 ℃. This will be used to determine the activity of intestinal amylase, lipase, and trypsin.\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eAbsorptive function measurement in the intestine\u003c/h2\u003e \u003cp\u003eSerum D-xylose activity and GSH uptake were determined using the methods of Chen et al\u003csup\u003e[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]\u003c/sup\u003e. On day 42, four broilers were selected per replicate. After 12 h of fasting and water deprivation, two broilers were given a 10% D-xylose solution by gavage at a dose of 1 mL/kg, and the other two broilers were given reduced GSH by gavage at a dose of 1 g/kg. 2 mL of blood was collected from a vein in the tip of the wing 1 h later, and 30 \u0026micro;L of 2% heparin anticoagulant was added. The blood was then centrifuged at 3000 rpm for 10 min at 4 ℃ to prepare the serum. The corresponding indexes were determined using D-xylose and GSH kits purchased from Nanjing Jianchang Bioengineering Institute.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eDetermination of apparent digestibility\u003c/h2\u003e \u003cp\u003eFresh manure (100 g) was collected daily during the test period. To fix nitrogen, 10% concentrated sulfuric acid was added, stirred well, and stored at -20 ℃ until analysis. The weight of the feces excreted by each group of broilers was measured and recorded. At the end of the test, the feed and feces of each group were mixed well and dried in an oven to prepare air-dried samples. The samples were ground and passed through a 40 mesh sieve. Then, they were analyzed for dry matter (DM), organic matter (OM), crude protein (CP), and crude ash (Ash) according to the procedures of the AOAC\u003csup\u003e[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]\u003c/sup\u003e. Finally, the apparent digestibility of OM, CP and Ash was calculated.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe study analyzed growth performance, digestive enzyme activity, intestinal absorption capacity, and apparent digestibility of broiler using one-way ANOVA with SPSS 19.0. The results are shown as the mean and standard deviation, with P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 indicating significant differences.\u003c/p\u003e \u003cp\u003eThe raw GC-MS data were preprocessed by Chroma TOF software (v4.34, LECO Inc., USA). Internal standard peaks and false positive peaks (including derivatization reagent peaks, column loss and noise) were removed, and redundancy and peak merging were performed to analyze the samples to obtain 370 metabolites. The normalized response intensities of the sample mass spectrometry peaks were imported into the SIM CA-P\u0026thinsp;+\u0026thinsp;14.0 software package, and the overall distribution among the samples, the stability of the analysis process and the overall differences in metabolic profiles among the groups were analyzed by principal component analysis (PCA) and partial least squares-discriminant analysis (PLS-DA) to identify the differentially abundant metabolites among the groups. In the PLS-DA analysis, variables with a variable importance in projection (VIP) greater than 1 were considered differential variables. The experimental analysis model was prevented from overfitting by a 7-cycle interaction validation analysis and a 200-cycle response ranking test analysis.\u003c/p\u003e \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e \u003ch2\u003eMetabolite identification\u003c/h2\u003e \u003cp\u003eThe metabolites that differed between the TN and CHS groups were screened by a combination of PLS-DA multidimensional analysis and t-test (VIP\u0026thinsp;\u0026gt;\u0026thinsp;1, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The GC-MS workstation software automatically compared the mass-to-charge ratio and abundance of the characteristic ion fragments of each substance detected in this experiment with the standard metabolites in the National Institute of Standards and Technology (NIST) database, and the standard selection criteria required a match of more than 70%.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section3\"\u003e \u003ch2\u003eMetabolic pathway enrichment analysis\u003c/h2\u003e \u003cp\u003eThe ID conversion function of the MBRole database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://csbg.cnb.csic.es/mbrole/\u003c/span\u003e\u003cspan address=\"http://csbg.cnb.csic.es/mbrole/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was used to obtain the substance IDs of the KEGG database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.genome.jp/kegg/pathway.html\u003c/span\u003e\u003cspan address=\"http://www.genome.jp/kegg/pathway.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) for the differential metabolites. The two databases\u0026rsquo; pathway analysis and enrichment functions were combined to identify the significantly different metabolic pathways exhibited in the jejuna of the TN and CHS groups. The metabolic pathway maps generated from the database and the histogram of metabolic pathway enrichment analysis during HS in broiler chickens were downloaded.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Animal Care and Use Committee of the Anhui Science and Technology University approved the animal treatment protocol used in this study (Protocol number 2023005). All experimental procedures were carried out following the guidelines mentioned in the ARRIVE guidelines.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the finding of this study are available from corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the Initiation Program of National Natural Science Foundation of China (No. 31702306), the Key Program of Scientific Research of Higher Education Institutions in Anhui Province (No. 2022AH051620), Major projects supported by Department of Education Anhui Province (No. 2023AH040282), and the Support Program for Excellent Young Talents in Universities and Colleges (No. gxyq2022056).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eY.J. Xiong and S.J. He conceptualized and supervised the study. S.A. Sun and B. Li performed the experiments. J.J. Yuan provided assistance with the animal experiments. Shiang Sun analyzed the data and wrote the paper. S.J. He provided revisions of important content. All authors read and approved the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe author would like to thank all teachers and students who dedicated their time to take part in this study. Also, we are grateful to Dr. Abulaiti, Adili for touching up and revising this paper.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eNajafi, P. et al. Environmental temperature and stocking density effects on acute phase proteins, heat shock protein 70, circulating corticosterone and performance in broiler chickens. \u003cem\u003eInt. J. 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Official methods of analysis.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"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":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"broiler, jejunum, growth performance, gas chromatography-mass spectrometry, digestive enzyme","lastPublishedDoi":"10.21203/rs.3.rs-6220478/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6220478/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eHeat stress (HS) is one of the major influencing factors limiting the development of poultry farming. To investigate the effect of chronic HS on the intestinal digestion and absorption function, we divided 80 broilers to control group (TN group), chronic HS group (CHS group), and then illustrated the effects using growth performance, gas chromatography-mass spectrometry (GC-MS)-based metabolomics, digestive and absorptive capacity, and apparent digestibility. Broilers in the CHS group were exposed to 12 h/day HS (32\u0026thinsp;\u0026plusmn;\u0026thinsp;1 ℃) for 14 consecutive days, and the rest of the time per day was maintained at 24\u0026thinsp;\u0026plusmn;\u0026thinsp;1 ℃, which was the same as that used for the TN group. The jejunum samples were collected at the end of the experiment, and tested for relevant indexes, and the GC-MS technique was applied to obtain two groups of broiler jejunum metabolic profiles. The study showed that HS reduced the average daily feed intake (ADFI), average daily grit (ADG), intestinal digestive enzyme activity, D-xylose and GSH absorption levels, apparent digestibility, and elevated feed conversion ratio (FCR). A total of 370 metabolites in the broiler jejunum were identified, and 43 up-regulated and 22 down-regulated metabolites were screened. Enrichment analysis of metabolic pathways revealed that eight metabolic pathways were significantly altered (mainly related to energy metabolism). These results indicate that changes in cellular metabolism in the jejunum when broilers are subjected to HS, decreased the activity of digestive enzymes in the jejunum, resulting in a decrease in digestibility and consequently affecting growth performance.\u003c/p\u003e","manuscriptTitle":"The effects of chronic heat stress on the growth performance, digestive and absorbtive-related parameters, and jejunal metabolomics in broilers","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-07 08:25:12","doi":"10.21203/rs.3.rs-6220478/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b3af543d-19c9-43e5-8219-1ab4d5f162d0","owner":[],"postedDate":"May 7th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":47301946,"name":"Biological sciences/Zoology"},{"id":47301947,"name":"Earth and environmental sciences/Climate sciences"}],"tags":[],"updatedAt":"2026-03-12T15:24:58+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-07 08:25:12","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6220478","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6220478","identity":"rs-6220478","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}