Effects of tannic acid on the immunity and intestinal health of broiler chickens with Clostridium perfringens-induced necrotic enteritis

Effects of tannic acid on the immunity and intestinal health of broiler chickens with Clostridium perfringens-induced necrotic enteritis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Effects of tannic acid on the immunity and intestinal health of broiler chickens with Clostridium perfringens-induced necrotic enteritis Huiping Xu, Jianyang Fu, Yimeng Luo, Peng Li, Bochen Song, Zengpeng Lv, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2283343/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 04 May, 2023 Read the published version in Journal of Animal Science and Biotechnology → Version 1 posted 5 You are reading this latest preprint version Abstract Background In broiler chickens, necrotic enteritis (NE) infection can reduce production performance. Tannic acid, as a kind of plant extract, has received extensive attention. However, the appropriate dosage of tannic acid in NE of broilers and the improvement effect on intestinal health are not very clear. In this study, we aimed to investigate the effects of different doses of tannic acid on the production performance, immunity, and intestinal health of broilers by constructing an NE model with C. perfringens infection and determining the appropriate dosage of tannic acid with regard to NE. Results Challenged birds showed significant reduction in body weights, villus heights, and the ratio of villus height to crypt depth (V/C) ( P < 0.05 ) and increase in the feed consumption gain ratio, intestinal lesion score, and crypt depth ( P < 0.05 ). NE infection significantly reduced the relative Bacteroides and Ligilactobacillus abundance ( P < 0.05 ) and increased the ratio of Firmicutes/Bacteroides and cecal content of C. perfringens ( P < 0.05 ). Challenged birds fed diets supplemented with tannic acid showed significantly increased mRNA expression of nutrient transport carriers and intestinal barrier genes and growth performance and reduced serum zonulin and endotoxin levels ( P < 0.05 ). Addition of tannic acid to the diet inhibited the inflammatory response by reducing the number of coccidia oocysts in feces and the content of C. perfringens in the cecum. Specifically, tannin acid reduced the serum levels of C reactive protein, myeloperoxidase, and specific IgY and ileal mucosal secretory immunoglobulin A (sIgA) levels in the ileal mucosa compared with those in the NE-infected birds. NE-infected birds fed diets supplemented with tannin acid also showed significantly increased relative Anaerocolumna , Thermoanaerobacterium , and Thermosinus abundance ( P < 0.05 ); their microbial composition and functional predictions were similar to those of the NC group. Conclusions Tannic acid in the diet alleviated NE by enhancing the intestinal barrier and absorption function. The recommended dietary tannic acid additive level is 500–750 mg/kg. Our study findings would be useful in reducing related economic losses in the broiler industry. tannic acid necrotic enteritis immunity intestinal health Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Background In the poultry industry, ensuring that the intestinal tracts of birds are healthy plays an important role in maximizing growth performance. Necrotic enteritis (NE) in broiler chickens, caused by Clostridium perfringens ( C. perfringens ), is one of the most prominent intestinal diseases in modern intensive farming. The destruction of the intestinal mucosal structure in broiler chickens with NE results in poor digestion and absorption of nutrients and reduced feed efficiency, causing huge losses for the industry [ 1 ]. In addition, C. perfringens can enter the human body through the food chain, threatening public health. Due to the limited use of antibiotic growth promoters (AGP) in the poultry industry, the incidence of NE in chickens has been increasing worldwide. Therefore, effective AGP alternatives are urgently required to prevent and control NE in broiler chickens. Tannins are water-soluble polyphenols and secondary metabolites in plants [ 2 ]. They are classified into two types according to their chemical structures—condensed tannins (CTs) and hydrolytic tannins (HTs). HTs are phenolic acid polyesters with a D-glucose core, such as gallic acid and ellagic acid. The relative molecular weight of HTs is usually 500–3000 Da, and they are easily hydrolyzed by acids, alkalis, and enzymes. CTs are composed of flavan-3-ol units connected by carbon-carbon double bonds, also called proanthocyanidins (catechins or epicatechins). CTs are the most prevalent and typical class of plant tannins, with relative molecular masses ranging from 1,900 to 28,000 Da; they are mainly found in legumes, trees, and shrubs [ 3 ]. Tannins have antibacterial, antiparasitic, antiviral, anti-inflammatory, antioxidant, antidiarrheal, and nutrient metabolism-modulating effects [ 4 ]. Dietary supplementation of broilers with tannins has been shown to improve growth performance [ 5 , 6 ] and reduce the abundance of C. perfringens , production of toxins such as alpha toxin [ 7 ], and the adverse effects of C. perfringens and Eimeria tenella in chickens [ 8 , 9 ]. The European Union approved tannins as a novel feed additive for livestock and poultry in 2016. However, it has been shown that the addition of tannins can result in negative effects, including reduction in production performance [ 10 ]. The differences in the results may be related to the tannic acid type or dosage used. Therefore, this study was conducted to investigate the modulating effects of different tannic acid additive levels on the intestinal health of broiler chickens co-infected with coccidia and C. perfringens to aid the development of more tailored tannic acid application strategies. Methods Tannic acid Tannic acid was provided by Hubei Chicheng Technology Development Co., Ltd. (Yichang City, Hubei Province, China). Tannic acid parameters were as follows: tannic acid ≥ 80.0%, moisture ≤ 10.0%, scorch residue ≤ 6.0%, total arsenic ≤ 5 ppm, and heavy metal content (as Pb) ≤ 10 ppm. Experimental Design, Birds, And Diets The experiment was carried out at the poultry experiment base of China Agricultural University (Zhuozhou City, Hebei Province, China). A total of 630 1-day-old Cobb 500 male broilers were weighed and randomly divided into six treatment groups according to the principle of similar body weight. There were seven replicate cages (1.0 × 0.7 × 0.38 m, length × width × height) per treatment and 15 chickens per replicate. They were grouped as follows: (i) negative control group (no tannin acid treatment or NE infection, NC group); (ii) positive control group (NE infection + 0 mg/kg tannin acid, PC group); (iii) PTA1 group (NE infection + 250 mg/kg tannic acid); (iv) PTA2 group (NE infection + 500 mg/kg tannic acid); (v) PTA3 group (NE infection + 750 mg/kg tannic acid); and (vi) PTA4 group (NE infection + 1000 mg/kg tannic acid). Broilers were reared in a room equipped with two-tiered battery cages. All chickens were vaccinated and managed (including light and temperature management) according to routine immunization and management programs of Cobb broilers. Furthermore, all chickens were provided feed and water ad libitum . Diets were formulated according to the Chinese chicken feeding standard (NY/T 33-2004) and were fed in the form of pellets; the nutritional information is summarized in Table 1 . Table 1 Ingredients and nutrient content of the chicken feed used during the trial Starter (day 1–14, %) Grower (day 15–28, %) Finisher (day 29–35, %) Ingredient Corn 54.12 58.00 61.79 Soybean meal 32.45 28.00 25.68 Corn gluten meal 5.00 4.00 2.50 Soybean oil 3.00 4.62 4.50 Flour 0.90 0.90 1.05 Calcium hydrogen phosphate 2.00 1.95 1.85 Stone powder 1.00 1.00 1.07 Sodium chloride 0.30 0.30 0.30 L-lysine hydrochloride, 78% 0.30 0.30 0.36 DL-Methionine, 98% 0.25 0.25 0.19 Threonine 0.10 0.10 0.10 Arginine 0.04 0.04 0.06 Choline chloride, 50% 0.20 0.20 0.20 Broilers - Mineral premix 1 0.20 0.20 0.20 Broilers - Vitamin premix 2 0.03 0.03 0.03 Phytase 10000 0.01 0.01 0.02 Zeolite 0.10 0.10 0.10 Total 100.00 100.00 100.00 Nutrient content 3 Metabolic energy, Mcal/kg 2.99 3.10 3.10 Crude protein, % 22.39 20.12 18.5 Lysine, % 1.29 1.17 1.14 Methionine, % 0.61 0.57 0.48 Cystine, % 0.93 0.86 0.75 Threonine, % 0.92 0.83 0.77 Calcium, % 1.08 1.05 1.03 Available phosphorus, % 0.44 0.42 0.40 1 per kg of trace element premixed feed: copper, 8 g; iron, 40 g; zinc, 55 g; manganese, 60 g; iodine, 750 mg; selenium, 150 mg; and cobalt, 250 mg. 2 per kg of vitamin premix feed: vitamin A, 50 million IU; vitamin D3, 12 million IU; vitamin E, 100,000 IU; vitamin K3, 10 g; vitamin B1, 8 g; vitamin B2, 32 g; vitamin B6, 12 g; vitamin B12, 100 mg; niacin, 150 g; D-pantothenic acid, 46 g; folic acid, 5 g; biotin, 500 mg. 3 Calculated values based on the experimental diet analysis C. perfringens challenge NE was induced in chickens according to a previously described method, with slight modifications [ 11 ]. On day 19, birds in the PC and PTA1-PTA4 groups were orally inoculated with 1 mL of a 25-fold dose of attenuated quadrivalent coccidia vaccine suspension (Foshan Standard Bio-Tech Co. Ltd., Foshan, China). Chickens in the NC group received 1 mL sterile PBS. Except for broilers in the NC group, chickens were orally gavaged with 1 mL C. perfringens type A CVCC52 (3 × 10 8 CFU/mL) per day on days 22–28. Uninfected birds received 1 mL sterile fluid medium. Growth Performance All broilers were weighed at 19, 28, and 35 d of age in replicates after 12 h of fasting. Body weight, feed consumption, and the feed consumption to weight gain ratio (F/G) were calculated at different experimental periods. Sample Collection One chicken with the average body weight was selected from each replicate at 28 and 35 days of age, and blood was collected from the wing vein, followed by intravenous injection of sodium pentobarbital at a dose of 30 mg/kg body weight. After the anesthesia, the chickens were bled through the jugular vein and sacrificed. The middle region (1 cm) of the ileum was excised and immediately fixed in 4% paraformaldehyde for intestinal morphological examination. Approximately 2 g of chyme from the middle ileum was collected aseptically into sterile tubes; the mucosa of the middle ileum was then gently scraped with a slide, collected in sterile tubes, and stored at − 80°C until further analysis. Intestinal Ne Lesion Scoring At 28 days of age, one chicken in each replicate was anesthetized and sacrificed. The intestinal cavity was opened, and chyme was removed to observe the lesions in the intestinal wall. The scoring rules for intestinal lesions described by Dahiya et al. were adopted [ 12 ]; the specific scoring criteria were as follows: 0 = normal intestinal appearance; 0.5 = severe congestion in serous surface and mesentery of the small intestine; 1 = the intestinal wall became thin and brittle, with red silting spots (more than 5); 2 = the intestinal wall appears needle-like necrosis or ulceration, and there is a small amount of gas in the intestinal cavity; 3 = flaky necrosis or ulceration of the intestinal wall, gas-filled intestines and small blood spots, and 1–2 cm long necrotic spots; 4 = diffuse necrosis, significant bleeding, and large intestinal gas. Fecal coccidia oocyst counts and determination of intestinal C. perfringens concentration On days 25–28, the feces of broilers were collected repeatedly, and fecal coccidia oocysts were counted. The feces collected from each replicate were mixed; then, 2 g feces was added to 58 mL saturated salt water, mixed thoroughly with a vortex shaker for 2 min, and sieved through a 40 mesh. The upper suspension was then added to a McMaster counting plate and allowed to stand for 3 min. Oocysts were counted under a light microscope (Leica, LEICA DM750), and the number of oocysts per gram of feces was calculated according to the following formula: oocyst content per gram of feces (opg) = number of oocysts in the counting chamber × 200. Cecal C. perfringens was counted as previously described [ 11 ]. Approximately 0.5 g of each sample was collected in 10 mL sterile plastic tubes, diluted with PBS to an initial 10 − 1 dilution, and serially diluted to prepare 10 − 1 to 10 − 5 dilutions. Each diluted sample of 100 µL was plated on tryptose-sulfite-cycloserine agar (TSC, CM 138; Beijing Land Bridge Technology Co., Ltd.). The number of C. perfringens was determined after anaerobic incubation at 37°C for 24 h. The opg and C. perfringens were then log 10 -transformed and counted. Intestinal Morphological Analyses And Observation The middle ileum was circumcised to a thickness of 5 µm and stained with hematoxylin and eosin. The height of the villi and depth of the crypt of the chicken ileum were measured according to the method described by Frankel et al. [ 13 ]. Briefly, 10 straight and structurally intact intestinal villi were selected from each intestinal section and measured. The mean value of each index was calculated to determine the ratio of villus height to crypt depth (V/C). Determination Of Serum Immunoglobulin, C-reactive Protein, Myeloperoxidase, And Ileal Mucosa Siga Levels The levels of natural serum IgA and IgM antibodies were determined using a chicken IgA enzyme-linked immunosorbent assay (ELISA) kit (Beijing Solaibao Technology Co., Ltd., SEKCN-0018) and chicken IgM ELISA kit (Beijing Solaibao Technology Co., Ltd., SEKCN-0128), respectively. C-reactive protein (CRP) (Shanghai Enzyme Link Biotechnology Co., Ltd., YJ036965) and myeloperoxidase (MPO) kits (Shanghai Enzyme Link Biotechnology Co., Ltd., YJ966521) were used to determine the serum levels of CRP and MPO, respectively. The tissue homogenate was prepared by taking 0.1 g of ileal mucosa at a weight: saline volume ratio of 1:9, and the supernatant was retained for subsequent analysis. The sIgA and total protein contents in the ileal mucosa were determined using a chicken sIgA ELISA kit (Shanghai Yuanmu Biotechnology Co., Ltd., YM-A3724) and a total protein quantification kit (Nanjing Jiancheng Institute of Biological Engineering), respectively. Serum immunoglobulin content was measured in mg/mL, and mucosal sIgA content was measured in µg/mg protein. The above-mentioned indexes were measured in strict accordance with the manufacturer's instructions. The antibody levels of C. perfringens in the serum were detected using an improved ELISA method. The methods referred to those described by Wu et al., with slight alterations[ 11 ]. C. perfringens was cultured to a concentration of 10 8 CFU/mL and centrifuged at 4000 rpm for 10 min; the thalli were collected and cleaned with 0.01 M sterile PBS solution thrice. The last time, the thalli were diluted with PBS and subjected to intermittent ultrasound over 10 times in an ice bath. When the bacterial solution was turbid and consistent, 9 ml PBS solution was added and incubated for more than 2 h. The supernatant was absorbed into another sterilized centrifuge tube. Protein concentration was measured using the total protein quantitative kit (Nanjing Jianguo Institute of Bioengineering) according to the manufacturer's instructions. Approximately 100 µL bacterial lysate of C. perfringens (40 µg/mL) was added to the enzyme-labeled plate and incubated overnight at 4°C. The 96-well plates were coated with 20 µg/mL albumin from bovine serum (BSA) and incubated overnight at 4°C. After washing five times with 200 µL of 1% BSA dissolved in PBS containing 0.05% Tween (PBST), the plates were incubated with the serum (1:50 dilution) at 37°C for 1.5 h. Then, the plate was washed and 100 µL diluted horseradish peroxidase-conjugated goat anti-chicken IgG (1:10,000; A30-104P, Bethyl Laboratories Inc., Montgomery, TX, USA) was added and incubated at 37°C for 30 min. After washing five times with PBST, 100 µL 0.05% tetramethylbenzidine was added to the plates and incubated at 37°C in the dark for 30 min. The reaction was terminated with 2 mol/L sulfuric acid. Readings were taken at 450 nm using an ELISA reader (SepctraMax i3x Platform, Molecular Devices, LLC). Serum Zonulin And Endotoxin (Et) Detection The venous blood of chicken wings was collected using common collection vessels (5 mL), and the serum was collected after centrifugation at 3500 rpm and 4°C for 10 min. Zonulin (Shanghai Fankewei Industrial Co., Ltd., F8139-A) and Endotoxin detection kits (Shanghai Meilian Biotechnology Co., Ltd., ml0122369-J) were then used according to the manufacturer’s instructions. Quantitative Reverse Transcription-pcr To Measure Mrna Expression In The Ileum The ileal tissues were collected, placed in RNase-free freezing tubes, and stored at − 80°C. Tissue samples (100 mg) were placed in a 2 mL centrifuge tube, 1 mL of Trizol (Invitrogen Life Technologies, Carlsbad, USA) extract was added, and total RNA was extracted according to the manufacturer’s instructions. The purity of the extracted RNA was checked using a nucleic acid spectrophotometer (AG 22331, Eppendorf, Hamburg, Germany), and samples with OD 260/OD 280 nm greater than 1.8 were used for subsequent processing. The samples were reverse-transcribed using a cDNA kit (Takara Biotechnology Co. Ltd., Beijing, China). The Applied Biosystems 7500 Fast Real-Time PCR System and SYBR Premix Ex Taq™ kit (Takara Biotechnology Co. Ltd., Beijing, China) were used to performed quantitative reverse transcription-PCR. All procedures were performed according to the manufacturer's instructions. Genes were quantified using β-actin as an internal reference, and the results were statistically analyzed according to the method described by Fu et al. [ 14 ]. The primer sequences for genes are shown in Table 2 . Table 2 Sequences of the oligonucleotide primers used for quantitative reverse transcription-PCR 1 Gene Primer sequences(5ʹ→3ʹ) NCBI serial number β-actin F-CAACACAGTGCTGTCTGGTGGTAC NM_205518.1 R-CTCCTGCTTGCTGATCCACATCTG Occludin F-ACGGCAGCACCTACCTCAA NM_205128.1 R-GGGCGAAGAAGCAGATGAG MUC2 F-TCCCCTGTTGAGGGAGAACTT XM_040673077.1 R-AGTGGTTGTACCTTCGGTGC ZO-1 F-CTTCAGGTGTTTCTCTTCCTCCTC XM_040680632.1 R-CTGTGGTTTCATGGCTGGATC PEPT1 F-TACGCATACTGTCACCATCA NM_204365.2 R-TCCTGAGAACGGACTGTAAT SGLT1 F-GATGTGCGGATACCTGAAGC NM_001293240.1 R-AGGGATGCCAACATGACTGA GLUT2 F-CCGCAGAAGGTGATAGAAGC NM_205129.1 R-ATTGTCCCTGGAGGTGTT I-FABP F-GAAGCAATGGGCGTGAATGTGATG NM_001007923.1 R-TTCGATGTCGATGGTACGGAAGTTG IL-1β F-ACTGGGCATCAAGGGCTA XM_046931582.1 R-GGTAGAAGATGAAGCGGGTC IL-6 F-CGCCCAGAAATCCCTCCTC NM_204628.2 R-AGGCACTGAAACTCCTGGTC TLR2 F-GATTGTGGACAACATCATTGACTC NM_001161650.3 R-AGAGCTGCTTTCAAGTTTTCCC TGF-β F-TCATCACCAGGACAGCGTTA NM_001031045.3 R-TGTGATGGAGCCATTCATGT F: upstream primer; R: downstream primers; ZO-1 : zonula occludens-1; PEPT1 , peptide-transporter 1; SGLT1 , recombinant sodium/glucose cotransporter 1; GLUT2 , recombinant glucose transporter 2; I-FABP : intestinal fatty acid binding protein; IL-1β, interleukin-1β; IL-6, interleukin-6; TLR2, toll like receptor 2; TGF-β, transforming growth factor-β. 1 The primers were synthesized by Shanghai Shenggong Biotechnology Co. Ltd. Ileal Microbiological Analysis The contents of middle ileal samples collected on d 28were used for 16S sequencing analysis, according to the method described by Zhang et al. [ 15 ]. First, fecal microbial DNA was extracted using a DNA extraction kit (QIAamp Fast DNA Stool Mini Kit, Qiagen Company, Dusseldorf, Germany), and then the purity of the DNA was tested using 1% agarose gel electrophoresis. All procedures were performed according to the manufacturer's instructions. Bacterial DNA was amplified using the following V3-V4 primers: 338 F (5ʹ-ACTCCTACGGGAGGCAGCA-3ʹ) and 806r (5ʹ-GGACTACHVGGGTWTCTAAT-3ʹ). In-machine sequencing was performed using HiSeq2500 PE250, and the sequences were analyzed by Beijing Nuohe Zhiyuan Bio-Information Technology Co. Ltd. The Qiime software (Qiime2-2019.7, Nature Biotechnology) was used to generate species abundance tables at different taxonomic levels. Analysis was then performed to identify biomarkers that were statistically different between groups and α, β diversity. Venn plots and Principal Co-ordinates Analysis (PCoA) plots were plotted using the cloud platform (Beijing Nuohe Zhiyuan Bio-Information Technology Co. Ltd.), and analysis of similarities (ANOSIM) and PICRUSt analyses were performed. Statistical analysis The data were analyzed using SPSS 26.0 (SPSS, Inc., Chicago, IL, USA). The effects of NE infection (NC vs. PC) were evaluated by the unpaired t-test. Challenged groups (PC, PTA1, PTA2, PTA3, and PTA4) were compared using one-way analysis of variance (ANOVA) and Duncan's multiple comparisons. The linear and quadratic effects of the different doses of tannic acid were tested by contrast. The results are shown as the mean ± SEM. Unpaired Student's t-test was used to compare the relative abundances of the phylum, genera, species and the Firmicutes/Bacteroidetes ratio between the two groups were performed. A P < 0.05 was considered statistically significant and a 0.05 ≤ P ≤ 0.1 was classified as a tendency. Using Pearson's correlation coefficient to evaluate the correlation analysis of the 28-day-old broiler growth performance, immunity, intestinal barrier, and other indicators of microbiota. Results Growth performance The growth performance data for the broiler chickens from the control and treatment groups used in this study are shown in Table 3 . The effect of tannic acid addition to the chicken diet showed a quadratic relationship with body weight and F/G. Additionally, it significantly increased the body weight at day 19 in the PTA1-PTA4 groups and markedly reduced the F/G during days 1–19 in the PTA2 and PTA3 groups ( P < 0.05 ), whereas the PC group showed a significant decrease in growth performance at day 28 and a significant increase in the F/G during days 1–28 and days 1–35( P < 0.05 ). On day 28, the PTA3 group showed a marked improvement in body weight and decrease in the F/G ( P < 0.05 ). On day 35, PTA2-PTA4 groups showed a significant increase in body weight, and the F/G ratio was significantly lower in the PTA2 and PTA3 groups during days 1–35( P < 0.05 ). Table 3 Effects of tannic acid on the production performance of broilers with NE NC 1 PC PTA1 PTA2 PTA3 PTA4 P-value 2 Linear 3 Quadratic 3 Body weight (g) d 19 745.1 ± 6.0 759.8 ± 7.5 c 794.3 ± 6.4 ab 801.2 ± 6.2 ab 815.1 ± 11.3 a 791.1 ± 5.6 b < 0.001 0.002 0.001 d 28 1486.6 ± 12.4 *** 1396.2 ± 8.8 b 1423.4 ± 7.9 ab 1430.7 ± 15.0 ab 1460.1 ± 19.5 a 1414.8 ± 15.2 b 0.043 0.105 0.025 d 35 2011.7 ± 29.7 1970.3 ± 12.0 b 2010.5 ± 25.0 ab 2036.2 ± 24.2 a 2050.3 ± 13.8 a 2043.1 ± 16.0 a 0.036 0.004 0.145 F/G d 1–19 1.330 ± 0.013 1.340 ± 0.016 a 1.310 ± 0.008 ab 1.270 ± 0.024 b 1.280 ± 0.018 b 1.300 ± 0.016 ab 0.039 0.055 0.012 d 1–28 1.390 ± 0.004 *** 1.440 ± 0.008 a 1.410 ± 0.007 b 1.420 ± 0.005 ab 1.410 ± 0.012 b 1.430 ± 0.005 a 0.017 0.612 0.009 d 1–35 1.530 ± 0.011 ** 1.570 ± 0.002 a 1.540 ± 0.010 ab 1.530 ± 0.010 b 1.530 ± 0.010 b 1.540 ± 0.004 ab 0.014 0.017 0.007 1 NC versus PC (unpaired t test): * 0.05 < P < 0.10 , ** P < 0.05 , *** P < 0.01 . 2 Overall P values obtained from ANOVA; 3 P values obtained using contrast trend analysis; different lower case letters in the same column indicate significant differences ( P < 0.05 ), data are the mean ± SEM, n = 7. NC: no tannin acid treatment or NE infection; PC: NE infection + 0 mg/kg tannic acid; PTA1: NE infection + 250 mg/kg tannic acid; PTA2: NE infection + 500 mg/kg tannic acid; PTA3: NE infection + 750 mg/kg tannic acid; PTA4: NE infection + 1000 mg/kg tannic acid. Intestinal morphology, lesion scores, and determination of cecal C. perfringens concentration The results of the intestinal lesion scoring are shown in Table 4 . While only a few chickens in the NC group showed slight intestinal congestion, all the chickens in the PC group showed gross macroscopic lesions. These included the thinning of the intestinal wall, flatulence, and localized bleeding. Addition of tannic acid improved the intestinal lesions, with a significant reduction in the ileal lesion score on day 28 in the PTA2 group ( P < 0.05 ). In this study (Table 4 ), NE infection significantly increased the number of C. perfringens in the cecums of broilers ( P < 0.05 ). Addition of tannic acid (500–1000 mg/kg) significantly reduced the number of C. perfringens in the cecums of broilers with NE ( P < 0.05 ). Table 4 Intestinal lesion scores and cecum C. perfringens concentrations in broilers with NE on 28 days NC 1 PC PTA1 PTA2 PTA3 PTA4 P-value 2 Linear 3 Quadratic 3 Intestinal lesion on d 28 Duodenum 0.36 ± 0.18 0.50 ± 0.19 0.57 ± 0.17 0.36 ± 0.18 0.43 ± 0.13 0.10 ± 0.07 0.267 0.064 0.383 Jejunum 0.21 ± 0.15 *** 0.86 ± 0.09 0.86 ± 0.09 0.65 ± 0.09 0.71 ± 0.10 0.50 ± 0.15 0.134 0.018 0.693 Ileum 0.21 ± 0.15 0.57 ± 0.17 ab 0.71 ± 0.15 a 0.14 ± 0.07 c 0.36 ± 0.09 abc 0.29 ± 0.10 bc 0.019 0.023 0.443 Cecum C. perfringens concentration d 28 2.75 ± 0.87 ** 6.00 ± 0.10 a 5.44 ± 0.34 ab 5.37 ± 0.15 b 5.33 ± 0.10 b 5.17 ± 0.18 b 0.053 0.007 0.262 1 NC versus PC (unpaired t test): * 0.05 < P < 0.10 , ** P < 0.05 , *** P < 0.01 . 2 Overall P values obtained from ANOVA; 3 P values obtained using contrast trend analysis; different lower case letters in the same column indicate significant differences ( P < 0.05 ), data are the mean ± SEM, n = 7. NC: no tannin acid treatment or NE infection; PC: NE infection + 0 mg/kg tannic acid; PTA1: NE infection + 250 mg/kg tannic acid; PTA2: NE infection + 500 mg/kg tannic acid; PTA3: NE infection + 750 mg/kg tannic acid; PTA4: NE infection + 1000 mg/kg tannic acid. Broilers in the NC group showed normal intestinal appearance, while those in the PC group showed obvious pathological changes in the villi, which were severely damaged, and the normal structure was destroyed. Intestinal lesions were alleviated by the addition of tannic acid to the feed (Fig. 1 ). The ileal villus height and V/C value significantly decreased, and the ileal crypt depth increased in the PC group on day 28 ( P < 0.05 ; TAble 5 ). On day 28, the ileal villus height was markedly increased in the PTA2–PTA4 groups, and the ileal crypt depth decreased in the PTA1, PTA3 and PTA4 groups( P < 0.05 ). In addition, the V/C value significantly increased in the PTA1–PTA4 groups on day 28 ( P < 0.05 ). Seven days after the infection, the ileal crypt depth was still markedly increased and V/C decreased in the PC group ( P < 0.05 ; Table 5 ). The ileal V/C values of the PTA1–PTA4 groups were significantly increased ( P < 0.05 ), but neither NE nor tannic acid treatment had any effect on the ileal villus height on day 35. Table 5 Effects of tannic acid on the morphology of the ileal midsection of broiler chickens with NE NC 1 PC PTA1 PTA2 PTA3 PTA4 P-value 2 Linear 3 Quadratic 3 d 28 Vill height µm 725.53 ± 21.88 *** 627.54 ± 13.31 b 661.86 ± 27.71 b 754.23 ± 28.68 a 769.97 ± 17.58 a 753.26 ± 23.13 a < 0.001 < 0.001 0.045 Crypt depth µm 108.14 ± 3.55 ** 123.09 ± 5.59 a 107.23 ± 4.13 b 113.97 ± 4.86 ab 106.97 ± 2.87 b 109.87 ± 2.67 b 0.059 0.052 0.139 V/C 6.72 ± 0.17 *** 5.15 ± 0.24 d 6.18 ± 0.15 c 6.64 ± 0.20 bc 7.21 ± 0.14 a 6.86 ± 0.14 ab < 0.001 < 0.001 < 0.001 d 35 Vill height µm 768.86 ± 44.27 738.36 ± 56.62 753.24 ± 31.83 739.62 ± 28.06 711.84 ± 15.71 734.47 ± 28.10 0.944 0.658 0.992 Crypt depth µm 108.26 ± 4.29 ** 130.62 ± 7.04 a 110.8 ± 2.86 b 102.9 ± 5.17 b 99.31 ± 2.95 b 104.97 ± 2.92 b < 0.001 < 0.001 0.003 V/C 7.08 ± 0.22 *** 5.67 ± 0.35 b 6.79 ± 0.17 a 7.24 ± 0.23 a 7.20 ± 0.24 a 7.00 ± 0.20 a < 0.001 < 0.001 0.002 1 NC versus PC (unpaired t test): * 0.05 < P < 0.10 , ** P < 0.05 , *** P < 0.01 . 2 Overall P values obtained from ANOVA; 3 P values obtained using contrast trend analysis; different lower case letters in the same column indicate significant differences ( P < 0.05 ), data are the mean ± SEM, n = 7. NC: no tannin acid treatment or NE infection; PC: NE infection + 0 mg/kg tannic acid; PTA1: NE infection + 250 mg/kg tannic acid; PTA2: NE infection + 500 mg/kg tannic acid; PTA3: NE infection + 750 mg/kg tannic acid; PTA4: NE infection + 1000 mg/kg tannic acid. Fecal Coccidia Oocyst Counts The coccidia oocyst counts are shown in Table 6 . The coccidia oocysts were not observed in the NC group (Fig. 2 ). Addition of tannic acid to the diet linearly reduced the number of fecal coccidia oocysts ( P < 0.05 ), and the numbers were significantly lower in the TA2 group at d 26 ( P < 0.05 ), TA3 group at d 26–28 ( P < 0.05 ), and TA4 group at d 25–28 ( P < 0.05 ) than those in the PC group. Table 6 Effects of tannic acid on coccidia oocysts number in feces of broilers with NE PC PTA1 PTA2 PTA3 PTA4 P-value 1 Linear 2 Quadratic 2 d 25 4.69 ± 0.03 a 4.6 ± 0.06 ab 4.59 ± 0.03 ab 4.59 ± 0.06 ab 4.49 ± 0.04 b 0.064 0.008 0.954 d 26 4.78 ± 0.04 a 4.75 ± 0.02 a 4.66 ± 0.02 b 4.61 ± 0.03 b 4.61 ± 0.01 b < 0.001 < 0.001 0.370 d 27 4.63 ± 0.03 a 4.54 ± 0.02 ab 4.56 ± 0.03 ab 4.49 ± 0.03 bc 4.43 ± 0.04 c 0.001 < 0.001 0.747 d 28 4.52 ± 0.04 a 4.50 ± 0.04 ab 4.43 ± 0.02 abc 4.41 ± 0.03 b 4.39 ± 0.03 c 0.027 0.002 0.789 1 Overall P-values obtained from ANOVA; 2 P-values obtained using contrast trend analysis; different lower-case letters in the same column indicate significant differences ( P < 0.05 ). Data are presented as mean ± SEM; n = 7. NC: no tannin acid treatment or NE infection; PC: NE infection + 0 mg/kg tannic acid; PTA1: NE infection + 250 mg/kg tannic acid; PTA2: NE infection + 500 mg/kg tannic acid; PTA3: NE infection + 750 mg/kg tannic acid; PTA4: NE infection + 1000 mg/kg tannic acid. Immune And Intestinal Barrier Function The changes in the levels of immune indicators in the serum and intestinal mucosa are shown in Table 7 . The results showed that on day 28, the levels of IgM and C. perfringens -specific antibodies IgY, CRP, and MPO in the serum and sIgA in the ileal mucosa were significantly higher in the PC group than in the NC group ( P < 0.05 ). Contrastingly, compared with that in the PC group, the addition of tannic acid to the feed significantly reduced the sIgA content in the ileal mucosa on day 28 ( P < 0.05 ). The levels of C. perfringens -specific antibodies IgY awas significantly lower in the PTA3 group than in the PC group on day 28 ( P < 0.05 ), and the levels of C. perfringens -specific antibodies IgY and MPO were remarkably lower in the PTA4 group than in the PC group on day 28 ( P < 0.05 ). On day 35, the levels of CRP and MPO in the serum of the PC group were markedly higher than those in the NC group ( P < 0.05 ). Addition of tannic acid had no significant effect on the levels of immunoglobulins, C. perfringens -specific antibodies IgY in the serum, and sIgA in the ileal mucosa on day 35; however, the serum levels of CRP and MPO were significantly reduced ( P < 0.05 ). Table 7 Effects of tannic acid on the serum immune indices of chickens with NE NC 1 PC PTA1 PTA2 PTA3 PTA4 P-value 2 Linear 3 Quadratic 3 d 28 IgA (mg/mL) 0.28 ± 0.04 0.34 ± 0.05 0.29 ± 0.03 0.3 ± 0.02 0.33 ± 0.03 0.28 ± 0.01 0.537 0.305 0.851 IgM (mg/mL) 0.08 ± 0.01 *** 0.16 ± 0.02 0.15 ± 0.01 0.18 ± 0.03 0.21 ± 0.02 0.15 ± 0.01 0.234 0.807 0.163 Serum-specific IgY antibody(OD 450 nm) 2.79 ± 0.05 ** 2.92 ± 0.03 a 2.87 ± 0.02 ab 2.85 ± 0.05 ab 2.79 ± 0.04 b 2.78 ± 0.03 b 0.044 0.003 0.637 CRP (µg/mL) 6.53 ± 0.76 *** 13.97 ± 1.28 12.64 ± 1.20 12.78 ± 0.70 11.50 ± 0.63 10.77 ± 1.04 0.224 0.024 0.952 MPO (pg/mL) 697.06 ± 65.48 *** 1291.64 ± 99.90 a 1305.64 ± 109.51 a 1132.20 ± 86.60 ab 1008.71 ± 103.05 ab 968.49 ± 76.27 b 0.053 0.004 0.872 sIgA (µg/mg prot) 30.42 ± 3.58 ** 52.52 ± 8.21 a 34.88 ± 3.78 b 35.13 ± 4.43 b 28.96 ± 3.19 b 28.07 ± 2.76 b 0.009 0.001 0.149 d 35 IgA (mg/mL) 0.38 ± 0.04 0.37 ± 0.05 0.36 ± 0.08 0.36 ± 0.08 0.35 ± 0.04 0.31 ± 0.03 0.956 0.497 0.737 IgM (mg/mL) 0.08 ± 0.01 0.08 ± 0.02 0.07 ± 0.02 0.08 ± 0.04 0.06 ± 0.003 0.049 ± 0.05 0.881 0.332 0.724 Serum-specific IgY antibody (OD 450 nm) 2.87 ± 0.04 2.88 ± 0.03 2.92 ± 0.02 2.88 ± 0.03 2.88 ± 0.02 2.90 ± 0.03 0.841 0.964 0.993 CRP (µg/mL) 9.02 ± 0.65 *** 14.00 ± 0.36 a 11.27 ± 0.66 b 10.16 ± 0.70 bc 9.48 ± 0.87 bc 8.59 ± 0.80 c < 0.001 < 0.001 0.128 MPO (pg/mL) 765.28 ± 50.28 *** 1570.16 ± 46.42 a 1009.84 ± 60.30 b 973.55 ± 65.72 b 871.87 ± 53.66 b 852.16 ± 57.78 b < 0.001 < 0.001 < 0.001 sIgA (µg/mg prot) 33.02 ± 4.45 38.44 ± 4.10 36.82 ± 2.67 35.72 ± 4.90 31.56 ± 3.13 29.74 ± 1.27 0.361 0.046 0.792 1 NC versus PC (unpaired t test): * 0.05 < P < 0.10 , ** P < 0.05 , *** P < 0.01 . 2 Overall P values obtained from ANOVA; 3 P values obtained using contrast trend analysis; different lower case letters in the same column indicate significant differences ( P < 0.05 ), data are the mean ± SEM, n = 7. NC: no tannin acid treatment or NE infection; PC: NE infection + 0 mg/kg tannic acid; PTA1: NE infection + 250 mg/kg tannic acid; PTA2: NE infection + 500 mg/kg tannic acid; PTA3: NE infection + 750 mg/kg tannic acid; PTA4: NE infection + 1000 mg/kg tannic acid. Serum zonulin and ET levels showed consistent results (Table 8 ). NE significantly increased serum zonulin and ET levels ( P < 0.01 ), while the addition of tannic acid significantly reduced serum zonulin and ET levels ( P < 0.05 ). A combined analysis of the zonulin and ET indices showed that the addition of tannic acid improved intestinal barrier function and reduced intestinal permeability. Table 8 Effects of tannic acid on serum zonulin and ET in broiler chickens with NE NC 1 PC PTA1 PTA2 PTA3 PTA4 P-value 2 Linear 3 Quadratic 3 d 28 Zonulin ng/L 1173.49 ± 26.79 *** 1618.44 ± 23.52 a 1470.91 ± 15.53 b 1342.39 ± 23.42 c 1308.53 ± 27.41 c 1307.13 ± 23.76 c < 0.001 < 0.001 < 0.001 ET EU/L 89.04 ± 3.15 *** 120.41 ± 2.86 a 105.59 ± 5.22 b 100.74 ± 3.54 b 96.05 ± 3.19 b 94.51 ± 2.92 b < 0.001 < 0.001 0.060 d 35 Zonulin ng/L 1115.82 ± 17.94 *** 1570.35 ± 25.15 a 1446.91 ± 21.36 b 1378.46 ± 15.37 bc 1316.14 ± 27.27 cd 1265.03 ± 27.26 d < 0.001 < 0.001 0.100 ET EU/L 86.11 ± 3.14 *** 115.91 ± 3.89 a 99.22 ± 3.85 b 102.75 ± 2.93 b 81.83 ± 2.20 c 94.44 ± 4.19 b < 0.001 < 0.001 0.014 1 NC versus PC (unpaired t test): * 0.05 < P < 0.10 , ** P < 0.05 , *** P < 0.01 . 2 Overall P values obtained from ANOVA; 3 P values obtained using contrast trend analysis; different lower case letters in the same column indicate significant differences ( P < 0.05 ), data are the mean ± SEM, n = 7. NC: no tannin acid treatment or NE infection; PC: NE infection + 0 mg/kg tannic acid; PTA1: NE infection + 250 mg/kg tannic acid; PTA2: NE infection + 500 mg/kg tannic acid; PTA3: NE infection + 750 mg/kg tannic acid; PTA4: NE infection + 1000 mg/kg tannic acid. mRNA expression of intestinal barrier gene , MUC2 , and nutrient transport carriers The results of intestinal barrier gene and MUC2 expression on day 28 (Fig. 3 a) showed that the expression of ZO-1 in the PC group was significantly reduced ( P < 0.05 ) and the mRNA expression of MUC2 and Occludin was significantly reduced ( P < 0.01 ) compared to that in the NC group. The addition of tannic acid to the feed significantly increased the expression of Occludin compared with that in the PC group ( P < 0.05 ); MUC2 expression was markedly increased in the PTA1 and PTA2 groups ( P < 0.05 ), and ZO-1 expression was significantly increased in the PTA3 group ( P < 0.05 ). The results for intestinal barrier genes and MUC2 mRNA expression on day 35 (Fig. 3 b) showed that MUC2 expression in the PC group was still significantly lower than that in the NC group ( P < 0.05 ). The expression of MUC2 was remarkably higher in the PTA2 group than in the PC group ( P < 0.05 ). Regarding the ileal nutrient transport carriers on day 28 (Fig. 3 c), the mRNA expression of intestinal I-FABP and PEPT1 was significantly lower in the PC group ( P < 0.01 ) and that of SGLT1 was lower in the PC group ( P < 0.05 ) than in the NC group. Compared with that in the PC group, the addition of tannic acid to the feed significantly increased the expression of SGLT1 in the PTA1-PTA4 groups( P < 0.05 ), I-FABP in the PTA1 group ( P < 0.05 ) and PEPT1 in the PTA2 group ( P < 0.05 ). The ileal nutrient transport carrier results on day 35 showed (Fig. 3 d) that the expression of PEPT1 in the PC group was remarkably lower than that in the NC group. The relative mRNA expression of SGLT1 was significantly higher in the PTA2 group than in the PC group on day 35 ( P 0.05 ). We found that the expression levels of IL-1β , TLR2 , and TGF-β under NE conditions were significantly increased on d 28 (Fig. 3 e) (P < 0.05) . After tannic acid supplementation, TLR2 mRNA expression levels in the PTA1 group were significantly increased compared with those in the PC group (P < 0.05 ). Tannic acid supplementation had no significant effect on cytokine expression levels. On d 35 (Fig. 3 f), compared with those in the NC group, IL-1β mRNA expression levels were significantly increased in the PC group (P < 0.05) , but tannic acid supplementation had no significant effect on cytokine expression levels. Ileal Microbiological Analysis To study the effect of tannic acid on the microbiota of the ileal midsection of broilers infected with NE, the changes in the ileal microorganisms were compared among the NC, PC, and PTA3 groups. There were 400 unique OTUs in the NC group, 288 in the NE group, and 183 in the PTA3 group (Fig. 4 a). Alpha diversity was measured using the ACE, Chao1, Simpson, and Shannon indices. Figure 4 b shows that NE did not significantly affect the microbial alpha diversity in mid-ileum microorganisms. The ACE and chao1 indices were significantly reduced after the addition of tannic acid ( P < 0.01 ), but there were no significant differences in the Shannon or Simpson indices. The principal component analysis of ileal microorganisms is shown in Fig. 4 c. As displayed in Table 9 , the results showed that there were remarkable differences in the composition and structure of the ileal microbiota between these groups ( P < 0.01 ). Table 9 ANOSIM for flora composition among the three treatments Treatment R value P-value NC-PC 0.799 0.003 PC-PTA3 0.238 0.008 R values range from − 1 to 1. Differences between groups were significant for R values > 0, and differences within groups were greater than those between groups for R values < 0. P < 0.05 indicates significant differences. At the phylum level (Fig. 5 a, b), NE infection significantly reduced the relative abundance of Anaerobacteria and increased the ratio of Firmicutes/Bacteroides ( P < 0.05 ). At the genus level (Fig. 5 c, d), Ligilactobacillus abundance was significantly reduced in the PC group ( P < 0.01 ), and addition of tannic acid significantly increased the relative abundance of Anaerocolumna , Thermoanaerobacterium , and Thermosinus ( P < 0.01 ). The effects of challenge on the intestinal Lactobacillus -associated flora were more pronounced at the species level (Fig. 5 e, f), with the abundance of Lactobacillus salivarius flora in the PC group significantly reduced ( P < 0.01 ); addition of tannic acid significantly increased the relative abundance of Thermosinus carboxydivorans ( P < 0.01 ). Prediction Of Bacterial Flora Function PICRUSt analysis was performed online using the microbial genome information from the Kyoto Encyclopedia of Genes and Genomes (KEGG) (Fig. 6 a, b). This analysis allowed for a comparison of the differences in functional profiles among the groups and revealed significantly different functional clustering of the gene pathways among the NC, PC, and PTA3 groups. The results showed that eight gene pathways differed among the three groups at level 1, mainly involving metabolic and disease-related gene pathways. The gut microbes of the NC group were mainly enriched in biological systems, cellular processes, and metabolic pathways; those of the PC group were mainly enriched in disease and genetic information processing pathways; while those of the PTA3 group were mainly enriched in environmental information processing signaling pathways. The results showed 35 different gene pathways at level 2. Among them, NE infection significantly downregulated the metabolic pathways of carbohydrates, lipids, and amino acids, and the addition of tannic acid upregulated the pathways related to the metabolism of these nutrients to some extent. Furthermore, we found that the metabolic pathways related to energy, nucleotides, cell growth and death, replication and repair, cancer, disease, and disease immunity were significantly elevated in the intestinal flora after challenge and that the addition of tannic acid downregulated these metabolic pathways to a certain extent. Indicator Correlation Analysis To explore the relationship between the effect of tannic acid on the gut microorganisms of broiler chickens with NE and the indices for growth performance and immunity, Pearson correlation analysis was conducted based on the above-shown microbial data. Ligilactobacillus showed a highly significant negative correlation ( P < 0.01 , Fig. 7 ) with IgM levels in the serum and a significant negative correlation ( P < 0.05 ) with CRP levels; Lactobacillus salivarius showed a highly significant positive correlation with body weight ( P < 0.01 ) and a highly significant negative correlation with the F/G, ET, MPO, and zonulin levels ( P < 0.01 ). The Firmicutes/Bacteroides ratio exhibited a negative correlation with body weight ( P < 0.05 ) and a significant positive correlation with serum ET levels and C. perfringens -specific antibodies ( P < 0.05 ); Thermoanaerobacterium showed a significant positive correlation with serum IgM levels ( P < 0.05 ); Thermosinus carboxydivorans and Thermosinus were significantly and positively correlated with GLUT2 expression levels ( P < 0.05 . The number of C. perfringens in the cecum was significantly positively correlated with CRP, ET, MPO, and Zonulin levels and the F/G ratio ( P < 0.01 ) and significantly positively correlated with IgY, sIgA, IgM, IL-1β and intestinal lesion score ( P < 0.05 ). In addition, the number of C. perfringens was negatively correlated with body weight ( P < 0.01 ). Discussion In this study, we investigated the modulating effects of different tannic acid additive levels on the intestinal health of broiler chickens co-infected with coccidia and C. perfringens . NE infection was found to significantly reduce the body weight and F/G of broilers, and their small intestines showed remarkable pathological changes, such as intestinal congestion, red bruises, thinning of the intestinal wall, and intestinal distension. The intestinal section analysis further showed damage to the villi structure in the PC group, which indicated the successful establishment of the necrotizing enteritis model and was consistent with the results of previous studies [ 15 , 16 ]. Regarding the intestinal barrier genes as well as nutrient transporters, most mRNA expression in the PC group showed negative effects. Previous studies have reported that NE reduces growth performance and elevated serum ET [ 15 ], which is consistent with our findings. In addition, the serum levels of zonulin levels were elevated, further indicating the successful establishment of NE model. Tannic acid addition to the feed had beneficial effects on the broilers with NE and resulted in reduced intestinal lesion scores, increased villus height, and improved intestinal morphology. Effects of tannic acid addition to the diet on the growth performance of broiler chickens with NE Results showed that tannic acid in the feed could ameliorate the growth performance degradation caused by NE, which is consistent with the results of previous studies. Yang et al. observed better F/G ratios when broilers were fed 7.5 to 15 mg/kg proanthocyanidins [ 17 ]. Wang et al. investigated the effects of addition of 5–80 mg/kg grape seed extracts to the coccidia-challenged diet and found that 10–20 mg/kg significantly improved the growth performance [ 18 ]; these results were consistent with those of this study. However, some studies have shown that adding 20 g/kg tannic acid significantly reduces body weight gain and the feed conversion efficiency in broilers [ 10 ].Jamroz et al. added 0.025–0.1% sweet chestnut tannin to the diet and found that < 0.05% had no effect on growth performance, whereas 0.1% significantly reduced growth performance [ 19 ]. These findings suggest that the biological effects of tannins are highly dose-dependent, and the inconsistent effects of tannins in the literature may be due to differences in their source, dose, extraction process, encapsulation, and basal diets. In the present study, the growth performance showed a quadratic curve that increased with the addition of tannic acid; 500–750 mg/kg tannic acid was found to be the optimal concentration for improving the growth performance. The reason dietary tannic acid improves the growth performance of NE may be that it enhances the intestinal barrier, improves intestinal morphology, balances microbiota, and improves nutrient absorption capacity. Effects Of Tannic Acid On The Intestinal Morphological Structures Of Broiler Chickens With Ne Infection A normal villus morphological structure is a prerequisite for the intestine to perform its absorption function. An increase in villus height may lead to enhanced nutrient absorption, and a lower crypt depth indicates a decrease in the metabolic cost of intestinal epithelial renewal. Our findings suggested that NE significantly increased crypt depth, indicating that there was faster tissue renewal for the villi, as needed in response to the inflammation caused by pathogens or their toxins, which is consistent with previous findings [ 11 , 20 ]. The addition of tannic acid significantly reduced the increase in the depth of intestinal crypts caused by NE and improved the V/C ratio, demonstrating the beneficial effects of tannic acid on the intestinal morphology. The V/C showed a quadratic curve change with the addition of tannic acid, which is consistent with our results regarding the growth performance, as 500–1000 mg/kg tannic acid was highly effective at ameliorating the intestinal morphology. Effects of tannic acid on the fecal coccidia oocyst excretion in broiler chickens with NE infection Examining coccidia oocyst shedding is an effective method to determine the level of coccidia infection [ 21 ]. The addition of tannic acid reduced the number of coccidia oocysts shed compared to that in the PC group, which is consistent with the results of previous reports [ 18 , 22 , 23 ]. The reduction in the number of oocysts in the feces indicated an increase in coccidia resistance in broilers. In the present study, the amount of excreted coccidia oocysts decreased linearly with an increase in tannic acid concentration. The decrease in the number of coccidia oocysts after tannic acid addition could be attributed to the modulation of intestinal microorganisms by tannic acid and its own antiparasitic biological properties [ 24 , 25 ]. Taken together, tannic acid supplementation reduced coccidia proliferation, significantly reduced the number of coccidia oocysts excreted in feces, accelerated coccidia clearance, and improved intestinal health. Effect Of Tannic Acid On The Immune Indexes In Broiler Chickens With Ne In this study, NE infection resulted in impaired intestinal morphology and elevated lesion scores, whereas the addition of tannic acid to the feed alleviated these changes. This process was accompanied by changes in the serum levels of IgM, MPO, CRP and C. perfringens -specific antibodies in broilers. IgM is a circulating antibody secreted by B cells that first responds to initial encounters with foreign antigens; however, IgM concentrations in the blood rapidly decline due to clearance, which is consistent with our study findings, as IgM levels were found to be elevated during the first days of infection with NE and did not change significantly seven days after the infection. CRP is an acute temporal protein that activates complement and enhances phagocytosis, and MPO is mainly found in myeloid cells; both of these are markers of the inflammatory response. In this study, the increased MPO and CRP activities in the serum of the PC group indicated that NE infection could activate immune cells in the blood and promote inflammation. The addition of tannic acid to the feed reduced the inflammatory response caused by NE and the concentration of C. perfringens -specific antibodies in the serum, which may be related to the anti-inflammatory and proliferation inhibitory properties of tannic acid [ 4 , 7 ]. The results of this experimental study showed that NE infection exerted long-term effects on broiler chickens, including reduced intestinal barrier function seven days after infection, continued high levels of MPO and CRP in the serum, and inferior growth performance compared to those in the NC group. In innate immunity, the intestinal mucosa is considered the first line of defense against pathogen infection, and mucosal immunity plays an important role in NE. The results of the present study showed that NE infection significantly damaged the ileum and stimulated the expression of sIgA in the intestinal mucosa, which is consistent with the results of a previous study [ 11 ]. We found that tannic acid reduced the sIgA secretion in the ileal mucosa induced by NE, which might be because tannic acid significantly reduced the number of coccidian merozoites and the colonization of C. perfringens . In addition, we observed that TLR2 was activated and inflammatory factor expression was increased in NE, consistent with previous studies [ 26 , 27 ]. We found that tannic acid addition reduced the number of C. perfringens and coccidia in the intestinal tract by stimulating TLR2 and thus activating the immune system, on the one hand, and by inhibiting the number of C. perfringens and coccidia, on the other hand, thus reducing the inflammatory response. Effects of tannic acid addition to the feed on mRNA expression of the gut barrier genes and nutrient transport carriers in chickens with NE The intestine plays key roles in defense against pathogens, host nutrient digestion, and absorption [ 28 ]. Numerous studies have shown that the mRNA expression of tight junctions and nutrient transport carriers decreases and nutrient digestion and absorption are diminished during intestinal inflammation [ 29 – 32 ]. Intestinal inflammation results in a reduction in mucin synthesis and the number of cupped cells, increasing the chances of further intestinal infection and bacterial translocation. In this study, NE led to a reduction in the mRNA expression of Occludin , ZO-1 , and MUC2 , causing an increase in intestinal permeability. However, the mRNA expression of intestinal tight junctions and MUC2 was increased with the addition of tannic acid compared to that in the PC group, indicating the positive effects of tannic acid in protection of chickens from NE. Zonulin is the only known regulator of tight intercellular junctions and is involved in the regulation of intestinal barrier function. Studies have also shown that zonulin levels in the serum are remarkably elevated in patients with irritable bowel syndrome or Crohn’s disease [ 33 ]. However, the changes in the sensitivity to zonulin in poultry intestinal diseases require further investigation. In the present study, NE led to a significant increase in zonulin levels in the serum, indicating that the intestinal barrier permeability was increased. ET is a gram-negative bacterial cell wall component, and serum ET levels are elevated in broiler chickens suffering from NE [ 34 , 35 ], which was also confirmed in our study. The addition of tannic acid to the feed increased the mRNA expression of Occludin , ZO-1 , and MUC2 and improved the integrity of the intestinal barrier, thereby reducing zonulin and ET levels. Nevertheless, the upregulation of intestinal tight junction-related pathways by tannins requires further investigation. I-FABP is a cytoplasmic protein located in mature cells at the top of intestinal villi, and it plays an important role in fatty acid uptake and metabolism. We observed that the mRNA expression of I-FABP was reduced in broiler chickens infected with NE, which was consistent with the results of a previous study [ 36 ]. However, it has also been shown that the mRNA expression of I-FABP does not change significantly in the dexamethasone-induced intestinal disorder model or infection with Campylobacter jejuni [ 37 , 38 ], which might be due to the different disease models and severity. In addition, we observed a decrease in the mRNA expression of PEPT1 and SGLT1 in broiler chickens infected with NE, which is consistent with the results of a previous study [ 39 ]. Addition of tannic acid improved the mRNA expression of nutrient transporter carriers and reduced the negative effects of NE on nutrient absorption in chickens. A comprehensive analysis revealed that growth performance, intestinal villus height, crypt depth, V/C, and nutrient transporter expression showed relatively consistent quadratic curve changes after tannic acid was added to the diet. The reason for this is that lower doses of tannins can reduce gastrointestinal peristalsis, thereby increasing the residence time of chyme in the gastrointestinal tract and improving nutrient digestibility and intestinal health. However, high doses of tannins exert a negative impact on growth performance by binding starch and protein in the feed, reducing digestive enzyme activity, and binding to intestinal wall proteins [ 40 , 41 ]. Effect Of Tannic Acid On The Ileal Microflora Of Broilers With Ne The intestinal microbiota forms a highly complex microecosystem. To further investigate the mechanisms by which tannic acid alleviates the intestinal damage caused by NE, we analyzed the microflora structure of the ileal microorganisms using 16S rRNA sequencing. The results of this study showed that the challenge did not have a significant effect on the microbial α-diversity, which is consistent with the results of previous studies [ 42 , 43 ]. We speculated that the possible reason for this result was that NE infection inhibited the proliferation of minor microorganisms in the broiler intestinal flora, which resulted in a convergence with the α-diversity of the NC group microbiota. However, NE has also been shown to result in lower or higher gut microbial α diversity [ 15 , 44 ], which may be related to the collection of different parts of the chyme. In the present study, addition of tannic acid significantly reduced the α-diversity but did not affect the balance abundance of the flora, which is consistent with a previous study [ 45 ]. It has been shown that low-abundance microbes lead to the formation of simpler metabolic networks, resulting in increased concentrations of specific components used to support host energy requirements [ 45 ]. Shabat et al. reported that a reduced microbiome abundance may be closely associated with an increased feed conversion efficiency, with specific enrichment and metabolic pathways of microbes leading to better energy and carbon delivery to the animal body [ 46 ], which could be one of the reasons tannic acid improves growth performance. The significant difference in β-diversity among the three groups indicated that the addition of tannins or challenges significantly altered the microbial community structures. Bacteroidetes are an important contributor to intestinal health, and this phylum plays an important role in breaking down complex molecules into simpler compounds and producing short-chain fatty acids that are beneficial for increased growth performance [ 47 , 48 ]. We found that NE led to a decrease in the abundance of Bacteroides , which is consistent with the results of a previous study [ 43 ]. The fermentation products of Bacteroidetes , such as Bacteroides fragilis , inhibit C. perfringens spore formation, and the decrease in Bacteroidetes may predispose the animal intestine to C. perfringens infection and gastroenteritis [ 49 ], which may be one of the reasons for the decrease in Bacteroidetes abundance in NE. Lately, several studies have suggested that stressed birds present a significantly increasing trend for the Firmicutes/Bacteroides ratio [ 50 , 51 ]. In this study, we found that the high Firmicutes/Bacteroidetes ratio caused by NE may be one of the reasons for dysbiosis of the gut microbial community. The results also showed that at the genus level, NE significantly reduced the abundance of the beneficial bacterium Ligilactobacillus , suggesting that NE infection inhibited the growth of beneficial intestinal bacteria. At the species level, NE significantly reduced the relative abundance of Lactobacillus salivarius , which is a probiotic in poultry that dates back more than 15 years. Furthermore, this bacterium can re-establish the proper microbial balance of bacteria by forming lactic and propionic acid, stimulate butyric acid-producing butyric acid production, and inhibit the production of pro-inflammatory cytokines [ 52 ]. In broilers, Lactobacillus_salivarius was found to improve growth performance, reduce the expression of inflammatory factors, and improve intestinal health [ 9 , 53 , 54 ]. In conclusion, in this study, we found that the intestinal flora of broiler chickens infected with NE was disturbed and the abundance of beneficial bacteria was reduced, leaving the intestine in an unhealthy state. The addition of tannic acid after the challenge significantly increased the relative abundance of the genus Anaerocolumna , which belongs to the Lachnospiraceae and can be metabolized by the fermentation of carbohydrates into acetate [ 55 ]. In addition, the relative abundance of Thermoanaerobacterium , which can secrete xylanase, degrade hemicellulose and even cellulose, and produce acetate and butyric acid via fermentation using xylan and others, was increased [ 56 ]. The genus Thermosinus includes gram-negative bacteria that can convert CO to carbon dioxide through a series of chemical reactions and can decompose organic substrates (glucose, sucrose, or lactose) to produce acetic acid as well as acetate, H 2 , and CO 2 during glucose fermentation [ 57 ]. Thus, it regulates the intestinal pH, nourishes butyric acid-producing bacteria, protects against pathogens, and contributes to intestinal health. In conclusion, the addition of tannins increases the abundance of short-chain fatty acid-producing-related flora, which may be one of the reasons for their beneficial impacts on intestinal health. In the PICRUSt analysis, we found that differences in the metabolic pathways were the most common in this study, especially in the PC group, where there were large metabolic differences when compared with the other two groups. The present study showed that nutrient metabolism in the intestinal flora of broilers with NE was generally downregulated, and the pathways related to intestinal flora metabolism were more enriched in the NC group, which was consistent with previous findings [ 43 ]. Bacteroidetes are the main carbohydrate-degrading bacteria in the intestine that contribute to the degradation of complex pectins. Therefore, the enrichment of the carbohydrate metabolic pathway in the NC group may be due to the high abundance of Bacteroidetes , which degrade complex polysaccharides and indigestible carbohydrates to generate short-chain fatty acids and maintain intestinal health [ 58 ]. Microbial catabolism of amino acids produces a large number of byproducts, including amines, phenols, indoles, and short-chain fatty acids [ 59 ], which can have beneficial or detrimental effects on epithelial cells, depending on the concentration of metabolic byproducts in the lumen. For example, aromatic amino acids are metabolized by colonies to produce tryptamine and indoles. Indoles play an important role in host defense by enhancing intestinal barrier function and downregulating the proinflammatory cytokines [ 60 ]. We speculated that the healthier gut in the NC group may be related to the products of carbohydrate and amino acid metabolism formed by the intestinal flora. Bacteroides , Propionibacterium , Fusobacterium , Lactobacillus , and Streptococcus play important roles in the protein hydrolysis process [ 61 ], and the downregulation of amino acid metabolic pathways in the PC group of microorganisms may be due to a decline in Bacteroides and Lactobacillus abundance. In the PC group, genes related to disease immunity, metabolic diseases, and energy metabolism were upregulated, which suggested that during the infection of chickens with C. perfringens , the intestinal flora competed with the host for energy, and more energy allocation was required for stimulation of the immune system, which may have resulted in the reduction in chicken growth performance. According to this study, the microbial communities in normal chickens were relatively healthier, and the ileal flora structure and function of the tannic acid groups were predicted to be similar to those of the NC group chickens. Therefore, we conclude that the addition of tannic acid could alleviate ileal microflora disorder in broiler chickens with NE. Taken together, the mechanism underlying the effect of tannic acid on intestinal health may involve changes in the intestinal microflora; however, relevant metabolomic analyses are still required to explore the effects of tannic acid on metabolites, which can further help us to establish the relationship among tannic acid, intestinal flora, metabolites, and intestinal health. To investigate the correlation between intestinal flora and growth performance, immunity, and mRNA expression of intestinal barrier genes and nutrient transport carriers, simple correlation analysis was performed. Correlation analysis can indicate the correlation between indicators but cannot determine a causal relationship. The results suggested that the abundance of Lactobacillus salivarius was positively correlated with growth performance and significantly negatively correlated with MPO, CRP, ET, and zonulin, which may be because Lactobacillus salivarius can inhibit inflammation [ 62 , 63 ] and improve intestinal barrier function [ 41 ]. The increase in the abundance of Thermosinus carboxydivorans and Thermosinus after the addition of tannic acid was significantly and positively correlated with the mRNA expression of GLUT2 , which suggested that the regulation of intestinal flora by tannic acid may be related to nutrient translocation and absorption. However, the specific mechanisms require further investigation. Conclusions The present study demonstrated that tannic acid supplementation is apparently effective in reducing NE in broiler chickens. The protective effects of dietary tannic acid supplementation against NE challenge were evidenced by the suppression of coccidia and C. perfringens colonization and invasion, regulation of intestinal flora, alleviation of inflammatory reaction, and improvement in intestinal barrier function and nutrient transport. As the amount of additive tannic acid increases, the body weight, mRNA expression of nutrient transport carriers, and villus height, V/C also increase in the form of a quadratic curve, while the intestinal barrier-related indices (Zonulin and ET content in serum), coccidia oocyst counts, cecum C. perfringens concentration, and immune indices in serum decrease linearly. After comprehensive consideration of the results, we recommend tannic acid supplementation to broiler feed at a dose of 500–750 mg/kg. Abbreviations BW: Body weight;CRP: C-reactive protein; ELISA: Enzyme-linked immunosorbent assay; ET: Endotoxin; F/G: The ratio of feed intake and body weight gain; GLUT2: Recombinant glucose transporter 2; I-FABP: Intestinal fatty acid binding protein; IgA: Immunoglobulin A; IgM: Immunoglobulin M; MPO: Myeloperoxidase; NE: Necrotic enteritis; PEPT1: Peptide-transporters 1; sIgA: Secretory immunoglobulin A; SGLT1: Recombinant sodium/glucose cotransporter 1; V/C: The ratio of villus height to crypt depth; ZO-1: Zonula occludens-1 Declarations Authors’ contributions YG designed the study; HX, JF, YL, PL, BS, and ZL performed the experiments; HX analyzed the data and wrote the manuscript; and YG revised the manuscript. All authors contributed to the data interpretation and approved the final version of the manuscript. Availability of data and materials All data generated or analyzed during this study are available from the corresponding author upon request. Datasets supporting the conclusions of this study are included in this article. The 16S gene sequencing data can be obtained from the following website: https://www.ncbi.nlm.nih.gov/sra/PRJNA897828; the accession number is PRJNA897828. Competing interests The authors declare that they have no competing interests. Consent for publication Not applicable. Ethics approval and consent to participate All experiments were approved by the Institutional Animal Care and Use Committee of the China Agricultural University. The animal welfare number is AW11112022-1-4. Funding This work was supported by the China Agriculture Research System program (CARS-41-G11). Acknowledgments The authors are grateful to the staff of the Department of Animal Science and Technology of the China Agricultural University, especially those involved in animal nutrition, for their valuable assistance in conducting the experiments. References Van Immerseel F, Rood JI, Moore RJ, Titball RW. 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Cite Share Download PDF Status: Published Journal Publication published 04 May, 2023 Read the published version in Journal of Animal Science and Biotechnology → Version 1 posted Editorial decision: Major revision 13 Dec, 2022 Reviewers agreed at journal 21 Nov, 2022 Reviewers invited by journal 21 Nov, 2022 Editor assigned by journal 17 Nov, 2022 First submitted to journal 16 Nov, 2022 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-2283343","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":153733350,"identity":"9a407701-ba70-402b-81a8-ac5541f2eb70","order_by":0,"name":"Huiping Xu","email":"","orcid":"","institution":"China Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Huiping","middleName":"","lastName":"Xu","suffix":""},{"id":153733351,"identity":"a9195206-b099-4eb7-9cf8-1c44076d63ce","order_by":1,"name":"Jianyang Fu","email":"","orcid":"","institution":"China Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Jianyang","middleName":"","lastName":"Fu","suffix":""},{"id":153733352,"identity":"5650aea1-9b2f-43ee-9631-04080b2fd133","order_by":2,"name":"Yimeng Luo","email":"","orcid":"","institution":"China Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Yimeng","middleName":"","lastName":"Luo","suffix":""},{"id":153733353,"identity":"3b544fb0-e495-461b-8059-3cfacf8ca5d6","order_by":3,"name":"Peng Li","email":"","orcid":"","institution":"Wuhan Polytechnic University","correspondingAuthor":false,"prefix":"","firstName":"Peng","middleName":"","lastName":"Li","suffix":""},{"id":153733354,"identity":"b07fe846-eb4e-49f4-8ac8-79659fa3cca4","order_by":4,"name":"Bochen Song","email":"","orcid":"","institution":"Shandong Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Bochen","middleName":"","lastName":"Song","suffix":""},{"id":153733355,"identity":"d12a76f6-5cb3-46b3-9af4-ff5c554960fb","order_by":5,"name":"Zengpeng Lv","email":"","orcid":"","institution":"China Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Zengpeng","middleName":"","lastName":"Lv","suffix":""},{"id":153733356,"identity":"4d4ae5e1-b71e-4bc5-ab25-230526620a47","order_by":6,"name":"Yuming Guo","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAvElEQVRIiWNgGAWjYPACGwjFQ4KWNNK1HCZBi3xE8jFp3pzz8vwzEhgfvG1jkDcnpMXwRlqaNO+224YzbiQwG85tYzDc2UBIy4wcM5CWBIYbCWzSvG0MCQYHiNNyLkH+RgL7b6K0yEuAtRxIMADawkyUFgOeZ8mWc7clG24887BZcs45CcMNBG1pTz544+02O3m548kHP7wps5EnbMsBBhYJCJOxAUhIEFAPsqWBgfkDYWWjYBSMglEwogEAY4U8jM4lc28AAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0001-8577-4803","institution":"China Agricultural University","correspondingAuthor":true,"prefix":"","firstName":"Yuming","middleName":"","lastName":"Guo","suffix":""}],"badges":[],"createdAt":"2022-11-17 08:25:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2283343/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2283343/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s40104-023-00867-8","type":"published","date":"2023-05-04T20:40:30+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":29450951,"identity":"2494da76-7103-4973-b98d-3a6a722d63d8","added_by":"auto","created_at":"2022-11-23 20:02:48","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":851489,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative photomicrographs of the cross sections of the middle ileum from chickens at d 28. The NC group shows normal physiological features of the intestinal villi, whereas the PC group shows severe pathological changes in the villi structure. The PTA1–PTA4 groups show mild pathological changes. NC: no tannin acid treatment or NE infection; PC: NE infection + 0 mg/kg tannic acid; PTA1: NE infection + 250 mg/kg tannic acid; PTA2: NE infection + 500 mg/kg tannic acid; PTA3: NE infection + 750 mg/kg tannic acid; PTA4: NE infection + 1000 mg/kg tannic acid. The scale is 200 μm.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-2283343/v1/28b4e8bfbd56f8de28803e4f.png"},{"id":29450952,"identity":"ae994e20-d3a8-4947-8daa-b6f89f92e3a9","added_by":"auto","created_at":"2022-11-23 20:02:48","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":735843,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative picture of the coccidia oocysts in the feces at d 28. No coccidia oocysts were found in the feces of the NC group. The number of coccidia oocysts gradually decreased with an increase of tannic acid additive levels. NC: no tannin acid treatment or NE infection; PC: NE infection + 0 mg/kg tannic acid; PTA1: NE infection + 250 mg/kg tannic acid; PTA2: NE infection + 500 mg/kg tannic acid; PTA3: NE infection + 750 mg/kg tannic acid; PTA4: NE infection + 1000 mg/kg tannic acid. The arrow indicates the coccidia oocysts.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-2283343/v1/f51877228a7aa59835731a86.png"},{"id":29450954,"identity":"7cd8f019-e917-4e46-9692-7d2569161702","added_by":"auto","created_at":"2022-11-23 20:02:48","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":229103,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of tannic acid on ileum gene transcription levels in broiler chickens with NE infection. (a) and (b) show the expression of genes related to the intestinal barrier and MUC2 in the ileum on d 28 and 35, respectively; (c) and (d) show the mRNA expression of genes related to nutrient transporter carriers in the ileum on d 28 and 35. (e) and (f) show the results of mRNA expression of genes related to immunity in the ileum on d 28 and 35. * and ** indicate \u003cem\u003e0.01\u0026lt; P\u0026lt; 0.05\u003c/em\u003eand \u003cem\u003eP \u0026lt; 0.01\u003c/em\u003e, respectively. NC: no tannin acid treatment or NE infection; PC: NE infection + 0 mg/kg tannic acid; PTA1: NE infection + 250 mg/kg tannic acid; PTA2: NE infection + 500 mg/kg tannic acid; PTA3: NE infection + 750 mg/kg tannic acid; PTA4: NE infection + 1000 mg/kg tannic acid. The error bars represent SEM; n = 7.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-2283343/v1/46a1b77694ddc1746cb9c9ef.png"},{"id":29452779,"identity":"73566629-e3ac-465e-8fd0-d07c60e4c7e4","added_by":"auto","created_at":"2022-11-23 20:18:48","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":84475,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of tannic acid on ileal microbial diversity in broiler chickens with NE. (a) Venn diagram showing the OTUs of the ileal microorganisms. (b) α-diversity diagram showing ileal microorganisms. (c) PCOA diagram. * and ** indicate \u003cem\u003e0.01\u0026lt; P\u0026lt; 0.05\u003c/em\u003e and \u003cem\u003eP \u0026lt; 0.01\u003c/em\u003e, respectively. NC: no tannin acid treatment or NE infection; PC: NE infection + 0 mg/kg tannic acid; PTA3: NE infection + 750 mg/kg tannic acid. The error bars represent SEM; n=7.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-2283343/v1/53cb85d3eb6eec667963272b.png"},{"id":29451914,"identity":"65386270-fe59-4db3-ac46-b7e0dd3ac585","added_by":"auto","created_at":"2022-11-23 20:10:48","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":227706,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of tannic acid on the relative abundance of microorganisms in the ileum of NE. (a), (c), and (e) show the abundance of the top fifteen species at the phylum, genus, and species levels, respectively; (b), (d), and (f) show the differential species at the phylum, genus, and species levels, respectively. * and ** indicate \u003cem\u003e0.01\u0026lt; P\u0026lt; 0.05\u003c/em\u003e and \u003cem\u003eP \u0026lt; 0.01\u003c/em\u003e, respectively. NC: no tannin acid treatment or NE infection; PC: NE infection + 0 mg/kg tannic acid; PTA3: NE infection + 750 mg/kg tannic acid. The error bars represent SEM; n = 7.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-2283343/v1/c32d43168e6528b6b5e78a09.png"},{"id":29450956,"identity":"576f8773-6eca-46e8-a992-2c31f9ed6a20","added_by":"auto","created_at":"2022-11-23 20:02:48","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":197459,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of tannic acid on the microflora in the mid-ileum of broiler chickens with NE. Results of the functional clustering at (a) level 1 and (b) level 2. NC: no tannin acid treatment or NE infection; PC: NE infection + 0 mg/kg tannic acid; PTA3: NE infection + 750 mg/kg tannic acid. N = 7.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-2283343/v1/f186f0d998d72e73678d251d.png"},{"id":29451911,"identity":"59562230-156e-479c-acdd-e38a52d1f0f2","added_by":"auto","created_at":"2022-11-23 20:10:48","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":81492,"visible":true,"origin":"","legend":"\u003cp\u003eCorrelation heat map for effect of tannic acid on the indices of broilers with NE. The graph demonstrates the Pearson correlation between the indicators. Squares are colored Pearson correlation coefficient R values. * and ** indicate \u003cem\u003e0.01\u0026lt; P\u0026lt; 0.05\u003c/em\u003e and \u003cem\u003eP \u0026lt; 0.01\u003c/em\u003e, respectively.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-2283343/v1/a9aba7b39b03ed8627d95e2b.png"},{"id":44728870,"identity":"ae7ac1f2-997f-4e86-9119-1bf96cfe5172","added_by":"auto","created_at":"2023-10-16 21:08:53","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3915594,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2283343/v1/196e5c0c-32d4-4401-ae41-edbfc8d76ff1.pdf"}],"financialInterests":"","formattedTitle":"Effects of tannic acid on the immunity and intestinal health of broiler chickens with Clostridium perfringens-induced necrotic enteritis","fulltext":[{"header":"Background","content":"\u003cp\u003eIn the poultry industry, ensuring that the intestinal tracts of birds are healthy plays an important role in maximizing growth performance. Necrotic enteritis (NE) in broiler chickens, caused by \u003cem\u003eClostridium perfringens\u003c/em\u003e (\u003cem\u003eC. perfringens\u003c/em\u003e), is one of the most prominent intestinal diseases in modern intensive farming. The destruction of the intestinal mucosal structure in broiler chickens with NE results in poor digestion and absorption of nutrients and reduced feed efficiency, causing huge losses for the industry [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. In addition, \u003cem\u003eC. perfringens\u003c/em\u003e can enter the human body through the food chain, threatening public health. Due to the limited use of antibiotic growth promoters (AGP) in the poultry industry, the incidence of NE in chickens has been increasing worldwide. Therefore, effective AGP alternatives are urgently required to prevent and control NE in broiler chickens.\u003c/p\u003e \u003cp\u003eTannins are water-soluble polyphenols and secondary metabolites in plants [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. They are classified into two types according to their chemical structures\u0026mdash;condensed tannins (CTs) and hydrolytic tannins (HTs). HTs are phenolic acid polyesters with a D-glucose core, such as gallic acid and ellagic acid. The relative molecular weight of HTs is usually 500\u0026ndash;3000 Da, and they are easily hydrolyzed by acids, alkalis, and enzymes. CTs are composed of flavan-3-ol units connected by carbon-carbon double bonds, also called proanthocyanidins (catechins or epicatechins). CTs are the most prevalent and typical class of plant tannins, with relative molecular masses ranging from 1,900 to 28,000 Da; they are mainly found in legumes, trees, and shrubs [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTannins have antibacterial, antiparasitic, antiviral, anti-inflammatory, antioxidant, antidiarrheal, and nutrient metabolism-modulating effects [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Dietary supplementation of broilers with tannins has been shown to improve growth performance [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e] and reduce the abundance of \u003cem\u003eC. perfringens\u003c/em\u003e, production of toxins such as alpha toxin [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], and the adverse effects of \u003cem\u003eC. perfringens\u003c/em\u003e and \u003cem\u003eEimeria tenella\u003c/em\u003e in chickens [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. The European Union approved tannins as a novel feed additive for livestock and poultry in 2016. However, it has been shown that the addition of tannins can result in negative effects, including reduction in production performance [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. The differences in the results may be related to the tannic acid type or dosage used. Therefore, this study was conducted to investigate the modulating effects of different tannic acid additive levels on the intestinal health of broiler chickens co-infected with coccidia and \u003cem\u003eC. perfringens\u003c/em\u003e to aid the development of more tailored tannic acid application strategies.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eTannic acid\u003c/h2\u003e \u003cp\u003eTannic acid was provided by Hubei Chicheng Technology Development Co., Ltd. (Yichang City, Hubei Province, China). Tannic acid parameters were as follows: tannic acid\u0026thinsp;\u0026ge;\u0026thinsp;80.0%, moisture\u0026thinsp;\u0026le;\u0026thinsp;10.0%, scorch residue\u0026thinsp;\u0026le;\u0026thinsp;6.0%, total arsenic\u0026thinsp;\u0026le;\u0026thinsp;5 ppm, and heavy metal content (as Pb)\u0026thinsp;\u0026le;\u0026thinsp;10 ppm.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eExperimental Design, Birds, And Diets\u003c/h3\u003e\n\u003cp\u003eThe experiment was carried out at the poultry experiment base of China Agricultural University (Zhuozhou City, Hebei Province, China). A total of 630 1-day-old Cobb 500 male broilers were weighed and randomly divided into six treatment groups according to the principle of similar body weight. There were seven replicate cages (1.0 \u0026times; 0.7 \u0026times; 0.38 m, length \u0026times; width \u0026times; height) per treatment and 15 chickens per replicate. They were grouped as follows: (i) negative control group (no tannin acid treatment or NE infection, NC group); (ii) positive control group (NE infection\u0026thinsp;+\u0026thinsp;0 mg/kg tannin acid, PC group); (iii) PTA1 group (NE infection\u0026thinsp;+\u0026thinsp;250 mg/kg tannic acid); (iv) PTA2 group (NE infection\u0026thinsp;+\u0026thinsp;500 mg/kg tannic acid); (v) PTA3 group (NE infection\u0026thinsp;+\u0026thinsp;750 mg/kg tannic acid); and (vi) PTA4 group (NE infection\u0026thinsp;+\u0026thinsp;1000 mg/kg tannic acid). Broilers were reared in a room equipped with two-tiered battery cages. All chickens were vaccinated and managed (including light and temperature management) according to routine immunization and management programs of Cobb broilers. Furthermore, all chickens were provided feed and water \u003cem\u003ead libitum\u003c/em\u003e. Diets were formulated according to the Chinese chicken feeding standard (NY/T 33-2004) and were fed in the form of pellets; the nutritional information is summarized in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eIngredients and nutrient content of the chicken feed used during the trial\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"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 \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eStarter (day 1\u0026ndash;14, %)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGrower (day 15\u0026ndash;28, %)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFinisher (day 29\u0026ndash;35, %)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eIngredient\u003c/b\u003e\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 \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCorn\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e54.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e58.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e61.79\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSoybean meal\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e32.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e28.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e25.68\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCorn gluten meal\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2.50\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSoybean oil\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4.62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e4.50\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eFlour\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.05\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCalcium hydrogen phosphate\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.85\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eStone powder\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.07\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSodium chloride\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.30\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eL-lysine hydrochloride, 78%\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.36\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eDL-Methionine, 98%\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.19\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eThreonine\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.10\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eArginine\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.06\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCholine chloride, 50%\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.20\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eBroilers - Mineral premix\u003c/b\u003e \u003csup\u003e\u003cb\u003e1\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.20\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eBroilers - Vitamin premix\u003c/b\u003e \u003csup\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.03\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePhytase 10000\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.02\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eZeolite\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.10\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTotal\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e100.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e100.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e100.00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eNutrient content\u003c/b\u003e \u003csup\u003e\u003cb\u003e3\u003c/b\u003e\u003c/sup\u003e\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 \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMetabolic energy, Mcal/kg\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2.99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3.10\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCrude protein, %\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e22.39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e20.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e18.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eLysine, %\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.14\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMethionine, %\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.48\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCystine, %\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.93\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.75\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eThreonine, %\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.92\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.77\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCalcium, %\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.08\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\u003e1.03\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAvailable phosphorus, %\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.40\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003e\u003csup\u003e1\u003c/sup\u003e per kg of trace element premixed feed: copper, 8 g; iron, 40 g; zinc, 55 g; manganese, 60 g; iodine, 750 mg; selenium, 150 mg; and cobalt, 250 mg.\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003e\u003csup\u003e2\u003c/sup\u003e per kg of vitamin premix feed: vitamin A, 50\u0026nbsp;million IU; vitamin D3, 12\u0026nbsp;million IU; vitamin E, 100,000 IU; vitamin K3, 10 g; vitamin B1, 8 g; vitamin B2, 32 g; vitamin B6, 12 g; vitamin B12, 100 mg; niacin, 150 g; D-pantothenic acid, 46 g; folic acid, 5 g; biotin, 500 mg.\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003e\u003csup\u003e3\u003c/sup\u003e Calculated values based on the experimental diet analysis\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eC. perfringens\u003c/span\u003e \u003cb\u003echallenge\u003c/b\u003e\u003c/p\u003e \u003cp\u003eNE was induced in chickens according to a previously described method, with slight modifications [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. On day 19, birds in the PC and PTA1-PTA4 groups were orally inoculated with 1 mL of a 25-fold dose of attenuated quadrivalent coccidia vaccine suspension (Foshan Standard Bio-Tech Co. Ltd., Foshan, China). Chickens in the NC group received 1 mL sterile PBS. Except for broilers in the NC group, chickens were orally gavaged with 1 mL \u003cem\u003eC. perfringens\u003c/em\u003e type A CVCC52 (3 \u0026times; 10\u003csup\u003e8\u003c/sup\u003e CFU/mL) per day on days 22\u0026ndash;28. Uninfected birds received 1 mL sterile fluid medium.\u003c/p\u003e\n\u003ch3\u003eGrowth Performance\u003c/h3\u003e\n\u003cp\u003eAll broilers were weighed at 19, 28, and 35 d of age in replicates after 12 h of fasting. Body weight, feed consumption, and the feed consumption to weight gain ratio (F/G) were calculated at different experimental periods.\u003c/p\u003e\n\u003ch3\u003eSample Collection\u003c/h3\u003e\n\u003cp\u003eOne chicken with the average body weight was selected from each replicate at 28 and 35 days of age, and blood was collected from the wing vein, followed by intravenous injection of sodium pentobarbital at a dose of 30 mg/kg body weight. After the anesthesia, the chickens were bled through the jugular vein and sacrificed. The middle region (1 cm) of the ileum was excised and immediately fixed in 4% paraformaldehyde for intestinal morphological examination. Approximately 2 g of chyme from the middle ileum was collected aseptically into sterile tubes; the mucosa of the middle ileum was then gently scraped with a slide, collected in sterile tubes, and stored at \u0026minus;\u0026thinsp;80\u0026deg;C until further analysis.\u003c/p\u003e\n\u003ch3\u003eIntestinal Ne Lesion Scoring\u003c/h3\u003e\n\u003cp\u003eAt 28 days of age, one chicken in each replicate was anesthetized and sacrificed. The intestinal cavity was opened, and chyme was removed to observe the lesions in the intestinal wall. The scoring rules for intestinal lesions described by Dahiya et al. were adopted [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]; the specific scoring criteria were as follows: 0\u0026thinsp;=\u0026thinsp;normal intestinal appearance; 0.5\u0026thinsp;=\u0026thinsp;severe congestion in serous surface and mesentery of the small intestine; 1\u0026thinsp;=\u0026thinsp;the intestinal wall became thin and brittle, with red silting spots (more than 5); 2\u0026thinsp;=\u0026thinsp;the intestinal wall appears needle-like necrosis or ulceration, and there is a small amount of gas in the intestinal cavity; 3\u0026thinsp;=\u0026thinsp;flaky necrosis or ulceration of the intestinal wall, gas-filled intestines and small blood spots, and 1\u0026ndash;2 cm long necrotic spots; 4\u0026thinsp;=\u0026thinsp;diffuse necrosis, significant bleeding, and large intestinal gas.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFecal coccidia oocyst counts and determination of intestinal\u003c/b\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eC. perfringens\u003c/span\u003e \u003cb\u003econcentration\u003c/b\u003e\u003c/p\u003e \u003cp\u003eOn days 25\u0026ndash;28, the feces of broilers were collected repeatedly, and fecal coccidia oocysts were counted. The feces collected from each replicate were mixed; then, 2 g feces was added to 58 mL saturated salt water, mixed thoroughly with a vortex shaker for 2 min, and sieved through a 40 mesh. The upper suspension was then added to a McMaster counting plate and allowed to stand for 3 min. Oocysts were counted under a light microscope (Leica, LEICA DM750), and the number of oocysts per gram of feces was calculated according to the following formula: oocyst content per gram of feces (opg)\u0026thinsp;=\u0026thinsp;number of oocysts in the counting chamber \u0026times; 200.\u003c/p\u003e \u003cp\u003eCecal \u003cem\u003eC. perfringens\u003c/em\u003e was counted as previously described [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Approximately 0.5 g of each sample was collected in 10 mL sterile plastic tubes, diluted with PBS to an initial 10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e dilution, and serially diluted to prepare 10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e to 10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e dilutions. Each diluted sample of 100 \u0026micro;L was plated on tryptose-sulfite-cycloserine agar (TSC, CM 138; Beijing Land Bridge Technology Co., Ltd.). The number of \u003cem\u003eC. perfringens\u003c/em\u003e was determined after anaerobic incubation at 37\u0026deg;C for 24 h. The opg and \u003cem\u003eC. perfringens\u003c/em\u003e were then log\u003csub\u003e10\u003c/sub\u003e-transformed and counted.\u003c/p\u003e\n\u003ch3\u003eIntestinal Morphological Analyses And Observation\u003c/h3\u003e\n\u003cp\u003eThe middle ileum was circumcised to a thickness of 5 \u0026micro;m and stained with hematoxylin and eosin. The height of the villi and depth of the crypt of the chicken ileum were measured according to the method described by Frankel et al. [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Briefly, 10 straight and structurally intact intestinal villi were selected from each intestinal section and measured. The mean value of each index was calculated to determine the ratio of villus height to crypt depth (V/C).\u003c/p\u003e\n\u003ch3\u003eDetermination Of Serum Immunoglobulin, C-reactive Protein, Myeloperoxidase, And Ileal Mucosa Siga Levels\u003c/h3\u003e\n\u003cp\u003eThe levels of natural serum IgA and IgM antibodies were determined using a chicken IgA enzyme-linked immunosorbent assay (ELISA) kit (Beijing Solaibao Technology Co., Ltd., SEKCN-0018) and chicken IgM ELISA kit (Beijing Solaibao Technology Co., Ltd., SEKCN-0128), respectively. C-reactive protein (CRP) (Shanghai Enzyme Link Biotechnology Co., Ltd., YJ036965) and myeloperoxidase (MPO) kits (Shanghai Enzyme Link Biotechnology Co., Ltd., YJ966521) were used to determine the serum levels of CRP and MPO, respectively. The tissue homogenate was prepared by taking 0.1 g of ileal mucosa at a weight: saline volume ratio of 1:9, and the supernatant was retained for subsequent analysis. The sIgA and total protein contents in the ileal mucosa were determined using a chicken sIgA ELISA kit (Shanghai Yuanmu Biotechnology Co., Ltd., YM-A3724) and a total protein quantification kit (Nanjing Jiancheng Institute of Biological Engineering), respectively. Serum immunoglobulin content was measured in mg/mL, and mucosal sIgA content was measured in \u0026micro;g/mg protein. The above-mentioned indexes were measured in strict accordance with the manufacturer's instructions.\u003c/p\u003e \u003cp\u003eThe antibody levels of \u003cem\u003eC. perfringens\u003c/em\u003e in the serum were detected using an improved ELISA method. The methods referred to those described by Wu et al., with slight alterations[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. \u003cem\u003eC. perfringens\u003c/em\u003e was cultured to a concentration of 10\u003csup\u003e8\u003c/sup\u003e CFU/mL and centrifuged at 4000 rpm for 10 min; the thalli were collected and cleaned with 0.01 M sterile PBS solution thrice. The last time, the thalli were diluted with PBS and subjected to intermittent ultrasound over 10 times in an ice bath. When the bacterial solution was turbid and consistent, 9 ml PBS solution was added and incubated for more than 2 h. The supernatant was absorbed into another sterilized centrifuge tube. Protein concentration was measured using the total protein quantitative kit (Nanjing Jianguo Institute of Bioengineering) according to the manufacturer's instructions. Approximately 100 \u0026micro;L bacterial lysate of \u003cem\u003eC. perfringens\u003c/em\u003e (40 \u0026micro;g/mL) was added to the enzyme-labeled plate and incubated overnight at 4\u0026deg;C. The 96-well plates were coated with 20 \u0026micro;g/mL albumin from bovine serum (BSA) and incubated overnight at 4\u0026deg;C. After washing five times with 200 \u0026micro;L of 1% BSA dissolved in PBS containing 0.05% Tween (PBST), the plates were incubated with the serum (1:50 dilution) at 37\u0026deg;C for 1.5 h. Then, the plate was washed and 100 \u0026micro;L diluted horseradish peroxidase-conjugated goat anti-chicken IgG (1:10,000; A30-104P, Bethyl Laboratories Inc., Montgomery, TX, USA) was added and incubated at 37\u0026deg;C for 30 min. After washing five times with PBST, 100 \u0026micro;L 0.05% tetramethylbenzidine was added to the plates and incubated at 37\u0026deg;C in the dark for 30 min. The reaction was terminated with 2 mol/L sulfuric acid. Readings were taken at 450 nm using an ELISA reader (SepctraMax i3x Platform, Molecular Devices, LLC).\u003c/p\u003e\n\u003ch3\u003eSerum Zonulin And Endotoxin (Et) Detection\u003c/h3\u003e\n\u003cp\u003eThe venous blood of chicken wings was collected using common collection vessels (5 mL), and the serum was collected after centrifugation at 3500 rpm and 4\u0026deg;C for 10 min. Zonulin (Shanghai Fankewei Industrial Co., Ltd., F8139-A) and Endotoxin detection kits (Shanghai Meilian Biotechnology Co., Ltd., ml0122369-J) were then used according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e\n\u003ch3\u003eQuantitative Reverse Transcription-pcr To Measure Mrna Expression In The Ileum\u003c/h3\u003e\n\u003cp\u003eThe ileal tissues were collected, placed in RNase-free freezing tubes, and stored at \u0026minus;\u0026thinsp;80\u0026deg;C. Tissue samples (100 mg) were placed in a 2 mL centrifuge tube, 1 mL of Trizol (Invitrogen Life Technologies, Carlsbad, USA) extract was added, and total RNA was extracted according to the manufacturer\u0026rsquo;s instructions. The purity of the extracted RNA was checked using a nucleic acid spectrophotometer (AG 22331, Eppendorf, Hamburg, Germany), and samples with OD 260/OD 280 nm greater than 1.8 were used for subsequent processing. The samples were reverse-transcribed using a cDNA kit (Takara Biotechnology Co. Ltd., Beijing, China). The Applied Biosystems 7500 Fast Real-Time PCR System and SYBR Premix Ex Taq\u0026trade; kit (Takara Biotechnology Co. Ltd., Beijing, China) were used to performed quantitative reverse transcription-PCR. All procedures were performed according to the manufacturer's instructions. Genes were quantified using β-actin as an internal reference, and the results were statistically analyzed according to the method described by Fu et al. [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. The primer sequences for genes are shown in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSequences of the oligonucleotide primers used for quantitative reverse transcription-PCR\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGene\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePrimer sequences(5ʹ\u0026rarr;3ʹ)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNCBI serial number\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eβ-actin\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eF-CAACACAGTGCTGTCTGGTGGTAC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNM_205518.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eR-CTCCTGCTTGCTGATCCACATCTG\u003c/p\u003e \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\u003e\u003cem\u003eOccludin\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eF-ACGGCAGCACCTACCTCAA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNM_205128.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eR-GGGCGAAGAAGCAGATGAG\u003c/p\u003e \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\u003e\u003cem\u003eMUC2\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eF-TCCCCTGTTGAGGGAGAACTT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eXM_040673077.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eR-AGTGGTTGTACCTTCGGTGC\u003c/p\u003e \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\u003e\u003cem\u003eZO-1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eF-CTTCAGGTGTTTCTCTTCCTCCTC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eXM_040680632.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eR-CTGTGGTTTCATGGCTGGATC\u003c/p\u003e \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\u003e\u003cem\u003ePEPT1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eF-TACGCATACTGTCACCATCA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNM_204365.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eR-TCCTGAGAACGGACTGTAAT\u003c/p\u003e \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\u003e\u003cem\u003eSGLT1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eF-GATGTGCGGATACCTGAAGC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNM_001293240.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eR-AGGGATGCCAACATGACTGA\u003c/p\u003e \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\u003e\u003cem\u003eGLUT2\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eF-CCGCAGAAGGTGATAGAAGC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNM_205129.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eR-ATTGTCCCTGGAGGTGTT\u003c/p\u003e \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\u003e\u003cem\u003eI-FABP\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eF-GAAGCAATGGGCGTGAATGTGATG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNM_001007923.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eR-TTCGATGTCGATGGTACGGAAGTTG\u003c/p\u003e \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\u003e\u003cem\u003eIL-1β\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eF-ACTGGGCATCAAGGGCTA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eXM_046931582.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eR-GGTAGAAGATGAAGCGGGTC\u003c/p\u003e \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\u003e\u003cem\u003eIL-6\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eF-CGCCCAGAAATCCCTCCTC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNM_204628.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eR-AGGCACTGAAACTCCTGGTC\u003c/p\u003e \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\u003e\u003cem\u003eTLR2\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eF-GATTGTGGACAACATCATTGACTC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNM_001161650.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eR-AGAGCTGCTTTCAAGTTTTCCC\u003c/p\u003e \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\u003e\u003cem\u003eTGF-β\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eF-TCATCACCAGGACAGCGTTA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNM_001031045.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eR-TGTGATGGAGCCATTCATGT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"3\"\u003eF: upstream primer; R: downstream primers; \u003cem\u003eZO-1\u003c/em\u003e: zonula occludens-1; \u003cem\u003ePEPT1\u003c/em\u003e, peptide-transporter 1; \u003cem\u003eSGLT1\u003c/em\u003e, recombinant sodium/glucose cotransporter 1; \u003cem\u003eGLUT2\u003c/em\u003e, recombinant glucose transporter 2; \u003cem\u003eI-FABP\u003c/em\u003e: intestinal fatty acid binding protein; IL-1β, interleukin-1β; IL-6, interleukin-6; TLR2, toll like receptor 2; TGF-β, transforming growth factor-β.\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"3\"\u003e\u003csup\u003e1\u003c/sup\u003e The primers were synthesized by Shanghai Shenggong Biotechnology Co. Ltd.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e\n\u003ch3\u003eIleal Microbiological Analysis\u003c/h3\u003e\n\u003cp\u003eThe contents of middle ileal samples collected on d 28were used for 16S sequencing analysis, according to the method described by Zhang et al. [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. First, fecal microbial DNA was extracted using a DNA extraction kit (QIAamp Fast DNA Stool Mini Kit, Qiagen Company, Dusseldorf, Germany), and then the purity of the DNA was tested using 1% agarose gel electrophoresis. All procedures were performed according to the manufacturer's instructions. Bacterial DNA was amplified using the following V3-V4 primers: 338 F (5ʹ-ACTCCTACGGGAGGCAGCA-3ʹ) and 806r (5ʹ-GGACTACHVGGGTWTCTAAT-3ʹ). In-machine sequencing was performed using HiSeq2500 PE250, and the sequences were analyzed by Beijing Nuohe Zhiyuan Bio-Information Technology Co. Ltd. The Qiime software (Qiime2-2019.7, Nature Biotechnology) was used to generate species abundance tables at different taxonomic levels. Analysis was then performed to identify biomarkers that were statistically different between groups and α, β diversity. Venn plots and Principal Co-ordinates Analysis (PCoA) plots were plotted using the cloud platform (Beijing Nuohe Zhiyuan Bio-Information Technology Co. Ltd.), and analysis of similarities (ANOSIM) and PICRUSt analyses were performed.\u003c/p\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe data were analyzed using SPSS 26.0 (SPSS, Inc., Chicago, IL, USA). The effects of NE infection (NC vs. PC) were evaluated by the unpaired t-test. Challenged groups (PC, PTA1, PTA2, PTA3, and PTA4) were compared using one-way analysis of variance (ANOVA) and Duncan's multiple comparisons. The linear and quadratic effects of the different doses of tannic acid were tested by contrast. The results are shown as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. Unpaired Student's t-test was used to compare the relative abundances of the phylum, genera, species and the Firmicutes/Bacteroidetes ratio between the two groups were performed. A \u003cem\u003eP \u003c 0.05\u003c/em\u003e was considered statistically significant and a \u003cem\u003e0.05\u0026thinsp;\u0026le;\u0026thinsp;P\u0026thinsp;\u0026le;\u0026thinsp;0.1\u003c/em\u003e was classified as a tendency. Using Pearson's correlation coefficient to evaluate the correlation analysis of the 28-day-old broiler growth performance, immunity, intestinal barrier, and other indicators of microbiota.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv class=\"Section2\" id=\"Sec15\"\u003e\n \u003ch2\u003eGrowth performance\u003c/h2\u003e\n \u003cp\u003eThe growth performance data for the broiler chickens from the control and treatment groups used in this study are shown in Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e. The effect of tannic acid addition to the chicken diet showed a quadratic relationship with body weight and F/G. Additionally, it significantly increased the body weight at day 19 in the PTA1-PTA4 groups and markedly reduced the F/G during days 1\u0026ndash;19 in the PTA2 and PTA3 groups (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e), whereas the PC group showed a significant decrease in growth performance at day 28 and a significant increase in the F/G during days 1\u0026ndash;28 and days 1\u0026ndash;35(\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e). On day 28, the PTA3 group showed a marked improvement in body weight and decrease in the F/G (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e). On day 35, PTA2-PTA4 groups showed a significant increase in body weight, and the F/G ratio was significantly lower in the PTA2 and PTA3 groups during days 1\u0026ndash;35(\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e).\u0026nbsp;\u003c/p\u003e\n \u003ctable border=\"1\" id=\"Tab3\" style=\"margin-right: calc(27%); width: 73%;\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003e\u003cstrong\u003eEffects of tannic acid on the production performance of broilers with NE\u003c/strong\u003e\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" colspan=\"2\" style=\"width: 1.698%;\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\" style=\"width: 2.0871%;\"\u003e\n \u003cp\u003eNC\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\" style=\"width: 1.7687%;\"\u003e\n \u003cp\u003ePC\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\" style=\"width: 1.8395%;\"\u003e\n \u003cp\u003ePTA1\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\" style=\"width: 1.8395%;\"\u003e\n \u003cp\u003ePTA2\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\" style=\"width: 1.7687%;\"\u003e\n \u003cp\u003ePTA3\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\" style=\"width: 1.8395%;\"\u003e\n \u003cp\u003ePTA4\u003c/p\u003e\n \u003c/th\u003e\n \u003cth style=\"width: 1.6626%;\"\u003eP-value\u003csup\u003e2\u003c/sup\u003e\u003cbr\u003e\u003c/th\u003e\n \u003cth align=\"left\" style=\"width: 11.449%;\"\u003e\n \u003cp\u003eLinear\u003csup\u003e3\u003c/sup\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" style=\"width: 40.681%;\"\u003e\n \u003cp\u003eQuadratic \u003csup\u003e3\u003c/sup\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" style=\"width: 1.698%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBody\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eweight (g)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\" style=\"width: 2.0871%;\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\" style=\"width: 1.7687%;\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\" style=\"width: 1.8395%;\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\" style=\"width: 1.8395%;\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\" style=\"width: 1.7687%;\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\" style=\"width: 1.8395%;\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\" style=\"width: 3.5728%;\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 11.449%;\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 40.681%;\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" style=\"width: 1.698%;\"\u003e\n \u003cp\u003e\u003cstrong\u003ed 19\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\" style=\"width: 2.0871%;\"\u003e\n \u003cp\u003e745.1\u0026thinsp;\u0026plusmn;\u0026thinsp;6.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\" style=\"width: 1.7687%;\"\u003e\n \u003cp\u003e759.8\u0026thinsp;\u0026plusmn;\u0026thinsp;7.5\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\" style=\"width: 1.8395%;\"\u003e\n \u003cp\u003e794.3\u0026thinsp;\u0026plusmn;\u0026thinsp;6.4\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\" style=\"width: 1.8395%;\"\u003e\n \u003cp\u003e801.2\u0026thinsp;\u0026plusmn;\u0026thinsp;6.2\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\" style=\"width: 1.7687%;\"\u003e\n \u003cp\u003e815.1\u0026thinsp;\u0026plusmn;\u0026thinsp;11.3\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\" style=\"width: 1.8395%;\"\u003e\n \u003cp\u003e791.1\u0026thinsp;\u0026plusmn;\u0026thinsp;5.6\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\" style=\"width: 3.5728%;\"\u003e\n \u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 11.449%;\"\u003e\n \u003cp\u003e0.002\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 40.681%;\"\u003e\n \u003cp\u003e0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" style=\"width: 1.698%;\"\u003e\n \u003cp\u003e\u003cstrong\u003ed 28\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\" style=\"width: 2.0871%;\"\u003e\n \u003cp\u003e1486.6\u0026thinsp;\u0026plusmn;\u0026thinsp;12.4\u003csup\u003e\u003cem\u003e***\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\" style=\"width: 1.7687%;\"\u003e\n \u003cp\u003e1396.2\u0026thinsp;\u0026plusmn;\u0026thinsp;8.8\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\" style=\"width: 1.8395%;\"\u003e\n \u003cp\u003e1423.4\u0026thinsp;\u0026plusmn;\u0026thinsp;7.9\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\" style=\"width: 1.8395%;\"\u003e\n \u003cp\u003e1430.7\u0026thinsp;\u0026plusmn;\u0026thinsp;15.0\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\" style=\"width: 1.7687%;\"\u003e\n \u003cp\u003e1460.1\u0026thinsp;\u0026plusmn;\u0026thinsp;19.5\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\" style=\"width: 1.8395%;\"\u003e\n \u003cp\u003e1414.8\u0026thinsp;\u0026plusmn;\u0026thinsp;15.2\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\" style=\"width: 3.5728%;\"\u003e\n \u003cp\u003e0.043\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 11.449%;\"\u003e\n \u003cp\u003e0.105\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 40.681%;\"\u003e\n \u003cp\u003e0.025\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" style=\"width: 1.698%;\"\u003e\n \u003cp\u003e\u003cstrong\u003ed 35\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\" style=\"width: 2.0871%;\"\u003e\n \u003cp\u003e2011.7\u0026thinsp;\u0026plusmn;\u0026thinsp;29.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\" style=\"width: 1.7687%;\"\u003e\n \u003cp\u003e1970.3\u0026thinsp;\u0026plusmn;\u0026thinsp;12.0\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\" style=\"width: 1.8395%;\"\u003e\n \u003cp\u003e2010.5\u0026thinsp;\u0026plusmn;\u0026thinsp;25.0\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\" style=\"width: 1.8395%;\"\u003e\n \u003cp\u003e2036.2\u0026thinsp;\u0026plusmn;\u0026thinsp;24.2\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\" style=\"width: 1.7687%;\"\u003e\n \u003cp\u003e2050.3\u0026thinsp;\u0026plusmn;\u0026thinsp;13.8\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\" style=\"width: 1.8395%;\"\u003e\n \u003cp\u003e2043.1\u0026thinsp;\u0026plusmn;\u0026thinsp;16.0\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\" style=\"width: 3.5728%;\"\u003e\n \u003cp\u003e0.036\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 11.449%;\"\u003e\n \u003cp\u003e0.004\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 40.681%;\"\u003e\n \u003cp\u003e0.145\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"17\" style=\"width: 2.8045%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eF/G\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" style=\"width: 1.698%;\"\u003e\n \u003cp\u003e\u003cstrong\u003ed 1\u0026ndash;19\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\" style=\"width: 2.0871%;\"\u003e\n \u003cp\u003e1.330\u0026thinsp;\u0026plusmn;\u0026thinsp;0.013\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\" style=\"width: 1.7687%;\"\u003e\n \u003cp\u003e1.340\u0026thinsp;\u0026plusmn;\u0026thinsp;0.016\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\" style=\"width: 1.8395%;\"\u003e\n \u003cp\u003e1.310\u0026thinsp;\u0026plusmn;\u0026thinsp;0.008\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\" style=\"width: 1.8395%;\"\u003e\n \u003cp\u003e1.270\u0026thinsp;\u0026plusmn;\u0026thinsp;0.024\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\" style=\"width: 1.7687%;\"\u003e\n \u003cp\u003e1.280\u0026thinsp;\u0026plusmn;\u0026thinsp;0.018\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\" style=\"width: 1.8395%;\"\u003e\n \u003cp\u003e1.300\u0026thinsp;\u0026plusmn;\u0026thinsp;0.016\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\" style=\"width: 3.5728%;\"\u003e\n \u003cp\u003e0.039\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 11.449%;\"\u003e\n \u003cp\u003e0.055\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 40.681%;\"\u003e\n \u003cp\u003e0.012\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" style=\"width: 1.698%;\"\u003e\n \u003cp\u003e\u003cstrong\u003ed 1\u0026ndash;28\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\" style=\"width: 2.0871%;\"\u003e\n \u003cp\u003e1.390\u0026thinsp;\u0026plusmn;\u0026thinsp;0.004\u003csup\u003e***\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\" style=\"width: 1.7687%;\"\u003e\n \u003cp\u003e1.440\u0026thinsp;\u0026plusmn;\u0026thinsp;0.008\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\" style=\"width: 1.8395%;\"\u003e\n \u003cp\u003e1.410\u0026thinsp;\u0026plusmn;\u0026thinsp;0.007\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\" style=\"width: 1.8395%;\"\u003e\n \u003cp\u003e1.420\u0026thinsp;\u0026plusmn;\u0026thinsp;0.005\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\" style=\"width: 1.7687%;\"\u003e\n \u003cp\u003e1.410\u0026thinsp;\u0026plusmn;\u0026thinsp;0.012\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\" style=\"width: 1.8395%;\"\u003e\n \u003cp\u003e1.430\u0026thinsp;\u0026plusmn;\u0026thinsp;0.005\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\" style=\"width: 3.5728%;\"\u003e\n \u003cp\u003e0.017\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 11.449%;\"\u003e\n \u003cp\u003e0.612\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 40.681%;\"\u003e\n \u003cp\u003e0.009\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" style=\"width: 1.698%;\"\u003e\n \u003cp\u003e\u003cstrong\u003ed 1\u0026ndash;35\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\" style=\"width: 2.0871%;\"\u003e\n \u003cp\u003e1.530\u0026thinsp;\u0026plusmn;\u0026thinsp;0.011\u003csup\u003e\u003cem\u003e**\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\" style=\"width: 1.7687%;\"\u003e\n \u003cp\u003e1.570\u0026thinsp;\u0026plusmn;\u0026thinsp;0.002\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\" style=\"width: 1.8395%;\"\u003e\n \u003cp\u003e1.540\u0026thinsp;\u0026plusmn;\u0026thinsp;0.010\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\" style=\"width: 1.8395%;\"\u003e\n \u003cp\u003e1.530\u0026thinsp;\u0026plusmn;\u0026thinsp;0.010\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\" style=\"width: 1.7687%;\"\u003e\n \u003cp\u003e1.530\u0026thinsp;\u0026plusmn;\u0026thinsp;0.010\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\" style=\"width: 1.8395%;\"\u003e\n \u003cp\u003e1.540\u0026thinsp;\u0026plusmn;\u0026thinsp;0.004\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\" style=\"width: 3.5728%;\"\u003e\n \u003cp\u003e0.014\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 11.449%;\"\u003e\n \u003cp\u003e0.017\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 40.681%;\"\u003e\n \u003cp\u003e0.007\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003csup\u003e1\u003c/sup\u003eNC versus PC (unpaired t test): *\u003cem\u003e0.05\u0026thinsp;\u0026lt;\u0026thinsp;P\u0026thinsp;\u0026lt;\u0026thinsp;0.10\u003c/em\u003e, **\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e, ***\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u003c/em\u003e. \u003csup\u003e2\u003c/sup\u003eOverall P values obtained from ANOVA; \u003csup\u003e3\u003c/sup\u003e P values obtained using contrast trend analysis; different lower case letters in the same column indicate significant differences (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e), data are the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM, n\u0026thinsp;=\u0026thinsp;7. NC: no tannin acid treatment or NE infection; PC: NE infection\u0026thinsp;+\u0026thinsp;0 mg/kg tannic acid; PTA1: NE infection\u0026thinsp;+\u0026thinsp;250 mg/kg tannic acid; PTA2: NE infection\u0026thinsp;+\u0026thinsp;500 mg/kg tannic acid; PTA3: NE infection\u0026thinsp;+\u0026thinsp;750 mg/kg tannic acid; PTA4: NE infection\u0026thinsp;+\u0026thinsp;1000 mg/kg tannic acid.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eIntestinal morphology, lesion scores, and determination of cecal\u003c/strong\u003e \u003cspan class=\"BoldItalic\" name=\"Emphasis\" type=\"BoldItalic\"\u003eC. perfringens\u003c/span\u003e \u003cstrong\u003econcentration\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eThe results of the intestinal lesion scoring are shown in Table \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e. While only a few chickens in the NC group showed slight intestinal congestion, all the chickens in the PC group showed gross macroscopic lesions. These included the thinning of the intestinal wall, flatulence, and localized bleeding. Addition of tannic acid improved the intestinal lesions, with a significant reduction in the ileal lesion score on day 28 in the PTA2 group (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e). In this study (Table \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e), NE infection significantly increased the number of \u003cem\u003eC. perfringens\u003c/em\u003e in the cecums of broilers (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e). Addition of tannic acid (500\u0026ndash;1000 mg/kg) significantly reduced the number of \u003cem\u003eC. perfringens\u003c/em\u003e in the cecums of broilers with NE (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e).\u003c/p\u003e\n \u003ctable border=\"1\" id=\"Tab4\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003e\u003cstrong\u003eIntestinal lesion scores and cecum\u003c/strong\u003e \u003cspan class=\"BoldItalic\" name=\"Emphasis\" type=\"BoldItalic\"\u003eC. perfringens\u003c/span\u003e \u003cstrong\u003econcentrations in broilers with NE on 28 days\u003c/strong\u003e\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eNC\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePC\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePTA1\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePTA2\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePTA3\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePTA4\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eP-value\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eLinear\u003csup\u003e3\u003c/sup\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eQuadratic \u003csup\u003e3\u003c/sup\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eIntestinal lesion on d 28\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eDuodenum\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.36\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.57\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.36\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.267\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.064\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.383\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eJejunum\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003csup\u003e***\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.86\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.86\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.65\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.71\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.134\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.018\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.693\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eIleum\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.57\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.71\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.36\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003csup\u003eabc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.29\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.019\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.023\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.443\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eCecum\u003c/strong\u003e \u003cspan class=\"BoldItalic\" name=\"Emphasis\" type=\"BoldItalic\"\u003eC. perfringens\u003c/span\u003e \u003cstrong\u003econcentration\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003ed 28\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.87\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.44\u0026thinsp;\u0026plusmn;\u0026thinsp;0.34\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.053\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.007\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.262\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"10\"\u003e\u003csup\u003e1\u003c/sup\u003eNC versus PC (unpaired t test): *\u003cem\u003e0.05\u0026thinsp;\u0026lt;\u0026thinsp;P\u0026thinsp;\u0026lt;\u0026thinsp;0.10\u003c/em\u003e, **\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e, ***\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u003c/em\u003e. \u003csup\u003e2\u003c/sup\u003eOverall P values obtained from ANOVA; \u003csup\u003e3\u003c/sup\u003e P values obtained using contrast trend analysis; different lower case letters in the same column indicate significant differences (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e), data are the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM, n\u0026thinsp;=\u0026thinsp;7. NC: no tannin acid treatment or NE infection; PC: NE infection\u0026thinsp;+\u0026thinsp;0 mg/kg tannic acid; PTA1: NE infection\u0026thinsp;+\u0026thinsp;250 mg/kg tannic acid; PTA2: NE infection\u0026thinsp;+\u0026thinsp;500 mg/kg tannic acid; PTA3: NE infection\u0026thinsp;+\u0026thinsp;750 mg/kg tannic acid; PTA4: NE infection\u0026thinsp;+\u0026thinsp;1000 mg/kg tannic acid.\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n \u003cp\u003eBroilers in the NC group showed normal intestinal appearance, while those in the PC group showed obvious pathological changes in the villi, which were severely damaged, and the normal structure was destroyed. Intestinal lesions were alleviated by the addition of tannic acid to the feed (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). The ileal villus height and V/C value significantly decreased, and the ileal crypt depth increased in the PC group on day 28 (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e; TAble \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e). On day 28, the ileal villus height was markedly increased in the PTA2\u0026ndash;PTA4 groups, and the ileal crypt depth decreased in the PTA1, PTA3 and PTA4 groups(\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e). In addition, the V/C value significantly increased in the PTA1\u0026ndash;PTA4 groups on day 28 (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e). Seven days after the infection, the ileal crypt depth was still markedly increased and V/C decreased in the PC group (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e; Table \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e). The ileal V/C values of the PTA1\u0026ndash;PTA4 groups were significantly increased (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e), but neither NE nor tannic acid treatment had any effect on the ileal villus height on day 35.\u003c/p\u003e\n \u003ctable border=\"1\" id=\"Tab5\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eEffects of tannic acid on the morphology of the ileal midsection of broiler chickens with NE\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eNC\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePC\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePTA1\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePTA2\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePTA3\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePTA4\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eP-value\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eLinear\u003csup\u003e3\u003c/sup\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eQuadratic \u003csup\u003e3\u003c/sup\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003ed 28\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eVill height \u0026micro;m\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e725.53\u0026thinsp;\u0026plusmn;\u0026thinsp;21.88\u003csup\u003e***\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e627.54\u0026thinsp;\u0026plusmn;\u0026thinsp;13.31\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e661.86\u0026thinsp;\u0026plusmn;\u0026thinsp;27.71\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e754.23\u0026thinsp;\u0026plusmn;\u0026thinsp;28.68\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e769.97\u0026thinsp;\u0026plusmn;\u0026thinsp;17.58\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e753.26\u0026thinsp;\u0026plusmn;\u0026thinsp;23.13\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt; \u0026nbsp;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.045\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eCrypt depth \u0026micro;m\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e108.14\u0026thinsp;\u0026plusmn;\u0026thinsp;3.55\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e123.09\u0026thinsp;\u0026plusmn;\u0026thinsp;5.59\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e107.23\u0026thinsp;\u0026plusmn;\u0026thinsp;4.13\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e113.97\u0026thinsp;\u0026plusmn;\u0026thinsp;4.86\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e106.97\u0026thinsp;\u0026plusmn;\u0026thinsp;2.87\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e109.87\u0026thinsp;\u0026plusmn;\u0026thinsp;2.67\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.059\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.052\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.139\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eV/C\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.72\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17\u003csup\u003e***\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.64\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.86\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003ed 35\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eVill height \u0026micro;m\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e768.86\u0026thinsp;\u0026plusmn;\u0026thinsp;44.27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e738.36\u0026thinsp;\u0026plusmn;\u0026thinsp;56.62\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e753.24\u0026thinsp;\u0026plusmn;\u0026thinsp;31.83\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e739.62\u0026thinsp;\u0026plusmn;\u0026thinsp;28.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e711.84\u0026thinsp;\u0026plusmn;\u0026thinsp;15.71\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e734.47\u0026thinsp;\u0026plusmn;\u0026thinsp;28.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.944\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.658\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.992\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eCrypt depth \u0026micro;m\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e108.26\u0026thinsp;\u0026plusmn;\u0026thinsp;4.29\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e130.62\u0026thinsp;\u0026plusmn;\u0026thinsp;7.04\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e110.8\u0026thinsp;\u0026plusmn;\u0026thinsp;2.86\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e102.9\u0026thinsp;\u0026plusmn;\u0026thinsp;5.17\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e99.31\u0026thinsp;\u0026plusmn;\u0026thinsp;2.95\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e104.97\u0026thinsp;\u0026plusmn;\u0026thinsp;2.92\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.003\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eV/C\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22\u003csup\u003e***\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.79\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.002\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"10\"\u003e\u003csup\u003e1\u003c/sup\u003eNC versus PC (unpaired t test): *\u003cem\u003e0.05\u0026thinsp;\u0026lt;\u0026thinsp;P\u0026thinsp;\u0026lt;\u0026thinsp;0.10\u003c/em\u003e, **\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e, ***\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u003c/em\u003e. \u003csup\u003e2\u003c/sup\u003eOverall P values obtained from ANOVA; \u003csup\u003e3\u003c/sup\u003e P values obtained using contrast trend analysis; different lower case letters in the same column indicate significant differences (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e), data are the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM, n\u0026thinsp;=\u0026thinsp;7. NC: no tannin acid treatment or NE infection; PC: NE infection\u0026thinsp;+\u0026thinsp;0 mg/kg tannic acid; PTA1: NE infection\u0026thinsp;+\u0026thinsp;250 mg/kg tannic acid; PTA2: NE infection\u0026thinsp;+\u0026thinsp;500 mg/kg tannic acid; PTA3: NE infection\u0026thinsp;+\u0026thinsp;750 mg/kg tannic acid; PTA4: NE infection\u0026thinsp;+\u0026thinsp;1000 mg/kg tannic acid.\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003ch3\u003eFecal Coccidia Oocyst Counts\u003c/h3\u003e\n\u003cp\u003eThe coccidia oocyst counts are shown in Table \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e. The coccidia oocysts were not observed in the NC group (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). Addition of tannic acid to the diet linearly reduced the number of fecal coccidia oocysts (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e), and the numbers were significantly lower in the TA2 group at d 26 (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e), TA3 group at d 26\u0026ndash;28 (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e), and TA4 group at d 25\u0026ndash;28 (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e) than those in the PC group.\u0026nbsp;\u003c/p\u003e\n\u003ctable border=\"1\" id=\"Tab6\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 6\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eEffects of tannic acid on coccidia oocysts number in feces of broilers with NE\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePC\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePTA1\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePTA2\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePTA3\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePTA4\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eP-value\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eLinear\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eQuadratic \u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003ed 25\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.69\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.59\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.59\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.49\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0.064\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.008\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.954\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003ed 26\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.78\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.370\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003ed 27\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.63\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.54\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.56\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.49\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.747\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003ed 28\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003eabc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.39\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0.027\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.002\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.789\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"10\"\u003e\u003csup\u003e1\u003c/sup\u003eOverall P-values obtained from ANOVA; \u003csup\u003e2\u003c/sup\u003e P-values obtained using contrast trend analysis; different lower-case letters in the same column indicate significant differences (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e). Data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM; n\u0026thinsp;=\u0026thinsp;7. NC: no tannin acid treatment or NE infection; PC: NE infection\u0026thinsp;+\u0026thinsp;0 mg/kg tannic acid; PTA1: NE infection\u0026thinsp;+\u0026thinsp;250 mg/kg tannic acid; PTA2: NE infection\u0026thinsp;+\u0026thinsp;500 mg/kg tannic acid; PTA3: NE infection\u0026thinsp;+\u0026thinsp;750 mg/kg tannic acid; PTA4: NE infection\u0026thinsp;+\u0026thinsp;1000 mg/kg tannic acid.\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n\u003c/table\u003e\n\u003ch3\u003eImmune And Intestinal Barrier Function\u003c/h3\u003e\n\u003cp\u003eThe changes in the levels of immune indicators in the serum and intestinal mucosa are shown in Table \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e. The results showed that on day 28, the levels of IgM and \u003cem\u003eC. perfringens\u003c/em\u003e-specific antibodies IgY, CRP, and MPO in the serum and sIgA in the ileal mucosa were significantly higher in the PC group than in the NC group (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e). Contrastingly, compared with that in the PC group, the addition of tannic acid to the feed significantly reduced the sIgA content in the ileal mucosa on day 28 (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e). The levels of \u003cem\u003eC. perfringens\u003c/em\u003e-specific antibodies IgY awas significantly lower in the PTA3 group than in the PC group on day 28 (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e), and the levels of \u003cem\u003eC. perfringens\u003c/em\u003e-specific antibodies IgY and MPO were remarkably lower in the PTA4 group than in the PC group on day 28 (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e). On day 35, the levels of CRP and MPO in the serum of the PC group were markedly higher than those in the NC group (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e). Addition of tannic acid had no significant effect on the levels of immunoglobulins, \u003cem\u003eC. perfringens\u003c/em\u003e-specific antibodies IgY in the serum, and sIgA in the ileal mucosa on day 35; however, the serum levels of CRP and MPO were significantly reduced (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e).\u003c/p\u003e\n\u003ctable border=\"1\" id=\"Tab7\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 7\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eEffects of tannic acid on the serum immune indices of chickens with NE\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eNC\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003ePC\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003ePTA1\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003ePTA2\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003ePTA3\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003ePTA4\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eP-value\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eLinear\u003csup\u003e3\u003c/sup\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eQuadratic \u003csup\u003e3\u003c/sup\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003ed 28\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"3\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eIgA (mg/mL)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003e0.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0.29\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.537\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.305\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.851\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eIgM (mg/mL)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003csup\u003e***\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003e0.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.234\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.807\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.163\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eSerum-specific IgY antibody(OD 450 nm)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.79\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003e2.92\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e2.87\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e2.85\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e2.79\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e2.78\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.044\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.003\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.637\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eCRP (\u0026micro;g/mL)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.76\u003csup\u003e***\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003e13.97\u0026thinsp;\u0026plusmn;\u0026thinsp;1.28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e12.64\u0026thinsp;\u0026plusmn;\u0026thinsp;1.20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e12.78\u0026thinsp;\u0026plusmn;\u0026thinsp;0.70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e11.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.63\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e10.77\u0026thinsp;\u0026plusmn;\u0026thinsp;1.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.224\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.024\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.952\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eMPO (pg/mL)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e697.06\u0026thinsp;\u0026plusmn;\u0026thinsp;65.48\u003csup\u003e***\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003e1291.64\u0026thinsp;\u0026plusmn;\u0026thinsp;99.90\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e1305.64\u0026thinsp;\u0026plusmn;\u0026thinsp;109.51\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e1132.20\u0026thinsp;\u0026plusmn;\u0026thinsp;86.60\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e1008.71\u0026thinsp;\u0026plusmn;\u0026thinsp;103.05\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e968.49\u0026thinsp;\u0026plusmn;\u0026thinsp;76.27\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.053\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.004\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.872\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003esIgA (\u0026micro;g/mg prot)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e30.42\u0026thinsp;\u0026plusmn;\u0026thinsp;3.58\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003e52.52\u0026thinsp;\u0026plusmn;\u0026thinsp;8.21\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e34.88\u0026thinsp;\u0026plusmn;\u0026thinsp;3.78\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e35.13\u0026thinsp;\u0026plusmn;\u0026thinsp;4.43\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e28.96\u0026thinsp;\u0026plusmn;\u0026thinsp;3.19\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e28.07\u0026thinsp;\u0026plusmn;\u0026thinsp;2.76\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.009\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.149\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003ed 35\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"3\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eIgA (mg/mL)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.38\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003e0.37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0.36\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0.36\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0.35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0.31\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.956\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.497\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.737\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eIgM (mg/mL)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003e0.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.003\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e0.049\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.881\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.332\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.724\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eSerum-specific IgY antibody (OD 450 nm)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.87\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003e2.88\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e2.92\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e2.88\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e2.88\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e2.90\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.841\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.964\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.993\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eCRP (\u0026micro;g/mL)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.65\u003csup\u003e***\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003e14.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.36\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e11.27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.66\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e10.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.70\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e9.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.87\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e8.59\u0026thinsp;\u0026plusmn;\u0026thinsp;0.80\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.128\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eMPO (pg/mL)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e765.28\u0026thinsp;\u0026plusmn;\u0026thinsp;50.28\u003csup\u003e***\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003e1570.16\u0026thinsp;\u0026plusmn;\u0026thinsp;46.42\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e1009.84\u0026thinsp;\u0026plusmn;\u0026thinsp;60.30\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e973.55\u0026thinsp;\u0026plusmn;\u0026thinsp;65.72\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e871.87\u0026thinsp;\u0026plusmn;\u0026thinsp;53.66\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e852.16\u0026thinsp;\u0026plusmn;\u0026thinsp;57.78\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003esIgA (\u0026micro;g/mg prot)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e33.02\u0026thinsp;\u0026plusmn;\u0026thinsp;4.45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003e38.44\u0026thinsp;\u0026plusmn;\u0026thinsp;4.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e36.82\u0026thinsp;\u0026plusmn;\u0026thinsp;2.67\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e35.72\u0026thinsp;\u0026plusmn;\u0026thinsp;4.90\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e31.56\u0026thinsp;\u0026plusmn;\u0026thinsp;3.13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e29.74\u0026thinsp;\u0026plusmn;\u0026thinsp;1.27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.361\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.046\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.792\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"16\"\u003e\u003csup\u003e1\u003c/sup\u003eNC versus PC (unpaired t test): *\u003cem\u003e0.05\u0026thinsp;\u0026lt;\u0026thinsp;P\u0026thinsp;\u0026lt;\u0026thinsp;0.10\u003c/em\u003e, **\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e, ***\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u003c/em\u003e. \u003csup\u003e2\u003c/sup\u003eOverall P values obtained from ANOVA; \u003csup\u003e3\u003c/sup\u003e P values obtained using contrast trend analysis; different lower case letters in the same column indicate significant differences (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e), data are the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM, n\u0026thinsp;=\u0026thinsp;7. NC: no tannin acid treatment or NE infection; PC: NE infection\u0026thinsp;+\u0026thinsp;0 mg/kg tannic acid; PTA1: NE infection\u0026thinsp;+\u0026thinsp;250 mg/kg tannic acid; PTA2: NE infection\u0026thinsp;+\u0026thinsp;500 mg/kg tannic acid; PTA3: NE infection\u0026thinsp;+\u0026thinsp;750 mg/kg tannic acid; PTA4: NE infection\u0026thinsp;+\u0026thinsp;1000 mg/kg tannic acid.\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n\u003c/table\u003e\n\u003cp\u003eSerum zonulin and ET levels showed consistent results (Table \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003e). NE significantly increased serum zonulin and ET levels (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u003c/em\u003e), while the addition of tannic acid significantly reduced serum zonulin and ET levels (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e). A combined analysis of the zonulin and ET indices showed that the addition of tannic acid improved intestinal barrier function and reduced intestinal permeability.\u0026nbsp;\u003c/p\u003e\n\u003ctable border=\"1\" id=\"Tab8\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 8\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eEffects of tannic acid on serum zonulin and ET in broiler chickens with NE\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eNC\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePC\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePTA1\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePTA2\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePTA3\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePTA4\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eP-value\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eLinear\u003csup\u003e3\u003c/sup\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eQuadratic \u003csup\u003e3\u003c/sup\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003ed 28\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eZonulin ng/L\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1173.49\u0026thinsp;\u0026plusmn;\u0026thinsp;26.79\u003csup\u003e***\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1618.44\u0026thinsp;\u0026plusmn;\u0026thinsp;23.52\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1470.91\u0026thinsp;\u0026plusmn;\u0026thinsp;15.53\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1342.39\u0026thinsp;\u0026plusmn;\u0026thinsp;23.42\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1308.53\u0026thinsp;\u0026plusmn;\u0026thinsp;27.41\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1307.13\u0026thinsp;\u0026plusmn;\u0026thinsp;23.76\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eET EU/L\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e89.04\u0026thinsp;\u0026plusmn;\u0026thinsp;3.15\u003csup\u003e***\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e120.41\u0026thinsp;\u0026plusmn;\u0026thinsp;2.86\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e105.59\u0026thinsp;\u0026plusmn;\u0026thinsp;5.22\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100.74\u0026thinsp;\u0026plusmn;\u0026thinsp;3.54\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e96.05\u0026thinsp;\u0026plusmn;\u0026thinsp;3.19\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e94.51\u0026thinsp;\u0026plusmn;\u0026thinsp;2.92\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.060\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003ed 35\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eZonulin ng/L\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1115.82\u0026thinsp;\u0026plusmn;\u0026thinsp;17.94\u003csup\u003e***\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1570.35\u0026thinsp;\u0026plusmn;\u0026thinsp;25.15\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1446.91\u0026thinsp;\u0026plusmn;\u0026thinsp;21.36\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1378.46\u0026thinsp;\u0026plusmn;\u0026thinsp;15.37\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1316.14\u0026thinsp;\u0026plusmn;\u0026thinsp;27.27\u003csup\u003ecd\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1265.03\u0026thinsp;\u0026plusmn;\u0026thinsp;27.26\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.100\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eET EU/L\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e86.11\u0026thinsp;\u0026plusmn;\u0026thinsp;3.14\u003csup\u003e***\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e115.91\u0026thinsp;\u0026plusmn;\u0026thinsp;3.89\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e99.22\u0026thinsp;\u0026plusmn;\u0026thinsp;3.85\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e102.75\u0026thinsp;\u0026plusmn;\u0026thinsp;2.93\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e81.83\u0026thinsp;\u0026plusmn;\u0026thinsp;2.20\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e94.44\u0026thinsp;\u0026plusmn;\u0026thinsp;4.19\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.014\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"10\"\u003e\u003csup\u003e1\u003c/sup\u003eNC versus PC (unpaired t test): *\u003cem\u003e0.05\u0026thinsp;\u0026lt;\u0026thinsp;P\u0026thinsp;\u0026lt;\u0026thinsp;0.10\u003c/em\u003e, **\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e, ***\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u003c/em\u003e. \u003csup\u003e2\u003c/sup\u003eOverall P values obtained from ANOVA; \u003csup\u003e3\u003c/sup\u003e P values obtained using contrast trend analysis; different lower case letters in the same column indicate significant differences (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e), data are the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM, n\u0026thinsp;=\u0026thinsp;7. NC: no tannin acid treatment or NE infection; PC: NE infection\u0026thinsp;+\u0026thinsp;0 mg/kg tannic acid; PTA1: NE infection\u0026thinsp;+\u0026thinsp;250 mg/kg tannic acid; PTA2: NE infection\u0026thinsp;+\u0026thinsp;500 mg/kg tannic acid; PTA3: NE infection\u0026thinsp;+\u0026thinsp;750 mg/kg tannic acid; PTA4: NE infection\u0026thinsp;+\u0026thinsp;1000 mg/kg tannic acid.\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003emRNA expression of intestinal barrier gene\u003c/strong\u003e, \u003cspan class=\"BoldItalic\" name=\"Emphasis\" type=\"BoldItalic\"\u003eMUC2\u003c/span\u003e, \u003cstrong\u003eand nutrient transport carriers\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe results of intestinal barrier gene and \u003cem\u003eMUC2\u003c/em\u003e expression on day 28 (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ea) showed that the expression of \u003cem\u003eZO-1\u003c/em\u003e in the PC group was significantly reduced (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e) and the mRNA expression of \u003cem\u003eMUC2\u003c/em\u003e and \u003cem\u003eOccludin\u003c/em\u003e was significantly reduced (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u003c/em\u003e) compared to that in the NC group. The addition of tannic acid to the feed significantly increased the expression of \u003cem\u003eOccludin\u003c/em\u003e compared with that in the PC group (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e); \u003cem\u003eMUC2\u003c/em\u003e expression was markedly increased in the PTA1 and PTA2 groups (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e), and \u003cem\u003eZO-1\u003c/em\u003e expression was significantly increased in the PTA3 group (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e). The results for intestinal barrier genes and \u003cem\u003eMUC2\u003c/em\u003e mRNA expression on day 35 (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eb) showed that \u003cem\u003eMUC2\u003c/em\u003e expression in the PC group was still significantly lower than that in the NC group (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e). The expression of \u003cem\u003eMUC2\u003c/em\u003e was remarkably higher in the PTA2 group than in the PC group (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e).\u003c/p\u003e\n\u003cp\u003eRegarding the ileal nutrient transport carriers on day 28 (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ec), the mRNA expression of intestinal \u003cem\u003eI-FABP\u003c/em\u003e and \u003cem\u003ePEPT1\u003c/em\u003e was significantly lower in the PC group (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u003c/em\u003e) and that of \u003cem\u003eSGLT1\u003c/em\u003e was lower in the PC group (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e) than in the NC group. Compared with that in the PC group, the addition of tannic acid to the feed significantly increased the expression of \u003cem\u003eSGLT1\u003c/em\u003e in the PTA1-PTA4 groups(\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e), \u003cem\u003eI-FABP\u003c/em\u003e in the PTA1 group (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e) and \u003cem\u003ePEPT1\u003c/em\u003e in the PTA2 group (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e). The ileal nutrient transport carrier results on day 35 showed (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ed) that the expression of \u003cem\u003ePEPT1\u003c/em\u003e in the PC group was remarkably lower than that in the NC group. The relative mRNA expression of \u003cem\u003eSGLT1\u003c/em\u003e was significantly higher in the PTA2 group than in the PC group on day 35 (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e). Neither NE infection nor tannic acid addition significantly affected the expression of \u003cem\u003eGLUT2\u003c/em\u003e (\u003cem\u003eP\u0026thinsp;\u0026gt;\u0026thinsp;0.05\u003c/em\u003e).\u003c/p\u003e\n\u003cp\u003eWe found that the expression levels of \u003cem\u003eIL-1\u0026beta;\u003c/em\u003e, \u003cem\u003eTLR2\u003c/em\u003e, and \u003cem\u003eTGF-\u0026beta;\u003c/em\u003e under NE conditions were significantly increased on d 28 (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ee) \u003cem\u003e(P\u0026thinsp;\u0026lt;\u0026thinsp;0.05)\u003c/em\u003e. After tannic acid supplementation, \u003cem\u003eTLR2\u003c/em\u003e mRNA expression levels in the PTA1 group were significantly increased compared with those in the PC group \u003cem\u003e(P\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e). Tannic acid supplementation had no significant effect on cytokine expression levels. On d 35 (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ef), compared with those in the NC group, \u003cem\u003eIL-1\u0026beta;\u003c/em\u003e mRNA expression levels were significantly increased in the PC group \u003cem\u003e(P\u0026thinsp;\u0026lt;\u0026thinsp;0.05)\u003c/em\u003e, but tannic acid supplementation had no significant effect on cytokine expression levels.\u003c/p\u003e\n\u003ch3\u003eIleal Microbiological Analysis\u003c/h3\u003e\n\u003cp\u003eTo study the effect of tannic acid on the microbiota of the ileal midsection of broilers infected with NE, the changes in the ileal microorganisms were compared among the NC, PC, and PTA3 groups. There were 400 unique OTUs in the NC group, 288 in the NE group, and 183 in the PTA3 group (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ea). Alpha diversity was measured using the ACE, Chao1, Simpson, and Shannon indices. Figure \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eb shows that NE did not significantly affect the microbial alpha diversity in mid-ileum microorganisms. The ACE and chao1 indices were significantly reduced after the addition of tannic acid (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u003c/em\u003e), but there were no significant differences in the Shannon or Simpson indices. The principal component analysis of ileal microorganisms is shown in Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ec. As displayed in Table \u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003e, the results showed that there were remarkable differences in the composition and structure of the ileal microbiota between these groups (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u003c/em\u003e).\u0026nbsp;\u003c/p\u003e\n\u003ctable border=\"1\" id=\"Tab9\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 9\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eANOSIM for flora composition among the three treatments\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTreatment\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eR value\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eP-value\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eNC-PC\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.799\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.003\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003ePC-PTA3\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.238\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.008\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eR values range from \u0026minus;\u0026thinsp;1 to 1. Differences between groups were significant for R values\u0026thinsp;\u0026gt;\u0026thinsp;0, and differences within groups were greater than those between groups for R values\u0026thinsp;\u0026lt;\u0026thinsp;0. \u003cem\u003eP\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.05 indicates significant differences.\u003c/p\u003e\n\u003cp\u003eAt the phylum level (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ea, b), NE infection significantly reduced the relative abundance of \u003cem\u003eAnaerobacteria\u003c/em\u003e and increased the ratio of \u003cem\u003eFirmicutes/Bacteroides\u003c/em\u003e (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e). At the genus level (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ec, d), \u003cem\u003eLigilactobacillus\u003c/em\u003e abundance was significantly reduced in the PC group (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u003c/em\u003e), and addition of tannic acid significantly increased the relative abundance of \u003cem\u003eAnaerocolumna\u003c/em\u003e, \u003cem\u003eThermoanaerobacterium\u003c/em\u003e, and \u003cem\u003eThermosinus\u003c/em\u003e (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u003c/em\u003e). The effects of challenge on the intestinal \u003cem\u003eLactobacillus\u003c/em\u003e-associated flora were more pronounced at the species level (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ee, f), with the abundance of \u003cem\u003eLactobacillus salivarius\u003c/em\u003e flora in the PC group significantly reduced (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u003c/em\u003e); addition of tannic acid significantly increased the relative abundance of \u003cem\u003eThermosinus carboxydivorans\u003c/em\u003e (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u003c/em\u003e).\u003c/p\u003e\n\u003ch3\u003ePrediction Of Bacterial Flora Function\u003c/h3\u003e\n\u003cp\u003ePICRUSt analysis was performed online using the microbial genome information from the Kyoto Encyclopedia of Genes and Genomes (KEGG) (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ea, b). This analysis allowed for a comparison of the differences in functional profiles among the groups and revealed significantly different functional clustering of the gene pathways among the NC, PC, and PTA3 groups. The results showed that eight gene pathways differed among the three groups at level 1, mainly involving metabolic and disease-related gene pathways. The gut microbes of the NC group were mainly enriched in biological systems, cellular processes, and metabolic pathways; those of the PC group were mainly enriched in disease and genetic information processing pathways; while those of the PTA3 group were mainly enriched in environmental information processing signaling pathways. The results showed 35 different gene pathways at level 2. Among them, NE infection significantly downregulated the metabolic pathways of carbohydrates, lipids, and amino acids, and the addition of tannic acid upregulated the pathways related to the metabolism of these nutrients to some extent. Furthermore, we found that the metabolic pathways related to energy, nucleotides, cell growth and death, replication and repair, cancer, disease, and disease immunity were significantly elevated in the intestinal flora after challenge and that the addition of tannic acid downregulated these metabolic pathways to a certain extent.\u003c/p\u003e\n\u003ch3\u003eIndicator Correlation Analysis\u003c/h3\u003e\n\u003cp\u003eTo explore the relationship between the effect of tannic acid on the gut microorganisms of broiler chickens with NE and the indices for growth performance and immunity, Pearson correlation analysis was conducted based on the above-shown microbial data. \u003cem\u003eLigilactobacillus\u003c/em\u003e showed a highly significant negative correlation (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u003c/em\u003e, Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e) with IgM levels in the serum and a significant negative correlation (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e) with CRP levels; \u003cem\u003eLactobacillus salivarius\u003c/em\u003e showed a highly significant positive correlation with body weight (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u003c/em\u003e) and a highly significant negative correlation with the F/G, ET, MPO, and zonulin levels (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u003c/em\u003e). The \u003cem\u003eFirmicutes/Bacteroides\u003c/em\u003e ratio exhibited a negative correlation with body weight (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e) and a significant positive correlation with serum ET levels and \u003cem\u003eC. perfringens\u003c/em\u003e-specific antibodies (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e); \u003cem\u003eThermoanaerobacterium\u003c/em\u003e showed a significant positive correlation with serum IgM levels (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e); \u003cem\u003eThermosinus carboxydivorans\u003c/em\u003e and \u003cem\u003eThermosinus\u003c/em\u003e were significantly and positively correlated with \u003cem\u003eGLUT2\u003c/em\u003e expression levels (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e. The number of \u003cem\u003eC. perfringens\u003c/em\u003e in the cecum was significantly positively correlated with CRP, ET, MPO, and Zonulin levels and the F/G ratio (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u003c/em\u003e) and significantly positively correlated with IgY, sIgA, IgM, IL-1\u0026beta; and intestinal lesion score (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e). In addition, the number of \u003cem\u003eC. perfringens\u003c/em\u003e was negatively correlated with body weight (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u003c/em\u003e).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we investigated the modulating effects of different tannic acid additive levels on the intestinal health of broiler chickens co-infected with coccidia and \u003cem\u003eC. perfringens\u003c/em\u003e. NE infection was found to significantly reduce the body weight and F/G of broilers, and their small intestines showed remarkable pathological changes, such as intestinal congestion, red bruises, thinning of the intestinal wall, and intestinal distension. The intestinal section analysis further showed damage to the villi structure in the PC group, which indicated the successful establishment of the necrotizing enteritis model and was consistent with the results of previous studies [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Regarding the intestinal barrier genes as well as nutrient transporters, most mRNA expression in the PC group showed negative effects. Previous studies have reported that NE reduces growth performance and elevated serum ET [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], which is consistent with our findings. In addition, the serum levels of zonulin levels were elevated, further indicating the successful establishment of NE model. Tannic acid addition to the feed had beneficial effects on the broilers with NE and resulted in reduced intestinal lesion scores, increased villus height, and improved intestinal morphology.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEffects of tannic acid addition to the diet on the growth performance of broiler chickens with NE\u003c/b\u003e \u003c/p\u003e \u003cp\u003eResults showed that tannic acid in the feed could ameliorate the growth performance degradation caused by NE, which is consistent with the results of previous studies. Yang et al. observed better F/G ratios when broilers were fed 7.5 to 15 mg/kg proanthocyanidins [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Wang et al. investigated the effects of addition of 5\u0026ndash;80 mg/kg grape seed extracts to the coccidia-challenged diet and found that 10\u0026ndash;20 mg/kg significantly improved the growth performance [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]; these results were consistent with those of this study. However, some studies have shown that adding 20 g/kg tannic acid significantly reduces body weight gain and the feed conversion efficiency in broilers [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].Jamroz et al. added 0.025\u0026ndash;0.1% sweet chestnut tannin to the diet and found that \u0026lt;\u0026thinsp;0.05% had no effect on growth performance, whereas 0.1% significantly reduced growth performance [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. These findings suggest that the biological effects of tannins are highly dose-dependent, and the inconsistent effects of tannins in the literature may be due to differences in their source, dose, extraction process, encapsulation, and basal diets. In the present study, the growth performance showed a quadratic curve that increased with the addition of tannic acid; 500\u0026ndash;750 mg/kg tannic acid was found to be the optimal concentration for improving the growth performance. The reason dietary tannic acid improves the growth performance of NE may be that it enhances the intestinal barrier, improves intestinal morphology, balances microbiota, and improves nutrient absorption capacity.\u003c/p\u003e\n\u003ch3\u003eEffects Of Tannic Acid On The Intestinal Morphological Structures Of Broiler Chickens With Ne Infection\u003c/h3\u003e\n\u003cp\u003eA normal villus morphological structure is a prerequisite for the intestine to perform its absorption function. An increase in villus height may lead to enhanced nutrient absorption, and a lower crypt depth indicates a decrease in the metabolic cost of intestinal epithelial renewal. Our findings suggested that NE significantly increased crypt depth, indicating that there was faster tissue renewal for the villi, as needed in response to the inflammation caused by pathogens or their toxins, which is consistent with previous findings [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. The addition of tannic acid significantly reduced the increase in the depth of intestinal crypts caused by NE and improved the V/C ratio, demonstrating the beneficial effects of tannic acid on the intestinal morphology. The V/C showed a quadratic curve change with the addition of tannic acid, which is consistent with our results regarding the growth performance, as 500\u0026ndash;1000 mg/kg tannic acid was highly effective at ameliorating the intestinal morphology.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEffects of tannic acid on the fecal coccidia oocyst excretion in broiler chickens with NE infection\u003c/b\u003e \u003c/p\u003e \u003cp\u003eExamining coccidia oocyst shedding is an effective method to determine the level of coccidia infection [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The addition of tannic acid reduced the number of coccidia oocysts shed compared to that in the PC group, which is consistent with the results of previous reports [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. The reduction in the number of oocysts in the feces indicated an increase in coccidia resistance in broilers. In the present study, the amount of excreted coccidia oocysts decreased linearly with an increase in tannic acid concentration. The decrease in the number of coccidia oocysts after tannic acid addition could be attributed to the modulation of intestinal microorganisms by tannic acid and its own antiparasitic biological properties [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Taken together, tannic acid supplementation reduced coccidia proliferation, significantly reduced the number of coccidia oocysts excreted in feces, accelerated coccidia clearance, and improved intestinal health.\u003c/p\u003e\n\u003ch3\u003eEffect Of Tannic Acid On The Immune Indexes In Broiler Chickens With Ne\u003c/h3\u003e\n\u003cp\u003eIn this study, NE infection resulted in impaired intestinal morphology and elevated lesion scores, whereas the addition of tannic acid to the feed alleviated these changes. This process was accompanied by changes in the serum levels of IgM, MPO, CRP and \u003cem\u003eC. perfringens\u003c/em\u003e-specific antibodies in broilers. IgM is a circulating antibody secreted by B cells that first responds to initial encounters with foreign antigens; however, IgM concentrations in the blood rapidly decline due to clearance, which is consistent with our study findings, as IgM levels were found to be elevated during the first days of infection with NE and did not change significantly seven days after the infection. CRP is an acute temporal protein that activates complement and enhances phagocytosis, and MPO is mainly found in myeloid cells; both of these are markers of the inflammatory response. In this study, the increased MPO and CRP activities in the serum of the PC group indicated that NE infection could activate immune cells in the blood and promote inflammation. The addition of tannic acid to the feed reduced the inflammatory response caused by NE and the concentration of \u003cem\u003eC. perfringens\u003c/em\u003e-specific antibodies in the serum, which may be related to the anti-inflammatory and proliferation inhibitory properties of tannic acid [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. The results of this experimental study showed that NE infection exerted long-term effects on broiler chickens, including reduced intestinal barrier function seven days after infection, continued high levels of MPO and CRP in the serum, and inferior growth performance compared to those in the NC group.\u003c/p\u003e \u003cp\u003eIn innate immunity, the intestinal mucosa is considered the first line of defense against pathogen infection, and mucosal immunity plays an important role in NE. The results of the present study showed that NE infection significantly damaged the ileum and stimulated the expression of sIgA in the intestinal mucosa, which is consistent with the results of a previous study [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. We found that tannic acid reduced the sIgA secretion in the ileal mucosa induced by NE, which might be because tannic acid significantly reduced the number of coccidian merozoites and the colonization of \u003cem\u003eC. perfringens\u003c/em\u003e. In addition, we observed that TLR2 was activated and inflammatory factor expression was increased in NE, consistent with previous studies [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. We found that tannic acid addition reduced the number of \u003cem\u003eC. perfringens\u003c/em\u003e and coccidia in the intestinal tract by stimulating TLR2 and thus activating the immune system, on the one hand, and by inhibiting the number of \u003cem\u003eC. perfringens\u003c/em\u003e and coccidia, on the other hand, thus reducing the inflammatory response.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEffects of tannic acid addition to the feed on mRNA expression of the gut barrier genes and nutrient transport carriers in chickens with NE\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe intestine plays key roles in defense against pathogens, host nutrient digestion, and absorption [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Numerous studies have shown that the mRNA expression of tight junctions and nutrient transport carriers decreases and nutrient digestion and absorption are diminished during intestinal inflammation [\u003cspan additionalcitationids=\"CR30 CR31\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Intestinal inflammation results in a reduction in mucin synthesis and the number of cupped cells, increasing the chances of further intestinal infection and bacterial translocation. In this study, NE led to a reduction in the mRNA expression of \u003cem\u003eOccludin\u003c/em\u003e, \u003cem\u003eZO-1\u003c/em\u003e, and \u003cem\u003eMUC2\u003c/em\u003e, causing an increase in intestinal permeability. However, the mRNA expression of intestinal tight junctions and \u003cem\u003eMUC2\u003c/em\u003e was increased with the addition of tannic acid compared to that in the PC group, indicating the positive effects of tannic acid in protection of chickens from NE.\u003c/p\u003e \u003cp\u003eZonulin is the only known regulator of tight intercellular junctions and is involved in the regulation of intestinal barrier function. Studies have also shown that zonulin levels in the serum are remarkably elevated in patients with irritable bowel syndrome or Crohn\u0026rsquo;s disease [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. However, the changes in the sensitivity to zonulin in poultry intestinal diseases require further investigation. In the present study, NE led to a significant increase in zonulin levels in the serum, indicating that the intestinal barrier permeability was increased. ET is a gram-negative bacterial cell wall component, and serum ET levels are elevated in broiler chickens suffering from NE [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], which was also confirmed in our study. The addition of tannic acid to the feed increased the mRNA expression of \u003cem\u003eOccludin\u003c/em\u003e, \u003cem\u003eZO-1\u003c/em\u003e, and \u003cem\u003eMUC2\u003c/em\u003e and improved the integrity of the intestinal barrier, thereby reducing zonulin and ET levels. Nevertheless, the upregulation of intestinal tight junction-related pathways by tannins requires further investigation.\u003c/p\u003e \u003cp\u003e \u003cem\u003eI-FABP\u003c/em\u003e is a cytoplasmic protein located in mature cells at the top of intestinal villi, and it plays an important role in fatty acid uptake and metabolism. We observed that the mRNA expression of \u003cem\u003eI-FABP\u003c/em\u003e was reduced in broiler chickens infected with NE, which was consistent with the results of a previous study [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. However, it has also been shown that the mRNA expression of \u003cem\u003eI-FABP\u003c/em\u003e does not change significantly in the dexamethasone-induced intestinal disorder model or infection with \u003cem\u003eCampylobacter jejuni\u003c/em\u003e [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e], which might be due to the different disease models and severity. In addition, we observed a decrease in the mRNA expression of \u003cem\u003ePEPT1\u003c/em\u003e and \u003cem\u003eSGLT1\u003c/em\u003e in broiler chickens infected with NE, which is consistent with the results of a previous study [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Addition of tannic acid improved the mRNA expression of nutrient transporter carriers and reduced the negative effects of NE on nutrient absorption in chickens. A comprehensive analysis revealed that growth performance, intestinal villus height, crypt depth, V/C, and nutrient transporter expression showed relatively consistent quadratic curve changes after tannic acid was added to the diet. The reason for this is that lower doses of tannins can reduce gastrointestinal peristalsis, thereby increasing the residence time of chyme in the gastrointestinal tract and improving nutrient digestibility and intestinal health. However, high doses of tannins exert a negative impact on growth performance by binding starch and protein in the feed, reducing digestive enzyme activity, and binding to intestinal wall proteins [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eEffect Of Tannic Acid On The Ileal Microflora Of Broilers With Ne\u003c/h3\u003e\n\u003cp\u003eThe intestinal microbiota forms a highly complex microecosystem. To further investigate the mechanisms by which tannic acid alleviates the intestinal damage caused by NE, we analyzed the microflora structure of the ileal microorganisms using 16S rRNA sequencing. The results of this study showed that the challenge did not have a significant effect on the microbial α-diversity, which is consistent with the results of previous studies [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. We speculated that the possible reason for this result was that NE infection inhibited the proliferation of minor microorganisms in the broiler intestinal flora, which resulted in a convergence with the α-diversity of the NC group microbiota. However, NE has also been shown to result in lower or higher gut microbial α diversity [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e], which may be related to the collection of different parts of the chyme. In the present study, addition of tannic acid significantly reduced the α-diversity but did not affect the balance abundance of the flora, which is consistent with a previous study [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. It has been shown that low-abundance microbes lead to the formation of simpler metabolic networks, resulting in increased concentrations of specific components used to support host energy requirements [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Shabat et al. reported that a reduced microbiome abundance may be closely associated with an increased feed conversion efficiency, with specific enrichment and metabolic pathways of microbes leading to better energy and carbon delivery to the animal body [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e], which could be one of the reasons tannic acid improves growth performance. The significant difference in β-diversity among the three groups indicated that the addition of tannins or challenges significantly altered the microbial community structures. \u003cem\u003eBacteroidetes\u003c/em\u003e are an important contributor to intestinal health, and this phylum plays an important role in breaking down complex molecules into simpler compounds and producing short-chain fatty acids that are beneficial for increased growth performance [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. We found that NE led to a decrease in the abundance of \u003cem\u003eBacteroides\u003c/em\u003e, which is consistent with the results of a previous study [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. The fermentation products of \u003cem\u003eBacteroidetes\u003c/em\u003e, such as \u003cem\u003eBacteroides fragilis\u003c/em\u003e, inhibit \u003cem\u003eC. perfringens\u003c/em\u003e spore formation, and the decrease in \u003cem\u003eBacteroidetes\u003c/em\u003e may predispose the animal intestine to \u003cem\u003eC. perfringens\u003c/em\u003e infection and gastroenteritis [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e], which may be one of the reasons for the decrease in \u003cem\u003eBacteroidetes\u003c/em\u003e abundance in NE. Lately, several studies have suggested that stressed birds present a significantly increasing trend for the \u003cem\u003eFirmicutes/Bacteroides\u003c/em\u003e ratio [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. In this study, we found that the high \u003cem\u003eFirmicutes/Bacteroidetes\u003c/em\u003e ratio caused by NE may be one of the reasons for dysbiosis of the gut microbial community.\u003c/p\u003e \u003cp\u003eThe results also showed that at the genus level, NE significantly reduced the abundance of the beneficial bacterium \u003cem\u003eLigilactobacillus\u003c/em\u003e, suggesting that NE infection inhibited the growth of beneficial intestinal bacteria. At the species level, NE significantly reduced the relative abundance of \u003cem\u003eLactobacillus salivarius\u003c/em\u003e, which is a probiotic in poultry that dates back more than 15 years. Furthermore, this bacterium can re-establish the proper microbial balance of bacteria by forming lactic and propionic acid, stimulate butyric acid-producing butyric acid production, and inhibit the production of pro-inflammatory cytokines [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. In broilers, \u003cem\u003eLactobacillus_salivarius\u003c/em\u003e was found to improve growth performance, reduce the expression of inflammatory factors, and improve intestinal health [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. In conclusion, in this study, we found that the intestinal flora of broiler chickens infected with NE was disturbed and the abundance of beneficial bacteria was reduced, leaving the intestine in an unhealthy state. The addition of tannic acid after the challenge significantly increased the relative abundance of the genus \u003cem\u003eAnaerocolumna\u003c/em\u003e, which belongs to the \u003cem\u003eLachnospiraceae\u003c/em\u003e and can be metabolized by the fermentation of carbohydrates into acetate [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. In addition, the relative abundance of \u003cem\u003eThermoanaerobacterium\u003c/em\u003e, which can secrete xylanase, degrade hemicellulose and even cellulose, and produce acetate and butyric acid via fermentation using xylan and others, was increased [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. The genus \u003cem\u003eThermosinus\u003c/em\u003e includes gram-negative bacteria that can convert CO to carbon dioxide through a series of chemical reactions and can decompose organic substrates (glucose, sucrose, or lactose) to produce acetic acid as well as acetate, H\u003csub\u003e2\u003c/sub\u003e, and CO\u003csub\u003e2\u003c/sub\u003e during glucose fermentation [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. Thus, it regulates the intestinal pH, nourishes butyric acid-producing bacteria, protects against pathogens, and contributes to intestinal health. In conclusion, the addition of tannins increases the abundance of short-chain fatty acid-producing-related flora, which may be one of the reasons for their beneficial impacts on intestinal health.\u003c/p\u003e \u003cp\u003eIn the PICRUSt analysis, we found that differences in the metabolic pathways were the most common in this study, especially in the PC group, where there were large metabolic differences when compared with the other two groups. The present study showed that nutrient metabolism in the intestinal flora of broilers with NE was generally downregulated, and the pathways related to intestinal flora metabolism were more enriched in the NC group, which was consistent with previous findings [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. \u003cem\u003eBacteroidetes\u003c/em\u003e are the main carbohydrate-degrading bacteria in the intestine that contribute to the degradation of complex pectins. Therefore, the enrichment of the carbohydrate metabolic pathway in the NC group may be due to the high abundance of \u003cem\u003eBacteroidetes\u003c/em\u003e, which degrade complex polysaccharides and indigestible carbohydrates to generate short-chain fatty acids and maintain intestinal health [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. Microbial catabolism of amino acids produces a large number of byproducts, including amines, phenols, indoles, and short-chain fatty acids [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e], which can have beneficial or detrimental effects on epithelial cells, depending on the concentration of metabolic byproducts in the lumen. For example, aromatic amino acids are metabolized by colonies to produce tryptamine and indoles. Indoles play an important role in host defense by enhancing intestinal barrier function and downregulating the proinflammatory cytokines [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. We speculated that the healthier gut in the NC group may be related to the products of carbohydrate and amino acid metabolism formed by the intestinal flora. \u003cem\u003eBacteroides\u003c/em\u003e, \u003cem\u003ePropionibacterium\u003c/em\u003e, \u003cem\u003eFusobacterium\u003c/em\u003e, \u003cem\u003eLactobacillus\u003c/em\u003e, and \u003cem\u003eStreptococcus\u003c/em\u003e play important roles in the protein hydrolysis process [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e], and the downregulation of amino acid metabolic pathways in the PC group of microorganisms may be due to a decline in \u003cem\u003eBacteroides\u003c/em\u003e and \u003cem\u003eLactobacillus\u003c/em\u003e abundance. In the PC group, genes related to disease immunity, metabolic diseases, and energy metabolism were upregulated, which suggested that during the infection of chickens with \u003cem\u003eC. perfringens\u003c/em\u003e, the intestinal flora competed with the host for energy, and more energy allocation was required for stimulation of the immune system, which may have resulted in the reduction in chicken growth performance. According to this study, the microbial communities in normal chickens were relatively healthier, and the ileal flora structure and function of the tannic acid groups were predicted to be similar to those of the NC group chickens. Therefore, we conclude that the addition of tannic acid could alleviate ileal microflora disorder in broiler chickens with NE. Taken together, the mechanism underlying the effect of tannic acid on intestinal health may involve changes in the intestinal microflora; however, relevant metabolomic analyses are still required to explore the effects of tannic acid on metabolites, which can further help us to establish the relationship among tannic acid, intestinal flora, metabolites, and intestinal health.\u003c/p\u003e \u003cp\u003eTo investigate the correlation between intestinal flora and growth performance, immunity, and mRNA expression of intestinal barrier genes and nutrient transport carriers, simple correlation analysis was performed. Correlation analysis can indicate the correlation between indicators but cannot determine a causal relationship. The results suggested that the abundance of \u003cem\u003eLactobacillus salivarius\u003c/em\u003e was positively correlated with growth performance and significantly negatively correlated with MPO, CRP, ET, and zonulin, which may be because \u003cem\u003eLactobacillus salivarius\u003c/em\u003e can inhibit inflammation [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e, \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e] and improve intestinal barrier function [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. The increase in the abundance of \u003cem\u003eThermosinus carboxydivorans\u003c/em\u003e and \u003cem\u003eThermosinus\u003c/em\u003e after the addition of tannic acid was significantly and positively correlated with the mRNA expression of \u003cem\u003eGLUT2\u003c/em\u003e, which suggested that the regulation of intestinal flora by tannic acid may be related to nutrient translocation and absorption. However, the specific mechanisms require further investigation.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThe present study demonstrated that tannic acid supplementation is apparently effective in reducing NE in broiler chickens. The protective effects of dietary tannic acid supplementation against NE challenge were evidenced by the suppression of coccidia and \u003cem\u003eC. perfringens\u003c/em\u003e colonization and invasion, regulation of intestinal flora, alleviation of inflammatory reaction, and improvement in intestinal barrier function and nutrient transport. As the amount of additive tannic acid increases, the body weight, mRNA expression of nutrient transport carriers, and villus height, V/C also increase in the form of a quadratic curve, while the intestinal barrier-related indices (Zonulin and ET content in serum), coccidia oocyst counts, cecum \u003cem\u003eC. perfringens\u003c/em\u003e concentration, and immune indices in serum decrease linearly. After comprehensive consideration of the results, we recommend tannic acid supplementation to broiler feed at a dose of 500\u0026ndash;750 mg/kg.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eBW: Body weight;CRP: C-reactive protein; ELISA: Enzyme-linked immunosorbent assay; ET: Endotoxin; F/G: The ratio of feed intake and body weight gain; GLUT2: Recombinant glucose transporter 2; I-FABP: Intestinal fatty acid binding protein; IgA: Immunoglobulin A; IgM: Immunoglobulin M; MPO: Myeloperoxidase; NE: Necrotic enteritis; PEPT1: Peptide-transporters 1; sIgA: Secretory immunoglobulin A; SGLT1: Recombinant sodium/glucose cotransporter 1; V/C: The ratio of villus height to crypt depth; ZO-1: Zonula occludens-1\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYG designed the study; HX, JF, YL, PL, BS, and ZL performed the experiments; HX analyzed the data and wrote the manuscript; and YG revised the manuscript. All authors contributed to the data interpretation and approved the final version of the manuscript.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are available from the corresponding author upon request. Datasets supporting the conclusions of this study are included in this article. The 16S gene sequencing data can be obtained from the following website: https://www.ncbi.nlm.nih.gov/sra/PRJNA897828; the accession number is PRJNA897828.\u0026nbsp;\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.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll experiments were approved by the Institutional Animal Care and Use Committee of the China Agricultural University. The animal welfare number is AW11112022-1-4.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the China Agriculture Research System program (CARS-41-G11).\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors are grateful to the staff of the Department of Animal Science and Technology of the China Agricultural University, especially those involved in animal nutrition, for their valuable assistance in conducting the experiments.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eVan Immerseel F, Rood JI, Moore RJ, Titball RW. Rethinking our understanding of the pathogenesis of necrotic enteritis in chickens. 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J Anim Sci Biotechnol. 2020;11:76. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/s40104-020-00488-5\u003c/span\u003e\u003cspan address=\"10.1186/s40104-020-00488-5\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-animal-science-and-biotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jasb","sideBox":"Learn more about [Journal of Animal Science and Biotechnology](http://jasbsci.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/jasb/default.aspx","title":"Journal of Animal Science and Biotechnology","twitterHandle":"@animalplantsci","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"tannic acid, necrotic enteritis, immunity, intestinal health","lastPublishedDoi":"10.21203/rs.3.rs-2283343/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2283343/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eIn broiler chickens, necrotic enteritis (NE) infection can reduce production performance. Tannic acid, as a kind of plant extract, has received extensive attention. However, the appropriate dosage of tannic acid in NE of broilers and the improvement effect on intestinal health are not very clear. In this study, we aimed to investigate the effects of different doses of tannic acid on the production performance, immunity, and intestinal health of broilers by constructing an NE model with \u003cem\u003eC. perfringens\u003c/em\u003e infection and determining the appropriate dosage of tannic acid with regard to NE.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eChallenged birds showed significant reduction in body weights, villus heights, and the ratio of villus height to crypt depth (V/C) (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e) and increase in the feed consumption gain ratio, intestinal lesion score, and crypt depth (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e). NE infection significantly reduced the relative \u003cem\u003eBacteroides\u003c/em\u003e and \u003cem\u003eLigilactobacillus\u003c/em\u003e abundance (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e) and increased the ratio of \u003cem\u003eFirmicutes/Bacteroides\u003c/em\u003e and cecal content of \u003cem\u003eC. perfringens\u003c/em\u003e (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e). Challenged birds fed diets supplemented with tannic acid showed significantly increased mRNA expression of nutrient transport carriers and intestinal barrier genes and growth performance and reduced serum zonulin and endotoxin levels (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e). Addition of tannic acid to the diet inhibited the inflammatory response by reducing the number of coccidia oocysts in feces and the content of \u003cem\u003eC. perfringens\u003c/em\u003e in the cecum. Specifically, tannin acid reduced the serum levels of C reactive protein, myeloperoxidase, and specific IgY and ileal mucosal secretory immunoglobulin A (sIgA) levels in the ileal mucosa compared with those in the NE-infected birds. NE-infected birds fed diets supplemented with tannin acid also showed significantly increased relative \u003cem\u003eAnaerocolumna\u003c/em\u003e, \u003cem\u003eThermoanaerobacterium\u003c/em\u003e, and \u003cem\u003eThermosinus\u003c/em\u003e abundance (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e); their microbial composition and functional predictions were similar to those of the NC group.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eTannic acid in the diet alleviated NE by enhancing the intestinal barrier and absorption function. The recommended dietary tannic acid additive level is 500\u0026ndash;750 mg/kg. Our study findings would be useful in reducing related economic losses in the broiler industry.\u003c/p\u003e","manuscriptTitle":"Effects of tannic acid on the immunity and intestinal health of broiler chickens with Clostridium perfringens-induced necrotic enteritis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-11-23 20:02:43","doi":"10.21203/rs.3.rs-2283343/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2022-12-13T22:06:11+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2022-11-21T08:28:44+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2022-11-21T08:04:52+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2022-11-17T10:39:20+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Animal Science and Biotechnology","date":"2022-11-17T03:23:55+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-animal-science-and-biotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jasb","sideBox":"Learn more about [Journal of Animal Science and Biotechnology](http://jasbsci.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/jasb/default.aspx","title":"Journal of Animal Science and Biotechnology","twitterHandle":"@animalplantsci","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e034f362-b572-4952-8832-3f71510a915b","owner":[],"postedDate":"November 23rd, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2023-10-16T20:56:30+00:00","versionOfRecord":{"articleIdentity":"rs-2283343","link":"https://doi.org/10.1186/s40104-023-00867-8","journal":{"identity":"journal-of-animal-science-and-biotechnology","isVorOnly":false,"title":"Journal of Animal Science and Biotechnology"},"publishedOn":"2023-05-04 20:40:30","publishedOnDateReadable":"May 4th, 2023"},"versionCreatedAt":"2022-11-23 20:02:43","video":"","vorDoi":"10.1186/s40104-023-00867-8","vorDoiUrl":"https://doi.org/10.1186/s40104-023-00867-8","workflowStages":[]},"version":"v1","identity":"rs-2283343","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2283343","identity":"rs-2283343","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}