Effects of betaine on ileal tissue and intestinal microbial metabolism in Tibetan sheep

Effects of betaine on ileal tissue and intestinal microbial metabolism in Tibetan sheep | 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 betaine on ileal tissue and intestinal microbial metabolism in Tibetan sheep Wei Gao, Zhenling Wu, Jiacheng Gan, Xianhua Zhang, Chengdi Shi, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7277025/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 04 Feb, 2026 Read the published version in AMB Express → Version 1 posted 11 You are reading this latest preprint version Abstract Research on betaine's role in Tibetan sheep ileal development and the microbiota-metabolite axis remains scarce, and the mechanism by which it enhances intestinal health through its function as a methyl donor has not yet been elucidated. This study evaluated the effects of 0.08% dietary betaine supplementation on 60 weaned male Tibetan lambs (2 months old, with a mean body weight of 17.72 ± 0.19 kg), which were randomly divided into a control group (Ctrl) and a betaine group (Bet), with 30 lambs in each group. After a 10-day adaptation period followed by a 90-day formal feeding period, 6 lambs from each group were randomly selected for slaughter. Results showed that betaine supplementation significantly increased ileal villus height and the villus height-to-crypt depth (VH/CD) ratio ( P < 0.05), enhanced total antioxidant capacity (T-AOC), reduced levels of malondialdehyde (MDA), lipopolysaccharide (LPS), and tumor necrosis factor-α (TNF-α) in the ileum, and increased Claudin-1 levels ( P < 0.05). It also raised total short-chain fatty acids (SCFAs), acetate, and propionate concentrations in the ileum, along with the relative abundance of Bifidobacterium and Aeriscardovia ( P < 0.05), and influenced arginine and proline metabolism as well as glycerophospholipid metabolism to enhance antioxidant and immune functions. 0.08% betaine can regulate ileal SCFA concentrations by modulating microbial composition and metabolic pathways, thereby supporting jejunal barrier function, providing a theoretical basis for its application as a functional feed additive. Betaine Tibetan sheep Ileal Microbiomics Metabolomics Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction The Qinghai-Tibet Plateau is the highest alpine ecological region in the world. Environmental factors such as high altitude, hypoxia, low temperature, strong ultraviolet radiation, and seasonal shortage of forage pose significant challenges to the physiological metabolism and production performance of local ruminants (Li et al., 2021 ).Tibetan sheep ( Ovis aries ) have evolved a variety of adaptive mechanisms during long-term natural selection and domestication, such as reducing basal metabolism, regulating fat mobilization, and modulating oxidative stress, so as to enhance their survival ability in extreme environments (Li et al., 2024 ). However, such adaptations are still accompanied by a decline in production performance, manifested as fluctuations in live weight during the cold season, low feed conversion efficiency, and digestive tract dysfunction, which restrict the benefits of their breeding (Wei et al., 2016 , Xu et al., 2017 ). Recent studies have shown that rumen microorganisms play a key role in energy metabolism and environmental adaptation of Tibetan sheep. Particularly in the cold season, the increased abundance of fiber-degrading bacteria promotes the utilization of cellulose in roughage (Liu et al., 2020 ). However, compared with the rumen, little is known about the response mechanism of the small intestine, especially the ileum, under plateau stress. As the distal part of the small intestine, the ileum is not only involved in nutrient absorption and bile acid reabsorption but also undertakes important functions of mucosal immunity and microbial interaction. Its structural and functional status is crucial to animal health (Chen et al., 2019 , Sun et al., 2021 ), which urgently requires in-depth research. Betaine, a natural derivative of trimethylglycine (Eklund et al., 2005 ), has dual functions as a methyl donor and an osmoprotectant, and is widely used in nutritional intervention for livestock and poultry (Abd El-Ghany and Babazadeh, 2022 , Yang et al., 2022 , Cheng et al., 2021 ). In animals, it can participate in methionine metabolism (Abd El-Ghany and Babazadeh, 2022 ), choline synthesis (Obeid, 2013 )and DNA methylation (Yang et al., 2020 )by providing methyl groups, which helps regulate lipid metabolism (Yang et al., 2021 ), enhance the antioxidant system (Wen et al., 2021a ), and maintain cellular osmotic homeostasis (Ratriyanto and Mosenthin, 2018 ). Studies have confirmed that betaine can improve intestinal morphology, barrier function, and antioxidant capacity in monogastric animals such as pigs and poultry (Wang et al., 2020 , Li et al., 2021 , Song et al., 2021 ). In ruminants, its application has shown multiple effects including increasing milk production, improving daily weight gain, and optimizing carcass composition. Moreover, meta-analyses have verified its stable synergistic effects under different environmental and breed conditions (Abhijith et al., 2024 ). In addition, betaine can alleviate stress responses by enhancing cellular antioxidant defense under heat stress conditions (Wen et al., 2021b ). Studies on Hu sheep (Dong et al., 2020 ), Merino sheep (DiGiacomo et al., 2023 ), and other breeds have further revealed its potential in regulating metabolic responses under special ecological conditions. However, current research mainly focuses on production performance and overall physiological indicators, and there is a lack of systematic analysis on the regulatory mechanisms of betaine in intestinal tissue, especially the microecology and metabolism of the small intestine. This limits the in-depth application of betaine in precise nutritional regulation. The ileum, as an important part of the small intestine, is not only a key site for terminal nutrient absorption but also a core hub for intestinal immune regulation and microorganism-metabolite interactions (Zhang et al., 2025 , Collins et al., 2024 ) The functional status of the ileum is collectively determined by intestinal mucosal integrity, the number of goblet cells, redox status, and microbial community structure (Yang and Yu, 2021 , Lin et al., 2022 ). Existing studies have shown that specific intestinal metabolites (such as SCFAs, secondary bile acids, etc.) participate in cellular signal transduction by acting on host receptors, exerting profound impacts on intestinal homeostasis (Visekruna and Luu, 2021 , Lin et al., 2023 ). The mechanism of action of betaine in the intestine has gradually attracted attention, including promoting villus development, increasing antioxidant enzyme activity, and regulating microbial diversity, which demonstrates its potential in improving intestinal function (Wang et al., 2018 , Al Sulaiman et al., 2024 ). However, there is currently a lack of systematic research based on the Tibetan sheep model under alpine and plateau ecological conditions to reveal the specific pathways and microscopic mechanisms of betaine in regulating ileal health. Based on this, this study took Tibetan sheep as the research object, combined with feeding and slaughter experiments, and comprehensively used histological observation, antioxidant index analysis, 16S rDNA sequencing and untargeted metabolomics technology to systematically evaluate the regulatory effects of betaine on the ileal structure, function and microorganism-metabolite interaction of Tibetan sheep. It aims to reveal its nutritional intervention mechanism under plateau stress conditions and provide theoretical support for efficient breeding. Materials and Methods This animal study was approved by the Institutional Animal Care and Use Committee of Qinghai University, China (Approval No.: QUA-2020-0710). Both the animal care and experimental protocols were reviewed and approved by the Committee. The study was conducted in compliance with local laws and regulations as well as institutional requirements. Experimental Design A feeding experiment was conducted at a Tibetan sheep breeding base in Qieji Township, Gonghe County, Qinghai Province (36°19′N, 99°41′E) from April 2024 to July 2024. Sixty 2-month-old plateau-type Tibetan sheep rams with similar body weights (17.72 ± 0.19 kg) and good body condition were selected and randomly divided into 2 groups: the control group (Ctrl) and the experimental group (Bet), with 30 sheep in each group (5 replicates, 6 sheep per replicate). The Ctrl group was fed a basal diet, while the Bet group was fed the basal diet supplemented with 0.08% betaine (on an air-dry basis). The basal diet consisted of a concentrate supplement and roughage at a concentrate-to-roughage ratio of 7:3. The roughage was a mixture of oat hay and oat silage in a 1:1 ratio on a dry matter basis. The betaine was provided by Jinan Tai Fei Animal Husbandry Technology Co., Ltd. with a purity of 98%. The composition and nutritional levels of the experimental diets are shown in Table 1 . The total experimental period was 100 days, including a 10-day pre-test period and a 90-day formal test period. Feeding was carried out at 8:00 and 17:00 daily, and the sheep had free access to feed and water during the experiment. The nutritional components in the feed used in this experiment, such as dry matter (Thiex et al., 2002a ), crude protein (Thiex et al., 2002b ), acid detergent fiber (HIfiFLHQF et al.), neutral detergent fiber (Hiraoka et al., 2012 ), Ca and P (Babos et al., 2018 ), were all determined with reference to relevant standards. Table 1 Composition of the basic diet. Item Ctrl Bet Corn 48.40 48.32 Wheat 9.00 9.00 Palm meal 16.00 16.00 Soybean meal 4.00 4.00 Rapeseed meal 15.00 15.00 Nacl 1.00 1.00 Limestone 1.00 1.00 Baking soda 1.00 1.00 1% Premix 1 0.60 0.60 4% Concentrate 4.00 4.00 Betaine 0.00 0.08 Total 100.00 100.00 Nutrient levels 2 Digestible energy (MJ·kg⁻¹) 12.66 12.65 Crude protein 13.69 13.69 Ether extract 4.61 4.61 Crude fiber 21.18 21.16 Neutral detergent fiber 21.18 21.18 Acid detergent fiber 14.72 14.72 Ca 0.85 0.85 P 0.41 0.41 Ctrl, basal diet; Bet, 0.08% betaine is added to the basal diet. 1 The premix provides the following per kilogram of the feed: Cu, 18 mg; Fe, 66 mg; Zn, 30 mg; Mn, 48 mg; Se, 0.36 mg; I, 0.6 mg; Co, 0.24 mg; vitamin A, 24,000 IU; vitamin D, 4,800 IU; vitamin E, 48 IU. 2 Digestible energy is a calculated value, while the others are measured values. Sample Collection At the end of the formal test period, 6 Tibetan sheep were randomly selected from each group and slaughtered at the slaughterhouse (n = 12). The Tibetan sheep were fasted for 12 hours. After slaughter, the intestines were quickly separated, and the ileocecal junction was clamped with hemostatic forceps. The collected ileal tissue samples were about 10 cm away from the ileocecal orifice, with a length of about 1 cm. Meanwhile, 4 mL of ileal contents were collected into a 5 mL cryopreservation tube, quickly frozen in liquid nitrogen, stored and transported in dry ice to the laboratory, and then preserved in a -80℃ refrigerator for subsequent untargeted metabolomics and 16S rDNA sequencing. Finally, the collected tissue samples were rinsed clean with physiological saline and placed in 4% paraformaldehyde for tissue observation. H&E staining The ileal tissues were fixed in 4% paraformaldehyde solution for no less than 48 hours, followed by gradient dehydration with ethanol and paraffin embedding. Finally, sections with a thickness of 3 µm were cut to prepare paraffin sections, which were then stained with hematoxylin-eosin (H&E) for analysis. The indicators measured included villus height (VH), villus width, crypt depth (CD), crypt number, muscular layer thickness, and the VH/CD ratio. A microscope (OLYMPUS, DP26, Tokyo, Japan) was used to observe the stained sections and collect images. Finally, Image-Pro Plus 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA) was employed to measure all indicators of the ileal tissues in the collected images at magnification levels of 500× and 200×. Determination of antioxidant capacity and lipopolysaccharide content The contents of total antioxidant capacity (T-AOC), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT), malondialdehyde (MDA) and the level of lipopolysaccharide (LPS) in ileal tissues were determined using ELISA kits (Shanghai Meilian Biotechnology Co., Ltd., Shanghai, China). Take 0.1 g of ileal tissue and add 900 µL of physiological saline. After crushing with a crusher (SM2000, Retsch, Haan, Germany), transfer to a low-temperature high-speed centrifuge (5430 R, Eppendorf, Hamburg, Germany) and centrifuge at 2500 × g for 15 minutes at 4°C to obtain the supernatant. The determination was carried out strictly in accordance with the steps provided in the kit, and finally the absorbance value was measured at a wavelength of 450 nm on an ELISA Analyzer (ELx808, BioTek, Winooski, VT, USA). Determination of SCFAs The ileal contents were preserved in dry ice and sent to Beijing Novogene Bioinformatics Technology Co., Ltd. (Beijing, China). The composition and content of SCFAs were quantitatively determined using an ultra-high performance liquid chromatography-tandem mass spectrometry system (Vanquish™ Flex UHPLC-TSQ Altis™, Thermo Scientific Corp., Germany). The sample extraction and determination procedures were carried out with reference to the method described by Yan et al. (Yan et al., 2022 ). Real Time Quantitative (RT-qPCR) Total RNA was extracted from ileal tissues using the Transzol Up kit (TRAN, Beijing, China), and its concentration was detected. Subsequently, the RNA was reverse-transcribed into cDNA using a kit. The subsequent fluorescent quantitative steps and qPCR reaction procedures were strictly performed according to the method described by Ji et al (Ji et al., 2024 ). The relative expression level of the target gene mRNA was calculated by the 2 −ΔΔCt method, and the primer sequences are shown in Table 2 . Table 2 Primers used in RT-qPCR. Items 1 Primer sequence (5’-3’) 2 Tm (℃) Product length NCBI Gene ID Claudin-1 F-CCTGCTGTGCTGCTCCTGTC 61.6 75bp 780473 R-GAAGGTGCTGGCTTGGGATAGG 61.4 Occludin F-CGAGAAGCGACCGTATCCAGAG 61.4 129bp 101118304 R-TCCAAGTTACCACTGCTGCTGTAG 59.6 ZO-1 F-GGGCAAGTTAAAGATGGTGGTTCAG 59.6 93bp 443200 R-GAGGCGTCAGCAGAGTGGATG 61.5 TNF-α F-ACGGCGTGGAGCTGAAAGAC 59.5 79bp 443540 R-CTGAAGAGGACCTGCGAGTAGATG 61.3 Muc-2 F-ACGACTCCTACGCCCTCCTG 61.6 130bp 780488 R-ACGCTGCCATCCGACTTGAAG 51.5 IL-6 F-TCTAATAACCACTCCAGCCACACAC 59.6 77bp 443406 R-TTGCGTTCTTTACCCACTCGTTTG 57.9 β-Actin F-AGCAAGCGTGGCATCCTAACC 59.5 87bp 443340 R-ATCTTCTCCATGTCGTCCCAGTTG 59.6 77bp 1 ZO-1, zonula occluden-1; MUC-2, mucoprotein-2; TNF-α, tumor necrosis factor-α; IL-6, interleukin-6. 2 F, forward primer; R, reverse primer. Analysis of microbial composition Microbial DNA in ileal contents was extracted using the DNA extraction kit from Novogene (Beijing, China), and the V3-V4 region of 16S rDNA was amplified by PCR with specific primers 341F (5′-CCTAYGGGRBGCASCAG-3′) and 806R (5′-GGACTACNNGGGTATCTAAT-3′). Sample reads were spliced using FLASH (Version 1.2.11) to generate Raw Tags data. Then, Cutadapt (Version 3.3) software was used to match the reverse primer sequences and remove redundant parts, and fastp (Version 0.23.1) software was employed to obtain Clean Tags (Bokulich et al., 2013 ). Subsequently, the Tags sequences were compared with the species annotation database, and chimeric sequences were detected and removed to obtain Effective Tags (Edgar et al., 2011 ). The SVG function of Perl (Version 5.26.2) software was used to draw the relative abundance distribution histogram. QIIME2 (Version 2022.02) software was used to calculate the Alpha diversity index. Principal coordinate analysis was performed using the ade4 package and ggplot2 package of R (Version 4.0.3) software. Untargeted metabolomics analysis The extraction of metabolites referred to the study by Want et al. (Want et al., 2013 ). The chromatographic column used was Hypersil Gold C18. The off-machine data were processed using Compound Discoverer 3.3 software(Thermo Fisher Scientific Inc., Waltham, Massachusetts, USA), with data processing based on the Linux system (CentOS version 6.6) as well as R (Version 4.0.3) and Python (Version 3.12.1) software. The identified metabolites were annotated using the KEGG database ( https://www.genome.jp/kegg/pathway.html ). For multivariate statistical analysis, after data transformation using metaX software, principal component analysis and orthogonal partial least squares discriminant analysis were performed to obtain VIP values; univariate analysis calculated P values and FC values based on t-tests, and the criteria for screening differential metabolites were VIP > 1, P 1.5. The R package ggplot2 was used to draw match plots and bubble plots, the corrplot package was used for correlation analysis, and the KEGG database was used to study metabolite functions and pathways. The enrichment of metabolic pathways was determined according to x/n > y/n and P < 0.05. Statistical analysis Experimental data were preprocessed using Microsoft Excel 2024, and all data were subjected to independent samples t-test using SPSS 26.0 software (IBM Corp., Armonk, NY, USA). Results were expressed as mean ± standard error. Graphs were plotted using GraphPad Prism 8.0 software, with P < 0.05 set as the criterion for judging significant differences with statistical significance. Wilcoxon rank-sum test was used for data α-diversity index analysis, Unweighted Unifrac distance was used for PCoA analysis, and NMDS was used to reflect the degree of differences between samples. Spearman rank correlation analysis was performed using the R language Psych package and R language Vegan package in R (Version 4.0.3) to calculate the correlations between ileal microbiota abundance, metabolite levels and short-chain fatty acids, antioxidant indices, tissue morphological parameters, ileal barrier function, and lipopolysaccharide levels. Results Histomorphological analysis Hematoxylin-eosin (H&E) stained sections intuitively showed the morphological changes in the ileal tissue of Tibetan sheep (Fig. 1 A). We observed that the ileal villus height and VH/CD ratio in the Bet group were significantly higher than those in the Ctrl group ( P 0.05) (Fig. 1 B). Antioxidant capacity and lipopolysaccharide content In terms of antioxidant capacity (Figs. 2 A-E), compared with the Ctrl group, the Bet group showed higher T-AOC activity ( P < 0.05) and lower MDA content ( P 0.05). Regarding LPS concentration (Fig. 2 F), the LPS level in the Bet group was significantly lower than that in the Ctrl group ( P < 0.05). Content of SCFAs As shown in Table 3 , dietary supplementation with betaine significantly increased the contents of acetic acid and propionic acid in the ileum of Tibetan sheep ( P < 0.05). There were no significant differences in the contents of isobutyric acid, butyric acid, 2-methylbutyrate, isovaleric acid, valeric acid, 2-methylvalerate, 3-methylvalerate, 4-methylvaleric acid, and hexanoic acid between the two groups ( P > 0.05). Table 3 The effects of adding 0.08% betaine on Short-chain Fatty Acids in Tibetan sheep. Items Ctrl Bet P- Value Acetic acid(ng/mL) 145752.63 ± 44506.93 b 527274.20 ± 12849.79 a 0.001 Propionic acid(ng/mL) 5399.60 ± 2262.09 b 43408.56 ± 3061.34 a 0.001 Isobutyric acid(ng/mL) 568.56 ± 76.00 14045.96 ± 5011.35 0.055 Butyric acid(ng/mL) 905.52 ± 387.62 36448.29 ± 10145.49 0.072 2-Methylbutyrate(ng/mL) 281.68 ± 133.19 9868.77 ± 3042.60 0.087 Isovaleric acid(ng/mL) 412.34 ± 219.24 16149.81 ± 5257.94 0.096 Valeric acid(ng/mL) 43.46 ± 12.58 1238.49 ± 435.96 0.111 2-Methylvalerate(ng/mL) 37.00 ± 6.39 24.56 ± 0.09 0.191 3-Methylvalerate(ng/mL) 42.82 ± 14.92 13.49 ± 0.94 0.188 4-Methylvaleric acid(ng/mL) 524.52 ± 352.91 183.46 ± 53.95 0.436 Hexanoic acid(ng/mL) 332.61 ± 40.28 386.69 ± 22.55 0.306 Ctrl, basal diet; Bet, 0.08% betaine is added to the basal diet. Data are expressed as mean ± SEM. If the superscripts of data in the same row are a and b respectively, it indicates that there is a significant difference between their means ( P < 0.05). Expression of ileal barrier-related genes Compared with the Ctrl group, dietary supplementation with betaine significantly increased the expression level of Claudin-1 gene in the jejunum of Tibetan sheep ( P < 0.05) and significantly decreased the expression level of TNF-α gene ( P 0.05) (Fig. 3 ). Microbiota As shown in the Venn diagram of Fig. 4 A, there were 1810 OTUs of ileal bacteria in Tibetan sheep of the control group, while 2509 OTUs were detected in those supplemented with betaine. Among these OTUs, 800 were shared by both groups. The number of ileal OTUs in Tibetan sheep supplemented with betaine was higher than that in the non-supplemented group. Principal Coordinate Analysis (PCoA) and Non-metric Multidimensional Scaling (NMDS) further revealed that the ileal bacterial communities of Tibetan sheep in the two groups clustered independently of each other (Fig. 4 B). In addition, there were no statistically significant differences in the Chao1 and Simpson indices, which reflect bacterial abundance and diversity, between the two groups ( P > 0.05) (Fig. 4 C). To better evaluate the effect of dietary betaine supplementation on the composition of the ileal bacterial community, we analyzed the percentage abundances at the phylum and genus levels. At the phylum level, Firmicutes , Euryarchaeota , and Actinobacteriota were the dominant groups, accounting for 97.61% (Ctrl) and 95.34% (Bet) of the total ileal bacteria, respectively (Fig. 4 D). At the genus level, the dominant genera in the Bet group included Acetitomaculum , Olsenella , and Christensenellaceae_R_7_group (Fig. 4 E). Among them, the relative abundances of Aeriscardovia and Bifidobacterium in the Bet group were significantly higher than those in the Ctrl group ( P < 0.05) (Fig. 4 F). As shown in Fig. 4 G, Acetitomaculum taxonomically belongs to Firmicutes with a high abundance, and it is associated through hierarchical branches such as Clostridia and Lachnospirales . Actinobacteriota is associated with Olsenella and Aeriscardovia , while Euryarchaeota is linked to Methanobrevibacter through methanogenic groups. Untargeted metabolomics A total of 847 metabolites were identified, which could be classified into Lipids and lipid-like molecules (44.51%), Organic acids and derivatives (20.54%), Organoheterocyclic compounds (11.92%), and Benzenoids (6.02%) (Fig. 5 A). The OPLS-DA score plot showed differences in ileal metabolites between the two groups of Tibetan sheep (Fig. 5 B). The OPLS-DA model revealed that the R2Y, R2X, and Q2Y values of the two groups were 0.997, 0.732, and 0.985, respectively, indicating that the model had strong explanatory power and significant clustering effect. Figure 5 C showed that the Q2 intercept of the regression line of the permutation test model was less than 0, indicating no overfitting. We used matchstick plots to display the differences in metabolites between the two groups of samples. A total of 14 metabolite markers were identified in the Ctrl and Bet groups (Fig. 5 D). Supplementary Table 1 showed the upregulated (LPA 22:5, PC 16:0_17:2, and L-arginine) and downregulated (Cer 18:1;2O/18:0, Cer 18:1;2O/24:2, SPB 19:0;2O, and (±)13-HpODE) metabolites in Ctrl vs. Bet. Through KEGG pathway enrichment analysis, 5 pathways including Fat digestion and absorption, Gap junction, ABC transporters, Arginine and proline metabolism, and Glycerophospholipid metabolism showed significant differences (Fig. 5 E). A variety of differential metabolites including LPA 22:5, PC 16:0_17:2, Cer 18:1;2O/18:0, Cer 18:1;2O/24:2, and L-arginine were mapped to the significantly different metabolic pathways. Correlation analysis The Spearman coefficient model was used to evaluate the correlations between ileal development indices, antioxidant capacity, lipopolysaccharide content, short-chain fatty acid content, metabolites, and the diversity and richness of the microbial community. The study found that Aeriscardovia was positively correlated with T-AOC, and negatively correlated with MDA, TNF-α, and LPS. Bifidobacterium was positively correlated with VH/CD, and negatively correlated with MDA, TNF-α, and LPS (Fig. 6 A). As shown in Fig. 6 B, Cer 18:1;2O/18:0, Cer 18:1;2O/24:2, and (±)13-HpODE were positively correlated with VH/CD, acetic acid, propionic acid, and butyric acid, while L-arginine was negatively correlated with acetic acid, propionic acid, butyric acid, and Claudin-1. The levels of acetic acid, propionic acid, butyric acid, isobutyric acid, 2-methylbutyrate, and isovaleric acid were also positively correlated with T-AOC, VH/CD, and Claudin-1, while negatively correlated with LPS and TNF-α (Fig. 6 C). The Mantel correlation analysis between differential microbiota and differential metabolites showed (Fig. 6 D) that the abundances of Aeriscardovia and Bifidobacterium were positively correlated with Cer 18:1;2O/18:0, Cer 18:1;2O/24:2, SPB 19:0;2O, and (±)13-HpODE, but negatively correlated with PC 16:0_17:2, L-arginine, and LPA 22:5. Discussion Villus height and the VH/CD ratio are key morphological indicators for evaluating intestinal absorption function (Farahat et al., 2021 ). In this study, it was found that intervention with 0.08% betaine significantly increased the ileal VH/CD ratio in Tibetan sheep, which is consistent with the results showing that betaine promotes villus development in models of rats under high-salt stress (Wang et al., 2018 , Sun et al., 2019 )and suckling piglets (Azad et al., 2022 ), suggesting that it may enhance the proliferation of intestinal epithelial cells by regulating the Wnt/β-catenin signaling pathway (Zhou et al., 2020 ). Betaine can effectively reverse the downward trend of tight junction proteins such as Occludin and Claudin-1 by inhibiting the LPS signaling pathway (Wu et al., 2020 ), and at the same time reduce the level of serum pro-inflammatory factor TNF-α, reconstructing the intestinal barrier defense system at the molecular network level (Perumal et al., 2025 ). In this study, betaine significantly downregulated LPS levels and upregulated Claudin-1 expression, indicating that it may block the “LPS-inflammation-barrier damage” vicious cycle by inhibiting the adhesion of pathogenic bacteria or enhancing the function of the intestinal mucus layer barrier. This mechanism has been verified in models of porcine intestinal epithelial cells (Arumugam et al., 2021 ), liver failure mice (Chen et al., 2020 , Zhao et al., 2022 ), and goslings (Yang et al., 2022 ), providing cross-species evidence for betaine in maintaining the intestinal mechanical barrier. Under oxidative stress, the reduction in antioxidant enzyme activity leads to the accumulation of reactive oxygen species (ROS), which triggers lipid peroxidation and the production of MDA, ultimately damaging the structure of intestinal epithelial cell membranes (Akanda et al., 2025 , Zhuang et al., 2019 , Rao, 2008 ). In this study, betaine significantly increased T-AOC and decreased MDA content, which is consistent with the findings in studies on broilers (Song et al., 2021 )and piglets (Xiong et al., 2023 )that betaine enhances antioxidant enzyme activity, suggesting that it maintains redox balance by scavenging ROS. It is noteworthy that ROS can enhance LPS-induced inflammatory cascades by activating the NF-κB pathway (Park et al., 2015 , Zhang and Igwe, 2018 ), while the inhibition of MDA by betaine may weaken the oxidative stress-inflammation cross-signaling (Shakeri et al., 2019 ), forming a “antioxidant-anti-inflammatory” synergistic protective mechanism. This holds important physiological significance for alleviating intestinal mucosal oxidative damage in high-altitude hypoxic environments. SCFAs produced by intestinal microbial metabolism are key hubs linking host-microbiota interactions (Silva et al., 2020 ). In this study, betaine significantly increased the concentrations of acetic acid and propionic acid in the ileum by enriching SCFA-producing dominant genera such as Bifidobacterium and Aeriscardovia . As the main energy substrate for intestinal epithelial cells, SCFAs can promote cell proliferation by activating GPR41/43 receptors, directly optimizing villus morphology and enhancing nutrient absorption capacity (Muralitharan et al., 2023 ). In addition, SCFAs reduce the release of LPS-induced pro-inflammatory factors such as TNF-α by inhibiting the NF-κB signaling pathway (Li et al., 2018 , Liu et al., 2012 ), and simultaneously upregulate the expression of the tight junction protein Claudin-1, improving intestinal function from multiple dimensions of “energy supply - inflammation inhibition - barrier reinforcement”. This is consistent with studies on rodent (Sun et al., 2023 ) and broiler models (Liao et al., 2020 ), indicating that betaine regulates host metabolism and immunity through the “microbiota-SCFAs” axis, forming a cascade effect of “proliferation of beneficial bacteria - production of metabolites - optimization of host functions”. It is worth emphasizing that acetic acid and propionic acid, as the main products of intestinal microbial fermentation, their increased concentrations not only reflect the optimization of the microbiota structure, but also directly participate in the regulation of host energy metabolism and oxidative stress, becoming key mediating factors for betaine to improve the ileal function of Tibetan sheep. Phylum-level analysis showed that Firmicutes , Euryarchaeota , and Actinobacteriota were the dominant bacterial phyla in the ileum of Tibetan sheep, accounting for more than 95% of the total. Although betaine supplementation did not significantly alter the α-diversity of the microbiota, it promoted the significant enrichment of key SCFA-producing genera ( Bifidobacterium and Aeriscardovia ) by regulating the abundance at the phylum level (e.g., increasing the proportion of Actinobacteriota ). Among them, Bifidobacterium decomposes carbohydrates through the unique “bifid shunt” metabolic pathway (Hald et al., 2016 )and efficiently produces acetic acid and lactic acid (Usta-Gorgun and Yilmaz-Ersan, 2020 ); Aeriscardovia , as a member of the Bifidobacteriaceae family, also has the ability to produce SCFAs (Simpson et al., 2004 ). Previous studies have confirmed that these two genera can improve intestinal health by inhibiting the NF-κB pathway and enhancing the activity of antioxidant enzymes (Lee et al., 2022 , Averina et al., 2021 , Farooq et al., 2023 ), which is consistent with the results of this study showing that SCFA levels were positively correlated with T-AOC and negatively correlated with MDA and LPS. These findings indicate that betaine reshapes the ileal microbial community by directionally enriching functional genera, and then improves host physiological functions through metabolite mediation, reflecting the precise regulatory relationship of “microbe-metabolite-host”. Metabolomics analysis revealed that betaine affects ileal function by regulating the “Arginine and proline metabolism” and “Glycerophospholipid metabolism” pathways. In arginine metabolism, betaine, as an efficient methyl donor, activates betaine-homocysteine methyltransferase to promote the production of S-adenosylmethionine (SAM) (Dobrijević et al., 2023 , Huang et al., 2016 ), thereby accelerating the conversion of L-arginine to nitric oxide (NO) and polyamines (Wu and Meininger, 2000 , Mato et al., 2008 ). NO exerts antioxidant effects by scavenging free radicals and inhibiting the NF-κB inflammatory pathway (Bogdan, 2001 , Nathan and Xie, 1994 ), while polyamines support the repair of villus structure by promoting the proliferation of intestinal epithelial cells and the synthesis of tight junction proteins (Rao et al., 2020), which is highly consistent with the phenotype of increased VH/CD ratio. On the other hand, the upregulation of ceramide metabolites (such as Cer 18:1;2O/18:0) is positively correlated with SCFAs, suggesting that they may synergize with SCFAs in barrier protection by enhancing membrane structural stability and inhibiting inflammatory signals. It is noteworthy that the downregulation of lysophosphatidic acid (LPA 22:5) may be related to betaine inhibiting phospholipase A2 activity and reducing oxidative stress-induced decomposition of membrane phospholipids (Murakami et al., 1999 , Craig, 2004 ), thereby blocking the LPA-mediated pro-inflammatory signaling pathway (Zhao and Natarajan, 2013 , Lin et al., 2010 , Xia et al., 2018 ). These metabolites form a molecular network for betaine to regulate ileal function through the cascade effect of “methyl donor-enzymatic reaction-signaling pathway”, providing a new explanation for the metabolic adaptation of Tibetan sheep in high-altitude environments. In this study, the interaction mechanism between differential intestinal microbiota and host metabolites was analyzed by constructing an association network, revealing that key genera such as Aeriscardovia and Bifidobacterium regulate the dynamic balance of lipid metabolism through multiple pathways. The results showed that Aeriscardovia and Bifidobacterium were significantly positively correlated with ceramide metabolites (Cer 18:1;2O/18:0, Cer 18:1;2O/24:2), which might affect the steady state of ceramide metabolism by regulating intestinal barrier function. As the core component of cell membrane lipids, abnormal metabolism of ceramides can directly trigger intestinal inflammation and epithelial cell apoptosis (Hannun and Obeid, 2008 ). It is noteworthy that the negative correlation between the above-mentioned genera and L-arginine suggests that they may competitively consume substrates through the arginine deiminase pathway, converting L-arginine into agmatine with immunomodulatory functions (Wu and Morris Jr, 1998 ). In terms of membrane lipid dynamic balance, the decrease in the level of phosphatidylcholine isomer PC 16:0_17:2 is closely related to changes in cell membrane fluidity (Van Meer et al., 2008 ). The study also found that intestinal flora forms a network regulation through metabolites such as SCFAs and secondary bile acids, which not only affects host lipid metabolism but also participates in the regulation of inflammatory responses (Tremaroli and Bäckhed, 2012 ). In conclusion, this study indicates that betaine significantly improves intestinal absorption function and antioxidant capacity by regulating the ileal microbial community and metabolites. This finding provides a theoretical basis for the development of precise nutritional strategies for plateau animal husbandry. Betaine can regulate the microbial community (including enriching SCFA-producing Bifidobacterium and Aeriscardovia , etc.), significantly increase the concentration of SCFAs in the ileum (especially acetate and propionate), and regulate metabolic pathways such as arginine and proline metabolism, and glycerophospholipid metabolism. Through the “microbiota-SCFAs-metabolite” cascade effect, it enhances the structure and function of the ileum (optimizing villus structure, strengthening barrier function, and alleviating oxidative stress and inflammatory response). The results of this study provide an important theoretical basis for the application of betaine supplementation in the nutritional management of Tibetan sheep, especially in improving the theory of intestinal stress regulation in high-altitude ruminants and developing precise nutritional strategies for plateau animal husbandry. Declarations Conflict of interest The authors state that no commercial or financial connections existed during the research that might be interpreted as a potential conflict of interest. Funding This research was funded by the project “Research and Integration of Production Technology for Special Tibetan Sheep in High Saline and Alkaline Habita” (Grant No. 2022-NK-169-3). Author Contribution Wei Gao: Conceptualization, Data curation, Formal analysis, Methodology, Software, Writing-original draft, Writing-review & editing. Zhenling Wu: Conceptualization, Data curation, Methodology, Validation. Jiacheng Gan: Resources, Formal Analysis, Writing-review & editing. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7277025","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":496947355,"identity":"362395c6-31b9-43a1-b53c-aafbbffb0052","order_by":0,"name":"Wei Gao","email":"","orcid":"","institution":"Qinghai University","correspondingAuthor":false,"prefix":"","firstName":"Wei","middleName":"","lastName":"Gao","suffix":""},{"id":496947356,"identity":"7cd24fec-9434-4863-abf2-8cac496639a5","order_by":1,"name":"Zhenling Wu","email":"","orcid":"","institution":"Qinghai University","correspondingAuthor":false,"prefix":"","firstName":"Zhenling","middleName":"","lastName":"Wu","suffix":""},{"id":496947357,"identity":"0e9552fa-f1ab-4b7a-9384-f66b2066b91f","order_by":2,"name":"Jiacheng Gan","email":"","orcid":"","institution":"Qinghai University","correspondingAuthor":false,"prefix":"","firstName":"Jiacheng","middleName":"","lastName":"Gan","suffix":""},{"id":496947358,"identity":"bae4dfbc-08ba-4f9b-9fa9-45185ebed927","order_by":3,"name":"Xianhua Zhang","email":"","orcid":"","institution":"Qinghai University","correspondingAuthor":false,"prefix":"","firstName":"Xianhua","middleName":"","lastName":"Zhang","suffix":""},{"id":496947359,"identity":"038bc55c-8172-4ead-8194-96570a1a35a3","order_by":4,"name":"Chengdi Shi","email":"","orcid":"","institution":"Qinghai University","correspondingAuthor":false,"prefix":"","firstName":"Chengdi","middleName":"","lastName":"Shi","suffix":""},{"id":496947361,"identity":"dca66de1-8ec8-4f10-a34a-a2794616fa81","order_by":5,"name":"Zhenglu Yang","email":"","orcid":"","institution":"Qinghai University","correspondingAuthor":false,"prefix":"","firstName":"Zhenglu","middleName":"","lastName":"Yang","suffix":""},{"id":496947362,"identity":"242dda1a-20fa-4b2f-beed-90ddbeadbb2b","order_by":6,"name":"Quyangangmao Su","email":"","orcid":"","institution":"Qinghai University","correspondingAuthor":false,"prefix":"","firstName":"Quyangangmao","middleName":"","lastName":"Su","suffix":""},{"id":496947363,"identity":"3d0cb4e6-e161-406f-8d83-1b82c86619fd","order_by":7,"name":"Shengzhen Hou","email":"","orcid":"","institution":"Qinghai University","correspondingAuthor":false,"prefix":"","firstName":"Shengzhen","middleName":"","lastName":"Hou","suffix":""},{"id":496947364,"identity":"13e31210-6e85-4beb-9af2-e4920a6bd25b","order_by":8,"name":"Lijuan Han","email":"","orcid":"","institution":"Qinghai University","correspondingAuthor":false,"prefix":"","firstName":"Lijuan","middleName":"","lastName":"Han","suffix":""},{"id":496947365,"identity":"36976319-7b60-44f4-aa81-5ef3309c3667","order_by":9,"name":"Linsheng Gui","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA2ElEQVRIiWNgGAWjYDACdgb2Dwk8EnL87I2NDz4QpYWZgY3hgYyFsWTP4WbDGcRqYXxgU5G44UZ6mzQHMTrkndmfPUjIkTCWnPmwQZqBwU5Ot4GAFsPDPOYGCWeAfpFObDAuYEg2NjtASEszD4NEYg/QltmJDckzGA4kbiOshf2BROI/icQNNw82HOYhRos8M4OZBDCQgd5nbGwmSosBM4+xAVALMJATmxlnGBDhF/n29ocPf/DUAaPy+PMfHyrs5AhqMUBVYEBAOdiWBiIUjYJRMApGwQgHAPBRQjbpwUpAAAAAAElFTkSuQmCC","orcid":"","institution":"Qinghai University","correspondingAuthor":true,"prefix":"","firstName":"Linsheng","middleName":"","lastName":"Gui","suffix":""}],"badges":[],"createdAt":"2025-08-02 09:08:30","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7277025/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7277025/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13568-026-02024-6","type":"published","date":"2026-02-04T15:57:01+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":88874209,"identity":"14f5b424-8f2e-4a18-86d0-617c120f98c8","added_by":"auto","created_at":"2025-08-12 09:49:18","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":4219942,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of betaine on the morphology of the ileal mucosa of Tibetan sheep. \u003cstrong\u003e(A)\u003c/strong\u003e Representative histological images of ileal sections stained with H\u0026amp;E (original magnifications 500× and 200× µm). \u003cstrong\u003e(B) \u003c/strong\u003eThe changes in villus height, villus width, crypt depth, crypt number, muscular layer thickness and villus height/crypt depth ratio (VH/CD). Ctrl, basal diet; Bet, 0.08% betaine is added to the basal diet. * \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7277025/v1/f86b8ada88d91cddb87dc6b7.png"},{"id":88873767,"identity":"01bfb8fe-86cb-4901-80a9-52afdb02042f","added_by":"auto","created_at":"2025-08-12 09:41:18","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1090345,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of betaine on ileal antioxidant capacity and LPS levels in Tibetan sheep. Antioxidant capacity includes \u003cstrong\u003e(A)\u003c/strong\u003e catalase (CAT), \u003cstrong\u003e(B)\u003c/strong\u003e glutathione peroxidase (GSH-Px), \u003cstrong\u003e(C)\u003c/strong\u003e malondialdehyde (MDA) content, \u003cstrong\u003e(D)\u003c/strong\u003e superoxide dismutase (SOD) and \u003cstrong\u003e(E)\u003c/strong\u003e total antioxidant capacity (T-AOC). \u003cstrong\u003e(F)\u003c/strong\u003e Lipopolysaccharide (LPS) levels. Ctrl, basal diet; Bet, 0.08% betaine is added to the basal diet. * \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7277025/v1/8b08773b8d2a2d2efa956edc.png"},{"id":88873768,"identity":"6ddab554-1024-4ab5-85a8-2eb5b5a54d10","added_by":"auto","created_at":"2025-08-12 09:41:18","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1082181,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of betaine on the ileal barrier of Tibetan sheep. Ctrl, basal diet; Bet, 0.08% betaine is added to the basal diet. * \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7277025/v1/ff0fcc9009dff95c4fcf2c91.png"},{"id":88874210,"identity":"09c7d2dd-cc07-45da-9231-20843f845d5d","added_by":"auto","created_at":"2025-08-12 09:49:18","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1975997,"visible":true,"origin":"","legend":"\u003cp\u003eMicrobial diversity in the ileum of Tibetan sheep. \u003cstrong\u003e(A)\u003c/strong\u003e Venn diagram of the ileal microorganisms in Tibetan sheep. \u003cstrong\u003e(B)\u003c/strong\u003e Alpha diversity of the ileal microorganisms in Tibetan sheep. \u003cstrong\u003e(C)\u003c/strong\u003e Beta diversity of the ileal microorganisms in Tibetan sheep. Composition of the ileal microbiota in sheep at the phylum\u003cstrong\u003e (D) \u003c/strong\u003eand genus \u003cstrong\u003e(E)\u003c/strong\u003e levels. \u003cstrong\u003e(F)\u003c/strong\u003e Box plot showing the changes of microorganisms at the genus level. \u003cstrong\u003e(G)\u003c/strong\u003e Sankey diagram of the ileal microorganisms in the Bet group of Tibetan sheep. Ctrl, basal diet; Bet, 0.08% betaine is added to the basal diet. * \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7277025/v1/87774643d4ff3b88dd1da72c.png"},{"id":88873770,"identity":"30807d18-f106-44f2-a470-d4856188135b","added_by":"auto","created_at":"2025-08-12 09:41:18","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1564689,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis results of ileal metabolites in Tibetan sheep.\u003cstrong\u003e(A)\u003c/strong\u003e Pie chart of metabolite classification. \u003cstrong\u003e(B)\u003c/strong\u003e Principal component analysis and \u003cstrong\u003e(C)\u003c/strong\u003e OPLS-DA score plot showing clusters with significant separation between groups. \u003cstrong\u003e(D)\u003c/strong\u003e Matchstick plot of differential metabolites. \u003cstrong\u003e(E)\u003c/strong\u003e KEGG functional enrichment analysis of differential metabolites. Up: metabolites are significantly up-regulated; down: metabolites are significantly down-regulated; Ctrl, basal diet; Bet, 0.08% betaine is added to the basal diet. * \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7277025/v1/92a1e61d8458a0f7eb229297.png"},{"id":88873772,"identity":"ca57374d-7831-4f45-b6da-bf3c7881bab3","added_by":"auto","created_at":"2025-08-12 09:41:19","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1598042,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCorrelation analysis. \u003c/strong\u003e(A)\u003cstrong\u003e Correlation analysis between ileal microorganisms and ileal tissue morphology, antioxidant capacity, lipopolysaccharide level, short-chain fatty acid content, and ileal barrier. \u003c/strong\u003e(B)\u003cstrong\u003e Correlation analysis between ileal metabolites and ileal tissue morphology, antioxidant capacity, lipopolysaccharide level, short-chain fatty acid content, and ileal barrier. \u003c/strong\u003e(C)\u003cstrong\u003e Correlation analysis between short-chain fatty acids and ileal tissue morphology, antioxidant capacity, lipopolysaccharide level, and ileal barrier. \u003c/strong\u003e(D)\u003cstrong\u003e Spearman correlation heatmap of metabolites and microorganisms. Red lines indicate positive correlations, while blue lines indicate negative correlations; the intensity of the color changes proportionally with the correlation value. Ctrl, basal diet; Bet, 0.08% betaine is added to the basal diet. * 0.01 \u0026lt; \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eP\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e ≤ 0.05; ** 0.001 \u0026lt; \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eP\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e ≤0.01; *** \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eP\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e ≤ 0.001.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-7277025/v1/39f172ef834b030f3d248773.png"},{"id":102234275,"identity":"9b1a03f6-5780-4861-9e14-497f50f7f9b1","added_by":"auto","created_at":"2026-02-09 16:08:56","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":12884458,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7277025/v1/eb0a179e-cd8f-4577-aa1d-5325c3eb221a.pdf"},{"id":88873765,"identity":"3023feb7-81f2-47fd-8358-c6d4c6a6d8f0","added_by":"auto","created_at":"2025-08-12 09:41:18","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":18506,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable1.docx","url":"https://assets-eu.researchsquare.com/files/rs-7277025/v1/037496a46eebeca226db8000.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Effects of betaine on ileal tissue and intestinal microbial metabolism in Tibetan sheep","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe Qinghai-Tibet Plateau is the highest alpine ecological region in the world. Environmental factors such as high altitude, hypoxia, low temperature, strong ultraviolet radiation, and seasonal shortage of forage pose significant challenges to the physiological metabolism and production performance of local ruminants (Li et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).Tibetan sheep (\u003cem\u003eOvis aries\u003c/em\u003e) have evolved a variety of adaptive mechanisms during long-term natural selection and domestication, such as reducing basal metabolism, regulating fat mobilization, and modulating oxidative stress, so as to enhance their survival ability in extreme environments (Li et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). However, such adaptations are still accompanied by a decline in production performance, manifested as fluctuations in live weight during the cold season, low feed conversion efficiency, and digestive tract dysfunction, which restrict the benefits of their breeding (Wei et al., \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2016\u003c/span\u003e, Xu et al., \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Recent studies have shown that rumen microorganisms play a key role in energy metabolism and environmental adaptation of Tibetan sheep. Particularly in the cold season, the increased abundance of fiber-degrading bacteria promotes the utilization of cellulose in roughage (Liu et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). However, compared with the rumen, little is known about the response mechanism of the small intestine, especially the ileum, under plateau stress. As the distal part of the small intestine, the ileum is not only involved in nutrient absorption and bile acid reabsorption but also undertakes important functions of mucosal immunity and microbial interaction. Its structural and functional status is crucial to animal health (Chen et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2019\u003c/span\u003e, Sun et al., \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), which urgently requires in-depth research.\u003c/p\u003e\u003cp\u003eBetaine, a natural derivative of trimethylglycine (Eklund et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2005\u003c/span\u003e), has dual functions as a methyl donor and an osmoprotectant, and is widely used in nutritional intervention for livestock and poultry (Abd El-Ghany and Babazadeh, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2022\u003c/span\u003e, Yang et al., \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e2022\u003c/span\u003e, Cheng et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In animals, it can participate in methionine metabolism (Abd El-Ghany and Babazadeh, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), choline synthesis (Obeid, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2013\u003c/span\u003e)and DNA methylation (Yang et al., \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e2020\u003c/span\u003e)by providing methyl groups, which helps regulate lipid metabolism (Yang et al., \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), enhance the antioxidant system (Wen et al., \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2021a\u003c/span\u003e), and maintain cellular osmotic homeostasis (Ratriyanto and Mosenthin, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Studies have confirmed that betaine can improve intestinal morphology, barrier function, and antioxidant capacity in monogastric animals such as pigs and poultry (Wang et al., \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2020\u003c/span\u003e, Li et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2021\u003c/span\u003e, Song et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In ruminants, its application has shown multiple effects including increasing milk production, improving daily weight gain, and optimizing carcass composition. Moreover, meta-analyses have verified its stable synergistic effects under different environmental and breed conditions (Abhijith et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). In addition, betaine can alleviate stress responses by enhancing cellular antioxidant defense under heat stress conditions (Wen et al., \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2021b\u003c/span\u003e). Studies on Hu sheep (Dong et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), Merino sheep (DiGiacomo et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), and other breeds have further revealed its potential in regulating metabolic responses under special ecological conditions. However, current research mainly focuses on production performance and overall physiological indicators, and there is a lack of systematic analysis on the regulatory mechanisms of betaine in intestinal tissue, especially the microecology and metabolism of the small intestine. This limits the in-depth application of betaine in precise nutritional regulation.\u003c/p\u003e\u003cp\u003eThe ileum, as an important part of the small intestine, is not only a key site for terminal nutrient absorption but also a core hub for intestinal immune regulation and microorganism-metabolite interactions (Zhang et al., \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e2025\u003c/span\u003e, Collins et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) The functional status of the ileum is collectively determined by intestinal mucosal integrity, the number of goblet cells, redox status, and microbial community structure (Yang and Yu, \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e2021\u003c/span\u003e, Lin et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Existing studies have shown that specific intestinal metabolites (such as SCFAs, secondary bile acids, etc.) participate in cellular signal transduction by acting on host receptors, exerting profound impacts on intestinal homeostasis (Visekruna and Luu, \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2021\u003c/span\u003e, Lin et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The mechanism of action of betaine in the intestine has gradually attracted attention, including promoting villus development, increasing antioxidant enzyme activity, and regulating microbial diversity, which demonstrates its potential in improving intestinal function (Wang et al., \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2018\u003c/span\u003e, Al Sulaiman et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). However, there is currently a lack of systematic research based on the Tibetan sheep model under alpine and plateau ecological conditions to reveal the specific pathways and microscopic mechanisms of betaine in regulating ileal health.\u003c/p\u003e\u003cp\u003eBased on this, this study took Tibetan sheep as the research object, combined with feeding and slaughter experiments, and comprehensively used histological observation, antioxidant index analysis, 16S rDNA sequencing and untargeted metabolomics technology to systematically evaluate the regulatory effects of betaine on the ileal structure, function and microorganism-metabolite interaction of Tibetan sheep. It aims to reveal its nutritional intervention mechanism under plateau stress conditions and provide theoretical support for efficient breeding.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e This animal study was approved by the Institutional Animal Care and Use Committee of Qinghai University, China (Approval No.: QUA-2020-0710). Both the animal care and experimental protocols were reviewed and approved by the Committee. The study was conducted in compliance with local laws and regulations as well as institutional requirements.\u003c/p\u003e\u003cp\u003e\u003cb\u003eExperimental Design\u003c/b\u003e\u003c/p\u003e\u003cp\u003eA feeding experiment was conducted at a Tibetan sheep breeding base in Qieji Township, Gonghe County, Qinghai Province (36\u0026deg;19\u0026prime;N, 99\u0026deg;41\u0026prime;E) from April 2024 to July 2024. Sixty 2-month-old plateau-type Tibetan sheep rams with similar body weights (17.72\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19 kg) and good body condition were selected and randomly divided into 2 groups: the control group (Ctrl) and the experimental group (Bet), with 30 sheep in each group (5 replicates, 6 sheep per replicate). The Ctrl group was fed a basal diet, while the Bet group was fed the basal diet supplemented with 0.08% betaine (on an air-dry basis). The basal diet consisted of a concentrate supplement and roughage at a concentrate-to-roughage ratio of 7:3. The roughage was a mixture of oat hay and oat silage in a 1:1 ratio on a dry matter basis. The betaine was provided by Jinan Tai Fei Animal Husbandry Technology Co., Ltd. with a purity of 98%. The composition and nutritional levels of the experimental diets are shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The total experimental period was 100 days, including a 10-day pre-test period and a 90-day formal test period. Feeding was carried out at 8:00 and 17:00 daily, and the sheep had free access to feed and water during the experiment. The nutritional components in the feed used in this experiment, such as dry matter (Thiex et al., \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2002a\u003c/span\u003e), crude protein (Thiex et al., \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2002b\u003c/span\u003e), acid detergent fiber (HIfiFLHQF et al.), neutral detergent fiber (Hiraoka et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), Ca and P (Babos et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), were all determined with reference to relevant standards.\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\u003eComposition of the basic diet.\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=\"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\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eItem\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCtrl\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eBet\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCorn\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e48.40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e48.32\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eWheat\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e9.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e9.00\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePalm meal\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e16.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e16.00\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSoybean meal\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e4.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e4.00\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRapeseed meal\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e15.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e15.00\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNacl\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"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\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLimestone\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\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBaking soda\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\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1% Premix\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.60\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.60\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e4% Concentrate\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e4.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e4.00\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBetaine\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.08\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTotal\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\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNutrient levels\u003csup\u003e2\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\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDigestible energy (MJ\u0026middot;kg⁻\u0026sup1;)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e12.66\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e12.65\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCrude protein\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e13.69\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e13.69\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEther extract\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e4.61\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e4.61\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCrude fiber\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e21.18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e21.16\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNeutral detergent fiber\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e21.18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e21.18\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAcid detergent fiber\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e14.72\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e14.72\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCa\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.85\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.85\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eP\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.41\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.41\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eCtrl, basal diet; Bet, 0.08% betaine is added to the basal diet.\u003c/p\u003e\u003cp\u003e\u003csup\u003e1\u003c/sup\u003eThe premix provides the following per kilogram of the feed: Cu, 18 mg; Fe, 66 mg; Zn, 30 mg; Mn, 48 mg; Se, 0.36 mg; I, 0.6 mg; Co, 0.24 mg; vitamin A, 24,000 IU; vitamin D, 4,800 IU; vitamin E, 48 IU.\u003c/p\u003e\u003cp\u003e\u003csup\u003e2\u003c/sup\u003eDigestible energy is a calculated value, while the others are measured values.\u003c/p\u003e\u003cp\u003e\u003cb\u003eSample Collection\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAt the end of the formal test period, 6 Tibetan sheep were randomly selected from each group and slaughtered at the slaughterhouse (n\u0026thinsp;=\u0026thinsp;12). The Tibetan sheep were fasted for 12 hours. After slaughter, the intestines were quickly separated, and the ileocecal junction was clamped with hemostatic forceps. The collected ileal tissue samples were about 10 cm away from the ileocecal orifice, with a length of about 1 cm. Meanwhile, 4 mL of ileal contents were collected into a 5 mL cryopreservation tube, quickly frozen in liquid nitrogen, stored and transported in dry ice to the laboratory, and then preserved in a -80℃ refrigerator for subsequent untargeted metabolomics and 16S rDNA sequencing. Finally, the collected tissue samples were rinsed clean with physiological saline and placed in 4% paraformaldehyde for tissue observation.\u003c/p\u003e\u003cp\u003e\u003cb\u003eH\u0026amp;E staining\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe ileal tissues were fixed in 4% paraformaldehyde solution for no less than 48 hours, followed by gradient dehydration with ethanol and paraffin embedding. Finally, sections with a thickness of 3 \u0026micro;m were cut to prepare paraffin sections, which were then stained with hematoxylin-eosin (H\u0026amp;E) for analysis. The indicators measured included villus height (VH), villus width, crypt depth (CD), crypt number, muscular layer thickness, and the VH/CD ratio. A microscope (OLYMPUS, DP26, Tokyo, Japan) was used to observe the stained sections and collect images. Finally, Image-Pro Plus 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA) was employed to measure all indicators of the ileal tissues in the collected images at magnification levels of 500\u0026times; and 200\u0026times;.\u003c/p\u003e\u003cp\u003e\u003cb\u003eDetermination of antioxidant capacity and lipopolysaccharide content\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe contents of total antioxidant capacity (T-AOC), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT), malondialdehyde (MDA) and the level of lipopolysaccharide (LPS) in ileal tissues were determined using ELISA kits (Shanghai Meilian Biotechnology Co., Ltd., Shanghai, China). Take 0.1 g of ileal tissue and add 900 \u0026micro;L of physiological saline. After crushing with a crusher (SM2000, Retsch, Haan, Germany), transfer to a low-temperature high-speed centrifuge (5430 R, Eppendorf, Hamburg, Germany) and centrifuge at 2500 \u0026times; g for 15 minutes at 4\u0026deg;C to obtain the supernatant. The determination was carried out strictly in accordance with the steps provided in the kit, and finally the absorbance value was measured at a wavelength of 450 nm on an ELISA Analyzer (ELx808, BioTek, Winooski, VT, USA).\u003c/p\u003e\u003cp\u003e\u003cb\u003eDetermination of SCFAs\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe ileal contents were preserved in dry ice and sent to Beijing Novogene Bioinformatics Technology Co., Ltd. (Beijing, China). The composition and content of SCFAs were quantitatively determined using an ultra-high performance liquid chromatography-tandem mass spectrometry system (Vanquish\u0026trade; Flex UHPLC-TSQ Altis\u0026trade;, Thermo Scientific Corp., Germany). The sample extraction and determination procedures were carried out with reference to the method described by Yan et al. (Yan et al., \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cb\u003eReal Time Quantitative (RT-qPCR)\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTotal RNA was extracted from ileal tissues using the Transzol Up kit (TRAN, Beijing, China), and its concentration was detected. Subsequently, the RNA was reverse-transcribed into cDNA using a kit. The subsequent fluorescent quantitative steps and qPCR reaction procedures were strictly performed according to the method described by Ji et al (Ji et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The relative expression level of the target gene mRNA was calculated by the 2\u003csup\u003e\u0026minus;ΔΔCt\u003c/sup\u003e method, and the primer sequences 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\u003ePrimers used in RT-qPCR.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" 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=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eItems\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePrimer sequence (5\u0026rsquo;-3\u0026rsquo;)\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTm (℃)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eProduct length\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eNCBI Gene ID\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eClaudin-1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eF-CCTGCTGTGCTGCTCCTGTC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e61.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e75bp\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e780473\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eR-GAAGGTGCTGGCTTGGGATAGG\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e61.4\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eOccludin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eF-CGAGAAGCGACCGTATCCAGAG\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e61.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e129bp\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e101118304\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eR-TCCAAGTTACCACTGCTGCTGTAG\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e59.6\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eZO-1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eF-GGGCAAGTTAAAGATGGTGGTTCAG\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e59.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e93bp\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e443200\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eR-GAGGCGTCAGCAGAGTGGATG\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e61.5\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eTNF-α\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eF-ACGGCGTGGAGCTGAAAGAC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e59.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e79bp\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e443540\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eR-CTGAAGAGGACCTGCGAGTAGATG\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e61.3\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eMuc-2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eF-ACGACTCCTACGCCCTCCTG\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e61.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e130bp\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e780488\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eR-ACGCTGCCATCCGACTTGAAG\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e51.5\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eIL-6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eF-TCTAATAACCACTCCAGCCACACAC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e59.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e77bp\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e443406\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eR-TTGCGTTCTTTACCCACTCGTTTG\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e57.9\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eβ-Actin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eF-AGCAAGCGTGGCATCCTAACC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e59.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e87bp\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e443340\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eR-ATCTTCTCCATGTCGTCCCAGTTG\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e59.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e77bp\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003csup\u003e1\u003c/sup\u003eZO-1, zonula occluden-1; MUC-2, mucoprotein-2; TNF-α, tumor necrosis factor-α; IL-6, interleukin-6.\u003c/p\u003e\u003cp\u003e\u003csup\u003e2\u003c/sup\u003eF, forward primer; R, reverse primer.\u003c/p\u003e\u003cp\u003e\u003cb\u003eAnalysis of microbial composition\u003c/b\u003e\u003c/p\u003e\u003cp\u003eMicrobial DNA in ileal contents was extracted using the DNA extraction kit from Novogene (Beijing, China), and the V3-V4 region of 16S rDNA was amplified by PCR with specific primers 341F (5\u0026prime;-CCTAYGGGRBGCASCAG-3\u0026prime;) and 806R (5\u0026prime;-GGACTACNNGGGTATCTAAT-3\u0026prime;). Sample reads were spliced using FLASH (Version 1.2.11) to generate Raw Tags data. Then, Cutadapt (Version 3.3) software was used to match the reverse primer sequences and remove redundant parts, and fastp (Version 0.23.1) software was employed to obtain Clean Tags (Bokulich et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Subsequently, the Tags sequences were compared with the species annotation database, and chimeric sequences were detected and removed to obtain Effective Tags (Edgar et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). The SVG function of Perl (Version 5.26.2) software was used to draw the relative abundance distribution histogram. QIIME2 (Version 2022.02) software was used to calculate the Alpha diversity index. Principal coordinate analysis was performed using the ade4 package and ggplot2 package of R (Version 4.0.3) software.\u003c/p\u003e\u003cp\u003e\u003cb\u003eUntargeted metabolomics analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe extraction of metabolites referred to the study by Want et al. (Want et al., \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). The chromatographic column used was Hypersil Gold C18. The off-machine data were processed using Compound Discoverer 3.3 software(Thermo Fisher Scientific Inc., Waltham, Massachusetts, USA), with data processing based on the Linux system (CentOS version 6.6) as well as R (Version 4.0.3) and Python (Version 3.12.1) software. The identified metabolites were annotated using the KEGG database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.genome.jp/kegg/pathway.html\u003c/span\u003e\u003cspan address=\"https://www.genome.jp/kegg/pathway.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). For multivariate statistical analysis, after data transformation using metaX software, principal component analysis and orthogonal partial least squares discriminant analysis were performed to obtain VIP values; univariate analysis calculated P values and FC values based on t-tests, and the criteria for screening differential metabolites were VIP\u0026thinsp;\u0026gt;\u0026thinsp;1, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, and FC\u0026thinsp;\u0026gt;\u0026thinsp;1.5. The R package ggplot2 was used to draw match plots and bubble plots, the corrplot package was used for correlation analysis, and the KEGG database was used to study metabolite functions and pathways. The enrichment of metabolic pathways was determined according to x/n\u0026thinsp;\u0026gt;\u0026thinsp;y/n and \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eExperimental data were preprocessed using Microsoft Excel 2024, and all data were subjected to independent samples t-test using SPSS 26.0 software (IBM Corp., Armonk, NY, USA). Results were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error. Graphs were plotted using GraphPad Prism 8.0 software, with \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 set as the criterion for judging significant differences with statistical significance. Wilcoxon rank-sum test was used for data α-diversity index analysis, Unweighted Unifrac distance was used for PCoA analysis, and NMDS was used to reflect the degree of differences between samples.\u003c/p\u003e\u003cp\u003eSpearman rank correlation analysis was performed using the R language Psych package and R language Vegan package in R (Version 4.0.3) to calculate the correlations between ileal microbiota abundance, metabolite levels and short-chain fatty acids, antioxidant indices, tissue morphological parameters, ileal barrier function, and lipopolysaccharide levels.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eHistomorphological analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eHematoxylin-eosin (H\u0026amp;E) stained sections intuitively showed the morphological changes in the ileal tissue of Tibetan sheep (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). We observed that the ileal villus height and VH/CD ratio in the Bet group were significantly higher than those in the Ctrl group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). There were no significant differences in ileal villus width, crypt depth, crypt number, or muscular layer thickness between the two groups of Tibetan sheep (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eAntioxidant capacity and lipopolysaccharide content\u003c/b\u003e\u003c/p\u003e\u003cp\u003eIn terms of antioxidant capacity (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-E), compared with the Ctrl group, the Bet group showed higher T-AOC activity (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and lower MDA content (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In addition, there were no significant differences in the activities of CAT, SOD, and GSH-Px between the two groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Regarding LPS concentration (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF), the LPS level in the Bet group was significantly lower than that in the Ctrl group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eContent of SCFAs\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAs shown in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, dietary supplementation with betaine significantly increased the contents of acetic acid and propionic acid in the ileum of Tibetan sheep (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). There were no significant differences in the contents of isobutyric acid, butyric acid, 2-methylbutyrate, isovaleric acid, valeric acid, 2-methylvalerate, 3-methylvalerate, 4-methylvaleric acid, and hexanoic acid between the two groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eThe effects of adding 0.08% betaine on Short-chain Fatty Acids in Tibetan sheep.\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=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eItems\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCtrl\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eBet\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eP-\u003c/em\u003eValue\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAcetic acid(ng/mL)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e145752.63\u0026thinsp;\u0026plusmn;\u0026thinsp;44506.93\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e527274.20\u0026thinsp;\u0026plusmn;\u0026thinsp;12849.79\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.001\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePropionic acid(ng/mL)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5399.60\u0026thinsp;\u0026plusmn;\u0026thinsp;2262.09\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e43408.56\u0026thinsp;\u0026plusmn;\u0026thinsp;3061.34\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.001\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eIsobutyric acid(ng/mL)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e568.56\u0026thinsp;\u0026plusmn;\u0026thinsp;76.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e14045.96\u0026thinsp;\u0026plusmn;\u0026thinsp;5011.35\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.055\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eButyric acid(ng/mL)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e905.52\u0026thinsp;\u0026plusmn;\u0026thinsp;387.62\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e36448.29\u0026thinsp;\u0026plusmn;\u0026thinsp;10145.49\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.072\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2-Methylbutyrate(ng/mL)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e281.68\u0026thinsp;\u0026plusmn;\u0026thinsp;133.19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e9868.77\u0026thinsp;\u0026plusmn;\u0026thinsp;3042.60\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.087\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eIsovaleric acid(ng/mL)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e412.34\u0026thinsp;\u0026plusmn;\u0026thinsp;219.24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e16149.81\u0026thinsp;\u0026plusmn;\u0026thinsp;5257.94\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.096\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eValeric acid(ng/mL)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e43.46\u0026thinsp;\u0026plusmn;\u0026thinsp;12.58\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1238.49\u0026thinsp;\u0026plusmn;\u0026thinsp;435.96\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.111\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2-Methylvalerate(ng/mL)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e37.00\u0026thinsp;\u0026plusmn;\u0026thinsp;6.39\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e24.56\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.191\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3-Methylvalerate(ng/mL)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e42.82\u0026thinsp;\u0026plusmn;\u0026thinsp;14.92\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e13.49\u0026thinsp;\u0026plusmn;\u0026thinsp;0.94\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.188\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e4-Methylvaleric acid(ng/mL)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e524.52\u0026thinsp;\u0026plusmn;\u0026thinsp;352.91\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e183.46\u0026thinsp;\u0026plusmn;\u0026thinsp;53.95\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.436\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHexanoic acid(ng/mL)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e332.61\u0026thinsp;\u0026plusmn;\u0026thinsp;40.28\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e386.69\u0026thinsp;\u0026plusmn;\u0026thinsp;22.55\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.306\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eCtrl, basal diet; Bet, 0.08% betaine is added to the basal diet. Data are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. If the superscripts of data in the same row are a and b respectively, it indicates that there is a significant difference between their means (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\u003cp\u003e\u003cb\u003eExpression of ileal barrier-related genes\u003c/b\u003e\u003c/p\u003e\u003cp\u003eCompared with the Ctrl group, dietary supplementation with betaine significantly increased the expression level of Claudin-1 gene in the jejunum of Tibetan sheep (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and significantly decreased the expression level of TNF-α gene (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). However, it had no significant effect on the expression levels of Occludin, ZO-1, Muc-2 and IL-6 genes (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eMicrobiota\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAs shown in the Venn diagram of Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, there were 1810 OTUs of ileal bacteria in Tibetan sheep of the control group, while 2509 OTUs were detected in those supplemented with betaine. Among these OTUs, 800 were shared by both groups. The number of ileal OTUs in Tibetan sheep supplemented with betaine was higher than that in the non-supplemented group. Principal Coordinate Analysis (PCoA) and Non-metric Multidimensional Scaling (NMDS) further revealed that the ileal bacterial communities of Tibetan sheep in the two groups clustered independently of each other (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). In addition, there were no statistically significant differences in the Chao1 and Simpson indices, which reflect bacterial abundance and diversity, between the two groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC).\u003c/p\u003e\u003cp\u003eTo better evaluate the effect of dietary betaine supplementation on the composition of the ileal bacterial community, we analyzed the percentage abundances at the phylum and genus levels. At the phylum level, \u003cem\u003eFirmicutes\u003c/em\u003e, \u003cem\u003eEuryarchaeota\u003c/em\u003e, and \u003cem\u003eActinobacteriota\u003c/em\u003e were the dominant groups, accounting for 97.61% (Ctrl) and 95.34% (Bet) of the total ileal bacteria, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). At the genus level, the dominant genera in the Bet group included \u003cem\u003eAcetitomaculum\u003c/em\u003e, \u003cem\u003eOlsenella\u003c/em\u003e, and \u003cem\u003eChristensenellaceae_R_7_group\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). Among them, the relative abundances of \u003cem\u003eAeriscardovia\u003c/em\u003e and \u003cem\u003eBifidobacterium\u003c/em\u003e in the Bet group were significantly higher than those in the Ctrl group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG, \u003cem\u003eAcetitomaculum\u003c/em\u003e taxonomically belongs to \u003cem\u003eFirmicutes\u003c/em\u003e with a high abundance, and it is associated through hierarchical branches such as \u003cem\u003eClostridia\u003c/em\u003e and \u003cem\u003eLachnospirales\u003c/em\u003e. \u003cem\u003eActinobacteriota\u003c/em\u003e is associated with \u003cem\u003eOlsenella\u003c/em\u003e and \u003cem\u003eAeriscardovia\u003c/em\u003e, while \u003cem\u003eEuryarchaeota\u003c/em\u003e is linked to \u003cem\u003eMethanobrevibacter\u003c/em\u003e through methanogenic groups.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eUntargeted metabolomics\u003c/b\u003e\u003c/p\u003e\u003cp\u003eA total of 847 metabolites were identified, which could be classified into Lipids and lipid-like molecules (44.51%), Organic acids and derivatives (20.54%), Organoheterocyclic compounds (11.92%), and Benzenoids (6.02%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). The OPLS-DA score plot showed differences in ileal metabolites between the two groups of Tibetan sheep (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). The OPLS-DA model revealed that the R2Y, R2X, and Q2Y values of the two groups were 0.997, 0.732, and 0.985, respectively, indicating that the model had strong explanatory power and significant clustering effect. Figure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC showed that the Q2 intercept of the regression line of the permutation test model was less than 0, indicating no overfitting. We used matchstick plots to display the differences in metabolites between the two groups of samples. A total of 14 metabolite markers were identified in the Ctrl and Bet groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). Supplementary Table\u0026nbsp;1 showed the upregulated (LPA 22:5, PC 16:0_17:2, and L-arginine) and downregulated (Cer 18:1;2O/18:0, Cer 18:1;2O/24:2, SPB 19:0;2O, and (\u0026plusmn;)13-HpODE) metabolites in Ctrl vs. Bet. Through KEGG pathway enrichment analysis, 5 pathways including Fat digestion and absorption, Gap junction, ABC transporters, Arginine and proline metabolism, and Glycerophospholipid metabolism showed significant differences (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). A variety of differential metabolites including LPA 22:5, PC 16:0_17:2, Cer 18:1;2O/18:0, Cer 18:1;2O/24:2, and L-arginine were mapped to the significantly different metabolic pathways.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eCorrelation analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe Spearman coefficient model was used to evaluate the correlations between ileal development indices, antioxidant capacity, lipopolysaccharide content, short-chain fatty acid content, metabolites, and the diversity and richness of the microbial community. The study found that \u003cem\u003eAeriscardovia\u003c/em\u003e was positively correlated with T-AOC, and negatively correlated with MDA, TNF-α, and LPS. \u003cem\u003eBifidobacterium\u003c/em\u003e was positively correlated with VH/CD, and negatively correlated with MDA, TNF-α, and LPS (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB, Cer 18:1;2O/18:0, Cer 18:1;2O/24:2, and (\u0026plusmn;)13-HpODE were positively correlated with VH/CD, acetic acid, propionic acid, and butyric acid, while L-arginine was negatively correlated with acetic acid, propionic acid, butyric acid, and Claudin-1.\u003c/p\u003e\u003cp\u003eThe levels of acetic acid, propionic acid, butyric acid, isobutyric acid, 2-methylbutyrate, and isovaleric acid were also positively correlated with T-AOC, VH/CD, and Claudin-1, while negatively correlated with LPS and TNF-α (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). The Mantel correlation analysis between differential microbiota and differential metabolites showed (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD) that the abundances of \u003cem\u003eAeriscardovia\u003c/em\u003e and \u003cem\u003eBifidobacterium\u003c/em\u003e were positively correlated with Cer 18:1;2O/18:0, Cer 18:1;2O/24:2, SPB 19:0;2O, and (\u0026plusmn;)13-HpODE, but negatively correlated with PC 16:0_17:2, L-arginine, and LPA 22:5.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eVillus height and the VH/CD ratio are key morphological indicators for evaluating intestinal absorption function (Farahat et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In this study, it was found that intervention with 0.08% betaine significantly increased the ileal VH/CD ratio in Tibetan sheep, which is consistent with the results showing that betaine promotes villus development in models of rats under high-salt stress (Wang et al., \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2018\u003c/span\u003e, Sun et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2019\u003c/span\u003e)and suckling piglets (Azad et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), suggesting that it may enhance the proliferation of intestinal epithelial cells by regulating the Wnt/β-catenin signaling pathway (Zhou et al., \u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Betaine can effectively reverse the downward trend of tight junction proteins such as Occludin and Claudin-1 by inhibiting the LPS signaling pathway (Wu et al., \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), and at the same time reduce the level of serum pro-inflammatory factor TNF-α, reconstructing the intestinal barrier defense system at the molecular network level (Perumal et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). In this study, betaine significantly downregulated LPS levels and upregulated Claudin-1 expression, indicating that it may block the \u0026ldquo;LPS-inflammation-barrier damage\u0026rdquo; vicious cycle by inhibiting the adhesion of pathogenic bacteria or enhancing the function of the intestinal mucus layer barrier. This mechanism has been verified in models of porcine intestinal epithelial cells (Arumugam et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), liver failure mice (Chen et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2020\u003c/span\u003e, Zhao et al., \u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), and goslings (Yang et al., \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), providing cross-species evidence for betaine in maintaining the intestinal mechanical barrier.\u003c/p\u003e\u003cp\u003eUnder oxidative stress, the reduction in antioxidant enzyme activity leads to the accumulation of reactive oxygen species (ROS), which triggers lipid peroxidation and the production of MDA, ultimately damaging the structure of intestinal epithelial cell membranes (Akanda et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2025\u003c/span\u003e, Zhuang et al., \u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e2019\u003c/span\u003e, Rao, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). In this study, betaine significantly increased T-AOC and decreased MDA content, which is consistent with the findings in studies on broilers (Song et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2021\u003c/span\u003e)and piglets (Xiong et al., \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2023\u003c/span\u003e)that betaine enhances antioxidant enzyme activity, suggesting that it maintains redox balance by scavenging ROS. It is noteworthy that ROS can enhance LPS-induced inflammatory cascades by activating the NF-κB pathway (Park et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2015\u003c/span\u003e, Zhang and Igwe, \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), while the inhibition of MDA by betaine may weaken the oxidative stress-inflammation cross-signaling (Shakeri et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), forming a \u0026ldquo;antioxidant-anti-inflammatory\u0026rdquo; synergistic protective mechanism. This holds important physiological significance for alleviating intestinal mucosal oxidative damage in high-altitude hypoxic environments.\u003c/p\u003e\u003cp\u003eSCFAs produced by intestinal microbial metabolism are key hubs linking host-microbiota interactions (Silva et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In this study, betaine significantly increased the concentrations of acetic acid and propionic acid in the ileum by enriching SCFA-producing dominant genera such as \u003cem\u003eBifidobacterium\u003c/em\u003e and \u003cem\u003eAeriscardovia\u003c/em\u003e. As the main energy substrate for intestinal epithelial cells, SCFAs can promote cell proliferation by activating GPR41/43 receptors, directly optimizing villus morphology and enhancing nutrient absorption capacity (Muralitharan et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In addition, SCFAs reduce the release of LPS-induced pro-inflammatory factors such as TNF-α by inhibiting the NF-κB signaling pathway (Li et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2018\u003c/span\u003e, Liu et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), and simultaneously upregulate the expression of the tight junction protein Claudin-1, improving intestinal function from multiple dimensions of \u0026ldquo;energy supply - inflammation inhibition - barrier reinforcement\u0026rdquo;. This is consistent with studies on rodent (Sun et al., \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) and broiler models (Liao et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), indicating that betaine regulates host metabolism and immunity through the \u0026ldquo;microbiota-SCFAs\u0026rdquo; axis, forming a cascade effect of \u0026ldquo;proliferation of beneficial bacteria - production of metabolites - optimization of host functions\u0026rdquo;. It is worth emphasizing that acetic acid and propionic acid, as the main products of intestinal microbial fermentation, their increased concentrations not only reflect the optimization of the microbiota structure, but also directly participate in the regulation of host energy metabolism and oxidative stress, becoming key mediating factors for betaine to improve the ileal function of Tibetan sheep.\u003c/p\u003e\u003cp\u003ePhylum-level analysis showed that \u003cem\u003eFirmicutes\u003c/em\u003e, \u003cem\u003eEuryarchaeota\u003c/em\u003e, and \u003cem\u003eActinobacteriota\u003c/em\u003e were the dominant bacterial phyla in the ileum of Tibetan sheep, accounting for more than 95% of the total. Although betaine supplementation did not significantly alter the α-diversity of the microbiota, it promoted the significant enrichment of key SCFA-producing genera (\u003cem\u003eBifidobacterium\u003c/em\u003e and \u003cem\u003eAeriscardovia\u003c/em\u003e) by regulating the abundance at the phylum level (e.g., increasing the proportion of \u003cem\u003eActinobacteriota\u003c/em\u003e). Among them, \u003cem\u003eBifidobacterium\u003c/em\u003e decomposes carbohydrates through the unique \u0026ldquo;bifid shunt\u0026rdquo; metabolic pathway (Hald et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2016\u003c/span\u003e)and efficiently produces acetic acid and lactic acid (Usta-Gorgun and Yilmaz-Ersan, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2020\u003c/span\u003e); \u003cem\u003eAeriscardovia\u003c/em\u003e, as a member of the \u003cem\u003eBifidobacteriaceae\u003c/em\u003e family, also has the ability to produce SCFAs (Simpson et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). Previous studies have confirmed that these two genera can improve intestinal health by inhibiting the NF-κB pathway and enhancing the activity of antioxidant enzymes (Lee et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2022\u003c/span\u003e, Averina et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2021\u003c/span\u003e, Farooq et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), which is consistent with the results of this study showing that SCFA levels were positively correlated with T-AOC and negatively correlated with MDA and LPS. These findings indicate that betaine reshapes the ileal microbial community by directionally enriching functional genera, and then improves host physiological functions through metabolite mediation, reflecting the precise regulatory relationship of \u0026ldquo;microbe-metabolite-host\u0026rdquo;.\u003c/p\u003e\u003cp\u003eMetabolomics analysis revealed that betaine affects ileal function by regulating the \u0026ldquo;Arginine and proline metabolism\u0026rdquo; and \u0026ldquo;Glycerophospholipid metabolism\u0026rdquo; pathways. In arginine metabolism, betaine, as an efficient methyl donor, activates betaine-homocysteine methyltransferase to promote the production of S-adenosylmethionine (SAM) (Dobrijević et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2023\u003c/span\u003e, Huang et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), thereby accelerating the conversion of L-arginine to nitric oxide (NO) and polyamines (Wu and Meininger, \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2000\u003c/span\u003e, Mato et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). NO exerts antioxidant effects by scavenging free radicals and inhibiting the NF-κB inflammatory pathway (Bogdan, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2001\u003c/span\u003e, Nathan and Xie, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e1994\u003c/span\u003e), while polyamines support the repair of villus structure by promoting the proliferation of intestinal epithelial cells and the synthesis of tight junction proteins (Rao et al., 2020), which is highly consistent with the phenotype of increased VH/CD ratio. On the other hand, the upregulation of ceramide metabolites (such as Cer 18:1;2O/18:0) is positively correlated with SCFAs, suggesting that they may synergize with SCFAs in barrier protection by enhancing membrane structural stability and inhibiting inflammatory signals. It is noteworthy that the downregulation of lysophosphatidic acid (LPA 22:5) may be related to betaine inhibiting phospholipase A2 activity and reducing oxidative stress-induced decomposition of membrane phospholipids (Murakami et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e1999\u003c/span\u003e, Craig, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2004\u003c/span\u003e), thereby blocking the LPA-mediated pro-inflammatory signaling pathway (Zhao and Natarajan, \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e2013\u003c/span\u003e, Lin et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2010\u003c/span\u003e, Xia et al., \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). These metabolites form a molecular network for betaine to regulate ileal function through the cascade effect of \u0026ldquo;methyl donor-enzymatic reaction-signaling pathway\u0026rdquo;, providing a new explanation for the metabolic adaptation of Tibetan sheep in high-altitude environments.\u003c/p\u003e\u003cp\u003eIn this study, the interaction mechanism between differential intestinal microbiota and host metabolites was analyzed by constructing an association network, revealing that key genera such as \u003cem\u003eAeriscardovia\u003c/em\u003e and \u003cem\u003eBifidobacterium\u003c/em\u003e regulate the dynamic balance of lipid metabolism through multiple pathways. The results showed that \u003cem\u003eAeriscardovia\u003c/em\u003e and \u003cem\u003eBifidobacterium\u003c/em\u003e were significantly positively correlated with ceramide metabolites (Cer 18:1;2O/18:0, Cer 18:1;2O/24:2), which might affect the steady state of ceramide metabolism by regulating intestinal barrier function. As the core component of cell membrane lipids, abnormal metabolism of ceramides can directly trigger intestinal inflammation and epithelial cell apoptosis (Hannun and Obeid, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). It is noteworthy that the negative correlation between the above-mentioned genera and L-arginine suggests that they may competitively consume substrates through the arginine deiminase pathway, converting L-arginine into agmatine with immunomodulatory functions (Wu and Morris Jr, \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). In terms of membrane lipid dynamic balance, the decrease in the level of phosphatidylcholine isomer PC 16:0_17:2 is closely related to changes in cell membrane fluidity (Van Meer et al., \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). The study also found that intestinal flora forms a network regulation through metabolites such as SCFAs and secondary bile acids, which not only affects host lipid metabolism but also participates in the regulation of inflammatory responses (Tremaroli and B\u0026auml;ckhed, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). In conclusion, this study indicates that betaine significantly improves intestinal absorption function and antioxidant capacity by regulating the ileal microbial community and metabolites. This finding provides a theoretical basis for the development of precise nutritional strategies for plateau animal husbandry.\u003c/p\u003e\u003cp\u003eBetaine can regulate the microbial community (including enriching SCFA-producing \u003cem\u003eBifidobacterium\u003c/em\u003e and \u003cem\u003eAeriscardovia\u003c/em\u003e, etc.), significantly increase the concentration of SCFAs in the ileum (especially acetate and propionate), and regulate metabolic pathways such as arginine and proline metabolism, and glycerophospholipid metabolism. Through the \u0026ldquo;microbiota-SCFAs-metabolite\u0026rdquo; cascade effect, it enhances the structure and function of the ileum (optimizing villus structure, strengthening barrier function, and alleviating oxidative stress and inflammatory response). The results of this study provide an important theoretical basis for the application of betaine supplementation in the nutritional management of Tibetan sheep, especially in improving the theory of intestinal stress regulation in high-altitude ruminants and developing precise nutritional strategies for plateau animal husbandry.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eConflict of interest\u003c/h2\u003e\u003cp\u003eThe authors state that no commercial or financial connections existed during the research that might be interpreted as a potential conflict of interest.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThis research was funded by the project \u0026ldquo;Research and Integration of Production Technology for Special Tibetan Sheep in High Saline and Alkaline Habita\u0026rdquo; (Grant No. 2022-NK-169-3).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eWei Gao: Conceptualization, Data curation, Formal analysis, Methodology, Software, Writing-original draft, Writing-review \u0026amp; editing. Zhenling Wu: Conceptualization, Data curation, Methodology, Validation. Jiacheng Gan: Resources, Formal Analysis, Writing-review \u0026amp; editing. Xianhua Zhang: Investigation, Validation, Writing-review \u0026amp; editing. Chengdi Shi: Investigation, Validation, Writing-review \u0026amp; editing. Zhenglu Yang: Formal Analysis, Resources, Writing-review \u0026amp; editing. Quyangangmao Su: Conceptualization, Resources. Lijuan Han: Project administration, Writing-review \u0026amp; editing. Shengzhen Hou: Methodology, Project administration, Writing-review \u0026amp; editing. Linsheng Gui: Formal Analysis, Resources, Validation, Writing-review \u0026amp; editing. All authors have reviewed and consented to the manuscript's published form. All authors reviewed the manuscript.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe data sets generated in this research are available in online storage platforms. Specifically, they can be accessed via NCBI SRA with the accession number PRJNA1295669 and OMIX with the accession number PRJCA044040.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eABD EL-GHANY, W. A. \u0026amp; BABAZADEH, D. 2022. Betaine: A potential nutritional metabolite in the poultry industry. \u003cem\u003eAnimals,\u003c/em\u003e 12\u003cstrong\u003e,\u003c/strong\u003e 2624.\u003c/li\u003e\n\u003cli\u003eABHIJITH, A., DUNSHEA, F. R., CHAUHAN, S. S., SEJIAN, V. \u0026amp; DIGIACOMO, K. 2024. A meta-analysis of the effects of dietary betaine on milk production, growth performance, and carcass traits of ruminants. \u003cem\u003eAnimals,\u003c/em\u003e 14\u003cstrong\u003e,\u003c/strong\u003e 1756.\u003c/li\u003e\n\u003cli\u003eAKANDA, K. M. M., MEHJABIN, S., HASAN, A. N. \u0026amp; PARVEZ, G. M. 2025. 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Resveratrol attenuates oxidative stress‐induced intestinal barrier injury through PI3K/Akt‐mediated Nrf2 signaling pathway. \u003cem\u003eOxidative medicine and cellular longevity,\u003c/em\u003e 2019\u003cstrong\u003e,\u003c/strong\u003e 7591840.\u003c/li\u003e\n\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":"amb-express","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ambe","sideBox":"Learn more about [AMB Express](http://amb-express.springeropen.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/AMBE/default.aspx","title":"AMB Express","twitterHandle":"@SpringerOpen","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Betaine, Tibetan sheep, Ileal, Microbiomics, Metabolomics","lastPublishedDoi":"10.21203/rs.3.rs-7277025/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7277025/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eResearch on betaine's role in Tibetan sheep ileal development and the microbiota-metabolite axis remains scarce, and the mechanism by which it enhances intestinal health through its function as a methyl donor has not yet been elucidated. This study evaluated the effects of 0.08% dietary betaine supplementation on 60 weaned male Tibetan lambs (2 months old, with a mean body weight of 17.72\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19 kg), which were randomly divided into a control group (Ctrl) and a betaine group (Bet), with 30 lambs in each group. After a 10-day adaptation period followed by a 90-day formal feeding period, 6 lambs from each group were randomly selected for slaughter. Results showed that betaine supplementation significantly increased ileal villus height and the villus height-to-crypt depth (VH/CD) ratio (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), enhanced total antioxidant capacity (T-AOC), reduced levels of malondialdehyde (MDA), lipopolysaccharide (LPS), and tumor necrosis factor-α (TNF-α) in the ileum, and increased Claudin-1 levels (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). It also raised total short-chain fatty acids (SCFAs), acetate, and propionate concentrations in the ileum, along with the relative abundance of \u003cem\u003eBifidobacterium\u003c/em\u003e and \u003cem\u003eAeriscardovia\u003c/em\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and influenced arginine and proline metabolism as well as glycerophospholipid metabolism to enhance antioxidant and immune functions. 0.08% betaine can regulate ileal SCFA concentrations by modulating microbial composition and metabolic pathways, thereby supporting jejunal barrier function, providing a theoretical basis for its application as a functional feed additive.\u003c/p\u003e","manuscriptTitle":"Effects of betaine on ileal tissue and intestinal microbial metabolism in Tibetan sheep","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-12 09:41:14","doi":"10.21203/rs.3.rs-7277025/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-11-17T14:57:29+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-16T10:44:15+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"177400515490155713987998429405816311813","date":"2025-11-16T10:36:02+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"284121137421438033968363071406265559784","date":"2025-11-04T10:11:08+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"19023592750438138102005255827842223876","date":"2025-11-01T07:06:31+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-02T01:17:04+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"119401002017848444655464003488741223863","date":"2025-08-17T07:55:32+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-08-07T06:04:03+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-08-06T07:04:11+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-08-06T06:59:32+00:00","index":"","fulltext":""},{"type":"submitted","content":"AMB Express","date":"2025-08-02T09:04:44+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"amb-express","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ambe","sideBox":"Learn more about [AMB Express](http://amb-express.springeropen.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/AMBE/default.aspx","title":"AMB Express","twitterHandle":"@SpringerOpen","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"153766d0-89c1-4f50-be0d-fdfc70963225","owner":[],"postedDate":"August 12th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-02-09T16:05:15+00:00","versionOfRecord":{"articleIdentity":"rs-7277025","link":"https://doi.org/10.1186/s13568-026-02024-6","journal":{"identity":"amb-express","isVorOnly":false,"title":"AMB Express"},"publishedOn":"2026-02-04 15:57:01","publishedOnDateReadable":"February 4th, 2026"},"versionCreatedAt":"2025-08-12 09:41:14","video":"","vorDoi":"10.1186/s13568-026-02024-6","vorDoiUrl":"https://doi.org/10.1186/s13568-026-02024-6","workflowStages":[]},"version":"v1","identity":"rs-7277025","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7277025","identity":"rs-7277025","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}