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

doi:10.21203/rs.3.rs-7277025/v1

Download PDF

Research Article

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

https://doi.org/10.21203/rs.3.rs-7277025/v1

This work is licensed under a CC BY 4.0 License

You are reading this latest preprint version

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

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.

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% Premix¹	0.60	0.60
4% Concentrate	4.00	4.00
Betaine	0.00	0.08
Total	100.00	100.00
Nutrient levels²
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.

¹The 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.

²Digestible 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.
Items¹	Primer sequence (5’-3’)²	Tm (℃)	Product length	NCBI Gene ID
Claudin-1	F-CCTGCTGTGCTGCTCCTGTC	61.6	75bp	780473
Claudin-1	R-GAAGGTGCTGGCTTGGGATAGG	61.4	75bp	780473
Occludin	F-CGAGAAGCGACCGTATCCAGAG	61.4	129bp	101118304
Occludin	R-TCCAAGTTACCACTGCTGCTGTAG	59.6	129bp	101118304
ZO-1	F-GGGCAAGTTAAAGATGGTGGTTCAG	59.6	93bp	443200
ZO-1	R-GAGGCGTCAGCAGAGTGGATG	61.5	93bp	443200
TNF-α	F-ACGGCGTGGAGCTGAAAGAC	59.5	79bp	443540
TNF-α	R-CTGAAGAGGACCTGCGAGTAGATG	61.3	79bp	443540
Muc-2	F-ACGACTCCTACGCCCTCCTG	61.6	130bp	780488
Muc-2	R-ACGCTGCCATCCGACTTGAAG	51.5	130bp	780488
IL-6	F-TCTAATAACCACTCCAGCCACACAC	59.6	77bp	443406
IL-6	R-TTGCGTTCTTTACCCACTCGTTTG	57.9	77bp	443406
β-Actin	F-AGCAAGCGTGGCATCCTAACC	59.5	87bp	443340
β-Actin	R-ATCTTCTCCATGTCGTCCCAGTTG	59.6	77bp	443340

¹ZO-1, zonula occluden-1; MUC-2, mucoprotein-2; TNF-α, tumor necrosis factor-α; IL-6, interleukin-6.

²F, 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 < 0.05, and FC > 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.

Histomorphological analysis

Hematoxylin-eosin (H&E) stained sections intuitively showed the morphological changes in the ileal tissue of Tibetan sheep (Fig. 1A). 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). There were no significant differences in ileal villus width, crypt depth, crypt number, or muscular layer thickness between the two groups of Tibetan sheep (P > 0.05) (Fig. 1B).

Antioxidant capacity and lipopolysaccharide content

In terms of antioxidant capacity (Figs. 2A-E), compared with the Ctrl group, the Bet group showed higher T-AOC activity (P < 0.05) and lower MDA content (P < 0.05). In addition, there were no significant differences in the activities of CAT, SOD, and GSH-Px between the two groups (P > 0.05). Regarding LPS concentration (Fig. 2F), 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). However, it had no significant effect on the expression levels of Occludin, ZO-1, Muc-2 and IL-6 genes (P > 0.05) (Fig. 3).

Microbiota

As shown in the Venn diagram of Fig. 4A, 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. 4B). 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. 4C).

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. 4D). At the genus level, the dominant genera in the Bet group included Acetitomaculum, Olsenella, and Christensenellaceae_R_7_group (Fig. 4E). 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. 4F). As shown in Fig. 4G, 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. 5A). The OPLS-DA score plot showed differences in ileal metabolites between the two groups of Tibetan sheep (Fig. 5B). 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 5C 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. 5D). 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. 5E). 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. 6A). As shown in Fig. 6B, 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. 6C). The Mantel correlation analysis between differential microbiota and differential metabolites showed (Fig. 6D) 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.

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.

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. Xianhua Zhang: Investigation, Validation, Writing-review & editing. Chengdi Shi: Investigation, Validation, Writing-review & editing. Zhenglu Yang: Formal Analysis, Resources, Writing-review & editing. Quyangangmao Su: Conceptualization, Resources. Lijuan Han: Project administration, Writing-review & editing. Shengzhen Hou: Methodology, Project administration, Writing-review & editing. Linsheng Gui: Formal Analysis, Resources, Validation, Writing-review & editing. All authors have reviewed and consented to the manuscript's published form. All authors reviewed the manuscript.

Data Availability

The 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.

ABD EL-GHANY, W. A. & BABAZADEH, D. 2022. Betaine: A potential nutritional metabolite in the poultry industry. Animals, 12, 2624.
ABHIJITH, A., DUNSHEA, F. R., CHAUHAN, S. S., SEJIAN, V. & DIGIACOMO, K. 2024. A meta-analysis of the effects of dietary betaine on milk production, growth performance, and carcass traits of ruminants. Animals, 14, 1756.
AKANDA, K. M. M., MEHJABIN, S., HASAN, A. N. & PARVEZ, G. M. 2025. Role of Reactive Oxygen Species, Superoxide Dismutases, and Inflammation in Association with Gut Microbiota and Inflammatory Bowel Disease. Inflammatory Bowel Disease and Gut Microbiota. Apple Academic Press.
AL SULAIMAN, A. R., ABUDABOS, A. M. & ALHOTAN, R. A. 2024. Protective influence of supplementary betaine against heat stress by regulating intestinal oxidative status and microbiota composition in broiler chickens. International Journal of Biometeorology, 68, 279-288.
ARUMUGAM, M. K., PAAL, M. C., DONOHUE, T. M., GANESAN, M., OSNA, N. A. & KHARBANDA, K. K. 2021. Beneficial effects of betaine: a comprehensive review. Biology, 10, 456.
AVERINA, O. V., POLUEKTOVA, E. U., MARSOVA, M. V. & DANILENKO, V. N. 2021. Biomarkers and utility of the antioxidant potential of probiotic lactobacilli and bifidobacteria as representatives of the human gut microbiota. Biomedicines, 9, 1340.
AZAD, M. A. K., GAO, Q., MA, C., WANG, K. & KONG, X. 2022. Betaine hydrochloride addition in Bama mini‐pigʼs diets during gestation and lactation enhances immunity and alters intestine microbiota of suckling piglets. Journal of the Science of Food and Agriculture, 102, 607-616.
BABOS, D. V., COSTA, V. C., SPERANCA, M. A. & PEREIRA-FILHO, E. R. 2018. Direct determination of calcium and phosphorus in mineral supplements for cattle by wavelength dispersive X-ray fluorescence (WD-XRF). Microchemical Journal, 137, 272-276.
BOGDAN, C. 2001. Nitric oxide and the immune response. Nature immunology, 2, 907-916.
BOKULICH, N. A., SUBRAMANIAN, S., FAITH, J. J., GEVERS, D., GORDON, J. I., KNIGHT, R., MILLS, D. A. & CAPORASO, J. G. 2013. Quality-filtering vastly improves diversity estimates from Illumina amplicon sequencing. Nature methods, 10, 57-59.
CHEN, M. L., TAKEDA, K. & SUNDRUD, M. S. 2019. Emerging roles of bile acids in mucosal immunity and inflammation. Mucosal Immunology, 12, 851-861.
CHEN, Q., WANG, Y., JIAO, F., SHI, C., PEI, M., WANG, L. & GONG, Z. 2020. Betaine inhibits Toll-like receptor 4 responses and restores intestinal microbiota in acute liver failure mice. Scientific reports, 10, 21850.
CHENG, Y., SONG, M., ZHU, Q., AZAD, M. A. K., GAO, Q. & KONG, X. 2021. Dietary betaine addition alters carcass traits, meat quality, and nitrogen metabolism of Bama mini-pigs. Frontiers in Nutrition, 8, 728477.
COLLINS, J. T., NGUYEN, A. & BADIREDDY, M. 2024. Anatomy, abdomen and pelvis, small intestine. StatPearls [Internet]. StatPearls Publishing.
CRAIG, S. A. 2004. Betaine in human nutrition. The American journal of clinical nutrition, 80, 539-549.
DIGIACOMO, K., SIMPSON, S., LEURY, B. J. & DUNSHEA, F. R. 2023. Dietary Betaine Impacts Metabolic Responses to Moderate Heat Exposure in Sheep. Animals, 13, 1691.
DOBRIJEVIĆ, D., PASTOR, K., NASTIĆ, N., ÖZOGUL, F., KRULJ, J., KOKIĆ, B., BARTKIENE, E., ROCHA, J. M. & KOJIĆ, J. 2023. Betaine as a functional ingredient: metabolism, health-promoting attributes, food sources, applications and analysis methods. Molecules, 28, 4824.
DONG, L., ZHONG, Z., CUI, H., WANG, S., LUO, Y., YU, L., LOOR, J. & WANG, H. 2020. Effects of rumen-protected betaine supplementation on meat quality and the composition of fatty and amino acids in growing lambs. Animal, 14, 435-444.
EDGAR, R. C., HAAS, B. J., CLEMENTE, J. C., QUINCE, C. & KNIGHT, R. 2011. UCHIME improves sensitivity and speed of chimera detection. Bioinformatics, 27, 2194-2200.
EKLUND, M., BAUER, E., WAMATU, J. & MOSENTHIN, R. 2005. Potential nutritional and physiological functions of betaine in livestock. Nutrition research reviews, 18, 31-48.
FARAHAT, M., IBRAHIM, D., KISHAWY, A., ABDALLAH, H., HERNANDEZ-SANTANA, A. & ATTIA, G. 2021. Effect of cereal type and plant extract addition on the growth performance, intestinal morphology, caecal microflora, and gut barriers gene expression of broiler chickens. Animal, 15, 100056.
FAROOQ, M. Z., WANG, X. & YAN, X. 2023. Effects of Aeriscardovia aeriphila on growth performance, antioxidant functions, immune responses, and gut microbiota in broiler chickens. Journal of Zhejiang University-SCIENCE B, 24, 1014-1026.
HALD, S., SCHIOLDAN, A. G., MOORE, M. E., DIGE, A., LAERKE, H. N., AGNHOLT, J., BACH KNUDSEN, K. E., HERMANSEN, K., MARCO, M. L. & GREGERSEN, S. 2016. Effects of arabinoxylan and resistant starch on intestinal microbiota and short-chain fatty acids in subjects with metabolic syndrome: a randomised crossover study. PLoS One, 11, e0159223.
HANNUN, Y. A. & OBEID, L. M. 2008. Principles of bioactive lipid signalling: lessons from sphingolipids. Nature reviews Molecular cell biology, 9, 139-150.
HIFIFLHQF, F., DQDO, E. U. P. & RI FIEUH, D. D. S. S. Animal feeding stuff: Global Standard for the Determination of Acid Detergent Fibre (ADF) and Lignin.
HIRAOKA, H., FUKUNAKA, R., ISHIKURO, E., ENISHI, O. & GOTO, T. 2012. Improvement and validation of the method to determine neutral detergent fiber in feed. Animal Science Journal, 83, 690-695.
HUANG, B.-X., ZHU, Y.-Y., TAN, X.-Y., LAN, Q.-Y., LI, C.-L., CHEN, Y.-M. & ZHU, H.-L. 2016. Serum betaine is inversely associated with low lean mass mainly in men in a Chinese middle-aged and elderly community-dwelling population. British Journal of Nutrition, 115, 2181-2188.
JI, Q., ZHANG, F., ZHANG, Y., SU, Q., HE, T., HOU, S. & GUI, L. 2024. Multi-Omics revealed Resveratrol and β-Hydroxy-β-methyl Butyric acid alone or in combination improved the jejunal function in Tibetan sheep. Antioxidants, 13, 892.
LEE, S.-Y., LEE, B.-H., PARK, J.-H., PARK, M.-S., JI, G.-E. & SUNG, M.-K. 2022. Bifidobacterium bifidum BGN4 paraprobiotic supplementation alleviates experimental colitis by maintaining gut barrier and suppressing nuclear factor kappa B activation signaling molecules. Journal of medicinal food, 25, 146-157.
LI, L.-L., MA, S.-K., PENG, W., FANG, Y.-G., DUO, H.-R., FU, H.-Y. & JIA, G.-X. 2021. Genetic diversity and population structure of Tibetan sheep breeds determined by whole genome resequencing. Tropical Animal Health and Production, 53, 174.
LI, M., VAN ESCH, B. C., WAGENAAR, G. T., GARSSEN, J., FOLKERTS, G. & HENRICKS, P. A. 2018. Pro-and anti-inflammatory effects of short chain fatty acids on immune and endothelial cells. European journal of pharmacology, 831, 52-59.
LI, X., HAN, B., LIU, D., WANG, S., WANG, L., PEI, Q., ZHANG, Z., ZHAO, J., HUANG, B. & ZHANG, F. 2024. Whole-genome resequencing to investigate the genetic diversity and mechanisms of plateau adaptation in Tibetan sheep. Journal of Animal Science and Biotechnology, 15, 164.
LIAO, X., SHAO, Y., SUN, G., YANG, Y., ZHANG, L., GUO, Y., LUO, X. & LU, L. 2020. The relationship among gut microbiota, short-chain fatty acids, and intestinal morphology of growing and healthy broilers. Poultry science, 99, 5883-5895.
LIN, M.-E., HERR, D. R. & CHUN, J. 2010. Lysophosphatidic acid (LPA) receptors: signaling properties and disease relevance. Prostaglandins & other lipid mediators, 91, 130-138.
LIN, P.-Y., STERN, A., PENG, H.-H., CHEN, J.-H. & YANG, H.-C. 2022. Redox and metabolic regulation of intestinal barrier function and associated disorders. International Journal of Molecular Sciences, 23, 14463.
LIN, S., WANG, S., WANG, P., TANG, C., WANG, Z., CHEN, L., LUO, G., CHEN, H., LIU, Y. & FENG, B. 2023. Bile acids and their receptors in regulation of gut health and diseases. Progress in lipid research, 89, 101210.
LIU, T., LI, J., LIU, Y., XIAO, N., SUO, H., XIE, K., YANG, C. & WU, C. 2012. Short-chain fatty acids suppress lipopolysaccharide-induced production of nitric oxide and proinflammatory cytokines through inhibition of NF-κB pathway in RAW264. 7 cells. Inflammation, 35, 1676-1684.
LIU, X., SHA, Y., DINGKAO, R., ZHANG, W., LV, W., WEI, H., SHI, H., HU, J., WANG, J. & LI, S. 2020. Interactions between rumen microbes, VFAs, and host genes regulate nutrient absorption and epithelial barrier function during cold season nutritional stress in Tibetan sheep. Frontiers in Microbiology, 11, 593062.
MATO, J. M., MARTINEZ-CHANTAR, M. L. & LU, S. C. 2008. Methionine metabolism and liver disease. Annu. Rev. Nutr., 28, 273-293.
MURAKAMI, M., KAMBE, T., SHIMBARA, S. & KUDO, I. 1999. Functional coupling between various phospholipase A2s and cyclooxygenases in immediate and delayed prostanoid biosynthetic pathways. Journal of Biological Chemistry, 274, 3103-3115.
MURALITHARAN, R. R., ZHENG, T., DINAKIS, E., XIE, L., BARBARO-WAHL, A., JAMA, H. A., NAKAI, M., PATTERSON, M., JOHNSON, C. & SALIMOVA, E. 2023. GPR41/43 regulates blood pressure by improving gut epithelial barrier integrity to prevent TLR4 activation and renal inflammation. bioRxiv, 2023.03. 20.533376.
NATHAN, C. & XIE, Q.-W. 1994. Nitric oxide synthases: roles, tolls, and controls. Cell, 78, 915-918.
OBEID, R. 2013. The metabolic burden of methyl donor deficiency with focus on the betaine homocysteine methyltransferase pathway. Nutrients, 5, 3481-3495.
PARK, J., MIN, J.-S., KIM, B., CHAE, U.-B., YUN, J. W., CHOI, M.-S., KONG, I.-K., CHANG, K.-T. & LEE, D.-S. 2015. Mitochondrial ROS govern the LPS-induced pro-inflammatory response in microglia cells by regulating MAPK and NF-κB pathways. Neuroscience letters, 584, 191-196.
PERUMAL, S. K., ARUMUGAM, M. K., OSNA, N. A., RASINENI, K. & KHARBANDA, K. K. 2025. Betaine regulates the gut-liver axis: a therapeutic approach for chronic liver diseases. Frontiers in Nutrition, 12, 1478542.
RAO, J. N., XIAO, L. & WANG, J.-Y. 2020. Polyamines in gut epithelial renewal and barrier function. Physiology, 35, 328-337.
RAO, R. 2008. Oxidative stress-induced disruption of epithelial and endothelial tight junctions. Frontiers in bioscience: a journal and virtual library, 13, 7210.
RATRIYANTO, A. & MOSENTHIN, R. 2018. Osmoregulatory function of betaine in alleviating heat stress in poultry. Journal of animal physiology and animal nutrition, 102, 1634-1650.
SHAKERI, M., COTTRELL, J. J., WILKINSON, S., ZHAO, W., LE, H. H., MCQUADE, R., FURNESS, J. B. & DUNSHEA, F. R. 2019. Dietary betaine improves intestinal barrier function and ameliorates the impact of heat stress in multiple vital organs as measured by evans blue dye in broiler chickens. Animals, 10, 38.
SILVA, Y. P., BERNARDI, A. & FROZZA, R. L. 2020. The role of short-chain fatty acids from gut microbiota in gut-brain communication. Frontiers in endocrinology, 11, 508738.
SIMPSON, P. J., ROSS, R. P., FITZGERALD, G. F. & STANTON, C. 2004. Bifidobacterium psychraerophilum sp. nov. and Aeriscardovia aeriphila gen. nov., sp. nov., isolated from a porcine caecum. International journal of systematic and evolutionary microbiology, 54, 401-406.
SONG, Y., CHEN, R., YANG, M., LIU, Q., ZHOU, Y. & ZHUANG, S. 2021. Dietary betaine supplementation improves growth performance, digestive function, intestinal integrity, immunity, and antioxidant capacity of yellow-feathered broilers. Italian Journal of Animal Science, 20, 1575-1586.
SUN, C.-Y., LIU, W.-C., XIAO, M., ZHAO, Z.-H. & AN, L.-L. 2019. Effects of graded levels of betaine supplementation on growth performance and intestinal morphology in indigenous young yellow feather broilers. Pakistan Journal of Zoology, 51, 23232328.
SUN, L., TAN, X., LIANG, X., CHEN, H., OU, Q., WU, Q., YU, X., ZHAO, H., HUANG, Q. & YI, Z. 2023. Maternal betaine supplementation mitigates maternal high fat diet-induced NAFLD in offspring mice through gut microbiota. Nutrients, 15, 284.
SUN, R., XU, C., FENG, B., GAO, X. & LIU, Z. 2021. Critical roles of bile acids in regulating intestinal mucosal immune responses. Therapeutic advances in gastroenterology, 14, 17562848211018098.
THIEX, N. J., EREM, T. V. & M, C. A. D. B. J. C. A. C. D. C. P. D. D. F. P. H. H. M. D. M. H. R. T. W. 2002a. Determination of water (moisture) and dry matter in animal feed, grain, and forage (plant tissue) by Karl Fischer titration: collaborative study. Journal of AOAC international, 85, 318-327.
THIEX, N. J., MANSON, H., ANDERSON, S., PERSSON, J.-Å. & M, C. A. S. B. E. B. G. B. D. E. C. E. S. F. G. F. E. H. C. H. C. K. M. L. L. M. H. M. J. R. 2002b. Determination of crude protein in animal feed, forage, grain, and oilseeds by using block digestion with a copper catalyst and steam distillation into boric acid: collaborative study. Journal of AOAC International, 85, 309-317.
TREMAROLI, V. & BäCKHED, F. 2012. Functional interactions between the gut microbiota and host metabolism. Nature, 489, 242-249.
USTA-GORGUN, B. & YILMAZ-ERSAN, L. 2020. Short-chain fatty acids production by Bifidobacterium species in the presence of salep. Electronic Journal of Biotechnology, 47, 29-35.
VAN MEER, G., VOELKER, D. R. & FEIGENSON, G. W. 2008. Membrane lipids: where they are and how they behave. Nature reviews Molecular cell biology, 9, 112-124.
VISEKRUNA, A. & LUU, M. 2021. The role of short-chain fatty acids and bile acids in intestinal and liver function, inflammation, and carcinogenesis. Frontiers in Cell and Developmental Biology, 9, 703218.
WANG, H., LI, S., FANG, S., YANG, X. & FENG, J. 2018. Betaine improves intestinal functions by enhancing digestive enzymes, ameliorating intestinal morphology, and enriching intestinal microbiota in high-salt stressed rats. Nutrients, 10, 907.
WANG, H., LI, S., XU, S. & FENG, J. 2020. Betaine improves growth performance by increasing digestive enzymes activities, and enhancing intestinal structure of weaned piglets. Animal Feed Science and Technology, 267, 114545.
WANT, E. J., MASSON, P., MICHOPOULOS, F., WILSON, I. D., THEODORIDIS, G., PLUMB, R. S., SHOCKCOR, J., LOFTUS, N., HOLMES, E. & NICHOLSON, J. K. 2013. Global metabolic profiling of animal and human tissues via UPLC-MS. Nature protocols, 8, 17-32.
WEI, C., WANG, H., LIU, G., ZHAO, F., KIJAS, J. W., MA, Y., LU, J., ZHANG, L., CAO, J. & WU, M. 2016. Genome-wide analysis reveals adaptation to high altitudes in Tibetan sheep. Scientific reports, 6, 26770.
WEN, C., CHEN, R., CHEN, Y., DING, L., WANG, T. & ZHOU, Y. 2021a. Betaine improves growth performance, liver health, antioxidant status, breast meat yield, and quality in broilers fed a mold-contaminated corn-based diet. Animal Nutrition, 7, 661-666.
WEN, C., LENG, Z., CHEN, Y., DING, L., WANG, T. & ZHOU, Y. 2021b. Betaine alleviates heat stress-induced hepatic and mitochondrial oxidative damage in broilers. The journal of poultry science, 58, 103-109.
WU, G. & MEININGER, C. J. 2000. Arginine nutrition and cardiovascular function. The Journal of nutrition, 130, 2626-2629.
WU, G. & MORRIS JR, S. M. 1998. Arginine metabolism: nitric oxide and beyond. Biochemical Journal, 336, 1-17.
WU, J., HE, C., BU, J., LUO, Y., YANG, S., YE, C., YU, S., HE, B., YIN, Y. & YANG, X. 2020. Betaine attenuates LPS-induced downregulation of Occludin and Claudin-1 and restores intestinal barrier function. BMC veterinary research, 16, 75.
XIA, Y., CHEN, S., ZHU, G., HUANG, R., YIN, Y. & REN, W. 2018. Betaine inhibits interleukin-1β production and release: potential mechanisms. Frontiers in Immunology, 9, 2670.
XIONG, L., WANG, K., SONG, M., AZAD, M. A. K., ZHU, Q. & KONG, X. 2023. Dietary betaine supplementation enhances colonic barrier function through the Nrf2/Keap1 and TLR4-NF-κB/MAPK signaling pathways and alters colonic microbiota in Bama mini-pigs. Antioxidants, 12, 1926.
XU, T., XU, S., HU, L., ZHAO, N., LIU, Z., MA, L., LIU, H. & ZHAO, X. 2017. Effect of dietary types on feed intakes, growth performance and economic benefit in Tibetan sheep and yaks on the Qinghai-Tibet Plateau during cold season. PloS one, 12, e0169187.
YAN, J., PAN, Y., SHAO, W., WANG, C., WANG, R., HE, Y., ZHANG, M., WANG, Y., LI, T. & WANG, Z. 2022. Beneficial effect of the short-chain fatty acid propionate on vascular calcification through intestinal microbiota remodelling. Microbiome, 10, 195.
YANG, S. & YU, M. 2021. Role of goblet cells in intestinal barrier and mucosal immunity. Journal of Inflammation Research, 3171-3183.
YANG, Y., JIANG, W., YANG, S., QI, F. & ZHAO, R. 2020. Transgenerational Inheritance of Betaine‐Induced Epigenetic Alterations in Estrogen‐Responsive IGF‐2/IGFBP2 Genes in Rat Hippocampus. Molecular Nutrition & Food Research, 64, 1900823.
YANG, Z., ASARE, E., YANG, Y., YANG, J., YANG, H. & WANG, Z. 2021. Dietary supplementation of betaine promotes lipolysis by regulating fatty acid metabolism in geese. Poultry Science, 100, 101460.
YANG, Z., YANG, J., ZHU, P., HAN, H., WAN, X., YANG, H. & WANG, Z. 2022. Effects of betaine on growth performance, intestinal health, and immune response of goslings challenged with lipopolysaccharide. Poultry Science, 101, 102153.
ZHANG, M., SHI, S., FENG, Y., ZHANG, F., XIAO, Y., LI, X., PAN, X., FENG, Y., LIU, D. & GUO, Y. 2025. Synthetic microbial community improves chicken intestinal homeostasis and provokes anti-Salmonella immunity mediated by segmented filamentous bacteria. The ISME Journal, wraf076.
ZHANG, Y. & IGWE, O. J. 2018. Exogenous oxidants activate nuclear factor kappa B through Toll-like receptor 4 stimulation to maintain inflammatory phenotype in macrophage. Biochemical Pharmacology, 147, 104-118.
ZHAO, N., YANG, Y., CHEN, C., JING, T., HU, Y., XU, H., WANG, S., HE, Y., LIU, E. & CUI, J. 2022. Betaine supplementation alleviates dextran sulfate sodium-induced colitis via regulating the inflammatory response, enhancing the intestinal barrier, and altering gut microbiota. Food & Function, 13, 12814-12826.
ZHAO, Y. & NATARAJAN, V. 2013. Lysophosphatidic acid (LPA) and its receptors: role in airway inflammation and remodeling. Biochimica et Biophysica Acta (BBA)-Molecular and Cell Biology of Lipids, 1831, 86-92.
ZHOU, J. Y., HUANG, D. G., ZHU, M., GAO, C. Q., YAN, H. C., LI, X. G. & WANG, X. Q. 2020. Wnt/β‐catenin‐mediated heat exposure inhibits intestinal epithelial cell proliferation and stem cell expansion through endoplasmic reticulum stress. Journal of cellular physiology, 235, 5613-5627.
ZHUANG, Y., WU, H., WANG, X., HE, J., HE, S. & YIN, Y. 2019. Resveratrol attenuates oxidative stress‐induced intestinal barrier injury through PI3K/Akt‐mediated Nrf2 signaling pathway. Oxidative medicine and cellular longevity, 2019, 7591840.

No competing interests reported.

SupplementaryTable1.docx

Download PDF

Editorial decision: Revision requested
17 Nov, 2025
Reviews received at journal
16 Nov, 2025
Reviewers agreed at journal
16 Nov, 2025
Reviewers agreed at journal
04 Nov, 2025
Reviewers agreed at journal
01 Nov, 2025
Reviews received at journal
01 Sep, 2025
Reviewers agreed at journal
17 Aug, 2025
Reviewers invited by journal
07 Aug, 2025
Editor assigned by journal
06 Aug, 2025
Submission checks completed at journal
06 Aug, 2025
First submitted to journal
02 Aug, 2025

You are reading this latest preprint version

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

Status:

Version 1

Abstract

Figures

Introduction

Materials and Methods

Statistical analysis

Results

Discussion

Declarations

Conflict of interest

Funding

Author Contribution

Data Availability

References

Additional Declarations

Supplementary Files

Status:

Version 1