Effects of dietary supplementation with 2-hydroxy-4-(methylthio) butanoic acid isopropyl ester on ileal microbiota and metabolism in Liaoning cashmere goats

Effects of dietary supplementation with 2-hydroxy-4-(methylthio) butanoic acid isopropyl ester on ileal microbiota and metabolism in Liaoning cashmere goats | 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 dietary supplementation with 2-hydroxy-4-(methylthio) butanoic acid isopropyl ester on ileal microbiota and metabolism in Liaoning cashmere goats Minjie Xi, Miaomiao Zhang, Peiyuan Sun, Jiali Jiang, Zhiqiang Zhong, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8062149/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Currently, rumen-protected methionine, such as 2-hydroxy-4-(methylthio)butanoic acid isopropyl ester (HMBi), is frequently employed in the ruminant breeding industry to reduce microbial degradation and subsequently enhance the methionine supply in an effective manner. However, there is a paucity of information regarding alterations in the microecological status of the small intestine with HMBi supplementation. Hence, the present study integrated multi-omics approaches to reveal the effects of HMBi on the microbial community, metabolites, and production performance in the ileum of Liaoning Cashmere goats. In this study, 14 female goats were assigned to be fed the control diets (CON, n = 7) and HMBi (n = 7) diets. After 67 days, the ileal digesta were sampled for measurements. Our results showed that the HMBi diets significantly increased ( P < 0.05) the cashmere length and growth rate, and tended to increase the ileal MCP concentration ( P = 0.085). Our results of ileal microbiota demonstrated that the HMBi supplementation decreased the abundance of cellulose-degrading bacteria (Cellulosilyticum, Lachnospiraceae_uncultured, Breznakia, etc.) and increased the abundance of beneficial bacteria (OTU9, OUT256, OTU657, etc.) in the ileum of goats. Our results of ileal metabolites also showed that, the content of HMBi degradation products (such as HMB and methionine) and some substance (such as taxifolin, Soyasaponin I, and tyrosol) enhanced the intestinal health in the ileum of goats were increased after HMBi feeding ( P < 0.05). Moreover, our Mantel tests showed that ileal microbiota and metabolites significantly contributed to the improved cashmere performance of goats ( P < 0.05). Overall, the above results revealed that the ileal microbiota and metabolism were involved in the process of HMBi regulating cashmere performance of goats. cashmere goats HMBi ileal digesta microbiota metabolome Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Methionine (Met) is the first limiting amino acid in ruminants such as cashmere goats, and has antioxidant, anti-inflammatory, immune-regulating and other functions(Gebeyew et al., 2021 ; Khan et al., 2022 ; Potts et al., 2023 ). Hence, Met supply is closely related to the health and production performance of ruminants. In modern ruminant breeding, the degradation of rumen microbiota has led to the increased use of rumen-protected methionine (RPM) as a means of reducing microbial degradation and subsequently increasing methionine supply more effectively. The 2-hydroxy-4-(methylthio) butanoic acid isopropyl ester (HMBi), a hydroxyl analog of Met, is often adopted to provide metabolisable Met for ruminants. Numerous studies indicated that HMBi supplementation elevated body weight gain, increased milk production, and improved milk components such as milk fat in cattles (Ordway et al., 2009 ; Osorio et al., 2013; Qin et al., 2022 ). Nevertheless, limited data suggested a potential effect of HMBi on cashmere growth of goats. One early research revealed that the HMBi adding in diets abled to enhance the growth rate of cashmere fibres(Feng et al., 2013b ), and also altered the rumen fermentation parameters (pH, ammonia-N concentration, and volatile fatty acids) in Liaoning cashmere goats(Feng et al., 2013a ). In general, the research on the effects of HMBi on ruminants is largely confined to the domains of production performance and the rumen, with no studies on the small intestine having been conducted to date. Besides the rumen, the microecological status in the small intestine is also crucial to the nutrient digestion and absorption and immune system for ruminants(Myer et al., 2016 ). For instance, early weaning significantly altered the ileal microbial structure and increased the mRNA expression of toll-like receptors and tight junction protein genes of Hu lambs(Li et al., 2018 ). Another study reported that the small intestinal microbiota and metabolite patterns with different residual feed intake phenotypes are very different in the Angus Heifers, suggesting that the micro-ecology is potentially important for the improvement of feed efficiency in ruminants(Liu et al., 2022 ). Previous studies demonstrated that ingested HMBi could be rapidly decomposed into 2-hydroxy-4-(methylthio) butanoic acid (HMB), and is metabolized in three ways: direct absorbed from the rumen wall, degraded by rumen microbiota and then synthesized into microbial proteins (MCP), or directly arrived at the small intestine(Lin et al., 2008 ). It has been demonstrated that HMBi has a biological activity of 40–58% in rumen(Graulet et al., 2004 ; Robert et al., 2001 ). Consequently, it is postulated that the HMBi supplementation may result in alterations to the substrates entering the intestine, thereby influencing the microecological status within the intestine. Therefore, our study utilized multi-omics approaches (16S rRNA sequencing and metabolomics) to reveal the effects of HMBi on ileal fermentation, microbial community and metabolites of Liaoning cashmere goats. Our results could offer a certain theoretical reference for the application of HMBi in Liaoning cashmere goats. Materials and methods Animal experiment, diets The experimental process was conducted according to the Institutional Animal Care and Use Committee of Shenyang Agricultural University (NO. 2021091501). Fourteen healthy female Liaoning cashmere goats (25.63 ± 0.87 kg body weight, around 8 months of age) were divided into the control group (CON, n = 7) and the HMBi (n = 7) groups. The goats in CON group was offered a basal diet, and the HMBi group was fed with 1.27% HMBi (MetaSmart; Adisseo Inc., Antony, France) in the basal diet. MetaSmart was offered as dry powder form and contained 57% HMBi, which is equivalent to 78% methionine (Osorio et al., 2013). The basal diets were formulated according to the recommendations of goats (NY/Y816-2004; Ministry of Agriculture of China, 2004). The ingredients and nutrient composition of the basal diet is listed in Table S1. All experimental goats were reared for 67 days, of which 7 days are the pre-feeding period. During the experiment, all animals were placed in individual pens. The feed intake, initial body weight, and final body weight (FBW) of every goat were determined, and the average daily gain (ADG) and the ratio of average daily feed intake to average daily gain (F/G) were calculated. Sample collection After 67 days of dietary treatments, all goats were slaughtered, and the ileum section was immediately dissected and isolated. The ileal digesta of experimental goats were collected after thorough mixing and homogenized, and stored in -80℃ until the further microbial and metabolomic analysis. The ileal tissue was flushed with ice-cold sterile saline to clear the adherent ileal content, and then quickly frozen in liquid nitrogen for gene quantification. Ileal fermentation parameters The measurement of volatile fatty acids (VFAs) used crotonic acid as an internal stand, and was performed on a gas chromatography (Agilent 7890B, CA, USA) according to the methods of Mao et al. (2007). The concentrations of ammonia (NH 3 -N) were determined as described by Chaney and Marbach (1962). The microbial crude protein (MCP) was analyzed according to the procedure reported by Makkar et al.(1982). Microbial DNA isolation and illumina sequencing Ileal samples were fully thawed and mixed, and bacterial DNA isolation was conducted using QIAamp DNA Stool Mini Kit (Qiagen, Hilden, Germany). After multiple extractions with phenol/chloroform, the DNA pellets were precipitated with ethanol and then redissolved using Tris-EDTA buffer. The isolated DNA was detected using NanoDrop 2000 spectrophotometer (Wilmington, DE, United States), only high-quality DNA were stored at -80℃ until subsequent analysis. The V3-V4 fragment of 16S rRNA genes were amplified using the following bacterial universal primers: 338F (5’-ACTCCTRCGGGAGGCAGCAG-3’) and 806R (5’-GGACTACCVGGGTATCTAAT-3’). The amplification was performed according to the following procedures: initial denaturation (95°C, 2 min), 25 cycles of amplification (95°C, 30 s; 55°C,30 s; 72°C, 30 s) and final extension (72°C, 5 min). Three replicates were set for each sample, and the amplified products of one sample were mixed and purified by Qiagen QIAquick PCR purification kit (Qiagen, Duesseldorf, Germany). The pair-end sequencing was performed on an Illumina Miseq platform (Majorbio, Shanghai, China) follow the manufacturer's recommended procedures. For data analyses, the paired-end raw sequences were demultiplexed, and filtered by conducting quality control. Sequences that did not meet the filtering criteria and were of low quality were removed from raw sequences. Sequences were screened for chimeras using UCHIME(Edgar et al., 2011 ), and unnormal gene sequences were removed. Operational taxonomic units (OTUs) clustering was performed using UPARSE at 97% identity. The most prevalent sequences within each OTU were aligned and annotated by SILVA (SSU138.1) database (Amato et al., 2013 ). Microbial richness and diversity were evaluated using Chao 1, Shannon and Simpson. Differences in ileal microbiota was estimated using principal coordinates analysis (PCoA). To further determine whether the microbial differences between the two groups reach a significant level, the permutation multivariate analysis (PERMANOVA) was adopted. LC-MS detection Ileal digesta samples were pretreated before the metabolomics analysis. Samples were fully thawed and mixed, then, 50 mg ileal samples were with 800 mL methanol. The mixture was ground (65HZ, 180 s), ultrasonicated (4°C, 30 min), and centrifuged (12000 rpm, 4°C, 15 min). Repeat the above-mentioned centrifugation for the collected supernatant. Dichlorophenylalanine was adopted as internal standard of LC-MS detection, and the detection was performed on a Waters ACQUITY UPLC® system (Waters Corp., USA). During the determination, the flow rate was kept at 0.3 ml/min, and the column temperature of instrument was kept at 40°C. The metabolome data was processed and annotated using Compound Discoverer 3.3 software (Thermo Fisher Scientific, Waltham, MA, USA). and the extracted data included retention time, peak intensity, sample names, and compound molecular weight were used for further analysis. Statistics of metabolomics data were mainly performed through SIMCA-P + 14.1 software (Umetrics, Umea, Sweden). Differences in ileal metabolites were estimated through the partial least squares discriminant analysis (PLS-DA) and orthogonal partial least-squared discriminant analysis (OPLS-DA). Variable importance in projection (VIP) scores in PLS-DA were harvested to measure the contributions and importance of metabolites. Statistical analysis Statistical calculations in the present study were carried out using SPSS (v23, SPSS Inc., Chicago, IL). Statistics of data on growth performance and cashmere performance was conducted utilizing the independent samples t test. Since the data on microbial phyla and genera and metabolomics did not conform to a normal distribution, the statistics of these data was performed using a Wilcoxon-Mann-Whitney U test. Characterized OTUs were identified using the linear discriminant analysis effect size (LEfSe) analysis. The metabolites with VIP > 1 (generated from PLS-DA analysis) and P ≤ 0.05 (generated from Wilcoxon-Mann-Whitney U test) were defined as ileal differential metabolites. The partial Mantel tests of cashmere performance, ileal fermentation, microbial community and metabolites were conducted by the “linkET” package in R (v3.5.0). Significant differences were declared at P ≤ 0.05. Results Production performance of goats The effects of HMBi on growth status of Cashmere goats was listed in the Table S2. The results suggested that the HMBi diets had no significant effects on FBW, ADG and F/G of Cashmere goats. The results regarding cashmere performance had been described in our previous study, and the results revealed that the HMBi group improved the cashmere length and cashmere growth rate ( P < 0.05), and also decreased the cashmere diameter of goats ( P < 0.05)(Xi et al., 2025 ). Ileal fermentation parameters The results of ileal fermentation parameters were listed in Table 1. The ileal MCP concentration tended to increase in the HMBi diets ( P = 0.085). However, the HMBi supplementation did not affect the levels of VFAs and NH 3 -N in the ileum of Liaoning Cashmere goats. Ileal microbial diversity The estimators of microbial richness and diversity were presented in Fig. 1. No differences were found in Chao 1, Shannon and Simpson of ileal microbiota between two dietary treatments. Next, the PCoA analysis (Fig. 1D) showed that there was no obvious separation in the distribution of ileal samples from two groups in the plots figures, and the PERMANOVA analysis also confirmed no statistical differences between the CON and HMBi group (PERMANOVA P = 0.402). Alterations in the structure of ileal microbiota between the CON and HMBi groups As shown in Fig. 2 A, we found that the abundance of 9 phyla were higher than 0.1%, and Firmicutes (average 81.37%) and Bacteroidota (average 7.92%) were the most abundance phyla in the ileal microbiota of goats. The results of statistical analysis revealed that the HMBi supplementation did not affect the microbial phyla in the ileal digesta of goats. At the genus level, Oscillospiraceae UCG-005 (average 15.21%), Eubacterium coprostanoligenes group_norank (average 10.87%), UCG-010_norank (average 6.42%), Christensenellaceae R-7 group (average 6.12%), and Monoglobus (average 4.05%) were the abundant genera in the ileal microbiota of goats (Fig. 2 B). Our statistical analysis (Fig. 2 C) indicated that the HMBi group decreased ( P < 0.05) the abundance of Lachnospiraceae_uncultured, Anaerorhabdus furcosa group, Breznakia, Meiothermus, and Cellulosilyticum in the ileal microbiota of goats. However, the HMBi supplementation did not affect other genera in the ileal microbiota of goats. To further evaluate the impacts of HMBi supplementation on microbial community, the LEfSe analysis was performed. The results showed that (Fig. 3 ), 13 OTUs, including 4 OTUs (OTU41, OTU295, OTU216, and OTU206) belonging to Christensenellaceae R-7 group , 2 OTUs (OTU99 and OTU324) belonging to Rikenellaceae RC9 gut group, 2 OTUs (OTU59 and OUT 241) belonging to the family Muribaculaceae, 2 OTUs (OTU322 and OTU355) belonging to the family Lachnospiraceae, OTU33 (G: Oscillospiraceae UCG-005), OTU167 (G: Ruminococcus), and OTU456 (F: Bacteroidales RF16 group) were enriched ( P < 0.05) in the CON group. Meanwhile, OTU361 (F: Ruminococcaceae), OTU657 (G: Monoglobus), OTU215 (G: Muribaculaceae), OTU256 (G: Blautia), OTU112 (G: Oscillospiraceae UCG-005), OTU188 (G: Treponema), and OTU9 (F: Eubacterium coprostanoligenes group) were enriched ( P < 0.05) in the HMBi group. Metabolic profiles in the ileum of goats fed the CON and HMBi diets Through the metabolomics analysis, as well as the quality control and identification, we totally harvested 603 valid ileal metabolites from both the CON and HMBi groups. Next, the results of PLS-DA and OPLS-DA analysis suggested that the sample plots from two groups were clustered together individually in the figures, suggesting the huge differences in ileal metabolites between these two groups (Fig. 4 A and 4 B). The ileal metabolites that with VIP and statistical P < 0.05 were defined as differential metabolites, and its distribution were presented in Fig. 4 C. Our results indicated that some ileal metabolites, such as phosphorylcholine, L-tyrosine methyl ester, dodecanoic acid, alpha-tocopherol acetate, and 1-Stearoylglycerophosphoglycerol were correlated with the CON diets, and some ileal compounds, such as 4-phenylbutyric acid, heptanoic acid, 4-hydroxybenzylalcohol, pentadecanoic acid, and L-malic acid were correlated with the HMBi diets. By sorting and classifying differential metabolites, our results suggested the HMBi supplementation altered the levels of 68 ileal metabolites (VIP > 1 and P < 0.05) (Fig. 5 ). Specifically, for amino acids, the HMBi diet increased ( P < 0.05) the level of methionine, ornithine, O-succinylhomoserine, L-2-aminoadipic acid, N-acetylalanine, and pyroglutamic acid, and decreased the level of tyrosine methyl ester in the ileum of goats ( P < 0.05). For fatty acids and lipids, the HMBi supplementation increased the content of FA 17:0, FA 9:1 + 1O, tetradecanedioic acid, pentadecanoic acid, heptanoic acid, and cholest-4-en-3-one ( P < 0.05), while decreased the content of elaidic acid, dodecanoic acid, 1-stearoylglycerophosphoglycerol, chaulmoogric acid, and glycodeoxycholic acid in the ileal digesta of goats ( P < 0.05). For alcohols, the content of taxifolin, 4-hydroxybenzylalcohol, and tyrosol in the goats fed HMBi diets were higher ( P < 0.05) than those fed the CON diets. For amines, the level of 4-methoxyaniline was higher ( P < 0.05), and the level of phosphorylcholine and phytosphingosine was lower ( P < 0.05) in the ileal digesta of goats fed HMBi diets than those fed the CON diets. In addition, the HMBi group also increased ( P < 0.05) the level of 10 organic acids including mono-methyl glutarate, malic acid, 4-phenylbutyric acid, 3-methylglutaconic acid, valeric acid, 1,4-cyclohexanedicarboxylic acid, N-acetylanthranilic acid, 4-toluic acid, methylmalonic acid, and suberylglycine than those in the CON group. Additionally, we also found HMBi supplementation significantly increased ( P < 0.05) the content of taxifolin, Soyasaponin I, (+)-ar-Turmerone, etc. in the ileal digesta of goats. We further conducted the enrichment analysis of these ileal differential metabolites identified in the CON and HMBi groups. Our results suggested these metabolites were enriched ( P < 0.05) in arginine biosynthesis, citrate cycle, pyruvate metabolism, and glutathione metabolism (Fig. 6 ). In addition, metabolites involved in the above pathways were ornithine, fumaric acid, malic acid, and pyroglutamic acid. The potential interactions among the cashmere performance, ileal fermentation, microbial community and metabolites To explore the potential mechanism by which HMBi improved the cashmere performance from an intestinal perspective, we conducted the partial Mantel tests. The results showed that (Fig. 7 A), the cashmere length and growth rate were negatively correlated with cashmere diameter ( P < 0.05). The cashmere length and growth rate were closely related to ileal microbiota and metabolites ( P < 0.05), among which ileal microbiota was more closely related ( P < 0.05). In addition, cashmere diameter tended to be related to ileal microbiota and metabolites. Next, we conducted the correlation network analysis of cashmere performance and ileal microbiota and metabolites. Methyl-containing metabolites attracted our attention, our results showed that (Fig. 7 B), cashmere length was negatively correlated with N,N-Dimethyltetradecylamine and tyrosine methyl ester, and positively correlated with 3'-O-methyluridine, 4-methoxyaniline, methylmalonic acid, mono-methyl glutarate, and 3-methylglutaconic acid ( P < 0.05); while cashmere diameter was negatively correlated with 3-methylglutaconic acid and methylmalonic acid ( P < 0.05). Moreover, cashmere length was negatively correlated with Lachnospiraceae_uncultured, Breznakia , Meiothermus, OTU33, OTU216, and OTU324 ( P < 0.05), and positively correlated with OTU9 and OTU361. Cashmere diameter was negatively related with OTU657 and OTU188, and positively related with OTU33 and OTU241 ( P < 0.05). Discussion Dietary HMBi supplementation increased the cashmere growth and decreased the cashmere diameter Met is the first limiting amino acid in ruminants such as cashmere goats, and has the potential effects of promoting hair follicle cell proliferation and differentiation, increasing keratin biosynthesis and accelerating hair growth(Galbraith, 2000 ). Consequently, Met is intimately associated with the cashmere performance of cashmere goats. Our results demonstrated that HMBi supplementation significantly increased the cashmere length and growth rate, and decreased the cashmere diameter. Partially consistent with our results, previous studies reported the HMBi could increase the growth rate, but did not affect the diameter of cashmere fibres(Feng et al., 2013b ). Our results indicated that HMBi could elevate the Met level reached the skin, promote the keratoprotein synthesis, and thereby improve the cashmere production and quality. Dietary HMBi supplementation did not affect ileal fermentation pattern, but increased MCP synthesis The microorganisms inhabited in the small intestine of animals have a certain ability to ferment nutrients and can also convert these nutrients into VFAs like rumen(Jiao et al., 2014 ). Our results showed that HMBi supplementation did not affect the VFAs and NH 3 -N in the ileal digesta of goats, which suggested that the ileal fermentation pattern remained unaltered following the HMBi supplementation. Nevertheless, in comparison to the CON group, the MCP concentration exhibited a tendency to increase following HMBi supplementation. Our findings indicated that the HMBi supplementation caused intestinal microbiota to use nutritional substrates to synthesize more MCP, and these MCP may contribute to host health. Dietary HMBi supplementation reduced the abundance of cellulose-degrading bacteria but increase the proliferation of probiotic bacteria As indicated by PCoA and PERMANOVA, our results demonstrated that no significant differences in the global microbiota were existed in the ileum of goats. Next, statistical analyses were performed at different taxonomic levels to reveal the further impacts of HMBi. Our results demonstrated that the HMBi supplementation resulted in a notable reduction in the abundance of bacteria with cellulose-degrading capabilities, such as Cellulosilyticum , Lachnospiraceae_uncultured, and Breznakia . The draft genome of Cellulosilyticum encoded a considerable number of cellulolytic enzymes, and species in this genus were postulated to play a key role in degrading cellulose and xylan(Cai et al., 2015 ). Lachnospiraceae members were common bacteria found in the intestines of humans and animals, and were uniquely suited to degrade some recalcitrant substrates, such as cellulose(Biddle et al., 2013 ; Strain et al., 2020 ). The genus Breznakia is frequently detected in the mammalian intestine, but the knowledge of these bacteria in this genus is very limited(Pacífico et al., 2021 ). One research reported two species ( B. pachnodae and B. blatticola ) in genus Breznakia abled to utilize a variety of substrates (including fructose, glucose, cellobiose, etc.) to ferment to produce formate, acetate, and ethanol(Tegtmeier et al., 2016 ). Hence, the above results suggested that the ability of intestinal bacteria to degrade cellulose was diminished following dietary supplementation with HMBi. In addition, previous studies reported that Met analogs or HMBi were able to stimulate the proliferation of cellulose-degrading bacteria in the rumen, such as Fibrobacter succinogenes and Ruminococcus flavefaciens (Firkins et al., 2015 ; Martin et al., 2013 ). Based on the above results on the rumen microbiota, we speculated that HMBi supplementation led to a substantial amount of cellulose to degraded in the rumen, and then a decrease in the cellulose reaching the small intestine, which further resulted in a decline in the cellulose-degrading bacteria in the small intestine. Furthermore, our results showed that OTU256 (G: Blautia ), OTU9 (F: Eubacterium coprostanoligenes group), and OTU657 (G: Monoglobus ) were enriched in the HMBi group. Blautia is an anaerobic bacterium with probiotic properties. The research reported the oral Blautia induced anti-inflammatory changes and thus reduced obesity and diabetes in mice (Hosomi et al., 2022 ). In addition, Blautia -derived acetate is vital for regulating host immunity, such as enhancing the stress of immune T-cells(Ye et al., 2023 ). We therefore speculated that HMBi induced enrichment of OTU256 (G: Blautia ) may have anti-inflammatory effects and benefit gut health in the present study. The Eubacterium coprostanoligenes group has been identified as a butyrate-producing bacterium, which is also capable of reducing cholesterol levels and protecting the intestinal mucosal barrier(Gérard, 2020 ). It had been reported that cholesterol homeostasis may be associated with the trigger of the nucleotide binding oligomerization domain like receptors protein 3 inflammatory vesicle signalling pathway in intestinal inflammation(Astorga et al., 2022 ). A further study demonstrated that the Eubacterium coprostanoligenes group enhanced intestinal integrity and barrier function by stimulating goblet cells to secrete mucin(Bai et al., 2024 ). Consequently, the increased relative abundance of OTU9 (F: Eubacterium coprostanoligenes group) suggested that the HMBi group may be able to enhance the barrier functions of the ileal mucosa of goats in the present study. Monoglobus spp. , often detected in the intestines of humans and animals, were reported to ferment dietary fibre and digest complex polysaccharides and may promote butyrate production(Lee et al., 2024 ). In addition, Monoglobus spp. , were reported to be correlated with Immunoglobulin A (IgA) levels in rats, and therefore, suggesting that it may possess prebiotic effects on intestinal health(de Morais et al., 2023 ). Overall, our study suggested the HMBi addition resulted in the proliferation of some probiotic bacteria in the ileum, which could generate some certain beneficial impacts on the intestinal health of ruminants. Dietary HMBi supplementation altered the ileal metabolic pattern and contribute to the intestinal health of goats We further evaluated the impacts of HMBi on ileal metabolites by LC-MS metabolomics analysis. In the present study, the results of PLS-DA and OPLS-DA showed the treatments had a significant effect on ileal metabolites. For specific changes, our results found that the HMBi supplementation resulted in higher content of 2-hydroxy-4-(methylthio) butanoic acid (VIP = 0.806, P 1.5) and Met (VIP = 1.279, P 1.5) in ileal digesta. Previous studies confirmed that approximately 50% of HMBi escaped absorption from the rumen wall and was degraded into HMB in the intestine to increase methionine supply(Graulet et al., 2005 ; Koenig et al., 2002 ). Our results demonstrated the above-mentioned degradation process of HMBi, but whether it could promote intestinal absorption of Met still require more studies. In addition, we postulated that the increase in ileal Met in the present study was partly related to microbial synthesis. O-Succinyhomoserine is an intermediate in the bacterial biosynthesis of Met via the trans-sulphation pathway(Brewster et al., 2021 ). Hence, the increase in ileal O-Succinyhomoserine may be indirect evidence that the HMBi diet promoted the synthesis of Met by intestinal bacteria in this study. Our results showed that the content of some substances (including taxifolin, Soyasaponin I, and tyrosol) enhanced intestine health significantly elevated in the ileal digesta after HMBi supplementation. Taxifolin was proved to have the ability to inhibit the expression of some inflammatory cytokines in the mouse colon and reduced intestinal inflammation and mucosal damage by promoting butyrate production(Li et al., 2022a ). Soyasaponin I is a representative component of soybeans that has been demonstrated to attenuate the inflammatory process in mice by inhibiting the pro-inflammatory cytokines or mediators, such as TNF-α, IL-1β, and NO. (Lee et al., 2010 ). Tyrosol is a biomolecule with various physiological functions such as antioxidant, anti-inflammatory and anti-apoptosis(Plotnikov and Plotnikova, 2021 ). Moreover, tyrosol has been demonstrated to enhance glutathione (GSH) levels and peroxidase activity, and reduce inflammation by decreasing TNF-α, IL-6, and TGF-β1 expression(Kutlu et al., 2021 ; Li et al., 2022b ). Therefore, the increased content of taxifolin, Soyasaponin I, and tyrosol in the ileum after HMBi supplementation indicated that the HMBi may alleviate intestinal inflammation and contribute to the intestinal health of goats. Moreover, our results suggested that ileal metabolites were enriched in arginine biosynthesis, citrate cycle, pyruvate metabolism, and glutathione metabolism, and metabolites involved in the above pathways were ornithine, fumaric acid, malic acid, and pyroglutamic acid. Ornithine can be converted to arginine via the ornithine cycle, resulting in the release of fumaric acid. Consequently, we speculated that the increase in fumaric acid content may be caused by the increase in ornithine content as shown in our result. Malic acid and fumaric acid are involved in the TCA, which is the final metabolic pathway and linkage between the three important nutrients (carbohydrates, lipids, and amino acids) and provides the energy for the host (Dashty, 2013 ). The increase in fumaric acid and malic acid content indicated that HMBi addition accelerated TCA process in ileal digesta in our study. In addition, pyroglutamic acid is an intermediate product of glutathione metabolism and is closely related to glutathione status(Ren et al., 2015 ). Therefore, due to the antioxidant effects of glutathione, the increase in pyroglutamic acid content implied that the intestinal antioxidant function of goats may be enhanced after HMBi supplementation in our study. Ileal microbiota and metabolism contributed to the improvement of cashmere performance after dietary HMBi supplementation in goats In recent years, gastrointestinal microbiota has received much attention, and has been proven to regulate animal growth performance, feed efficiency, and meat quality, etc. by manipulating gastrointestinal microbiota(Chen et al., 2022 ; Gardiner et al., 2020 ). Nevertheless, the linkage between the cashmere performance and intestinal microbiota remains unconfirmed. In the present study, to further explore whether the intestinal micro-ecological environment was involved in HMBi regulating the cashmere performance of goats, we conducted the Mantel tests. Our results showed that the cashmere length and growth rate were significantly and cashmere diameter was tended to be related to ileal microbiota and metabolites. These results indicated that the intestinal microbiota partly contributed to cashmere performance like some production traits such as meat quality. The cashmere length and diameter are important indicators of cashmere quality, and hence, we next the specific connections between the cashmere performance and ileal microbiota and metabolites. Among these connections, the methyl-containing metabolites attracted our attention. Our study revealed that cashmere length was negatively related with N,N-Dimethyltetradecylamine and tyrosine methyl ester, and positively related with 3'-O-methyluridine, 4-methoxyaniline, methylmalonic acid, mono-methyl glutarate, and 3-methylglutaconic acid; while cashmere diameter was negatively correlated with 3-methylglutaconic acid and methylmalonic acid. As we all known that, Met serves as methyl donors and participates in many reactions, such as DNA methylation, and glutathione synthesis(Chandler and White, 2017 ). The above results suggested that the HMBi supplementation lead to an increase in methyl donors in the intestinal tract of goats, which may be one of the reasons for improving cashmere performance in the present study. In addition, we also found some connections between cashmere performance and ileal microbiota, we speculated that these microbiota were partly related to methyl metabolism, but due to the limited information, more studies were still needed to confirm. Next, we will conduct systematic studies on the intestinal transport and absorption of nutrients to further confirm the intestinal mechanism by which HMBi promotes cashmere growth in Liaoning cashmere goats. Conclusion Our study integrated 16S rRNA sequencing and LC-MS metabolomics to reveal that HMBi supplementation changed the microbial community and metabolism pattern in the ileum, subsequently improving the cashmere performance of goats. The proposed framework of how HMBi affected the cashmere performance of Liaoning cashmere goats through the intestinal micro-ecological environment was shown in Fig. 8 . In general, the HMBi increased the cashmere length and growth rate, and decreased the cashmere diameter of goats. The HMBi supplementation resulted in a reduction of cellulose-degrading bacteria (including Cellulosilyticum , Lachnospiraceae_uncultured, and Breznakia ), and an increase of beneficial bacteria (such as OTU9, OUT256, and OTU657) in the ileum of goats. Moreover, the HMBi supplementation increased the content of HMBi degradation products (such as HMB and Met), and also increased some substance enhanced the intestinal health (such as taxifolin, Soyasaponin I, and tyrosol) in the ileum of goats. These findings provide further insight into the relationship between the intestinal micro-ecological environment and animal production performance. More in-depth studies should be conducted on the connections between cashmere performance and intestinal micro-ecological environment from the perspective of nutrient transport and absorption, and develop nutritional measures to improve cashmere performance by regulating the intestinal microecological environment. Declarations Competing interests The authors declare no conflicts of interest. Author Contribution R.Z. and M.X. designed the experiment, M.X., M.Z., P.S., J.J., Z.Z., L.L., and Y.Z. performed the experiment and collected the samples, M.X. and M.Z. conducted laboratory analyses, M.X. and R.Z. analyzed the data and wrote the manuscript, R.Z. and Y.C. revised the manuscript. Acknowledgement This work was supported by the Liaoning Provincial Department of Education Funding for Fundamental Research Projects (JYTQN2023305). Data Availability None of the data were deposited in an official repository. The data/models that support the study findings are available to reviewers, or available from the authors upon request. References Amato KR, Yeoman CJ, Kent A, Righini N, Carbonero F, Estrada A, Gaskins R, Stumpf H, Yildirim RM, Torralba S, M (2013) Habitat degradation impacts black howler monkey (Alouatta pigra) gastrointestinal microbiomes. ISME J 7:1344–1353 Astorga J, Gasaly N, Dubois-Camacho K, De la Fuente M, Landskron G, Faber KN, Urra FA, Hermoso MA (2022) The role of cholesterol and mitochondrial bioenergetics in activation of the inflammasome in IBD. Front Immunol 13:1028953 Bai D, Zhao J, Wang R, Du J, Zhou C, Gu C, Wang Y, Zhang L, Zhao Y, Lu N (2024) Eubacterium coprostanoligenes alleviates chemotherapy-induced intestinal mucositis by enhancing intestinal mucus barrier. Acta Pharm Sin B 14:1677–1692 Biddle A, Stewart L, Blanchard J, Leschine S (2013) Untangling the genetic basis of fibrolytic specialization by Lachnospiraceae and Ruminococcaceae in diverse gut communities. Diversity 5:627–640 Brewster JL, Pachl P, McKellar JLO, Selmer M, Squire CJ, Patrick WM (2021) Structures and kinetics of Thermotoga maritima MetY reveal new insights into the predominant sulfurylation enzyme of bacterial methionine biosynthesis. J Biol Chem 296:100797 Cai S, Shao N, Dong X (2015) Cellulosilyticum. Bergey's Manual of Systematics of Archaea and Bacteria, 1–4 Chandler TL, White HM (2017) Choline and methionine differentially alter methyl carbon metabolism in bovine neonatal hepatocytes. PLoS ONE 12:e0171080 Chen B, Li D, Leng D, Kui H, Bai X, Wang TJFiM (2022) Gut microbiota and meat quality. Front Microbiol 13:951726 Dashty M (2013) A quick look at biochemistry: carbohydrate metabolism. Clin Biochem 46:1339–1352 de Morais VN, Gomes MJC, Grancieri M, Moreira LdPD, Toledo RCL, Costa NMB, da Silva BP, Martino HSD (2023) Chia (Salvia hispanica L.) flour modulates the intestinal microbiota in Wistar rats fed a high-fat and high-fructose diet. Food Rev Int 172:113095 Edgar RC, Haas BJ, Clemente JC, Quince C, Knight R (2011) UCHIME improves sensitivity and speed of chimera detection. Bioinformatics 27:2194–2200 Feng Y, Cong Y, Deng H, Guo D, Liu HJRBdZ (2013a) Effects of 2-hydroxy-4 (methylthio) butanoic acid isopropyl ester on rumen fermentation in cashmere goats. Rev Bras Zootecn 42:342–346 Feng Y, Sun Y, Deng H, Cong Y (2013b) Promotion of cashmere growth by sulfur supplements in cashmere goats. Rev Bras Zootecn 42:792–797 Firkins JL, Fowler C, Devillard E, Bequette B (2015) Kinetics of microbial methionine metabolism in continuous cultures administered different methionine sources. J Dairy Sci 98:1178–1194 Galbraith H (2000) Protein and sulphur amino acid nutrition of hair fibre-producing Angora and Cashmere goats. Livest Prod Sci 64:81–93 Gardiner GE, Metzler-Zebeli BU, Lawlor PG (2020) Impact of Intestinal Microbiota on Growth and Feed Efficiency in Pigs: A Review, vol 8. Microorganisms, p 1886 Gebeyew K, Yang C, He Z, Tan Z (2021) Low-protein diets supplemented with methionine and lysine alter the gut microbiota composition and improve the immune status of growing lambs. Appl Microbiol Biotechnol 105:8393–8410 Gérard P (2020) Gastrointestinal Tract: Microbial Metabolism of Steroids. In: Goldfine H (ed) Health Consequences of Microbial Interactions with Hydrocarbons, Oils, and Lipids. Springer International Publishing, Cham, pp 389–399 Graulet B, Richard C, Robert J (2005) Methionine availability in plasma of dairy cows supplemented with methionine hydroxy analog isopropyl ester. J Dairy Sci 88:3640–3649 Graulet B, Richard C, Robert JC (2004) The isopropyl ester of methionine hydroxy-analogue is absorbed through the rumen wall in the cow.J. Anim Feed Sci 13:269–272 Hosomi K, Saito M, Park J, Murakami H, Shibata N, Ando M, Nagatake T, Konishi K, Ohno H, Tanisawa K, Mohsen A, Chen YA, Kawashima H, Natsume-Kitatani Y, Oka Y, Shimizu H, Furuta M, Tojima Y, Sawane K, Saika A, Kondo S, Yonejima Y, Takeyama H, Matsutani A, Mizuguchi K, Miyachi M, Kunisawa J (2022) Oral administration of Blautia wexlerae ameliorates obesity and type 2 diabetes via metabolic remodeling of the gut microbiota. Nat Commun 13:4477 Jiao J, Wang P, He Z, Tang S, Zhou C, Han X, Wang M, Wu D, Kang J, Tan Z (2014) In vitro evaluation on neutral detergent fiber and cellulose digestion by post-ruminal microorganisms in goats. J Sci Food Agr 94:1745–1752 Khan MZ, Liu S, Ma Y, Ma M, Ullah Q, Khan IM, Wang J, Xiao J, Chen T, Khan A, Cao Z (2022) Overview of the effect of rumen-protected limiting amino acids (methionine and lysine) and choline on the immunity, antioxidative, and inflammatory status of periparturient ruminants. Front Immunol 13:1042895 Koenig K, Rode L, Knight C, Vázquez-Añón M (2002) Rumen degradation and availability of various amounts of liquid methionine hydroxy analog in lactating dairy cows. J Dairy Sci 85:930–938 Kutlu T, Özkan H, Guvenç M (2021) Tyrosol retards induction of fibrosis in rats. J Food Biochem 45 Lee H, Kim M, Masaki T, Ikuta K, Iwamoto E, Nishihara K, Nonaka I, Ashihara A, Baek Y, Lee SJSR (2024) Assessing the impact of three feeding stages on rumen bacterial community and physiological characteristics of Japanese Black cattle. Sci Rep 14:4923 Lee IA, Park YJ, Yeo HK, Han MJ, Kim DH (2010) Soyasaponin I attenuates TNBS-Induced colitis in mice by inhibiting NF-κB pathway. J Agric Food Chem 58:10929–10934 Li C, Wang W, Liu T, Zhang Q, Wang G, Li F, Li F, Yue X, Li T (2018) Effect of early weaning on the intestinal microbiota and expression of genes related to barrier function in lambs. Front Microbiol 9:1431 Li W, Zhang L, Xu Q, Yang W, Zhao J, Ren Y, Yu Z, Ma L (2022a) Taxifolin Alleviates DSS-Induced Ulcerative Colitis by Acting on Gut Microbiome to Produce Butyric Acid. Nutrients 14. Li X, Wei T, Li J, Yuan Y, Wu M, Chen F, Deng ZY, Luo T (2022b) Tyrosol Ameliorates the Symptoms of Obesity, Promotes Adipose Thermogenesis, and Modulates the Composition of Gut Microbiota in HFD Fed Mice. Mol Nutr Food Res 66:e2101015 Lin Y, Wang Z, Zhou A (2008) Isopropyl 2-hydroxy-4-methylthiobutyrate (HMBi) and 2-hydroxy-4-methylthiobutyric acid (HMB) in ruminants. China Dairy Cattle, 11–15 Liu Y, Liu C, Wu H, Meng Q, Zhou Z (2022) Small intestine microbiome and metabolome of high and low residual feed intake Angus heifers. Front Microbiol 13:862151 Martin C, Mirande C, Morgavi DP, Forano E, Devillard E, Mosoni P (2013) Methionine analogues HMB and HMBi increase the abundance of cellulolytic bacterial representatives in the rumen of cattle with no direct effects on fibre degradation. Anim Feed Sci Technol 182:16–24 Myer P, Wells J, Smith T, Kuehn L, Freetly H (2016) Microbial community profiles of the jejunum from steers differing in feed efficiency. J Anim Sci 94:327–338 Ordway R, Boucher S, Whitehouse NL, Schwab CG, Sloan BJJo.d.s (2009) Effects of providing two forms of supplemental methionine to periparturient Holstein dairy cows on feed intake and lactational performance. J Dairy Sci 92:5154–5166 Osorio J, Ji P, Drackley JK, Luchini D, Loor JJ J.J.o.d.s., 2013. Supplemental Smartamine M or MetaSmart during the transition period benefits postpartal cow performance and blood neutrophil function. J Dairy Sci 96, 6248–6263 Pacífico C, Hartinger T, Stauder A, Schwartz-Zimmermann HE, Reisinger N, Faas J, Zebeli Q (2021) Supplementing a clay mineral-based feed additive modulated fecal microbiota composition, liver health, and lipid serum metabolome in dairy cows fed starch-rich diets. Front Vet Sci 8:714545 Plotnikov MB, Plotnikova TM (2021) Tyrosol as a Neuroprotector: Strong Effects of a Weak Antioxidant. Curr Neuropharmacol 19:434–448 Potts SB, Brady KM, Scholte CM, Moyes KM, Sunny NE, Erdman RA (2023) Rumen-protected choline and methionine during the periparturient period affect choline metabolites, amino acids, and hepatic expression of genes associated with one-carbon and lipid metabolism. J Dairy Sci 106:4559–4579 Qin X, Zhang D, Qiu X, Zhao K, Zhang S, Liu C, Lu L, Cui Y, Shi C, Chen Z, Hao R, Li Y, Yang S, Wang L, Wang H, Cao B, Su H (2022) 2-Hydroxy-4-(Methylthio) Butanoic Acid Isopropyl Ester Supplementation Altered Ruminal and Cecal Bacterial Composition and Improved Growth Performance of Finishing Beef Cattle. Front Nutr 9:833881 Ren W, Yin J, Gao W, Chen S, Duan J, Liu G, Li T, Li N, Peng Y, Yin Y (2015) Metabolomics study of metabolic variations in enterotoxigenic Escherichia coli-infected piglets. RSC Adv 5:59550–59555 Robert J, Sloan B, Etave G, Bouza BJJDS (2001) Influence of length and ramification of the alcohol radical of esters of methionine and of 2-hydroxy-4 (methylthio) butanoic acid on methionine bioavailability. J Dairy Sci 84:34 Strain CR, Collins KC, Naughton V, McSorley EM, Stanton C, Smyth TJ, Soler-Vila A, Rea MC, Ross PR, Cherry P (2020) Effects of a polysaccharide-rich extract derived from Irish-sourced Laminaria digitata on the composition and metabolic activity of the human gut microbiota using an in vitro colonic model. Eur J Nutr 59:309–325 Tegtmeier D, Riese C, Geissinger O, Radek R, Brune A (2016) Breznakia blatticola gen. nov. sp. nov. and Breznakia pachnodae sp. nov., two fermenting bacteria isolated from insect guts, and emended description of the family Erysipelotrichaceae. Syst Appl Microbiol 39:319–329 Xi M, Jiang J, Wang B, Wang Y, Di M, Cong Y, Zhang R (2025) Alterations in Methionine Cycle and Wnt/MAPK Signaling Associated with HMBi-Induced Cashmere Growth in Goats. Int J Mol Sci 26:1663 Ye L, Hou Y, Hu W, Wang H, Yang R, Zhang Q, Feng Q, Zheng X, Yao G, Hao H (2023) Repressed Blautia-acetate immunological axis underlies breast cancer progression promoted by chronic stress. Nat Commun 14:6160 Tables Table 1 Effects of 2-Hydroxy-4-(methylthio)butanoic acid isopropyl ester (HMBi) Items Group SEM P value CON HMBi Acetate, mmol/L 34.96 32.17 3.292 0.689 Propionate, mmol/L 7.78 6.68 0.759 0.493 Butyrate, mmol/L 3.81 3.14 0.605 0.602 Isobutyrate, mmol/L 0.26 0.28 0.021 0.772 Valerate, mmol/L 1.47 1.64 0.164 0.622 Isovalerate, mmol/L 0.20 0.20 0.022 0.900 Toal VFA, mmol/L 48.48 44.11 4.682 0.659 NH 3 -N, mg/dL 9.35 9.03 0.664 0.820 MCP, mg/dL 17.83 19.90 0.572 0.085 Additional Declarations No competing interests reported. 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17:53:35","extension":"html","order_by":21,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":136632,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8062149/v1/7af160b44ee916d9de55e027.html"},{"id":96755862,"identity":"66acd0b6-66a5-4562-a1c3-a1b1178ce7b5","added_by":"auto","created_at":"2025-11-25 17:53:35","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":440139,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of 2-Hydroxy-4-(methylthio)butanoic acid isopropyl ester (HMBi) supplementation on ileal microbial richness and diversity of goats. \u003c/strong\u003e(A) the Chao 1 index; (B) the Shannon index; (C) the Simpson index; (D) the principal coordinate analysis (PCoA) of ileal microbiota.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8062149/v1/441a29d2be5069a97c2dba3c.jpeg"},{"id":96755820,"identity":"dae0e4a1-868c-4f69-bbe9-32618f349f8a","added_by":"auto","created_at":"2025-11-25 17:53:34","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":182622,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of 2-Hydroxy-4-(methylthio)butanoic acid isopropyl ester (HMBi) supplementation on ileal microbial composition at the phylum and genus level.\u003c/strong\u003e (A) the dominated phyla in the ileal microbiota of goats; (B) the dominated genera in the ileal microbiota of goats; (C) The microbial genera with significant differences between the CON and HMBi groups. Only the abundant genera (the relative abundance \u0026gt;0.01%) were subjected to the statistical analysis.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8062149/v1/5a225f71dd0fb2a6946cafae.png"},{"id":96914023,"identity":"4d8167a1-eb94-4271-b706-d3823a580e37","added_by":"auto","created_at":"2025-11-27 14:05:13","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":385134,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe linear discriminant analysis effect size (LEfSe) analysis of ileal microbiota at the OTU level.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8062149/v1/02d3c2770f794eff70e09a0e.png"},{"id":96755833,"identity":"5c5b3c95-a6d1-46c5-9b24-fecd7c498230","added_by":"auto","created_at":"2025-11-25 17:53:35","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":314622,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe ileal metabolome of goats fed the CON and HMBi diets.\u003c/strong\u003e (A) Partial least squares discriminant analysis (PLS-DA) analysis. (B) orthogonal partial least-squares discriminant (OPLS-DA) analysis. (C) PLS-DA loading plots of ileal metabolites from the CON and HMBi groups. CON, control. HMBi, 2-Hydroxy-4-(methylthio)butanoic acid isopropyl ester.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8062149/v1/d581df034fd6fda4e3e86af4.png"},{"id":96915712,"identity":"1a6c053e-4965-4ac3-82e7-2bc188a7f68e","added_by":"auto","created_at":"2025-11-27 14:07:34","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":399688,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHeatmap of ileal differential metabolites between the CON and HMBi groups. \u003c/strong\u003eCON, control. HMBi, 2-Hydroxy-4-(methylthio)butanoic acid isopropyl este\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8062149/v1/bc11bbb6c2431c4ab0bc30a7.png"},{"id":96755823,"identity":"d11e6147-db04-4501-83ff-2d08e714f842","added_by":"auto","created_at":"2025-11-25 17:53:34","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":363880,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe enrichment analysis of ileal differential metabolites between the CON and HMBi groups.\u003c/strong\u003e CON, control. HMBi, 2-Hydroxy-4-(methylthio)butanoic acid isopropyl ester.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8062149/v1/3706d6209ca1502e0aba8c4d.png"},{"id":96755865,"identity":"86e3289d-e084-448d-9776-ed146ad36d08","added_by":"auto","created_at":"2025-11-25 17:53:36","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":538281,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe potential interactions among the cashmere performance, ileal fermentation, microbial community and metabolites.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) the contributions of ileal microbiota and metabolites to cashmere performance and ileal fermentation. The altered microbiota (genus and OTUs) and metabolites were used in Mantel tests. (B) the correlations between the cashmere performance (cashmere length and diameter) and altered microbiota and metabolites.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-8062149/v1/eac8cebaba5c471548878336.png"},{"id":96915720,"identity":"c7660f8c-ef8c-4706-a2cc-0bce2b31a7d5","added_by":"auto","created_at":"2025-11-27 14:07:35","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":66976,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe proposed schematic diagram of the overall effects of HMBi on the ileal microecological environment of Liaoning cashmere goats.\u003c/strong\u003e HMBi, 2-Hydroxy-4-(methylthio)butanoic acid isopropyl ester\u003c/p\u003e","description":"","filename":"Onlinefloatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-8062149/v1/eac574f3332f81b6bc5100e3.png"},{"id":99313508,"identity":"283cdb22-9d09-4557-8670-a7d0234e3593","added_by":"auto","created_at":"2025-12-31 16:20:15","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4017411,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8062149/v1/866c01fa-9f74-4aa5-b43f-128fb0c80593.pdf"},{"id":96914836,"identity":"a932a3b3-a93f-4a02-a471-b386e24ca005","added_by":"auto","created_at":"2025-11-27 14:06:27","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":14943,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFileforWMB.docx","url":"https://assets-eu.researchsquare.com/files/rs-8062149/v1/af3f3978b4b54201bce8c55d.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Effects of dietary supplementation with 2-hydroxy-4-(methylthio) butanoic acid isopropyl ester on ileal microbiota and metabolism in Liaoning cashmere goats","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMethionine (Met) is the first limiting amino acid in ruminants such as cashmere goats, and has antioxidant, anti-inflammatory, immune-regulating and other functions(Gebeyew et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Khan et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Potts et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Hence, Met supply is closely related to the health and production performance of ruminants. In modern ruminant breeding, the degradation of rumen microbiota has led to the increased use of rumen-protected methionine (RPM) as a means of reducing microbial degradation and subsequently increasing methionine supply more effectively.\u003c/p\u003e\u003cp\u003eThe 2-hydroxy-4-(methylthio) butanoic acid isopropyl ester (HMBi), a hydroxyl analog of Met, is often adopted to provide metabolisable Met for ruminants. Numerous studies indicated that HMBi supplementation elevated body weight gain, increased milk production, and improved milk components such as milk fat in cattles (Ordway et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Osorio et al., 2013; Qin et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Nevertheless, limited data suggested a potential effect of HMBi on cashmere growth of goats. One early research revealed that the HMBi adding in diets abled to enhance the growth rate of cashmere fibres(Feng et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2013b\u003c/span\u003e), and also altered the rumen fermentation parameters (pH, ammonia-N concentration, and volatile fatty acids) in Liaoning cashmere goats(Feng et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2013a\u003c/span\u003e). In general, the research on the effects of HMBi on ruminants is largely confined to the domains of production performance and the rumen, with no studies on the small intestine having been conducted to date.\u003c/p\u003e\u003cp\u003eBesides the rumen, the microecological status in the small intestine is also crucial to the nutrient digestion and absorption and immune system for ruminants(Myer et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). For instance, early weaning significantly altered the ileal microbial structure and increased the mRNA expression of toll-like receptors and tight junction protein genes of Hu lambs(Li et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Another study reported that the small intestinal microbiota and metabolite patterns with different residual feed intake phenotypes are very different in the Angus Heifers, suggesting that the micro-ecology is potentially important for the improvement of feed efficiency in ruminants(Liu et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Previous studies demonstrated that ingested HMBi could be rapidly decomposed into 2-hydroxy-4-(methylthio) butanoic acid (HMB), and is metabolized in three ways: direct absorbed from the rumen wall, degraded by rumen microbiota and then synthesized into microbial proteins (MCP), or directly arrived at the small intestine(Lin et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). It has been demonstrated that HMBi has a biological activity of 40\u0026ndash;58% in rumen(Graulet et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Robert et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). Consequently, it is postulated that the HMBi supplementation may result in alterations to the substrates entering the intestine, thereby influencing the microecological status within the intestine.\u003c/p\u003e\u003cp\u003eTherefore, our study utilized multi-omics approaches (16S rRNA sequencing and metabolomics) to reveal the effects of HMBi on ileal fermentation, microbial community and metabolites of Liaoning cashmere goats. Our results could offer a certain theoretical reference for the application of HMBi in Liaoning cashmere goats.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eAnimal experiment, diets\u003c/h2\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003cp\u003eThe experimental process was conducted according to the Institutional Animal Care and Use Committee of Shenyang Agricultural University (NO. 2021091501). Fourteen healthy female Liaoning cashmere goats (25.63\u0026thinsp;\u0026plusmn;\u0026thinsp;0.87 kg body weight, around 8 months of age) were divided into the control group (CON, n\u0026thinsp;=\u0026thinsp;7) and the HMBi (n\u0026thinsp;=\u0026thinsp;7) groups. The goats in CON group was offered a basal diet, and the HMBi group was fed with 1.27% HMBi (MetaSmart; Adisseo Inc., Antony, France) in the basal diet. MetaSmart was offered as dry powder form and contained 57% HMBi, which is equivalent to 78% methionine (Osorio et al., 2013). The basal diets were formulated according to the recommendations of goats (NY/Y816-2004; Ministry of Agriculture of China, 2004). The ingredients and nutrient composition of the basal diet is listed in Table S1. All experimental goats were reared for 67 days, of which 7 days are the pre-feeding period. During the experiment, all animals were placed in individual pens. The feed intake, initial body weight, and final body weight (FBW) of every goat were determined, and the average daily gain (ADG) and the ratio of average daily feed intake to average daily gain (F/G) were calculated.\u003c/h2\u003e\u003c/div\u003e\n\u003ch3\u003eSample collection\u003c/h3\u003e\n\u003cp\u003eAfter 67 days of dietary treatments, all goats were slaughtered, and the ileum section was immediately dissected and isolated. The ileal digesta of experimental goats were collected after thorough mixing and homogenized, and stored in -80℃ until the further microbial and metabolomic analysis. The ileal tissue was flushed with ice-cold sterile saline to clear the adherent ileal content, and then quickly frozen in liquid nitrogen for gene quantification.\u003c/p\u003e\n\u003ch3\u003eIleal fermentation parameters\u003c/h3\u003e\n\u003cp\u003eThe measurement of volatile fatty acids (VFAs) used crotonic acid as an internal stand, and was performed on a gas chromatography (Agilent 7890B, CA, USA) according to the methods of Mao et al. (2007). The concentrations of ammonia (NH\u003csub\u003e3\u003c/sub\u003e-N) were determined as described by Chaney and Marbach (1962). The microbial crude protein (MCP) was analyzed according to the procedure reported by Makkar et al.(1982).\u003c/p\u003e\n\u003ch3\u003eMicrobial DNA isolation and illumina sequencing\u003c/h3\u003e\n\u003cp\u003eIleal samples were fully thawed and mixed, and bacterial DNA isolation was conducted using QIAamp DNA Stool Mini Kit (Qiagen, Hilden, Germany). After multiple extractions with phenol/chloroform, the DNA pellets were precipitated with ethanol and then redissolved using Tris-EDTA buffer. The isolated DNA was detected using NanoDrop 2000 spectrophotometer (Wilmington, DE, United States), only high-quality DNA were stored at -80℃ until subsequent analysis.\u003c/p\u003e\u003cp\u003eThe V3-V4 fragment of 16S rRNA genes were amplified using the following bacterial universal primers: 338F (5\u0026rsquo;-ACTCCTRCGGGAGGCAGCAG-3\u0026rsquo;) and 806R (5\u0026rsquo;-GGACTACCVGGGTATCTAAT-3\u0026rsquo;). The amplification was performed according to the following procedures: initial denaturation (95\u0026deg;C, 2 min), 25 cycles of amplification (95\u0026deg;C, 30 s; 55\u0026deg;C,30 s; 72\u0026deg;C, 30 s) and final extension (72\u0026deg;C, 5 min). Three replicates were set for each sample, and the amplified products of one sample were mixed and purified by Qiagen QIAquick PCR purification kit (Qiagen, Duesseldorf, Germany). The pair-end sequencing was performed on an Illumina Miseq platform (Majorbio, Shanghai, China) follow the manufacturer's recommended procedures.\u003c/p\u003e\u003cp\u003eFor data analyses, the paired-end raw sequences were demultiplexed, and filtered by conducting quality control. Sequences that did not meet the filtering criteria and were of low quality were removed from raw sequences. Sequences were screened for chimeras using UCHIME(Edgar et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), and unnormal gene sequences were removed. Operational taxonomic units (OTUs) clustering was performed using UPARSE at 97% identity. The most prevalent sequences within each OTU were aligned and annotated by SILVA (SSU138.1) database (Amato et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Microbial richness and diversity were evaluated using Chao 1, Shannon and Simpson. Differences in ileal microbiota was estimated using principal coordinates analysis (PCoA). To further determine whether the microbial differences between the two groups reach a significant level, the permutation multivariate analysis (PERMANOVA) was adopted.\u003c/p\u003e\n\u003ch3\u003eLC-MS detection\u003c/h3\u003e\n\u003cp\u003eIleal digesta samples were pretreated before the metabolomics analysis. Samples were fully thawed and mixed, then, 50 mg ileal samples were with 800 mL methanol. The mixture was ground (65HZ, 180 s), ultrasonicated (4\u0026deg;C, 30 min), and centrifuged (12000 rpm, 4\u0026deg;C, 15 min). Repeat the above-mentioned centrifugation for the collected supernatant. Dichlorophenylalanine was adopted as internal standard of LC-MS detection, and the detection was performed on a Waters ACQUITY UPLC\u0026reg; system (Waters Corp., USA). During the determination, the flow rate was kept at 0.3 ml/min, and the column temperature of instrument was kept at 40\u0026deg;C.\u003c/p\u003e\u003cp\u003eThe metabolome data was processed and annotated using Compound Discoverer 3.3 software (Thermo Fisher Scientific, Waltham, MA, USA). and the extracted data included retention time, peak intensity, sample names, and compound molecular weight were used for further analysis. Statistics of metabolomics data were mainly performed through SIMCA-P\u0026thinsp;+\u0026thinsp;14.1 software (Umetrics, Umea, Sweden). Differences in ileal metabolites were estimated through the partial least squares discriminant analysis (PLS-DA) and orthogonal partial least-squared discriminant analysis (OPLS-DA). Variable importance in projection (VIP) scores in PLS-DA were harvested to measure the contributions and importance of metabolites.\u003c/p\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eStatistical calculations in the present study were carried out using SPSS (v23, SPSS Inc., Chicago, IL). Statistics of data on growth performance and cashmere performance was conducted utilizing the independent samples t test. Since the data on microbial phyla and genera and metabolomics did not conform to a normal distribution, the statistics of these data was performed using a Wilcoxon-Mann-Whitney U test. Characterized OTUs were identified using the linear discriminant analysis effect size (LEfSe) analysis. The metabolites with VIP\u0026thinsp;\u0026gt;\u0026thinsp;1 (generated from PLS-DA analysis) and \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.05 (generated from Wilcoxon-Mann-Whitney U test) were defined as ileal differential metabolites. The partial Mantel tests of cashmere performance, ileal fermentation, microbial community and metabolites were conducted by the \u0026ldquo;linkET\u0026rdquo; package in R (v3.5.0). Significant differences were declared at \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.05.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eProduction performance of goats\u003c/h2\u003e\u003cp\u003eThe effects of HMBi on growth status of Cashmere goats was listed in the Table S2. The results suggested that the HMBi diets had no significant effects on FBW, ADG and F/G of \u003cem\u003eCashmere\u003c/em\u003e goats. The results regarding cashmere performance had been described in our previous study, and the results revealed that the HMBi group improved the cashmere length and cashmere growth rate (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and also decreased the cashmere diameter of goats (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05)(Xi et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eIleal fermentation parameters\u003c/h2\u003e\u003cp\u003eThe results of ileal fermentation parameters were listed in Table\u0026nbsp;1. The ileal MCP concentration tended to increase in the HMBi diets (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.085). However, the HMBi supplementation did not affect the levels of VFAs and NH\u003csub\u003e3\u003c/sub\u003e-N in the ileum of Liaoning Cashmere goats.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eIleal microbial diversity\u003c/h2\u003e\u003cp\u003eThe estimators of microbial richness and diversity were presented in Fig.\u0026nbsp;1. No differences were found in Chao 1, Shannon and Simpson of ileal microbiota between two dietary treatments. Next, the PCoA analysis (Fig.\u0026nbsp;1D) showed that there was no obvious separation in the distribution of ileal samples from two groups in the plots figures, and the PERMANOVA analysis also confirmed no statistical differences between the CON and HMBi group (PERMANOVA \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.402).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eAlterations in the structure of ileal microbiota between the CON and HMBi groups\u003c/h2\u003e\u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, we found that the abundance of 9 phyla were higher than 0.1%, and Firmicutes (average 81.37%) and Bacteroidota (average 7.92%) were the most abundance phyla in the ileal microbiota of goats. The results of statistical analysis revealed that the HMBi supplementation did not affect the microbial phyla in the ileal digesta of goats.\u003c/p\u003e\u003cp\u003eAt the genus level, Oscillospiraceae UCG-005 (average 15.21%), Eubacterium coprostanoligenes group_norank (average 10.87%), UCG-010_norank (average 6.42%), \u003cem\u003eChristensenellaceae R-7 group\u003c/em\u003e (average 6.12%), and \u003cem\u003eMonoglobus\u003c/em\u003e (average 4.05%) were the abundant genera in the ileal microbiota of goats (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Our statistical analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003eC) indicated that the HMBi group decreased (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) the abundance of Lachnospiraceae_uncultured, \u003cem\u003eAnaerorhabdus furcosa group, Breznakia, Meiothermus, and Cellulosilyticum\u003c/em\u003e in the ileal microbiota of goats. However, the HMBi supplementation did not affect other genera in the ileal microbiota of goats.\u003c/p\u003e\u003cp\u003eTo further evaluate the impacts of HMBi supplementation on microbial community, the LEfSe analysis was performed. The results showed that (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003e), 13 OTUs, including 4 OTUs (OTU41, OTU295, OTU216, and OTU206) belonging to \u003cem\u003eChristensenellaceae R-7 group\u003c/em\u003e, 2 OTUs (OTU99 and OTU324) belonging to Rikenellaceae RC9 gut group, 2 OTUs (OTU59 and OUT 241) belonging to the family Muribaculaceae, 2 OTUs (OTU322 and OTU355) belonging to the family Lachnospiraceae, OTU33 (G: Oscillospiraceae UCG-005), OTU167 (G: Ruminococcus), and OTU456 (F: Bacteroidales RF16 group) were enriched (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in the CON group. Meanwhile, OTU361 (F: Ruminococcaceae), OTU657 (G: Monoglobus), OTU215 (G: Muribaculaceae), OTU256 (G: Blautia), OTU112 (G: Oscillospiraceae UCG-005), OTU188 (G: Treponema), and OTU9 (F: Eubacterium coprostanoligenes group) were enriched (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in the HMBi group.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eMetabolic profiles in the ileum of goats fed the CON and HMBi diets\u003c/h2\u003e\u003cp\u003eThrough the metabolomics analysis, as well as the quality control and identification, we totally harvested 603 valid ileal metabolites from both the CON and HMBi groups. Next, the results of PLS-DA and OPLS-DA analysis suggested that the sample plots from two groups were clustered together individually in the figures, suggesting the huge differences in ileal metabolites between these two groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eA and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). The ileal metabolites that with VIP and statistical \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were defined as differential metabolites, and its distribution were presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eC. Our results indicated that some ileal metabolites, such as phosphorylcholine, L-tyrosine methyl ester, dodecanoic acid, alpha-tocopherol acetate, and 1-Stearoylglycerophosphoglycerol were correlated with the CON diets, and some ileal compounds, such as 4-phenylbutyric acid, heptanoic acid, 4-hydroxybenzylalcohol, pentadecanoic acid, and L-malic acid were correlated with the HMBi diets.\u003c/p\u003e\u003cp\u003eBy sorting and classifying differential metabolites, our results suggested the HMBi supplementation altered the levels of 68 ileal metabolites (VIP\u0026thinsp;\u0026gt;\u0026thinsp;1 and \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Specifically, for amino acids, the HMBi diet increased (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) the level of methionine, ornithine, O-succinylhomoserine, L-2-aminoadipic acid, N-acetylalanine, and pyroglutamic acid, and decreased the level of tyrosine methyl ester in the ileum of goats (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). For fatty acids and lipids, the HMBi supplementation increased the content of FA 17:0, FA 9:1\u0026thinsp;+\u0026thinsp;1O, tetradecanedioic acid, pentadecanoic acid, heptanoic acid, and cholest-4-en-3-one (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), while decreased the content of elaidic acid, dodecanoic acid, 1-stearoylglycerophosphoglycerol, chaulmoogric acid, and glycodeoxycholic acid in the ileal digesta of goats (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). For alcohols, the content of taxifolin, 4-hydroxybenzylalcohol, and tyrosol in the goats fed HMBi diets were higher (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) than those fed the CON diets. For amines, the level of 4-methoxyaniline was higher (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and the level of phosphorylcholine and phytosphingosine was lower (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in the ileal digesta of goats fed HMBi diets than those fed the CON diets. In addition, the HMBi group also increased (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) the level of 10 organic acids including mono-methyl glutarate, malic acid, 4-phenylbutyric acid, 3-methylglutaconic acid, valeric acid, 1,4-cyclohexanedicarboxylic acid, N-acetylanthranilic acid, 4-toluic acid, methylmalonic acid, and suberylglycine than those in the CON group. Additionally, we also found HMBi supplementation significantly increased (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) the content of taxifolin, Soyasaponin I, (+)-ar-Turmerone, etc. in the ileal digesta of goats.\u003c/p\u003e\u003cp\u003eWe further conducted the enrichment analysis of these ileal differential metabolites identified in the CON and HMBi groups. Our results suggested these metabolites were enriched (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in arginine biosynthesis, citrate cycle, pyruvate metabolism, and glutathione metabolism (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003e). In addition, metabolites involved in the above pathways were ornithine, fumaric acid, malic acid, and pyroglutamic acid.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eThe potential interactions among the cashmere performance, ileal fermentation, microbial community and metabolites\u003c/h2\u003e\u003cp\u003eTo explore the potential mechanism by which HMBi improved the cashmere performance from an intestinal perspective, we conducted the partial Mantel tests. The results showed that (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e7\u003c/span\u003eA), the cashmere length and growth rate were negatively correlated with cashmere diameter (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The cashmere length and growth rate were closely related to ileal microbiota and metabolites (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), among which ileal microbiota was more closely related (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In addition, cashmere diameter tended to be related to ileal microbiota and metabolites.\u003c/p\u003e\u003cp\u003eNext, we conducted the correlation network analysis of cashmere performance and ileal microbiota and metabolites. Methyl-containing metabolites attracted our attention, our results showed that (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e7\u003c/span\u003eB), cashmere length was negatively correlated with N,N-Dimethyltetradecylamine and tyrosine methyl ester, and positively correlated with 3'-O-methyluridine, 4-methoxyaniline, methylmalonic acid, mono-methyl glutarate, and 3-methylglutaconic acid (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05); while cashmere diameter was negatively correlated with 3-methylglutaconic acid and methylmalonic acid (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Moreover, cashmere length was negatively correlated with Lachnospiraceae_uncultured, \u003cem\u003eBreznakia\u003c/em\u003e, Meiothermus, OTU33, OTU216, and OTU324 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and positively correlated with OTU9 and OTU361. Cashmere diameter was negatively related with OTU657 and OTU188, and positively related with OTU33 and OTU241 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eDietary HMBi supplementation increased the cashmere growth and decreased the cashmere diameter\u003c/h2\u003e\u003cp\u003eMet is the first limiting amino acid in ruminants such as cashmere goats, and has the potential effects of promoting hair follicle cell proliferation and differentiation, increasing keratin biosynthesis and accelerating hair growth(Galbraith, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). Consequently, Met is intimately associated with the cashmere performance of cashmere goats. Our results demonstrated that HMBi supplementation significantly increased the cashmere length and growth rate, and decreased the cashmere diameter. Partially consistent with our results, previous studies reported the HMBi could increase the growth rate, but did not affect the diameter of cashmere fibres(Feng et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2013b\u003c/span\u003e). Our results indicated that HMBi could elevate the Met level reached the skin, promote the keratoprotein synthesis, and thereby improve the cashmere production and quality.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003eDietary HMBi supplementation did not affect ileal fermentation pattern, but increased MCP synthesis\u003c/h2\u003e\u003cp\u003eThe microorganisms inhabited in the small intestine of animals have a certain ability to ferment nutrients and can also convert these nutrients into VFAs like rumen(Jiao et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Our results showed that HMBi supplementation did not affect the VFAs and NH\u003csub\u003e3\u003c/sub\u003e-N in the ileal digesta of goats, which suggested that the ileal fermentation pattern remained unaltered following the HMBi supplementation. Nevertheless, in comparison to the CON group, the MCP concentration exhibited a tendency to increase following HMBi supplementation. Our findings indicated that the HMBi supplementation caused intestinal microbiota to use nutritional substrates to synthesize more MCP, and these MCP may contribute to host health.\u003c/p\u003e\u003cp\u003e\u003cb\u003eDietary HMBi supplementation reduced the abundance of cellulose-degrading bacteria but increase the proliferation of probiotic bacteria\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAs indicated by PCoA and PERMANOVA, our results demonstrated that no significant differences in the global microbiota were existed in the ileum of goats. Next, statistical analyses were performed at different taxonomic levels to reveal the further impacts of HMBi. Our results demonstrated that the HMBi supplementation resulted in a notable reduction in the abundance of bacteria with cellulose-degrading capabilities, such as \u003cem\u003eCellulosilyticum\u003c/em\u003e, Lachnospiraceae_uncultured, and \u003cem\u003eBreznakia\u003c/em\u003e. The draft genome of \u003cem\u003eCellulosilyticum\u003c/em\u003e encoded a considerable number of cellulolytic enzymes, and species in this genus were postulated to play a key role in degrading cellulose and xylan(Cai et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Lachnospiraceae members were common bacteria found in the intestines of humans and animals, and were uniquely suited to degrade some recalcitrant substrates, such as cellulose(Biddle et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Strain et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The genus \u003cem\u003eBreznakia\u003c/em\u003e is frequently detected in the mammalian intestine, but the knowledge of these bacteria in this genus is very limited(Pac\u0026iacute;fico et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). One research reported two species (\u003cem\u003eB. pachnodae\u003c/em\u003e and \u003cem\u003eB. blatticola\u003c/em\u003e) in genus \u003cem\u003eBreznakia\u003c/em\u003e abled to utilize a variety of substrates (including fructose, glucose, cellobiose, etc.) to ferment to produce formate, acetate, and ethanol(Tegtmeier et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Hence, the above results suggested that the ability of intestinal bacteria to degrade cellulose was diminished following dietary supplementation with HMBi. In addition, previous studies reported that Met analogs or HMBi were able to stimulate the proliferation of cellulose-degrading bacteria in the rumen, such as \u003cem\u003eFibrobacter succinogenes\u003c/em\u003e and \u003cem\u003eRuminococcus flavefaciens\u003c/em\u003e(Firkins et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Martin et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Based on the above results on the rumen microbiota, we speculated that HMBi supplementation led to a substantial amount of cellulose to degraded in the rumen, and then a decrease in the cellulose reaching the small intestine, which further resulted in a decline in the cellulose-degrading bacteria in the small intestine.\u003c/p\u003e\u003cp\u003eFurthermore, our results showed that OTU256 (G: \u003cem\u003eBlautia\u003c/em\u003e), OTU9 (F: Eubacterium coprostanoligenes group), and OTU657 (G: \u003cem\u003eMonoglobus\u003c/em\u003e) were enriched in the HMBi group. \u003cem\u003eBlautia\u003c/em\u003e is an anaerobic bacterium with probiotic properties. The research reported the oral \u003cem\u003eBlautia\u003c/em\u003e induced anti-inflammatory changes and thus reduced obesity and diabetes in mice (Hosomi et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In addition, \u003cem\u003eBlautia\u003c/em\u003e-derived acetate is vital for regulating host immunity, such as enhancing the stress of immune T-cells(Ye et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). We therefore speculated that HMBi induced enrichment of OTU256 (G: \u003cem\u003eBlautia\u003c/em\u003e) may have anti-inflammatory effects and benefit gut health in the present study. The Eubacterium coprostanoligenes group has been identified as a butyrate-producing bacterium, which is also capable of reducing cholesterol levels and protecting the intestinal mucosal barrier(G\u0026eacute;rard, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). It had been reported that cholesterol homeostasis may be associated with the trigger of the nucleotide binding oligomerization domain like receptors protein 3 inflammatory vesicle signalling pathway in intestinal inflammation(Astorga et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). A further study demonstrated that the Eubacterium coprostanoligenes group enhanced intestinal integrity and barrier function by stimulating goblet cells to secrete mucin(Bai et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Consequently, the increased relative abundance of OTU9 (F: Eubacterium coprostanoligenes group) suggested that the HMBi group may be able to enhance the barrier functions of the ileal mucosa of goats in the present study. \u003cem\u003eMonoglobus spp.\u003c/em\u003e, often detected in the intestines of humans and animals, were reported to ferment dietary fibre and digest complex polysaccharides and may promote butyrate production(Lee et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). In addition, \u003cem\u003eMonoglobus spp.\u003c/em\u003e, were reported to be correlated with Immunoglobulin A (IgA) levels in rats, and therefore, suggesting that it may possess prebiotic effects on intestinal health(de Morais et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Overall, our study suggested the HMBi addition resulted in the proliferation of some probiotic bacteria in the ileum, which could generate some certain beneficial impacts on the intestinal health of ruminants.\u003c/p\u003e\u003cp\u003e\u003cb\u003eDietary HMBi supplementation altered the ileal metabolic pattern and contribute to the intestinal health of goats\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWe further evaluated the impacts of HMBi on ileal metabolites by LC-MS metabolomics analysis. In the present study, the results of PLS-DA and OPLS-DA showed the treatments had a significant effect on ileal metabolites. For specific changes, our results found that the HMBi supplementation resulted in higher content of 2-hydroxy-4-(methylthio) butanoic acid (VIP\u0026thinsp;=\u0026thinsp;0.806, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, and FC\u0026thinsp;\u0026gt;\u0026thinsp;1.5) and Met (VIP\u0026thinsp;=\u0026thinsp;1.279, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, and FC\u0026thinsp;\u0026gt;\u0026thinsp;1.5) in ileal digesta. Previous studies confirmed that approximately 50% of HMBi escaped absorption from the rumen wall and was degraded into HMB in the intestine to increase methionine supply(Graulet et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Koenig et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). Our results demonstrated the above-mentioned degradation process of HMBi, but whether it could promote intestinal absorption of Met still require more studies. In addition, we postulated that the increase in ileal Met in the present study was partly related to microbial synthesis. O-Succinyhomoserine is an intermediate in the bacterial biosynthesis of Met via the trans-sulphation pathway(Brewster et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Hence, the increase in ileal O-Succinyhomoserine may be indirect evidence that the HMBi diet promoted the synthesis of Met by intestinal bacteria in this study.\u003c/p\u003e\u003cp\u003eOur results showed that the content of some substances (including taxifolin, Soyasaponin I, and tyrosol) enhanced intestine health significantly elevated in the ileal digesta after HMBi supplementation. Taxifolin was proved to have the ability to inhibit the expression of some inflammatory cytokines in the mouse colon and reduced intestinal inflammation and mucosal damage by promoting butyrate production(Li et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2022a\u003c/span\u003e). Soyasaponin I is a representative component of soybeans that has been demonstrated to attenuate the inflammatory process in mice by inhibiting the pro-inflammatory cytokines or mediators, such as TNF-α, IL-1β, and NO. (Lee et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Tyrosol is a biomolecule with various physiological functions such as antioxidant, anti-inflammatory and anti-apoptosis(Plotnikov and Plotnikova, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Moreover, tyrosol has been demonstrated to enhance glutathione (GSH) levels and peroxidase activity, and reduce inflammation by decreasing TNF-α, IL-6, and TGF-β1 expression(Kutlu et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Li et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2022b\u003c/span\u003e). Therefore, the increased content of taxifolin, Soyasaponin I, and tyrosol in the ileum after HMBi supplementation indicated that the HMBi may alleviate intestinal inflammation and contribute to the intestinal health of goats.\u003c/p\u003e\u003cp\u003eMoreover, our results suggested that ileal metabolites were enriched in arginine biosynthesis, citrate cycle, pyruvate metabolism, and glutathione metabolism, and metabolites involved in the above pathways were ornithine, fumaric acid, malic acid, and pyroglutamic acid. Ornithine can be converted to arginine via the ornithine cycle, resulting in the release of fumaric acid. Consequently, we speculated that the increase in fumaric acid content may be caused by the increase in ornithine content as shown in our result. Malic acid and fumaric acid are involved in the TCA, which is the final metabolic pathway and linkage between the three important nutrients (carbohydrates, lipids, and amino acids) and provides the energy for the host (Dashty, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). The increase in fumaric acid and malic acid content indicated that HMBi addition accelerated TCA process in ileal digesta in our study. In addition, pyroglutamic acid is an intermediate product of glutathione metabolism and is closely related to glutathione status(Ren et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Therefore, due to the antioxidant effects of glutathione, the increase in pyroglutamic acid content implied that the intestinal antioxidant function of goats may be enhanced after HMBi supplementation in our study.\u003c/p\u003e\u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003e\u003cb\u003eIleal microbiota and metabolism contributed to the improvement of cashmere performance after dietary HMBi supplementation in goats\u003c/b\u003e\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eIn recent years, gastrointestinal microbiota has received much attention, and has been proven to regulate animal growth performance, feed efficiency, and meat quality, etc. by manipulating gastrointestinal microbiota(Chen et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Gardiner et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Nevertheless, the linkage between the cashmere performance and intestinal microbiota remains unconfirmed. In the present study, to further explore whether the intestinal micro-ecological environment was involved in HMBi regulating the cashmere performance of goats, we conducted the Mantel tests. Our results showed that the cashmere length and growth rate were significantly and cashmere diameter was tended to be related to ileal microbiota and metabolites. These results indicated that the intestinal microbiota partly contributed to cashmere performance like some production traits such as meat quality.\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003c/p\u003e\u003cp\u003eThe cashmere length and diameter are important indicators of cashmere quality, and hence, we next the specific connections between the cashmere performance and ileal microbiota and metabolites. Among these connections, the methyl-containing metabolites attracted our attention. Our study revealed that cashmere length was negatively related with N,N-Dimethyltetradecylamine and tyrosine methyl ester, and positively related with 3'-O-methyluridine, 4-methoxyaniline, methylmalonic acid, mono-methyl glutarate, and 3-methylglutaconic acid; while cashmere diameter was negatively correlated with 3-methylglutaconic acid and methylmalonic acid. As we all known that, Met serves as methyl donors and participates in many reactions, such as DNA methylation, and glutathione synthesis(Chandler and White, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The above results suggested that the HMBi supplementation lead to an increase in methyl donors in the intestinal tract of goats, which may be one of the reasons for improving cashmere performance in the present study. In addition, we also found some connections between cashmere performance and ileal microbiota, we speculated that these microbiota were partly related to methyl metabolism, but due to the limited information, more studies were still needed to confirm. Next, we will conduct systematic studies on the intestinal transport and absorption of nutrients to further confirm the intestinal mechanism by which HMBi promotes cashmere growth in Liaoning cashmere goats.\u003c/p\u003e\u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eOur study integrated 16S rRNA sequencing and LC-MS metabolomics to reveal that HMBi supplementation changed the microbial community and metabolism pattern in the ileum, subsequently improving the cashmere performance of goats. The proposed framework of how HMBi affected the cashmere performance of Liaoning cashmere goats through the intestinal micro-ecological environment was shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e8\u003c/span\u003e. In general, the HMBi increased the cashmere length and growth rate, and decreased the cashmere diameter of goats. The HMBi supplementation resulted in a reduction of cellulose-degrading bacteria (including \u003cem\u003eCellulosilyticum\u003c/em\u003e, Lachnospiraceae_uncultured, \u003cem\u003eand Breznakia\u003c/em\u003e), and an increase of beneficial bacteria (such as OTU9, OUT256, and OTU657) in the ileum of goats. Moreover, the HMBi supplementation increased the content of HMBi degradation products (such as HMB and Met), and also increased some substance enhanced the intestinal health (such as taxifolin, Soyasaponin I, and tyrosol) in the ileum of goats. These findings provide further insight into the relationship between the intestinal micro-ecological environment and animal production performance. More in-depth studies should be conducted on the connections between cashmere performance and intestinal micro-ecological environment from the perspective of nutrient transport and absorption, and develop nutritional measures to improve cashmere performance by regulating the intestinal microecological environment.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflicts of interest.\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\n\u003cp\u003eR.Z. and M.X. designed the experiment, M.X., M.Z., P.S., J.J., Z.Z., L.L., and Y.Z. performed the experiment and collected the samples, M.X. and M.Z. conducted laboratory analyses, M.X. and R.Z. analyzed the data and wrote the manuscript, R.Z. and Y.C. revised the manuscript.\u003c/p\u003e\n\u003ch2\u003eAcknowledgement\u003c/h2\u003e\n\u003cp\u003eThis work was supported by the Liaoning Provincial Department of Education Funding for Fundamental Research Projects (JYTQN2023305).\u003c/p\u003e\n\u003ch2\u003eData Availability\u003c/h2\u003e\n\u003cp\u003eNone of the data were deposited in an official repository. The data/models that support the study findings are available to reviewers, or available from the authors upon request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAmato KR, Yeoman CJ, Kent A, Righini N, Carbonero F, Estrada A, Gaskins R, Stumpf H, Yildirim RM, Torralba S, M (2013) Habitat degradation impacts black howler monkey (Alouatta pigra) gastrointestinal microbiomes. ISME J 7:1344\u0026ndash;1353\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAstorga J, Gasaly N, Dubois-Camacho K, De la Fuente M, Landskron G, Faber KN, Urra FA, Hermoso MA (2022) The role of cholesterol and mitochondrial bioenergetics in activation of the inflammasome in IBD. Front Immunol 13:1028953\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBai D, Zhao J, Wang R, Du J, Zhou C, Gu C, Wang Y, Zhang L, Zhao Y, Lu N (2024) Eubacterium coprostanoligenes alleviates chemotherapy-induced intestinal mucositis by enhancing intestinal mucus barrier. Acta Pharm Sin B 14:1677\u0026ndash;1692\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBiddle A, Stewart L, Blanchard J, Leschine S (2013) Untangling the genetic basis of fibrolytic specialization by Lachnospiraceae and Ruminococcaceae in diverse gut communities. Diversity 5:627\u0026ndash;640\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBrewster JL, Pachl P, McKellar JLO, Selmer M, Squire CJ, Patrick WM (2021) Structures and kinetics of Thermotoga maritima MetY reveal new insights into the predominant sulfurylation enzyme of bacterial methionine biosynthesis. J Biol Chem 296:100797\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCai S, Shao N, Dong X (2015) Cellulosilyticum. Bergey's Manual of Systematics of Archaea and Bacteria, 1\u0026ndash;4\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChandler TL, White HM (2017) Choline and methionine differentially alter methyl carbon metabolism in bovine neonatal hepatocytes. PLoS ONE 12:e0171080\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChen B, Li D, Leng D, Kui H, Bai X, Wang TJFiM (2022) Gut microbiota and meat quality. Front Microbiol 13:951726\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDashty M (2013) A quick look at biochemistry: carbohydrate metabolism. Clin Biochem 46:1339\u0026ndash;1352\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ede Morais VN, Gomes MJC, Grancieri M, Moreira LdPD, Toledo RCL, Costa NMB, da Silva BP, Martino HSD (2023) Chia (Salvia hispanica L.) flour modulates the intestinal microbiota in Wistar rats fed a high-fat and high-fructose diet. Food Rev Int 172:113095\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eEdgar RC, Haas BJ, Clemente JC, Quince C, Knight R (2011) UCHIME improves sensitivity and speed of chimera detection. Bioinformatics 27:2194\u0026ndash;2200\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFeng Y, Cong Y, Deng H, Guo D, Liu HJRBdZ (2013a) Effects of 2-hydroxy-4 (methylthio) butanoic acid isopropyl ester on rumen fermentation in cashmere goats. Rev Bras Zootecn 42:342\u0026ndash;346\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFeng Y, Sun Y, Deng H, Cong Y (2013b) Promotion of cashmere growth by sulfur supplements in cashmere goats. Rev Bras Zootecn 42:792\u0026ndash;797\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFirkins JL, Fowler C, Devillard E, Bequette B (2015) Kinetics of microbial methionine metabolism in continuous cultures administered different methionine sources. J Dairy Sci 98:1178\u0026ndash;1194\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGalbraith H (2000) Protein and sulphur amino acid nutrition of hair fibre-producing Angora and Cashmere goats. Livest Prod Sci 64:81\u0026ndash;93\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGardiner GE, Metzler-Zebeli BU, Lawlor PG (2020) Impact of Intestinal Microbiota on Growth and Feed Efficiency in Pigs: A Review, vol 8. Microorganisms, p 1886\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGebeyew K, Yang C, He Z, Tan Z (2021) Low-protein diets supplemented with methionine and lysine alter the gut microbiota composition and improve the immune status of growing lambs. Appl Microbiol Biotechnol 105:8393\u0026ndash;8410\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eG\u0026eacute;rard P (2020) Gastrointestinal Tract: Microbial Metabolism of Steroids. In: Goldfine H (ed) Health Consequences of Microbial Interactions with Hydrocarbons, Oils, and Lipids. Springer International Publishing, Cham, pp 389\u0026ndash;399\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGraulet B, Richard C, Robert J (2005) Methionine availability in plasma of dairy cows supplemented with methionine hydroxy analog isopropyl ester. J Dairy Sci 88:3640\u0026ndash;3649\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGraulet B, Richard C, Robert JC (2004) The isopropyl ester of methionine hydroxy-analogue is absorbed through the rumen wall in the cow.J. Anim Feed Sci 13:269\u0026ndash;272\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHosomi K, Saito M, Park J, Murakami H, Shibata N, Ando M, Nagatake T, Konishi K, Ohno H, Tanisawa K, Mohsen A, Chen YA, Kawashima H, Natsume-Kitatani Y, Oka Y, Shimizu H, Furuta M, Tojima Y, Sawane K, Saika A, Kondo S, Yonejima Y, Takeyama H, Matsutani A, Mizuguchi K, Miyachi M, Kunisawa J (2022) Oral administration of Blautia wexlerae ameliorates obesity and type 2 diabetes via metabolic remodeling of the gut microbiota. Nat Commun 13:4477\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJiao J, Wang P, He Z, Tang S, Zhou C, Han X, Wang M, Wu D, Kang J, Tan Z (2014) In vitro evaluation on neutral detergent fiber and cellulose digestion by post-ruminal microorganisms in goats. J Sci Food Agr 94:1745\u0026ndash;1752\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKhan MZ, Liu S, Ma Y, Ma M, Ullah Q, Khan IM, Wang J, Xiao J, Chen T, Khan A, Cao Z (2022) Overview of the effect of rumen-protected limiting amino acids (methionine and lysine) and choline on the immunity, antioxidative, and inflammatory status of periparturient ruminants. Front Immunol 13:1042895\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKoenig K, Rode L, Knight C, V\u0026aacute;zquez-A\u0026ntilde;\u0026oacute;n M (2002) Rumen degradation and availability of various amounts of liquid methionine hydroxy analog in lactating dairy cows. J Dairy Sci 85:930\u0026ndash;938\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKutlu T, \u0026Ouml;zkan H, Guven\u0026ccedil; M (2021) Tyrosol retards induction of fibrosis in rats. J Food Biochem 45\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLee H, Kim M, Masaki T, Ikuta K, Iwamoto E, Nishihara K, Nonaka I, Ashihara A, Baek Y, Lee SJSR (2024) Assessing the impact of three feeding stages on rumen bacterial community and physiological characteristics of Japanese Black cattle. Sci Rep 14:4923\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLee IA, Park YJ, Yeo HK, Han MJ, Kim DH (2010) Soyasaponin I attenuates TNBS-Induced colitis in mice by inhibiting NF-κB pathway. J Agric Food Chem 58:10929\u0026ndash;10934\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLi C, Wang W, Liu T, Zhang Q, Wang G, Li F, Li F, Yue X, Li T (2018) Effect of early weaning on the intestinal microbiota and expression of genes related to barrier function in lambs. Front Microbiol 9:1431\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLi W, Zhang L, Xu Q, Yang W, Zhao J, Ren Y, Yu Z, Ma L (2022a) Taxifolin Alleviates DSS-Induced Ulcerative Colitis by Acting on Gut Microbiome to Produce Butyric Acid. Nutrients 14.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLi X, Wei T, Li J, Yuan Y, Wu M, Chen F, Deng ZY, Luo T (2022b) Tyrosol Ameliorates the Symptoms of Obesity, Promotes Adipose Thermogenesis, and Modulates the Composition of Gut Microbiota in HFD Fed Mice. Mol Nutr Food Res 66:e2101015\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLin Y, Wang Z, Zhou A (2008) Isopropyl 2-hydroxy-4-methylthiobutyrate (HMBi) and 2-hydroxy-4-methylthiobutyric acid (HMB) in ruminants. China Dairy Cattle, 11\u0026ndash;15\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLiu Y, Liu C, Wu H, Meng Q, Zhou Z (2022) Small intestine microbiome and metabolome of high and low residual feed intake Angus heifers. Front Microbiol 13:862151\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMartin C, Mirande C, Morgavi DP, Forano E, Devillard E, Mosoni P (2013) Methionine analogues HMB and HMBi increase the abundance of cellulolytic bacterial representatives in the rumen of cattle with no direct effects on fibre degradation. Anim Feed Sci Technol 182:16\u0026ndash;24\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMyer P, Wells J, Smith T, Kuehn L, Freetly H (2016) Microbial community profiles of the jejunum from steers differing in feed efficiency. J Anim Sci 94:327\u0026ndash;338\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eOrdway R, Boucher S, Whitehouse NL, Schwab CG, Sloan BJJo.d.s (2009) Effects of providing two forms of supplemental methionine to periparturient Holstein dairy cows on feed intake and lactational performance. J Dairy Sci 92:5154\u0026ndash;5166\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eOsorio J, Ji P, Drackley JK, Luchini D, Loor JJ J.J.o.d.s., 2013. Supplemental Smartamine M or MetaSmart during the transition period benefits postpartal cow performance and blood neutrophil function. J Dairy Sci 96, 6248\u0026ndash;6263\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePac\u0026iacute;fico C, Hartinger T, Stauder A, Schwartz-Zimmermann HE, Reisinger N, Faas J, Zebeli Q (2021) Supplementing a clay mineral-based feed additive modulated fecal microbiota composition, liver health, and lipid serum metabolome in dairy cows fed starch-rich diets. Front Vet Sci 8:714545\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePlotnikov MB, Plotnikova TM (2021) Tyrosol as a Neuroprotector: Strong Effects of a Weak Antioxidant. Curr Neuropharmacol 19:434\u0026ndash;448\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePotts SB, Brady KM, Scholte CM, Moyes KM, Sunny NE, Erdman RA (2023) Rumen-protected choline and methionine during the periparturient period affect choline metabolites, amino acids, and hepatic expression of genes associated with one-carbon and lipid metabolism. J Dairy Sci 106:4559\u0026ndash;4579\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eQin X, Zhang D, Qiu X, Zhao K, Zhang S, Liu C, Lu L, Cui Y, Shi C, Chen Z, Hao R, Li Y, Yang S, Wang L, Wang H, Cao B, Su H (2022) 2-Hydroxy-4-(Methylthio) Butanoic Acid Isopropyl Ester Supplementation Altered Ruminal and Cecal Bacterial Composition and Improved Growth Performance of Finishing Beef Cattle. Front Nutr 9:833881\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRen W, Yin J, Gao W, Chen S, Duan J, Liu G, Li T, Li N, Peng Y, Yin Y (2015) Metabolomics study of metabolic variations in enterotoxigenic Escherichia coli-infected piglets. RSC Adv 5:59550\u0026ndash;59555\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRobert J, Sloan B, Etave G, Bouza BJJDS (2001) Influence of length and ramification of the alcohol radical of esters of methionine and of 2-hydroxy-4 (methylthio) butanoic acid on methionine bioavailability. J Dairy Sci 84:34\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eStrain CR, Collins KC, Naughton V, McSorley EM, Stanton C, Smyth TJ, Soler-Vila A, Rea MC, Ross PR, Cherry P (2020) Effects of a polysaccharide-rich extract derived from Irish-sourced Laminaria digitata on the composition and metabolic activity of the human gut microbiota using an in vitro colonic model. Eur J Nutr 59:309\u0026ndash;325\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTegtmeier D, Riese C, Geissinger O, Radek R, Brune A (2016) Breznakia blatticola gen. nov. sp. nov. and Breznakia pachnodae sp. nov., two fermenting bacteria isolated from insect guts, and emended description of the family Erysipelotrichaceae. Syst Appl Microbiol 39:319\u0026ndash;329\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eXi M, Jiang J, Wang B, Wang Y, Di M, Cong Y, Zhang R (2025) Alterations in Methionine Cycle and Wnt/MAPK Signaling Associated with HMBi-Induced Cashmere Growth in Goats. Int J Mol Sci 26:1663\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYe L, Hou Y, Hu W, Wang H, Yang R, Zhang Q, Feng Q, Zheng X, Yao G, Hao H (2023) Repressed Blautia-acetate immunological axis underlies breast cancer progression promoted by chronic stress. Nat Commun 14:6160\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable 1 Effects of 2-Hydroxy-4-(methylthio)butanoic acid isopropyl ester (HMBi)\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cdiv \u003e\n \u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"515\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"bottom\" style=\"width: 248px;\"\u003e\n \u003cp\u003eItems\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"bottom\" style=\"width: 133px;\"\u003e\n \u003cp\u003eGroup\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" valign=\"bottom\" style=\"width: 67px;\"\u003e\n \u003cp\u003eSEM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" valign=\"bottom\" style=\"width: 67px;\"\u003e\n \u003cp\u003e\u003cem\u003eP\u0026nbsp;\u003c/em\u003evalue\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 63px;\"\u003e\n \u003cp\u003eCON\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 71px;\"\u003e\n \u003cp\u003eHMBi\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 248px;\"\u003e\n \u003cp\u003eAcetate, mmol/L\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 63px;\"\u003e\n \u003cp\u003e34.96\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 71px;\"\u003e\n \u003cp\u003e32.17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 67px;\"\u003e\n \u003cp\u003e3.292\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 67px;\"\u003e\n \u003cp\u003e0.689\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 248px;\"\u003e\n \u003cp\u003ePropionate, mmol/L\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 63px;\"\u003e\n \u003cp\u003e7.78\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 71px;\"\u003e\n \u003cp\u003e6.68\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 67px;\"\u003e\n \u003cp\u003e0.759\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 67px;\"\u003e\n \u003cp\u003e0.493\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 248px;\"\u003e\n \u003cp\u003eButyrate, mmol/L\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 63px;\"\u003e\n \u003cp\u003e3.81\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 71px;\"\u003e\n \u003cp\u003e3.14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 67px;\"\u003e\n \u003cp\u003e0.605\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 67px;\"\u003e\n \u003cp\u003e0.602\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 248px;\"\u003e\n \u003cp\u003eIsobutyrate, mmol/L\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 63px;\"\u003e\n \u003cp\u003e0.26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 71px;\"\u003e\n \u003cp\u003e0.28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 67px;\"\u003e\n \u003cp\u003e0.021\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 67px;\"\u003e\n \u003cp\u003e0.772\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 248px;\"\u003e\n \u003cp\u003eValerate, mmol/L\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 63px;\"\u003e\n \u003cp\u003e1.47\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 71px;\"\u003e\n \u003cp\u003e1.64\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 67px;\"\u003e\n \u003cp\u003e0.164\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 67px;\"\u003e\n \u003cp\u003e0.622\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 248px;\"\u003e\n \u003cp\u003eIsovalerate, mmol/L\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 63px;\"\u003e\n \u003cp\u003e0.20\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 71px;\"\u003e\n \u003cp\u003e0.20\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 67px;\"\u003e\n \u003cp\u003e0.022\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 67px;\"\u003e\n \u003cp\u003e0.900\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 248px;\"\u003e\n \u003cp\u003eToal VFA, mmol/L\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 63px;\"\u003e\n \u003cp\u003e48.48\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 71px;\"\u003e\n \u003cp\u003e44.11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 67px;\"\u003e\n \u003cp\u003e4.682\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 67px;\"\u003e\n \u003cp\u003e0.659\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 248px;\"\u003e\n \u003cp\u003eNH\u003csub\u003e3\u003c/sub\u003e-N, mg/dL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 63px;\"\u003e\n \u003cp\u003e9.35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 71px;\"\u003e\n \u003cp\u003e9.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 67px;\"\u003e\n \u003cp\u003e0.664\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 67px;\"\u003e\n \u003cp\u003e0.820\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 248px;\"\u003e\n \u003cp\u003eMCP, mg/dL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 63px;\"\u003e\n \u003cp\u003e17.83\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 71px;\"\u003e\n \u003cp\u003e19.90\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 67px;\"\u003e\n \u003cp\u003e0.572\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 67px;\"\u003e\n \u003cp\u003e0.085\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"cashmere goats, HMBi, ileal digesta, microbiota, metabolome","lastPublishedDoi":"10.21203/rs.3.rs-8062149/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8062149/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCurrently, rumen-protected methionine, such as 2-hydroxy-4-(methylthio)butanoic acid isopropyl ester (HMBi), is frequently employed in the ruminant breeding industry to reduce microbial degradation and subsequently enhance the methionine supply in an effective manner. However, there is a paucity of information regarding alterations in the microecological status of the small intestine with HMBi supplementation. Hence, the present study integrated multi-omics approaches to reveal the effects of HMBi on the microbial community, metabolites, and production performance in the ileum of Liaoning Cashmere goats. In this study, 14 female goats were assigned to be fed the control diets (CON, n\u0026thinsp;=\u0026thinsp;7) and HMBi (n\u0026thinsp;=\u0026thinsp;7) diets. After 67 days, the ileal digesta were sampled for measurements. Our results showed that the HMBi diets significantly increased (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) the cashmere length and growth rate, and tended to increase the ileal MCP concentration (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.085). Our results of ileal microbiota demonstrated that the HMBi supplementation decreased the abundance of cellulose-degrading bacteria (Cellulosilyticum, Lachnospiraceae_uncultured, Breznakia, etc.) and increased the abundance of beneficial bacteria (OTU9, OUT256, OTU657, etc.) in the ileum of goats. Our results of ileal metabolites also showed that, the content of HMBi degradation products (such as HMB and methionine) and some substance (such as taxifolin, Soyasaponin I, and tyrosol) enhanced the intestinal health in the ileum of goats were increased after HMBi feeding (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Moreover, our Mantel tests showed that ileal microbiota and metabolites significantly contributed to the improved cashmere performance of goats (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Overall, the above results revealed that the ileal microbiota and metabolism were involved in the process of HMBi regulating cashmere performance of goats.\u003c/p\u003e","manuscriptTitle":"Effects of dietary supplementation with 2-hydroxy-4-(methylthio) butanoic acid isopropyl ester on ileal microbiota and metabolism in Liaoning cashmere goats","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-25 17:53:25","doi":"10.21203/rs.3.rs-8062149/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"14c11c61-69b2-4136-8aa7-023088a299d3","owner":[],"postedDate":"November 25th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-12-26T00:23:32+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-25 17:53:25","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8062149","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8062149","identity":"rs-8062149","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}