Synergistic Regulation of Selenium Yeast and Vitamin E on the Rumen Microbiota-VFA-Liver Axis in Lambs

Synergistic Regulation of Selenium Yeast and Vitamin E on the Rumen Microbiota-VFA-Liver Axis in Lambs | 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 Synergistic Regulation of Selenium Yeast and Vitamin E on the Rumen Microbiota-VFA-Liver Axis in Lambs Shangwu Ma, Yuzhu Sha, Xiaoyong Ma, Haibo Wang, Shengguo Zhao, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8416285/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 19 Mar, 2026 Read the published version in BMC Microbiology → Version 1 posted 14 You are reading this latest preprint version Abstract Background Lambs in intensive farming face susceptibility to environmental and physiological stress, which can adversely affect their health and growth. Although selenium and vitamin E are recognized for their strong antioxidant and stress-alleviating properties, the combined effects of these substances in ruminants, especially within the regulatory framework of the "rumen-liver" axis, remain inadequately elucidated. This study sought to explore the regulatory pathways within this axis by evaluating lamb growth, immunity, antioxidative capacity, rumen function, liver transcriptome, and their interrelationships. Results The average daily weight gain, growth hormone levels, and immunoglobulin concentrations in the MIX group of lambs were significantly elevated, accompanied by an enhancement in serum antioxidant capacity ( P < 0.05). Concurrently, the rumen environment was notably improved, as evidenced by a reduction in pH and ammonia nitrogen levels, an increase in volatile fatty acid production, and a rise in the abundance of beneficial bacteria, including Firmicutes and Prevotella. Further analysis of the liver transcriptome indicated that differentially expressed genes were enriched in immune and antioxidant-related pathways, such as arachidonic acid metabolism. Correlation analysis suggested that rumen microorganisms may influence liver gene expression via volatile fatty acid metabolism. Conclusion The combined addition of selenium yeast and Vitamin E to the diet enhances antioxidant and immune capabilities through the "microbiotic-VFA-liver" axis, thereby synergistically improving the growth and health of lambs. Immunity Antioxidant Microbiota VFA Transcriptome Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction The healthy and rapid growth of fattening lambs represents a primary objective of modern, efficient sheep farming. However, under intensive farming conditions, factors such as early weaning, high-concentrate diets, and environmental changes frequently trigger stress responses in lambs, thereby disrupting their oxidative and immune balance. This disruption adversely impacts their growth performance and limits both farming profitability and animal welfare.[ 1 , 2 ] Consequently, the pursuit of green and safe nutritional regulation methods to bolster the stress resistance of lambs and promote their healthy growth has emerged as a significant focus in animal nutrition research. Selenium yeast (SY), an organic selenium source, offers high bioavailability and low toxicity, playing a crucial role in key antioxidant enzymes like glutathione peroxidase (GSH-PX) and actively contributing to the body's REDOX regulation. [ 3 ] Recent research indicates that selenium yeast significantly mitigates oxidative stress in ruminants. Maurya [ 4 ] demonstrated its ability to notably decrease oxidative and metabolic stress in Barbari goats exposed to heat stress, reducing lipid peroxidation reactions and enhancing overall antioxidant levels. Furthermore, SY has been shown to optimize rumen fermentation function and microbial composition, [ 5 ] indirectly influencing nutrient digestion and utilization. Vitamin E (VE), a fat-soluble antioxidant, effectively scavenges free radicals, safeguards cell membrane integrity, and exerts anti -inflammatory and immune-boosting properties. [ 6 ] Previous studies have demonstrated that the combined application of these two factors can enhance animal growth performance, improve antioxidant and immune functions, and promote rumen fermentation in ruminants. [ 7 , 8 ] However, the overall regulatory mechanisms in lambs, particularly through the "rumen-liver" axis, require further investigation. The rumen serves as the primary site for nutrient metabolism in ruminants; its microbial community structure, fermentation function, and morphological development directly influence nutrient digestion and absorption. [ 9 , 10 ] The liver is the core organ for material metabolism and detoxification, and it is also the key target for the physiological functions of selenium and VE. [ 11 , 12 ] We speculate that yeast selenium and vitamin E may promote the efficient production and utilization of volatile fatty acids by regulating rumen fermentation patterns and microbial composition, while enhancing related metabolic pathways such as antioxidants and immunity in the liver, thereby systematically improving the health level and growth performance of lambs. Based on this, this study took Hu sheep generation weaned male lambs as the subjects. By adding SY and VE alone and in combination to the diet, the effects of these additives on the growth performance, blood immunity and antioxidant indicators, rumen fermentation characteristics and tissue morphology, microbial community structure, and liver transcriptome of the lambs were systematically evaluated. The aim is to reveal the synergistic mechanism of yeast selenium and vitamin E in enhancing the stress resistance and promoting healthy growth of lambs, providing theoretical basis and technical support for precise nutrition and green and healthy breeding of ruminants. Material and Method Experimental Design Twenty-four healthy and well-in condition weaned male lambs were selected for the experiment, with an average body weight of (20.39 ± 1.52) kg. All the lambs are sourced from a large-scale and well-managed farm in Linxia City, Gansu Province. Before the experiment began, the lambs had completed routine deworming, brucellosis testing and vaccination to ensure a consistent initial health status. Twenty-four lambs were randomly divided into 4 groups according to their weight, with 6 lambs in each group, and were raised in separate pens. CON group: Basal diet, SY group: basal diet + 0.6 mg/kg yeast selenium, VE group: basal diet + 200 IU/kg vitamin E, MIX group: basal diet + 0.6 mg/kg yeast selenium + 200 IU/kg vitamin E. The addition amount was in accordance with the studies of Suganthi [ 13 ] and Nurlatifah [ 14 ]. The experiment consists of a 7-day pre-test period and a 60-day formal test period. The automatic feeding trough is used to ensure free feeding and drinking. During the experiment, the environmental conditions, feeding procedures and daily management of each group remained consistent. The basic diet used in the experiment was formulated in accordance with the "Lamb Rearing Standards of the Ministry of Agriculture of the People's Republic of China". The specific formula and nutritional levels are shown in Table 1 . Sample Collection After the trial period ended, the lambs fasted for 12 hours. Blood samples were collected from the jugular vein, centrifuged at 3500 r/min and 4°C for 10 minutes, and the serum was separated. After it was stored at -80°C for the determination of hormones, antioxidants and immune indicators. After blood collection, the lambs were food-producing animals. Slaughter was conducted humanely at a licensed abattoir in full compliance with the national standard NY/T 3469 − 2019, which mandates effective stunning to ensure unconsciousness prior to exsanguination. samples were aseptically collected by the research team members. Liver sample: Aseptically collect left lobe tissue of the liver (0.5-1 g), immediately place it in a cryotube containing RNA protective solution and store it at -80°C for transcriptome sequencing; Rumen tissue: Rumen abdominal capsule tissue (1 cm³) was collected and fixed with 4% paraformaldehyde for 24 hours for tissue morphology analysis; Rumen fluid: Collect the contents of the rumen abdominal capsule, filter them through 4 layers of sterile gauze, and then aliquot: ① Measure the pH on-site with a portable pH meter (Leici PHS-3C) (repeat three times and take the average value); ② Store at -2℃ for the determination of volatile fatty acids (VFAs) and NH₃-N; ③ Store at -80℃ for microbial DNA extraction. Table 1 Basic diet formulation Ingredient content(%) Nutrient level (%) Corn stalk 15.00 digestive energy DE/(MJ/kg) 10.21 Corn 32.00 CP 14.81 Wheat bran 6.00 EE 3.50 Soybean meal(43%) 6.00 Ash 9.00 Sunflower seed shells 12.00 CF 14.60 Spraying corn skin 8.00 NDF 33.20 DDGS 5.00 ADF 21.50 NaHCO3 0.60 Ca 1.30 Molasses 5.00 TP 0.60 Pumpkin Seed Meal 5.20 NaCl 0.50 Limestone 0.60 Soybean oil 0.60 Premix a 4.00 Total 100.00 a Composition (per kg of dry matter): 100,000–500 000 IU of vitamin A, 50,000–200 000 IU of vitamin D3, Fe 1500–7000 mg, Cu 300–750 mg, Mn 1000–5000 mg, Zn 1500–4000 mg, I 20–30 mg, Co 8–35 mg. Determination of growth performance At the beginning and end of the formal trial period, sheep in each group were fasting for 12 hours and then weighed (with an accuracy of 0.01 kg). The feed consumption at each stage was recorded, and the average daily gain, average daily feed intake and feed-to-weight ratio were calculated. Average daily gain (ADG) = (final weight - initial weight)/number of feeding days Average daily feed intake (ADFI) = stage feed intake/number of feeding days Material-to-weight ratio (F/G) = ADFI/ADG. Serum hormone determination The levels of serum GH, glucagon and IGF-1 were determined by ELISA. The operation strictly followed the instructions of the kit. The absorbance at 450 nm was measured by the microplate reader (Thermo Multiskan FC), and the concentration was calculated by the standard curve method (n = 6). Antioxidant and immunoglobulin indicators The total antioxidant capacity of serum (T-AOC), glutathione peroxidase (GSH-PX), superoxide dismutase (SOD), malondialdehyde (MDA), and catalase (CAT) indicators were determined by colorimetry. The levels of IgA, IgG and IgM in serum were determined by ELISA, with quantitative analysis at 450 nm absorbance (n = 6). Characteristics of Gastric Juice Fermentation NH₃-N, Take 5 mL of rumen fluid, add 10 mL of 0.2 mol/L HCl and mix well. Centrifuge at 3500 r/min for 10 minutes, and measure the supernatant by colorimetry at 420 nm. [ 15 ] VFA, Take 1 mL of rumen fluid, add 0.2 mL of 25% metaphosphoric acid (containing 0.8% tobacic acid), centrifuge at 12000 r/min for 10 minutes, and the supernatant is filtered through a 0.22 µm filter membrane. Then, determine it by gas chromatography (Shimadzu GC-2010 Plus). Chromatographic conditions DB-FFAP capillary column (30 m×0.25 mm×0.25 µm), column temperature 40℃ (3 min) →5℃/min to 180℃ (5 min), detector (FID) 250℃, injection port 220℃, carrier gas (N₂) flow rate 1.0 mL/min The injection volume is 1 µL (split ratio 10:1). [ 16 ] Preparation and observation of HE sections of rumen tissue The fixed rumen epithelial tissue was dewatered, paraffin embedded, sectured (5 µm), stained with HE, observed under an optical microscope (Nikon Eclipse Ni-U), and the papillary length, width and muscular layer thickness were determined using Image Pro Plus 6.0 software (10 fields were randomly selected for each sample). Repeat three times and take the average. Rumen microbial 16S rRNA sequencing DNA extraction The total microbial DNA of the tumor fluid was extracted using the MN NucleoSpin 96 Soil kit. The DNA concentration (≥ 50 ng/µL) and purity (A260/A280 = 1.8-2.0) were detected by NanoDrop 2000, and the integrity was verified by 1% agarose gel electrophoresis. PCR amplification PCR amplification was performed on the V3-V4 region of the 16S rRNA gene with primers 338F (5 '-ACTCCTACGGGAGGCAGCAG-3') and 806R (5 '-GGACTACHVGGGTWTCTAAT-3'). The reaction system (25 µL) : 2×Taq Plus Master Mix 12.5 µL, upstream and downstream primers (10 µmol/L) each 0.5 µL, DNA template 1 µL, enzyme-free water 10.5 µL. Reaction procedure: Pre-denaturation at 95℃ for 5 minutes, 30 cycles ( 95℃ for 30 seconds, 55℃ for 30 seconds, 72℃ for 30 seconds), final extension at 72℃ for 10 minutes. Sequencing and analysis, After the PCR products were purified, they were sequenced on the Illumina MiSeq platform (2×300 bp double-ended sequencing). After merging the original data with FLASH v1.2.7, filtering with Trimmomatic v0.33, and de-chimerization with UCHIME v4.2, Usearch v11.0 clustered OTUs with 97% similarity and annotated species based on the Silva 138 database. Bioinformatics analysis on BMKCloud ( www.biocloud.net ) was used to analyze Alpha diversity (Chao1, Shannon, Simpson, ACE index) and Beta diversity (PCoA analysis). The Metastats software was used to test the differences in species abundance between groups ( P < 0.05). [ 17 ] Liver transcriptomic analysis RNA extraction: Total RNA from liver tissue was extracted using TRIzol reagent. The quality was verified by 1% agarose gel electrophoresis (28S/18S ≈ 2:1) and NanoDrop 2000 (A260/A280 = 1.8-2.0, A260/A230 > 1.5). Library construction and sequencing For each sample, 1 µg of RNA was taken. The cDNA Library was constructed using the Illumina TruSeq Stranded mRNA Library Prep Kit and sequenced using the Illumina NovaSeq 6000 platform (2×150 bp double-ended sequencing). Data analysis: The Fastp software filtered the raw data to obtain Clean reads. HISAT2 was aligned to the sheep reference genome (Oar_rambouillet_v1.0), and StringTie was used to calculate the gene expression level (FPKM value). Bioinformatics analysis on BMKCloud ( www.biocloud.net ) was used to analyze DESeq2 screens differentially expressed genes (DEGs), with the criteria being log₂FC ≥ 2 and P < 0.05. Functional enrichment analysis was conducted between the GO database and the KEGG database, and Fisher's exact test determined significance ( P < 0.05).[ 18 ] Statistical Analysis Statistical analysis was conducted using IBM SPSS Statistics 26.0 software: Growth performance, hormones, antioxidants, immunity and rumen fermentation parameters were analyzed by one-way ANOVA. Duncan’s multiple comparison tests were used to test the differences between groups P < 0.05 was considered significant, P < 0.01 was considered extremely significant. The correlation between rumen microorganisms and physiological indicators was analyzed using Spearman correlation analysis. The correlation coefficients (r) ranged from − 1 to 1. r > 0 and < 0 represented positive and negative correlations, respectively. The |r| value denoted the degree of correlation among variables. In particular, r = − 1, 0, and 1 reflected a completely negative correlation, nocorrelation and a completely positive correlation, respectively Results The Effects of adding SY and VE on the growth, development and antioxidant immunity of Lambs The addition of SY, VE and their MIX to the diet has certain effects on the growth, antioxidation and immunity of lambs. Compared with the CON group (Fig. 1), the average daily gain (ADG) in the MIX group was significantly increased ( P 0.05). The contents of growth hormone (GH) and insulin growth factor-1 (IGF-1) in the MIX group were significantly increased ( P < 0.05). The glucagon (GCG) in the MIX group was extremely significantly higher than that in the CON group ( P < 0.01), and significantly higher than that in the SY group and the VE group ( P < 0.05). Analysis of serum antioxidant indicators revealed that the SOD activity in the MIX group was significantly higher than that in the CON group and the SY group ( P < 0.05). The activity of GSH-PX in the serum of the SY group and the MIX group was significantly higher than that of the CON group ( P < 0.05). The CAT activity in the MIX group was significantly higher than that in the SY group and the VE group, and extremely significantly higher than that in the CON group ( P < 0.01). Among the serum immune indicators, the contents of IgA, IgG and IgM in the MIX group were significantly higher than those in the CON group, SY group and VE group ( P < 0.05), with the CON group having the lowest content. Effects of adding SY and VE on Rumen Fermentation Function and epithelial Structure of Lambs MIX group was both the pH value of rumen fluid ( P = 0.014) and the concentration of ammonia nitrogen ( P = 0.013) were significantly reduced (Table 2 ). The Total volatile fatty acid (VFA) production in the SY group and the MIX group was significantly higher than that in the CON group and the VE group, and the contents of acetic acid and butyric acid in these two groups (SY group and MIX group) were also significantly higher than those in the CON group and the VE group ( P < 0.05). Analysis of the histological morphology of rumen epithelium (Fig. 2 , Table 3 ) showed that the length of rumen papillae in the MIX group and the VE group was significantly higher than that in the CON group and the SY group ( P < 0.05); The muscular layer thickness of the SY group and the MIX group was significantly lower than that of the CON group and the VE group ( P < 0.05). The rumen nipple width in the MIX group was significantly higher than that in the CON group ( P < 0.05). Table 2 Rumen Fermentation Parameters of Lambs Item Treatment SEM P-Value CON SY VE MIX pH 6.68 a 6.64 a 6.68 a 6.51 b 0.022 0.014 NH 3 -N,mg/100mL 10.20 a 10.20a 9.10ab 8.32 b 0.282 0.013 Total VFA,mmol/L 29.99 b 36.53 a 29.08 b 38.56 a 4.681 0.004 Acetic acid,mmol/L 17.10 b 21.00 a 18.23 b 21.12 a 1.972 0.014 Propionic acid ,mmol/L 7.37 9.41 6.56 10.05 2.074 0.217 Isobutyric acid ,mmol/L 0.72 0.65 0.56 0.80 0.127 0.250 Butyric acid,mmol/L 2.20 b 3.38 a 2.15 b 3.49 a 0.774 0.028 Isovaleric acid ,mmol/L 1.36 1.33 1.01 1.49 0.217 0.121 Pentanoic acid ,mmol/L 0.70 0.76 0.60 0.94 0.153 0.120 Acetic/Propionic ratio 2.33 2.23 2.79 2.10 0.130 0.320 Table 2 The same lowercase letters (a,b,c) indicate no significant difference P > 0.05, while different lowercase letters indicate a significant difference P < 0.05. Table 3 Histological Morphological Characteristics of rumen epithelium Item Treatment SEM P-Value CON SY VE MIX Papilla length,µm 2289.14 b 2318.41 b 3257.56 a 3167.46 a 160.229 0.011 Papilla width,µm 449.85 b 491.65 ab 518.45 ab 554.99 a 50.127 0.075 Muscle thickness,µm 1125.35 a 733.65 b 1138.89 a 857.41 b 61.729 0.013 Table 3 The same lowercase letters (a,b) indicate no significant difference P > 0.05, while different lowercase letters indicate a significant difference P < 0.05. Effects of adding SY and VE on the structure and Function of rumen microorganisms in Lambs As shown in (Fig. 3), PCoA analysis revealed certain differences in rumen microbiota among the treatment groups. Among them, the CON group, SY group, VE group and MIX group had 3322, 2892, 2857 and 3264 OTUs respectively, and the total number of OTUs in each group was 278. The results of Alpha diversity analysis showed that the ACE and Chao1 indices in the MIX group were significantly higher than those in the CON group and the VE group. Moreover, the Simpson index of the MIX group was significantly higher than that of the VE group ( P < 0.05). Analysis of microbial species composition revealed that the abundance of Bacteroidota was the highest at the phylum level in each group, followed by Firmicutes. The relative abundance of Firmicutes in the MIX group was significantly higher than that in the CON group ( P < 0.05). Horizontal analysis revealed that the relative abundance of Prevotella was the highest, followed by uncultured_rumen_bacterium. Moreover, the relative abundance of Prevotella in the MIX group was significantly higher than that in the CON group, SY group and VE group (P < 0.05). LEfSe analysis revealed the differential biomarkers among different treatment groups: the SY group had Asteroleplasma, UCG_005, and Succinivibrionaceae_UCG_001; VE group Blvii28_wastewater, Williamwhitmaniaceae, Syntrophococcus, etc. The MIX group has UCG_001, unclassified_UCG, unclassified Bacteria, while the CON group does not. Further differential microbiota KEGG function analysis revealed (Fig. 4 ) that compared with the CON group, the Endocrine and metabolic diseases in the MIX group were decreased, while the Infectious diseases in the SY group were: Bacterial and Environmental adaptation increased, while Xenobiotics, biodegradation and metabolism in the SY group decreased. Rumen microbial fermentation metabolism driven by SY and VE is related to lamb growth, antioxidation and immunity To further reveal the correlation between the changes in rumen microbial fermentation metabolism after the addition of SY and VE and the growth, antioxidation and immunity of lambs (Fig. 4 A and B), it was found through correlation analysis that ADG was significantly positively correlated with Succinivibrio ( P < 0.05) Growth hormone GCG was significantly positively correlated with [Eubacterium] ruminantium group, Prevotella, and SP3-e08 ( P < 0.05), while it was significantly negatively correlated with Unclassified Prevotellaceae ( P < 0.05). Both GH and IGF-1 were significantly negatively correlated with Prevotellaceae UCG-001 ( P < 0.05). Among the immune indicators, the contents of IgA, IgG and IgM were significantly negatively correlated with Prevotellaceae UCG-001 ( P < 0.05). Among them, IgM was significantly positively correlated with the [Eubacterium] ruminantium group ( P < 0.05). Among the antioxidant indicators, CAT was significantly negatively correlated with UCG-004, Rikenellaceae RC9 gut group, and Succiniclasticum ( P < 0.05), and significantly positively correlated with Mitsuokella ( P < 0.05). Rumen volatile fatty acids (VFA) were significantly correlated with the growth performance, antioxidant capacity and immune function of lambs (Fig. 4 C and D). Among them, ADG and IgA were significantly positively correlated with rumen pH value ( P < 0.05). T-AOC was significantly positively correlated with NH 3 -N ( P < 0.05), and MDA was significantly negatively correlated with NH₃-N ( P < 0.05). GCG, IGF-1, IgG and IgM were significantly positively correlated with TVFA, Acetic, Propionic, Isovaleric and pentanoic acid ( P < 0.05). GH, IGF-1 were significantly positively correlated with Isobutyric ( P < 0.05), and ADFI was significantly negatively correlated with isobutyric acid ( P < 0.05). GH, GCG, IGF-1 and MDA were significantly positively correlated with Butyric ( P < 0.05). GH and IGF-1 were significantly positively correlated with isovaleric acid ( P < 0.05). GCG and IgM were significantly negatively correlated with A/P ( P < 0.05). Figure 5 Correlation analysis of rumen microbial fermentation metabolism in lambs with their growth, antioxidation and immunity. * P < 0.05, ** P < 0.01. Effects of adding SY and VE on the transcriptional levels of Lamb livers SY and VE regulate liver degenes and are associated with lamb growth, antioxidation and immunity Based on liver transcriptome sequencing data, this study conducted a weighted gene co-expression network analysis (WGCNA) on the phenotypic indicators related to the growth performance, antioxidant capacity and immune function of lambs for the differentially expressed genes screened out. The results showed that there was a significant and strong correlation between the Megrey module and IgG, GCG, and IGF-1 ( P < 0.05). KEGG functional enrichment analysis of differentially expressed genes (DEGs) in the Meblue module indicated significant enrichment in immune metabolism-related pathways, specifically Biosynthesis of amino acids, Carbon metabolism, Metabolism, Arachidonic acid metabolism, and Biosynthesis of unsaturated fatty acids and Bile secretion pathways. Additionally, DEGs in the MEturquoise module were predominantly enriched in Pancreatic secretion, Arachidonic acid metabolism, Antigen processing and presentation, and pathways closely associated with immune regulation and material metabolism, such as Toll-like receptor signaling pathways. Figure 7 WGCNA analysis of differentially expressed genes and growth, antioxidant and immune indicators of lambs.(A) WGCNA module diagram, (B) Gene-phenotype clustering diagram,(C)KEGG enrichment diagram (Meblue),༈D༉KEGG enrichment (MEturquoise). SY and VE drive rumen microbiota-VFA-liver mRNA interactions in lambs Through the interaction analysis of rumen microbiota - VFA-liver mRNA, it was found that pH was significantly positively correlated with Succinivibrionaceae UCG001 and Succinivibrio ( P < 0.05). The ratio of A/P was significantly negatively correlated with Succiniclasticum ( P < 0.05). Isovaleric, Propionic and Pentanoic were all significantly positively correlated with Succiniclasticum ( P < 0.05). TVFA was significantly positively correlated with [Eubacterium]_ ruminantium_group ( P < 0.05). Butyric was significantly positively correlated with [Eubacterium]_ ruminantium_group and SP3-e08 ( P < 0.05), and significantly negatively correlated with Prevotellaceae UCG001 ( P < 0.05). To further reveal the association between VFA, related functional microorganisms and liver gene expression, weighted gene co-expression network analysis (WGCNA) was conducted on the three, and a total of three core gene modules significantly associated with VFA and corresponding microorganisms were identified. Among them, the MEgrey module was significantly correlated with SP3_e08, NH3-N, TVFA, Acetic, Propionic, Butyric, Pentanoic ( P <0.05). The Meblue module was significantly positively correlated with [Eubacterium]_ruminantium_group and Succiniclasticum ( P <0.05). Conduct GO functional enrichment analysis on DEGs in the Meblue module ( P <0.05), the results showed that the genes of this module were mainly enriched in immune metabolism-related functional items, including immune system process, cell killing, structural molecule activity, etc. The Meblue module was significantly positively correlated with [Eubacterium]_ruminantium_group and Succiniclasticum ( P <0.05). Conduct GO functional enrichment analysis on DEGs in the Meblue module (FDR<0.05), the results showed that the genes of this module were mainly enriched in immune metabolism-related functional items, including immune system process, cell killing, structural molecule activity, etc. Figure 8 Analysis of the interaction between rumen microbiota, VFA and liver mRNA in lambs. A: Heat map illustrating the correlation among the top 20 rumen bacterial genera and volatile fatty acid parameters. B: Gene-phenotype clustering map displaying a: Prevotellaceae_UCG_001, b: Succinivibrionaceae_UCG_001, c: Succinivibrio, d: [Eubacterium]_ruminantium_group, e: Succiniclasticum, and f: SP3_e08. C: Plot of WGCNA modules. D: Plot of GO enrichment (Meblue). E: Map of GO Enrichment (MEturquoise). Discussion SY and VE are excellent antioxidants. This study systematically explored the effects of adding SY, VE, and their mixed addition (MIX) to the diet on the growth and development, antioxidant immune function, rumen fermentation and epithelial structure, rumen microbial community, and liver transcriptome of lambs. It also revealed the regulation of the interaction network of rumen microbiota - VFA - liver mRNA on the growth and development and antioxidant immunity of lambs. The improvement of growth performance is the core objective of livestock and poultry nutrition research, and the ADG, and feed conversion ratio (F/G) are the key indicators for evaluating growth performance. [ 19 ] In this study, the ADG in the MIX group was significantly increased and the decrease in F/G was the largest. GH and IGF-1 were significantly elevated. GH and IGF-1 are core endocrine factors regulating the growth and development of animals. They can promote animal growth by facilitating cell proliferation and differentiation, accelerating protein synthesis and energy metabolism. [ 20 ] Meanwhile, the GCG in the MIX group also increased extremely significantly, further demonstrating that the growth performance of the lambs in the MIX group was improved. GCG can activate enzymes related to glucose metabolism, promote the decomposition of liver glycogen into glucose, and provide sufficient energy substrates for the growth of lambs. [ 21 ] The improvement of growth performance is closely related to the enhancement of the body's antioxidant capacity and immune function. The antioxidant system is an important barrier for maintaining the normal and stable state of cells. SOD, GSH-PX, and CAT jointly constitute the defense system for eliminating reactive oxygen species in the body. [ 22 , 23 ] The activities of SOD, GSH-PX and CAT in the MIX group were significantly higher than those in the control group and the group added alone, indicating that SY and VE have a synergistic antioxidant effect. This result is consistent with the research conclusion of Wang, [ 24 ] that SY as an essential component of GSH-PX, directly enhances the ability of the enzymatic antioxidant system. VE as a fat-soluble antioxidant, mainly targets the cell membrane and can preferentially eliminate free radicals in the lipid peroxidation chain reaction, protect the integrity of the membrane structure, and jointly maintain the REDOX homeostasis of lambs. [ 25 , 26 ] The contents of IgA, IgG and IgM in humoral immunity can directly reflect the immune capacity of the body. The contents of the three immunoglobulins in the MIX group were significantly higher than those in other groups, which may be closely related to the improvement of the antioxidant system in the MIX group. Oxidative stress can damage the activity of immune cells. The powerful antioxidant capacity of the MIX group can alleviate oxidative damage to immune cells, promote the proliferation and differentiation of B lymphocytes and the secretion of antibodies. [ 27 ] Collectively, the elevated circulating growth hormone levels, enhanced antioxidant capacity, and improved immune defense function in lambs synergistically promoted their health status and feed conversion efficiency, thereby contributing to the enhancement of overall growth performance. The phenotypic changes are related to rumen microorganisms. As the core organ for the digestion and absorption of nutrients in ruminants, the stability of fermentation parameters and the integrity of epithelial structure in the rumen directly affect feed utilization rate, [ 28 ] and the synergistic effect of SY and VE shows a significant optimization effect in this process. The pH value of rumen fluid and the concentration of NH 3 -N in the MIX group were significantly decreased, indicating that the mixed addition could optimize the rumen fermentation environment. An appropriate rumen pH (6.2–6.8) is the guarantee of microbial activity. The pH of the MIX group was maintained at 6.51, which not only avoided the risk of acidosis, It also provides conditions for the synergistic effect of fiber-decomposing bacteria and acid-producing bacteria. [ 29 ] A decrease in NH 3 -N concentration indicates an improvement in the efficiency of rumen microorganisms in degrading feed proteins and synthesizing microbial proteins, reducing nitrogen source waste. [ 30 ] This is consistent with the research findings of Alhidary [ 31 ], the combined addition of selenium and VE can regulate nitrogen metabolism efficiency of sheep. Another important feature of the MIX group was the significant increase in the contents of total volatile fatty acids (TVFA), acetic acid and butyric acid. TVFA is the main source of energy supply for lambs, among which acetic acid can participate in fat synthesis and butyric acid is the substrate for ATP generation. [ 32 , 33 ] The increase in TVFA production in the MIX group directly provided an energy basis for the increase in ADG, while the enrichment of acetic acid and butyric acid further promoted rumen epithelial development. The morphological changes of rumen epithelial structure are closely related to the efficiency of nutrient absorption. The length and width of rumen papillae in the MIX group increased significantly, which can effectively expand the absorption area of rumen epithelium. A reduction in muscle layer thickness may decrease the resistance to the diffusion of nutrients from the rumen to the bloodstream, and together they enhance the absorption efficiency of nutrients such as VFA.[ 34 ] This result echoes the optimization of rumen fermentation parameters in the MIX group, indicating that SY and VE significantly enhanced the rumen's digestive and absorptive functions through the pathway of "improving the fermentation environment - increasing VFA yield - optimizing epithelial structure". The optimization of rumen fermentation function essentially depends on the stability of the microbial community structure and functional synergy. [ 35 ] The synergistic effect of SY and VE significantly reshaped the characteristics of the rumen microbial community in lambs. The diversity and composition stability of the rumen microbial community are the basis for its digestive function. The ACE and Chao1 indices in the MIX group were significantly higher than those in the control group and the VE group, and the Simpson index was significantly higher than that in the VE group. This indicates that mixed addition can improve the richness and uniformity of the rumen microbial community and enhance the stability of the community. OTU analysis showed that there were only 278 OTUs in each group, while the MIX group had a larger number of OTUs, indicating that the synergistic effect of SY and VE could introduce or enrich specific functional microorganisms and reshape the rumen microbial community structure. At the phylum level, Bacteroidota and Firmicutes were the dominant rumen microbiota, and the relative abundance of Firmicutes in the MIX group was significantly increased. Bacteroidota and Firmicutes are closely related to the degradation of feed carbohydrates. [ 36 ] Particularly, Firmicutes contain a large number of fiber- decomposing bacteria and butyric acid-producing bacteria. An increase in their abundance can promote the degradation of cellulose and hemicellulose as well as the generation of butyric acid. [ 37 ] This is consistent with the increased results of TVFA and butyric acid content in the MIX group in this study. At the genus level, Prevotella was significantly enriched in the MIX group. Microorganisms of the Prevotella genus can secrete various glycoside hydrolases, which efficiently degrade starch, cellulose and pectin in feed. [ 38 ] The increase in its abundance further verified the enhanced feed digestion efficiency in the MIX group. The differential biomarkers revealed by LEfSe analysis provide clues for understanding the functional differences of microorganisms. The microbiota such as UCG_001 and unclassified_UCG_010 enriched in the MIX group may be involved in VFA metabolism and antioxidant synthesis. The specific microbiota functions of the SY group and the VE group are relatively single, which also explains the dominant effect of the mixed addition group. KEGG functional analysis revealed that the "endocrine and metabolic diseases" related pathways in the MIX group were decreased, indicating that it could reduce the risk of metabolic disorders by regulating microbial functions. However, the increase in the "environmental adaptation" pathways in the SY group might be related to the stress adaptation of microorganisms when SY was added alone. [ 39 ] To explain the mechanism of action of SY and VE at the molecular level, this study further carried out liver transcriptomic analysis. PCA analysis indicated that there was a significant segregation trend in the gene expression profiles among different treatment groups, which provided a molecular basis for explaining their different physiological effects. Analysis of differentially expressed genes (DEGs) revealed that the SY group and the VE group were mainly down-regulated genes, while the gap in the number of up-regulated and down-regulated genes in the MIX group narrowed. This indicates that the combination of the two may alleviate the transcriptional suppression of a single intervention, demonstrating functional complementarity and synergy, which is consistent with the conclusion of Sivertsen. [ 40 ] In the GO enrichment analysis, the VE group was specifically enriched in the "membrane encapsulation cavity" item, which was consistent with its role in scavenging membrane oxidation free radicals. The MIX group enriched the most genes in "biological regulation", further supporting the synergistic regulation of the two in biological processes. KEGG pathway enrichment revealed that the SY group was specifically enriched in "niacin and nicotinamide metabolism", which might be related to the promotion of NAD(P) + synthesis by SY and the enhancement of GSH-PX activity; [ 41 ]The VE group is enriched in the "phagosome" pathway, enhancing macrophage-mediated innate immunity; The MIX group was significantly enriched in "arachidonic acid metabolism", a pathway that generates lipid mediators such as prostaglandins and leukotrienes, which play a core role in regulating inflammation and immunity, [ 42 ] indicating that SY and VE may systematically coordinate antioxidant and immune homeostasis by regulating lipid signaling networks. The functional remodeling of rumen microorganisms further forms an interactive regulatory network with the host liver gene expression through the metabolic product VFA, jointly mediating the synergistic regulatory effect of SY and VE. The results of correlation analysis indicated that rumen microbiota, VFA were associated with lamb growth, antioxidant and immune indicators. ADG was significantly positively correlated with Succinivibrio, and Succinivibrio could participate in the metabolism of succinic acid and propionic acid. [ 43 ] An increase in its abundance could promote the generation of VFA. And then provide energy for growth. GCG and IgM were significantly positively correlated with the [Eubacterium] ruminantium group. Studies have shown that this microbiota is related to immunity, [ 44 ] and it may synergistically affect growth and immune function by regulating hormone secretion and antibody production. As a microbial metabolic product, VFA plays a key signaling role in the "microbiota-host" interaction. The significant positive correlation between TVFA, acetic acid, butyric acid, etc. in the MIX group and indicators such as GH, IGF-1, IgG indicates that VFA is not only an energy source, but also can regulate the endocrine system and the immune system. It affects the growth performance of lambs, which is related to Yu's discovery that supplementing selenium and vitamin E can improve growth performance and oxidative stress status. [ 25 ] As the metabolic and immune regulatory center, the liver's transcriptome response further explains the host's molecular adaptation to nutritional intervention. The differential genes in the MIX group were significantly enriched in pathways such as "hematopoietic cell lineage" and "arachidonic acid metabolism". WGCNA analysis further identified three gene modules closely related to growth and immune indicators. Among them, the MEgrey module had a strong correlation with IgG, GCG, IGF-1, etc., indicating that liver gene expression is directly involved in hormone and immune regulation. Conclusion This study indicates that the combined addition of 0.6 mg of SY and 200 IU of VE in the diet has a significantly better regulatory effect on lambs than the group with the addition alone, and it is an efficient nutritional regulation protocol. Mixed addition can increase the average daily gain (ADG) and reduce the feed-to-weight ratio (F/G) by up-regulating the secretion of GH, IGF-1 and GCG. At the same time, it collaboratively enhances the body's antioxidant and immune defense capabilities, and builds a positive cycle of "growth - antioxidant - immunity". In terms of rumen function, it can optimize the fermentation environment and enhance the efficiency of nutrient absorption by promoting the development of rumen papillae and reducing the thickness of the muscular layer. The core mechanism lies in the synergistic remodeling of the rumen microbiota by SY and VE (enriching functional flora such as Firmicutes and Prevotella), and mediating the coordinated regulation of growth, metabolism and immunity through the "Microorganism -VFA-Liver" interaction network. Abbreviations SY Selenium yeast VE Vitamin E ADG Average daily gain ADFI Average daily feed intake GH growth hormone IGF-1 insulin growth factor-1 GCG glucagon T-AOC total antioxidant capacity of serum GSH-PX glutathione peroxidase SOD superoxide dismutase MDA malondialdehyde CAT catalase F/G Material-to-weight ratio NH3-N Ammonia nitrogen TVFA Total volatile fatty acid VFA Volatile fatty acid OTUs Operational taxonomic units LEfSe LDA effect size PcoA Principal coordinates analysis KEGG Kyoto Encyclopedia of Genes and Genomes Declarations Acknowledgements We sincerely thank everyone and every enterprise that has provided assistance for this research. Author Contribution Shangwu M: investigation, validation, formal analysis, writing–review & editing. Yuzhu S: investigation, formal analysis, data analysis, software, writing–review and editing. Xiaoyong M, and Haibo W: supervision, conceptualization, methodol ogy, writing–original draft. Shengguo Z and Ting J: supervision, conceptualiza tion, methodology, writing–review and editing. All authors read and approved the final version of the manuscript. Funding The work was supported by the Open Competition Projects to Select the Guanghe County Cattle and Sheep Industry Quality Improvement and Efficiency Enhancement Technology Demonstration Project (LXZC21520230202) and Gansu Provincial Modern Cold-Arid Characteristic Agriculture Cattle Industry Technology System (GSARS01). Data Availability All the raw data were submitted to the NCBI Sequence Read Archive (SRA) database (Accession number: PRJNA-1135556). 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Cite Share Download PDF Status: Published Journal Publication published 19 Mar, 2026 Read the published version in BMC Microbiology → Version 1 posted Editorial decision: Revision requested 05 Jan, 2026 Reviews received at journal 03 Jan, 2026 Reviewers agreed at journal 31 Dec, 2025 Reviews received at journal 29 Dec, 2025 Reviewers agreed at journal 28 Dec, 2025 Reviewers agreed at journal 27 Dec, 2025 Reviewers agreed at journal 26 Dec, 2025 Reviewers agreed at journal 26 Dec, 2025 Reviewers agreed at journal 26 Dec, 2025 Reviewers invited by journal 26 Dec, 2025 Editor assigned by journal 26 Dec, 2025 Editor invited by journal 26 Dec, 2025 Submission checks completed at journal 25 Dec, 2025 First submitted to journal 25 Dec, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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02:58:19","extension":"xml","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":140275,"visible":true,"origin":"","legend":"","description":"","filename":"9adc19e729c843d48050c96f0caca22f1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8416285/v1/411326a4b8ae09f0ad15fde3.xml"},{"id":99197689,"identity":"de6a4d48-c0d7-4494-91d3-36d71a90f2a4","added_by":"auto","created_at":"2025-12-30 02:58:18","extension":"html","order_by":20,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":152333,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8416285/v1/9962938ead497a8345956093.html"},{"id":99197671,"identity":"ee466d77-dea9-41fa-bd31-9e0db7e5ff6e","added_by":"auto","created_at":"2025-12-30 02:58:17","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":153249,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of growth and development, serum growth hormone and antioxidant immune indicators of lambs. The same lowercase letters (a,b,c) indicate no significant difference \u003cem\u003eP\u003c/em\u003e\u0026gt; 0.05, while different lowercase letters indicate a significant difference \u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8416285/v1/17049b31850df0f3ef7b7322.png"},{"id":99317785,"identity":"d891ec85-8ea7-491e-8e9c-33db391eb104","added_by":"auto","created_at":"2025-12-31 16:30:42","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":631042,"visible":true,"origin":"","legend":"\u003cp\u003eHistological features of rumen epithelium in lambs. (A) CON, (B) SY, (C) VE, (D) MIX. Cut at 500μm.The same lowercase letters (a,b,c) indicate no significant difference \u003cem\u003eP\u003c/em\u003e\u0026gt; 0.05, while different lowercase letters indicate a significant difference \u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8416285/v1/c451645cb074d90dbd5a644e.png"},{"id":99197672,"identity":"e77ebc38-1866-4610-982a-dd572a30fef6","added_by":"auto","created_at":"2025-12-30 02:58:17","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":192561,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of the diversity and composition of rumen microorganisms in lambs. (A) PCoA analysis, (B) OTU-Venn analysis,(C-F) ACE, Chao, Simpson and Shannon indices，(G) phylum level microorganisms, (H) genus level microorganisms, (I) LEfSe analysis. The same lowercase letters (a,b,c) indicate no significant difference\u003cem\u003e P\u003c/em\u003e\u0026gt; 0.05, while different lowercase letters indicate a significant difference\u003cem\u003e P\u003c/em\u003e\u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8416285/v1/ef8591dbcc722cfe09da63a8.png"},{"id":99316936,"identity":"cadc89e2-8d8b-47ba-bd24-35f8d4882058","added_by":"auto","created_at":"2025-12-31 16:29:28","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":64275,"visible":true,"origin":"","legend":"\u003cp\u003eFunctional analysis of differential microbiota KEGG in the rumen of lambs.The same lowercase letters (a,b,c) indicate no significant difference\u003cem\u003e P\u003c/em\u003e\u0026gt; 0.05, while different lowercase letters indicate a significant difference\u003cem\u003e P\u003c/em\u003e\u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8416285/v1/466f578ba7a68238884bf6bb.png"},{"id":99197684,"identity":"73d1ec83-8f5e-45b9-b1e4-e7c2906cc9e3","added_by":"auto","created_at":"2025-12-30 02:58:18","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":406317,"visible":true,"origin":"","legend":"\u003cp\u003eCorrelation analysis of rumen microbial fermentation metabolism in lambs with their growth, antioxidation and immunity. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8416285/v1/ce51bef1f92a0e41208a5017.png"},{"id":99197688,"identity":"c466719e-b1a4-4341-be2c-662f8d2cd48d","added_by":"auto","created_at":"2025-12-30 02:58:18","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":262618,"visible":true,"origin":"","legend":"\u003cp\u003eTranscriptome analysis of Lamb liver. A shows PCA, B is the Venn plot of gene expression, C-E are the volcano plots of differential gene expression between SY, VE and MIX and the CON group respectively, F-H are the KEGG enrichment plots of differential expression between SY, VE and MIX and the CON group respectively.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-8416285/v1/7f2fc8cadd9ab6546e06813e.png"},{"id":99197679,"identity":"0ba5d1bd-7b9a-4e8f-bb5d-b33dc5984056","added_by":"auto","created_at":"2025-12-30 02:58:18","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":339778,"visible":true,"origin":"","legend":"\u003cp\u003eWGCNA analysis of differentially expressed genes and growth, antioxidant and immune indicators of lambs.(A) WGCNA module diagram, (B) Gene-phenotype clustering diagram,（C）KEGG enrichment diagram (Meblue),（D）KEGG enrichment (MEturquoise).\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-8416285/v1/ba87a62d6e089e962de695c7.png"},{"id":99316222,"identity":"ca3fa00b-11b2-4471-bb9b-e9554371ea92","added_by":"auto","created_at":"2025-12-31 16:27:54","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":463175,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of the interaction between rumen microbiota, VFA and liver mRNA in lambs. A: Heat map illustrating the correlation among the top 20 rumen bacterial genera and volatile fatty acid parameters. B: Gene-phenotype clustering map displaying a: Prevotellaceae_UCG_001, b: Succinivibrionaceae_UCG_001, c: Succinivibrio, d: [Eubacterium]_ruminantium_group, e: Succiniclasticum, and f: SP3_e08. C: Plot of WGCNA modules. D: Plot of GO enrichment (Meblue). E: Map of GO Enrichment (MEturquoise).\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-8416285/v1/35db9c489500b8700f1dc29b.png"},{"id":99197691,"identity":"8b5084c5-4c96-42d2-8b58-7e366d9d3d46","added_by":"auto","created_at":"2025-12-30 02:58:18","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":642214,"visible":true,"origin":"","legend":"\u003cp\u003e\"Microorganism-VFA-Liver\" regulatory mechanism diagram\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-8416285/v1/9924ce883f8be1d75208499d.png"},{"id":105223354,"identity":"7ef8a679-f861-426d-817c-a7e1871253d6","added_by":"auto","created_at":"2026-03-23 16:04:53","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4154313,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8416285/v1/7a57b6cc-ebc3-46df-8fd5-1bb2ba37220e.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Synergistic Regulation of Selenium Yeast and Vitamin E on the Rumen Microbiota-VFA-Liver Axis in Lambs","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe healthy and rapid growth of fattening lambs represents a primary objective of modern, efficient sheep farming. However, under intensive farming conditions, factors such as early weaning, high-concentrate diets, and environmental changes frequently trigger stress responses in lambs, thereby disrupting their oxidative and immune balance. This disruption adversely impacts their growth performance and limits both farming profitability and animal welfare.[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e] Consequently, the pursuit of green and safe nutritional regulation methods to bolster the stress resistance of lambs and promote their healthy growth has emerged as a significant focus in animal nutrition research.\u003c/p\u003e \u003cp\u003eSelenium yeast (SY), an organic selenium source, offers high bioavailability and low toxicity, playing a crucial role in key antioxidant enzymes like glutathione peroxidase (GSH-PX) and actively contributing to the body's REDOX regulation. [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e] Recent research indicates that selenium yeast significantly mitigates oxidative stress in ruminants. Maurya [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e] demonstrated its ability to notably decrease oxidative and metabolic stress in Barbari goats exposed to heat stress, reducing lipid peroxidation reactions and enhancing overall antioxidant levels. Furthermore, SY has been shown to optimize rumen fermentation function and microbial composition, [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e] indirectly influencing nutrient digestion and utilization. Vitamin E (VE), a fat-soluble antioxidant, effectively scavenges free radicals, safeguards cell membrane integrity, and exerts anti -inflammatory and immune-boosting properties. [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e] Previous studies have demonstrated that the combined application of these two factors can enhance animal growth performance, improve antioxidant and immune functions, and promote rumen fermentation in ruminants. [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] However, the overall regulatory mechanisms in lambs, particularly through the \"rumen-liver\" axis, require further investigation. The rumen serves as the primary site for nutrient metabolism in ruminants; its microbial community structure, fermentation function, and morphological development directly influence nutrient digestion and absorption. [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] The liver is the core organ for material metabolism and detoxification, and it is also the key target for the physiological functions of selenium and VE. [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] We speculate that yeast selenium and vitamin E may promote the efficient production and utilization of volatile fatty acids by regulating rumen fermentation patterns and microbial composition, while enhancing related metabolic pathways such as antioxidants and immunity in the liver, thereby systematically improving the health level and growth performance of lambs.\u003c/p\u003e \u003cp\u003eBased on this, this study took Hu sheep generation weaned male lambs as the subjects. By adding SY and VE alone and in combination to the diet, the effects of these additives on the growth performance, blood immunity and antioxidant indicators, rumen fermentation characteristics and tissue morphology, microbial community structure, and liver transcriptome of the lambs were systematically evaluated. The aim is to reveal the synergistic mechanism of yeast selenium and vitamin E in enhancing the stress resistance and promoting healthy growth of lambs, providing theoretical basis and technical support for precise nutrition and green and healthy breeding of ruminants.\u003c/p\u003e"},{"header":"Material and Method","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eExperimental Design\u003c/h2\u003e \u003cp\u003eTwenty-four healthy and well-in condition weaned male lambs were selected for the experiment, with an average body weight of (20.39\u0026thinsp;\u0026plusmn;\u0026thinsp;1.52) kg. All the lambs are sourced from a large-scale and well-managed farm in Linxia City, Gansu Province. Before the experiment began, the lambs had completed routine deworming, brucellosis testing and vaccination to ensure a consistent initial health status. Twenty-four lambs were randomly divided into 4 groups according to their weight, with 6 lambs in each group, and were raised in separate pens. CON group: Basal diet, SY group: basal diet\u0026thinsp;+\u0026thinsp;0.6 mg/kg yeast selenium, VE group: basal diet\u0026thinsp;+\u0026thinsp;200 IU/kg vitamin E, MIX group: basal diet\u0026thinsp;+\u0026thinsp;0.6 mg/kg yeast selenium\u0026thinsp;+\u0026thinsp;200 IU/kg vitamin E. The addition amount was in accordance with the studies of Suganthi [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] and Nurlatifah [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. The experiment consists of a 7-day pre-test period and a 60-day formal test period. The automatic feeding trough is used to ensure free feeding and drinking. During the experiment, the environmental conditions, feeding procedures and daily management of each group remained consistent. The basic diet used in the experiment was formulated in accordance with the \"Lamb Rearing Standards of the Ministry of Agriculture of the People's Republic of China\". The specific formula and nutritional levels are shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSample Collection\u003c/h3\u003e\n\u003cp\u003eAfter the trial period ended, the lambs fasted for 12 hours. Blood samples were collected from the jugular vein, centrifuged at 3500 r/min and 4\u0026deg;C for 10 minutes, and the serum was separated. After it was stored at -80\u0026deg;C for the determination of hormones, antioxidants and immune indicators. After blood collection, the lambs were food-producing animals. Slaughter was conducted humanely at a licensed abattoir in full compliance with the national standard NY/T 3469\u0026thinsp;\u0026minus;\u0026thinsp;2019, which mandates effective stunning to ensure unconsciousness prior to exsanguination. samples were aseptically collected by the research team members. Liver sample: Aseptically collect left lobe tissue of the liver (0.5-1 g), immediately place it in a cryotube containing RNA protective solution and store it at -80\u0026deg;C for transcriptome sequencing; Rumen tissue: Rumen abdominal capsule tissue (1 cm\u0026sup3;) was collected and fixed with 4% paraformaldehyde for 24 hours for tissue morphology analysis; Rumen fluid: Collect the contents of the rumen abdominal capsule, filter them through 4 layers of sterile gauze, and then aliquot: ① Measure the pH on-site with a portable pH meter (Leici PHS-3C) (repeat three times and take the average value); ② Store at -2℃ for the determination of volatile fatty acids (VFAs) and NH₃-N; ③ Store at -80℃ for microbial DNA extraction.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eBasic diet formulation\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIngredient\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003econtent(%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003eNutrient level (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCorn stalk\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e15.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003edigestive energy DE/(MJ/kg)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e10.21\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCorn\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e32.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e14.81\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWheat bran\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e6.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eEE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3.50\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSoybean meal(43%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e6.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAsh\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e9.00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSunflower seed shells\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e12.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e14.60\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSpraying corn skin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e8.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNDF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e33.20\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDDGS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eADF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e21.50\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNaHCO3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.30\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMolasses\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.60\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePumpkin Seed Meal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5.20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNaCl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.50\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLimestone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSoybean oil\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePremix\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e100.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003csup\u003ea\u003c/sup\u003eComposition (per kg of dry matter): 100,000\u0026ndash;500 000 IU of vitamin A, 50,000\u0026ndash;200 000 IU of vitamin D3, Fe 1500\u0026ndash;7000 mg, Cu 300\u0026ndash;750 mg, Mn 1000\u0026ndash;5000 mg, Zn 1500\u0026ndash;4000 mg, I 20\u0026ndash;30 mg, Co 8\u0026ndash;35 mg.\u003c/p\u003e\n\u003ch3\u003eDetermination of growth performance\u003c/h3\u003e\n\u003cp\u003eAt the beginning and end of the formal trial period, sheep in each group were fasting for 12 hours and then weighed (with an accuracy of 0.01 kg). The feed consumption at each stage was recorded, and the average daily gain, average daily feed intake and feed-to-weight ratio were calculated.\u003c/p\u003e \u003cp\u003eAverage daily gain (ADG) = (final weight - initial weight)/number of feeding days\u003c/p\u003e \u003cp\u003eAverage daily feed intake (ADFI)\u0026thinsp;=\u0026thinsp;stage feed intake/number of feeding days\u003c/p\u003e \u003cp\u003eMaterial-to-weight ratio (F/G)\u0026thinsp;=\u0026thinsp;ADFI/ADG.\u003c/p\u003e\n\u003ch3\u003eSerum hormone determination\u003c/h3\u003e\n\u003cp\u003eThe levels of serum GH, glucagon and IGF-1 were determined by ELISA. The operation strictly followed the instructions of the kit. The absorbance at 450 nm was measured by the microplate reader (Thermo Multiskan FC), and the concentration was calculated by the standard curve method (n\u0026thinsp;=\u0026thinsp;6).\u003c/p\u003e\n\u003ch3\u003eAntioxidant and immunoglobulin indicators\u003c/h3\u003e\n\u003cp\u003eThe total antioxidant capacity of serum (T-AOC), glutathione peroxidase (GSH-PX), superoxide dismutase (SOD), malondialdehyde (MDA), and catalase (CAT) indicators were determined by colorimetry. The levels of IgA, IgG and IgM in serum were determined by ELISA, with quantitative analysis at 450 nm absorbance (n\u0026thinsp;=\u0026thinsp;6).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eCharacteristics of Gastric Juice Fermentation\u003c/h2\u003e \u003cp\u003eNH₃-N, Take 5 mL of rumen fluid, add 10 mL of 0.2 mol/L HCl and mix well. Centrifuge at 3500 r/min for 10 minutes, and measure the supernatant by colorimetry at 420 nm. [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] VFA, Take 1 mL of rumen fluid, add 0.2 mL of 25% metaphosphoric acid (containing 0.8% tobacic acid), centrifuge at 12000 r/min for 10 minutes, and the supernatant is filtered through a 0.22 \u0026micro;m filter membrane. Then, determine it by gas chromatography (Shimadzu GC-2010 Plus). Chromatographic conditions DB-FFAP capillary column (30 m\u0026times;0.25 mm\u0026times;0.25 \u0026micro;m), column temperature 40℃ (3 min) \u0026rarr;5℃/min to 180℃ (5 min), detector (FID) 250℃, injection port 220℃, carrier gas (N₂) flow rate 1.0 mL/min The injection volume is 1 \u0026micro;L (split ratio 10:1). [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePreparation and observation of HE sections of rumen tissue\u003c/h3\u003e\n\u003cp\u003eThe fixed rumen epithelial tissue was dewatered, paraffin embedded, sectured (5 \u0026micro;m), stained with HE, observed under an optical microscope (Nikon Eclipse Ni-U), and the papillary length, width and muscular layer thickness were determined using Image Pro Plus 6.0 software (10 fields were randomly selected for each sample). Repeat three times and take the average.\u003c/p\u003e\n\u003ch3\u003eRumen microbial 16S rRNA sequencing\u003c/h3\u003e\n\u003cp\u003eDNA extraction The total microbial DNA of the tumor fluid was extracted using the MN NucleoSpin 96 Soil kit. The DNA concentration (\u0026ge;\u0026thinsp;50 ng/\u0026micro;L) and purity (A260/A280\u0026thinsp;=\u0026thinsp;1.8-2.0) were detected by NanoDrop 2000, and the integrity was verified by 1% agarose gel electrophoresis. PCR amplification PCR amplification was performed on the V3-V4 region of the 16S rRNA gene with primers 338F (5 '-ACTCCTACGGGAGGCAGCAG-3') and 806R (5 '-GGACTACHVGGGTWTCTAAT-3'). The reaction system (25 \u0026micro;L) : 2\u0026times;Taq Plus Master Mix 12.5 \u0026micro;L, upstream and downstream primers (10 \u0026micro;mol/L) each 0.5 \u0026micro;L, DNA template 1 \u0026micro;L, enzyme-free water 10.5 \u0026micro;L. Reaction procedure: Pre-denaturation at 95℃ for 5 minutes, 30 cycles ( 95℃ for 30 seconds, 55℃ for 30 seconds, 72℃ for 30 seconds), final extension at 72℃ for 10 minutes. Sequencing and analysis, After the PCR products were purified, they were sequenced on the Illumina MiSeq platform (2\u0026times;300 bp double-ended sequencing). After merging the original data with FLASH v1.2.7, filtering with Trimmomatic v0.33, and de-chimerization with UCHIME v4.2, Usearch v11.0 clustered OTUs with 97% similarity and annotated species based on the Silva 138 database. Bioinformatics analysis on BMKCloud (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ewww.biocloud.net\u003c/span\u003e\u003cspan address=\"http://www.biocloud.net\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was used to analyze Alpha diversity (Chao1, Shannon, Simpson, ACE index) and Beta diversity (PCoA analysis). The Metastats software was used to test the differences in species abundance between groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eLiver transcriptomic analysis\u003c/h2\u003e \u003cp\u003eRNA extraction: Total RNA from liver tissue was extracted using TRIzol reagent. The quality was verified by 1% agarose gel electrophoresis (28S/18S\u0026thinsp;\u0026asymp;\u0026thinsp;2:1) and NanoDrop 2000 (A260/A280\u0026thinsp;=\u0026thinsp;1.8-2.0, A260/A230\u0026thinsp;\u0026gt;\u0026thinsp;1.5). Library construction and sequencing For each sample, 1 \u0026micro;g of RNA was taken. The cDNA Library was constructed using the Illumina TruSeq Stranded mRNA Library Prep Kit and sequenced using the Illumina NovaSeq 6000 platform (2\u0026times;150 bp double-ended sequencing). Data analysis: The Fastp software filtered the raw data to obtain Clean reads. HISAT2 was aligned to the sheep reference genome (Oar_rambouillet_v1.0), and StringTie was used to calculate the gene expression level (FPKM value). Bioinformatics analysis on BMKCloud (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ewww.biocloud.net\u003c/span\u003e\u003cspan address=\"http://www.biocloud.net\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was used to analyze DESeq2 screens differentially expressed genes (DEGs), with the criteria being log₂FC\u0026thinsp;\u0026ge;\u0026thinsp;2 and \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Functional enrichment analysis was conducted between the GO database and the KEGG database, and Fisher's exact test determined significance (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eStatistical analysis was conducted using IBM SPSS Statistics 26.0 software: Growth performance, hormones, antioxidants, immunity and rumen fermentation parameters were analyzed by one-way ANOVA. Duncan\u0026rsquo;s multiple comparison tests were used to test the differences between groups \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered significant, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01 was considered extremely significant. The correlation between rumen microorganisms and physiological indicators was analyzed using Spearman correlation analysis. The correlation coefficients (r) ranged from \u0026minus;\u0026thinsp;1 to 1. r\u0026thinsp;\u0026gt;\u0026thinsp;0 and \u0026lt;\u0026thinsp;0 represented positive and negative correlations, respectively. The |r| value denoted the degree of correlation among variables. In particular, r\u0026thinsp;=\u0026thinsp;\u0026minus;\u0026thinsp;1, 0, and 1 reflected a completely negative correlation, nocorrelation and a completely positive correlation, respectively\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eThe Effects of adding SY and VE on the growth, development and antioxidant immunity of Lambs\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe addition of SY, VE and their MIX to the diet has certain effects on the growth, antioxidation and immunity of lambs. Compared with the CON group (Fig.\u0026nbsp;1), the average daily gain (ADG) in the MIX group was significantly increased (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and the feed conversion ratio (F/G) decreased the most (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). The contents of growth hormone (GH) and insulin growth factor-1 (IGF-1) in the MIX group were significantly increased (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The glucagon (GCG) in the MIX group was extremely significantly higher than that in the CON group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), and significantly higher than that in the SY group and the VE group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAnalysis of serum antioxidant indicators revealed that the SOD activity in the MIX group was significantly higher than that in the CON group and the SY group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The activity of GSH-PX in the serum of the SY group and the MIX group was significantly higher than that of the CON group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The CAT activity in the MIX group was significantly higher than that in the SY group and the VE group, and extremely significantly higher than that in the CON group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Among the serum immune indicators, the contents of IgA, IgG and IgM in the MIX group were significantly higher than those in the CON group, SY group and VE group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), with the CON group having the lowest content.\u003c/p\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eEffects of adding SY and VE on Rumen Fermentation Function and epithelial Structure of Lambs\u003c/h2\u003e \u003cp\u003eMIX group was both the pH value of rumen fluid (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.014) and the concentration of ammonia nitrogen (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.013) were significantly reduced (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The Total volatile fatty acid (VFA) production in the SY group and the MIX group was significantly higher than that in the CON group and the VE group, and the contents of acetic acid and butyric acid in these two groups (SY group and MIX group) were also significantly higher than those in the CON group and the VE group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Analysis of the histological morphology of rumen epithelium (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003e, Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) showed that the length of rumen papillae in the MIX group and the VE group was significantly higher than that in the CON group and the SY group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05); The muscular layer thickness of the SY group and the MIX group was significantly lower than that of the CON group and the VE group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The rumen nipple width in the MIX group was significantly higher than that in the CON group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eRumen Fermentation Parameters of Lambs\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eItem\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c5\" namest=\"c2\"\u003e \u003cp\u003eTreatment\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eSEM\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cem\u003eP-Value\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCON\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSY\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eVE\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMIX\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003epH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6.68\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.64\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6.68\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6.51\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.022\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.014\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNH\u003csub\u003e3\u003c/sub\u003e-N,mg/100mL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10.20\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10.20a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e9.10ab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8.32\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.282\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.013\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal VFA,mmol/L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e29.99\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e36.53\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e29.08\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e38.56\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e4.681\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.004\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAcetic acid,mmol/L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e17.10\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e21.00\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e18.23\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e21.12\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.972\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.014\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePropionic acid ,mmol/L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7.37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e9.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e10.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e2.074\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.217\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIsobutyric acid ,mmol/L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.127\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.250\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eButyric acid,mmol/L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.20\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.38\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.15\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.49\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.774\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.028\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIsovaleric acid ,mmol/L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.217\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.121\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePentanoic acid ,mmol/L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.153\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.120\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAcetic/Propionic ratio\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.79\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.130\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.320\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e The same lowercase letters (a,b,c) indicate no significant difference \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05, while different lowercase letters indicate a significant difference \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eHistological Morphological Characteristics of rumen epithelium\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eItem\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c5\" namest=\"c2\"\u003e \u003cp\u003eTreatment\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eSEM\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cem\u003eP-Value\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCON\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSY\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eVE\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMIX\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePapilla length,\u0026micro;m\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2289.14\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2318.41\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3257.56\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3167.46\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e160.229\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.011\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePapilla width,\u0026micro;m\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e449.85\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e491.65\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e518.45\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e554.99\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e50.127\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.075\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMuscle thickness,\u0026micro;m\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1125.35\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e733.65\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1138.89\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e857.41\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e61.729\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.013\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e The same lowercase letters (a,b) indicate no significant difference \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05, while different lowercase letters indicate a significant difference \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEffects of adding SY and VE on the structure and Function of rumen microorganisms in Lambs\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAs shown in (Fig.\u0026nbsp;3), PCoA analysis revealed certain differences in rumen microbiota among the treatment groups. Among them, the CON group, SY group, VE group and MIX group had 3322, 2892, 2857 and 3264 OTUs respectively, and the total number of OTUs in each group was 278. The results of Alpha diversity analysis showed that the ACE and Chao1 indices in the MIX group were significantly higher than those in the CON group and the VE group. Moreover, the Simpson index of the MIX group was significantly higher than that of the VE group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Analysis of microbial species composition revealed that the abundance of Bacteroidota was the highest at the phylum level in each group, followed by Firmicutes. The relative abundance of Firmicutes in the MIX group was significantly higher than that in the CON group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Horizontal analysis revealed that the relative abundance of Prevotella was the highest, followed by uncultured_rumen_bacterium. Moreover, the relative abundance of Prevotella in the MIX group was significantly higher than that in the CON group, SY group and VE group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). LEfSe analysis revealed the differential biomarkers among different treatment groups: the SY group had Asteroleplasma, UCG_005, and Succinivibrionaceae_UCG_001; VE group Blvii28_wastewater, Williamwhitmaniaceae, Syntrophococcus, etc. The MIX group has UCG_001, unclassified_UCG, unclassified Bacteria, while the CON group does not. Further differential microbiota KEGG function analysis revealed (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e4\u003c/span\u003e) that compared with the CON group, the Endocrine and metabolic diseases in the MIX group were decreased, while the Infectious diseases in the SY group were: Bacterial and Environmental adaptation increased, while Xenobiotics, biodegradation and metabolism in the SY group decreased.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eRumen microbial fermentation metabolism driven by SY and VE is related to lamb growth, antioxidation and immunity\u003c/b\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further reveal the correlation between the changes in rumen microbial fermentation metabolism after the addition of SY and VE and the growth, antioxidation and immunity of lambs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e4\u003c/span\u003eA and B), it was found through correlation analysis that ADG was significantly positively correlated with Succinivibrio (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) Growth hormone GCG was significantly positively correlated with [Eubacterium] ruminantium group, Prevotella, and SP3-e08 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), while it was significantly negatively correlated with Unclassified Prevotellaceae (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Both GH and IGF-1 were significantly negatively correlated with Prevotellaceae UCG-001 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Among the immune indicators, the contents of IgA, IgG and IgM were significantly negatively correlated with Prevotellaceae UCG-001 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Among them, IgM was significantly positively correlated with the [Eubacterium] ruminantium group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Among the antioxidant indicators, CAT was significantly negatively correlated with UCG-004, Rikenellaceae RC9 gut group, and Succiniclasticum (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and significantly positively correlated with Mitsuokella (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Rumen volatile fatty acids (VFA) were significantly correlated with the growth performance, antioxidant capacity and immune function of lambs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e4\u003c/span\u003eC and D). Among them, ADG and IgA were significantly positively correlated with rumen pH value (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). T-AOC was significantly positively correlated with NH\u003csub\u003e3\u003c/sub\u003e-N (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and MDA was significantly negatively correlated with NH₃-N (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). GCG, IGF-1, IgG and IgM were significantly positively correlated with TVFA, Acetic, Propionic, Isovaleric and pentanoic acid (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). GH, IGF-1 were significantly positively correlated with Isobutyric (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and ADFI was significantly negatively correlated with isobutyric acid (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). GH, GCG, IGF-1 and MDA were significantly positively correlated with Butyric (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). GH and IGF-1 were significantly positively correlated with isovaleric acid (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). GCG and IgM were significantly negatively correlated with A/P (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003eFigure 5 Correlation analysis of rumen microbial fermentation metabolism in lambs with their growth, antioxidation and immunity. *\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eEffects of adding SY and VE on the transcriptional levels of Lamb livers\u003c/h2\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eSY and VE regulate liver degenes and are associated with lamb growth, antioxidation and immunity\u003c/h2\u003e \u003cp\u003eBased on liver transcriptome sequencing data, this study conducted a weighted gene co-expression network analysis (WGCNA) on the phenotypic indicators related to the growth performance, antioxidant capacity and immune function of lambs for the differentially expressed genes screened out. The results showed that there was a significant and strong correlation between the Megrey module and IgG, GCG, and IGF-1 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). KEGG functional enrichment analysis of differentially expressed genes (DEGs) in the Meblue module indicated significant enrichment in immune metabolism-related pathways, specifically Biosynthesis of amino acids, Carbon metabolism, Metabolism, Arachidonic acid metabolism, and Biosynthesis of unsaturated fatty acids and Bile secretion pathways. Additionally, DEGs in the MEturquoise module were predominantly enriched in Pancreatic secretion, Arachidonic acid metabolism, Antigen processing and presentation, and pathways closely associated with immune regulation and material metabolism, such as Toll-like receptor signaling pathways.\u003c/p\u003e \u003cp\u003eFigure 7 WGCNA analysis of differentially expressed genes and growth, antioxidant and immune indicators of lambs.(A) WGCNA module diagram, (B) Gene-phenotype clustering diagram,(C)KEGG enrichment diagram (Meblue),༈D༉KEGG enrichment (MEturquoise).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eSY and VE drive rumen microbiota-VFA-liver mRNA interactions in lambs\u003c/h2\u003e \u003cp\u003eThrough the interaction analysis of rumen microbiota - VFA-liver mRNA, it was found that pH was significantly positively correlated with Succinivibrionaceae UCG001 and Succinivibrio (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The ratio of A/P was significantly negatively correlated with Succiniclasticum (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Isovaleric, Propionic and Pentanoic were all significantly positively correlated with Succiniclasticum (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). TVFA was significantly positively correlated with [Eubacterium]_ ruminantium_group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Butyric was significantly positively correlated with [Eubacterium]_ ruminantium_group and SP3-e08 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and significantly negatively correlated with Prevotellaceae UCG001 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). To further reveal the association between VFA, related functional microorganisms and liver gene expression, weighted gene co-expression network analysis (WGCNA) was conducted on the three, and a total of three core gene modules significantly associated with VFA and corresponding microorganisms were identified. Among them, the MEgrey module was significantly correlated with SP3_e08, NH3-N, TVFA, Acetic, Propionic, Butyric, Pentanoic (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05). The Meblue module was significantly positively correlated with [Eubacterium]_ruminantium_group and Succiniclasticum (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05). Conduct GO functional enrichment analysis on DEGs in the Meblue module (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05), the results showed that the genes of this module were mainly enriched in immune metabolism-related functional items, including immune system process, cell killing, structural molecule activity, etc. The Meblue module was significantly positively correlated with [Eubacterium]_ruminantium_group and Succiniclasticum (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05). Conduct GO functional enrichment analysis on DEGs in the Meblue module (FDR\u0026lt;0.05), the results showed that the genes of this module were mainly enriched in immune metabolism-related functional items, including immune system process, cell killing, structural molecule activity, etc.\u003c/p\u003e \u003cp\u003eFigure 8 Analysis of the interaction between rumen microbiota, VFA and liver mRNA in lambs. A: Heat map illustrating the correlation among the top 20 rumen bacterial genera and volatile fatty acid parameters. B: Gene-phenotype clustering map displaying a: Prevotellaceae_UCG_001, b: Succinivibrionaceae_UCG_001, c: Succinivibrio, d: [Eubacterium]_ruminantium_group, e: Succiniclasticum, and f: SP3_e08. C: Plot of WGCNA modules. D: Plot of GO enrichment (Meblue). E: Map of GO Enrichment (MEturquoise).\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eSY and VE are excellent antioxidants. This study systematically explored the effects of adding SY, VE, and their mixed addition (MIX) to the diet on the growth and development, antioxidant immune function, rumen fermentation and epithelial structure, rumen microbial community, and liver transcriptome of lambs. It also revealed the regulation of the interaction network of rumen microbiota - VFA - liver mRNA on the growth and development and antioxidant immunity of lambs. The improvement of growth performance is the core objective of livestock and poultry nutrition research, and the ADG, and feed conversion ratio (F/G) are the key indicators for evaluating growth performance. [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] In this study, the ADG in the MIX group was significantly increased and the decrease in F/G was the largest. GH and IGF-1 were significantly elevated. GH and IGF-1 are core endocrine factors regulating the growth and development of animals. They can promote animal growth by facilitating cell proliferation and differentiation, accelerating protein synthesis and energy metabolism. [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] Meanwhile, the GCG in the MIX group also increased extremely significantly, further demonstrating that the growth performance of the lambs in the MIX group was improved. GCG can activate enzymes related to glucose metabolism, promote the decomposition of liver glycogen into glucose, and provide sufficient energy substrates for the growth of lambs. [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] The improvement of growth performance is closely related to the enhancement of the body's antioxidant capacity and immune function. The antioxidant system is an important barrier for maintaining the normal and stable state of cells. SOD, GSH-PX, and CAT jointly constitute the defense system for eliminating reactive oxygen species in the body. [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] The activities of SOD, GSH-PX and CAT in the MIX group were significantly higher than those in the control group and the group added alone, indicating that SY and VE have a synergistic antioxidant effect. This result is consistent with the research conclusion of Wang, [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] that SY as an essential component of GSH-PX, directly enhances the ability of the enzymatic antioxidant system. VE as a fat-soluble antioxidant, mainly targets the cell membrane and can preferentially eliminate free radicals in the lipid peroxidation chain reaction, protect the integrity of the membrane structure, and jointly maintain the REDOX homeostasis of lambs. [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] The contents of IgA, IgG and IgM in humoral immunity can directly reflect the immune capacity of the body. The contents of the three immunoglobulins in the MIX group were significantly higher than those in other groups, which may be closely related to the improvement of the antioxidant system in the MIX group. Oxidative stress can damage the activity of immune cells. The powerful antioxidant capacity of the MIX group can alleviate oxidative damage to immune cells, promote the proliferation and differentiation of B lymphocytes and the secretion of antibodies. [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] Collectively, the elevated circulating growth hormone levels, enhanced antioxidant capacity, and improved immune defense function in lambs synergistically promoted their health status and feed conversion efficiency, thereby contributing to the enhancement of overall growth performance.\u003c/p\u003e \u003cp\u003eThe phenotypic changes are related to rumen microorganisms. As the core organ for the digestion and absorption of nutrients in ruminants, the stability of fermentation parameters and the integrity of epithelial structure in the rumen directly affect feed utilization rate, [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] and the synergistic effect of SY and VE shows a significant optimization effect in this process. The pH value of rumen fluid and the concentration of NH\u003csub\u003e3\u003c/sub\u003e-N in the MIX group were significantly decreased, indicating that the mixed addition could optimize the rumen fermentation environment. An appropriate rumen pH (6.2\u0026ndash;6.8) is the guarantee of microbial activity. The pH of the MIX group was maintained at 6.51, which not only avoided the risk of acidosis, It also provides conditions for the synergistic effect of fiber-decomposing bacteria and acid-producing bacteria. [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] A decrease in NH\u003csub\u003e3\u003c/sub\u003e-N concentration indicates an improvement in the efficiency of rumen microorganisms in degrading feed proteins and synthesizing microbial proteins, reducing nitrogen source waste. [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e] This is consistent with the research findings of Alhidary [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], the combined addition of selenium and VE can regulate nitrogen metabolism efficiency of sheep. Another important feature of the MIX group was the significant increase in the contents of total volatile fatty acids (TVFA), acetic acid and butyric acid. TVFA is the main source of energy supply for lambs, among which acetic acid can participate in fat synthesis and butyric acid is the substrate for ATP generation. [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e] The increase in TVFA production in the MIX group directly provided an energy basis for the increase in ADG, while the enrichment of acetic acid and butyric acid further promoted rumen epithelial development. The morphological changes of rumen epithelial structure are closely related to the efficiency of nutrient absorption. The length and width of rumen papillae in the MIX group increased significantly, which can effectively expand the absorption area of rumen epithelium. A reduction in muscle layer thickness may decrease the resistance to the diffusion of nutrients from the rumen to the bloodstream, and together they enhance the absorption efficiency of nutrients such as VFA.[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e] This result echoes the optimization of rumen fermentation parameters in the MIX group, indicating that SY and VE significantly enhanced the rumen's digestive and absorptive functions through the pathway of \"improving the fermentation environment - increasing VFA yield - optimizing epithelial structure\". The optimization of rumen fermentation function essentially depends on the stability of the microbial community structure and functional synergy. [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e] The synergistic effect of SY and VE significantly reshaped the characteristics of the rumen microbial community in lambs. The diversity and composition stability of the rumen microbial community are the basis for its digestive function. The ACE and Chao1 indices in the MIX group were significantly higher than those in the control group and the VE group, and the Simpson index was significantly higher than that in the VE group. This indicates that mixed addition can improve the richness and uniformity of the rumen microbial community and enhance the stability of the community. OTU analysis showed that there were only 278 OTUs in each group, while the MIX group had a larger number of OTUs, indicating that the synergistic effect of SY and VE could introduce or enrich specific functional microorganisms and reshape the rumen microbial community structure. At the phylum level, Bacteroidota and Firmicutes were the dominant rumen microbiota, and the relative abundance of Firmicutes in the MIX group was significantly increased. Bacteroidota and Firmicutes are closely related to the degradation of feed carbohydrates. [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e] Particularly, Firmicutes contain a large number of fiber- decomposing bacteria and butyric acid-producing bacteria. An increase in their abundance can promote the degradation of cellulose and hemicellulose as well as the generation of butyric acid. [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e] This is consistent with the increased results of TVFA and butyric acid content in the MIX group in this study. At the genus level, Prevotella was significantly enriched in the MIX group. Microorganisms of the Prevotella genus can secrete various glycoside hydrolases, which efficiently degrade starch, cellulose and pectin in feed. [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e] The increase in its abundance further verified the enhanced feed digestion efficiency in the MIX group. The differential biomarkers revealed by LEfSe analysis provide clues for understanding the functional differences of microorganisms. The microbiota such as UCG_001 and unclassified_UCG_010 enriched in the MIX group may be involved in VFA metabolism and antioxidant synthesis. The specific microbiota functions of the SY group and the VE group are relatively single, which also explains the dominant effect of the mixed addition group. KEGG functional analysis revealed that the \"endocrine and metabolic diseases\" related pathways in the MIX group were decreased, indicating that it could reduce the risk of metabolic disorders by regulating microbial functions. However, the increase in the \"environmental adaptation\" pathways in the SY group might be related to the stress adaptation of microorganisms when SY was added alone. [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eTo explain the mechanism of action of SY and VE at the molecular level, this study further carried out liver transcriptomic analysis. PCA analysis indicated that there was a significant segregation trend in the gene expression profiles among different treatment groups, which provided a molecular basis for explaining their different physiological effects. Analysis of differentially expressed genes (DEGs) revealed that the SY group and the VE group were mainly down-regulated genes, while the gap in the number of up-regulated and down-regulated genes in the MIX group narrowed. This indicates that the combination of the two may alleviate the transcriptional suppression of a single intervention, demonstrating functional complementarity and synergy, which is consistent with the conclusion of Sivertsen. [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e] In the GO enrichment analysis, the VE group was specifically enriched in the \"membrane encapsulation cavity\" item, which was consistent with its role in scavenging membrane oxidation free radicals. The MIX group enriched the most genes in \"biological regulation\", further supporting the synergistic regulation of the two in biological processes. KEGG pathway enrichment revealed that the SY group was specifically enriched in \"niacin and nicotinamide metabolism\", which might be related to the promotion of NAD(P)\u0026thinsp;+\u0026thinsp;synthesis by SY and the enhancement of GSH-PX activity; [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]The VE group is enriched in the \"phagosome\" pathway, enhancing macrophage-mediated innate immunity; The MIX group was significantly enriched in \"arachidonic acid metabolism\", a pathway that generates lipid mediators such as prostaglandins and leukotrienes, which play a core role in regulating inflammation and immunity, [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e] indicating that SY and VE may systematically coordinate antioxidant and immune homeostasis by regulating lipid signaling networks.\u003c/p\u003e \u003cp\u003eThe functional remodeling of rumen microorganisms further forms an interactive regulatory network with the host liver gene expression through the metabolic product VFA, jointly mediating the synergistic regulatory effect of SY and VE. The results of correlation analysis indicated that rumen microbiota, VFA were associated with lamb growth, antioxidant and immune indicators. ADG was significantly positively correlated with Succinivibrio, and Succinivibrio could participate in the metabolism of succinic acid and propionic acid. [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e] An increase in its abundance could promote the generation of VFA. And then provide energy for growth. GCG and IgM were significantly positively correlated with the [Eubacterium] ruminantium group. Studies have shown that this microbiota is related to immunity, [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e] and it may synergistically affect growth and immune function by regulating hormone secretion and antibody production. As a microbial metabolic product, VFA plays a key signaling role in the \"microbiota-host\" interaction. The significant positive correlation between TVFA, acetic acid, butyric acid, etc. in the MIX group and indicators such as GH, IGF-1, IgG indicates that VFA is not only an energy source, but also can regulate the endocrine system and the immune system. It affects the growth performance of lambs, which is related to Yu's discovery that supplementing selenium and vitamin E can improve growth performance and oxidative stress status. [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] As the metabolic and immune regulatory center, the liver's transcriptome response further explains the host's molecular adaptation to nutritional intervention. The differential genes in the MIX group were significantly enriched in pathways such as \"hematopoietic cell lineage\" and \"arachidonic acid metabolism\". WGCNA analysis further identified three gene modules closely related to growth and immune indicators. Among them, the MEgrey module had a strong correlation with IgG, GCG, IGF-1, etc., indicating that liver gene expression is directly involved in hormone and immune regulation.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study indicates that the combined addition of 0.6 mg of SY and 200 IU of VE in the diet has a significantly better regulatory effect on lambs than the group with the addition alone, and it is an efficient nutritional regulation protocol. Mixed addition can increase the average daily gain (ADG) and reduce the feed-to-weight ratio (F/G) by up-regulating the secretion of GH, IGF-1 and GCG. At the same time, it collaboratively enhances the body's antioxidant and immune defense capabilities, and builds a positive cycle of \"growth - antioxidant - immunity\". In terms of rumen function, it can optimize the fermentation environment and enhance the efficiency of nutrient absorption by promoting the development of rumen papillae and reducing the thickness of the muscular layer. The core mechanism lies in the synergistic remodeling of the rumen microbiota by SY and VE (enriching functional flora such as Firmicutes and Prevotella), and mediating the coordinated regulation of growth, metabolism and immunity through the \"Microorganism -VFA-Liver\" interaction network.\u003c/p\u003e"},{"header":"Abbreviations","content":" \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Taba\" border=\"1\"\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eSY\u003c/div\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003eSelenium yeast\u003c/div\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eVE\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003eVitamin E\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eADG\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003eAverage daily gain\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eADFI\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003eAverage daily feed intake\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eGH\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003egrowth hormone\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eIGF-1\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003einsulin growth factor-1\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eGCG\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003eglucagon\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eT-AOC\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003etotal antioxidant capacity of serum\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eGSH-PX\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003eglutathione peroxidase\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eSOD\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003esuperoxide dismutase\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eMDA\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003emalondialdehyde\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eCAT\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003ecatalase\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eF/G\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003eMaterial-to-weight ratio\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eNH3-N\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003eAmmonia nitrogen\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eTVFA\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003eTotal volatile fatty acid\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eVFA\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003eVolatile fatty acid\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eOTUs\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003eOperational taxonomic units\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eLEfSe\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003eLDA effect size\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003ePcoA\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003ePrincipal coordinates analysis\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eKEGG\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003eKyoto Encyclopedia of Genes and Genomes\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003cbr/\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe sincerely thank everyone and every enterprise that has provided assistance for this research.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eShangwu M: investigation, validation, formal analysis, writing\u0026ndash;review \u0026amp; editing. Yuzhu S: investigation, formal analysis, data analysis, software, writing\u0026ndash;review and editing. Xiaoyong M, and Haibo W: supervision, conceptualization, methodol ogy, writing\u0026ndash;original draft. Shengguo Z and Ting J: \u0026nbsp; supervision, conceptualiza tion, methodology, writing\u0026ndash;review and editing. All authors read and approved the final version of the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe work was supported by the Open Competition Projects to Select the Guanghe County Cattle and Sheep Industry Quality Improvement and Efficiency Enhancement Technology Demonstration Project (LXZC21520230202) and Gansu Provincial Modern Cold-Arid Characteristic Agriculture Cattle Industry Technology System (GSARS01).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the raw data were submitted to the NCBI Sequence Read Archive (SRA) database (Accession number: PRJNA-1135556).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll studies involving animal were carried out in accordance with the regulations for the Administration of Affairs Concerning Experimental Animal (Ministry of Science and Technology, China; revise in June 2004), and sample collection protocols were approved by the Livestock Care Committee of Gansu Agricultural University (Approval No. GSAU-Eth-AST- 2023-035).We confrm that the study was conducted in accordance with ARRIVE guidelines.Informed consent was obtained from all owners of the animals involved in this study.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot Applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eVera N, Guti\u0026eacute;rrez C, Williams P. 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Effects of Dietary Vitamin E on Fertility Functions in Poultry Species. Int J Mol Sci. 2015;16:9910\u0026ndash;21.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang W, Huang Y, Zhang J, Zhang Y, Guo D, Guo C, Duan J, Liu P. Immunoregulation and Antioxidant Activities of a Novel Acidic Polysaccharide from Radix Paeoniae Alba. Glycoconj J. 2020;37:361\u0026ndash;71.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang J, Wang D, Wang Z, Sun L, Hao R, Yin Z, Chen L, Zhang W, Kang S, Zhao C, Pan Q, Zhang H. Effects of Yeast β-Glucan on Fermentation Parameters, Microbial Community Structure, and Rumen Epithelial Cell Function in High-Concentrate-Induced Yak Rumen Acidosis in Vitro. Int J Biol Macromol. 2025;314:144441.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSardinha L, Rodrigo S, Limede A, Soares L, Fernando A, Toledo A, Gasparina J, Assis R, Goulart R, Ferreira E, Pires A, Polizel D. Levels of Physically Effective Neutral Detergent Fiber from Forage and Narasin Inclusion: Performance, Carcass Traits, Meat Quality, Gastrointestinal Fermentation, and Rumen Histology in Feedlot Lambs. J Anim Sci. 2025;103:235.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang J, Wang L, Yang L, Zhang X, Qiao S. Different Dietary Starch Patterns in Low-Protein Diets: Effect on Nitrogen Efficiency, Nutrient Metabolism, and Intestinal Flora in Growing Pigs. J Anim Sci Biotechnol. 2022;13:78.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAlhidary IA, Shini S, Jassim RA, Abudabos AM, Gaughan JB. Effects of selenium and vitamin E on performance, physiological response, and selenium balance in heat-stressed sheep. J Anim Sci. 2015;93:576\u0026ndash;88.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKallie E, Jacob M, Ryznar R. The Interplay between Gut Microbiota, Short-Chain Fatty Acids, and Implications for Host Health and Disease. Gut Microbes. 2024;16:2393270.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGao X, Sha Y, Liu W, Pan X, Liu X, Liu Y, He J, Wang J, Liu S, Zhang Z. Sex Differences in Rumen Fermentation and Microbiota of Tibetan Goat. Microb Cell Fact. 2022;21:55.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSha Y, Han Y, Liu X, Zhang S, He J, Wang J, Liu S, Liu W, Shi B. Rumen Epithelial Development- and Metabolism-Related Genes Regulate Their Micromorphology and VFAs Mediating Plateau Adaptability at Different Ages in Tibetan Sheep. Int J Mol Sci. 2022;23:16078.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu K, Zhang Y, Yu Z, Xu Q, Zhang N, Zhang S, Hua G, Wang J. Ruminal Microbiota\u0026ndash;Host Interaction and Its Effect on Nutrient Metabolism. Anim Nutr. 2021;7:49\u0026ndash;55.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePinnell LJ, Arquimides AR, Wolfe CA, Weinroth MD, Metcalf JL, Delmore RJ, Belk KE, Morley PS, Engle TE. Bacteroidetes and Firmicutes Drive Differing Microbial Diversity and Community Composition among Micro-Environments in the Bovine Rumen. Front Veterinary Sci. 2022;9:897996.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGharechahi J, Vahidi MF, Sharifi G, Ariaeenejad S, Ding X, Han J, Salekdeh GH. Lignocellulose Degradation by Rumen Bacterial Communities: New Insights from Metagenome Analyses. Environ Res. 2023;229:115925.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMhuantong T, Tangphatsornruang T. Comparative Analysis of Microbial Profiles in Cow Rumen Fed with Different Dietary Fiber by Tagged 16S rRNA Gene Pyrosequencing. Curr Microbiol. 2013;67:130\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHuwiler V, Stephan M, Stanga Z, M\u0026uuml;hlebach S, Trepp R, Bally L, Bano A. Selenium Supplementation in Patients with Hashimoto Thyroiditis: A Systematic Review and Meta-Analysis of Randomized Clinical Trials. Thyroid. 2024;34:295\u0026ndash;313.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSivertsen T, Vie E, Bernhoft A, Baustad B. Vitamin E and Selenium Plasma Concentrations in Weanling Pigs under Field Conditions in Norwegian Pig Herds. Acta Vet Scand. 2007;49:47\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBoronovskiy SE, Kopylova VS, Nartsissov YR. Metabolism and Receptor Mechanisms of Niacin Action. Cell Tissue Biology. 2024;18:128\u0026ndash;47.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJung Y, Kim J, Lee J, Ryu S. Bacteriophages JEP7 and PBC2, which target foodborne pathogens, elicit cytokine responses in mammalian cells. Food Sci Biotechnol. 2025;2:42\u0026ndash;3.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang Y, Lu Y, Sun J, Zhang W, Guo Z, Mei Q. Arachidonic Acid Metabolism Health Disease MedComm. 2023;4:e47.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBerardesca E, Cameli N. Vitamin E supplementation in inflammatory skin diseases. Dermatol Ther. 2021;34:e15160.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcro","sideBox":"Learn more about [BMC Microbiology](http://bmcmicrobiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/mcro","title":"BMC Microbiology","twitterHandle":"#bmcmicrobiology","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Immunity, Antioxidant, Microbiota, VFA, Transcriptome","lastPublishedDoi":"10.21203/rs.3.rs-8416285/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8416285/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eLambs in intensive farming face susceptibility to environmental and physiological stress, which can adversely affect their health and growth. Although selenium and vitamin E are recognized for their strong antioxidant and stress-alleviating properties, the combined effects of these substances in ruminants, especially within the regulatory framework of the \"rumen-liver\" axis, remain inadequately elucidated. This study sought to explore the regulatory pathways within this axis by evaluating lamb growth, immunity, antioxidative capacity, rumen function, liver transcriptome, and their interrelationships.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe average daily weight gain, growth hormone levels, and immunoglobulin concentrations in the MIX group of lambs were significantly elevated, accompanied by an enhancement in serum antioxidant capacity (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Concurrently, the rumen environment was notably improved, as evidenced by a reduction in pH and ammonia nitrogen levels, an increase in volatile fatty acid production, and a rise in the abundance of beneficial bacteria, including Firmicutes and Prevotella. Further analysis of the liver transcriptome indicated that differentially expressed genes were enriched in immune and antioxidant-related pathways, such as arachidonic acid metabolism. Correlation analysis suggested that rumen microorganisms may influence liver gene expression via volatile fatty acid metabolism.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThe combined addition of selenium yeast and Vitamin E to the diet enhances antioxidant and immune capabilities through the \"microbiotic-VFA-liver\" axis, thereby synergistically improving the growth and health of lambs.\u003c/p\u003e","manuscriptTitle":"Synergistic Regulation of Selenium Yeast and Vitamin E on the Rumen Microbiota-VFA-Liver Axis in Lambs","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-30 02:58:10","doi":"10.21203/rs.3.rs-8416285/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-01-05T05:33:04+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-04T03:41:05+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"276048893706195850833086490824160432779","date":"2025-12-31T11:49:30+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-30T03:23:56+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"142356109914626304564164256258046965390","date":"2025-12-28T10:36:52+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"157329916075287673561484598284469988184","date":"2025-12-27T13:45:24+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"312444059700697425309012192903324957577","date":"2025-12-27T00:01:37+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"32757827390563772538469713773710907692","date":"2025-12-26T17:42:41+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"249239477478012914175557292841676053263","date":"2025-12-26T11:14:08+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-12-26T07:40:42+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-26T07:37:59+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-12-26T07:28:34+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-12-25T09:33:53+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Microbiology","date":"2025-12-25T09:26:55+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcro","sideBox":"Learn more about [BMC Microbiology](http://bmcmicrobiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/mcro","title":"BMC Microbiology","twitterHandle":"#bmcmicrobiology","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"8ca644a0-3991-418b-8621-cb10081e546e","owner":[],"postedDate":"December 30th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-03-23T16:01:29+00:00","versionOfRecord":{"articleIdentity":"rs-8416285","link":"https://doi.org/10.1186/s12866-026-04785-3","journal":{"identity":"bmc-microbiology","isVorOnly":false,"title":"BMC Microbiology"},"publishedOn":"2026-03-19 15:58:17","publishedOnDateReadable":"March 19th, 2026"},"versionCreatedAt":"2025-12-30 02:58:10","video":"","vorDoi":"10.1186/s12866-026-04785-3","vorDoiUrl":"https://doi.org/10.1186/s12866-026-04785-3","workflowStages":[]},"version":"v1","identity":"rs-8416285","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8416285","identity":"rs-8416285","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}