Dietary L-leucine supplementation improves ruminal fermentation parameters and epithelium development in fattening Angus beef cattle

Dietary L-leucine supplementation improves ruminal fermentation parameters and epithelium development in fattening Angus beef cattle | 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 Dietary L-leucine supplementation improves ruminal fermentation parameters and epithelium development in fattening Angus beef cattle Jishan An, Yu Ge, Huitian He, Hao Ge, Jing Li, Zhiqing Li, Lei Liu, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5408088/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 23 Apr, 2025 Read the published version in Journal of Animal Science and Biotechnology → Version 1 posted 5 You are reading this latest preprint version Abstract Background Leucine can play a crucial role in regulating rumen fermentation, rumen bacterial composition, and nutrient degradation, however, most of these findings are derived from in vitro rumen fermentation results. In this study, the effects of L -leucine (Leu) on rumen fermentation parameters, rumen epithelium development, amino acid metabolism, rumen bacterial communities and metabolites in beef cattle were investigated. Twenty-four Angus cows of similar initial weight (575.5 ± 22.1 kg) were randomly assigned to 2 treatments with 6 replicate pens (2 cattle per pen). They were fed a basal diet or a basal diet supplemented with 6.0 g/100 kg BW per day of L -Leu for 120 days. Results (1) Leu increased the ruminal concentrations of total volatile fatty acid (VFA) ( P = 0.017), propionate ( P = 0.023), iso-valerate ( P = 0.001), branched-chain volatile fatty acid (BCVFA) ( P = 0.01) at 4 h post-feeding, and tended to increase acetate ( P = 0.083) and decrease the ammonia-N (NH 3 -N) concentration ( P = 0.055), but it did not affect ruminal pH ( P > 0.1). Leu also increased microbial crude protein (MCP) ( P = 0.026) at 4 h post-feeding, but decreased MCP at 8 h post-feeding ( P < 0.05). (2) Supplementation with L -Leu increased the ruminal concentrations of phenylalanine ( P = 0.011), lysine ( P = 0.034), and tyrosine ( P = 0.033), and decreased the cystine concentrations ( P = 0.010). (3) Leu increased the thickness of stratum spinosum and basal ( P < 0.05), while decreased the thickness of stratum granulosum. (4) Leu up-regulated the relative mRNA expression of genes involved in tight junction proteins ( P < 0.05) and VFA absorption and metabolism ( P < 0.01) in the rumen epithelium, and this upregulation was positively correlated with ruminal isovalerate and BCVFA concentrations ( P 0.05), but differential bacterial biomarkers (LEfSe, LDA>2) were positively or negatively correlated with ruminal MCP, NH 3 -N, and BCVFA concentrations ( P 1.5) were primarily enriched in the amino acid metabolism pathway ( P < 0.05). Conclusions Dietary supplementation with L -Leu improved rumen fermentation parameters and patterns, promoted epithelial development, and enhanced rumen epithelium VFA absorption and metabolism in beef cattle. Leucine Beef cattle Rumen fermentation Rumen epithelial Ruminal microbiome and metabolome Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction In ruminants, rumen fermentation is an effective way to extract dietary nutrients and is one of the key indicators of changes in rumen function. For functional amino acid nutrition strategies in ruminants, rumen-protected amino acids are applied in beef cattle production due to the rumen’s ability to degrade feed crude protein and amino acids [ 1 – 3 ]. Even so, consistent results have not been obtained, and the role of functional amino acids in rumen development is largely overlooked. Leucine (Leu), the most abundant of the branched-chain amino acids (BCAAs), has seen only limited progress in rumen function research [ 4 ]. Leu also is an essential functional amino acid for beef cattle, which are primarily derived from rumen microbial crude protein (MCP), rumen undegraded feed protein, and dietary supplements such as L -type crystalline Leu [ 5 , 6 ]. Results from in vitro rumen fermentation experiments found that the removal of all BCAAs resulted in suppressed growth of ruminal microorganisms, decreased fiber degradation, and reduced rumen MCP and volatile fatty acid (VFA) production [ 7 – 9 ], indicating a direct effect of BCAAs on rumen microorganisms and fermentation parameters. Available studies have confirmed that Leu and iso-valerate can be interconverted in the rumen, with iso-valerate serving as an essential growth factor for numerous rumen fiber-degrading microorganisms, which can alter rumen fermentation patterns, improve VFA concentrations, and enhance MCP synthesis [ 7 , 9 – 11 ]. Generally, high levels of VFA in rumen fluid can affect rumen microecology and rumen function, while a healthy rumen epithelium can dynamically balance VFA absorption and metabolism [ 12 ]. However, limited research has been conducted examining the impact of BCAAs and branched-chain volatile fatty acid (BCVFAs) on rumen epithelial cells, it is hypothesized that BCAAs and their metabolites (VFA) may promote the morphology and development of these cells [ 13 , 14 ], further in vivo animal experiments are necessary to validate this hypothesis. Additionally, increased MCP synthesis in rumen fluid and moved into the lower digestive tract with digesta, and this may be an effective way to improve leucine bioavailability for ruminants [ 2 , 15 ], but it remains to be verified in beef cattle. To the best of our knowledge, research on Leu has primarily focused on dairy ruminants using post-ruminal and jugular infusion methods, with more attention given to intestinal digestive enzymes and their effects on dairy products [ 4 ]. Even fewer studies have reported on the effects of Leu rumen fermentation function in beef cattle. Therefore, based on our previous findings comparing the effects of rumen-protected and rumen-unprotected Leu on in vitro rumen fermentation [ 7 ] and Leu improve average daily gain of beef cattle [ 16 ], and the findings of others [ 9 , 17 ]. We hypothesized that L-Leu facilitates rumen fermentation and rumen epithelium development to improve the growth performance of beef cattle. The experiment aimed to study the effects of dietary supplementation with L -Leu on rumen fermentation parameters, rumen epithelium development, rumen bacterial communities and metabolites, and amino acid metabolism in beef cattle. Materials and methods Animal experimental design Twenty-four fattening black Angus cows with similar initial body weights (24 months old, 575.5 ± 22.1 kg) were randomly assigned to 2 treatment groups with 6 replicates (pens) per treatment and 2 cows per pen (21 m 2 per animal). The dietary treatment groups were fed a basal diet (CON) or a basal diet supplemented with 6.0 g/100 kg BW per day of L -Leu (Leu) based on the initial average body weight of per group. The basal total mixed ration (TMR), with a concentrate-to-forage ratio of 70:30, was formulated to meet or exceed the Nutrient Requirements of Beef Cattle (Nutrient requirements of beef cattle, 2015) (Table 1 ). The total experimental period lasted 135 days. During the 15-day adaptation period, cattle were gradually transitioned to the high-concentrate diet by replacing10% of the pre-experimental diet with the new diet each day, based on total dry matter intake. Throughout the 120-day treatment period, each group of animals received its corresponding dietary treatment. All cattle were fed fresh feed daily at 08:00 and 17:00 and had free access to fresh water. Table 1 Composition and nutrient contents of total mixed rations (DM basis). Item Content Composition of concentrate supplement, % Ingredient, % Corn 72.20 Rice straw 30.00 Rice bran 2.00 Concentrate supplement 70.00 Soybean meal 13.30 Total 100 Soybean oil 1.00 Analyzed chemical composition 1 ,% Wheat bran 6.00 Crude protein 2 12.01 Salt 1.00 Metabolism energy 3 , MJ/kg 11.57 Limestone 1.00 Calcium 0.46 Montmorillonite 0.20 Phosphorus 0.32 CaHPO 4 0.50 Neutral detergent fiber 30.07 NaHCO 3 1.00 Acid detergent fiber 14.36 Premix 4 1.80 Total digestible nutrients 3 78.90 Total 100 Leucine 0.60 Performed in duplicate on a composite derived from 4 samples (one sample collected per 30 day). 1 Chemical composition: metabolism energy and total digestible nutrients were a calculated value, while other values were measured. 2 Calculated as nitrogen × 6.25. 3 Total digestible nutrients and Metabolism energy was calculated according to the Nutrient Requirements of Beef Cattle (Nutrient requirements of beef cattle, 2015). 4 Premix contained the following ingredients per kilogram of diet: vitamin A ≥ 120 kIU; vitamin D3 ≥ 100 kIU; vitamin E ≥ 300 IU; vitamin K3 ≥ 1250 mg; Mn ≥ 1500 mg; Zn ≥ 2000 mg; Cu ≥ 1500 mg; Fe ≥ 4000 mg; I ≥ 12 mg; Co ≥ 20 mg; Se ≥ 10 mg. L -Leucine (food grade, ≥ 98% purity) was purchased from Hebei Huayang Biotechnology Co., Ltd. The supplement level of L -Leu used in this study was based on previously published literature [ 18 , 19 ] and our previous in vitro rumen study [ 7 ]. In this study, the precisely weighed L -Leu product was first mixed with a small amount of TMR and provided to each cow in a small basin for consumption, after which the remaining TMR was offered. Sample collection During the last 3 days of the experimental period, 6 cows were randomly selected from each treatment group to conduct rumen fluid sampling, and subsequent slaughter, with each cow serving as the experimental unit. TMR diets were collected at 30-day intervals during the treatment period. Rumen fluid The rumen fluid was collected from each cow using an esophageal tube at 2 h before the morning feeding, as well as 4 and 8 h post-feeding, over a period of 3 days. The first tube of rumen fluid was discarded. Next, the second tube of rumen fluid, approximately 100 mL, was taken as the sample. The pH of the rumen fluid was immediately measured using a pH meter (Seven2Go; Mettler Toledo Technology Co. Ltd., Shanghai, China). The rumen fluid sample was then filtered through four layers of cheesecloth, dispensed into eight 5 mL RNA- and DNA-enzyme-free cryopreservation tubes, and stored at -80 ℃ for rumen fermentation parameters, free amino acids, metabolomics, and metagenomics analysis [ 20 ]. Rumen epithelial tissue and intestinal contents On day 121, after 12 h fast, the cattle were slaughtered at a commercial slaughterhouse following the commercial slaughter procedures [ 21 ]. The rumen was immediately separated, and its contents were removed. Subsequently, two segments of epithelial tissue from the ventral sac of the rumen were collected after removing the muscular and serosal layers, then immediately washed in ice-cold phosphate-buffered saline solution until clear. One ruminal epithelial sample was fixed in 4% paraformaldehyde for histomorphology analysis. Another epithelial sample was cut into pieces, divided into three 5 mL RNA- and DNA- enzyme-free cryopreservation tubes, and stored in liquid nitrogen for RNA extraction and analysis [ 12 , 22 ]. Chemical analyses Chemical analyses of TMR diet The chemical composition of the TMR was analyzed according to the guidelines outlined by the Association of Official Analytical Chemists (AOAC, 2005). The dry matter (DM; 105°C), crude protein (CP; No. 988.05), and ether extract (EE; No. 922.06) in the TMR diet were analyzed. Total nitrogen was analyzed using the Dumas combustion method (D60; Hanon Technology Development Co., Ltd., Shandong, China), and CP was calculated using a 6.25 nitrogen-to-protein conversion factor. EE content was determined using a Soxhlet apparatus with petroleum ether as the extraction solvent. The neutral detergent fiber (NDF) in TMR diet was analyzed following the procedures described by [ 23 ], with the inclusion of heat-stable alpha-amylase. Acid detergent fiber (ADF) was analyzed using an Ankom A200i fiber analyzer (Ankom Technology, Macedon, NY, USA). Also, the contents of calcium (No. 977.29) and phosphorus (No. 995.11) were analyzed with the AOAC methods. Ruminal fermentation parameters analysis Ruminal volatile fatty acids (VFA) contents were analyzed using a gas chromatograph (GC-8600, Agilent Technologies Inc., USA) equipped with a DB-WAX UI column (30 m × 0.25 mm × 0.25 µm) following the procedures described by [ 24 ]. Ruminal ammonia nitrogen (NH 3 -N) concentration was analyzed using a microplate reader (Multiskan FC, Thermo Fisher Scientific, New York, USA) based on the method of [ 25 ]. The microbial crude protein (MCP) concentration in rumen fluid was analyzed using the colorimetric method of [ 26 ]. Ruminal free amnio acid The free amino acids (FAAs) in the rumen liquid were determined using an automatic amino acid analyzer (L-8900, Hitachi Technologies, Inc., Tokyo, Japan). Briefly, the rumen liquid was centrifuged at 12,000 r/min for 15 minutes at 4°C, and an aliquot of the supernatant was mixed (1:1) with a 10% trichloroacetic acid solution and vortexed for 1 minute. Afterward, the supernatant was collected after centrifugation at 12,000 r/min for 15 min at 4°C, filtered with a 0.22 µm filter membrane, and transferred into an autosampler vial [ 7 ]. Metagenomics analysis in rumen fluid samples and data analysis The quality and quantity of total genomic DNA from rumen fluid were assessed using 1% agarose gel electrophoresis. The extracted DNA samples were fragmented to an average size of approximately 350 bp using a Covaris M220 (Gene Company Limited, China) for paired-end (PE) library construction. A PE library was constructed using TruSeq DNA Sample Prep Kit according to the manufacturer’s instructions (Illumina). PE sequencing was performed using the Illumina HiSeq 4000 platform at Majorbio Bioinformatics Technology Co. Ltd. (Shanghai, China). Adapter sequences were removed from the 3′ and 5′ ends of the paired-end Illumina read using SeqPrep (version 1.1; https://github.com/jstjohn/ SeqPr ep). The quality control of each dataset was performed using Sickle (version 1.33) to trim low-quality bases (quality scores < 20), and remove short reads (< 50 bp) and “N” records. The filtered reads were de novo assembled for each sample using Megahit (v1.0.6). MetaGene was used to predict open reading frames (ORFs) from the assembled contigs with the length > 300 bp. Assembled contigs were then pooled and non-redundancies were constructed based on the identical contigs using CD-HIT with 95% identity. The information on the abundance of individual genes in different samples was counted and normalized to obtain the gene abundance table. Species composition analysis was based on reads using DIAMOND, compared to the NCBI NR database and combined with RefSeq parsing. Taxonomic profiles were conducted at the domain, phylum, genus, and species levels, with relative abundances calculated. A PCoA based on Bray-Curtis dissimilarity matrices at the species level was also performed [ 27 ]. The predicted nonredundant gene sets were compared with the functional annotation databases the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Carbohydrate-Active enZymes (CAZy), and the overall number of nonredundant genes annotated, as well as the number in each sample, was counted [ 28 ]. Rumen fluid metabolites analysis by metabolomics Metabolome analysis was conducted using ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) (Ren et al., 2022). Approximately 100 µL of the rumen fluid samples were transferred into centrifuge tubes (1.5 mL) and mixed with 300 µL of methanol and 10 µL of internal standard (2.8 mg/mL, DL-o-Chlorophenylalanine). Then, the mixture was vortexed for 30 s (Votex-5, Kylin-Bell Lab Instruments Co, LTD, Haimen, China), kept for 1 hour, and centrifuged at 13,000 × g and 4°C for 15 min. In the end, 200 µL of supernatant was transferred into a vial for liquid chromatography-mass spectrometry (LC-MS) analysis. The data were analyzed through the free online platform of majorbio cloud platform (cloud. majorbio.com). Orthogonal partial least squares discriminate analysis (OPLS-DA) with minimal supervision was conducted to reduce and classify the collected metabolomics data. The model’s validity was evaluated using model parameters R 2 X, R 2 Y, and Q 2 , which provide information on the interpretability and predictability, respectively, and help avoid the risk of over-fitting. Statistically significant differences among groups were identified with VIP value greater than 1 and P value less than 0.05. The differential metabolites were further identified and validated by KEGG. Enrichment analysis of the metabolic pathways was performed based on the differential metabolites through KEGG pathway database. RNA extraction and measured of rumen epithelial tissue The total RNA from the rumen epithelial tissue across all samples was extracted using an RNA extraction kit (AG21024, Accurate Biology, Changsha, China) according to the manufacturer’s instructions. A spectrophotometer (NanoDrop 2000; Thermo Fisher Scientific, USA) was then used to quantify the RNA concentration, and the integrity of the RNA samples was evaluated using a 1.0% agarose-formaldehyde gel electrophoresis. Afterward, the concentration of each RNA sample was adjusted to 500 ng/µL per sample based on account of optical density and then stored at − 80°C. A total of 1 µg high-quality RNA per sample was reverse-transcribed using a Prime Script RT reagent Kit with a gDNA Eraser (AG11707, Accurate Biology, Changsha, China) according to the manufacturer’s instructions. The primers for monocarboxylate transporter isoform 1 ( MCT1 ), monocarboxylate transporter isoform 4 ( MCT4 ), putative anion transporter isoform 1 ( PAT1 ), downregulated in adenoma ( DRA ), sodium/proton exchanger isoform 1, 2, and 3 ( NHE1, NHE2, NHE3 , respectively), and beta-hydroxybutyrate dehydrogenase, isoform 1 and 2 ( BDH-1 , BDH-2 ), 3-hydroxy-3-methylglutaryl-CoA lyase ( HMGCL ), 3-hydroxy-3-methylglutaryl-CoA synthase isoform 1 and 2 ( HMGCS1, HMGCS2 ), claudin 1 ( CLDN1 ), occludin ( OCLN ), and zonula occluden 1 ( ZO1 ), and glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ) were used as described in the published literature (Supplementary Table S1 ). The primer sequences and amplification size of these primers were cited from [ 29 , 30 ]. The primer sequences were identified and designed using National Center for Biotechnology Information (NCBI). All primers were synthesized by Sangon Biotech Co., Ltd (Shanghai, China). A BioRad CFX-96 real-time PCR system with fluorescence detection of SYBR green dye was used to perform quantitative real-time PCR (qRT-PCR) of the target genes and GAPDH. The amplification conditions were as follows: 95 ℃ for 30 seconds followed by 40 cycles of 5 seconds at 95 ℃ and 30 seconds at 60 ℃ (AG11701, Accurate Biology, Changsha, China). All measurements were performed in triplicate. The quantification results showed that these primers had no stray peaks or non-specific amplification. The mRNA expression level of GAPDH (a housekeeping gene) was used to normalize the relative amount of each studied mRNA, and the 2 −ΔΔCT method was used to analyze the data. Measurements of rumen epithelial papilla morphology Five samples of rumen epithelium from the ventral sac of each cow were dehydrated, paraffin-embedded, sliced, and stained with hematoxylin and eosin. The thickness of the total epithelia stratum corneum, stratum granulosum, stratum spinosum, and stratum basal was measured using Image-Pro Express 6.0 software (Media Cybernetics, Bethesda, MD) [ 31 ]. Statistical analysis Data on rumen fermentation parameters, ruminal amino acid composition, rumen papillae morphology, and the mRNA expression levels of genes in the rumen epithelium were analyzed using a linear mixed model (SPSS 19.0 software, SPSS Inc., Chicago, IL, USA). Data visualization was performed using GraphPad Prism software (version 8.0.2). In the mixed model, Leu treatment was the fixed effect, and individual cattle were the random term. In the rumen microbiome analysis, the non-parametric Mann-Whitney test was used to assess alpha-diversity and the relative abundance of the microbiota. The correlations between rumen fermentation parameters and rumen microbiota or epithelial function genes were calculated using Spearman's correlation test, implemented on Majorbio Cloud Platform ( https://cloud.majorbio.com/page/tools/ ). LEfSe analysis was performed using the online LEfSe analysis tool. In the rumen metabolite analysis, the VIP value from multivariable analysis OPLS-DA and the P value from the univariable analysis t -test were used to screen significantly differential metabolites ( R software, Version 1.6.2). Metabolic pathway and enrichment analyses were then conducted on Metabo Analyst 3.0 using the differential metabolites. Differences were considered significant when P < 0.05, and tendencies noted were 0.05 < P < 0.10. The results are presented as mean values with the standard error of the mean (SEM). Results Rumen fermentation parameters Table 2 indicates that supplementing with L -Leu increased the ruminal concentrations of total VFA ( P = 0.017), propionate ( P = 0.023), iso-valerate ( P = 0.001), BCVFA ( P = 0.01), and MCP ( P = 0.026) at 4 h post-feeding, and tended to increase the acetate concentration ( P = 0.083) and decrease the NH 3 -N concentration ( P = 0.055). At 8 h post-feeding, the concentrations of MCP were decreased ( P = 0.01), the concentrations of total VFA and individual VFAs showed no significant effect ( P > 0.1). No significant differences were found in the ruminal pH at 2 h before morning feeding and at 4 and 8 h post-feeding ( P > 0.1). Table 2 Effects of L -Leu on rumen fermentation in beef cattle ( n = 6). Item Time (h) 1 Treatment 2 SEM 3 P -value CON Leu pH 2 7.29 7.31 0.046 0.829 4 6.44 6.30 0.080 0.379 8 6.49 6.60 0.064 0.413 VFA, m M Total VFA 2 67.49 70.17 3.563 0.726 4 123.32 a 148.41 b 5.626 0.017 8 135.95 139.95 4.336 0.667 Acetate (A) 2 48.09 49.91 2.351 0.718 4 90.47 103.97 3.913 0.083 8 94.02 95.70 3.053 0.797 Propionate (P) 2 10.37 10.30 0.922 0.969 4 19.06 a 25.56 b 1.513 0.023 8 23.16 23.14 0.965 0.988 Butyrate 2 5.74 6.50 0.337 0.278 4 10.63 14.74 1.809 0.276 8 15.15 16.62 1.516 0.649 Valerate 2 0.58 0.62 0.031 0.579 4 0.78 0.88 0.514 0.380 8 1.07 1.10 0.077 0.850 Iso-valerate 2 1.35 1.55 0.135 0.477 4 1.30 a 2.20 b 0.148 0.001 8 1.41 2.04 0.200 0.127 Iso-butyrate 2 1.33 1.27 0.055 0.583 4 1.05 1.04 0.070 0.956 8 1.13 1.34 0.090 0.239 BCVFA 2 3.27 3.45 0.200 0.683 4 3.15 a 4.13 b 0.211 0.010 8 3.61 4.48 0.324 0.190 A:P 2 4.71 5.94 0.751 0.437 4 5.21 4.09 0.514 0.296 8 4.09 4.18 0.135 0.775 NH 3 -N (mg/dL) 2 31.19 30.48 1.336 0.805 4 19.31 14.64 1.246 0.055 8 13.83 14.64 0.808 0.641 MCP (mg/dL) 2 16.49 18.71 0.888 0.226 4 23.77 a 27.36 b 0.850 0.026 8 38.45 a 31.54 b 1.475 0.010 Different superscript letters within the same row indicate significant differences ( P < 0.05). VFA = volatile fatty acids; BCVFA = branched chain VFA; A:P = ratio of acetate to propionate; NH 3 -N = ammonia nitrogen; MCP = microbial crude protein. 1 2: 2h before morning feeding; 4/8: 4 /8h post-feeding. 2 CON: control group; Leu: 6.0 g /100 kg BW·d L -Leu group. 3 Standard error of the mean. Free amino acids concentrations in ruminal Table 3 indicates that supplementing with L -Leu increased the ruminal concentrations of phenylalanine ( P = 0.011), lysine ( P = 0.034), and tyrosine ( P = 0.033), and deceased the concentrations of cystine ( P = 0.010). It also tended to increase the concentrations of threonine ( P = 0.058), isoleucine ( P = 0.064), total essential amino acids ( P = 0.085), and histidine ( P = 0.066). No significant differences were found for other amino acid concentrations ( P > 0.1). Table 3 Effects of L -Leu on ruminal free amino acids concentration at 4 h post-feeding in beef cattle ( n = 6). Item (µg/mL) Treatment 1 SEM 2 P -value CON Leu Methionine 1.02 1.35 0.127 0.222 Threonine 3.40 5.09 0.462 0.058 Valine 3.18 3.53 0.232 0.495 Isoleucine 1.90 2.97 0.297 0.064 Leucine 3.45 3.22 0.468 0.831 Phenylalanine 2.42 a 3.64 b 0.279 0.011 Lysine 5.97 a 10.96 b 1.266 0.034 Total EAA 2 21.35 30.79 2.772 0.085 Asparagine 2.85 4.17 0.429 0.129 Cystine 0.92 a 0.55 b 0.084 0.010 Serine 2.11 2.61 0.197 0.234 Glutamine 15.46 20.95 2.354 0.275 Glycine 1.49 1.86 0.137 0.190 Alanine 5.11 5.82 0.400 0.414 Tyrosine 2.56 a 3.60 b 0.261 0.033 Histidine 0.05 0.17 0.032 0.066 Arginine 0.79 0.67 0.132 0.692 Total NEAA 31.36 40.43 3.790 0.261 Total BCAA 8.53 9.73 0.860 0.530 Total AA 52.71 71.22 6.479 0.167 Different superscript letters within the same row indicate significant differences ( P < 0.05). BCAA: branched chain amino acids; EAA: essential amino acids; NEAA: no essential amino acids. 1 CON: control group; Leu: 6.0 g /100 kg BW·d L -Leu group. 2 Standard error of the mean. Rumen epithelium gene expression The relative mRNA expression of genes related to VFA absorption and metabolism in the rumen epithelium and tight junction protein is shown in Fig. 1 and Table S2. Supplementing L -Leu increased the relative mRNA expression of the MCT1 ( P < 0.01), NHE2 ( P < 0.01), NHE3 ( P < 0.01), DRA ( P < 0.05), BDH2 ( P < 0.01), HMGCL ( P < 0.01), HMGCS1 ( P < 0.01), HMGCS2 ( P < 0.01), CLDN1 ( P < 0.05), OCLN ( P < 0.05), and ZO1 ( P 0.1). Morphological structure of rumen epithelium The results showed that in Table 4 and Fig. 2 , supplementing L -Leu increased the thickness of stratum spinosum and basal ( P 0.1). Table 4 Effects of L -Leu on ruminal papillae morphology structure in beef cattle ( n = 6). Item Treatment 1 SEM 2 P -value CON Leu Total epithelia, µm 309.55 353.32 13.79 0.116 Stratum corneum, µm 9.33 9.22 0.240 0.827 Stratum germinativum, µm 14.50 a 10.82 b 0.800 0.012 Stratum spinosum and basal, µm 81.46 a 108.62 b 6.479 0.028 Different superscript letters within the same row indicate significant differences ( P < 0.05). 1 CON: control group; Leu: 6.0 g /100 kg BW·d L -Leu group. 2 Standard error of the mean. Ruminal microbial community Metagenome sequencing generated 45.84 ± 0.44 million raw reads. After quality control and removal of host genes, 45.44 ± 0.44 million clean reads were retained (Table S3). The Good’s coverage of all samples was greater than 0.99, indicating that the metagenomic sequencing data were sufficient and high accuracy. No difference was observed among the alpha-diversity indices, including observed species, Shannon and Chao indexes ( P > 0.05) (Fig. S1 A, B). Furthermore, supplementing with L -Leu did not affect the community structure, as determined using principal coordinates analysis (PCoA) and non-metric multidimensional scaling (NMDS) (Fig. S1 C, D). At the phylum level, the dominant phyla were Bacteroidota and Bacillota , accounting for 47.26% and 43.9% of total reads, respectively (Fig. 3 A; Table 5 ). Based on Welch’s t-test, with P -value threshold of less than 0.05, a total of 8 differential phyla were identified (Fig. S2 A). Among these differential phyla, 5 had higher abundance in the Leu groups, while 3 was lower. At the species level, the most predominant species were Bacteroidates-bacterium (17.95%) and Prevotella_sp (13.27%) in the rumen (Fig. 3 B; Table 5 ). Based on Welch’s t-test, a total of 15 differential species were identified (Fig. S2 B). Moreover, the relative proportions of Ruminococcus and Clostridia , belonging to the genus of fiber-degrading bacteria, showed a slight increase in the Leu treatment group. Linear discriminant analysis effect size (LEfSe) was used to screen the main specific microorganisms between the two groups, and we found that the relative abundance of Mammaliicoccus_sciuri , unclassified_g_Mammaliicoccus , Aerococcus_urinaeequi , Prevotellaceae_bacterium , Staphylococcus_equorum , and Aerococcus_viridans was greater in the CON group, while Thermoguttaceae_bacterium was greater in the Leu group (Fig. 3 C, LDA > 2). Table 5 Effects of L -Leu on the relative abundances of the rumen bacterial community at phylum and species in beef cattle ( n = 6). Item Treatment 1 SEM 2 P -value CON Leu Dominant at phylum (%, top 5) Bacteroidota 52.50 49.12 1.939 0.410 Bacillota 40.05 42.56 2.124 0.580 Pseudomonadota 2.06 2.19 0.274 0.831 Spirochaetota 1.16 1.48 0.205 0.452 unclassified_d__Bacteria 1.09 1.08 0.030 0.876 Differential (%, top 3) Planctomycetota 0.05 0.09 0.009 0.031 Candidatus_Melainabacteria 0.006 0.013 0.001 0.013 Myxococcota 0.003 0.002 0.001 0.032 Dominant at species (%, top 5) Bacteroidates-bacterium 17.56 16.63 0.918 0.638 Prevotella_sp. 15.55 13.93 1.269 0.548 Paludibacteraceae_bacterium 7.09 7.72 1.288 0.820 Clostridia_bacterium 6.88 7.53 0.306 0.308 Lachnospiraceae_bacterium 6.35 6.00 0.292 0.571 Differential (%, top 3) Mammaliicoccus_sciuri 0.31 0.15 0.036 0.024 unclassified_g__Mammaliicoccus 0.20 0.10 0.024 0.026 Prevotellaceae_bacterium 0.10 0.06 0.007 0.002 Different superscript letters within the same row indicate significant differences ( P < 0.05). 1 CON: control group; Leu: 6.0 g /100 kg BW·d L -Leu group. 2 Standard error of the mean. Rumen microbiome function Through metagenomic sequencing, 71.92% of the unique genes derived from the rumen microflora were classified into KEGG pathways, and 11.96% into CAZymes. Based on the KEGG database for functional annotation of metagenomic data, Welch’s t-test ( P < 0.05) revealed significant differences in two pathways: biosynthesis of secondary metabolites and nonribosomal peptide structures between the two groups (Fig. 4 A). The function of CAZymes helps explore the contribution of microorganisms to carbohydrate metabolism. We found the highest percentages in four major classes: carbohydrate-binding modules (CMB), polysaccharide lyases (PL), glycoside hydrolases (GH), and glycosyltransferases (GTs) at level A (Fig. 4 B). At level B, based on Welch’s t-test, the following differential enzyme families were found to be higher in the Leu group ( P < 0.05): CBM66, GT2-Chitin-synth-2, PL10, PL17-2, GH13-15, and GT63. Meanwhile, GH5-45, CBM40, GH121, GH126, GT42, and GH5-43 were found to be higher in the CON group ( P < 0.05). Metabolites analysis of ruminal fluid After rigorous quality screening and identification, we obtained 58 reliable metabolites across all samples (Fig. 5 A). Combined with statistical analysis and the VIP values obtained from the OPLS-DA analysis (Fig. 5 B, C), the top 30 of these differential rumen metabolites were further classified according to their properties, which are mainly distributed among organic acids, lipids, and hormones and transmitters (Fig. S3). According to the pathway topology analysis, 5 metabolic pathways were significantly enriched by differential metabolites (Fig. 5 D, P < 0.05), including biotin metabolism, biosynthesis of cofactors, arginine biosynthesis, arginine and proline metabolism, alanine, and aspartate and glutamate metabolism. Correlations between rumen fermentation parameters, rumen bacterial biomarkers, and rumen epithelium gene expression To explore potential microbial functions, Spearman correlations were constructed between the microorganism biomarkers and rumen fermentation parameters (Fig. 6 A). The MCP content was negatively correlated with Aerococcus_urinaeequi , Aerococcus_viridans , Mammaliicoccus_sciuri , Prevotellaceae_bacterium , Staphylococcus_equorum , and unclassified_g_Mammaliicoccus ( P < 0.05). The NH 3 -N content was positively correlated with Aerococcus_urinaeequi , Aerococcus_viridans , and Prevotellaceae_bacterium ( P < 0.05), while negatively correlated with Cytophagales_bacterium and Thermoguttaceae_bacteriu m ( P < 0.05). The concentrations of isovalerate and BCVFA were negatively correlated with Aerococcus_urinaeequi , Aerococcus_viridans , Prevotellaceae_bacterium , and Staphylococcus_equorum ( P < 0.05). The Spearman correlation analysis shows that the concentrations of total VFA, propionate, and isovalerate were positively correlated with the relative expression levels of ZO1 , COLD1 , HMGCS1 , HMGCS2 , HMGCL , and BDH2 ( P < 0.01) (Fig. 6 B). Additionally, the concentrations of isovalerate and BCVFA were positively correlated with the relative expression levels of DRA , NHE3 , NHE2 , and MCT1 ( P < 0.01). A negative correlation was also found between the NH 3 -N content and the relative expression levels of HMGCS2 , HMGCL , BDH1 , NHE1 , and MCT1 ( P < 0.05). However, the MCP content was positively correlated with the relative expression levels of HMGCS2 , BDH2 , NHE1 , and MCT1 ( P < 0.05). Discussion Rumen fermentation parameters Dietary crude protein or amino acids can be extensively degraded into peptides, AA, CO 2 , and NH 3 by ruminal microorganisms. Like other essential amino acids, L -Leu is degraded during rumen fermentation [ 7 ], however, what it is degraded into and its effect on the rumen function in beef cattle is rarely reported. The results of the present experiment showed that supplementing with L -Leu increased the concentrations of total volatile fatty acids (VFA), propionate, iso-valerate, and branched-chain volatile fatty acid (BCVFA) at 4 h post-feeding but did not affect the dynamics of rumen fluid pH, similar to findings by others [ 9 , 32 , 33 ]. This suggests that supplementing with L -Leu potentially balances ruminal pH through VFA uptake and metabolism and improve rumen fermentation patterns. Available studies have confirmed that leucine and iso-valerate are directionally converted into each other in the rumen [ 5 , 34 ]. Additionally, iso-valerate promotes the growth of fiber-degrading bacteria and microbial crude protein (MCP) synthesis [ 7 , 11 ], and may potentially promote the development of rumen epithelium. The results of the present experiment also showed that supplementing with L -Leu increased the MCP concentration at 4 h post-feeding, while deceased the ruminal ammonia nitrogen (NH 3 -N), which is consistent with our previous in vitro rumen fermentation results [ 7 ], suggesting that ruminal NH 3 -N can be used for MCP synthesis. Ruminal MCP accounts for 50 to 80% of total absorbable protein, and Leu is an important component in MCP [ 35 ]. MCP is absorbed into blood and moves into the lower digestive tract with digesta, which may be an effectively pathway for increasing the free Leu level in serum. Additionally, the carbohydrate-active enzyme (CAZyme) database analysis showed that supplementing with L -Leu increased the relative abundance of CBM66, GH13_15, GT2_Chitin_synth_2, GT63, PL10, and PL17_2 in the present experiment. Available studies have confirmed that polysaccharide lyases (PL) catalyze the breaking of glycosidic bonds within the polysaccharide molecules through the β -elimination mechanism to produce oligosaccharides, and the carbohydrate-binding module (CBM) plays an important role in enhancing the catalytic activity of additional CAZymes [ 36 ], while the GH13 family, also known as the α -amylase family, is the largest family of glycoside hydrolases in CAZymes [ 37 ]. This suggests that Leu can enhance the degradation of non-structural carbohydrate materials by regulating CAZyme abundance in the rumen. This may partly explain how Leu promoted the ruminal propionate fermentation pattern. Rumen microorganism composition and metabolites The changes of rumen fermentation parameters and fermentation patterns were closely related to the structure of rumen microbes and their metabolites [ 38 ]. The results of the present experiment showed that supplementing with L -Leu did not affect the richness and diversity (Chao1 and Shannon index) of the rumen bacterial community in beef cattle. At the phylum level, the dominant ruminal bacteria were found to be Bacteroidota and Bacillota , and Leu slightly increased the relative abundance of Bacteroidota , but the effect was not significant. This suggesting that it has the potential to increase the number of bacteria associated with the degradation of non-fiber materials (crude protein). These changes are similar to the effects of other essential amino acids [ 20 , 39 ]. Additionally, the relative abundance of Planctomycetota , Candidatus_Melainabacteria , and Thermotogota at the phylum level was increased by Leu. Combined with the results of the linear discriminant analysis effect size (LEfSe), it was found that Mammaliicoccus_sciuri , Aerococcus_urinaeequi , Staphylococcus_equorum , and Aerococcus_viridans belong to the Bacillota phylum, while Thermoguttaceae_bacterium belongs to the Planctomycetota phylum. Available studies have confirmed that the Planctomycetota phylum can effectively degrade fiber feeds [ 40 , 41 ], and the Bacillota phylum can reduce the growth of pathogenic bacteria. The results of Spearman correlation analysis showed that Bacillota phylum was negatively correlated with MCP, iso-valerate, and BCVFA concentrations, whereas it was positively correlated with NH 3 -N, suggesting that Leu can improve the ruminal NH 3 -N utilization, MCP synthesis, and VFA production by altering the relative abundance of these bacteria biomarkers [ 7 , 35 ]. The results of the present experiment showed that supplementing with L -Leu upregulated and downregulated 31 and 27 differential rumen metabolites, respectively. Additionally, the top 30 of these differential metabolites were mainly distributed among organic acids, lipids, and hormones and transmitters, and the metabolic pathways were mainly enriched in amino acid metabolism. This suggests that Leu can alter ruminal metabolites through the amino acid metabolism pathway. The results of the present experiment also showed that supplementing with L -Leu increased the concentration of essential amino acids (phenylalanine, lysine, threonine, isoleucine) and nonessential amino acids (cystine, tyrosine, histidine) in the rumen. These changes were similar to the results of our previous in vitro bovine rumen fermentation study [ 7 ]. Moreover, ruminal amino acids and VFAs, as metabolites of feed degradation by rumen microorganisms, can in turn affect rumen microorganism growth [ 42 , 43 ]. Therefore, leucine treatment, rumen microorganisms, and rumen metabolites form a virtuous cycle that improves rumen fermentation. Rumen epithelial development Although Leu treatment increased the ruminal concentrations of total VFA and individual VFAs in beef cattle, it still dynamically balanced the rumen fluid pH within the normal range, which is closely related to regulating the rate of absorption and metabolism of VFAs by the rumen epithelium [ 22 , 31 ]. Considering that the concentrate-to-roughage ratio of the experimental diet was 7:3, the integrity of the rumen epithelial papillae is extremely important for the absorption of VFA. The results of the present experiment showed that supplementing with L -Leu increased the mRNA expression levels of CLDN1 , OCLN , and ZO1 , which are involved in barrier function and tight junctions. VFA absorption and metabolism are the most important physiological functions of the ruminal epithelium. The results of the present experiment also showed that supplementing with L -Leu increased the mRNA expression levels of MCT1 , NHE2 , NHE3 , DRA , BDH2 , HMGCL , HMGCS1 , and HMGCS2 . Available studies have confirmed that MCT1 and DRA can improve VFA absorption, NHE2 and NHE3 can regulate the intracellular pH of rumen papillae, and BDH2 , HMGCL , HMGCS1 , and HMGCS2 are associated with cholesterol synthesis and ketogenesis in the ruminal epithelium [ 22 , 29 , 31 ]. The ketogenesis effect and cholesterol biosynthesis are the primary pathways of VFA metabolism in rumen epithelium cells [ 44 ], providing energy for ruminants. This explains how leucine maintains the dynamic balance of rumen fluid pH even at high levels of VFA. Moreover, Spearman correlations analysis shows that the concentrations of VFAs, particularly isovalerate and BCVFA, were positively correlated with the relative expression levels of genes involved in VFA absorption and metabolism in rumen papillae. This suggests that Leu can improve barrier function and enhance VFA absorption and metabolism by improving rumen fermentation function. In rumen epithelium papillae, the stratum corneum and stratum granulosum cells have the function of absorption and transportation of nutrients, while the stratum spinosum and stratum basal cells have the function of ruminal VFA metabolism and renewal and repair papillae cell [ 31 ]. The results of the present experiment also showed that supplementing with L -Leu increased the thickness of the stratum spinosum and stratum basal, while decreased the thickness of the stratum granulosum. This suggests that ruminal VFA can be transported to the stratum spinosum more quickly, improve VFA metabolism and enhance papillae barrier ability, potentially providing more energy for beef cattle [ 29 , 44 ]. Previous study reported that dietary supplementation with branched-chain VFA (iso-butyrate, iso-valerate) can stimulate rumen development in young ruminants [ 45 , 46 ]. Therefore, we suggest that iso-valerate, as an intermediate of leucine, has a role in promoting rumen development in mature ruminants. Additionally, the thickness of stratum corneum was not significant difference in the present study, so Leu does not negative affect the absorption of nutrients. Based on the results of changes in the morphologic structure of the rumen epithelium, confirmed again that dietary supplementation with Leu can improve the rumen development of mature beef cattle. Meanwhile, this favorable result may also explain to some extent the mechanism by which Leu increased the average daily weight gain of beef cattle in our previous results [ 16 ]. Conclusion Overall, dietary supplementation with L -Leu improved the rumen fermentation parameters, promoted the propionate fermentation pattern, increased MCP synthesis, and enhanced rumen epithelial function in beef cattle. Supplementing with L -Leu enhanced the relative mRNA expression levels of genes involved in barrier function and VFA absorption and metabolism in rumen papillae by enhancing total VFA, propionate, isovalerate and BCVFA production. This study provides a novel understanding of the effect of L -Leu on the rumen function and development in fattening beef cattle and a relevant reference for the application form of rumen-unprotected Leu. Abbreviations AA amino acid ADF acid detergent fiber A:P acetate to propionate ratio BCAA branched-chain amino acid BDH-1/2 beta-hydroxybutyrate dehydrogenase, isoform 1/ 2 BW body weight BCVFA branched-chain volatile fatty acid CAZys carbohydrate-active enzymes CP crude protein Ca calcium CLDN1 claudin 1 DM dry matter DRA downregulated in adenoma EAA essential amino acid EE ether extract FAA free amino acid GAPDH glyceraldehyde-3-phosphate dehydrogenase HMGCL 3-hydroxy-3-methylglutaryl-CoA lyase HMGCS1/2 3-hydroxy-3-methylglutaryl-CoA synthase isoform 1 and 2 KEGG kyoto encyclopedia of genes and genomes LEfSe linear discriminant analysis effect size Leu leucine MCP microbial crude protein MCT1/4 monocarboxylate transporter isoform 1/4 NDF neutral detergent fiber NH 3 -N ammonia-N NHE1/2/3 sodium/proton exchanger isoform 1/2/3 OCLN occludin PAT1 putative anion transporter isoform 1 P phosphorus qRT-PCR quantitative real-time PCR TAA total amino acids TMR total mixed ration VFA volatile fatty acid ZO1 zonula occluden 1 Declarations Acknowledgments We really appreciate all the supports from the funding agencies and all the participants. Supplementary data Supplementary data to this article can be found online at Author contributions Jishan An: Methodology, Investigation, Data curation, Formal analysis, Visualization, Writing-Original. Huitian He, Jing Li, Zhiqing Li, and Hao Ge: Investigation, Data curation. Lei Liu, Zuo Wang, Yu Ge, Xinyi Lan, Anwei Cheng, and Weijun Shen: Methodology, Writing-Review & Editing, Funding acquisition. Fachun Wan: Conceptualization, Methodology, Supervision, Writing-Review & Editing, Supervision, Funding acquisition. The authors read and approved the final manuscript. Funding This work is supported by the National Key R&D Program (Grant No. 2022YFD1301101-1), National Natural Science Foundation of China (Grant No. 32172758), and Key Research and Development Program of Ningxia (Grant No. 2024BBF01008). Ethics approval and consent to participate The animal experiment protocol for the present study was approved by the Hunan Agricultural University Institutional Animal Care and Use Committee (Protocol number: 20220317). Consent for publication Not applicable. Competing interests No conflict of interest Author details 1 College of Animal Science and Technology, Hunan Agricultural University, Changsha, Hunan 410128, P. R. China. 2 College of Veterinary Medicine, Hunan Agricultural University, Changsha, Hunan 410128, P. R. China. 3 College of Food Science and Technology, Hunan Agricultural University, Changsha, Hunan 410128, P. R. China. 4 Yuelushan Laboratory, Changsha 410128, China References Yepes FL, Mann S, Overton T, Ryan C, Bristol L, Granados G, Nydam D, Wakshlag J. Effect of rumen-protected branched-chain amino acid supplementation on production-and energy-related metabolites during the first 35 days in milk in Holstein dairy cows. 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Effects of isovalerate supplements on morphology and functional gene expression of rumen mucosa in pre-and post-weaning dairy calves. Animal . 2018, 12(3):491-500. https://doi.org/10.1017/s175173111700194x. Supplementary Files 1107Supplementalfiles.docx Cite Share Download PDF Status: Published Journal Publication published 23 Apr, 2025 Read the published version in Journal of Animal Science and Biotechnology → Version 1 posted Editorial decision: Major revision 30 Dec, 2024 Reviewers agreed at journal 16 Nov, 2024 Reviewers invited by journal 14 Nov, 2024 Editor assigned by journal 08 Nov, 2024 First submitted to journal 06 Nov, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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An","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA0klEQVRIiWNgGAWjYBACNobDB4z//PjPw8/MfIA4LXyMxxIKeHuY5STb2xKI0yLHfMbgAw8bs7FBzxkDIh3GdixxgwQPG5DI+XjjDYOdnG4DIS08hw8bGFjwJG6XyN1sOYch2djsACEtEsfSDBJ4JBJ3zsjdJs3DcCBxG0Et8m/MfxxgM0jccCPnGZFaGM4YGDawJRgbnDnDRqyWYwnGjD0HQIFsbDnHgAi/yDcAo5LhxwFQVD688abCTo6gFhQgwUNk1CBrIVXHKBgFo2AUjAgAAOcGQzG1LGRsAAAAAElFTkSuQmCC","orcid":"","institution":"Hunan Agricultural University College of Animal Science and Technology","correspondingAuthor":true,"prefix":"","firstName":"Jishan","middleName":"","lastName":"An","suffix":""},{"id":377984995,"identity":"0d4316da-06d0-4b34-b72d-651d82925746","order_by":1,"name":"Yu Ge","email":"","orcid":"","institution":"Hunan Agricultural University College of Animal Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Yu","middleName":"","lastName":"Ge","suffix":""},{"id":377984996,"identity":"5edc80bf-f067-406a-9fb7-a8a443e874a1","order_by":2,"name":"Huitian He","email":"","orcid":"","institution":"Hunan Agricultural University College of Animal Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Huitian","middleName":"","lastName":"He","suffix":""},{"id":377984997,"identity":"900a91d5-d273-49c5-8005-4b39244803d2","order_by":3,"name":"Hao Ge","email":"","orcid":"","institution":"Hunan Agricultural University College of Animal Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Hao","middleName":"","lastName":"Ge","suffix":""},{"id":377984998,"identity":"d10db813-5fc4-4479-8f8e-dd510b21fcf8","order_by":4,"name":"Jing Li","email":"","orcid":"","institution":"Hunan Agricultural University College of Animal Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Jing","middleName":"","lastName":"Li","suffix":""},{"id":377984999,"identity":"c946549c-3e51-485c-b986-3b3b5ccb5fe8","order_by":5,"name":"Zhiqing Li","email":"","orcid":"","institution":"Hunan Agricultural University College of Animal Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Zhiqing","middleName":"","lastName":"Li","suffix":""},{"id":377985000,"identity":"991596a5-fee0-4f16-a7a8-bec505c9bc79","order_by":6,"name":"Lei Liu","email":"","orcid":"","institution":"Hunan Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Lei","middleName":"","lastName":"Liu","suffix":""},{"id":377985001,"identity":"aad5e31c-01d4-499f-9158-1c594368b9cf","order_by":7,"name":"Zuo Wang","email":"","orcid":"","institution":"Hunan Agricultural University College of Animal Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Zuo","middleName":"","lastName":"Wang","suffix":""},{"id":377985002,"identity":"c5d7824f-5d2c-4421-b831-7b9056d53f67","order_by":8,"name":"Xinyi Lan","email":"","orcid":"","institution":"Hunan Agricultural University College of Animal Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Xinyi","middleName":"","lastName":"Lan","suffix":""},{"id":377985003,"identity":"bfbf50e1-fe3d-4fa7-ba73-a7cab3e4d929","order_by":9,"name":"Weijun Shen","email":"","orcid":"","institution":"Hunan Agricultural University College of Animal Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Weijun","middleName":"","lastName":"Shen","suffix":""},{"id":377985004,"identity":"f70c20be-0f37-430e-aede-4c17b01f43c5","order_by":10,"name":"Anwei Cheng","email":"","orcid":"","institution":"Hunan Agricultural University College of Food Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Anwei","middleName":"","lastName":"Cheng","suffix":""},{"id":377985005,"identity":"b1b819eb-291e-4465-af76-909b2a332bda","order_by":11,"name":"Fachun Wan","email":"","orcid":"https://orcid.org/0000-0001-8051-3663","institution":"Hunan Agricultural University College of Animal Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Fachun","middleName":"","lastName":"Wan","suffix":""}],"badges":[],"createdAt":"2024-11-07 08:25:45","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5408088/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5408088/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s40104-025-01190-0","type":"published","date":"2025-04-23T15:57:43+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":71226379,"identity":"cbbab9e6-e444-4264-ab9c-e532b5b6ea6e","added_by":"auto","created_at":"2024-12-12 10:00:33","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":131151,"visible":true,"origin":"","legend":"\u003cp\u003eThe effects of dietary supplementation \u003cem\u003eL\u003c/em\u003e-leucine on messenger RNA (mRNA) expression of genes involved in rumen epithelial (\u003cem\u003en\u003c/em\u003e=6). (A) The relative expression of genes related to VFA absorption in rumen papillae. (B) The relative expression of genes related to VFA metabolism in rumen papillae. (C) The relative expression of genes related to integrity in rumen papillae. Quantitative RT-PCR results were expressed as relative mRNA expression and the data were analyzed by the 2\u003csup\u003e−ΔΔCT\u003c/sup\u003e method. Significant correlations are shown with ** (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05) and *** (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01). MCT1/4 = monocarboxylate transporter isoform 1/4, PAT1 = putative anion transporter isoform 1, DRA = downregulated in adenoma, NHE1/2/3 = sodium/proton exchanger isoform 1/2/3, BDH-1/2 = beta-hydroxybutyrate dehydrogenase-1/2, HMGCL = 3-hydroxy-3-methylglutaryl-CoA lyase, HMGCS 1/2 = 3-hydroxy-3-methylglutaryl-CoA synthase isoform 1/2. CON: control group; Leu: 6.0 g /100 kg BW·d\u003cem\u003e L\u003c/em\u003e-Leu group.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5408088/v1/679877eaa14d27420a08e573.png"},{"id":71224752,"identity":"0073b74c-4d17-4d72-b574-87f8ce8111cf","added_by":"auto","created_at":"2024-12-12 09:52:33","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":769962,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of Leu on ruminal papillae morphology in beef cattle (\u003cem\u003en\u003c/em\u003e=6). Representative rumen epithelial visual graph (A, C) and micrograph (B, D) of the beef cattle between CON) and Leu. CON: control group; Leu: 6.0 g /100 kg BW·d\u003cem\u003e L\u003c/em\u003e-Leu group. Visual images of the rumen tissues were taken with a camera. Images are obtained through a light micrograph of rumen tissue at a magnification of 10 × and 30 × objective lens.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5408088/v1/f406fdaf1a533134a4aa81df.png"},{"id":71224750,"identity":"94cf2faf-5373-4fe2-8792-696c6482c272","added_by":"auto","created_at":"2024-12-12 09:52:33","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":268697,"visible":true,"origin":"","legend":"\u003cp\u003eResults of metagenomic sequencing of the rumen bacteria in the CON and Leu (\u003cem\u003en\u003c/em\u003e=6). (A) Differences in bacterial phylum levels. (B) Differences in bacterial species levels. (C) The significantly differential microorganisms based on the linear discriminant analysis effect size (LEfSe) cladogram in metagenomic sequencing. The differences are represented by the color of the group, “CON” means basal diet group (A1-A6); “Leu” means basal diet supplementation with 6.0 g/100 kg BW·d \u003cem\u003eL\u003c/em\u003e-Leu group (B1-B6). \u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, \u003csup\u003e***\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5408088/v1/c6ff5a35ace0f886a70739fe.png"},{"id":71224748,"identity":"cc642c42-32f0-4f78-809e-ca4893029cc8","added_by":"auto","created_at":"2024-12-12 09:52:33","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":164844,"visible":true,"origin":"","legend":"\u003cp\u003eResults of metagenomic sequencing of the rumen bacteria function in the CON and Leu (\u003cem\u003en\u003c/em\u003e=6). (A) Prediction of microbial functional differences based on KEGG database. (B) Comparisons of the abundance of CAZymes genes of rumen microbiomes by the Welch’s t-test in metagenomic sequencing. The differences are represented by the color of the group, “CON” means basal diet group; “Leu” means basal diet supplementation with 6.0 g/100 kg BW·d \u003cem\u003eL\u003c/em\u003e-Leu group. \u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, \u003csup\u003e***\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-5408088/v1/4edd2988314d51284bf579af.png"},{"id":71224753,"identity":"fe5744c2-17c5-4bc6-a66b-82f4f06b76a9","added_by":"auto","created_at":"2024-12-12 09:52:33","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":205673,"visible":true,"origin":"","legend":"\u003cp\u003eResults of rumen metabolites analysis (\u003cem\u003en\u003c/em\u003e=5). (A) The ruminal differential metabolites as affected by Leu. (B) Principal component analysis (OPLS-DA) of rumen metabolites. (C) The Top 30 rumen differential metabolites by KEGG compound database (CON/Leu, variable importance in the projection [VIP] \u0026gt; 1.0, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05). (D) Metabolic pathways were analyzed based on different metabolites (Impact \u0026gt; 0.2, \u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05). The differences are represented by the color of the group, “CON” means basal diet group; “Leu” means basal diet supplementation with 6.0 g/100 kg BW·d \u003cem\u003eL\u003c/em\u003e-Leu group.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-5408088/v1/37291d938ee6129ca6312b56.png"},{"id":71224751,"identity":"f9e4dafd-5fc6-469f-ab8b-b406a915f407","added_by":"auto","created_at":"2024-12-12 09:52:33","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":232074,"visible":true,"origin":"","legend":"\u003cp\u003eThe Spearman correlations between the rumen fermentation parameters, rumen bacterial biomarkers, and rumen epithelium gene expression. (A) Spearman correlation analysis of differential species and rumen fermentation parameters. (A) Spearman correlation analysis of rumen fermentation parameters and genes involved in VFA absorption and metabolism of rumen papilla.\u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, \u003csup\u003e***\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001. VFA = volatile fatty acid, BCVFA = branched chain VFA, A:P = ratio of acetate to propionate, NH\u003csub\u003e3\u003c/sub\u003e-N = ammonia nitrogen, MCP = microbial crude protein, MCT1/4 = monocarboxylate transporter isoform 1/4, PAT1 = putative anion transporter isoform 1, DRA = downregulated in adenoma, NHE1/2/3 = sodium/proton exchanger isoform 1/2/3, BDH-1/2 = beta-hydroxybutyrate dehydrogenase-1/2, HMGCL = 3-hydroxy-3-methylglutaryl-CoA lyase, HMGCS 1/2 = 3-hydroxy-3-methylglutaryl-CoA synthase isoform 1/2, CLDN1 = claudin 1, OCLN = occludin, ZO1 = zonula occluden 1.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-5408088/v1/20789f632cdf0e2c92b4c5f3.png"},{"id":81569659,"identity":"ad269ccf-1ffe-4fd6-98a2-b394d8827042","added_by":"auto","created_at":"2025-04-28 16:09:45","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3329681,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5408088/v1/f142f6d9-821e-4809-8045-800116034fbd.pdf"},{"id":71224754,"identity":"0b99b17d-d38c-413a-9d7e-f322ed3bf687","added_by":"auto","created_at":"2024-12-12 09:52:33","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":10384531,"visible":true,"origin":"","legend":"","description":"","filename":"1107Supplementalfiles.docx","url":"https://assets-eu.researchsquare.com/files/rs-5408088/v1/eab95d611c055108c8da2f81.docx"}],"financialInterests":"","formattedTitle":"Dietary L-leucine supplementation improves ruminal fermentation parameters and epithelium development in fattening Angus beef cattle","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIn ruminants, rumen fermentation is an effective way to extract dietary nutrients and is one of the key indicators of changes in rumen function. For functional amino acid nutrition strategies in ruminants, rumen-protected amino acids are applied in beef cattle production due to the rumen\u0026rsquo;s ability to degrade feed crude protein and amino acids [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Even so, consistent results have not been obtained, and the role of functional amino acids in rumen development is largely overlooked.\u003c/p\u003e \u003cp\u003eLeucine (Leu), the most abundant of the branched-chain amino acids (BCAAs), has seen only limited progress in rumen function research [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Leu also is an essential functional amino acid for beef cattle, which are primarily derived from rumen microbial crude protein (MCP), rumen undegraded feed protein, and dietary supplements such as \u003cem\u003eL\u003c/em\u003e-type crystalline Leu [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Results from \u003cem\u003ein vitro\u003c/em\u003e rumen fermentation experiments found that the removal of all BCAAs resulted in suppressed growth of ruminal microorganisms, decreased fiber degradation, and reduced rumen MCP and volatile fatty acid (VFA) production [\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], indicating a direct effect of BCAAs on rumen microorganisms and fermentation parameters. Available studies have confirmed that Leu and iso-valerate can be interconverted in the rumen, with iso-valerate serving as an essential growth factor for numerous rumen fiber-degrading microorganisms, which can alter rumen fermentation patterns, improve VFA concentrations, and enhance MCP synthesis [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Generally, high levels of VFA in rumen fluid can affect rumen microecology and rumen function, while a healthy rumen epithelium can dynamically balance VFA absorption and metabolism [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. However, limited research has been conducted examining the impact of BCAAs and branched-chain volatile fatty acid (BCVFAs) on rumen epithelial cells, it is hypothesized that BCAAs and their metabolites (VFA) may promote the morphology and development of these cells [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], further \u003cem\u003ein vivo\u003c/em\u003e animal experiments are necessary to validate this hypothesis. Additionally, increased MCP synthesis in rumen fluid and moved into the lower digestive tract with digesta, and this may be an effective way to improve leucine bioavailability for ruminants [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], but it remains to be verified in beef cattle.\u003c/p\u003e \u003cp\u003eTo the best of our knowledge, research on Leu has primarily focused on dairy ruminants using post-ruminal and jugular infusion methods, with more attention given to intestinal digestive enzymes and their effects on dairy products [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Even fewer studies have reported on the effects of Leu rumen fermentation function in beef cattle. Therefore, based on our previous findings comparing the effects of rumen-protected and rumen-unprotected Leu on \u003cem\u003ein vitro\u003c/em\u003e rumen fermentation [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] and Leu improve average daily gain of beef cattle [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], and the findings of others [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. We hypothesized that L-Leu facilitates rumen fermentation and rumen epithelium development to improve the growth performance of beef cattle. The experiment aimed to study the effects of dietary supplementation with \u003cem\u003eL\u003c/em\u003e-Leu on rumen fermentation parameters, rumen epithelium development, rumen bacterial communities and metabolites, and amino acid metabolism in beef cattle.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimal experimental design\u003c/h2\u003e \u003cp\u003eTwenty-four fattening black \u003cem\u003eAngus\u003c/em\u003e cows with similar initial body weights (24 months old, 575.5\u0026thinsp;\u0026plusmn;\u0026thinsp;22.1 kg) were randomly assigned to 2 treatment groups with 6 replicates (pens) per treatment and 2 cows per pen (21 m\u003csup\u003e2\u003c/sup\u003e per animal). The dietary treatment groups were fed a basal diet (CON) or a basal diet supplemented with 6.0 g/100 kg BW per day of \u003cem\u003eL\u003c/em\u003e-Leu (Leu) based on the initial average body weight of per group. The basal total mixed ration (TMR), with a concentrate-to-forage ratio of 70:30, was formulated to meet or exceed the Nutrient Requirements of Beef Cattle (Nutrient requirements of beef cattle, 2015) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The total experimental period lasted 135 days. During the 15-day adaptation period, cattle were gradually transitioned to the high-concentrate diet by replacing10% of the pre-experimental diet with the new diet each day, based on total dry matter intake. Throughout the 120-day treatment period, each group of animals received its corresponding dietary treatment. All cattle were fed fresh feed daily at 08:00 and 17:00 and had free access to fresh water.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComposition and nutrient contents of total mixed rations (DM basis).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\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=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eItem\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eContent\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c5\" namest=\"c3\"\u003e \u003cp\u003eComposition of concentrate supplement, %\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"1\" nameend=\"c6\" namest=\"c6\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eIngredient, %\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003eCorn\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e72.20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c6\" namest=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRice straw\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e30.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003eRice bran\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c6\" namest=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eConcentrate supplement\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e70.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003eSoybean meal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e13.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c6\" namest=\"c6\"\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=\"left\" colname=\"c2\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003eSoybean oil\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c6\" namest=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eAnalyzed chemical composition \u003csup\u003e1\u003c/sup\u003e,%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003eWheat bran\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c6\" namest=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCrude protein \u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e12.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003eSalt\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c6\" namest=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMetabolism energy \u003csup\u003e3\u003c/sup\u003e, MJ/kg\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e11.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003eLimestone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c6\" namest=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCalcium\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003eMontmorillonite\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c6\" namest=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePhosphorus\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCaHPO\u003csub\u003e4\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e0.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNeutral detergent fiber\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e30.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003eNaHCO\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c6\" namest=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAcid detergent fiber\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e14.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003ePremix \u003csup\u003e4\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c6\" namest=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal digestible nutrients \u003csup\u003e3\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e78.90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003eTotal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c6\" namest=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLeucine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c6\" namest=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003ePerformed in duplicate on a composite derived from 4 samples (one sample collected per 30 day).\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003e\u003csup\u003e1\u003c/sup\u003e Chemical composition: metabolism energy and total digestible nutrients were a calculated value, while other values were measured.\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003e\u003csup\u003e2\u003c/sup\u003e Calculated as nitrogen \u0026times; 6.25.\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003e\u003csup\u003e3\u003c/sup\u003e Total digestible nutrients and Metabolism energy was calculated according to the Nutrient Requirements of Beef Cattle (Nutrient requirements of beef cattle, 2015).\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003e\u003csup\u003e4\u003c/sup\u003e Premix contained the following ingredients per kilogram of diet: vitamin A\u0026thinsp;\u0026ge;\u0026thinsp;120 kIU; vitamin D3\u0026thinsp;\u0026ge;\u0026thinsp;100 kIU; vitamin E\u0026thinsp;\u0026ge;\u0026thinsp;300 IU; vitamin K3\u0026thinsp;\u0026ge;\u0026thinsp;1250 mg; Mn\u0026thinsp;\u0026ge;\u0026thinsp;1500 mg; Zn\u0026thinsp;\u0026ge;\u0026thinsp;2000 mg; Cu\u0026thinsp;\u0026ge;\u0026thinsp;1500 mg; Fe\u0026thinsp;\u0026ge;\u0026thinsp;4000 mg; I\u0026thinsp;\u0026ge;\u0026thinsp;12 mg; Co\u0026thinsp;\u0026ge;\u0026thinsp;20 mg; Se\u0026thinsp;\u0026ge;\u0026thinsp;10 mg.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eL\u003c/em\u003e-Leucine (food grade, \u0026ge; 98% purity) was purchased from Hebei Huayang Biotechnology Co., Ltd. The supplement level of \u003cem\u003eL\u003c/em\u003e-Leu used in this study was based on previously published literature [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] and our previous \u003cem\u003ein vitro\u003c/em\u003e rumen study [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. In this study, the precisely weighed \u003cem\u003eL\u003c/em\u003e-Leu product was first mixed with a small amount of TMR and provided to each cow in a small basin for consumption, after which the remaining TMR was offered.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSample collection\u003c/h3\u003e\n\u003cp\u003eDuring the last 3 days of the experimental period, 6 cows were randomly selected from each treatment group to conduct rumen fluid sampling, and subsequent slaughter, with each cow serving as the experimental unit. TMR diets were collected at 30-day intervals during the treatment period.\u003c/p\u003e\n\u003ch3\u003eRumen fluid\u003c/h3\u003e\n\u003cp\u003eThe rumen fluid was collected from each cow using an esophageal tube at 2 h before the morning feeding, as well as 4 and 8 h post-feeding, over a period of 3 days. The first tube of rumen fluid was discarded. Next, the second tube of rumen fluid, approximately 100 mL, was taken as the sample. The pH of the rumen fluid was immediately measured using a pH meter (Seven2Go; Mettler Toledo Technology Co. Ltd., Shanghai, China). The rumen fluid sample was then filtered through four layers of cheesecloth, dispensed into eight 5 mL RNA- and DNA-enzyme-free cryopreservation tubes, and stored at -80 ℃ for rumen fermentation parameters, free amino acids, metabolomics, and metagenomics analysis [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eRumen epithelial tissue and intestinal contents\u003c/h3\u003e\n\u003cp\u003eOn day 121, after 12 h fast, the cattle were slaughtered at a commercial slaughterhouse following the commercial slaughter procedures [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The rumen was immediately separated, and its contents were removed. Subsequently, two segments of epithelial tissue from the ventral sac of the rumen were collected after removing the muscular and serosal layers, then immediately washed in ice-cold phosphate-buffered saline solution until clear. One ruminal epithelial sample was fixed in 4% paraformaldehyde for histomorphology analysis. Another epithelial sample was cut into pieces, divided into three 5 mL RNA- and DNA- enzyme-free cryopreservation tubes, and stored in liquid nitrogen for RNA extraction and analysis [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eChemical analyses\u003c/h3\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eChemical analyses of TMR diet\u003c/h2\u003e \u003cp\u003eThe chemical composition of the TMR was analyzed according to the guidelines outlined by the Association of Official Analytical Chemists (AOAC, 2005). The dry matter (DM; 105\u0026deg;C), crude protein (CP; No. 988.05), and ether extract (EE; No. 922.06) in the TMR diet were analyzed. Total nitrogen was analyzed using the Dumas combustion method (D60; Hanon Technology Development Co., Ltd., Shandong, China), and CP was calculated using a 6.25 nitrogen-to-protein conversion factor. EE content was determined using a Soxhlet apparatus with petroleum ether as the extraction solvent. The neutral detergent fiber (NDF) in TMR diet was analyzed following the procedures described by [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], with the inclusion of heat-stable alpha-amylase. Acid detergent fiber (ADF) was analyzed using an Ankom A200i fiber analyzer (Ankom Technology, Macedon, NY, USA). Also, the contents of calcium (No. 977.29) and phosphorus (No. 995.11) were analyzed with the AOAC methods.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eRuminal fermentation parameters analysis\u003c/h3\u003e\n\u003cp\u003eRuminal volatile fatty acids (VFA) contents were analyzed using a gas chromatograph (GC-8600, Agilent Technologies Inc., USA) equipped with a DB-WAX UI column (30 m \u0026times; 0.25 mm \u0026times; 0.25 \u0026micro;m) following the procedures described by [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Ruminal ammonia nitrogen (NH\u003csub\u003e3\u003c/sub\u003e-N) concentration was analyzed using a microplate reader (Multiskan FC, Thermo Fisher Scientific, New York, USA) based on the method of [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. The microbial crude protein (MCP) concentration in rumen fluid was analyzed using the colorimetric method of [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eRuminal free amnio acid\u003c/h3\u003e\n\u003cp\u003eThe free amino acids (FAAs) in the rumen liquid were determined using an automatic amino acid analyzer (L-8900, Hitachi Technologies, Inc., Tokyo, Japan). Briefly, the rumen liquid was centrifuged at 12,000 r/min for 15 minutes at 4\u0026deg;C, and an aliquot of the supernatant was mixed (1:1) with a 10% trichloroacetic acid solution and vortexed for 1 minute. Afterward, the supernatant was collected after centrifugation at 12,000 r/min for 15 min at 4\u0026deg;C, filtered with a 0.22 \u0026micro;m filter membrane, and transferred into an autosampler vial [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eMetagenomics analysis in rumen fluid samples and data analysis\u003c/h2\u003e \u003cp\u003eThe quality and quantity of total genomic DNA from rumen fluid were assessed using 1% agarose gel electrophoresis. The extracted DNA samples were fragmented to an average size of approximately 350 bp using a Covaris M220 (Gene Company Limited, China) for paired-end (PE) library construction. A PE library was constructed using TruSeq DNA Sample Prep Kit according to the manufacturer\u0026rsquo;s instructions (Illumina). PE sequencing was performed using the Illumina HiSeq 4000 platform at Majorbio Bioinformatics Technology Co. Ltd. (Shanghai, China). Adapter sequences were removed from the 3\u0026prime; and 5\u0026prime; ends of the paired-end Illumina read using SeqPrep (version 1.1; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/jstjohn/\u003c/span\u003e\u003cspan address=\"https://github.com/jstjohn/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e SeqPr ep). The quality control of each dataset was performed using Sickle (version 1.33) to trim low-quality bases (quality scores\u0026thinsp;\u0026lt;\u0026thinsp;20), and remove short reads (\u0026lt;\u0026thinsp;50 bp) and \u0026ldquo;N\u0026rdquo; records. The filtered reads were de novo assembled for each sample using Megahit (v1.0.6). MetaGene was used to predict open reading frames (ORFs) from the assembled contigs with the length\u0026thinsp;\u0026gt;\u0026thinsp;300 bp. Assembled contigs were then pooled and non-redundancies were constructed based on the identical contigs using CD-HIT with 95% identity. The information on the abundance of individual genes in different samples was counted and normalized to obtain the gene abundance table. Species composition analysis was based on reads using DIAMOND, compared to the NCBI NR database and combined with RefSeq parsing. Taxonomic profiles were conducted at the domain, phylum, genus, and species levels, with relative abundances calculated. A PCoA based on Bray-Curtis dissimilarity matrices at the species level was also performed [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe predicted nonredundant gene sets were compared with the functional annotation databases the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Carbohydrate-Active enZymes (CAZy), and the overall number of nonredundant genes annotated, as well as the number in each sample, was counted [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eRumen fluid metabolites analysis by metabolomics\u003c/h2\u003e \u003cp\u003eMetabolome analysis was conducted using ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) (Ren et al., 2022). Approximately 100 \u0026micro;L of the rumen fluid samples were transferred into centrifuge tubes (1.5 mL) and mixed with 300 \u0026micro;L of methanol and 10 \u0026micro;L of internal standard (2.8 mg/mL, DL-o-Chlorophenylalanine). Then, the mixture was vortexed for 30 s (Votex-5, Kylin-Bell Lab Instruments Co, LTD, Haimen, China), kept for 1 hour, and centrifuged at 13,000 \u0026times; \u003cem\u003eg\u003c/em\u003e and 4\u0026deg;C for 15 min. In the end, 200 \u0026micro;L of supernatant was transferred into a vial for liquid chromatography-mass spectrometry (LC-MS) analysis. The data were analyzed through the free online platform of majorbio cloud platform (cloud. majorbio.com).\u003c/p\u003e \u003cp\u003eOrthogonal partial least squares discriminate analysis (OPLS-DA) with minimal supervision was conducted to reduce and classify the collected metabolomics data. The model\u0026rsquo;s validity was evaluated using model parameters R\u003csup\u003e2\u003c/sup\u003eX, R\u003csup\u003e2\u003c/sup\u003eY, and Q\u003csup\u003e2\u003c/sup\u003e, which provide information on the interpretability and predictability, respectively, and help avoid the risk of over-fitting. Statistically significant differences among groups were identified with VIP value greater than 1 and \u003cem\u003eP\u003c/em\u003e value less than 0.05. The differential metabolites were further identified and validated by KEGG. Enrichment analysis of the metabolic pathways was performed based on the differential metabolites through KEGG pathway database.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eRNA extraction and measured of rumen epithelial tissue\u003c/h2\u003e \u003cp\u003eThe total RNA from the rumen epithelial tissue across all samples was extracted using an RNA extraction kit (AG21024, Accurate Biology, Changsha, China) according to the manufacturer\u0026rsquo;s instructions. A spectrophotometer (NanoDrop 2000; Thermo Fisher Scientific, USA) was then used to quantify the RNA concentration, and the integrity of the RNA samples was evaluated using a 1.0% agarose-formaldehyde gel electrophoresis. Afterward, the concentration of each RNA sample was adjusted to 500 ng/\u0026micro;L per sample based on account of optical density and then stored at \u0026minus;\u0026thinsp;80\u0026deg;C. A total of 1 \u0026micro;g high-quality RNA per sample was reverse-transcribed using a Prime Script RT reagent Kit with a gDNA Eraser (AG11707, Accurate Biology, Changsha, China) according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e \u003cp\u003eThe primers for monocarboxylate transporter isoform 1 (\u003cem\u003eMCT1\u003c/em\u003e), monocarboxylate transporter isoform 4 (\u003cem\u003eMCT4\u003c/em\u003e), putative anion transporter isoform 1 (\u003cem\u003ePAT1\u003c/em\u003e), downregulated in adenoma (\u003cem\u003eDRA\u003c/em\u003e), sodium/proton exchanger isoform 1, 2, and 3 (\u003cem\u003eNHE1, NHE2, NHE3\u003c/em\u003e, respectively), and beta-hydroxybutyrate dehydrogenase, isoform 1 and 2 (\u003cem\u003eBDH-1\u003c/em\u003e, \u003cem\u003eBDH-2\u003c/em\u003e), 3-hydroxy-3-methylglutaryl-CoA lyase (\u003cem\u003eHMGCL\u003c/em\u003e), 3-hydroxy-3-methylglutaryl-CoA synthase isoform 1 and 2 (\u003cem\u003eHMGCS1, HMGCS2\u003c/em\u003e), claudin 1 (\u003cem\u003eCLDN1\u003c/em\u003e), occludin (\u003cem\u003eOCLN\u003c/em\u003e), and zonula occluden 1 (\u003cem\u003eZO1\u003c/em\u003e), and glyceraldehyde-3-phosphate dehydrogenase (\u003cem\u003eGAPDH\u003c/em\u003e) were used as described in the published literature (Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The primer sequences and amplification size of these primers were cited from [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. The primer sequences were identified and designed using National Center for Biotechnology Information (NCBI). All primers were synthesized by Sangon Biotech Co., Ltd (Shanghai, China). A BioRad CFX-96 real-time PCR system with fluorescence detection of SYBR green dye was used to perform quantitative real-time PCR (qRT-PCR) of the target genes and GAPDH. The amplification conditions were as follows: 95 ℃ for 30 seconds followed by 40 cycles of 5 seconds at 95 ℃ and 30 seconds at 60 ℃ (AG11701, Accurate Biology, Changsha, China). All measurements were performed in triplicate. The quantification results showed that these primers had no stray peaks or non-specific amplification. The mRNA expression level of GAPDH (a housekeeping gene) was used to normalize the relative amount of each studied mRNA, and the 2\u003csup\u003e\u0026minus;ΔΔCT\u003c/sup\u003e method was used to analyze the data.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eMeasurements of rumen epithelial papilla morphology\u003c/h2\u003e \u003cp\u003eFive samples of rumen epithelium from the ventral sac of each cow were dehydrated, paraffin-embedded, sliced, and stained with hematoxylin and eosin. The thickness of the total epithelia stratum corneum, stratum granulosum, stratum spinosum, and stratum basal was measured using Image-Pro Express 6.0 software (Media Cybernetics, Bethesda, MD) [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eData on rumen fermentation parameters, ruminal amino acid composition, rumen papillae morphology, and the mRNA expression levels of genes in the rumen epithelium were analyzed using a linear mixed model (SPSS 19.0 software, SPSS Inc., Chicago, IL, USA). Data visualization was performed using GraphPad Prism software (version 8.0.2). In the mixed model, Leu treatment was the fixed effect, and individual cattle were the random term.\u003c/p\u003e \u003cp\u003eIn the rumen microbiome analysis, the non-parametric Mann-Whitney test was used to assess alpha-diversity and the relative abundance of the microbiota. The correlations between rumen fermentation parameters and rumen microbiota or epithelial function genes were calculated using Spearman's correlation test, implemented on Majorbio Cloud Platform (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://cloud.majorbio.com/page/tools/\u003c/span\u003e\u003cspan address=\"https://cloud.majorbio.com/page/tools/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). LEfSe analysis was performed using the online LEfSe analysis tool. In the rumen metabolite analysis, the VIP value from multivariable analysis OPLS-DA and the \u003cem\u003eP\u003c/em\u003e value from the univariable analysis \u003cem\u003et\u003c/em\u003e-test were used to screen significantly differential metabolites (\u003cem\u003eR\u003c/em\u003e software, Version 1.6.2). Metabolic pathway and enrichment analyses were then conducted on Metabo Analyst 3.0 using the differential metabolites. Differences were considered significant when \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, and tendencies noted were 0.05\u0026thinsp;\u0026lt;\u0026thinsp;\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.10. The results are presented as mean values with the standard error of the mean (SEM).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eRumen fermentation parameters\u003c/h2\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e indicates that supplementing with \u003cem\u003eL\u003c/em\u003e-Leu increased the ruminal concentrations of total VFA (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.017), propionate (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.023), iso-valerate (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.001), BCVFA (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.01), and MCP (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.026) at 4 h post-feeding, and tended to increase the acetate concentration (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.083) and decrease the NH\u003csub\u003e3\u003c/sub\u003e-N concentration (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.055). At 8 h post-feeding, the concentrations of MCP were decreased (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.01), the concentrations of total VFA and individual VFAs showed no significant effect (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.1). No significant differences were found in the ruminal pH at 2 h before morning feeding and at 4 and 8 h post-feeding (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.1).\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\u003eEffects of \u003cem\u003eL\u003c/em\u003e-Leu on rumen fermentation in beef cattle (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\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=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\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\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eTime (h) \u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003eTreatment \u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eSEM \u003csup\u003e3\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e-value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCON\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLeu\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003epH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.046\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.829\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.080\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.379\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6.60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.064\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.413\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eVFA, m\u003c/b\u003e\u003cb\u003eM\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eTotal VFA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e67.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e70.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.563\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.726\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e123.32\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e148.41\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5.626\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.017\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e135.95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e139.95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.336\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.667\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eAcetate (A)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e48.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e49.91\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.351\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.718\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e90.47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e103.97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.913\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.083\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e94.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e95.70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.053\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.797\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003ePropionate (P)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10.37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.922\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.969\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e19.06\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e25.56\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.513\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.023\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e23.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e23.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.965\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.988\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eButyrate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.337\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.278\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10.63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e14.74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.809\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.276\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e15.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e16.62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.516\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.649\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eValerate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.031\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.579\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.514\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.380\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.077\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.850\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eIso-valerate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e 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colname=\"c4\"\u003e \u003cp\u003e4.13\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.211\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.010\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.324\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.190\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eA:P\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e 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\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.641\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eMCP\u003c/p\u003e \u003cp\u003e(mg/dL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e16.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e18.71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.888\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.226\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e23.77\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e27.36\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.850\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.026\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e38.45\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e31.54\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.475\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.010\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003eDifferent superscript letters within the same row indicate significant differences (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003eVFA\u0026thinsp;=\u0026thinsp;volatile fatty acids; BCVFA\u0026thinsp;=\u0026thinsp;branched chain VFA; A:P\u0026thinsp;=\u0026thinsp;ratio of acetate to propionate; NH\u003csub\u003e3\u003c/sub\u003e-N\u0026thinsp;=\u0026thinsp;ammonia nitrogen; MCP\u0026thinsp;=\u0026thinsp;microbial crude protein.\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003e\u003csup\u003e1\u003c/sup\u003e 2: 2h before morning feeding; 4/8: 4 /8h post-feeding.\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003e\u003csup\u003e2\u003c/sup\u003e CON: control group; Leu: 6.0 g /100 kg BW\u0026middot;d \u003cem\u003eL\u003c/em\u003e-Leu group.\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003e\u003csup\u003e3\u003c/sup\u003e Standard error of the mean.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eFree amino acids concentrations in ruminal\u003c/h2\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e indicates that supplementing with \u003cem\u003eL\u003c/em\u003e-Leu increased the ruminal concentrations of phenylalanine (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.011), lysine (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.034), and tyrosine (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.033), and deceased the concentrations of cystine (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.010). It also tended to increase the concentrations of threonine (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.058), isoleucine (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.064), total essential amino acids (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.085), and histidine (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.066). No significant differences were found for other amino acid concentrations (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.1).\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\u003eEffects of \u003cem\u003eL\u003c/em\u003e-Leu on ruminal free amino acids concentration at 4 h post-feeding in beef cattle (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"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 \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eItem\u003c/p\u003e \u003cp\u003e(\u0026micro;g/mL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eTreatment \u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eSEM \u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e-value\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\u003eLeu\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMethionine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.127\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.222\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eThreonine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.462\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.058\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eValine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.232\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.495\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIsoleucine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.297\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.064\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLeucine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.468\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.831\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePhenylalanine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.42\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.64\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.279\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.011\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLysine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5.97\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10.96\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.266\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.034\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal EAA \u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e21.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e30.79\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.772\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.085\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAsparagine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.85\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.429\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.129\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCystine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.92\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.55\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.084\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.010\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSerine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.197\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.234\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGlutamine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e15.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e20.95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.354\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.275\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGlycine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.137\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.190\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAlanine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.400\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.414\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTyrosine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.56\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.60\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.261\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.033\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHistidine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.032\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.066\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eArginine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.79\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.132\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.692\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal NEAA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e31.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e40.43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.790\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.261\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal BCAA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e9.73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.860\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.530\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal AA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e52.71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e71.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6.479\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.167\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003eDifferent superscript letters within the same row indicate significant differences (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003eBCAA: branched chain amino acids; EAA: essential amino acids; NEAA: no essential amino acids.\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003e\u003csup\u003e1\u003c/sup\u003e CON: control group; Leu: 6.0 g /100 kg BW\u0026middot;d \u003cem\u003eL\u003c/em\u003e-Leu group.\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003e\u003csup\u003e2\u003c/sup\u003e Standard error of the mean.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eRumen epithelium gene expression\u003c/h2\u003e \u003cp\u003eThe relative mRNA expression of genes related to VFA absorption and metabolism in the rumen epithelium and tight junction protein is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Table S2. Supplementing \u003cem\u003eL\u003c/em\u003e-Leu increased the relative mRNA expression of the \u003cem\u003eMCT1\u003c/em\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), \u003cem\u003eNHE2\u003c/em\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), \u003cem\u003eNHE3\u003c/em\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), \u003cem\u003eDRA\u003c/em\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), \u003cem\u003eBDH2\u003c/em\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), \u003cem\u003eHMGCL\u003c/em\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), \u003cem\u003eHMGCS1\u003c/em\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), \u003cem\u003eHMGCS2\u003c/em\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), \u003cem\u003eCLDN1\u003c/em\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), \u003cem\u003eOCLN\u003c/em\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and \u003cem\u003eZO1\u003c/em\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). No differences were found in the mRNA expression of \u003cem\u003eMCT4\u003c/em\u003e, \u003cem\u003ePAT1\u003c/em\u003e, \u003cem\u003eNHE1\u003c/em\u003e, and \u003cem\u003eBDH1\u003c/em\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.1).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eMorphological structure of rumen epithelium\u003c/h2\u003e \u003cp\u003eThe results showed that in Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, supplementing \u003cem\u003eL\u003c/em\u003e-Leu increased the thickness of stratum spinosum and basal (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), while decreased the thickness of stratum granulosum. No significant differences were found for the thickness of total epithelia and stratum corneum (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.1).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffects of \u003cem\u003eL\u003c/em\u003e-Leu on ruminal papillae morphology structure in beef cattle (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"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 \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=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eTreatment \u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eSEM \u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e-value\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\u003eLeu\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal epithelia, \u0026micro;m\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e309.55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e353.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e13.79\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.116\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStratum corneum, \u0026micro;m\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e9.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e9.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.240\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.827\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStratum germinativum, \u0026micro;m\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e14.50\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10.82\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.800\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.012\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStratum spinosum and basal, \u0026micro;m\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e81.46\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e108.62\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6.479\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.028\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003eDifferent superscript letters within the same row indicate significant differences (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003e\u003csup\u003e1\u003c/sup\u003e CON: control group; Leu: 6.0 g /100 kg BW\u0026middot;d \u003cem\u003eL\u003c/em\u003e-Leu group.\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003e\u003csup\u003e2\u003c/sup\u003e Standard error of the mean.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eRuminal microbial community\u003c/h2\u003e \u003cp\u003eMetagenome sequencing generated 45.84\u0026thinsp;\u0026plusmn;\u0026thinsp;0.44\u0026nbsp;million raw reads. After quality control and removal of host genes, 45.44\u0026thinsp;\u0026plusmn;\u0026thinsp;0.44\u0026nbsp;million clean reads were retained (Table S3). The Good\u0026rsquo;s coverage of all samples was greater than 0.99, indicating that the metagenomic sequencing data were sufficient and high accuracy. No difference was observed among the alpha-diversity indices, including observed species, Shannon and Chao indexes (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05) (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e A, B). Furthermore, supplementing with \u003cem\u003eL\u003c/em\u003e-Leu did not affect the community structure, as determined using principal coordinates analysis (PCoA) and non-metric multidimensional scaling (NMDS) (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e C, D). At the phylum level, the dominant phyla were \u003cem\u003eBacteroidota\u003c/em\u003e and \u003cem\u003eBacillota\u003c/em\u003e, accounting for 47.26% and 43.9% of total reads, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA; Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Based on Welch\u0026rsquo;s t-test, with \u003cem\u003eP\u003c/em\u003e-value threshold of less than 0.05, a total of 8 differential phyla were identified (Fig. S2 A). Among these differential phyla, 5 had higher abundance in the Leu groups, while 3 was lower. At the species level, the most predominant species were \u003cem\u003eBacteroidates-bacterium\u003c/em\u003e (17.95%) and \u003cem\u003ePrevotella_sp\u003c/em\u003e (13.27%) in the rumen (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB; Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Based on Welch\u0026rsquo;s t-test, a total of 15 differential species were identified (Fig. S2 B). Moreover, the relative proportions of \u003cem\u003eRuminococcus\u003c/em\u003e and \u003cem\u003eClostridia\u003c/em\u003e, belonging to the genus of fiber-degrading bacteria, showed a slight increase in the Leu treatment group. Linear discriminant analysis effect size (LEfSe) was used to screen the main specific microorganisms between the two groups, and we found that the relative abundance of \u003cem\u003eMammaliicoccus_sciuri\u003c/em\u003e, \u003cem\u003eunclassified_g_Mammaliicoccus\u003c/em\u003e, \u003cem\u003eAerococcus_urinaeequi\u003c/em\u003e, \u003cem\u003ePrevotellaceae_bacterium\u003c/em\u003e, \u003cem\u003eStaphylococcus_equorum\u003c/em\u003e, and \u003cem\u003eAerococcus_viridans\u003c/em\u003e was greater in the CON group, while \u003cem\u003eThermoguttaceae_bacterium\u003c/em\u003e was greater in the Leu group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, LDA\u0026thinsp;\u0026gt;\u0026thinsp;2).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffects of \u003cem\u003eL\u003c/em\u003e-Leu on the relative abundances of the rumen bacterial community at phylum and species in beef cattle (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"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 \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=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eTreatment \u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eSEM \u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e-value\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\u003eLeu\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e \u003cp\u003eDominant at phylum (%, top 5)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eBacteroidota\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e52.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e49.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.939\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.410\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eBacillota\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e40.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e42.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.124\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.580\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ePseudomonadota\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.274\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.831\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eSpirochaetota\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.205\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.452\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eunclassified_d__Bacteria\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.030\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.876\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e \u003cp\u003eDifferential (%, top 3)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ePlanctomycetota\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.009\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.031\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCandidatus_Melainabacteria\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.006\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.013\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.013\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eMyxococcota\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.003\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.002\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.032\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e \u003cp\u003eDominant at species (%, top 5)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eBacteroidates-bacterium\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e17.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e16.63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.918\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.638\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ePrevotella_sp.\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e15.55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e13.93\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.269\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.548\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ePaludibacteraceae_bacterium\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.288\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.820\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eClostridia_bacterium\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6.88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.306\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.308\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eLachnospiraceae_bacterium\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.292\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.571\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e \u003cp\u003eDifferential (%, top 3)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eMammaliicoccus_sciuri\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.036\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.024\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eunclassified_g__Mammaliicoccus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.024\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.026\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ePrevotellaceae_bacterium\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.007\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.002\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003eDifferent superscript letters within the same row indicate significant differences (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003e\u003csup\u003e1\u003c/sup\u003e CON: control group; Leu: 6.0 g /100 kg BW\u0026middot;d \u003cem\u003eL\u003c/em\u003e-Leu group.\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003e\u003csup\u003e2\u003c/sup\u003e Standard error of the mean.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eRumen microbiome function\u003c/h2\u003e \u003cp\u003eThrough metagenomic sequencing, 71.92% of the unique genes derived from the rumen microflora were classified into KEGG pathways, and 11.96% into CAZymes. Based on the KEGG database for functional annotation of metagenomic data, Welch\u0026rsquo;s t-test (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) revealed significant differences in two pathways: biosynthesis of secondary metabolites and nonribosomal peptide structures between the two groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). The function of CAZymes helps explore the contribution of microorganisms to carbohydrate metabolism. We found the highest percentages in four major classes: carbohydrate-binding modules (CMB), polysaccharide lyases (PL), glycoside hydrolases (GH), and glycosyltransferases (GTs) at level A (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). At level B, based on Welch\u0026rsquo;s t-test, the following differential enzyme families were found to be higher in the Leu group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05): CBM66, GT2-Chitin-synth-2, PL10, PL17-2, GH13-15, and GT63. Meanwhile, GH5-45, CBM40, GH121, GH126, GT42, and GH5-43 were found to be higher in the CON group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eMetabolites analysis of ruminal fluid\u003c/h2\u003e \u003cp\u003eAfter rigorous quality screening and identification, we obtained 58 reliable metabolites across all samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Combined with statistical analysis and the VIP values obtained from the OPLS-DA analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, C), the top 30 of these differential rumen metabolites were further classified according to their properties, which are mainly distributed among organic acids, lipids, and hormones and transmitters (Fig. S3). According to the pathway topology analysis, 5 metabolic pathways were significantly enriched by differential metabolites (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), including biotin metabolism, biosynthesis of cofactors, arginine biosynthesis, arginine and proline metabolism, alanine, and aspartate and glutamate metabolism.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eCorrelations between rumen fermentation parameters, rumen bacterial biomarkers, and rumen epithelium gene expression\u003c/h2\u003e \u003cp\u003eTo explore potential microbial functions, Spearman correlations were constructed between the microorganism biomarkers and rumen fermentation parameters (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). The MCP content was negatively correlated with \u003cem\u003eAerococcus_urinaeequi\u003c/em\u003e, \u003cem\u003eAerococcus_viridans\u003c/em\u003e, \u003cem\u003eMammaliicoccus_sciuri\u003c/em\u003e, \u003cem\u003ePrevotellaceae_bacterium\u003c/em\u003e, \u003cem\u003eStaphylococcus_equorum\u003c/em\u003e, and \u003cem\u003eunclassified_g_Mammaliicoccus\u003c/em\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The NH\u003csub\u003e3\u003c/sub\u003e-N content was positively correlated with \u003cem\u003eAerococcus_urinaeequi\u003c/em\u003e, \u003cem\u003eAerococcus_viridans\u003c/em\u003e, and \u003cem\u003ePrevotellaceae_bacterium\u003c/em\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), while negatively correlated with \u003cem\u003eCytophagales_bacterium\u003c/em\u003e and \u003cem\u003eThermoguttaceae_bacteriu\u003c/em\u003em (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The concentrations of isovalerate and BCVFA were negatively correlated with \u003cem\u003eAerococcus_urinaeequi\u003c/em\u003e, \u003cem\u003eAerococcus_viridans\u003c/em\u003e, \u003cem\u003ePrevotellaceae_bacterium\u003c/em\u003e, and \u003cem\u003eStaphylococcus_equorum\u003c/em\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe Spearman correlation analysis shows that the concentrations of total VFA, propionate, and isovalerate were positively correlated with the relative expression levels of \u003cem\u003eZO1\u003c/em\u003e, \u003cem\u003eCOLD1\u003c/em\u003e, \u003cem\u003eHMGCS1\u003c/em\u003e, \u003cem\u003eHMGCS2\u003c/em\u003e, \u003cem\u003eHMGCL\u003c/em\u003e, and \u003cem\u003eBDH2\u003c/em\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Additionally, the concentrations of isovalerate and BCVFA were positively correlated with the relative expression levels of \u003cem\u003eDRA\u003c/em\u003e, \u003cem\u003eNHE3\u003c/em\u003e, \u003cem\u003eNHE2\u003c/em\u003e, and \u003cem\u003eMCT1\u003c/em\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). A negative correlation was also found between the NH\u003csub\u003e3\u003c/sub\u003e-N content and the relative expression levels of \u003cem\u003eHMGCS2\u003c/em\u003e, \u003cem\u003eHMGCL\u003c/em\u003e, \u003cem\u003eBDH1\u003c/em\u003e, \u003cem\u003eNHE1\u003c/em\u003e, and \u003cem\u003eMCT1\u003c/em\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). However, the MCP content was positively correlated with the relative expression levels of \u003cem\u003eHMGCS2\u003c/em\u003e, \u003cem\u003eBDH2\u003c/em\u003e, \u003cem\u003eNHE1\u003c/em\u003e, and \u003cem\u003eMCT1\u003c/em\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cdiv id=\"Sec26\" class=\"Section2\"\u003e \u003ch2\u003eRumen fermentation parameters\u003c/h2\u003e \u003cp\u003eDietary crude protein or amino acids can be extensively degraded into peptides, AA, CO\u003csub\u003e2\u003c/sub\u003e, and NH\u003csub\u003e3\u003c/sub\u003e by ruminal microorganisms. Like other essential amino acids, \u003cem\u003eL\u003c/em\u003e-Leu is degraded during rumen fermentation [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], however, what it is degraded into and its effect on the rumen function in beef cattle is rarely reported. The results of the present experiment showed that supplementing with \u003cem\u003eL\u003c/em\u003e-Leu increased the concentrations of total volatile fatty acids (VFA), propionate, iso-valerate, and branched-chain volatile fatty acid (BCVFA) at 4 h post-feeding but did not affect the dynamics of rumen fluid pH, similar to findings by others [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. This suggests that supplementing with \u003cem\u003eL\u003c/em\u003e-Leu potentially balances ruminal pH through VFA uptake and metabolism and improve rumen fermentation patterns. Available studies have confirmed that leucine and iso-valerate are directionally converted into each other in the rumen [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Additionally, iso-valerate promotes the growth of fiber-degrading bacteria and microbial crude protein (MCP) synthesis [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], and may potentially promote the development of rumen epithelium. The results of the present experiment also showed that supplementing with \u003cem\u003eL\u003c/em\u003e-Leu increased the MCP concentration at 4 h post-feeding, while deceased the ruminal ammonia nitrogen (NH\u003csub\u003e3\u003c/sub\u003e-N), which is consistent with our previous \u003cem\u003ein vitro\u003c/em\u003e rumen fermentation results [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], suggesting that ruminal NH\u003csub\u003e3\u003c/sub\u003e-N can be used for MCP synthesis. Ruminal MCP accounts for 50 to 80% of total absorbable protein, and Leu is an important component in MCP [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. MCP is absorbed into blood and moves into the lower digestive tract with digesta, which may be an effectively pathway for increasing the free Leu level in serum.\u003c/p\u003e \u003cp\u003eAdditionally, the carbohydrate-active enzyme (CAZyme) database analysis showed that supplementing with \u003cem\u003eL\u003c/em\u003e-Leu increased the relative abundance of CBM66, GH13_15, GT2_Chitin_synth_2, GT63, PL10, and PL17_2 in the present experiment. Available studies have confirmed that polysaccharide lyases (PL) catalyze the breaking of glycosidic bonds within the polysaccharide molecules through the \u003cem\u003eβ\u003c/em\u003e-elimination mechanism to produce oligosaccharides, and the carbohydrate-binding module (CBM) plays an important role in enhancing the catalytic activity of additional CAZymes [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], while the GH13 family, also known as the \u003cem\u003eα\u003c/em\u003e-amylase family, is the largest family of glycoside hydrolases in CAZymes [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. This suggests that Leu can enhance the degradation of non-structural carbohydrate materials by regulating CAZyme abundance in the rumen. This may partly explain how Leu promoted the ruminal propionate fermentation pattern.\u003c/p\u003e \u003cdiv id=\"Sec27\" class=\"Section3\"\u003e \u003ch2\u003eRumen microorganism composition and metabolites\u003c/h2\u003e \u003cp\u003eThe changes of rumen fermentation parameters and fermentation patterns were closely related to the structure of rumen microbes and their metabolites [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. The results of the present experiment showed that supplementing with \u003cem\u003eL\u003c/em\u003e-Leu did not affect the richness and diversity (Chao1 and Shannon index) of the rumen bacterial community in beef cattle. At the phylum level, the dominant ruminal bacteria were found to be \u003cem\u003eBacteroidota\u003c/em\u003e and \u003cem\u003eBacillota\u003c/em\u003e, and Leu slightly increased the relative abundance of \u003cem\u003eBacteroidota\u003c/em\u003e, but the effect was not significant. This suggesting that it has the potential to increase the number of bacteria associated with the degradation of non-fiber materials (crude protein). These changes are similar to the effects of other essential amino acids [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Additionally, the relative abundance of \u003cem\u003ePlanctomycetota\u003c/em\u003e, \u003cem\u003eCandidatus_Melainabacteria\u003c/em\u003e, and \u003cem\u003eThermotogota\u003c/em\u003e at the phylum level was increased by Leu. Combined with the results of the linear discriminant analysis effect size (LEfSe), it was found that \u003cem\u003eMammaliicoccus_sciuri\u003c/em\u003e, \u003cem\u003eAerococcus_urinaeequi\u003c/em\u003e, \u003cem\u003eStaphylococcus_equorum\u003c/em\u003e, and \u003cem\u003eAerococcus_viridans\u003c/em\u003e belong to the \u003cem\u003eBacillota\u003c/em\u003e phylum, while \u003cem\u003eThermoguttaceae_bacterium\u003c/em\u003e belongs to the \u003cem\u003ePlanctomycetota\u003c/em\u003e phylum. Available studies have confirmed that the \u003cem\u003ePlanctomycetota\u003c/em\u003e phylum can effectively degrade fiber feeds [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e], and the \u003cem\u003eBacillota\u003c/em\u003e phylum can reduce the growth of pathogenic bacteria. The results of Spearman correlation analysis showed that \u003cem\u003eBacillota\u003c/em\u003e phylum was negatively correlated with MCP, iso-valerate, and BCVFA concentrations, whereas it was positively correlated with NH\u003csub\u003e3\u003c/sub\u003e-N, suggesting that Leu can improve the ruminal NH\u003csub\u003e3\u003c/sub\u003e-N utilization, MCP synthesis, and VFA production by altering the relative abundance of these bacteria biomarkers [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe results of the present experiment showed that supplementing with \u003cem\u003eL\u003c/em\u003e-Leu upregulated and downregulated 31 and 27 differential rumen metabolites, respectively. Additionally, the top 30 of these differential metabolites were mainly distributed among organic acids, lipids, and hormones and transmitters, and the metabolic pathways were mainly enriched in amino acid metabolism. This suggests that Leu can alter ruminal metabolites through the amino acid metabolism pathway. The results of the present experiment also showed that supplementing with \u003cem\u003eL\u003c/em\u003e-Leu increased the concentration of essential amino acids (phenylalanine, lysine, threonine, isoleucine) and nonessential amino acids (cystine, tyrosine, histidine) in the rumen. These changes were similar to the results of our previous \u003cem\u003ein vitro\u003c/em\u003e bovine rumen fermentation study [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Moreover, ruminal amino acids and VFAs, as metabolites of feed degradation by rumen microorganisms, can in turn affect rumen microorganism growth [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Therefore, leucine treatment, rumen microorganisms, and rumen metabolites form a virtuous cycle that improves rumen fermentation.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003eRumen epithelial development\u003c/h2\u003e \u003cp\u003eAlthough Leu treatment increased the ruminal concentrations of total VFA and individual VFAs in beef cattle, it still dynamically balanced the rumen fluid pH within the normal range, which is closely related to regulating the rate of absorption and metabolism of VFAs by the rumen epithelium [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Considering that the concentrate-to-roughage ratio of the experimental diet was 7:3, the integrity of the rumen epithelial papillae is extremely important for the absorption of VFA. The results of the present experiment showed that supplementing with \u003cem\u003eL\u003c/em\u003e-Leu increased the mRNA expression levels of \u003cem\u003eCLDN1\u003c/em\u003e, \u003cem\u003eOCLN\u003c/em\u003e, and \u003cem\u003eZO1\u003c/em\u003e, which are involved in barrier function and tight junctions. VFA absorption and metabolism are the most important physiological functions of the ruminal epithelium. The results of the present experiment also showed that supplementing with \u003cem\u003eL\u003c/em\u003e-Leu increased the mRNA expression levels of \u003cem\u003eMCT1\u003c/em\u003e, \u003cem\u003eNHE2\u003c/em\u003e, \u003cem\u003eNHE3\u003c/em\u003e, \u003cem\u003eDRA\u003c/em\u003e, \u003cem\u003eBDH2\u003c/em\u003e, \u003cem\u003eHMGCL\u003c/em\u003e, \u003cem\u003eHMGCS1\u003c/em\u003e, and \u003cem\u003eHMGCS2\u003c/em\u003e. Available studies have confirmed that \u003cem\u003eMCT1\u003c/em\u003e and \u003cem\u003eDRA\u003c/em\u003e can improve VFA absorption, \u003cem\u003eNHE2\u003c/em\u003e and \u003cem\u003eNHE3\u003c/em\u003e can regulate the intracellular pH of rumen papillae, and \u003cem\u003eBDH2\u003c/em\u003e, \u003cem\u003eHMGCL\u003c/em\u003e, \u003cem\u003eHMGCS1\u003c/em\u003e, and \u003cem\u003eHMGCS2\u003c/em\u003e are associated with cholesterol synthesis and ketogenesis in the ruminal epithelium [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. The ketogenesis effect and cholesterol biosynthesis are the primary pathways of VFA metabolism in rumen epithelium cells [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e], providing energy for ruminants. This explains how leucine maintains the dynamic balance of rumen fluid pH even at high levels of VFA. Moreover, Spearman correlations analysis shows that the concentrations of VFAs, particularly isovalerate and BCVFA, were positively correlated with the relative expression levels of genes involved in VFA absorption and metabolism in rumen papillae. This suggests that Leu can improve barrier function and enhance VFA absorption and metabolism by improving rumen fermentation function.\u003c/p\u003e \u003cp\u003eIn rumen epithelium papillae, the stratum corneum and stratum granulosum cells have the function of absorption and transportation of nutrients, while the stratum spinosum and stratum basal cells have the function of ruminal VFA metabolism and renewal and repair papillae cell [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. The results of the present experiment also showed that supplementing with \u003cem\u003eL\u003c/em\u003e-Leu increased the thickness of the stratum spinosum and stratum basal, while decreased the thickness of the stratum granulosum. This suggests that ruminal VFA can be transported to the stratum spinosum more quickly, improve VFA metabolism and enhance papillae barrier ability, potentially providing more energy for beef cattle [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Previous study reported that dietary supplementation with branched-chain VFA (iso-butyrate, iso-valerate) can stimulate rumen development in young ruminants [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Therefore, we suggest that iso-valerate, as an intermediate of leucine, has a role in promoting rumen development in mature ruminants. Additionally, the thickness of stratum corneum was not significant difference in the present study, so Leu does not negative affect the absorption of nutrients. Based on the results of changes in the morphologic structure of the rumen epithelium, confirmed again that dietary supplementation with Leu can improve the rumen development of mature beef cattle. Meanwhile, this favorable result may also explain to some extent the mechanism by which Leu increased the average daily weight gain of beef cattle in our previous results [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eOverall, dietary supplementation with \u003cem\u003eL\u003c/em\u003e-Leu improved the rumen fermentation parameters, promoted the propionate fermentation pattern, increased MCP synthesis, and enhanced rumen epithelial function in beef cattle. Supplementing with \u003cem\u003eL\u003c/em\u003e-Leu enhanced the relative mRNA expression levels of genes involved in barrier function and VFA absorption and metabolism in rumen papillae by enhancing total VFA, propionate, isovalerate and BCVFA production. This study provides a novel understanding of the effect of \u003cem\u003eL\u003c/em\u003e-Leu on the rumen function and development in fattening beef cattle and a relevant reference for the application form of rumen-unprotected Leu.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eAA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eamino acid\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eADF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eacid detergent fiber\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eA:P\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eacetate to propionate ratio\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eBCAA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ebranched-chain amino acid\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eBDH-1/2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ebeta-hydroxybutyrate dehydrogenase, isoform 1/ 2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eBW\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ebody weight\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eBCVFA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ebranched-chain volatile fatty acid\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eCAZys\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ecarbohydrate-active enzymes\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eCP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ecrude protein\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eCa\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ecalcium\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eCLDN1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eclaudin 1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eDM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003edry matter\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eDRA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003edownregulated in adenoma\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eEAA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eessential amino acid\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eEE\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eether extract\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eFAA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003efree amino acid\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eGAPDH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eglyceraldehyde-3-phosphate dehydrogenase\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eHMGCL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e3-hydroxy-3-methylglutaryl-CoA lyase\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eHMGCS1/2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e3-hydroxy-3-methylglutaryl-CoA synthase isoform 1 and 2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eKEGG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ekyoto encyclopedia of genes and genomes\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eLEfSe\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003elinear discriminant analysis effect size\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eLeu\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eleucine\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eMCP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003emicrobial crude protein\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eMCT1/4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003emonocarboxylate transporter isoform 1/4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eNDF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eneutral detergent fiber\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eNH\u003csub\u003e3\u003c/sub\u003e-N\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eammonia-N\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eNHE1/2/3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003esodium/proton exchanger isoform 1/2/3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eOCLN\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eoccludin\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ePAT1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eputative anion transporter isoform 1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ephosphorus\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eqRT-PCR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003equantitative real-time PCR\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eTAA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003etotal amino acids\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eTMR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003etotal mixed ration\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eVFA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003evolatile fatty acid\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eZO1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ezonula occluden 1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe really appreciate all the supports from the funding agencies and all the participants.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary data\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSupplementary data to this article can be found online at\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJishan An: Methodology, Investigation, Data curation, Formal analysis, Visualization, Writing-Original. Huitian He, Jing Li, Zhiqing Li, and Hao Ge: Investigation, Data curation. Lei Liu, Zuo Wang, Yu Ge, Xinyi Lan, Anwei Cheng, and Weijun Shen: Methodology, Writing-Review \u0026amp; Editing, Funding acquisition. Fachun Wan: Conceptualization, Methodology, Supervision, Writing-Review \u0026amp; Editing, Supervision, Funding acquisition. The authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work is supported by the National Key R\u0026amp;D Program (Grant No. 2022YFD1301101-1), National Natural Science Foundation of China (Grant No. 32172758), and Key Research and Development Program of Ningxia (Grant No. 2024BBF01008).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe animal experiment protocol for the present study was approved by the Hunan Agricultural University Institutional Animal Care and Use Committee (Protocol number: 20220317).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo conflict of interest\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor details\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e1\u003c/sup\u003e College of Animal Science and Technology, Hunan Agricultural University, Changsha, Hunan 410128, P. R. China. \u003csup\u003e2\u0026nbsp;\u003c/sup\u003eCollege of Veterinary Medicine, Hunan Agricultural University, Changsha, Hunan 410128, P. R. China. \u003csup\u003e3\u003c/sup\u003e College of Food Science and Technology, Hunan Agricultural University, Changsha, Hunan 410128, P. R. China.\u003csup\u003e\u0026nbsp;4\u003c/sup\u003e Yuelushan Laboratory, Changsha 410128, China\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eYepes FL, Mann S, Overton T, Ryan C, Bristol L, Granados G, Nydam D, Wakshlag J. Effect of rumen-protected branched-chain amino acid supplementation on production-and energy-related metabolites during the first 35 days in milk in Holstein dairy cows. J Dairy Sci.\u003cem\u003e\u0026nbsp;\u003c/em\u003e2019, 102(6):5657-5672. https://doi.org/10.3168/jds.2018-15508.\u003c/li\u003e\n \u003cli\u003eGilbreath KR, Nawaratna GI, Wickersham TA, Satterfield MC, Bazer FW, Wu G. 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Effects of isovalerate supplements on morphology and functional gene expression of rumen mucosa in pre-and post-weaning dairy calves. Animal\u003cem\u003e.\u0026nbsp;\u003c/em\u003e2018, 12(3):491-500. https://doi.org/10.1017/s175173111700194x.\u003cstrong\u003e\u003c/strong\u003e\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-animal-science-and-biotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jasb","sideBox":"Learn more about [Journal of Animal Science and Biotechnology](http://jasbsci.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/jasb/default.aspx","title":"Journal of Animal Science and Biotechnology","twitterHandle":"@animalplantsci","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Leucine, Beef cattle, Rumen fermentation, Rumen epithelial, Ruminal microbiome and metabolome ","lastPublishedDoi":"10.21203/rs.3.rs-5408088/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5408088/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground \u003c/strong\u003eLeucine can play a crucial role in regulating rumen fermentation, rumen bacterial composition, and nutrient degradation, however, most of these findings are derived from \u003cem\u003ein vitro\u003c/em\u003e rumen fermentation results. In this study, the effects of \u003cem\u003eL\u003c/em\u003e-leucine (Leu) on rumen fermentation parameters, rumen epithelium development, amino acid metabolism, rumen bacterial communities and metabolites in beef cattle were investigated. Twenty-four \u003cem\u003eAngus\u003c/em\u003e cows of similar initial weight (575.5 ± 22.1\u0026nbsp;kg) were randomly assigned to 2 treatments with 6 replicate pens (2 cattle per pen). They were fed a basal diet or a basal diet supplemented with 6.0 g/100 kg BW per day of \u003cem\u003eL\u003c/em\u003e-Leu for 120 days.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e (1) Leu increased the ruminal concentrations of total volatile fatty acid (VFA) (\u003cem\u003eP\u003c/em\u003e = 0.017), propionate (\u003cem\u003eP\u003c/em\u003e = 0.023), iso-valerate (\u003cem\u003eP\u003c/em\u003e = 0.001), branched-chain volatile fatty acid (BCVFA) (\u003cem\u003eP\u003c/em\u003e = 0.01) at 4 h post-feeding, and tended to increase acetate (\u003cem\u003eP\u003c/em\u003e = 0.083) and decrease the ammonia-N (NH\u003csub\u003e3\u003c/sub\u003e-N) concentration (\u003cem\u003eP\u003c/em\u003e = 0.055), but it did not affect ruminal pH (\u003cem\u003eP\u003c/em\u003e \u0026gt; 0.1). Leu also increased microbial crude protein (MCP) (\u003cem\u003eP\u003c/em\u003e = 0.026) at 4 h post-feeding, but decreased MCP at 8 h post-feeding (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05). (2) Supplementation with \u003cem\u003eL\u003c/em\u003e-Leu increased the ruminal concentrations of phenylalanine (\u003cem\u003eP\u003c/em\u003e = 0.011), lysine (\u003cem\u003eP\u003c/em\u003e = 0.034), and tyrosine (\u003cem\u003eP\u003c/em\u003e = 0.033), and decreased the cystine concentrations (\u003cem\u003eP\u003c/em\u003e = 0.010). (3) Leu increased the thickness of stratum spinosum and basal (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05), while decreased the thickness of stratum granulosum. (4) Leu up-regulated the relative mRNA expression of genes involved in tight junction proteins (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05) and VFA absorption and metabolism (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01) in the rumen epithelium, and this upregulation was positively correlated with ruminal isovalerate and BCVFA concentrations (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01). (5) \u003cem\u003eL\u003c/em\u003e-Leu did not affect the diversity and richness of ruminal microbes (\u003cem\u003eP \u003c/em\u003e\u0026gt; 0.05), but differential bacterial biomarkers (LEfSe, LDA\u0026gt;2) were positively or negatively correlated with ruminal MCP, NH\u003csub\u003e3\u003c/sub\u003e-N, and BCVFA concentrations (\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001), and differential bacterial metabolites (OPLS-DA, VIP\u0026gt;1.5) were primarily enriched in the amino acid metabolism pathway (\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e Dietary supplementation with \u003cem\u003eL\u003c/em\u003e-Leu improved rumen fermentation parameters and patterns, promoted epithelial development, and enhanced rumen epithelium VFA absorption and metabolism in beef cattle.\u003c/p\u003e","manuscriptTitle":"Dietary L-leucine supplementation improves ruminal fermentation parameters and epithelium development in fattening Angus beef cattle","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-12-12 09:52:28","doi":"10.21203/rs.3.rs-5408088/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2024-12-30T20:59:53+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2024-11-16T05:13:29+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-11-14T07:02:36+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-11-08T06:52:46+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Animal Science and Biotechnology","date":"2024-11-07T03:24:50+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-animal-science-and-biotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jasb","sideBox":"Learn more about [Journal of Animal Science and Biotechnology](http://jasbsci.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/jasb/default.aspx","title":"Journal of Animal Science and Biotechnology","twitterHandle":"@animalplantsci","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"345f33f9-62fe-40c1-85d0-77df1a40ca0b","owner":[],"postedDate":"December 12th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-04-28T16:02:16+00:00","versionOfRecord":{"articleIdentity":"rs-5408088","link":"https://doi.org/10.1186/s40104-025-01190-0","journal":{"identity":"journal-of-animal-science-and-biotechnology","isVorOnly":false,"title":"Journal of Animal Science and Biotechnology"},"publishedOn":"2025-04-23 15:57:43","publishedOnDateReadable":"April 23rd, 2025"},"versionCreatedAt":"2024-12-12 09:52:28","video":"","vorDoi":"10.1186/s40104-025-01190-0","vorDoiUrl":"https://doi.org/10.1186/s40104-025-01190-0","workflowStages":[]},"version":"v1","identity":"rs-5408088","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5408088","identity":"rs-5408088","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}