Effects of rutin on in vitro rumen microbial composition and urea utilization in sheep

Abstract

This in vitro study aimed to evaluate rutin as a potential feed additive for improving nitrogen metabolism in the rumen. Two experiments were conducted. In Experiment 1, the effects of rutin (0, 7.5, 10, 12.5, and 15% of substrate dry matter) on fermentation were assessed. The 12.5% rutin dose (RT3) yielded the most favorable outcomes: it reduced ammonia nitrogen (NH₃-N) and pH while increasing microbial crude protein (MCP) after 12 h. RT3 also increased propionate proportion and decreased the acetate-to-propionate ratio, along with the proportions of acetate, isobutyrate, butyrate, valerate, isovalerate, and hexanoate. Microbial community analysis showed a significant increase in Patescibacter, Selenomonas, and Syntrophocococcus, while Spirochaetota and Prevotella decreased.

In Experiment 2, a 3 × 4 factorial design explored the interaction between urea (2.5, 5, 7.5%) and rutin (7.5, 10, 12.5, 15%). The combination of 2.5% urea and 12.5% rutin produced the most pronounced synergistic effect: it significantly inhibited urease activity, reduced NH₃-N, and increased MCP. This combination also increased propionate while decreasing butyrate, isobutyrate, valerate, isovalerate, and hexanoate. Microbial analysis revealed enrichment of NK4A214_group and Selenomonas, with inhibition of Prevotella. The results suggest that rutin holds promise as a urease inhibitor to improve urea utilization in ruminants. Further in vivo studies are necessary to confirm these effects and to evaluate the long-term impacts of rutin on animal health and performance.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12866-026-04904-0.

Introduction

Nitrogen represents a significant cost component in ruminant feed, yet its utilization is often inefficient, with a substantial portion being excreted rather than incorporated into animal products. This inefficiency, coupled with land constraints and variable production efficiency of conventional protein sources like soybean meal, contributes to ongoing challenges in securing sustainable protein feed resources [1, 2]. To overcome this problem, solutions such as partially substituting soybean meal nitrogen with higher nitrogen forage [3, 4] and different protein meals [5] have been investigated. Furthermore, the development of additives that boost nitrogen usage efficiency is another way to reduce the dependency on soybean meal as a protein source [6].

Urea stands out as a viable non-protein nitrogen (NPN) source due to its high nitrogen content (46.7%, approximately 6.8 times that of soybean meal), low cost, and wide availability [7, 8]. Ruminants can uniquely utilize NPN, as rumen microbes convert it into microbial crude protein (MCP), which is then used by the host animal [9]; [10]. Existing studies have generally shown that urea, as a feed supplement, exerts positive effects on ruminants. The addition of urea can enhance the digestibility of nutrients, improve animal production performance and carcass yield [11]. For instance, when 10 g/kg urea (on a dry matter basis) is added to the diet of fattening lambs to replace 75% of soybean meal, it does not reduce nutrient utilization efficiency, rumen fermentation efficiency or animal production performance. Furthermore, the supplementation of urea (10–15 g/kg DM) can also increase the digestibility of dry matter, organic matter and crude protein, and elevate the concentrations of rumen microbial nitrogen, ammonia nitrogen and VFAs in sheep [12, 13].

However, a key limitation is the rapid hydrolysis of urea to ammonia in the rumen, which often outpaces microbial uptake, leading to nitrogen loss and potential toxicity [14]. Enhancing urea-N utilization efficiency is therefore crucial for improving ruminant production economics and reducing environmental nitrogen load. Therefore, strategies to slow urea hydrolysis and improve its synchrony with microbial demand are crucial for enhancing ruminant production efficiency and reducing environmental nitrogen pollution. One prominent approach is the dietary inclusion of urease inhibitors [15]. Rutin (3′,4′,5,7-tetrahydroxyflavone-3-rutinoside) is a flavonol glycoside found in numerous edible plants, recognized for its broad pharmacological properties such as anti-inflammatory, antioxidant, and hepatoprotective effects [16–18]. Notably, rutin has demonstrated a strong binding affinity and inhibitory activity against urease [19]. Despite this known interaction, its specific effects on urea metabolism and rumen fermentation in ruminants remain unexplored.

Therefore, this study was conducted to investigate the effects of rutin on rumen fermentation parameters, microbial community structure, and urease activity through in vitro fermentation, aiming to provide a theoretical foundation for the development of novel, natural urease inhibitors to improve nitrogen utilization in ruminant production.

Materials and methods

Diets and plant extracts

Shanghai Yuanye Biotechnology Co., Ltd. provides rutin with a 95% purity, and Jiangsu Kelundo Food Ingredients Co., Ltd. provides urea. Table 1 presents the ingredients and chemical composition of the basal diet. Feed samples were analyzed in duplicate according to Association of Official Analytical Chemists (AOAC) methods [20] (for dry matter (method 934.01), crude protein (method 934.01), ash (method 930.05), calcium (method 927.02), and phosphorus (method 984.27). Neutral detergent fiber and acid detergent fiber was analyzed using an ANKOM A2000i fiber analyzer (ANKOM Technology, Macedon, NY, USA) following the methodology of Van Soest et al. [21].

Table 1.

Ingredients and nutrient levels of the basal diet (air-dry basis, %)

Items	Content, %
Ingredients
Corn grain	37.30
Soybean meal	6.00
Cottonseed meal	5.00
Corn germ meal	21.00
Rice bran meal	10.50
Corn stover	14.80
Limestone	1.00
Salt	0.60
Vitamin-mineral premix1	1.80
Dicalcium phosphate	0.50
Bentonite	1.50
Total	100.00
Nutrient levels
Dry matter, %	91.80
Crude protein, %	15.74
Neutral detergent fiber, %	41.57
Acid detergent fiber, %	14.98
Ash, %	8.13
Calcium, %	1.15
Phosphorous, %	0.43
Metabolizable energy, MJ/kg2	9.42

¹ Premix provided the following per kilogram of diet: Copper: 300 mg; Iodine: 18 mg; Zinc: 2500 mg; Iron: 3100 mg; Manganese: 2300 mg; Selenium: 10 mg; Cobalt: 25 mg; Vitamin A: 320,000 IU; Vitamin D3: 64,000 IU; Vitamin E: 860 mg; Vitamin K3: 58 mg

² Metabolizable energy was a calculated value, while the others were measured values

In vitro experiments

This study was divided into a 2-step in vitro rumen fermentation assay: one batch of treated systems was used to evaluate the effects of rutin on fermentation parameters, gas production, and the microbiome (Exp. 1), and one batch of treated systems was used to assess the response effects of the combination of rutin and urea (Exp. 2). All rumen fluid required for fermentation for each trial was collected under controlled conditions in the same abattoir (e.g. rumen fluid was collected from five slaughtered kidlets at a time that were fed a total mixed diet, non-emergency slaughtered, in good health, and the rumen fluid was sampled within 20 min of slaughter) and placed in closed thermos flasks, refluxed with carbon dioxide, and maintained at a temperature of 39 ℃ Celsius within 30 min. The substrate used for all in vitro fermentations was the basal diet detailed in Table 1.

In vitro fermentation of rutin-treated (Exp. 1)

Rumen in vitro fermentation was conducted using a completely randomized design. Samples were collected at 6 h and 12 h, with four replicates per treatment group per time point. The treatment groups included: blank control group (CTL, no rutin addition) and four rutin-treated groups (designated as RT1, RT2, RT3, and RT4), with each group supplemented with rutin at one of the following doses: 7.50%, 10%, 12.50%, or 15%, respectively, based on 1 g of substrate dry matter. These doses were selected based on preliminary experiments, which identified 7.5% as the minimum effective dose and 15% as the upper limit before inhibition occurs, thereby covering the full dose-response spectrum. Following the method by Menke and Steingass (1988) [22], rumen inoculum was mixed with buffer solution at a 1:2 ratio (vol/vol) under continuous CO₂ infusion. Approximately 60 mL of the resulting artificial rumen medium was dispensed into 100 mL serum bottles containing 1 g of substrate. Bottles were sealed airtight and incubated in an oscillating water bath at 39 °C. After the cultivation is completed, the bottle is placed in an ice bath at -10 ℃ to stop fermentation, Collect pH, ammonia nitrogen (NH₃-N), microbial protein (MCP), and volatile fatty acids (VFAs) samples at 6 h and 12 h. The cumulative gas production (GP) was recorded in real-time by the automated trace gas recording system (AGRS-III, Beijing, China) [23].

Study on the combination effect of rutin and urea in rumen fermentation system (Exp. 2)

A combined in vitro fermentation experiment of rutin and urea was conducted, with fermentation terminated at 12 h. The experiment was designed as a 3 × 4 two-factor randomized design (four replicates per group) and used the same equipment and methods as Exp. 1. The two factors and their respective levels were set as follows: Factor A (urea concentration, based on substrate dry matter mass) with three levels: 2.50%, 5%, and 7.50%; Factor B (rutin concentration, based on substrate dry matter mass) with three levels: 5%, 10%, 12.50%, and 15%. These urea concentrations were specifically selected for the combination study with rutin. Preliminary experiments indicated that outside this range (below 2.5% or above 7.5%), no significant interaction between urea and rutin was observed. Therefore, the range of 2.5%–7.5% urea was chosen to ensure that any interactive effects of the urea-rutin combination on rumen fermentation could be effectively captured. All possible combinations of the two factors were included as treatment groups. From the designed urea-rutin combinations, the “5% urea + 12.5% rutin” group (based on the dry matter mass of the substrate) was selected as the key treatment group. In addition, a blank control group (CTL, no urea or rutin added) was included—these two groups were used to compare the fermentation response and elucidate the interactive effect of urea and rutin in the rumen system.

Determination of urease activity

Urease activity was measured following the method of Yu et al. (2015) with minor modifications [24]: briefly, cell-free extracts were first prepared by centrifuging the fermentation fluid at 12,000 × g for 15 min at 4 °C, then collecting the supernatant; 0.5 mL of this cell-free extract was mixed with 0.5 mL of urea solution (50 mmol/L) and incubated at 37 °C for 30 min. Subsequently, 1.5 mL of phenol-sodium nitroprusside solution and 1.5 mL of NaClO-NaOH solution were added sequentially. The mixed solution was incubated at 37 ℃ for another 30 min, and the absorbance was measured at 625 nm using a spectrophotometer. One unit of urease activity was defined as the amount of enzyme required to produce 1.0 nmol of NH₃-N per milligram of crude enzyme protein per minute.

Sample analysis

The pH was immediately measured after sample collection (PHS-3 C; Shanghai, China). Place the samples in a 10 mL centrifuge tube and centrifuge at 4500 r·min^− 1 for 15 min. The NH₃-N concentration was analyzed according to Ma et al. (2015) [25]. MCP concent was quantified using the Coomassie brilliant blue method [26]. Additionally, the VFAs were analyzed using gas chromatography (Agilent Tech nologies 7820 A, Santa Clara, CA, USA), in accordance with the procedure outlined by [27].

S rDNA amplification, sequencing, and analysis of fermentation liquid

Genomic DNA was extracted from rumen fluid microbiota using an Omega Biotek kit (Norcross, GA, USA) and visualized by 1.0% agarose gel electrophoresis. The V3-V4 regions of bacterial 16 S rRNA genes were amplified via PCR with universal primers 338 F/806R. PCR reactions contained: 15 µL Phusion High-Fidelity PCR Master Mix (New England Biolabs, Ipswich, MA, USA), 0.2 µM primers, and 10 ng DNA template. Amplification conditions comprised: initial denaturation at 98 °C (1 min); 30 cycles of denaturation at 98 °C (10 s), annealing at 50 °C (30 s), and extension at 72 °C (30 s); final extension at 72 °C (5 min). PCR products were electrophoresed on 2% agarose gels. Qualified amplicons were bead-purified, enzyme-quantified, and pooled equimolarly based on concentration. Pooled products were re-electrophoresed (2% agarose), target bands excised, and DNA recovered using a TianGen universal purification kit (Beijing, China). Purified libraries were sequenced on the Illumina MiSeq platform.The raw sequencing data were processed in QIIME2 (version 2022.2), where demultiplexed sequences were denoised, quality-filtered, and merged using the DADA2 pipeline to generate amplicon sequence variants (ASVs). Taxonomic assignment of ASVs was performed against the SILVA database (version 138) using a pre-trained naive Bayes classifier. Microbial diversity was assessed at the ASV level. Alpha diversity indices, including Chao1 richness estimator and Shannon diversity index, were calculated to evaluate within-sample diversity. Beta diversity was assessed using Principal Coordinate Analysis (PCoA) based on Bray-Curtis distances to visualize microbial community structure differences among groups.To identify taxa with significant changes in relative abundance between groups, differential abundance analysis was performed using both Linear discriminant analysis Effect Size (LEfSe) and the Wilcoxon rank-sum test.

Statistical analysis

All data were analyzed using SPSS software (version 26.0, SPSS, Inc., Chicago, IL, USA) and GraphPad Prism 10 (GraphPad software, La Jolla, CA, USA). Firstly, the Shapiro Wilk test was used to verify the normality of the distribution of all data. The data is presented as mean ± standard error of mean (SEM), with a statistical significance of P < 0.05. For Experiment 1, the data that met the normality hypothesis were analyzed using one-way analysis of variance (ANOVA), and orthogonal polynomial comparisons were applied to test the linear and quadratic effects of rutin dose. If the normality assumption is violated, non parametric Kruskal Wallis H test is used for inter group comparison. For Experiment 2, a two-way ANOVA was performed on the data to evaluate the main effects and interactions of urea and rutin. For these two experiments, Tukey’s HSD test or Dunn’s Bonferroni correction test were used for post hoc pairwise comparison after significant analysis of variance or Kruskal Wallis results. In addition, Spearman rank correlation analysis was conducted to explore the relationship between rumen fermentation parameters and relative abundance of core microbial communities. All bar charts were constructed and visualized using GraphPad Prism 10.

Results

Effects of RT on fermentation (Exp. 1)

Table 2 indicates that RT supplementation in the basal diet exerted no significant effects (ANOVA, P > 0.05) on pH, MCP, GP, and Urease following 6 h fermentation, whereas it significantly decreased NH₃-N concentration (ANOVA, P < 0.001). Moreover, the alteration in NH₃-N concentration demonstrated significant linear and quadratic responses (Linear, P < 0.001; Quadratic, P = 0.022). After 12 h of fermentation, pH values in RT2, RT3, and RT4 groups decreased linearly (ANOVA, P < 0.001; Linear, P < 0.001) compared with the control group, though all values remained within the normal physiological range (5.92–5.99); NH₃-N concentration exhibited significant linear and quadratic reductions (ANOVA, P < 0.001; Linear, P < 0.001; Quadratic, P < 0.001); whereas MCP content showed a tendency toward linear increase (ANOVA, P = 0.001; Linear, P < 0.001). Urease activity decreased linearly and quadratically in response to elevated RT concentrations (ANOVA, P = 0.009; Linear, P < 0.001; Quadratic, P < 0.001).

Table 2.

Effects of different fermentation times and RT addition on the pH Value, NH₃-N Concentration, BCP Content, Gas Production and Urease activity of fermentation liquor

Items	CTL	RT1	RT2	RT3	RT4	SEM	P-Valve
							ANOVA	Linear	Quadratic
6 h
pH	6.33	6.34	6.39	6.33	6.38	0.01	0.13	0.236	0.912
NH3-N (mg/dL)	21.58a	20.29b	20.17b	19.64b	20.22b	0.17	< 0.001	< 0.001	0.022
MCP (mg/dL)	30.19	32.38	31.51	34.48	31.28	0.52	0.082	0.147	0.266
GP (mL)	99.30	99.35	101.95	95.61	100.56	0.91	0.207	0.890	0.457
Urease (nmol/min/mg)	15.04	12.22	13.73	13.38	11.18	0.51	0.164	< 0.001	< 0.001
12 h
pH	6.03a	5.99ab	5.96b	5.92c	5.96bc	0.01	< 0.001	< 0.001	0.864
NH3-N (mg/dL)	28.54a	25.42b	25.05bc	24.72c	24.44c	0.35	< 0.001	< 0.001	< 0.001
MCP (mg/dL)	28.72c	30.61c	31.76bc	35.31a	34.00ab	0.66	0.001	< 0.001	0.503
GP (mL)	163.55	156.60	159.00	151.15	156.11	2.04	0.409	0.130	0.329
Urease (nmol/min/mg)	10.74a	5.94b	3.46b	3.22b	3.87b	0.81	0.009	< 0.001	< 0.001

NH₃-N  Ammonia nitrogen, MCP  Microbial protein, GP  Gas production. SEM Standard error of the mean. CTL, RT1, RT2, RT3 and RT4 were substrates supplemented with 0%, 7.5%, 10%, 12.5% and 15% of RT based on dry matter weight, respectively. ANOVA=contrast between CTL, RT1, RT2, RT3 and RT4 (^{a, b, c} Means with different superscripts in the same row are different (P < 0.05)). Linear = linear effect of RT addition; Quadratic = quadratic effect of RT addition

Based on the observed dose-response pattern in Table 2, the RT3 group was selected for subsequent VFAs analysis. As shown in Table 3, compared with the CON group, the RT3 group exhibited no significant differences (P > 0.05) in total VFAs and heptanoic acid concentrations. However, it significantly reduced the molar proportions of acetate, isobutyrate, butyrate, isovalerate, caproate and A: P (P < 0.05), while significantly increasing the proportion of propionate (P < 0.05).

Table 3.

Composition and production of VFAs under CON and RT3

Items	CON	RT3	SEM	P-Valve
Total VFAs (mmol/L)	9.77	10.02	0.12	0.314
Acetate (%)	54.45	51.84	0.54	0.002
Propionate (%)	31.56	36.71	0.99	<0.001
Isobutyrate (%)	0.83	0.63	0.04	<0.001
Butyrate (%)	10.02	8.34	0.32	<0.001
Valerate (%)	2.00	1.59	0.08	<0.001
Isovalerate (%)	0.89	0.71	0.04	<0.001
Caproate (%)	0.21	0.15	0.02	0.048
Heptanoic acid (%)	0.03	0.03	0.01	0.629
A: P	1.73	1.41	0.06	<0.001

VFAs  Volatile fatty acids, A/P  Acetate: propionate. SEM Standard error of the mean. CTL and RT3 were substrates supplemented with 0% and 12.5% of RT based on dry matter weight, respectively

Effect of RT3 on rumen microbiota (Exp. 1)

Changes in microbiota diversity in the rumen

Illumina sequencing generated 845,014 raw tags (mean 105,627 per sample). Post-processing through size selection, quality control, and chimera elimination retained 802,174 high-quality tags, yielding a mean of 100,272 valid sequences per sample with 98.09–98.47% data retention efficiency. Alpha diversity analysis (Fig. 1) revealed no statistically significant differences (P > 0.05) in Chao1, Shannon and Simpson indices between experimental groups. This confirms adequate sequencing depth for microbial profiling while demonstrating that dietary RT3 supplementation did not alter ruminal bacterial richness or diversity in the studied cohort.

Fig. 1.

Changes in bacterial alpha diversity. a Chao 1 index, (b) Shannon index, and (c) Simpson index. CTL = without supplementation, RT3 = supplementation with 12.5% of RT based on dry matter weight

Effect of RT3 on microbiota composition in the rumen

A Venn diagram illustrated the shared and unique ASVs between the two groups, identifying a total of 1843 ASVs (Fig. 2a). Principal co-ordinates analysis (PCoA) revealed distinct microbial community structures between the groups (Fig. 2b). Furthermore, analysis of phylum level composition identified Bacteroidetes, Firmicutes, and Proteobacteria as the predominant phyla (Fig. 2c; supplementary material S1, 2). The results indicated that Prevotellaceae, Prevotella_sp_DJF_CP65, Prevotella and Prevotella_7 in the CTL group, Rikenellaceae, Selenomonadaceae, Veillonellales_Selenomonadales, Rikenellaceae_RC9_gut_group and Selenomonas in the RT3 group possessed relatively high abundance (Fig. 2e). Moreover, we overlapped the top 10 genera with high abundance observed at the genus level and the top 20 genera identified by simper analysis using a Venn diagram, which revealed 8 genera shared among the groups (Fig. 2d, f-j). We analyzed the composition and distribution of these shared genera among the groups. Among these, the relative abundances of Prevotella and Prevotella_7 increased in the CTL group and dropped in the RT3 group. Conversely, the relative abundances of Rikenellaceae_RC9_gut_group, Selenomonas, Syntrophococcus and Succinivibrio dropped in the RT3 group, but increased in the CTL group (Fig. 2h).

Fig. 2.

RT3 altered the microbial community structure in the in vitro rumen fermentation liquor. a Venn Graph. b Principal coordinates analysis (PCoA) was performed to calculate beta diversity on a distance matrix of Bray–Curtis indices. c Changes of fermentation liquor microbial composition at the phylum level (TOP 10). d Abundance analysis of the fermentation liquor microbiota at the genus level (TOP 10). e Linear discriminant analysis effect size (LEfSe) comparison analysis between the groups. f Simper analysis (top 10 at the genus level). g Venn diagram analysis of differential genera. The differential genera screened by the top 10 genera and the importance top 10 genera from Simper analysis are presented in a Venn diagram, with the coincidence part indicating the potential biomarkers. h Relative abundance analysis of common differential genera. CTL = without supplementation, RT3 = supplementation with 12.5% of RT based on dry matter weight

At the genus level, Spearman correlation analysis was used to examine the correlation between rumen differential microorganisms and fermentation parameters. Correlation analysis demonstrated that NH₃-N, butyrate, isovalerate, isobutyrate, and valerate exhibited positive correlations with Prevotella_7 and Prevotella but negative correlations with Succinivibrio, Selenomonas, and Syntrophococcus (P < 0.05); pH showed a positive correlation with Prevotella and negative correlations with Selenomonas, Syntrophococcus, and Lachnospiraceae_NK3A20_group (P ＜ 0.05); the A:P positively correlated with Prevotella_7 but inversely correlated with Succinivibrio, Selenomonas, and Syntrophococcus (P ＜ 0.05); caproate concentration was positively associated with Prevotella and negatively associated with Syntrophococcus and Lachnospiraceae_NK3A20_group (P ＜ 0.05); while both MCP and propionate displayed positive correlations with Prevotella_7/Prevotella and negative correlations with Selenomonas, Syntrophococcus, and Lachnospiraceae_NK3A20_group (P ＜ 0.05) (Fig. 3).

Fig. 3.

Correlation of the rumen fermentation parameters with the microbial community. Positive and negative correlations are shown in red and blue, respectively. * P < 0.05, ** P < 0.01, *** P < 0.001

Effects of the combined use of rutin and urea on fermentation (Exp. 2)

The results of the two-way ANOVA for the 3 × 4 factorial design are presented in Table 4. Urea supplementation exhibited significant main effects on all measured fermentation parameters and urease activity (P < 0.001). Similarly, rutin supplementation showed significant main effects on NH₃-N concentration and MCP content (P < 0.001). Most importantly, a significant urea × rutin interaction was observed for MCP content and urease activity (P = 0.028 and P < 0.001, respectively). To elucidate the nature of these significant interactions, the effects of rutin at each urea level were further examined. For MCP, the positive effect of rutin was most pronounced at the low urea level (2.5%). Specifically, the combination of 2.5% urea + 12.5% rutin yielded the highest MCP content, which was significantly greater than most other combinations (P < 0.05). In contrast, at higher urea levels (5% and 7.5%), the enhancing effect of rutin on MCP was diminished or absent. For urease activity, the inhibitory effect of rutin was most effective at the medium urea level (5%). The 5% urea + varying rutin groups collectively exhibited the lowest urease activity, which was significantly different from most groups except 2.5% urea + 12.5% rutin. Regarding the main effects and parameters without significant interaction, the results demonstrated clear dose-dependent patterns. Increasing urea levels linearly elevated pH and NH₃-N concentration across all rutin doses (P < 0.05). Conversely, increasing rutin levels generally reduced NH₃-N concentration across all urea doses.

Table 4.

The effect of different levels of rutin and urea combination on rumen fermentation parameters and urease activity

Treatment		pH	NH3-N (mg/dL)	MCP (mg/dL)	Urease (nmol/min/mg)
Urea(%)	Rutin(%)
2.50	7.50	6.34f	32.39d	33.44bc	9.34bc
	10	6.36f	31.60de	33.74abc	8.80de
	12.50	6.37ef	31.84de	35.45a	8.45ef
	15	6.39def	31.51e	32.82bc	8.78de
5	7.50	6.46cde	41.71c	29.25e	7.46g
	10	6.47cd	41.31c	29.92e	8.26f
	12.50	6.51bc	41.45c	29.66e	8.22f
	15	6.50bc	40.90c	30.77de	8.21f
7.50	7.50	6.58ab	50.53a	31.92cd	9.68ab
	10	6.60ab	49.97ab	30.77de	9.87a
	12.50	6.62a	49.70b	34.25ab	9.55ab
	15	6.67a	49.25b	32.47bcd	9.01cd
	SEM	0.017	1.125	0.317	0.105
P-Value	Urea	<0.001	<0.001	<0.001	<0.001
	rutin	0.140	0.001	0.008	0.061
	Interaction	0.966	0.853	0.028	<0.001

^a–f Means within a column with different superscripts differ (P < 0.05). NH₃-N  Ammonia nitrogen, MCP  microbial protein, SEM Standard error of the mean

In summary, based on the factorial analysis, the combination of 2.5% urea and 12.5% rutin was selected for further investigation because it uniquely optimized multiple key parameters: it maintained a low NH₃-N concentration, produced the highest MCP content among all interactions, and sustained low urease activity comparable to the most inhibitory combinations. This profile suggests an optimal synergy for promoting nitrogen incorporation into microbial protein while controlling ammonia release.

Consequently, this selected combination (UR + RT: 2.5% urea + 12.5% rutin) was compared against the basal control (CTL) and a urea-only control (UR: 2.5% urea) in a subsequent focused analysis (Table 5). Compared with CTL, UR significantly increased pH, NH₃-N, and MCP while decreasing urease activity (P < 0.05). More importantly, compared with the UR group, the UR + RT combination significantly increased the propionate proportion (P = 0.001) and significantly decreased NH₃-N concentration, urease activity, and the proportions of several branched-chain and long-chain fatty acids (P < 0.05), demonstrating the added benefit of rutin in modulating fermentation patterns.

Table 5.

The effect of rutin and urea combination on rumen fermentation parameters and urease activity

Items	Groups			SEM	P-value
	CTL	UR	UR + RT		CTL vs. UR	UR vs. UR + RT
pH	6.27	6.39	6.35	0.02	0.020	0.090
NH3-N	21.29	34.25	31.57	1.84	<0.001	0.008
MCP	32.10	35.86	35.66	0.68	0.019	0.796
Urease (nmol/min/mg)	8.48	9.93	8.64	0.24	0.021	0.004
Total VFAs (mmol/L)	8.01	7.96	8.13	0.09	0.859	0.554
Acetate (%)	58.36	58.69	58.12	0.19	0.473	0.254
Propionate (%)	26.76	26.73	28.86	0.33	0.939	0.001
Isobutyrate (%)	0.68	0.65	0.60	0.01	0.017	0.001
Butyrate (%)	10.74	10.55	9.39	0.19	0.221	<0.001
Valerate (%)	2.34	2.31	2.14	0.03	0.336	0.002
Isovalerate (%)	0.69	0.66	0.58	0.02	0.077	<0.001
Caproate (%)	0.41	0.39	0.28	0.02	0.548	0.031
Heptanoic acid (%)	0.03	0.02	0.04	0.01	0.262	0.070
A: P	2.18	2.20	2.01	0.03	0.751	0.003

NH₃-N  Ammonia nitrogen, MCP  Microbial protein, VFAs  Volatile fatty acids, A/P  Acetate, propionate, SEM Standard error of the mean. CTL =without supplementation, UR= supplementation with 2.5% of UR based on dry matter weight, UR + RT = supplementation with 2.5% of UR and 12.5% RT based on dry matter weight

Effect of RT3 on rumen microbiota (Exp. 2)

We analyzed the microbial composition in fermentation liquid samples from treated with UR and UR + RT using 16 S rRNA gene sequencing technology. Firstly, alpha diversity indicators including Chao1, Shannon index and Simpson index were employed to assess the rich ness and diversity of individual taxa (Fig. 4). The exposure to both UR and UR + RT significantly affected the diversity of the fermentation broth microbiota, as evidenced by higher microbial community abundances and diversities shown by the UR and UR + RT groups compared to the Con group and the considerably larger trend in alpha diver sity indices shown by the UR group compared to the UR + RT group. Principal co-ordinates analysis (PCoA) revealed differences in microbial structure among the groups. (Fig. 5a). In addition, the Heatmap (Fig. 5b; supplementary material S3) clearly showed the abundance of the top 10 abundant microbial species in each sample at the phylum level and the differences in the abundance of each species between samples. Bacteroidota and Firmicutes were significantly different among the three groups. The heatmap (Fig. 5c, e; supplementary material S4) displayed distinct abundance patterns of the top 10 microbial genera across experimental groups. Fretibacterium exhibited higher relative abundance in UR + RT compared to other groups, while Prevotella_7 and Selenomonas showed increased abundance in UR relative to both CTL and UR + RT. Christensenellaceae_R-7_group and Rikenellaceae_RC9_gut_group demonstrated reduced abundance in UR and UR + RT groups versus CTL. Concurrent enrichment of Succiniclasicum and Lachnospiraceae_NK3A20_group was observed specifically in UR + RT samples. Conversely, CTL samples maintained elevated abundance of Candidatus_Saccharimonas and NK4A214_group relative UR and UR + RT treatment groups. The results indicated that Bacteroidota, Bacteroidales, Bacteroidia, Prevotellaceae and Prevotella in the UR group, Firmicutes, Clostridia, Lachnospiraceae and Lachnospirales in the UR + RT group possessed relatively high abundance(Fig. 5d).

Fig. 4.

Changes in bacterial alpha diversity. a Chao 1 index, b Shannon index, and c Simpson index. CTL =without supplementation, UR= supplementation with 2.5% of UR based on dry matter weight, UR + RT = supplementation with 2.5% of UR and 12.5% RT based on dry matter weight. *P < 0.05, ***P < 0.001, ns = not significant

Fig. 5.

UR and UR + RT altered the microbial community structure in the in vitro rumen fermentation liquor. a principal coordinates analysis (PCoA) was performed to calculate beta diversity on a distance matrix of Bray–Curtis indices. b Changes of fermentation liquor microbial composition at the phylum level (TOP 10). c Abundance analysis of the fermentation liquor microbiota at the genus level (TOP 10). d Linear discriminant analysis effect size (LEfSe) comparison analysis between the groups. e Comparison of MetagenomeSeq analysis at the genus level. CTL =without supplementation, UR= supplementation with 2.5% of UR based on dry matter weight, UR + RT = supplementation with 2.5% of UR and 12.5% RT based on dry matter weight

As show in Fig. 6, Prevotella was significantly positively correlated with isobutyrate, butyrate, valerate, isovalerate, caproate, and A: P, while significantly negatively correlated with propionate (P < 0.05). Rikenellaceae_RC9_gut_group showed a significant positive correlation with NH₃-N and MCP, and a significant negative correlation with isobutyrate, butyrate, valerate, and isovalerate (P < 0.05). Selenomonas was significantly positively correlated with propionate, and significantly negatively correlated with isobutyrate, butyrate, valerate, isovalerate, caproate, and A: P (P < 0.05).

Fig. 6.

Correlation of the rumen fermentation parameters with the microbial community. Positive and negative correlations are shown in red and blue, respectively. * P < 0.05, ** P < 0.01, *** P < 0.001

Discussion

Rumen urease is crucial for utilizing NPN sources in feed; however, its high activity also compromises the utilization efficiency of urea-based feeds in ruminants [28]. Enhancing the efficiency of urea nitrogen conversion into ruminal microbial nitrogen is significant for ruminant nutrition, with the potential to improve protein synthesis and reduce nitrogen waste. In this study, we investigated the effects of rutin on in vitro ruminal fermentation parameters, urease activity, and microbial community composition in Exp. 1. Subsequently, in Exp. 2, we examined the effects of rutin combined with urea on these same parameters.

Effect of rutin on rumen fermentation and microbiota (Exp. 1)

Rumen fermentation parameters

The results of Exp. 1 demonstrated that supplementation with varying doses of rutin significantly reduced NH₃-N concentration after both 6 h and 12 h of fermentation. Furthermore, after 12 h of fermentation, rutin supplementation induced a dose-dependent increase in MCP content, while significantly decreasing pH values and urease activity. NH₃-N concentration exhibited a strong correlation with MCP content, reflecting the proteolytic capacity and absorption efficiency of ruminal microorganisms, while also serving as a key precursor for MCP synthesis. Previous studies have consistently demonstrated that most flavonoids tend to reduce NH₃-N levels [29, 30] yet enhance MCP production [31–33], which aligns with the findings of the present study. This inhibited urease activity, thereby curtailing NH₃-N generation and enhancing microbial urea utilization, which collectively improved microbial protein anabolism [34].

VFAs, particularly acetate, propionate, and butyrate, constitute the predominant end-products of anaerobic microbial fermentation in the rumen. These VFAs provide 70%-80% of the metabolic energy required for ruminant physiological functions [35]. Critically, VFA concentrations serve as dual indicators: not only reflecting the efficacy of ruminal fermentation, but also significantly contributing to fluctuations in ruminal pH. Acetate constitutes a principal end-product of ruminal microbial metabolism, arising primarily from cellulose and hemicellulose breakdown. As the dominant precursor for ruminant fatty acid biosynthesis, it plays an essential role in milk fat and body adipose formation [36]. In contrast, propionate serves as the primary substrate for lipogenesis and lactose production, with hepatic gluconeogenesis converting this acid into glucose for energy provision. Crucially, elevated ruminal propionate concentrations proportionally enhance metabolic energy availability [37]. Our results indicate that after 12 h of fermentation with 12.5% rutin supplementation (RT3), acetate proportion and the A/P were significantly reduced, concomitantly with a marked increase in propionate proportion. This is also confirmed by the results of Kamra et al. (2006) [38] and Xiao et al. (2025) [39]. That is, the addition of flavonoids can reduce A/P and increase propionate proportion.

Microbial community modulation

In Exp. 1, there was no significant difference in alpha diversity between the RT3 group and the control group, as demonstrated by Oskoueian et al. (2013) [40]. According to the results of PCoA analysis, the bacterial community structure of RT3 group was significantly changed compared with the CTL group. The results of this study are consistent with previous reports and may be related to the antibacterial effects of flavonoids on certain bacterial phyla and genera. Firmicutes, Bacteroidetes, Proteobacteria, Actinobacteria, and Patescibacteria represented the core bacterial phyla in ruminal fluid, exhibiting dynamic abundance shifts during digestion [41]. In this study, Firmicutes and Bacteroidetes functioned synergistically in carbohydrate and protein hydrolysis and synthesis [42], establishing them as the dominant taxa. Proteobacteria, Actinobacteria, and Patescibacteria followed in descending order of abundance. At the genus level, Succiniclasticum, Prevotella, and Prevotella_7 were identified as the dominant taxa, consistent with our findings. Melchior et al. (2019) reported that Prevotella became dominant following the in vitro supplementation of flavonoids [43]. Compared with the CTL, the RT3 group significantly elevated the relative abundances of the cellulolytic bacteria Rikenellaceae_RC9_gut_group, Selenomonas, and Succinivibrio, whereas it markedly reduced those of the proteolytic taxa Prevotella and Prevotella_7. Xu et al. (2023) [44] study found that the number of fiber decomposing bacteria Ruminococcus flavefaciens and Ruminobacter amylophilus increased significantly after chickpea germ a was added to the feed. Based on these observed shifts in microbial community structure, particularly the increase in putative cellulolytic bacteria, we hypothesize that rutin may create a microbial environment more conducive to fiber utilization through modulation of rumen flora. The complex interaction between rutin and cellulose degradation process highlighted the potential of rutin to improve rumen function and increase fiber degradation rate. These findings are of great significance for improving rumen fiber degradation rate and rumen function in ruminants. In addition, studies have shown that flavonoids can reduce the abundance of peptide decomposing bacteria and amino acid decomposing bacteria, indicating that they can promote protein degradation [45]. Prevotella is a kind of bacteria involved in protein decomposition and ammonia production. The reduction of proteolytic bacteria, especially Prevotella, may be related to the antimicrobial properties of flavonoids, the reduction of urea decomposing bacteria, and the inhibition of urease activity by rutin.

Effects of rutin combined with urea on rumen fermentation and microbiota (Exp. 2)

Synergistic effect of rutin and urea on rumen fermentation parameters

Urea combined with a urease inhibitor is commonly used as a food supplement to give degradable nitrogen and to act as a solution for the quick hydrolysis of urea in rumen. The results of Experiment 1 showed that rutin could reduce urease activity, increase MCP synthesis and reduce proteolytic bacteria. As an important member of flavonoids, the urease inhibition effect of rutin is consistent with the common mechanism of flavonoids. The hydroxyl group in its molecular structure can chelate with the nickel ion in the active center of urease, and form hydrogen bonds or hydrophobic interactions with amino acid residues near the active site, thereby blocking the catalytic hydrolysis of urea by urease [46, 47]. In addition, the electron donating group (hydroxyl) on the benzene ring of rutin further enhances its urease inhibitory activity, which is consistent with the research results on the relationship between the structure and activity of flavonoids [48, 49].Therefore, in Experiment 2, the study of rutin combined with urea found that the interaction of urease inhibitor (rutin) and urea successfully inhibited the hydrolysis of urea and the formation of ammonia, which was consistent with previous in vivo and in vitro studies [50, 51]. In our study, urease activity decreased by 12.99% in the UR + RT group compared with the UR group. The CTL of dietary urea hydrolysis aims to enhance the incorporation of nitrogen in rumen MCP, which is the main source of metabolic proteins absorbed by the ruminant gut [52]. However, our results showed that reducing the rate of urea hydrolysis did not improve the nitrogen utilization efficiency of MCP synthesis. Existing studies have shown that the optimal rumen NH₃-N concentration produced by maximum MCP is different: 12.8 mg/100 ml in lactating Holstein cattle fed urea supplemented corn silage [53], while 6.29 mg/100 ml (3.7 mmol) in the in vitro system [54]. In our study, the high crude protein diet may provide enough ammonia (more than 12.74 mg/100 ml in CTL group) to saturate the microbial demand, which may explain the comparable MCP production between different treatments.

Synergistic effect of rutin and urea on microbial community regulation

Rumen microbial ecosystems exhibit unusually high population density, significant diversity, and complex interspecific interactions [55]. Previous in vitro studies have shown that nitrogen availability, especially ammonia concentration, significantly regulates rumen bacterial diversity [56], because microbial populations exhibit a dose-dependent response to ammonia levels [57]. The results of alpha diversity study showed that compared with the control group, the UR group significantly increased the Chao 1 index; Compared with the other two groups, the UR + RT group significantly increased Shannon, Chao 1 and Simpson indexes. PCoA results showed that there were significant changes in community structure in CTL, UR and UR + RT groups, and the relative abundance of proteolytic bacterium Prevotella was significantly reduced in UR + RT group compared with UR group. Possible explanations for these results are: (I) changes in urease activity levels; (II) flavonoids inhibit the growth of pathogenic bacteria, and then promote the growth of other microorganisms to compete with them. Flavonoids, as plant derived secondary metabolites, have natural antibacterial activity and can affect community structure by regulating competition among microorganisms [58], which is consistent with the regulatory effect of rutin on rumen microbial communities. It was reported that the degradation rate of flavonoids increased with the increase of flavonoid dose in a specific concentration range [58]. Therefore, more microbial flavonoid degradation products are produced.

By utilizing NH₃-N for microbial protein synthesis, intestinal cellulolytic bacteria play a crucial role in rumen microbial ecology [59]. Their growth showed a dose-dependent response to NH₃-N concentration [60]. Although excessive ammonia produced by urea hydrolysis and dietary protein degradation typically inhibits these bacterial populations, the addition of 12.5% RT regulates this effect by slowing down ammonia release, which is consistent with the mechanism by which flavonoids reduce ammonia production and protect the gut microbiota environment by inhibiting urease activity [61, 62].

Increased diversity of the microbiota generally confers greater ecosystem stability [63], benefiting host animals. Although urease inhibitors such as RT are expected to optimize rumen microbial communities during urea supplementation, microbial adaptation may lead to ammonia rebound over time [64]. The urease inhibition effect of flavonoids depends on their structural integrity and functional group substitution characteristics [65]. In long-term applications, attention may need to be paid to the interaction between their structural stability and microbial metabolism. Therefore, it is necessary to conduct long-term in vivo studies to fully evaluate the effectiveness of RT, and developing improved RT based inhibitors is an important direction for future research.

Conclusions

In summary, rutin has an inhibitory effect on rumen urease activity, improves nitrogen utilization efficiency, promotes microbial protein synthesis, and improves rumen fermentation by regulating the abundance of beneficial bacteria Selenomonas and Lachnospiraceae NK3A20_group in the rumen. When 12.5% rutin is combined with 2.5% urea, rutin can also inhibit urease activity, reduce the number of protein degrading bacteria, increase the abundance of beneficial bacteria, promote rumen digestion and absorption capacity, and positively regulate rumen fermentation. It should be noted that these doses are intended for mechanism exploration, and future in vivo studies will focus on practical low doses. Future research should explore the in vivo application of rutin as a feed additive in combination with NPN for ruminant nutrition, in order to fully investigate its benefits on production performance, growth metabolism, and livestock product quality.

Abbreviations

NH₃

N Ammonia nitrogen

MCP

Microbial crude protein

GP

Gas production

VFAs

Volatile fatty acids

A/P

Acetate: propionate

RT

Rutin

UR

Urea

Authors’ contributions

JQ and YW secured funding to support the execution of the study. YH coordinated the recruitment process and oversaw the collection of biological samples, along with the acquisition of all demographic and survey-related data. LG and XC carried out the 16 S rRNA gene sequencing and preliminary processing. SS conducted the metabolomics experiments, developed the overarching study concept, and played a central role in organizing the dataset. SS also performed all statistical analyses, interpreted the results, and created the data visualizations. SS drafted the initial manuscript, with editorial and structural input from WW. Both WW and YW critically reviewed and revised the manuscript for intellectual content. All authors reviewed and approved the f inal version of the manuscript for submission.

Funding

This study was financially supported by Talent Project in the Field of Science and Technology Innovation of Hohhot (2022RC - Industrial Research Institute − 4), Inner Mongolia Autonomous Region “Talents Revitalizing Inner Mongolia” Project for Teams (2025TYL10), Scientific Research Special Project for First-Class Disciplines of the Education Department of Inner Mongolia Autonomous Region (YLXKZX-NND-007).

Data availability

The 16 S rRNA gene-sequences data supporting the conclusions of this article are deposited at the National Center for Biotechnology Information (BioProject SRA [PRJNA1321475](https:/www.ncbi.nlm.nih.gov/bioproject/PRJNA1321475) .

Declarations

Ethics approval and consent to participate

Rumen fluid was obtained from a commercial abattoir. The slaughter procedures and animal handling prior to slaughter complied with the national standard Guidelines for the Welfare of Slaughter Animals (GB/T 42304 − 2023). As the samples were collected post-mortem from animals slaughtered for routine meat production, and no animals were raised or euthanized specifically for this experiment, ethical approval from an institutional animal care and use committee was not required.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.