Introduction

In recent years, the rumen and intestinal microbiomes have become the focus of significant scientific interest due to their pivotal role in animal health and disease, with numerous studies highlighting their essential contribution to animal physiology1. For ruminants, the rumen is a complex and crucial ecosystem that contains a wide range of functional microorganisms, which are capable of fermenting human-inedible plant-based biomass to form volatile fatty acids (VFAs). Research has verified that VFAs could serve as a major nutrient source for host animal growth as well as production, particularly in meat and milk and exert beneficial effects on gastrointestinal membrane integrity, glucose and lipid metabolism of animals as well as host health1,2,3,4,5,6. The composition of the rumen and gut microbiome is susceptible to nutritional changes, medication or chronic diseases7. Therefore, modifying the gastrointestinal bacterial composition to a ‘healthier’ or a ‘better’ microbiome has become an attractive strategy for improving animal health. In addition to dietary modifications, this could be achieved by supplementing nutrients or additives, such as extracts of the medicinal plant or itself containing active ingredients, or probiotics or their alternatives, including bacteriocins and antimicrobial metabolites to manage microorganisms to benefit for the host7. In addition, the rearing of livestock will certainly result in the production of waste matter, i.e., feces. As the fecal microbiota has a relationship with gastrointestinal microbes8 and volatile odor, mainly odour, can affect the farm surroundings and pollute the human living environment9, it is necessary to understand the composition of fecal microbes and reduce the emission of fecal odor gas. Mulberry leaves (MLs) are not only the main feed for silkworms of all time, but are also a traditional Chinese medicinal plant, cultivated in China for more than four millennia10. MLs are rich in nutrients, including edible fiber, and a variety of active ingredients, mainly including flavonoids, polysaccharides, polyphenols, alkaloids. These components have ever been reported to exhibit antihyperglycemic, anti-inflammatory, antioxidative effects, and immunostimulatory activities, as well as modulation effects on the rumen and gut microbiota and metabolites11,12,13. A previous study highlighted that the antihyperlipidemic effect of mulberry-derived 1-deoxynojirimycin (1-DNJ) is conferred by indole-3-propionic acid produced by the gut microbiota14. It is therefore important to explore the regulatory effects of MLs on the rumen and fecal microbiota as well as rumen fermentation, and to investigate whether MLs could reduce the emission of fecal odor gas, which is crucial for the optimization of livestock health.

Materials and methods15

Preparation and measurement of chemical indexes of fermented mulberry leaves and dried mulberry leaves15

MLs were harvested in July 2021 at the Institute of Sericulture and Silk in Zhouzhi, Shaanxi Province, China. The collection of wild MLs adhered to the guidelines and legislation of China. One half was sun-dried for seven days, lightly rubbed, and then sealed in woven bags. The prepared dried mulberry leaves (DMLs) were stored in a dry, dark place for use in the feeding experiment. The other half with 65.03% moisture content after wilted by sun-shine for a half day, is smashed a little and vacuum sealed in special fermentation bags to ferment with 5% Lactobacillus plantarum inoculation at room temperature (27.5–28 ℃) for thirty days in a dry, dark place for preparation of fermented mulberry leaves (FMLs). Here, 5% is adding 5 mL of bacterial culture suspension to 100 g of MLs and the concentration of bacterial culture suspension is 1 × 108 CFU/mL. Before feeding experiments, the pH, crude protein, crude fiber, and gross energy of FMLs and DMLs are determined according to Cui et al. (2022). And their contents are shown in Table S1. FMLs are deemed qualified without aflatoxin B1 detected at a minimum checked value of 0.1 µg/kg by Huayan Testing Group Co., Ltd in Xi’an City, Shaanxi Province (Detection number: SP202115450).

Experimental design and feeding diets15

Animal experiments were conducted on six-month-old healthy female mutton sheep (white-headed Suffolk sheep♂×Hu sheep♀) weighing 30.41 kg on average in the Ring County Mukang Fengmao grass industry Association (Huan County, Qingyang City, Gansu Province, China). The animals were randomly assigned to group control (Con) fed with a normal control diet (n = 18), group treatment 1 (TR1) fed with an experimental diet with FMLs (n = 18) and group treatment 2 (TR2) fed with an experimental diet with DMLs (n = 18) and then treated for an experiment of fifty days. Three sheep were kept in a pen with six pens in each group. Before feeding experiment, the animals underwent an acclimatization period of six days to obtain an appropriate feed intake, during which they were allowed unlimited access to their corresponding experimental diet and tap water. The experimental sheep were housed in sheepfolds and given self-help feeding in three groups every day. The ingredients and chemical composition of three experimental diets are shown in Table S2. The chemical compositions of the three experimental diets were determined by Ulanqab Yima Agriculture and Animal Husbandry Technology Co., Ltd. (Ulanqab City, Inner Mongolia, China). At the end of the experiment, animals died under anesthesiarumen. Afterwards, fluid and feces of each group were collected for 6 sheep, and 1 sheep randomly selected from each pen. As there were not enough hands and some samples were not collected during the sampling process, the ultimately sample size of the rumen fluid was respectively 6 (Con), 5 (TR1), and 4 (TR2) and Feces sample size is respectively 6 (Con), 6 (TR1), 4 (TR2). The study in this manuscript is in accordance with ARRIVE guidelines.

Sample collection

Fresh ruminal fluids were thoroughly mixed, filtered through four layers of gauze, transferred into a 2 mL frozen storage tube and immediately stored in liquid nitrogen to be taken to the laboratory for further analysis, including microbiota and metabolites analysis. Similarly, fresh feces samples were collected directly by massaging the rectum, wrapped in tin foil, and immediately stored in liquid nitrogen to be taken to the laboratory for further analysis, including microbiota and metabolites analysis.

Ruminal volatile fatty acids, digestive enzymes and microbial proteins

VFAs in rumen fluid were determined by Agilent 7890B gas chromatograph and 7000D mass spectrometry (GC-MS/MS). DB-FFAP column parameters: 30 m length ×0.25 mm, i.e. ×0.25 μm film thickness, J&W Scientific, USA. Helium was used as a carrier gas at a flow rate of 1.2mL/min. The dose was 2µL by separate injection. The oven temperature was initially held at 90 °C for 1 min, then increased to 100 °C at 25 °C/min, subsequently increased to 150 °C at 20 °C/min for 0.6 min, and eventually increased to 200 °C at 25 °C/min for 0.5 min, running for 3 min. All samples were analyzed in multiple response monitoring mode. The injector inlet and conveying line temperature were 200℃ and 230℃ respectively16,17.

The contents of pectinase, acidic xylanase (ACX), lipase, carboxymethyl cellulase and α-amylase were measured respectively using commercially available kits (M1707B, M1731B, M1004B, M1733B, M1104B; Suzhou Yanxi Biological Technology Co., Ltd.). The contents of protozoan protein, bacterial protein and microbial protein were obtained using the commercially available kit (M1808A; Suzhou Yanxi Biological Technology Co., Ltd.).

Volatile fecal metabolites

The dry matter (DM) was determined by drying fresh feces in an oven at 65 ℃ for 24 h. The contents of inorganic ammonia nitrogen (NH3-N) and hydrogen sulfide (H2S) were determined using commercially available kits (M0213B; Suzhou Yanxi Biological Technology Co., Ltd. and G0133F; Suzhou Gris Biotechnology Co., Ltd.).

The organic volatile metabolites of feces were determined by GC-MS and 1 g (1 mL) of the powder was transferred immediately to a 20 mL head-space vial (Agilent, Palo Alto, CA, USA), containing NaCl saturated solution, to inhibit any enzyme reaction. The vials were sealed using crimp-top caps with TFE-silicone headspace septa (Agilent). At the time of SPME analysis, each vial was placed in 60 °C for 5 min, then a 120 μm DVB/CWR/PDMS fibre (Agilent) was exposed to the sample headspace for 15 min at 100 °C. After sampling, the desorption of VOCs from the fiber coating was carried out in the injection port of the GC apparatus (Model 8890; Agilent) at 250 °C for 5 min in the splitless mode. The identification and quantification of VOCs was carried out using an Agilent Model 8890 GC and a 7000D mass spectrometer (Agilent), equipped with a 30 m × 0.25 mm × 0.25 μm DB-5MS (5% phenyl-polymethylsiloxane) capillary column. Helium was used as the carrier gas at a linear velocity of 1.2 mL/min. The injector temperature was kept at 250 °C and the detector at 280 °C. The oven temperature was programmed from 40 °C (3.5 min), increasing at 10 °C/min to 100 °C, at 7 °C/min to 180 °C, at 25 °C/min to 280 °C, hold for 5 min. Mass spectra were recorded in electron impact (EI) ionisation mode at 70 eV. The quadrupole mass detector, ion source, and transfer line temperatures were set, respectively, at 150, 230 and 280 °C. Mass spectra were scanned in the range m/z 50–500 amu at 1s intervals. Identification of volatile compounds was achieved by comparing the mass spectra with the data system library and linear retention index18.

16s sequencing of the bacterial community

Total microbial genomic DNA was extracted from ruminal and fecal samples using the E.Z.N.A.® soil DNA kit (Omega Bio-tek, Norcross, GA, U.S.) according to manufacturer’s instructions. The quality and concentration of DNA were determined by 1.0% agarose gel electrophoresis and a NanoDrop® ND-2000 spectrophotometer (Thermo Scientific Inc., USA) and kept at -80 ℃ prior to further use. The hypervariable region V3-V4 of the bacterial 16 S rRNA gene was amplified with primer pairs 338 F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R(5′-GGACTACHVGGGTWTCTAAT-3′)19 by an ABI GeneAmp® 9700 PCR thermocycler (ABI, CA, USA). The PCR reaction mixture including 4 µL 5 × Fast Pfu buffer, 2 µL 2.5 mM dNTPs, 0.8 µL each primer (5 µM), 0.4 µL Fast Pfu polymerase, 10 ng of template DNA, and ddH2O to a final volume of 20 µL. The cycling conditions for PCR amplification were as follows: initial denaturation at 95 ℃ for 3 min, followed by 27 cycles of denaturation at 95 ℃ for 30 s, annealing at 55 ℃ for 30 s and extension at 72 ℃for 45 s, and single extension at 72 ℃ for 10 min, and end at 4 ℃. All samples were amplified in triplicate. The PCR product was extracted from 2% agarose gel and purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA) according to manufacturer’s instructions and quantified using Quantus™ Fluorometer (Promega, USA). Purified amplicons were pooled in equimolar amounts and paired-end sequenced on an Illumina MiSeq PE300 platform/NovaSeq PE250 platform (Illumina, San Diego, USA) according to the standard protocols by Majorbio Bio-Pharm Technology Co. Ltd. (Shanghai, China). After demultiplexing, the resulting sequences were quality filtered with fastp (0.19.6)20 and merged with FLASH ( v1.2.11)21. Then the high-quality sequences were de-noised using DADA222 or Deblur23 plugin in the Qiime224 (version 2020.2) pipeline with recommended parameters, which obtains single nucleotide resolution based on error profiles within samples. DADA2 (or Deblur)-denoised sequences are usually called amplicon sequence variants (ASVs).

Statistical analysis

Statistical analysis was performed using SPSS 22.0 software (IBM-SPSS Statistics, IBM Corporation, Armonk, NY, United States). Data were evaluated using a one-way ANOVA followed by Turkey’s multiple range tests for physiological and biochemical indices. Significance was declared if p < 0.05. Additionally, the data for 16s sequencing of bacteria are provided by majorbio and analyzed using majorbio cloud platform (https://cloud.majorbio.com/). Histograms and metabolic maps are plotted, respectively using Graphpad Prism 8 and Adobe Illustrator CS6.

Results

Rumen fermentation and bacterial community

According to Cui, et al.15, shown in Table S3, both FMLs and DMLs significantly increased ADFI (p < 0.05), which suggests MLs are a delicious feed for promotion of animal consumption. In addition, ADG, daily gain (25–50d) and serum growth hormone levels were significantly improved in both MLs-treatment groups (p < 0.05) in the study. Furthermore, FMLs feeding resulted in a significant increase in the final body weight (p < 0.05).

Fig. 1
figure 1

Metabolites in the rumen. Fermentation parameters (A), the map for the biosynthetic pathway of VFAs (B) and digestion characteristics (C).

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As shown in Fig. 1A, compared to group Con, treatment with FMLs (TR1) reduced NH3-N content in rumen fluid, while there was a significant increase in treatment with DMLs (TR2) VFAs in rumen fluid increased (Con < TR1 < TR2), including acetate (p > 0.05), prpionate (p < 0.05), isobutyrate (p > 0.05), butyrate (p > 0.05), isovalerate (p > 0.05), valerate (p > 0.05), caproate (p > 0.05) and total VFA (p > 0.05). Referenced to RUSSELL and HESPELL25, the hand-painted map of the biosynthetic pathway in VFAs in the rumen was constructed in Fig. 1B. VFAs in the rumen were primarily produced through the degradation of indigestible carbohydrates (dietary fiber) and the fermentation of pyruvate. Acetic acid, butyric acid, caproic acid, valerate acid were mainly produced from acetyl CoA, while propionic acid is mainly produced by the metabolism of succinic acid and lactic acid. The end products of rumen fermentation were primarily acetic acid, propionic acid and butyric acid (RUSSELL & HESPELL, 1981). Figure 1C illustrates the contents of digestive enzymes and microbial proteins in rumen fluid. It was obvious that the contents of pectinase, lipase, protozoan protein, bacterial protein and total microbial protein were increased significantly (p < 0.05) with MLs treatments, while there were insignificant variations in the contents of ACX, carboxymethyl cellulose, and α-amylase (p > 0.05).

Fig. 2
figure 2

Bacterial community in rumen. PCoA of community composition (A); bar diagram of bacterial community in phylum level (B) and genus level (C). *p < 0.05, **p < 0.01.

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Principal coordinates analysis (PCoA) is a non-binding data dimension reduction analysis method, usually used to study the similarity or difference of samples and groups in bacterial communities. Obviously, little differentiation was emerged in between-group TR1 vs. TR2, while MLs treatments significantly distinguished group TR1 and TR2 from group Con in bacterial communities (Fig. 2A). The bacterial community at the phylum level of different groups is displayed in Fig. 2B. Bacteroidota and Firmicutes were the main phylums in the rumen, with Bacteroidota representing nearly 70% of the total bacterial population. Notably, FMLs treatment reduced the relative abundance of Bacteroidota. Figure 2C gave a presentation on the relative abundance of the top 17 genus-level bacteria. The abundance of Prevotella, the most abundant genus, increased, while that of Rikenellaceae_RC9_gut_group ranking the first was reduced with MLs treatments (p > 0.05). Furthermore, FMLs treatment could significantly improve the relative abundance of norank_f_Bacteroidales_UCG-001, Fibrobacter, and Ruminococcus (p < 0.05). DMLs could significantly increase the relative abundance of Ruminococcus (p < 0.01).

Fig. 3
figure 3

Spearman correlation heatmap of rumen microflora and metabolites. *p < 0.05, **p < 0.01. L1, NH3-N; L2, acetate; L3, propionate; L4, Isobutyrate; L5, butyrate; L6, Isovalerate; L7, valerate; L8, caproate; L9, Total VFA; L10, pectinase; L11, ACX; L12, lipase; L13, carboxymethyl cellulose; L14, α-amylase.

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Correlation heat-map analysis (Fig. 3) presented that Prevotella and Fibrobacter had a significantly positive correlation with α-amylase (p < 0.01).Respectively, Succiniclasticum had a significant positive correlation with acetic acid (p < 0.05), isobutyric acid (p < 0.01), isovaleric acid (p < 0.05) and valerate acid (p < 0.05). Saccharofermentans could significantly increase the content of acid xylanase (p < 0.01); norank_f_Bacteroidales_UCG-001 and NK4A214_group could significantly improve the contents of valeric acid and pectinase respectively (p < 0.05). However, norank_f_Muribaculaceae, UCG-002, Butyrivibrio, and Norank_F_Muribaculaceae could significantly reduce the contents of caproic acid, pectinase, α-amylase and NH3-N respectively (p < 0.05). In addition, it was speculated that lipase content might be correlated with Prevotella, norank_f_F082, Succiniclasticum, Saccharofermentans, norank_f_Bacteroidales_UCG-001, Lachnospiraceae_AC2044_group, and Fibrobacter (p > 0.05).

Fecal volatile metabolites and bacterial community

Fig. 4
figure 4

Volatile metabolites in feces. Inorganic volatile metabolites (A) and organic volatile metabolites (B).

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As observed in Fig. 4A and B, MLs treatment could significantly decrease the contents of odorous H2S and harmful Benzene, 1, 2, 3, 5-tetramethyl, while significantly increasing 5-heptene, 2-one, 6-methyl- (p < 0.05). Additionally, although fecal DM and octadecane with MLs treatment was lessened, there was no significant difference among the three groups (p > 0.05). Ultimately, NH3-N, 3-methyl indole (i.e. skatole), p-Cresol and DL-Lactamide, propyl ether were dramatically boosted by DMLs treatment, compared to FMLs treatment and the control without added MLs.

Fig. 5
figure 5

Bacterial community in feces. PCoA of community composition (A); bar diagram of bacterial community in phylum level (B) and genus level (C). *p < 0.05, **p < 0.01.

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PCoA (Fig. 5A) displayed notable differences in bacterial community structure across treatment groups, there was a small variation between FMLs and DMLs treatment groups, while there existed a large variation in MLs treatment groups and the control group without MLs. As exhibited in Fig. 5B, consistent with the rumen, the bacteria at the phylum level in feces were mainly Firmicutes and Bacteroidota. However, the variation was less in Bacteroidota than Firmicutes with the proportion of up to 80%. Figure 5C gave a presentation on the relative abundance of the top 15 genus-level bacteria. It was evident that MLs supplementation could significantly increase the abundance of Unclassified_f_Lachnospiraceae (p < 0.05), while significantly decrease the abundance of Christensenellaceae_R-7_group and NK4A214_group (p < 0.05).

Fig. 6
figure 6

Spearman correlation heatmap of fecal bacterial community and volatile metabolites. *p < 0.05, **p < 0.01. F1, DM; F2, H2S; F3, NH3-N; F4, Indole; F5, Indole, 3-methyl-; F6, Diehyl carbonate; F7, p-Cresol; F8, 5-Hepten-2-one, 6-methyl-; F9, DL-Lactamide, propyl ether; F10, Benzene, 1, 2, 3, 5-tetramethyl; F11, Octadecane.

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Spearman correlation analysis (Fig. 6) revealed that Christensenellaceae_R-7_group, similar to NK4A214_group and in contrast with Unclassified_f_Lachnospiraceae, had a remarkable negative correlation with p-Cresol, 5-Hepten-2-one, 6-methyl- and DL-Lactamide, propyl ether, while positively correlated with benzene, 1, 2, 3, 5-tetramethyl and Octadecane. Apparently, the abundance of UCG-002 was positively correlated with the content of indole (p < 0.05) and norank_f_Eubacterium_coprostanoligenes_group was positively correlated with the content of diehyl carbonate (p < 0.05). Bacteroides was negatively correlated with DL-Lactamide and propyl ether (p < 0.05) and norank_f_F082 was negatively correlated with 5-Hepten-2-one, 6-methyl- (p < 0.05).

Discussion

Rumen bacteria and digestion

The rumen hosts a diverse and extensive bacterial community, essential for the degradation of structural carbohydrates such as cellulose, hemicellulose, and lignin in feed. These microbial activities facilitate the production of pyruvate and synthesize rumen VFAs through the fermentation of pyruvate as energy source for animals themselves and rumen microorganisms. Additionally, VFAs play a critical role in the normal development of rumen epithelium, the integrity of the intestinal membrane, local intestinal immunity, and microbial-intestine-health. Recent studies have shown that VFAs, produced by gastrointestinal fermentation of fibers in the diet, also have the ability to regulate appetite through the release of intestinal hormones; to produce ATP, which provides energy for the body; to stimulate the expression of mucin and compact junction protein, protect mucosal barrier and promote intestinal immunity; to promote the production of regulatory immune T, fight against inflammation; to regulate glucolipid metabolism and so on1,2,3,4,5,6. In this study, an increasing trend in VFAs production was recorded across all three groups (Con < TR1 < TR2), suggesting that the addition of MLs could promote the production of VFAs to further guarantee the body’s nutritional supply and immune health. The highest content of VFAs in DMLs treatment may be attributed to the elevated content of soluble carbohydrates and polysaccharides in DMLs10. Notably, propionic acid between groups (Con vs. TR2) reaches an extremely significant level. According to Cueva, et al.26, we inferred that the increase in propionic acid was mainly due to the possibility that MLs could promote fermentation pathway of odd-chain VFAs. Additionally, Bedford, et al.27 proposed that the contents of acetic acid and propionic acid are related to feed intake and can play a role in appetite regulation. Moreover, propionic acid plays a crucial role in anti-inflammation, immunity and regulation of lipid metabolism (for instance, preventing obesity)2,3. Therefore, the increase of propionic acid can enhance the dietary intake of animals and reduce the body’s deposition of fat. In addition, rumen fermentation also produces methane, heat, and ammonia, which represent the loss of energy and nitrogen during the utilization of nutrients in animals25. In this study, compared with the control group without MLs, FMLs group could reduce the NH3-N content, while DMLs group could increase the NH3-N content. Notably, the NH3-N content of FMLs is significantly lower than that of DMLs. The above indicates that MLs fermentation could reduce the nitrogen loss during its utilization by animals, thus promoting the digestion and absorption of MLs protein, which is beneficial to MLs utilization and animal production. Abundant bacteria, fungi, archaea and protozoa live in the rumen, which together constitute a complex rumen micro-ecosystem28,29. These rumen microbes produce various digestive enzymes, that play an important role in the digestion process. The activities of digestive enzymes is primarily influenced by the type and number of rumen microbiota as well as their metabolism30. Pectin, widely found in plant cell walls29, can be degraded by pectinase, resulting in smaller molecules that can be used by ruminants31. Many bacteria in the rumen can produce lipase, which can hydrolyze fatty acids, particularly long-chain fatty acids32. This study demonstrates that the addition of MLs, especially DMLs, significantly increases pectinase and lipase activities in rumen of mutton sheep. It suggests that MLs supplementation aids ruminants utilize plant-based feed and enhance lipid metabolism. Furthermore, microorganisms in the rumen can synthesize microbial proteins to satisfy the demand for protein themselves and those of animals. In this study, it is also realised that supplementation with MLs can improve protozoal protein, bacterial protein, and microbial total protein in the rumen, and thus provide more protein for the growth, development and production of animals. This effect can be attributed to the high protein content of MLs, which is comparable to alfalfa, often considered the “king of protein feeds”10.

Jami, et al.33 found that the microbial community is closely linked to the production traits of ruminants, highlighting the importance to study the composition of microbial community. Bacteroidota and Firmicutes are the two primarily groups of beneficial bacteria in the gastrointestinal tract of animals. An increase in Bacteroidota is associated to weightlessness34, therefore suggesting that the decrease in Bacteroidota in FMLs group may be related to the promoting-growth effect. Consistent with the results of Tian, et al.35, the most abundant bacteria genera in the three groups are Prevotella and Rikenellaceae_RC9_gut_group, respectively. Prevotella is a diverse and dominant genus of Gram-negative anaerobic bacteria and well known as one of main dietary fiber fermenters36. Chen, et al.37 discovered that the Prevotella spp. are competent producers of short-chain fatty acid propionate from arabinoxylans and fructo-oligosaccharides in vitro. And Galvez, et al.38 identified the efficiency of specific strains in degrading these polysaccharides is linked with dominance patterns of Prevotella and distinct Prevotella spp. compete in vivo for similar plant-derived polysaccharides.Additionally, Tett, et al.36 clarifies that the higher the diversity of Prevotella spp. and other fiber degraders, the more advantageous the fermentation ability of the microbiome will be for the benefit of the human gut. In our study, the relative abundance of Prevotella in the MLs treatment was more than double that of the control group. The current most likely interpretation is that MLs contain rich dietary fiber and active polysaccharides, which can promote the expansion of Prevotella spp., This, in turn, may help lower propionic acid content and enhance the efficiency of rumen fermentation. Rikenellaceae_RC9_gut_group have been implicated in a high-fat diet39 or dietary polyphenols40, even in mental illness41. Thus, either low-fat and high-fiber diet or polyphenols in MLs resulted in stark reductions in the abundance of Rikenellaceae_RC9_gut_group, which might protect livestock against illness. As observed by Tian, et al.35 the ruminal norank_f_Bacteroidales_UCG-001 increased in growing goats fed with basal diet and 1 g/d purple corn pigment, which is rich in anthocyanin. The marked increase in norank_f_Bacteroidales_UCG-001 can be explained by the most anthocyanin-rich FMLs and promote dietary fiber fermentation to benefit animals. In addition, the increase in valerate and the reduction in NH3-N are significantly related to the expanded relative abundance of norank_f_Bacteroidales_UCG-001, evidenced by spearman correlation analysis in this study. The cellulolytic microbiota, including Fibrobacter and Ruminococcus, contributes mainly to the provision of energy and health to the host and favor its proliferation though the metabolization of the D-glucose complex units chains into VFA in the gastrointestinal tract of both omnivorous and herbivorous animals42,43. In this regard, the significantly increased relative abundance of Fibrobacter and Ruminococcus after FMLs supplementation is probably attributed to high accessible cellulose, a complex polysaccharide composed of linked D-glucose units and constructs major component of plant cell walls, together with hemicellulose and pectin42. Concurrently, we observed a significant increase in pectinase activity, which may facilitate the degradation of pectin. Moreover, a previous study has also reported that intervention with metformin or berberine could increase the number of VFA-producing bacteria (for example, Ruminococcus) and restore the intestinal VFA content to relieve intestinal inflammation in db/db mice44. Consequently, alkaloids in MLs might also explain for stark increase in the abundance of Ruminococcus in rumen fluid after MLs treatments. The Correlation analysis in our study reveals that Fibrobacter and Prevotella are extremely positively correlated with α-amylase, while no significant fluctuation in α-amylase occurs. this warrants further investigations.

Thus, MLs themselves provide high-quality dietary fiber for animals. Meanwhile, MLs could regulate rumen bacterial community and promote the production of volatile fatty acids, so as to ensure the supply of nutrients and promote immune health. This regulation further affects the metabolism of glucose and lipid and contributing to the reduction of fat deposition.

Fecal bacteria and odors

Malodours are usually indicators that protect humans from potential illness caused by infection through contaminated food and matter45,46. These odours are generally attributed to the evolution of different smell-causing substances (volatile compounds) arising from the anaerobic decomposition of the faecal matter46,47. Sulphur and nitrogen containing compounds, particularly NH3 and H2S, are of particular importance, as they are the primary odorous substances and possess a distinctive odor that is easily noticeable even at small concentrations(H2S = 0.005ppm; NH3 = 0.05ppm)48,49. In addition, skatole, p-cresol, some carboxylic acids, phenol and indole have also been associated with farmyard manure-like odours46,50. In this study, compared to DMLs treatment, the production of fecal volatile compounds, especially odor gas (H2S, NH3-N, p-Cresol, Indole, 3-methyl-) can be reduced by the addition of FMLs, which is comparable to that of group control without supplementation with MLs. Additionally, MLs supplementations also raise the levels in aromatic gas, while lessening the release of thermal gas.

Earlier studies showed that Lachnospiraceae was found to be present at smaller abundances in the cecum of weaned piglets suffering from deoxynivalenol-induced feed refusal and in the feces of diarrheic pigs than that of normal pigs51,52. Additionally, Lachnospiraceae was more enriched in animals that exhibited greater feed intake51,53. Therefore, the increase in Lachnospiraceae observed with the addition of MLs in this study will promote the increase of animal feed intake and protect animal health. Moreover, the correlation analysis in this study revealed that the increase number of Lachnospiraceae in the MLs groups may result in a reduction in the content of DM and harmful odor compounds (benzene, 1, 2, 3, 5-tetrmethyl, octadecane) in feces. The reduction of DM in feces may improve the digestion of nutrients in animals, thus reducing the amount of feces excretion; Benzene, 1, 2, 3, 5-tetramethyl, octadecane are harmful gas, the reduction of the gas is beneficial in optimizing the livestock environment and reducing the emission of environmental pollution gas. Tang, et al.54 observed a decrease in fecal Christensenellaceae_R-7_group in the fermented complete feed group compared to the basal diet group. This suggests that, fermented fodder feeding will lead to a lessened fecal Christensenellaceae_R-7_group. A previous study aimed to investigate the effects of different supplementation levels of stevia residues in high-fiber diets on the fecal microorganisms of pregnant sows, followed by a significantly increased abundance of beneficial bacteria, such as Christensenellaceae_R-7_group55. The notable reduction of fecal Christensenellaceae_R-7_group in mutton sheep fed with high- fiber MLs in our study might b as a result of the antibacterial activity of MLs. Given that the fecal NK4A214_group is one of markers in between-group variance in murine gavage fed with reuterin supernatant acting as an alternative to live probiotics to manage dysbiosis7, it is plausible that MLs may be able to function as an alternative to affect fecal bacterial community because of an obvious between-group (with MLs or without MLs) change in fecal NK4A214_group. Conte, et al.56 demonstrates that Christensenellaceae_R-7_group and Ruminococcaceae_NK4A214_group in bovine rumen are related to lipid metabolism. The correlation analysis in our study reveals that the decreases of the fecal Christensenellaceae_R-7_group and NK4A214_group in MLs supplementation may contribute the reduction in production of detrimental Benzene, 1, 2, 3, 5-tetramethyl and Octadecane to lessen the pollution gas. Nowadays, uncertain functions of Christensenellaceae_R-7_group and NK4A214_group are present and further research is needed to verify the roles in affecting fecal metabolism of ruminants.

In this regard, MLs-supplementation aids in the regulation of fecal microbial community, potentially reducing DM excretion and the emission of fecal smelly or harmful volatile gases. This regulation promotes the absorption of animal nutrients and optimizes the livestock environment, and fosters healthy breeding as well as environmental protection. Notably, FMLs treatment was found to be more effective than DMLs in terms of reducing the emission of polluting gases.

Conclusion

The present study revealed that added mulberry leaves could promote absorption of animal’s nutrients and immune health. Furthermore, the addition of mulberry leaves, especially fermented mulberry leaves, contributed to optimize the livestock environment and strengthen environmental protection. The findings from the study provide suggestions for the forage applications of mulberry leaves in animal husbandry production.