Mining Novel Specific Rumen Bacteria to Enhance the Bioactive Function of Mulberry Leaf in Promoting Host Antioxidant Activity and Immunomodulatory


 Background: Rumen is a natural fermentation system and the microorganisms inside can effectively utilize plant bioresource and interact with host metabolism. Here, analysis of rumen microbiome, together with animal performance and serum metabolism in a lamb model were performed to identify the potential use of mulberry leaf silage (MS) to replace alfalfa silage (AS) as a new functional feed resource and to mining the novel specific mulberry leaf associated rumen bacteria interact with host metabolism. Results: The lambs fed with MS diet showed improved antioxidant capacity and immune function compared to those fed AS diet. The MS diet significantly altered rumen microbiota α- and β-diversity and taxonomic composition. Microbial analysis revealed that Bifidobacterium, Lactobacillus and Schwartzia were enhanced, and Ruminococcaceae UCG-010 and Lachnospiraceae_XPB1014_group were down-regulated in the rumen of MS group. A strong association was also found between these rumen microbial taxa and host antioxidant capacity and immunomodulatory. Conclusion: These findings indicated that mulberry leaf silage can be a high-quality feed source or bioactive pharmaceutical that is responsible for ruminants health benefits by modifying the rumen microbial community with potentially enhanced probiotics and inhibited biohydrogenation and methane emission.

studies found that high-quality mulberry leaf silage can be achieved by additives [1,10,13], but did not refer to the utilization of mulberry leaf silage. Rumen is a main position for the degradation of dietary nutrients in ruminants. The rumen microbiome is emerging as a cross bridge interacted with dietary utilization, host metabolism and phenotype changes, as shown by the association between rumen microbiome and the host metabolism [14][15][16].
Our questions focused on speci c microbial species establishment in the rumen ecosystem induced by mulberry leaf silage, which might interact with the host metabolism in promoting antioxidant activity and immunomodulatory. Here, this study was to assess the mulberry leaf silage that could be a high-quality feed source with important biological activity and bio-feed resources for ruminants, and then to mining the associated rumen microbiome accounting for its bioactive function.

Growth and slaughter performance
As shown in Table 1, DM intake, average daily gain (ADG), body weight (BW), and the ratio between DM intake and ADG were similar between the two diets. The slaughter BW, carcass weight, and dressing percentage were similar between the two groups. The weight of head and spleen in alfalfa silage diet (AS) group lambs were greater than those in mulberry leaf silage diet (MS) group (P < 0.05). The weight of kidney in AS group lambs had a tendency to be lower than that in MS group (P = 0.053).

Meat quality characteristics
Meat quality characteristics were similar between the two diets, as shown in Table 1. Compared with the AS diet group, the lightness (L*) value at 45 min had a tendency to be lower in the MS diet group compare to AS group (P = 0.10).
Nutrients metabolism, antioxidant activity, and immune response Overall, serum lipid metabolite levels including the concentrations of cholesterol, triacylglycerol, and the high density lipoprotein cholesterol (HDL) and low density lipoprotein cholesterol were un-affected by the MS diet ( Table 2). The serum blood urea nitrogen (BUN) concentration was greater in the MS group than in the AS group (P < 0.01) but not for the serum albumin and globulin. As shown in Table 2, the activity of serum catalase (CAT), glutathione peroxidase (GSH-PX), superoxide dismutase (SOD), and total antioxidant capacity (T-AOC) were signi cantly greater in the MS group than in the AS group (P < 0.05). The content of malondialdehyde (MDA)was signi cantly lower in the MS group than in the AS group (P < 0.05). Compared to the AS group, the serum interferon-γ (IFN-γ) concentration was signi cantly increased in the MS group (P < 0.05). No signi cant difference was found for the tumor necrosis factor-α (TNF-α) concentration between the two groups.

Fermentation characteristics
As shown in Table 3, rumen pH value and the concentrations of ammonia-N and total volatile fatty acids were similar between the AS and MS groups. The molar proportion of the isobutyrate had a tendency to be lower in the MS group than in the AS group (P = 0.09).

Change in ruminal bacterial communities
The Good's coverage of all samples was above 0.99. The Chao, Sobs, Shannon, and Ace indexes of bacterial richness and diversity were different between AS and MS (Table S2). The NMDS plots (Fig. 1A) showed that the clouds derived from the AS and MS data were clearly separated from each other with a signi cant stress value (0.026). There were 1,046 OTUs that were identi ed in both of the AS and MS groups, while 973 and 478 speci c OTUs were observed in AS and MS groups, respectively (Fig. 1B). The anosim (analysis of similarities) based on bray-curtis distances showed signi cant different between the two groups (P = 0.007).

Discussion
The biomass yield of fresh mulberry leaves is approximately 25 to 30 tons/ha/year, and the mulberry leaf is rich in protein (15-35%) [3]. Therefore, mulberry leaves might be used as an excellent protein or feed supplement in animals [3,17]. In the current study, we found that the animal growth performance was maintained when mulberry leaf silage replaced alfalfa silage, which might be due to their similar protein level and digestible nutrients [18,19]. Furthermore, we also found that the mulberry leaf silage displays stronger antioxidant activities and immunomodulatory, which has not been reported in sheep. Oxidative stress, induced by reactive oxygen species and free radicals, is usually regarded as one of the causes of cell damage and even disease during nowadays high-intensive farming conditions, which inhibits ruminant growth and induces production loss [20,21]. Free radicals can be eliminated by antioxidant enzymes such as SOD, GSH-PX, CAT, etc. [22], thus the activities of antioxidant enzymes are a re ection of the antioxidative function of animal. The supplementation of exogenous antioxidants or utilizing plant source rich in antioxidants could be effective to prevent oxidative stress and improve animal health [21,23]. It was reported mulberry leaves could be a source of natural antioxidants to combat oxidative stress in livestock production due to its scavenging of free radicals in animal feed [7]. Flavonoids and phenolic acids, presenting in mulberry leaf, were the large number of phytochemicals [24]. It was also found that the mulberry leaf is rich polyphenols, such as neochlorogenic acid, chlorogenic acid, rutin, quercetin, astragalin, and kaempferol [25], and the mulberry bioactive compounds showed the antioxidative capacity and anti-in ammatory effects [13,26]. The anti-oxidant effect of mulberry leaf avonoids was associated with increased SOD, CAT and GSH-PX expression, as well as reduced MDA levels [27,28]. It has been also reported that mulberry leaves could bene t animals through the enhancement of serum T-AOC and the activities of CAT and SOD [7]. In the present study, the activity of serum CAT, GPX-PX, SOD, and T-AOC were increased and the serum MDA was reduced after lambs fed with mulberry leaf silage, con rming the improved antioxidant status in the Tan-lambs due to the preserved bioactive components in mulberry leaf silage [13].
The effective of mulberry leaf or its bioactive compounds in improving antioxidant capacity and immune function has been found in many previous studies what we talked above and was con rmed in the current study. To date, there have been few reports on the relationship between the effect roles of mulberry leaf silage on ruminal microbial community and the potential microbial targeted terminal function in antioxidant activity and immunomodulatory. In a previous study, the ruminal microbiota composition of nishing steers was not changed by partial replacement of corn grain and cotton seed meal with ensile mulberry leaves [29]. However, Tan et al. [30] reported that the inclusive of mulberry leaf in diet can not only stimulate the animal growth, but manipulate the ruminal microorganisms in cattle. In the current study, we found several genera bacteria were enhance or decreased by the feeding of mulberry leaf silage, of these changed bacteria, several of them also showed signi cantly correlated with host serum metabolism. The Prevotella accounts to the abundant genera in sheep rumen in the present study, which was also shown in the Hu-sheep [31]. It was also reported that the Prevotella genus has the ability in proteolytic [32]. It was found that ruminal Prevotella species may affect the amino acids metabolism in dairy cow [16]. We also found the improved serum BUN in MS group. Thus, the nitrogen utilization e ciency might be reduced by decreasing the abundance of Prevotella_1 in Tan-lambs fed with mulberry leaf silage.
In the current study, rumen Ruminococcac-eae_UCG_010 and Lachnospiraceae_XPB1014_group were down-regulated by feeding lambs mulberry leaf silage, which were also negatively correlated with serum antioxidant parameters. Ruminococcaceae UCG-010 is associated with ruminal biohydrogenation [33]. The abundances of Lachnospiraceae_XPB1014_group were negatively correlated with body fat weight in nishing pigs, indicating its function in lipid metabolism [34]. Thus, the reduced abundance of Ruminococcac-eae_UCG_010 and Lachnospiraceae_XPB1014_group in MS group indicated the inhibited lipid biohydrogenation in rumen. Thus, the mulberry leaf silage may have the ability to prevent the ruminal biohydrogenation by decreasing Ruminococcaceae UCG-010 and Lachnospiraceae_XPB1014_group.
It was also found that the Schwartzia was increased in the MS group compared to the AS group. The mulberry leaf avonoid showed the ability in methane inhibiting in sheep [35]. Schwartzia was found to be negatively correlated with methane emissions [36]. Thus, the detected increased abundance of Schwartzia in the MS group from the current study and its positively correlated with antioxidant function might be due to the roles of Schwartzia in competing with methanogenic bacteria. Thus, these results may suggest the inhibited methane emission when lambs fed with mulberry leaf silage.
The immunoreaction is closely related to health of animals [37]. IFN-γ is involved in the initiation and regulation of the immune response [38]. It was found that water extracts of mulberry leaf mitigate in ammation through the interactions among insulin signalling pathway and TNF-α [39]. However, the current study did not nd the changes of serum glucose and TNF-α, but with increased serum IFN-γ concentration shown in the current study. Mulberry leaves silage probably contains polyphenols and polysaccharides that could exert similar effects, as polyphenols and polysaccharides can inhibit in ammatory processes by improving serum IFN-γ concentration or affecting microbiota [40,41]. In addition, cumulative evidences indicate that mulberry leaf polysaccharides have immunomodulating activity, which has been assessed on many different kind cells and macrophage-dependent immune system responses [42]. Here, we show, to our knowledge for the rst time, the ruminal Bi dobacterium and Lactobacillus genera were increased by feeding mulberry leaf silage in sheep which were also positively correlated with the serum IFN-γ concentration in the current study. It was found the bacterial supernatants of the Bi dobacterium and Lactobacillus genera, are able to affect ghrelin receptor (growth hormone secretagogue receptor-1a) signaling, which plays a crucial role in maintaining energy balance and metabolism, and modulating food intake, motivation, reward, and mood in human being [43]. Thus, in total, we estimated that the improved level of Bi dobacterium and Lactobacillus genera in rumen of MS group might contribute to the improved healthy level and status of lambs fed with mulberry leaf silage.
The increased Bi dobacterium in rumen of lamb were associated with the improved contents of meat nutrients comprising alpha-linolenic acid, conjugated linoleic acid and eicosapentaenoic acid [44]. Lactobacilli and Bi dobacterium are considered by the European Food Safety Authority, indicating they can be considered possible prebiotics for improvement of gut health [45]. Bi dobacteria was abundant in the digestive tract of humans, which has assumed health-promoting activities [46]. Vlková et al. [47] also found that the supplementation of Bi dobacterium strains promoted rumen health for calves in the milkfeeding period. On the other hand, Bi dobacteria can utilize a variety of iso avones, mono-and oligosaccharide, and polysaccharides [45]. Mulberry leaf contains abundant polysaccharides and other plant secondary metabolites [41,48], thus the increased abundance of rumen Bi dobacteria in the MS group might due to the feeding of mulberry leaf silage. Our ndings for the increased abundance of rumen Bi dobacteria in MS group compared to AS group and its highly positively correlation with serum IFN-γ seem to indicate the bene cial effects of genus Bi dobacterium on Tan-sheep.
Additionally, except for the described potentially differential functional bacterial, we observed that Fibrobacter, Lachnospiraceae_AC2044_group, Prevotellaceae UCG-003, and rumen_bacterium_YS3 were negatively correlated with the enhancement of serum antioxidant characteristics and immune function. In addition, we also found the increased abundance of Howardella, Pelagibacterium, Protochlamydia, Shuttleworthia, Advenella, and Planctomicrobium were achieved in the MS group. However, we did not nd the related function of these bacteria. More attention should be paid in detecting their function in further studies by screening and culture methods, as most of their potential function of them in restricting rumen health and host health is still unknown. In total, our research provides a preliminary view of the e cient utilization of mulberry leaf silage in lambs, and we detect several potential mulberry leaf silageassociated rumen bacteria that may interact with the host metabolism and account for the enhanced antioxidant capacity and immunomodulatory.

Silage preparation
Alfalfa and mulberry were cultivated and harvested on Wuzhong, Ningxia Hui Autonomous Region (37.99°N, 106.20°E). The fresh alfalfa and mulberry leaf were collected and chopped to 2-3 cm using a forage cutter (Lingong Machinery, Shandong, China). Then, both of the alfalfa and mulberry leaf were mixed with 1% glucose and ensiled with 1 × 10 6 colony forming units (cfu)/g of fresh material Lactobacillus plantarum (GenBank accession number: WCFS1) [49]. Both of these two kind silages were fermented well after 60 d ensiling based on the sensory evaluation and pH value measured by a pH meter (PHS-3C, Shanghai Leijun Experimental Instrument Co., Ltd., Shanghai, China) after the bale silage opening. Then, the ingredients were sampled and analyzed. The dry matter (DM) content of the alfalfa silage and mulberry leaf silage were 32.9 and 28.7%, respectively. The concentrations of crude protein (CP), neutral detergent ber (NDF), acid detergent ber (ADF), ash, and ether extract (EE) in alfalfa silage were 15 (Table S1). The animals in each diet group were randomly placed in 4 pens with 5 lambs in each pen. The two groups lambs were both fed with a basal total mixed ration diet. The AS and MS diets contain (DM basis), 14.6 and 14.7% of CP, 31.1 and 32.4% of NDF, 17.8% and 18.2% of ADF, 4.70% and 4.94% of EE, 6.20 and 6.15% of ash, respectively. The feed intake was recorded daily and was measured based on the difference between the amount of feed deliveries and the remaining. The experiment lasted for 80 days, including a 20 days' adaptation and 60 days' formal feeding. All the lambs had ad libitum access to feed and water.
The BW of each lambs were recorded every 20 days. On the day 80 (the end of the experiment), six lambs in each group were then selected for blood sampling and slaughtering based on the average BW. Blood was collected from the jugular vein of the sheep to separate serum (centrifuged for 10 min at 3,000 × g). After slaughtering, the weight of carcass, head, skin, limbs, internal organs (heart, liver, spleen, lung, kidney) were measured and recorded. All the collected samples were then stored at liquid nitrogen for subsequent analysis.
The detail information of the meat quality detection was according to a previous study of Liang et al. [50]. In brief, body fat (assessed as GR) value was assessed by measuring the thickness at the 12th/13th rib intersection 110 mm away from the midline using a vernier caliper. The pH value and meat colour (redness (a*), yellowness (b*), and lightness (L*), psychometric chroma (c* = (a 2 + b 2 ) 0.5 ), and Hue angle (H* = arctan (b*/a*) × (180/π)) at 45 min and 24 h were measured. Each sample was determined three times at different positions on the meat surface and the average value was obtained. Serum metabolite concentrations, including total protein, albumin, BUN, triacylglycerol, total cholesterol, high density lipoprotein cholesterol, low density lipoprotein cholesterol, immunoglobin G, immunoglobin A, and immunoglobin M were measured using an automatic analyzer (Kehua ZY KHB-1280, Shanghai, China) with a commercial kits (Shanghai Kehua Biological Technology Co. Ltd., Shanghai, China) [51]. The activities of antioxidant enzymes such as SOD, CAT, GSH-PX, T-AOC and MDA, and the concentrations of IFN-γ and TNF-α were determined by commercial kits (Jiancheng Biological Technology Co. Ltd., Nanjing, China) following the manufacturer's instructions.
Rumen uid was collected from the ventral part of the rumen by straining the ruminal content through four layers of cheesecloth. Rumen uid pH was measured immediately. Rumen uid samples were then stored at liquid nitrogen for subsequent analysis. The concentrations of volatile fatty acids were measured by gas chromatography (Trace 1300; Thermo Fisher Scienti c Co., Ltd., Shanghai, China). The ammonia nitrogen was determined by the method described in Broderick and Kang [52].
Pro ling of rumen bacterial community diversity Microbial DNA from rumen uid was extracted by a HiPure Stool DNA Kit (Magen, Guangzhou, China) according to manufacturer's protocols. The 16S rDNA V3-V4 region of the ribosomal RNA gene were ampli ed by PCR using primers 341F: CCTACGGGNGGCWGCAG; 806R: GGACTACHVGGGTATCTAAT following the procedure described by Sun et al. [53]. The amplicons were puri ed by the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, U.S.). Then it was quanti ed by ABI StepOnePlus Real-Time PCR System (Life Technologies, Foster City, USA).
Puri ed amplicons were pooled in equimolar and paired-end sequenced (PE250) on an Illumina platform (Illumina Novaseq 6000 sequencing) according to the standard protocols. Raw reads were further ltered using FASTP to get high quality clean reads [54]. The noisy sequences of raw tags were ltered by QIIME (version 1.9.1) [55]. Then, chimeric tags were removed using UCHIME algorithm. The nally obtained effective tags were clustered into operational taxonomic units (OTUs) of ≥ 97 % similarity. Based on the SILVA database (version 132), the representative sequences were assigned to organisms by a naive Bayesian model using RDP classi er (Version 2.2). The abundance statistics of each taxonomy were constructed using a Perl script and visualized by SVG. The alpha index including Chao, Simpson, Sobs, Shannon, and Ace were calculated in QIIME. Unweighted non-metric multi-dimensional scaling (NMDS) was generated in R project Vegan package (version 2.5.3). Statistical analysis of Wilcoxon rank test and Anosim test was calculated in R project Vegan package (version 2.5.3). The bacterial community comparison between the AS and MS groups was calculated by Wilcoxon rank test in R project Vegan package (version 2.5.3). Pearson correlation coe cient between environmental factors and species and between blood parameters and species was calculated in R project psych package (version 1.8.4). Network of these correlation coe cients were generated using igraph package (version 1.1.2) in R project.

Statistical analyses
The animal performance, blood parameters, and rumen fermentation characteristics data were analyzed for a completely random design using the PROC MIXED procedure of SAS (version 9.4, SAS Institute Inc., Cary, NC). The means of each treatments are presented as least squares means and statistical signi cance was de ned at P < 0.05, and the trends were declared at 0.05 ≤ P ≤ 0.10.
We here identify a potent effect of mulberry leaf silage-associated ruminal microbiota interact with host metabolism and physiology. The enriched ruminal Bi dobacterium, Lactobacillus, and Schwartzia, and the down-regulated ruminal Ruminococcaceae UCG-010 and Lachnospiraceae_XPB1014_group in the MS group lambs might contribute to the bioconversion and bioactive function of mulberry leaf silage in rumen. Furthermore, these speci c mulberry leaf silage-associated bacteria were associated with the enhanced animal antioxidant capacity or immunomodulatory. Altogether, evidence from the results of the current study suggest that the naturally mulberry leaf silage could be an alternative functional supplement in maintaining animal health by rescheduling the rumen bacterial community.

Supplementary information
Supplementary information accompanies this paper at Additional le 1: Table S1. Ingredients and nutrient composition of the total mixed ration containing alfalfa silage (AS) and mulberry leaf silage (MS) as the main forage. Table S2

Availability of data and materials
The data sets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. The raw reads of 16S rRNA sequencing data have been deposited into the NCBI Sequence Read Archive (SRA) database (Accession Number: SRP265735).

Ethical approval and consent to participate
The animals used in this study were approved by the Animal Care Committee of China Agricultural University (Beijing, China; approval no. AW30129102-1-1; approval date: 26 October 2019). The detail experimental procedures were following the university's guidelines for animal research.

Consent for publication
Our study does not contain data from any individual person. Hence this section is not applicable.        The core speci c bacterial biomarker. Signi cantly different genus bacteria between alfalfa silage (AS) and mulberry leaf silage (MS) treatments (B) were tested by Wilcoxon rank-sum test with P-value of < 0.05 (A). LEfSe (Linear discriminant analysis Effect Size) determined using the Wilcoxon rank sum test (p < 0.05) with a linear discriminant analysis (LDA) score analysis shows differentially abundant bacteria communities between alfalfa silage (AS) and mulberry leaf silage (MS) treatments (B); The cladogram shows the taxonomic levels represented by rings with six layers from the inside of this plot to the outside, corresponding to six levels of taxonomy (kingdom, phylum, class, order, family, and genus). Each node (small circle) represents a taxon (C). Green and red columns or nodes represent the bacteria with the signi cant higher relative abundance in MS and AS group, respectively.

Supplementary Files
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