Effects of wilting and additives on fermentation characteristics, microbial composition, metabolome, and ruminal degradation properties of mulberry silage

Effects of wilting and additives on fermentation characteristics, microbial composition, metabolome, and ruminal degradation properties of mulberry silage | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Effects of wilting and additives on fermentation characteristics, microbial composition, metabolome, and ruminal degradation properties of mulberry silage Fangshu Di, Jian Gao, Jing Ma, Xi Wang, Yufei Jiang, Shixiu Qiu, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7345171/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 10 Jan, 2026 Read the published version in BMC Microbiology → Version 1 posted 13 You are reading this latest preprint version Abstract Background Optimizing the processing technology of mulberry silage is a prerequisite for enhancing the utilization efficiency of mulberry resources. This study examined effects of wilting pretreatment and silage additives on mulberry silage fermentation, microbiota, metabolites, and ruminal degradation. Lactobacillus plantarum (LP), organic acids (OA), and a control treatment without additives were applied to unwilted (73% moisture) or wilted (62% moisture) mulberry forage. Results Wilting significantly enhanced lactic acid and crude protein (CP) contents, and lowered pH ( P < 0.05). Adding OA or LP additives reduced pH and increased CP content ( P < 0.05). LP treatment further reduced ammonia nitrogen and pH, improved lactic acid content ( P < 0.05). Pre-wilted mulberry inoculated with LP showed further reductions in acetic acid and neutral detergent fiber contents ( P < 0.05). LP treatment enriched Lactiplantibacillus and suppressed Enterococcus ( P < 0.05). Lactiplantibacillus was strongly correlated with lactic acid, CP, and beneficial metabolites L-arginine and salicin ( P < 0.05). These metabolites were enriched in the phosphotransferase system and arginine biosynthesis pathways ( P < 0.05). Wilting improved DM digestibility while reducing methane and ammonia nitrogen level. LP treatment reduced ruminal ammonia nitrogen level ( P < 0.05). Pre-wilted mulberry inoculated with LP further increased microbial protein content ( P < 0.05). Conclusion In conclusion, combining wilting pretreatment and LP inoculant offers an effective strategy to enhance silage quality. Mulberry silage Wilting Silage additives Microbial composition Metabolites Ruminal degradation Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction In recent years, with the rapid development of the livestock industry, the demand for livestock products has increased dramatically, and the shortage of feed resources has become an important factor constraining the development of the industry [ 1 ] . To meet the challenge of insufficient supply of feed resources, it is urgent to develop more available forage resources for animal feed. Mulberry ( Morus alba L.) is considered a protein feed resource with great potential due to its high annual yield, rich crude protein content, and abundance of various bioactive compounds, which is expected to effectively alleviate the feed shortage problem [ 2 , 3 ] . However, biomass harvesting from mulberry trees is seasonal in nature and thus requires drying or ensiling of mulberry for long-term preservation. However, there are some potential limitations of haymaking that can lead to nutrient loss and its storage process can be affected by weather changes. Therefore, silage can better preserve the nutrient content of mulberry and become a more suitable preservation method compared to the traditional haymaking method [ 4 ] . Ensiling is based on the principle of anaerobic fermentation, fresh forage is sealed and preserved, and soluble carbohydrates are converted into organic acids mainly lactic acid by lactic acid bacteria fermentation to inhibit spoilage microorganisms, thus realizing the purpose of long-term preservation of forage. [ 5 , 6 ] . Therefore, the success of silage depends to a large extent on whether the lactic acid bacteria in the raw material can multiply and ferment rapidly in an anaerobic environment. However, a high moisture content, high buffer capacity, and insufficient number of LAB colonizing the surface of mulberry leaves may result in incomplete fermentation of mulberry silage [ 7 , 8 ] . Therefore, wilting and additives are commonly used to achieve the desired silage fermentation quality [ 9 – 11 ] . Wilting is commonly applied in forage ensiling to reduce moisture content. This method inhibits microbial growth and prevents proteolysis by undesirable microorganisms like Clostridium , thereby supporting fermentation [ 12 , 13 ] . In addition, the selection and application of additives also play an important role in regulating the fermentation process. Studies have shown that the rational use of additives can promote the rapid formation of acidic environment, so as to optimize the fermentation characteristics of silage. Among them, organic acid additives can inhibit the proliferation of spoilage bacteria and other harmful microorganisms through direct acidification, enhance aerobic stability and improve the overall quality of silage [ 5 ] . The addition of lactic acid bacteria can rapidly expand the base number of lactic acid bacteria in silage, make it become the dominant bacteria in silage, inhibit the activity of aerobic microorganisms, and improve the quality of silage fermentation [ 14 ] . It is worth noting that the silage process is accompanied by a complex microbial metabolic process, and the dynamic changes of its microbial community structure and metabolite profiles are important indicators for measuring the fermentation quality and revealing the fermentation mechanism [ 12 ] . Therefore, in-depth investigation of the microecological characteristics of mulberry silage and its regulatory pathways is of great significance for improving silage quality and realizing efficient utilization of feed resources. In view of this, this study evaluates the effects of wilting and additives on silage fermentation parameters, microbiota composition, metabolites, rumen degradation properties, with a view to providing theoretical basis for the optimization of mulberry silage technology and its application in livestock production. Materials and methods Materials Mulberry forage (cultivar Chuansisang 1) was harvested at an average height of 1.0-1.2 m with a stubble height of 10–20 cm at the experimental site in Chengdu City, Sichuan Province, China (103.49°E, 30.42°N). Mulberry raw material contained 27.4% dry matter (DM), 18.9% crude protein (CP), 34.4% neutral detergent fiber (NDF), 24.7% acid detergent fiber (ADF), and 8.4% water-soluble carbohydrates (WSC), DM basis, respectively. The silage additives, including of organic acids and lactic acid bacteria, were supplied by Jilongda Biotechnology Co., Ltd. (Sichuan, China). Experimental Design and Treatment. The wilting pretreatment (wilted vs. fresh mulberry with 62% vs. 73% moisture content) and silage additive, including control, Lactobacillus plantarum (LP) and organic acids (OA) treatments, were evaluated in a completely randomized design with 6 replications using a 2 × 3 factorial arrangement. Half of the collected mulberries are exposed to the sun for 3 h and the other half are kept fresh [ 16 ] . The entire mulberry plant was severed into segments measuring 1–2 cm, subsequently subjected to uniform treatment involving one of three silage additives: no additive control was treated with 1 mL distilled water per kilogram of fresh matters; LP, applied at a rate of 1 × 10 8 cfu per g of fresh matter (1 mL/kg per kilogram) dissolved in 1 mL of distilled water and then sprayed uniformly on the mulberries; and a mixture of OA comprising ammonium propionate, formic acid, and propionic acid in a 9:7:1 ratio, applied at 2 mL per kg of fresh matter diluted with 1 mL distilled water before inoculation to equalize total moisture addition across treatments [ 16 ] . The additives were sprayed uniformly onto the mulberries at the designated dosages, thoroughly mixed, and then packed into specialized polyethylene vacuum bags (500 g per bag). A total of 36 silage samples (6 replicates × 2 moisture treatments × 3 additive treatments) were immediately sealed using a vacuum sealer and stored at 25–28 ℃ for 60 days. These comprised six experimental groups: fresh control (FC), fresh OA-treated (FO), fresh LP -inoculated (FL), wilted control (WC), wilted OA-treated (WO), and wilted LP -inoculated (WL). Samples after ensiling were taken for later analysis. Fermentation quality. After 60 d of ensiling, the silage bags were opened and 20 g of silage sample from each bag were mixed with distilled water (180 mL) stored in a 4 ℃ refrigerator for 24 h before filtering through 4 layers of medical gauze. The pH value of the filtrate was immediately checked using a pH meter (Sartorius PB-30L, Beijing, China). NH 3 -N content was quantified via phenol-hypochlorite colorimetry with ninhydrin detection [ 17 ] . The analysis of volatile fatty acids (VFAs) and lactic acid were conducted using gas chromatography (Agilent 7890B, Santa Clara, USA) in accordance with the methodologies delineated by Wang et al. [ 16 ] . Chemical analysis. Silage samples were analyzed for chemical composition based on DM. DM concentrations were measured by drying at 65°C for 48 h [ 16 ] . Then, the samples ground through a 1.0 mm screen for nutrient analyses. The ash (942.05), ether extract (EE, 920.39), and CP (984.13) contents were analyzed using the AOAC procedures [ 18 ] . The NDF, ADF, and acid detergent lignin (ADL) were determined using ANKOM 2000 Fiber Analyzer (Ankom Technology Corp., Macedon, NY, USA), as described by Van Soest et al. [ 19 ] . The WSC content was analyzed by Arthur Thomas using anthrone reagent colorimetry [ 20 ] . Bacterial community of mulberry silage. Microbial genomic DNA was extracted from silage samples (3 randomly selected biological replicates) using the E.Z.N.A.® Soil DNA Kit (Omega Bio-tek, Norcross, GA, USA) following the manufacturer's protocol. The concentration and purity of the extracted DNA were determined using a NanoDrop 2000 UV–vis Spectrophotometer (Thermo Scientific, Wilmington, USA), and the quality of the extracted DNA was assessed using 1% agarose gel electrophoresis. The bacterial 16S rRNA gene were amplified using primers 27F (5’-AGRGTTYGATYMTGGCTCAG-3’) and 1492R(5’-RGYTACCTTGTTACGACTT-3’). The PCR amplification program was referred to Song et al. [ 21 ] . Amplicons were purified using AMPure® PB beads (Pacific Biosciences, CA, USA) and quantified via Qubit 4.0 (Thermo Fisher Scientific, USA). SMRTbell libraries constructed from equimolar pools (Prep Kit 3.0, Pacific Biosciences) were sequenced on Sequel IIe (Pacific Biosciences) via Bio-Pharm Technology Co. Ltd. (Shanghai, China). Raw reads were processed in QIIME2 (v2020.2) with DADA2 to generate single-nucleotide resolution ASVs, which were taxonomically classified against SILVA 16S rRNA (v138) using QIIME2's Naive Bayes classifier. Mulberry silage metabolomics analysis. Silage metabolites were profiled via metabolomics. Samples (50 mg) were homogenized in 400 µL ice-cold methanol: water (4:1, v/v) with 0.02 mg/mL L-2-chlorophenylalanine (internal standard). After settling at -10°C, samples were processed using a Wonbio-96c high-throughput tissue crusher (Wanbo Biotechnology, Shanghai) at 50 Hz for 6 min, followed by ultrasonication at 40 kHz for 30 min at 5°C. Subsequent protein precipitation was performed at -20°C for 30 min. Supernatants collected after centrifugation (13,000 × g, 4°C, 15 min) were subjected to LC-MS/MS analysis. (n = 6, wilted LP treatment n = 4). Pooled QC samples ensured analytical reproducibility. The metabolites in the supernatant were analyzed by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS, UHPLC-Q Exactive, Thermo fisher, USA). The UHPLC-MS/MS workflow followed the method of Wang et al. [ 22 ] . In Vitro rumen fermentation. Rumen fluid collection and buffered inoculum preparation followed the methods of Menke et al. [ 23 ] and Wang et al. [ 16 ] . Rumen contents were collected 2 h post-morning feeding from four ruminally fistulated Jianzhou goat (39.32 kg ± 2 kg on average). Equal aliquots from each animal were combined, transported in pre-warmed anaerobic containers, and processed within 30 min of collection. All goats were fed twice daily (08:00 and 17:30) with diet consisting of 32.7% corn grain, 20.5% alfalfa hay, 11.3% soybean meal, 3% wheat bran, 2% vitamin and mineral premix, and 1% sodium bicarbonate. Artificial buffer was prepared according to Menke et al. [ 23 ] . The rumen fluid was then mixed with buffer (1:2 v/v) under continuous CO 2 supply. After thorough mixing, approximately 1,000 mg of mulberry silage and 100 mL of the resulting inoculum were added to 250 mL culture bags (Hangzhou Anstey Technology Co., Ltd., Hangzhou, China) and sealed anaerobically. Afterward, The samples were incubated at 39°C for 48 h at 45 r/min [ 24 ] . Total gas production (TGP) was measured using a graduated syringe, with 2 blank bags containing only rumen fluid to account for background gas. NH 3 -N and VFAs measurements were made as described above. After 48 h of incubation, the bags were washed with tap water, then dried in an oven at 105°C for 48 h to determine the DM digestibility (DMD) [ 16 ] .Methane (CH 4 ) content was determined via flame ionization GC with a 19095P-Q04 capillary column (30 m × 0.53 mm × 40.00 µm; Agilent), as described by Chen et al. [ 25 ] . Microbial protein (MCP) content was determined by the method of Makkar et al. [ 26 ] . Data analysis. The data was analyzed using the PROC GLM program in SAS 9.4 software, with the model specified as Y ijk = µ + E i +A j +E i ×A j +e ijk . where Y ijk = observed value, µ = mean value, E i = wilting pretreatment, (i = 2, respectively, for the wilted and fresh); A j = additive inoculation (j = 3, respectively, the control, LP and OA). E×A ij = interaction between environmental conditions and additive inoculation and e ijk =error. Duncan’s test was used for pairwise mean comparisons. The significant level was set at α = 0.05. Microbial data was analyzed using Majorbio platform ( https://cloud.majorbio.com ). Principal coordinate analysis (PCoA) based on the Bray–Curtis distance algorithm (R package, version 3.6.1). The alpha diversity based on the ASV level colony abundance table (Supplementary Table 1) and community composition the top 10 genera in relative abundance (Supplementary Table 2) were evaluated via two-way ANOVA using SAS 9.4 software. LC-MS metabolomics raw data was analyzed by Progenesis QI software (Waters Corporation, Milford, USA). Metabolites were identified using the Human Metabolome Database ( http://www.hmdb.ca/ ), METLIN Metabolite Database ( https://metlin.scripps.edu/ ), and Majorbio database, and then analyzed on the Majorbio cloud platform. We calculated Variable Importance in Projection (VIP) scores using Principal Component Analysis (PCA) and Partial Least Squares Discriminant Analysis (PLS-DA). The significant differences among metabolites between treatments were tested by Welch’s two-sample t-test based on the criteria of VIP values > 1.0, P 1.5. The metabolic pathway diagram was drawn referring to the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Spearman correlation analysis assessed relationships among key microbial genera, differential metabolites, and fermentation properties, with results visualized using the heatmap function (R package, version 2.8.2) Results Fermentation quality of mulberry silage. As shown in Table 1 , Adding OA and LP treatments increased acetate content (1.78%, 2.55% vs 1.14%, P < 0.05) in fresh mulberry. Addition of OA or LP also increased acetic acid content compared to pre-wilted mulberry without any additives (2.60%, 2.10% vs 1.29%, P < 0.05). Lactic acid (3.21% vs 2.86%, DM basis) and pH (4.35 vs 4.42) were higher ( P < 0.05) in pre-dried mulberry silage compared to fresh mulberry. Inoculation with LP decreased the NH 3 -N content of silage from 2.17–1.06%, decreased pH from 4.47 to 4.31, and increased the lactic acid content from 2.62 to 3.60% compared to mulberry without any additives ( P < 0.05). Inoculation with OA reduced pH from 4.47 to 4.38 compared to control ( P < 0.05). No butyrate was detected in all groups except the fresh control group, and propionate was only detected in the fresh mulberry inoculated with OA treatment. Table 1 Fermentation quality of whole-plant mulberry silage ((% Dry Matter Basis except for ammonia-N) Moisture Additives 1 pH NH 3 -N(%TN) 2 Lactic acid Acetate Propionate Butyrate Fresh 4.42 a 1.82 2.86 b 1.82 0.47 0.96 Wilted 4.35 b 1.72 3.21 a 1.82 - - SEM 0.012 0.051 0.116 0.058 NA NA Control 4.47 a 2.17 a 2.62 b 1.21 ND 0.96 OA 4.38 b 2.08 a 2.88 b 2.32 0.47 - LP 4.31 c 1.06 b 3.60 a 1.92 - - SEM 0.015 0.062 0.142 0.063 NA NA Fresh Control 4.46 2.24 2.62 1.14 d - 0.96 Fresh OA 4.40 2.16 2.63 2.55 c 0.47 - Fresh LP 4.36 1.07 3.34 1.78 a - - Wilted Control 4.44 2.10 2.63 1.29 d - - Wilted OA 4.36 2.01 3.14 2.10 b 0.065 - Wilted LP 4.25 1.05 3.86 2.08 b NA - SEM 0.015 0.084 0.194 0.089 NA NA P -value Moisture < 0.001 0.156 0.044 0.968 NA NA Additives < 0.001 < 0.001 < 0.001 < 0.001 NA NA M×A 0.071 0.735 0.35 0.002 NA NA 1 OA: organic acid treatment consisted of a mixture of ammonium propionate, formic acid, and propionic acid at 9:7:1 ratio, 2 mL/kg of fresh matter; LP: Lactobacillus plantarum treatment, 108 cfu of Lactobacillus plantarum per g of fresh matter. 2 NH 3 -N: Ammonia nitrogen, TN: total nitrogen; -: not detected; NA: not available due to non-detectable data. a−d Means within a column with different superscripts differ ( P < 0.05). Chemical characteristics of mulberry silage. As shown in Table 2 , both OA and LP additives reduced WSC content in pre-wilted silage ( P < 0.05). OA and LP additives significantly reduced the ash content of fresh mulberries ( P < 0.05). Inoculation with OA reduced ash content compared to pre-wilted mulberries without any additives ( P < 0.05). Adding OA reduced NDF in fresh mulberries, while adding OA and LP reduced NDF in pre-wilted mulberries ( P < 0.05). Wilting pretreatment significantly decreased ADF and ADL, while increasing DM, CP and EE contents ( P < 0.05). Both adding OA and LP decreased ADF and ADL content and increased CP and EE content ( P < 0.05). Table 2 Chemical composition of whole-plant mulberry silage (% Dry Matter Basis) Moisture Additives 1 DM 2 Ash 2 CP 2 EE 2 NDF 2 ADF 2 ADL 2 WSC 2 Fresh 26.42 b 13.49 16.61 b 3.87 b 30.56 20.38 a 5.67 a 4.43 Wilted 38.00 a 13.41 16.98 a 4.13 a 29.23 19.59 b 4.87 b 3.53 SEM 0.145 0.052 0.046 0.081 0.268 0.200 0.665 0.155 Control 32.14 13.87 16.48 c 3.72 b 30.85 20.96 a 6.07 a 4.42 OA 32.09 13.39 16.86 b 4.00 a 29.26 19.25 b 5.27 b 4.20 LP 32.40 13.08 17.05 a 4.29 a 29.59 19.75 b 4.47 c 3.34 SEM 0.270 0.063 0.057 0.099 0.324 0.244 0.081 0.198 Fresh Control 26.27 14.17 a 16.34 3.69 31.41 a 21.16 6.47 4.51 ab Fresh OA 26.34 13.33 bc 16.57 3.98 29.35 bc 19.56 5.57 4.59 a Fresh LP 26.64 12.97 d 16.92 3.97 30.92 a 20.43 4.96 4.21 ab Wilted Control 38.00 13.57 b 16.62 3.77 30.28 a 20.77 5.66 4.32 ab Wilted OA 37.84 13.46 bc 17.14 4.02 29.17 b 18.92 4.97 3.80 b Wilted LP 38.16 13.20 cd 17.19 4.61 28.25 b 19.07 3.98 2.47 c SEM 0.245 0.086 0.077 0.134 0.065 0.332 0.11 0.260 P -value Moisture < 0.001 0.274 < 0.001 0.029 0.441 0.009 < 0.001 < 0.001 Additives 0.458 < 0.001 < 0.001 0.002 0.001 < 0.001 < 0.001 0.002 M×A 0.861 < 0.001 0.117 0.090 0.003 0.387 0.278 0.039 1 OA: organic acid treatment consisted of a mixture of ammonium propionate, formic acid, and propionic acid at 9:7:1 ratio, 2 mL/kg of fresh matter; LP: Lactobacillus plantarum treatment, 108 cfu of Lactobacillus plantarum per g of fresh matter. 2 DM: dry matter; Ash: crush ash; CP: crude protein; EE: Ether Extract; NDF: neutral detergent fiber with ash correction; ADF: acid detergent fiber; ADL: acid detergent lignin; WSC: water soluble carbohydrate. a−c Means within a column with different superscripts differ ( P 0.99), indicating that most bacterial communities were identified correctly. Regardless of the presence of additives, the levels of ACE, Chao1 and Sobs decreased in pre-wilted silages. ( P < 0.05) (Supplementary Table S1 ). Silage microbial communities differed between those with and without additives, as indicated by a decline in the Shannon index with LP inoculation ( P < 0.05) (Supplementary Table 1). The PCoA plots showed that in fresh silage, the treatment groups were clearly separated; in pre-wilted silage, the control and LP groups were clearly separated and there was no clear separation between the other treatment groups (Fig. 1 A). At the phylum level, the bacterial community in mulberry silages was dominated by Firmicutes (84.43 ~ 92.75%), Proteobacteria (2.47 ~ 6.35%), and Cyanobacteria (2.61 ~ 8.46%) (Fig. 1 B). The most abundant genera in all silages were Lactiplantibacillus (30.19 ~ 68.62%), Levilactobacillus (4.15 ~ 24.17%), Weissella (0.90 ~ 14.24%), and unclassified_p__Cyanobacteria (2.56 ~ 8.38%) (Fig. 1 C). In LP-treated silage, the abundances of Lactiplantibacillus increased, while Enterococcus decreased ( P < 0.05). Adding LP resulted in an increase in unclassified_p_Cyanobacteria in fresh silage ( P < 0.05). Adding LP resulted in undetectable level of Complanilactobacillus in silages (Fig. 1 D, Supplementary Table 2). Analyses of the metabolite profiles. A total of 4638 metabolites were identified in the silage, of which 168 were annotated by the KEGG compound database at the compounds with biological roles class level, mainly including 39 lipids, 31 peptides, 19 organic acids, 18 carbohydrates, and 17 hormones and transmitters (Fig. 2 A). PLS-DA results showed minimal variation among biological replicates, with replicates clustering tightly within their respective treatment groups (Fig. 2 B), suggesting the sufficient reproducibility and reliability of the experiment. The metabolite compositions in the fresh silages with additives were separated from the control, whereas groups of the LP and the control were separated from each other in pre-wilted silage, indicating that adding OA and LP have a great influence on the metabolites in mulberry silage. Differential metabolites were identified according to the following criteria VIP values > 1.0, P 1.5 (Supplementary file 1). The FO group showed 73 metabolites up-regulated and 24 metabolites down-regulated relative to the FC group (Fig. 2 A). Among these substances, 15-hydroxynorandrosten-3,17-dione glucuronide was downregulated while ascorbic acid was upregulated, both enriched in ascorbate and aldarate metabolism pathway (ko00053). Concomitantly, L-cysteine showed upregulation whereas (R)-pantoate was downregulated, with these metabolites significantly enriched in pantothenate and CoA biosynthesis pathway (ko00770). Additionally, salicin and ascorbic acid were upregulated, both enriched to phosphotransferase system pathway (PTS) (ko02060) ( P < 0.05; Fig. 3 A). The FL group showed 151 up-regulated and 122 down-regulated metabolites relative to FC group (Fig. 2 B). Among these substances, 15-hydroxynorandrosten-3,17-dione glucuronide was down-regulated and ascorbic acid up-regulated, both enriched in ascorbate and aldarate metabolism pathway (ko00053). Concomitantly, salicin, chitobiose, and ascorbic acid were upregulated, both enriched to phosphotransferase system pathway (PTS) (ko02060) Additionally, L-arginine and citrulline showed significant up-regulation and were enriched in arginine biosynthesis pathway (ko00220) ( P < 0.05; Fig. 3 B). The FO group had 73 up-regulated and 20 down-regulated metabolites compared to the FL group (Fig. 2 C). Indoxyl and 5–hydroxyindoleacetylglycine were up-regulated, both enriched to tryptophan metabolism pathway (ko00380) (( P < 0.05; Fig. 3 C). The WO group showed upregulation of 4 differential metabolites and downregulation of 5 differential metabolites relative to the WC group (Fig. 2 D). Ascorbic acid was significantly upregulated and enriched in the ascorbate and aldarate metabolism pathway (ko00053) and PTS pathway (ko02060) ( P < 0.05; Fig. 3 D). Compared to the WC group, the WL group showed 38 up-regulated and 36 down-regulated metabolites (Fig. 2 E). 15-hydroxynorandrosten-3,17-dione glucuronide was downregulated while ascorbic acid was upregulated, both enriched in ascorbate and aldarate metabolism pathway (ko00053) ( P < 0.05). Meanwhile, cis-aconitic acid was up-regulated, tending to affect the citric acid cycle pathway (ko00020) ( P = 0.060) (Fig. 3 E). There were 32 differential metabolites was up-regulated and 25 differential metabolites were down-regulated in WO group compared with the WC group (Fig. 2 F). N-hydroxy-L-tyrosine was downregulated, tending to affect the cyanoamino acid metabolism pathway (ko00460). N-formyliminyl-glutamic acid was upregulated, tending to affect the histidine metabolism pathway (ko00340) ( P < 0.1; Fig. 3 F). Correlation of microbial, chemical composition, fermentation characteristics and metabolites Lactiplantibacillus positively correlated with CP and lactic acid contents, while negatively correlating with pH, acetic acid, NH 3 -N, ash, WSC, and ADL contents. Conversely, Enterococcus was positively correlated with pH, acetic acid, and ADL contents, whereas exhibiting negative correlations with CP and lactic acid contents. Companilactobacillus positively correlated with pH, acetic acid, NH 3 -N, ash, and WSC but negatively correlated with EE, CP, and lactic acid contents ( P < 0.05; Fig. 4 A). Lactiplantibacillus demonstrated positive correlations with L-arginine, salicin, and cis-aconitic acid yet negative correlation with N-hydroxy-L-tyrosine ( P < 0.05). In contrast, Enterococcus and Companilactobacillus exhibited consistent positive correlations with N-hydroxy-L-tyrosine, 15-hydroxynorandrostene-3,17-dione glucuronide, and (R)-pantoate, while displaying inverse negative correlations with L-arginine, salicin, cis-aconitic acid, citrulline, and ascorbic acid ( P < 0.05; Fig. 4 B). In Vitro ruminal fermentation of mulberry silage. As shown in Table 3 , inoculating LP enhanced MCP content in fresh mulberry. The addition of OA and LP increased MCP content compared to pre-wilted mulberries without any additives, with the LP treatment having higher MCP content than the OA treatment. The wilting pretreatment increased the pH, DMD, and acetate content, whereas a decreased NH 3 -N and CH 4 production, acetate, acetate to propionate content ( P < 0.05). Adding OA or LP additives helped to increased TGP content ( P < 0.05). Adding LP also reduced NH 3 -N content ( P < 0.05). Adding OA also increased isobutyrate content ( P < 0.05). Table 3 In vitro rumen fermentation characteristics of whole-plant mulberry silage. Moisture Additives 1 DMD 2 (%) TGP 2 (mL/g) CH 4 2 (mL/g) NH 3 -N 2 (mg/dL) MCP 2 (mg/ml) TVFA 2 (mL/g) Acetate (mmol/L) Propionate (mmol/L) Butyrate (mmol/L) Isobutyrate (mmol/L) Valerate (mmol/L) Isovalerate (mmol/L) A/P 2 Fresh 45.57 b 216.00 20.76 a 19.38 a 0.41 50.56 41.49 a 7.75 2.43 0.68 0.84 1.02 5.44 a Wilted 47.63 a 218.36 19.67 b 18.37 b 0.69 53.05 37.90 b 7.99 2.26 0.67 0.75 0.85 4.75 b SEM 0.555 1.201 0.246 0.243 0.007 1.276 1.097 0.219 0.117 0.024 0.047 0.067 0.093 Control 47.48 210.14 b 20.39 19.49 a 0.61 51.46 39.93 7.83 2.31 0.66 b 0.80 ab 0.97 5.16 OA 45.64 220.08 a 19.95 18.96 ab 0.67 50.96 38.09 7.910 2.60 0.77 a 0.91 a 1.08 4.89 LP 46.69 221.31 a 20.31 18.18 b 0.68 53.00 41.08 7.87 2.13 0.59 b 0.67 b 0.76 5.23 SEM 0.679 1.471 0.301 0.298 0.008 1.564 1.344 0.249 0.144 0.030 0.058 0.082 0.213 Fresh Control 47.46 207.55 21.09 20.17 0.58 d 49.10 43.13 7.45 2.35 0.67 0.83 1.07 5.85 Fresh OA 43.54 220.17 20.56 19.27 0.60 d 48.91 41.05 8.01 2.76 0.76 1.07 1.24 5.27 Fresh LP 45.73 220.28 20.64 18.71 0.66 bc 53.68 40.30 7.79 2.20 0.60 0.69 0.76 5.19 Wilted Control 47.50 212.73 19.69 18.80 0.64 c 53.82 36.74 8.21 2.28 0.64 0.77 0.87 4.47 Wilted OA 47.74 220.00 19.34 18.64 0.69 b 53.02 35.12 7.81 2.45 0.78 0.81 0.91 4.51 Wilted LP 47.66 222.34 19.98 17.66 0.75 a 52.31 41.85 7.95 2.06 0.58 0.66 0.76 5.27 SEM 0.961 2.081 0.426 0.436 0.011 2.211 1.900 0.351 0.203 0.024 0.081 0.116 0.301 P -value Moisture 0.022 0.191 0.008 0.012 < 0.001 0.194 0.039 0.442 0.316 0.747 0.181 0.086 0.027 Additives 0.200 < 0.001 0.560 0.029 < 0.001 0.643 0.319 0.977 0.103 0.005 0.041 0.053 0.589 M×A 0.138 0.478 0.671 0.685 < 0.001 0.349 0.104 0.439 0.830 0.839 0.562 0.378 0.099 1 OA: organic acid treatment consisted of a mixture of ammonium propionate, formic acid, and propionic acid at 9:7:1 ratio, 2 mL/kg of fresh matter; LP: Lactobacillus plantarum treatment, 108 cfu of Lactobacillus plantarum per g of fresh matter. 2 DMD: DM digestibility; TGP: total gas production; CH 4 : methane; TVFA: total volatile fatty acids; NH 3 -N: ammonia nitrogen; MCP: microbial protein; A/P: acetate to propionate. a−d Means within a column with different superscripts differ ( P < 0.05). Discussion Effects of wilting pretreatment and additives on silage fermentation. Our study showed that both LP and OA increased acetic acid production in silage, which was consistent with whole-plant Corn Silage [ 27 ] . It suggests that LP addition promoted lactic acid bacteria to utilize water-soluble carbohydrates, with large levels of lactic acid and small levels of acetic acid being produced. In contrast, OA supplementation favored heterofermentative fermentation, yielding higher acetic acid concentrations and proliferation of acetic acid bacteria. Wilting pretreatment improved fermentation quality via decreased pH and increased lactic acid content. Other forages have similar benefits such a Moringa oleifera leaves [ 8 ] and king grass [ 28 ] . The reason could be that wilting directly reduced the microorganism on the mulberry, while lactic acid accumulation during silage induced by lactic acid bacteria decreased the pH of the silage and subsequently inhibited harmful microorganisms [ 29 ] . Adding OA could also improve the silage fermentation, but it was not as effective as adding LP in the present study, since OA mainly acts as preservatives by lowering pH, thereby achieving an antibacterial effect [ 30 ] . NH 3 -N is another important index for assessing the quality of silage [ 5 ] . This is due to the fact that proteins in silage are mainly hydrolyzed to NH 3 -N, which reduces the nutritive value of the silage [ 12 ] . The results suggested that both adding OA and LP significantly reduced the NH 3 -N level in silages, indicating that inoculation of these additives effectively inhibited protein hydrolysis induced by plant proteases and/or protein hydrolyzing microorganisms by rapid acidification [ 31 , 32 ] . Moreover, butyric acid was only found in the fresh silage control treatment, likely because wilting promoted lactic acid fermentation while inhibiting butyric acid bacteria colonization [ 33 ] . Effects of wilting pretreatment and additives on chemical composition. It was found that WSC content was reduced in wilted mulberry with LP treatment, which was consistent with stylosanthes silage [ 34 ] . The shift in WSC was caused by the acid hydrolysis of fiber fractions, which released WSC content. This was then used by lactic acid bacteria to produce organic acids [ 35 ] . The NDF content was lower in LP and OA treatments versus untreated wilted mulberry silage, likely due to organic acids hydrolyzing digestible cell walls [ 36 ] . Both wilting and additives increased CP and EE content, though OA treatment was less effective than LP treatment. These practices may reduce aerobic nutrient degradation by inhibiting protein breakdown and spoilage microbial activity, retaining more nutrients, consistent with highland alfalfa silage [ 16 ] . In summary, wilting and LP inoculation had more significant positive effects on the fermentation quality and nutrient preservation of mulberry silage. Effects of wilting pretreatment and additives on silage bacterial community. The main phyla of adhering bacteria in mulberry silage were Firmicutes, Proteobacteria, and Cyanobacteria, consistent with the findings by Si et al. [ 37 ] . A large number of cyanobacterial phyla were also observed in our study, which may be related to the raw material characteristics. During ensiling, Firmicutes and Proteobacteria were the dominant genera in all silages, which was consistent with the previous study [ 38 ] . Firmicutes and Proteobacteria were the most common phyla in silage, owing to their ability to adapt to anaerobic and acidic environments [ 38 ] . The high abundance of the dominant genus Lactiplantibacillus indicated its rapid environmental adaptation, substrate utilization, and lactic acid production during fermentation, enabling it to gain competitive superiority, aligning with the findings by Xu et al. [ 39 ] , who identified Lactiplantibacillus as a core functional genus in silage systems. Meanwhile, Levilactobacillus and Weissella appeared in some groups, indicating that interactions among lactic acid bacteria may affect fermentation quality. Notably, LP treatment boosted Lactiplantibacillus abundance and reduced Enterococcus. This is likely due to Enterococcus 's less efficient glycolysis and limited acid tolerance, leading to replacement by dominant bacteria during mid-late silage stages [ 40 ] . This suppression mechanism effectively mitigated the negative impacts of Enterococcus on fermentation, thereby enhancing the overall fermentation quality of silages, aligning with prior research [ 7 , 41 ] . Effects of wilting pretreatment and additives on silage metabolomic profiles. Metabolomics revealed treatment-specific modulation of silage fermentation pathways. Co-activation of ascorbate and aldarate metabolism and PTS emerged as a core metabolic signature across treatments. Ascorbate and aldarate metabolism linked to carbohydrate metabolism while enhancing antioxidant defenses through up-regulation of ascorbic acid, which is essential for mitigating oxidative stress during the curing process [ 13 ] . Complementarily, the PTS, a microbial sugar transport system leveraging phosphorylation cascades to regulate enzymatic activity [ 42 ] , facilitated efficient uptake of substrates like salicin. This co-activation shows how microbes adapt to use carbohydrates better. Adding OA and LP suppress gram-negative bacteria and enrich acid-tolerant taxa using the PTS pathway to accelerate glycolysis for lactic acid fermentation [ 43 ] . Adding OA to fresh silage activated pantothenate and CoA biosynthesis, reflected by L-cysteine upregulation and (R)-pantoate downregulation. This redirects metabolic flux toward CoA synthesis, sustaining acyl metabolism and energy generation for preservation [ 44 ] . Adding LP to fresh mulberry enriched the arginine biosynthesis with metabolites such as L-arginine and citrulline in silages, pointing to lactic acid bacteria-driven proteolytic regulation. LP inoculants enhanced nitrogen retention by redirecting metabolic flux towards arginine synthesis, reducing ammonia accumulation and improving silage protein quality [ 45 ] . The enrichment of tryptophan metabolism such as indoxyl derivatives suggests that adding OA to fresh silage accelerates the breakdown of aromatic amino acids compared to inoculation of LP, with a potential impact on silage aroma [ 46 ] . Nevertheless, different results were observed between adding OA and LP after wilting pretreatment. Wilting altered LP functionality, tending to affect the citric acid cycle to regulate microbial energy metabolism and influence fermentation efficiency [ 47 ] . After wilting, adding OA treatment compared to inoculation of LP predominantly influenced the cyanogenic amino acid metabolism and histidine metabolism, thereby exerting an impact on the dynamic nitrogen metabolism during ensiling [ 15 ] . Analysis of the correlation between microbial and silage characteristics and metabolites in mulberry silage. Our correlation analyses revealed diverse regulatory roles of LP in substrate utilization and metabolite transformation during fermentation of mulberry silages. Adding LP promoted Lactiplantibacillus to dominate the microbial community. This was concurrent with decreases in silage pH and NH 3 -N levels, an increase in lactic acid production, and enhanced crude protein preservation. This suggests that Lactiplantibacillus appears to be responsible for the rapid conversion of WSC to lactic acid, rapidly reduces the pH, thereby inhibiting proteolysis and spoilage [ 48 – 50 ] . Other forages, such as oat [ 51 ] and Italian ryegrass [ 52 ] , have shown Similarly beneficial effects. Metabolomic data further supported this, showing that the abundance of Lactiplantibacillus demonstrated positive correlations with L-arginine, salicin, and cis-aconitic acid. Salicylic acid and cis-aconitic acid are enriched in the PTS and the citric acid cycle, which are implicated in carbohydrate metabolism [ 42 , 47 ] . L-arginine was associated with nitrogen retention [ 45 ] . In contrast, both Enterococcus and Companilactobacillus were associated with suboptimal fermentation outcomes. Enterococcus was positively correlated with pH, acetic acid, and ADL, whereas exhibiting negative correlations with CP and lactic acid. Companilactobacillus showed positive correlations with pH, acetic acid, NH 3 -N and WSC but negative correlation with CP and lactic acid. Enterococcus and Companilactobacillus exhibited consistent positive correlations with N-hydroxy-L-tyrosine, 15-hydroxynorandrostene-3,17-dione glucuronide, and (R)-pantoate, while displaying inverse negative correlations with L-arginine, salicin, cis-aconitic acid, citrulline, and ascorbic acid. Our results suggested that Enterococcus and Companilactobacillus , may utilize the pantothenate and CoA biosynthesis and ascorbate and aldarate pathways to convert WSC to acetic acid and degrade arginine, leading to a reduction in CP, which suggests that Enterococcus faecalis and Companilactobacillus have a potential role in disrupting amino acid metabolism and possibly leading to protein degradation in silage. Studying how microorganisms affect silage quality and metabolite changes can help us to understand the fermentation mechanism of silage and thus obtain high-quality silage through regulation. Effects of wilting pretreatment and additives on in Vitro ruminal fermentation of mulberry silage. The wilting pretreatment improved DM digestibility, whereas a decreased NH 3 -N and CH 4 production, as also observed in highland alfalfa silage [ 16 ] . The increasing DM digestibility observed in wilted silage corresponded to higher DM content. Moreover, this effect may be attributed to reduced acetate concentration, as acetic acid serves as a direct precursor for CH 4 formation by methanogens. Direct cleavage of acetic acid to CH 4 and CO₂ via the acetyl-CoA pathway is one of the most prominent methanogenic pathways in anaerobic systems [ 53 ] . Adding OA significantly increased isobutyrate content, which may have activated branched-chain fatty acid synthesizing flora supporting fiber degradation [ 54 ] , while LP addition may have reduced NH 3 -N concentration by promoting NH 3 -N utilization efficiency [ 55 ] . Notably, adding OA and LP increased MCP production, suggesting that both can promote microbial protein synthesis by modulating microbial activity. However, further research is required to elucidate how wilted and additive-treated mulberry silage modulates ruminal microbiota and to evaluate its effects on animal performance. Conclusion The results showed that, to varying degrees, wilting pretreatment and silage additives can improve the quality and nutritive value of mulberry silage. Combining wilting pretreatment with LP inoculation increased the lactic acid content, reduced the NH 3 -N content and enriched Lactiplantibacillus . Metabolomics showed that the combination activated ascorbate metabolism, the PTS, and arginine synthesis, promoted the preservation of silage nutrients. Wilting and adding LP significantly increased MCP content, wilting reduced CH 4 emissions and NH 3 -N contents. This suggest that combining wilting and LP inoculation improves silage quality by managing the microbiology and metabolism more effectively. This provides a theoretical basis for improving feed efficiency and has practical applications in production. Abbreviations OA Organic acids LP Lactobacillus plantarum DM dry matter FM fresh matter NDF neutral detergent fiber ADF acid detergent fiber ADL acid detergent lignin CP crude protein EE ether extract WSC water soluble carbohydrate NH 3 -N ammonia nitrogen TN total nitrogen DMD DM digestibility TGP total gas production CH 4 methane TVFA total volatile fatty acids A/P Acetate/ Propionate MCP microbial protein Declarations Authors’ contributions D: Writing original draft. M and J: performed the experiments. W: data analysis and methodology. G: review and editing. Q: funding acquisition. S: conceptualization, funding acquisition, review and editing. All authors have read and agreed to the published version of the manuscript. Funding This study was funded by the Innovation Team Development Funds for SichuanMeat Goat and Sheep (SCCXTD-2024-14), and the Young and Middle-aged Talents Project of the National Ethnic Affairs Commission (Beijing, China). Data availability The raw sequencing files and associated metadata have been deposited in the NCBI Sequence Read Archive (PRJNA1304251). Ethics approval and consent to participate The animals used for ruminal fluid collection were approved by the Animal Management Committee of Southwest Minzu University (SMU, Chengdu, China; approval code: SMU-202401162). This study did not involve any endangered or protected animal species. 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Supplementary Files SupplementaryTableS1S3.docx SupplementaryfIile1.xlsx Cite Share Download PDF Status: Published Journal Publication published 10 Jan, 2026 Read the published version in BMC Microbiology → Version 1 posted Editorial decision: Revision requested 24 Nov, 2025 Reviewers agreed at journal 27 Oct, 2025 Reviews received at journal 22 Oct, 2025 Reviewers agreed at journal 08 Oct, 2025 Reviews received at journal 29 Sep, 2025 Reviewers agreed at journal 24 Sep, 2025 Reviews received at journal 14 Sep, 2025 Reviewers agreed at journal 05 Sep, 2025 Reviewers invited by journal 04 Sep, 2025 Editor invited by journal 02 Sep, 2025 Editor assigned by journal 02 Sep, 2025 Submission checks completed at journal 02 Sep, 2025 First submitted to journal 11 Aug, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7345171","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":512969928,"identity":"e9e3aedb-39a3-4419-bb96-7aa3eed491c5","order_by":0,"name":"Fangshu Di","email":"","orcid":"","institution":"Southwest Minzu University","correspondingAuthor":false,"prefix":"","firstName":"Fangshu","middleName":"","lastName":"Di","suffix":""},{"id":512969929,"identity":"b9cab97f-93a6-41b3-8749-0c78fc458b46","order_by":1,"name":"Jian Gao","email":"","orcid":"","institution":"Southwest Minzu University","correspondingAuthor":false,"prefix":"","firstName":"Jian","middleName":"","lastName":"Gao","suffix":""},{"id":512969930,"identity":"d52593e3-4ed8-42d0-856c-716ae448be2b","order_by":2,"name":"Jing Ma","email":"","orcid":"","institution":"Southwest Minzu University","correspondingAuthor":false,"prefix":"","firstName":"Jing","middleName":"","lastName":"Ma","suffix":""},{"id":512969931,"identity":"6ce35091-d26e-440a-8c48-264db08697cb","order_by":3,"name":"Xi Wang","email":"","orcid":"","institution":"Southwest Minzu University","correspondingAuthor":false,"prefix":"","firstName":"Xi","middleName":"","lastName":"Wang","suffix":""},{"id":512969932,"identity":"1a4b3eb2-7165-4213-a06a-d0578ee00c7c","order_by":4,"name":"Yufei Jiang","email":"","orcid":"","institution":"Southwest Minzu University","correspondingAuthor":false,"prefix":"","firstName":"Yufei","middleName":"","lastName":"Jiang","suffix":""},{"id":512969933,"identity":"5c3302c7-60d4-47bc-8999-fc64e8d6d1e3","order_by":5,"name":"Shixiu Qiu","email":"","orcid":"","institution":"Chengdu Academy of Agriculture and Forestry Sciences","correspondingAuthor":false,"prefix":"","firstName":"Shixiu","middleName":"","lastName":"Qiu","suffix":""},{"id":512969934,"identity":"4af23ea2-6f87-4788-82c5-f1ea2d5ae253","order_by":6,"name":"Haitao Shi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA50lEQVRIiWNgGAWjYDACCcY2BgYDIIO9ASpygDgtQD08MKWEtTCwMYCtkUggUov87Oa2Bz8K/sjzSz5/Js27g0GO70YC4+cCPFoM7hxsN+wxMDCcOTvHTJr3DIOx5I0EZukZ+LRIJLZJ8BgYMG64ncN2m7eNIXHDjQQ2Zh58DpuR2Cb5x8DAfsPN489AWuoJamG4kdgmDbQFaDiDGUhLggEhLQYgLTIGxskze3LMf85tkzCceeZhszR+h6U/k3zzR862n/34Y4O3bTbyfMeTD37G6zA0IAHEjA0kaBgFo2AUjIJRgA0AAFvQSWi4aS2TAAAAAElFTkSuQmCC","orcid":"","institution":"Southwest Minzu University","correspondingAuthor":true,"prefix":"","firstName":"Haitao","middleName":"","lastName":"Shi","suffix":""}],"badges":[],"createdAt":"2025-08-11 10:08:39","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7345171/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7345171/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12866-025-04669-y","type":"published","date":"2026-01-10T15:58:11+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":91093540,"identity":"b03190ab-3322-428e-9cfa-2a94e8a2f9b1","added_by":"auto","created_at":"2025-09-11 13:39:14","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":164250,"visible":true,"origin":"","legend":"\u003cp\u003eMicrobial community analysis of mulberry silage. (\u003cstrong\u003eA\u003c/strong\u003e) Principal coordinates analysis (PCoA) based on Bray-Curtis distance; (\u003cstrong\u003eB\u003c/strong\u003e) Relative abundance of bacterial composition in mulberry silage at the phylum level; (\u003cstrong\u003eC\u003c/strong\u003e) Relative abundance of bacterial composition in mulberry silage at the genus level; (\u003cstrong\u003eD\u003c/strong\u003e) Bubble map shows the bacterial communities with abundance of the top 10 most abundant genera. FC: fresh control; FO: fresh OA; FL: fresh LP; WC: wilted control; WO: wilted OA; WL: wilted LP.\u003c/p\u003e\n\u003cp\u003eNotes: \u003csup\u003ea-b \u003c/sup\u003eMeans within a row with different superscripts differ in community composition with abundance of the top 10 most abundant genera in Fig.1D. Bubble color represents its phylum level classification and bubble size its relative abundance.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7345171/v1/2365ef5834e86111c81df410.jpg"},{"id":91094053,"identity":"5d3b00b5-4aab-491e-b0bd-88c0f7e137be","added_by":"auto","created_at":"2025-09-11 13:47:14","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":124610,"visible":true,"origin":"","legend":"\u003cp\u003eMetabolite analysis of mulberry silage. (\u003cstrong\u003eA\u003c/strong\u003e) Statistical mapping of KEGG compound classification profiles; (\u003cstrong\u003eB\u003c/strong\u003e) The PLS-DA model assessed the differences among the treatment groups; (\u003cstrong\u003eC\u003c/strong\u003e) Bar graph showing up-regulated and down-regulated metabolites in different tratments. FC: fresh control; FO: fresh OA; FL: fresh LP; WC: wilted control; WO: wilted OA; WL: wilted LP.\u003c/p\u003e\n\u003cp\u003eNotes: KEGG compound classification profiles were based on compounds with biological roles; The significant differences among metabolites between treatments were tested by Welch’s two-sample t-test based on the criteria of VIP values \u0026gt; 1.0, minimum fold change \u0026gt; 1.5 and \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7345171/v1/80f89b2361b8caa4781ec5ea.jpg"},{"id":91093544,"identity":"120a2628-ec9c-4c62-9131-0b9b7fc3a160","added_by":"auto","created_at":"2025-09-11 13:39:14","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":59234,"visible":true,"origin":"","legend":"\u003cp\u003eKEGG pathway enrichment analysis of differentially accumulated metabolites. (\u003cstrong\u003eA\u003c/strong\u003e) pathway enrichment in FC vs FO; (\u003cstrong\u003eB\u003c/strong\u003e) pathway enrichment in FC vs FL; (\u003cstrong\u003eC\u003c/strong\u003e) pathway enrichment in FO vs FL; (\u003cstrong\u003eD\u003c/strong\u003e) pathway enrichment in WC vs WO; (\u003cstrong\u003eE\u003c/strong\u003e) pathway enrichment in WC vs WL. (\u003cstrong\u003eF\u003c/strong\u003e) pathway enrichment in WO vs WL. FC: fresh control; FO: fresh OA; FL: fresh LP; WC: wilted control; WO: wilted OA; WL: wilted LP. KO01064: Biosynthesis of alkaloids derived from ornithine, lysine and nicotinic acid; KO00945: Stilbenoid, diarylheptanoid and gingerol biosynthesis; KO1063: Biosynthesis of alkaloids derived from shikimate pathway; KO00130: Ubiquinone and other terpenoid-quinone biosynthesis; KO01053: Biosynthesis of siderophore group nonribosomal peptides.\u003c/p\u003e\n\u003cp\u003eNotes: Differential metabolites between treatment groups were enriched in the KEGG pathway after screening according to the screening criteria of VIP values \u0026gt; 1.0, minimum fold change \u0026gt; 1.5 and \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; The x-axis represents the enrichment factor, while the y-axis represents the \u003cem\u003eP\u003c/em\u003e-values; Bubble size indicates the number of annotated KEGG compound IDs.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7345171/v1/0689e63c74764724f30dc808.jpg"},{"id":91093541,"identity":"f0bda42f-80d9-4fc8-b9cf-7627a6b64f57","added_by":"auto","created_at":"2025-09-11 13:39:14","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":107239,"visible":true,"origin":"","legend":"\u003cp\u003eCorrelation of key microbial genera, differential metabolites, and silage characteristics. (\u003cstrong\u003eA\u003c/strong\u003e) Heatmap of the correlation analysis between the key genera and fermentation properties; (\u003cstrong\u003eB\u003c/strong\u003e) Heatmap of the correlation analysis between the key genera and key differential metabolites.\u003c/p\u003e\n\u003cp\u003eNotes: R-values are shown in different colors in the graph, with red indicating positive correlation (0 \u0026lt; \u003cem\u003er\u003c/em\u003e \u0026lt; 1) and blue indicating negative correlation (-1 \u0026lt; \u003cem\u003er\u003c/em\u003e\u0026lt; 0). The p-values are marked with * 0.01 \u0026lt; \u003cem\u003eP\u003c/em\u003e ≤ 0.05; ** 0.001 \u0026lt; \u003cem\u003eP\u003c/em\u003e ≤ 0.01.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7345171/v1/1503ad9caf79908f3ae67642.jpg"},{"id":100069557,"identity":"653d092c-7b90-464e-b9aa-73fcec65f3bc","added_by":"auto","created_at":"2026-01-12 16:14:56","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2016367,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7345171/v1/a04f08e6-abc7-43f6-a431-0e94adaec57f.pdf"},{"id":91094961,"identity":"6028e72d-927a-40e2-b7cd-fc46a29d6771","added_by":"auto","created_at":"2025-09-11 13:55:14","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":32327,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTableS1S3.docx","url":"https://assets-eu.researchsquare.com/files/rs-7345171/v1/72e71f52596ac288f599bca3.docx"},{"id":91093546,"identity":"48f1ae56-6e20-40c7-a7cc-c5b512c4a68a","added_by":"auto","created_at":"2025-09-11 13:39:14","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":86901,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryfIile1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7345171/v1/f09b8e6c89f26ec982eb6477.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Effects of wilting and additives on fermentation characteristics, microbial composition, metabolome, and ruminal degradation properties of mulberry silage","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIn recent years, with the rapid development of the livestock industry, the demand for livestock products has increased dramatically, and the shortage of feed resources has become an important factor constraining the development of the industry\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. To meet the challenge of insufficient supply of feed resources, it is urgent to develop more available forage resources for animal feed. Mulberry (\u003cem\u003eMorus alba\u003c/em\u003e L.) is considered a protein feed resource with great potential due to its high annual yield, rich crude protein content, and abundance of various bioactive compounds, which is expected to effectively alleviate the feed shortage problem\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. However, biomass harvesting from mulberry trees is seasonal in nature and thus requires drying or ensiling of mulberry for long-term preservation. However, there are some potential limitations of haymaking that can lead to nutrient loss and its storage process can be affected by weather changes. Therefore, silage can better preserve the nutrient content of mulberry and become a more suitable preservation method compared to the traditional haymaking method\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eEnsiling is based on the principle of anaerobic fermentation, fresh forage is sealed and preserved, and soluble carbohydrates are converted into organic acids mainly lactic acid by lactic acid bacteria fermentation to inhibit spoilage microorganisms, thus realizing the purpose of long-term preservation of forage.\u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. Therefore, the success of silage depends to a large extent on whether the lactic acid bacteria in the raw material can multiply and ferment rapidly in an anaerobic environment. However, a high moisture content, high buffer capacity, and insufficient number of LAB colonizing the surface of mulberry leaves may result in incomplete fermentation of mulberry silage\u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. Therefore, wilting and additives are commonly used to achieve the desired silage fermentation quality\u003csup\u003e[\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. Wilting is commonly applied in forage ensiling to reduce moisture content. This method inhibits microbial growth and prevents proteolysis by undesirable microorganisms like \u003cem\u003eClostridium\u003c/em\u003e, thereby supporting fermentation\u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. In addition, the selection and application of additives also play an important role in regulating the fermentation process. Studies have shown that the rational use of additives can promote the rapid formation of acidic environment, so as to optimize the fermentation characteristics of silage. Among them, organic acid additives can inhibit the proliferation of spoilage bacteria and other harmful microorganisms through direct acidification, enhance aerobic stability and improve the overall quality of silage\u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e. The addition of lactic acid bacteria can rapidly expand the base number of lactic acid bacteria in silage, make it become the dominant bacteria in silage, inhibit the activity of aerobic microorganisms, and improve the quality of silage fermentation\u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. It is worth noting that the silage process is accompanied by a complex microbial metabolic process, and the dynamic changes of its microbial community structure and metabolite profiles are important indicators for measuring the fermentation quality and revealing the fermentation mechanism\u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e. Therefore, in-depth investigation of the microecological characteristics of mulberry silage and its regulatory pathways is of great significance for improving silage quality and realizing efficient utilization of feed resources.\u003c/p\u003e\u003cp\u003eIn view of this, this study evaluates the effects of wilting and additives on silage fermentation parameters, microbiota composition, metabolites, rumen degradation properties, with a view to providing theoretical basis for the optimization of mulberry silage technology and its application in livestock production.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eMaterials\u003c/h2\u003e\u003cp\u003eMulberry forage (cultivar Chuansisang 1) was harvested at an average height of 1.0-1.2 m with a stubble height of 10\u0026ndash;20 cm at the experimental site in Chengdu City, Sichuan Province, China (103.49\u0026deg;E, 30.42\u0026deg;N). Mulberry raw material contained 27.4% dry matter (DM), 18.9% crude protein (CP), 34.4% neutral detergent fiber (NDF), 24.7% acid detergent fiber (ADF), and 8.4% water-soluble carbohydrates (WSC), DM basis, respectively. The silage additives, including of organic acids and lactic acid bacteria, were supplied by Jilongda Biotechnology Co., Ltd. (Sichuan, China).\u003c/p\u003e\u003cp\u003e\u003cb\u003eExperimental Design and Treatment.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe wilting pretreatment (wilted vs. fresh mulberry with 62% vs. 73% moisture content) and silage additive, including control, \u003cem\u003eLactobacillus plantarum\u003c/em\u003e (LP) and organic acids (OA) treatments, were evaluated in a completely randomized design with 6 replications using a 2 \u0026times; 3 factorial arrangement. Half of the collected mulberries are exposed to the sun for 3 h and the other half are kept fresh\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. The entire mulberry plant was severed into segments measuring 1\u0026ndash;2 cm, subsequently subjected to uniform treatment involving one of three silage additives: no additive control was treated with 1 mL distilled water per kilogram of fresh matters; LP, applied at a rate of 1 \u0026times; 10\u003csup\u003e8\u003c/sup\u003e cfu per g of fresh matter (1 mL/kg per kilogram) dissolved in 1 mL of distilled water and then sprayed uniformly on the mulberries; and a mixture of OA comprising ammonium propionate, formic acid, and propionic acid in a 9:7:1 ratio, applied at 2 mL per kg of fresh matter diluted with 1 mL distilled water before inoculation to equalize total moisture addition across treatments\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. The additives were sprayed uniformly onto the mulberries at the designated dosages, thoroughly mixed, and then packed into specialized polyethylene vacuum bags (500 g per bag). A total of 36 silage samples (6 replicates \u0026times; 2 moisture treatments \u0026times; 3 additive treatments) were immediately sealed using a vacuum sealer and stored at 25\u0026ndash;28 ℃ for 60 days. These comprised six experimental groups: fresh control (FC), fresh OA-treated (FO), fresh LP -inoculated (FL), wilted control (WC), wilted OA-treated (WO), and wilted LP -inoculated (WL). Samples after ensiling were taken for later analysis.\u003c/p\u003e\u003cp\u003e\u003cb\u003eFermentation quality.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAfter 60 d of ensiling, the silage bags were opened and 20 g of silage sample from each bag were mixed with distilled water (180 mL) stored in a 4 ℃ refrigerator for 24 h before filtering through 4 layers of medical gauze. The pH value of the filtrate was immediately checked using a pH meter (Sartorius PB-30L, Beijing, China). NH\u003csub\u003e3\u003c/sub\u003e-N content was quantified via phenol-hypochlorite colorimetry with ninhydrin detection\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. The analysis of volatile fatty acids (VFAs) and lactic acid were conducted using gas chromatography (Agilent 7890B, Santa Clara, USA) in accordance with the methodologies delineated by Wang et al.\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003cb\u003eChemical analysis.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eSilage samples were analyzed for chemical composition based on DM. DM concentrations were measured by drying at 65\u0026deg;C for 48 h \u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. Then, the samples ground through a 1.0 mm screen for nutrient analyses. The ash (942.05), ether extract (EE, 920.39), and CP (984.13) contents were analyzed using the AOAC procedures \u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e. The NDF, ADF, and acid detergent lignin (ADL) were determined using ANKOM 2000 Fiber Analyzer (Ankom Technology Corp., Macedon, NY, USA), as described by Van Soest et al.\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. The WSC content was analyzed by Arthur Thomas using anthrone reagent colorimetry\u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003cb\u003eBacterial community of mulberry silage.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eMicrobial genomic DNA was extracted from silage samples (3 randomly selected biological replicates) using the E.Z.N.A.\u0026reg; Soil DNA Kit (Omega Bio-tek, Norcross, GA, USA) following the manufacturer's protocol. The concentration and purity of the extracted DNA were determined using a NanoDrop 2000 UV\u0026ndash;vis Spectrophotometer (Thermo Scientific, Wilmington, USA), and the quality of the extracted DNA was assessed using 1% agarose gel electrophoresis. The bacterial 16S rRNA gene were amplified using primers 27F (5\u0026rsquo;-AGRGTTYGATYMTGGCTCAG-3\u0026rsquo;) and 1492R(5\u0026rsquo;-RGYTACCTTGTTACGACTT-3\u0026rsquo;). The PCR amplification program was referred to Song et al.\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e. Amplicons were purified using AMPure\u0026reg; PB beads (Pacific Biosciences, CA, USA) and quantified via Qubit 4.0 (Thermo Fisher Scientific, USA). SMRTbell libraries constructed from equimolar pools (Prep Kit 3.0, Pacific Biosciences) were sequenced on Sequel IIe (Pacific Biosciences) via Bio-Pharm Technology Co. Ltd. (Shanghai, China). Raw reads were processed in QIIME2 (v2020.2) with DADA2 to generate single-nucleotide resolution ASVs, which were taxonomically classified against SILVA 16S rRNA (v138) using QIIME2's Naive Bayes classifier.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMulberry silage metabolomics analysis.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eSilage metabolites were profiled via metabolomics. Samples (50 mg) were homogenized in 400 \u0026micro;L ice-cold methanol: water (4:1, v/v) with 0.02 mg/mL L-2-chlorophenylalanine (internal standard). After settling at -10\u0026deg;C, samples were processed using a Wonbio-96c high-throughput tissue crusher (Wanbo Biotechnology, Shanghai) at 50 Hz for 6 min, followed by ultrasonication at 40 kHz for 30 min at 5\u0026deg;C. Subsequent protein precipitation was performed at -20\u0026deg;C for 30 min. Supernatants collected after centrifugation (13,000 \u0026times; g, 4\u0026deg;C, 15 min) were subjected to LC-MS/MS analysis. (n\u0026thinsp;=\u0026thinsp;6, wilted LP treatment n\u0026thinsp;=\u0026thinsp;4). Pooled QC samples ensured analytical reproducibility. The metabolites in the supernatant were analyzed by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS, UHPLC-Q Exactive, Thermo fisher, USA). The UHPLC-MS/MS workflow followed the method of Wang et al.\u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003cb\u003eIn Vitro\u003c/b\u003e \u003cb\u003erumen fermentation.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eRumen fluid collection and buffered inoculum preparation followed the methods of Menke et al.\u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e and Wang et al.\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. Rumen contents were collected 2 h post-morning feeding from four ruminally fistulated Jianzhou goat (39.32 kg\u0026thinsp;\u0026plusmn;\u0026thinsp;2 kg on average). Equal aliquots from each animal were combined, transported in pre-warmed anaerobic containers, and processed within 30 min of collection. All goats were fed twice daily (08:00 and 17:30) with diet consisting of 32.7% corn grain, 20.5% alfalfa hay, 11.3% soybean meal, 3% wheat bran, 2% vitamin and mineral premix, and 1% sodium bicarbonate. Artificial buffer was prepared according to Menke et al.\u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e. The rumen fluid was then mixed with buffer (1:2 v/v) under continuous CO\u003csub\u003e2\u003c/sub\u003e supply. After thorough mixing, approximately 1,000 mg of mulberry silage and 100 mL of the resulting inoculum were added to 250 mL culture bags (Hangzhou Anstey Technology Co., Ltd., Hangzhou, China) and sealed anaerobically. Afterward, The samples were incubated at 39\u0026deg;C for 48 h at 45 r/min \u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e. Total gas production (TGP) was measured using a graduated syringe, with 2 blank bags containing only rumen fluid to account for background gas. NH\u003csub\u003e3\u003c/sub\u003e-N and VFAs measurements were made as described above. After 48 h of incubation, the bags were washed with tap water, then dried in an oven at 105\u0026deg;C for 48 h to determine the DM digestibility (DMD) \u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e.Methane (CH\u003csub\u003e4\u003c/sub\u003e) content was determined via flame ionization GC with a 19095P-Q04 capillary column (30 m \u0026times; 0.53 mm \u0026times; 40.00 \u0026micro;m; Agilent), as described by Chen et al.\u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e. Microbial protein (MCP) content was determined by the method of Makkar et al.\u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003eData analysis.\u003c/h2\u003e\u003cp\u003eThe data was analyzed using the PROC GLM program in SAS 9.4 software, with the model specified as Y\u003csub\u003eijk\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;\u0026micro;\u0026thinsp;+\u0026thinsp;E\u003csub\u003ei\u003c/sub\u003e+A\u003csub\u003ej\u003c/sub\u003e+E\u003csub\u003ei\u003c/sub\u003e\u0026times;A\u003csub\u003ej\u003c/sub\u003e+e\u003csub\u003eijk\u003c/sub\u003e. where Y\u003csub\u003eijk\u003c/sub\u003e = observed value, \u0026micro;\u0026thinsp;=\u0026thinsp;mean value, E\u003csub\u003ei\u003c/sub\u003e = wilting pretreatment, (i\u0026thinsp;=\u0026thinsp;2, respectively, for the wilted and fresh); A\u003csub\u003ej\u003c/sub\u003e = additive inoculation (j\u0026thinsp;=\u0026thinsp;3, respectively, the control, LP and OA). E\u0026times;A\u003csub\u003eij\u003c/sub\u003e = interaction between environmental conditions and additive inoculation and e\u003csub\u003eijk\u003c/sub\u003e=error. Duncan\u0026rsquo;s test was used for pairwise mean comparisons. The significant level was set at α\u0026thinsp;=\u0026thinsp;0.05.\u003c/p\u003e\u003cp\u003eMicrobial data was analyzed using Majorbio platform (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://cloud.majorbio.com\u003c/span\u003e\u003cspan address=\"https://cloud.majorbio.com\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Principal coordinate analysis (PCoA) based on the Bray\u0026ndash;Curtis distance algorithm (R package, version 3.6.1). The alpha diversity based on the ASV level colony abundance table (Supplementary Table\u0026nbsp;1) and community composition the top 10 genera in relative abundance (Supplementary Table\u0026nbsp;2) were evaluated via two-way ANOVA using SAS 9.4 software.\u003c/p\u003e\u003cp\u003eLC-MS metabolomics raw data was analyzed by Progenesis QI software (Waters Corporation, Milford, USA). Metabolites were identified using the Human Metabolome Database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.hmdb.ca/\u003c/span\u003e\u003cspan address=\"http://www.hmdb.ca/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), METLIN Metabolite Database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://metlin.scripps.edu/\u003c/span\u003e\u003cspan address=\"https://metlin.scripps.edu/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), and Majorbio database, and then analyzed on the Majorbio cloud platform. We calculated Variable Importance in Projection (VIP) scores using Principal Component Analysis (PCA) and Partial Least Squares Discriminant Analysis (PLS-DA). The significant differences among metabolites between treatments were tested by Welch\u0026rsquo;s two-sample t-test based on the criteria of VIP values\u0026thinsp;\u0026gt;\u0026thinsp;1.0, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fold Change\u0026thinsp;\u0026gt;\u0026thinsp;1.5. The metabolic pathway diagram was drawn referring to the Kyoto Encyclopedia of Genes and Genomes (KEGG) database.\u003c/p\u003e\u003cp\u003eSpearman correlation analysis assessed relationships among key microbial genera, differential metabolites, and fermentation properties, with results visualized using the heatmap function (R package, version 2.8.2)\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eFermentation quality of mulberry silage.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAs shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Adding OA and LP treatments increased acetate content (1.78%, 2.55% vs 1.14%, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in fresh mulberry. Addition of OA or LP also increased acetic acid content compared to pre-wilted mulberry without any additives (2.60%, 2.10% vs 1.29%, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Lactic acid (3.21% vs 2.86%, DM basis) and pH (4.35 vs 4.42) were higher (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in pre-dried mulberry silage compared to fresh mulberry. Inoculation with LP decreased the NH\u003csub\u003e3\u003c/sub\u003e-N content of silage from 2.17\u0026ndash;1.06%, decreased pH from 4.47 to 4.31, and increased the lactic acid content from 2.62 to 3.60% compared to mulberry without any additives (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Inoculation with OA reduced pH from 4.47 to 4.38 compared to control (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). No butyrate was detected in all groups except the fresh control group, and propionate was only detected in the fresh mulberry inoculated with OA treatment.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eFermentation quality of whole-plant mulberry silage ((% Dry Matter Basis except for ammonia-N)\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"8\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMoisture\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAdditives\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003epH\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNH\u003csub\u003e3\u003c/sub\u003e-N(%TN)\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eLactic acid\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eAcetate\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003ePropionate\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003eButyrate\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFresh\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e4.42\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.82\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e2.86\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1.82\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.47\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.96\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eWilted\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e4.35\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.72\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e3.21\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1.82\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSEM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.012\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.051\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.116\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.058\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eControl\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e4.47\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2.17\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e2.62\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1.21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eND\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.96\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e4.38\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2.08\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e2.88\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e2.32\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.47\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLP\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e4.31\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.06\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e3.60\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1.92\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSEM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.015\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.062\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.142\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.063\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFresh\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eControl\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e4.46\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2.24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e2.62\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1.14\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.96\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFresh\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e4.40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2.16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e2.63\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e2.55\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.47\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFresh\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLP\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e4.36\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.07\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e3.34\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1.78\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eWilted\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eControl\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e4.44\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2.10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e2.63\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1.29\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eWilted\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e4.36\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e3.14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e2.10\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.065\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eWilted\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLP\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e4.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e3.86\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e2.08\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSEM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.015\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.084\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.194\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.089\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eP\u003c/em\u003e-value\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMoisture\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.156\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.044\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.968\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAdditives\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eM\u0026times;A\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.071\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.735\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.35\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.002\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"8\"\u003e\u003csup\u003e1\u003c/sup\u003eOA: organic acid treatment consisted of a mixture of ammonium propionate, formic acid, and propionic acid at 9:7:1 ratio, 2 mL/kg of fresh matter; LP: \u003cem\u003eLactobacillus plantarum\u003c/em\u003e treatment, 108 cfu of \u003cem\u003eLactobacillus plantarum\u003c/em\u003e per g of fresh matter.\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"8\"\u003e\u003csup\u003e2\u003c/sup\u003eNH\u003csub\u003e3\u003c/sub\u003e-N: Ammonia nitrogen, TN: total nitrogen; -: not detected; NA: not available due to non-detectable data. \u003csup\u003ea\u0026minus;d\u003c/sup\u003e Means within a column with different superscripts differ (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eChemical characteristics of mulberry silage.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAs shown in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, both OA and LP additives reduced WSC content in pre-wilted silage (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). OA and LP additives significantly reduced the ash content of fresh mulberries (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Inoculation with OA reduced ash content compared to pre-wilted mulberries without any additives (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Adding OA reduced NDF in fresh mulberries, while adding OA and LP reduced NDF in pre-wilted mulberries (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Wilting pretreatment significantly decreased ADF and ADL, while increasing DM, CP and EE contents (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Both adding OA and LP decreased ADF and ADL content and increased CP and EE content (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eChemical composition of whole-plant mulberry silage (% Dry Matter Basis)\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"10\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMoisture\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAdditives\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eDM\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eAsh\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eCP\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eEE\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eNDF\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003eADF\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c9\"\u003e\u003cp\u003eADL\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c10\"\u003e\u003cp\u003eWSC\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFresh\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e26.42\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e13.49\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e16.61\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e3.87\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e30.56\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e20.38\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e5.67\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e4.43\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eWilted\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e38.00\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e13.41\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e16.98\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e4.13\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e29.23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e19.59\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e4.87\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" 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colname=\"c2\"\u003e\u003cp\u003eLP\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e38.16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e13.20\u003csup\u003ecd\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e17.19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e4.61\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e28.25\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e19.07\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e3.98\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e2.47\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSEM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.245\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.086\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.077\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.134\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.065\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.332\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0.260\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eP\u003c/em\u003e-value\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMoisture\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.274\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.029\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.441\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.009\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAdditives\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.458\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.002\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0.002\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eM\u0026times;A\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.861\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.117\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.090\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.003\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.387\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.278\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0.039\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"10\"\u003e\u003csup\u003e1\u003c/sup\u003eOA: organic acid treatment consisted of a mixture of ammonium propionate, formic acid, and propionic acid at 9:7:1 ratio, 2 mL/kg of fresh matter; LP: \u003cem\u003eLactobacillus plantarum\u003c/em\u003e treatment, 108 cfu of \u003cem\u003eLactobacillus plantarum\u003c/em\u003e per g of fresh matter.\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"10\"\u003e\u003csup\u003e2\u003c/sup\u003eDM: dry matter; Ash: crush ash; CP: crude protein; EE: Ether Extract; NDF: neutral detergent fiber with ash correction; ADF: acid detergent fiber; ADL: acid detergent lignin; WSC: water soluble carbohydrate. \u003csup\u003ea\u0026minus;c\u003c/sup\u003e Means within a column with different superscripts differ (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eMicrobial community analysis.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eEstimates of the α-diversity showed that all samples had adequate coverages (\u0026gt;\u0026thinsp;0.99), indicating that most bacterial communities were identified correctly. Regardless of the presence of additives, the levels of ACE, Chao1 and Sobs decreased in pre-wilted silages. (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Silage microbial communities differed between those with and without additives, as indicated by a decline in the Shannon index with LP inoculation (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Supplementary Table\u0026nbsp;1). The PCoA plots showed that in fresh silage, the treatment groups were clearly separated; in pre-wilted silage, the control and LP groups were clearly separated and there was no clear separation between the other treatment groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). At the phylum level, the bacterial community in mulberry silages was dominated by Firmicutes (84.43\u0026thinsp;~\u0026thinsp;92.75%), Proteobacteria (2.47\u0026thinsp;~\u0026thinsp;6.35%), and Cyanobacteria (2.61\u0026thinsp;~\u0026thinsp;8.46%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). The most abundant genera in all silages were \u003cem\u003eLactiplantibacillus\u003c/em\u003e (30.19\u0026thinsp;~\u0026thinsp;68.62%), \u003cem\u003eLevilactobacillus\u003c/em\u003e (4.15\u0026thinsp;~\u0026thinsp;24.17%), \u003cem\u003eWeissella\u003c/em\u003e (0.90\u0026thinsp;~\u0026thinsp;14.24%), and unclassified_p__Cyanobacteria (2.56\u0026thinsp;~\u0026thinsp;8.38%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). In LP-treated silage, the abundances of \u003cem\u003eLactiplantibacillus\u003c/em\u003e increased, while \u003cem\u003eEnterococcus\u003c/em\u003e decreased (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Adding LP resulted in an increase in unclassified_p_Cyanobacteria in fresh silage (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Adding LP resulted in undetectable level of \u003cem\u003eComplanilactobacillus\u003c/em\u003e in silages (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD, Supplementary Table\u0026nbsp;2).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eAnalyses of the metabolite profiles.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eA total of 4638 metabolites were identified in the silage, of which 168 were annotated by the KEGG compound database at the compounds with biological roles class level, mainly including 39 lipids, 31 peptides, 19 organic acids, 18 carbohydrates, and 17 hormones and transmitters (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). PLS-DA results showed minimal variation among biological replicates, with replicates clustering tightly within their respective treatment groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB), suggesting the sufficient reproducibility and reliability of the experiment. The metabolite compositions in the fresh silages with additives were separated from the control, whereas groups of the LP and the control were separated from each other in pre-wilted silage, indicating that adding OA and LP have a great influence on the metabolites in mulberry silage. Differential metabolites were identified according to the following criteria VIP values\u0026thinsp;\u0026gt;\u0026thinsp;1.0, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fold Change\u0026thinsp;\u0026gt;\u0026thinsp;1.5 (Supplementary file 1). The FO group showed 73 metabolites up-regulated and 24 metabolites down-regulated relative to the FC group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Among these substances, 15-hydroxynorandrosten-3,17-dione glucuronide was downregulated while ascorbic acid was upregulated, both enriched in ascorbate and aldarate metabolism pathway (ko00053). Concomitantly, L-cysteine showed upregulation whereas (R)-pantoate was downregulated, with these metabolites significantly enriched in pantothenate and CoA biosynthesis pathway (ko00770). Additionally, salicin and ascorbic acid were upregulated, both enriched to phosphotransferase system pathway (PTS) (ko02060) (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). The FL group showed 151 up-regulated and 122 down-regulated metabolites relative to FC group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Among these substances, 15-hydroxynorandrosten-3,17-dione glucuronide was down-regulated and ascorbic acid up-regulated, both enriched in ascorbate and aldarate metabolism pathway (ko00053). Concomitantly, salicin, chitobiose, and ascorbic acid were upregulated, both enriched to phosphotransferase system pathway (PTS) (ko02060) Additionally, L-arginine and citrulline showed significant up-regulation and were enriched in arginine biosynthesis pathway (ko00220) (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). The FO group had 73 up-regulated and 20 down-regulated metabolites compared to the FL group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Indoxyl and 5\u0026ndash;hydroxyindoleacetylglycine were up-regulated, both enriched to tryptophan metabolism pathway (ko00380) ((\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). The WO group showed upregulation of 4 differential metabolites and downregulation of 5 differential metabolites relative to the WC group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Ascorbic acid was significantly upregulated and enriched in the ascorbate and aldarate metabolism pathway (ko00053) and PTS pathway (ko02060) (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Compared to the WC group, the WL group showed 38 up-regulated and 36 down-regulated metabolites (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). 15-hydroxynorandrosten-3,17-dione glucuronide was downregulated while ascorbic acid was upregulated, both enriched in ascorbate and aldarate metabolism pathway (ko00053) (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Meanwhile, cis-aconitic acid was up-regulated, tending to affect the citric acid cycle pathway (ko00020) (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.060) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). There were 32 differential metabolites was up-regulated and 25 differential metabolites were down-regulated in WO group compared with the WC group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). N-hydroxy-L-tyrosine was downregulated, tending to affect the cyanoamino acid metabolism pathway (ko00460). N-formyliminyl-glutamic acid was upregulated, tending to affect the histidine metabolism pathway (ko00340) (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.1; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eCorrelation of microbial, chemical composition, fermentation characteristics and metabolites\u003c/h3\u003e\n\u003cp\u003e\u003cem\u003eLactiplantibacillus\u003c/em\u003e positively correlated with CP and lactic acid contents, while negatively correlating with pH, acetic acid, NH\u003csub\u003e3\u003c/sub\u003e-N, ash, WSC, and ADL contents. Conversely, \u003cem\u003eEnterococcus\u003c/em\u003e was positively correlated with pH, acetic acid, and ADL contents, whereas exhibiting negative correlations with CP and lactic acid contents. \u003cem\u003eCompanilactobacillus\u003c/em\u003e positively correlated with pH, acetic acid, NH\u003csub\u003e3\u003c/sub\u003e-N, ash, and WSC but negatively correlated with EE, CP, and lactic acid contents (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). \u003cem\u003eLactiplantibacillus\u003c/em\u003e demonstrated positive correlations with L-arginine, salicin, and cis-aconitic acid yet negative correlation with N-hydroxy-L-tyrosine (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In contrast, \u003cem\u003eEnterococcus\u003c/em\u003e and \u003cem\u003eCompanilactobacillus\u003c/em\u003e exhibited consistent positive correlations with N-hydroxy-L-tyrosine, 15-hydroxynorandrostene-3,17-dione glucuronide, and (R)-pantoate, while displaying inverse negative correlations with L-arginine, salicin, cis-aconitic acid, citrulline, and ascorbic acid (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eIn Vitro\u003c/b\u003e \u003cb\u003eruminal fermentation of mulberry silage.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAs shown in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, inoculating LP enhanced MCP content in fresh mulberry. The addition of OA and LP increased MCP content compared to pre-wilted mulberries without any additives, with the LP treatment having higher MCP content than the OA treatment. The wilting pretreatment increased the pH, DMD, and acetate content, whereas a decreased NH\u003csub\u003e3\u003c/sub\u003e-N and CH\u003csub\u003e4\u003c/sub\u003e production, acetate, acetate to propionate content (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Adding OA or LP additives helped to increased TGP content (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Adding LP also reduced NH\u003csub\u003e3\u003c/sub\u003e-N content (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Adding OA also increased isobutyrate content (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003e\u003cem\u003eIn vitro\u003c/em\u003e rumen fermentation characteristics of whole-plant mulberry silage.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"15\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c13\" colnum=\"13\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c14\" colnum=\"14\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c15\" colnum=\"15\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMoisture\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAdditives\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eDMD\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\u003cp\u003e(%)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eTGP\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\u003cp\u003e(mL/g)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eCH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\u003cp\u003e(mL/g)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eNH\u003csub\u003e3\u003c/sub\u003e-N\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\u003cp\u003e(mg/dL)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eMCP\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\u003cp\u003e(mg/ml)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003eTVFA\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\u003cp\u003e(mL/g)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c9\"\u003e\u003cp\u003eAcetate (mmol/L)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c10\"\u003e\u003cp\u003ePropionate\u003c/p\u003e\u003cp\u003e(mmol/L)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c11\"\u003e\u003cp\u003eButyrate\u003c/p\u003e\u003cp\u003e(mmol/L)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c12\"\u003e\u003cp\u003eIsobutyrate\u003c/p\u003e\u003cp\u003e(mmol/L)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c13\"\u003e\u003cp\u003eValerate\u003c/p\u003e\u003cp\u003e(mmol/L)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c14\"\u003e\u003cp\u003eIsovalerate\u003c/p\u003e\u003cp\u003e(mmol/L)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c15\"\u003e\u003cp\u003eA/P\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFresh\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e45.57\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e216.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e20.76\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e19.38\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.41\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e50.56\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e41.49\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e7.75\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e2.43\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e0.68\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e0.84\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e\u003cp\u003e1.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c15\"\u003e\u003cp\u003e5.44\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eWilted\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e47.63\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e218.36\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e19.67\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e18.37\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.69\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e53.05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e37.90\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e7.99\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e2.26\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e0.67\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e0.75\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e\u003cp\u003e0.85\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c15\"\u003e\u003cp\u003e4.75\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSEM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.555\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.201\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.246\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.243\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.007\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e1.276\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e1.097\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e0.219\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e0.117\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e0.024\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e0.047\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e\u003cp\u003e0.067\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c15\"\u003e\u003cp\u003e0.093\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eControl\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e47.48\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e210.14\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e20.39\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e19.49\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.61\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e51.46\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e39.93\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e7.83\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e2.31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e0.66\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e0.80\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e\u003cp\u003e0.97\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c15\"\u003e\u003cp\u003e5.16\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e45.64\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e220.08\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e19.95\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e18.96\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.67\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e50.96\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e38.09\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e7.910\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e2.60\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e0.77\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e0.91\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e\u003cp\u003e1.08\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c15\"\u003e\u003cp\u003e4.89\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLP\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e46.69\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e221.31\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e20.31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e18.18\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.68\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e53.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e41.08\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e7.87\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e2.13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e0.59\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e0.67\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e\u003cp\u003e0.76\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c15\"\u003e\u003cp\u003e5.23\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSEM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.679\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.471\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.301\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.298\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.008\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e1.564\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e1.344\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e0.249\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e0.144\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e0.030\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e0.058\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e\u003cp\u003e0.082\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c15\"\u003e\u003cp\u003e0.213\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFresh\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eControl\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e47.46\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e207.55\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e21.09\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e20.17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.58\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e49.10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e43.13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e7.45\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e2.35\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e0.67\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e0.83\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e\u003cp\u003e1.07\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c15\"\u003e\u003cp\u003e5.85\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFresh\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e43.54\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e220.17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e20.56\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e19.27\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.60\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e48.91\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e41.05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e8.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e2.76\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e0.76\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e1.07\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e\u003cp\u003e1.24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c15\"\u003e\u003cp\u003e5.27\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFresh\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLP\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e45.73\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e220.28\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e20.64\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e18.71\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.66\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e53.68\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e40.30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e7.79\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e2.20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e0.60\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e0.69\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e\u003cp\u003e0.76\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c15\"\u003e\u003cp\u003e5.19\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eWilted\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eControl\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e47.50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e212.73\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e19.69\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e18.80\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.64\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e53.82\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e36.74\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e8.21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e2.28\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e0.64\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e0.77\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e\u003cp\u003e0.87\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c15\"\u003e\u003cp\u003e4.47\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eWilted\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e47.74\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e220.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e19.34\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e18.64\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.69\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e53.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e35.12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e7.81\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e2.45\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e0.78\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e0.81\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e\u003cp\u003e0.91\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c15\"\u003e\u003cp\u003e4.51\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eWilted\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLP\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e47.66\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e222.34\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e19.98\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e17.66\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.75\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e52.31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e41.85\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e7.95\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e2.06\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e0.58\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e0.66\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e\u003cp\u003e0.76\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c15\"\u003e\u003cp\u003e5.27\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSEM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.961\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2.081\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.426\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.436\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.011\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e2.211\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e1.900\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e0.351\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e0.203\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e0.024\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e0.081\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e\u003cp\u003e0.116\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c15\"\u003e\u003cp\u003e0.301\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eP\u003c/em\u003e-value\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c15\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMoisture\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.022\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.191\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.008\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.012\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0.194\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.039\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e0.442\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e0.316\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e0.747\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e0.181\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e\u003cp\u003e0.086\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c15\"\u003e\u003cp\u003e0.027\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAdditives\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.200\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.560\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.029\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0.643\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.319\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e0.977\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e0.103\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e0.005\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e0.041\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e\u003cp\u003e0.053\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c15\"\u003e\u003cp\u003e0.589\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eM\u0026times;A\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.138\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.478\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.671\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.685\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0.349\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.104\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e0.439\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e0.830\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e0.839\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e0.562\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e\u003cp\u003e0.378\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c15\"\u003e\u003cp\u003e0.099\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"15\"\u003e\u003csup\u003e1\u003c/sup\u003eOA: organic acid treatment consisted of a mixture of ammonium propionate, formic acid, and propionic acid at 9:7:1 ratio, 2 mL/kg of fresh matter; LP: \u003cem\u003eLactobacillus plantarum\u003c/em\u003e treatment, 108 cfu of \u003cem\u003eLactobacillus plantarum\u003c/em\u003e per g of fresh matter.\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"15\"\u003e\u003csup\u003e2\u003c/sup\u003eDMD: DM digestibility; TGP: total gas production; CH\u003csub\u003e4\u003c/sub\u003e: methane; TVFA: total volatile fatty acids; NH\u003csub\u003e3\u003c/sub\u003e-N: ammonia nitrogen; MCP: microbial protein; A/P: acetate to propionate. \u003csup\u003ea\u0026minus;d\u003c/sup\u003e Means within a column with different superscripts differ (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003e\u003cb\u003eEffects of wilting pretreatment and additives on silage fermentation.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eOur study showed that both LP and OA increased acetic acid production in silage, which was consistent with whole-plant Corn Silage\u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e. It suggests that LP addition promoted lactic acid bacteria to utilize water-soluble carbohydrates, with large levels of lactic acid and small levels of acetic acid being produced. In contrast, OA supplementation favored heterofermentative fermentation, yielding higher acetic acid concentrations and proliferation of acetic acid bacteria. Wilting pretreatment improved fermentation quality via decreased pH and increased lactic acid content. Other forages have similar benefits such a \u003cem\u003eMoringa oleifera\u003c/em\u003e leaves\u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e and king grass\u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e. The reason could be that wilting directly reduced the microorganism on the mulberry, while lactic acid accumulation during silage induced by lactic acid bacteria decreased the pH of the silage and subsequently inhibited harmful microorganisms\u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e. Adding OA could also improve the silage fermentation, but it was not as effective as adding LP in the present study, since OA mainly acts as preservatives by lowering pH, thereby achieving an antibacterial effect\u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e. NH\u003csub\u003e3\u003c/sub\u003e-N is another important index for assessing the quality of silage\u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e. This is due to the fact that proteins in silage are mainly hydrolyzed to NH\u003csub\u003e3\u003c/sub\u003e-N, which reduces the nutritive value of the silage\u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e. The results suggested that both adding OA and LP significantly reduced the NH\u003csub\u003e3\u003c/sub\u003e-N level in silages, indicating that inoculation of these additives effectively inhibited protein hydrolysis induced by plant proteases and/or protein hydrolyzing microorganisms by rapid acidification\u003csup\u003e[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e. Moreover, butyric acid was only found in the fresh silage control treatment, likely because wilting promoted lactic acid fermentation while inhibiting butyric acid bacteria colonization\u003csup\u003e[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003cb\u003eEffects of wilting pretreatment and additives on chemical composition.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eIt was found that WSC content was reduced in wilted mulberry with LP treatment, which was consistent with stylosanthes silage\u003csup\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/sup\u003e. The shift in WSC was caused by the acid hydrolysis of fiber fractions, which released WSC content. This was then used by lactic acid bacteria to produce organic acids\u003csup\u003e[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/sup\u003e. The NDF content was lower in LP and OA treatments versus untreated wilted mulberry silage, likely due to organic acids hydrolyzing digestible cell walls\u003csup\u003e[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]\u003c/sup\u003e. Both wilting and additives increased CP and EE content, though OA treatment was less effective than LP treatment. These practices may reduce aerobic nutrient degradation by inhibiting protein breakdown and spoilage microbial activity, retaining more nutrients, consistent with highland alfalfa silage\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. In summary, wilting and LP inoculation had more significant positive effects on the fermentation quality and nutrient preservation of mulberry silage.\u003c/p\u003e\u003cp\u003e\u003cb\u003eEffects of wilting pretreatment and additives on silage bacterial community.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe main phyla of adhering bacteria in mulberry silage were Firmicutes, Proteobacteria, and Cyanobacteria, consistent with the findings by Si et al.\u003csup\u003e[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]\u003c/sup\u003e. A large number of cyanobacterial phyla were also observed in our study, which may be related to the raw material characteristics. During ensiling, Firmicutes and Proteobacteria were the dominant genera in all silages, which was consistent with the previous study\u003csup\u003e[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]\u003c/sup\u003e. Firmicutes and Proteobacteria were the most common phyla in silage, owing to their ability to adapt to anaerobic and acidic environments\u003csup\u003e[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]\u003c/sup\u003e. The high abundance of the dominant genus \u003cem\u003eLactiplantibacillus\u003c/em\u003e indicated its rapid environmental adaptation, substrate utilization, and lactic acid production during fermentation, enabling it to gain competitive superiority, aligning with the findings by Xu et al.\u003csup\u003e[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]\u003c/sup\u003e, who identified \u003cem\u003eLactiplantibacillus\u003c/em\u003e as a core functional genus in silage systems. Meanwhile, \u003cem\u003eLevilactobacillus\u003c/em\u003e and \u003cem\u003eWeissella\u003c/em\u003e appeared in some groups, indicating that interactions among lactic acid bacteria may affect fermentation quality. Notably, LP treatment boosted \u003cem\u003eLactiplantibacillus\u003c/em\u003e abundance and reduced Enterococcus. This is likely due to \u003cem\u003eEnterococcus\u003c/em\u003e's less efficient glycolysis and limited acid tolerance, leading to replacement by dominant bacteria during mid-late silage stages\u003csup\u003e[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]\u003c/sup\u003e. This suppression mechanism effectively mitigated the negative impacts of \u003cem\u003eEnterococcus\u003c/em\u003e on fermentation, thereby enhancing the overall fermentation quality of silages, aligning with prior research\u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003cb\u003eEffects of wilting pretreatment and additives on silage metabolomic profiles.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eMetabolomics revealed treatment-specific modulation of silage fermentation pathways. Co-activation of ascorbate and aldarate metabolism and PTS emerged as a core metabolic signature across treatments. Ascorbate and aldarate metabolism linked to carbohydrate metabolism while enhancing antioxidant defenses through up-regulation of ascorbic acid, which is essential for mitigating oxidative stress during the curing process\u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. Complementarily, the PTS, a microbial sugar transport system leveraging phosphorylation cascades to regulate enzymatic activity\u003csup\u003e[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]\u003c/sup\u003e, facilitated efficient uptake of substrates like salicin. This co-activation shows how microbes adapt to use carbohydrates better. Adding OA and LP suppress gram-negative bacteria and enrich acid-tolerant taxa using the PTS pathway to accelerate glycolysis for lactic acid fermentation\u003csup\u003e[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]\u003c/sup\u003e. Adding OA to fresh silage activated pantothenate and CoA biosynthesis, reflected by L-cysteine upregulation and (R)-pantoate downregulation. This redirects metabolic flux toward CoA synthesis, sustaining acyl metabolism and energy generation for preservation\u003csup\u003e[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]\u003c/sup\u003e. Adding LP to fresh mulberry enriched the arginine biosynthesis with metabolites such as L-arginine and citrulline in silages, pointing to lactic acid bacteria-driven proteolytic regulation. LP inoculants enhanced nitrogen retention by redirecting metabolic flux towards arginine synthesis, reducing ammonia accumulation and improving silage protein quality\u003csup\u003e[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]\u003c/sup\u003e. The enrichment of tryptophan metabolism such as indoxyl derivatives suggests that adding OA to fresh silage accelerates the breakdown of aromatic amino acids compared to inoculation of LP, with a potential impact on silage aroma\u003csup\u003e[\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]\u003c/sup\u003e. Nevertheless, different results were observed between adding OA and LP after wilting pretreatment. Wilting altered LP functionality, tending to affect the citric acid cycle to regulate microbial energy metabolism and influence fermentation efficiency\u003csup\u003e[\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]\u003c/sup\u003e. After wilting, adding OA treatment compared to inoculation of LP predominantly influenced the cyanogenic amino acid metabolism and histidine metabolism, thereby exerting an impact on the dynamic nitrogen metabolism during ensiling\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003cb\u003eAnalysis of the correlation between microbial and silage characteristics and metabolites in mulberry silage.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eOur correlation analyses revealed diverse regulatory roles of LP in substrate utilization and metabolite transformation during fermentation of mulberry silages. Adding LP promoted \u003cem\u003eLactiplantibacillus\u003c/em\u003e to dominate the microbial community. This was concurrent with decreases in silage pH and NH\u003csub\u003e3\u003c/sub\u003e-N levels, an increase in lactic acid production, and enhanced crude protein preservation. This suggests that \u003cem\u003eLactiplantibacillus\u003c/em\u003e appears to be responsible for the rapid conversion of WSC to lactic acid, rapidly reduces the pH, thereby inhibiting proteolysis and spoilage\u003csup\u003e[\u003cspan additionalcitationids=\"CR49\" citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]\u003c/sup\u003e. Other forages, such as oat\u003csup\u003e[\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]\u003c/sup\u003e and Italian ryegrass\u003csup\u003e[\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]\u003c/sup\u003e, have shown Similarly beneficial effects. Metabolomic data further supported this, showing that the abundance of \u003cem\u003eLactiplantibacillus\u003c/em\u003e demonstrated positive correlations with L-arginine, salicin, and cis-aconitic acid. Salicylic acid and cis-aconitic acid are enriched in the PTS and the citric acid cycle, which are implicated in carbohydrate metabolism\u003csup\u003e[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]\u003c/sup\u003e. L-arginine was associated with nitrogen retention\u003csup\u003e[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]\u003c/sup\u003e. In contrast, both \u003cem\u003eEnterococcus\u003c/em\u003e and \u003cem\u003eCompanilactobacillus\u003c/em\u003e were associated with suboptimal fermentation outcomes. \u003cem\u003eEnterococcus\u003c/em\u003e was positively correlated with pH, acetic acid, and ADL, whereas exhibiting negative correlations with CP and lactic acid. \u003cem\u003eCompanilactobacillus\u003c/em\u003e showed positive correlations with pH, acetic acid, NH\u003csub\u003e3\u003c/sub\u003e-N and WSC but negative correlation with CP and lactic acid. \u003cem\u003eEnterococcus\u003c/em\u003e and \u003cem\u003eCompanilactobacillus\u003c/em\u003e exhibited consistent positive correlations with N-hydroxy-L-tyrosine, 15-hydroxynorandrostene-3,17-dione glucuronide, and (R)-pantoate, while displaying inverse negative correlations with L-arginine, salicin, cis-aconitic acid, citrulline, and ascorbic acid. Our results suggested that \u003cem\u003eEnterococcus\u003c/em\u003e and \u003cem\u003eCompanilactobacillus\u003c/em\u003e, may utilize the pantothenate and CoA biosynthesis and ascorbate and aldarate pathways to convert WSC to acetic acid and degrade arginine, leading to a reduction in CP, which suggests that Enterococcus faecalis and \u003cem\u003eCompanilactobacillus\u003c/em\u003e have a potential role in disrupting amino acid metabolism and possibly leading to protein degradation in silage. Studying how microorganisms affect silage quality and metabolite changes can help us to understand the fermentation mechanism of silage and thus obtain high-quality silage through regulation.\u003c/p\u003e\u003cp\u003e\u003cb\u003eEffects of wilting pretreatment and additives on\u003c/b\u003e \u003cb\u003ein Vitro\u003c/b\u003e \u003cb\u003eruminal fermentation of mulberry silage.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe wilting pretreatment improved DM digestibility, whereas a decreased NH\u003csub\u003e3\u003c/sub\u003e-N and CH\u003csub\u003e4\u003c/sub\u003e production, as also observed in highland alfalfa silage\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. The increasing DM digestibility observed in wilted silage corresponded to higher DM content. Moreover, this effect may be attributed to reduced acetate concentration, as acetic acid serves as a direct precursor for CH\u003csub\u003e4\u003c/sub\u003e formation by methanogens. Direct cleavage of acetic acid to CH\u003csub\u003e4\u003c/sub\u003e and CO₂ via the acetyl-CoA pathway is one of the most prominent methanogenic pathways in anaerobic systems\u003csup\u003e[\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]\u003c/sup\u003e. Adding OA significantly increased isobutyrate content, which may have activated branched-chain fatty acid synthesizing flora supporting fiber degradation\u003csup\u003e[\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]\u003c/sup\u003e, while LP addition may have reduced NH\u003csub\u003e3\u003c/sub\u003e-N concentration by promoting NH\u003csub\u003e3\u003c/sub\u003e-N utilization efficiency\u003csup\u003e[\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]\u003c/sup\u003e. Notably, adding OA and LP increased MCP production, suggesting that both can promote microbial protein synthesis by modulating microbial activity. However, further research is required to elucidate how wilted and additive-treated mulberry silage modulates ruminal microbiota and to evaluate its effects on animal performance.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe results showed that, to varying degrees, wilting pretreatment and silage additives can improve the quality and nutritive value of mulberry silage. Combining wilting pretreatment with LP inoculation increased the lactic acid content, reduced the NH\u003csub\u003e3\u003c/sub\u003e-N content and enriched \u003cem\u003eLactiplantibacillus\u003c/em\u003e. Metabolomics showed that the combination activated ascorbate metabolism, the PTS, and arginine synthesis, promoted the preservation of silage nutrients. Wilting and adding LP significantly increased MCP content, wilting reduced CH\u003csub\u003e4\u003c/sub\u003e emissions and NH\u003csub\u003e3\u003c/sub\u003e-N contents. This suggest that combining wilting and LP inoculation improves silage quality by managing the microbiology and metabolism more effectively. This provides a theoretical basis for improving feed efficiency and has practical applications in production.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003eOA\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eOrganic acids\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003eLP\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eLactobacillus plantarum\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003eDM\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003edry matter\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003eFM\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003efresh matter\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003eNDF\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eneutral detergent fiber\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003eADF\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eacid detergent fiber\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003eADL\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eacid detergent lignin\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003eCP\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ecrude protein\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003eEE\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eether extract\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003eWSC\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ewater soluble carbohydrate\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003eNH\u003c/b\u003e\u003csub\u003e\u003cb\u003e3\u003c/b\u003e\u003c/sub\u003e\u003cb\u003e-N\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eammonia nitrogen\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003eTN\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003etotal nitrogen\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003eDMD\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eDM digestibility\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003eTGP\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003etotal gas production\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003eCH\u003c/b\u003e\u003csub\u003e\u003cb\u003e4\u003c/b\u003e\u003c/sub\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003emethane\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003eTVFA\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003etotal volatile fatty acids\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003eA/P\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eAcetate/ Propionate\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003eMCP\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003emicrobial protein\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eD: Writing original draft. M and J: performed the experiments. W: data analysis and methodology. G: review and editing. Q: funding acquisition. S: conceptualization, funding acquisition, review and editing.\u0026nbsp;All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was funded by the Innovation Team Development Funds for SichuanMeat Goat and Sheep (SCCXTD-2024-14), and the Young and Middle-aged Talents Project of the National Ethnic Affairs Commission (Beijing, China).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe raw sequencing files and associated metadata have been deposited in the NCBI Sequence Read Archive (PRJNA1304251).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe animals used for ruminal fluid collection were approved by the Animal Management Committee of Southwest Minzu University (SMU, Chengdu, China; approval code: SMU-202401162). This study did not involve any endangered or protected animal species. Individual oral/written informed consent was obtained from all animal owners for the use of samples, and written consent was also obtained for the participation of their animals in the study.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eMa J, Fan X, Wu T, Zhou J, Huang H, Qiu T, et al. Lactic Acid Bacteria and Cellulase Improve the Fermentation Characteristics, Aerobic Stability and Rumen Degradation of Mixed Silage Prepared with Amaranth and Rice Straw. 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Trop Anim Health Pro. 2022;54(5):261.\u003c/li\u003e\n\u003cli\u003eMarcelli V, Osimani A, Aquilanti L. Research progress in the use of lactic acid bacteria as natural biopreservatives against Pseudomonas spp. in meat and meat products: A review. Food Res Int. 2024;196:115129.\u003c/li\u003e\n\u003cli\u003eWang X, Liu H, Wang Y, Lin Y, Ni K, Yang F. Effects of lactic acid bacteria and cellulase additives on the fermentation quality, antioxidant activity, and metabolic profile of oat silage. Bioresour Bioprocess. 2024d;11(1):92.\u003c/li\u003e\n\u003cli\u003eLi Y, Wang LL, Yu YS, Panyavong X, Wu LZ, Kim JG. Forage quality and fermentation dynamics of silages of Italian ryegrass (Lolium multiflorum Lam.) wilted for varying periods. Anim Biosci. 2024b;37(12):2091-100.\u003c/li\u003e\n\u003cli\u003eTsigkou K, Zagklis D, Parasoglou M, Zafiri C, Kornaros M. Proposed protocol for rate-limiting step determination during anaerobic digestion of complex substrates. Bioresour Technol. 2022;361:127660.\u003c/li\u003e\n\u003cli\u003eMitchell KE, Wenner BA, Lee C, Park T, Socha MT, Kleinschmit DH, et al. Supplementing branched-chain volatile fatty acids in dual-flow cultures varying in dietary forage and corn oil concentrations. I: Digestibility, microbial protein, and prokaryotic community structure. J Dairy Sci. 2023;106(11):7530-47.\u003c/li\u003e\n\u003cli\u003eLi S, Zeng H, Wang C, Han Z. Effect of Methionine Hydroxy Analog on Hu Sheep Digestibility, Rumen Fermentation, and Rumen Microbial Community In Vitro. Metabolites. 2023;13(2):169.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcro","sideBox":"Learn more about [BMC Microbiology](http://bmcmicrobiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/mcro","title":"BMC Microbiology","twitterHandle":"#bmcmicrobiology","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Mulberry silage, Wilting, Silage additives, Microbial composition, Metabolites, Ruminal degradation","lastPublishedDoi":"10.21203/rs.3.rs-7345171/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7345171/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eOptimizing the processing technology of mulberry silage is a prerequisite for enhancing the utilization efficiency of mulberry resources. This study examined effects of wilting pretreatment and silage additives on mulberry silage fermentation, microbiota, metabolites, and ruminal degradation. \u003cem\u003eLactobacillus plantarum\u003c/em\u003e (LP), organic acids (OA), and a control treatment without additives were applied to unwilted (73% moisture) or wilted (62% moisture) mulberry forage.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eWilting significantly enhanced lactic acid and crude protein (CP) contents, and lowered pH (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Adding OA or LP additives reduced pH and increased CP content (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). LP treatment further reduced ammonia nitrogen and pH, improved lactic acid content (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Pre-wilted mulberry inoculated with LP showed further reductions in acetic acid and neutral detergent fiber contents (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). LP treatment enriched \u003cem\u003eLactiplantibacillus\u003c/em\u003e and suppressed \u003cem\u003eEnterococcus\u003c/em\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). \u003cem\u003eLactiplantibacillus\u003c/em\u003e was strongly correlated with lactic acid, CP, and beneficial metabolites L-arginine and salicin (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). These metabolites were enriched in the phosphotransferase system and arginine biosynthesis pathways (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Wilting improved DM digestibility while reducing methane and ammonia nitrogen level. LP treatment reduced ruminal ammonia nitrogen level (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Pre-wilted mulberry inoculated with LP further increased microbial protein content (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e\u003cp\u003eIn conclusion, combining wilting pretreatment and LP inoculant offers an effective strategy to enhance silage quality.\u003c/p\u003e","manuscriptTitle":"Effects of wilting and additives on fermentation characteristics, microbial composition, metabolome, and ruminal degradation properties of mulberry silage","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-11 13:39:09","doi":"10.21203/rs.3.rs-7345171/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-11-24T06:08:17+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"310965012710292655052765822878343196000","date":"2025-10-27T15:20:58+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-22T10:08:12+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"137708710594663581900468054841057185286","date":"2025-10-09T01:17:51+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-29T14:25:42+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"28511023370456764151922442593605112099","date":"2025-09-25T03:42:06+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-14T08:13:17+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"78970125758639366782303793336007214535","date":"2025-09-05T07:49:15+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-05T02:45:17+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-09-02T18:23:11+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-02T09:06:11+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-02T09:05:05+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Microbiology","date":"2025-08-11T10:06:58+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcro","sideBox":"Learn more about [BMC Microbiology](http://bmcmicrobiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/mcro","title":"BMC Microbiology","twitterHandle":"#bmcmicrobiology","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3ebe50a7-741f-45e1-b358-b2f4d4d9aea2","owner":[],"postedDate":"September 11th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-01-12T16:08:10+00:00","versionOfRecord":{"articleIdentity":"rs-7345171","link":"https://doi.org/10.1186/s12866-025-04669-y","journal":{"identity":"bmc-microbiology","isVorOnly":false,"title":"BMC Microbiology"},"publishedOn":"2026-01-10 15:58:11","publishedOnDateReadable":"January 10th, 2026"},"versionCreatedAt":"2025-09-11 13:39:09","video":"","vorDoi":"10.1186/s12866-025-04669-y","vorDoiUrl":"https://doi.org/10.1186/s12866-025-04669-y","workflowStages":[]},"version":"v1","identity":"rs-7345171","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7345171","identity":"rs-7345171","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}