Effects of Verbena and Polygonum cuspidatum on growth performance, immune functions, cecal microbiota, and brain metabolites in Sansui ducks

Effects of Verbena and Polygonum cuspidatum on growth performance, immune functions, cecal microbiota, and brain metabolites in Sansui ducks | 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 Verbena and Polygonum cuspidatum on growth performance, immune functions, cecal microbiota, and brain metabolites in Sansui ducks Yongcai Zhu, Qiaoqun Wu, Linli Luo, Shenglin Yang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6522380/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Verbena and Polygonum cuspidatum, recognized for their antioxidant and immunomodulatory properties, have demonstrated potential benefits. However, the specific mechanisms by which these herbs impact poultry health, particularly regarding alterations in gut microbiota and brain metabolite profiles, remain insufficiently investigated. This study aimed to investigate the effects of Verbena and Polygonum cuspidatum supplementation on the growth performance, immune function, cecal microbiota, and brain metabolites in Sansui ducks. Methods A total of 216 one-day-old ducks were randomly assigned to three treatment groups for a 35-day trial, each with 6 replicates of 12 ducks. The ducks were fed a basal diet (T3), a basal diet supplemented with 40mg/kg Verbena (T1), and a basal diet supplemented with 40mg/kg Polygonum cuspidatum (T2). Results The results showed that both herbs significantly increased body weight, IgG, and IgM levels, while decreasing the feed conversion ratio (FCR) compared to the control group (T3) (P < 0.05) Notably, Verbena supplementation increased the relative abundance of beneficial bacteria, such as Bacteroidetes and Saccharibacteria , and significantly decreased the relative abundance of pathogenic bacteria such as Actinobacteria ompared to the control group (P < 0.05). Polygonum cuspidatum treatment increased the relative abundance of Megamonas compared to the control group (P < 0.05). Additionally, Verbena treatment increased the concentration of Gln and decreased the concentrations of GABA, Tyr, and Ach compared to the control group (P < 0.05). Polygonum cuspidatum treatment increased 5-HIAA concentration compared to the control group (P < 0.05). Further correlation analysis highlighted a significant link between gut microbiota ( Villanella, Anaerosporobacter, Anaerofustis , and Flavonifracter ) changes and brain metabolites (GABA, Ach, and Glu), suggesting the potential influence of these herbs through the microbiota-gut-brain axis. Conclusion This study provides novel insights into how dietary herbal supplements can improve poultry health by modulating both gut and brain biochemistry, offering a promising natural alternative to antibiotics in poultry production. Verbena Polygonum cuspidatum duck growth performance brain metabolite microbiota Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Antibiotics are commonly employed in the poultry industry to enhance growth and improve feed efficiency by promoting intestinal health [ 1 ]. However, the widespread and prolonged use of antibiotics has led to the rise of resistance, and the presence of antibiotic residues in poultry meat and waste poses direct or indirect risks to both human and animal health, and countries are taking antibiotics more seriously [ 2 ]. Therefore, these concerns have prompted an increasing interest in finding natural and efficient alternatives to antibiotics, particularly those that can enhance intestinal health and promote growth performance [ 3 ]. Natural feed additives, including herbal supplements, have shown promise as potential alternatives to antibiotics in poultry. Previous studies have indicated that the beneficial effects of Chinese herbs on poultry include promoting growth [ 4 ], enhancing immunity [ 5 ], and regulating intestinal microbiota [ 6 ]. Among these, Verbena and Polygonum cuspidatum , two Chinese herbs, are also utilized as feed supplements owing to their distinct biological activities, including antioxidant, anti-inflammatory, antibacterial, and growth-promoting properties. Studies have indicated that bioactive compounds in these herbs, including flavonoids, verbascoside (from Verbena ), and resveratrol (from Polygonum cuspidatum ), play crucial roles in promoting growth, improving feed efficiency, and maintaining intestinal health in animals [ 7 ]. Several studies have indicated that verbascoside acid is responsible for multiple beneficial properties of Verbena , like its antioxidant, anti-inflammatory, and antineoplastic properties [ 8 ]. Previous studies have shown that pigs fed a diet enriched with verbascoside had improved growth and feed efficiency [ 9 ]. In addition, Studies have demonstrated that dietary resveratrol supplementation can enhance the immune response, modulate the intestinal microbiota, and improve feed efficiency in poultry [ 10 , 11 ]. However, the precise mechanisms through which these herbal supplements exert their effects on poultry performance and gut health, particularly the connection between gut microbiota and brain metabolites, remain underexplored. The bidirectional communication between the gut microbiota and the brain is termed the microbiota–gut–brain axis. The gut microbiota is known to influence brain function through the production of metabolites, which in turn affect various physiological processes, including immune modulation, behavior, and metabolism [ 12 ]. While studies have primarily focused on the gut’s role in influencing metabolic processes, less is known about how dietary supplements like Verbena and Polygonum cuspidatum affect this microbiome-brain communication. It is hypothesized that herbal supplementation could impact the composition of the gut microbiota, which may subsequently alter the levels of brain metabolites involved in neurotransmission and neurophysiological processes. In addition, gut bacteria use primarily γ-aminobutyric acid (GABA), dopamine (DA), norepinephrine (NE), serotonin (5-HT), and histamine (His) to communicate with the central nervous system (CNS), which can significantly influence the brain’s cognitive and stress responses [ 13 ]. However, the underlying mechanisms of communication between the gut microbiota and the brain remain unclear. This study aims to investigate the efficacy of Verbena and Polygonum cuspidatum supplementation on growth performance, immunity, cecal microbiota, and brain metabolites in Sansui ducks. Specifically, the study examines the alterations in the gut microbiota and the associated changes in brain metabolite concentrations, shedding light on the potential mechanisms by which these herbal supplements influence poultry health and performance. By integrating growth data, gut microbiome analysis, and brain metabolite profiling, this research uncovers the complex interplay between diet, gut ecology, and brain biochemistry, contributing novel insights into the potential of herbal supplementation as a natural alternative to antibiotics in poultry production. Results Growth Performance and Serum Immunoglobulins The effect of Verbena and Polygonum cuspidatum supplementation in duck diets on growth performance and serum immunoglobulins is shown in Table 2. Dietary supplementation with Verbena and Polygonum cuspidatum significantly increased final body weight and IgG and IgM levels while decreasing the feed conversion ratio compared to the ducks fed with the control diets (P < 0.05). Microbial Composition of the Cecum The composition of the microbial community in the cecal digesta using 16S rRNA amplicon sequencing is shown in Figure 1. The Chao index estimates the species’ richness (i.e., the number of species present) in a treatment. Simpson's index measures microbial richness or evenness, while Shannon entropy measures species richness and the community's evenness in a sample or within a treatment. The Chao index, Simpson index, and Shannon index in the alpha diversity among the groups were not significantly different (Figure 1A). The Venn diagram (Figure 1B) shows that 3 groups contained a total of 340 shared OTUs, while the T3, T2, and T1 groups had 343, 386, and 647 unique OTUs, respectively. The PCoA by Unweighted UniFrac distance indicated that the control group was separated from the other groups (Figure 1C). Additionally, Bacteroidetes and Firmicutes were the major phylum species across all groups (Figure 2A). Further analysis of the phylum composition of the top 25 ranked groups showed that a total of 5 differential bacteria were identified in each group, with a significant increase in the relative abundance of Bacteroidetes , Spirochaetae and Saccharibacteria in the Verbena supplemented group compared to control group, and a significant decrease in the relative abundance of Fimicutes and Actinobacteria (P < 0.05) (Figure 2B). Taxon-based analysis at the genus level revealed Bacteroides , Fusobacterium , and Megamonas as the predominant genera (Figure 2C). The Collinsella , Subdoligranulum , Peptoclostridium , and Sellimonas were significantly enriched (P < 0.05) in the Verbena supplemented group at the genus level as compared to the control group, while supplementation with Polygonum cuspidatum significantly increased the relative abundance of Megamonas compared to the control group (P < 0.05). LEfSe Analysis of Species To identify the specific bacteria that were characteristic of the 3 groups, LEfSe was used (expressed as values of linear discriminant analysis) in further evaluating the differences in bacterial composition among the different dietary treatments (Figure 3). At the phylum level, a great abundance of Firmicutes and Actinobacteria in the control (T3) group, and Spirochaetae , Saccharibacteria , and Verrucomicrobia in the Verbena supplemented (T1) group were detected. At the genus level, Collinsella , Peptoclostridium , Subdoligranulum , Sellimonas , Intestinomonas , Anaerofustis , Anaerosporobacter , Bifidobacteriales , and Enterorhabdus in the control (T3) group, and Megamonas , Catenisphaera , Anaeroplasma , and Eubacterium in the Polygonum cuspidatum (T2) group, and Treponema , Succinatimonas , and Ruminoccus in the Verbena supplemented (T1) group were detected. Effects of Verbena and Polygonum cuspidatum on the level of the B rain M etabolites of Sansui ducks The effect of dietary Verbena and Polygonum cuspidatum supplementation on the concentrations of brain metabolites in Sansui ducks is presented in Figure 4. Tukey’s multiple comparison tests indicated that dietary supplementation with Verbena significantly reduced the GABA and Tyr concentrations (P < 0.05), and both Verbena and Polygonum cuspidatum supplementation significantly increased the Gln concentrations compared to the control group (P < 0.05). Based on Tukey’s multiple comparison tests, dietary supplementation with Verbena and Polygonum cuspidatum significantly decreased Glu and Ach concentrations (P < 0.05), while Polygonum cuspidatum supplementation significantly increased 5-HIAA concentration compared to the control group (P < 0.05). Correlation between B rain M etabolite C oncentrations and C ecal M icrobiota in Sansui ducks To find out the correlation between brain metabolites and cecal microbiota, we performed a Spearman correlation analysis between the top 40 differential bacteria and brain metabolite concentrations in Sansui ducks treated with Verbena and P olygonum cuspidatum, as shown in Figure 5. The level of Glu showed a positive correlation with 5 bacterial genera ( Intestinimonas, Peptoclostridium, Tyzzerella, Lactobacillus , and Gallibacterium ) and a negative correlation with 3 major bacterial genera ( Elusimicrobium, Coprococcus , and Synergistes ). The level of Ach showed a positive correlation with 2 main bacterial genera ( Fusobacterium and Flavonifracter ) and a negative correlation with 3 bacterial genera ( Olsenella, Megasphaera, and Shuttleworthia ). The level of Gln had no negative correlation with all bacterial genera but showed a positive correlation with 4 bacterial genera ( Enterococcus, Gallibacterium, Veillonella, and Anaerosporobacter ). Moreover, the GABA level showed a positive correlation with 4 bacterial genera ( Veillonella, Anaerosporobacter, Anaerofustis, and Flavonifracter ) and a negative correlation with Weissella and Ruminococcus . The level of 5-HIAA showed a positive correlation with Anaerofustis and a negative correlation with 3 bacterial genera ( Weissella, Staphylococcus, and Coprobacter ). Discussion In traditional herbal medicine, Verbena and Polygonum cuspidatum are polyphenolic plants that have been reported to possess antioxidant and immunostimulant properties [ 14 ]. Our results showed that ducks fed Verbena and Polygonum cuspidatum significantly increased final body weight and decreased feed conversion ratio (FCR), indicating improved feed efficiency. These improvements in growth performance align with previous studies on Verbena and Polygonum cuspidatum bioactive substances, such as flavonoids and verbascoside from Verbena, and resveratrol from Polygonum cuspidatum, which are known to enhance nutrient absorption, modulate gut health, and reduce oxidative stress in poultry [ 7 ], which also shows that it might be ascribed to these bioactive components from Verbena and Polygonum cuspidatum improved overall growth performance in poultry. Owing to the crucial role in the immune system, immunoglobulins are commonly used to evaluate the immune status of animals, and three main types, IgA, IgG, and IgM, are present in birds, which could resist the intrusion of a variety of pathogens and toxins [ 15 ]. The IgG is the major avian systemic antibody that acts upon infection with an immunoglobulin that occurs as a second antibody reaction after IgM production [ 16 ]. Our results showed that Verbena and Polygonum cuspidatum enhanced the immune status, evidenced by elevated serum levels of IgG and IgM, suggesting that these herbs may exert immunomodulatory effects. Similar increases in IgG and IgM concentrations upon treatment with Lemon Verbena and resveratrol have been confirmed in broilers [ 17 ]. The bioactive components from Verbena and Polygonum cuspidatum have been shown to promote humoral immunity by activating lymphocyte proliferation and antibody production [ 18 ]. Although IgA levels did not change significantly, the increase in IgG and IgM supports the hypothesis that these herbal supplements may enhance mucosal and systemic immune responses, possibly contributing to better disease resistance and growth. The gut microbiota plays vital roles in the host's immunological, physiological, nutritional, and metabolic homeostasis [ 19 ]. The Chao1, Shannon, and Simpson indices reflect the richness and diversity of microbiota. In our study, despite no significant differences in alpha diversity indices (Chao1, Shannon, and Simpson), the beta diversity (PCoA analysis) clearly showed that the microbial communities in the control group were distinctly separated from those in the Verbena and Polygonum cuspidatum groups. These observations are consistent with those of several studies on resveratrol supplementation [ 20 ]. This suggests that although supplementation with both herbal sources altered the gut microbiota, it did not significantly affect microbial diversity. A potential explanation could be that the duration of supplementation (35 days) was insufficient to observe more pronounced changes in microbial richness, or the ducks may have an inherently stable gut microbiota that is resilient to short-term dietary changes [ 21 ]. Alterations in the composition of the microbiota were also detected in ducks treated with Verbena and Polygonum cuspidatum. In this study, Firmicutes and Bacteroidetes were identified as the dominant phyla in the cecal contents of Sansui ducks, a result that aligns with previous reports on the microbiota of ducks [ 22 ]. Studies have demonstrated that Firmicutes negatively affect growth performance and intestinal barrier function, but Bacteroidetes exert a positive influence in these areas [ 23 ]. Bacteroidetes are crucial for carbohydrate fermentation and demonstrate inhibitory effects on pathogen colonization [ 24 ]. Furthermore, the Bacteroidetes / Firmicutes ratio serves as a key indicator of microbiota functionality [ 25 ]. Our results indicated that supplementation with Verbena significantly increased Bacteroidetes abundance, while decreasing Firmicutes abundance compared to the control group, thereby increasing the Bacteroidetes / Firmicutes ratio, a result that is consistent with previous studies [ 26 ]. Given the increased Bacteroidetes / Firmicutes ratio, alongside the elevated final body weight and decreased feed conversion ratio, we proposed that the changes in the Bacteroidetes / Firmicutes ratio induced by Verbena supplementation may be responsible for the observed improvement in growth performance. In addition, supplementation with Verbena significantly increased Saccharibacteria abundance, while decreasing Actinobacteria abundance compared to the control group. Actinobacteria are associated with dysbiosis and inflammation [ 27 ]. On the other hand, Actinobacteria have the ability to effectively degrade cellulose, hemicellulose, and lignin [ 28 ], indicating supplementation with Verbena enhances immunity and feed efficiency. To our best knowledge, the role of Saccharibacteria (a candidate bacterial phylum) in the duck intestinal microbiota remains inadequately understood [ 29 ]. At the genus level, supplementation with Verbena significantly increased the abundance of Collinsella , Subdoligranulum , Peptoclostridium , and Sellimonas , while supplementation with Polygonum cuspidatum had a higher Megamonas abundance, compared to the control group in this study, which is consistent with previous findings [ 29 ]. Collinsella primarily produces gases in the intestine, a process that has been linked to abnormal lipid metabolism and type 2 diabetes [ 30 ]. Subdoligranulum belongs to the Ruminococcaceae family. Previous studies have shown that Subdoligranulum has the ability to produce butyrate, which plays a key role in promoting intestinal health by supplying energy to host cells and maintaining the integrity of the intestinal barrier [ 31 ]. Similarly, studies suggest that Peptoclostridium plays a role in modulating immune responses, reducing inflammation, and stimulating the production of butyrate, a key factor in maintaining intestinal health [ 32 ]. Megamonas have also been shown to utilize amino acids or carbohydrates to produce acetic acid, which plays a crucial role in intestinal energy supply, maintenance of the intestinal mucosal barrier, and regulation of intestinal motility [ 33 , 34 ]. These findings indicate that supplementation with Verbena or Polygonum cuspidatum promotes intestinal microbiota balance by increasing the abundance of beneficial bacteria and decreasing pathogenic bacterial populations, thereby enhancing intestinal health and indirectly improving production performance. The LEfSe analysis further substantiated these shifts, identifying Treponema , Succinatimonas , and Ruminococcus as biomarkers of the Verbena group. Treponema is involved in fiber degradation [ 35 ], while Succinatimonas can ferment glucose and other carbohydrates to generate SCFAs, especially acetate and succinate, that can benefit enterocyte development [ 36 ]. Moreover, Ruminococcus is involved in dietary fiber fermentation, producing butyrate, which plays a critical role in reducing inflammation and maintaining the health of the gut epithelium [ 37 ]. These taxa suggest enhanced fermentation capacity, immunity, and microbial efficiency in ducks receiving Verbena. Moreover, we further analyzed the changes in the levels of 10 brain metabolites related to neurotransmitter function. Supplementation with Verbena resulted in higher levels of Gln, while reducing the levels of GABA, Tyr, Glu, and Ach. In addition, supplementation with Polygonum cuspidatum increased the levels of Gln and 5-HIAA while decreasing Glu and Ach. GABA and Glu are important inhibitory neurotransmitters in the central nervous system [ 38 ]. Moreover, it has been reported that the GABA neurotransmitter stimulates food intake in chicks by interacting with the GABAergic system via GABA A receptors [ 39 ]. The reduction of GABA may be an important reason for the loss of appetite in ducks during brooding. In addition, Gln, as an important source of energy for intestinal cell metabolism, regulates the intestinal barrier function through reducing gut permeability [ 40 ]. Ach, as the primary neurotransmitter, is associated with cognition, learning, and memory in the neuromuscular and sensory systems for most vertebrates. AchE plays a critical role in Ach degradation and regulating cholinergic nervous transmission [ 41 ]. A previous study showed that following exposure to 100 µg/L PS-NPs, an increased content of Ach chloride accelerates AchE activity, which can lead to neurotoxicity and negative health effects in aquatic organisms [ 40 ]. Verbena supplementation increased Gln while decreasing GABA, Glu, and Ach, which may contribute to applying energy and altered food intake behavior, possibly due to the increase in Gln and reduction in GABA. On the other hand, the increased level of Gln in Verbena supplementation decreased intestinal permeability and may be associated with decreases in the phylum content of Firmicutes [ 40 ]. In addition, increased 5-HIAA, the main serotonin metabolite, in the Polygonum cuspidatum group suggests that this herb may enhance serotonin turnover. This aligns with the known effect of resveratrol (from Polygonum cuspidatum) on serotonergic pathways, including its antidepressant-like effects in mammals [ 42 ]. Polygonum cuspidatum increased 5-HIAA, with decreases in Glu and Ach, which could influence cognition and memory. The alterations in Ach and GABA levels might also have implications for the overall health and neurophysiological balance in ducks. Further studies are needed to explore the precise mechanisms through which these neurotransmitter changes affect growth and production performance in poultry. Neurotransmitters are essential endogenous chemical messengers that govern fundamental nervous system functions, including communication via the brain-gut axis [ 43 ]. As such, they can directly influence the activity and composition of the gut microbiota community [ 44 ]. The correlation analysis demonstrates that, upon exposure to Verbena and Polygonum cuspidatum, 3 altered metabolites (GABA, Ach, and Glu) involved in neurotransmitter production and function were highly related to changes in the microbial groups, including Veillonella , Anaerosporobacter , Anaerofustis , Fusobacterium , Flavonifracter , Intestinimonas , Peptoclostridium , and Lactobacillus , as compared to the control group. Veillonella and Anaerofustis are known to produce SCFAs and lactate, which can influence neurochemical production in the gut epithelium and enteric neurons [ 45 ]. Previous studies found that Flavonifractor produces butyrate through lysine fermentation, which can reduce intestinal inflammation and improve intestinal barrier function [ 35 ]. In this study, Ach was positively correlated with Flavonifractor , indicating that acetylcholine concentration may be related to intestinal inflammation. Lactobacillus are well-studied for their capacity to produce GABA directly from glutamate, a phenomenon observed in both mice [ 46 ]. Previous studies have explored the interactions between brain metabolites, including neurotransmitters, and gut microbiota, such as Proteobacteria , Firmicutes , and Bacteroidetes [ 47 ]. Taken together, the relationship between metabolites involved in neurotransmitter function and the intestinal microbiome provides insight into the underlying pathways connecting the brain and intestine that are impacted by Verbena and Polygonum cuspidatum supplementation in ducks. Conclusion To the best of our knowledge, this study represents the first to report that has linked Verbena and Polygonum cuspidatum to both gut microbiota and brain metabolites in ducks. Dietary supplementation with both herbal sources in Sansui ducks enhanced growth performance and immune function (evidenced by elevated IgG and IgM levels), improved the composition of gut microbial community, and significantly impacted the brain metabolite (GABA, Gln, Glu, Tyr, Ach, and 5-HIAA) profile. The strong correlations between gut bacterial taxa ( Veillonella , Anaerofustis , Fusobacterium , Flavonifracter , and Lactobacillus ) and brain metabolites (GABA, Ach, and Glu ) suggest that these herbs may influence duck physiology through the gut-brain axis. These findings contribute novel insights into how dietary botanicals can shape animal health beyond gut-level changes, potentially impacting behavior, stress response, and neural function in poultry. Moreover, this study is also distinguished by its integration of growth data, gut microbiome, and brain metabolite analysis, which uncovers a complex interplay between diet, gut ecology, and brain biochemistry. Materials and methods Birds, E xperimental D esign, and D iets Herbs (Verbena and Polygonum cuspidatum powder) were purchased from Ju Chun Tang Chinese Herbal Medicine Sales Co., LTD., Bozhou City, Anhui Province, China. The feeding process was mixed in powder form. Verbena (powder obtained from dried Verbena roots) is rich in flavonoids and verbascoside [48] . Polygonum cuspidatum (powder obtained from dried Polygonum cuspidatum roots) is rich in resveratrol [49]. A total of 216 Sansui ducks (1-day-old) with similar body weight were supplied by Sanyuan Agricultural Development Co., Ltd., Guizhou, China. Ducks were randomly allocated to 3 groups with 6 replicates per group and 12 ducks per replicate, using a completely randomized design. The dietary treatments were the control (T3) group: a basal diet; Verbena (T1) group: a basal diet with 40 mg/kg Verbena; Polygonum cuspidatum (T2) group: a basal diet with 40 mg/kg Polygonum cuspidatum. The composition and nutrient levels of the diets are shown in Table 1. Diets were formulated to meet nutritional requirements according to the recommendations of the National Research Council [50], containing 2,800 kcal of metabolizable energy/kg and 15% crude protein, and were maintained on a 16-h light cycle daily, with water available ad libitum. At the end of 35 days of age, six ducks were randomly selected from each of the three groups following a 12-hour fasting period. The pen number and individual body weight of each duck were recorded prior to their slaughter. Approximately 3 mL of blood was collected from the subwing vein using a vacuum coagulation tube, allowed to stand at 25°C for 2 hours, and then placed on ice. After coagulation, the serum was separated and centrifuged at 3,000 rpm for 10 minutes to eliminate impurities. The resulting supernatant was transferred to a clean centrifuge tube and stored at -20°C for further analysis. Then, the ducks were euthanized with an overdose intravenous sodium pentobarbital injection (50 mg/kg), and brains were isolated, rinsed in ice-cold phosphate-buffered saline (PBS) to remove blood and other contaminants, immediately frozen in liquid nitrogen, and then stored at –80°C for further analysis. The cecum contents samples were collected, quickly frozen in liquid nitrogen, and stored in a refrigerator at –20°C for subsequent analyses. Determination of Immunoglobulin Levels in the Serum An enzyme-linked immunosorbent assay (ELISA) was used to determine the levels of immunoglobulin (Ig)A, IgG, and IgM, levels in the serum. All commercial ELISA kits used for testing were purchased from Nanjing Jiancheng Bioengineering Institute. The procedures followed the provided instructions. Bacterial DNA Extraction and 16S rRNA Gene Sequencing DNA was extracted from the contents of the cecum using the QIAamp DNA Stool Mini Kit (catalog NO. 51504, Qiagen, CA, USA) following the manufacturer's instructions. The V3-V4 region of the bacterial 16S rRNA gene was PCR amplified using primers 338F (5′-ACTCCTACGGGAGGCAGCA-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′). All PCR products have been recovered using the AxyPrep DNA gel recovery kit and quantified using the FTC-3000TM real-time PCR instrument. After obtaining the DNA fragment sequence, the QIAamp DNA Stool Mini Kit was used to construct the library, Illumina's Miseq PE300 platform was used for sequencing, and the sequencing was completed at Shanghai Weiji Biotechnology Co., Ltd. (Shanghai, China). For 16S rRNA gene analysis, raw sequence data were subjected to quality control using QIIME2 (https://qiime2.org/), which removed low-quality sequences and potential contaminants. UPARSE software was used to cluster operational taxonomic units (OTUs) based on 97% sequence similarity. Alpha diversity of the microbial communities was assessed using Chao1, Shannon, and Simpson indices in QIIME2. Beta diversity was evaluated through principal coordinate analysis (PCoA). Differences in relative bacterial abundance were determined using the nonparametric Kruskal-Wallis sum-rank test. Bacterial biomarkers distinguishing the microbial communities across all groups were identified using linear discriminant analysis (LDA) effect size (LEfSe) (LDA > 2.5, P < 0.05). The co-occurrence of microbial communities was analyzed for the top 40 genera based on significant Spearman correlations ( P < 0.05). Brain M etabolite A nalysis Accurately weigh 15 mg of sample into a 2 mL EP tube, accurately add 200 μL of 10% formic acid methanol solution-ddH 2 O (1:1.V/V) solution, add 50 mg of glass beads; put it into a high-throughput tissue grinder and oscillate at 60 Hz for 1 min, repeat twice; centrifuge at 12000 rpm and 4 ℃ for 5 min, take 50 μL of supernatant, accurately add 50 μL of dual isotope internal standard with a concentration of 100 ppb, vortex and oscillate for 30 s, filter the supernatant through a 0.22 μm membrane, and add the filtrate to the detection bottle (detection of low-content substances). Take 10 μL of the original supernatant, add 490 μL of 10% formic acid methanol solution-ddH 2 O (1:1. V/V) solution, vortex and oscillate for 30 s, take 100 μL of the diluted sample, add 100 μL of 100 ppb dual isotope internal standard, vortex and oscillate for 30 s, filter the supernatant through a 0.22 μm membrane, and add the filtrate to the detection bottle (detection of high-content substances) vial for liquid chromatography-mass spectrometry (LC-MS) analysis. Weigh appropriate amounts of 10 brain metabolites (4-Aminobutyric acid [GABA], Glutamine [Gln], Glutamic acid [Glu], Histamine [His], L-Histidine [L-His], Tyramine [Tyr], Tryptamine [Trp], Acetylcholine [Ach], Norepinephrine [NE], and 5-Hydroxyindoleacetic acid [5-HIAA]) standards and prepare single standard stock solutions with 10% formic acid in methanol. Take appropriate amounts of each stock solution to prepare mixed standards, dilute each to the appropriate concentration with 10% formic acid in methanol, and prepare working standard solutions. For LC-MS analysis, chromatographic conditions: chromatographic column: ACQUITY UPLC BEH C18 column (2.1×100 mm, 1.7 μm, Waters, USA), injection volume 5 μL, column temperature 40 ℃, mobile phase A-10% methanol-water (containing 0.1% formic acid), B-50% methanol-water (containing 0.1% formic acid). Gradient elution conditions are 0-1 min, 20-100% B; 1-7 min, 100% B; 7-7.5 min, 100-20% B; 7.5-11 min, 20% B. Flow rate 0.4 mL/min. Mass spectrometry conditions: electrospray ionization (ESI) source, positive ionization mode. The ion source temperature is 500 °C, the ion source voltage is 5500 V, the collision gas is 6 psi, the curtain gas is 30 psi, and the nebulizer gas and auxiliary gas are 50 psi. Multiple reaction monitoring (MRM) scanning was used. Statistical Analysis Data for growth performance, immunoglobulin levels, and brain metabolite concentrations were analysed using SPSS software (version 27.0). All data values were stated as mean ± SEM. One-way analysis of variance (ANOVA) and Tukey's post hoc test were employed to determine the significance of mean differences. P-values were considered statistically different at P < 0.05. Alpha diversity analysis was calculated based on the Chao index, Shannon index, and Simpson index using QIIME2 (v1.9.1). Beta diversity was calculated based on the unweighted UniFrac distance, and statistical comparisons among groups were performed with permutational ANOVA. Figures were generated in GraphPad Prism 10.0 software (GraphPad Software Inc., San Diego, CA). Spearman rank correlation analysis assessed the relationship between microbiota and other brain metabolites. Abbreviations FCR Feed conversion ratio IgA Immunoglobulin A IgG Immunoglobulin G IgM Immunoglobulin M OUT Operational taxonomic unit GABA γ-Aminobutyric acid Gln Glutamine Glu Glutamic acid His Histamine L-His L-Histidine Tyr Tyramine Trp Tryptamine Ach Acetylcholine NE Norepinephrine 5-HIAA 5-Hydroxyindoleacetic acid Declarations Acknowledgments The authors gratefully acknowledge the financial support provided by the General Program of the National Natural Science Foundation of China (Grant No. 31960682). Funding This work was funded by the General Program of the National Natural Science Foundation of China (No.31960682). Author i nformation Authors and Affiliations School of Animal Technology and Innovation, Institute of Agricultural Technology, Suranaree University of Technology, Nakhon Ratchasima, 30000, Thailand Yongcai Zhu & Linli Luo College of Animal Science, Guizhou University, Guiyang, China Qiaoqun Wu & Shenglin Yang Author Contributions YZ: Data curation, Investigation, Validation, Writing – original draft. QW: Investigation, Writing – original draft. LL: Data curation, Investigation, Writing – original draft. SY: Data curation, Investigation, Supervision, Writing – original draft, Writing – review & editing. Corresponding authors Correspondence to Shenglin Yang Ethics declarations Ethics approval and consent to participate All animal experimental procedures were approved by the Ethics Committee of Guizhou University (permit No. EAE-GZU-2022-E032) and performed in accordance with the Regulations for the Administration of Affairs Concerning Experimental Animals (State Council, P. R. China, 2017, Revision). All methods were performed in accordance with the relevant guidelines/regulations of the Guizhou University Animal Care and Use Committee. For the animal trial, day-old ducks were sourced from a leading and reliable commercial supplier. The supplier provided full informed consent for their ducks to be used in this animal trial; they reviewed an informed consent form, were given the opportunity to ask questions, and signed the form. Consent for publication Not applicable. Competing interests The authors declare that they have no competing financial interests or personal relationships that may have influenced the work reported in this study. Additional information Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 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The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. References Zhu Q, Sun P, Zhang B, Kong L, Xiao C, Song Z. Progress on Gut Health Maintenance and Antibiotic Alternatives in Broiler Chicken Production. Front Nutr. 2021;8:692839. Hernando-Amado S, Coque TM, Baquero F, Martínez JL. Antibiotic Resistance: Moving From Individual Health Norms to Social Norms in One Health and Global Health. Front Microbiol. 2020;11:1914. Ayalew H, Zhang H, Wang J, Wu S, Qiu K, Qi G. Potential Feed Additives as Antibiotic Alternatives in Broiler Production. Front Vet Sci. 2022;9:916473. Sadeghi G, Karimi A, Shafeie F, Vaziry A, Farhadi D. The Effects of purslane ( Portulaca oleracea L. ) powder on growth performance, carcass characteristics, antioxidant status, and blood metabolites in broiler chickens. Livest Sci. 2016;184:35–40. 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Zhao Y, Li X, Sun S, Chen L, Jin J, Liu S, et al. Protective role of dryland rearing on netting floors against mortality through gut microbiota-associated immune performance in Shaoxing ducks. Poult Sci. 2019;98:4530–8. Li L, Lv X, Han X, Sun C, An K, Gao W, et al. Effect of Dietary Bacillus licheniformis Supplementation on Growth Performance and Microbiota Diversity of Pekin Ducks. Front Vet Sci. 2022;9:832141. Van Hul M, Le Roy T, Prifti E, Dao MC, Paquot A, Zucker J-D, et al. From correlation to causality: the case of Subdoligranulum . Gut Microbes. 2020;12:1849998. Chang C, Gu Z, Du L, Guo J, Yang Y, Wu Z. Effects of L-β-Galactoglucan Supplementation on Growth Performance, Palatability, and Intestinal Microbiota in Adult Beagle Dogs. Metabolites. 2025;15:160. Biasato I, Ferrocino I, Grego E, Dabbou S, Gai F, Gasco L, et al. Yellow Mealworm Inclusion in Diets for Heavy-Size Broiler Chickens: Implications for Intestinal Microbiota and Mucin Dynamics. Animals. 2020;10:1909. Feng J, Li Z, Ma H, Yue Y, Hao K, Li J, et al. Quercetin alleviates intestinal inflammation and improves intestinal functions via modulating gut microbiota composition in LPS-challenged laying hens. Poult Sci. 2023;102:102433. Rychlik I. Composition and Function of Chicken Gut Microbiota. Animals. 2020;10:103. Dworkin M, Falkow S, Rosenberg E, Schleifer K-H, Stackebrandt E, editors. The Prokaryotes. New York, NY: Springer New York; 2006. Lakshmanan AP, Al Zaidan S, Bangarusamy DK, Al-Shamari S, Elhag W, Terranegra A. Increased Relative Abundance of Ruminoccocus Is Associated with Reduced Cardiovascular Risk in an Obese Population. Front Nutr. 2022;9:849005. Dai SF, Gao F, Zhang WH, Song SX, Xu XL, Zhou GH. Effects of dietary glutamine and gamma-aminobutyric acid on performance, carcass characteristics and serum parameters in broilers under circular heat stress. AFST. 2011;168:51–60. Jonaidi H, Noori Z. Neuropeptide Y-induced feeding is dependent on GABAA receptors in neonatal chicks. J Comp Physiol A. 2012;198:827–32. Teng M, Zhao X, Wang C, Wang C, White JC, Zhao W, et al. Polystyrene Nanoplastics Toxicity to Zebrafish: Dysregulation of the Brain–Intestine–Microbiota Axis. ACS Nano. 2022;16:8190–204. Sarter M, Parikh V, Howe WM. Phasic acetylcholine release and the volume transmission hypothesis: time to move on. Nat Rev Neurosci. 2009;10:383–90. Mallick K, Banerjee S. Plants Affecting Serotonergic Neurotransmission. In: Dhara AK, Mandal SC, editors. Role of Herbal Medicines. Singapore: Springer Nature Singapore; 2023. p. 211–29. Dicks LMT. Gut Bacteria and Neurotransmitters. Microorganisms. 2022;10:1838. Liu S, da Cunha AP, Rezende RM, Cialic R, Wei Z, Bry L, et al. The Host Shapes the Gut Microbiota via Fecal MicroRNA. Cell Host & Microbe. 2016;19:32–43. Zhang S-M, Huang S-L. The Commensal Anaerobe Veillonella dispar Reprograms Its Lactate Metabolism and Short-Chain Fatty Acid Production during the Stationary Phase. Microbiol Spectr. 2023;11:e03558-22. Bibi A, Zhang F, Shen J, Din AU, Xu Y. Behavioral alterations in antibiotic-treated mice associated with gut microbiota dysbiosis: insights from 16S rRNA and metabolomics. Front Neurosci. 2025;19:1478304. Chen T, You Y, Xie G, Zheng X, Zhao A, Liu J, et al. Strategy for an Association Study of the Intestinal Microbiome and Brain Metabolome Across the Lifespan of Rats. Anal Chem. 2018;90:2475–83. Sánchez-Marzo N, Lozano-Sánchez J, Cádiz-Gurrea MDLL, Herranz-López M, Micol V, Segura-Carretero A. Relationships Between Chemical Structure and Antioxidant Activity of Isolated Phytocompounds from Lemon Verbena. Antioxidants. 2019;8:324. Zhang J, Zhou L, Zhang P, Liu T, Yang G, Lin R, et al. Extraction of polydatin and resveratrol from Polygonum cuspidatum root: Kinetics and modeling. Food and Bioprod Process. 2015;94:518–24. Applegate TJ, Angel R. Nutrient requirements of poultry publication: History and need for an update. J Appl Poultry Res. 2014;23:567–75. Tables Table 1. Composition and nutrient levels of the experimental diets (air-dry basis). Ingredients Nutrient Level 2 Corn 55.75 CP (%) 18.10 Soybean meal 27.40 ME (MJ/kg) 10.65 Wheat bran 1.50 Crude fibre (%) 3.07 RaPeseed cake 4.00 Ca (%) 3.37 CaHPO 4 2.75 P (%) 0.63 Limestone 7.25 Lysine (%) 0.92 NaCl 0.35 Methionine (%) 0.27 Premix 1 1.00 Total 100.00 1 Pemix provided per kilogram of diet: vitamin A, 4000 IU, vitamin E, 20 mg; vitamin K3, 2 mg; vitamin B1, 3.5 mg; vitamin B12, 0.01 mg; niacin, 50 mg; folic acid, 1.0 mg; Cu, 10 mg; Fe, 80 mg; Mn, 60 mg; Zn, 60 mg; I, 0.4 mg; Se, 0.2 mg; Calcium Pantotherate, 10 mg; Pyridoxol, 2.5 mg; biotin, 0.1 mg. 2 ME is calculated, and the rest are measured values. T able 2. Effect of Verbena and Polygonum cuspidatum on growth performance and plasma immunoglobulins of Sansui ducks 1 . Item Groups T 3(Control) T1 ( Verbena ) T2 ( Polygonum cuspidatum ) P- value Body weight, g/duck 205.67 b 211.90 a 214.11 a <0.001 1 to 35 d Feed intake, g/ducks 1 to 35 d 790.40 798.61 792.32 0.307 Feed conversion ratio 2.52 a 2.39 b 2.40 b 0.001 1 to 35 d Serum immunoglobulins IgA (g/L) 0.82 0.85 0.92 0.112 IgG (g/L) 1.22 b 1.31 a 1.33 a 0.004 IgM (g/L) 0.93 b 1.07 a 1.13 a 0.038 1 T3: feed with the basic diet; T2: feed with basic diet and 40 mg/kg Polygonum cuspidatum; T1: feed with basic diet and 40 mg/kg Verbena. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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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-6522380","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":455741449,"identity":"443c4636-50d2-4537-a0ad-0af7d2b326c1","order_by":0,"name":"Yongcai Zhu","email":"","orcid":"","institution":"Suranaree University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Yongcai","middleName":"","lastName":"Zhu","suffix":""},{"id":455741450,"identity":"59f85130-9ba3-4661-be7e-abff23a7ebe5","order_by":1,"name":"Qiaoqun Wu","email":"","orcid":"","institution":"Guizhou University","correspondingAuthor":false,"prefix":"","firstName":"Qiaoqun","middleName":"","lastName":"Wu","suffix":""},{"id":455741451,"identity":"e918de01-2134-49a2-aae1-4a94463f565e","order_by":2,"name":"Linli Luo","email":"","orcid":"","institution":"Suranaree University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Linli","middleName":"","lastName":"Luo","suffix":""},{"id":455741452,"identity":"ea95d736-0576-494f-98d5-fd21594b2dfe","order_by":3,"name":"Shenglin Yang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA50lEQVRIiWNgGAWjYPACCQYDBsbGB0AWDx8JWpibDUBa2Ii2x4CBvU0CxCCoxeB47+EXP2os7M0lEtsqv+bYybAxMD98dAOfljPn0ix7jkkwW85IbLstuy0Z6DA2Y+McfFpu5JgZM7BJsBncAGqR3MYM1MLDJk1Yyz8JHpCWYslt9URpMX7M2CYhAdLC+HHbYcJaJM+cMWPs7ZMwMDjzsFmacdtxHjZmAn7hO95j/OHHtzp7g+PpDz/+3FZtz8/e/PAxPi0KB4B+h3GYecAkHuUgIN/AwPwBxmH8QUD1KBgFo2AUjEwAANZHRqYfwbXiAAAAAElFTkSuQmCC","orcid":"","institution":"Guizhou University","correspondingAuthor":true,"prefix":"","firstName":"Shenglin","middleName":"","lastName":"Yang","suffix":""}],"badges":[],"createdAt":"2025-04-24 15:53:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6522380/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6522380/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":82619852,"identity":"c4e5847e-c03c-46f5-bced-a572a97f32df","added_by":"auto","created_at":"2025-05-13 12:11:16","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":271365,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of dietary supplementation with Verbena and Polygonum cuspidatum on diversity of the cecal microbiota in Sansui ducks. (A) Chao index, Shannon index, and Simpson index. (B) Venn diagram based on the OTU level. (C) Principal coordinate analysis (PCoA) based on Unweighted UniFrac. T3: feed with the basic diet; T2: feed with basic diet and 40 mg/kg Polygonum cuspidatum; T1: feed with basic diet and 40 mg/kg Verbena.\u003c/p\u003e","description":"","filename":"ExtractPage21.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6522380/v1/e23efbceb4231ef2725bb369.jpg"},{"id":82619853,"identity":"cd86807e-90f8-4a76-b709-b6a53f29cc61","added_by":"auto","created_at":"2025-05-13 12:11:16","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":346422,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of dietary supplementation with Verbena and Polygonum cuspidatum on composition of the cecal microbiota in Sansui ducks. (A) Microbial composition at the phylum level. (B) Significantly abundant microbiota at the phylum level. (C) Microbial composition at the genus level. (D) Significantly abundant microbiota at the genus level. T3: feed with the basic diet; T2: feed with basic diet and 40 mg/kg Polygonum cuspidatum; T1: feed with basic diet and 40 mg/kg Verbena.\u003c/p\u003e","description":"","filename":"ExtractPage22.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6522380/v1/610846c44fd4a4c359c69db1.jpg"},{"id":82622343,"identity":"921d5fbf-2a9e-42c7-b419-560ca8f69a31","added_by":"auto","created_at":"2025-05-13 12:27:16","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":491947,"visible":true,"origin":"","legend":"\u003cp\u003eLinear discriminant analysis (LDA) effect size (LEfSe) of intestinal microbiota (LDA \u0026gt; 2.5, P \u0026lt; 0.05).\u003c/p\u003e\n\u003cp\u003eT3: feed with the basic diet; T2: feed with basic diet and 40 mg/kg Polygonum cuspidatum; T1: feed with basic diet and 40 mg/kg Verbena.\u003c/p\u003e","description":"","filename":"ExtractPage23.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6522380/v1/8c2cca98651ad9eaf93f8c38.jpg"},{"id":82619854,"identity":"a469227b-ecd0-476a-9a19-d625993dad98","added_by":"auto","created_at":"2025-05-13 12:11:16","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":135806,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of dietary supplementation with Verbena and Polygonum cuspidatum on brain metabolites in Sansui ducks.\u003c/p\u003e\n\u003cp\u003eT3: feed with the basic diet; T2: feed with basic diet and 40 mg/kg Polygonum cuspidatum; T1: feed with basic diet and 40 mg/kg Verbena.\u003c/p\u003e","description":"","filename":"ExtractPage24.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6522380/v1/d6aa499ac32b96f077d94f80.jpg"},{"id":82619862,"identity":"ea29cbb2-5805-424b-b3d6-756bcc56818c","added_by":"auto","created_at":"2025-05-13 12:11:16","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":748230,"visible":true,"origin":"","legend":"\u003cp\u003eSpearman's correlation analysis between the abundances of cecal microbiota and hypothalamic metabolite concentrations.\u003c/p\u003e\n\u003cp\u003eSignificant correlations are noted by 0.01 \u0026lt; P ≤ 0.05*, 0.001 \u0026lt;\u003cem\u003e \u003c/em\u003eP ≤ 0.01**.\u003c/p\u003e","description":"","filename":"ExtractPage25.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6522380/v1/0275743767e9d7f512c45f03.jpg"},{"id":86941374,"identity":"f5f1b69d-ee5a-4c2f-91fa-f2efcfc34e96","added_by":"auto","created_at":"2025-07-17 11:53:43","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2939803,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6522380/v1/c7198a55-0b2a-49f3-8792-79516d6e5b7e.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Effects of Verbena and Polygonum cuspidatum on growth performance, immune functions, cecal microbiota, and brain metabolites in Sansui ducks","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAntibiotics are commonly employed in the poultry industry to enhance growth and improve feed efficiency by promoting intestinal health [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. However, the widespread and prolonged use of antibiotics has led to the rise of resistance, and the presence of antibiotic residues in poultry meat and waste poses direct or indirect risks to both human and animal health, and countries are taking antibiotics more seriously [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Therefore, these concerns have prompted an increasing interest in finding natural and efficient alternatives to antibiotics, particularly those that can enhance intestinal health and promote growth performance [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eNatural feed additives, including herbal supplements, have shown promise as potential alternatives to antibiotics in poultry. Previous studies have indicated that the beneficial effects of Chinese herbs on poultry include promoting growth [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], enhancing immunity [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], and regulating intestinal microbiota [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Among these, \u003cem\u003eVerbena\u003c/em\u003e and \u003cem\u003ePolygonum cuspidatum\u003c/em\u003e, two Chinese herbs, are also utilized as feed supplements owing to their distinct biological activities, including antioxidant, anti-inflammatory, antibacterial, and growth-promoting properties. Studies have indicated that bioactive compounds in these herbs, including flavonoids, verbascoside (from \u003cem\u003eVerbena\u003c/em\u003e), and resveratrol (from \u003cem\u003ePolygonum cuspidatum\u003c/em\u003e), play crucial roles in promoting growth, improving feed efficiency, and maintaining intestinal health in animals [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Several studies have indicated that verbascoside acid is responsible for multiple beneficial properties of \u003cem\u003eVerbena\u003c/em\u003e, like its antioxidant, anti-inflammatory, and antineoplastic properties [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Previous studies have shown that pigs fed a diet enriched with verbascoside had improved growth and feed efficiency [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. In addition, Studies have demonstrated that dietary resveratrol supplementation can enhance the immune response, modulate the intestinal microbiota, and improve feed efficiency in poultry [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. However, the precise mechanisms through which these herbal supplements exert their effects on poultry performance and gut health, particularly the connection between gut microbiota and brain metabolites, remain underexplored.\u003c/p\u003e \u003cp\u003eThe bidirectional communication between the gut microbiota and the brain is termed the microbiota\u0026ndash;gut\u0026ndash;brain axis. The gut microbiota is known to influence brain function through the production of metabolites, which in turn affect various physiological processes, including immune modulation, behavior, and metabolism [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. While studies have primarily focused on the gut\u0026rsquo;s role in influencing metabolic processes, less is known about how dietary supplements like \u003cem\u003eVerbena\u003c/em\u003e and \u003cem\u003ePolygonum cuspidatum\u003c/em\u003e affect this microbiome-brain communication. It is hypothesized that herbal supplementation could impact the composition of the gut microbiota, which may subsequently alter the levels of brain metabolites involved in neurotransmission and neurophysiological processes. In addition, gut bacteria use primarily γ-aminobutyric acid (GABA), dopamine (DA), norepinephrine (NE), serotonin (5-HT), and histamine (His) to communicate with the central nervous system (CNS), which can significantly influence the brain\u0026rsquo;s cognitive and stress responses [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. However, the underlying mechanisms of communication between the gut microbiota and the brain remain unclear.\u003c/p\u003e \u003cp\u003eThis study aims to investigate the efficacy of \u003cem\u003eVerbena\u003c/em\u003e and \u003cem\u003ePolygonum cuspidatum\u003c/em\u003e supplementation on growth performance, immunity, cecal microbiota, and brain metabolites in Sansui ducks. Specifically, the study examines the alterations in the gut microbiota and the associated changes in brain metabolite concentrations, shedding light on the potential mechanisms by which these herbal supplements influence poultry health and performance. By integrating growth data, gut microbiome analysis, and brain metabolite profiling, this research uncovers the complex interplay between diet, gut ecology, and brain biochemistry, contributing novel insights into the potential of herbal supplementation as a natural alternative to antibiotics in poultry production.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eGrowth Performance and Serum Immunoglobulins\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe effect of Verbena and Polygonum cuspidatum supplementation in duck diets on growth performance and serum immunoglobulins is shown in Table 2. Dietary supplementation with Verbena and Polygonum cuspidatum significantly increased final body weight and IgG and IgM levels while decreasing the feed conversion ratio compared to the ducks fed with the control diets (P \u0026lt; 0.05).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMicrobial Composition of the Cecum\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe composition of the microbial community in the cecal digesta using 16S rRNA amplicon sequencing is shown in Figure 1. \u0026nbsp;The Chao index estimates the species\u0026rsquo; richness (i.e., the number of species present) in a treatment. Simpson\u0026apos;s index measures microbial richness or evenness, while Shannon entropy measures species richness and the community\u0026apos;s evenness in a sample or within a treatment. The Chao index, Simpson index, and Shannon index in the alpha diversity among the groups were not significantly different (Figure 1A). The Venn diagram (Figure 1B) shows that 3 groups contained a total of 340 shared OTUs, while the T3, T2, and T1 groups had 343, 386, and 647 unique OTUs, respectively. The PCoA by Unweighted UniFrac distance indicated that the control group was separated from the other groups (Figure 1C).\u003c/p\u003e\n\u003cp\u003eAdditionally, \u003cem\u003eBacteroidetes\u003c/em\u003e and\u003cem\u003e\u0026nbsp;Firmicutes\u003c/em\u003e were the major phylum species across all groups (Figure 2A). \u0026nbsp;Further analysis of the phylum composition of the top 25 ranked groups showed that a total of 5 differential bacteria were identified in each group, with a significant increase in the relative abundance of \u003cem\u003eBacteroidetes\u003c/em\u003e, \u003cem\u003eSpirochaetae\u003c/em\u003e and \u003cem\u003eSaccharibacteria\u003c/em\u003e in the Verbena supplemented group compared to control group, and a significant decrease in the relative abundance of\u003cem\u003e\u0026nbsp;Fimicutes\u0026nbsp;\u003c/em\u003eand \u003cem\u003eActinobacteria\u003c/em\u003e (P \u0026lt; 0.05) (Figure 2B). Taxon-based analysis at the genus level revealed \u003cem\u003eBacteroides\u003c/em\u003e, \u003cem\u003eFusobacterium\u003c/em\u003e, and \u003cem\u003eMegamonas\u003c/em\u003e as the predominant genera (Figure 2C). \u0026nbsp;The \u003cem\u003eCollinsella\u003c/em\u003e, \u003cem\u003eSubdoligranulum\u003c/em\u003e, \u003cem\u003ePeptoclostridium\u003c/em\u003e, and \u003cem\u003eSellimonas\u003c/em\u003e were significantly enriched (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in the \u003cem\u003eVerbena\u003c/em\u003e supplemented group at the genus level as compared to the control group, while supplementation with Polygonum cuspidatum\u003cem\u003e\u0026nbsp;\u003c/em\u003esignificantly increased the relative abundance of\u003cem\u003e\u0026nbsp;Megamonas\u003c/em\u003e compared to the control group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLEfSe Analysis of Species\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo identify the specific bacteria that were characteristic of the 3 groups, LEfSe was used (expressed as values of linear discriminant analysis) in further evaluating the differences in bacterial composition among the different dietary treatments (Figure 3). At the phylum level, a great abundance of \u003cem\u003eFirmicutes\u003c/em\u003e and \u003cem\u003eActinobacteria\u003c/em\u003e in the control (T3) group, and \u003cem\u003eSpirochaetae\u003c/em\u003e, \u003cem\u003eSaccharibacteria\u003c/em\u003e, and \u003cem\u003eVerrucomicrobia\u003c/em\u003e in the Verbena supplemented (T1) group were detected. At the genus level, \u003cem\u003eCollinsella\u003c/em\u003e, \u003cem\u003ePeptoclostridium\u003c/em\u003e, \u003cem\u003eSubdoligranulum\u003c/em\u003e, \u003cem\u003eSellimonas\u003c/em\u003e, \u003cem\u003eIntestinomonas\u003c/em\u003e, \u003cem\u003eAnaerofustis\u003c/em\u003e, \u003cem\u003eAnaerosporobacter\u003c/em\u003e, \u003cem\u003eBifidobacteriales\u003c/em\u003e, and \u003cem\u003eEnterorhabdus\u003c/em\u003e in the control (T3) group, and \u003cem\u003eMegamonas\u003c/em\u003e, \u003cem\u003eCatenisphaera\u003c/em\u003e, \u003cem\u003eAnaeroplasma\u003c/em\u003e, and\u003cem\u003e\u0026nbsp;Eubacterium\u003c/em\u003e in the Polygonum cuspidatum (T2) group, and \u003cem\u003eTreponema\u003c/em\u003e, \u003cem\u003eSuccinatimonas\u003c/em\u003e, and \u003cem\u003eRuminoccus\u003c/em\u003e in the \u003cem\u003eVerbena\u003c/em\u003e supplemented (T1) group were detected.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffects of Verbena and Polygonum cuspidatum on the level of the\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eB\u003c/strong\u003e\u003cstrong\u003erain\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eM\u003c/strong\u003e\u003cstrong\u003eetabolites\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;of Sansui ducks\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe effect of dietary Verbena and Polygonum cuspidatum supplementation on the concentrations of brain metabolites in Sansui ducks is presented in\u0026nbsp;Figure 4.\u0026nbsp;Tukey\u0026rsquo;s multiple comparison tests indicated that dietary supplementation with Verbena significantly reduced the GABA and Tyr concentrations (P \u0026lt; 0.05), and both Verbena and Polygonum cuspidatum supplementation significantly increased the Gln concentrations compared to the control group (P \u0026lt; 0.05). Based on Tukey\u0026rsquo;s multiple comparison tests, dietary supplementation with Verbena and Polygonum cuspidatum significantly decreased Glu and Ach concentrations (P \u0026lt; 0.05), while Polygonum cuspidatum supplementation significantly increased 5-HIAA concentration compared to the control group (P \u0026lt; 0.05).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorrelation between\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eB\u003c/strong\u003e\u003cstrong\u003erain\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eM\u003c/strong\u003e\u003cstrong\u003eetabolite\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eC\u003c/strong\u003e\u003cstrong\u003eoncentrations and\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eC\u003c/strong\u003e\u003cstrong\u003eecal\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eM\u003c/strong\u003e\u003cstrong\u003eicrobiota in Sansui ducks\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo find out the correlation between brain metabolites and cecal microbiota, we performed a Spearman correlation analysis between the top 40 differential bacteria and brain metabolite concentrations in Sansui ducks treated with Verbena and \u003cem\u003eP\u003c/em\u003eolygonum cuspidatum, as shown in Figure 5. The level of Glu showed a positive correlation with 5 bacterial genera (\u003cem\u003eIntestinimonas, Peptoclostridium, Tyzzerella, Lactobacillus\u003c/em\u003e,\u003cem\u003e\u0026nbsp;\u003c/em\u003eand \u003cem\u003eGallibacterium\u003c/em\u003e) and a negative correlation with 3 major bacterial genera (\u003cem\u003eElusimicrobium, Coprococcus\u003c/em\u003e,\u003cem\u003e\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;Synergistes\u003c/em\u003e). The level of Ach showed a positive correlation with 2 main bacterial genera (\u003cem\u003eFusobacterium\u003c/em\u003e and\u003cem\u003e\u0026nbsp;Flavonifracter\u003c/em\u003e) and a negative correlation with 3 bacterial genera (\u003cem\u003eOlsenella, Megasphaera,\u0026nbsp;\u003c/em\u003eand \u003cem\u003eShuttleworthia\u003c/em\u003e). \u0026nbsp;The level of Gln had no negative correlation with all bacterial genera but showed a positive correlation with 4 bacterial genera (\u003cem\u003eEnterococcus, Gallibacterium, Veillonella,\u003c/em\u003e and \u003cem\u003eAnaerosporobacter\u003c/em\u003e). Moreover, the GABA level showed a positive correlation with 4 bacterial genera (\u003cem\u003eVeillonella, Anaerosporobacter, Anaerofustis,\u003c/em\u003e and \u003cem\u003eFlavonifracter\u003c/em\u003e) and a negative correlation with \u003cem\u003eWeissella\u003c/em\u003e and \u003cem\u003eRuminococcus\u003c/em\u003e. The level of 5-HIAA showed a positive correlation with \u003cem\u003eAnaerofustis\u003c/em\u003e and a negative correlation with 3 bacterial genera (\u003cem\u003eWeissella, Staphylococcus,\u003c/em\u003e and \u003cem\u003eCoprobacter\u003c/em\u003e).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn traditional herbal medicine, Verbena and Polygonum cuspidatum are polyphenolic plants that have been reported to possess antioxidant and immunostimulant properties [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Our results showed that ducks fed Verbena and Polygonum cuspidatum significantly increased final body weight and decreased feed conversion ratio (FCR), indicating improved feed efficiency. These improvements in growth performance align with previous studies on Verbena and Polygonum cuspidatum bioactive substances, such as flavonoids and verbascoside from Verbena, and resveratrol from Polygonum cuspidatum, which are known to enhance nutrient absorption, modulate gut health, and reduce oxidative stress in poultry [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], which also shows that it might be ascribed to these bioactive components from Verbena and Polygonum cuspidatum improved overall growth performance in poultry.\u003c/p\u003e \u003cp\u003eOwing to the crucial role in the immune system, immunoglobulins are commonly used to evaluate the immune status of animals, and three main types, IgA, IgG, and IgM, are present in birds, which could resist the intrusion of a variety of pathogens and toxins [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. The IgG is the major avian systemic antibody that acts upon infection with an immunoglobulin that occurs as a second antibody reaction after IgM production [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Our results showed that Verbena and Polygonum cuspidatum enhanced the immune status, evidenced by elevated serum levels of IgG and IgM, suggesting that these herbs may exert immunomodulatory effects. Similar increases in IgG and IgM concentrations upon treatment with Lemon Verbena and resveratrol have been confirmed in broilers [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The bioactive components from Verbena and Polygonum cuspidatum have been shown to promote humoral immunity by activating lymphocyte proliferation and antibody production [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Although IgA levels did not change significantly, the increase in IgG and IgM supports the hypothesis that these herbal supplements may enhance mucosal and systemic immune responses, possibly contributing to better disease resistance and growth.\u003c/p\u003e \u003cp\u003eThe gut microbiota plays vital roles in the host's immunological, physiological, nutritional, and metabolic homeostasis [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The Chao1, Shannon, and Simpson indices reflect the richness and diversity of microbiota. In our study, despite no significant differences in alpha diversity indices (Chao1, Shannon, and Simpson), the beta diversity (PCoA analysis) clearly showed that the microbial communities in the control group were distinctly separated from those in the Verbena and Polygonum cuspidatum groups. These observations are consistent with those of several studies on resveratrol supplementation [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. This suggests that although supplementation with both herbal sources altered the gut microbiota, it did not significantly affect microbial diversity. A potential explanation could be that the duration of supplementation (35 days) was insufficient to observe more pronounced changes in microbial richness, or the ducks may have an inherently stable gut microbiota that is resilient to short-term dietary changes [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAlterations in the composition of the microbiota were also detected in ducks treated with Verbena and Polygonum cuspidatum. In this study, \u003cem\u003eFirmicutes\u003c/em\u003e and \u003cem\u003eBacteroidetes\u003c/em\u003e were identified as the dominant phyla in the cecal contents of Sansui ducks, a result that aligns with previous reports on the microbiota of ducks [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Studies have demonstrated that \u003cem\u003eFirmicutes\u003c/em\u003e negatively affect growth performance and intestinal barrier function, but \u003cem\u003eBacteroidetes\u003c/em\u003e exert a positive influence in these areas [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. \u003cem\u003eBacteroidetes\u003c/em\u003e are crucial for carbohydrate fermentation and demonstrate inhibitory effects on pathogen colonization [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Furthermore, the \u003cem\u003eBacteroidetes\u003c/em\u003e/\u003cem\u003eFirmicutes\u003c/em\u003e ratio serves as a key indicator of microbiota functionality [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Our results indicated that supplementation with Verbena significantly increased \u003cem\u003eBacteroidetes\u003c/em\u003e abundance, while decreasing \u003cem\u003eFirmicutes\u003c/em\u003e abundance compared to the control group, thereby increasing the \u003cem\u003eBacteroidetes\u003c/em\u003e/\u003cem\u003eFirmicutes\u003c/em\u003e ratio, a result that is consistent with previous studies [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Given the increased \u003cem\u003eBacteroidetes\u003c/em\u003e/\u003cem\u003eFirmicutes\u003c/em\u003e ratio, alongside the elevated final body weight and decreased feed conversion ratio, we proposed that the changes in the \u003cem\u003eBacteroidetes\u003c/em\u003e/\u003cem\u003eFirmicutes\u003c/em\u003e ratio induced by Verbena supplementation may be responsible for the observed improvement in growth performance. In addition, supplementation with Verbena significantly increased \u003cem\u003eSaccharibacteria\u003c/em\u003e abundance, while decreasing \u003cem\u003eActinobacteria\u003c/em\u003e abundance compared to the control group. \u003cem\u003eActinobacteria\u003c/em\u003e are associated with dysbiosis and inflammation [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. On the other hand, \u003cem\u003eActinobacteria\u003c/em\u003e have the ability to effectively degrade cellulose, hemicellulose, and lignin [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], indicating supplementation with Verbena enhances immunity and feed efficiency. To our best knowledge, the role of \u003cem\u003eSaccharibacteria\u003c/em\u003e (a candidate bacterial phylum) in the duck intestinal microbiota remains inadequately understood [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. At the genus level, supplementation with Verbena significantly increased the abundance of \u003cem\u003eCollinsella\u003c/em\u003e, \u003cem\u003eSubdoligranulum\u003c/em\u003e, \u003cem\u003ePeptoclostridium\u003c/em\u003e, and \u003cem\u003eSellimonas\u003c/em\u003e, while supplementation with Polygonum cuspidatum had a higher \u003cem\u003eMegamonas\u003c/em\u003e abundance, compared to the control group in this study, which is consistent with previous findings [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. \u003cem\u003eCollinsella\u003c/em\u003e primarily produces gases in the intestine, a process that has been linked to abnormal lipid metabolism and type 2 diabetes [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. \u003cem\u003eSubdoligranulum\u003c/em\u003e belongs to the Ruminococcaceae family. Previous studies have shown that \u003cem\u003eSubdoligranulum\u003c/em\u003e has the ability to produce butyrate, which plays a key role in promoting intestinal health by supplying energy to host cells and maintaining the integrity of the intestinal barrier [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Similarly, studies suggest that \u003cem\u003ePeptoclostridium\u003c/em\u003e plays a role in modulating immune responses, reducing inflammation, and stimulating the production of butyrate, a key factor in maintaining intestinal health [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. \u003cem\u003eMegamonas\u003c/em\u003e have also been shown to utilize amino acids or carbohydrates to produce acetic acid, which plays a crucial role in intestinal energy supply, maintenance of the intestinal mucosal barrier, and regulation of intestinal motility [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. These findings indicate that supplementation with Verbena or Polygonum cuspidatum promotes intestinal microbiota balance by increasing the abundance of beneficial bacteria and decreasing pathogenic bacterial populations, thereby enhancing intestinal health and indirectly improving production performance.\u003c/p\u003e \u003cp\u003eThe LEfSe analysis further substantiated these shifts, identifying \u003cem\u003eTreponema\u003c/em\u003e, \u003cem\u003eSuccinatimonas\u003c/em\u003e, and \u003cem\u003eRuminococcus\u003c/em\u003e as biomarkers of the \u003cem\u003eVerbena\u003c/em\u003e group. \u003cem\u003eTreponema\u003c/em\u003e is involved in fiber degradation [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], while \u003cem\u003eSuccinatimonas\u003c/em\u003e can ferment glucose and other carbohydrates to generate SCFAs, especially acetate and succinate, that can benefit enterocyte development [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Moreover, \u003cem\u003eRuminococcus\u003c/em\u003e is involved in dietary fiber fermentation, producing butyrate, which plays a critical role in reducing inflammation and maintaining the health of the gut epithelium [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. These taxa suggest enhanced fermentation capacity, immunity, and microbial efficiency in ducks receiving Verbena.\u003c/p\u003e \u003cp\u003eMoreover, we further analyzed the changes in the levels of 10 brain metabolites related to neurotransmitter function. Supplementation with Verbena resulted in higher levels of Gln, while reducing the levels of GABA, Tyr, Glu, and Ach. In addition, supplementation with Polygonum cuspidatum increased the levels of Gln and 5-HIAA while decreasing Glu and Ach. GABA and Glu are important inhibitory neurotransmitters in the central nervous system [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Moreover, it has been reported that the GABA neurotransmitter stimulates food intake in chicks by interacting with the GABAergic system via GABA\u003csub\u003eA\u003c/sub\u003e receptors [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. The reduction of GABA may be an important reason for the loss of appetite in ducks during brooding. In addition, Gln, as an important source of energy for intestinal cell metabolism, regulates the intestinal barrier function through reducing gut permeability [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Ach, as the primary neurotransmitter, is associated with cognition, learning, and memory in the neuromuscular and sensory systems for most vertebrates. AchE plays a critical role in Ach degradation and regulating cholinergic nervous transmission [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. A previous study showed that following exposure to 100 \u0026micro;g/L PS-NPs, an increased content of Ach chloride accelerates AchE activity, which can lead to neurotoxicity and negative health effects in aquatic organisms [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Verbena supplementation increased Gln while decreasing GABA, Glu, and Ach, which may contribute to applying energy and altered food intake behavior, possibly due to the increase in Gln and reduction in GABA. On the other hand, the increased level of Gln in Verbena supplementation decreased intestinal permeability and may be associated with decreases in the phylum content of \u003cem\u003eFirmicutes\u003c/em\u003e [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. In addition, increased 5-HIAA, the main serotonin metabolite, in the Polygonum cuspidatum group suggests that this herb may enhance serotonin turnover. This aligns with the known effect of resveratrol (from Polygonum cuspidatum) on serotonergic pathways, including its antidepressant-like effects in mammals [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Polygonum cuspidatum increased 5-HIAA, with decreases in Glu and Ach, which could influence cognition and memory. The alterations in Ach and GABA levels might also have implications for the overall health and neurophysiological balance in ducks. Further studies are needed to explore the precise mechanisms through which these neurotransmitter changes affect growth and production performance in poultry.\u003c/p\u003e \u003cp\u003eNeurotransmitters are essential endogenous chemical messengers that govern fundamental nervous system functions, including communication via the brain-gut axis [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. As such, they can directly influence the activity and composition of the gut microbiota community [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. The correlation analysis demonstrates that, upon exposure to \u003cem\u003eVerbena\u003c/em\u003e and Polygonum cuspidatum, 3 altered metabolites (GABA, Ach, and Glu) involved in neurotransmitter production and function were highly related to changes in the microbial groups, including \u003cem\u003eVeillonella\u003c/em\u003e, \u003cem\u003eAnaerosporobacter\u003c/em\u003e, \u003cem\u003eAnaerofustis\u003c/em\u003e, \u003cem\u003eFusobacterium\u003c/em\u003e, \u003cem\u003eFlavonifracter\u003c/em\u003e, \u003cem\u003eIntestinimonas\u003c/em\u003e, \u003cem\u003ePeptoclostridium\u003c/em\u003e, and \u003cem\u003eLactobacillus\u003c/em\u003e, as compared to the control group. \u003cem\u003eVeillonella\u003c/em\u003e and \u003cem\u003eAnaerofustis\u003c/em\u003e are known to produce SCFAs and lactate, which can influence neurochemical production in the gut epithelium and enteric neurons [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Previous studies found that \u003cem\u003eFlavonifractor\u003c/em\u003e produces butyrate through lysine fermentation, which can reduce intestinal inflammation and improve intestinal barrier function [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. In this study, Ach was positively correlated with \u003cem\u003eFlavonifractor\u003c/em\u003e, indicating that acetylcholine concentration may be related to intestinal inflammation. \u003cem\u003eLactobacillus\u003c/em\u003e are well-studied for their capacity to produce GABA directly from glutamate, a phenomenon observed in both mice [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Previous studies have explored the interactions between brain metabolites, including neurotransmitters, and gut microbiota, such as \u003cem\u003eProteobacteria\u003c/em\u003e, \u003cem\u003eFirmicutes\u003c/em\u003e, and \u003cem\u003eBacteroidetes\u003c/em\u003e [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Taken together, the relationship between metabolites involved in neurotransmitter function and the intestinal microbiome provides insight into the underlying pathways connecting the brain and intestine that are impacted by Verbena and Polygonum cuspidatum supplementation in ducks.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eTo the best of our knowledge, this study represents the first to report that has linked Verbena and Polygonum cuspidatum to both gut microbiota and brain metabolites in ducks. Dietary supplementation with both herbal sources in Sansui ducks enhanced growth performance and immune function (evidenced by elevated IgG and IgM levels), improved the composition of gut microbial community, and significantly impacted the brain metabolite (GABA, Gln, Glu, Tyr, Ach, and 5-HIAA) profile. The strong correlations between gut bacterial taxa (\u003cem\u003eVeillonella\u003c/em\u003e, \u003cem\u003eAnaerofustis\u003c/em\u003e, \u003cem\u003eFusobacterium\u003c/em\u003e, \u003cem\u003eFlavonifracter\u003c/em\u003e, and \u003cem\u003eLactobacillus\u003c/em\u003e) and brain metabolites (GABA, Ach, and Glu\u003cb\u003e)\u003c/b\u003e suggest that these herbs may influence duck physiology through the gut-brain axis. These findings contribute novel insights into how dietary botanicals can shape animal health beyond gut-level changes, potentially impacting behavior, stress response, and neural function in poultry. Moreover, this study is also distinguished by its integration of growth data, gut microbiome, and brain metabolite analysis, which uncovers a complex interplay between diet, gut ecology, and brain biochemistry.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cstrong\u003eBirds,\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eE\u003c/strong\u003e\u003cstrong\u003experimental\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eD\u003c/strong\u003e\u003cstrong\u003eesign, and\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eD\u003c/strong\u003e\u003cstrong\u003eiets\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHerbs (Verbena and Polygonum cuspidatum powder) were purchased from Ju Chun Tang Chinese Herbal Medicine Sales Co., LTD., Bozhou City, Anhui Province, China. The feeding process was mixed in powder form.\u0026nbsp;Verbena (powder obtained from dried Verbena roots) is rich in flavonoids and verbascoside [48]\u003csub\u003e.\u003c/sub\u003e Polygonum cuspidatum (powder obtained from dried Polygonum cuspidatum roots) is rich in resveratrol [49].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eA total of 216 Sansui ducks (1-day-old) with similar body weight were supplied by Sanyuan Agricultural Development Co., Ltd., Guizhou, China. Ducks were randomly allocated to 3 groups with 6 replicates per group and 12 ducks per replicate, using a completely randomized design. The dietary treatments were the control (T3) group: a basal diet; Verbena (T1) group: a basal diet with 40 mg/kg Verbena; Polygonum cuspidatum (T2) group: a basal diet with 40 mg/kg Polygonum cuspidatum. The composition and nutrient levels of the diets are shown in\u0026nbsp;Table 1. Diets were formulated to meet nutritional requirements according to the recommendations of the National Research Council [50], containing 2,800 kcal of metabolizable energy/kg and 15% crude protein, and were maintained on a 16-h light cycle daily, with water available ad libitum. At the end of 35 days of age, six ducks were randomly selected from each of the three groups following a 12-hour fasting period. The pen number and individual body weight of each duck were recorded prior to their slaughter. Approximately 3 mL of blood was collected from the subwing vein using a vacuum coagulation tube, allowed to stand at 25°C for 2 hours, and then placed on ice. After coagulation, the serum was separated and centrifuged at 3,000 rpm for 10 minutes to eliminate impurities. The resulting supernatant was transferred to a clean centrifuge tube and stored at -20°C for further analysis. Then, the ducks were euthanized with an overdose intravenous sodium pentobarbital injection (50 mg/kg), and brains were isolated, rinsed in ice-cold phosphate-buffered saline (PBS) to remove blood and other contaminants, immediately frozen in liquid nitrogen, and then stored at –80°C for further analysis. The cecum contents samples were collected, quickly frozen in liquid nitrogen, and stored in a refrigerator at –20°C for subsequent analyses. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetermination of Immunoglobulin Levels in the Serum\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAn enzyme-linked immunosorbent assay (ELISA) was used to determine the levels of immunoglobulin (Ig)A, IgG, and IgM, levels in the serum. \u0026nbsp;All commercial ELISA kits used for testing were purchased from Nanjing Jiancheng Bioengineering Institute. The procedures followed the provided instructions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBacterial DNA Extraction and 16S rRNA Gene Sequencing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDNA was extracted from the contents of the cecum using the QIAamp DNA Stool Mini Kit (catalog NO. 51504, Qiagen, CA, USA) following the manufacturer's instructions. The V3-V4 region of the bacterial 16S rRNA gene was PCR amplified using primers 338F (5′-ACTCCTACGGGAGGCAGCA-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′). All PCR products have been recovered using the AxyPrep DNA gel recovery kit and quantified using the FTC-3000TM real-time PCR instrument. After obtaining the DNA fragment sequence, the QIAamp DNA Stool Mini Kit was used to construct the library, Illumina's Miseq PE300 platform was used for sequencing, and the sequencing was completed at Shanghai Weiji Biotechnology Co., Ltd. (Shanghai, China). For 16S rRNA gene analysis, raw sequence data were subjected to quality control using QIIME2 (https://qiime2.org/), which removed low-quality sequences and potential contaminants. UPARSE software was used to cluster operational taxonomic units (OTUs) based on 97% sequence similarity. Alpha diversity of the microbial communities was assessed using Chao1, Shannon, and Simpson indices in QIIME2. Beta diversity was evaluated through principal coordinate analysis (PCoA). Differences in relative bacterial abundance were determined using the nonparametric Kruskal-Wallis sum-rank test. Bacterial biomarkers distinguishing the microbial communities across all groups were identified using linear discriminant analysis (LDA) effect size (LEfSe) (LDA \u0026gt; 2.5, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05). The co-occurrence of microbial communities was analyzed for the top 40 genera based on significant Spearman correlations (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBrain\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eM\u003c/strong\u003e\u003cstrong\u003eetabolite\u003c/strong\u003e\u003cstrong\u003eA\u003c/strong\u003e\u003cstrong\u003enalysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAccurately weigh 15 mg of sample into a 2 mL EP tube, accurately add 200 μL of 10% formic acid methanol solution-ddH\u003csub\u003e2\u003c/sub\u003eO (1:1.V/V) solution, add 50 mg of glass beads; put it into a high-throughput tissue grinder and oscillate at 60 Hz for 1 min, repeat twice; centrifuge at 12000 rpm and 4 ℃ for 5 min, take 50 μL of supernatant, accurately add 50 μL of dual isotope internal standard with a concentration of 100 ppb, vortex and oscillate for 30 s, filter the supernatant through a 0.22 μm membrane, and add the filtrate to the detection bottle (detection of low-content substances). Take 10 μL of the original supernatant, add 490 μL of 10% formic acid methanol solution-ddH\u003csub\u003e2\u003c/sub\u003eO (1:1. V/V) solution, vortex and oscillate for 30 s, take 100 μL of the diluted sample, add 100 μL of 100 ppb dual isotope internal standard, vortex and oscillate for 30 s, filter the supernatant through a 0.22 μm membrane, and add the filtrate to the detection bottle (detection of high-content substances) vial for liquid chromatography-mass spectrometry (LC-MS) analysis. Weigh appropriate amounts of 10 brain metabolites (4-Aminobutyric acid [GABA], Glutamine [Gln], Glutamic acid [Glu], Histamine [His], L-Histidine [L-His], Tyramine [Tyr], Tryptamine [Trp], Acetylcholine [Ach], Norepinephrine [NE], and 5-Hydroxyindoleacetic acid [5-HIAA]) standards and prepare single standard stock solutions with 10% formic acid in methanol. Take appropriate amounts of each stock solution to prepare mixed standards, dilute each to the appropriate concentration with 10% formic acid in methanol, and prepare working standard solutions.\u0026nbsp;For\u0026nbsp;LC-MS analysis, chromatographic conditions: chromatographic column: ACQUITY UPLC BEH C18 column (2.1×100 mm, 1.7 μm, Waters, USA), injection volume 5 μL, column temperature 40 ℃, mobile phase A-10% methanol-water (containing 0.1% formic acid), B-50% methanol-water (containing 0.1% formic acid). Gradient elution conditions are 0-1 min, 20-100% B; 1-7 min, 100% B; 7-7.5 min, 100-20% B; 7.5-11 min, 20% B. Flow rate 0.4 mL/min.\u0026nbsp;Mass spectrometry conditions: electrospray ionization (ESI) source, positive ionization mode. The ion source temperature is 500 °C, the ion source voltage is 5500 V, the collision gas is 6 psi, the curtain gas is 30 psi, and the nebulizer gas and auxiliary gas are 50 psi. Multiple reaction monitoring (MRM) scanning was used.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData for growth performance, immunoglobulin levels, and brain metabolite concentrations were analysed using SPSS software (version 27.0). All data values were stated as mean ± SEM. One-way analysis of variance (ANOVA) and Tukey's post hoc test were employed to determine the significance of mean differences. P-values were considered statistically different at \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05. Alpha diversity analysis was calculated based on the Chao index, Shannon index, and Simpson index using QIIME2 (v1.9.1). Beta diversity was calculated based on the unweighted UniFrac distance, and statistical comparisons among groups were performed with permutational ANOVA. \u0026nbsp;Figures were generated in GraphPad Prism 10.0 software (GraphPad Software Inc., San Diego, CA). Spearman rank correlation analysis assessed the relationship between microbiota and other brain metabolites.\u0026nbsp;\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eFCR \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Feed conversion ratio\u003c/p\u003e\n\u003cp\u003eIgA \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Immunoglobulin A\u003c/p\u003e\n\u003cp\u003eIgG \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Immunoglobulin G\u003c/p\u003e\n\u003cp\u003eIgM \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Immunoglobulin M\u003c/p\u003e\n\u003cp\u003eOUT \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Operational taxonomic unit\u003c/p\u003e\n\u003cp\u003eGABA \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026gamma;-Aminobutyric acid\u003c/p\u003e\n\u003cp\u003eGln \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Glutamine\u003c/p\u003e\n\u003cp\u003eGlu \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Glutamic acid\u003c/p\u003e\n\u003cp\u003eHis \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Histamine\u003c/p\u003e\n\u003cp\u003eL-His \u0026nbsp; \u0026nbsp; \u0026nbsp; L-Histidine\u003c/p\u003e\n\u003cp\u003eTyr \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Tyramine\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTrp \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Tryptamine\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAch \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Acetylcholine\u003c/p\u003e\n\u003cp\u003eNE \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Norepinephrine\u003c/p\u003e\n\u003cp\u003e5-HIAA \u0026nbsp;5-Hydroxyindoleacetic acid\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eAcknowledgments\u003c/p\u003e\n\u003cp\u003eThe authors gratefully acknowledge the financial support provided by the General Program of the National Natural Science Foundation of China (Grant No. 31960682).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was funded by\u0026nbsp;the General Program of the National Natural Science Foundation of China\u0026nbsp;(No.31960682).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ei\u003c/strong\u003e\u003cstrong\u003enformation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors and Affiliations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSchool of Animal Technology and Innovation, Institute of Agricultural Technology, Suranaree University of Technology, Nakhon Ratchasima, 30000, Thailand\u003c/p\u003e\n\u003cp\u003eYongcai Zhu \u0026amp; Linli Luo\u003c/p\u003e\n\u003cp\u003eCollege of Animal Science, Guizhou University, Guiyang, China\u003c/p\u003e\n\u003cp\u003eQiaoqun Wu \u0026amp; Shenglin Yang\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYZ: Data curation, Investigation, Validation, Writing – original draft. QW: Investigation, Writing – original draft. LL: Data curation, Investigation, Writing – original draft. SY: Data curation, Investigation, Supervision, Writing – original draft, Writing – review \u0026amp; editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorresponding authors\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCorrespondence to Shenglin Yang\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal experimental procedures were approved by the Ethics Committee of Guizhou University (permit No. EAE-GZU-2022-E032) and performed in accordance with the Regulations for the Administration of Affairs Concerning Experimental Animals (State Council, P. R. China, 2017, Revision). All methods were performed in accordance with the relevant guidelines/regulations of the Guizhou University Animal Care and Use Committee. For the animal trial, day-old ducks were sourced from a leading and reliable commercial supplier. The supplier provided full informed consent for their ducks to be used in this animal trial; they reviewed an informed consent form, were given the opportunity to ask questions, and signed the form.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing financial interests or personal relationships that may have influenced the work reported in this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePublisher’s note\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSpringer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRights and permissions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOpen Access\u003c/strong\u003e This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third-party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eZhu Q, Sun P, Zhang B, Kong L, Xiao C, Song Z. Progress on Gut Health Maintenance and Antibiotic Alternatives in Broiler Chicken Production. 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Microorganisms. 2020;8:1715.\u003c/li\u003e\n\u003cli\u003eHao Y, Ji Z, Shen Z, Wu Y, Zhang B, Tang J, et al. Effects of Total Dietary Fiber on Cecal Microbial Community and Intestinal Morphology of Growing White Pekin Duck. Front Microbiol. 2021;12:727200.\u003c/li\u003e\n\u003cli\u003eYang J-Y, Chen S-Y, Wu Y-H, Liao Y-L, Yen G-C. Ameliorative effect of buckwheat polysaccharides on colitis via regulation of the gut microbiota. Int J Biol Macromol. 2023;227:872\u0026ndash;83.\u003c/li\u003e\n\u003cli\u003eOladokun S, Alizadeh M, Mallick AI, Fazel F, Doost JS, Blake K, et al. Influenza a virus subtype H9N2 infection induces respiratory microbiota dysbiosis in chickens via type-I interferon-mediated mechanisms. FEMS Microbes. 2025;6:xtaf001.\u003c/li\u003e\n\u003cli\u003eSteger K, Jarvis \u0026Aring;, Vasara T, Romantschuk M, Sundh I. Effects of differing temperature management on development of Actinobacteria populations during composting. Res Microbiol. 2007;158:617\u0026ndash;24.\u003c/li\u003e\n\u003cli\u003eZhao Y, Li X, Sun S, Chen L, Jin J, Liu S, et al. Protective role of dryland rearing on netting floors against mortality through gut microbiota-associated immune performance in Shaoxing ducks. Poult Sci. 2019;98:4530\u0026ndash;8.\u003c/li\u003e\n\u003cli\u003eLi L, Lv X, Han X, Sun C, An K, Gao W, et al. Effect of Dietary Bacillus licheniformis Supplementation on Growth Performance and Microbiota Diversity of Pekin Ducks. Front Vet Sci. 2022;9:832141.\u003c/li\u003e\n\u003cli\u003eVan Hul M, Le Roy T, Prifti E, Dao MC, Paquot A, Zucker J-D, et al. From correlation to causality: the case of \u003cem\u003eSubdoligranulum\u003c/em\u003e. Gut Microbes. 2020;12:1849998.\u003c/li\u003e\n\u003cli\u003eChang C, Gu Z, Du L, Guo J, Yang Y, Wu Z. Effects of L-\u0026beta;-Galactoglucan Supplementation on Growth Performance, Palatability, and Intestinal Microbiota in Adult Beagle Dogs. Metabolites. 2025;15:160.\u003c/li\u003e\n\u003cli\u003eBiasato I, Ferrocino I, Grego E, Dabbou S, Gai F, Gasco L, et al. Yellow Mealworm Inclusion in Diets for Heavy-Size Broiler Chickens: Implications for Intestinal Microbiota and Mucin Dynamics. Animals. 2020;10:1909.\u003c/li\u003e\n\u003cli\u003eFeng J, Li Z, Ma H, Yue Y, Hao K, Li J, et al. Quercetin alleviates intestinal inflammation and improves intestinal functions via modulating gut microbiota composition in LPS-challenged laying hens. Poult Sci. 2023;102:102433.\u003c/li\u003e\n\u003cli\u003eRychlik I. Composition and Function of Chicken Gut Microbiota. Animals. 2020;10:103.\u003c/li\u003e\n\u003cli\u003eDworkin M, Falkow S, Rosenberg E, Schleifer K-H, Stackebrandt E, editors. The Prokaryotes. New York, NY: Springer New York; 2006.\u003c/li\u003e\n\u003cli\u003eLakshmanan AP, Al Zaidan S, Bangarusamy DK, Al-Shamari S, Elhag W, Terranegra A. Increased Relative Abundance of Ruminoccocus Is Associated with Reduced Cardiovascular Risk in an Obese Population. Front Nutr. 2022;9:849005.\u003c/li\u003e\n\u003cli\u003eDai SF, Gao F, Zhang WH, Song SX, Xu XL, Zhou GH. Effects of dietary glutamine and gamma-aminobutyric acid on performance, carcass characteristics and serum parameters in broilers under circular heat stress. AFST. 2011;168:51\u0026ndash;60.\u003c/li\u003e\n\u003cli\u003eJonaidi H, Noori Z. Neuropeptide Y-induced feeding is dependent on GABAA receptors in neonatal chicks. J Comp Physiol A. 2012;198:827\u0026ndash;32.\u003c/li\u003e\n\u003cli\u003eTeng M, Zhao X, Wang C, Wang C, White JC, Zhao W, et al. Polystyrene Nanoplastics Toxicity to Zebrafish: Dysregulation of the Brain\u0026ndash;Intestine\u0026ndash;Microbiota Axis. ACS Nano. 2022;16:8190\u0026ndash;204.\u003c/li\u003e\n\u003cli\u003eSarter M, Parikh V, Howe WM. Phasic acetylcholine release and the volume transmission hypothesis: time to move on. Nat Rev Neurosci. 2009;10:383\u0026ndash;90.\u003c/li\u003e\n\u003cli\u003eMallick K, Banerjee S. Plants Affecting Serotonergic Neurotransmission. In: Dhara AK, Mandal SC, editors. Role of Herbal Medicines. Singapore: Springer Nature Singapore; 2023. p. 211\u0026ndash;29.\u003c/li\u003e\n\u003cli\u003eDicks LMT. Gut Bacteria and Neurotransmitters. Microorganisms. 2022;10:1838.\u003c/li\u003e\n\u003cli\u003eLiu S, da Cunha AP, Rezende RM, Cialic R, Wei Z, Bry L, et al. The Host Shapes the Gut Microbiota via Fecal MicroRNA. Cell Host \u0026amp; Microbe. 2016;19:32\u0026ndash;43.\u003c/li\u003e\n\u003cli\u003eZhang S-M, Huang S-L. The Commensal Anaerobe Veillonella dispar Reprograms Its Lactate Metabolism and Short-Chain Fatty Acid Production during the Stationary Phase. Microbiol Spectr. 2023;11:e03558-22.\u003c/li\u003e\n\u003cli\u003eBibi A, Zhang F, Shen J, Din AU, Xu Y. Behavioral alterations in antibiotic-treated mice associated with gut microbiota dysbiosis: insights from 16S rRNA and metabolomics. Front Neurosci. 2025;19:1478304.\u003c/li\u003e\n\u003cli\u003eChen T, You Y, Xie G, Zheng X, Zhao A, Liu J, et al. Strategy for an Association Study of the Intestinal Microbiome and Brain Metabolome Across the Lifespan of Rats. Anal Chem. 2018;90:2475\u0026ndash;83.\u003c/li\u003e\n\u003cli\u003eS\u0026aacute;nchez-Marzo N, Lozano-S\u0026aacute;nchez J, C\u0026aacute;diz-Gurrea MDLL, Herranz-L\u0026oacute;pez M, Micol V, Segura-Carretero A. Relationships Between Chemical Structure and Antioxidant Activity of Isolated Phytocompounds from Lemon Verbena. Antioxidants. 2019;8:324.\u003c/li\u003e\n\u003cli\u003eZhang J, Zhou L, Zhang P, Liu T, Yang G, Lin R, et al. Extraction of polydatin and resveratrol from Polygonum cuspidatum root: Kinetics and modeling. Food and Bioprod Process. 2015;94:518\u0026ndash;24.\u003c/li\u003e\n\u003cli\u003eApplegate TJ, Angel R. Nutrient requirements of poultry publication: History and need for an update. J Appl Poultry Res. 2014;23:567\u0026ndash;75.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable 1.\u003c/strong\u003e Composition and nutrient levels of the experimental diets (air-dry basis).\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"470\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 156px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eIngredients\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 83px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 165px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNutrient Level\u003csup\u003e2\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 156px;\"\u003e\n \u003cp\u003eCorn\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 83px;\"\u003e\n \u003cp\u003e55.75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 165px;\"\u003e\n \u003cp\u003eCP (%)\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e18.10\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 156px;\"\u003e\n \u003cp\u003eSoybean meal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 83px;\"\u003e\n \u003cp\u003e27.40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 165px;\"\u003e\n \u003cp\u003eME (MJ/kg)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e10.65\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 156px;\"\u003e\n \u003cp\u003eWheat bran\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 83px;\"\u003e\n \u003cp\u003e1.50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 165px;\"\u003e\n \u003cp\u003eCrude fibre (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e3.07\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 156px;\"\u003e\n \u003cp\u003eRaPeseed cake\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 83px;\"\u003e\n \u003cp\u003e4.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 165px;\"\u003e\n \u003cp\u003eCa (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e3.37\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 156px;\"\u003e\n \u003cp\u003eCaHPO\u003csub\u003e4\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 83px;\"\u003e\n \u003cp\u003e2.75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 165px;\"\u003e\n \u003cp\u003eP (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e0.63\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 156px;\"\u003e\n \u003cp\u003eLimestone\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 83px;\"\u003e\n \u003cp\u003e7.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 165px;\"\u003e\n \u003cp\u003e\u003ca href=\"file:///D%3A/Program%2520Files%2520(x86)/Dict/8.9.6.0/resultui/html/index.html#/javascript%3A;\"\u003eLysine\u003c/a\u003e (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e0.92\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 156px;\"\u003e\n \u003cp\u003eNaCl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 83px;\"\u003e\n \u003cp\u003e0.35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 165px;\"\u003e\n \u003cp\u003e\u003ca href=\"file:///D%3A/Program%2520Files%2520(x86)/Dict/8.9.6.0/resultui/html/index.html#/javascript%3A;\"\u003eMethionine\u003c/a\u003e (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e0.27\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 156px;\"\u003e\n \u003cp\u003ePremix\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 83px;\"\u003e\n \u003cp\u003e1.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 165px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 156px;\"\u003e\n \u003cp\u003eTotal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 83px;\"\u003e\n \u003cp\u003e100.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 165px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003csup\u003e1\u0026nbsp;\u003c/sup\u003ePemix provided per kilogram of diet: vitamin A, 4000 IU, vitamin E, 20 mg; vitamin K3, 2 mg; vitamin B1, 3.5 mg; vitamin B12, 0.01 mg; niacin, 50 mg; folic acid, 1.0 mg; Cu, 10 mg; Fe, 80 mg; Mn, 60 mg; Zn, 60 mg; I, 0.4 mg; Se, 0.2 mg; Calcium Pantotherate, 10 mg; Pyridoxol, 2.5 mg; biotin, 0.1 mg.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e2\u0026nbsp;\u003c/sup\u003eME is calculated, and the rest are measured values.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eT\u003c/strong\u003e\u003cstrong\u003eable 2.\u0026nbsp;\u003c/strong\u003eEffect of Verbena and Polygonum cuspidatum on growth performance and plasma immunoglobulins of Sansui ducks\u003csup\u003e1\u003c/sup\u003e.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"603\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 158px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eItem\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 353px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGroups\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 158px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eT 3(Control)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eT1 (\u003c/strong\u003e\u003cstrong\u003eVerbena\u003c/strong\u003e\u003cstrong\u003e)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eT2 (\u003c/strong\u003e\u003cstrong\u003ePolygonum cuspidatum\u003c/strong\u003e\u003cstrong\u003e)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eP-\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003evalue\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 158px;\"\u003e\n \u003cp\u003eBody weight, g/duck\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003e205.67\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003e211.90\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003e214.11\u003csup\u003ea\u003c/sup\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 158px;\"\u003e\n \u003cp\u003e1 to 35 d\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 158px;\"\u003e\n \u003cp\u003eFeed intake, g/ducks\u003c/p\u003e\n \u003cp\u003e1 to 35 d\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003e790.40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003e798.61\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003e792.32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e\u0026nbsp;0.307\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 158px;\"\u003e\n \u003cp\u003eFeed conversion ratio\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003e2.52\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003e2.39\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003e2.40\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 92px;\"\u003e\n \u003cp\u003e\u0026nbsp;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 158px;\"\u003e\n \u003cp\u003e1 to 35 d\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"4\" style=\"width: 444px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 158px;\"\u003e\n \u003cp\u003eSerum immunoglobulins\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 92px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 158px;\"\u003e\n \u003cp\u003eIgA (g/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003e0.82\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003e0.85\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003e0.92\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e\u0026nbsp;0.112\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 158px;\"\u003e\n \u003cp\u003eIgG (g/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003e1.22\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003e1.31\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003e1.33\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e0.004\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 158px;\"\u003e\n \u003cp\u003eIgM (g/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003e0.93\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003e1.07\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003e1.13\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e\u0026nbsp; 0.038\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003csup\u003e1\u003c/sup\u003eT3: feed with the basic diet; T2: feed with basic diet and 40 mg/kg Polygonum cuspidatum; T1: feed with basic diet and 40 mg/kg Verbena.\u0026nbsp;\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Verbena, Polygonum cuspidatum, duck, growth performance, brain metabolite, microbiota","lastPublishedDoi":"10.21203/rs.3.rs-6522380/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6522380/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eVerbena and Polygonum cuspidatum, recognized for their antioxidant and immunomodulatory properties, have demonstrated potential benefits. However, the specific mechanisms by which these herbs impact poultry health, particularly regarding alterations in gut microbiota and brain metabolite profiles, remain insufficiently investigated. This study aimed to investigate the effects of Verbena and Polygonum cuspidatum supplementation on the growth performance, immune function, cecal microbiota, and brain metabolites in Sansui ducks.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eA total of 216 one-day-old ducks were randomly assigned to three treatment groups for a 35-day trial, each with 6 replicates of 12 ducks. The ducks were fed a basal diet (T3), a basal diet supplemented with 40mg/kg Verbena (T1), and a basal diet supplemented with 40mg/kg Polygonum cuspidatum (T2).\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe results showed that both herbs significantly increased body weight, IgG, and IgM levels, while decreasing the feed conversion ratio (FCR) compared to the control group (T3) (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) Notably, Verbena supplementation increased the relative abundance of beneficial bacteria, such as \u003cem\u003eBacteroidetes\u003c/em\u003e and \u003cem\u003eSaccharibacteria\u003c/em\u003e, and significantly decreased the relative abundance of pathogenic bacteria such as \u003cem\u003eActinobacteria\u003c/em\u003e ompared to the control group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Polygonum cuspidatum treatment increased the relative abundance of \u003cem\u003eMegamonas\u003c/em\u003e compared to the control group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Additionally, Verbena treatment increased the concentration of Gln and decreased the concentrations of GABA, Tyr, and Ach compared to the control group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Polygonum cuspidatum treatment increased 5-HIAA concentration compared to the control group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Further correlation analysis highlighted a significant link between gut microbiota (\u003cem\u003eVillanella, Anaerosporobacter, Anaerofustis\u003c/em\u003e, and \u003cem\u003eFlavonifracter\u003c/em\u003e) changes and brain metabolites (GABA, Ach, and Glu), suggesting the potential influence of these herbs through the microbiota-gut-brain axis.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThis study provides novel insights into how dietary herbal supplements can improve poultry health by modulating both gut and brain biochemistry, offering a promising natural alternative to antibiotics in poultry production.\u003c/p\u003e","manuscriptTitle":"Effects of Verbena and Polygonum cuspidatum on growth performance, immune functions, cecal microbiota, and brain metabolites in Sansui ducks","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-13 12:11:11","doi":"10.21203/rs.3.rs-6522380/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b5ad604d-38a7-4cdc-8360-ff91f9ed2d04","owner":[],"postedDate":"May 13th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-07-17T11:53:17+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-13 12:11:11","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6522380","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6522380","identity":"rs-6522380","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}