Vanillin Improves Antioxidant Status, Immunity and Gut Microbiota in Broilers

1. Introduction

Antibiotics have served as essential growth promoters in livestock and poultry production for decades. Their multifunctional benefits in animal husbandry include protecting animal health by inhibiting pathogenic microorganisms, enhancing growth performance, optimizing physiological parameters, and improving nutrient absorption and feed efficiency. These collective effects have profoundly contributed to modern animal production systems (Abd et al., 2022). However, the widespread application of antibiotics in animal production has generated substantial challenges, particularly the development of antimicrobial resistance (AMR) in pathogenic bacteria. This emerging threat now represents one of the most urgent global public health concerns facing the livestock and poultry industries (Silveira et al., 2021).

In recent years, significant progress has been made in developing antibiotic-alternatives, with promising substitutes including plant extracts, probiotics, prebiotics, antimicrobial peptides (AMPs), etc. (Zhu et al., 2021). Among these, plant extracts have attracted particular attention due to some advantages, such as abundant source availability, high nutritional value, diverse bioactive properties, low or no toxicity, minimal risk of inducing AMR, and negligible residue accumulation (Chen et al., 2022). These attributes make phytogenic additives as prime candidates for replacing antibiotics in animal production. Many studies have demonstrated their multifaceted benefits in broiler production, including potent antioxidant, anti-inflammatory, antimicrobial, immunomodulatory, and gut microbiota-modulating effects (Peng et al., 2022, Abdel-Latif et al., 2021, Al-Kahtani et al., 2022). Notably, dietary supplementation with thymol and carvacrol significantly improves antioxidant capacity, as evidenced by increased serum and hepatic superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities, along with reduced malondialdehyde (MDA) levels (Hashemipour et al., 2013). Carvacrol essential oil exerts anti-inflammatory effects through dual mechanisms: suppressing pro-inflammatory cytokines and the Toll-like receptors (TLRs)/ Nuclear factor-κB (NF-κB) signaling pathway, thereby alleviating lipopolysaccharide (LPS)-induced inflammation (Liu et al., 2019). Flavonoids promote immune organ development and strengthen stimulate immune-stress responses (Yang et al., 2017), while Glycyrrhiza polysaccharides regulate immune overactivation and bolster host defense mechanisms (Li et al., 2009). Similarly, Lycium barbarum polysaccharides help maintain immune homeostasis by regulating anti-inflammatory response (Peng et al., 2014). Furthermore, oregano essential oil and organic acid blends profoundly influence gut microbiota composition and function. These additives significantly improve cecal short-chain fatty acid (SCFA) levels and induce a marked gut microbial shift, particularly increased the Firmicutes-to-Bacteroidetes ratio. Such microbial remodeling enhances lipid metabolism and fat deposition, ultimately improving weight gain and overall growth performance in broilers (Li, 2020).

Vanillin, a bioactive phenolic compound primarily derived from Vanilla planifolia beans, has gained widespread recognition as both a flavoring agent and therapeutic molecule. As one of the most extensively used food additives, vanillin imparts its characteristic aroma to various products including baked goods (e.g., biscuits and cakes), confectionery (e.g., chocolate, candy, chewing gum), and dairy desserts (s e.g., ice cream and milk tea) (Graf et al., 2016, Bagdas et al., 2024, Zhang et al., 2014, Bezerra et al., 2019). Additionally, vanillin exhibits remarkable pharmacological properties, particularly antimicrobial activity (Patterson et al., 2015). The compound demonstrates favorable pharmacokinetics, characterized by rapid systemic absorption and blood-brain barrier permeability, along with an excellent safety profile (Ho et al., 2011). Its therapeutic potential is further evidenced by potent antioxidant and anti-inflammatory properties. In vitro studies have shown its capacity to suppresses LPS-induced inflammatory responses in THP-1 monocytes, while in vivo studies confirm its efficacy in alleviating mastitis in pregnant BALB/c mice (Zhao et al., 2019; Guo et al., 2019). Vanillin’s therapeutic applications extend to gastrointestinal inflammation, where it attenuates trinitrobenzene sulfonic acid (TNBS)-induced colitis by modulation of the NF-κB signaling pathway (Wu et al., 2009, Ben et al., 2017). Recent evidence suggests additional protective effects against Escherichia coli-induced infectious colitis (Wang et al., 2024a). Notably, vanillin has been shown to alleviate potassium bromate (KBrO₃)-induced depressive-like behaviors in mice, mediated through its antioxidant capacity and suppression of pro-inflammatory cytokines, including tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β) (Ben et al., 2017a; Ben et al., 2018, Ben et al., 2016). These findings collectively highlight vanillin’s multi-target pharmacological profile and therapeutic potential.

Despite its well-documented pharmacological properties, the potential applications of vanillin in broilers remain largely unexplored. This study systematically evaluates the effects of dietary vanillin supplementation on broiler health, including antioxidant capacity, anti-inflammatory responses, immune function, and intestinal microbiota composition. Our findings address a significant knowledge gap in poultry nutrition while providing a scientific basis for developing vanillin as a novel, plant-derived alternative to conventional growth promoters in sustainable poultry production systems.

2. Materials and Methods

2.1. Animals, Diets, and Experimental Design

The experimental animals used in this study were purchased from the Dingjiazhuang Breeding Farm (Beijing Dapa Zhengda Co., Ltd.). All animal experiments and housing conditions were approved by the Experimental Animal Ethics Committee of the Institute of Feed Research of the Chinese Academy of Agricultural Sciences (No. IFR CAAS20241213).

A total of 60 one-day-old Kebao broilers were randomly distributed into five experimental groups (n=12 replicates per group). The control group received a basal diet, while treatment groups were supplemented with either 0.1%, 0.2%, 0.4% food-grade vanillin, or 20% chlortetracycline (CTC) premix as a positive control. All diets were formulated to meet or exceed NRC (1994) nutritional requirements across two growth phases: starter (days 1-21) and grower (days 22-42). The detailed dietary formulation and nutritional compositions are provided in Table 1. Upon trial initiation, broilers were housed in multi-tier cages with ad libitum access to feed and water, with twice-daily scheduled feeding. Environmental conditions were strictly controlled, with temperature maintained at 34-36℃ during the first week and gradually reduced by 2-3℃ weekly until reaching a stable temperature of 23±2℃. Standard husbandry practices included daily manure removal and ventilation to maintain optimal hygiene and air quality through the experimental period.

2.2. Growth Performance Assessment

Broilers were subjected to a 12-hour fasting period (with free access to water) prior to body weight measurement on days 21 and 42. Feed intake was recorded weekly through each experimental phase. Growth performance parameters including average daily feed intake (ADFI), average body weight (ABW), and feed conversion ratio were calculated for both the starter (days 1-21) and grower (days 22-42) phases.

2.3. Biological Sample Collection

At the end of each experimental phase (days 21 and 42), six healthy broilers per group were randomly selected for blood sample collection. Venous blood (5-6 mL) was aseptically collected from the pterygoid vein using sterile syringes and transferred to anticoagulant-free tubes. After 2-h clotting at room temperature, samples were centrifuged at 4,000 × g for 15 min at 4℃ to obtain serum. The serum was aliquoted into sterile tubes and stored at -20℃.

Following complete exsanguination, systematic necropsies were performed under aseptic conditions. Primary immune organs (such as thymus, spleen, and Bursa of Fabricius) and liver were dissected, weighed, and examined for gross pathology. Liver tissue samples were harvested from standardized lobar locations using sterile instruments, frozen in liquid nitrogen, and stored at -80℃ for further analysis.

The small intestine was carefully segmented into duodenum, jejunum, and ileum. From each bird, a representative 2-cm mid-duodenal segment was collected using the following preservation protocols: frozen in liquid nitrogen for gene expression, and fixed in 4% paraformaldehyde at 4 °C for 24 h for histomorphological analysis.

2.4. Serum Biomarker Analysis

Serum biomarkers were analyzed using commercial ELISA kits following manufacturers’ protocols. Humoral immune response was assessed by measuring immunoglobulin levels (such as IgA, IgG, and IgM) using chicken-specific ELISA kits. Inflammatory cytokine profiles were characterized by quantifying pro-inflammatory mediators (such as IL-1β, TNF-α, and IL-6) and anti-inflammatory cytokine IL-10 were with chicken-specific ELISA kits. Oxidative stress parameters were evaluated by determining MDA levels and the enzymatic activities of SOD and GSH-Px through corresponding ELISA kits. All analyses were performed in triplicate.

2.5. Duodenum Histopathological Analysis

For histological analysis, duodenal tissue samples were fixed in 4% paraformaldehyde at 4 °C for 24 h, then processed through a standardized protocol involving graded ethanol dehydration (70-100%), paraffin embedding, and sectioning at 4 µm thickness using a rotary microtome. The tissue sections were then dewaxed in xylene, rehydrated through a graded alcohol series, and stained with hematoxylin and eosin (H&E) following established protocols, including hematoxylin differentiation in 1% acid alcohol, bluing in 0.2% ammonia water, and final dehydration prior to mounting with neutral balsam. Histomorphological evaluation was performed using a fluorescence microscopy equipped with a digital imaging system.

Quantitative morphometric analysis was performed to measure key intestinal parameters, including crypt depth (CD), intestinal wall thickness (IWT), villus height (VH), and the villus height-to-crypt depth ratio (VH:CD). A comprehensive pathological scoring system was conducted to evaluate mucosal integrity (epithelial continuity and villus structure), glandular architecture (crypt morphology and dilation), cellular components (goblet cell density and inflammatory infiltration), pathological changes (such as edema, hemorrhage, crypt abscess formation, and epithelial dysplasia).

2.6. 16S rRNA Sequencing of Cecal Microbiota

Cecal content samples (≥2 g per biological replicate) were aseptically collected and immediately frozen in liquid nitrogen prior to storage at -80℃ for subsequent microbial analysis. Total genomic DNA was extracted using the EZNA Mag-Bind Soil DNA Kit Soil DNA Kit (Omega Bio-tek, M5635-02, USA) following the manufacturer’s protocol, including mechanical lysis and proteinase K digestion to ensure maximal cellular disruption and DNA yield. DNA concentration and purity were determined using the Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific, USA) with fluorescence measurement. All DNA extracts were aliquoted and stored at -80℃ until PCR amplification.

The V3-V4 hypervariable regions of bacterial 16S rRNA genes were amplified using universal primers of 338F (5’-CCTACGGGNGGCWGCAG-3’) and 806R (5’-GACTACHVGGGTATCTAATCC-3’). PCR amplification was performed under the following conditions: initial denaturation at 94℃ for 3 min, followed by 20 cycles of denaturation (94℃, 30 s), annealing (45℃, 20 s), and extension (72℃, 30 s). An additional 20 cycles were then conducted with modified conditions: 94℃ for 20 s, 55℃ for 20 s, and 72℃ for 30 s. The reaction was completed with a final extension at 72℃ for 5 min.

PCR products were purified using the QIAquick Gel Extraction Kit (Qiagen, Germany) and quantified using the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher Scientific, USA). Purified amplicons were then submitted to paired-end sequencing on the Illumina platform (Sangon Biotech, Shanghai, China).

2.7. Statistical Analysis

Microbial community analysis was performed using QIIME and R (version 3.5.1). Alpha diversity (Shannon index) and bacterial abundance were compared across groups using Kruskal-Wallis tests, followed by pairwise Mann-Whitney U tests with false discovery rate (FDR) correction. Beta diversity was assessed using UniFrac distance metrics and visualized via principal coordinate analysis (PCoA). To identify differentially abundant taxa, we performed STAMP (version 2.1.3) and LEfSe (version 1.1.0) analysis. Microbial co-occurrence networks were constructed using SparCC (version 1.1.0), which calculates correlation coefficients and p-values between operational taxonomic units (OTUs).

All statistical analyses were performed in GraphPad Prism (version 9.0). Data are presented as mean ± standard deviation (SD). Group differences were evaluated using one-way ANOVA with post-hoc least significant difference (LSD) tests, and statistically significance was set as p-value < 0.05.

3. Results

3.1. Effects of Vanillin on Body Weight and Immune Organs in Broilers

Dietary supplementation with CTC had no significant effect on body weight gain in Kebao broilers at 21 or 42 days of age (p>0.05) (Table 2). In contrast, vanillin supplementation (0.4%) did not significantly affect the final body weight of 21-day-old broilers (p>0.05) but significantly increased the weight of 42-day-old birds (p<0.05). Notably, lower vanillin doses (0.1% and 0.2%) also showed a tendency to enhance final weight in 42-day-old broilers, suggesting a potential dose-dependent growth-promoting effect. Additionally, vanillin supplementation consistently improved average daily gain (ADG) and reduced the feed-to-gain ratio (F/G) during the early, late, and overall experimental periods, further supporting its beneficial effects on growth performance.

As shown in Table 3, dietary supplementation with 0.2% and 0.4% vanillin significantly decreased the relative pancreas weight in 21-day-old broilers compared to the control group (p<0.05). Additionally, 0.4% vanillin reduced the relative spleen weight, whereas 0.1% vanillin significantly increased the relative weights of both the Bursa of Fabricius and spleen (p<0.05). By 42 days of age, all experimental groups exhibited lower Bursa of Fabricius weights compared to 21-day-old birds. Notably, both CTC and vanillin treatments at various dosages significantly decreased the relative Bursa of Fabricius weight relative to the control group (p<0.05).

3.2. Effects of Vanillin on Serum Anti-inflammatory Factors in Broilers

Serum concentrations of inflammatory mediators are shown in Figure 1. At 21 days of age, dietary vanillin supplementation significantly reduced IL-6 levels compared to the control group (p<0.05), with the 0.1% concentration exhibiting the strongest suppression. Notably, 0.1% vanillin also significantly decreased proinflammatory cytokines (IL-1β and TNF-α; p<0.001) while increasing the anti-inflammatory cytokine IL-10. By 42 days of age, all vanillin dosages significantly modulated both pro- and anti-inflammatory cytokine expression. Specifically, 0.1% vanillin significantly reduced serum IL-6 and TNF-α levels. Compared to 21-day-old birds, all experimental groups exhibited higher IL-10 levels, with vanillin-supplemented groups showing a significantly greater increase than both the control and antibiotic-treated groups (p<0.05). These findings indicate that dietary vanillin supplementation exerts potent anti-inflammatory properties in broilers, with the 0.1% concentration demonstrating optimal efficacy across both growth stages (21 and 42 days). Notably, the anti-inflammatory effects showed progressive enhancement with prolonged supplementation, suggesting a time-dependent potentiation of vanillin’s immunomodulatory efficacy.

3.3. Effects of Vanillin on Antioxidant Factors in Broilers

Dietary vanillin supplementation significantly modulated serum antioxidant enzyme profiles (Figure 2). The 0.4% Vanillin treatment elicited a marked 35% rise in SOD activity. (p<0.001), whereas the 1% Vanillin supplementation significantly decreased MDA levels by 29% (p<0.05). By 42 days of age, all vanillin-supplemented groups exhibited significantly enhanced GSH-Px activity compared to the control group (p<0.001). Notably, the 0.1% vanillin treatment group showed the most substantial GSH-Px induction, demonstrating a dose-dependent regulation pattern in antioxidant enzyme activity.

3.4. Immunomodulatory Effects of Vanillin in Broilers

At 21 days of age, broilers receiving 0.4% vanillin dietary supplementation showed a tendency for increased serum IgA levels compared to the control group (p<0.05) (Figure 3). By day 42, the 0.4% vanillin treatment exhibited significantly higher concentrations of both IgA (23%) and IgM (83%) relative to the CTC group (p<0.05). These results suggest that dietary vanillin supplementation may enhance humoral immunity more effectively than CTC in broilers, as evidenced by its significant effect on immunoglobulin production.

3.5. Effects of Vanillin on Duodenal Morphology in Broilers

Dietary supplementation with 0.1% and 0.4% vanillin significantly reduced duodenal crypt depth (p<0.05) (Table 4). Notably, the 0.1% vanillin treatment demonstrated superior effects by increasing the villus height-to-crypt depth (VH:CD) ratio by 53% (p<0.05), suggesting improved intestinal absorptive capacity. Histopathological analysis revealed well-preserved intestinal architecture across all groups, with the exception of the 0.4% vanillin group, which exhibited mild epithelial and glandular disruption. In contrast, the 0.1% vanillin group maintained intact mucosal surfaces characterized by clearly visible folds, normal glandular morphology, and complete absence of pathological features including inflammatory infiltrates or hyperplastic changes in villi and crypts. These results indicate that 0.1% vanillin supplementation optimally enhances intestinal morphology through simultaneous enhancement of villus architecture and crypt structure while maintaining tissue homeostasis-benefits that were attenuated at higher concentrations.

Figure 4. Effect of Vanillin on Duodenal Morphology in Broilers at 42 d of Age.

Figure 4. Effect of Vanillin on Duodenal Morphology in Broilers at 42 d of Age.

3.6. Effects of Vanillin on Cecal Microbiota in Broilers

Microbial diversity analysis revealed distinct clustering patterns among treatment groups (Figure 5A). While no significant differences in Alpha diversity were observed across groups, biologically notable trends emerged. The CTC group showed reduced Alpha diversity compared to controls, whereas both 0.1% and 0.2% vanillin supplementation maintained Alpha diversity at levels comparable to the control group.

Principal component analysis (PCA) demonstrated significant microbial community restructuring (Figure 5B). The CTC-treated group formed a distinct cluster separate from controls, indicating marked microbiota alteration. In contrast, all vanillin-supplemented groups (0.1% VA-1, 0.2% VA-2, and 0.4% VA-4) exhibited microbiota profiles that clustered closely with the control group, with considerable distribution overlap. These findings highlight vanillin’s superior ability to maintain cecal microbial homeostasis compared to antibiotics. Particularly, the 0.1% vanillin concentration optimally preserved both microbial diversity and community structure, suggesting its potential as an antibiotic alternative for broiler gut health management.

No significant differences were observed in the relative abundance of bacterial taxa at the phylum level among groups (Figure 6A). Five dominant phyla consistently comprised the cecal microbiota: Bacillota, Bacteroidota, Pseudomonadota, Cyanobacteriota, and Verrucomicrobiota. The control group exhibited a typical composition of Bacillota (79.59%), Bacteroidota (18.12%), Pseudomonadota (1.91%), and Cyanobacteriota (0.16%). A similar distribution was observed in the CTC group (Bacillota: 76.00%; Bacteroidota: 19.29%; Pseudomonadota: 3.67%; Cyanobacteriota: 0.57%). Vanillin-supplementation induced dose-dependent shifts in cecal microbiota composition (Figure 6A). The VA-1 group (0.1% vanillin) maintained a profile comparable to controls (Bacillota: 79.32%; Bacteroidota: 16.15%), while higher vanillin concentrations progressively increased Pseudomonadota abundance (VA-2: 21.13%; VA-4: 23.32%) with corresponding reductions in Bacillota (VA-2: 74.90%; VA-4: 73.99%). This shift was reflected in the Firmicutes/Bacteroidota ratio, which displayed a dose-dependent decline: 4.91 (VA-1), 3.55 (VA-2), and 3.17 (VA-4), compared to 4.39 (Control) and 3.94 (CTC group). Notably, CTC supplementation significantly increased Pseudomonadota abundance, whereas 0.1% vanillin optimally enriched both Bacillota and Bacteroidota while maintaining microbial equilibrium. These findings suggest that low-dose vanillin (0.1%) more effectively modulates broiler cecal microbiota than higher vanillin concentrations or antibiotic treatment, demonstrating its potential as a targeted intervention for gut health management.

Genus-level analysis revealed 10 dominant microbial taxa, including Alistipes, unclassified_Clostridia, unclassified_Christensenellaceae, Faecalibacterium, Bacteroides, Negativibacillus, Mediterraneibacter, unclassified_Oscillospiraceae, unclassified_Ruminococcaceae, and UCG-005 (Figure 6B). Notably, the VA-1 group (0.1% vanillin) exhibited a significant enrichment of lactic acid bacteria compared to other treatment groups. While the CTC-treated (Positive) group showed significant microbial shifts in Parabacteroides and Blautia abundance (p<0.05) (Figure 6C), the VA-1 group showed a distinct probiotic profile with significantly elevated levels of beneficial genera Negativibacillus and CHKCI001 (p<0.05) (Figure 6D). These findings demonstrate that 0.1% vanillin supplementation effectively promotes the proliferation of probiotic populations while counteracting the microbiota disturbances induced by antibiotic treatment.

4. Discussion

The development and functional integrity of avian immune organs, particularly the thymus, spleen, and Bursa of Fabricius, serves as fundamental indicators of immunological competence in poultry. These primary and secondary lymphoid organs collectively mediate both cellular and humoral immune responses, playing key roles in pathogen defense and immune homeostasis maintenance. Extensive research has established a strong positive correlation between immune organ indices and overall immunocompetence in avian species (Wang et al., 2017). Previous studies have demonstrated that probiotic interventions, including Lactobacillus casei KL1 (Chen et al., 2020) and Lactobacillus plantarum (Wang et al., 2023), can significantly enhance immune organ development as measured by growth indices. Our results advance this understanding by demonstrating that dietary supplementation with 0.1% vanillin induced marked increases in organ indices for the pancreas (16%), Bursa of Fabricius (78%), and spleen (38%) in 21-day-old broilers compared to control groups (Table 3). These results suggest that vanillin may act as a potent immunomodulatory compound capable of stimulating immune organ development in poultry.

The cellular antioxidant defense system, primarily mediated by key enzymes such as SOD and GSH-Px, constitutes a critical mechanism for maintain redox homeostasis and preventing oxidative damage. This defense cascade operates through sequential reactions: SOD serves as the primary antioxidant enzyme, catalyzes the conversion of superoxide anions (O₂⁻) into molecular oxygen (O₂) and hydrogen peroxide (H₂O₂). The resulting H₂O₂ is subsequently efficiently detoxified to water through the synergistic action of GSH-Px and catalase (CAT) (Falkowskaet al., 2015). Notably, the selenium-dependent GSH-Px plays an essential role in preserving cell membrane integrity by directly reducing lipid hydroperoxides and preventing peroxidative damage to polyunsaturated fatty acids (PUFAs) (Luo et al., 2003). When oxidative stress overwhelms, accumulated O₂⁻ initiates lipid peroxidation chain reactions, producing MDA as a stable byproduct (Lin et al., 2019). As a well-established biomarker, MDA concentrations accurately quantify oxidative stress severity in biological systems (Fu et al., 2013). Our findings demonstrate that dietary vanillin supplementation significantly enhances the avian antioxidant defense system in a time-dependent manner. Specifically, GSH-Px activity increased by 28% (42 days) compared to controls (p<0.05). SOD activity rose by 35% in 21-day-old broilers; serum MDA concentrations decreased by 29% (Figure 2), indicating reduced lipid peroxidation. The results suggest that vanillin exerts its potent antioxidant effects through multiple mechanisms: i) direct enhancement of key antioxidant enzymes, ii) effective protection against membrane lipid peroxidation, and iii) maintenance of optimal cellular redox status. The demonstrated efficacy, particularly during the critical early growth phase, positions vanillin as a highly promising natural alternative to synthetic antioxidants in poultry nutrition, offering a sustainable solution for oxidative stress management during early growth phases.

The avian immune system features a sophisticated immunoglobulin network comprising five functionally distinct classes (IgG, IgM, IgA, IgE, and IgD) that work synergistically to provide comprehensive immune protection (Adaptation Physiology et al., 2018). These immunoglobulins exhibit specialized functional distribution: IgG and IgM primarily mediate systemic immunity as circulating antibodies, whereas IgA plays a crucial role in mucosal defense at epithelial surfaces. Our findings demonstrate that dietary supplementation with 0.1% vanillin significant modulated immunoglobulin profiles in 42-day-old broilers (Figure 3), including a 19% increase in serum IgA and a remarkable 56% elevation in IgM, while causing an 11% reduction in IgG levels. This distinctive modulation pattern, which parallels the effects reported for L. plantarum supplementation (Wang et al., 2023), suggests vanillin may promote a more balanced immune response by inflammatory responses. Such immunomodulatory properties could be particularly valuable in poultry production systems, as they may optimize pathogen defense mechanisms without compromising growth performance through excessive inflammatory reactions.

The inflammatory response in poultry is highly regulated by a dynamic balance between pro- and anti-inflammatory cytokines, where any disruption of this equilibrium can lead to pathological conditions. Under homeostatic conditions, a precisely controlled equilibrium maintains immune vigilance while preventing excessive activation. However, pathogenic challenges trigger a cytokine storm characterized by the overexpression of key mediators including IL-1β, IL-6, and TNF-α, the latter playing a particularly pivotal role in initiating inflammatory cascades through NF-κB activation (Wen-Jye and Wen-Chen, 2005). Such inflammatory dysregulation has been mechanistically associated with intestinal barrier dysfunction due to the disruption of tight junction proteins (Rana et al., 2009). Our findings demonstrate that vanillin supplementation exerts potent immunomodulatory effects, with the 0.1% concentration showing optimal efficacy. Specifically, pro-inflammatory cytokines (such as IL-6, IL-1β, and TNF-α) were significantly attenuated (p<0.05), while anti-inflammatory IL-10 was upregulated in 42-day-old broilers (Figure 1). These results are consistent with the established anti-inflammatory mechanisms of phytogenic compounds (such as plant essential oils and organic acids), which act through modulation of the NF-κB and MAPK pathways (Wei et al., 2021; Nie et al., 2024, Zhe et al., 2008). These findings suggest that vanillin holds promise as a natural alternative for managing inflammation in poultry production (Figure 1).

The structural integrity of the avian small intestine represents a pivotal factor governing physiological performance, as it coordinates crucial functions including nutrient absorption, immune regulation, and microbial homeostasis. Histomorphological analysis of intestinal integrity, particularly through quantitative assessment of VH and CD, provides fundamental insights into intestinal functional capacity, with the VH/CD ratio serving as an established biomarker for intestinal maturity and absorptive efficiency (Yang et al., 2019). Our results demonstrate that dietary supplementation with 0.1% vanillin significantly enhanced duodenal morphology, increasing the VH/CD ratio by 53% compared to control groups (p<0.05) (Table 4). These results are consistent with previous documenting the effects of phytogenic growth promoters (Ou et al., Yang et al., 2019, Fu et al., 2021), supporting vanillin’s role as an effective modulator of intestinal morphogenesis.

Lactic acid bacteria (LAB), particularly Lactobacillus species, have emerged as functionally versatile probiotics with demonstrated efficacy in diverse animals. These beneficial microorganisms exert multifaceted physiological effects, including the production of antimicrobial peptides, stimulation of digestive enzymes, and enhancement of growth performance through modulation of the gut microbiota. Documented benefits include improved weight gain in yaks (Wang et al., 2022), enhanced nutrient digestibility in canines (Wang et al., 2024c), and significant reinforcement of intestinal barrier function in murine models (Kim et al., 2021). In broilers, Lactobacillus supplementation has been shown to improve body weight gain, boost serum antioxidant capacity, and strength intestinal barrier integrity (Wang et al., 2024b). Equally noteworthy is Negativibacillus, a phylogenetically distinct member of Bacillota, characterized by its unique Gram-negative endospore formation. Although research on this genus remains limited, emerging evidence indicates that its abundance positively correlates with microecological complexity indices (Wang et al., 2025). Notably, increased Negativibacillus populations have been associated with greater villus height development in broilers, suggesting a potential role in intestinal morphogenesis (Wang et al., 2025).

Emerging evidence indicates that increased relative abundance of CHKCI001 in laying hen feces is significantly associated with enhanced egg production performance and improved feed conversion efficiency (Deng et al., 2022). Supporting this observation, Yang et al. (2024) demonstrated that dietary supplementation with Polygonum polysaccharides effectively elevates CHKCI001 abundance in broiler intestinal microbiota, subsequently enhancing gastrointestinal function-a finding consistent with our current resuls. Importantly, our genus-level analysis revealed that 0.1% vanillin supplementation significantly increased the relative abundance of Lactobacillus, Negativibacillus, and CHKCI001 compared to the blank control group (Figure 6). These collective findings suggest that vanillin may serve as a promising alternative to traditional prebiotics for targeted microbiome modulation in poultry production systems. Given its demonstrated effects on performance-associated microbial taxa, vanillin supplementation holds particular promise for operations aiming to reduce antibiotic reliance while maintaining optimal production metrics.

5. Conclusions

This study demonstrates that dietary vanillin supplementation significantly enhanced broiler growth performance through increased body weight. Vanillin exhibited potent immunomodulatory effects, characterized by significant downregulation of proinflammatory cytokines and upregulation of anti-inflammatory mediators. Furthermore, vanillin administration enhanced antioxidant capacity and immune function. Moreover, it improved intestinal morphology and favorably regulated gut microbiota composition. The optimal supplementation level was identified as 0.1% vanillin. These findings establish vanillin as a promising feed additive, demonstrating significant potential for enhancing sustainability in poultry production systems.