Dietary Ecliptae herba improved growth performance and meat quality possibly associated with gut microbiota and metabolomic changes in Lingshan chickens

1 Introduction

Poultry meat serves as a cornerstone of the global food supply, delivering high-quality animal protein to sustain a continuously expanding population (Chen et al., 2025). The Lingshan chicken, an economic indigenous breed native to Guangxi, China (Xu et al., 2018), is highly prized for its tender meat texture and rich flavor (Deng et al., 2025). However, compared with commercial fast-growing broilers, Lingshan chickens exhibit a prolonged growth cycle and lower feed conversion efficiency. This poses a challenge for improving growth performance and feed efficiency while maintaining their prized meat quality. Also, antibiotic growth promoters have long been used in poultry production to improve growth performance and feed efficiency. However, their widespread use has contributed to the emergence of antimicrobial resistance and raised public health concerns, leading to the progressive global ban on dietary antibiotic growth promoters (AGPs) (Abdelzaher et al., 2026; Zhang et al., 2026). Consequently, exploiting safe and effective natural feed additivessuch as E. herba has become a top priority for the sustainable development of indigenous poultry industries.

The gut microbiota plays a pivotal role in regulating host immunity, maintaining metabolic homeostasis, and suppressing excessive inflammation (Budden et al., 2024; Song et al., 2024; Xiang et al., 2026). A homeostatic intestinal microenvironment not only participates in nutrient digestion and immune regulation but also generates various bioactive substances including short-chain fatty acids, bile acid derivatives, and amino acid metabolites that systemically modulate host metabolism (Pang et al., 2025). Moreover, the composition and functional activity of the gut microbiota directly dictate feed utilization efficiency in poultry (Deng et al., 2025). Given this profound influence, targeted modulation of the microbial community structure has emerged as a key strategy to improve poultry production. Accumulating evidence indicates that natural plants and their bioactive constituents can effectively optimize gut microbiota composition (Tian et al., 2024; Xue et al., 2025), reinforce intestinal barrier integrity, and exert potent immunomodulatory, anti-inflammatory, antibacterial, and antioxidant effects (Tian et al., 2024; Wu et al., 2025; Xue et al., 2025). For instance, dietary supplementation with extracts of turmeric (Abdelzaher et al., 2026), Hypericum perforatum L. (Seraji-Kopkan et al., 2026), or Portulaca oleracea L. (Zhang et al., 2026) significantly improved growth performance and meat quality in broilers and Wenchang chickens by modulating the gut microbiota. Similarly, Zataria multiflora Boiss., Gundelia tournefortii L., and Glycyrrhiza glabra L. effectively enhanced antioxidant capacity and intestinal morphology (Abaszadeh et al., 2026). Given their high safety and multi-target regulatory properties, natural herbs such as E. herba are increasingly recognized as promising antibiotic alternatives for driving green farming systems (Xiang et al., 2025; Zhang et al., 2026).

A well-known traditional Chinese medicine, E. herba is derived from the dried aerial parts of Eclipta prostrata L (Tian et al., 2023). It possesses a diverse phytochemical profile rich in coumestans, triterpenes, thiophenes, steroids, and flavonoids (Feng et al., 2019), which underpins its extensive pharmacological repertoire, including potent immunomodulatory, antioxidant, hypolipidemic, and anti-inflammatory activities (You et al., 2015; Tian et al., 2023; Xu et al., 2025). Despite its well-documented therapeutic efficacy in human health management (Tao et al., 2022), the utilization of E. herba in livestock and poultry husbandry remains unknown, and its strategic value as a natural feed additive has yet to be fully exploited. Therefore, this study aimed to evaluate how dietary inclusion of E. herba powder at varying doses influences the growth performance, meat quality traits, gut microbiota, and metabolome of Lingshan chickens during a 49-day feeding regimen.

2 Materials and methods

2.1 Preparation of E. herba

E. herba (dried aerial parts of Eclipta prostrata L.) was purchased from Guangxi Deruntang Traditional Chinese Medicine Technology Co., Ltd. The botanical identity was confirmed by a qualified herbalist. After washing, the material was dried at 60 °C, ground, and passed through a 60-mesh sieve. The resulting powder was added to the basal diet as a top-dressing without replacing any ingredient to achieve final inclusion levels of 0.2%, 0.4%, and 0.6%.

2.2 Chickens, experimental design and bird management

Seventy-two 60-day-old healthy Lingshan chickens with similar body condition (average initial body weight: 1089.04 ± 42.61 g, range: 1000.61–1172.71 g; purchased from Guangxi Lingshan Xinfeng Poultry Breeding Co., Ltd.) were randomly allocated to four treatment groups, each consisting of three replicates with six birds per replicate. The groups were as follows: a basal diet control group (Con) and three groups fed the basal diet supplemented with 0.2%, 0.4%, or 0.6% E. herba powder (designated as 0.2% MHL, 0.4% MHL, and 0.6% MHL, respectively). The experimental period lasted 49 days.

The experiment was conducted in a closed poultry house under floor-rearing conditions. All birds had free access to feed and water, and the ambient temperature was maintained at 18-25 °C, relative humidity at 55–65%, and a 16 h light/8 h dark cycle was provided throughout the experimental period. Temperature and humidity were monitored daily using a digital thermo-hygrometer and maintained by an automatic ventilation and heating system. The composition of the basal diet is presented in Table 1. The animal experimental protocol was reviewed and approved by the Animal Experimental Ethics Committee of Guangxi Vocational University of Agriculture (Approval No. 2025064). All procedures were performed in strict accordance with the Regulations for the Administration of Affairs Concerning Experimental Animals and the institutional animal ethics guidelines of Guangxi Vocational University of Agriculture.

Table 1. Ingredient composition and calculated nutrient contents of the basal diet.

① The premix provided the following per kilogram of basal diet: vitamin A (retinyl acetate, 500,000 IU/g), 0.675 mg; vitamin D₃ (cholecalciferol, 500,000 IU/g), 0.24 mg; vitamin E (DL-α-tocopheryl acetate, 50%), 30.00 mg; vitamin K₃ (menadione sodium bisulfite, 96%), 1.25 mg; thiamine hydrochloride (vitamin B₁, 99%), 0.91 mg; riboflavin (vitamin B₂, 80%), 4.69 mg; pyridoxine hydrochloride (vitamin B₆, 99%), 2.27 mg; vitamin B₁₂ (cyanocobalamin, 1%), 1.50 mg; nicotinamide (99%), 24.24 mg; D-calcium pantothenate (99%), 7.30 mg; folic acid (99%), 0.76 mg; D-biotin (2%), 33.75 mg; Fe (ferrous sulfate monohydrate, 30%), 120.00 mg; Mn (manganese sulfate monohydrate, 31%), 154.84 mg; Zn (zinc sulfate monohydrate, 35%), 109.71 mg; I (potassium iodide, 75%), 0.47 mg; Se (sodium selenite, 45%), 0.23 mg; Co (cobalt sulfate monohydrate, 32%), 0.48 mg. Carrier (zeolite powder/rice husk powder) was added to achieve a premix inclusion of 2.00% (20.00 g/kg diet).② All nutrient levels were calculated values.

2.3 Growth performance measurement

On the mornings of day 1 and day 49, birds were weighed before feeding. Feed offered and refused was recorded daily for each replicate pen. Average daily gain (ADG), average daily feed intake (ADFI), and feed conversion ratio (FCR) were calculated as follows:

ADG (g/bird/day) = (final body weight − initial body weight)/49 days

ADFI (g/bird/day) = total feed consumed per pen/(number of birds per pen × 49 days)

FCR = ADFI/ADG

2.4 Meat quality determination

For meat quality assessment, two birds per pen were randomly selected at the end of the feeding trial, and the mean value of these two birds was used to represent the pen. The pen served as the experimental unit (n = 3 per treatment group). After slaughter, breast and thigh muscles were immediately dissected. Meat quality traits were measured as follows:

• pH: At 45–60 min postmortem, the pH of the left breast and thigh muscles was measured using a portable pH meter equipped with a metal probe. Three points were evenly distributed along the midline of the long axis of the breast muscle, and the probe was inserted approximately 1 cm beneath the muscle surface. Readings were recorded after stabilization, and the average of three measurements was used.
• Meat color: At 45–60 min postmortem, the lightness (L), redness (a), and yellowness (b*) of the left breast muscle were measured using an NH310 colorimeter. Three random locations on the medial side near the bone were selected, and the average of three measurements was recorded.
• Drip loss: Within 45 min postmortem, a muscle block of 3.0 cm × 3.0 cm × 4.0 cm was cut along the direction of the muscle fibers and weighed (W1, g). The sample was suspended in a sealed plastic bag with the muscle fibers oriented vertically and the sample not touching the bag walls, and stored at 4 °C for 24 h. After removal, the surface exudate was gently blotted with filter paper, and the sample was reweighed (W2, g). Drip loss (%) was calculated as [(W1 − W2)/W1] × 100%.
• Cooking loss: The muscle sample obtained after the 24-h drip loss measurement was weighed (M1, g), placed in a cooking bag, and a thermometer probe was inserted from top to bottom into the center of the meat block. The sample was heated in a water bath at 80.0 °C until the core temperature reached 70.0 °C. After cooling to room temperature under running water, the surface moisture was blotted dry with filter paper, and the sample was weighed (M2, g). Cooking loss (%) was calculated as [(M1 − M2)/M1] × 100%.
• Shear force: After the cooking loss measurement, meat cores of approximately 2.5–3 cm in length were cut along the direction of the muscle fibers using a circular corer, ensuring uniform thickness as much as possible. The meat core was placed in the blade slot of a tenderness analyzer, and the shear force was measured by cutting perpendicularly to the muscle fibers. The average of three measurements was recorded.

All procedures were performed in accordance with the Chinese agricultural industry standards NY/T 1333–2007 (Determination of livestock and poultry meat quality), NY/T 1180–2006 (Determination of meat tenderness—Shear force method), and NY/T 2793–2015 (Objective evaluation methods for edible quality of meat) (China MoAotPsRo, 2006; China MoAotPsRo, 2007; China MoAotPsRo, 2015).

2.5 16S rRNA sequencing and analysis

For 16S rRNA sequencing and untargeted metabolomics analyses, cecal samples were collected from the Con group and the 0.4% E. herba group (hereafter referred to as the MHL group). The 0.4% level was selected because it showed consistent improvements in growth performance and meat quality in the phenotypic trial. Six biological replicates were included per group, obtained by randomly selecting two birds from each of the three replicate pens.

After slaughter, the abdominal cavity was aseptically opened, and cecal contents were collected, snap-frozen in liquid nitrogen, and stored at −80 °C. Total genomic DNA was extracted from cecal contents using a Magnetic Stool DNA Kit (DP712, Tiangen Biotech, Beijing, China) according to the manufacturer’s instructions. A negative control (nuclease-free water) was included during both DNA extraction and PCR amplification to monitor potential contamination. DNA purity and concentration were assessed using a Qubit fluorometer (Invitrogen, USA) and agarose gel electrophoresis (Xiang et al., 2026).

The V3–V4 hypervariable region of the bacterial 16S rRNA gene was amplified with primers 341F (5′-CCTACGGGNGGCWGCAG-3′) and 805R (5′-GACTACHVGGGTATCTAATCC-3′). PCR was performed in 25 μL reactions containing 5–50 ng DNA template, 2.5 μL each primer (1 μM), 12.5 μL Phusion Hot Start Flex 2× Master Mix, and nuclease-free water to volume. The cycling conditions were: initial denaturation at 98 °C for 30 s; 32 cycles of 98 °C for 10 s, 54 °C for 30 s, and 72 °C for 45 s; and a final extension at 72 °C for 10 min. PCR products were purified with AMPure XP beads (Beckman Coulter, USA), quantified by Qubit, and evaluated using Illumina library quantification kits (Kapa Biosciences, USA). The amplicon library was paired-end sequenced (2 × 300 bp) on a DNBSEQ-G99 platform at LC-Bio Technology Co., Ltd (Hangzhou, China).

Raw paired-end reads were processed using QIIME2 (version 2025.7). Adapters and primers were removed with cutadapt, and paired reads were merged using VSEARCH. Low-quality sequences were filtered based on Q-score, and chimeric sequences were identified and removed using VSEARCH against the RDP database. DADA2 was then applied for denoising, generating amplicon sequence variants (ASVs). Taxonomy was assigned to ASVs using the Naive Bayes classifier in QIIME2 against the SILVA database (release 138.2) with a confidence threshold of 0.7. To standardize sequencing depth, the ASV table was rarefied to the minimum sample depth prior to downstream analysis. Negative controls yielded no detectable amplification products, indicating negligible contamination.

2.6 Untargeted metabolomics analysis

Cecal contents were mixed with pre-cooled 80% methanol, extracted by low-temperature ultrasonication, and incubated at −20 °C for 30 min to precipitate proteins. After centrifugation at 12,000 rpm for 5 min at 4 °C, the supernatant was dried under nitrogen flow and freeze-dried. The residue was redissolved in methanol and centrifuged, and the resulting supernatant was subjected to UPLC-HRMS analysis. A pooled quality control (QC) sample was prepared by mixing equal aliquots of all sample supernatants.

Chromatographic separation was performed on an ACQUITY UPLC HSS T3 column (100 mm × 2.1 mm, 1.8 µm, Waters) using a gradient of water (5 mmol/L ammonium acetate + 5 mmol/L acetic acid) and acetonitrile at a flow rate of 0.35 mL/min. The gradient program was as follows: 0–0.8 min, 2% B; 0.8–2.8 min, 2%–70% B; 2.8–5.0 min, 70%–90% B; 5.0–5.5 min, 90%–100% B; 5.5–7.5 min, 100% B; 7.5–7.6 min, 100%–2% B; 7.6–10.0 min, 2% B. Mass spectrometric detection was performed on a Thermo Orbitrap Exploris 120 instrument operated in both positive and negative electrospray ionization modes. Full-scan acquisition was performed at a resolution of 60,000 over m/z 70–1050, and data-dependent acquisition of the top 4 ions was performed at a resolution of 15,000.

Raw data were processed using XCMS, CAMERA, and metaX in R. Metabolites were annotated by matching accurate masses against KEGG and HMDB databases (mass tolerance < 10 ppm), and identities were further verified by isotopic distribution patterns and an in-house MS/MS spectral library. Only features with relative standard deviation (RSD) < 30% in QC samples were retained for subsequent analysis. Differential metabolites were identified using P < 0.05, fold change > 1.2, and VIP ≥ 1. KEGG pathway enrichment analysis was performed using hypergeometric tests.

2.7 Statistical analysis

Except for the omics data, all data were organized in Excel and analyzed using SPSS 26.0. Graphs were generated with GraphPad Prism 10 (GraphPad Software, San Diego, CA, USA). For growth performance and meat quality traits, the pen served as the experimental unit (n = 3 pens per treatment group); for meat quality, each pen value was derived from the mean of two randomly selected birds. For 16S rRNA sequencing and untargeted metabolomics, individual birds served as the observational unit (n = 6 per group; two birds per pen). Results are expressed as mean ± standard deviation (Mean ± SD). A one-way fixed-effects model was used with treatment as the fixed factor. Data were analyzed by one-way analysis of variance (One-way ANOVA), followed by Tukey’s HSD post hoc test for multiple comparisons among all treatment groups. A P value < 0.05 was considered statistically significant.

3 Results

3.1 Growth performance of Lingshan chickens

The growth performance of Lingshan chickens fed diets supplemented with E. herba is summarized in Table 2. No differences in average initial body weight and ADFI were observed among the groups (P > 0.05). The 0.4% and 0.6% treatment groups exhibited higher average final body weight and ADG, and lower FCR (P < 0.05). These results indicate that dietary supplementation with 0.4% and 0.6% E. herba can increase ADG and reduce FCR in Lingshan chickens.

Table 2. Effects of E. herba on growth performance of Lingshan chickens.

Data are presented as pen means ± SD (n = 3 pens per treatment group; each pen contained 6 birds). Means were compared among all treatment groups using one-way ANOVA followed by Tukey’s HSD test. Asterisks indicate significant differences versus the Con group only (*P < 0.05; **P < 0.01). ADG, average daily gain; ADFI, average daily feed intake; FCR, feed conversion ratio.

3.2 Meat quality of Lingshan chickens

The effects of dietary E. herba supplementation on the breast and thigh meat quality of Lingshan chickens are shown in Tables 3 and 4. In breast muscle, the 0.4% and 0.6% treatment reduced drip loss and cooking loss (P < 0.05), whereas L, a, b, and pH₄₅min did not differ (P* > 0.05). In thigh muscle, the same treatment increased a* and reduced drip loss and cooking loss (P < 0.05), with no differences in the remaining meat quality indicators (P > 0.05). Collectively, these findings demonstrate that 0.4% and 0.6% E. herba can effectively improve the meat quality of Lingshan chickens by reducing drip loss and cooking loss in both breast and thigh muscles, and by enhancing thigh muscle redness (a*).

Table 3. Effects of E. herba on breast meat quality of Lingshan chickens.

Values are presented as pen means ± SD (n = 3 pens per treatment group; two birds per pen were sampled and averaged for each pen). Means were compared among all treatment groups using one-way ANOVA followed by Tukey’s HSD test. Asterisks indicate significant differences versus the Con group only (* P < 0.05; ** P < 0.01). L: lightness; a: redness; b*: yellowness; pH₄₅min: pH at 45 min postmortem; Shear force (N): meat tenderness measured by Warner-Bratzler shear force in Newtons; Drip loss (%): percentage of weight loss after 24 h hanging at 4 °C; Cooking loss (%): percentage of weight loss after heating in a water bath at 80.0 °C until the core temperature reached 70.0 °C, cooling to room temperature, and blotting dry.

Table 4. Effects of E. herba on thigh meat quality of Lingshan chickens.

Values are presented as pen means ± SD (n = 3 pens per treatment group; two birds per pen were sampled and averaged for each pen). Means were compared among all treatment groups using one-way ANOVA followed by Tukey’s HSD test. Asterisks indicate significant differences versus the Con group only (*P < 0.05; **P < 0.01). L: lightness; a: redness; b*: yellowness; pH₄₅min: pH at 45 min postmortem; Shear force (N): meat tenderness measured by Warner-Bratzler shear force in Newtons; Drip loss (%): percentage of weight loss after 24 h hanging at 4 °C; Cooking loss (%): percentage of weight loss after heating in a water bath at 80.0 °C until the core temperature reached 70.0 °C, cooling to room temperature, and blotting dry.

3.3 Gut microbiota of chickens

To further characterize the regulatory effects of dietary E. herba on the gut microecology, the intestinal microbial community structure was systematically analyzed. Venn diagram analysis showed that the Con and MHL groups shared 302 core ASVs, with few unique ASVs detected in either group (Figure 1A), indicating that E. herba supplementation did not disrupt the core gut microbiota. PCoA based on Bray–Curtis distance combined with PERMANOVA confirmed that the overall microbial community structure of the MHL group was separated from that of the Con group (P < 0.05) (Figure 1B), suggesting that E. herba substantially reshaped the gut microbiota composition. At the phylum level (Figure 1C), both groups were dominated by Bacteroidota and Bacillota; MHL treatment increased the relative abundance of Synergistota while decreasing the proportion of potentially pathogenic phyla such as Campylobacterota. At the genus level (Figure 1D), MHL upregulated beneficial genera such as Bacillus and Cloacibacillus, while downregulating inflammation-associated genera such as Paraprevotella and Negativibacillus. LEfSe analysis further identified specific biomarker taxa (Figure 1E): the Con group was enriched in pathogen-related taxa represented by Helicobacter pullorum, whereas the MHL group was specifically enriched in symbiotic bacteria with anti-inflammatory and digestion-promoting functions, including Phascolarctobacterium, Synergistota, and Bacillus.

In summary, dietary E. herba optimized the gut microbial community while maintaining core microbiota stability, promoting the enrichment of beneficial taxa such as Bacillus, Phascolarctobacterium, and Synergistota, and inhibiting the colonization of Campylobacterota, Helicobacter, and inflammation-associated bacteria, thereby contributing to intestinal microecological homeostasis and probiotic effects.

3.4 Intestinal metabolic profiles of Lingshan chickens

PCA showed that samples from the Con and MHL groups formed distinct clusters (Figure 2A), indicating a clear separation of metabolic profiles between the two groups. Volcano plot analysis identified 109 up-regulated and 93 down-regulated differential metabolites (Figure 2B). The heatmap of the top 30 differential metabolites visually revealed distinct metabolite expression patterns, with color gradient differences reflecting that MHL treatment substantially altered the abundance of key metabolites such as amino acids, phenols, and terpenoids (Figure 2C). KEGG enrichment analysis showed that differential metabolites were enriched in pathways including Metabolic pathways (ko01100), Protein digestion and absorption (ko04974), Biosynthesis of amino acids (ko01230), D-Amino acid metabolism (ko00470), ABC transporters (ko02010), Mineral absorption (ko04978), Aminoacyl-tRNA biosynthesis (ko00970), and Biosynthesis of plant secondary metabolites (ko01060) (Figure 2D). GSEA further confirmed the overall upregulation of three core pathways (Figure 2E): Biosynthesis of amino acids, Protein digestion and absorption, and Biosynthesis of plant secondary metabolites. Among them, the Protein digestion and absorption pathway showed the highest enrichment score, indicating the strongest activation. The interaction network of the top 30 differential metabolites revealed extensive associations between amino acid metabolism and plant secondary metabolite-related metabolites, collectively forming a core metabolic network under E. herba regulation (Figure 2F).

These results indicate that dietary E. herba induced substantial changes in the intestinal metabolic profile, characterized by significant enrichment of the Biosynthesis of amino acids and Protein digestion and absorption pathways, as well as the Biosynthesis of plant secondary metabolites pathway.

4 Discussion

The present study demonstrates that dietary E. herba supplementation improves the growth performance and meat quality of Lingshan chickens, with the 0.4% and 0.6% inclusion levels exerting the optimal effects. Crucially, the observed improvement in growth performance was driven by enhanced feed utilization efficiency rather than increased feed intake (Dittoe et al., 2022). While ADFI remained unaltered across all groups, the FCR was significantly reduced, indicating that E. herba enhanced the intestinal capacity for nutrient digestion and absorption. This phenotypic improvement is strongly supported by the gut microbiota profiling, which revealed that E. herba enriched beneficial genera, such as Bacillus and Cloacibacillus, while suppressing inflammation-associated taxa, including Paraprevotella and Negativibacillus. Bacillus, a well-recognized probiotic (Lu et al., 2024), secretes various digestive enzymes that facilitate the degradation of dietary macromolecules, particularly proteins (Yang et al., 2021; Chen et al., 2024), whereas Cloacibacillus is closely linked to nitrogen utilization efficiency (Khan et al., 2021; Geng et al., 2022). Conversely, the downregulation of Paraprevotella and Negativibacillus, which typically proliferate during intestinal inflammation, suggests that E. herba mitigated a suboptimal gut health status (Wan et al., 2023; Hou et al., 2024). This microbial optimization was accompanied by metabolic changes, as evidenced by the enrichment of the protein digestion and absorption pathway in the KEGG analysis, with GSEA confirming the highest enrichment score among all differential pathways. Although such enrichment indicates altered metabolite profiles related to protein metabolism, it does not directly demonstrate improved protein digestion or nutrient absorption. Collectively, these observations suggest a potential association between the enrichment of beneficial bacteria and changes in the protein digestion and absorption pathway, which may be related to the improved feed efficiency observed in this study, but causal relationships remain to be established. Similar microbe–metabolite associations have been reported in poultry, where beneficial bacteria such as Bacillus were correlated with enhanced amino acid metabolism and protein utilization (Liang and Song, 2023; Qiu et al., 2025), However, direct statistical correlations between the specific bacterial taxa and the differential metabolites were not calculated in the present study, and therefore this inferred gut microbiota–metabolite relationship should be regarded as a hypothesis requiring further validation.

With regard to meat quality, the reductions in drip loss and cooking loss observed in this study may be related to the antioxidant properties of the bioactive constituents in E. herba, such as flavonoids and coumestans, which have been shown to scavenge free radicals and protect cell membranes from oxidative damage (You et al., 2015; Tian et al., 2023; Xu et al., 2025). However, oxidative stress markers and antioxidant enzyme activities in muscle were not measured in the present study. Similarly, the increased thigh muscle redness might be associated with a potential delay in myoglobin oxidation, but this interpretation remains speculative and requires direct experimental evidence (Richards, 2013; Su et al., 2024). Notably, this improvement in meat quality occurred concurrently with the enhancement of growth performance, suggesting that the active components of E. herba may simultaneously target muscle tissue and the gut microbiota, thereby synergistically elevating the overall production traits of Lingshan chickens through multiple physiological dimensions.

Nevertheless, several limitations of this study should be acknowledged, which also outline promising directions for future research. First, only three supplementation levels were evaluated. Although the 0.6% dose exerted significant beneficial effects, whether higher doses would provide additional performance gains or trigger potential adverse effects remains unclear; thus, expanding the dosage range in future studies is warranted to precisely determine the optimal inclusion level. Second, while the present study elucidated the regulatory mechanisms of E. herba at both the gut microbiota and metabolomic levels, direct mathematical or biological associations between specific bacterial genera and differential metabolites have not yet been established. The definitive causal relationships between the altered microbiota and metabolic pathways still require verification via fecal microbiota transplantation or mono-colonization experiments (Mullish et al., 2018; Ran et al., 2024). Third, only untargeted metabolomics was employed to screen differential profiles, without targeted quantitative validation of key metabolites (Zhou et al., 2025; Wang et al., 2026); consequently, the absolute concentration changes of crucial metabolites within the protein digestion and absorption and amino acid biosynthesis pathways remain to be confirmed. Finally, because E. herba possesses a complex mixture of multiple bioactive compound classes, it remains obscured whether the observed improvements in growth performance and meat quality are driven by a singular dominant monomer or by the synergistic cross-talk of multiple constituents. Future research should integrate bioassay-guided fractionation with targeted metabolomics to accurately identify key bioactive monomers, conduct absolute quantification of pathway-specific metabolites, and decipher their intricate interactions with specific gut microbes, thereby advancing the application of E. herba from empirical utilization toward a precision feeding strategy.

5 Conclusions

Dietary supplementation with E. herba effectively improved the growth performance and meat quality of Lingshan chickens, with the 0.4% and 0.6% supplementation levels showing the most pronounced effects. These improvements may be associated with changes in gut microbiota composition and metabolic pathways such as amino acid biosynthesis and protein digestion and absorption. This study provides scientific evidence supporting the potential application of E. herba as a natural feed additive in sustainable poultry production.

Correction note

This article has been corrected with minor changes. These changes do not impact the scientific content of the article.

Generative AI statement

The author(s) declared that generative AI was not used in the creation of this manuscript.

Any alternative text (alt text) provided alongside figures in this article has been generated by Frontiers with the support of artificial intelligence and reasonable efforts have been made to ensure accuracy, including review by the authors wherever possible. If you identify any issues, please contact us.

Publisher’s note

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

Footnotes

Edited by: Doctor Mziwenkosi Nhlanhla Mthiyane, North-West University, South Africa

Reviewed by: Yuanxiao Li, Henan University of Science and Technology, China

Selim Esen, Balikesir Republic of Turkey Ministry of Agriculture and Forestry, Türkiye